Analytical Methods for Glycerol

Analytical Methods for Glycerol

M. R. F. Ashworth

Organische und Instrumentelle Analytik Universitat des Saarlandes 66 Saarbriicken, Germany

Edited and with a final chapter by A. A. Newman

1 9 7 9

ACADEMIC PRESS

London New York San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

ACADEMIC PRESS INC. (LON D ON ) LTD 24/28 Oval Road

London NW1

United States edition published by ACADEMIC PRESS INC.

I l l Fifth Avenue New York, New York 10003

Copyright © 1979 by ACADEMIC PRESS INC. (LONDON) LTD

All Rights Reserved

N o part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

Library of Congress Catalog Card Number: 78-68218 ISBN: 0-12-065050-9

Printed in Great Britain by Page Bros (Norwich) Ltd, Mile Cross Lane, Norwich

Preface

Arguably, glycerol is one of the most important compounds in the list of useful chemicals, and its world production is between 300,000 and 400,000 tonnes per annum. It is a major technical raw material with literally hundreds of applications. Direct uses are, for example, as a humectant in tobacco; a component of therapeutic and cosmetic preparations; a preservative for foodstuffs; a solvent for dyes; a plasticiser for cellophane; an antifreeze agent. It is also used to prepare esters, including: "monostearate", as an emulsi-fying agent, employed with foodstuffs such as margarine and ice cream; triacetate, "acetin", used as a solvent, e.g. for film; trinitrate, "nitro-glycerine", used both medicinally and as an explosive; glycerol dichloro-hydrins, as intermediates in the preparation of epichlorohydrin; glycero-phosphate salts, used as tonics. Further, it plays an absolutely vital role in the biochemistry of most living organisms, and is listed in the pharmacopoeas of all nations.

It was felt that these varied and extensive uses justified an attempt to com-pile in detail a work devoted to the detection, identification, determination and separation of glycerol in analytical procedures. The chemical and physical information is concentrated in Chapter 1. Analytical work on the important naturally occurring and synthetic materials containing combined glycerol is treated in Chapters 2, 3 and 4, always trying to keep the glycerol moiety in mind. Chapter 5 deals with the analysis of glycerol samples, especially from the point of view of pharmacopoiea specifications. The important methods for enzymic determination of glycerol are given in Chapter 6. In this virtually complete survey of the available analytical methods, it should be possible to find a procedure best suited to any particular situation.

The literature cited in this volume covers the period up to the end of 1976.

M. R. F. Ashworth A. A. Newman

1

Glycerol

1.1. O X I D A T I O N M E T H O D S

Glycerol can be oxidised to many products, e.g. glyceraldehyde, glyceric acid, tartronic acid, dihydroxyacetone, mesoxalic acid, formic acid, and formaldehyde, and of course carbon dioxide and water. Some of these oxidations are stoichiometrically uniform, well-defined, and controllable and are thus adaptable to analytical, especially quantitative, aims.

Analytical information is classified below in alphabetical order of reagent.

1.1.1. B r o m i n e

Bromine is a classical reagent, yielding a mixture of glyceraldehyde and dihydroxyacetone, known as glycerose. Dihydroxyacetone can be dehydrated with concentrated sulphuric acid to methylglyoxal:

C H 2 O H

I CHOH

C H 2 O H l

CRO

CHOH ° > +

C H 2 O H CH2OH C H 3

' - H , 0 I c=o — ^ c = o I I

C H 2 O H CHO

These carbonyl compounds yield colours with various reagents, pre-l

2 GLYCEROL 1.1

dominantly phenols, which are the bases of detection and quantitative determinations.

Deniges (1909, 1910) tested for glycerol by heating 0-1 g or less of sample with 10 ml of 0*3 % bromine water for 20 min, then boiling out excess bromine and showing that the product(s) gave colours with reagents such as codeine, salicylic acid, resorcinol, thymol, and (3-naphthol; the first two were the most sensitive reagents. Others basing analytical methods on the blue colour yielded with codeine have been: de Coquet (1928), for wine glycerol, who also heated the sample for 20 min with bromine water on the boiling water bath and then added 10% alcoholic codeine solution and finally concen-trated sulphuric acid; Helweg-Mikkelsen (1948), with a similar procedure, boiling off the excess of bromine for 5 min and then treating an aliquot with a solution of 0-5 g of codeine in 10 ml of 96% alcohol and sulphuric acid; and, in a later publication (1949), using bromide-bromate-2N-sulphuric acid so as to give 2-5-4 mol of bromine per mol of glycerol. Ka (1940) tested the method and claimed that a bromine oxidation period of 25 min and a colour development of 20-25 min were best; excess of bromine water appeared to make no difference.

Resorcinol has been a popular reagent for demonstrating the presence of the carbonyl compounds derived from glycerol by reaction with bromine. For example, Ohl (1938) detected glycerol in textile sizing agents through the blood-red colour yielded by heating an extract with bromine water until colourless and then mixing the product with 5% alcoholic resorcinol and concentrated sulphuric acid. Cunha (1939) employed a similar procedure to estimate glycerol in wines. Jones (1947) detected glycerol in preservatives by oxidising with bromine water, boiling off the excess of reagent, cooling, and adding concentrated sulphuric acid and then 0-5% resorcinol to give a wine-red colour. Weigel (1955) also based determinations of glycerol (as a measure of the lipid fraction of neutral fats, lecithin, and cephalin) on the bromine oxidation-resorcinol reaction, quoting, however, an orange to violet product. Javicoli and Mattei (1956) detected and determined glycerol in faeces according to the same principle.

Pyrocatechol as final component was used by Ghimicescu (1935) for micro-colorimetric determination of glycerol in wine. He oxidised with bromine water in a sealed tube, removed the excess of bromine with zinc, and ultimately treated an aliquot of the resulting solution with 5% pyro-catechol solution and concentrated sulphuric acid, heating for 5 min on the water bath for colour development. Ghimicescu et al. (1963) evidently slightly modified this procedure for micro-determination in the presence of sugar and tartaric acid. After the bromination, Arreguine (1936) added concentrated sulphuric acid and 1 % alcoholic veratrole (which presumably functions like pyrocatechol, of which it is the dimethyl ether).

1.1 OXIDATION METHODS 3

Thomas and Micsa (1924) described tests for polyalcohols based on heating with bromine water, then removing the excess of bromine in a current of air and finally adding a solution of 2-hydroxynaphthalene-3,6-disulphonic acid in concentrated sulphuric acid; glycerol gave a greenish-blue colour with a yellow ring. Salzer and Weber (1950) gave this test among others for polyalcohols; they also quoted the use of guaiacol as phenol component.

Fiirst (1948a) used a reagent of 2,7-dihydroxynaphthalene in concentrated sulphuric acid as phenol reagent, obtaining a reddish-violet colour; and Bonino (1952) recorded a violet product using Bertrand's orcinol reagent after bromine oxidation of glycerol.

de Prada (1934) employed carbazole-sulphuric acid as final reagent in a colorimetric micro-determination of glycerol in beverages.

Conclusions other than observation or estimation of colour appear to be extremely rare. Juhlin (1938) determined glycerol in aqueous solutions by treating a sample containing 20-40 mg (neutralised to Methyl Orange) with 10 ml of 0-1 % bromine water for 15 min. He then determined the unreacted bromine by adding 10ml of 10% potassium iodide and 50-100 ml of water and titrating liberated iodine with thiosulphate. One molecule of bromine evidently reacted to yield one molecule of dihydroxyacetone.

Bacila (1949) detected glycerol by oxidation with bromine water to dihydroxyacetone, conversion of this into methylglyoxal with sulphuric acid, distillation of the methylglyoxal, and reaction of it with iodine-alkali to give iodoform:

C H 3 - C O — C H O + 3I 2 - • C I 3 — C O — C H O + 3HI( + OH~)

CI 3 —CO—CHO + O H " CHI3 + CHO—COO"

1.1.2. C e r i u m ( I V )

Cuthill and Atkins (1938) seem to have been the first to apply cerium(IV) oxidation to glycerol determination. They refluxed the sample for 1 h with eerie sulphate in acid solution and back-titrated unused reagent with ferrous iron to Xylene Cyanol FF indicator. They stated that 1 mol of glycerine reacted with 8 mol of cerium(IV) and presumed that tartronic acid was the end-product of oxidation. Fulmer et al (1940) used this principle of eerie sulphate oxidation for estimating glycerol in fermentation media in the presence of dextrose, back-titrating with ferrous iron to erioglaucin or phenanthroline, and determining the glucose through a separate titration with copper(II). Mull (1943) likewise determined glycerol and pentoses together by oxidation with excess eerie sulphate in sulphuric acid solution (for 45 min on the boiling water bath) and back-titrating with Mohr salt to

4 GLYCEROL 1.1

o-phenanthroline, and determined pentose alone through copper(II) titra-tion. More recently, Rao and Gopala Rao (1972) determined glycerol (also ethylene glycol or mannitol) by 45 min heating at 50-60°C with a 1 -5-2-fold excess of ammonium hexanitratocerate, ( N H 4 ) 2 [ C e ( N 0 3 ) 6 ] , in 0 5 N nitric acid and back-titrating with iron(II) to ferroin after adding sulphuric acid.

Smith and Duke (1941) studied the reaction and its stoichiometry, suggest-ing a procedure with excess ammonium hexanitratocerate in the presence of perchloric acid. They heated for 15 min at 50°C (not more than 60°C) and back-titrated with standard sodium oxalate to nitroferroin. In the presence of sulphuric acid instead of perchloric acid, temperatures of 90-100°C were necessary. They reported also a 1:8 reaction stoichiometry with formic acid as end-product, according to:

C H 2 O H

I CHOH + 3 H 2 0 + 8 C e 4 + - > 3 H C O O H + 8H + + 8Ce 3 +

I C H 2 O H

In later work (1943) they discussed reaction mechanisms for the cerium(IV) oxidation of glycerol and other polyols, comparing with periodate oxidation. Silverman (1947) determined glycerol in soap, employing oxidation for 12-13 min at 50°C with cerium(IV) in perchloric acid solution (18% in the final solution) and also back-titrating with oxalate to nitroferroin. Guardia (1950) reviewed the use of eerie perchlorate in determinations of many materials, including crude glycerol, based on the work of Smith and co-workers.

Other investigations or reviews have been made by Michalski and Stapor (1966) who compared cerium(IV) oxidation of alcohols (methanol, ethanol, ethylene glycol, glycerol, etc.) with permanganate, dichromate, and periodate oxidations, and by.Misantone (1966) who recommended back titration with ferrous iron to o-phenanthroline and found cerium(IV) to be superior to permanganate for glycerol and other materials.

Guilbault and McCurdy (1961) studied catalysis by silver(I)-manganese(II) of the oxidation of polyols with cerium(IV) and were able to suggest a much faster procedure. They heated a 3-25 mg sample with reagent in 2 0 - 6 5 % excess + catalyst + 72% perchloric acid for 3 -5 min at 95°C until the solution was red (from permanganate). The solution was then cooled immediately, 6F sulphuric acid added, and they back-titrated with standard iron(II) to ferroin.

Gordon (1951) determined unused cerium(IV) by estimating the residual amount of an oxidisable triphenylmethane dye which it destroyed. He


accomplished this by measuring the diminution in light absorbance of the dye (e.g. at 628 nm for Fast Green FCF). His procedure was empirical, requiring standard conditions because of the competition between two reactions, the one quoted above and another with only four cerium(lV) molecules:

C H 2 O H

CHOH + H 2 0 + 4 C e 4 + - ^ 2 H C H O + HCOOH + 4 C e 3 + + 4H +

I C H 2 O H

For glycerol, Gordon oxidised 1-10 jig amounts for 15 min at 165°C. Sharma and Mehrotra (1955) analysed glycerol-ethylene glycol mixtures

by using two oxidation procedures with cerium(IV). In one, formic acid was the end-product (30 min heating), and in the other, carbon dioxide and water (70 min, in presence of additional sulphuric acid and a drop of 1 % chromium(III) sulphate catalyst). Khan and Bose (1969) also touch on the subject of formic acid decomposition by more drastic treatment. They carried out two oxidations with ammonium hexanitratocerate in perchloric acid at 10°C in sun or ultraviolet light for 3h or 90 min, respectively, with reagent excesses of 100 and 1000%. They determined unused reagent iodometrically. This enabled them to determine compounds that yield formic acid on oxidation in the dark (e.g. glycerol, also ethylene glycol and methanol) in the presence of those that do not (e.g. ethanol or benzyl alcohol) by employing two sets of oxidation conditions, one with and one without photochemical decomposition.

Sand and Huber (1967) were able to titrate directly 0-5-5 mg amounts of glycerol in aqueous solution 2F in perchloric acid, using ammonium hexanitratocerate. They employed constant-current (100 |iA) potentiometry at 80°C (70°C was too low).

Knappe et al (1964) detected glycerol and other polyols in TLC by spraying with ammonium hexanitratocerate reagent in nitric acid, + Is ,iV-dimethylphenylenediamine reagent (1 + 10) or + N,N,N',AT-tetra-methyl-4,4'-diaminodiphenylmethane (tetrabase) reagent (then 1 +1), and heating at 105°C for 10 or 5 min respectively. This gave white or pale blue zones on a blue background of oxidation products of the organic bases.

1.1.3. Ch lo ramine T

Afanas'ev (1949) reported the ready reactivity of polyols with chloramine T at 80-90°C in dilute sulphuric acid, enabling a titrimetric determination to

B

6 GLYCEROL 1.1

be carried out. Glycerol reacted with 7 mol of reagent. Balwant Singh et al (1953b) published work on determinations of about a dozen compounds, including glycerol, by oxidation with excess chloramine T in acid solution to give carbon dioxide. They estimated the unused reagent by adding potassium iodide and titrating the liberated iodine with thiosulphate:

C H 2 O H

I CHOH + / 7 A r S 0 2 N C l + 3 H 2 0 - * 3 C 0 2 + 7 A r S 0 2 N H 2 + 7CT

I

C H 2 O H

1.1.4. C h r o m i u m ( V I )

Chromium(VI) as dichromate is one of the great standard oxidising agents and has been used extensively in quantitative methods for many oxidisable organic compounds, including glycerol. The comparative ease of use is neutralised by this rather too ready reaction. Oxidation of glycerol is generally to carbon dioxide, according to:

C H 2 O H

I 3 CHOH + 7 C r 2 0 . 2 ~ + 5 6 H + - * 9 C 0 2 + 4 0 H 2 O + 14Cr 3 +

I C H 2 O H

A vast amount of older literature describes the influence of factors such as the concentrations of the dichromate, sample, and acid (usually sulphuric) components of the medium, reaction temperature, and time and even order of mixing of the reaction partners. Summing up, it can be said that it is not difficult to obtain reliable results on fairly pure aqueous solutions of glycerol but that impurities and other components of glycerol-containing samples interfere through their own susceptibility to oxidation. It is not possible here to do more than give a very brief summary of the basic methods used.

Fairly concentrated sulphuric acid medium is customary. Tortelli and Ceccherelli (1913, 1914) used a 50% acid reagent, and Kellner (1922, 1924) stated that the minimum sulphuric acid concentration for complete oxidation of glycerol was about 32 % (density 1 -23). Reaction periods used to be 2-3 h with the older macro-methods but more recently 5-20 min appear more usual.

The earliest investigators, e.g. Legler (1885) and Cross and Bevan (1887), gravimetrically determined the carbon dioxide product through the increase in weight of an absorbent. This procedure was used by some later workers,


e.g. Fachini (1923) and Pramme (1931) for glycerol in greases after saponifi-cation. Neale (1926) also determined glycerol in sized cotton materials via the carbon dioxide but used a gas volumetric method.

In the classical procedure of Hehner (1887, 1889), oxidising agent was used in measured excess, and the unused was determined after complete reaction. Hehner oxidised for 2 h on the boiling water bath and then back-titrated with ferrous ammonium sulphate (Mohr salt) to an external indicator of ferri-cyanide, responding to the first excess of titrant. Others who back-titrated with a ferrous reagent include: Richardson and Jaffe (1898); Ferre and Bourges (1928) and Semichon and Flanzy (1930) for wine glycerol; Fuchs (1942) on technical products. (These investigators determined unused dichromate by adding excess ferrous reagent and completing the titration with permanga-nate); Randa (1937) on soaps and spent lyes; Procter and Gamble Co. (1937); Launer and Tomimatsu (1953) who made use of the heat of dilution of sulphuric acid; Karpov (1960) for drugs; Damyanov et al (1970) for spent lyes, lard, and crude technical glycerol, back-titrating coulometrically with ferrous iron.

Thivoile and Raveux (1941, 1942) (also Raveux, 1943) oxidised in con-centrated nitric acid and back-titrated with the less usual ferrocyanide. Erdey et al. (1955) back-titrated with ascorbic acid to Variamine Blue in their dichromate oxidation procedure for numerous compounds, including glycerol.

The other standard procedure for determining unused dichromate is to add potassium iodide and titrate the liberated iodine with thiosulphate. This was used by, for example, Steinfels (1910, 1915); Hoyt and Pemberton (1922) in the presence of sugar; Bennett (1924); and Tschirch (1951) in a semimicro adaptation of the Steinfels method.

Colorimetric and spectrophotometric procedures were introduced later. These are based on the colours and light absorbance of unused yellow dichromate and of green chromium(III) product. Johnson and Ladyn (1944) determined glycerol in kettle soap by boiling for 2 min with excess dichromate and 50% sulphuric acid and comparing the colour of the solution with those of standards from known amounts of glycerol. Englis and Wollerman (1952) oxidised for 20 min on the boiling water bath, then determined unreacted dichromate through the light absorbance at 350 nm of the 50-fold diluted solution, or alternatively, determined the chromium(III) through absorbance measurements at 587 nm of the solution 4-4N in sulphuric acid. Almost at the same time, Cardone and Compton (1952) also carried out absorbance measurements of unused dichromate from oxidations, including that of glycerol, at 349 nm and studied the effect of chromium(III) and sulphuric and phosphoric acids on the values. For glycerol, they tested oxidation periods at 100°C of 30, 60, and 120 min, finding that 30 min gave complete recovery.

8 GLYCEROL 1.1

Sargent and Rieman (1956, 1957) oxidised 1-250 |ig amounts of glycerol and other polyols for 15 min at 100°C and then evaluated the chromium(III) formed in equivalent amounts by absorbance measurements in 50% sul-phuric acid at 610 nm. They claimed that this was better than determination via unused dichromate.

A less usual method for determining unused dichromate is based on the violet colour of its complex with 1,5-diphenylcarbazide. Reese and Williams (1954) oxidised a 10 ml sample with 10 ml of 1 % potassium dichromate and 20 ml of cone, sulphuric acid for 5 min at 100°C, and then treated a 5 ml aliquot of the cooled and diluted (to 21) solution with 2.9 ml of 6 N sulphuric acid and 1 0 ml of saturated diphenylcarbazide in 95% ethanol, evaluating the complex at 540 nm within 10 min.

Bordas and de Raczkowsky (1896) directly titrated glycerol in 0 1 % sulphuric acid at the boiling point with dichromate reagent, taking as end-point the colour change from blue-green to yellow-green. This naturally demands some experience. Nicloux (1897, 1903) also conducted similar titrations of glycerol, methanol, formic acid, and formaldehyde.

Detection of glycerol through the blue-green colour yielded by oxidation with excess dichromate is not specific, as stated by, for example, Rosen-thaler (1939). The dichromate-sulphuric acid reagent has nevertheless been used, for instance by Prey et al (1962), for visualising glycerol (and ethylene glycol) on silica gel G thin layers as white spots on a yellow-brown back-ground.

Many comparative studies of glycerol methods have been undertaken, most of them to assess the relative merits of the dichromate and esterification "acetin" methods (see Section 1.2.2.A). Only a few citations from this extensive literature are made. Garrigues (1897) preferred the Hehner method for soap lyes but acetylation for crude glycerol. Tortelli and Ceccherelli (1913, 1914) preferred the Hehner procedure. Grosso (1946) stated that the determination of glycerol in industrial products was exact but tedious. Helweg-Mikkelsen (1949) preferred dichromate oxidation to acetylation, density, and refractive index determination for glycerol in galenical preparations. Habicht (1950), in a review of the dichromate and acetylation methods, improved the former by modifying the preliminary treatment.

1.1.5. C o p p e r ( l l l )

Beck (1951) titrated numerous polyols, including glycerol, with his percuprate reagent made up from cupric sulphate, potassium hydroxide, and an oxidising agent such as potassium persulphate, iodate, or periodate. These were studies, rather than quantitative determinations.

Bonner (1960) used a similar reagent, potassium periodate cuprate (KPR),


for visualisation on paper chromatograms. It yielded white spots on a brownish background with oxidisable compounds. He prepared it by adding first potassium periodate, then concentrated potassium hydroxide, to aqueous cupric sulphate. Then potassium persulphate was added at intervals, the solution boiled, cooled, decanted, and treated with additional alkali. Bourne et al. (1963) visualised carbohydrates and related compounds on paper chromatograms with this reagent. Glycerol was among the reagents.

1.1.6. G o l d ( l l l )

Srivastava and Saxena (1967) determined some glycols by boiling with excess gold trichloride and alkali for 3 h, yielding gold metal and formalde-hyde. After filtering, they estimated unused gold(III) by adding acid and a measured excess of ferrocyanide and finally back-titrating with eerie sulphate to iV-phenylanthranilic acid indicator. Their examples did not include glycerol but the method, even though not very specific, would probably be applicable to this compound also.

1.1.7. H y p o h a l i t e

Neuberg and Mandel (1916) quoted an early test for small amounts of gly-cerol (0-1-1 % solutions). They boiled with sodium hypochlorite to yield glycerose (see p. 1), then demonstrated its presence by adding concentrated hydrochloric acid and a little orcinol. The hot solution gave a violet or green-blue colour. Alber (1929) oxidised with bromine-sodium carbonate, destroyed excess bromine with sulphur dioxide after 10 min, and then showed that the reaction product(s) reduced Fehling solution.

Hypohalite has been little used in quantitative determination of glycerol. Szahlender (1933) found that many hydroxy-compounds, including glycerol, were quantitatively oxidised by sodium hypobromite in strongly alkaline solution in red light. He completed the procedure by iodometric estimation of unused hypobromite. Oxalic acid, surprisingly, was suggested as reaction product. Similar oxidation with excess sodium hypobromite and iodometric estimation of excess reagent was carried out also by Cuthill and Atkins (1938) for glycerol (90 min oxidation, converting into carbon dioxide) and by Umar and Badar-ud-Din (1966) for glycerol, nitrite, glucose, and hypo-phosphite.

Verma and Grover (1971) analysed mixtures of glycerol and dihydroxy-acetone by titration with periodate (which reacts with both) and with sodium hypobromite (which reacts only with the former).

10 GLYCEROL 1.1

1.1.8. lodate

This powerful but unspecific oxidising agent has found only limited use with glycerol. Chaumeil (1902) determined glycerol in aqueous solution by oxidation with iodic acid-sulphuric acid (approximate end-concentrations of 6 and 60% respectively), distilling the iodine formed into 20% potassium iodide solution, and then titrating with thiosulphate. The reaction equation was given as:

C H 2 O H I

5 CHOH + 7 I 2 0 5 -> 1 5 C 0 2 + 2 0 H 2 O + 7I 2

C H 2 O H

Morello (1938) described the application of Chaumeil's method to deter-mine glycerol in different types of cosmetic.

Strebinger and Streit (1924) also determined glycerol by oxidation with excess potassium iodate-conc. sulphuric acid, heating at 170°C until no more iodine vapours were evolved. However, they estimated unused iodate by adding potassium iodide and titrating the liberated iodine with thio-sulphate.

1.1.9. Iod ine

Iodine, as vapour or spray reagent in solution, is a well-known visualising agent for many compounds in chromatography. It functions more through forming loose addition products with many classes of compound rather than through any oxidative activity. However, it is convenient to mention examples of its use for polyols at this point. These are in any case rare. Salzer and Weber (1950) mention it among many other reagents for detecting polyols. Nadirov et al. (1971) used iodine vapour to detect various polyols on thin layers of mixtures with alumina (1-3 jig of glycerol); and Constantinescu and Enache (1974) quoted it (and dichromate) for visualisation of glycerol and diethylene glycol and their acetate esters on silica gel thin layers.

Iodine has been much more used to visualise glycerides (see Chapter 4, Section 4.8.2).

1.1.10. Lead( IV )

The first analytical use of lead(IV) for glycerol was evidently that of Buchanan et al (1950) who visualised carbohydrates and polyols on paper chromato-grams by several methods, including spraying with 1 % lead tetraacetate


in benzene. After allowing to evaporate at room temperature they observed white zones on a brown background of lead dioxide formed from atmos-pheric hydrolysis of unused reagent. This principle was applied by others, such as: Gross (1955) in paper electrophoresis of polyols; Michl (1955); Wright (1963) in thin layer chromatography of tobacco humectants; and Dallas and Stewart (1967) who observed pale orange-yellow on brownish-orange background.

The visualisation on paper chromatograms and electrophoregrams was claimed to be improved by following the lead tetraacetate spray with a second of rosaniline base in a solvent such as acetic acid-acetone ( 1 + 9 ) (Sampson et al., 1961). Concentrations of 003-0-05 % are used. The rosaniline is apparently oxidised by the lead dioxide to a pale grey product as back-ground, whereas it yields red spots with the aldehydes obtained by fission of the polyols with the lead(IV). BergeFson et al (1961) used this reagent com-bination in TLC of polyols, although they found an ammoniacal silver reagent to be more effective.

More recently, polyols, including glycerol, have been detected by a combined reagent of saturated lead tetraacetate in acetic acid and dichloro-fluorescein, yielding in a few minutes orange-red spots on a light background. Tanner and Duperrex (1968) applied this to detect glycerol and other polyols from beverages on thin-layer chromatograms, carrying out also estimations of the peak areas fluorescing in radiation of 350 nm wavelength. Savage and Wagstaffe (1973) similarly detected glycerol and butane-2,3-diol in products of alcoholic fermentation on thin-layer chromatograms through their fluorescence in light of this wavelength. Their method was used also by Carballido and Valdehita (1975).

Some reactive 1,2-diols have been titrated directly with a lead(IV) reagent but glycerol is not among them. Indirect titrations have been described, however. Berka et al (1962) and Zyka and Berka (1962) determined glycerol and some other compounds by reaction with excess 0-05M lead tetra-acetate and back-titrating potentiometrically with standard 0-05M hydro-quinone. The best reaction conditions for glycerol were in a final concentra-tion of 80% acetic acid containing potassium acetate and with 30 min reaction time. Evidently formaldehyde and formic acid are first formed and the latter is subsequently further oxidised to carbon dioxide. The overall equation is thus:

C H 2 O H I

CHOH + 3Pb(OCOCH 3 ) 4 + H 2 0 - * 2 H C H O + C 0 2

CH OH + 3Pb(OCOCH 3 ) 2 + 6CH3COOH

12 GLYCEROL 1.1

Berka et al (1963) applied this to determine glycerol in pharmaceutical preparations, using a reaction time of 45-50 min. In a later end-point variant, Berka and Holada (1969) carried out reaction with lead tetraacetate-acetic acid-potassium acetate for 30 min, then added solid sodium pyrophosphate, N a 4 P 2 0 7 , manganese(II) sulphate, and 4N sulphuric acid and titrated with the hydroquinone to a pink colour; they then added diphenylamine and continued titration until the violet colour changed to yellow. Presumably the manganese(II) is oxidised by unreacted lead(IV) to permanganate, then titrated with the hydroquinone.

Procedures other than back-titration of unused lead(IV) are known also. Berka (1963) determined glycerol (9 mg) among several polyols by oxidising 5 ml of solution with a mixture of 1 g of potassium acetate and 10 ml of 0 1 N lead tetraacetate in glacial acetic acid for 30 min. He determined the lead(II) reduction product by reacting with a zinc-EDTA complex and titrating the liberated zinc with 0-025M EDTA to Eriochrome Black T. Unused lead(IV) was previously hydrolysed to insoluble lead dioxide with 80 ml of boiling water, and filtered. Aleksandrov and Berka (1967) determined unused lead(IV) by this hydrolysis to colloidal lead dioxide which was estimated turbidi-metrically; and they also estimated formaldehyde reaction product from the polyols colorimetrically after reaction with chromotropic acid in sulphuric acid solution [cf. Section 1.1.14.C.2(a)].

Benson and Fletcher (1966) carried out kinetic analyses of glycol mixtures by cleavage with lead(IV) at 40 and 50°C, following the disappearance of the reagent by absorbance measurements at 320 nm; glycerol was not among their examples but application must be possible.

1.1 .11 . M a n g a n a t e

Polak et al (1962) described some oxidations with potassium manganate, including that of glycerol. This needs at least 3 h at 60°C for total oxidation to carbon dioxide and water. They treated the sample solution with alkali until it was 1-3M in alkali, then added 250 mg of manganate. After the heating period, the mixture was cooled, potassium iodide and sulphuric acid were added, and the iodine liberated by unused reagent was titrated with thiosulphate.

1.1.12. M e r c u r y ( l l )

Nessler reagent has been used to detect polyols, evidently oxidising and liberating mercury metal. Salzer and Weber (1950) mention it among the reagents for polyols tested by them but it has found little favour.


1.1.13. O x y g e n

Ravich et al (1939) determined the purity of glycerol samples by measuring the heat of combustion in a bomb with oxygen. They compared the values obtained with those from known sample amounts.

1.1.14. Per iodate

Periodate is undoubtedly the most used reagent for detection and determina-tion of glycerol. The reaction is:

C H . O H I

CHOH + 2 H K X — 2 H C H O + HCOOH + 2 H I 0 3 + H 2 0 I

C H 2 O H

All compounds containing a 1,2-diol group react. These include glycols, sugars, and sugar alcohols, some of which are often present in glycerol samples.

The reaction has been exploited analytically in several ways: detection or determination of unused periodate reagent; detection or determination of a reaction product (formic acid, formaldehyde, or iodate).

A. D E T E C T I O N O F U N U S E D P E R I O D A T E R E A G E N T

This principle utilises the oxidising properties of the reagent but it is then important to prevent oxidation by the iodate always present as a reaction product.

The best-known and most used scheme is due to Cifonelli and Smith (1954) for polyols and sugars. They sprayed paper chromatograms first with aqueous metaperiodate and, after an adequate reaction time (1-2 min for glycerol), followed with a spray of benzidine-acetone-hydrochloric acid. Unused periodate oxidises the benzidine to a blue compound as background. The polyols and sugars have reduced the periodate to iodate which is not capable of oxidising benzidine. Their positions therefore appear as colourless zones on the blue background. Many others have applied this in paper chromatography; Gordon et al (1956); Bean and Porter (1959) who immersed the paper first in saturated benzidine hydrochloride in ethanol, heated for 3-10 min at over 120°C, and then dipped into freshly prepared saturated aqueous potassium periodate-acetone (1 + 4) and allowed to dry; Lindberg and Swan (1960) in electrophoretic studies of polyols in germanate buffer; Vasyunina et al (1962); Waldi and Lange (1963); Siegel et al (1964) for polyglycerol mixtures, also estimating with the help of spot

14 GLYCEROL 1.1

areas; Cotte and Guillot (1967); Garegg and Lindstrom (1971) in paper electrophoretic studies of complex formation between diphenylboronic acid and polyols; Abdilaev et al. (1974) for polyols resulting from hydrogenolysis of glucose.

The principle was extended naturally to thin-layer chromatography also, for example: BergePson et al. (1961) on silica gel; Dyatlovitskaya et al. (1962) on cellulose; Knappe et al (1964) on various layers; Seher (1964) on kieselguhr G; Jaworski et al (1969) for glycerol in the presence of oligo-glycerols on silica gel; Ehrhardt and Sucker (1970) after separation on boric acid-buffered silica gel G; Nadirov et al (1971) on mixed layers containing alumina; and Talipov et al (1972) on Silufol U V 2 5 4 for hydrogenation pro-ducts of monosaccharides.

Bean and Porter (1959) also replaced benzidine by p-anisidine in their procedure, mentioned above, of spraying first with the amine, observing then a purple background. Knappe et al. (1964) used N,iV,iV ' ,Ar-tetramethyl-4,4'-diaminodiphenylmethane (tetrabase) as organic base, observing dirty yellow zones on dark violet. Papin and Udiman (1975) also visualised polyols (C 3 to C 7 ) using tetrabase, obtaining white zones on a sky-blue background after heating to 50°C

Periodate can be demonstrated in the presence of iodate also by treating with iodide-starch at a near neutral pH. The reaction:

IO" + 21" + 2 H + - * I 0 3 - + I 2 + H 2 0

takes place and the iodine formed yields a blue product with the starch component. The end result is again white zones of substance on a blue back-ground, as described by Metzenberg and Mitchell (1954) and Okhuma and Miyauchi (1962), for example.

B. D E T E R M I N A T I O N O F U N U S E D P E R I O D A T E R E A G E N T

Two different principles are utilised in this quantitative application of the oxidation.

1. A differential procedure, based on the reactions:

I 0 4 " + 71" + 8 H + - > 4 I 2 + 4 H 2 0

I 0 3 " + 51" + 6 H + - * 3 I 2 + 3 H 2 0

In a control without polyol sample, the periodate yields 4 mol of iodine, which is titrated with thiosulphate. The sample consumes some periodate, yielding iodate which liberates only 3 mol of iodine on subsequent addition of iodide and eventual acidification. The difference from the thiosulphate titration of the control is a measure of consumed periodate and hence of polyol.


2. A procedure based on the same principle as the visualisation method of Metzenberg and Mitchell (1954), cited above. At pH values near 7, periodate reacts with iodide to yield an equivalent amount of iodine and iodate:

I 0 4 " + 21" + 2 H + - * I 2 + I 0 3 " + H 2 0

Under these conditions, iodate liberates no iodine, so the iodine formed is a direct measure of periodate, and this circumvents the differential method.

1. Differential Method This was the basis of the first quantitative adaptation, of Malaprade (1928), who oxidised for 2-3 h at room temperature with potassium periodate (K 4 I 2 0 9 ) - su lphur ic acid, then added potassium iodide and titrated with thiosulphate. Some of the many articles describing the application of this method are: Amerine and Dietrich (1943) for glycerol in wine, titrating with arsenic(III) at the end; Reznikov and Farber (1953), using a reaction time of 10 min and final titration with thiosulphate as in all following quoted examples; Hintermaier (1955), also with 10min reaction; Teodorescu et al. (1957), modifying the method of Amerine and Dietrich by titrating with thio-sulphate; Smirnova and Eskina (1962) for glycerol in wine, again reacting for 10 min and preferring the method to chromium(VI) oxidation because alcohols and carbonyl compounds and others in the wine do not interfere; Patterson (1963) for glycerol in tobacco, oxidising for 40 min; Ghimicescu et al (1963) in a micro-determination of glycerol in the presence of sugars and tartaric acid and oxidising for 20 min; Doihara et al (1966) who examined and modified Patterson's method for glycerol in tobacco; and de Kuck et al (1967) using an oxidation period of 30 min.

2. Iodometric Back-titration at Near-neutral pH This technique has found considerable use. The first reference appears to be that of Fleury and Fatome (1935) for glycerol in aqueous solution, who oxidised at room temperature with periodic acid; after 15 min, they added sodium hydrogen carbonate, potassium iodide, and excess standard arsenite solution; after a further 15 min, they back-titrated unused arsenite with iodine. Fatome (1935) applied the procedure to determine wines and, later (1936), glycerol in galenical and apotherapic preparations. Ferre and Michel (1938) studied the method, noting that wine contains other substances that reduce periodate and suggesting a new pretreatment to ensure their more complete removal. Others applying the procedure to wines, after removal of sugars etc. are: Vasconcellos (1946) for port and lighter wines (20 min oxidation period); Thaler and Roos (1950); Bouzigues (1953) who used the Vasconcellos method. The method has been applied to other samples also, for example, by: Voris et al (1940) in blood, oxidising for 20-30 min; Bruening

16 GLYCEROL 1.1

(1946) in vanishing creams; Polak and Wilkosz (1959) in the presence of aniline; Novak (1960) in stills; Paulssen and Waaler (1962) in injection solutions (10 min oxidation); Hromatka and Stainer (1962) in vinegar mash and vinegars; Khadeev and Mukhamedzhanova (1968) who determined glycerol (also mannitol and threonine) by 50-60 min oxidation with periodate, then adding sodium hydrogen carbonate and potassium iodide and back-titrating amperometrically with standard arsenite. Hartman (1960) employed a 30 min reaction time and buffered with borate instead of the otherwise almost universally used sodium hydrogen carbonate.

3. Other Determinations of Unused Periodate Jankauskas and Norkus (1971) oxidised polyols with excess sodium periodate in 0-1-1N sulphuric acid for 2-3 min, then added alkali to yield a 0-2-1N solution; after adding 3-4 drops of 0-2% potassium ruthenate catalyst they back-titrated unused periodate potentiometrically with arsenite.

Linevich et al (1972) based their automatic recording of elution curves of polyols (e.g. glycerol, mannitol) in gel chromatography on Sephadex G-10 on oxidation of the effluent with periodate and determination of its absorbance at 222*5 nm in a flow-through cuvette.

An acidimetric method was used by Dal Nogare and Oemler (1952). They oxidised a 50 ml sample with 50 ml of 0-1M sodium periodate for 20 min and then determined unused periodate by titrating sodium paraperiodate to disodium paraperiodate with 0-1N sodium hydroxide:

N a I 0 4 + 2 H 2 0 —• N a H 4 I 0 6 (sodium paraperiodate)

N a H 4 I 0 6 + N a O H - * N a 2 H 3 I 0 6 + H 2 0

The end-point of this titration corresponds to an inflection at pH 9-7, rendered sharper by cooling to 0°C. They employed a mixed indicator of thymolphthalein and 1-naphtholbenzein with colour change from yellow-orange to greyish-blue. Glycerol of course yields formic acid in the oxidation; they allowed for this by oxidising a separate 50 ml aliquot with periodate, removing unused reagent with ethylene glycol, and titrating at room temperature with sodium hydroxide to Methyl Red and subtracting the value.

Bark et al (1976) titrated sorbitol in dilute sulphuric acid with 0-4N sodium periodate in 0-01M sulphuric acid using thermometric end-point indication. Application of this direct titration to glycerol may be possible.

C. D E T E C T I O N A N D D E T E R M I N A T I O N O F A R E A C T I O N P R O D U C T

Four reaction products, formaldehyde, formic acid, iodate, and water, can theoretically be detected or determined in analytical methods for glycerol.


There appears to be no example of water being detected or determined but the other three, especially the two organic products, have been the target of many analytical applications.

1. Formic Acid Detection of glycerol via the periodate oxidation product, formic acid, has not been often used. Frehden and Furst (1939) published spot tests for phar-maceuticals and detected polyhydroxy compounds by oxidising with excess periodic acid to formic acid which they then oxidised further to carbon dioxide using bromine. They demonstrated the presence of the carbon dioxide through the turbidity yielded with barium hydroxide solution and were thus able to detect down to 2-5 |ig of glycerol. Feigl (1966d) quoted this method.

Orchin (1943) detected glycerol by mixing 5 ml of sample, rendered just alkaline to Methyl Red, with 5 ml of ~ 0-05N periodic acid, likewise just alkaline to Methyl Red; an immediate colour change to red showed the formation of formic acid.

A paper chromatographic visualisation due to Buchanan et al. (1950) also evidently depends on formic acid formation. They sprayed the dried chromatogram with 2 % aqueous sodium metaperiodate, then, after 7 min, removed unused periodate with 10% aqueous ethylene glycol and finally added 5% potassium iodide. Triols yielded blue spots, presumably due to the action of the formic acid on the mixture of iodide and iodate (from original periodate oxidation):

51" + I 0 3 " + 6 H + -> 3I 2 + 3 H 2 0

Formic acid has been determined in many analytical methods for glycerol, especially in the presence of diols which, of course, do not yield this product.

Malaprade (1937) introduced the method, using a sample brought just to the colour change of Methyl Red and allowing to react for 20 min with excess sodium periodate. After adding cone, potassium nitrate to precipitate unused periodate (which otherwise interferes) he titrated the formic acid with alkali. He also used solid potassium periodate as reagent, shaking for 20 min and then titrating formic acid as before. Neither ethanol nor ethylene glycol interfered. Other subsequent investigators also used Methyl Red, e.g. Bradford et al. (1942), and in a number of determinations of glycerol in the presence of diols, such as: Allen et al. (1940) (in the presence of ethylene and diethylene glycols); Hoepe and Treadwell (1942) (of ethylene and propylene glycols); Shupe (1943) (of ethylene and propylene glycols in cosmetics, as done also by Bruening, 1946 and 1947, in collaborative studies); Hoepe (1943) (of butane-2,3-diol); Pohle and Mehlenbacher (1947) (of propylene and trimethylene glycols); Bruening (1950) (glycerol as impurity in propylene

18 GLYCEROL 1.1

glycol from vanilla extracts); Griffin (1954) (of propylene glycol in desiccated coconut); Mathers and Pro (1954) (of propylene glycol in foods and medici-nals); and Janowska (1968) (of methyl oleate and hydrogenated palm oil).

Glycerol in other materials was determined according to this principle also by Keppel (1949, 1953) for added glycerol in egg and egg products; and Roussos (1964) (glycerol from hydrolysed sucroglycerols).

Newburger and Bruening (1947) preferred Bromocresol Purple or p-Chlo-rophenol Red, and gave an improved procedure using the former and also employing propylene glycol to destroy unused periodate before alkali titration; they used a reaction time of 1 h. Bond (1949) confirmed this in glycerol determinations in the presence of ethylene and propylene glycols in a clear-type skin lotion. Colson (1950,1951) too used propylene glycol and Bromocresol Purple, as did Hintermaier (1955) who oxidised with periodate for only 5 min. Ludwicki and Sobiczewska (1963), in determinations of glycerol in pharmaceutical preparations such as suppositories, ointments, and extracts, also reacted for 5 min and employed Bromocresol Purple.

On the other hand, Erskine et al (1953) used Phenol Red as indicator and, after 30 min oxidation in the dark, destroyed unused periodate with ethylene glycol before final titration with sodium hydroxide in a nitrogen atmosphere. Sporek and Williams (1954) determined glycerol in fermentation solutions after column chromatographic separation, by oxidation for 7 min with neutral potassium periodate, removing unused reagent also with ethylene glycol, and titrating like Erskine et al to Phenol Red. Lloyd (1962) used their method to determine glycerol in fruit pastilles. The method of Erskine et al was used by Lazarus and Newlove (1955) to analyse mixtures of glycerol and trimethylene glycol.

Pohle and Mehlenbacher (1947) concluded with potentiometric titration, as did Golova et al (1974) (to pH 7-4) in analyses of the products of reaction of allyl alcohol and peracetic acid.

Hartman (1956) adopted a slightly different procedure, determining the formic acid through the iodine that it liberates from iodide-iodate mixtures (corresponding to the visualisation method of Buchanan et al, mentioned early in this section). He treated the neutral sample (about 1 g of glycerol) with a measured amount of formic acid (5 ml of 1 % solution), made up to 250 ml, and oxidised a 100 ml aliquot for 10 min with 50 ml of 6% sodium periodate. He then reduced unreacted periodate with 10 ml of 50% propylene glycol and, after 5 min, added 25 ml of 15 % potassium iodide and 50 ml of 0 1 N sodium thiosulphate. Evidently, the iodine, liberated from the acid-iodide-iodate mixture, consumes some of the thiosulphate, which is measured by back-titration with 0-1N iodine. A blank titration without sample gave a correction for the deliberately added formic acid (to accelerate iodine development from iodide-iodate).


In more recent years, small discrepancies in results focused attention on possible losses of the relatively volatile formic acid which would yield low figures. These losses would be accentuated by the heat developed in the exo-thermic reaction between glycerol and periodate (temperature rises of several degrees). Antonin (1967) determined the glycerol content of technical grade glycerols and observed that use of a diluted sample reduces this temperature rise and hence the partial pressure of the formic acid. He stated that reliable results are obtained only if the final pH of sample and blank is 8 1 . Mormont and co-workers (Mormont and Gillet, 1967; Mormont et a/., 1969; Mormont, 1971,1972) devoted particular attention to this problem, suggesting addition of sodium formate to both test solution and blank, and titrating the former to end pH 8 and the latter to pH 6-5. This was because the blank contains only strong acids. Barbour and Devine (1971) say that Mormont's procedure needs further testing but they appear to be in general agreement. They also tried to limit loss of formic acid by sealing the apparatus and cooling with tap water during the reaction.

Determination via the formic acid product is the basis of glycerol assay in many world pharmacopoeias (see Chapter 5).

2. Formaldehyde Many procedures are known for detecting and determining formaldehyde quantitatively and some of these have been used as the concluding stage for glycerol and other polyols after prior oxidation with periodate. This principle is less specific than that via formic acid since 1,2-glycols also yield form-aldehyde. The information is classified according to the reagent used with the formaldehyde.

(a) With chromotropic acid. This reagent, l,8-dihydroxynaphthalene-3,6-disulphonic acid, is the most used in formaldehyde determination as the last stage in glycerol estimation. This includes also glycerol derived from com-bined glycerol, e.g. in glycerides (see Chapter 3, Section 3.1.1.B.2). The probable reaction is formation of a hydroxydiphenylmethane derivative:

OH OH

+ HCHO

j ^ S 0 3 H

CH

H 0 3 S

Absorption maximum ca. 570 nm

SO3H HO3S SO3H

O OH

20 GLYCEROL 1.1

The water elimination is accomplished by sulphuric acid in which the chromotropic acid is dissolved.

Lambert and Neish (1950) described a method for rapid determination of glycerol in fermentation solutions, in the presence of sugars and butane-2,3-diol. They carried out quantitative oxidation of 0-2-0-8 mg of glycerol in 20 ml of water with 5 ml of 0-1M sodium periodate + 1 ml of 10N sulphuric acid for 5 min and then removed unused oxidation agent by reaction for 5-10 min with a large excess of arsenic(III) (5 ml of 1M reagent). After adding 10 ml of a 0-2 % solution of chromotropic acid in ca. 20N sulphuric acid to an aliquot (1 ml from 200) of the reaction mixture, they heated for 30 min at 100°C and evaluated the coloured solution at 570 nm. The influence of glucose was stated to be minimised by the short oxidation time. Neish (1950) applied the method to glycerol after separation from other materials by column chromatography. Some others employing this principle with reduction of iodate and unused periodate by arsenic(III) are: Henry (1957) who used Neish's method for determining glycerol and propylene glycol in nuts; Vecher and Ulitina (1958) for glycerol and butane-2,3-diol; Biesold and Strack (1958) for glycerol in blood; Jackson and Ramamurti (1958) for micro-determination of glycerol in Acetobacter culture media, first destroying metabolites with cerium(IV) and finally evaluating at 580 nm; Bergner and Meyer (1960) for glycerol in wines and juices, after separation by ascending PC; Mizsei et al (1964) for glycerol in fermentation liquors, evaluating at 580 nm and removing interfering sugar alcohols beforehand; Caruso and Falanghe (1968), using Lambert and Neish's method for glycerol and other polyols in the presence of fructose, for which they applied a correction. Levy and McGee (1964) found that protein interfered with the determination, probably binding some of the formaldehyde. Carballido and Valdehita (1975) determined polyols (C 3 to C 7 ) after TLC separation, by oxidation with periodate, removal of oxidising agent, and then colour reaction with chromotropic acid, evaluating at 540 nm.

Peynaud and Charpentie (1954) determined glycerol in musts and wines by a similar procedure, oxidising with periodate for 15 min but then removing iodate and periodate with sulphur dioxide; they used 0-25% chromotropic acid in cone, sulphuric acid and evaluated the violet colour at 540 nm. Smullin et al (1958) reduced with stannous chloride in determination of polyols, and measured final absorbance at 570 nm. Another reducing agent for iodate and periodate removal is ascorbic acid, used by Loseva et al (1970) in a modification of the method of Neish.

Numerous compounds yield formaldehyde by oxidation or hydrolysis, and Gronsberg (1969) studied the determination of some via the colour reaction with chromotropic acid; his examples included glycerol.

Sawicki et al (1967) mention the use of another hydroxynaphthalene-


sulphonic acid to estimate formaldehyde derived from periodate oxidation of various compounds, including glycerol, in their determination. This reagent, J acid (6-amino-l-hydroxynaphthalene-3-sulphonic acid) was used in 001 % solution in cone, sulphuric acid. The aqueous sample (1 ml) was oxidised for 15 min with 0-2 ml of 0*5% periodic acid in water-sulphuric acid (2*50 + 1), then treated with excess arsenite (0-4 ml of 2 % solution) in hydrochloric acid-water (1 + 20). After 2 min, 5 ml of the reagent solution was added and the mixture heated for 5 min at 100°C before cooling in ice. The fluorescence at 520 nm (excitation at 462 nm) was evaluated 10-25 min later.

(b) With phenylhydrazine-oxidising agent. The use of phenylhydrazine for colorimetric determination of formaldehyde dates back to the work of Schryver (1910). Part of the reagent condenses with the aldehyde to yield a hydrazone, and another part undergoes oxidation with an extra component of the reagent mixture to give a diazonium salt:

C 6 H 5 N H N H 2 + HCHO C 6 H 5 N H N = C H 2 + H 2 0

C 6 H 5 N H N H 2 ^ [ C 6 H 5 N = N ] + + 3 H + + 4e

The diazonium salt couples with the hydrazone to give a coloured formazan:

C 6 H 5 N H — N

[ C 6 H 5 N = N ] + + C 6 H 5 N H N = C H 2 C H + H +

/ C 6 H 5 N = N

This coloured product has generally been evaluated spectrophotometrically at 520-530 nm. The customary oxidising agent is ferricyanide.

Ramsay and Stewart (1941) applied the method to determine glycerol in studies of phospholipids. Desnuelle and Naudet (1945) determined glycerol, ethylene glycol, and propylene glycol. Bailey (1959) adapted the method to a micro-determination of glycerol (also mannitol and sorbitol) in biological fluids, destroying excess periodate and iodate with arsenite before carrying out the reaction of colour development. Marshev (1964) based his micro-method on that of Bailey, modifying to ensure complete protein precipitation from biological samples, such as blood, and to find the best temperature for oxidation and colorimetry. Laforest and Combrisson (1968) also determined glycerol and sorbitol in blood in this way, likewise removing iodate and periodate with arsenic(III). Isai and Vas'kovskii (1969) applied the method to polyalcohol determination.

Examples of the application of this principle to determine combined glycerol in glycerides, etc. are given in Chapter 3 (Section 3.1.1.B).

22 GLYCEROL 1.1

(c) With Schiff reagent The colour reaction with the classical Schiff reagent has been used to visualise polyols, including glycerol, after oxidation with periodate. Buchanan et al. (1950) sprayed paper chromatograms with 2% aqueous sodium metaperiodate and allowed them to stand for 7 min at 60°C in a nitrogen atmosphere. After passing through sulphur dioxide to remove iodine, they sprayed with Schiff reagent (2 % rosaniline, decolorised with sulphur dioxide and diluted). Coloured zones were observed after 3 h although acceleration was possible by heating to 60°C. Feigl (1966d) suggested a similarly functioning spot test, allowing 1 drop of sample to react for 5 min with 1 drop of 5% potassium periodate and 1 drop of 10% sulphuric acid, then reducing unused periodate with saturated sulphurous acid and adding Schiff reagent which gave red to blue colours within a few minutes; this enabled down to 2-5 pg of glycerol to be detected.

Conacher and Rees (1966) visualised ethylene and propylene glycols and glycerol in TLC also with the help of periodate oxidation and subsequent treatment with Schiff reagent to yield dark purple spots.

(d) With sulphite or hydrogen sulphite. The formation of a bisulphite com-pound is the basis of a quantitative aldehyde determination and has been applied to determine formaldehyde resulting from the periodate oxidation of glycerol. Elving et al. (1948) distilled pretreated fermentation residues with ca. 0-5N periodic acid and collected formaldehyde from glycerol oxidation. After bringing the distillate to pH 9-3 (pH-meter) they added 12% sodium sulphite solution:

H

HCHO — S 0 3

2 " + H 2 0 - * H C — S 0 3 " + OH"

OH

The liberated alkali was titrated with 0 1 N hydrochloric acid to the original pH. Peynaud (1948) also employed a similar method to determine glycerol in wine.

Maros and Schulek (1959,1960) based their determinations of polyols and sugars on the sulphite procedure. After periodate oxidation they added sodium sulphite to destroy unused reagent as seen by the discharge of the yellow-brown iodine colour. They then added further sulphite to react with the formaldehyde, performing this under a pentane layer to limit atmos-pheric oxidation. After 10 min, they removed excess sulphite with iodine and decomposed the bisulphite compound by adding potassium cyanide or

OXIDATION METHODS 23

hydroxylamine in alkaline solution:

H C — S 0 3 - + C N " + HC—CN + S 0 3

2 "

OH OH

H

H C — S O 3 - + N H 2 O H ^ H C = N O H + H 2 0 + H S 0 3 "

I OH

Finally, they titrated the liberated sulphite or hydrogen sulphite with standard iodine.

(e) With dimedone (5,5-dimethylcyclohexane-l93-dione). As well as deter-mining glycerol through periodate oxidation and titration of the formic acid formed and through estimation of unused oxidising agent, Mead and Bartron (1948) determined the formaldehyde from 20 min oxidation of ca. 10 ~ 4 mol of sample with about 10% excess of saturated aqueous potassium periodate. They then added a borate buffer and potassium iodide, and titrated the iodine with arsenite. They added a 10% excess of dimedone in alcohol at pH 4, warmed to 60°C, and cooled in ice for complete precipitation. The precipitate was collected and weighed.

Separation as a dimedone derivative was used also by Rauschenbach and Lamprecht (1966) to isolate formaldehyde from periodate cleavage of glycerol containing 1 4 C .

(f) With 3-methylbenzothiazolin-2-one hydrazone (MBTH). Pays et al. (1967) and Sawicki et al. (1967) used the MBTH reagent to determine formaldehyde from polyol oxidation. They removed unused oxidising agent with arsenic(III), then treated with reagent and ferric chloride and evaluated the coloured product, formed according to the reactions:

1.1

GLYCEROL 1.1

followed by a coupling oxidative reaction between these two compounds to

Pays et al. used 2-76% MBTH in 0-1N hydrochloric acid and 5% ferric chloride in water, and left the reaction mixture for 30 min before extracting with ether and evaluating the aqueous layer at 630 or 660 nm. Sawicki et al. treated 1-6 ml of reaction mixture with 1ml of 0-8% aqueous reagent, heating for 3 min on a boiling water bath, then adding 2 ml of 0-5 % aqueous ferric chloride. After 15-25 min they read the absorbance at 654 nm.

This method has been used also to determine glycerol from glycerides (see Chapter 3, Section 3.1.1.B.4).

(g) With phloroglucinol. Rebelein (1957) determined glycerol in wine by periodate oxidation to formaldehyde, then estimated colorimetrically through reaction with phloroglucinol-sodium hydroxide. Pure solutions gave good results but correction was needed for losses due to sugar precipi-tation with barium hydroxide from the sample. Acids such as tartaric, citric, lactic, succinic, and malic did not influence the results.

(h) With silver. Aldehydes reduce silver reagents to silver metal, a property that has had a vast analytical application. It appears, however, to have been seldom used to detect formaldehyde from oxidation of polyols. Yamada et al. (1975) detected non-reducing saccharides (glycerol, erythritol, maltitol, etc.) in paper chromatography by spraying the dry chromatogram with 1 % sodium periodate in 50% acetone and, after 2 min, with saturated silver nitrate-95% acetone (1 + 100). This yielded bleached spots on a pale yellow background. Spraying with 1 % sodium hydroxide after 5 min gave black spots, presumably of silver metal. The authors then bleached the background by dipping in 5% sodium thiosulphate. They stated that this periodate-silver ion combination had not been previously reported and that more compact spots were obtained with periodate solution in acetone.

(i) With acety lace tone-ammonium salt. Formaldehyde reacts with acetyl-acetone in the presence of ammonium salts to yield 3,5-diacetyl-l,4-dihydro-lutidine which has an absorption maximum in the 400-420 nm region and

give:

C = N — N = C H—N=N—C

N H 3 H

CH—CO CO—CH 3 - 3 H 2 0

CH 3 CO—CH 2

0 = C H ,

24


also fluoresces, so quantitative determination is possible (Nash, 1953). This principle has been widely applied to determine glycerol after prior oxidation with periodate but almost all examples are of determination of glycerol derived from glycerides, which are described with fuller detail in Chapter 3 (Section 3.1.1.B.3). de Freitas (1967) applied the method to deter-mine 0-05-5 jig amounts of glycerol (and glucose) in plasma. After PC he oxidised with periodate and reacted with ammoniacal acetylacetone, estimating the dihydrolutidine fluorometrically

(j) With permanganate. Borisovich et al. (1970) detected glycerol and other polyols after TLC on alumina by allowing to react for 6-7 min with 1 % sodium periodate and then treating with 1 % potassium permanganate. This yielded bright yellow zones on a pink background, presumably due to reduction of the permanganate with formaldehyde (and formic acid).

(k) Using polarography. One of the methods of Elving et al. (1948) for determining glycerol in fermentation residues was based on polarography of the formaldehyde yielded in a distillate obtained after periodate oxidation [see Section (d)]. They used a 0 1 N lithium hydroxide-001N lithium chloride mixture as supporting electrolyte and measured wave heights at —1.40 and —1.63 V (vs. mercury pool anode). The difference between these wave height values was proportional to formaldehyde concentration.

Jaworski et al. (1969) also determined glycerol in the presence of oligo-glycerols by thin-layer chromatography, followed by polarography between — 1.4 and — 2 V in lithium hydroxide solution containing gelatin.

Orlova et al. (1976) oxidised glycerol with 0-035M periodic acid and then determined the formaldehyde (also iodic acid, see below) by oscillopolaro-graphy, measuring the height of the reduction current peak for — 1-7 V (vs. SCE). This was rectilinearly related to the glycerol concentration in 1M lithium hydroxide. Unreacted periodate was precipitated as lithium salt.

3. Iodate The visualisation procedure of Buchanan et al. (1950) and the quantitative determination of Hartman (1956) depend on formic acid formation and also on the presence of iodate reaction product. They have been described in Section 1. Buchanan et al. (1950) visualised triols as blue spots, yielded after oxidation with neutral periodate and subsequent addition of iodide. The formic acid-iodate mixture is reduced by this iodide to give iodine:

6 H + + I 0 3 " + 51" - * 3 I 2 + 3 H 2 0

In Hartman's method for glycerol, the iodine is titrated with thiosulphate. Burnel et al. (1971) determined glycerol and some other compounds by

oxidation with dipotassium paraperiodate (K 2 H 3 I0 6 ) - su lphur ic acid for 30 min and then iodometric estimation of the iodate formed. To prevent

26 GLYCEROL 1.1

interference by residual periodate, they masked it with a 30-fold excess of molybdate, as sodium salt in a trichloroacetate buffer of pH 3.

The differing solubilities in water of silver iodate and silver periodate have been utilised analytically. The former has a solubility at 10°C of only 0 003 g per 100 ml of water. Feigl (1966a) quotes a spot test for detecting polyols and other compounds which are oxidised by periodate. It depends on precipita-tion of silver iodate. He used a reagent made up from 2 ml of cone, nitric acid, 2 ml of 10% silver nitrate, and 25 ml of 2% potassium periodate; silver periodate is not precipitated from this solution. On mixing 1 drop of test solution (in water or dioxan) with 1 drop of reagent, a white or yellow turbidity (of silver iodate) is the positive response. Down to 2-5 \ig of glycerol can be detected.

Pesez (1956) published a quantitative procedure based on this. His reagent consisted of 4-5 g of potassium periodate in 500 ml of water, to which were added 50 ml of nitric acid (d 1 -33) and 250 ml of 0-1N silver nitrate, the whole being made up to 1 litre. Aqueous samples of polyols (including glycerol) and other compounds were allowed to react with this reagent at room temperature for times ranging from 10 min to 24 h (30 min for glycerol). He then filtered off silver iodate and back-titrated unused silver ion in the filtrate with thiocyanate. This enabled him to estimate the amount of iodate formed and hence of original polyol.

Oles and Siggia (1974) determined |imol amounts of polyols by oxidation for 10-30 min with periodic acid, adding nitric acid and precipitating iodate with silver nitrate. They filtered the precipitate at —10 to — 15°C to minimise its solubility, dissolved it in ammonium hydroxide, and determined the silver content through atomic absorption.

Orlova et al (1976) determined glycerol by periodate oxidation and oscillopolarographic evaluation of iodic acid through the height of the reduction current peak at — 1 -25 V (vs. SCE). Reaction and polarographic conditions were the same as those quoted above in Section 2(k) for form-aldehyde evaluation according to the same principle.

1.1.15. Permanganate

This classical oxidising agent has been used both for detection and for quantitative determination of glycerol and other polyols.

Permanganate can be used in either acid or alkaline solution and, as expected, various reaction products result. Total oxidation to carbon dioxide and water demands 7 atoms of oxygen per molecule of glycerol:

C 3 H 8 0 3 + 7 0 3 C 0 2 + 4 H 2 0


It appears possible, however, to obtain organic reaction products, for example:

C H 2 O H | COOH

CHOH + 6 0 - H + C 0 2 + 3 H 2 0 | COOH

C H 2 O H

in alkaline solution. Acetic acid and formaldehyde can also be end-products. The permanganate can yield manganate, manganese dioxide, and manganous salts.

A. D E T E C T I O N

Qualitative oxidation with permanganate usually gives decoloration (in acid solution) or brown manganese dioxide (in alkaline solution). This is, of course, not specific for polyalcohols. It has, however, been used in visualisa-tion on chromatograms, generally in neutral or alkaline solution. Pale spots on a purple background are usually obtained, the purple changing gradually into brown. Sometimes, unused permanganate reagent is carefully removed by washing, so that the pale or brownish spots appear on an almost colourless background. Some examples may be quoted.

Gordon et al (1956) visualised carbohydrates and related compounds on paper chromatograms using neutral 0-01M permanganate, obtaining pale spots on a mauve background. Hay et al (1963) used 0-5% potassium permanganate in IN sodium hydroxide, heating for 30-120 s at 100°C for detecting sugars and especially polyalcohols on silica gel G layers. These appeared as bright yellow spots on purple, fading on standing to white zones on brown. Grasshof (1963) used a 1 % potassium permanganate spray and obtained light spots on violet. Knappe et al (1964) visualised many polyols with a permanganate-sodium carbonate reagent which gave yellow zones on a pink background Dallas and Stewart (1967) visualised glycerol and poly-glycerols on mixed thin layers of kieselguhr G and silica gel, containing calcium chloride, by spraying with 0 1 6 % potassium permanganate in acetone; this gave yellow spots on a mauve background. De Simone and Vicedomini (1968) used a spray of potassium permanganate and sodium carbonate to detect polyols on silica gel layers impregnated with lead nitrate. Glycerol and also mannitol, glucose, and maltose were visualised on silica gel G layers with 0-35% potassium permanganate in I N potassium hydroxide by Tateo (1970). Knappe et al (1964) detected polyols in TLC by spraying with alkaline permanganate and then immediately with iV,iV-dimethyl-p-phenylenediamine, subsequently heating at 105°C for 5 min to obtain yellow zones on the dark violet oxidation product of the amine.

28 GLYCEROL 1.1

Mixed periodate-permanganate reagents were used by Lemieux and Bauer (1954) and Bergel'son et al. (1961) for polyols on paper chromatograms. The former authors' reagent was 2% aq. sodium periodate-1 % potassium permanganate in 2% aq. sodium carbonate (1+4). After leaving at room temperature and then washing under the tap they obtained brown spots on an almost white background.

Rather more specific tests are based on oxidation to formaldehyde, which is then detected. An early example is due to Kolthoff (1924) who treated the sample for 10 min with 4N phosphoric acid and 3 % potassium perman-ganate. After adding acetic and dilute sulphuric acids he then tested with the Schiff reagent which demonstrated through a reddish-violet colour that formaldehyde from glycerol oxidation was present. Feigl (1966e) mixed 1 drop of sample, of 5 % phosphoric acid, and of 5 % potassium permanganate. After 1 min, the solution was decolorised with sodium hydrogen sulphite and then 4 ml of 12N sulphuric acid and a little powdered chromotropic acid were added. Formaldehyde was shown by a yellow solution fluorescing green.

B. D E T E R M I N A T I O N

Quantitative determination of glycerol has been achieved by evaluating permanganate consumption, or one of the reaction products, oxalic acid, formaldehyde, or manganese dioxide.

1. Evaluation of Reagent Consumption The standard procedure in former times was oxidation with a measured excess of reagent and back-titration of the unused amount. Lalieu (1881), for example, used alkaline conditions (ca. 4% potassium hydroxide) and allowed the sample to stand for 24 h with excess IN potassium permanganate. After acidification he back-titrated with oxalic acid. Although Lenz (1885) stated that the results were highly inaccurate and depended on the amount of reagent in excess, similar procedures with alkaline permanganate reagent were subsequently employed. Among some more recent publications may be mentioned those of the following: Marconi (1952), who reacted for 15-20 min at room temperature then added sulphuric acid and excess standard oxalic acid and concluded with permanganate titration; Balwant Singh et al. ^1953a), who oxidised for 10 min at 25°C using the conditions of Stamm's method (1934, 1935), whereby permanganate is reduced only to manganate which is then precipitated as barium salt, the final titration stage being potentiometric using sodium formate; and Schroder (1960) who refluxed for 1 h with potassium permanganate-potassium hydroxide, then acidified with sulphuric acid, added manganese(II) sulphate as catalyst and excess oxalic acid, and finally back-titrated with permanganate.


Oxidation with excess (and concluding back-titration) has been performed also in acid solution, for example by Oliveri and Spica (1890) who added standard permanganate to the sample plus sulphuric acid at 100°C until the solution was coloured, then back-titrated with oxalic acid. Gailhat (1902) refluxed the sample for 25 min with manganese(II) sulphate, sulphuric acid, and excess standard permanganate, cooled, added excess oxalic acid, and also titrated finally with permanganate. Ravenna (1928) oxidised the glycerol sample for 2 h with permanganate and sulphuric acid on the water bath and then back-titrated with oxalic acid. In a study of the photochemical reaction of glycerol with ferric iron, Loury (1959) determined the glycerol by oxidation with excess permanganate and back-titration with ferrous-iron.

Procedures of direct titration of glycerol with permanganate are very rare. As a further part of his study of oxidisability of glycols in connection with their toxicity, Reif (1951) titrated a mixture of a 10% aqueous solution of the glycol (including glycerol as example) and acetic acid in 2:1 ratio with 01 % potassium permanganate, waiting for decoloration before adding further titrant, and continuing for 5 min. This is, of course, not a complete titration to determine the glycol but merely gives relative values of oxidisability from the titration volumes. Singh and Sharma (1970) titrated low concentrations ( 1 0 - 2 to 5 x 1 0 _ 5 N solutions) of some inorganic compounds and also ascorbic, formic, and acetic acids and glycerol in sulphuric acid medium with permanganate, using an amperometric end-point indication with a polarised platinum micro-electrode and silver-silver chloride electrode at 25°C.

2. Determination of Oxalic Acid Permanganate oxidation to yield oxalic acid as reaction product, followed by estimation of this acid, has also been a popular procedure for determining glycerol. The classical procedure of Benedikt and Zsigmondy (1885) is based on this principle. They added potassium hydroxide to the sample and then 5% potassium permanganate at room temperature until the solution was blue to black. They then heated to boiling, causing manganese dioxide to separate and the solution to become red. After decoloration with sulphur dioxide they filtered and acidified the filtrate with acetic acid. They then brought to the boil, precipitated the oxalic acid as calcium salt, and esti-mated the calcium oxalate either by titration in acid solution with perman-ganate or by ignition and alkalimetry. Mangold (1891) likewise treated the sample plus potassium hydroxide with an excess (ca. 50%) of potassium permanganate, allowing the mixture to stand for 30 min. He decolorised excess permanganate with hydrogen peroxide, the excess of which was destroyed by boiling. The solution was acidified with sulphuric acid and he then titrated the oxalic acid with permanganate. A more recent example of the use of this principle is due to Ibrahim and Taha (1962) who determined

30 GLYCEROL 1.1

glycerol in pharmaceutical preparations by Mangold's method and also by a modification in which excess permanganate was decolorised with methanol instead of hydrogen peroxide and the oxalic acid was quantitatively precipi-tated as its calcium salt.

Berka and coworkers (Berka and Zavesky, 1969; Berka and Dusic, 1970) developed a new method of determining the oxalic acid derived from oxida-tion of organic substances, notably glycerol, by potassium permanganate-sodium carbonate. Berka and Zavesky oxidised a 20ml sample (ca. 0-005M in glycerol) with a mixture of 20 ml of 0 1 N potassium permanganate and 20 ml of 2M sodium carbonate, refluxing for 20 min. They then reduced unused reagent by adding 0-5M formic acid, filtered off the manganese dioxide, and added to the filtrate containing the oxalic acid an excess of the pyrophosphate complex of manganese(III), [ M n ( H 2 P 2 0 7 ) 3 ] . This oxidises the oxalic acid, and the unreacted complex was titrated after 30 min with standard hydroquinone to a pink colour. They then added diphenylamine indicator and completed titration with the hydroquinone to a colour change from violet to yellow. Any excess formic acid does not interfere in the oxida-tion of the oxalic acid. In the second cited publication, unused pyrophosphate complex was reduced also by ascorbic acid to manganese(II) which Berka and Dusic titrated with Complexon III to Eriochrome Black T. They oxidised a 5 ml sample with 15 ml of 0 1 M K M n 0 4 and 1 g of N a 2 C 0 3 for 90 min at 90°C.

3. Determination of Manganese Dioxide In a publication subsequent to those just mentioned, Berka and Pauleova (1971) determined glycerol (also ethylene glycol and mannitol) by boiling for 1 h with 0-1N potassium permanganate and 2M sodium carbonate. After cooling and adding enough sulphuric acid to achieve a 2N concentration, they waited for 15 min, added 0*5M manganese(II) sulphate, and filtered off the manganese dioxide formed. This was dissolved in saturated pyro-phosphate-4N sulphuric acid-0*5M manganese sulphate (6 + 2 + 1 ) , yielding the pyrophosphate complex of manganese(III). They titrated this with hydroquinone as described above.

4. Determination of Formaldehyde This method has been less frequently used. Diemair et al. (1940) determined glycerol in wine by oxidation to formaldehyde, then removed unused reagent with oxalic acid and developed colour with the Schiff reagent (fuchsin-sulphur dioxide) which they evaluated photometrically, von Fellenberg (1943) used a similar procedure for micro-determination of glycerol, also in sweet and dry wines.


1.1.16. Si lver

The use of a silver-containing reagent for visualising polyols on chrom-atograms appears to date back to 1950. Hough in that year used for the purpose 5% silver nitrate containing excess ammonium hydroxide which gave dark brown spots on heating and enabled him to detect amounts down to 1 jxg. Such a reduction of silver ion was surprising. Shortly afterwards, Trevelyan et al. (1950) used a reagent prepared by diluting 0-1 ml of saturated aqueous silver nitrate to 20 ml with acetone and then adding water until the silver salt just redissolved. After spraying and drying the chromatogram, they then sprayed with 0-5N sodium hydroxide in aqueous ethanol to give black spots. On immersing the paper strip in 6N ammonium hydroxide and washing and drying, black or dark brown zones on a white background were obtained with polyols. Sperlich (1952) showed that the presence of at least two hydroxyl groups is necessary for response to the silver reagent.

The combined silver-ammonium hydroxide reagent found regular use in the subsequent paper chromatography of polyols. Bergner and Sperlich (1953b) detected traces of glycerol from fat splitting by spraying with ammoniacal silver nitrate and heating for 15 min at 100°C. Smullin et al. (1958) detected glycerol on a reference strip by spraying with a combined reagent of ca.10% silver nitrate and cone, ammonia-water (1+2) , then heating for 5-10 min at 100°C to obtain black spots on a light background. Popiel (1961) used Hough's method to visualise carbohydrates and polyols in paper electro-phoresis. Beer (1961) used the Tollens ammoniacal silver reagent, also heating for 15 min at 100°C to visualise polyols and monosaccharides. A similar procedure was employed by Kroller (1963) to identify glycerol in tobacco additives after paper chromatography. Hennies and Eckert (1963) used 0-1N silver nitrate-5N ammonium hydroxide (1 + 1 ) and heated for 5-10 min at 105°C to visualise polyols, including glycerol, on paper chrom-atograms in a study of cosmetics. Patterson (1963) treated Whatman No. 1 paper with an aqueous acetone solution of silver nitrate and, after develop-ment, exposed it to ammonia vapour, heated for 15 min at 95-100°C, and then washed with ammonium hydroxide, thus detecting glycerol as a dark spot. Siegel et al. (1964) detected glycerol in polyglycerol mixtures after PC as a dark grey zone by spraying with ammoniacal silver nitrate and heating at 80°C for 30-60 min. Datta and Ghosh (1965) used circular PC on paper impregnated with silver nitrate to separate glycerol from complex pharma-ceutical preparations, and detected 5-10 jig amounts as brown spots by exposing to ammonia vapour, drying, heating to 80-90°C, and washing with 10% ammonium hydroxide and water before re-drying. Ammoniacal silver nitrate was used also by Udalova (1966) with planimetric semi-quantitative estimation, to detect glycerol in the deproteinised culture fluid of Bacillus

32 GLYCEROL 1.1

brevis var. GB; by Cotte and Guillot (1967) for detecting polyols after ascending PC; and by de Freitas (1967) for glycerol and glucose in plasma after PC on Whatman No. 1 paper.

Examples can be quoted also from thin-layer chromatography. Bergel'son et al. (1961) used 5% silver nitrate-25% ammonia to detect down to 2 \ig of glycerol on silica gel, and preferred this method to other detections such as periodate or lead(IV). Seher (1964) detected glycerol derived from the hydrolysis of some non-ionic surface-active agents by spraying the kieselguhr G layer with 0 1 N silver nitrate-5N ammonium hydroxide (1 + 1) and then heating for 10-20 min at 100-105°C to yield brown zones on a pale back-ground. Nadirov et al. (1971) also detected various polyols on alumina-containing thin layers with the ammoniacal silver nitrate reagent among several. Venturini (1972) sprayed silica gel G layers with a solution of 2 g of silver nitrate in 3 ml of 25% ammonia, diluted to 100 ml with ethanol, to visualise glycerol from liqueurs and syrups, heating finally for 10 min at 140°C to obtain brownish-black spots.

The silver nitrate-sodium hydroxide reagent has also been used chrom-atographically. Palleroni and Vega (1954) separated wine glycerol by PC and visualised it by treating with a solution of 0-1 ml of saturated aqueous silver nitrate in acetone, drying, then spraying with 0-5N sodium hydroxide in ethanol to yield a dark brown spot. The principle of Trevelyan et al. was employed also by Olley (1956) in PC to detect glycerol, and by Lindberg and Swan (1960) and Popiel (1961) to visualise carbohydrates and polyols in paper electrophoretic studies of the influence of germanate and tellurate buffers. Hromatka (1962) combined ethanolic sodium hydroxide and silver nitrate to visualise glycerol after its separation from dihydroxyacetone in PC. Zajic (1962) sprayed with silver nitrate and alkali to detect poly glycerols after PC. Guerra Salazar (1962) visualised polyols and sugars after PC using acetonic silver nitrate and ethanolic sodium hydroxide. The Trevelyan reagent was employed also by Lees and Weigel (1964) for polyols on paper chromatograms containing sodium stannate.

1.1.17. Vanadate

Vanadate is a too powerful and unspecific oxidising agent to be especially valuable in analytical work but some examples of its use with glycerol among other alcohols may be cited. Pozzi-Escot (1938) detected glycerol by heating it with vanadium pentoxide to 180-200°C, cooling, and testing for methyl-glyoxal. This was done by adding the reaction mixture to a solution of 1 mg of phloroglucinol in a few ml of sulphuric acid, which yields a yellow or orange colour. Resorcinol or thymol can be used instead of phloroglucinol, but are less sensitive. The author preferred vanadium(V) to bromine.


West and Skoog (1959) determined glycerol in dilute aqueous solution by oxidation with acid vanadate reagent to formic acid through heating for 1 h on the water bath:

C 3 H 5 ( O H ) 3 + 8 V 0 2

+ + 8H + 3HCOOH + 8 V 0 2 + + 5 H 2 0

After cooling the solution they back-titrated with ferrous iron to the colour change from violet to green of the indicator N-phenylanthranilic acid. Conditions have to be carefully chosen; too high an acid concentration and too large an excess of reagent give high results, probably through further oxidation. They found that sulphuric acid of about 4-3N was best.

Eremina and Gurevich (1961) determined compounds, including glycerol, in organic pharmaceutical preparations, by oxidation with a 3-6-5-fold excess of ammonium vanadate and sulphuric acid. After heating for up to several hours on the water bath, they diluted and back-titrated also with ferrous iron (Mohr salt) to the same indicator.

Mention may be made here of a test for alcohols with a reagent con-taining vanadium(V). This reagent was prepared by Buscarons et al (1949) from 8-hydroxyquinoline (oxine) and ammonium vanadate, and is evidently a phenol ester of orthovanadic acid. It is black-green but yields a red product with alcohols, probably a solvate:

Buscarons et al treated a sample of 2-5 ml with 0-5-1 ml of aqueous ammonium vanadate (containing 20-30 mg of vanadium/1) and then added 2-5% oxine in 6% acetic acid until the vanadate was completely precipitated. Alcohols, including glycerol, gave a colour change to red within some minutes. Feigl (1966b) quoted a slightly modified test by warming the reaction mixture to 60°C on the water bath. However, this is not a particu-larly sensitive test for glycerol. Feigl gives 0-5 mg as the smallest detectable amount, probably because of its poor solubility in the benzene solution reagent employed by him.

More recently Izquierdo and Lacort (1972) replaced the oxine by 5,7-dichloro-8-hydroxy-2-methylquinoline. They mixed 1 ml of a 0-22 % solution of this base in 0-1M hydrochloric acid with 1 ml of 01 % ammonium vanadate and 1 ml of benzene or carbon tetrachloride. On shaking, colour was observed in the organic phase with alcohol samples. Glycerol yielded a green colour.

TAT (ROH)

Black- or grey-green Red

34 GLYCEROL 1.2

1.2. ESTERIF ICAT ION M E T H O D S

Glycerol can be converted into mono-, di-, and tri-esters. Triesters with a single acid component are the easiest to prepare, and this reaction is the basis of standard analytical procedures.

1.2 .1 . Ident i f i ca t ion of Glycero l

A standard method for identifying organic compounds is to convert them into suitable derivatives which are then examined. The melting point determination of solid derivatives is the commonest realisation of this principle. Alcohols, including glycerol, are often esterified to this end. The triesters of the lower aliphatic acids are liquid at ordinary temperatures but the tribenzoate is solid (m.p. 72°C) and is easily prepared using customary techniques, for example with excess benzoyl chloride in pyridine solution. This information, together with melting points of other suitable esters, such as the tri-p-nitrobenzoate, is available in standard handbooks of qualitative analysis for organic compounds. Two such identifications of glycerol in technical materials may be mentioned: by Smith (1926) for glycerol in cotton cloth and sized yarns; by Bennett and Streeter (1959) from a laboratory preparation of soap.

Urethanes are also esters prepared for identification of hydroxy-com-pounds. Standard works include examples of these also; for example, that of Pesez and Poirier (1953) quotes melting points for urethanes of glycerol derived from a-naphthyl and p-nitrophenyl isocyanates and from p-bromo-benzazide:

O—R

I R—OH + A r — N = C = 0 A r — N H — C = 0

R—OH + Ar— C O - N 3 - + A r — N H — C = 0 + N 2

I O—R

The Handbook of Tables (1974) gives also the melting point of the urethane from phenyl isocyanate. Chapman (1926) identified glycerol from tobacco as its urethane from a-naphthyl isocyanate.

Identification of esters with the help of reference data other than melting points is naturally also possible. For example, Gasparic and Borecky (1961) prepared the 3,5-dinitrobenzoate esters of some hydroxy-compounds and submitted them to TLC using various mobile phases. They quoted Rf values

1.2 ESTERIFICATION METHODS 35

for identification purposes for the mono-, di-, and tri-esters of glycerol. Dallas (1970) also carried out TLC of poly glycerols (including glycerol itself) on silica gel G-kieselguhr (1+1) containing calcium chloride, using ethyl acetate-isopropanol-water (110 + 61 + 29), then spraying with 60% (vol.) acetic anhydride in pyridine to esterify, and chromatographing at right angles with cyclohexane-diethyl ether-ethanol-formic acid (10+102 + 6 + 2) as mobile phase.

Goodlett (1965) characterised hydroxy-compounds through the downfield shift of NMR peaks (of hydrogen a to the hydroxyl group) on esterification with ketene or trichloromethyl isocyanate. Glycerol was in fact not among his examples but he worked on two diols and the method must be applicable to glycerol also. This applies also to the work of Jung et al (1970) who converted polyalcohols into trifluoroacetate esters before carrying out 1 9 F N M R studies.

Esterification followed by gas chromatography is dealt with in Section I.2.2.E.

1.2.2. Quantitative Determination

Two broad types of determination depend on esterification of glycerol, followed by: estimation of the ester formed or of the consumption of acyla-tion agent; gas chromatography of the product.

A . E S T I M A T I O N O F T H E E S T E R F O R M E D

This is the classical esterification method for glycerol determination, carried out with acetic anhydride and hence often referred to as the "acetin" method. It appears to date back to the work of Benedikt and Cantor in 1888. They determined hydroxyl compounds, including glycerol, by refluxing with acetic anhydride and anhydrous sodium acetate. For glycerol, 60-90 min were necessary because three groups have to be esterified. After allowing to cool, they diluted the reaction mixture and titrated the clear solution with alkali to phenolphthalein indicator. A measured excess of standard alkali was then added, the glycerin triacetate saponified by boiling for 15 min, and finally the unused alkali back-titrated with standard hydrochloric acid to the same indicator. From the alkali consumption the amount of ester and hence of glycerol could be evaluated.

This remained a standard procedure for many years, despite certain sources of error and the fact that it is applicable only to samples with fairly high glycerol content and not to very dilute solutions. Interference by carbon dioxide in the titrations was referred to, for example, by Berth (1928). More serious, however, is the risk of hydrolysis of the ester during the neutralisation of unused acetylation agent and the acetic acid formed in this

36 GLYCEROL 1.2

reaction. This was noted, for instance, by Frey (1929) and Andrews (1933). In his report on glycerol analysis to the committee of the American Oil Chemists' Society in 1935, Andrews recommended reducing this hydrolysis by chilling the reaction mixture in ice. Procedures were published containing details to be strictly observed, as by Testorelli et al. (1955) for example.

B. D I F F E R E N T I A L M E T H O D

In 1901 Verley and Bolsing introduced their differential procedure for determining hydroxyl groups through esterification. This largely superseded the acetin method. They used a mixed reagent of acetic anhydride and pyridine (approximately 1 + 7). A measured amount of it was added to the sample and a second, equal aliquot was mixed with water which rapidly decomposes acetic anhydride to acetic acid, especially in the presence of pyridine. The two reactions are:

( C H 3 C O ) 2 0 + ROH CH3COOR + CH3COOH (Sample)

After 15 min at 100°C for complete esterification, they carefully hydrolysed the unused anhydride and then titrated the final solutions from reaction with the sample and with water alone. The difference between the alkali titrations corresponded to the ester formed and hence to the amount of alcohol (glycerol) in the sample. Any free acetic acid in the anhydride reagent was titrated each time and thus cancelled out in the difference. This procedure is

Differential Procedures for the Determination of Glycerol (and Other Alcohols)

( C H 3 C O ) 2 0 + H 2 0 2CH3COOH (Control)

TABLE 1.1

Reagent Reaction conditions* References

1-5M Acetyl chloride in toluene

20min/60 ± 1°C Smith and Bryant (1935)

Acetic anhydride-pyridine (1 + 10)

Acetyl chloride (no solvent)

3-6 h/boiling Wilson and Hughes (1939)

Christensen et al. (1941) 20 min/40°C

24 h/room temp. Peterson et al. (1943)

30-40 min/steam bath Pohle and Mehlenbacher (1946)

1 h/100°C (sealed bottle)

Elving and Warshowsky (1947)

TABLE 1.1—continued

Reagent Reaction conditions* References

Acetyl chloride-toluene ca. 1M

2M Acetic anhydride in ethyl acetate or pyridine, containing HCl6 4 catalyst

0-5M Pyromellitic anhydride in tetrahydrofuran

0-2M 3,5-Dinitrobenzoyl chloride in pyridine

20 g Toluene-p-sulphonyl chloride -I- 100 ml pyridine

0-2M 3-Nitrophthalic anhydride in dimethyl-formamide

20 % Acetic anhydride in toluene

10% o-Sulphobenzoic anhydride in dioxan

0-5M o-Sulphobenzoic anhydride in dioxan or of succinic anhydride in pyridine

1-35M Acetic anhydride and 0006M H C 1 0 4 in ethyl acetate

10% Acetic anhydride in pyridine.

Also with acetic anhydride-ethyl acetate-toluene-p-sulphonic acid (33 + 100 + 4 g)

2 h/reflux

5-7 min/room temp. (Titrated to Cresol Red-Thymol Blue or potent to pH 9-8)

+ pyridine and 2 min/ 100°; most THF then boiled off

5-15 min/room temp (Titrated with tetra-butylammonium hydroxide, yellow to red

Reflux (2 h still gave only 82% recovery)

+ triethylamine and 10+1 min/room temp, (gave only ca. 82 % recovery; even 2 h gave only 95%) (Titrated with tetrabutyl-ammonium hydroxide to thymolphthalein)

4 h/reflux

2-3 h/reflux (nearly 90 % reaction)

+ pyridine and 15min/100°C

15min/21°C

Johnson (1948)

Fritz and Schenk (1959)

Siggia et al (1961)

Robinson et al (1961)

Mesnard et al (1963)

Floria et al (1964)

2h/100°C

20min/50 + 1°C

Kartha and Kidwai (1965)

Iyer and Mathur (1965)

Narang et al (1965)

Zelenetskaya et al. (1968)

IUPAC methods (1973)

* The final titration was with alkali to phenolphthalein indicator, except in the three cases where it is stated otherwise.

c

38 GLYCEROL 1.2

faster because the ester product does not have to be quantitatively hydrolysed in a following determining step. But care is needed here too during the removal of unused anhydride to ensure that the ester does not undergo hydrolysis.

The original procedure of Verley and Bolsing has undergone many modifications, a dominating aim being acceleration. Different acetylating agents, solvents, media, and catalysts have been suggested. A selection of procedures, mostly of relatively recent date, is given in Table 1.1. They all include glycerol or a similar polyol among the examples tested.

From Table 1.1 can be seen the various anhydrides and acid chlorides that have been employed, and also the catalysts, such as the base pyridine and the acid perchloric acid. Reaction times have thereby been reduced to a few minutes. Results with glycerol were often low (ca. 97 %) but this could often be traced to impurities, notably water, as shown by control determinations using other procedures.

C . E S T I M A T I O N O F T H E A N H Y D R I D E C O N S U M E D

Pesez (1954) developed a non-differential method, using a reagent prepared from 5 ml of propionic anhydride and 30 ml of acetic acid, containing 1 g of toluene-p-sulphonic acid as catalyst. He treated a 0-5-1-5 mmol sample with 2 ml of reagent for 2 h at room temperature or for 20 min at ca. 100°C on the steam bath. Unused anhydride was then reacted with excess (25 ml) standard 0-9% aniline (ca. 0-1N) in benzene solution and 30 ml of glacial acetic acid; after 5 min the residual aniline from this was back-titrated with 0-1N perchloric acid-acetic acid to Crystal Violet indicator:

( C 2 H 5 C O ) 2 0 + C 6 H 5 N H 2 - * C 2 H 5 C O N H C 6 H 5 + C 2 H 5 C O O H

D . D I R E C T E S T E R I F I C A T I O N W I T H C A R B O X Y L I C A C I D

This principle is seldom used. Bryant et al. (1940) determined alcohols, including glycerol, by reaction in dioxan with acetic acid containing dissolved boron trifluoride as catalyst for the esterification:

CH3COOH + R O H - * C H 3 C O O R + H 2 0

After 2 h at 67 + 2°C they added pyridine and titrated the water formed using the Karl Fischer reagent. A blank yielded the water content of the components without esterification.

The qualitative work of Frehden and Huang (1937) may also be mentioned here. They published a test for glycerol amounts down to 5 \ig in which they heated it with some crystals of hydrated oxalic acid. Carbon dioxide is liberated according to the reactions:

1.2 ESTERIFICATION METHODS 39

C H 2 O H C H 2 0 — C O — C O O H C H 2 0 — C O H I COOH | I

CHOH + | - + C H O H - • C H O H + C 0 2

| COOH | I C H 2 O H C H 2 O H C H 2 O H

Monooxalate Monoformate

This carbon dioxide was detected with filter paper, moistened with phenol-phthalein and sodium carbonate solution, which is then decolorised. Care must be taken because oxalic acid itself yields carbon dioxide on stronger heating.

Alternatively, the monoformate ester product can be detected through the hydroxamic acid ester test. This consists of warming with free hydroxylamine (from a salt, such as the hydrochloride, and alkali) to give a hydroxamic acid which, after acidification, yields a brown-violet chelate complex with ferric chloride:

RCOOR' + N H 2 O H R C = 0 + R'OH

I N H — O H

R C = 0 3 | + F e 3 + -+RC=0^ / O H N + 3H +

N H O H I ^ F e - I N H O / \ " 0 = C R

6 O II H C—N R

Brown-violet

This enabled down to 40 \ig of glycerol to be detected. Feigl (1966g) adopted these reactions as spot tests in his book.

E. A C Y L A T I O N , F O L L O W E D B Y G A S C H R O M A T O G R A P H Y O F T H E

P R O D U C T

The high boiling point and low volatility of glycerol and other polyols render them difficultly amenable to gas chromatography. A logical approach is to convert them into less polar and more volatile derivatives. Esters are among the more easily prepared derivatives of this sort.

Most frequently used has been the triacetate, b.p. 258°C. In recent years, since trifluoroacetic anhydride has become more readily available, trifluoro-acetates have been often prepared and submitted to gas chromatography. Table 1.2 summarises information about some examples.

TABLE 1.2

Gas Chromatography of Glycerol Esters

Sample Acylation Chromatographic details References

Glycerol in tobacco humectants

Polyols freed from alkyd resins by aminolysis with B u N H 2

Polyols, including glycerol

Free glycerol in blood serum

Glycerol from LiAlH 4

reduction of glycerides

Glycerol and diglycerol in presence of each other

Polyols, including glycerol, from reduction of mono-saccharides

A c 2 0 at 20°C on Impregnated Celite 545; CHCI3 extract; or, if N 2 gas 0-5-10% humectant, direct on tobacco with BF 3 -dioxan catalyst

90 min reflux with A c 2 0 , unused decom-posed with water, and ester extracted with chloroform

10%Carbowax 20M on Chromosorb W; He gas; 30 to 225°C;heat conductivity detector

5 % Polyoxyethylene glycol on Embacel; FID

Puschmann and Miller (1961)

Esposito and Swann (1961) Esposito (1962)

Mlejnek (1963)

3-4 h reflux with A c 2 0 -b e n z e n e - H 2 S 0 4 (600 + 1100 + 5); benzene distilled off below 115°C; + water, and ester extracted with CC1 4

1 h heating with A c 2 0 , 8 % Butanediol succinate Jellum and then + excess EtOH and on Chromosorb W; FID Bjornstad 20min/70°C;EtOAc (1964) and unused EtOH boiled off; glycerol acetate extracted with ether for GLC

Reacted with A c 2 0 ; 10% Polyethylene Holla et ah excess removed with glycol succinate) on 60-80 (1964) EtOH mesh Gas-Chrom P;

He gas; 170-200°C; heat conductivity detector

0-25 % Silicone high-vacuum grease or 0-5 %

2 h reflux with A c 2 0 ; excess distilled off at 100°C in vacuo; + water silicone rubber gum and extracted with ether SE-30 on 0177 mm glass

beads; 207°C; Ar-ionisa-tion detector

15 % Poly(diethylene glycol succinate) on 0-25-0-4 mm Sterchamol; H 2 ; 200°C; heat conducti-vity detector

(also Horrocks, 1961; Horrocks and Cornwell, 1962)

Hartman (1964)

25 min reflux with A c 2 0 , excess removed with boiling water; extracted with dichloroethane

Anzhele et al. (1964)


Sample Acylation

Polyols, + A c 2 0 - E t O A c -including H 0 0 4 ; unused glycerol decomposed with

MeOH and acetic acid; product adsorbed on alumina

Polyols 4 h with pyr id ine-Ac 2 0 (3 + 1) on water bath; evaporated in N 2 ; residue for GLC

Glycerol in Tribenzoate formed incubation of with benzoyl chloride adipose tissue

Polyols from Heated overnight with N a B H 4 ( C F 3 C O ) 2 0 + trace of reduction of pyridine sugars

Polyols freed 2-5 h reflux with A c 2 0 from alkyd resins by aminolysis with phenyl-ethylamine

Polyol base Refluxed with A c 2 0 -compounds in toluene-p-sulphonic polyurethanes acid; product extracted

with ether

Polyols from Acetylated cat. hydro-genation of carbohydrates

Polyols from Acetylated aminolysis of alkyd resins with poly-ethylene poly-amine

Chromatographic details References

10% Reoplex 400 or 10% Vaver et al. PEG on 60-80 mesh (1967) Chromosorb W at 140-150°C; or on 7% high-vacuum lubricant at 125°C; FID

Stainless steel column; Sen et al. 5 % S E - 3 0 o n 80-100 (1967) mesh AW Chromosorb W ; N 2 ; 100 to 275°C at 4°/min, then kept there; FID

4 % SE-30 on 80-90 mesh Decroix et al Anakrom ABS; N 2 ; 180 (1968) to 250°C at 10°/min; FID

Open column coated Shapira (1969) with FS 1265 (a trifluoro-propylmethyl poly-siloxane); He; 160°C; FID

Carbowax 20M or sili- de la Court cone rubber on Diatoport et al. (1969) S; He; 68 to 225°C at 8°/min or 150 to 200° at 6°/min, respectively; FID

Stainless steel column; Tsuji and 25% Apiezon L on 60-80 Konishi (1971) mesh AW Chromosorb W; He; 180 to 260°C at 6°/min

5% SE-30 on AW D M C S Zelikman Chromaton N ; N 2 : FID et al. (1973)

Silicone SE-30 on Lushchik Chromosorb et al. (1974)

42 GLYCEROL


1.2

Sample Acylation Chromatographic details References

Glycerol and other poly-hydroxy compounds in aged distilled spirits

Acetylated with acetic anhydride-pyridine (1 + l ) for4h /115°C.

Acetates on 3 % ECNSS- Black and M on 100-120 mesh Gas- Andreasen C h r o m Q ; N 2 ; 190°C. (1974)

Trifluoroacetylated with Trifluoroacetates on 3 % anhydride-ethyl acetate XF-1105 on same carrier; (1 + 1) for several hours N 2 ; 100 to 180°C at at room temp. 2°/min. Both FID.

Studies have also been carried out on the esters of polyols, for example by Jung et al. (1970) who measured retention times of trifluoroacetates on various columns.

1.3. ETHER F O R M A T I O N

Ether formation, like esterification, has been applied to polyols to convert them into more volatile derivatives, and thus render them more suitable for gas chromatography.

1.3 .1 . T r imethy ls i ly la t ion

Almost without exception the trimethylsilyl ethers have been used for this purpose. In what appears to be the first application of this sort, Smith and Carlsson (1963) prepared such ethers by 10-15 h treatment at 100-150°C with excess trimethylsilyl chloride in pyridine:

R—OH + ( C H 3 ) 3 S i C l - * R — O — S i ( C H 3 ) 3 + HC1 (+pyridine)

This technique was superseded by using a solution of hexamethyldisilazane, (CH 3 ) 3 SiSi(CH 3 ) 3 , and trimethylsilyl chloride in pyridine, or, less often, dimethyl sulphoxide which enabled reaction times to be reduced to as little as 30 min (e.g. Blum and Koehler, 1968). Other trimethylsilylation reagents are trimethylsilyldiethylamine, ( C H 3 ) 3 S i N ( C 2 H 5 ) 2 , and N,0-bis(trimethyl-silyl)acetamide, ( C H 3 ) 3 S i N = C ( C H 3 ) O S i ( C H 3 ) 3 , this last named also with the trifluoro group, C F 3 , attached to carbon (Esposito and co-workers, 1968, 1969). Internal standard polyols are of course subject to the same trimethylsilylation.

Table 1.3 contains examples of gas chromatography of such ethers of glycerol and other polyols. Summarised chromatographic information is given but preparative details of the silylation are not.

TABLE 1.3

Gas Chromatography of Trimethylsilyl Ethers of Polyols

Sample Chromatographic details References

Polyols

Metabolites in urine and tissue containing OH

Diols, triols

Humectants in dentifrices (e.g. glycerol, propylene glycol, sorbitol)

Polyols from periodate oxidation of a polysaccharide

Identification of glycerol, ribitol, and anhydroribitol

Glycerol in lipids (vegetable oils)

1 4 C-labelled sugar and polyols; study of recovery in GLC

Polyols (silyl derivatives formed on column by subsequent injection of reagent)

20 % SE-30 or Apiezon M on 60-80 mesh Smith and Celite; He gas; 184°C; thermistor detector Carlsson

(1963)

1 % or 10% F-60 (a polysiloxane) on 80-100 Dalgliesh mesh Gas-Chrom P; N 2 gas; 30 to 260°C et al (1966) at 2 ° / m i n ; H 2 - F I D

10 % Silicone SKT-V or SKT polymer on Vaver et al Chromosorb W; Ar gas; 100°C; p- (1967) ionisation detector

6% SE-30 (methylsilieone polymer) on Blum and silanised Chromosorb W; 75 to 225°C.at Koehler 6°/min (glycerol eluted at 130°C); FID (1968)

20% SF 96 on 60-80 mesh Diatoport S; Dutton et al 130°C for 6 min, then to 220°C at 3°/min; (1968) thermal cond. detector. Some simpler mixtures on shorter column; 70 to 230° at 10°/min

3-5 % SE-52 on 85-100 mesh silanised Gregory (1968) Diatomite C; 130°C

2 % SE-30 on Chromosorb W; H 2 - N 2 - Rajiah et al compressed air (2 + 5 + 20); 100°C until (1968) glycerol ether eluted, then to 110° at 5°/min, then isothermal; H-ionisation detector

2 % SF-96-50, 2 % Carbowax 20M, or 2 % Jansen and SF-96-50 + 0-005 % Igepal on acid- Baglan (1968) and base-washed 100-200 mesh Chromosorb G; SF columns at 225 or 250°C, Carbowax at 105, 135, 170, or 200°C; Ar gas; electron-capture detector; effluent led into naphthalene-dioxan scintillation solution for 1 4 C counting; ca. 80% glycerol recovery

20 % Silicone grease on 60-80 mesh Esposito Chromosorb W; He gas; 40 to 250°C at (1968) 4°/min; thermal conductivity detector. 6 and 10 ft columns good, 4 ft and less gave poorer yields



Polyols from oils and alkyd resins after aminolysis with' butylamine

Polyols from rigid polyurethane foam

Products of hexose oxidation

Glycerol (also mono-and di-glycerides) in food products

Glycerol and sugars from beer

Polyol humectants in tobacco and tobacco smoke

Products from N a B H 4 reduction of glycopeptides and related materials

Tested on polyols and carbohydrates in aqueous solution

Compounds, including glycerol, in tobacco smoke

Glycerol and mono-saccharides

Polyols

Method of Esposito (1968), with temp. Esposito and programming from 100 to 300°C at Swann (1969) 6°/min

1-5 % SE-52 on AW DMCS Chromosorb G; Corbett et al He gas; 70 to 380°C at 8°/min (1969)

3-7 m of 1 % GESE 52 or 4-3 m of 3 % PPE Verhaar and (polyphenyl ether, 5-ring) on 60-80 mesh De Wilt (1969) AW-DMCS Chromosorb W, or 2-0 m PO 17 (OV 17) (50% phenyl-substituted methyl silicone) on unknown support; Ar gas; 175, 200, or 175°C, resp.; hydrogen-FID

Glass column; 3 % OV-1 on 80-100 mesh Blum and Chromosorb W; 50 to 300°C at 12°/min; Koehler H 2 - F I D (1970)

Stainless steel column; 1 % OV-17 on 80- Parker and 100 mesh Chromosorb W-HP; N 2 ; Richardson 3 min/45°, then 15°/min to 300°C, held (1970) there until final peak; dual-FID

5 % SE-100 on 60-80 mesh Chromosorb W; Carugno N 2 ; 17 min/130°, then 2-5°/min to et al (1971) 200°C; FID; 98-6 % recovery of glycerol

Glass columns; 3-8 % SE-30 on 80-100 mesh Hughes and Diatoport S; N 2 ; 70 to 160°C at 2°/min Clamp (1972) or 110 to 200° at l°/min; FID

OV-17 SCOT column (Perkin-Elmer Weiss and 900 GC); N 2 ; 100 to 225°C at 4°/min; FID Tambawala

(1972)

4 % OV-101 on 80-100 mesh Chromosorb Guerin et al G; 10 min/70°, then 5°/min to 250°C; (1974) dual hydrogen-FID

Stainless steel columns; 3 % JXR or 3 % Black and XE-60 on 100-120 mesh Gas-Chrom Q; Andreasen N 2 ; 100to 200°Cor 85 to 180°Cat (1974) 2°/min; FID: quantitative with erythritol standard

3 % SE-30 on AW DMCS Chromosorb W; Knappe and N 2 ; 80 to 220°C at 5°/min; FID Miessner

(1976)

1.3 ETHER FORMATION 45

Gregory (1968) also subjected the trimethylsilyl ethers of glycerol, ribitol, and anhydroribitol to TLC on silica gel F 2 5 4 , using benzene for development and visualising by inspection in UV or by charring with 50% sulphuric acid at 150°C.

1.3.2. Other Ether Format ion Procedures

Mason and Waller (1964) used 2,2-dimethoxypropane in a transesterification procedure of fats and oils, also of pure glycerides, with methanolic hydrogen chloride. This yields the isopropylidene derivative from the liberated glycerol:

C H 2 O H C H 3

I I CHOH + ( C H 3 0 ) 2 C I I

C H 2 O H C H 3

Although the primary aim of the work was gas-chromatographic determina-tion of the methyl esters of the fatty acids, the isopropylideneglycerol served as a convenient marker for measuring their retention times. In a following publication of Mason et al. (1964), the determination of glycerol as iso-propylidene derivative was mentioned. They used an aluminium column containing 14-5% of ethylene glycol succinate (EGS) on 100-110 mesh Anakrom, type A, with helium carrier gas and at various temperatures.

Whyte (1973) analysed mixtures of products from reduction by tetra-hydroborate of aldoses, using methylsulphinyl carbanion in dimethyl sulphoxide, following with methyl iodide to complete methylation. He then carried out gas chromatography in aluminium columns containing various packings: 10% SE-30 at 140°C; 1% OV-17 at 130°C; 3 % QF-1 at 110°C; and 1 -5 % SE-60 -h 1*5% EGS at 130°C; the first and last were on 60-80 mesh Chromosorb W, the second and third on Gas-Chrom Q. The carrier gas was nitrogen, and hydrogen-flame detection was used. Of the compounds studied, glycerol had the shortest retention time.

C H 2 0 | C(CH 3 ) 2 + 2 C H 3 O H

CHO '

I C H 2 O H

46 GLYCEROL 1.4

1.4. D E H Y D R A T I O N A N D DETECTION OR D E T E R M I N A T I O N OF T H E A C R O L E I N F O R M E D

The dehydration of glycerol to yield acrolein is a well-known reaction :

C H 2 O H C H 2

' - 2 H , 0 "

CHOH — C H I

C H 2 O H CHO

This reaction distinguishes glycerol from other polyols and has found some analytical application. The usual dehydrating agent is potassium hydrogen sulphate; boric and phosphoric acids and salts and even direct pyrolysis have also been used analytically. Various procedures have been adopted to detect, identify, or determine the resulting acrolein. The chemical methods are based on its olefinic, aldehyde, or carbonyl group.

1.4 .1 . Pungent O d o u r of A c r o l e i n

The simplest test for acrolein is through its acrid smell. Chapman (1926) tested for glycerol in tobacco by heating with potassium hydrogen sulphate, and Soule (1929) dropped the sample on to a piece of hot iron, to observe the odour. Some pharmacopoeias quote this test (see Chapter 5).

1.4.2. React ion of the C = C G r o u p t o f o r m Ep ihydr ina ldehyde

Taufel and Thaler (1933) detected glycerol by distilling with phosphoric acid and shaking several drops of distillate for 1 min with 1 drop of 3 % hydrogen peroxide and 1 ml of cone, hydrochloric acid. This evidently yields epi-hydrinaldehyde, C H 2 — C H — C H O , with the acrolein in the distillate. The

O authors demonstrated its presence through the red to violet colour yielded within 30 min with a 0-15% phloroglucinol solution in ether. The same authors (1934) applied this to detect glycerol in foods, drugs, and cosmetics.

1.4.3. A l d e h y d e R e d u c i n g React ions

Standard reagents have been used here. Kataoka (1934) heated the sample with 20 times its weight of potassium hydrogen sulphate, carrying the evolved

1.4 DEHYDRATION A N D DETECTION OR DETERMINATION 47

gases with carbon dioxide into Bertrand copper(II) reagent for sugars and noting the cuprous oxide precipitate. The Schiff reagent of decolorised fuchsin was used, for instance: by Alfend (1932, 1933) on eggs and egg products which were first heated with potassium sulphate; by Ohl (1938) for glycerol in sizing agents on rayon and staple rayon, after heating with potassium hydrogen sulphate; and by Levin et al (1939) for glycerol m anti-freeze materials. The Nessler mercury(II) reagent can also be used, although Rosenthaler (1953) criticised it (and other similar reducing tests) as being too unspecific. The Tollens silver reagent likewise detects aldehydes and was used by Heiduschka and Englert (1921) to determine glycerol in wine. They heated a concentrated extract at 320°C in the presence of ammonia and passed the acrolein formed into excess standard silver nitrate; after heating the mixture until there was only a faint smell of ammonia they titrated unused silver with thiocyanate to ferric iron indicator.

1.4.4. React ion w i t h N i t r o p r u s s i d e - B a s e

Feigl (1937) described a spot test for glycerol, heating a drop of sample in a tube with finely powdered potassium hydrogen sulphate and testing the issuing vapours with a paper moistened with 5% sodium nitroprusside-20% piperidine or morpholine (1 + 1). Acrolein gives a deep gentian blue colour. Salzer and Weber (1950) mention the test, but convert the glycerol into acrolein with sulphuric acid.

1.4.5. C o n d e n s a t i o n React ions of the C = 0 G r o u p

A. W I T H 0 - D I A N I S I D I N E

Feigl (1937) tested for acrolein in the issuing vapours from the test described above through the brown-red to yellow colour given with paper moistened with a saturated solution of o-dianisidine in glacial acetic acid; this is probably a Schiff base resulting from condensation of the amino group with the carbonyl group in the acrolein.

B. W I T H H Y D R A Z I N E S

Formation of hydrazones is one of the classical methods of identification of aldehydes and ketones, based on the determination of the melting points of these derivatives. It is clearly applicable also for identifying the acrolein obtained from glycerol. For example, Jambor and Demeny (1936) detected glycerol in leather by converting it into aceolein and preparing the p-nitro-phenylhydrazone. An interesting variant from melting point determination is described by Opfer-Schaum (1944) who heated a drop of the sample in a test tube with potassium hydrogen sulphate to 180°C, holding over the mouth of

48 GLYCEROL 1.4

the tube a slide carrying a drop of a s o l u t i o n ^ p-nitrophenylhydrazine in 15 % acetic acid. Acrolein was then identified through the eutectic temperature of its p-nitrophenylhydrazone, thus prepared, with acetone p-nitrophenyl-hydrazone, prepared from a drop of aqueous acetone and the same reagent in excess.

Ganassini (1930) utilised hydrazone formation in a colour test. He heated the sample with potassium hydrogen sulphate, boron trioxide, or phos-phorus pentoxide to give acrolein, reacting this with phenylhydrazine and converting the phenylhydrazone into phenylpyrazoline which yielded a violet colour with acidic oxidising agents.

^ 6 H 5 C 6 H 5

N H \ . N L H 2 C N H 2 ^ H 2 C ^

HC CHO H 2 C - -CH

C. P Y R I D I N E R I N G F O R M A T I O N

A test of Sanchez (1944) probably depends on formation of a pyridine ring from acrolein. He heated the sample with diammonium hydrogen phosphate and potassium hydrogen sulphate until yellowish vapours were observed. The residue probably contained 3-methylpyridine, formed according to the reaction

H C ^ C H = C H 2 2 H 2 o r ^ ^ C H

CHO CHO

N H 3

The residue was dissolved in ethanol and treated with bromine cyanide, splitting the pyridine ring to a glutacondialdehyde monoenolate:

OrCHj

^ N

+ BrCN + 2 0 H ~ -*• CH 3

N H 2 C N + B r - + O C H — C H = C H — C = C H O

1 - O C H = C H — C H = C — C H O

C H 3

He identified this last product by heating with benzidine hydrochloride to give the well known red condensation product.


D . S K R A U P R E A C T I O N

The Skraup synthesis of quinolines depends on reaction of aromatic amines with glycerol in the presence of sulphuric acid and an oxidising agent such as nitrobenzene. The acrolein from the glycerol reacts with the amine and ring closure follows:

Stempel (1949) based a detection of glycerol on this, evaporating a mixture of 5-10 ml of sample, 0-5 ml of aniline, 0-5 ml of nitrobenzene, and 1 ml of concentrated sulphuric acid to a syrup at 135°C. After cooling and diluting with 50 ml of water he added 20 ml of 5 % sodium nitrite (presumably to destroy unused amine) and then heated for 30 min on a steam bath. About 30 ml were then distilled off, a few drops of I N hydrochloric acid added to this distillate, the solution was concentrated to ca. 5 ml, and potassium tetraiodomercurate, K 2 H g I 4 , added. This detected quinoline amounts as little as 1 mg of glycerol through the white precipitate of quinoline mercuric iodide, (quinoline) 2(HgI) 3 . 2HI.

Reichard and Gspahn (1954) applied this principle quantitatively to deter-mine glycerol in wine and liquors, boiling the sample for 2 h with aniline sulphate, sodium m-nitrobenzenesulphonate, and concentrated sulphuric acid. They then rendered the mixture alkaline with potassium hydroxide and steam-distilled the quinoline into 10 % hydrochloric acid, finally precipitating with potassium iodide-mercuric chloride reagent for gravimetric estimation. Sucrose had to be precipitated with barium hydroxide if present in more than 20g/l concentrations. Grohmann and Muhlberger (1956) applied this method to grape must, wine, and dessert wine, finding it better with low glycerol contents to add some before reaction and to deduct this added amount from the result.

Eisenbrand and Raisch (1960) adopted a fluorometric conclusion for deter-mining glycerol concentrations of 0-5-1 mg/ml. They mixed 1 ml of sample with 1-1 ml of cone, sulphuric acid and 0-25 mg of a mixture of 20 g of aniline sulphate and 5 g of sodium m-nitrobenzenesulphonate, and heated for 30 min at 15O-160°C. After cooling and adding alkali they steam-distilled into 0-1N sulphuric acid and determined the quinoline through its fluoresc-ence at 420 nm (excited at 313 nm). The results agreed well with those from other methods, such as periodate oxidation and then reaction of the form-aldehyde with phloroglucinol.

Feigl (1966f) gives a test for down to 0-5 jig of glycerol which is based on

50 GLYCEROL 1.4

the Skraup reaction. Two drops of 2 % alcoholic o-aminophenol are evapo-rated at 110°C in a micro test tube. One drop of test solution and then four drops of 1 % arsenic acid (as oxidising agent) in cone, sulphuric acid are added and the mixture is heated for 15 min at 140°C. After cooling he adds five drops of concentrated sodium hydroxide, one drop of 2N magnesium sulphate, and three drops of cone, ammonium hydroxide. This yields a bluish-green fluorescence in ultraviolet light. From the aminophenol, 8-hydroxyquinoline is formed and this, precipitated as magnesium salt, fluoresces. Mendelsohn and Antonis (1961) used this principle to determine glycerol derived from serum triglycerides. They evaporated 1 -6 % o-amino-phenol in acetone and heated this with the sample and 0-6 % arsenic acid in cone, sulphuric acid for 15 min at 140°C. After cooling and adding magnesium sulphate and 28 % ammonia they evaluated the fluorescence intensity.

E. W I T H A R O M A T I C C O M P O U N D S

In some tests for glycerol an aromatic reagent (generally a phenol or carbonyl compound) in concentrated acid (often sulphuric) is used, yielding charac-teristic and intense colours. It is probable in many cases that the acid dehydrates the glycerol to acrolein which then participates in a condensation reaction with the aromatic nucleus.

1. Carbonyl Compounds Bally and Scholl (1911) showed that acrolein reacts with an throne to give benzan throne:

This dimerises in cone, sulphuric acid to give violet-red dibenzanthrone or green isodibenzanthrone.

Schutz (1938) based detection of as little as 2 [ig of glycerol per ml in parch-ment paper extract on this. The aqueous extract was heated to 170°C with 0-1 % anthrone-conc. sulphuric acid to give a reddish-yellow solution with marked orange fluorescence, intensified by adding sulphuric acid. Radley (1950) applied the anthrone-conc. sulphuric acid reagent to detect, through fluorescence, microgram amounts of compounds such as glycerol, glycol derivatives, and tartaric acid, for example in industrial and foodstuff investi-gations. Kwon and Watts (1963) observed that malonaldehyde, croton-aldehyde, acrolein, and glycerol all yielded violet-red pigments, of absorption


maximum 510 nm, with anthrone-conc. sulphuric acid. Colours formed by sugar alcohols (glycerol, sorbitol, mannitol, dulcitol, etc.) on heating for 1 h on the water bath with 0*15 % anthrone in cone, sulphuric acid were reported by Graham (1963). He cooled the solutions in ice and estimated the colour intensities, which were highest with glycerol.

Lyne et al. (1968) determined 10-75 jag amounts of glycerol by heating 1 ml of a solution in 85% sulphuric acid for 15 min at 120°C with 1 ml of 0-1 % anthrone in the same acid. After cooling and diluting with 5 ml of 98 % sulphuric acid they evaluated the fluorescence intensity at 575 nm, exciting with light of 350-500 nm. They confirmed that the method is based on dehydration of the glycerol to acrolein and subsequent formation of benz-anthrone, and found that it was especially good for chromatographic spots. Sugars interfere as expected.

2. Phenols Phenols have been especially popular as components of colour reagents for glycerol. Christensen (1962) carried out a systematic investigation of the colours yielded from 3 ml of various alcohols and 5 mg or 5 jxl of 17 different phenols on mixing in 5 ml of cone, sulphuric acid. The best responses from glycerol were olive with a- and P-naphthols, blue-yellow with thymol, and purple with resorcinol. Bauer-Moll (1960) also quotes characteristic colour reactions and absorption spectra yielded by glycerol in cone, sulphuric acid with phenols such as (3-naphthol, thymol, salicylic acid, guaiacol, resorcinol, and gallic acid, as well as with codeine.

Dallas and Stewart (1967) and Dallas (1970) visualised glycerol on thin-layer chromatograms with a reagent prepared from 0-5 g of thymol + 5 ml of cone, sulphuric acid, diluted with 95 % ethanol. On heating at 120°C grey-purple spots were yielded on a mauve background. This was originally a reagent for detecting carbohydrates.

Mulliken (1904) quoted a test for glycerol in which 1 drop of sample in 2 ml of water is mixed with 5 drops of 1 % aqueous pyrogallol and 2 ml of cone, sulphuric acid. On boiling for 20-25 s, cooling, and diluting to 20 ml with alcohol, a purplish-red colour is given, fading in a few minutes. Ethylene glycol gives the same colour, thus casting doubt on the intermediate forma-tion of acrolein. In an analytical scheme for investigation of foodstuffs, and cosmetic and pharmaceutical preparations, Kroiler (1949) tested for glycerol in aqueous solution by adding fresh 5 % pyrogallol in cone, sulphuric acid and gently warming to yield a dark cherry red coloration; glycols gave a different, pale yellow colour. Salzer and Weber (1950) included pyrogallol-sulphuric acid (and vanillin-sulphuric acid) among detection reagents for polyols.

Hovey and Hodgins (1937) detected glycerol in dilute aqueous solution in

52 GLYCEROL 1.4

the presence of ethylene glycol by mixing 3 ml of sample with 3 ml of freshly prepared 10% aqueous pyrocatechol and 6 ml of cone, sulphuric acid. They heated for 30 s at 140-145°C, obtaining an orange-red colour. Other phenolic components were found to be much less satisfactory. They presumed that acrolein was first formed and then reacted further, but found that acrolein itself gave a purple precipitate with the reagent. Harvey and Higby (1951) adapted this qualitative test to the determination of glycerol. They heated 1 ml of sample with 1 ml of freshly prepared 10 % aqueous pyrocatechol and 4 m l of 3:1 sulphuric acid for 10min at 140-148°C, and evaluated the red colour at 510 nm or with a green filter.

Rosenthaler and Vegezzi (1954) suggested an improved detection of glycerol by dehydrating to acrolein in a tube and testing the issuing vapours for acrolein with paper impregnated with a solution of 4-hexylresorcinol in trichloroacetic acid. This gives a green or blue-green colour later turning red. Cohen and Altshuller (1961) used the 4-hexylresorcinol-trichloroacetic acid reagent to determine acrolein itself, though not as a product from glycerol dehydration; they evaluated the coloured product spectrophoto-metrically at 605 nm.

Fiirst (1948a) was able to detect as little as 15 jig of glycerol by heating 1-2 drops of test solution with 2 drops of 1 % naphthalene-2,7-diol in cone, sulphuric acid for 20-25 min on a steam bath; this gave a yellow to reddish-yellow solution, fluorescing deep green. He applied this (1948b) to detect glycerol in pharmaceutical preparations and cosmetics, first precipitating any sugars with calcium hydroxide and finally heating with phosphoric acid or boric acid in an air current, and detecting the acrolein dehydration product with a 0-01 % solution of the naphthalenediol in cone, sulphuric acid.

Bogs (1967) distinguished and identified glycerol and some common glycols by mixing 0-2 ml of sample with 2-0 ml of cone, sulphuric acid and then adding 01 ml of guaiacol RL (as quoted in the German Pharmacopoeia, DAB VII), finally diluting with an equal volume of water. He recorded the colour, also after 1 min and 5 min on the boiling water bath. The clearest distinctions were after heating for 1 min: ethylene glycol, pink; propylene glycol, dark red; triethylene glycol, red; glycerol, orange.

3. Other Aromatic Reagents Radley (1944) found that various compounds, e.g. ethylene glycol, tartaric acid, formaldehyde, and glycerol, gave fluorescent products with 0001 % acenaphthene-5-carboxylic acid in cone, sulphuric acid. Possibly first-formed acrolein condenses with one or both of the methylene groups.

Some aromatic aldehydes have been employed in acid solution as colour reagents for glycerol, and a likely explanation here also is initial dehydration to acrolein which can undergo aldol addition with the aromatic component.

1.5 REACTION WITH HYDRIODIC ACID Y I E L D I N G ISOPROPYL IODIDE 53

Godin (1954) used a 1:1 mixture of 1 % ethanolic vanillin and 3 % aqueous perchloric acid as a spray reagent for detecting 30 \ig amounts of glycerol and other sugar alcohols on paper chromatograms; on heating for 3 - 4 min at 85°C, pale blue or lilac spots on a pale sandy-coloured background were obtained. Graham (1965) found that sugar alcohols, including glycerol, yield orange, pink, or red colours on heating with cyclic aldehydes, thiourea, and cone, sulphuric acid in the Komarowsky reaction. Good sensitivities were obtained with p-hydroxy- and p-dimethylamino-benzaldehydes, and Graham adapted this quantitatively. Interfering carbohydrates had to be removed beforehand. Bleiweis and Coleman (1969) visualised glycerol, ribitol, and some sugars in TLC by spraying with the Morgan-Elson reagent of anis-aldehyde-sulphuric acid and then heating for 3-10 min at 100°C.

1.5. R E A C T I O N W I T H H Y D R I O D I C A C I D Y I E L D I N G I S O P R O P Y L I O D I D E

On heating with hydriodic acid, glycerol yields isopropyl iodide according to the reaction:

C H 2 O H C H 3

I I CHOH + 5HI « • CHI + 2I 2 + 3 H 2 0 I I

C H 2 O H C H 3

Zeisel and Fanto (1903) utilised this to determine glycerol. They boiled 5 ml of sample (not more than 5% glycerol) with 15 ml of hydriodic acid of density 1-9 (ca. 68 % acid), sweeping out the reaction products with a current of carbon dioxide. Most iodine and hydriodic acid were retained by a reflux condenser and returned to the reaction mixture, but any traces escaping were absorbed by a paste of red phosphorus and water. The gas stream containing the isopropyl iodide was led into 4% silver nitrate in ca. 90% ethanol, precipitating the double salt Ag l . A g N 0 3 . They filtered this off and heated a suspension in water for 30 min at ca. 100°C, yielding silver iodide that was easily filtered off; it was dried at 120-130°C and weighed. A reaction time of about 2 h was adequate for aqueous glycerol solutions. Zeisel and Fanto applied their method to determine glycerol in various wines and juices. Others used it also, for example: Ripper and Wohack (1916) for wines; Chapman (1926) for tobacco; Lacroix and Kropacsy (1928) for beer; and for glycerides (see Chapter 3, Section 3.1.7).

Criticisms of the method were expressed and improvements suggested. N o detailed discussion of this is made here and only a few references are given.

54 GLYCEROL 1.5

Neumann (1917) prevented interference of sulphur-containing substances by including cadmium sulphate in the wash solution to take out hydrogen sulphide derived from these by the reduction with hydriodic acid. Camillo Marchi (1923) found that allyl and propylene iodides were formed and escaped partly unchanged unless an acid such as acetic, propionic, succinic, or tartaric was included in the reaction mixture; the presence of acetic acid permitted hydriodic acid of density 1-71 (ca. 57%) to be used, and an im-proved wash solution contained slightly acidified lead acetate with the red phosphorus suspension.

In the early 1930s, the iodometric conclusion of Viebock and co-workers (1930) replaced the argentometric procedure; for example, von Bruchhausen (1934) observed that the new procedure was less sensitive to hydrogen sulphide and phosphine and of course had a more favourable conversion factor. In this procedure, the alkyl iodide is oxidised by bromine to iodate in two stages:

R—1 + Br 2 IBr + R—Br

IBr + 2Br 2 + 3 H 2 0 - > H I 0 3 + 5HBr

After removing unused bromine with formic acid, which does not reduce the iodic acid, the pH of the solution is lowered by adding sulphuric acid; on addition of iodide, iodine is liberated according to the well-known reaction:

H I 0 3 + 5 H I - * 3 I 2 + 3 H 2 0

This iodine is titrated in the usual way with thiosulphate. Modifications of this procedure too were introduced in later years. For

instance, Franzen et al. (1954) studied the reaction with thiosulphate wash solution (as used by Blix, 1937, for example) and found that there was negligible reaction between a 5% solution and isopropyl iodide; this contrasts with the observation of some reaction with ethyl iodide and extensive reaction with methyl iodide. The use of the thiosulphate wash solution does not therefore impair the accuracy of the results.

Kirsten and Nilsson (1960) investigated the reaction of some functional groups with hydriodic acid, using acid of density 1-70, of density 1-96, and a third reagent of density 1*70 containing red phosphorus, phenol, and propionic acid. With glycerol they obtained low results (ca. 70% recovery) with all three reagents. The results with the first-named reagent did not worsen appreciably when long (up to 24 h) digestion periods were used. But a digestion period of 24 h reduced recovery to only 16-2% using the second reagent and to 7-3 % using the third. This clearly casts doubt on the reliability of the method.

1.6 REACTION WITH INORGANIC CATIONS A N D ANIONS 55

Anderson and Zaidi (1963) determined 1,2-diols by refluxing with 55% hydriodic acid (density 1 -7) in nitrogen for ca. 30 min and then estimating the reaction products ethylene and ethyl iodide by IR measurements. It may be possible to determine glycerol in a parallel way although they do not mention this.

1.6. R E A C T I O N W I T H I N O R G A N I C C A T I O N S A N D A N I O N S

The formation of derivatives of polyols, including glycerol, with certain cations and anions has been known for many years. In most of these deriva-tives, hydrogen atoms of the hydroxyl groups are replaced, and links formed between the oxygen atoms and the metal or non-metal of the ion. The products may be covalent or ionic. Best-known are probably the derivatives of copper(II) and of borate. These and others are considered under the headings below.

1.6 .1 . W i t h C o p p e r ( l l )

These have been utilised in quantitative methods for glycerol, which depend on formation of soluble derivatives. These are separated and their copper content is estimated, giving a measure of the original amount of glycerol. Most work has been empirical and without reference to a definite equation or composition of product(s). A compound C 3 H 5 0 3 C u N a is quoted in Beilstein, probably to be formulated:

This may be a principal product. Other polyols can yield similar products and interfere with glycerol determination.

Probably the earliest method is that of Muter (1881) who treated a 1 g sample with 50 ml of cone. (1:1) potassium hydroxide and then added dilute copper sulphate until a permanent precipitate of cupric hydroxide was formed. After making up to a definite volume and allowing the suspension to stand until a clear supernatant was yielded, he removed an aliquot of this clear solution, added nitric acid and ammonium hydroxide, and titrated with potassium cyanide to decoloration. He corrected for the slight solubility of cupric hydroxide in the alkali by carrying out a blank without glycerol

C H 2 0 ,

C H 2 0 " N a +

56 GLYCEROL 1.6

sample. The potassium cyanide was standardised by repeating the procedure with a sample of pure glycerol.

This basic principle was retained by subsequent investigators, for example Wagenaar (1911) who treated the sample with an excess of alkali and copper salt; after a suitable interval (12 h in Wagenaar's method) an aliquot of clear supernatant is removed for estimating the copper content, generally iodo-metrically, by adding potassium iodide and sulphuric acid and titrating with thiosulphate:

2 C u 2 + + 2 r - * 2 C u + + I 2

Attention was later devoted to particular features in attempts to improve results. These included the composition of the reaction medium (added methanol or ethanol) and excess of copper(II) salt. For example, Schoorl (1939) said that too little excess copper gave incomplete formation of deriva-tive, and too much gave low results, probably as a result of adsorption of the derivative on the precipitate of cupric hydroxide. Later workers deter-mined the copper in solution by spectrophotometry at 635 nm or by com-parison with coloured standards. The experimental details of some pro-cedures are summarised in Table 1.4.

TABLE 1.4

Determination of Glycerol with Copper Reagents

Conditions for formation of copper-containing derivative

Concluding stage References

Sample ( < 10 ml containing max. of 800 mg glycerol) + water to give 10 ml, -I- 10 ml 30% NaOH + 60 ml ethanol + 10% aq. C u C l 2 . H 2 0 with shaking to faint permanent turbidity of Cu(OH) 2

10 ml sample (max. of 500 mg glycerol) + 10 ml 7-5N NaOH + 60 ml methanol + 10% CuCl 2 until no more formation of precipitate; equal volume of the CuCl 2

then added in excess; made up to 100 ml with methanol

Modification of SchoorFs procedure, using alcoholic CuCl 2 and adding only 0-5 ml excess after attainment of slight permanent precipitate; kept at 20°C; volume made up with alcohol

Centrifuged; copper Bertram and content of supernatant Rutgers (1938) determined iodometrically

Centrifuged; super-natant concentrated to a few ml and evaluated iodo-metrically

As in Schoorl's method

Schoorl (1939)

Andrews et al. (1941)


Conditions for formation of copper-containing derivative

Concluding stage References

10 ml sample (1-2-1-7 mg glycerol) + 10 ml 20% NaOH + 60 ml ethanol + 8 drops 10% CuCl 2 in ethanol, shaking after each addition until permanent turbidity; then + 10 ml CuCl 2 in excess, shaken for 2 min, and made up to 100 ml with ethanol

Max. of 10 ml neutral sample (100-800 mg glycerol) + 10 ml 30% N a O H + 60 ml ethanol containing 5 % methanol + 10% CuCl 2 in ethanol to permanent turbidity; made up to 100 ml with ethanol

Method of Bertram and Rutgers (1938) for glycerol extracted from leather

10 ml sample (18-64 mg glycerol) + 10 ml 21 % NaOH + 60 ml ethanol + 6-0 ml 10 % C u C l 2 . 2 H 2 0 in 95 % ethanol added with swirling; shaken for 2 min, then made up to 100 ml with ethanol

10 ml sample (50-400 mg glycerol) + 10 ml 7-5M N a O H + 60 ml 95 % ethanol + 1M CuCl 2 in small amounts shaking 1 min after each addition until slight permanent precipitate; + 2 0 ml 95% ethanol, shaken 1 min, more CuCl 2 if needed to yield permanent precipitate

5 ml sample (0-5-40 mg glycerol) + 7-5 ml 10M N a O H + 50 ml 95 % ethanol, then treated with 1M CuCl 2

as in their other publication

5 ml sample + 1 ml H 2 0 + 0-5ml0-5M C u S 0 4 + 0-6 ml 25 % NaOH (for glycerol in salt solutions)

5 ml 5 % C u S 0 4 + either 3-5 or 5 ml 7M KOH + 0-5-4-5 ml polyol (containing 10 mg of a binary mixture of glycerol with xylitol or sorbitol), diluted to 50 ml

Centrifuged 12-15 min; supernatant evaluated spectro-photometrically

Filtered; aliquot estimated iodo-metrically

Centrifuged; copper(II) estimated at 635 nm

Filtered; copper determined iodo-metrically

Filtered; Cu(II) determined spectro-photometrically at 635 nm

Centrifuged 5 min at 1500 rpm; colour of supernatant compared with standards

Centrifuged 5 min; absorbance of each solution determined with red filter; composition derived from the two results

Whyte (1946)

Bore (1950)

Seligsberger (1950)

Spagnolo (1953)

McAloren and Reynolds (1965a)

McAloren and Reynolds (1965b)

Lisetskaya et al. (1971)

Talipov et al (1972)

58 GLYCEROL 1.6

It is perhaps surprising that copper(II)-impregnated thin layers do not appear to have been used to separate polyols from other compounds or from one another.

1.6.2. W i t h Other Cat ions

Few other cations appear to have been used in analytical procedures for glycerol. De Simone and Vicedomini (1968) separated polyols through TLC on silica gel H prepared containing lead nitrate (slurry of 30 g of adsorbent and 70 ml of 0-1M salt), activated by heating for 30 min at 110°C. They used ammoniacal ethanolic solvents and found that retardation was greater for compounds containing more adjacent hydroxyl groups, e.g. glycerol or erythritol, especially when the solvent contained water. These compounds form more stable complexes, for which the authors give the equilibrium reaction:

Pb(OH) 3 " + P ( p o l y o l ) ^ P b P " + n H 2 0

It is interesting to note that in 1880 Morawski reported an attempt to deter-mine glycerol by heating 2-3 g of sample with 50-60 g of lead oxide (both accurately weighed) at 130°C to constant weight. The change in weight from the original lead oxide was related to formation of the product C 3 H 6 0 3 P b , presumably

C H 2 0

I ^ P b C H O ^

C H 2 O H

Dallas and Stewart (1967) separated poly glycerols and glycerol on kieselguhr-silica gel layers prepared by slurrying with 0 0 4 5 M calcium chloride, using ethyl acetate-isopropanol-water (110 + 61+29) as solvent mixture. Impregnations with metal salts were employed also by Turgel' et al. (1972) for separating and analysing reaction mixtures from isoprene synthesis which included diols and triols (not, in fact, glycerol itself). They carried out TLC on alumina containing nickel sulphate, silver nitrate, or calcium chloride. The best separations of diols and of diols from triols were on

1.6 REACTION WITH INORGANIC CATIONS A N D ANIONS 59

alumina containing ca. 1% calcium chloride. Their best separation of two pentane triols was on the other two layers, using chloroform-ethanol (3 + 1).

1.6.3. With Borate

Polyols form complexes with certain anions, of which, as already stated, borate is the best-known:

H O

H O OH H O H O O \ Y O O v / \ v / \ *«/ / \y \ B + B X - 2 ^ * X B X

/ \ / / \ / \ / \ / H O OH H O H O O O O

Such complex formation influences the migration of polyols (including carbohydrates, of course) in chromatographic and electrophoretic pro-cedures. This has been studied and employed to separate polyols from other compounds and from one another. Hockenhull (1953) used borate-impreg-nated Whatman No. 4 paper in a 15 h ascending separation of polyols, with n-butanol-pyridine-water (3 + 2 +1-5 or 4 + 1 + 1). Gross (1955) separated polyols in 0-05M borax, pH 9-2, by paper electrophoresis at 80V/cm. Sargent (1956) also separated polyols (e.g. ethylene, propylene, and diethylene glycols, glycerol) by anion-exchange chromatography of their borate com-plexes. Kroiler (1963) identified tobacco additives (glycol, glycerol) in an acetone extract by PC of the borate complexes, using propanol-25% ammonia (7 + 3) as mobile phase. Ikawa et al. (1966) identified glycerol in bacterial cell walls after ultimate PC using a mobile phase of isopropanol-5% boric acid (7 + 1).

Some examples of the use of borate in thin-layer chromatography can also be quoted. Prey et al. (1962) separated glycerol, ethylene glycol, mannitol, and sorbitol on silica gel G using butanol-water (9 + 1) as mobile phase. They found that the separation of the first two was improved by impregnation with 0-1N boric acid. Ehrhardt and Sucker (1970) used TLC on silica gel G, buffered with boric acid, to separate polyols and sugars, their solvent being propanol-methyl ethyl ketone-water-ethyl acetate ( 2 0 + 1 0 + 1 0 + 1 ) .

Borate complexes with polyols have been mentioned in reviews by Foster (1957) (in connection with zone electrophoresis) and Weigel (1963) (paper electrophoresis).

Organic borates in which one or more hydroxyl groups are replaced by an organic group (usually by the phenyl group) have also been studied. These cannot form the double complex anion like borate and react only

60 GLYCEROL 1.6

according to:

C 6 H 5 OH H O C 6 H , O V / \ \ / \

B + X->- B X / \ / / \ /

C 6 H 5 OH HO C 6 H 5 O

Diphenylboric acid

Garegg and Lindberg (1961) found that sugar mobilities at pH 7 in the presence of phenylboric acid differed little from those in the presence of borate, despite this cdmplexation limitation. Bourne et al (1963) carried out PC of carbohydrates and related compounds on Whatman No. 1 paper, in the absence and in the presence of phenylboric acid (0*55%), visualising them with periodate-cuprate and rosaniline. Garegg and Lindstrom (1971) studied the complexes between polyols and diphenylborate buffer at pH 10 and also reported that mobilities were very similar to those in the presence of borate itself. They carried out paper electrophoresis on Whatman 3MM paper, at 20 V/cm for 2-5 h, visualising with the periodate-benzidine reagent (Section 1.1.14. A).

Barker et al (1973) used poly(4-vinylphenylboric acid) resins as chromato-graphic packings to fractionate carbohydrates, with water as eluent. They studied the effect of pH and temperature.

The complexes resulting from reaction of polyols and boric acid are stronger acids than boric acid itself. This has been utilised in analytical chemistry to enable boric acid to be titrated and also to detect glycerol (and other polyols). Borax solution, of pH 9-2, turns phenolphthalein purple. On adding a polyol, this colour disappears almost instantaneously. Feigl (1966c) profits from the more strongly acidic complex in a more sophisticated test. He prepares the Griess test reagents: 2-9g of sodium sulphanilate + 0-7g of sodium nitrite in 30 ml of water; and 1-8 g of oc-naphthylamine in 40 ml of dioxan or ethanol. A mixture of one drop of each shows no reaction until acidified. Then nitrous acid is formed, diazotising the sulphanilic acid, and the diazonium salt couples with the naphthylamine to yield a red or orange product. With the very weak boric acid, colour appears only after 10-15 min. With boric acid plus a few drops of a 1,2-diol, colour formation is within seconds.

1.6.4. W i t h Other A n i o n s

Weigel's review (1963), mentioned above, refers also to complex formation between polyols and numerous other anions, such as arsenite, stannate, germanate (including the work of Lindberg and Swan, 1960; Everest and

1.6 P U R E L Y PHYSICAL METHODS 61

Harrison, 1960; and Popiel, 1961), tellurate (work of Roy, 1967; and Popiel, 1961), tungstate and molybdate.

These other anions have found some use in chromatography. For instance, Lees and Weigel (1964) carried out electrophoresis of polyols on Whatman 3MM paper using 2% aqueous sodium stannate (pH 11-5). They observed that migration was controlled primarily by complex formation and that threo groups yielded more stable complexes than erythro groups. Guerra Salazar (1962) impregnated Whatman No. 1 paper with 2% ammonium molybdate at pH 5 and chromatographed sugars and other polyols in butanol-pyridine-water (6 + 4 + 3) for 26 h.

Plsko (1958) described a test closely similar to that based on the decolora-tion of the phenolphthalein alkaline colour but he used sodium tungstate or molybdate instead of borax. This decoloration served as a test for compounds such as sugars, sorbitol, and glycerol.

1.7. PURELY P H Y S I C A L M E T H O D S

There is relatively little published work on detection and determination of glycerol by purely physical methods. Some information is summarised below. Probably such methods have been often used in routine work and it was not felt worthy of publication.

1.7 .1 . Refract ive Index

The property of refractive index has been used for many years to determine glycerol concentrations in aqueous solution. Glycerol has the value of 1 -4746 at 20°C (Handbook of Chemistry and Physics, 1972/73a) for the usual sodium light, conveniently far removed from the value for water. The curve relating glycerol concentration and refractive index is not linear but it is well established. In glycerol determinations in various materials, an aqueous extract is made and potentially interfering substances such as salts are removed by standard procedures including precipitation, extraction, or distillation before finally measuring the refractive index of the extract and reading off the glycerol amount from the calibration curve; sometimes correction is made for the influence of other materials instead of separation.

von Schltitter (1932), for example, used this to determine glycerol in threads and films after water extraction; Kulikov (1940), in water extracts of lacquers; Matsumoto (1940), in soap waste liquors, correcting for sodium chloride and carbonate which were determined volumetrically in separate experiments; Larmer (1956), in the presence of low boric acid concentrations for which a correction was made after determination by titration with alkali;

62 GLYCEROL 1.7

Aleksandrovich and Kuritsina (1962), in cellophane; and Zamyshevskaya and Yaroshinskaya (1965), in cellophane film which they cut into squares and extracted with water for 10 min at 18-20°C.

The relation between refractive index and concentration of glycerol in some pharmaceutical preparations was given by Szigetvtfry and Kuttel (1963). Refractive indices for 24 compounds, including glycerol, were quoted by Belikovskii (1972) who also gave equations for analysis of binary mixtures of these compounds. Values of the refractive index (and also absorption in the ultraviolet) were given for 5 -80% aqueous solutions of polyols (ethylene glycol, sucrose, glycerol) by Krivacic and Urry (1971) although they made no direct suggestion of analytical use.

Mares (1957) stated that the degree of etherification in the formation of polyglycerols from glycerol can be followed simply by refractive indices.

Paquet (1946) reviewed the use of refractive index to determine glycerol in aqueous solutions.

In recent years refractive index measurements have been used regularly to monitor eluates from column chromatographic and ion-exchange pro-cedures. Examples in which glycerol has figured are given in Tables 1.8 and 1.9.

1.7.2. Densi ty , Surface Tens ion , and Crit ical S o l u t i o n Temperature

Very little can be reported under this heading. Guseva (1954) analysed glycerol-water mixtures through their density, correcting for ash and organic matter, and claimed that the method was as accurate as oxidation with dichromate. The density of glycerol at 20°C is 1-2613 (Handbook of Chemistry and Physics, 1972/73a) which is clearly different from that of water. The property of critical mixing temperature was utilised by Kartnig and Kren (1963) to determine polyols in mixtures with water. They employed nitro-methane, -propane, and -benzene, and benzene as solvents.

Other properties have been used in conjunction with refractive index. For example, Lopez and Casares (1948) used the Gladstone constant, (n — l)/d, where n is the refractive index and d the density, to determine the glycerol content of commercial glycerol. This has the value of 0-3749 for glycerol and 0-3390 for water, and they were able to interpolate on the straight line relating the constant to composition. Fischer and Kolmayr (1955) assayed glycerol-glycol-water mixtures with the help of a nomograph of refractive index values and critical mixing temperatures with acetophenone in the 60-220°C range. Jasinski and Grabowska (1955) analysed some prescription mixtures such as glycerol-phenol-water and glycerol-borax-water using refractive index, density, and surface tension data. They evidently extended this work, and their later publication (1956) included the example of glycerol-ethanol-water.

1.7 PURELY PHYSICAL METHODS 63

1.7.3. V iscos i ty

Glycerol has a viscosity of 2330 cP at 15°C, 1490 cP at 20°C, and 954 cP at 25°C, values taken from the Handbook of Chemistry and Physics (1972/73b). N o other commonly encountered organic compound—and certainly no other likely component of technical glycerol—has a viscosity anywhere near this. For water the corresponding values are 1-139, 1-002, and 0-8904 cP, respectively. In view of this, it is very surprising that this property in particular has not found much use. As mentioned above, analyses through viscosity values may indeed be extensive but judged as too obvious to be worthy of publication.

The only work that can be quoted is that of Haendel (1973) who analysed mixtures of glycerol and glycol using a capillary viscometer, measuring the flow times at 10° intervals between +90° and +10°C. Viscosity measure-ments were employed also to determine the water content of glycerol. Betteridge and Ruzicka (1976) tested glycerol-water mixtures in their "flow injection analyser". The sample is injected into a stream of 0-001 % Bromo-thymol Blue in dilute borax buffer (pH 9-2). The viscosity of the sample plug, which depends on the glycerol content, determines the degree of mixing with the dye solution which is shown through the reduction in absorbance (at 620 nm) of this solution further down the line. The authors observed this correlation and suggested the possibility of analysis of glycerol-water mixtures.

1.7.4. Infrared S p e c t r o p h o t o m e t r y

Infrared measurements appear to have been used only rarely in analytical work on glycerol. Shay et al (1954) identified the polyol component (in-cluding glycerol) in polyester hydrolysates, recording the IR spectra between 700 and 1500 c m - 1 .

Hasegawa et al (1964) analysed anti-icing additives in jet fuel by com-paring IR absorption at 1130 c m - 1 of sample and of sample after extraction with water, which removes the ethylcellosolve and glycerol additives. Binary mixtures of brake fluid components (ethanol, butanol, isoamyl alcohol, and glycerol) were analysed by Gordienko et al (1964) using IR data; they measured at 992 c m - 1 for glycerol determination. Sushkov et al (1973) determined the glycerol content of regenerated cellophane film by recording the IR spectrum between 800 and 1000 c m - 1 and evaluating the intensity of the bands at 860 c m " 1 (methylene groups) and 895 c m - 1

(hydrocellulose); they related the glycerol content to D860/D895.

64 GLYCEROL 1.7

1.7.5. Nuclear M a g n e t i c Resonance

Although NMR has been applied with analytical aims to alcohols, polyols appear very rarely among the examples. Sawyer and Brannan (1966) carried out NMR studies in deuterium oxide of polyols, also hydroxy-acids, quoting chemical shifts for the a,P, and y hydrogens of glycerol, but did not mention any possible analytical use. Arapbaev et al (1971) monitored glycerol-water mixtures with the help of NMR measurements.

1.8. N O N - C H R O M A T O G R A P H I C S E P A R A T I O N M E T H O D S

Classical methods for separation of glycerol from other compounds, often with the aim of determining it, include extraction and distillation. Both of these have been used for probably longer than a century and, even if some-what superseded by chromatographic procedures, merit treatment here.

1.8 .1 . So lvent Extract ion

In early methods glycerol was often detected, identified, or determined in a sample by direct extraction with an appropriate solvent. It was then identified or determined in this extract by conventional methods, or the extract was evaporated to leave a residue identified as glycerol or weighed in a simple gravimetric procedure.

However, glycerol is highly polar, and this limits the number of available solvents. Water is, of course, a natural choice since it is miscible in all pro-portions with glycerol. It can be used to separate glycerol when the sample contains no other water-soluble material that can interfere. Examples are the work of Schutz (1938) who thus extracted glycerol from parchment paper; Kulikov (1940), from lacquers; and Wright (1963), from tobacco. These three concluded with, respectively, detection using anthrone-conc. sulphuric acid (Section 1.4.5.E.1), measurement of refractive index (Section 1.7.1), and TLC (Section 1.9.3). Water is difficult to remove quantitatively, so the simple gravimetric principle mentioned above cannot be applied with accuracy.

Shukoff and Schestakoff (1905) mixed their glycerol-containing sample with powdered anhydrous sodium sulphate, yielding a mass which they then extracted with dry acetone in a Soxhlet apparatus. They claimed reasonably quantitative extraction of the glycerol which could be determined from the residual weight after easy evaporation of the volatile solvent. This extraction principle was used by numerous subsequent investigators in this domain, for example: Fachini and Dorta (1910) for crude glycerol and aqueous

1.8 NON-CHROMATOGRAPHIC SEPARATION METHODS 65

glycerol extracts; Chapman (1926) for tobacco; Lazar and Meyling (1938) for manufactured tobacco; Tasman and Smith (1943) for food products; Druce (1952) for traces of glycerol in the presence of sugars; Procopio and Antona (1960) for sweet and aromatic wines; and Venturini (1972) for liqueurs and syrups.

Occasionally acetone seems to have been used without the sodium sulphate, for example: by Kai Ho and Tzu-Hui Cheng (1933), for egg yolk; by Friedmann and Raab (1963), for polyols in tobacco humectants (Soxhlet extraction for 4 h , evaporation to dryness and freeing from waxes by washing with benzene); by Kroiler (1963), also for glycol and glycerol in tobacco additives; by Doihara et al. (1965), extracting polyols from cigarette smoke with 98-7% acetone; and by Nisbet and Schmeller (1970), who also extracted glycerol from tobacco with aqueous acetone. Monterumici (1932) removed fat from tinctures and extracts with ligroin and then refluxed with dry acetone containing 10% absolute alcohol, finally evaporating the solvent, drying at 70°C for 1 h, and weighing the residue.

Methanol and ethanol have also been used to extract polyols from tobacco materials, especially in more recent work where a chromatographic stage has followed. Thus, Cardini et al. (1967) extracted glycerol-containing pharmaceutical preparations with 95% ethanol; Nishi et al. (1969) also used ethanol; Venturini (1972) extracted glycerol from 5 g samples of liqueurs and syrups with ethanol + 10 g of anhydrous sodium sulphate; Giles (1970) shook for 1 h with methanol; Williams (1971) also employed methanol; and Carugno et al. (1971) extracted for 5 h with methanol. In all these cases, the extract was subjected to gas chromatography.

A mixture of ethanol and diethyl ether was used by, for example: Bohanes (1935), to extract glycerol from dye suspensions for printed fabrics, adding absolute ethanol and then ether; Diemair et al. (1940), in determination of glycerol (and lactic acid) in wine; Amerine and Dietrich (1943), also for wine, using a mixture of 3 parts of ether to 2 parts of alcohol in which mannitol was claimed to be insoluble; Fiirst (1948b), for pharmaceutical preparations and cosmetics; and Mathers and Pro (1954), for foods and medicinals.

Seligsberger (1950) extracted glycerol and other polyols from leather in a Soxhlet apparatus for 6-12 h with propyl acetate. According to Jacquin and Tavernier (1952), glycerol is about six times more soluble in propyl acetate than in diethyl ether (ca. 4-33 and 0-77 g/1 respectively).

Many samples, for example of wines, contain sugars which have solubilities similar to those of glycerol. They are thus extracted as well and can interfere in many determinations, for example those using periodate. Various pro-cedures of precipitation have been suggested over the years, such as with barium hydroxide-alcohols, lead acetate, or silver nitrate (e.g. work of Hoepe, 1943). This preparation of the sample and prior removal of eventual

66 GLYCEROL 1.8

interfering materials falls rather outside the scope of this monograph. In any case, the problems associated with this separation have largely vanished with the advent of chromatography.

1.8.2. D is t i l la t ion

Because of its high boiling point it is difficult to purify glycerol by distillation and hence to estimate it in this way. Even under reduced pressure there is a tendency to decomposition to give acrolein. Lower boiling impurities should normally be removable by distillation but here too there is a danger of co-distillation of the glycerol itself. Water is commonly present in glycerol samples but, although its boiling point lies nearly 200° below that of glycerol, is it not possible to remove it quantitatively by distillation without some loss of glycerol.

Steam distillation has been used in attempts to profit analytically from this volatility. This enables glycerol to be freed from many otherwise inter-fering materials, and the glycerol-water distillate can be subjected to analysis as a binary mixture, or to determination of the glycerol content by a chemical procedure. Janssens (1906) described an apparatus for heating samples at 200°C and passing through steam, finally estimating glycerol in the distillate through density measurements or dichromate oxidation, von Fellenberg (1931) also removed glycerol from wine by steam distillation, finding that 10 min at 115°C sufficed. Okuhara and Yokotsuka (1958) separated glycerol from soy sauce by adding excess water and steam distilling under reduced pressure (20 mm Hg). They determined the glycerol in the distillate through periodate oxidation. In their opinion this was better than precipitating impurities or extraction with solvents. In a patent of Kyowa Chemicals (1967), steam distillation below 200°C and at ca. 35 mm pressure was suggested to separate glycerol from other triols such as butane-1,2,3- and -1,2,4-triols.

Some other entrainers have been suggested also, usually to remove the glycerol but also to take out water. For example, Schaefer (1937) determined glycol or glycerol in dilute (ca. 5 %) aqueous solutions containing oxidisable impurities by adding pyridine and distilling until the temperature reached 110°C. This removed most water without loss of polyol. He was then able to determine the polyol in the more concentrated solution using an acetylation procedure. Thivoile and Raveux (1942) replaced steam distillation by entrainment with ethanol to separate glycerol from complex media.

Hydrocarbon entrainers, such as limonene, appear to have been most used for separating glycerol. According to the tables of Horsley (1947) d-limonene and thymene give the lowest-boiling azeotropes (below 180°C) with glycerol, but these contain only 1 % of the polyol. Turpentine was used for this purpose for glycerol (and other polyols) by Palfray et al. (1946). Metayer (1947) tested

1.9 CHROMATOGRAPHIC METHODS 67

several entrainers for polyols; he found that decalin was better than spirit of turpentine, especially for glycerol and water in creams, and that d-pinane was slower than turpentine. Decalin was used by Griffin (1954) to separate glycerol and propylene glycol from desiccated coconut, distilling at ca. 200°C; he then used two periodate oxidation procedures on the distillate to determine the sum of these two polyols and then the glycerol alone. Mathers and Pro (1954) used a light mineral oil to distil propylene glycol and glycerol from an extract of foods and medicinals, following then also with a periodate oxidation and titration of formic acid product to determine the glycerol.

Rosenberger and Shoemaker (1957) analysed three-component mixtures of water, ethylene glycol, and glycerol by using three separate entrainers. Distillation with benzene removed only water; with tetrachloroethylene, glycol and water; and with ( + )-limonene, all three. Several hours distillation were necessary.

Polyols, especially glycerol, have been extensively studied chromatographic-ally. Under this heading are included procedures without prior chemical treatment such as the ester and ether formation described in Sections 1.2 and 1.3. Citations here are almost entirely limited to work on glycerol or on polyols which included the example of glycerol; in rare cases other publica-tions are quoted here also but only where it is evident that the procedure would apply by analogy to glycerol. A convenient classification is into the various chromatographic techniques.

1.9 .1 . Gas C h r o m a t o g r a p h y

The amount of direct gas chromatographic work on glycerol and polyols is surprising in view of their low volatility. Most of the analytical work has been performed on tobacco samples (polyols, especially glycerol, are popular

1.9. C H R O M A T O G R A P H I C M E T H O D S

TABLE 1.5

Gas Chromatography of Glycerol and Other Polyols

Sample Gas chromatographic details References

Propane-1,3-diol in glycerol

Polyester Reoplex 400 + 60-85 mesh Clifford (1960) Celite (1 + 3); 120°C; Ar carrier gas; Ar-ionisation detector

Propane-1,3-diol in glycerol

25 % Reoplex 400 on 60-100 mesh Embacel Murray and kieselguhr; 150°C; N 2 gas; H 2 - F I D Williams (insensitive to water) (1961)



Mixtures of glycerol with mono-, di-, and tri-ethylene glycols

Tobacco humectants (4 h Soxhlet acetone extract, including glycerol and glycols)

Polyols

Glycols (ethylene, propylene, diethylene), glycerol

Polyols in cigarette smoke, extracted with 98-7% acetone; aqueous extract used for GLC

- C 3 alcohols in mixtures with alkanes and O-containing compounds

Glycerol in pharm. analysis; extracted with 95 % ethanol

Glycerol in cosmetics (aqueous samples first dehydrated by adding 2-methoxyethanol and evaporating to 1 ml)

Glycerol in cosmetics

Stainless steel column; polyphenyl ether-Car bo wax (10% + 2%) on Fluoropak 80; 75 to 200°C at 15°/min; He gas; thermal conductivity detector (F and M model 500)

5 % Carbowax 1500 on Haloport F; 50 to 200°C at 9°/min, then constant; F and M model 300

30% Polyoxyethylene glycol 10000 on Celite 545; 138-145°C; He gas; thermal cond. detector

25 % Acetylated dextrin on Celite 545; 220°C; He gas; thermal cond. detector

Stainless steel column; 3 % PEG 3000 on 100-180 mesh Shimalite Q; 120 to 210°C at 5°/min; He gas

27% Triethanolamine on Inza clay; 85°C; He gas (glycerol gave poor results)

Stainless steel column; 15% Carbowax 20M on Chromosorb W, pretreated with 5 % KOH; 200°C; N 2 gas; differential flame ionisation (Perkin-Elmer FII)

In absence of water on a copper column of 5 % sucrose diacetate hexa-isobutyrate on silanised acid-washed Chromosorb G; ca. 200°C; He gas; thermal cond. detector

Collaborative study of the method of Gross and Jones (1967); impregnation bled excessively at 200°C, hence used at 17O-180°C

Ghanayem and Swann (1962)

Friedman and Raab(1963)

Balakhontseva and Poltinina (1964)

Balakhontseva and Poltinina (1965)

Doihara et al. (1965)

Kolesnikova et al. (1965)

Cardini et al. (1967)

Gross and Jones (1967)

Gross (1967)



Firebrick (50 mesh) treated Nonaka with 20 % KF solution at room temp.; (1967) 130°C; H 2 Q carrier gas; F I D

Many compound classes (alcohols, hydrocarbons, carbonyl compounds, polyols e.g. ethylene and propylene glycols, glycerol)

Diols, glycerol Copper column of 10 % poly(diethylene Vaver et al. glycol succinate) (LAC-3R-728) on (1968) silanised 60-80 mesh Chromosorb W; 3 min/80°C, then 4°/min to 185°C; Ar gas; FID; butane- 1,4-diol internal standard

Firebrick treated with phosphoric acid Nonaka (for glycerol); H 2 0 carrier gas (1968)

25 % Acetylated dextrin on Chromo- Kochnova sorb W; 225°C; He gas et al (1969)

2-5 % Polyoxyethylene glycol 4000 Filatova et al. on Chromosorb 102 (also Chromosorb (1969) 102 without impregnation); 215-220°C; H 2 gas; thermal cond. detector

Glass columns; 120-200 mesh Polypak Dooms et al. 1; 250°C or programmed from 150 to (1969) 250°C (glycerol emerges at 180°C); N 2 gas; FID

20% l,2,3,4,5,6-Hexa-(2-cyanoethoxy) Usmanskaya hexane on Chromosorb W at 220°C, or on et al (1970) Polysorb 1 at 178°C; He gas; thermal cond. detector

8 % Poly(ethylene glycol succinate) on Melkonyan 0-25-0-4 mm Armenian tuff; 100-180°C; (1970) H 2 gas; FID

Glycerol, propane- Copper column; 5% Carbowax 2 0 M - Giles 1,2-and-1,3-diols in terephthalic acid on 60-80 mesh (1970) MeOH extract of Diatoport S-Chromosorb 101 (1 + 1); Williams tobacco (collaborative 90 to 240°C at 15°/min; He gas; thermal (1971) studies) cond. detector; internal standard anethole

(Giles) or butane-1,3-diol (Williams)

Polyols, fatty acids, phenols, amines

Binary mixtures of various polyols (glycols, glycerol)

Glycols, glycerol from hydrogenation of monosaccharides

Polyols (glycerol; C 5 and C 6 sugar alcohols) in biological e.g. fermentation, media

Glycols and glycerol from hydrogenation of sugars

Diols, glycerol

D

70 GLYCEROL


1.9


Ethylene and propylene glycols, glycerol

Glycerol, some halides and halo-alcohols

Polyols (glycerol, diethylene glycol) in tobacco smoke

Glycerol, diethylene glycol, trimethylol-propane, also from ester hydrolysates

Diols and glycerol in flavour bases and flavoured wines

Porous polymers, especially Polysorb-1; 100 to 220°C at 5°/min; He gas; thermal cond. detector; cyclohexanol internal standard

Stainless steel column; 4 % polyoxy-ethylene glycol on NaCl; H 2 gas; FID

Porapak P: 140 to 230°C at 5°/min; N 2 gas

Maksimenko et al. (1973)

Tomi et al. (1974)

Sakagami and Fukuzumi (1974)

15 cm column; 15% Apiezon L on AW- Tsarfin and DMCS Chromaton N + 35 cm of 10% Kharchenkova 1,2,3,4,5,6-hexa-(2-cyanoethoxy)hexane (1975) on same support; 142°C; Ar gas; FID

Three columns: 100-120 mesh Chromosorb Martin et al. 101 at 220°C; 2 % SP-1000 on 100-120 mesh (1975) Chromosorb 101 at 210°C; 10% SP-1000 on 100-120 mesh Chromosorb W at 170°C; FID; third column best

humectants), cosmetics, and products of energetic hydrogenation of mono-saccharides. Table 1.5 contains a selection of examples with summarised information.

Some investigations may be quoted separately. Kiselev and Yashin (1966) tested a column of activated carbon at 220-245°C for the separation of Cx

to C 3 alcohols, including glycols, glycerol, and propane-1,3-diol. Assmann et al. (1967) prepared liquid phases from poly(vinylformal) of 14-8 % hydroxy-vinyl content, t-butyl alcohol, and acrylonitrile, giving as general formula for these products:

- C H C H 2 \ / — C H C H 2 - \

O C H 2 C H 2 C N / y \ O C O C H 3

These could be used at temperatures up to 300°C without bleeding, and the authors quote examples of polyol separation (e.g. glycerol, and ethylene and propylene glycols) on a column of 20% liquid phase on 0-09-0-25 mm Celite at temperatures programmed from 140 to 290°C at 12°/min and using hydrogen gas and flame ionisation detection.


Levins and Ottenstein (1967) studied the effect of the tubing material on the gas chromatography of propylene glycol, glycerol, vanillin, and ethyl-Vanillin. They used a column of 5% Carbowax 20M or of FFAP on 80-100 mesh AW-DMCS Chromosorb G. Symmetrical peaks were obtained in copper and glass columns. Aluminium and stainless steel tubes showed adsorption. Symmetrical peaks could be obtained with the aluminium tubes by pretreatment with 5 % FFAP in chloroform and then evaporation of the solvent to give an inner layer.

1.9.2. Paper Chromatography

A wide variety of samples has been subjected to paper chromatography. Food (wines, vinegar), tobacco (humectants), and pharmaceutical prepara-tions (cosmetics) constitute typical domains, as observed in gas chromato-graphy also. Most problems are of separation of polyols (including sugars) from one another. Table 1.6 contains some summarised information.

TABLE 1.6

Paper Chromatography of Glycerol and Other Polyols


Polyols Whatman No. 1; various solvent mixtures, all containing water and n-butanol, with ethanol, benzene, pyridine, acetic acid;

visualised with ammoniacal A g +

Hough (1950)

Polyols, carbohydrates

Whatman No. 1; descending, with n-butanol-acetic acid-water (4 + 1 + 5) or n-butanol-acetic acid-water-conc. HC1 (20 + 5 + 25 + 1); visualisation with periodate-Schiff reagent, periodate-iodide, or Pb(IV)

Borate-impregnated Whatman No. 4; ascending, n-butanol-pyridine-water (6 + 4 + 3) or (4 + 1 + 1 ) ; visualised with indicators (Methyl Red, Phenol Red)

Buchanan et al (1950)

Polyols, carbohydrates

Hockenhull (1953)

Polyols from foods Rf values given with lower alcohols, chloroform-ethanol mixtures and water-saturated ether

Bergner and Sperlich (1953a)

Wine glycerol Whatman No. 1; descending, upper layer butanol-acetic acid-water (4 + 1 + 5 ) mobile phase and lower layer in chamber; visualised with Ag + -a lka l i

Palleroni and Vega (1954)



Carbohydrates and related compounds

Glycerol

Polyols, e.g. glycols and glycerol

Glycerol and other polyols up to C 6

Polyols, sugars

Glycerol and dihydroxyacetone in vinegar and vinegar malts

Polyols, C 2 - C 6

Polyglycerols, including glycerol

Polyols and sugars

Glycerol and C 2 - C 6

polyols in cosmetics

Glycerol in tobacco

Whatman No. 4; ascending, isopropanol- Gordon et al. pyridine-acetic acid-water (8 + 8 + 1 + 4 ) ; (1956) visualised with periodate-benzidine or K M n 0 4

Mobile phase n-propanol-water (4 + 1); Olley (1956) visualised with Ag + -a lkal i

Mobile phase acetone-chloroform (9 + 1) Tupalska and chamber moistened with water- (1957) chloroform-acetone (20 + 4 + 1)

Whatman No. 1; water-saturated butanols; Smullin et al. visualised with ammoniacal A g + ; also (1958) eluted with water and determined with periodate

Mobile phase n-butanol-acetic acid-water Cerutti and (400 + 100 + 247); visualised with Cresol Vezzini (1961) R e d - N H 4 O H , drying, and inspection in UV

Mobile phase ethyl acetate-acetic Hromatka acid-water (10 + 1-3 + 1); visualised with (1962) Ag +

Mobile phase pyridine-ethyl acetate- Vasyunina water (2 + 7 + 1); visualised with et al. (1962) periodate-benzidine

Whatman No. 1; descending, butanol- Zajic (1962) acetic acid-water (12 + 3 + 4); visualised with Ag + -a lkal i (spot areas measured)

Whatman No. 1 impregnated with 2 % Guerra ammonium molybdate at pH 5; butanol- Salazar pyridine-water (6 + 4 + 3); visualised with (1962) Ag + -a lka l i

Schleicher-Schull 2043; ethyl acetate- Hennies and ethanol-water ( 1 2 + 1 2 + 1), butanol- Eckert (1963) acetic acid-water (4 + 1 + 5), or best, chloroform-ethanol (4 + 1); visualised with 0 1 N - A g N O 3 - 5 N - N H 4 O H ( l + 1) spray, then 5-10min/105°C

Whatman No. 1 (impregnated with Patterson A g N 0 3 - a c e t o n e for ultimate detection); (1963) n-butanol saturated with water (ascending and descending)



Glycol, glycerol as tobacco additives

Glycerol-dihydroxy-acetone

Carbohydrates and related compounds

Poly glycerols

Glycerol in complex pharm. preparations

Glycerol in bacterial cell walls

Glycerol deter-mination in culture fluid of Bac. brevis var GB

Glycerol and glucose in plasma

Polyols

Boric acid complex of acetone extract; Kroller

Polyols from glucose hydrogenolysis

propanol-25% N H 3 (7 + 3); visualised with (1963) ammoniacal Ag +

Mobile phase phenol-xylene (1 + 1) three- Waldi and quarters saturated with water; detected Lange (1963) with periodate-benzidine

Whatman No. 1 with or without 0-55% Bourne et al. phenylboric acid; ethyl acetate-acetic acid- (1963) water (9 + 2 + 2); visualised with periodato-cuprate and rosaniline

Whatman 3MM; descending, water- Siegel et al. saturated butanol; detected with periodate- (1964) benzidine (spot areas estimated) or ammoniacal Ag +

Circular PC; water-saturated butanol; Datta and detected with ammoniacal A g + Ghosh (1965)

Mobile phase isopropanol-5 % boric Ikawa et al. acid(7 + l) (1966)

Ultimate PC using water-pyridine- Udalova benzene-butanol (3 + 3 + 1 + 5) or (1966) butanol-acetic acid-water (5 + 1 + 2 ) ; detected with ammoniacal A g + , evaluating spot areas planimetrically

Whatman No. 1; propanol-ethyl acetate- de Freitas 10% formic acid (7 + 1 + 2 ) ; visualised (1967) with ammoniacal A g +

Ascending PC; various solvent mixtures Cotte and containing ethanol, butanol, acetic acid, Guillot (1967) ethyl acetate, and water; visualised with ammoniacal A g + , periodate-benzidine, or iodobismuthate; many Rf values quoted

Mobile phase isopropanol-toluene-ethyl Abdilaev acetate-25% N H 3 (70 + 45 + 20 + 20, et al. (1974) 65 + 45 + 20 + 16-5, or 58 + 3 0 + 13 + 15); detected with periodate-benzidine

74 GLYCEROL 1.9

1.9.3. Th in Layer C h r o m a t o g r a p h y

The types of problem tackled by thin layer chromatography are closely

similar to those investigated by paper chromatography—food, tobacco, and

pharmaceutical samples, and separations of polyols, including sugars, from

one another. Here, too, some information is giver! in summarised form in

Table 1.7.

TABLE 1.7

Thin Layer Chromatography of Glycerol and Other Polyols


Compounds with 1,2-diol groups, including glycerol

Polyols

Glycerol, ethylene glycol

Tobacco humectants (glycerol, glycols, sorbitol) (aqueous extract)

Glycerol and sugars and other polyols

Sugars and polyols

Glycerol and poly-glycerols

Silica gel; methanol-chloroform (1 + 9), 96 % ethanol-acetic acid-water (240 + 1 + 40), or ethanol-25 % N H 3 -water (85 + 5 + 10), detecting down to 2-4, 4, and 3 \ig glycerol; visualised with ammoniacal A g + , periodate-benzidine, periodate-permanganate, and Pb(IV)

Cellulose; many solvent mixtures, many containing alcohols and water, some with ethyl acetate and pyridine; visualised with periodate-benzidine

Silica gel G; butanol-water (9 + 1); separation improved in presence of boric acid; visualised with C r 2 0 7

2 " - H 2 S 0 4

Silica gel; acetone; detected with Pb(IV)

Silica gel G; n-butanol-acetic acid-diethyl ether-water (9 + 6 + 3-1-1); detected with H 2 S 0 4 or alkaline K M n 0 4

Mg silicate; propanol-water-chloroform (6 + 2 + 1) or propanol-water-butanol (2 + 1 + 1); detected with 1 % K M n 0 4 as light spots on a violet background

Kieselguhr G; ethyl acetate-isopropanol-water (65 + 22-7 + 12-3); visualised with ammoniacal A g + or periodate-benzidine

BergePson et al (1961)

Dyatlovitskaya et al (1962)

Prey et al (1962)

Wright (1963)

Hay et al (1963)

Grasshof (1963)

Seher (1964)



Technically important polyols (17)

Alumina, silica gel G, kieselguhr G - Knappe et al. Ultramid IC (6 + 1 ) ; chloroform- (1964) toluene-formic acid (80 + 17 + 3), butanol, saturated with 1-5N N H 4 O H , and chloroform, resp.; visualised with many agents including permanganate, periodate, hexanitratocerate

Sugars, sugar alcohols Silica gel G prepared with phosphate buffer Waldi (1965) (glycerol and C 6 )

Poly glycerols and glycerol itself

Glycerol and other polyols

Polyols

Glycerol, ribitol, and some sugars

Glycerol in presence of oligoglycerols

Glycerol, mannitol, glucose, maltose

of pH 5; butanol-acetone-water (4 + 5 + 1 ) ; detected with periodate-benzidine, then ammoniacal A g +

Kieselguhr G (Merck)-silica gel Dallas and (Whatman SG 41) (1 + 1), prepared in Stewart (1967) 0-5% sodium metabisulphite; ethyl acetate-isopropanol-acetone-methanol-water (50 + 15 + 15 + 4 + 16); or same adsorbent prepared in 0 0 4 5 N CaCl 2 , with mobile phase ethyl acetate-isopropanol-water (110 + 61 + 2 9 ) ; visualised with thymol in ethanol-sulphuric acid and other agents

Silica gel H-kieselguhr G (25 + 15); 60 vol Tanner and of dioxan-amyl acetate-isopropanol- Duperrex water (20 + 60 + 20 + 8), 40 vol of (1968) isopropanol-ethyl acetate-water (54 + 7 + 8), and 1 vol of anhydrous acetic acid; visualised with Pb(IV)-4,5-dichloro-fluorescein, spot areas estimated through fluorescence at 350 nm

Silica gel impregnated with P b ( N 0 3 ) 2 ; De Simone ethanol saturated with N H 3 , or various and Vice-e thanol -NH 4 OH-water mixtures; detected domini (1968) with alkaline K M n 0 4

Silica gel impregnated with phosphate; Bleiweis and chloroform-acetic acid-water (10 + 7 + 1), Coleman then at right angles with acetone-butanol- (1969) water (5 + 4 + 1); visualised with an i sa ldehyde -H 2 S0 4

Silica gel; three times with water-saturated Jaworski et al. butanol; visualised with periodate- (1969) benzidine, and H C H O product ultimately determined polarographically

Silica gel G; ethanol-isobutanol-water Tateo (1970) (6 + 3 + 1); detected with alkaline K M n 0 4



Glycols, glycerol, erythritol, sorbitol

Polyols, sugars (in adjuvants)

Glycerol with free fatty acids and glycerides

Mixture of polyols

Polyols

Ethanolic extract of glycerol from liqueurs and syrups

Ointment ingredients (glycerol, starch)

Glycerol and butane-2,3-diol from alcohol fermentation

Glycerol and acetate esters; diethylene glycol and acetate

esters

Alumina; various solvent mixtures; best in 2 steps, chloroform-ethanol (4 + 1), then chloroform-acetone-acetic acid (6 + 3 + 1), or butanol-acetone-water (4 + 5 + 1); visualised with periodate-K M n 0 4

Silica gel G buffered with boric acid; propanol-butanone-water-ethyl acetate (20 + 10 + 10 + 1); detection with periodate-benzidine

Silica gel G prepared from 7-2 % oxalic acid; diethyl ether-hexane (1 + 1); visualised by charring with H 2 S 0 4

Various layers: alumina-bentonite clay (1 + 1), alumina-kieselguhr (2 + 1), alumina-gypsum (1 + 1 or 2 + 1); various solvents, all containing a lower-alcohol component; detected with iodine vapour, periodate-benzidine, ammoniacal Ag + . Also quantitative determination on Sorb-1 (alumina - gypsum) with iso-propanol-toluene (3 + 4), visualisation with iodine, and estimation of spot size

Silufol UV 254; NH 4 OH-pyridine (9 + 16); visualised with periodate-benzidine

Silica gel G-kieselguhr G (1 + 1); butanol-water (9 + 1); visualised with ammoniacal Ag +

Silica gel G 2 5 4 or Silufol UV 254; acetone-chloroform-25 % N H 3

(8 + 1 + 1) for these two components; chloroform-acetic acid-pet. ether (4 + 1 + 10) for these two in presence of propyl p-hydroxybenzoate, stearic acid, menthol, and cholesterol

Kieselguhr G-silica gel G (2 + 1 ) ; water-saturated butanone (separates glycerol from sugars); detected with Pb(IV)-dichlorofluorescein.

Silica gel; toluene-acetone-methanol-acetic acid (14 + 5 + 1 + 0-3); detected with iodine vapour or C r 2 0 7

2 _ - H 2 S 0 4

Borisovich et al (1970)

Ehrhardt and Sucker (1970)

Saracco and Gay (1971)

Nadirov et al (1971)

Talipov et al (1972)

Venturini (1972)

Sarsunova (1973)

Savage and Wagstaffe (1973)

Constantinescu and Enache (1974)



Glycerol in vinegar

Polyols from C 3

to C,

About 30 compounds tested, including glycerol

Based on method of Savage and Wagstaffe Carballido (1973); determined by periodate oxidation and Valdehita and ultimate colorimetry with (1975) chromotropic acid

Silica gel 60; 13 solvent systems Papin and (containing an alcohol, such as ethanol Udiman or a propanol, plus acetone, ethyl acetate, (1975) water, or 0 1 M boric acid); visualised with periodate oxidation and spraying with tetrabase

Silica gel (0-23 mm) without organic binder; Shanfield exposed 5 s to a gaseous electrical et al. (1976) discharge, then heated to 130°C and fluorescence in UV (365 nm) observed within 1-2 min (1 ug glycerol gave medium intensity)

1.3.4. Column and Liquid Chromatography

Some examples of the application of column, including liquid, chromato-graphy have been collected in Table 1.8. The samples are of similar origin and the problems are similar to those subjected to the previously mentioned chromatographic procedures.

TABLE 1.8

Column and Liquid Chromatography of Glycerol and Other Polyols


Glycerol and ethylene Frontal adsorption analysis (Claesson) on Wetterholm glycol mixtures highly active Hoganas MK carbon, using (1946)

water solvent

Separation of acetoin, Column of Celite 535; top 15% wetted Neish (1950) glycerol, and butane- with the developing solvent ethyl acetate or 2,3-diol from other periodate-reacting substances

benzene-butanol (1 + 3); remaining 85% wetted with water-solvent equilibrium mixture; glycerol ultimately determined in eluate through periodate oxidation and H C H O reaction with chromotropic acid




Glycerol in sulphite fermentation solutions

Column of coarse cellulose powder (2-5 g) supporting alumina (5 g). Aqueous sample mixed with sodium sulphite, sodium acetate, acetic acid (these help retention of sugars and other impurities), and alumina, and brought on to the column; eluted with acetone containing 5 % water and 0-05 % acetic acid. Glycerol oxidised with periodate, and the formic acid formed titrated with NaOH

Method of Sporek and Williams (1954), modified by increasing the amount of water for solution and the length and amount of columnar alumina

Silica gel column; used for glycerol and high M.W. polyethylene glycols, methanol-chloroform (1 + 2); 10 ml eluate amounts evaporated and residues weighed

Cellulose column capped with cellulose-charcoal; diethyl ether-95 % ethanol (2 + 1); aliquots determined by oxidation with excess periodate and estimation of unused

Cellulose column; butanol saturated with 1*1 % ammonia; any ethylene glycol removed with 450 ml eluent, glycerol with 600 ml, sucrose with 860-1500 ml; glycerol determined by periodate oxidation

Separation of glycerol Cellulose column; benzene-butanol from sugar alcohols in (1 + 3) saturated with water; glycerol fermentation liquors ultimately determined by periodate

oxidation and reaction of HCHO with chromotropic acid

Glycerol in fruit pastilles

Non-ionic surface-active agents and related materials

Glycerol in extracts of tobacco with water

Glycerol-sucrose mixtures

Glycerol in tobacco

Brake fluid analysis; includes glycerol, many glycols, and the esters of these polyols

Modification of method of Patterson (1963); final determination by periodate oxidation and titration of HCOOH formed.

Gel permeation chromatography on 10 5 -100 A porous polystyrene beads; tetrahydrofuran containing 0-1 % hydro-quinone; refractometric monitoring

Williams (1953) Sporek and Williams (1954)

Lloyd (1962)

Rosen (1963)

Patterson (1963)

Loiacono (1964)

Mizsei et al. (1964)

Doihara et al. (1966)

Lambert (1970)



Five polyols

Polyols and related compounds

Ethylene, propylene and butane-2,3-glycols, glycerol; alcohol mixtures in cosmetics; glycerol and glycols in tobacco, essences and wine

Used to compare gel permeation properties Brown and of a highly cross-linked hydroxyethyl- Chitumbo cellulose in bead form with Sephadex LH (1972) 20; dimethylformamide solvent

High-pressure liquid chromatography on Belue (1974) silica gel in stainless steel columns; butanone-water-acetone (85 + 10 + 5); e.g. Porasil A for the N

example of glycollic aldehyde, ethylene glycol, glycerol, and isoerythritol

Liquid chromatography on glass column of Stahl et al microcrystalline (20-40 urn) cellulose; (1975) ethyl acetate-propanol-water (121 + 49 + 30) at 3-5 atm; differential refractometric detection

1.9.5. Paper Electrophoresis

Some examples of paper electrophoresis in the presence of various complexing anions have been quoted earlier (Section 1.6.3). These are briefly mentioned here. Gross (1955) separated polyols in this way, in the presence of borate (pH 9.2). Lindberg and Swan (1960) compared paper electrophoretic mobilities of many polyols in germanate buffer, pH 10-7, at 40°C and 25-30 V/cm with those found in borate. A similar comparison was made by Popiel (1961), for various polyols (glycerol, glycols, sugars, and sugar alcohols) in paper ionophoresis on Whatman No. 3 paper at 12 V/cm, between tellurate and germanate at pH 8-11 and borate and germanate at pH 10-7 under the conditions of Lindberg and Swan. Lees and Weigel (1964) studied paper electrophoresis of polyols in the presence of sodium stannate at pH 11 -5.

1.9.6. Ion Exchange

As in the chromatographic procedures tabulated in brief form above, most applications of ion exchange to glycerol have been to separate it from other polyols, notably sugars, and to determine it in fluids such as wine, sea water, or urine. Table 1.9 contains some information.


TABLE 1.9

Ion Exchange Methods applied to Glycerol and Other Polyols


Tested Dowex 50 and Dowex 1 with water eluent. Organic mixture best with Dowex 50 in H + form

Glycerol, triethylene glycol, in presence of sucrose and phenol; also separation of polyols from NaCl

Diol-glycerol Dowex 1-X8 columns; 0-02 M or 0-925M mixtures sodium borate; effluents determined by

Cr(VI) oxidation and evaluation spectro-photometrically at 610 nm

H + -form of 100-200 mesh Dowex 50-X 12; 60°C; preheated, demineralised

products of glucose or water; monitored refractometrically sorbitol

Separation of glycerol from hydrogenation

Glycerol in technical material

Separation of glycerol and NaCl in crude glycerol lye

Glycerol and other polyols from hydrogenation of sugars

Glycerol, xylitol, ethylene glycol mixtures

Polyols, carbonyl compounds, and sugars

Glycerol, butane-2,3-diol in wines

Diluted solution passed through column of Wofatit KPS 200-Wofatit L 165 (1 + 1-5) (strongly acid and strongly basic ion-exchangers); percolate monitored refractometrically

Column of anionic exchanger S-8TM containing incorporated acrylic acid; eluted with water; glycerol comes first; studied effect of conditions

Dowex-50 with 12% cross-linking, in H + -form, 200-300 mesh; 60°C; water elution; refractometric monitoring

Dowex 20-X12 resin, also as Ca 2 + -form which gave inverted order of elution; also inefficient, glycols only slowly desorbed

Study of separations on anion and cation exchangers with ethanol-water mixtures; good on Li + -, N a + - , and K + -forms of Dowex 50W-X8 at 75° with 80-97 % ethanol; very good for polyols on Li + ; carbonyl compounds-polyols on basic T5C (sulphate form) at 40°C, with 80-96 % ethanol

Passed through anionic exchanger Amberlite IRA-400, then cationic Amberlite IRA-120; ultimately determined via periodate oxidation

Wheaton and Baumann (1953)

Sargent and Rieman (1957)

Clark (1958)

Kopecky et al (1960)

Kopecky (1966)

Datagov and Balandin (1966a)

Datagov and Balandin (1966b)

Samuelson and Stromberg (1968)

Castino and Usseglio-Tomasset (1968)



Propylene glycol On Dowex-1 resin in sulphate form; 50°C; glycerol, glycollic acid, 86 % ethanol erythritol, xylitol

Desalted and evaporated sample in 85 % ethanol on anion exchanger in sulphate form; eluted with the 85% ethanol; traces of glycerol and glycols found, determined with periodate

Tested Fe(III)-Amberlyst 15 resins, 50 -100 mesh; absolute methanol; polyols more strongly bound than corresponding sugars; retention increases with M.W. and no. o f - O H

E.g. ethanolamines, Metal-loaded ion-exchangers, e.g. glycerol, iV-dimethyl- Amberlite CG-50-Ni, 100-200 mesh, which monoethanolamine in does not retain glycerol; eluted with mixtures N H 4 O H (0-34 and 0-23M); monitored

refractometrically

Sea water content of monosaccharides

Many substances, including poly-alcohols, sugars

Glycerol, sugars, and ethanol in wine

High-pressure ion-exchange chromato-graphy of the deacidified sample on a tantalum column of Aminex A 6 in the H + -form; 200 atm; water eluent; monitored refractometrically; sugars first eluted, then glycerol, then ethanol

Barker et al. (1968)

Joseffson (1970)

Shaw and Walton (1972)

Shimomura et al. (1973)

Rapp et al. (1975)

Jandera and Churacek (1970, 1974) have published reviews of liquid chromatography of organic substances, including alcohols and polyols, on ion exchangers.

REFERENCES

Abdilaev, B., Talipov, Sh. T. and Nadirov, N. K. (1974). Gidroliz Lesokhim. Promst 12 (Chem. Abs.Sl, 130621).

Afanas'ev, B. N. (1949). Zav. Lab. 15, 1271. Alber, H. (1929). Mikrochem. 7, 21. Aleksandrov, A. and Berka, A. (1967). Nauch. Jr. Vissh. Pedagog. Inst. Plovdiv. Mat.,

Fiz. Khim. Biol. 5, 91 (Anal. Chem. 79R (1970)). Aleksandrovich, I. F. and Kuritsina, G. N. (1962). Khim. Volokna 57 (Chem. Abs. 62,

2907). Alfend, S. (1932). J. Ass. Off. Agric. Chem. 15, 331.


82 GLYCEROL

Alfend, S. (1933). J. Ass. Off. Agric. Chem. 16, 293. Allen, N., Charbonnier, H. Y. and Coleman, R. M. (1940). Ind. Eng. Scl, Anal. Ed. 12,

384. Amerine, M. A. and Dietrich, W. C (1943) J. Ass. Off Agric. Chem. 26, 408. Anderson, D. M. W. and Zaidi, S. S. H. (1963) Talanta 10, 691. Andrews, J. T. R. (1933) Oil and Soap 10, 71. Andrews, J. T. R. (1935). Oil and Soap 12, 90. Andrews, J. T. R. et al. (1941). Oil and Soap 18, 14. Antonin, K. (1967). Chem. Prum. 17, 45. Anzhele, P. G., Vasyunina, N. A., Balandin, A. A. and Leibnitz, F. (1964). Molekul.

Khromatogr., Akad. Nauk SSSR, Inst. Fiz. Khim. 61 (Chem. Abs. 62, 8394). Arapbaev, A., Toropchanin, A. M. and Khabibov, E. Kh. (1971). Metody Prob.

Opred. Vlazhnosti 98 (Chem. Abs. 78, 52332). Arreguine, V. (1936). AnaLAsoc. Quim. Argent. 24, 613. Assmann, K., Serfas, O. and Geppert, G. (1967). J. Chromatogr. 26, 495. Bacila, M. (1949). Arquiv. Biol, e Tecnoi, Inst. Biol, e Pesquisas Tecnol. 4, 33 (Chem.

Abs. 48, 1201). Bailey, M. (1959). J. Lab. Clin. Med. 54, 158. Balakhontseva, V. N. and Poltinina, R. M. (1964). Zh. Anal. Khim. 19, 757. Balakhontseva, V. N. and Poltinina, R. M. (1965). Zh. Anal. Khim. 20, 739. Bally, O. and Scholl, R. (1911). Ber. 44, 1656. Balwant Singh, Apar Singh and Gurdas Singh (1953a). / . Indian Chem. Soc. 30, 488. Balwant Singh, Apar Singh and Mohan Singh (1953b). Res. Bull. East Panjab Univ.

No. 30, 55 (Chem. Abs. 48, 4370). Barbour, R. F. and Devine, J. (1971). Analyst (London) 96, 288. Bark, L. S., Griffin, D. and Prachuabpaibul, P. (1976). Analyst (London) 101, 306. Barker, S. A., How, M. J., Peplow, P. V. and Somers, P. J. (1968). Anal. Biochem. 26,

219. Barker, S. A., Hatt, B. W., Somers, P. J. and Woodbury, R. R. (1973). Carbohyd. Res.

26, 55. Bauer-Moll (1960). "Die Organische Analyse", 4th Ed., Leipzig, p. 94. Bean, R. C. and Porter, G. G. (1959). Anal. Chem. 31, 1929. Beck, G. (1951). Mikrochemie ver. Mikrochim. Acta 38, 152. Beer, J. Z. (1961). Talanta 8, 809. Belikovskii, Ya. E. (1972). Farmatsiya (Moscow) 21, 62. Belue, G. P. (1974). / . Chromatogr. 100, 233. Benedikt, R. and Cantor, M. (1888). Z. Angew. Chem. 1, 460. Benedikt, R. and Zsigmondy, R. (1885). Chem.-Ztg. 9, 975. Bennett, H. B. (1924). J. Chem. Soc. 125, 1971. Bennett, G. W. and Streeter III, R. (1959). Proc. Penna Acad. Sci. 33, 94 (Chem. Abs.

54, 993). Benson, D. and Fletcher, N. (1966). Talanta 13, 1207. Bergel'son, L. D., Dyatlovitskaya, E. V. and Voronkova, V. V. (1961). Dokl. Akad.

Nauk SSSR 141, 84. Bergner, K. G. and Meyer, H. (1960). Deut. Lebensm.-Rundschau 56,49. Bergner, K. G. and Sperlich, H. (1953a). Z. Lebensm.-Untersuch. u. Forsch. 97, 253. Bergner, K. G. and Sperlich, H. (1953b). Deut. Apoth.-Ztg. 93, 676. Berka, A. (1963). Z. Anal. Chem. 195, 263. Berka, A. and Dusic, Z. (1970). Acta Pharm. Jugoslav. 20, 171. Berka, A. and Holada, K. (1969). Cesk. Farm. 18, 12.

REFERENCES 83

Berka, A. and Pauleova, B. (1971). Cesk. Farm. 20, 376. Berka, A. and Zavesky, Z. (1969). Cesk. Farm. 18, 9. Berka, A., Dvorak, V. and Zyka, J. (1962). Mikrochim. Acta 541. Berka, A., Fara, M. and Zyka, J. (1963). Cesk. Farm. 12, 366. Berth, O. (1928). Chemiker-Ztg. 52, 597, 619, 737. Bertram, S. H. and Rutgers, R. (1938). Rec. Trav. Chim. 57, 681. Betteridge, D. and Ruzicka, J. (1976). Talanta 23, 409. Biesold, D. and Strack, E. (1958). Z. Physiol. Chem. 311, 115. Black, R. A. and Andreasen, A. A. (1974). / . Ass. Off. Anal. Chemists 57, 111. Bleiweis, A. S. and Coleman, S. E. (1969). Anal. Biochem. 29, 343. Blix, G. (1937). Mikrochim. Acta 1, 75. Blum, J. and Koehler, W. R. (1968). J. Gas. Chromatogr. 6, 120. Blum, J. and Koehler, W. R. (1970). Lipids 5, 601. Bogs, H. U. (1967). Pharm. Zentralh. 106, 7. Bohanes, A. (1935). Chem. Obzor 10, 28. Bond, H. R. (1949). J. Ass. Off. Agric. Chem. 32, 606. Bonino, R. C. d'A. de C. (1952). Rev. Asoc. Bioquim. Argent. 17, 273. Bonner, T. G. (1960). Chem. Ind. (London) 345. Bordas, F. and de Raczkowsky, S. (1896). Compt. Rend. 123, 1071. Bore, P. (1950). Bull. Mens. ITERG 4, 168 (Chem. Abs. 44, 6171). Borisovich, I. G., Orobinskaya, L. N. and Vasyunina, N. A. (1970). Izv. Akad. Nauk

SSSR, Ser. Khim. 2361. Bourne, E. J., Lees, E. M. and Weigel, H. (1963). J. Chromatogr. 11, 253. Bouzigues, L. (1953). Ann. Inst. Natl. Recherche Agron., Ser. E, Ann. Technol. Agr. 2,

51 (Chem. Abs. 50,6743). Bradford, P., Pohle, W. D., Gunther, J. K. and Mehlenbacher, V. C. (1942). Oil and

Soap 19, 189. Brown, W. and Chitumbo, K. (1972). Chemica Scripta 2, 88. von Bruchhausen, F. (1934). Z. Untersuch. Lebensm. 68, 32. Bruening, C. F. (1946). J. Ass. Off. Agric. Chem. 29, 29. Bruening, C. F. (1947). J. Ass. Off. Agric. Chem. 30, 507. Bruening, C. F. (1950). / . Ass. Off. Agric. Chem. 33, 103. Bryant, W. M. D., Mitchell, J. and Smith, D. M. (1940). J. Amer. Chem. Soc. 62, 1. Buchanan, J. G , Dekker, C. A. and Long, A. G. (1950). J. Chem. Soc. 3162. Burnel, D., Hutin, H. F. and Malaprade, L. (1971). Chim. Anal. (Paris) 53, 230. Buscarons, F., Marin, J. L. and Claver, J. J. (1949). Anal. Chim. Acta 3, 310, 417; also

(1953). Anales Real Soc. Espah. Fis. y Quim. 49B, 367. Camillo Marchi (1923). Staz. Sper. Agrar. Ital. 56, 231 (Chem. Abs. 18, 2667). Carballido, A. and Valdehita, M. T. (1975). Anal. Bromatol. 27, 103. Cardini, C , Quercia, V. and Calo, A. (1967). Boll. Chim. Farm. 106, 459. Cardone, M. J. and Compton, J. (1952). Anal. Chem. 24, 1903. Carugno, N., Rossi, S. and Lionetti, G. (1971). Beitr. Tabakforsch. 6, 79 (Chem. Abs.

76, 70328; Anal. Abs. 22, 4251). Caruso, J. G. B. and Falanghe, H. (1968). Appl. Microbiol. 16, 1807 (Chem. Abs.70,

26315). Castino, M. and Usseglio-Tomasset, L. (1968). Riv. Viticolt. Enol. 21, 465 (Chem. Abs.

70, 86213). Cerutti, G. and Vezzini, W. (1961). Chim. e Ind. 43, 784. Chapman, A. C. (1926). Analyst (London) 51, 382. Chaumeil, A. (1902). Bull. Soc. Chim. 27, 629.

84 GLYCEROL

Christensen, G. M. (1962). Anal. Chem. 34, 1030. Christensen, B. E., Pennington, L. and Dimick, K. P. (1941). Ind. Eng. Chem., Anal.

Ed. 13, 821. Cifonelli, J. A. and Smith, F. (1954). Anal. Chem. 26, 1132. Clark, R. T. (1958). Anal. Chem. 30, 1356. Clifford, J. (1960). Analyst (London) 85, 475. Cohen, I. R. and Altshuller, A. P. (1961). Anal. Chem. 33, 726. Colson, R. (1950). Oleagineux 5, 701. Colson, R. (1951). Ind. Parfum. 6, 115. Conacher, H. B. S. and Rees, D. I. (1966). Analyst (London) 91, 55. Constantinescu, T. and Enache, S. (1974). Farmacia (Bucharest) 22, 179. de Coquet, C. (1928). Soap 5, 235 (Chem. Abs. 24, 4580). Corbett, G. E., Hughes, W. and Morris Jones, R. G (1969). J. Appl. Polym. Sci. 13,

1297. Cotte, J. and Guillot, B. (1967). Gattefosse ST PA Notice No. OL 0072 (Chem. Abs.

68, 46030). de la Court, F. H., van Cassel, N. J. P. and van der Valk, J. A. M. (1969). Farbe Lack

75,218. Cross, C. F. and Bevan, E. J. (1887). Chem. News 55, 2. Cunha, V. (1939). Arquiv. hig. saude publica (Sao Paulo) 4, No. 6, 65 (Chem. Abs. 34,

1440). Cuthill, R. and Atkins, C. (1938). J. Soc. Chim. Ind. 57, 89. Dalgliesh, C. E., Horning, E C , Horning, M. G, Knox, K. L. and Yarger, K. (1966).

Biochem. J. 101, 792. Dallas, M. S. J. (1970). J. Chromatogr. 48, 225. Dallas, M. S. J. and Stewart, M. F. (1967). Analyst (London) 92, 634. Dal Nogare, S. and Oemler, A. N. (1952). Anal. Chem. 24, 902. Damyanov, S., Mitev, S. and Damyanov, N. (1970). God. Vissh. Khimiko-tekhnol.

Inst., Sofia 15, 245 (Chem. Abs. 79, 7153). Datagov, N. S. and Balandin, A. A. (1966a). Izv. Akad. Nauk SSSR, Ser. Khim. 1308. Datagov, N. S. and Balandin, A. A. (1966b). Izv. Akad. Nauk SSSR, Ser. Khim. 1315. Datta, D. D. and Ghosh, D. (1965). J. Pharm. 27, 265. Decroix, G. A. R., Gobert, J. G. and De Deurwaerder, R. (1968). Anal Biochem. 25,

523. Deniges, G. (1909). Bull. Soc. Chim. (Paris) [4] 5, 421. Deniges, G. (1910). Bull. Trav. Pharm. Bordeaux 49, 105. De Simone, V. and Vicedomini, M. (1968). J. Chromatogr. 37, 538. Desnuelle, P. and Naudet, M. (1945). Bull Soc. Chim. 12, 871. Diemair, W., Riffart, H. and Mollenkopf, K. (1940). Z. Anal Chem. 119, 189. Doihara, T., Kobashi, U., Sugawara, S. and Kaburaki, Y. (1965). Nippon Senbai

Kosha Chuo Kenkyusho Kenkyu Hokoku No. 107, 141 (Chem. Abs. 64, 20217). Doihara, T., Kobashi, U., Sugawara, S. and Kaburaki, Y. (1966). Nippon Senbai

Kosha Chuo Kenkyusho Kenkyu Hokoku No. 108, 155 (Chem. Abs. 66, 62561). Dooms, L., Declerck, D. and Verachtert, H. (1969). J. Chromatogr. 42, 349. Druce, S. (1952). Mfg. Chemist 23, 187. Dutton, G. G. S., Gibney, K. B., Jensen, G. D. and Reid, P. E. (1968). J. Chromatogr.

36, 152. Dyatlovitskaya, E. V., Voronkova, V. V. and Bergel'son, L. D. (1962). Dokl Akad.

Nauk SSSR 145, 325. Ehrhardt, L. and Sucker, H. (1970). Pharm. Ind. (Berlin) 32, 92.

REFERENCES 85

Eisenbrand, J. and Raisch, M. (1960). Deut. Lebensm.-Rundschau 56, 257; also Z. Anal. Chem. Ill, 1.

Elving, P. J. and Warshowsky, B. (1947). Anal. Chem. 19, 1006. Elving, P. J., Warshowsky, B., Shoemaker, E. and Margolit, J. (1948). Anal. Chem. 20,

25. Englis, D. T. and Wollerman, L. A. (1952). Anal. Chem. 24, 1983. Erdey, L., Bodor, E. and Papay, M. (1955). Acta Chim. Acad. Sci. Hung. 5, 235. Eremina, Z. I. and Gurevich, V. G. (1961). Farm. Zhur. (Kiev) 16, 13. Erskine, J. W. B., Strouts, C. R. N., Walley, G. and Lazarus, W. (1953). Analyst

(London) 78, 630. Esposito, G. G. (1962). Anal. Chem. 34, 1173. Esposito, G. G. (1968). Anal. Chem. 40, 1902. Esposito, G. G. and Swann, M. H. (1961). Anal. Chem. 33, 1854. Esposito, G. G. and Swann, M. H. (1969). Anal. Chem. 41, 1118. Everest, D. A. and Harrison, J. C. (1960). J. Chem. Soc. 1745. Fachini, S. (1923). Chem. Trade J. 73, 127 (Chem. Abs. 17, 3149). Fachini, S. and Dorta, G. (1910). Ger. Seifenfabrikant 1205; Z. Anal. Chem. 51, 779. Fatome, M. (1935). Ann. Fermentations 1, 291. Fatome, M. (1936). J. Pharm. Chim. 23, 23. Feigl, F. (1937). Mikrochim. Acta 1, 127. Feigl, F. "Spot Tests in Organic Analysis", 7th Ed., Elsevier: (1966a), p. 128; (1966b),

p. 174; (1966c), p. 315; (1966d), p. 316-7; (1966e), p. 413; (1966f), p. 417; (1966g), p. 418.

von Fellenberg, T. (1931). Mitt. Lebensm. Hyg. 22, 231. von Fellenberg, T. (1943). Mitt. Lebensm. Hyg. 34, 344. Ferre, L. and Bourges, J. (1928). Chimie et Ind., Spec. No. 775 (Chem. Abs. 22, 4713). Ferre, L. and Michel, A. (1938). Ann. Fals. 31, 85. Filatova, T. N., Vasyunina, N. A. and Kuznetsova, L. A. (1969). Izv. Akad. Nauk

SSSR, Ser. Khim. 2581. Fischer, R. and Kolmayr, F. (1955). Pharm. Zentralh. 94, 8. Fleury, P. and Fatome, M. (1935). J. Pharm. Chim. 21, 247; also Ann. Fermentations

1, 285. Floria, J. A., Dobratz, I. W. and McClure, J. H. (1964). Anal. Chem. 36, 2053. Foster, A. B. (1957). Adv. Carboh. Chem. 12, 81. Franzen, F., Eysell, K. and Hack, H. (1954). Mikrochim. Acta 708. Frehden, O. and Fiirst, K. (1939). Mikrochemie 26, 36. Frehden, O. and Huang, Ch-H. (1937): Mikrochim. Acta 2, 20. de Freitas, A. S. W. (1967). Can. J. Biochem. 45, 1041. Frey, O. (1929). Wiss. Mitt. Osterreich. Heilmittelstelle No. 9, 23 (Chem. Abs. 25, 4728). Friedman, R. L. and Raab, W. J. (1963). Anal. Chem. 35, 67. Fritz, J. S. and Schenk, G. H. (1959). Anal. Chem. 31, 1808. Fuchs, P. (1942). Chemiker-Ztg. 66, 73. Fiirst, W. (1948a). Mikrochemie ver. Mikrochim. Acta 34, 25. Fiirst, W. (1948b). Scient. Pharm. 16, 85. Fulmer, E. I., Hickey, R. J. and Underkofler, L. A. (1940). Ind. Eng. Chem., Anal. Ed.

12, 729. Gailhat, J. (1902). Monit. Scient. [4 ] , 16, 1, 89. Ganassini, D. (1930). Arch. 1st. Biochim. Ital. 2, 239. Garegg, P. J. and Lindberg, B. (1961). Acta Chem. Scand. 15, 1913. Garegg, P. J. and Lindstrom, K. (1971). Acta Chem. Scand. 25, 1559.

86 GLYCEROL

Garrigues, W. E. (1897). J. Amer. Chem. Soc. 19, 181. Gasparic, J. and Borecky, J. (1961). J. Chromatogr, 5, 466. Ghanayem, I. and Swann, W. B. (1962). Anal. Chem. 34, 1847. Ghimicescu, G. L. (1935). Ann. Sci. Univ. Jassu 21, 346 (Chem. Abs. 30, 1939). Ghimicescu, G. L., Musteata-Ghimicescu, C. and Dumbrava, E. (1963). Acad. Rep.

Pop. Romine, Stud. Cercetari Chim. 11, 105. Giles, J. A. (1970). J. Ass. Off Anal. Chemists 53, 655. Godin, P. (1954). Nature (London) 174,134. Golova, B. M., Motovilyak, L. V., Politanskii, S. F., Stepanov, M. V. and Chelyadin,

V. T. (1974). Zav. Lab. 40, 1192. Goodlett, V. W. (1965). Anal. Chem. 37, 431. Gordienko, T. A., Gordon, B. E., MaPnev, A. F. and Puchkovskaya, G. A. (1964).

Zh. Prikl. Spektroskopii 1, 88. Gordon, H. T. (1951). Anal. Chem. 23, 1853. Gordon, H. T„ Thornburg, W. and Werum, L. N. (1956). Anal. Chem. 28, 849. Graham, H. D. (1963). J. Food Sci. 28, 440. Graham, H. D. (1965). J. Food Sci. 30, 846. Grasshof, H. (1963). Deut. Apotheker-Ztg. 103, 1396. Gregory, N. L. (1968). / . Chromatogr. 36, 342. Griffin, J. C. M. (1954). J. Ass. Off Agric. Chem. 37, 874. Grohmann, H. and Muhlberger, F. H. (1956). Z. Lebensm.-Untersuch. u. Forsch. 103,

177. Gronsberg, E. Sh. (1969). Nov. Obi Prom.-Sanit. Khim. 8 (Chem. Abs. 72, 35436). Gross, D. (1955). Nature (London) 176, 362. Gross, F. C. (1967). J. Ass. Off Anal. Chemists 50, 1292. Gross, F. C. and Jones, J. H. (1967). J. Ass. Off Anal. Chemists 50, 1287. Grosso, J. C. (1946). Rev. Fac. Cienc. Quim. (Univ. Nacl. La Plata) 19, 61 (Chem. Abs.

41, 1577). Guardia, C. C. (1950). Afinidad 27, 289, 454. Guerin, M. R., Olerich, G. and Horton, A. D. (1974). J. Chromatogr. Sci. 12, 385. Guerra Salazar, G. E. (1962). An Asoc. Quim. Argent. 50, 59. Guilbault, G. G. and McCurdy jr., W. H. (1961). Anal. Chem. 33, 580. Guseva, M. G. (1954). Maslob.-Zhirovaya Prom. 19, No. 8, 27 (Chem. Abs. 49, 5004). Habicht, L. (1950). Fette u. Seifen 52,174. Haendel, M. (1973). Apotheker-prakt. Pharm.-Tech. Assistent 19, 73 (Chem. Abs. 80,

124661). Handbook of Chemistry and Physics, 53rd Ed., Chem. Rubber Co., Akron (1972/73a),

p. C312;(1972/73b), p. F40. Handbook of Tables for Organic Compound Identification (1974). 3rd Ed., CRC,

p. 86. Hartman, L. (1956). Analyst (London) 81, 67. Hartman, L. (I960). Fette, Seifen, Anstrichm. 62, 271. Hartman, L. (1964). / . Chromatogr. 16, 223. Harvey, S. C. and Higby, V. (1951). Arch. Biochem. 30, 14. Hasegawa, K., Kajikawa, M., Kawaguchi, M. and Nishijima, T. (1964). Bull Japan

Petr. Inst. 6, 19. Hay, G. W., Lewis, B. A. and Smith F. (1963). / . Chromatogr. 11, 479. Hehner, O. (1887). Analyst (London) 12, 44; also (1889). J. Soc. Chem. Ind. 8, 4. Heiduschka, A. and Englert, F. (1921). Z. Anal Chem. 60, 161. Helweg-Mikkelsen, V. (1948). Analyst (London) 73, 447.

REFERENCES 87

Helweg-Mikkelsen, V. (1949). Arch. Pharm. Chemi 56, 123. Hennies, J. and Eckert, B. (1963). Pharmazie 18, 127. Henry, A. M. (1957). J. Ass. Off. Agric. Chem. 40, 775. Hintermaier, A. (1955). Fette, Seifen, Anstrichm. 57, 11. Hockenhull, D. J. D. (1953). Nature (London) 171, 982. Hoepe, G. (1943). Helv. Chim. Acta 26,1931. Hoepe, G. and Treadwell, W. D. (1942). Helv. Chim. Acta 25, 353. Holla, K. S., Horrocks, L. A. and Cornwell, D. G. (1964). J. Lipid Res. 5, 263. Horrocks, L. A. (1961). Diss. Abs. 21, 2876. Horrocks, L. A. and Cornwell, D. G. (1962). J. Lipid Res. 3, 165. Horsley, L. (1947). Anal. Chem. 19, 544; also (1949). ibid. 21, 831. Hough, L. (1950). Nature (London) 165, 400. Hovey, A. G. and Hodgins, T. S. (1937). Ind. Eng. Chem., Anal. Ed. 9, 509. Hoyt, L. F. and Pemberton, H. V. (1922). Ind. Eng. Chem. 14, 54, 340. Hromatka, O. (1962). Branntweinwirtschaft 102, 703. Hromatka, O. and Stainer, J. (1962). Branntweinwirtschaft 102, 507. Hughes, K. W. and Clamp, J. R. (1972). Biochim. Biophys. Acta 264, 418. Ibrahim, M. A. El-F. and Taha, A. M. (1962). J. Pharm. Sci. U. Arab. Rep. 3, 167. Ikawa, M., Morrow, J. W. and Harney, S. J. (1966). J. Bacteriol. 92, 812. Isai, S. V. and Vas'kovskii, V. E. (1969). Izv. Sib. Otdel. Akad. Nauk SSSR, Ser. Khim.

Nauk 142 (Anal. Abs. 18, 4091). IUPAC Sub-committee (1973). Pure Appl. Chem. 33, 411. Iyer, V. and Mathur, N. K. (1965). Anal. Chim. Acta 33, 554. Izquierdo, A. and Lacort, G. (1972). Inform. Quim. Anal. 26, 192 (Chem. Abs. 78,

52373). Jackson, C. P. and Ramamurti, K. (1958). J. Sci. Food Agr. 9, 787. Jacquin, P. and Tavernier, J. (1952). Inds. Agr. et Aliment (Paris) 69, 497 (Chem. Abs.

47, 7364). Jambor, N. and Demeny, Z. (1936). Collegium 74 (Chem. Abs. 30, 4353). Jandera, P. and Churacek, J. (1970). Chem. Listy 64, 1301; also (1974). J. Chromatogr.

95, 55. Jankauskas, J. and Norkus, P. (1971). Zh. Anal. Khim. 26, 1425. Janowska, T. (1968). Tluszce, Sradki Piorace, Kosmet. 12, 94 (Chem. Abs. 70, 16906). Jansen, E. F. and Baglan, N. C. (1968). J. Chromatogr. 38, 18. Janssens, L. C. (1906). Seifensieder-Ztg. 33, 286; Z. Anal. Chem. 54, 593. Jasinski, T. and Grabowska, I. (1955). Acta Polon. Pharm. 12, 233. Jasinski, T. and Grabowska, I. (1956). Dissertationes Pharm. 8, 99 (Chem. Abs. 51,

11654). Javicoli, A. and Mattei, F. (1956). Arch. Ital. Sci. Med. Trop. e Parassitol. 37, 33

(Chem. Abs. 50, 11476). Jaworski, M., Bogaczek, J. and Walczyk, K. (1969). Chem. Anal. (Warsaw) 14, 313. Jellum, E. and Bjornstad, P. (1964). J. Lipid Res. 5, 314. Johnson, B. L. (1948). Anal. Chem. 20, 777. Johnson, K. E. and Ladyn, H. W. (1944). Oil and Soap 21,141. Jones, C. N. (1947). J. Ass. Off. Agr. Chem. 30, 486. Joseffson, B. O. (1970). Anal. Chim. Acta 52,65. Juhlin, O. (1938). Z. Anal. Chem. 113, 339. Jung, G , Voelter, W. and Breitmeier, E. (1970). Mikrochim. Acta 850. Ka, H. (1940). Dept. Ind. Sci. Res. Manchoukuo 4, 141 (Chem. Abs. 34, 6898). Kai Ho and Tzu-Hui Cheng (1933). J. Chinese Chem. Soc. 1, 190 (Chem. Abs. 28,

1415).

88 GLYCEROL

Karpov, O. N. (1960). Aptech. Delo 9, 28. Kartha, A. R. S. and Kidwai, M. A. (1965). Indian J. Chem. 3, 371. Kartnig, T. and Kren, G. (1963). Sci. Pharm. 31, 128. Kataoka, E. (1934). J. Biochem. (Japan) 19, 15 (Chem. Abs. 28, 2300). Kellner, J. (1922). Z. Deut. Oel-Fett-Ind. 42, 345. Kellner, J. (1924). Z. Deut. Oel-Fett-Ind. 44, 13. Keppel, G E. (1949). J. Ass. Off. Agr. Chem. 32, 506; also (1953) ibid. 36, 195. Khadeev, V. A. and Mukhamedzhanova, D. (1968). Tr. Tashkent Gos. Univ. No.

323, WKChem. Abs. 72,18165). Khan, I. and fiose, S. (1969). Indian J. Appl. Chem. 32, 165. Kirsten, W. J. and Nilsson, K. (1960). Mikrochim. Acta 983. Kiselev, A. V. and Yashin, Ya, I. (1966). Zh. Fiz. Khim. 40, 603. Knappe, E. and Miessner, N. (1976). Proc. 13th Congr., Fed. Ass. Tech. Inds. Paints,

Vern, Emaux, Encres Imprim. Eur. Continent, Cannes, p. 316 (Anal. Abs. 32, 1063). Knappe, E., Peteri, D. and Rohdewald, I. (1964). Z. Anal. Chem. 199, 270. Kochnova, Z. A., Sorokin, M. F., Grafkin, B. N. and Shabanova, N. P. (1969).

Lakokrasoch. Mater, ikh Primen. 25 (Chem. Abs. 72, 13272). Kolesnikova, L. P., Simonyants, E. G. and Kryukov, Yu. B. (1965). Zav. Lab. 31, 1330. Kolthoff, I. M. (1924). Pharm. Weekblad 61, 1497. Kopecky, A. (1966). Chem. Zvesti 20,274. Kopecky, A., Klozar, V. and Krejcar, E. (1960). Prum. Potravin 11, 438 (Anal. Abs.

8, 1074). Krivacic, J. R. and Urry, D. W. (1971). Anal. Biochem. 43,240. Kroller, E. (1949). Deut. Lebensm.-Rundschau 45, 46. Kroller, E. (1963). Deut. Lebensm.-Rundschau 59, 317. de Kuck, J. G , Macchi, R. A. and Crespo, F. (1967). Rev. Argent. Grasas Aceit 9,

32 (Anal. Abs. 16, 2203). Kulikov, A. M. (1940). Org. Chem. Ind. (USSR) 7, 521 (Chem. Abs. 35, 6473). Kwon, T-W. and Watts, B. M. (1963). Anal. Chem. 35, 733. Kyowa Chemicals (1967). Brit. Patent 1,150,490; applied May 26 (Anal. Abs. 17,

3503). Lacroix, H. and Kropacsy, S. (1928). Wochschr. Brau. 45, 490 (Chem. Abs. 23,

1987). Laforest, J. and Combrisson, A. (1968). Rev. Fr. Transfus. 11, 23 (Chem. Abs. 69,

103717). Lalieu, M. (1881). "Manuel d'Oxalimetrie", Brussels. Lambert, A. (1970). J. Appl. Chem. 20, 307. Lambert, M. and Neish, A. C. (1950). Can. J. Res. 28B, 83. Larmer, J. (1956). Farmdcia 25,151 (Chem. Abs. 51, 3931). Launer, H. F. and Tomimatsu, Y. (1953). Anal. Chem. 25, 1767. Lazar, O. and Meyling, A. H. (1938). J. S. African Chem. Inst. 21, 8. Lazarus, W. and Newlove, T. H. (1955). Analyst (London) 80, 276. Lees, E. M. and Weigel, H. (1964). / . Chromatogr. 16, 360. Legler, L. (1885). Rep. Anal. Chem. 6, 631. Lemieux, R. U. and Bauer, H. F. (1954). Anal. Chem. 26, 920. Lenz, W. (1885). Z. Anal. Chem. 24, 37. Levin, H , Uhrig, K. and Stehr, E. (1939). Ind. Eng. Chem., Anal. Ed. 11, 134. Levins, R. J. and Ottenstein, D. M. (1967). J. Gas Chromatogr. 5, 539. Levy, R. S. and McGee, E. D. (1964). J. Lipid Res. 5, 265. Lindberg, B. and Swan, B. (1960). Acta Chem. Scand. 14, 1043.

REFERENCES 89

Linevich, L. I., Erygki, G. D. and Ambartsumyan, E. R. (1972). Prikl. Biokhim. Mikrobiol. 8, 622 (Chem. Abs. 78, 1641).

Lisetskaya, G. S., Olefirenko, T. L. and Starodub, O. M. (1971). Metody Anal Khim.-Reaktiv.Prep.No. 19, 81 (Chem. Abs. 77, 134879).

Lloyd, W. J. W. (1962). Analyst (London) 87, 62. Loiacono, M. (1964). Rass. Chim. 16, 126 (Chem. Abs. 62, 32). Lopez, R. C. and Casares, C. (1948). Inform. Quim. Anal. (Madrid) 2, 111. Loseva, N. L., Abramova, L. A. and Domaradskii, I. V. (1970). Lab Delo 116. Loury, M. (1959). Fette, Seifen, Anstrichm. 61, 961. Ludwicki, H. and Sobiczewska, M. (1963). Farm. Polska 19, 228. Lushchik, V. L, Zlobina, V. R. and Gomozova, V. G. (1974). Lakokrasoch. Mater.

ikh Primen. 46 (Chem. Abs. 81, 38172). Lyne, F. A , Radley, J. A. and Taylor, M. B. (1968). Analyst (London) 93, 186. McAloren, J. T. and Reynolds, G. F. (1965a). Anal. Chim. Acta 32, 170. McAloren, J. T. and Reynolds, G. F. (1965b). Anal. Chim. Acta 32, 227. Maksimenko, O. A., Zyukova, L. A., Ignat'eva, E. V. and Fedorovich, R. M. (1973).

Zh. Anal. Khim. 28, 1588. Malaprade, L. (1928). Bull. Soc. Chim. France 43, 683. Malaprade, L. (1937). Bull. Soc. Chim. France [5] 4, 906. Mangold, C. (1891). Z. Angew. Chem. 4, 400. Marconi, M. (1952). Chimica (Milan) 7, 336. Mares, E. (1957). Prum. Potravin. 8, 147 (Anal. Abs. 5, 1546). Maros, L. and Schulek, E. (1959). Magy. Kern. Folyoirat 65, 361; also Acta Chim.

Acad. Sci. Hung. 20, 358. Maros, L. and Schulek, E. (1960). Ann. Univ. Sci. Budapest Rolando Eotvos Nominatae,

Sect. Chim. 2, 227, 247. Marshev, P. M. (1964). Lab. Delo 601. Martin, G. E., Dyer, R. H. and Figert, D. N. (1975). / . Ass. Off Anal. Chemists 58,

1147. Mason, M. E. and Waller, G. R. (1964). Anal. Chem. 36, 583. Mason, M. E., Eager, M. E. and Waller, G. R. (1964). Anal. Chem. 36, 587. Mathers, A. P. and Pro, M. J. (1954). J. Ass. Off. Agr. Chem. 37, 869. Matsumoto, T. (1940). Repts. Chem. Res. Prefect. Inst. Adv. Ind. Tokyo No . 3,1 (Chem.

Abs. 35, 7746). Mead, J. F. and Bartron, E. A. (1948). J. Amer. Chem. Soc. 70,1286. Melkonyan, S. A. (1970). Sb. Nauch. Tr., Erevan. Arm. Gos. Pedagog. Inst. Khim.

No. 1, 71 (Chem. Abs. 11, 69822). Mendelsohn, D. and Antonis, A. (1961). / . Lipid Res. 2, 45. Mesnard, P., Gibirila, B. and Bertucat, M. (1963). Chim. Anal. (Paris) 45, 491. Metayer, G. (1947). Ann. Pharm. Franc. 5, 369. Metzenberg, R. L. and Mitchell, H. K. (1954). J. Amer. Chem. Soc. 16,4187. Michalski, E. and Stapor, M. (1966). Lodz. Tow. Nauk Wydz. Ill Acta Chim. 11, 25

(Chem. Abs. 66, 72271). Michl, H. (1955). Monatsh. 83, 737. Misantone, R. (1966). Carriere Farm. 21, 454 (Chem. Abs. 66, 88686). Mizsei, A., Igloy, M. and Veress, G. (1964). Magy. Kern. Lapya 19, 503 (Anal. Abs.

13, 355). Mlejnek, O. (1963). Chem. Prumysl 13, 105. Monterumici, R. (1932). Boll. Chim. Farm. 71, 757. Morawski, T. (1880). J. Prakt. Chem. (NF) [2] 22, 416.

90 GLYCEROL

Morello, J. (1938). Parfumerie Moderne 32, 55, 57 (Chem. Abs. 32, 9397). Mormont, R. (1971). Talanta 18, 1171. Mormont, R. (1972). Chem. Ind. (London) 128. Mormont, R. and Gillet jr., A. C. (1967). Ind. Chim. Beige 32 (Spec. No. Part 3) 735

(Chem. Abs. 70, 69453). Mormont, R., Gillet jr. A. C. and Heinerth, E. (1969). Talanta 16, 701. Mull, R. P. (1943). Arch. Biochem. 2, 425. Mulliken, S. P. (1904). "Identification of Pure Organic Compounds", Wiley, New

York, Test No . 816, p. 169. Murray, W. J. and Williams, A. F. (1961). Analyst (London) 86, 849. Muter, J. (1881). Analyst (London) 6, 41. Nadirov, N. K., Freze, N. A., Ozerova, S. N., Knyazev, V. N., Divnenko, Z. A. and

Abdilaev, B. V. (1971). Khim. Khim. Tekhnol. No . 2, 24 (Chem. Abs. 79,121626). Narang, C. K., Iyer, V. and Mathur, N. K. (1965). Microchem. J. 9, 408. Nash, T. (1953). Biochem. J. 55, 416. Neale, S. M. (1926). J. Text. Inst. 17, 511 (Chem. Abs. 21, 499). Neish, A. C. (1950). Can. J. Res. 28B, 535. Neuberg, C. and Mandel, J. A. (1916). Z.d. Verein deut. ZuckAnd. p. 6; Z. Anal.

Chem. 56, 407. Neumann, R. (1917). Z. Angew. Chem. 30, 234. Newburger, S. H. and Bruening, C F. (1947). J. Ass. Off Agr. Chem. 30, 651. Nicloux, M. (1897). Bull. Soc. Chim. France 17, 453,839; also (1903). ibid. 29, 245. Nisbet, M. A. and Schmeller, S. (1970). Tob. Sci. 14, 145 (Chem. Abs. 74, 10529). Nishi, H., Yoshitani, H. and Kamachi, T. (1969). Nippon Sembai Kosha Chuo

Kenkyusho Kenkyu Hokoku No. I l l , 85 (Chem. Abs. 73, 73979). Nonaka, A. (1967). Japan Analyst 16, 1166; also (1968). ibid. 17, 91. Novak, L. (1960), Kvasny Prumsyl 6, 109 (Chem. Abs. 54, 15822). Ohkuma, S. and Miyauchi, C. (1962). Seikagaku 34, 166 (Chem. Abs. 57, 4962). Ohl, F. (1938). Kunstseide u. Zellwolle 20, 230 (Chem. Abs. 32, 6872). Okuhara, A. and Yokotsuka, T. (1958). J. Agr. Chem. Soc. Japan 32,138 (Chem. Abs.

54, 10231). Oles, P. J. and Siggia, S. (1974). Anal. Chem. 46, 2197. Oliveri, V. and Spica, P. (1890). Le Stazione Sperimente Agric. Ital. 19, 34. Olley, J. (1956). Biochem. J. 62, 107. Opfer-Schaum, R. (1944). Mikrochemie ver. Mikrochim. Acta 31, 330. Orchin, M. (1943). J. Ass. Off. Agr. Chem. 26, 99. Orlova, I. Yu., Sennikov, G. P. and Al'perovich, M. A. (1976). Zh. Anal. Khim. 31,595. Palfray, L., Sabetay, S. and Libmann, G. (1946). Compt. Rend. 223, 247. Palleroni, N. J. and Vega, R. (1954). Univ. Nacl. Cuyo, Fac. Cienc. Agrar. Biol. Tec.

No. 5/6, 3 (Chem. Abs. 49, 11235). Papin, J-P. and Udiman, M. (1975). J. Chromatogr. 115, 267. Paquet, C. (1946). Inds. Corps Gras 2, 272 (Chem. Abs. 41, 297). Parker, W. E. and Richardson, P. J. (1970). J. Inst. Brew. 76, 191. Patterson, S. J. (1963). Analyst (London) 88, 387. Paulsson, R. B. and Waaler, T. (1962). Pharm: Helv. Acta 37, 125. Pays, M., Malangeau, P. and Bourdon, R. (1967). Ann. Pharm. Frang. 25, 29. Pesez, M. (1954). Bull Soc. Chim. France 1231. Pesez, M. (1956). Bull Soc. Chim. France 148. Pesez, M. and Poirier, P. (1953). "Methodes et Recherches de l'Analyse Organique",

Masson, Paris, Vol. II, p. 109.

REFERENCES 91

Peterson, J. W., Hedberg, K. W. and Christensen, B. E. (1943). Ind. Eng. Chem., Anal. Ed. 15, 225.

Peynaud, E. (1948). Ann. Fals. et Fraudes 41, 384. Peynaud, E. and Charpentie, Y. (1954). Ann. Fals. et Fraudes 47, 85. Plsko, V. (1958). Chem. Zvesti 12, 312 (Chem. Abs. 52, 15343). Pohle, W. D. and Mehlenbacher, V. C. (1946). Oil and Soap 23, 48. Pohle, W. D. and Mehlenbacher, V. C. (1947). J. Amer. Oil Chem. Soc. 24, 155. Polak, F. and Wilkosz, L. (1959). Chem. Anal. (Warsaw) 4, 947. Polak, H. L., Pronk, H. F. and den Boef, G. (1962). Z. Anal. Chem. 189, 411. Popiel, W. J. (1961). Chem. Ind. (London) 434. Pozzi-Escot, E. (1938). Bull. Assoc. Chem. 55, 353; also Rev. Cienc. (Peru) N o . 423,

127 (Chem. Abs. 32, 5334, 7682). de Prada, L. (1934). Anales Farm. Bioquim. 5, 98 (Chem. Abs. 30, 7775). Pramme, M. H. (1931). Ind. Eng. Chem., Anal. Ed. 3, 232. Prey, V., Berbalk, H. and Kausz, M. (1962). Mikrochim. Acta 449. Procopio, M. and Antona, M. (1960). Riv. Viticolt. Enol. 13, 242 (Chem. Abs. 60,

13844). Procter and Gamble Co. (1937). Ind. Eng. Chem., Anal. Ed. 9, 514. Puschmann, H. and Miller, J. E. (1961). Z. Lebensm.-Untersuch. u. Forsch. 114, 297. Radley, J. A. (1944). Analyst (London) 69, 15. Radley, J. A. (1950). J. Sci. Food Agr. 1, 222. Rajiah, A., Subbaram, M. R. and Achaya, K. T. (1968). J. Chromatogr. 38, 35. Ramsay, W. N. M. and Stewart, C. P. (1941). Biochem. J. 35, 39. Randa, E. (1937). Oil and Soap 14, 7. Rao, B. M. and Gopala Rao, G. (1972). Z. Anal. Chem. 258, 368. Rapp, A., Bachmann, O. and Ziegler, A. (1975). Deut. Lebensm.-Rundschau 71, 345. Rauschenbach, F. and Lamprecht, W. (1966). Z. Physiol. Chem. 346, 290. Ravenna, A. (1928). Zymol. Chim. Col. e Zucch. 3, 174 (Chem. Abs. 23, 1593). Raveux, R. (1943). Ann. Chim. Anal. 25, 70, 95 (Chem. Abs. 40, 1117). Ravich, B., Popov, M. M. and Klynchevich, E. S. (1939). J. Appl. Chem. (USSR) 12,

1571. Rebelein, H. (1957). Z. Lebensm.-Untersuch. u. Forsch. 105, 296. Reese, H. D. and Williams, M. B. (1954). Anal. Chem. 26, 568. Reichard, O. and Gspahn, H. (1954). Z. Anal. Chem. 141, 252. Reif, G. (1951). Pharmazie 6, 149. Reznikov, I. G. and Farber, E. L. (1953). Masloboino-Zhirov. Prom. 18, No. 5, 13

(Chem. Abs. 47, 9863). Richardson, F. W. and Jaffe, A. (1898). / . Soc. Chem. Ind. 17, 330. Ripper, M. and Wohack, F. (1916). Z. fur Landw. Versuchsw. Osterreich 19, 372;

Z. Anal. Chem. 56,163. Robinson, W. T., Cundiff, R. H. and Markunas, P. C. (1961). Anal. Chem. 33,

1030. Rosen, M. J. (1963). Anal. Chem. 35, 2074. Rosenberger, H. M. and Shoemaker, C. J. (1957). Anal. Chem. 29, 100. Rosenthaler, L. (1939). Pharm. Acta Helv. 14, 218. Rosenthaler, L. (1953). Pharm. Ztg. ver. Apoth.-Ztg. 89, 883. Rosenthaler, L. and Vegezzi, G. (1954). Z. Lebensm.-Untersuch. u. Forsch. 99, 352. Roussos, M. (1964). Chem. Phys. Appl. Surface Act. Subs., Proc. 4th Int. Congr.

(Publ. 1967) 1, 449 (Chem. Abs. 70, 98140). Roy, G. L., La Ferriere, A. L. and Edwards, J. O. (1957). / . Inorg. Nucl. Chem. 4, 106.

92 GLYCEROL

Sakagami, H. and Fukuzumi, T. (1974). Nippon Sembai Kosha Chuo Kenkyushi, Kenkyu, Hokoku 116, 61 (Chem. Abs. 84, 71657).

Salzer, F. and Weber, G. (1950). Z. Lebensm.-Untersuch. u. Forsch. 91, 174. Sampson, K., Schild, F. and Wicker, R. J. (1961). Chem. Ind. (London) 82. Samuelson, O. and Stromberg, H. (1968). Acta Chem. Scand. 22,1252. Sanchez, J. A. (1944). Rev. Asoc. Bioquim. Argent. 10, 63 (Chem. Abs. 39, 476). Sand, J. R. and Huber, C. O. (1967). Talanta 14, 1309. Saracco, G. B. and Gay, M. (1971). Riv. Ital. Sostanze Grasse 48, 319 (Anal. Abs. 22,

2714). Sargent, R. N. (1956). Dissert. Abs. 16, 1809. Sargent, R. and Rieman III, W. (1956). Anal. Chim. Acta 14, 381. Sargent, R. and Rieman III, W. (1957). Anal. Chim. Acta 16, 144. Sarsunova, M. (1973). Cesk. Farm. 22, 259. Savage, R. I. and Wagstaffe, P. J. (1973). Ann. Fals. Expert. Chim. 66, 246. Sawicki, E., Schumacher, R. and Engel, C. R. (1967). Microchem. J. 12, 377. Sawyer, D. T. and Brannan, J. R. (1966). Anal. Chem. 38, 192. Schaefer, W. E. (1937). Ind. Eng. Chem., Anal Ed. 9, 449. von Schlatter, A. (1932). Kunstseide 14, 367. Schoorl, N. (1939). Pharm. Weekbladl6, 111. Schroder, E. (1960). Plaste u. Kautschuk 7, 167. Schryver, S. B. (1910) Proc. Roy. Soc. 82B, 226. Schutz, F. (1938). Papier-Fabr. 36 (Tech. 77. 55; Chem. Abs. 32, 2744). Seher, A. (1964). Fette, Seifen, Anstrichm. 66, 371. Seligsberger, L. (1950). / . Amer. Leather Chem. Ass. 45, 770 (Chem. Abs. 45, 4075). Semichon, L. and Flanzy, M. (1930). Ann. Fals. 23, 583. Sen, N., Keating, M. and Barrett, C. B. (1967). J. Gas. Chromatogr. 5, 269. Shanfield, H , Hsu, F. and Martin, A. J. P. (1976). J. Chromatogr. 126, 457. Shapira, J. (1969). Nature (London) 222, 792. Sharma, N. N. and Mehrotra, R. C. (1955). Anal Chim. Acta 13, 419. Shaw, V. and Walton, H. F. (1972). J. Chromatogr. 68, 267. Shay, J. F , Skilling, S. and Stafford, R. W. (1954). Anal. Chem. 26, 652. Shimomura, K. Hsu, T-J. and Walton, H. F. (1973). Anal. Chem. 45, 501. Shukoff, A. A. and Schestakoff, P. J. (1905). Z. Angew. Chem. 18, 295. Shupe, I. S. (1943). / . Ass. Off. Agr. Chem. 26, 249. Siegel, H., Bullock, A. B. and Carter, G. B. (1964). Anal Chem. 36, 502. Siggia, S., Hanna, J. G. and Culmo, R. (1961). Anal Chem. 33, 900. Silverman, L. (1947). J. Amer. Oil Chem. Soc. 24, 410. Singh, D. and Sharma, S. (1970). Indian J. Chem. 8, 192. Smirnova, A. P. and Eskina, N. A. (1962). Vinodelie i Vinogradarstvo SSSR 22, No. 8,

15 (Chem. Abs. 58, 11925). Smith, G. (1926). J. Text. Inst. 17, 87T. Smith, D. M. and Bryant, W. M. D. (1935). / . Amer. Chem. Soc. 57, 61. Smith, B. and Carlsson, O. (1963). Acta Chem. Scand. 17, 455. Smith, G. F. and Duke, F. R. (1941). Ind. Eng. Chem., Anal Ed. 13, 558. Smith, G. F. and Duke, F. R. (1943). Ind. Eng. Chem., Anal. Ed. 15, 120. Smullin, C F., Hartmann, L. and Stetzler, R. S. (1958). J. Amer. Oil Chem. Soc. 35, 179. Soule, S. (1929). Chemist-Analyst 18, 8. Spagnolo F. (1953). Anal Chem. 25, 1566. Sperlich, H. (1952). Dissertation, Stuttgart. Sporek, K. and Williams, A. F. (1954). Analyst (London) 79, 63.

R E F E R E N C E S 93

Srivastava, V. N. P. and Saxena, O. C. (1967). Microchem. J. 12, 435. Stahl, E., Laub, E. and Woller, R. (1975). Z. Anal. Chem. 275, 257. Stamm, H. (1934). Z. Angew. Chem. 47, 191. Stamm, H. (1935). Z. Angew. Chem. 48, 710. Steinfels, W. (1910). Seifensiederz. 37, 793; Z. Anal. Chew..51,515. Steinfels, W. (1915). Seifensiederz. 42, 721; Z. Anal. Chem. 55, 60. Stempel, B. (1949). Z. Anal. Chem. 129, 232. Strebinger, R. and Streit, J. (1924). Z. Xmi/. Ctem. 64, 136. Sushkov, V. I., Aleksandrovich, I. F., Rozenberg, A. Ya., Kim, Y. P. and Gusev, S. S.

(1973). Khim. Volokna 15, 64 (Chem. Abs. 79, 106247). Szahlender, K. (1933). Magy. Gydgy. Tars. Fries. 9, 125 (Chem. Abs. 27, 3897). Szigetvary, F. and Kuttel, D. (1963). Gyogyszereszet 7, 19 (Chem. Abs. 60, 5275). Taufel, K. and Thaler, H. (1933). Z. Anal. Chem. 95, 235. Taufel, K. and Thaler, H. (1934). Pharm. Ztg. 79, 341. Talipov. S. T., Abdilaev, B., Knyazev, V. N. and Freze, N. A. (1972). Izv. Vyssh.

Ucheb. Zaved., Khim. Khim. Tekhnol. 15, 1592 (Chem. Abs. 78, 66663). Talipov, S. T., Nadirov, N. K., Freze, N. A. and Abdilaev, B. V. (1972). Nauch. Tr.

Tashkent Univ. No. 419, 108 (Chem. Abs. 79, 121633; Anal. Abs. 25, 2319). Tanner, H. and Duperrex, M. (1968). Fruchtsaft. 13, 98 (Chem.Abs. 69, 105110;

Anal. Abs. 17, 3042). Tasman, A. and Smith, L. (1943). Chem. Weekblad 40,32. Tateo, F. (1970). Sci. Aliment. 16, 150, 189 (Chem. Abs. 73, 119288, 137130). Teodorescu, S. C , Tomescu, F. C. and Iliescu, L. V. (1957). Acad. Rep. Pop. Romine

Bui. Stiint., Sect. Biol Stiint. Agr. (Sec. Agron) 9, 19 (Chem. Abs. 52,4099). Testorelli, A. J. A., Saguier, R. A. and Bayer, J. R. (1955). Anales Direc. Nacl. Quim.

(Buenos Aires) 8, No. 16, 60 (Chem. Abs. 52, 8853). Thaler, H. and Roos, W. (1950). Z. Anal. Chem. 131, 24. Thivolle, L. and Raveux, R. (1941). Trav. Membres Soc. Chim. Biol. 23, 1445 (Chem.

Abs. 39, 2083). Thivolle, L. and Raveux, R. (1942). Trav. Membres Soc. Chim. Biol. 24, 1066. Thomas, P. and Micsa, A. (1924). Bui. Soc. Stiinte Cluj 2, 222 (Chem. Abs. 19, 3074). Tomi, P., Iuonas, E. and Pop, F. (1974). Rev. Chim. (Bucharest) 25, 761 (Chem. Abs.

82, 128970). Tortelli, M. and Ceccherelli, A. (1913). Chemiker-Ztg. 37, 1505, 1573; also (1914).

ibid. 38, 2, 28, 46. Trevelyan, W. E., Procter, D. P. and Harrison, J. S. (1950). Nature (London) 166, 444. Tsarfin, Ya. A. and Kharchenkova, V. D. (1975). Zh. Anal. Khim. 30, 391. Tschirch, E. (1951). Seifen-Ole-Fette-Wachse 11, 333. Tsuji, K. and Konishi, K. (1971). Analyst (London) 96, 457. Tupalska, M. (1957). Roczn. Panst. Zakl. Hig. 8, 349 (Chem. Abs. 52, 3264). Turgel', E. O., Kuznetsova, E. V., Rudoi, S. A. and Skop, S. L. (1972). Zh. Anal. Khim.

27, 1194. Udalova, T. P. (1966). Vestn. Mosk. Univ., Ser. VI 21, 88 (Chem. Abs. 67, 681). Umar, M. and Badar-ud-Din (1966). Pakistan J. Sci. Res. 18, 15. Usmanskaya, A. A., Makarenkova, R. M., Sokolov, N. M. and Zhavoronkov, M. M.

(1970). Zh. Anal. Khim. 25, 1211. Vasconcellos e Lencastre, A. de Q. (1946). Anais Inst. Vinho Porto No. 7, 31 (Chem.

Abs. 42, 3131). Vasyunina, N. A., Balandin, A. A., Mamatov, Yu. and Pustovaya, L. M. (1962).

Gidrol. i Lesokh. Prom. 15, No. 1, 13 (Chem. Abs. 57,1558).

94 GLYCEROL

Vaver, V. A., Ushakov, A. N. and Bergel'son, L. D. (1967). Izv. Akad. Nauk SSSR, Ser. Khim. 1187 (Chem. Abs. 67, 105837; Anal. Abs. 15, 6708).

Vaver, V. A , Ushakov, A. N. and Bergel'son, L. D. (1968). Izv. Akad. Nauk. SSSR, Ser. Khim. 400 (Chem. Abs. 69, 8328; Anal. Abs. 16, 3061).

Vecher, A. S. and Ulitina, O. A. (1958). Izv. Vyssh. Ucheb. Zav. Pishchev. Tekhnol. No. 1, 103 (Chem. Abs. 53, 8531):

Venturini, A. (1972). Boll. Lab. Chim. Prov. 23, 333. Verhaar, L. A. T. and De Wilt, H. G. J. (1969). J. Chromatogr. 41, 168. Verley, A. and Bolsing, F. (1901). Ber. 34, 3354. Verma, P. S. and Grover, K. S. (1971). Vijnqna Parishad Anusandhan Patrika 14, 13

(Chem. Abs. 11, 13762). Viebock, F. and Brecher, C. (1930). Ber. 63, 3207. Viebock, F. and Schwappach, A. (1930). Ber. 63, 2818. Voris, L., Ellis, G. and Maynard, L. A. (1940). J. Biol. Chem. 133, 491. Wagenaar, M. (1911). Pharm. Weekblad 48,497; Z. Anal. Chem. 51,515. Waldi, D. (1965). / . Chromatogr. 18, 417. Waldi, D. and Lange, H. R. (1963). Naturwiss. 50, 126. Weigel, W. (1955). Fette, Seifen, Anstrichsm. 57,486. Weigel, H. (1963). Adv. Carboh. Chem. 18, 61. Weiss, A. H. and Tambawala, H. (1972). J. Chromatogr. Sci. 10, 120. West, D. M. and Skoog, D. A. (1959). Anal. Chem. 31, 586. Wetterholm, A. (1946). Harold Nordensen Anniv. Vol. 460 (Chem. Abs. 43, 5325). Wheaton, R. M. and Baumann, W. G. (1953). Ann. N.Y. Acad. Sci. 57, 159. Whyte, L. K. (1946). Oil and Soap 23, 323. Whyte, J. N. C. (1973). J. Chromatogr. 87, 163. Williams, A. F. (1953). Nature (London) 171, 655. Williams, J. F. (1971). J. Ass. Off. Anal. Chemists 54, 560. Wilson, H. N. and Hughes, W. C (1939). J. Soc. Chem. Ind. 58, 74. Wright, J. (1963). Chem. Ind. (London) 1125. Yamada, T., Hisamatsu, M. and Taki, M. (1975). J. Chromatogr. 103, 390. Zajic, J. (1962). Sb. Vysoke Skoly Chem.-Technol v. Praze, Oddil Fak. Potravin.

Technol. 6, 179 (Chem. Abs. 61, 15358). Zamyshevskaya, N. N. and Yaroshinskaya, N. P. (1965). Khim. Volokna 67 (Chem.

Abs. 64, 6881). Zeisel, S. and Fanto, R. (1903). Z. Anal. Chem. 42, 549. Zelenetskaya, A. A., Voronseva, I. M. and Markina, A. B. (1968). Tr. Vses. Nauch.

Issled. Inst. Sint. Nat. Dush. Veshch. 8, 342 (Anal. Abs. 17, 2164). Zelikman, Z. L., Tkachenko, S. E., Mamina, N. A., Fedenova, A. A. and Glazman,

R. A. (1973). Tr. Krasnodar. Politekh. Inst. 49, 23 (Chem. Abs. 80, 152570). Zyka, J. and Berka, A. (1962). Mickrochem. J. Symp. Ser. 2, 789.

2

Glycerol Compounds: Introduction, and Methods

Based on Residual Hydroxyl Groups

Glycerol is combined in many naturally occurring and synthetic materials, as esters or ethers. Best known are the fats and oils which are glycerol esters of higher fatty acids, saturated (such as palmitic and stearic) and unsaturated (such as oleic, linoleic, and linolenic). The fats and oils consist almost entirely of triesters in which all three glycerol hydroxyl groups are esterified, although not necessarily with the same acid moiety. Mono- and di-esters are also encountered in fresh plant and animal materials but not usually in more than small amounts.

Also found in nature but only comparatively rarely are alkyl diglycerides:

CH 2 —O—R I

CH—O—COR' I

C H 2 — O — C O R "

in which a long-chain alkyl group, R, is attached through an ether linkage to the 1-position of L-glycerol which is esterified in the other two positions. Related to these, but present in nature only in traces, are the neutral plas-

95

96 GLYCEROL COMPOUNDS: INTRODUCTION A N D METHODS

The residual hydrogen atom of the phosphate group can be replaced in ether structures by various groups, such as:

Choline

— C H 2 — C H 2 — N ( C H 3 ) 3

(Lecithin)

Ethanolamine

—CH 2 —C (Cephalin)

Serine

C H 2 — C H 2 — N H 3 — C H 2 — C H — N H 3

Glycerol

— C H 2

I CHOH I

C H 2 O H

COOH

OH OH

All these compounds occur in animal and plant tissues. An ether linkage may be found in position 3 of 1,2-diacyl-sn-glycerols,

with sugar moieties, especially with galactose and digalactose (yielding, respectively, monogalactosyl and digalactosyl diglycerides). Other sugar moieties, e.g. glucose, mannose, or rhamnose, are also known in bacteria.

Sulphoquinovosyl diglycerides, "sulpholipids", are found only in plant tissues; in them, the — C H 2 O H group of the sugar moiety is replaced by the — C H 2 — S 0 3 H group.

malogens. These contain a vinyl ether group in the 1-position of L-glycerol:

CH 2 — O — C H = C H — R I

CH—O—CO—R' I

CH 2 —O—CO—R"

The so-called complex lipids include the glycerophosphatides (also called phosphoglycerides), built up from glycerol, long-chain fatty acids, and phosphoric acid:

CH 2 —O—COR I

CH—O—COR' | (free acid = phosphatidic acid)

C H 2 — O — P — O — H ^ \

O O - H +

INTRODUCTION 97

Among synthetic glycerol-containing materials may be mentioned glycerophosphoric acid:

CH7—OH I

CH—OH

I C H , — O — P — O H

O O" H +

This is best known through its salts (sodium, calcium) which are used as tonics.

Two well-known synthetic materials are "nitroglycerine", the trinitrate ester of glycerol, used as an explosive and medicament, and certain alkyd resins ("glyptals"). These last-named are polyesters of dibasic acids (usually phthalic or maleic) with polyols containing two or more hydroxyl groups, glycerol being a prominent representative; it can form three-dimensional polyesters.

Most analytical problems in connection with these compounds and classes concern detection, identification, determination, or separation of (a) indi-vidual glycerol-containing compounds; (b) totals of compound classes containing combined glycerol; (c) the glycerol, fatty acid, phosphoric acid, nitric acid, etc. in these compounds or classes.

In a book devoted to glycerol, certain limitations have to be imposed on the content. Under (a) and (b) are treated only those methods that depend, at least to some extent, on a chemical reaction of the glycerol moiety or on physical properties conferred or determined by it. Determinations of gly-cerides via, for example, the fatty acid moiety are not dealt with. Under (c) are considered only those methods with analytical response or evaluation of the glycerol content.

An attempt is made to classify the material into three broad categories; some overlapping is unavoidable. The remainder of this chapter is devoted to methods based on residual glycerol hydroxyl groups. Chapter 3 deals with methods based on release of glycerol from its combined state in the sample, followed by its analytical evaluation. Chapter 4 describes methods based on probable participation of the complete molecules of the sample (thereby including the glycerol moiety).

The methods based on residual glycerol hydroxyl groups are for only the mono- and di-esters and -ethers. Many of these methods involve reaction of the free hydroxyl group(s). A few purely physical methods also fall into

98 GLYCEROL COMPOUNDS: INTRODUCTION A N D METHODS 2.1

this category, for example infrared procedures in which the absorbance associated with the hydroxyl groups is measured.

The decrease in polarity with progressive removal of the hydroxyl groups, in the order glycerol, mono-esters or -ethers, di-esters or -ethers, tri-esters or -ethers, permits successful use of various physical methods of separation. Prominent among these are chromatographic procedures which have indeed been applied widely to glyceride mixtures. A selection of examples is given.

A distinction may be made between methods for the hydroxyl groups in general and those for the 1,2-diol grouping. The latter include the selective oxidation methods with periodate and lead(IV), and also formation of iso-propylidene groups and of complexes with borate and similar anions. Their application is restricted to 1-monoglycerides and 1-ethers and analogous derivatives possessing the 1,2-diol group. The former methods are typified by esterification and ether formation, mostly followed by gas chromato-graphy.

2 . 1 . O X I D A T I O N W I T H PERIODATE

Oxidation with periodate is the most widely used chemical method for detecting and, especially, determining glycerol compounds containing a 1,2-diol group. As seen in Chapter 1, several qualitative and quantitative procedures are possible.

2 . 1 . 1 . Use of Excess Reagent and De tec t ion or Determina t ion of the

U n u s e d Reagent

In most quantitative periodate procedures for 1-monoglycerides and similar compounds, a measured amount of reagent in excess is used and the unreacted part is estimated by one of the procedures mentioned in Chapter 1, Section 1.1.14.B. Oxidation conditions (solvent) and times have varied from several hours to some minutes.

Fleury and Paris (1933) determined 1-glycerophosphate by oxidising for 10 min, then adding potassium iodide and excess standard arsenite in the presence of sodium hydrogen carbonate. Under these near-neutral condi-tions, periodate is reduced only to iodate:

I 0 4 " + 21" + 2 H + - * I 0 3 - + I 2 + H 2 0

The liberated iodine is consumed by the arsenite, the unused part of which can afterwards be titrated; the authors used iodine for this. Ivanoff (1945) similarly determined 1-monoglycerides in glyceride mixtures by dissolving

2.1 OXIDATION WITH PERIODATE 99

the sample in 97-98 % ethanol and oxidising with acid periodate for 15-20 h in the dark. After adding sodium hydrogen carbonate, potassium iodide, and excess arsenite, he left for 15 min and finished by back-titrating with iodine.

Kruty et al. (1954) concluded by estimating unreacted periodate in sodium hydrogen carbonate-potassium iodide solution, but by direct titra-tion with arsenite. They determined 1-monoglyceride + free glycerol by treating the sample in dimethylformamide-chloroform (1 + 19) with methan-olic periodic acid. Glycerol alone was determined on an aqueous extract of a chloroform solution of the sample, and the monoglyceride amount ob-tained by difference. Monostearin was determined by Pedersen (1953) by oxidising the sample in t-butanol-water (10 + 1) with periodic acid for 4 h and then adopting the back-titration principle of Fleury and Paris. Baur and Distler (1966) also adopted this principle to determine monoglycerides.

Others have preferred the differential method with back-titration of iodine liberated from unused periodate in acid solution, whereby it is re-duced to the iodide stage. Pohle et al. (1945) determined 1-monoglycerides by mixing a 50 ml aliquot of sample solution in chloroform with 50 ml of 0-27% periodic acid ( H 5 I 0 6 ) in acetic acid-water (19 + 1). After 30 min at room temperature they added 20 ml of 15% aqueous potassium iodide, left for 1 min, and then titrated the iodine with 0-1N thiosulphate after having diluted with 100 ml of water. The customary control was carried out under similar conditions in the absence of sample. Pohle and Mehlenbacher (1950) used practically the same method after previously extracting free glycerol with water. Handschumaker and Linteris (1947) determined 1-mono-glycerides in fats and oils likewise by oxidation in acid solution [sample in acetic acid-chloroform (2 + 1) + reagent in 80% acetic acid] and estimating unused reagent under acid conditions. These authors, also Desnuelle et al. (1948) whose procedure was closely similar, used shorter oxidation times than Pohle and co-workers. The method of Handschumaker and Linteris was employed by Kummerow and Daubert (1950) for fats and oils and by Doadrio and Montequi (1952) for technical monoglycerides. Montequi and Doadrio (1952) modified the procedure slightly by washing several times with 15% sodium sulphate to remove free glycerol. Hartman's (1956) procedure for determining 1-monoglycerides in technical material is also very similar. He took the sample in pyridine-acetic acid-chloroform (1 + 1 + 8) and a reagent of potassium periodate, acetic acid, and sulphuric acid, and allowed to react for 30 min in the dark before adding aqueous potassium iodide and titrating as usual with thiosulphate.

Pohle et al. (1957) reported a comparison between the methods of Kruty et al. (1954), of Pohle and Mehlenbacher (1950), and a method in which the monoglyceride and free glycerol are partitioned into chloroform and water, respectively; the last named was found best. Becker and Krull (1958) also


compared the methods of Kruty et al. and of Pohle and Mehlenbacher, and preferred the former. Franzke and Strandt (1967) critically examined some methods for glyceride determination, including those of Pohle and Mehlen-bacher.

The work of Guernet et al. (1973) may be mentioned at this point. They tested quaternary ammonium periodates (e.g. cetyltrimethylammonium, cetylpyridinium, and cetylbenzyldimethylammonium) for oxidising in organic solvents (such as chloroform, pyridine, ethanol, or acetic acid) compounds that are poorly soluble in water. The unused reagent was removed with excess arsenic(III), the unreacted part of which was then back-titrated with iodine to a yellow colour, or potentiometrically. Their examples in-cluded glycerol stearate.

The periodate-aromatic amine reagent (see Chapter 1, Section 1.1.14.A), based on the oxidation of the amine to coloured products by residual period-ate, has been used also to detect glycerol 1-mono-compounds. Wawszkiewicz (1961) detected on Whatman No. 1 paper many phosphate esters of polyols, including glycerol-1-phosphate, by reaction with 0-lM-sodium periodate-acetone (1 + 19), drying for 3 min and then dipping into a benzidine-acetic acid-water-acetone reagent to obtain yellow zones on a blue-green to grey-green background after drying. Another example is the use by Halvarson and Qvist (1974) of potassium periodate oxidation and then treatment with anisidine to yield white spots on a coloured background in ths detection of monoglycerides on a reference silica gel thin layer.

2.1.2. Isomer isat ion of Glycero l 2 - M o n o e s t e r s

Migration of acyl groups in glycerol esters has been known for a relatively long time, since early investigations by Fischer (1920). Daubert and King (1938) found that aliphatic and aromatic 2-monoesters of glycerol were transformed into the 1-isomers in a short time at room temperature in 0-1N hydrochloric acid or ammonium hydroxide. Analytical interest in this was probably aroused after the development of the periodate oxidation which would be applicable to the 2-esters also if they could be quantitatively isomerised. In a study of diose phosphates, Fleury and Courtois (1941) noted that the 2-glycerophosphate could be converted into the 1-form in hot dilute acid.

Martin (1953) reported that the 2-monoglycerides (of fatty acids) are iso-merised to the 1 -compounds in chloroform solution with 56 % perchloric acid. The equilibrium mixture contained about 90 % of 1-monoester and he used a correction factor of 1-15 to allow for this incomplete conversion. Jensen and Morgan (1959) applied isomerisation with perchloric acid to determine both types of monoglyceride in milk. Bertoni et al. (1963) studied the efficiency of

2.1 OXIDATION WITH PERIODATE 101

the perchloric acid catalyst for determination of total monoglyceride. They found that an acid concentration above 0-02% (of 56% acid) yielded high results. More recently, Franzke and Strandt (1967) studied the isomerisa-tion in acetic acid-chloroform ( 1 + 2 ) with 56% perchloric acid, likewise employing a factor of 1-15.

2.1.3. De tec t ion and D e t e r m i n a t i o n of A l d e h y d e React ion Products

Emmerie (1953) oxidised glycerol 1-ethers (with long-chain alcohols con-taining 14, 16, and 18 C atoms) using a slight excess of periodic acid for 4 h at room temperature. He separated the reaction products by ascending paper chromatography on Whatman No. 1 impregnated with paraffin or cetyl acetate, using aqueous lower alcohols as mobile phase and visualising with the Schiff reagent. Shaw (1968) detected glycerophosphatides on silicic acid layers as purple zones by spraying with 1 % sodium periodate and then, after 5-10 min, with 1 % pararosaniline hydrochloride decolorised with sulphur dioxide. The Schiff reagent and periodate were used also by Qureshi and Waheed (1972) in their analytical work on the 1-monoglycerides in fats; and by Hack and Helmy (1975) to detect glycerol ethers.

Quantitative methods may be based on the formaldehyde or other aldehyde product:

C H 2 O H HCHO

I + CHOH + H I 0 4 -> CHO + H I O , + H 2 0 I I

C H 2 0 — X C H 2 0 — X

Dowse and Saunders (1956) determined 1-monoglycerides in chloroform by treating for 30 min with propionic acid and aqueous periodic acid, stopping the reaction by adding stannous chloride, removing chloroform in a current of air, and steam-distilling the formaldehyde. They estimated this product in an aliquot of distillate using the colour reaction with chromotropic acid in sulphuric acid. Jensen and Morgan (1959) applied the same principle to determine milk monoglycerides (butyrin, olein, laurin, palmitin). Mono-glyceride emulsifiers in ice cream were determined by Schmidt (1963) by extraction with ether, periodate oxidation, and colour reaction with chromo-tropic acid also.

Karnovsky and Rapson (1946) determined glycerol ethers in natural fats by periodate oxidation, and then precipitation and gravimetric assay of the formaldehyde as its condensation product with dimedone.

Szonyi and Sparrow (1964) oxidised 1-monoglycerides in benzene solution with periodic acid in acetic acid-water (19 + 1) for 30 min, then removing the

E


2.2. O X I D A T I O N W I T H L E A D ( I V )

Wormith and Rae (1941) used the other standard oxidation reagent for 1,2-diol groups, lead(IV), in quantitative analytical work. They determined 1-glycerophosphates by treating the aqueous solution with dilute hydro-chloric acid and excess lead tetraacetate in acetic acid (under these conditions the loss of reagent through hydrolysis is negligible). After 6 h at room temperature they added sodium acetate and potassium iodide, and titrated with thiosulphate the iodine liberated from unused reagent; a control titra-tion of reagent in sodium dihydrogen phosphate solution was carried out in the same way. The authors claimed that the end-points were sharper and the blank correction was smaller than in the periodate method.

The reagent has also been used for detection, e.g. of 1-monoglycerides by Mangold et al. (1955); spraying a 1 % solution in benzene yielded white zones on brown with down to 50-100 ug on paper chromatograms.

2.3. F O R M A T I O N OF I S O P R O P Y L I D E N E DERIVATIVES

Hanahan et al. (1963) converted glycerol 1-ethers into isopropylidene deri-vatives in 95% yield by reaction at room temperature in dry acetone, 001M in perchloric acid catalyst. They were then able to separate the derivatives by GLC.

Wood et al. (1969b) studied the O- and 5-alkyl ethers of glycerol, using chromatographic (gas and thin-layer) methods and infrared and nuclear magnetic resonance procedures. They also prepared the isopropylidene derivatives of these compounds and separated these by the same chromato-graphic techniques. Gas chromatography using several liquid phases, e.g. 15% poly(ethylene glycol succinate) on 80-100 mesh Gas-Pack WAB at 175°C, gave complete resolution of mixtures of the O- and 5-compounds; thin-layer chromatography, e.g. on silica gel G using hexane-diethyl ether (9 + 1 ) , separated compound classes but not individuals. Blank and Snyder

excess oxidising agent by adding potassium iodide and decomposing the released iodine with thiosulphate. They then treated the organic phase with trichloroacetic acid and 2,4-dinitrophenylhydrazine for 30 min at 60°C, extracted the hydrazone product with hexane and evaluated it spectrophoto-metrically at 340 nm. Closely similar is the method of Gelman and Gilbertson (1969) for glycerol 1-ethers. They oxidised with periodate, converted the alkoxy-aldehyde into the p-nitrophenylhydrazone, and evaluated it at 380 nm.

2.4 COMPLEX FORMATION WITH BORON COMPOUNDS 103

(1969) were also able to separate O-alkylglycerols from alkane-l,2-diols as isopropylidene derivatives bv GLC and by TLC on silica gel G.

2.4. C O M P L E X F O R M A T I O N W I T H B O R O N C O M P O U N D S

1-Monoglycerides and similar monosubstituted glycerol derivatives are able to form cyclic products with borate and organic borate anions, and this has been utilised for their separation just as with glycerol and other polyols:

C H 2 — O — X H O OH C H 2 — O — X

i

f \ - / - H 2 0 I

HOH + — 2 — + C H — O ^ - ^ O H

C H 2 O H H O OH C H 2 — o / \ ) H

(with possible reaction with a second glyceride molecule^

Thus, Thomas et al. (1965) separated monoglyceride isomers on layers pre-pared by slurrying 25 g of silica gel G with 50 ml of ca. 0-4N boric acid. They used various solvents, chloroform-acetone mixtures (from 19 + 1 to 1 + 1) also containing 0-5% acetic acid and up to 6% methanol. Visualisation was achieved with saturated chromic oxide in 70% sulphuric acid, followed by heating for 25 min at 200°C. They carried out densitometric evaluation of the spots of separated compounds. Wachs (1967) separated mono- and di-glyce-rides on silica gel G impregnated with boric acid, using chloroform-acetone-acetic acid (188 + 12 4- 1), and this method was recommended by Schewe and Coutelle (1970). Sahasrabudhe (1967) used silica gel containing 4% boric acid to separate fatty acid esters of glycerol and polyglycerols, his mobile phase being benzene-methanol (8 + 3). Biernoth (1968) also separated mono- and di-glycerides on boric acid-impregnated silica gel G; for the former he used also chloroform-acetone (24 + 1), and for the latter, petroleum ether-diethyl ether (73 + 27). After visualising with sulphuryl chloride vapour and heating, he too evaluated the charred compound zones. Blank and Snyder (1969) carried out TLC separation of Oalkyl-glycerols and alkane-l,2-diols on silica gel G impregnated with boric acid or, also, arsenite. Mono- and di-glycerides and esters of propylene glycol in margarine and shortening were separated and determined by Kanematsu et al (1972), employing TLC conditions essentially identical to those of Thomas et al mentioned above; they used the chloroform-acetone ( 1 9 + 1 ) mobile phase but detected with 2',7'-dichlorofluorescein, ultimately collecting the zones and submitting them to trimethylsilylation and subsequent GLC.


2.5. ESTERIF ICATION

As described in Chapter 1, glycerol and other polyols are converted into the more volatile esters which lend themselves better to gas chromatographic separation (p. 39). This can naturally be applied equally well to mono-and di-glycerides and glycerol ethers. For example, Blomstrand and Giirtler (1959) converted glycerol monoethers into diacetates by reaction with acetic acid-pyridine for 12 h at 100°C. They then carried out GLC on LHR-IR 296 (a polar polyester) + 100-140 mesh AW alkali-treated Celite ( 1 + 4 ) at a column temperature of 218°C using an argon ionisation detector. Acetate and trifluoroacetate esters of long-chain monoethers were prepared by Wood and Snyder (1966) and submitted to GLC which enabled them to separate 1- and 2-isomers. Wood et al. (1969a) converted dialkyl, alkyl acyl, and diacyl derivatives of glycerol into acetates and carried out GLC on these. They used a column of 1 % OV-1 on Gas-Chrom Q, programmed from 150 to 275°C at 5° min. Uehara et al. (1971) converted mono- and di-glycerides into acetate esters for GLC. They and the previously mentioned research groups also prepared trimethylsilyl ethers for GLC (see below, Section 2.6.1). Kuksis (1971, 1972) prepared acetates from diglycerides using acetic an-hydride-pyridine (10 + 1), and then separated these esters by GLC on

Boron-containing reagents have been used also in a prior step before gas chromatography. For example, Kresze and Schauffelhut (1967) prepared phenylboronate esters from polyols and glycerides. Monoglycerides required 4 h reflux with the calculated amount of phenylboronic anhydride in dry acetone. They carried out the GLC on 10% SE-30 on Chromosorb W, programming the column temperature from 225 to 310°C at 2°/min; they employed helium carrier gas and thermal conductivity detection. Green-halgh and Wood (1973) studied the GLC of boron-containing compounds on a column of 3 % OV-17 on 100-120 mesh Gas-Chrom Q. They used nitrogen carrier gas and a three-electrode EFID with a caesium bromide annulus. They suggested the use of boronate (butyl- and phenyl-boronates) as suitable derivatives of, for instance, monoglycerides. Hartman and Esteves (1976) determined monoglycerides in crude oils and fats by making use of complex formation with borate. They extracted glycerol from a hexane solution of the sample using 5 % acetic acid, and then extracted the residual hexane solution 3 times with 90% acetic acid saturated with boric acid. Periodate oxidation using the differential procedure followed on their extract. They claimed enrichment of 1-monoglycerides, saying that the boric acid reduces solubility of free fatty acids and diglycefrides at least as well as the acetonitrile used by Halvarson and Qvist (1974).

2.5 ESTERIFICATION 105

stabilised polyester liquid phases supported on 60-80 mesh Gas-Chrom Q at 28O-300°C with helium carrier gas and hydrogen-FID detector. Fischer (1974) carried out a chromatographic investigation of technical glycerol monostearate (containing mono-, di-, and tri-stearates). He prepared the trifluoroacetates and separated these on a column of 10% OV-17 on 60-80 mesh AW DMCS Chromosorb W; the column temperature was programmed from 150 to 320°C at 8°/min and he used helium carrier gas and FID with hydrogen. He was thus able to separate the monoesters from one another and from the diesters.

In a different type of gas chromatographic application of esterification, Mclnnes et al. (1960) treated monoglycerides (1- or 2-) in chloroform with methanesulphonyl chloride in pyridine at 0°C. The products yielded over-night could be converted into allyl esters of the fatty acids by heating with sodium iodide in acetone for 2 h at 100°C:

C H 2 0 — C O R C H 2 0 — C O R C H 2 0 — C O R

I C H 3 S 0 2 C 1 I + N a I I

CHOH • C H O — S O X H . • CH I I II

C H 2 O H C H 2 0 — S 0 2 C H 3 C H 2

They extracted these allyl esters with ether and finally subjected them to chromatography on Apiezon M-Celite at 240°C with helium carrier gas and thermal conductivity detection.

A final thin-layer chromatographic step may follow esterification. Malins et al. (1964) prepared nitrate esters of glycerides, glycerol ethers, and other hydroxy-compounds by reaction for 10 min with acetyl nitrate (from acetic anhydride -I- some drops of 70% nitric acid). They carried out TLC of these esters on silica gel G, using hexane-diethyl ether (85 + 15) as mobile phase and visualising with iodine vapour or 2 ,,7 /-dichlorofluorescein. The products were characterised and determined by infrared spectrophotometry (utilising bands from the nitrate moiety, however). In the work cited above, Wood et al. (1969a) also converted dialkyl, alkyl acyl, and diacyl derivatives of glycerol into acetates and then subjected these to TLC on silica gel G with the mobile phase hexane-diethyl ether-methanol (80 + 20 + 5), visualising by charring with chromic oxide-sulphuric acid.

Timmen et al. (1970) prepared derivatives of partial glycerides and hydroxy-triglycerides in milk lipids by reaction with pyruvoyl chloride-2,6-dinitro-

106 GLYCEROL COMPOUNDS: INTRODUCTION AND METHODS

phenylhydrazine in trimethylenediamine:

ROH CH3COCOCl

R O — C O — C O C H 3 2, 6 DNPH

N O RO—CO—C—CH

II N—NH-

After removing the unused reagent they carried out column chromatography on magnesia and finally submitted the coloured fractions to TLC on silica gel H.

Acetylation of hydroxyl groups in organic matter was carried out by Mlejnek (1955), using acetic anhydride in acetic-sulphuric acids for 1 h at 60°C. He then titrated the water formed in the esterification reaction with the Karl Fischer reagent and obtained good results with the mono- and di-glycerides of linseed oil.

The classical differential method was applied to glycerol monoethers by Stross and Stuckey (1950). They refluxed with 15% acetic anhydride in pyridine for 2 h, then added water and titrated with sodium hydroxide to phenolphthalein. A control was similarly treated and the difference in the alkali titrations was a measure of the amount of diol (see Chapter 1, Section 1.2.2.B).

2 . 6 . 1 . T r imethy ls i l y la t ion

Conversion of hydroxyl groups into trimethylsiloxyl groups in order to increase volatility and hence applicability of gas chromatographic methods has predictably been used with mono- and di-glycerides and other com-pounds containing one or two free glycerol hydroxyl groups. Some com-ments about the preparation of these derivatives were made in Chapter 1, Section 1.3, which apply essentially to the compounds considered here. Table 2.1 contains summaries of information about the subsequent chromato-graphic separation. As expected, most of the work has been on mono- and di-glycerides.

Prada et al (1970) blocked the free hydroxyl groups of mono- and di-glycerides by trimethylsilylation, so that they no longer interfered with the gas chromatography of the triglycerides in lipid mixtures. They carried out

2.6. ETHER F O R M A T I O N

2.6

TABLE 2.1

Gas Chromatography of Trimethylsilyl Ethers of Glycerol Derivatives


Mono- and di-glycerides

1- and 2-Mono-glycerides

15% SE-30 on Diatoport W; 150 to 345°C at 5°/min; He gas; thermal cond. detector, also via NMR spectra

Capillary coated with Apiezon L, with temp, programming from 180 to 240°C; or Cu tube containing 20 % poly(diethylene glycol succinate) on Chromosorb W at 215°C; He gas; H 2 - F I D

Long chain ( C 1 2 - C 1 8 ) Less well separated than as di(trifluoro-monoethers of acetate) esters (see p. 104) glycerol


Glycerol, poly-glycerols, and their fatty acid esters

5% SE-30 on Chromosorb W; 150 to 340°C at 4°/min; N 2 gas; FID (separated 1,2- and 1,3-diglycerides)

Stainless steel columns; 3 % JXR on Gas-Chrom Q; 120 to 325°C at 10°/min; He gas dual FID

1,2- and 1,3-Distearin and -diolein, derived from phosphatidyl serines on treatment with silylation reagents

Monoglycerides (palmitin, stearin, myristicin, olein); mixed alkyl ethers ( C 1 4 , C 1 6 , C 1 8 ) of glycerol

Mono- and di-glycerides with C 2 -C 1 8 groups

Glass column; 1 % SE-30 on 100-120 mesh Gas-Chrom P; coupled with mass spectrometry

Kresze et al. (1965)

Wood et al. (1965)

Wood and Snyder (1966)

Kresze and Schauffelhut (1967)

Sahasrabudhe (1967) Sahasrabudhe and Legari (1967)

Casparrini * et al. (1968)

Various columns; best in glass columns with 5 % JXR methylsilicone at 225°C, 5 % Apiezon L at 240°C, and 3 % XE-60 at 185°C, all on Gas-Chrom Q; also 20% poly(diethylene glycol succinate) on 80-100 mesh Diatoport W at 200°C; He gas; FID

Stainless steel column; 3 % QF-1 on AW D M C S Chromosorb W or of 10% SE-30 on Gas-Chrom Q; isothermal at temps, from 140 to 330°C or programmed from 110 to 330°C at 2-6°/min; N 2 gas; FID

Mono- and di- 3 % OV-1 on 60-80 mesh Gas-Chrom Q; glycerides in glyceride 110 to 400°C at 4°/min; He gas; FID mixtures in partly hydrolysed seed oil

Rumsby (1968)

Watts and Dils(1968)

Tallent and Kleiman (1968)



Synthetic diglycerides, Glass column; 1-5% XE-60 on silanised e.g. dipalmitin, Chromosorb W; 225°C increased at 3°/min; distearin He gas; coupled with MS

Isomeric glycerol mono-, di-, and tri-lactates

Lactylated mono-glycerides

Mono- and di-glycerides among other compounds (sterols, sterol esters)

Glycerophosphatides (evidently yielding Me 3 Si ethers of diglycerides during the short heating period of the preparation)

Dialkyl, alkyl acyl, and diacyl glycerols

Glycerol, mono- and di-glycerides, in various food products

Glycerol nitrates in aged double-base propellants

Natural diglycerides

Diglycerides (separation of 1,2 and 1,3)

Glycerophosphatides (yielding diglyceride products as in ref. of Horning et al, 1969)

Stainless steel column; 3 % SE-52 on 80 -100 mesh Gas-Chrom Z; 100 to 300°C at 6-7°/min; He gas; FID, also MS

Stainless steel columns; 2 % SE-30 on Aeropak 30; 175 to 375°C at 6°/min; N 2

gas; FID

Stainless steel or glass columns pretreated with M e 2 S i C l 2 ; SE-30, SE-52, or OV-17 on AW-silylated 100-120 mesh Celite; 150 to 350°C at 3-6°/min; FID

Glass column; 1 % SE-30 on 100-120 mesh Gas-Chrom P; 250 or 270 to 300°C at 2° /min;FID

1 % OV-1 on 100-120 mesh Gas-Chrom Q; 150 to 275°C at 5°/min

Glass column; 3 % OV-1 on 80-100 mesh Chromosorb W; 50 to 300°C at 12°/min; H 2 - F I D

Column of 2-5% OV-17 + 2-5% QF-1 on 60-80 mesh Gas-Chrom Q; 70 to 230°C at 10°/min;He gas; FID

Column of 10% EGSS-X or ECNSS-M on 100-120 mesh Gas-Chrom Q; isothermally at 250 or 270°C; He or N 2 gas; H 2 - F I D

Glass column; 3 % OV-1 on 100-120 mesh Gas-Chrom Q; 298°C; Ar gas; Ar-ion detector with Ra source

1 % SE-30 or OV-17 on 80-100 mesh Gas-Chrom P; programmed at 1 or 2°/niin from 230, 250, or 270°C; FID

Barber et al (1968)

Brandt et al (1968)

Neckermann and Noznick (1968)

Wood (1969)

Horning et al (1969)

Wood et al (1969a)

Blum and Koehler (1970)

Trowell (1970)

Kuksis(1971)

O'Brien and Klopfenstein (1971)

Horning et al (1971)

2.6 ETHER FORMATION 109


Natural diglycerides

Monoglycerides in fats and oils

Monoacyl-, mono-alky 1-glycerols (e.g. from edible oils)

Monoacyl-, mono-alkyl-glycerols

Satd. and unsatd. diacylglycerols

3 % Cyclohexane dimethanol succinate (HI- Kuksis (1972) EFF-8BP) or of neopentyl glycol succinate (HI-EFF-3BP) on 60-80 mesh Gas-Chrom Q at 28O-300°C (isothermal); also on mixtures of the two polyesters with 5-10% JXR methylsilicone on 100-120 mesh Gas-Chrom Q; He gas; H 2 - F I D

Stainless steel column; 3 % OV-1 on AW Halvarson DMCS Chromosorb W; 175-200°C and Qvist (isothermal); N 2 gas; FID (1974)

Glass column with 3 % SILAR 5CP Myher and (cyanopropylphenylsiloxane) on 100-200 Kuksis (1974) mesh Gas-Chrom Q up to 270°C; or with 3 % EGSS-X on the same support below 250°C; or stainless steel column with 3 % OV-1 on the same support at 170°C, programmed upwards at 10°/min.

As Myher and Kuksis (1974) but with MS Myher et al. identification (1974)

Glass column; 3 % SILAR 5CP on 100-120 Myher and mesh Gas-Chrom Q; 270°C; He gas; FID Kuksis (1975)

the GLC on dual stainless steel columns containing 3 % silicone JXR on

Gas-Chrom Q, programming temperature from 150°C at 6°/min and using

nitrogen carrier gas and FID.

2.6.2. Other Ether F o r m a t i o n React ions

These are rare methods but an example is the work of Hallgren and Larsson

(1959). They converted glycerol monoethers (with long-chain groups) into

dimethyl ethers with diazomethane-boron trifluoride according to Muller and

Rundel (1958) and separated these first from monomethyl ethers and the un-

changed residues through column chromatography on alumina, eluting with

25% diethyl ether in petroleum ether. They then separated the dimethyl

ethers by GLC on a column impregnated with silicone grease or Reoplex 400

at 247°C in helium carrier gas (Perkin-Elmer 116 apparatus).



2.7. I N F R A R E D S P E C T R O P H O T O M E T R Y

The infrared spectra of mono- and di-glycerides, in contrast to those of tri-glycerides, contain certain bands attributed to the hydroxyl groups, e.g. in the 3300-3700 c m " 1 and also the 1000-1100 c m - 1 domains. Some examples may be quoted of the analytical application of these bands.

Susi et al. (1961) analysed mixtures of 1- and 2-monoglycerides through infrared absorption data of the overtone of the O—H stretching vibration at ca. 7000 c m " l . This was found to be virtually independent of the chain length of the esterifying fatty acid group. They gave monopalmitins and mono-stearins as examples. Susi et al. (1963) studied the O—H stretching modes in the fundamental and first overtone regions of diglycerides. They discussed the analytical prospects with the example of distearins and were able to deter-mine the 1,2-distearin in the 1,3-compound through IR data at 3531 and 7116 cm" 1 , using solutions in carbon tetrachloride.

Murphy (1962) determined the hydroxyl number of alkyd resins by infra-red measurements at 3550 c m " 1 in dichloromethane solution and sodium chloride cuvettes, correcting for water content and hydroxyl groups of any organic acids present.

Lueck and Kohn (1965) determined hydroxyl groups in mono- and di-glycerides in butterfat through IR measurements at 3750 c m " 1 . Maruta and Iwama (1965) also determined mono- and di-glycerides but with the help of absorption data at 1050 c m " 1 (primary alcohol groups) and 1120 c m " 1

(secondary groups); their results agreed with those from chemical analyses and they were able to determine the monoglycerides in rapeseed oil to within less than 1 -8 %.

The IR spectra of alkyd resins were studied by Nagakura et al (1968). They were able to show the presence of polyols through characteristic absorption bands in the 1500-1360, 1350-1040, and 1000-900 c m " 1 regions. They were also able to show whether an alkyd resin was composed of polyols containing primary or secondary hydroxyl groups.

Chapman (1965) reviewed infrared spectra of lipids, including glycerides, and Freeman (1968) reviewed the applications of IR absorption spectra in analysis of lipids. These articles contain useful information but are concerned more with problems such as chain length, degree of unsaturation, extent of chain branching, etc.


2.8. N U C L E A R M A G N E T I C R E S O N A N C E

Warren and Zarembo (1970) used N M R to analyse mixtures of 1,2- and 1,3-diglycerides. They employed 8 % sample solutions in deuterochloroform containing 3 % chloroform, at 60 MHz. The NMR spectra of the glycerides differ markedly in the region 3*6-4-4 ppm (220-260 c/s). They determined the 1,2-compound via its duplet at 3-75 ppm (corresponding to the two 3-protons).

2.9. O T H E R O X I D A T I O N S

Oxidation with classical agents has evidently seldom been performed but Karpov (1964) determined the glycerophosphates of calcium, sodium, and iron by refluxing for 1 h with a dichromate-sulphuric acid reagent and then back-titrating with 0-1N ferrous ammonium sulphate to phenylanthranilic acid as indicator. Such methods are, however, too unspecific to be useful.

2.10 T H E R M O G R A V I M E T R Y

A thermogravimetric analysis of a fatty acid monoglyceride was performed by Lorant (1966). It yielded a maximum at 255°C (loss of 1 molecule of water to yield an epoxide) and then at 310°C (formation of acrolein). However, prospects of analytical use appear small.

2 . 1 1 . N O N - C H R O M A T O G R A P H I C S E P A R A T I O N M E T H O D S

2 . 1 1 . 1 . Thermal D i f f u s i o n

Seelbach and Quackenbush (1957) fractionated glyceride types by thermal diffusion in an apparatus made up of parallel steel plates with a thermal gradient from one plate heated with low-pressure steam to the other cooled with tap water. Oils were allowed to diffuse for 72 h and they found that triglycerides tended to move towards the top, and glycerol and monogly-cerides to the bottom. They checked this by chemical analysis using saponifi-cation, periodate oxidation, and other procedures. Commercial monoolein was thereby shown to contain some di- and tri-olein and also free acid and glycerol.


2.11.2. Urea Complexes

During the 1950s considerable work was carried out on the formation of urea adducts with molecules containing unbranched chains of more than about 6 carbon atoms. Since the naturally occurring glycerides contain linear fatty acid units of 14 to 18 carbon atoms, attempts were made to separate them according to this principle. Of course, the participating moiety of the glyceride molecule is the fatty acid part; further, most work was aimed at separation according to chain length, degree of unsaturation, or degree of branching. Nevertheless, some publications may be justifiably cited here of separations of mono- from di-glycerides.

Roncero et al (1952) and Bradley et al (1955) observed that diglycerides formed urea complexes more readily than did monoglycerides. [Surprisingly, Heckles and Dunlap (1955) came to the opposite conclusion!] Aylward and Wood (1956) tested the fractionation of commercial monostearin by stepwise crystallisation with urea in methanol. This led to preferential precipitation of distearin. By using more urea, they obtained higher yields of monostearin but of lower purity.

Mehta and Shah (1957) studied the glycerolysis of coconut, sesame, and linseed oils and tested the urea separation method on technical mono-glycerides of sesame oil and lauric acid. They mixed 1 part of sample with 3 parts of urea and 13-5 parts (all by weight) of methanol, heated to a clear solution, and left overnight. They then filtered, and decomposed the adducts with a hot acid solution of salt, extracting the glycerides with chloroform. After evaporating the chloroform they analysed the residue. The same authors (1958) fractionated technical grade monoglycerides by dissolving 15 g of sample in 30 ml of methanol and dissolving 60 g of urea by warming. The solution was kept overnight at 27-30°C, causing crystals of the adduct to separate. These were filtered off and decomposed with warm acidified water. The emulsion formed was broken with salt and the fatty portion dissolved in chloroform. They dried this solution over sodium sulphate, evaporated the solvent, and noted the yield of residue. This was analysed also for hydroxyl groups by the acetylation method of Pohle and Mehlenbacher (1946) (see Table 1.1) and for monoglycerides by the periodate oxidation method of the same authors (1950) (cf. p. 17). They concentrated the filtrate by distilling off some methanol, obtaining further precipitated adduct. This was filtered off, decomposed, measured, and analysed as before. They repeated this after further successive concentration stages and showed that the adducts were formed more readily with diglycerides than with monoglycerides.


2.11.3. Fract ional D is t i l la t ion and Extract ion

As mentioned early in this chapter, the decrease in polarity in passing from a monoalkyl or monoacyl glycerol to a di- and tri-compound should lend itself to separation based on the ensuing differences in solubility and volatility. These two themes are discussed briefly below.

A. F R A C T I O N A L D I S T I L L A T I O N

The decrease in polarity from mono- to di- to tri-compound lowers the boiling point through progressive removal of the hydroxyl groups capable of association, but this effect is compensated by the increase in molecular weight. The boiling points (at 760 mm) of the methyl and ethyl ethers of glycerol, taken from Ullmann (1957), are given in Table 2.2.

TABLE 2.2

Boiling Points of Glycerol Methyl and Ethyl Ethers

Compound B.p. (°C) Compound B.p. (°C)

1-Methyl 220 1,2,3-Trimethyl 148 2-Methyl 232 1-Ethyl 220 1,2-Dimethyl 180 1,3-Diethyl 191-2 1,3-Dimethyl 169 1,2,3-Triethyl 181

Fractionation of the methyl ethers into mono-, di-, and tri-compounds would not be difficult; that of the ethyl ethers would be less easy on account of the molecular weight compensation. With increasing size of substituent the chances become even less.

Boiling points for esters of glycerol are difficult to compare because they have been measured at various pressures. The values for formate esters, again taken from Ullmann, are in Table 2.3. It can be seen from it that the

TABLE 2.3

Boiling Points of Glycerol Formates and Acetates

Compound B.p. (°C)/mm Hg Compound B.p. (°C)/mm Hg

2-Formate 154-7/10 1 - Acetate 129-131/3 1,2-Diformate 151-3/17 1,2-Diacetate 140-2/12 1,3-Diformate 144-6/11 1,3-Diacetate 280/760 Triformate 258-9/760 Triacetate 258-9/760

114 GLYCEROL C O M P O U N D S : INTRODUCTION A N D METHODS 2.11

effect of the removal of hydroxyl groups is almost completely compensated by that of the acyl groups introduced. Fractionation into mono-, di-, and tri-glyceride classes thus becomes highly problematic. Further, a straight-forward mixture of the mono-, di-, and tri-acyl derivatives of glycerol with one acid moiety is not encountered so often in practice. Glyceride samples contain acyl components of various chain lengths which interfere with a clear-cut fractionation into the three classes. At best, a partial enrich-ment can be accomplished.

B. M O L E C U L A R D I S T I L L A T I O N

Molecular distillation has been used to separate glyceride types by, for instance. Privett etal. (1961), employing the apparatus of Booy and Waterman (1949), taken over by Paschke et al (1954). Samples of from 0*5 to 2 g were taken at a pressure below 10" 3 mm. Free fatty acids, glycerol, and simple esters distilled at 75-80°C, monoglycerides at 145-150°C, and diglycerides at 200-210°C. The operation took about 24 h.

C. F R A C T I O N A L E X T R A C T I O N

The polarity change from trihydroxy-compound (glycerol) to trialkyl or triacyl compound should be accompanied by decreasing solubility in polar solvents such as water and lower alcohols, and by increasing solubility in non-polar solvents such as hydrocarbons and halides. As with the distilla-tion, sharp separations are rarely possible. Thus, although the 1 -monoacetate and 1,3-diacetate of glycerol are easily soluble in water, the triacetate also yields an approximately 7 % (w/v) aqueous solution. Likewise, the solutions of the acetate esters in, say, benzene do not show marked concentration differences. The compensating influence of additional groups makes itself felt here also. In addition, the likely presence of various acyl (or alkyl) moieties complicates the solubility sequence. Thus, glycerol-1-monostearate is only very slightly soluble in ethanol, less soluble in fact than is glycerol triacetate, quoted in Beilstein as miscible in all proportions with this solvent.

Many attempts have been made to achieve at least some enrichment of one of the classes. Water is generally used to remove free glycerol and mono-glycerides from glyceride mixtures, even though diglycerides will then be removed to some small extent. Many other solvents and mixtures have been tested and claims made for their power. Three recent examples may be given. Halvarson and Qvist (1974) removed monoglycerides from a 30 ml hexane solution of fats and oils by three extractions with 15 ml portions of a hexane-saturated acetonitrile. Hartman and Esteves (1976) claimed at least as good a selective removal of monoglycerides by using 90 % acetic acid saturated with boric acid (profiting naturally from the complex formation with 1,2-diol groups—a theme treated above in Section 2.4). Schmid and Otteneder (1976)

2.12 CHROMATOGRAPHY OF MIXTURES 115

extracted monoglyceride emulsifiers from alimentary pastes with water-saturated butanol.

Schlenk and Ener (1959) quote a more exotic example. They studied the solubilities of various lipids in sulphur dioxide at low temperatures. Some values for glyceride samples are given in Table 2.4. The authors stated that fractionation of glycerides is possible, based on these solubility differences.

TABLE 2.4

Solubilities of Glycerides in Liquid Sulphur Dioxide

Glyceride Temp. (°C) Solubility (mg/100 g)

Tripalmitin 21 8 Triolein - 2 7 , - 1 7 27, 350 resp. Trilinolein - 3 3 1-25 1,3-Dipalmitin 16-5 32 1-Monopalmitin - 3 . 5 43

2.12. C H R O M A T O G R A P H Y OF M I X T U R E S OF G L Y C E R O L M O N O - , D I - , A N D TRI -ESTERS OR -ETHERS

The literature contains many examples of the chromatography of mixtures of the three glyceride types, either alone or in lipid samples which may con-tain further compounds such as free fatty acids, free glycerol, sterols and their esters, g lycerophosphates , etc. Gradient procedures have been widely used to separate these mixtures of compounds of highly differing polarity.

A selection of relevant publications is given in the following pages., It is convenient, as always, to take as separate headings the various practical chromatographic techniques.

2 . 1 2 . 1 . T h i n Layer C h r o m a t o g r a p h y

Thin-layer procedures evidently predominate, and examples of these are given in Table 2.5.

TABLE 2.5

Thin Layer Chromatography of Glycerol Esters or Ethers


Mono-, di-, and tri- Silica gel G with diethyl ether-Skellysolve Privett et al glycerides F mixtures; mono- best with (9+1) , di- best (1961)

with (3 + 7), and tri- with (1 + 9 ) ; detected by spraying with 50% H 2 S 0 4 and charring; quantitative densitometric evaluation



Mono-, di-, and tri-glycerides


Mono-, di-, and tri-glycerides of higher fatty acids

Lipids, including glycerides, also fatty acids and methyl esters

Isomeric mono-glycerides

Glycerides and sugar esters

Lipids in blood serum, including glyceride types, cholesterol, and esters

Glycerides, in study of rearrangement

Serum lipids, including glycerides, cholesterol and esters, fatty acids, phosphatides

Fine mesh (below 240) silicic acid -I-10% C a S 0 4 with gradient technique using diethyl ether-pet. ether increasing from 10 to 60%; visualised with 0-2% dichloro-fluorescein or with cone. H 2 S 0 4 and charring at 120°C

Silica gel, also impregnated with A g N 0 3

for unsaturated glycerides; petroleum ether-benzene (1+4) ; extracted with ether, saponified, and glycerol estimated by periodate

Floridin AS with heptane or CC1 4 con-taining 10-20% dioxan; oleins best at 20°C, satd. glycerides at 3O-40°C; detected with iodine vapour (also molybdophosphate)

Silica gel without C a S 0 4 binder; diisopropyl ether-acetic acid (96 + 4) in first step, then further development with light petroleum-diethyl ether—acetic acid (90 + 10 + 1)

Silica gel GF containing ca. 2-5 % of boric acid; chloroform-acetone mixtures (see Section 2.4); charred with C r 2 0 ^ ~ - H 2 S 0 4

and evaluated densitometrically

Silica gel containing 15% C a S 0 4 ; chloroform-methanol-acetic acid-water ( 8 0 + 1 0 + 8 + 2); sprayed with 2% H 2 S 0 4

in formalin after preheating 15 min at 150°C

Silica g e l - C a S 0 4 (6 + 1); heptane-ethanol-ethyl acetate (80 + 20+1-5); detected with 2 % ethanolic molybdophosphate and heating; densitometry

Kieselguhr impregnated with liquid paraffin; acetone-methanol ( 4 + 1 or 7 + 4); detected with iodine vapour, then 1 % a-naphthodextrin, giving blue zones on white

Silica gel G; hexane-ether-acetic acid (80 + 2 0 + 1 ) ; detected with 0-2 % dichloro-fluorescein, located in UV; scraped off and determined

Rybicka (1962)

Jurriens et al. (1964)

Pokorny and Herodek (1964)

Skipski et al. (1965)

Thomas et al. (1965)

Ranny (1966)

Baryshkov (1966)

Chakrabarty et al. (1966)

Chiarioni et al. (1966)




Additives, coatings, surface-active materials in paper, extracted by light petroleum


Mono-, di-, and tri-glycerides, also sterols, higher alcohols, hydro-carbons

Mono-, di-glycerides

and tri-


Simple lipid classes

Mobile phase hexane-ether (7 + 3) or, in absence of free fatty acids, ether-petroleum ether (3 + 7); detected with 0-02 % 2',7-di-chlorofluorescein and inspection in UV

Silica gel G; triglycerides with hexane-ether (8 + 2) or hexane-ether-acetic acid (90 + 10+1) ; diglycerides with hexane-ether-acetic acid (70 + 30 + 2); diglycerides and monoglycerides, also oxidised lipids and resin acids with benzene-methanol (9 + 1)

Silica gel containing 12% gypsum, mobile phase light petroleum-ether-acetic acid (50 + 5 0 + 1 ) ; detected with K M n 0 4 - H 2 S 0 4 ; zones scraped off, hydrolysed with alkali, and glycerol estimated via H I 0 4 oxidation and colour reaction of HCHO with chromotropic acid

Silica gel G; petroleum ether-ether-acetic acid (80 + 2 0 + 1 ) ; visualised with 60% H 2 S 0 4 or 10% H 3 P 0 4 , then drying at 130°C

Reaction products from glycerol (and other polyol) adipates with excess adipic acid

Silica gel G; hexane-ether (7 + 3) or, in absence of free fatty acids, light petroleum-ether (7 + 3); detection via hydroxamic acid ester test

Silica gel G impregnated with boric acid; chloroform-acetone-acetic acid (188 + 12 + 1)

Two-stage TLC on silica gel; diisopropyl ether-acetic acid (19 + 1), then pet. ether-ether-acetic acid ( 9 0 + 1 0 + 1); detected by Rhodamine 6G; tri- and di-glycerides eluted from scraped-off zones with ether, monoglycerides with chloroform-methanol (4 + 1); glycerides (and hydrocarbons) finally determined using IR

Mobile phase moist ether-acetic acid (100+1)

Ceglowska et al. (1966)

Broniatowski (1967)

Franzke et al. (1967a)

Popov et al. (1967)

Grynberg et al. (1967)

Wachs(1967)


Loehnert and Schoellner (1968)




Food emulsifiers, including mono-glycerides of lactic, tartaric, citric, and fatty acids

Glycerides in ointment bases, suppositories

Lipid classes in amniotic fluid and serum after extraction with chloroform-methanol (2 + 1)

Mono-, di-, and tri-nitrate esters of glycerol, with 1 4 C

Glycerol alk-l-enyl ethers from LiAlH 4

reduction of lipids in bovine heart muscle

Mono-, di-, and tri-glycerides in pharm. emulsifiers and solubilisers

Lipids

Silica gel G-boric acid; chloroform- Biernoth acetone (19 + 1 ) for mono-, and pet. ether- (1968) ether (73 + 27) for di-glycerides; on silica gel G using petrol, ether-ether-acetic acid (96 + 4 + 0-35) for triglycerides; detected with S 0 2 C 1 2 and heating 25 min/220°C, with densitometry

Silica gel containing potassium hydrogen Gernert oxalate; chloroform containing 1 % (1968) methanol; detected with 2',7'-dichloro-fluorescein, then inspection in UV

Fluorescent layers using pet. ether-ether Febure and (7 + 3), then (3 + 7), and finally ether alone, Aiache (1968) separating tri-, then di-, and mono-glycerides; separated, saponified, and the glycerol determined

Silica gel G; first ether-benzene-ethanol- Biezinski et al. acetic acid (200 + 250 + 1 0 +1) , dried, and (1968) then in same direction with light petroleum-ether-acetic acid ( 9 0 + 1 0 + 1 ) ; visualised with 50% H 2 S 0 4 and heating at ca. 260°C until no more fumes (ca. 8 min); densito-metry evaluation. Three glyceride classes separated

Silica gel G; various solvents; isomers Crew and separated in ethyl acetate-toluene (1 + 1), DiCarlo water-saturated ethyl acetate, and ethyl (1968) acetate-n-heptane (9 + 1); detected via radioactivity

Silica gel; hexane-diethyl ether (1 + 4) Baumann et al. (1969)

Silica gel; pet. ether (40-60°)-ether-acetic Neissner acid (60 + 60 + 1 ) ; detected with 2\T- (1969) dichlorofluorescein and inspection in UV light of 254 or 366 nm

Silica gel H mixed with 10% Mg silicate; Fewster et al. light petroleum-ether (7 + 3) or chloroform (1969) for diglycerides-cholesterol; visualised by spray with Cu acetate in 8 % H 3 P 0 4 and heating 20min/180°C; densitometric evaluation



Products of fat digestion (three glyceride types, free fatty acids, cholesterol and esters, phospholipids)

Serum lipids (extract in ether-ethanol (3 + 1))

Glyceride types, free fatty acids, cholesterol and its esters

Mono-, di-, and tri-glycerides in model mixtures and in soya bean oil

Milk lipids (glycerides, free fatty acids, cholesterol acetate)

Glycerides, free fatty acids, sterols and esters

Glycerol 1-mono-, 1,2- and 1,3-di-, and tri-nitrates

Silica gel G; ether-light petroleum (40-60°)- Manners acetic acid (130 + 7 0 + 1 ) and then further et al. (1969) to top of plate with ether-light petroleum (3 + 47); detected with 5% H 2 S 0 4

containing 0-1 % K 2 C r 2 0 . and heating to 250°C

Silica gel; chloroform-methanol-formic Wildgrube acid-water (65 + 35 + 2 + 4), then in the et al (1969) same direction with n-hexane-ether-acetic acid (160 + 40 + 3); separated glyceride types and phospholipids; visualisation and densitometry using molybdophosphate.

Silica gel G; benzene-acetic acid (88 +12) Schewe and (no separation of 1- and 2-monoglycerides); Coutelle (1970) method of Wachs (1967) better for separating mono- and di-glycerides; detected with iodine vapour

Silica gel F 2 5 4 ; light petroleum (40-60°)- Berner (1970) ether (13 + 7); located with Ultraphor WT (366 nm)

Silica gel G prepared in 0 0 1 M N a 2 C 0 3 Duthie and suspension; hexane-ethyl acetate-formic Atherton acid (175 + 25 + 2); monoglycerides remain (1970) near origin, triglycerides separated from acids; sprayed with 50% H 2 S 0 4 and charred at 120°C

Adsorbosil-5 silica gel; hexane-ether- Sallee and methanol (18 + 6 + 1 ) to move mono- Adams (1970) glycerides away, then, after drying, with light petroleum; detected by spray with 20% N H 4 H S 0 4 and charring 90 min/170°C

Silica gel F 2 5 4 ; benzene-ethyl acetate Rosseel et al. (4 + 1) for tri- and both di-nitrates, and (1970) (2 + 5) for the mononitrate; visualised via the nitrate moiety by spraying with 1 % diphenylamine-ethanol and UV irradiation for 10 min (yellow-green on light tan); densitometry



Mono-, di-, and tri-glycerides and free fatty acids in biological samples

Serum lipids; including glycerides; chloroform-ethanol extract

Glycerol, mono-, di-, and tri-glycerides, free fatty acids

Mono-, di-, and tri-glycerides (palmitins); palmitic acid and other esters of it

Mono- and di-glycerides; propylene glycol esters (in margarine and shortening)

Mammalian neutral lipids (glycerides, cholesterol and esters, phospholipids)

Neutral lipids (glycerides, cholesterol esters, fatty acid methyl esters)

Silica gel H R - C a S 0 4 (83 + 17); light Marzo et al petroleum-ether-methanol-acetic acid (1971) (180 + 14 + 4 + 1); detected with iodine vapour, zones eluted into cone. H 2 S 0 4 and heated 15 min/200°C; evaluated at 375 nm colour stable for 6 days

Silica gel H; chloroform-methanol-acetic Wildbrube acid-water (65 + 35 + 2 + 4) (separating and Erb monoglycerides and phospholipids), then in (1971) the same direction with n-hexane-ether-acetic acid (80 + 20 + 1-5) (separating di- and tri-glycerides, cholesterol and esters, free fatty acids); detected by H 2 S 0 4 charring with densitometry

Layer of silica gel G-oxalic acid; n-hexane- Saracco and ether (1 + 1); detected by spray with 50% Gay (1971) H 2 S 0 4 and heating 1 h/180°C; densito-metric assay

Silica gel; ether-acetic acid (99 + 1), air Wiklund and drying, then with hexane-ether-acetic acid Eliasson (85 + 15 + 1); detected with satd. (1972) K 2 C r 2 0 180°

in 70% H 2 S Q 4 and heating to

Silica gel containing borate; chloroform-acetone (19 + 1); visualised with 2',7'-dichlorofluorescein followed by UV inspection; spots collected and ultimately converted into trimethylsilyl ethers for GLC

Glass fibre paper impregnated with silica gel G; isooctane-benzene-acetic acid-acetone (1000 + 300 + 1 + 3); detected by spray with 50% H 2 S 0 4 and charring

Silica gel G; light petroleum (30-60°)-ether-acetic acid (30 + 10 + 1), drying, and then ether-light petroleum-acetic acid (70 + 30 + 1) at right angles; detected by spraying with satd. N a 2 C r 2 0 7 in 80% H 2 S Q 4 and charring 30-60 min/ca. 140°C

Kanematsu et al (1972)

Pocock et al (1972)

Palmer et al (1972)



Partial esters of polyols, including glycerol, with palmitic, oleic, and lauric acids

Acyl and alkyl glycerols, analogues of naturally occurring compounds

Serum lipids

Silica gel; light petroleum (40-60°C)-ether- Neissner acetic acid (60 + 60 + 1 ) (1972)

Acetate esters of glycerol and diethylene glycol

Monoglycerides in fats and oils (coconut and palm kernel oil)

Technical glycerol monostearate (containing all three types)

Lipids (standard of mono-, di-, and tri-olein, cholesterol and its palmitate)

Oxidised frying oils, thus containing oxidised and partial glycerides

Lipid fractions, including phospho-lipids, cholesterol, mono-, di-, and tri-glycerides

Systematic study of many (11) solvent Snyder (1973) mixtures on silica gel G; contained 4 0 -100 % of hexane, chloroform, or ether, and up to 10% of water, acetic acid, or N H 4 O H

Method of Wildgrube et al. (1969) but Wildgrube detection by spraying with 20 % H 2 S 0 4 et al. (1973) and heating 90 min/180°C; quant, densitometry

Silica gel; toluene-acetone-methanol- Constantin-acetic acid (14 + 5 + 1 +0-3); detected with escu and iodine vapour or K 2 C r 2 0 7 - H 2 S 0 4 Enache (1974)

Two two oils mentioned, on silica gel Halvarson (Alufolie, Merck; sheets); ether-petroleum and Qvist ether (40-60°) (3 + 2); located by periodate- (1974) anisidine on reference zone; removed, silylated, and subjected to GLC

Silica gel; chloroform-acetone (19 + 1); Fischer (1974) visualised with 0 0 1 % aqueous Rhodamine B

Silica gel G; light petroleum-ether-acetic Mlekusch acid (90 + 1 0 + 1 ) ; detected by charring et al. (1974) with H 2 S 0 4 ; glycerides then located through yellow-green fluorescence

Silica gel G; benzene-ether (99 + 1 ) ; Freeman oxidised and partial glycerides remain near (1974) start, unoxidised triglycerides migrate; detected with K 2 C r 2 0 7 - H 2 S 0 4 spray, heating 15 min/180° C; zone areas compared

Silica gel containing C u S 0 4 ; pet. ether Korolczuk and (40-60°)-ether-acetic acid (80 + 20 + 1 ) ; Kwasniewska detected by heating 1 h/180°C, giving brown (1974) copper oxides


Samples Chromatographic details References

1-Mono-, 1,2- and 1,3-di-, and tri-palmitin

Minor components in smokeless powders including nitro-glycerine

Glycerol ethers, obtained from neutral and phosphatide glycerol ethers in amniotic fluid by reduction with "vitride"

Lipid classes, including cholesterol and esters, all glyceride types, fatty acids

Comparison of TLC, column, and gel chromatography on glyceride mixtures (column and gel conditions at end of Table 2.7)

Monoglycerides in alimentary pastes (extract in water-saturated butanol)

Silica gel G; pet. ether-ether-formic acid (120 + 80 + 3); sprayed with ammonium s u l p h a t e - H 2 S 0 4 and heated 4 h/187°C; densitometry evaluation of charred zones (study of charring)

Silica gel GF254; benzene, benzene-pet. ether(40-60°)-€thyl acetate (12 + 12 + 1), benzene-pet. ether(40-60°) (1 + 1), benzene-chloroform (1 + 1), or chloroform; detected via nitrate moiety (with vanillin-acetic acid-phosphoric acid, orange-brown on heating), with N ( C H 3 ) 4 O H - a c e t o n e -ethanol, or with sulphanilic acid-naphthyl-amine

Gelman ITLC type SG glass fibre sheet; isooctane-isopropyl acetate-isopropanol (50 + 2 + 1); separated ethers determined with periodate-Schiff reagent

Wathelet et al. (1975)

Archer (1975)

Hack and Helmy(1975)

Silica gel G; heptane-pet. ether-ether- Kabara and acetic acid (60 + 20 + 20 + 1); visualised on a Chen (1976) reference layer with iodine vapour; zones on the main layer scraped off, heated 30 min/64 + 2°C, cooled, 5 m l . H 2 S 0 4

added, mixed, heated 15 min/185 + 2°C, cooled, decanted, and evaluated at 375 nm

Silufol UV-254 bonded with starch; light Coupek et al. petroleum (40 + 60°)-ether-acetone (1976) (80 + 1 9 + 1 for less polar glycerides, 45 + 50 + 5 for more polar)

Evaporated extract on silica gel G; pet. Schmid and ether-ether-acetic acid (50 + 50 +1 ) ; Otteneder monoglycerides removed from plate and (1976) determined fluorimetrically with dichloro-fluorescein

TABLE 2.5~continued


Biological lipids, Silica gel G; lower 3 cm of layer using Chabard et al. including mono-, di-, silica gel G slurried with 0-4N N a O H ; (1976) and tri-glycerides; successively with ether-hexane-benzene all containing 1 4 C (11 + 64-3), hexane-benzene (4 +1) , and

ether-benzene-acetic acid (31 + 10 + 9); located through radioactivity and counted

2.12.2. Paper C h r o m a t o g r a p h y

Some examples of the application of paper chromatography to separate mono-, di-, and tri-glycerides or other esters from other materials or from one another are given in Table 2.6.

TABLE 2.6

Paper Chromatography of Glycerol Ester or Ether Mixtures


Many phosphates, including glycerol 1 -and 2-phosphates

Glycerol-1- and -2-phosphates

Glycerol-1-, -2-, and -1,2-phosphates


Whatman No. 3; ascending, 69 % aqueous Wade and butyric acid and 0-85 % NaOH (pH 3-5); Morgan detected via phosphate moiety (1955)

Whatman No. 1 paper, pretreated with Dierick et al. EDTA or oxine (to reduce tailing); n- (1956) propanol-NH 4OH(<* 0-91)-water (6 + 3 + 1), propanol-acetic acid-water (8 + 1 +1) , or isopropanol-pyridine-water-ethyl acetate ( 2 + 1 +1 +2); best on non-treated paper with nitromethane-pyridine-water ( 5 + 6 + 4); detected via phosphate moiety

Whatman No. 1, treated with 1 % oxalic Wawszkiewicz acid; ascending with ammonium acetate- (1961) e thanol -NH 4 -EDTA, then at right angles with ethanol-butanol-water-NH 4 -EDTA-picric acid (EDTA to reduce tailing, picric acid to suppress dissociation of the phosphate group); detected by washing out picric acid with acetone dipping, then periodate-benzidine method

Glass fibre filter paper impregnated with Ory (1961) silicic acid; isooctane containing 2 or 5% ether; visualised by H 2 S 0 4 charring



Fatty acids, mono-, di-, and tri-glycerides

Glycerides, sucrose esters

Glycerides from glycerolysis of linseed and cottonseed oils

Mammalian lipids (phospholipids, glycerides, fatty acids, cholesterol esters)

Glycerol ethers, obtained from neutral and phosphatide glycerol ethers in amniotic fluid by reduction with "vitride"

Silicone oil-impregnated paper; various solvents, mostly with acetic acid-water plus methanol, methyl acetate, kerosine; visualised with iodine vapour and alcoholic solution, K M n 0 4 , and 0-5 % aqueous Rhodamine B

Whatman No. 3 impregnated with Na si l icate-NH 4 Cl; benzene-isopropanol or cyclohexane-isopropanol (benzene alone separated di- and tri-glycerides but not mono-; isopropanol addition effected this); detected with 0-01 % Rhodamine B in 0-025M K 2 H P 0 4

Paper impregnated with octyl acetate-benzene; 75% methanol saturated with A g N 0 3 , or with octyl alcohol; visualised with K M n 0 4 after removal of methanol and Ag +

Glass fibre paper impregnated with silica gel; ascending, isooctane-benzene-acetic acid-acetone ( 1 0 0 0 + 3 0 0 + 1 + 3); detected with 50% H 2 S 0 4 and charring

Whatman SG-81 paper; diisobutyl ketone-acetic acid-water (60 +3 5 + 6); separated ethers determined with periodate-Schiff reagent

Hiroyama (1961)

Ranny (1966)

Novitskaya and Vereshchagin (1969)

Pocock et al. (1972)

Hack and Helmy (1975)

2.12.3. C o l u m n and L i q u i d C h r o m a t o g r a p h y

Under this heading are included the classical separations and the more

recent "liquid" chromatography especially under pressure. Most examples of

this latter type are aimed at fractionation into groups of lipids rather than

discrete separation-of glycerol mono-, di-, and tri-derivatives. Examples are

given in Table 2.7; most are of glycerides, as expected.

TABLE 2.7

Column Chromatography of Glycerol Ester or Ether Mixtures



Mono-, di-, and tri-glycerides after removing fatty acids on Amberlite IRA-400

Mono-, di-, and tri-stearin mixture containing mineral oil

Mono-, di-, and tri-glycerides in mono-glyceride concentrates

Mono-, di-, and tri-glycerides and mono-and di-esters of ethylene and poly-ethylene glycols

Separation of mono-and di-laurin from trilaurin

Lipids in bovine blood serum, extracted with chloroform-methanol (2 + 1)

Mono- separated from the others on silicic Borgstrom acid; heptane and 80% ethanol (1951)

Reversed phase on silicone-treated kiesel- Savary and guhr; cyclohexane-95 % ethanol-water Desnuelle (5 + 15 + 2) (replacement of ethanol by (1954) acetone sometimes used to reduce isomerisation of 2-mono- and 1,2-di-glycerides); detected at 232 nm by added "indicator" of glyceride containing diene fatty acid absorbing at this wavelength

Davison No. 922 silica gel, 200 mesh; Revin et al. successively with 16 % diisopropyl ether in (1957) isooctane, diisopropyl ether, 70 % diethyl ether in isooctane, and 20 % ethanol in diisopropyl ether, eluting in turn the mineral oil, tri-, di-, and mono-stearin; eluates evaporated dry and residues weighed

Davison No. 923, 100-200 mesh silica gel Quinlin and containing 5 % water; successively with Weiser (1958) 200 ml of benzene, of benzene-ether (9+1) , and of ether, to yield tri-, di-, and mono-glycerides; these solvents prevent isomerisation; eluates evaporated in nitrogen; residues weighed

Benzene-wetted silica gel; in succession (for Papariello the glycerides) 40 % diisopropyl ether in et al (1960) isooctane, 70 % diethyl ether in isooctane, and 20 % ethanol in diisopropyl ether, removing tri-, di-, and mono-glycerides, resp.; eluates evaporated and residues weighed

Rubber powder; methanol, which elutes Trowbridge the first two compounds et al. (1964)

Silica gel G under nitrogen pressure; 1 % Brown and ether in pet. ether (cholesterol esters), 10 % Stull (1966) ethyl acetate in pet. ether (triglycerides and cholesterol), ether (free fatty acids and mono- and di-glycerides), and finally acetic acid-methanol-ether ( 4 + 9 + 2 7 ) for phospholipids



Mono-, di-, and tri-glycerides as emulsifiers in shortenings

Glyceride types in fat emulsions


Lactylated mono-glycerides

Brake fluids, including glycerol and other polyols and mono-, di-, and tri-esters of glycerol

1,2- and 1,3-Diglycerides (part of a study of neutral hydroxy-lipids)

Model mixture of monoolein, 1,3-di-and tri-palmitin, cholesterol, and palmitic acid

Lipid classes, including 1-mono-olein, 1,3-diolein, and triolein

Silica gel; benzene, benzene -I- 10% ether, Distler and and ether alone; eluates evaporated and Baur (1966) residues weighed

Silica gel; isooctane-benzene (3 + 7), Ingraito and isooctane-ether (8 + 7), and ethanol- Boari (1967) diisopropyl ether (1+4) , removing tri-, di-, and mono-glycerides, resp.; fractions evaporated, heated 1 h/50°C, and weighed

Method of Quinlin and Weiser (1958) on Franzke et al. silica gel containing 15 % water (1967b)

Silica gel G, containing 5-6 % water; in Neckermann succession 250 ml of benzene, of 10 % ether and Noznick in benzene, and of ether alone, removing tri-, (1968) di-, and then mono-glycerides; glycerol and polyglycerols then eluted with 250 ml of methanol; eluates evaporated and residues weighed

Gel permeation chromatography with Lambert polystyrene beads from 100 to 10 5 A; THF (1970) containing 0 1 % hydroquinone at 38°C at 1 ml/min; refractometric response

Gel permeation chromatography on Calderon Sephadex SR 25/100 containing and Baumann Sephadex LH-20; ethanol eluent; fraction (1970) contents determined gravimetrically

Poragel 60A or 200A; 2-5 to 10 % Lawrence aqueous acetone at 30, 47, or 62°C; (1973) moving wire FID; studied effect of factors

Gradient elution adsorption chromato- Stolyhwo and graphy on Corasil II, modified by Privett (1973) treatment with N H 4 O H ; pentane, ether, chloroform, methanol containing 8 % N H 4 O H as gradient; (oleic acid-glyceride mixture separated with pentane, diluted continuously with pentane containing 25% ether); FID.



Neutral lipids, including mono-stearin, distearin, and tripalmitin

Glycerine nitrates in waste waters from manufacture (dichloromethane extract)

Silicic acid, 100 mesh, with slightly over 8-6% water content; pet. ether (65-75°) with concave gradient of ether; effluent monitored by H 2 - F I D after solvent evaporation; fractions evaporated and residue weighed

Dupont 830 apparatus with stainless steel column containing 30-44 urn Vydac adsorbent; dichloromethane-hexane (3 + 2); 32 atm pressure; phthalimide internal standard; refractometric detection and evaluation

Mono-, di-, and tri- Durapak OPN-Porasil C (36-75 urn); stearin in technical gradient elution with isooctane-benzene; glycerol monostearate detected with moving wire system and

pyrolysis-FID; eluates evaporated and residues weighed

Glyceride-based lubricants (e.g. mono-stearin, 1,2-di-palmitin, triolein, and myristin)

Lipids, including 1-mono- and di-palmitin, tristearin, also palmitic acid, cholesterol

Explosive propellants (Soxhlet extracted with dichloro-methane)

Serum lipids, in chloroform-methanol (2 + 1) extract

Dupont 830 apparatus with stainless steel column containing Zorbax-Sil; ether-n-hexane, (1 + 19), then (1 + 3), and finally (4 + 1); ca. 80 atm pressure; differential refractometry

Varian MicroPak SH-10 column with pre-column of Merkosorb SI-60; hexane-chloroform (9 + 1) with linear ethanol gradient of 3 %/min up to 70 % in original solvent; moving wire FID

Evaporated extract + acetanilide in 1,1-dichloroethane brought on to Vydac 30-43 um silica column; eluting with same solvent at ca. 30 atm.; monitored by UV absorption at 254 nm, recording peak areas

Activated silica gel HR 60; eluted at 100 ml/h with complex gradient from light petroleum, ether, ethyl acetate, methanol, acetic acid, and water; control analysis with moving wire detector; order of elution; hydrocarbons, cholesterol esters, triglycerides, free fatty acids, cholesterol, diglycerides, then phospholipids

Cavina et al. (1973)

Chandler et al (1974)

Fischer (1974)

Sinsel et al (1975)

Kiuchi et al. (1975)

Dalton et al (1975)

Vandamme et al (1975)

128 GLYCEROL C O M P O U N D S : INTRODUCTION A N D METHODS 2.12


Comparison of TLC and column and gel chromatographic separation of glycerides (last-named method found easiest and fastest for analysing emulsifier monoglycerides)

Column chromatography according to Quinlin and Weiser (1958), after removing free glycerol with water from sample extract in ether. Gel chromatography on styrene-divinylbenzene copolymer S-Gel 832, exclusion limit of M.W. ca. 1000; tetrahydrofuran at room temp.; differential UV and refractometric monitoring.

Coupek et al. (1976)

2.12.4. Gas C h r o m a t o g r a p h y

There appear to be remarkably few examples of direct gas chromatographic

separation and determination of mono-, di-, and tri-esters or -ethers of

glycerol. Mares (1969) reported the separation of synthetic mixtures of 1,2-

diglycerides, triglycerides, and saturated and unsaturated fatty acids in glass

columns containing 10% DEGS on acid-washed Chromosorb W. Rosseel

and Bogaert (1972) carried out GLC of glycerol nitrate esters (1-mono-, 1,2-

and 1,3-di-, and tri-) and nitrate esters also of isosorbide and isomannide in

glass columns containing 3 % XE-60 or 3-5 % QF-1 on 60-80 mesh Gas-Chrom

Q, at 150°C or (second column) 110°C (then with FID) or 120°C (then with

ECD).

REFERENCES

Archer, A. W. (1975). J. Chromatogr. 108, 401. Aylward, F. and Wood, P. D. S. (1956). Nature (London) 177, 146. Barber, M., Chapman, J. R. and Wolstenholme, W. A. (1968). Int. J. Mass Spectrom.

Ion. Phys. 98. Baryshkov, Yu. A. (1966). Lab. Delo 615. Baumann, W. J., Schmid, H. H. O. and Mangold, H. K. (1969). J. Lipid Res. 10, 132. Baur, F. J. and Distler, E. (1966). / . Ass. Off. Agric. Chem. 49, 816. Becker, E. and Krull, L. (1958). Fette, Seifen, Anstrichm. 60, 449. Berner, G. (1970). Z. Lebensm.-Unters.-Forsch. 141, 318. Bertoni, M. H., Rotsztein de Cymeryng, F. and Cattaneo, P. (1963). Rev. Argent.

Grasas Aceites 5, 3. Biernoth, G. (1968). Fette, Seifen, Anstrichm. 70, 402. Beizinski, J. J., Pomerance, W. and Goodman, J. (1968). / . Chromatogr. 38, 148. Blank, M. L. and Snyder, F. (1969). Biochim. Biophys. Acta 187, 154. Blomstrand, B. and Gurtler, J. (1959). Acta Chem. Scand. 13, 1466.


REFERENCES 129

Blum, J. and Koehler, W. R. (1970). Lipids 5, 601. Booy, H. and Waterman, H. I. (1949). Anal. Chim. Acta 3, 440. Borgstrom, B. (1951). Acta Physiol. Scand. 30, 231. Bradley, T. F., Mueller, A. C. and Shokal, E. C. (1955). U.S. Patent 2,700,036,

Jan. 18. Brandt, P. E., Krog, N., Lauridsen, J. B. and Tolboe, O. (1968). Acta Chem. Scand.

22, 1691. Broniatowski, A. (1967). Sv. Papperstidn, 70, 234. Brown, W. H. and Stull, J. W. (1966). / . Dairy Sci. 49, 636. Calderon, M. and Baumann, W. J. (1970). / . Lipid Res. 11, 167. Casparrini, G., Hornung, M. G. and Hornung, E. C. (1968). Anal. Letters 1, 481. Cavina, G , Moretti, G , Mollica, A., Moretta, L. and Siniscalchi, P. (1973). / . Chro-

matogr. 44, 493. Ceglowska, K., Schumann, K. and Szczepariska, H. (1966). Tluszcze, Sradki Piorace,

Kosmet. 10, 157 (Chem. Abs. 67, 45063). Chabard, J.-L., Vedrine, F., Godeneche, D., Petit, J. and Berger, J.-A. (1976). / .

Chromatogr. 121, 295. Chakrabarty, M. M., Bhattacharyya, D. and Gupta, A. (1966). J. Chromatogr. 22, 84. Chandler, C. D., Gibson, G. R. and Bolleter, W. T. (1974). / . Chromatogr. 100, 185. Chapman, D. (1965). J. Am. Oil Chem. Soc. 42, 353. Chiarioni, T., Ronchi, F., Sasso, G. F., Mancini, P. and Nardi, E. (1966). Med. Clin.

Sper. 16, 209. Constantinescu, T. and Enache, S. (1974). Farmacia (Bucharest) 22, 179. Coupek, J., Pokorny, S., Mares, E., Zezulkova, L., Nguyen-Thien Luan and Pokorny,

J. (1976). J. Chromatogr. 120, 411. Crew, M. C. and DiCarlo, F. J. (1968). / . Chromatogr. 35, 506. Dalton, R. W., Chandler, C. D. and Bolleter, W. T. (1975). J. Chromatogr. Sci. 13, 40. Daubert, B. F. and King, C. G. (1938). / . Amer. Chem. Soc. 60, 3003. Desnuelle, P., Naudet, M. and Rouzier, J. (1948). Biochim. Biophys. Acta 2, 561. Dierick, W., Stockx, J. and Vandendriessche, L. (1956). Naturmssenschaften 43, 82. Distler, E. and Baur, F. J. (1966). / . Amer. Oil Chem. Soc. 49, 812. Doadrio, A. and Montequi, R. (1952). Anales Real Soc. Espan. Fis. y Quim. 48B, 69. Dowse, C. M. and Saunders, J. A. (1956). Biochem. J. 62, 455. Duthie, A. H. and Atherton, H. V. (1970). / . Chromatogr. 51, 319. Emmerie, A. (1953). Rec. Trav. Chim.12, 893. Febure, P. and Aiache, J. M. (1968). Ann. Pharm. Franc. 26, 463. Fewster, M. E., Burns, B. J. and Mead, J. F. (1969). / . Chromatogr. 43, 120. Fischer, E. (1920). Ber. 53, 1621. Fischer, R. (1974). Chromatographia 7, 207. Fleury, P. and Courtois, J. (1941). Bull. Soc. Chim. (Paris) 8, 397. Fleury, P. and Paris, R. (1933). J. Pharm. Chim. 18, 470; also Compt. Rend. 196, 1416. Franzke, C. and Strandt, F. (1967). Ndhrung 11, 501. Franzke, C , Heims, K. O. and Vollgraf, I. (1967a). Ndhrung 11, 515. Franzke, C , Kretzschmann, I., Rustow, B. and Rugenstein, H. (1967b). Pharmazie 22,

487. Freeman, I. P. (1974). Chem. Ind. (London) 623. Freeman, N. K. (1968). J. Amer. Oil Chem. Soc. 45, 798. Gelman, R. A. and Gilbertson, J. R. (1969). Anal. Biochem. 31, 463. Gernert, F. (1968). Z. Lebensm.-Unters.-Forsch. 138, 216. Greenhalgh, R. and Wood, P. J. (1973). / . Chromatogr. 82, 410.

130 GLYCEROL C O M P O U N D S : INTRODUCTION A N D METHODS

Grynberg, H., Beldowicz, M., Ceglowska, K. and Szczepanska, H. (1967). Chem. Anal. (Warsaw) 12, 495.

Guernet, M., Espinassou, E. and Hamon, M. (1973). Ann. Pharm. Franc. 31, 343. Hack, M. H. and Helmy, F. M. (1975). / . Chromatogr. 107, 155. Hallgren, B. and Larsson, S. O. (1959). Acta Chem. Scand. 13, 2147. Halvarson, H. and Qvist, O. (1974). Amer. Oil Chem. Soc. 51, 162. Hanahan, D. J., Ekholm, J. and Jackson, C. M. (1963). Biochemistry 2, 630. Handschumaker, E. and Linteris, L. (1947). J. Amer. Oil Chem. Soc. 24, 143. Hartman, L. (1956). Analyst (London) 81, 67. Hartman, L. and Esteves, W. (1976). / . Amer. Oil. Chem. Soc. 53, 584. Heckles, T. S. and Dunlap, L. H. (1955). / . Amer. Oil Chem. Soc. 32, 224. Hiroyama, O. (1961). Nippon Nogei Kagaku Kaishi 35, 372. Horning, M. G., Casparrina, G. and Horning, E. C. (1969). J. Chromatogr. Sci. 7, 267. Horning, M. G., Murakami, S. and Horning, E. C. (1971). Amer. J. Clin. Nutr. 24,1086. Ingraito, D. and Boari, A. (1967). G. Med. Mil. 117, 567 (Chem. Abs. 68, 93410; Anal.

Abs. 15, 6185). Ivanoff, N. (1945). Bull. Mat. Grasses Inst. Coloniale Marseille 29, 45 (Chem. Abs. 40,

5361). Jensen, R. G. and Morgan, M. E. (1959). J. Dairy Sci. 42, 232. Jurriens, G., de Vries, B. and Schouten, L. (1964). J. Lipid Res. 5, 267. Kabara, J. J. and Chen, J. S. (1976). Anal. Chem. 48, 814. Kanematsu, H., Maruyama, T., Niiya, I., Imamura, M. and Kawakita,~H. (1972).

Eiyo To Shokuryo 25, 46 (Chem. Abs. 77, 3877). Karnovsky, M. L. and Rapson, W. S. (1946). J. Soc. Chem. Ind. 65, 138. Karpov, O. N. (1964). Tr. Vses. Nauch.-Issled. Inst. Khim. Reaktivov Osobo Chist.

Khim. Veshchestv. No. 26, 273 (Chem. Abs. 66, 82149). Kiuchi, K., Ohta, T. and Ebine, H. (1975). / . Chromatogr. Sci. 13, 461. Korolczuk, J. and Kwa^niewska, I. (1974). / . Chromatogr. 88, 428. Kresze, G. and Schauffelhut, F. (1967). Z. Anal. Chem. 229, 401. Kresze, G , Bederke, K. and Schauffelhut, F. (1965). Z. Anal. Chem. 209, 329. Kruty, M., Segur, J. B. and Miner jr., C. S. (1954). / . Amer. Oil Chem. Soc. 31, 466. Kuksis, A. (1971). Can. J. Biochem. 49, 1245. Kuksis, A. (1972). / . Chromatogr. Sci. 10, 53. Kummerow, F. A. and Daubert, B. F. (1950). J. Amer. Oil Chem. Soc. 27, 100. Lambert, A. (1970). J. Appl. Chem. (London) 20, 307. Lawrence, J. G. (1973). / . Chromatogr. 84, 299. Loehnert, P. and Schoellner, R. (1968). Plaste Kaut. 15, 520. Lorant, B. (1966). Seifen-Ole-Fette-Wachse 92, 617. Lueck, H. and Kohn, R. (1965). Milchmssenschaft 20, 455. Mclnnes, A. G , Tattrie, N. H. and Kales, M. (1960). / . Amer. Oil Chem. Soc. 37, 7. Malins, D. C , Wekell, J. C. and Houle, C. R. (1964). Anal. Chem. 36, 658. Mangold, H. K., Lamp, B. C. and Schlenk, H. (1955). / . Amer. Chem. Soc. 77, 6070. Manners, M. J., Kidder, D. E. and Parsons, P. M. (1969). J. Chromatogr. 43, 276. Mares, P. (1969). Sbornik Lek. 71, 166 (Chem. Abs. 72, 50746) Martin, J. B. (1953). / . Amer. Chem. Soc. 75, 5483. Maruta, S. and Iwama, F. (1965). Kogyo Kagaku Zasshi 68, 2137. Marzo, A., Ghirardi, P., Sardini, D. and Meroni, G. (1971). Clin. Chem. 17, 145. Mehta, T. N. and Shah, S. N. (1957). / . Amer. Oil Chem. Soc. 34, 587. Mehta, T. N. and Shah, S. N. (1958). / . Amer. Oil Chem. Soc. 35, 482. Mlejnek, O. (1955).' Chem. Zvesti 9, 27.

REFERENCES 131

Mlekusch, W., Truppe, W. and Paletta, B. (1974) J. Chromatogr. 93, 183. Montequi, R. and Doadrio, A. (1952). Inform. Quim. Anal. (Madrid) 6, 31. Miiller, E. and Rundel, W. (1958). Angew. Chem.70, 105. Murphy, J. F. (1962). Appl. Spectroscopy 16, 139. Myher, J. J. and Kuksis, A. (1974). Lipids 9, 382. Myher, J. J. and Kuksis, A. (1975). / . Chromatogr. Sci. 13, 138. Myher, J. J., Marai, L. and Kuksis, A. (1974). I Lipid Res. 15, 586. Nagakura, M., Ogawa, Y. and Yoshitomi, Y. (1968). Shikizai Kyokaishi 41, 542 (Chem.

Abs.70, 68925). Neckermann, E. F. and Noznick, P. P. (1968). J. Amer. Oil Chem. Soc. 45, 845. Neissner, R. (1969). Pharm. Ind. 31, 724. Neissner, R. (1972). Fette, Seifen, Anstrichm. 74, 198. Novitskaya, G. V. and Vereschchagin, A. G. (1969). J. Chromatogr. 40, 422. O'Brien, J. F. and Klopfenstein, W. E. (1971). Chem. Phys. Lipids 6, 1. Ory, R. L. (1961). / . Chromatogr. 5, 153. Palmer, A. A., Kintzios, J. A. and Papadopoulos, N. M. (1972). / . Chromatogr. Sci.

10, 107. Papariello, G. J., Chulkaratara, S., Higuchi, T., Martin, J. E. and Kuceski, V. P. (1960).

Amer. Oil Chem. Soc. 37, 396. Paschke, R. F., Kerns, J. and Wheeler, D. H. (1954). J. Amer. Oil Chem. Soc. 31, 5. Pedersen, V. (1953). Arch. Pharm. Chemi 60, 498. Pocock, D. M.-E., Rafal, S. and Vost, A. (1972). / . Chromatogr. Sci. 10, 72. Pohle, W. D. and Mehlenbacher, V. C. (1946). Oil and Soap 23, 48. Pohle, W. D. and Mehlenbacher, V. C. (1950). / . Amer. Oil Chem. Soc. 27, 54. Pohle, W. D., Mehlenbacher, V. C. and Cook, J. H. (1945). Oil and Soap 22, 115. Pohle, W. D., Bolley, D. S., Marmor, R. A., Privett, O. S., Rini, S. J. and Walker, W. D.

(1957). / . Amer. Oil Chem. Soc. 34, 301. Pokorny, J. and Herodek, O. (1964). Sb. Vys. Sk. Chem.-Techriol. v Praze, Potrav.

Technol. 8, 87 (Anal. Abs. 13, 1542). Popov, A., Chobanov, D. and Stefanov, K. (1967). Maslo-Sapunena Prom., Byul 3, 3

(Chem. Abs. 68, 88410). Prada, D., Carracedo, C. F., Montenegro, L. and Prieto, A. (1970). Grasas Aceites

(Seville) 21, 261. Privett, O. S., Blank, M. H. and Lindberg, W. O. (1961). J. Amer. Oil Chem. Soc. 38,312. Quinlin, P. and Weiser jr., H. J. (1958). / . Amer. Oil Chem. Soc. 35, 325. Qureshi, M. I. and Waheed, S. (1972). Pak. J. Sci. Ind. Res. 15, 323. Ranny, M. (1966). Abh. Deut. Akad. Wiss. Berlin, Kl. Chem., Geol, Biol. 216 (Chem.

Abs. 68, 65512). Revin, J. L., Meyer, R. J. and Higuchi, T. (1957). / . Amer. Oil Chem. Soc. 34, 261. Roncero, A. V., Fiestas, J., Mazuelos, F. and Foreno, J. M. (1952). Fette, Seifen,

Anstrichm. 54, 550. Rosseel, M. T. and Bogaert, M. G. (1972). / . Chromatogr. 64, 364. Rosseel, M. T., Bogaert, M. G. and Maerman, E. J. (1970). J. Chromatogr. 53, 263. Rumsby, M. G. (1968). / . Chromatogr. 34, 461. Rybicka, S. M. (1962). Chem. Ind. (London) 308. Sahasrabudhe, M. R. (1967). / . Amer. Oil Chem. Soc. 44, 376. Sahasrabudhe, M. R. and Legari, J. J. (1967). / . Amer. Oil Chem. Soc. 44, 379. Sallee, T. L. and Adams, G. M. (1970). / . Chromatogr. 51, 545. Saracco, G. B. and Gay, M. (1971). Riv. Ital. Sostanze Grasse 48, 319. Savary, P. and Desnuelle, P. (1954). Bull. Soc. Chim. France 21, 936.

132 GLYCEROL C O M P O U N D S : INTRODUCTION A N D METHODS

Schewe, T. and Coutelle, C. (1970). Acta Biol. Med. Ger. 24, 223. Schlenk, H. and Ener, M. A. (1959). J. Amer. Oil Chem. Soc. 36, 145. Schmid, M. J. and Otteneder, H. (1976). Getreide, Mehl, Brot 30, 62. Schmidt, H. E. (1963). Fette, Seifen, Anstrichm. 65, 488. Seelbach, C. W. and Quackenbush, F. W. (1957). J. Amer. Oil Chem. Soc. 34, 603. Shaw, N. (1968). Biochim. Biophys. Acta 164, 435. Sinsel, J. A , LaRue, B. M. and McGraw, L. D. (1975). Anal. Chem. 41, 1987. Skipski, V. P., Smolowe, A. F., Sullivan, R. C. and Barclay, M. (1965). Biochim.

Biophys. Acta 106, 386. Skipski, V. P., Good, J. J., Barclay, M. and Reggio, R. B. (1968). Biochim. Biophys.

Acta 152, 10. Snyder, F. (1973). / . Chromatogr. 82, 7. Stolyhwo, A. and Privett, O. S. (1973). / . Chromatogr. Sci. 11, 20. Stross, P. and Stuckey, R. E. (1950). / . Pharm. Pharmacol. 2, 549. Susi, H., Morris, S. G. and Scott, W. E. (1961). J. Amer. Oil Chem. Soc. 38, 199. Susi, H , Morris, S. G., Zell, T. E. and Scott, W. E. (1963). J. Amer. Oil Chem. Soc. 40,

329. Szonyi, C. and Sparrow, K. (1964). / . Amer. Oil Chem. Soc. 41, 535. Tallent, W. H. and Kleiman, R. (1968). J. Lipid Res. 9, 146. Thomas III, A. E., Scharoun, J. E. and Ralston, H. (1965). J. Amer. Oil Chem. Soc. 42,

789. Timmen, H., Dimick, P. S., Patton, S. and Pohanka, D. S. (1970). Milchwissenschaft

25,217. Trowbridge, J. R., Herrick, A. B. and Bauman, R. A. (1964). J. Amer. Oil Chem. Soc.

41, 306. Trowell, J. M. (1970). Anal. Chem. 42, 1440. Uehara, R., Horii, T., Katayama, K. and Tomita, Y. (1971). Yukagaku 20, 174 (Chem.

Abs. 74, 127959). Ullmann (1957) "Encyclopadie der Technischen Chemie", 3rd. Ed., Vol. 8. Vandamme, D., Declercq, B., Denoo, A., Blaton, V. and Peeters, H. (1957). Anal.

Biochem. 69, 29. Wachs, W. (1967). Tenside 4, 40. Wade, H. E. and Morgan, D. M. (1955). Biochem. J. 60, 264. Warren, R. J. and Zarembo, J. E. (1970). / . Pharm. Sci. 59, 840. Wathelet, J.-P., Claustriaux, J.-J. and Severin, M. (1975). J. Chromatogr. 110, 157. Watts, R. and Dils, R. (1968). Biochem. J. 110, 51P. Wawszkiewicz, E. J. (1961). Anal. Chem. 33, 252. Wiklund, B. and Eliasson, R. (1972). J. Chromatogr. 68, 153. Wildbrube, H. J. and Erb, W. (1971). Verh. Deut. Ges. Inn. Med. 11, 588. Wildgrube, H. J, Erb, W. and Boehle, E. (1969). Z. Klin. Chem. Klin. Biochem. 1, 514. Wildgrube, H. J., Erb, W. and Boehle, E. (1973). Fette, Seifen, Anstrichm. 75, 168. Wood, P. S. (1969). Column. 3, 7. Wood, R. and Snyder, F. (1966). Lipids 1, 62. Wood, R., Raju, P. K. and Reiser, R. (1965). J. Amer. Oil Chem. Soc. 42, 161. Wood, R., Baumann, W. J., Snyder, F. and Mangold, H. K. (1969a). J. Lipid Res. 10,

128. Wood, R., Piantadosi, C. and Snyder, F. (1969b). J. Lipid Res. 10, 370. Wormith, D. J. and Rae, J. J. (1941). / . Amer. Chem. Soc. 63, 2523.

3

Glycerol Compounds: Methods Based on Release of

Glycerol from the Sample

The methods discussed in this chapter have two stages, namely, the degrada-tion to release glycerol and then the subsequent analytical application to this freed glycerol. Analytical procedures for glycerol have already been generally discussed in Chapter 1. Notable is the accent on periodate oxidation pro-cedures for this freed glycerol, and in particular those in which formaldehyde, formed in this oxidation, is converted into a dihydrolutidine product, then evaluated through its light absorbance or fluorescence [see Chapter 1, Section 1.1.14.C.2(i)]. This procedure has been rarely used directly for free glycerol in samples.

The two stages are generally discrete, and often the glycerol product is partly cleaned up before the second stage. Occasionally, however, the two steps are telescoped, for example when the analytical reagent for the glycerol contains a component that effects degradation.

Classification of information according to broad headings of the degrada-tion procedure is most convenient.

Hydrolytic breakdown, usually under alkaline conditions, is the most popular method, especially for esters:

3 . 1 . H Y D R O L Y S I S

C H 2 0 — X C H , O H

C H O — X + 3 0 H I from ethers)

C H 2 O H

I (carboxylate from C H O H + 3 X O - esters; alkoxide

C H 2 0 — X

133

F

134 GLYCEROL COMPOUNDS 3.1

The customary fission agent is alkali hydroxide in a lower alcohol solvent (methanol, ethanol, isopropanol) or in aqueous alcohol, in which the sample is soluble. Temperature ranges from room temperature to 70-80°C, with reaction durations up to about 30 min, depending on temperature and concentration. After hydrolysis, organic solvent is removed, water added if necessary, and fatty acids (from glycerides) are precipitated by acidification. These are filtered off or extracted with a solvent that does not dissolve glycerol (e.g. a lower alkane such as hexane or heptane), and the glycerol in the residual aqueous solution is submitted to an analytical procedure.

Any polar components of the samples, for instance phospholipids, are separated in an early stage, usually by adsorption from the sample extract on to silicic acid or Zeolite in a chromatographic procedure.

Transesterification with alkoxides has found some use with glycerides. Sodium derivatives of the lower alcohols have been the most employed:

CFLO—COR C H 2 O H I I

CHO—COR + 3 C H 3 O H -> CHOH + 3RCOOCH, I I

C H 2 0 — C O R C H 2 O H Some analytical reagents for glycerol that are acid or alkaline may effect

hydrolysis of glycerol esters or ethers in situ. The examples, relatively rare, are conveniently grouped under this heading.

The classification below is based on the subsequent procedure for the

released glycerol.

3 . 1 . 1 . O x i d a t i o n w i t h Periodate

A. D E T E R M I N A T I O N O F U N U S E D R E A G E N T

Periodate oxidation followed by determination of unused reagent has evidently been only rarely used. Examples are the work of Troy and Bell (1946) on the determination of mono-, di-, and tri-glycerides, and of den Otter (1947) on monoglyceride in emulsifiers.

B. D E T E R M I N A T I O N O F F O R M A L D E H Y D E

1. With Phenylhydrazine-Oxidising Agent The principle of this method is given in Chapter 1, Section 1.1.14.C.2(b).

Biggs (1954) determined glycerides in citronella oil by first saponifying for 1 h with 0-5N methanolic potassium hydroxide (he preferred methanol to ethanol because subsequent evaporation of solvent can be performed at a lower temperature, thus minimising loss of glycerol). After adding saturated aqueous sodium chloride and acidifying with sulphuric acid, he evaporated the alcohol on a water bath and extracted three times with ether to remove

3.1 HYDROLYSIS 135

fatty acids. The glycerol was oxidised with 1*25% potassium periodate in IN sulphuric acid for 10min; he then added 1% phenylhydrazine hydro-chloride and, after a further 10 min, 1 % ferric ammonium sulphate followed by 6N sulphuric acid. The resulting red colour is stable for at least 15 min and was evaluated at 520 nm.

Randrup (1960) hydrolysed plasma triglycerides in an extract in hexane with barium hydroxide overnight at 85°C. He then acidified with hydro-chloric acid, removing the liberated fatty acids in the hexane, and treated the aqueous phase for 10 min with a periodic acid reagent and then with phenyl-hydrazine hydrochloride also for 10 min. Ferricyanide as oxidising agent was then added, followed by cooling in ice and addition of concentrated hydro-chloric acid within 4 min. He read the absorbance at 530 nm at 11-15 min from the moment of addition of the concentrated acid. Jover (1963) deter-mined serum glycerides by saponification with alcoholic potassium hydrox-ide, removing fatty acids with hexane and then applying Randrup's method to the glycerol. Buckley et al. (1966) determined serum triglycerides also, using a modification of the Randrup-Jover procedure. After extracting the sample with chloroform-methanol (2 + 1) and removing phospholipids with silicic acid, they heated the evaporated residue for 30 min at 60°C with 4 % potassium hydroxide in 95 % ethanol. Following this operation the mixture was acidified with hydrochloric acid and the fatty acid was extracted with hexane. Subsequently 0-16% potassium metaperiodate, 1*82% phenyl-hydrazine hydrochloride, and 4-5 % potassium ferricyanide were successively introduced into the aqueous layer, with successive waits of 10 min in the dark (twice) and 5 min in the ice bath. After a further addition of hydrochloric acid and a final waiting period of 10 min the glycerol was evaluated at 540 nm. Torjescu et al. (1966) used this procedure of Buckley et al. to estimate blood serum triglycerides.

Lloyd and Goldrick (1968) hydrolysed an aliquot of extracted plasma glycerides in diethyl ether using ethanolic potassium hydroxide at 60°C for 1 h. They then used essentially the same procedure as Randrup for glycerol determination.

Tixier et al. (1974) determined triglycerides in blood serum according to the same principle of hydrolysis with potassium hydroxide and then reaction of the formaldehyde with phenylhydrazine in the presence of an oxidising agent in acid solution, finally evaluating at 530 nm.

2. With Chromotropic Acid The probable reaction between the formaldehyde and chromotropic acid in sulphuric acid medium is given in Chapter 1, Section 1.1.14.C.2(a). It is one of the most used procedures for determination (and detection) of glycerol.

Possibly the first application to glycerides is due to Stewart (1954) who


extracted blood samples with ether-ethanol, ultimately saponifying and oxidising with periodate. He distilled the formaldehyde into sodium sulphite, developed colour with chromotropic acid, and evaluated the absorbance at 540 nm.

Van Handel et al. (1957) developed a procedure for serum triglycerides which was accepted as a standard method for this compound class. They hydrolysed the glycerides in a 1 ml aliquot by heating for 15 min at 60-70°C with 0-5 ml of ethanolic potassium hydroxide, and then acidified with 0-5 ml of 0-2N sulphuric acid and evaporated off ethanol on the boiling water bath for 15 min. They determined the liberated glycerol according to the method of Lambert and Neish (1950). They stated that the 5 min oxidation with sodium periodate-sulphuric acid brought about no oxidation of any glucose present. After removing oxidising agent with arsenite they developed colour by heating for 30 min at 100°C with a 0-2% solution of chromotropic acid in ca. 20N sulphuric acid and evaluating at 570 nm. This procedure was employed with some modifications by numerous later investigators. Carlson and Wadstrom (1959) and Van Handel and Ordway (1961) separated phospholipids on zeolite. Chlouverakis et al. (1963) hyrolysed with meth-a n o l s instead of ethanolic potassium hydroxide. Vanzetti and Denegri (1964) saponified for 30 min at 37°C with 2% potassium hydroxide before ulti-mately oxidising with 0-5 % sodium periodate in the presence of acetic and hydrochloric acids for 9 min; they removed oxidising agent with lead(II) acetate and then precipitated excess lead with sodium sulphate before developing colour. Lofland (1964) found erratic results when using iso-propanol in the original solvent in the application of the AutoAnalyzer in the determination of serum glycerides.

Christophe and Matthijs (1964) hydrolysed glycerides with ethanolic potassium hydroxide for 30 min at 60-70°C, acidified with 2N sulphuric acid, and extracted interfering lipids with petroleum ether. They then oxidised with periodate, removed the liberated iodine with hydroxylamine at 70°C, developed colour with a chromotropic acid reagent, and evaluated at 570 nm. Bandyopadhyay (1968) scraped off triglyceride zones from silica gel-silver nitrate thin layers and applied the Van Handel method, hydro-lysing with 0-4% ethanolic potassium hydroxide for 30 min at 60-70°C. Wessels (1973) also removed separated triglyceride zones from thin layers and hydrolysed with 0-4% ethanolic potassium hydroxide for 80 min at 70°C before colorimetry using the chromotropic acid method at 570 nm; he quoted the example of a palm kernel oil. Revutskii et al. (1975), who adsorbed phospholipids on silicic acid, and Kawade (1962) employed modified Van Handel procedures.

In a clinical study of serum lipids, Sordi (1960) saponified glycerides by heating at 65°C with alcoholic potassium hydroxide. He oxidised with

3.1 HYDROLYSIS 137

sodium periodate-sulphuric acid for 10 min at room temperature, also removing iodate and periodate with arsenic(III). His chromotropic acid reagent was slightly more concentrated (1%). Blankenhorn et al. (1961) separated polar compounds on Florisil columns and hydrolysed blood glycerides by heating for 30 min at 55°C with 4 % ethanolic potassium hydroxide before applying the Lambert-Neish method to glycerol deter-mination. Chin et al. (1971) modified their procedure to determine serum and plasma triglycerides. Franzke et al. (1967) saponified serum triglycerides with 0-4 % ethanolic potassium hydroxide for 15 min at 70°C after having separated them from mono- and di-glycerides using TLC. Medvedev and Kalantar (1972) hydrolysed for 30 min at 70°C, then oxidising the glycerol with sodium periodate-sulphuric acid for 15 min; they removed oxidising compounds with thiosulphate and then developed colour with a chromo-tropic acid reagent by heating at 100°C for 45 min.

An ethanolic potassium hydroxide-barium hydroxide reagent was used at ca. 80°C by Haux and Natelson (1971) to hydrolyse serum triglycerides after phospholipids had been removed from a chloroform extract with silica gel. Subsequent acidification with sulphuric acid liberated the fatty acids and precipitated barium sulphate, on which they were adsorbed. An aliquot of 0-5 ml of the resulting acid solution was treated for 10 min with 0T ml of 0-02M sodium periodate, and unused periodate and iodate were removed with 01 ml of 0-2M arsenite. They developed colour with 3 ml of ca. 0-2% chromotropic acid in ca. 20N sulphuric acid and heating for 30'min at 105-110°C. They measured absorbance at 575 nm.

Renkonen (1962) determined phosphatide glycerol by heating for 48 h with 2N hydrochloric acid in a sealed tube, conditions under which the glycerol is not decomposed; its amount was determined via the chromotropic acid colour reaction. Hanahan et al. (1957) refluxed phospholipids for the same period with 6N acid and recorded about 70% destruction of the glycerol, estimated likewise through periodate oxidation and colour forma-tion with chromotropic acid; they consequently changed to the 2N hydro-chloric acid.

3. With Acetylacetone-Ammonium Salt The reaction of formaldehyde with acetylacetone in the presence of ammo-nium salts has been mentioned in Section L1.14.C.2(i). The product, 3,5-diacetyl-l,4-dihydrolutidine, can be determined via its absorption in the 400-420 nm region, or via its fluorescence in the vicinity of 500 nm.

The method is relatively new and the first application to determine glycerides after, preliminary hydrolysis evidently dates back to Kessler and Lederer (1965). They estimated glycerides fluorimetrically in serum samples after extraction with isopropanol and hydrolysis with potassium hydroxide.


Some subsequent investigators adopted this procedure and attempted to automate it, e.g. Block and Jarrett (1968). Cramp and Robertson (1968) used a modified version, hydrolysing with 1 % potassium hydroxide in 50% isopropanol and then oxidising with 5 mM sodium periodate in 2M acetic acid. They then reacted the formaldehyde with acetylacetone in 2M ammo-nium acetate (at pH 6) at 58°C before final fluorimetric evaluation. Royer and Ko (1969, 1972) extracted glycerides from plasma samples with nonane and removed phospholipids on silicic acid, Florisil, zeolite, or Doucil, before transesterifying with sodium methoxide-isopropanol. They estimated fluorescence at 510 nm from excitation at 405 nm. In their second quoted publication they reported automatic extraction and determination.

Claude et al. (1969) also used the method of Kessler and Lederer, extracting with isopropanol and using zeolite to remove polar lipids. Lemaur and Le Palec (1970) extracted with isopropanol in the presence of zeolite and saponified with potassium hydroxide before fluorimetric (and also colori-metric) determination.

Goedicke and Gerike (1972) commented on the high blank values when unused periodate is not destroyed after oxidation. The customary procedure of adding iodide and removing liberated iodine with thiosulphate or sulphite led to fluorescence quenching, and they suggested adding rhamnose directly to the reaction mixture after glycerol oxidation. This reduces periodate excess and is itself oxidised to acetaldehyde which does not interfere in the subsequent formaldehyde determination. They hydrolysed glyceride with potassium hydroxide in isopropanol for 1 h at 60°C and oxidised the glycerol for 10 min at room temperature with sodium periodate-hydrochloric acid. After treating with rhamnose they reacted the formaldehyde with acetyl-acetone-ammonium acetate (at pH 5-6, slightly more acidic than used by Cramp and Robertson) for 30 min at 60°C. They diluted with isopropanol and determined fluorescence intensity at 485 nm with excitation at 405 nm, the evaluating wavelength thus being slightly shorter than that used by Royer and Ko. Townsend and Singer (1974) determined triglyceride (with or without previous TLC separation) by hydrolysing with sodium ethoxide-isopropanol for 2 min, oxidising with potassium periodate-sulphuric acid at 0°C for 90 min, and adding ammonium acetate, rhamnose, and acetyl-acetone in isopropanol. After heating for 30 min at 40° C the fluorescence of the product was evaluated at 512 nm (excitation at 422 nm).

The colorimetric method appears to have been more often used than the fluorimetric. Dunsbach (1966) used extraction and oxidation conditions of Van Handel et al. (1957) but determined the formaldehyde by reaction for 20 min at 60°C with acetylacetone-ammonium acetate; he extracted the coloured product into amyl alcohol and measured light absorbance at 400-420 nm. Sardesai and Manning (1968) proceeded similarly to determine

3.1 HYDROLYSIS 139

triglycerides in plasma and tissue, evaluating at 412 nm. The Van Handel procedure was employed also by Matsumiya et al. (1970) for serum tri-glycerides, using this alternative conclusion.

Fletcher (1968) saponified serum triglycerides for 15 min at 70°C with 5 % potassium hydroxide after having removed phospholipids in the usual way on zeolite from an isopropanol-water mixture. He determined the absorb-ance of the end-product at 405 nm after 30 min reaction at 50°C. Fletcher's method was used in the colorimetric variant of Lemaur and Le Palec (see above) (1970) and of Foster and Dunn (1973), for example, although they separated polar components on alumina instead of zeolite. Neri and Frings (1973) modified Fletcher's procedure, hydrolysing for 5 min at room tempera-ture and carrying out the final colour-yielding reaction for 15 min at 6 0 -70°C. Mendez et al. (1975) used the saponification, oxidation, and colori-metric steps of Neri and Frings also for serum glycerides, extracting these by the method of Gottfried and Rosenberg (1973) (see below).

Others who used alkali hydroxides for hydrolysis may be mentioned, for example: Mill et al. (1970) for serum triglycerides, with 1% potassium hydroxide at 60°C and final evaluation at 410 nm with the help of the Auto Analyzer; Smernoff (1971) patented a procedure for triglycerides (and cholesterol) in blood serum and plasma, depending on extraction with isopropanol, removal of polar components with alumina, hydrolysis with potassium hydroxide, oxidation with sodium periodate-acetic acid, and colorimetric estimation of the dihydrolutidine reaction product at 405 nm; Sobotka (1971) removed phospholipids with silica gel G from chloroform solution and evaluated at 420 nm; Agradi et al. (1972) for plasma triglycerides and cholesterol, saponified with 2 % potassium hydroxide in isopropanol at 50°C and carried out final measurement at 408 nm; Edwards et al. (1972) extracted neutral lipids, including triglycerides, with isopropanol in the presence of zeolite, Lloyd's reagent, cupric sulphate, and calcium hydroxide to remove phospholipids and carbohydrates before saponifying with alcoholic potassium hydroxide and converting into the dihydrolutidine; Gottfried and Rosenberg (1973) for serum triglycerides, extracted the sample with heptane instead of the more customary nonane, and hydrolysed with aqueous potassium hydroxide at the higher temperature of 70°C; Giegel et al. (1975) partitioned serum lipids between isopropanol-water and nonane (extracting over 99 % of the triglycerides according to recoveries of 3H-olein), then hydrolysed by treatment for less than 5 min with sodium hydroxide and oxidised with sodium periodate for 1-2 min at 25°C before final absorbance measurement. Others using the method for serum tri-glycerides are Phuaphairoj and Chindavanig (1975) who extracted like Royer and Ko (1969), saponified for only 1 min, and developed colour for 1 min at 100°C instead of 60°C; and Schirardin and Bauer (1976) who


removed phospholipids on alumina, saponified with potassium hydroxide, and evaluated finally at 405 nm.

Alkoxides have found use as transesterifying agents-. The work of Royer and Ko (1969, 1972), in which sodium methoxide-isopropanol was used, has been mentioned earlier among the procedures with fluorimetric evaluation. Lartillot and Vogel (1970) extracted serum triglycerides with isopropanol-heptane (7 + 4), hydrolysed with sodium methoxide-isopropanol, and measured absorbance at 410 nm. Soloni (1971) extracted serum triglycerides by partition between nonane-isopropanol (4 + 7) and dilute sulphuric acid, and hydrolysed the upper phase with sodium ethoxide-isopropanol for 15 min at 60°C; after 10 min oxidation with sodium periodate-dilute sulphuric acid, the oxidising agent was destroyed with arsenite, the formalde-hyde reacted with acetylacetone-ammonium acetate for 10 min at 60°C, and the dihydrolutidine evaluated at 415 nm. After a similar partition. Rojkin and Repetto (1972) transesterified serum triglycerides with potassium ethoxide-s-butanol. They oxidised the glycerol with periodic acid-0-7N-sulphuric acid and treated the formaldehyde formed with an acetylacetone reagent, 1M in ammonium acetate, 0*038M in sodium arsenite; and 0-005M in manganese(II) ethylene-bis(dithidcarbamate) as catalyst; after 5 min at 55-65°C they measured absorbance at 410nm. In a patent, Fried and Hoeflmayr (1973) suggest using sodium acetylacetonate instead of the diketone itself. After partition between isopropanol-heptane (7 + 4) and 0-08N sulphuric acid, they treated the organic phase with 0-1M sodium methoxide in isopropanol and then oxidised with sodium periodate-2-5 % acetic acid. A solution of sodium acetylacetonate in 15% ammonium acetate was then added, the mixture left for 20 min at 37°C, and the colour compared with standards. Holub (1973) also used nonane-isopropanol (4 + 7) as organic extraction solvent for serum triglycerides before transesterifying with sodium meth-oxide in nonane-isopropanol (1+1), oxidising, and evaluating at 420 nm. Erikson and Biggs (1973) shook for 2 min 0-5 ml of sample with 2 ml of heptane and 4 5 m l of isopropanol-004N-sulphuric acid (7 + 2), mixed 0-2 ml of upper phase with 3 ml of 0 1 5 % sodium methoxide in isopropanol and warmed for 5 min at 60°C. After oxidising with 01 ml of 0-06M periodic acid in 0-88M acetic acid they added 1 ml of 0-073M acetylacetone in ca. 2M ammonium acetate, and, after 15 min at 60°C, measured absorbance at 410 nm. Hoeflmayr and Fried (1974) modified the method of Lartillot and Vogel (1970) to determine serum and liver tissue triglycerides, decomposing with sodium methoxide and finally evaluating absorbance at 415 nm. Kruse-Jarres (1975) used the semi-automatic method with the Auto Analyzer for triglyceride determination, with sodium ethoxide as transesterifying agent and final measurement also at 415nm. Chambon et al. (1975) also applied the technique of Lartillot and Vogel to determine levels of serum

3.1 HYDROLYSIS

glyceride. Demacker et al (1975) extracted like Royer and Ko (1969) and carried out their colorimetric estimation at 420 nm.

Nakamura et al (1973) found high results when using commercial iso-propanol, acetic acid, and acetylacetone, and stressed the importance of purifying these reagents.

4. With 3-Methylbenzothiazolin-2-one hydrazone (MBTH) This reagent was used by Pays et al (1967) and Sawicki et al (1967) to deter-mine formaldehyde derived from periodate oxidation of glycerol and other compounds [see Chapter 1, Section 1.1.14.C.2(f)]. The former authors suggested the application to glycerides also, and quoted a procedure for triolein as test compound. They refluxed it with potassium carbonate in aqueous ethanol for 30 min at 60°C, then cooled and acidified with 6 % acetic acid. After removing the alcohol on a boiling water bath they added dichloro-methane and again refluxed at 60°C for 30 min. Water was then added to the extract to dissolve the glycerol, and the solution was oxidised with periodate, with subsequent destruction of unused reagent with sodium arsenite-potassium carbonate and development of colour with MBTH in hydro-chloric acid in the presence of the oxidising agent ferric chloride.

Malangeau and Pays (1967) applied the procedure to determine glycerides in blood after having adsorbed polar lipids on zeolite. Neeley et al (1972) adapted the principle for use with the AutoAnalyzer, carrying out on-line hydrolysis of triglycerides with potassium hydroxide and measuring absorb-ance at 670 nm, a slightly longer wavelength than that used by the other authors. They removed polar compounds with activated alumina from isopropanol solution.

5. With 4-Amino-3-hydrazino-5-mercapto-l,2,4-triazole Nishimura et al (1973) hydrolysed serum triglycerides with potassium hydroxide for 15 min at 37°C and then oxidised with sodium periodate in acetic acid also for 15 min at 37°C. They then added the title reagent which reacted with the formaldehyde oxidation product to yield 3-mercapto-s-triazolo[4,3-£]-s-tetrazine which they evaluated spectrophotometrically at 550 nm.

H H

SH N H 2 H 2 N

3 . 1 . 2 . Format ion of I sopropy l idene G r o u p s

Mason and Waller (1964) determined fat and oil glycerides by treating with a mixture of dry benzene-dimethoxypropane-methanolic hydrochloric acid

141


(at least 10%) (14 + 1 + 5 ) . Pure triglycerides were left for 30 min at room temperature, fats and oils overnight. This yielded methyl esters of the fatty acids and isopropylidene-glycerol, derived from the action of dimethoxy-propane on glycerol liberated by hydrolysis (Chapter 1, Section 1.3.2.). They determined these products by GLC, mostly on 60-80 mesh Chromosorb W impregnated with 15% of Lac-3R-728, or on 100-110 mesh type A Anakrom with 14-5% poly(ethylene glycol succinate) impregnation. With both columns they used helium carrier gas and various column temperatures.

Mason et al. (1964) used benzene-dimethoxypropane-methanol-2-0N-sodium methoxide in methanol (10 + 4 + 5 + 1 ) for glycerides in oils and fats, allowing it to react for 5 min and then adding a slight excess of methanolic hydrochloric acid and leaving for ca. 50 min. They then carried out GLC of the methyl esters of the fatty acids and the isopropylidene-glycerol using the second type of column mentioned above under the first publication.

3.1.3. Other O x i d a t i o n s

These are less specific than the periodate method.

A. C H R O M I U M ( V I )

Pramme (1931) determined glycerides in grease by hydrolysing, and then oxidising the glycerol with dichromate to carbon dioxide which he absorbed in Ascarite tubes and estimated through weight increase. He hydrolysed 10 g samples by boiling with 75 ml of 10N sulphuric acid. The fatty acids remained dissolved in the mineral oil part of the sample, and the glycerol entered the aqueous layer. This was oxidised by boiling for 10 min with 100 ml of 50% sulphuric acid saturated with potassium dichromate.

B. B R O M I N E

Ohl (1938) detected glycerides in sizing agents on rayon and staple rayon by hydrolysing with alcoholic potassium hydroxide and then treating the liberated glycerol with bromine reagent to yield "glycerose" (see p. 1) which he demonstrated through the colour with resorcinol.

C. P E R M A N G A N A T E

Fat-soluble ester glycerol in lymph was determined by Freeman and Friedemann (1935) in an extract of the lymph by first hydrolysing for 15 min on the boiling water bath with 5N sodium hydroxide. After neutralising the solution by addition of the equivalent amount of 10N sulphuric acid, they cooled the mixture and precipitated proteins and fatty acids with a mercuric chloride-copper sulphate reagent followed by a calcium hydroxide suspen-

3.1 HYDROLYSIS 143

sion. The precipitate was filtered off and aliquots of filtrate were oxidised by heating with alkali and excess 0 0 2 N standard permanganate for 20 min on the boiling water bath, converting the glycerol into oxalic acid and carbon dioxide (Chapter 1, Section 1.1.15). After cooling for 10 min and acidifying with ION sulphuric acid (the oxalic acid being oxidised) they determined unused permanganate by adding potassium iodide and titrating the liberated iodine with 0-02N thiosulphate.

D. SILVER(I )

Bergner and Sperlich (1953) detected fats on paper (Schleicher and Schull 2043a) by spotting, then adding a drop of saturated aqueous potassium

hydroxide and leaving for 2 h or overnight. They then developed chromato-graphically with chloroform-ethanol (7 + 3) and demonstrated the presence of glycerol by spraying with 25% aq. N H 3 - 5 % silver nitrate (11 + 9 ) and heating for 15 min at 100°C to give grey zones.

3.1.4. D e h y d r a t i o n

Glycerol derived from its compounds, such as glycerides, can be dehydrated to acrolein which is detectable or determinable in various ways (Chapter 1, Section 1.4). Most reagents used for dehydration are acidic, and liberate glycerol from its compounds, enabling direct application to these also. Feigl (1937) heated the glyceride sample in an ignition tube with potassium hydrogen sulphate and detected the acrolein through the colour formed with sodium nitroprusside-piperidine or -morpholine (deep gentian blue) or with o-dianisidine in glacial acetic acid (brown-red to yellow) on filter paper in the mouth of the tube (cf. Section 1.4.4). Sanchez(1944) heated glycerophosphate samples with diammonium hydrogen phosphate and potassium hydrogen sulphate which evidently hydrolysed and then converted the acrolein formed into 3-methylpyridine (cf. Section 1.4.5.C). After ring fission with bromine cyanide to glutaconic aldehyde monoenolate, he reacted this with benzidine hydrochloride to give a red product.

In other cases, a separate degradation or hydrolysis stage for the glycerol derivative has been employed. For example, Ohl (1938), whose work was mentioned above (Section 3.1.3.B), also tested for glycerol, derived from alkaline hydrolysis of glycerides, by heating with potassium hydrogen sulphate and detecting acrolein through its odour (Section 1.4.1) or by colour reaction with Schiff reagent (Section 1.4.B). Fiirst (1948) hydrolysed glyceride samples first with alcoholic potassium hydroxide and then heated the liberated glycerol with vitreous phosphoric acid or boric acid in a current of air, detecting acrolein through the lilac colour yielded with 001 %, 2,7-dihydroxynaphthalene in cone, sulphuric acid (cf. Chapter 1, end of Section 1.4.5.E.2).


Mendelsohn and Antonis (1961) separated serum triglycerides from phosphatides by adsorbing the latter on silicic acid. They hydrolysed the evaporated extract for 30 min at 6O-70°C with methanolic potassium hydroxide and acidified with sulphuric acid. After removing the fatty acids with petroleum ether they tested for glycerol hydrolysis product by heating at 140°C with o-aminophenol and 0*6% arsenic acid in sulphuric acid, which yields 8-hydroxyquinoline through the Skraup reaction. On cooling and adding magnesium sulphate and 28% ammonia they observed and evaluated the fluorescence of the magnesium salt derived from the 8-hydroxy-quinoline (cf. end of Section 1.4.5.D).

Bauer-Moll (1960) quote the acrolein test for glyceride detection in fats. The sample is heated in an ignition tube with about twice its mass of potassium hydrogen sulphate, whereby acrolein can be recognised through its characteristic acrid odour, or, alternatively, by passing the issuing vapours over a Schiff reagent prepared from mixing 220 ml of aqueous sulphur dioxide, 3 ml of cone, sulphuric acid, and 30 ml of 0-1 % fuchsin. The initial red product turns indigo blue on heating on the water bath. Their book also contains reference to Rosenthaler's method (1956) for glyceride detection, by heating with zinc chloride (or alone) and testing for acrolein by the lilac colour given with filter paper or glass wool saturated with a hexylresorcinol reagent (cf. Section 1.4.5.E.2).

3.1.5. Ester i f icat ion

Some investigators have determined glycerol derivatives (esters) by hydrolysis and then acetylation to acetate ester which was estimated by GLC.

Hartman (1964) hydrolysed glycerides in the usual way with methanolic or ethanolic potassium hydroxide, removed most alcohol by evaporation in vacuo, then acidified and extracted the fatty acids with chloroform. The aqueous layer was concentrated and refluxed with acetic anhydride for 2 h. The triacetin yielded was ultimately subjected to GLC on a column of 0-25 % silicone high-vacuum grease or 0-5% silicone rubber gum SE-30 on glass beads of 0-177 mm diameter at 207°C using an argon ionisation detector.

Vaver et al. (1972) determined the glycerol moiety in phospholipids in a similar way. They cautiously hydrolysed for 30 min at 40°C with potassium hydroxide-methanol, separating the fatty acid moieties through ion ex-change. The glycerol was then acetylated by heating for 5 h at 150°C with acetic acid-acetic anhydride (3 + 2) in a sealed ampoule. After final extraction and evaporation they carried out GLC on a column of 10% polyethylene glycol succinate) on Chromosorb W using flame ionisation detection.

Tsuji and Konishi (1971) identified polyhydroxy-base compounds in polyurethane polyesters by cleavage for 2-5 h at 130°C with toluene-p-

3.1 HYDROLYSIS 145

sulphonic acid-acetic anhydride (ca. 5 + 6). The acetate ester products were extracted with ether and gas-chromatographed on a stainless steel column with 25% Apiezon L on acid-washed 60-80 mesh Chromosorb W, using helium carrier gas and programming column temperature from 180 to 260°C at 6°/min.

3.1.6. Ether Format ion

Determination of glycerol-containing compounds by release of glycerol, followed by conversion of this glycerol into an ether and its estimation, appears to be very rare. An example is the work of Rayah et al. (1968). They determined triglycerides, also in vegetable oils, by saponifying for 2 h with 6 % methanolic potassium hydroxide and then preparing the trimethylsilyl ether of the glycerol thus formed. This was subjected to GLC using stainless steel columns containing 60-80 mesh Chromosorb W impregnated with 2 % of SE-30, with nitrogen carrier gas and flame ionisation detector. They held the column temperature at 100°C until the glycerol ether emerged, and then raised it at 5°/min to 110°C, maintaining it there until the standard, hexa-decane, emerged.

3.1.7. React ion w i t h H y d r i o d i c A c i d

Zeisel and Fanto (1902), who adapted the Zeisel method to glycerol (see Chapter 1, Section 1.5) by estimating argentometrically the isopropyl iodide derived from it, tested the determination of glycerides directly with the hydriodic acid reagent, functioning also as hydrolysis agent. Their results were, however, always low. Willstatter and Madinaveitia (1912) improved the procedure by using hydriodic acid of density 1-8 instead of the customary 1*7, and also using smaller samples of 0-15-0-35 g. They needed 2-3 h for quantitative results with tristearin and triolein. Later workers were also able to carry out the direct method, e.g. Flaschentrager (1920) with fat samples of ca. 20 m g . Blix (1937) reported a glycerol micro-determination in fats and phosphatides by heating for 3-5 h at 120-135°C, although he used acid of density 1 -7. He employed the Viebock bromine oxidation method to estimate the isopropyl iodide. Bradbury (1951) preferred prior hydrolysis with 50% potassium hydroxide before treatment with hydriodic acid in propionic acid solvent.

3.1.8. Infrared S p e c t r o p h o t o m e t r y

Shay et al. (1954) identified the polyol component in polyesters through the IR spectrum after hydrolysis. They saponified a 10 g sample (in a little benzene


or acetone) with 250 ml of ca. 0-5N potassium hydroxide in absolute ethanol (time and temperature not given), then filtered off the insoluble dipotassium salts of the acids, just acidified the filtrate with cone, hydrochloric acid, and re-filtered to remove potassium chloide. The filtrate was concentrated and extracted with ether. The aqueous layer was ultimately brought to dryness and a thin film of the residue between rock-salt plates was prepared. They recorded the IR spectrum in the range 700-1 500 c m " 1 .

3.1.9. C h r o m a t o g r a p h i c M e t h o d s

Some examples may be given of the application of chromatography after a hydrolysis stage.

For example, Tawn and May (1957) applied PC of polyols (and of dibasic acids) to the analysis of alkyd resins. They saponified the resin sample with a mixture of alcoholic potassium hydroxide and benzene. After removing the acid moieties, they subjected the polyol mixture (including glycerol) to paper chromatography using a mobile phase of n-butanol saturated with 1-5N aqueous ammonium hydroxide and visualised the polyols with alkaline permanganate obtaining yellow spots.

Fijolka et al. (1963, see also Fijolka and Radowitz, 1965) hydrolysed polyesters in the conventional way and analysed the dicarboxylic acid and polyol products with the help of paper chromatography. They were able to separate glycerol and pentaerythritol with pyridine-butanol-water (5 + 2 +2), visualising with ammoniacal silver nitrate or isovanillin-toluene-p-sulphonic acid in ethanol.

Another example of the application of paper chromatography comes from the publication of Kochnova et al. (1969). They studied the composition of terephthalates of polyols, by saponifying with potassium hydroxide, filtering precipitated potassium salts and removing excess potassium hydroxide with carbon dioxide. The resulting polyol mixture was submitted to PC with a solvent of benzene-acetone-water (10 + 1 0 +1). They used also GCL (see below p. 147).

Thin-layer chromatography was used by Seher (1964) for non-ionic surface-active agents which he hydrolysed first with ethanolic potassium hydroxide. He extracted the fatty acids with ether or petroleum ether and finally chro-matographed the residue on kieselguhr G using ethyl acetate-isopropanol-water (65 + 22-7 + 123) for 45 min. Visualisation was effected by the periodate-benzidine method, giving white spots on a blue background, or by spraying with 0* 1 N-silver nitrate-5N-ammonium hydroxide (1 + 1) and heat-ing for 10-20 min at 100-105°C to yield brown zones on a pale background. He was thus able to separate glycerol from di-, tri-, and tetra-glycerols.

3.2 AMINOLYSIS 147

Neissner (1972) also applied TLC after acid hydrolysis of partial esters of many polyols, including glycerol, with palmitic, oleic, or lauric acids. The alcohol components were separated on silica gel with a mobile phase of acetone-water-saturated butanone (1 + 10).

As mentioned above, Kochnova et al (1969) used GLC also to separate the polyols derived from hydrolysis of their esters with terephthalic acid. For this they employed a column of acetylated dextrin-Chromosorb W (1 +3) , at 225°C with helium carrier gas. Tsarfin and Kharchenkova (1975) also determined glycerol (and trimethylolpropane) in complex esters, both as residual monomers and in combination, with the help of GLC. For the latter determination they hydrolysed, and carried out GLC on a combined column of 15 cm length of 15% Apiezon L on AW-DMCS Chromaton N + 35 cm length of 10% l,2,3,4,5,6-hexakis-(2-cyanoethoxy)-hexane on the same support. Their column was at 142°C, carrier gas was argon, and detection was through flame ionisation.

3.2. A M I N O L Y S I S

Aminolysis offers an alternative to hydrolysis of glycerol-containing esters:

RCOOR' + R ' — N H 2 RCONHR" + R — O H

If R" is a moderately large group, the resulting acid amides are poorly soluble in water, which facilitates separation of the water-soluble glycerol (and other polyols).

The principle was originally introduced by Kappelmeier and coworkers for alkyd resins (Kappelmeier et al, 1954; Kappelmeier, 1955) who refluxed for 3 h with 2-phenylethylamine. Kappelmeier and Mostert (1957, 1958) applied it to determine polyols in paint media. They precipitated penta-erythritol from the refluxed mixture by adding chloroform. On cooling, trimethylolethane separated out. The filtrate from this contained trimethylol-propane and glycerol, and they were able to determine the latter via specific oxidation with periodate.

The same aminolytic agent was used by De la Court et al (1969) to deter-mine polyols in alkyd resins. After 1-4 h reflux with 2-phenylethylamine they acetylated the liberated alcohols by refluxing for 2-5 h with acetic anhydride. Finally they subjected the acetate esters thus prepared to GLC on Diatoport S impregnated with Carbowax 20M or silicone rubber, pro-gramming column temperature from 68 to 225°C at 8°/min or from 150 to 290°C at 6°/min, respectively. Helium carrier gas and flame ionisation detection were used.


2-Phenylethylamine is suggested in the IUPAC Sub-Committee's recom-mendation (1973) for analysis of alkyd resins. After 2 h reflux of a 0-5—1 g resin sample with 4 ml of the amine, 25 ml of acetic anhydride are added to the cooled resin and the mixture is heated for 2-5 h. A gas chromatographic stage follows, on 60-80 mesh silanised Chromosorb W with 10% Carbowax 20M, programmed at 8°/min from 70 to 225°C and with helium carrier gas.

Schroder and Thinius (1960) used monoethanolamine, refluxing a 2-5 g sample of alkyd resin with 7-5 ml of amine for 1 h. After separating the amides they determined total polyols by refluxing for 2 h with 20 ml of 0-5N potassium permanganate and 5 ml of 30% potassium hydroxide. They then added 10 ml of 1:1 sulphuric acid, 22 ml of 0-5N oxalic acid, and 10 ml of 5 % manganous sulphate, and finally back-titrated unused oxalic acid with 0-1N permanganate. They determined glycerol by oxidising for 1 h with periodate and titrating the formic acid yielded with standard alkali to Methyl Red indicator. Wicek (1968) obtained "monoethanolamine indices" by heating a 0-2-2 g fat sample with an equal weight of monoethanolamine for 1 h at 110°C and for a further 1 h after the mixture had become homogeneous. He then titrated the colloidal solution with hydrochloric acid to Bromophenol Blue. A control was performed on amine without the sample, and the difference in acid titration, the "index", gave a measure of amide formed and hence of glycerol. He applied the method later to lecithin (1970). Lushchik et al (1971) also used monoethanolamine with alkyd and polyester resins. The C 2 - C 5

polyol products were acetylated with acetic anhydride, and the acetates submitted to gas chromatography on a column of 5 % poly(ethylene glycol adipate) on Chromosorb carrier at 100-220°C, with nitrogen carrier gas and a flame ionisation detector.

Despite its lower boiling point, n-butylamine was used by Esposito and Swann (1961) to detect and characterise polyols in alkyd resins. They refluxed for 1 h, acetylated the liberated polyols by 90 min reflux with acetic anhydride, and extracted the acetate esters into chloroform after having added water. These were chromatographed on 10% Carbowax 20M or 20% silicone rubber on 60-80 mesh Chromosorb W in a copper tube, at 240 or 300°C, respectively, using a thermal conductivity detector. The same authors later (1969) aminolysed alkyd resins, and oils such as linseed and soybean, with n-butylamine in butane-1,4-diol, and subsequently converted the freed polyols into their trimethylsilyl ethers. They chromatographed these on 20% silicone grease (DC-11) on 60-80 mesh Chromosorb W in a copper tube with helium carrier gas and thermal conductivity detection.

Sestrienkova and Singliar (1972) studied the aminolysis procedure for alkyd resins, followed by acetylation, extraction with chloroform, and GLC. They found the order of effectiveness of the amines to be: ethanolamine > 2-phenylethylamine > butylamine.

3.3 REDUCTION 149

Mlejnek and Cveckova (1974) heated polyester samples with 30% aqueous hydrazine hydrate for 1 h at 50°C in sealed ampoules. They subsequently determined the liberated polyols by trimethylsilylation and gas chromato-graphy in an aluminium column with 5 % SE-30 on acid-washed Chromosorb W of 0-20-0-25 mm diameter. They programmed column temperature from 80 to 220°C at 5°/min and employed a dual flame ionisation detector.

3.3. R E D U C T I O N

Reduction of esters with powerful agents such as lithium aluminium hydride yields the alcohol component and alcohols from the fatty acid moiety; for example, with glycerides:

C H 2 0 — C O R C H 2 O H I I

CHO—COR + 1 2 H - * C H O H + 3 R C H 2 O H I I

C H 2 0 — C O R C H 2 O H

Horrocks and co-workers (Horrocks, 1961; Horrocks and Cornwell, 1962; Holla et al, 1964) then acetylated with excess acetic anhydride, claiming quantitative conversion in 1 h. After removing unused anhydride by refluxing with ethanol, they finally carried out GLC of the products derived from glycerol esters. The GLC conditions quoted by Holla et al, for example, were: 10% polyethylene glycol succinate) on 60-80 mesh Gas-Chrom P at 170-200°C using methyl eicosonate as standard.

Schmid et al (1975) reduced ether lipids also with lithium aluminium hydride in diethyl ether, and destroyed any excess reagent with water. The resulting alkyl and alk-l-enyl ethers of glycerol were separated by TLC on silica gel, using hexane-diethyl ether (1 +4) as mobile phase. In this example, the fission is not of the glycerol-ether bond, however. Similar is the work of Hack and Helmy (1975) who reduced glycerophosphatides in human amniotic fluid with "vitride", i.e. sodium dihydrobis-(2-methoxyethoxy)alumi-nate. They separated the alkyl and alkenyl glycerol ether products by chromatography on silicic acid-impregnated Whatman SG-8 paper using diisobutyl ketone-acetic acid-water (60 + 35 + 6), or on Gelman ITLC type silica gel-impregnated glass fibre sheets with a mobile phase of isooctane-isopropyl acetate-isopropanol (50 + 2 + 1). They visualised the glycerol ethers with the periodate-Schiff reagent method.


3.4. T H E R M A L C L E A V A G E

Weigel (1972) determined glycerophosphatides via their glycerol content.

Since these compounds do not release glycerol quantitatively by acid or

alkaline hydrolysis, he carried out thermal cleavage by heating for 10 min at

255-260°C. The glycerol was then determined by oxidation with sodium

periodate in aqueous acetic acid and the reaction of the formaldehyde product

with an acetylacetone-ammonium acetate-isopropanol reagent for 15 min

at 60°C. He evaluated the dihydrolutidine derivative at 412 nm [cf. Chapter 1,

Section 1.1.14.C.2(i); this chapter, Section 3.1.1.B.3].

REFERENCES

Agradi, E., Sirtori, C. and Sisti, G. (1972). Boll. Soc. Ital. Biol. Sper. 48, 128. Bandyopadhyay, C. (1968). J. Chromatogr. 37, 123. Bauer-Moll (1960). "Die Organische Analyse", 4th Ed., Leipzig, p. 470. Bergner, K. G. and Sperlich, H. (1953). Deut. Apoth.-Z. 93,676. Biggs, A. I. (1954). Anal. Chem. 26,602. Blankenhorn, D. H., Rouser, G. and Weimer, T. J. (1961). J. Lipid Res. 2,281. Blix, G. (1937). Mikrochim. Acta 1, 75. Block, W. D. and Jarrett jr., K. J. (1968). Amer. J. Med. Technol. 35, 93. Bradbury, R. B. (1951). Mikrochemie ver. Mikrochim. Acta 38, 114. Buckley, G. E., Cutler, J. M. and Little, J. A. (1966). Can. Med. Assoc. J. 94, 886. Carlson, L. A. and Wadstrom, L. B. (1959). Clin. Chim. Acta 4,197. Chambon, A., Landivier, J., Couvert, J. P. and Fonbonne, R. (1975). Pharm Biol.

9, 177. Chin, H. P., Abd El-Meguid, S. S. and Blankenhorn, D. H. (1971). Clin. Chim. Acta 31,

381. Chlouverakis, C , Hanley, T. and Butterfield, W. J. H. (1963). Guys Hosp. Rept. 112,

193 (Chem. Abs. 59, 13109; Anal. Abs. 11, 3847). Christophe, A. and Matthijs, C. (1964). Bull. Soc. Chim. Beiges 73, 592. Claude, J. R., Corre, F. and Warnet, J. M. (1969). Archs. Mai. Coeur, Revue Athero-

sclerose 11, 16 (Anal. Abs. 19, 497). Cramp, D. G. and Robertson, G. (1968). Anal. Biochem. 25, 246. De la Court, F. H., Van Cassel, N. J. P. and Van der Valk, J. A. M. (1969). Farbe,

Lack IS, 218. Demacker, P. N. M., van Oppenraay, J. B. H. A., Baadenhuijsen, H. and Jansen, A. D.

(1975). Clin. Chim. Acta 64, 45. Dunsbach, F. (1966). Z. Klin. Chem. 4,262. Edwards, L., Falkowski, C , Chilcote, M. E., Hirsch, R. L. and Mather, A. (1972).

Stand. Methods Clin. Chem. 7, 69. Erikson, J. M. and Biggs, H. G. (1973). / . Chem. Educ. 50, 631. Esposito, G. G. and Swann, M. H. (1961). Anal. Chem. 33, 1854. Esposito, G. G. and Swann, M. H. (1969). Anal. Chem. 41, 1118. Feigl, F. (1937). Mikrochim. Acta 1, 127.

REFERENCES 151

Fijolka, P. and Radowitz, W. (1965). Plaste Kautsch. 12, 207. Fijolka, P., Radowitz, W. and Runge, F. (1963) Plaste Kautsch. 10, 521. Flaschentrager, B. (1920). Mikrochemie, Pregl-Festschr. 89. Fletcher, M. J. (1968). Clin. Chim. Acta 22, 393. Foster, L. B. and Dunn, R. T. (1973). Clin. Chem. 19, 338. Franzke, C , Heims, K. O. and Vollgraf, I. (1967) Ndhrung 11, 515. Freeman, S. and Friedmann,T. E. (1935). / . Biol. Chem. 108,471. Fried, R. and Hoeflmayr, J. (1973). German Patent No. 2,139,163, Feb. 15th (Chem.

AbsJS, 133113). Fiirsj, W. (1948). Scient. Pharm. 16, 85. Giegel, J. L., Ham, A. B. and Clema, W. (1975). Clin. Chem. (Winston-Salem, N.C.)

21,1575. Goedicke, W. and Gerike, U. (1972). Mikrochim. Acta 603. Gottfried, S. P. and Rosenberg, B. (1973). Clin. Chem. 19, 1077. Hack, M. H. and Helmy, F. M. (1975). J. Chromatogr. 107, 155. Hanahan, D. J., Dittmer, J. C. and Warashina, E. (1957). / . Biol. Chem. 228, 685. Hartman, L. (1964). J. Chromatogr. 16, 223. Haux, P. and Natelson, S. (1971). Microchem. J. 16, 68. Hoeflmayr, J. and Fried, R. (1974). Arzneimittelforsch. 24, 904. Holla, K. S., Horrocks, L. A. and Cornwell, D. G. (1964). J. Lipid Res. 5, 263. Holub, W. R. (1973). Clin. Chem. 19, 1391. Horrocks, L. A. (1961). Diss. Abs. 9, 2876. Horrocks, L. A. and Cornwell, D. G. (1962). / . Lipid Res. 3, 165. IUPAC Sub-Committee (1973). Pure Appl. Chem. 33, 411. Jover, A. (1963). J. Lipid Res. 4, 228. Kappelmeier, C. P. A. (1955). Fette u. Seifen 57, 229. Kappelmeier, C. P. A. and Mostert, J. (1957). Verjkroniek 30, 48. Kappelmeier, C. P. A. and Mostert, J. (1958). Verjkroniek 31, 61. Kappelmeier, C. P. A., Mostert, J. and Boon, J. F. (1954). Verjkroniek 27, 291. Kawade, M. (1962). Mie Med. J. 11, 399 (Chem. Abs. 58, 742; Anal. Abs. 10, 1529). Kessler, G. and Lederer, H. (1965). Technicon Symp. 2nd N.Y. London 341 (Chem.

Abs. 67, 40942); also Clin. Chem. 11, 809. Kochnova, Z. A., Sorokin, M. F., Grafkin, B. N. and Shabanova, N. P. (1969).

Lasokrasoch. Mater, ikh Primen. 25 (Chem. Abs. 72, 13272). Kruse-Jarres, J. D. (1975). Arztl. Lab. 21, 140. Lambert, M. and Neish, A. C. (1950). Can. J. Res. 28B, 83. Lartillot, S. and Vogel, C. (1970). Feuill. Biol. 11, 39. Lemaur, R. and Le Palec, J. P. (1970). Ann. Pharm. Franc. 28, 257. Lloyd, M. R. and Goldrick, R. B. (1968). Med. J. Austr. 2, 493. Lofland jr., H. B. (1964). Anal. Biochem. 9, 393. Lushchik, V. I., Zlobina, V. R. and Gomazova, V. G. (1971). Lakokrasoch. Mater ikh

Primen. 41 (Chem. Abs. 75, 77584). Malangeau, P. and Pays, M. (1967). Ann. Biol. Clin. (Paris) 25, 854. Mason, M. E. and Waller, G. R. (1964). Anal. Chem. 36, 583. Mason, M. E., Eager, M. E. and Waller, G. R. (1964). Anal. Chem. 36, 587. Matsumiya, K., Arao, M., Nakamura, M., Okishio, T. and Omori, K. (1970). Rinsho

Byori 18, 383 (Chem. Abs. 73, 95313). Medvedev, I. K. and Kalantar, I. L. (1972). Nov. Metody Modij. Biokhim. Fiziol.

Issled. Zhivatnovod. No. 2, 44 (Chem. Abs. 81, 60280). Mendelsohn, D. and Antonis, A. (1961). / . Lipid Res. 2, 45.

152 GLYCEROL C O M P O U N D S

Mendez, J., Franklin, B. and Ganaghan, H. (1975). Clin. Chem. (Winston-Salem, N.C.) 21, 768.

Mill, F., Lawn, G , Phillips, R. and O'Malley, J. A. (1970). / . Amer. Med. Technol. 32, 693.

Mlejnek, O. and Cveckova, L. (1974). / . Chromatogr. 94, 135. Nakamura, M., Matsumiya, K., Okishio, T. and Omori, K. (1973). Rinsho Byori 21,

279 (Chem. Abs. 79, 89043). Neeley, W. E., Goldman, G. E. and Cupas, C. A. (1972). Clin. Chem. 18, 1350. Neissner, R. (1972). Fette, Seifen, Anstrichm. 74, 198. Neri, B. P. and Frings, C. S. (1973). Clin. Chem. 19, 1201. Nishimura, T., Imai, T., Okawa, S. and Tomita, S. (1973). Rinsho Byori 21, 839 (Chem.

Abs. M, 142625). Ohl, F. (1938). Kunstseide u. Zellwolle 20, 230 (Chem. Abs. 32, 6872). den Otter, H. P. (1947). Chem. Weekblad 43, 345. Pays, M., Malangeau, P. and Bourdon, R. (1967). Ann. Pharm. Franc. 25, 29. Phuaphairoj, S. and Chindavanig, S. (1975). J. Med. Assoc. Thailand 58, 547 (Chem.

Abs. 84, 86369). Pramme, M. H. (1931). Ind. Eng. Chem., Anal. Ed. 3, 232. Randrup, A. (1960). Scand. J. Clin, and Lab. Invest. 12, 1. Rayah, A., Subbaram, M. R. and Achaya, K. T. (1968). / . Chromatogr. 38, 35. Renkonen, O. (1962). Biochim. Biophys. Acta 56, 367. Revutskii, E. L., Tsiomik, V. A., Solovtsova, K. M. and Bronshtein, V. N. (1975).

Vrach. Delo 5, 51 (Chem. Abs. 83, 143890). Rojkin, M. L. and Repetto, J. R. (1972). Rev. Asoc. Bioquim. Argent. 37, 177. Rosenthaler, L. (1956). Pharm. Z. 101, 150. Royer, M. E. and Ko, H. (1969). Anal. Biochem. 29, 405. Royer, M. E. and Ko, H. (1972). Biochem. Med. 6, 144. Sanchez, J. A. (1944). Rev. Asoc. Bioquim. Argent. 10, 63. Sardesai, V. M. and Manning, J. A. (1968). Clin. Chem. 14, 156. Sawicki, E., Schumacher, R. and Engel, C. R. (1967). Michrochem. J. 12, 377. Schirardin, H. and Bauer, M. (1976). Feuill. Biol. 17, 63. Schmid, H. H. O., Bandi, P. C. and Kwei Lee Su (1975). / . Chromatogr. Sci. 13, 478. Schroder, E. and Thinius, K. (1960). Deut. Farben-Z. 14, 144, 189. Seher, A. (1964). Fette, Seifen, Anstrichm. 66, 371. Sestrienkova, M. and Singliar, M. (1972). Petrochimia 12, 94. Shay, J. F., Skilling, S. and Stafford, R. W. (1954). Anal. Chem. 26, 652. Smernoff, R. B. (1971). German Patent No. 2,113,762, Oct. 14th (Chem. Abs. 16,11795). Sobotka, J. (1971). Vnitr. Lek. 17, 600 (Chem. Abs. 16, 11726). Soloni, F. G. (1971). Clin. Chem. 17, 529. Sordi, A. (1960). Arch. Studia Fisiopatal. Clin. Ricambio 24, 448 (Chem. Abs. 57,

11473). Stewart, R. D. (1954). Can. J. Biochem. and Physiol. 32, 679. Tawn, A. R. H. and May, G. J. (1957). / . Oil Colour Chem. Ass. 40, 528. Tixier, M., Claude, J., Bessas, D. and Martin, M. J. (1974). Ann. Biol. Clin. (Paris) 32, 53. Torjescu, V., Valeanu, M. and Torjescu, A. (1966). Viata Med. 15, 1201 (Chem. Abs.

70, 103600). Townsend, de W. and Singer, L. (1976). Michrochem. J. 21, 385. Troy, A. and Bell, A. C. (1946). Am. Perfumer 48, 54 (Chem. Abs. 40, 5667). Tsarfin, Ya. A. and Kharchenkova, V. D. (1975). Zh. Anal. Khim. 30, 391. Tsuji, K. and Konishi, K. (1971). Analyst (London) 96, 457.

REFERENCES 153

Van Handel, E. and Ordway, R. (1961). Clin. Chem. 7, 249. Van Handel, E., Zilversmit, D. B. and Bowman, K. (1957). J. Lab. Clin. Med. 50, 152. Vanzetti, G. and Denegri, E. (1964). Giorn. Biochim. 13, 405. Vaver, V. A., Kolesova, N. P. and Tsirenna, M. L. (1972). Khim. Prir. Soedin. 158

(Anal. Abs. 24, 1007). Weigel, W. (1972). Z. Physiol. Chem. 353, 113. Wessels, H. (1973). Fette, Seifen, Anstrichm. 75,478. Wicek, H. B. (1968). Oleagineux 23, 113; also (1970). ibid. 25, 25, 473, 537. Willstatter, R. and Madinaveitia, A. (1912). Ber. 45, 2825. Zeisel, S. and Fanto, Z. (1902). Z.f. Landw. Versuchsw. Osterreich 5, 729.

4

Glycerol Compounds: Methods based on Probable

Participation of the Complete Molecules of the Sample

Most of the methods given under this heading are for lipids or phospho-lipids, comprising triglycerides, glycerophosphatides, cholesterol and its esters, as well as sugar glycerides and further compounds containing com-bined glycerol but no free glycerol hydroxyl groups. Such a participation must necessarily include that of the glycerol moiety, which therefore affords some justification for inclusion here. Methods of determination include nephelometry, differential thermal analysis, viscometry, and mass spectro-metry, but the great majority of quoted procedures are of separation based on solvent extraction and, in particular, on chromatography. The ubiquity and importance of these last-named procedures demanded some discussion here, although it must be admitted that, in many cases, a relation to the glycerol moiety is slender!

4 . 1 . N E P H E L O M E T R Y

Stone and Thorp (1966) measured the light scattering intensity (LSI) of sera, using a micronephelometer and red light of wavelength 680 nm. They fractionated the diluted sera suspensions by filtering through cellulose ester membranes of different pore diameters (01 and 0-45 pm) and measured the LSI values after filtration. Among other results they found a correlation

155

156 GLYCEROL C O M P O U N D S 4.2

between the LSI and serum triglyceride levels in fasting subjects. In attempts to estimate triglyceride concentrations in serum and plasma samples, this principle was applied by subsequent investigators, e.g. Hollender et al. (1970), Buckley et al. (1970), and Hori et al. (1970). Helman et al. (1971) used filters of 0-45 and 0-05 um diameter and compared the results for triglyceride estimation with those from the Van Handel method (Chapter 3, Section 3.1.1.B.2). Baldi and Scuccimarra (1973) and Trappe et al. (1975) compared their values from the nephelometric method of Stone and Thorp with those using an enzymic procedure. Ruys et al. (1975) filtered serum samples through a membrane (450 nm), reading the nephelometric values after 10 min; they confirmed a correlation between LSI and triglyceride concentration in serum samples, although Baer (1974) reported technical difficulties in carrying out the nephelometric determination. The method can probably be regarded as nothing more than rough and empirical. Dean (1974) com-pared a nephelometric and an enzymic method for triglycerides.

4.2. C R I T I C A L S O L U T I O N T E M P E R A T U R E

Measurements of critical solution temperature can often be used to analyse binary mixtures when a suitable liquid is known. No examples of the appli-cation of this principle to binary glyceride mixtures have been found but the work of Maeda (1966) may be quoted. He measured the lower critical solution temperatures of mixtures of water and ethers of glycerol with poly-ethylene glycol) and with poly(propylene glycol):

CH 2 0 (CH 2 — C H R — 0 ) n — H

C H O ( C H 2 — C H R — 0 ) n — H R = H or C H 3

CH 2 0 (CH 2 — C H R — 0 ) n — H

He found a relationship between concentration and the critical solution temperature at various molecular weight levels. For example at the molecular weight level of 3000, a critical solution temperature of 7°C was found for a concentration of 18*2%, and a temperature of 34°C for a concentration of 55-8 %. The work does not appear to have had an analytical aim but it would evidently be possible to analyse aqueous solutions in this way.

Schmid et al. (1966) characterised glycerol ethers (alkyl, dialkyl, and tri-alkyl), 1-alkoxy-diglycerides and 1,2-dialkoxy-glycerides through their critical solution temperatures with nitromethane and with acetonitrile.

4.5 INFRARED SPECTROPHOTOMETRY 157

4.3. D I F F E R E N T I A L T H E R M A L A N A L Y S I S

This method is applied rather to investigate the melting and polymorphic properties of fats, but a more analytical example may be given. Golborn (1969) heated lard samples from - 4 0 to + 100°C at 8°/min and recorded the thermogram. This showed melting peaks due to the component triglycerides. Golborn quoted a list of the melting points of 15 triglycerides, ranging from + 5 to + 50°C, and was able to correlate the melting peaks with certain of these values of particular components.

4.4. M A S S S P E C T R O M E T R Y

Hites (1970) described the quantitative analysis of triglyceride mixtures by mass spectrometry. The sample of, for example, olive arachis, or maize oil was sprayed directly into the ion source. He measured the molecular ions ( M + ) and the (M — 18) + ions over a m/e range of 700-900.

Most mass spectrometric studies have attempted identification of the fatty acid moieties. Although it does not fall within the scope of this book, two examples of this modern method can justifiably be quoted. Klein (197.1) recorded MS of the diacyl-glycerylphosphorylcholines containing different fatty acid groups (dipalmitoyl, dioleoyl, oleoylstearoyl) at 250°C and 70 eV; the differences in fragmentation pattern could serve for identification. Wood (1974) used field desorption MS for analysing intact phospholipids, e.g. dipalmitoyl-L-a-lecithin. The method was used to distinguish this from the dimyristoyl and distearoyl analogues.

4.5. I N F R A R E D S P E C T R O P H O T O M E T R Y

Infrared measurements have not been used much for qualitative or quanti-tative analytical purposes with glycerol derivatives. Most work appears to have been performed on nitroglycerine and is, in any case, evidently based on absorption bands associated with the nitrate group and therefore remote from qualification for inclusion here. A summary is given nevertheless of some infrared determinations of nitroglycerine and also of triacetin as a propellant component in explosives.

Pristera (1953) determined these two compounds among propellant com-ponents, employing the 601 p (1665 c m - 1 ) band for the nitrate ester in di-chloroethane solution and the 7-75 p (1290 cm" 1 ) band in chloroform for it in the presence of triacetin, dinitrotoluene, and ethyl centralite. He estimated


the triacetin in this solvent at 5 7 2 ( 1 7 6 5 c m " 1 ) . Castelli et al. (1954) also used the 1660 and 1760 c m " 1 maxima to determine nitroglycerine and tri-acetin. Tippett (1963) examined nitroglycerine explosives in the infrared, obtaining characteristic spectra in methanol and chloroform solutions. He too carried out quantitative work at 1270 and 1650 c m " 1 , using a solution of the sample in chloroform. Sinha et al. (1970) extracted propellants with ether, evaporated the solvent, and took up the residue in chloroform; they estimated nitroglycerine through its absorption at 7-75 (1290 cm" 1 ) .

Macke (1968) separated nitroglycerine and its stabilisers and decomposi-tion products by TLC, then scraped off the zones and finally measured IR absorption in dichloroethane solution, his chosen wavenumbers being 1660 for nitroglycerine and 1745 for triacetin.

Pizzoli et al. (1967) determined lecithin and triglycerides in soya beans and eggs after fractionation with ethanol, using IR measurements on carbon tetrachloride solutions at 8-6 and 9-35 ji (1160 and 1070 cm" 1 ) . They used the equations:

,4(8-6 u) = 0-653T + 0-465L

,4(9-35 u) = 0-1487 + 0-749L

where T and L are the concentrations of triglyceride and lecithin.

4.6. S O L V E N T E X T R A C T I O N

Many of the compound classes listed at the beginning of Chapter 2, loosely referred to as lipids, occur in the animal and plant samples which are those principally encountered. Solvent extraction has been used for many years to separate these lipids from other, mostly highly polar, material such as sugars, proteins, amino-acids, and salts. This separation, a form of clean-up, can then be followed by separation of the different lipid classes, e.g. by chromato-graphy, or by detection or determination of a particular class by a specific method. Interest here is of course confimed to the glycerol-containing compounds.

Many solvents and solvent mixtures have been tried. Some procedures are evidently direct extraction but others are partition procedures. The dis-tinction is not always sharp because water in the sample can probably change an intended extraction into a partition procedure.

A detailed discussion of solvent extraction would occupy far more space than can be allotted here but a few remarks may be permitted.

The standard mixture for extracting lipids in general from biological material is chloroform-methanol (2 -I- 1). In the so-called Folch procedure

4.6 SOLVENT EXTRACTION 159

(Folch et al, 1957) this extract is freed from many contaminants by shaking with a salt solution, yielding two layers, the lower of which consists of about 86% chloroform and 1 % water. It contains the purified lipid mixture.

Examples of the use of this extraction medium are legion. At random can be mentioned its use to extract glycerides from serum by Carlson and Wadstrom (1959), Jover (1963), Young and Eastman (1963), and Torjescu et al (1968), who also extracted phospholipids and cholesterol; to extract tissue by Hack and Ferrans (1959) (for plasmalogens) and by Abdel-Latif and Chang (1966), Gione and Orning (1966), Klemig and Lempert (1970), Heyneman et al (1972), and Vandamme et al (1975) (for phospholipids).

Some investigators have used a chloroform-methanol mixture of different composition. Examples are given in Table 4.1.

TABLE 4.1

Extraction with Chloroform-Methanol Mixtures

Proportion, Sample Extracted Reference chloroform + methanol

32 + 14 Blood plasma Triglycerides

+ 5 + 1 + 1 + 1 + 6 4- 7

Serum Blood serum Rat liver Serum Blood, faeces Plant tissue

Lipids Lipids Lipids Lipids Lipids Sulphoquinovosyl diglycerides; mono-and di-galactosyl diglycerides

Medvedev and Kalantar (1972) Egge et al (1970) Baryshkov(1966) Katyal et al (1969) Chedid et al (1972) Amenta (1970) Roughan and Batt (1968)

Other solvents and mixtures have also found application, and Table 4.2 summarises some examples.

TABLE 4.2

Extraction of Lipids with Other Solvents

Solvent Sample Extracted Reference

Chloroform Serum Triglycerides Altmann et al (1967) Kasum oil Glycerides Kundu (1969)

Triglycerides Sobotka (1971) Alkyd Oligomeric adipates Loehnert and Schoellner resins of trimethylolpropane (1968)

and glycerol


Solvent Sample Extracted Reference

Chloroform-abs. ethanol

Ethanol-ether (1 + 3 )

Ethanol-ether (3 + 1)

Ethanol-ether

Ethanol-acetone

5 % Ethanol in diisopropyl ether

Isopropanol

Isopropanol-heptane (7 + 4)

Isopropanol-nonane (7 + 4)

Serum Lipids

Serum Lipids

Blood Triglycerides serum

Total Triglycerides and free lipids fatty acids

Blood Glycerides

Plasma Triglycerides

Serum Triglycerides Triglycerides

Triglycerides and cholesterol

Blood plasma, serum Neutral Triglycerides lipids Serum Triglycerides Biological Triglycerides samples

Serum Triglycerides (claimed free of phospholipids)

Serum, Triglycerides liver tissue

Plasma Glycerides

Serum Triglycerides Serum Triglycerides

Wildgrube and Erb (1971)

Wildgrube et al. (1969)

Young and Eastman (1963)

Fosbrooke and Tamir (1968)

Blankenhorn et al. (1961)

Laurell(1966)

Matsumiya et al. (1970) Lemaur and Le Palec (1970) Smernoff(1971)

Edwards et al. (1972)

Nishimura et al. (1973) Miyashita et al. (1974)

Lartillot and Vogel (1970)

Hoeflmayr and Fried (1974)

Royer and Ko(1969)

Holub(1973) Yamada and Miyazaki (1975)

Zuckerman and Natelson (1948) extracted triglycerides with chloroform

in the presence of IN sulphuric acid, claiming that soluble substances such as

glucose were thus wholly removed in the acid layer. Haux and Natelson

(1971) used this in a micro-determination of serum glycerides. Dilute sulphuric

acid has been used also in combination with isopropanol and an alkane,

whereby triglycerides are extracted into the upper layer. Thus Soloni (1971),

Rojkin et al. (1973), and Sone et al (1973) treated serum samples with dilute


4.6 SOLVENT EXTRACTION 161

sulphuric acid and nonane-isopropanol (4 + 7). Fried and Hoeflmayr (1973) used heptane instead of nonane, treating 0*2 ml of blood serum with 0*4 ml of 0-08N sulphuric acid and extracting with 2-2 ml of isopropanol-heptane (7 + 4), ultimately saponifying the triglycerides in a 0*05 ml aliquot of the upper layer. In another example of the use of heptane, Erikson and Biggs (1973) added to 0-5 ml of sample 2 ml of heptane and 4-5 ml of iso-propanol-004N-sulphuric acid (7 + 2), shook for 2 min, and then used a 0*2 ml aliquot of the upper layer for triglyceride estimation. Giegel et al. (1975) studied the partition of serum triglycerides in the system water-isopropanol-nonane and found that over 99 % was extracted into the nonane-containing phase, using 3 H-olein as test substance.

Another example of a system alcohol-water-alkane is the extraction of serum glycerides (also cholesterol and its esters) by Galanos et al. (1964) with a mixture of petroleum ether and 87% ethanol (3 + 1).

Mention must be made of the frequently employed separation of the polar phospholipids from lipid extracts by means of adsorption on active agents. This may be done in order to remove them during a determination of glyce-rides, or in order to determine them after subsequent desorption. Probably silicic acid is the most popular substrate, and among those who used this adsorption agent are: Nicolaysen and Nygaard (1963); Ignatowska (1964); Laurell (1966); Galletti (1967); Royer and Ko (1969); Montet et al. (1970); Sobotka (1971); and Medvedev and Kalantar (1972). Zeolite was used by Altmann et al. (1967), Lemaur and Le Palec (1970), and Edwards et al. (1972). Those employing alumina include Smernoff (1971) and Miyashita et al. (1974), and Florisil was the adsorption agent of Ryan and Rasho (1967) and Royer and Ko (1969).

Various solvents have been used to extract nitroglycerine (glycerol tri-nitrate) from samples containing it as an explosive or as a pharmaceutical. Marvillet (1958) extracted the trinitrate from propellants with dichloro-methane which was also used by Trowell (1970) to extract glycerol nitrates from aged double base propellants. He used also diethyl ether for this, a solvent likewise employed to determine glycerol trinitrate in propellant blocks or cakes through the loss in weight caused by this extraction (Anony-mous, 1958). Christos and Spinetti (1973) used Halon 2330 (trichlorotri-fluoroethane) to extract glycerol trinitrate and ethylene glycol dinitrate from dynamite explosive oils.

Schwartzman (1956) mixed powdered nitroglycerine tablets with water and extracted the ester with carbon disulphide (ready for IR estimation); Soeterboek and Van Thiel (1973) preferred extraction with glacial acetic acid for glycerol trinitrate in tablets. This compound (also ethylene glycol dinitrate) in blood was extracted from blood samples with hexane after having added sodium chloride, by Williams et al. (1966), whereas Alley and


Dykes (1972b) extracted nitroglycerine from pharmaceutical preparations using 1,2-dichloroethane. Bogaert and Rosseel (1972) and Rosseel and Bogaert (1973) extracted nitroglycerine from blood plasma using ethyl acetate before subjecting to GLC.

4.7. C H R O M A T O G R A P H Y

4 . 7 . 1 . Gas C h r o m a t o g r a p h y

Examples of gas chromatography applied directly to glycerol-containing compounds, i.e. without prior degradation or modification of the molecule, are evidently restricted almost entirely to glycerol esters of nitric acid and fatty acids. Much of the work carried out on triglycerides has been devoted to separations based on the number of carbon atoms, degree of unsaturation, or another structural feature related to the fatty acid moiety, and therefore falling outside the intended scope of this book. However, as in other places, the importance of the gas chromatographic method justifies quotation here of at least some work and this is summarised in Table 4.3.

TABLE 4.3

Gas Chromatography of Glycerol-containing Compounds


Triglycerides; also oils, lard, etc.

Natural triglycerides

Fully esterified polyol nitrates

Triglycerides

Stainless steel columns; SE-30 (ca. 3 % after Huebner pretreatment) on 100-120 mesh Gas-Chrom (1961) P; ca. 120 to 370°C depending on sample; He gas; thermal conductivity detector

Stainless steel columns; 2-25 % SE-30 on Kuksis and 60-80 mesh Chromosorb W; 200 to 325°C McCarthy at 3°/min; N 2 gas; FID (No evidence of (1962) fragmentation even at the highest temp.)

Stainless steel columns; 10% ethylene Camera and glycol succinate on 40-60 mesh acid- Pravisani washed Celite C22ak; 145 or 150°C; He (1964) gas; hot wire detector

Studied factors for quantitative GLC. Litchfield et al. Efficient was 3 % JRX or SE-30 on 100-120 (1965) mesh Gas-Chrom Q in glass or stainless steel tubes; 170 to 325°C at 2°/min (steel) or 4°/min (glass), depending on sample; He better than N 2



Many nitrate esters and nitro compounds

Nitroglycerine, chloroglyceryl dinitrate

Traces of glycerol trinitrate and ethylene glycol dinitrate in blood

Triglycerides

Triglycerides

Triglycerides

Triglycerides

Triglycerides in cocoa butter

Alkyl polynitrates (application to air pollution)

Triglycerides, also natural fats and oils (lard, beef tallow, whale oil, butter, margarine, cheese)

10% Silicone grease E301 on Celite C22ak; Camera et al. two columns, one at 150°C, one at 180°C; (1965) He gas (Decomposition limits general use)

Aluminium column; 3 % SE-30 on 50-60 Fossel (1965) mesh Anakrom 4B 15; 130 and 100°C column respectively

Hexane extract submitted to GLC on 10 % Williams et al. silicone elastomer on Embacel; 140°C; N 2 (1966) gas; electron-capture detector

Aluminium or stainless steel column; 5 % Kuksis and JXR or SE-30 on 60-80 mesh silanised Ludwig (1966) Chromosorb W; programmed from 150 to 350°C; H 2 - F I D

4 % Silicone SE-52 on 60-80 mesh H D M S Lefort et al. Chromosorb; 260 to 340°C at l ^ ° / m i n ; (1966) N 2 gas; FID. Trapped fractions subjected to IR, TLC, and GLC on 15 % SE 52 on same support at 320°C

2 % JXR (methylsilicone) on 60-80 mesh Sato et al. Shimalite W; 200 to 325°C at 4°/min; N 2 (1966) g a s ; H 2 - F I D

Stainless steel tubes; 3 % JXR on 100-200 Kuksis and mesh silanised Gas-Chrom Q; 5% SE-30 Breckenridge on 60-80 mesh Chromosorb W; 3-2 % JXR (1966) or SE-30 on Chromosorb W treated with dichlorodimethylsilane; from 200 to 350°C; N 2 gas; FID

5 % SE-30 on Chromosorb W; 250 to Avancini et al. 3 3 0 ° C ; N 2 ; F I D (1966)

10% Igepal CO-880 on siliconised Camera and Chromosorb P; 160°C (for nitroglycerine); Pravisani N 2 gas; electron capture detector (1967)

Tested JXR, OV-1, OV-17, and Versamid Sato et al. (Versamid 900 best for thermal stability and (1967) separation). Natural fats on glass column; 2 % OV-17 on 80-100 mesh Shimalite W; 200 to 320°C; H 2 - F I D



Lipid extracts (with chloroform-methanol)

Triglycerides after prior column chromatography

Triglycerides

Triglycerides in milk products

Triglycerides from rat adipose tissue

Triglycerides; also in fats and oils, e.g. coconut and palm oil

Triglycerides in coconut oil

Natural triglycerides, also labelled with

Adipose tissue triglycerides from various animal species

Nitrate esters of glycerol, isosorbide, and isomannide

Nitrate esters etc. in nitrocellulose base propellants

Plasma nitroglycerine

Stainless steel column; 3 % JXR or 1 % Kuksis et al. OV-17 on 100-200 mesh Gas-Chrom Q; (1967) 100 to 325°C at 4~6°/min; N 2 gas; FID, peaks confirmed by TLC

3 % JXR on Gas-Chrom P; 200 or 320°C Nickell and (according to chain length); He gas Privett (1967)

Stainless steel columns; 3 % QF-1 on AW- Watts and Dils DMCS Chromosorb W, or 10 % SE-30 on (1968) Gas-Chrom Q; isothermally at temps, from 140 to 330°, or 110 to 330°C at 2-6°/min; N 2

gas; FID

Glass columns; OV-17 on 80-100 mesh Matsui et al. Shimalite W; 200 to 300°C at 4°/min; N 2 (1969) gas; FID

1 % JXR on 100-120 mesh Gas-Chrom Q; Bezard and 250 to 360°C at 3°/min; N 2 gas Bugaut (1969)

3 % JXR on Gas-Chrom Q; 175 to 210°C Carracedo and at 2-6°/min; N 2 gas; FID Prieto (1969)

Stainless steel column; 1 % JXR on 100-120 Bugaut and mesh Gas-Chrom Q; 280 to 380°C at Bezard (1970) 3°/min; N 2 gas; FID

Glass column with 3 % JXR on 100-120 Breckenridge mesh Gas-Chrom Q; dual H 2 - F I D and Kuksis

(1970)

Glass column; 15 % polyethylene glycol Boyne and adipate) or 7% Apiezon L on 85-100 mesh Duncan (1970) AW-Celite 545 at 180 or 185°C, resp.; Ar gas; 9 0 S r detector

Glass column; 3 % XE-60 or 3-5 % QF-1 Rosseel and on-£0=80 mesh Gas-Chrom Q; 110, 120, Bogaert (1972) 150°C; N 2 gas; tritium electron-capture detector or FID

3-8 % OV-101 or 11 % OV-225 on Gas- Alley and Chrom Q; or 2-5% OV-201 on Dykes (1972a) Chromosorb W-HP; 70 to 220°C at 6°/min;He gas; FID

Extract in ethyl acetate submitted to GLC Bogaert and as in method of Rosseel and Bogaert (1972) Rosseel (1972)



Nitroglycerine in pharm. preparations

Triglycerides in rat adipose tissue (prior column and TLC)

Triglycerides (also hydrocarbons and cholesterol esters)

Nitroglycerine and isosorbide dinitrate in blood plasma

Fish lipid triglycerides (e.g. carp and trout oil)

Triglycerides of rat adipose tissue after TLC fractionation

Triglycerides, e.g. in coconut and arachis oils

Triglycerides

Triglycerides (study of detection of adulteration of cottonseed oil)

Nitrate esters etc. in nitrocellulose base propellants

Triglycerides

Extract in dichloroethane; stainless steel columns; 3 % UCW-98 on 80-100 mesh Gas-Chrom Q; 70°C for 1 min, then to 130°C at 15°/min and kept there until standard (diethyl phthalate) eluted; He gas; dual FID

Stainless steel column; JXR on Gas-Chrom Q; 240 to 340°C at 3°/min; N 2 gas

Tested carborane-siloxane copolymers, Dexsil 300 GC; up to 350°C; in glass capillaries; FID

Extract in ethyl acetate; glass columns; 3-5 % QF-1 on 60-80 mesh Gas-Chrom Q; 120°C; N 2 gas; tritium electron capture detector

Glass columns; OV-17 on 80-100 mesh Shimalite W; 280-340°C

JXR on 100-120 mesh Gas-Chrom Q; 240 to 350°C at 3°/min

3 % SE-30 on 100-120 mesh AW-DMCS Chromosorb G; 200 to 360°C at 2°/min; N 2 gas; FID

1 % OV-1 on 80-100 mesh Chromosorb W; 200 to 330°C at 4°/min, then held at 330°; He gas; MS study of fatty acid moieties

1 % OV-17 on AW-DMCS 80-100 mesh Chromosorb W; 200 to 300°C at 10°/min; N 2 gas; FID

3-8% OV-101 or 1-1 % OV-225 on silylated Gas-Chrom Q, or 2-5 % OV-210 on silylated Chromosorb W(HP); He gas; FID

Stainless steel columns; 3 % JXR on 100-120 mesh Gas-Chrom Q; 150 to 300°C at 4°/min; N 2 gas; FID

Alley and Dykes (1972b)

Bezard and Bugaut(1972)

Novotny et al. (1972)

Rosseel and Bogaert (1973)

Matsui et al. (1973)

Bugaut and Bezard (1973)

Eckert(1973)

Murata and Takahashi (1973)

Imai et al. (1974)

Dykes and Alley (1974)

Parodi(1975)

G



Moderately polar compounds, e.g. tri-glycerides, esters of cholesterol

Tested polyphenyl ether sulphone as liquid Schwartz et al. phase at 200 to 400°C. Triglycerides on (1975) 1 % Poly S-179 on 100-120 mesh Gas-Chrom Q; e.g. 250 to 375°C at 10°/min or, for coconut oil, 250 to 350°C at 6°/min; FID

Kuksis has written reviews on the application of gas chromatography to neutral glycerides (1967), to lipids (1971), and to glycerophosphatides, glycolipids, and sphingolipids (1973). Two reviews are the work of Viswana-than: on gas chromatography coupled with mass spectrometry in separation and characterisation of polar lipids (including glycerophosphatides) (1974a); and on gas and thin-layer chromatography of alkoxy-lipids (1974b). Shehata and De Man (1971) reviewed the use of adsorption and partition chromato-graphy and gas-liquid chromatography in separation and quantitative ana-lysis of triglycerides, and Isoda (1973) also published a review on the appli-cation of gas chromatography to glycerides.

4.7.2. Paper Chromatography

Table 4.4 contains some summarised data on paper chromatographic separations of glycerol-containing compounds. Two features stand out: (1) many separations of triglycerides have been carried out with reverse-phase PC, on paper impregnated with non-polar materials such as higher alkanes or silicone oils; (2) mixtures of phosphatides or phospholipids in general have been separated on paper impregnated with silica in some form.

To save space, visualisation details are not given in the table. These are dealt with separately in Section 4.8.

TABLE 4.4

Paper Chromatography of Glycerol-containing Compounds


Phosphatidyl-choline, Acetone-methanol (4+1) , then at right Amelung and -ethanolamine, and angles with water-saturated phenol. Serine Bohm (1954) -serine compound prevented from migration by

Cu(II).




Phospholipids (e.g. phosphatidyl-choline, -ethanol-amine, lysolecithin)

1 - and 2-Glycero-phosphates

Glass-fibre paper, impregnated with silicic Dieckert and acid; methanol-diethyl ether (1 + 1 ) Rieser (1956)

Phosphatides

1 - and 2-Glycero-phosphates

Plasmalogens

Synthetic and natural glycerides

Glycerides, e.g. in soya oil

Phosphatides (after removal of tri-glycerides and cholesterol)

Fatty acid esters of alcohols, including glycerol

Whatman No. 1, treated with EDTA or 1 % oxine (to reduce tailing); n-propanol-conc. N H 4 O H - w a t e r (6 + 3 + 1), propanol-acetic acid-water (8 + 1 +1) , or isopropanol-pyridine-water-ethyl acetate (2 + 1 + 1 + 2 ) ; also best with nitromethane-pyridine-water (5 + 6 + 4), then on untreated paper

Paper impregnated with silicic acid; e.g. diisobutyl ketone-acetic acid-water (40 + 30 + 7) and dibutyl ether-acetic acid-chloroform-water (40 + 35 + 6 + 5); also 2-dimensional, first with chloroform-methanol (3 + 1 ) containing 2 % water, or with the second solvent above, then with first solvent

Alumina-impregnated paper; descending, methano l -NH 4 OH (60 + 5)

Paper impregnated with sodium silicate; diisobutyl ketone-acetic acid-water (40 + 23 + 3); at 2° to prevent hydrolysis by the acetic acid

Schleicher and Schiill paper 2040b or 2043b impregnated with undecane or solid paraffin; various mobile phases, e.g. acetic acid, 90 % acetic acid, and mixtures of isopropanol, water, and acetic acid

Paper impregnated with silicone oil; acetone-acetonitrile

Formaldehyde-treated paper; n-butanol-acetic acid-water ( 4 + 1 + 5) or this mixture-diethyl ether (20 + 5)

Dierick et al (1956)

Marinetti and Stolz(1956)

Urakami and Kakutani (1957)

Hack and Ferrans(1959)

Kaufmann and Makus(1959)

Kaufmann and Schnurbusch (1959)

H or hammer et al (1959)

Paper impregnated with silicone oil and Kaufmann and using acetone-water (85 + 15); or Grothues impregnated with undecane and using acetic (1961) acid-water (97 + 3)



Triglycerides Paper impregnated with silicone oil; Hirayama two-stage technique with acetone-acetic (1961) acid ( 1 + 4 )

Unsaturated tri- Whatman No. 3 paper impregnated with Michalec glycerides of various 0-5 % liquid paraffin in diethyl ether; et al. (1961) fats and oils acetic acid-chloroform-liquid paraffin

(16 + 13 + 1)

Phosphatides Paper impregnated with silicic acid; Hack and 2,6-dimethylheptan-4-one with methanol- Leatherman water (100 + 25 + 4), with acetic acid- (1961) water (25 + 10 + 2), or with pyridine-water ( 2 0 + 1 5 + 2)

Phosphatidyl-choline, Glass fibre paper impregnated with silicic Cornatzer -ethanolamine,-serine, acid; diisobutyl ketone-acetic acid-water- et al. sphingomyelin, etc. benzene (160 + 50 + 8 + 7) (1962)

Phosphatidyl-choline Whatman No. 1 paper impregnated with Zn Collier (1962) and -ethanolamine salt; dibutyl ether-propionic acid (2 + 1),

satd. with ZnCl 2

Whatman No. 1 paper; propanol-conc. Zajac (1962) N H 4 O H - w a t e r (7 + 2 + 1), then at right angles with propanol-formic acid-water (7 + 1 + 2) or phenol-water (4 + 1), all ascending; or second stage descending with propanol-formic acid-water-pyridine (35 + 5 + 1 0 + 1 ) giving best results

Explosives, including Completely acetylated paper; butanol- Krien (1963) nitroglycerine acetic acid at 20°C

Glycerides Paper impregnated with gasoline; Trippel (1964) acetonitrile-propionitrile

Phospholipids Whatman No. 3 saturated with 40% Letters (1964) HCHO-acetic acid (20 + 1); butanol-acetic acid-water ( 4 + 1 + 5 ) ; then in second dimension on Schleicher and Schull No. 289 impregnated with silicic acid (first sheet clamped on to second); diisobutyl ketone-acetic acid-water (80 + 50 + 7)

Triglycerides Glass fibre paper; pyridine-water ( 4 + 1 ) Swartout and Gross (1964)

Phosphatidyl-serine, -ethanolamine, and -threonine



Phosphatidyl-serine, -choline, -ethanol-amine, etc.

Triolein (and oleic acid labelled with 1 3 1 J )

Serum triglycerides

Triglycerides (and methyl esters of unsaturated acids)

Blood serum lipids

Glycerides

Phospholipids

Schleicher and Schull No. 2043b impregnated with silicic acid; diisobutyl ketone-pyridine-water (100 + 74 + 11)

Whatman 3MM soaked in 5% C u S 0 4 , dried, then in 5 % N a O H ; chloroform-benzene (2 + 1 ) , ascending

Glass fibre paper; isooctane-isopropyl acetate (100 + 1 to 3)

Paper impregnated with dodecane; 70-100% methanol saturated with A g N 0 3 and dodecane

Paper impregnated with silica; diisobutyl ketone-heptane (4 + 96) or diethyl ether-benzene (5 + 95)

Whatman No. 1 paper impregnated with liquid paraffin; acetone-methanol (4 + 1)

Whatman No. 3 paper impregnated with silicic acid; ascending, chloroform-methanol-ethanol-water (60 + 8 + 2 + 1 )

Monoglycerides (from Paper impregnated with octyl acetate; glycerolysis of oils) 75 % methanol saturated with A g N 0 3

and octyl acetate

Phospholipids (e.g. phosphatidyl-serine, -ethanolamine, -choline)

Phospholipids, extracted from canine body fluids

Phosphatides in sugar beet

Paper treated with silica gel; diisobutyl ketone-acetic acid-water (40 + 30 + 7)

Paper pretreated with sodium silicate and washed with HC1 and water; dibutyl ether-acetic acid-chloroform-water (80 + 70 + 12 + 11)

Silica gel paper; 2-dimensional PC with THF-diisobutyl ketone-water-conc. N H 4 O H (90 + 5 + 4 + 2), then diisobutyl ketone-isobutyl methyl ketone-butanone-chloroform-98 % formic acid-acetic acid-water (10 + 8 + 8 + 100 + 10 + 10 + 1)

Scrignar (1964)

Anghileri (1964)

Pinter et al. (1964)

Vereshchagin (1965)

Stajner (1966)


Mezesova (1967)

Novitskaya and Vereshchagin (1969)

Kilroe-Smith (1969)

Karagezyan (1969)

Beiss(1969)


4.7.3. T h i n Layer C h r o m a t o g r a p h y

There are many applications of thin layer chromatography to the two main problems mentioned in the introductory words to this chapter, namely, the separation of biological lipids into the principal classes, and the separation from one another of individual triglycerides or phospholipids. Tables below summarise some of the publications.

An attempt has been made to distinguish between work devoted primarily to triglyceride separation (Table 4.5) and that concerned with the phospho-lipids (Table 4.6). The boundary is diffuse, however.

It will be noted that some publications on triglycerides discuss separation according to the number of carbon atoms or degree of unsaturation (then using layers impregnated with a silver salt). These are features pertaining to the fatty acid moiety, and therefore there are comparatively few such examples given here. Work on phospholipids is included only where glycerol-con-taining examples (such as phosphatidyl-choline, -serine, -ethanolamine, -inositol, -glycerol, or cardiolipins) are involved.

In general, the samples subjected to TLC were obtained by prior treat-ment, mostly solvent extraction (see Section 4.6).

Table 4.7 summarises thin layer chromatographic procedures for other glycerol-containing compounds of non-biological origin, which in practice means glycerol nitrates.

As in Table 4.4, the visualisation procedures are not included in the tables, in the interest of saving space; exceptions are made only for quanti-tative methods. The visualisation methods are discussed separately in Section 4.8.

TABLE 4.5

Thin Layer Chromatography of Triglycerides and Other Neutral Lipids


Synthetic and natural triglyceride mixtures

Triglycerides and cholesterol esters

Kieselguhr G impregnated with 5 % tetradecane in ether; acetone-acetonitrile (4 + 1), saturated to 80% with the tetradecane. Also with silicone oil impregnation, then with methanol-acetonitrile-propionitrile (10 + 8 + 3)

Silicic acid-gypsum, impregnated with 0-5 % paraffin oil in ether; acetic acid

Oils (characterisation) Silica gel + 15 % C a S 0 4 ; chloroform-including glycerides) benzene (7 + 3)

Kaufmann et al (1961)

Michalec et al (1962)

Crump (1962)



Triglycerides

Glycerides

Triglycerides, free fatty acids

Triglycerides

Serum triglycerides

Triglycerides from fats and oils

Triglycerides

Glycerides

Silica gel impregnated with 7-5 % petroleum fraction of b.p. 24O-250°C; acetone-acetonitrile (4 + 1), satd. with the impregnant. Also tetradecane impregnation

Silica gel G - A g N 0 3 (4 + 1); 0-5 % acetic acid in chloroform

Gypsum impregnated with un- or tetra-decane; acetone-acetonitrile ( 4 + 1 ) for triglycerides

Silica g e l - A g N 0 3 ; CCl 4 -chloroform-acetic acid (120 + 80 + 1) plus 0-4% ethanol

Silica gel G (containing Rhodamine B); diethyl ether-acetic acid-hexane (25 + 2 + 73)

Silica gel G - A g N 0 3 ; benzene-ether (4 + 1); fractions further separated on kieselguhr impregnated with liquid paraffin; acetone-acetonitrile (4 + 1), 80% saturated with the impregnant; tetradecane instead of paraffin with satd. triglycerides

Silica gel containing A g N 0 3 ; benzene

Kieselguhr impregnated with 8 % paraffin oil in gasoline; acetone-acetic acid (7 + 3)

Glycerides in olive oil Method of Kwapniewski and Sliwiok (1964)

Triglycerides

Serum lipids (fatty acids^ cholesterol and esters, triglycerides)

Silica gel G with partial A g N 0 3 content; benzene-ether (8 + 2), then at right angles with ether until all zones in area free from A g N 0 3 , and finally developed with benzene-pet. ether (7 + 3)

Silica gel; hexane-ether-acetic acid (80 + 20 + 1 '5li triglycerides determined by extraction with hexane-ether (1 + 1), saponification, and glycerol estimation through periodate oxidation

Kaufmann and Das (1962)

Barrett et al (1962)

Kaufmann and Khoe(1962)

Barrett et al (1963)

Krell and Hashim(1963)

Kaufmann and Wessels(1964)

Jurriens (1964)

Kwapniewski and Sliwiok (1964)

Sliwiok and Kwapniewski (1965)

Kaufmann and Mukherjee (1965)

Whitner et al (1965)



Triglycerides (and free fatty acids) in plasma

Natural glycerides, e.g. of groundnut, sesame, cottonseed, safflower, linseed, and mustard oils

Glycerides

Glycerides, fatty acids, alcohols, and other esters

Silica gel G; light petroleum (30-60°)-ether-acetic acid (82 + 18 + 1); charred zones reproduced, cut out, and weighed in quant, method

Kieselguhr impregnated with liquid paraffin; acetone-methanol (4 + 1 or 7 + 4), saturated with the paraffin; developed twice or more

Silica gel G containing A g N 0 3 ; benzene; zones extracted with ether and ultimately weighed

Silica gel with 15 % C a S 0 4 ; chloroform-acetic acid (99 + 1) for separating the different classes

Triglycerides and fatty Silanised silica gel G containing A g N 0 3

acid methyl esters

Triglycerides

Triglycerides in fat

Triglycerides in plasma

Triglycerides and free fatty acids

Triglycerides

Sugar glycerides (and sucrose esters)

water-acetonitrile-ethanol-acetone (8 + 2 + 18 + 72) satd. with A g N 0 3 , also 7 + 2 + 8 + 83

Silica gel G - A g N 0 3 ; chloroform-acetic acid (199 + 1); ultimate quantitative determination by ether extraction, hydrolysis, and periodate oxidation of the glycerol

Silica gel containing 12-5 % A g N 0 3 ; chloroform

Silica gel G; heptane-ether (4 + 1)

Silica gel G containing 001 % Rhodamine 6G; hexane-ether-acetic acid (80 + 20 + 1)

Silicic acid impregnated with 8 % hexadecane; nitroethane satd. with the hexadecane

Silica gel; isopropanol-cyclohexane (8 + 92 to 10 + 90), also chloroform-methanol-acetic acid-water (80 + 10 + 8 + 2). Position isomers with chloroform-methanol-acetic acid (85 + 5 + 10). Glycerides with same components but 9 4 + 4 — 3

Schlierf and Wood (1965)


Amat et al. (1966)

Rao and Sreenivasan (1966)

Ord and Bamford(1967)

Bandyopadhyay (1968)

Persmark and Toregard (1968)

Laurell(1968)

Fosbrooke and Tamir (1968)

Litchfield (1968)

Ranny (1968)



Glycerides of kusum (kusam) oil

Triglycerol esters of cyclopentane fatty acids

Triglycerides

Triglycerides

Faecal triglycerides

Triglycerides from liver biopsy

Neutral fat in serum

Triglycerides, free fatty acids, cholesterol and its esters

Triglycerides

Neutral lipids

Triglycerides of rat adipose tissue

Triglycerides, free fatty acids cholesterol, and esters

Silica gel G; n-hexane-ether-acetic acid Kundu (1969) (75 + 25 + 1); isolated triglycerides separated then on silica gel G impregnated with A g N 0 3 , using chloroform-acetic acid (100 + 0-2)

Kieselguhr G impregnated with 5 % paraffin Bandi and oil in hexane; acetone-acetonitrile Mangold (7 + 3) (1969)

Silica gel G - A g N 0 3 ; benzene or benzene- Wessels and ether (95 + 5, 80 + 20, 70 + 30) or Rajagopal chloroform containing 0-8 % methanol. (1969) Also on kieselguhr G impregnated with 7-5 % liquid paraffin, then acetone-acetonitrile (8 + 2), 80% satd. with the paraffin

Silica gel G - A g N 0 3 ; benzene-cyclohexene Burns et al. or cyclooctene (cis) (1969)

Silica gel G; light petroleum-acetic acid- Thompson ether (89 + 1 + 10) et al. (1969)

Silica gel G; light petroleum (60-70°) Jaross and -ether-acetic acid (80 + 20 + 1) Freimuth

(1970)

Silica gel; heptane-butanone-acetic Dabels (1970) acid (85 + 15 + 1)

Silica gel G; hexane-ether-acetic Vioque et al. acid (49 + 1 + 0, 9 + 1 + 0, 88 + 12 + 1, (1970) 50 + 50 + 1)

Rubberised filter paper; acetone-acetic acid Vereshchagin satd. with hydrocarbons of solar oil (1971)

Silica gel G containing Rhodamine 6G; Roch and chloroform-acetic acid (24 + 1), then light Grossberg petroleum (3O-60°)-ether-acetic acid (1971) (90 + 10 + 1)

Silica gel G with 5 % A g N 0 3 ; hexane- Bezard and ether-methanol-acetic acid Bugaut (1972) (158 + 40 + 2 + 1)

Silica gel G; light petroleum containing Romans and 1 % acetic acid and either 12-5 or 37 % ether Palmer (1972)



Triglycerides and free fatty acids in blood (esterified with diazomethane)

Triglycerides (saturated and unsaturated)

Acetates of glycerol and diethylene glycol

Triglycerides

Acetodiacylglycerols in milk fat lipids

Serum triglycerides

Triglycerides

Triglycerides

Glycerides

Silica gel; hexane-ether (9 + 1) Vioque et al. (1973)

Silica g e l - A g N 0 3 , then reversed phase on Wessels (1973) kieselguhr G-liquid paraffin, using acetone-acetonitrile (4 + 1), satd. to 80% with the paraffin

Silica gel; toluene-acetone-methanol- Constan-acetic acid (14 + 5 + 1 + 0 * 3 ) tinescu and

Enache(1974)

Microcrystalline cellulose with Haworth incorporated transition metal salts, e.g. et al. (1975) of Ag, Fe, Co, Cd, Al, Zn, and Hg. CdCl 2 and ZnCl 2 gave good separations in some solvent systems (7 tried)

Silica gel G; hexane-ethyl acetate Parodi (1975) (22 + 3)

Silica gel sheet (Merck 5711); chloroform Mantel et al. added on top of the serum sample; sheet (1975) dried in cold air; developed with pet. ether (6O~80°)-ether-acetic acid (90 + 1 0 + 1 )

Silica gel G - A g N 0 3 in "sandwich Utrilla et al. chamber", using benzene (1976)

Silica gel G - A g N 0 3 , in open vessel; Chobanov light petroleum-acetone (25 + 1) et al. (1976)

Silufol UV-254 bonded with starch; Coupek light petroleum (40-60°)-ether-acetone et al. (1976) (80 + 19 + 1) for less polar, (45 + 50 + 5) for more polar compounds

TABLE 4.6

Thin Layer Chromatography of Phospholipids


Plasma

Lipid components

Silica gel; chloroform-methanol-water Habermann ( 6 5 + 25 + 4) etal. (1961)

Silica gel containing dichlorofluorescein; Brown and hexane-ether-acetic acid-methanol Johnston (90 + 20 + 2 + 3) (1962)



Phospholipids

Phosphatidyl compounds

Serum fractions

Liver phospholipids

Brain phospholipids

Serum


Phosphatides

Lipids

Phosphatidyl compounds, etc.

Extracts of egg yolk, blood serum, tissue

Sardines

Phospholipids

Silicic a c i d - C a S 0 4 (50 + 1); chloroform- Vogel et al. methanol-water (80 + 25 + 1) (1962)

Silica gel G slurried with water or 0-01M Skipski et al. Na acetate or carbonate; chloroform- (1962) methanol-acetic acid-water (65 + 25 + 8 + 4)

Alumina without binder; pet. ether-ether Vacikova et al. (95 + 5) (1962)

Silica gel without C a S 0 4 binder; Skipski et al. chloroform-methanol-acetic acid-water (1963) (50 + 25 + 7 + 3)

Silica gel G; chloroform-methanol- Horrocks N H 4 O H ( 6 5 + 25 + 4) (1963)

Silica gel G; chloroform-methanol-water Robinson and (65 + 25 + 4) Phillips (1963)

Silica gel, without gypsum binder and Skipski et al. slurried with N a 2 C 0 3 ; chloroform- (1964) methanol-acetic acid-water (25 + 15 + 4 + 2)

Silica gel G; diisobutyl ketone-formic acid- Thiele and water ( 4 0 + 1 5 + 2) Wober (1964)

Silica gel G (10 % moisture); methanol- Crider et al. acetic acid-water (8 + 1 + 1) for the (1964) phospholipids

Silica gel G, slurried with 0 0 1 M N a 2 C 0 3 ; Abramson and 2-dimensional, chloroform-methanol- Blecher (1964) acetic acid-water (250 + 74 + 19 + 3), then chloroform-methanol-7M N H 4 O H (230 + 90 + 15)

Silicic a c i d - C a S 0 4 ; chloroform-methanol- Musil (1965) acetic acid-water (60 + 8 + 2 + 1)

Silica gel G; chloroform-methanol-water (65 + 25 + 4)

El-Nockrashy and Mahfouz (1965)

Silica gel H or G, or silica gel H - M g silicate Angelelli (9 + 1); 1-dimensional, chloroform-methanol-water (65 + 25 + 4) or chloroform-methanol-acetic acid-water (65 + 25 + 8 + 4); 2-dimensional (for polar lipids), first solvent mixture, then butanol-acetic acid-water (3 + 1 + 1 )

et al. (1966)



Plasma phospholipids Silica gel G; chloroform-methanol-water (65 + 25 + 4)

Clinical separation of phosphatidyl-ethanolamine from urine

Phospholipid classes

Phospholipids

Brain phospholipids, also with 3 2 P

Many lipid classes

Serum

Lipids (phosphatidyl-ethanolamine as model)

Lipids, including phospholipids

Human hair from previous TLC separation

Liver tissue

Lipid classes

Lipid classes from blood serum

Plant sulpholipid and galactolipids (after initial column chromatography)

Gione and Orning(1966)

Dittmann (1966)

Cellulose; propanol-water (3 + 2) containing tris-HCl, then at right angles with isopropanol-water (7 + 3)

Silica gel-Mg silicate (9 + 1), 2-dimensional Rouser et al. with chloroform-methanol-water (1966) (65 + 25 + 4) and butanol-acetic acid-water (3 + 1 + 1), and with chloroform-methanol -28%NH 3 (13 + 7 + 1) and chloroform-acetone-methanol-acetic acid-water (10 + 4 + 2 + 2 + 1) Silica gel G or H; light petroleum- Kaufmann ether-acetic acid (80 + 20 + 3) or et al. (1966) chloroform-methanol-water (65 + 25 + 4)

Silica gel G; chloroform-methanol-acetic Abdel-Latif acid-water (25 + 15 + 4 + 2) and then further and Chang with butanol-pyridine-water (9 + 1 + 4) (1966)

Silica gel G; ether-benzene-ethanol-acetic Freeman and acid (40 + 50 + 2 + 0-2), then in same West (1966) direction ether-hexane (6 + 94)

Silica gel G; hexane-ether-acetic acid Chiarioni et al. (80 + 20 + 1) (1966)

Silica gel G; chloroform-methanol-water Neskovic (14 + 6 + 1) (1967)

Silica gel; chloroform-methanol-water Guimaraes (65 + 25 + 4) (1967)

Silica gel G; chloroform-methanol-6N Singh and N H 4 O H (65 + 30 + 5), and then (30 + 65 + 5) Gershbein at right angles (1967)

Kieselguhr G; pet. ether-ether-acetic acid Holczabek (90 + 10 + 1) (1967)

Silica gel; 2-stage development with Skipski et al. diisopropyl ether-acetic acid (24 +1), then (1968) pet. ether-ether-acetic acid (90 + 1 0 + 1 )

Silica gel D O ; chloroform, then hexane- Van Gent chloroform (3 + 1 ) in same direction (1968)

Silica gel G or H; chloroform-methanol- Roughan and acetic acid-water (85 + 1 5 + 1 0 + 3) (for Batt (1968) purification)



Phospholipids

Various rat tissues

Rat liver

Phosphatides of human placenta during pregnancy

Lipid classes in amniotic fluid and serum

Phospholipids of insects

Lipid classes

Lipids of rat plasma and tissue

Fats (phosphatidyl-choline and -ethanolamine)

A l u m i n a - C s S 0 4 (9 + 1); chloroform- Venkata Rao methanol-water-pyridine-aq. N H 3 et al. (1968) (130 + 554-8-1-4-1-4) and, in 2-dimensional work, then chloroform-methanol-water (30 + 20 + 3)

Silica gel G; 88 % liquefied phenol-water- Kennan et al. 28 % N H 3 (83 + 16 + 1); lecithin and (1968) sphingomyelin separated by further TLC using chloroform-methanol-28%NH 3

(14 + 6 + 1)

Silica gel H-Florisil (9 + 1); chloroform- Neskovic and methanol-30%NH 3 -water(140 + 50 + 7 + 3), Kostic (1968) then in same direction after drying, using chloroform-methanol-acetic acid-water (320 + 40 + 8 + 3)

M N Kieselgel N-HR/DC; first chloroform- Delruck et al. methanol-water (65 + 25 + 4), then (1968) chloroform-methanol-acetic acid (65 + 25 + 8) at right angles

Silica gel G; ether-benzene-ethanol-acetic Biezinski et al. acid (200 + 250 + 1 0 +1) , then dried and (1968) continued with light petroleum-ether-acetic acid ( 9 0 + 1 0 + 1)

Silica gel with glass fibre support; chloro- Yurkiewicz form to separate neutral lipids (moved with (1968) front) from phospholipids (remained at origin); dried, then further TLC with isobutyl methyl ketone-acetic acid-water (50 + 2 0 + 1 )

Silica gel G; chloroform-methanol-N H 4 O H - w a t e r (150 + 60 + 8 +1) . Silica gel H; chloroform-methanol-water-acetic acid (50 + 30 + 4 + 8)

Silica gel G; hexane-ether-acetic acid (60 + 4 0 + 1 , 90 + 10 + 1, and 30 + 70 + 1) to separate neutral lipids; phospholipids fractionated with chloroform-methanol-7M N H 4 O H ( 1 1 5 + 75 + 7-5)

Silica gel G; chloroform-methanol-water Kaufmann (65 + 25 + 4) and Mukherjee

(1969)

Nutter and Privett (1968)

Misra(1968)



Serum lipids

Erythrocytes

Serum lipids

Blood

Lipids

Phospholipids

Lipids of rat liver

Rat brain lipids

Phospholipids

Phospholipids

Silica gel; chloroform-methanol-formic acid-water (65 + 35 + 2 + 4), then n-hexane-ether-acetic acid (160 + 40 + 3) in same direction for phospholipids

Silica gel HR; chloroform-methanol-acetic acid-water (30 + 1 5 + 4 + 2)

Silica gel G; chloroform-methanol-water-acetic acid (65 + 45 + 8 + 1)

Silica gel containing 1 % alkaline Mg silicate or 2 % Mg hydroxyapatite; chloroform-methanol-7M N H 4 O H (90 + 54 + 1), then at right angles with chloroform-methanol-acetic acid-water (40 + 20 + 6 + 1)

Silica gel HR; chloroform-methanol-water (13 + 6 + 1); phospholipid zones slurried with chloroform-methanol (5 + 1) and applied to second layer, using mobile phase chloroform-methanol-acetic acid-water (80 + 40 + 5 + 7)

Silica gel CH + 10% C a S 0 4 ; chloroform-methanol-water (65 + 25 + 4)

Silica gel G; hexane-ether-acetic acid (60 + 40 +1) , then (90 + 1 0 + 1 ) to separate neutral lipids; phospholipids on N a 2 C 0 3 -impregnated silica gel; chloroform-methano l -NH 4 OH solvents

Silica gel; e.g. chloroform-methanol-2 5 % N H 3 (14 + 6 + 1), then at right angles with chloroform-methanol-acetic acid-water (250 + 74 + 1 9 + 3); also single system of chloroform-methanol-25 % N H 3

(70 + 30 + 5)

Silica gel G treated with borate buffer pH 8; chloroform-methanol-water (65 + 25 + 4); or 2-dimensional, chloroform-methanol-7N N H 3 (12 + 7 + 1 and 7 + 12 + 1) Silica gel H - B a C 0 3 (5 + 3); ether-ethanol-water ( 1 0 + 1 0 + 1 ) , then at 90° with isobutanol. Also silica gel G; chloroform-methanol-benzylamine-water


Brockmann and Gercken (1969)

Williams et al. (1969)

Broekhuyse (1969)

lacono and Ishikawa (1969)

Kahovcova and Odavic (1969)

Katyal et al. (1969)

Roozemond (1969)

Bunn et al. (1969)

Srivastava and Rastogi(1969)



Plasma

Phospholipids

Yeast

Malarial parasites

Lipid classes

Amenta (1970)

Neskovic et al (1970)

(20 + 1 8 + 1 + 2), chloroform-methanol-isobutylamine-water (15 + 14 + 1+2) , and chloroform-ethanol-methanol-dipropyl-amine-water (50 + 1 6 + 1 8 + 5 + 4); and on alumina, using chloroform-methanol-acetic acid-water ( 1 1 + 7 + 6 + 1)

Silica gel H-silica gel G (77 + 9); Kunz and chloroform-butanol-propanol-ethanol- Kosin (1970) methanol-acetic acid (22 + 26 + 2-5 + 5-5 + 9-4 + 10-5) containing many alkali halides and other salts in very small amounts, claimed to give more effective separation (into 9 main fractions)

Silica gel G; hexane-ether-acetic acid (65 + 25 + 1 or 9 0 + 1 0 + 1)

Silica gel H-Florisil (9 + 1); pyridine-acetone-chloroform-20 %NH 3 -water (30 + 1 5 + 10 + 1 +1) , then in same direction with chloroform-acetone-methanol-acetic acid-water (130 + 70 + 22 + 8 + 3) (first separates glycolipids from phospholipids, then latter resolved

Adsorbosil-5 (a silica gel); THF-methanol Adams and (3 + 1), then in same direction with Sallee (1970) chloroform-methanol-4M N H 4 O H (75 + 37 + 7)

Silica gel G - C a S 0 4 (3 + 1); chloroform- Klemig and methanol-acetic acid-water Lempert (150 + 50 + 2 + 5) or chloroform-methanol- (1970) water (60 + 20 + 1) in first direction, then with chloroform-methanol-28%NH 3

(15 + 5 + 1) at right angles

Silica gel H or H-HR; chloroform- Getz et al methanol-acetic acid-water (52 + 20 + 7 + 3) (1970) then at right angles with chloroform-methanol-40 % C H 3 N H 2 - w a t e r (13 + 7 + 1 + 1)

Silica gel; chloroform-methanol-water De Zeeuw (55 + 40 + 5), "vapour programmed" from et al (1970) 52% to saturation in 10 min at 23°C

Silica gel G; pet. ether-ether-acetic acid Nicolosi et al (78 + 20 + 2), then turned 180° and run in (1971) the reverse direction with chloroform-methanol-acetic acid-water (65 + 20 + 6 + 4)

Lipids in blood and faeces

Glycolipids in animal lipid mixtures

Serum



Epidermal lipids

Human phospholipids

Lipids (neutral, phospho-)

Serum lipids

Tissue

Bacterial lipids

Mammalian lipids

Lipids

Lecithin in serum lipids

Phospholipids, also with 3 H and 1 4 C

Horse erythrocyte plasma

Silica gel G, slurried with 0-01M N a 2 C 0 3 ; Lee and chloroform-methanol-water (13 + 6 + 1 ) Mezei (1971)

Silica gel; 2-dimensional, chloroform- Rastogi et al. methanol-water (65 + 25 + 4) and butanol- (1971) acetic acid-water (3 + 1 + 1 ) or with chloroform-methanol-7N N H 3 ( 1 2 + 7 + 1 a n d 7 + 12 + l)

Silica gel G; chloroform-methanol- Nardi et al N H 4 O H (14 + 6 + 1 ) (1971)

Silica gel G; with 4 developments, turning Pollack et al 90° after each; chloroform-ethanol- (1971) trimethyl borate ( 1 0 0 + 1 + 6 ) , benzene-ethyl acetate-trimethyl borate (125 + 25 + 9), heptane-benzene (3 + 2), and heptane alone

Silica gel; pet. ether-ether-acetic acid Torres et al (85 + 15 + 2), separating into classes (1971)

Silica gel H or HR; chloroform-methanol- Blond et al acetic acid-water (50 + 30 +11 + 5) (1971)

Silica gel P F 2 5 4 , slurried with 0-2 % Na Minnikin and acetate; chloroform-methanol-water Abdol-(65 + 25 + 4) or chloroform-acetic acid- rahimzadeh methanol-water (80 + 18 + 12 + 5) (1971)

Glass fibre paper impregnated with silica Pocock et al. gel G; isooctane-benzene-acetic acid- (1972) acetone (1000 + 300 + 1 + 3)

Silica gel KSK, slurried with 10-15 % Svetashev and gypsum; 2-dimensional, chloroform- Vas'kovskii methanol -28%NH 3 (13 + 7 + 1), then (1972) chloroform-acetone-methanol-acetic acid-water ( 1 0 + 4 + 2 + 2 + 1 )

Silica gel paper; chloroform-methanol- Stott (1972) water (65 + 2 5 + 4 ) Silica gel; chloroform-methanol-NH 4 OH Chapman (140 + 50 + 7), then at right angles with (1972) chloroform-methanol-acetone-acetic acid-water ( 1 0 + 2 + 4 + 2 + 1 )

Silica gel; chloroform-methanol-water Webb and (9 + 9 + 2) Mettrick

(1972)

Silica gel; chloroform-methanol-water Heyneman (60 + 25 + 4) etal{\912)



Phospholipids

Lipid classes

Neutral lipid classes, e.g. in plasma

Lipids

Lipids

Rat and pork liver lipids

Lipids of Tetra-hymena pyriformis

Serum

Neutral and phospholipids

Sphingomyelin/ lecithin ratio in amniotic fluid

Mlekusch et al (1973)

Hubmann (1973)

Silica gel G; light petroleum-ether ( 1 4 - 1 ) , Ilinov and then at right angles with chloroform- Kharizanova methanol-water (65 + 25 + 4) (1972)

Silica gel F 2 5 4 ; light petroleum-ether- Gasbarro acetic acid (170 + 30 + 1 ) (1972)

Silica gel G-silica gel H (ca. 1+10) ; Kunz (1973) opposite edge of plate first developed for 40 s with hexane, then normal development with ether-benzene-heptane-light petroleum (b.p. below 40°) (22 + 20 + 1 4 + 1 9 , containing small amounts of lower alcohols and fatty acids)

Silica gel G containing ammonium sulphate; ether-benzene-ethanol-acetic acid (200 + 250 + 10 + 1), dried, and then light petroleum-ether-acetic acid (90 + 10+1)

Silica gel; chloroform-methanol-water (65 + 25+4) , then further with n-hexane-ether-acetic acid (85 + 20 + 2) until solvent front at the top edge. Turned 90° and developed with latter mixture. Silica gel scraped away on one side and further developed with n-butanol-acetic acid-water (6 + 2 + 2)

Silica gel (Eastman No. 6061); benzene- French and hexane (15 + 85) to move cholesterol esters Andersen away, then hexane-ether-acetic acid (1973) (69 + 29 + 2)

Silica gel F 2 5 4 ; chloroform-methanol- Viswanathan cone. N H 4 O H (13 + 7 + 1) (1973)

Silica gel H; chloroform-methanol-formic Wildgrube acid-water (65 + 35 + 2 + 4), then further et al. (1973) with hexane-ether-formic acid (160 + 40 + 3)

Silica gel impregnated with Na silicate; Althaus and successively, chloroform-methanol-water- Neuhoff acetic acid (65 + 35 + 3-6 + 2), same mixture (1973) + acetone (60 + 25 + 5-3 + 1 0 + 5), and finally hexane-ether-acetic acid (70 + 3 0 + 1 )

ITLC Type SG (Gelman); dichloro- Blass et al. methane-ethanol-water (34 + 8 + 1) (1973, 1974)



Amniotic fluid

Sphingomyelin/ lecithin ratio in amniotic fluid

Sphingomyelin, lysolecithin in phospholipids

Amniotic fluid

Lipids

Lipid classes (6)

Lipids

Biological lipids with 1 4 C

Lipid classes

Phospholipids

Glycerophosphatide and derivatives

Lecithin/sphingo-myelin in amniotic fluid

Lecithin/sphingo-myelin

Silica gel; chloroform-methanol-25 % N H 3 Verder and (14 + 6 + 1) Clausen (1974)

Silica gel; chloroform-methanol-25 % N H 3 Verhoeven and (65 + 30 + 4) Merkus(1974)

Silica gel G; chloroform-methanol-conc. Hoffman et al N H 3 (65 + 35 + 8) (1974)

Silica gel; chloroform-methanol-water Casu et al (70 + 22 + 3) (1974)

Silica gel; methanol (3 cm), then isooctane- Segura and ether-acetic acid (75 + 25 + 2) Gotto (1974)

Silica gel G; ether-acetic acid-pet. ether Hojnacki and (100 + 3 + 97) (separated phospholipids, Smith (1974) sterols, and free fatty acids)

Silica gel G or H; chloroform-methanol- Teichman water (65 + 2 5 + 4 ) et al (1974)

Silica gel G, lowest 3 cm being from silica Chabard et al gel G slurried with 0-4N N a O H ; (1976) successively ether-hexane-benzene ( 1 1 + 6 + 3), hexane-benzene (4 + 1), and ether-benzene-acetic acid (31 + 1 0 + 9); bands for different glyceride classes, cholesterol and esters, fatty acids, and phospholipids

Silica gel G; heptane-pet. ether-ether- Kabara and acetic acid (60 + 20 + 20 + 1) Chen (1976)

Silica gel G; chloroform-methanol-acetic Ishida-acid-water (25 + 1 5 + 4 + 1 ) for lecithin and Ichimasa et al cholesterol, and chloroform-methanol- (1976) water (65 + 25 + 4) for phosphatidyl-inositol and -glycerol

Silica gel G with boric acid impregnation; Poorthuis chloroform-methanol-water-conc. N H 4 O H et al (1976) (70 + 30 + 3 + 2), then at right angles, chloroform-methanol-water (13 + 7 + 1)

Silica gel; dichloromethane-ethanol-water Sass et al (103 + 24 + 3) (1976)

Gelman ITLC-SG sheet; dichloromethane- Cusick (1976) ethanol-water (35 + 8 + 1 ) or chloroform-methano l -NH 4 OH (170 + 20 + 3) in a "sandwich" chamber

TABLE 4.7

Thin Layer Chromatography of Nitrate Esters of Glycerol and Other Polyols


Aromatic and aliphatic nitrates, nitro compounds, amines, and their derivatives

Propellants

Double-base propellants, including nitroglycerine

Nitroglycerine, resorcinol in double-base propellant

Some nitro compounds and nitrate esters

Propellants

Aromatic nitro compounds, aliphatic nitrates

Propellants

Explosive nitrate

esters

Nitrate esters of 5 alcohols

Nitroglycerine and related compounds

Nitroglycerine and its stabilisers (e.g. triacetin)

Nitrate esters of glycerol and pentaerythritol (cardiovascular drugs)

Silicic acid-starch; trichloroethylene-acetone (8 + 2)

Silica gel-gypsum (85 + 15); benzene-pet. ether (1 + 1)

Silica gel G; benzene

Silica gel; benzene-ether (4 + 1 )

Silica gel G; benzene-pet. ether (40-60°}-methanol (40 + 30 + 5 or 40 + 50 +10)

MN-300 acetylcellulose; butanol-acetic

acid-water (4 + 1 + 1 )

Silica gel G; pet. ether-ethyl acetate

Silica gel; pet. ether-diisopropyl ether (7 + 3)

Mg silicate and alumina; toluene, chlorobenzene, xylene, and pet. ether-dichloroethane (4 + 1)

Silica gel G slurried with water or N a O H ; CC1 4, CHC1 3 , CH 2 C1 2 , ether, ether-CCl 4

(1 + 1), CCl 4 -acetone ( 4 + 1 ) , or C C 1 4 -ethyl acetate (2 + 1)

Silica gel G; benzene

Silica gel; benzene-ethyl acetate (17 + 3)

Silufol UV-254; ether-CCl 4 (5 + 1)

Prat and Forestier (1963)

Sinha et al (1964)

Del Campo (1965)

Kohlbeck (1965)

Boehm(1966)

Del Campo (1966)

Boehm(1967)

Ripper (1967)

Parihar et al (1967)

Barnes (1967)

Lloyd (1967)

Macke(1968)

Schwaisch and Beyrich(1970)



Dynamite residues Silica gel; xylene-hexane (3 + 2) Kempe and Tannert (1972)

Smokeless powders Silica gel G F 2 5 4 ; benzene, benzene-pet. Archer (1975) ether (40-60°)-ethyl acetate (12 + 12 + 1), benzene-pet. ether (1 + 1), benzene-chloroform (1 +1) , or chloroform

4.7.4. C o l u m n C h r o m a t o g r a p h y

Classical column chromatography and newer techniques, such as high

performance liquid chromatography and gel-permeation chromatography,

have found considerable use for separating neutral lipid classes and also

individuals within a class. Many methods are based on gradient elution.

In Table 4.8 are collected together some miscellaneous examples of rela-

tively recent application to biological samples and also to nitro-glycerine

and alkyd resins.

TABLE 4.8

Column Chromatography of Glycerol-containing Compounds


Nitroglycerine with nitrocellulose and other explosive components

Propellant explosives (ether-soluble part)

Phospholipid classes

Serum lipids

Serum lipids

Rabbit skin lipids

Silicic acid-Celite 535 (4 + 1); dichloro- Schroeder methane-ligroin (1948)

Silica gel-Celite; mostly light petroleum Ovenston (40-50°) + benzene or ether (1949)

Silica; chloroform-methanol (4 + 1,3 + 2, Hanahan et al. and 1 + 4 in succession); 5 fractions (1957)

Silicic acid-Celite (2 + 1); successively Freeman et al. chloroform-hexane (1 +19), chloroform (1957) methanol

Silicic acid-Celite; successively, dichloro- Nelson and methane, acetone, dichloromethane- Freeman methanol (65 + 35), giving 2 fractions, and (1959) 95 % methanol

Silicic acid; graded from chloroform- Schwarz et al. methanol (19 + 1) to methanol (1960)




Blood

Wheat flour lipids

Brain lipids

Intestinal mucosa of the fasting rat

Rat brain

Florisil; glycerides eluted with chloroform- Blankenhorn acetone-water (99 -1-99 + 2) et al. (1961)

Silicic acid; concave gradient from Wren and chloroform to methanol Elliston (1961)

Alumina; successively, chloroform- Davison and methanol (49 + 1 , then 1 +1) , chloroform- Wajda (1962) methanol-water (7 + 7 +1) , and ethanol-chloroform-water (5 + 2 + 2) (Triglycerides in 1 st, phosphatidyl-choline in 2nd)

Silicic acid; various chloroform-methanol Clement and mixtures Di Constanzo

(1963)

KSK silica gel; chloroform-methanol Dvorkin et al. (9 + 1, 4 + 1 , 3 + 1, and finally 1 + 4), giving (1963) fractions

Human cerebrospinal Silicic acid, eluting cholesterol and ester Shin (1963) fluid

Blood serum

Phosphatidyl-serine and -ethanolamine

Triglycerides (model mixtures); palm oil

Triglycerides, e.g. in coconut and linseed oils

Lipids

Lipids

with light petroleum, and phospholipids with methanol

Silicic acid; chloroform eluant

Ammonium silicate; latter eluted with chloroform-methanol, former with absolute methanol

Silica (with some Celite) impregnated with A g N 0 3 ; light petroleum containing up to 90% benzene, then with 20% ether and pure ether

Rubber powder; methanol-acetone (1 + 1 )

Silicic acid; eluting neutral lipids with chloroform, glycosyl diglycerides with chloroform + increasing amounts of acetone, and phosphatides with chloroform + increasing methanol

Silicic acid; eluting hydrocarbons with pet. ether + 1 % ether, triglycerides with 4 % ether, phospholipids with methanol-ether ( 1 + 3 , then 1 + 1 )

Young and Eastman (1963)

Spitzer and Balint (1963)

de Vries(1964)

Trowbridge et al. (1964)

Vorbeck and Marinetti (1965)

Mano(1965)



Lipids of bovine blood serum

Lung tissue phospholipids and glycerides

Triglycerides

Spinach leaves (most phospholipids precipitated from extract)

Lipids

Serum lipids

Acidic phospholipids and phosphatidyl-ethanolamine

Tissues, brain lipids

Silica gel G; triglycerides eluted with 10% Brown and ethyl acetate in pet. ether, phospholipids Stull (1966) with ether-methanol-acetic acid (27-1-9-1- 4)

DEAE-cellulose; benzene-acetone (9 + 1) Gluck et al. and benzene-ether (1 +4) to elute (1966) glycerides, chloroform-methanol (7 + 3) and ethyl acetate-ether (1 + 1) for non-acidic phospholipids, and ethyl acetate-methanol ( 1+1 ) containing 0-05 % aq. N H 3 for phosphatidyl-serine and -inositol and polyglycerophosphate

Celite, treated with (CH 3 ) 2 SiCl 2 ; Nickell and heptane-acetonitrile (17 + 3) separated Privett (1967) according to chain length and unsaturation

Sephadex LH-20; chloroform (remaining Helmsing phospholipids and monogalactosyl (1967) diglycerides), chloroform-methanol ( 1 0 + 1 ) (digalactosyl diglycerides), finally methanol

100-200 or 200-325 mesh silicic acid; pet. Karmen (1967) ether containing 5 then 25 % ether, and finally pure ether, giving fractions containing cholesterol esters, triglycerides, and phospholipids

Silicic acid; successively, Skellysolve B Leeder and (chiefly n-hexane) with 1 then 4 % ether, Clark (1967) ether, methanol; eluted cholesterol ester, triglycerides, free cholesterol, and phospholipids

Silica gel H; THF-methylal-methanol- Gray (1967) water (10 + 6 + 4 + 1 )

DEAE-cellulose (acetate); chloroform (for Rouser (1968) cholesterol), chloroform-methanol (9 + 1) (for lecithin etc.), chloroform-methanol (7 + 3) (for phosphatidylethanolamine), and chloroform-methanol ( 4 + 1 ) containing 28% N H 3 in 0-5M ammonium acetate (for acidic lipids)



E.g. triglycerides, waxes, cholesterol esters, free fatty acids

Lipids (e.g. fatty acids, triglycerides, phospholipids)

Plant tissue sulpholipid and galactolipids

Various compound classes, including triglycerides in synthetic mixtures with hydrocarbons and alcohols

Serum lipids


Neutral lipids and phospholipids

Polar lipids

Triglycerides of neatsfoot and sperm whale oils

Phospholipids

Hydroxypropyl-Sephadex (from Ellingboe Sephadex + N a O H + propylene oxide); et al (1968) reversed-phase, heptane-acetone-water (4 + 15 + 1) or heptane-isopropanol-water (4 + 20 4- 6) for triglycerides and free fatty acids

Sephadex LH-20; chloroform or 20% Downey et al methanol in chloroform containing 1-25 % (1968) water

DEAE-cellulose; neutral lipids eluted with Roughan and chloroform-methanol (3 4- 2), acidic lipids Batt (1968) (including sulpholipid) with chloroform-methanol-conc. N H 4 O H (24 + 16-1-3)

Gel permeation chromatography on Bonbaugh Styragel (cross-linked polystyrene beads of et al (1968) 35 urn, equivalent to 180000 theoretical plates); eluted with tetrahydrofuran

100-200 mesh silicic acid; cholesterol esters Moline and eluted with benzene-hexane (1 + 3 ) , then Barron (1969) free cholesterol and triglycerides with chloroform and phospholipids with chloroform-methanol

Sephadex LH 20; chloroform or Addison and chloroform containing 0-2 % acetic acid Ackman (1969)

DEAE-Sephadex LH-20 in formate form; Dittmer (1969) chloroform-methanol (1 + 1) eluted simple and neutral complex lipids; chloroform-methanol (1 + 1) with concentration gradient of ammonium formate up to 0-02M eluted acidic complex lipids

Hydroxyapatite; successively, chloroform, Slomiany and acetone-methanol (9 + 1, 7 + 3, 1 + 1, Horowitz finally 3 + 7), then ether-ethanol-0-04N (1970) K O H ( 1 0 + 7 + 5)

Silica gel; light petroleum containing Barr et al 3-5 % ether to separate the triglycerides (1970) from the rest

HPLC on silicic acid; chloroform, Montet et al methanol, ethanol, water in various (1970) proportions



Phospholipids in citrus juice

Triglycerides in milk fat

Phospholipids

Phospholipids in tissue

Lipid classes

Nitroglycerine in tablets

Alkyd resins

Triglyceride fraction from butter, margarine

DEAE-cellulose; chloroform-methanol Vandercook (9 + 1 , 7 4- 3), acetic acid-chloroform et al. (1970) (6 + 1), and finally chloroform-methanol (4 + 1) containing 28 % NH 3 /1 and 0-01M ammonium acetate, to elute, successively, lecithin and other compounds, phosphatidyl-ethanolamine, -serine, -inositol, sulphatide, cardiolipin, and phosphatidic acid

Silicic acid (Bio-Rad 200-325 mesh); Shehata et al ether-pet. ether (3 + 97) (1971)

Alumina (Brockmann IV); lysolecithin (and Lutura and sphingomyelin) eluted with chloroform- Sheltawy methanol-water (25 + 25 + 4), (1972) phosphatidylethanolamine with same solvents (10 + 10 + 3), phosphatidylinositol and cardiolipin with chloroform-ethanol-0-07M ammonium nitrate (pH 51) (18 + 25 + 7), phosphatidylserine with chloroform-ethanol- 0-07M ammonium acetate (pH 7-8) (18 + 25 + 7)

Silicic acid; concave gradient of Cavina et al chloroform-methanol (1972)

Gradient elution adsorption chromato- Stolyhwo and graphy on Corasil II, modified by Privett (1973) treatment with N H 4 O H ; continuous series of gradient changes of pentane, ether, chloroform, then methanol containing 8 % N H 4 O H

Celite 545; eluted with isooctane

Gel permeation chromatography on porous cross-linked polystyrene-gel; tetra-hydrofuran as eluant

Hydroxyalkylpropyl-Sephadex ( C 1 5 - C 1 8 ) at 40°C; linear gradient of isopropanol-chloroform-heptane-water (115 + 15 + 2 + 35) and heptane-acetone-water ( 4 + 1 5 + 1) (separated C 9 - C 5 0 glycerides in 24 h; unsaturated eluted faster than saturated)

Soeterboek and Van Thiel (1973)

Christensen and Fink-Jensen (1973)

Lindqvist et al (1974)

4.8 LIPID REAGENTS 189


Fatty acids, methyl esters, triglycerides

Oils from wastewater

Serum lipids

Unchanged glycerides in used frying fats

Triglycerides, organo-chloroxenobiotics

Lecithin/ sphingomyelin in extracts of blood, liver, brain, and amniotic fluid

High performance reversed-phase liquid Pei et al. chromatography on chemically bonded (1975) reversed-phase 35-44 um Vydac; methanol-water eluants

Gel permeation chromatography on Ohkita (1975) Styragel; tetrahydrofuran (3 peaks: mineral oil, waxes, and triglycerides)

Activated silica gel HR 60; eluted at Vandamme 100 ml/h with complex gradient from light et al. (1975) petroleum-ether-ethyl acetate-methanol, acetic acid, and water; elution order hydrocarbons, cholesterol esters, triglycerides, fatty acids, cholesterol, diglycerides, then phospholipids

Merckogel SI 50 (36-75 um) or Merck Aitzetmiiller Kieselgel 60 (40-63 um); unchanged and Guhr triglycerides eluted with heptane, then (1976) non-volatile products of oxidation, polymerisation, and hydrolysis with heptane-diisopropyl ether (4 + 1)

HPLC on silica gel; n-hexane eluted latter, Rohleder et al. then acetone for triglycerides; also (1976) sometimes with alumina

HPLC on Mikropak SI-10 silica gel; Jungalwala acetonitrile-methanol-water(65 + 21 + 14), et al (1976) monitoring by UV absorption at 203 nm

4.8. L IP ID REAGENTS

Reagents have been developed and suggested for lipids in general. These

are either aggressive reagents which char the molecule and are really uni-

versal reagents for practically all organic compounds, or less destructive

agents, such as iodine vapour or certain dyes, the use of which is restricted

largely to the less polar compound classes. Examples of the use of some of

these reagents are given in the following pages. Almost all of them are em-

ployed in chromatographic visualisation with frequent adaptation to quanti-

tative procedures. In his review of the TLC of lipids, Mangold (1961) gives a

useful list of some of these visualisation agents.



4 . 8 . 1 . Charr ing Procedures

Oxidising agents are needed for charring. However, the conditions must be chosen so as to lead to a visible product and not to carbon dioxide and water.

Sulphuric acid is the most used reagent, with dichromate-sulphuric acid next. Some other reagents, such as sulphuryl chloride, copper(II) salts, ammonium sulphate, and phosphoric acid, have found occasional use.

A. S U L P H U R I C A C I D

Some examples of the use of sulphuric acid in the detection of glycerol-containing lipids as charred products on thin layers after TLC are given in Table 4.9. These illustrate several points: the application to all the biological compound classes; the wide range of sulphuric acid concentrations employed, from 5% to the undiluted acid; an equally wide range of charring tempera-tures, from 110 to 500°C.

Scanning with a densitometer is the principal quantitative adaptation of charring. The work of Gartzke and Nolte (1973) may be mentioned here. They studied the densitometric evaluation of charred phospholipids, tri-glycerides, free fatty acids, and cholesterol esters on thin layers and gave "blackening" factors for the evaluation of these classes.

Marsh and Weinstein (1966) applied charring with cone, sulphuric acid to quantitative spectrophotometric lipid determination. In this method, the sample is heated with 2 ml of cone, sulphuric acid for 15 min at 200 ± 2°C, and then cooled in water for 15 s and in ice for 5 min. Water is then added and, after leaving for 10 min, the absorbance is evaluated at 375 nm. This principle was applied by, for example, Dabels (1970), Marzo et al (1971), and Kabara and Chen (1976) to glycerides and other lipids after removal from the thin layer. In another quantitative method, copies of the charred zones were cut out and weighed.

TABLE 4.9

Detection of Lipids by Charring with Sulphuric Acid

Sample Layer Charring conditions References

Phospholipids Glass fibre paper, impregnated with silicic acid

Hot sulphuric acid Dieckert and Reiser (1956)

Phosphatidyl Glass fibre paper, compounds impregnated with

silicic acid

Sulphuric acid, densitometry

Muldrey et al (1959)



Glycerides

Oils

Glass fibre paper, impregnated with silicic acid

Silica gel + 1 5 % CaSO A

Serum phospholipids


Phospholipids Silica gel H from lung tissue

Plasmalogens Silica gel H

Mono-, di, and Silica gel G tri-glycerides, sterols, higher alcohols, hydrocarbons, etc.

Lipids, e.g. Silica gel D O from blood serum (sterol and esters, free fatty acids, total phospho-lipids, glycerides)

Phosphatidyl Silica gel H-Florisil compounds (9 + 1) from rat liver

Sulphuric acid, and heating to charring

Ory (1961)

Cone. H 2 S 0 4 , 15 min/ 120°C giving dark brown fluorescing zones

18N H 2 S 0 4 , several min/ 110°C

Crump (1962)

Silica gel G

Phospholipids Silica gel + N a 2 C 0 3 Sulphuric acid

Phospholipids Silica gel + N a 2 C 0 3

Lipids Silica gel G

Silica gel G

4 0 % H 2 S O 4 (no further details in original)

1 0 % H 2 S O 4 , 15 min/ 200°C; densitometry

50% H 2 S 0 4 , heated to 250°C; spots copied with Verifax copier, cut out, and weighed


18N H 2 S 0 4 , lh/180°C

60% H 2 S 0 4 , heated at 130°C

1 0 % H 2 S O 4 , 30-60 s/ 100°C, then 15 min/ 200°C; densitometry

Robinson and Phillips (1963)

Abramson and Blecher(1964)


Lines (1965)

Schlierf and Wood (1965)

Gluck et al. (1966)

Owens (1966)

Popov et al. (1967)


Van Gent (1968)

Neskovic and Kostic(1968)



Lipid classes in Silica gel G amniotic fluid and serum

Neutral lipids Silica gel G

Phospholipids Silica gel CH + 10% C a S 0 4

Phospholipids Silica gel H - B a C 0 3 , silica gel G, or alumina

Triglycerides, Silica gel G free fatty acids, cholesterol and esters

5 0 % H 2 S O 4 , heated at ca. Biezinski et al. 260° until no more fumes (1968) {ca. 8 min); densitometry

5 0 % H 2 S O 4 , heated to 220°C during ca. 30 min, kept there for 10 min; densitometry

5 0 % H 2 S O 4 , 1 h/180°C

5 0 % H 2 S O 4 , 5 min/ 120°C

5 0 % H 2 S O 4 , heated at 250°C until black; densitometry

Triglycerides, free fatty acids, phospholipids, cholesterol and esters

Lipids from wheat flour

Serum lipids (phospholipids, triglycerides, cholesterol and esters, free fatty acids)

Serum lipids (triglycerides)

Silica gel G

Silica gel

Method of Van Gent (1968)


Silica gel

1 0 % H 2 S O 4 ; densitometry

5 0 % H 2 S O 4 , 10 min/ 120°C; triglyceride zone removed and determined by Marsh and Weinstein's (1966) photometric method

Downing (1968)

Kahovcova and Odavic (1969)

Srivastava and Rastogi (1969)

Vioque et al. (1970)

Postma and Stroes (1970)

Clayton et al. (1970)

Egge et al. (1970)

Dabels(1970)



Mono-, di-, and tri-, glycerides, free fatty acids, glycerol

Lipids

Mono-, di, and tri-glycerides; free fatty acids

Mammalian neutral lipids (glycerides, free fatty acids, cholesterol esters, phospholipids)

Serum lipids (e.g. phospho-lipids, glycerides)

Phospholipids, neutral lipids

Silica gel G + oxalic acid

5 0 % H 2 S O 4 , l h /180°C; densitometry

Silica gel G

Silica gel H R - C a S 0 4

(83 + 17)

Glass fibre paper, impregnated with silica gel G

Silica gel H

Silica gel, impregnated with Na silicate

Zone + cone. H 2 S 0 4 , 15 min/200°; colour evaluated at 375 nm

50% H 2 S 0 4 , charred at 500°C(ca. 15 min);


Sulphuric acid (no details in original)

Silica gel G

Lipids

Lipids (glyceride types with cholesterol and ester)

Serum Silica gel sheet triglycerides (Merck 5711)

Lipid classes Silica gel G

Compared photometric charring method of Marsh and Weinstein (1966) with others

5 0 % H 2 S O 4 , 7-5 min/ 150°C; zones fluoresce yellow-green in 366 nm radiation


Scraped-off zones evaluated according to photometric charring method of Marsh and Weinstein (1966)

Saracco and Gay (1971)

Pollack et al. (1971)

Marzo et al. (1971)

Pocock et al. (1972)


Althaus and Neuhoff(1973)

Kritchevsky et al. (1973)

Mlekusch et al. (1974)

Mantel et al. (1975)

Kabara and Chen (1976)


B. D I C H R O M A T E - S U L P H U R I C A C I D

The remarks made above about the examples of charring with sulphuric acid

in Table 4.9 apply to those of charring with dichromate-sulphuric acid in

Table 4.10. Dichromate concentrations vary from 01 % to saturated which is

about 5-10% at room temperature. It is surprising that this more powerful

oxidising agent does not fail by virtue of yielding carbon dioxide. Probably

this danger has kept the charring temperatures at lower values than those

used with sulphuric acid alone.

TABLE 4.10

Detection of Lipids by Charring with Dichromate-Sulphuric Acid


Mono-, di-, (Not given in original) and tri-glycerides

Triglycerides Silica gel

Partial Silica gel G glycerides in fats

Mono- Silica gel GF -I- boric glycerides acid

Phospholipids Silica gel-Mg silicate ( 9 + 1 )

Phospholipids Silica gel

Lipids Silica gel-Mg silicate (9 + 1)

Lipid classes, Silica gel G including tri-glycerides and phospholipids

Neutral lipids

Satd. K 2 C r 2 0 7 in 80% H 2 S 0 4 ; 2 5 min/180°C

Satd. K 2 C r 2 0 7 in 70% H 2 S O 4 ; 2 5 m i n / 1 8 0 ° C ; densitometry

K 2 C r 2 0 7 - H 2 S 0 4 at 180°C

Satd. C r 0 3 in 70% H 2 S 0 4 ; 2 5 min/200°C; densitometry

0 - 6 % K 2 C r 2 O 7 in 55% H 2 S O 4 ; 3 0 min/180°C

Acidic dichromate; densitometry

0-6% K 2 C r 2 0 7 in 55% H 2 S 0 4 ; 20-30 min/ ca. 185°C; viewed in light of 360 nm (claimed to give higher sensitivity)

Satd. K 2 C r 2 0 7 in 70% H 2 S 0 4 ; 4 5 min/200°C; densitometry

Dichromate, then densitometry

Privett and Blank (1962)

Kaufmann and Mukherjee (1965)

Naudet et al. (1965)

Thomas et al. (1965)

Rouser et al.

(1966)

Morin(1966)

Nelson and Booth (1967)

Nutter and Privett (1968)

Kanno and Hirabayashi (1969)



Glycerides, Silica gel G phospholipids, free fatty acids, cholesterol and esters

Faecal triglycerides

Lipids in blood and faeces

Silica gel G

Silica gel G

Triglycerides Silica gel G from liver biopsy specimen

Esters and Anosil B and S ether-esters of (silica gel with 15 % glycerol and MgO, with and ethylene glycol without C a S 0 4 , resp.)

Neutral lipids Silica gel G

Lipid classes

Glycerides in frying oils

Silica gel G C a S Q 4

Silica gel G

15%

0 - l % K 2 C r 2 O 7 in 5% H 2 S 0 4 ; heated to 250°C

Satd. K 2 C r 2 0 7 in 7 0 % H 2 S O 4 ; 2 5 min/ 180°C; densitometry

Zones removed and heated with K 2 C r 2 0 7 -H 2 S 0 4 ; absorbance evaluated at 430 nm

C r 0 3 - H 2 S 0 4 , then densitometry

Satd. K 2 C r 2 0 7 in 7 0 % H 2 S O 4 ; 2 0 min/ 210°C

Manners et al. (1969)

Thompson et al. (1969)

Amenta (1970)

Jaross and Freimuth (1970)

Kaufmann et al. (1971)

Satd. N a 2 C r 2 0 7 in Palmer et al. 80% H 2 S 0 4 ; 30-60 min/ (1972) ca. 140°C

Satd. K 2 C r 2 0 7 in 70% H 2 S 0 4 ; 2 5 min/180°C; densitometry and planimetry

K 2 C r 2 0 7 - H 2 S 0 4

(satd. K 2 C r 2 0 7 -I- cone. H 2 S 0 4 until deep red; then + equal vol. water); 15 min/180°C; measured relative areas of charred

Wiklund and Eliasson(1972)

Freeman (1974)

C. S U L P H U R Y L C H L O R I D E

Jones et al. (1966) criticised the procedure of spraying before charring, and

suggested that exposure to volatile reagents such as sulphur trioxide or

sulphuryl chloride would overcome the unevenness. They exposed test

lipids to the vapour of the latter for 2 min, and then held the TL plate over

a steaming water bath for 30 s to hydrolyse to sulphuric acid. The lipids

charred rapidly on heating to 200°C.



Others using this reagent have slightly varied the details. Biernoth (1968) separated mono-, di-, and tri-glycerides by TLC on silica gel, then exposed them to sulphuryl chloride vapour for 3 min, heated on a steam bath for 1 min, and finally heated for 25 min at 220°C. He evaluated the dark zones densito-metrically. Fedeli and Camurati (1969) used this procedure also for various lipid classes (sterols, triterpenes, methyl esters, and glycerides) separated in TLC, but with 45 min exposure, 2 min steaming, and 20 min heating at 110°C; they too carried out densitometry'evaluation. Chobanov et al. (1976) visualised triglycerides on thin layers by placing in a sulphuryl chloride atmosphere for 30 min and heating for 10-15 min at 150-180°C.

D . A M M O N I U M S U L P H A T E

Ammonium sulphate acts effectively like sulphuric acid. It has been used in a spray reagent and as additive to a thin layer in chromatography. Adams and Sallee (1970) visualised serum phospholipids on Adsorbosil-5 silica gel by spraying with 20% ammonium hydrogen sulphate and heating at 170°C for 90 min. Densitometric evaluation of the dark zones was stated to be possible. The same authors (Sallee and Adams, 1970) applied the procedure to neutral lipid classes on the same layers. Sundler and Akesson (1973) sprayed a reagent solution of the same composition, then heated at 120°C, to detect phosphatidyl-ethanolamines on silica gel H containing 17*5% of silver nitrate.

Walker (1971) prepared thin layers from silica gel G slurried with 1% and 10% ammonium sulphate solutions, and tested visualisation of polar and non-polar lipids by heating for 20 min at 200°C. This proved satisfactory, the 10% impregnation being more sensitive. Truppe et al. (1972) used a slurry of silica gel with 10% ammonium sulphate to prepare thin layers for separating triglycerides; they heated at 150°C for 25, 45, or 85 min to obtain products which could be evaluated through their yellow-green fluorescent emission at 450 nm. Fluorescence at 495 nm was used by Mlekusch et al. (1973) to determine serum lipids (phospholipids, free and esterified cholesterol triglycerides) on thin layers of silica gel G + ammonium sulphate after heating for 25 min at 150°C. A recent patent of Smith (1974) deals with separation of many lipids (including triglycerides) on silica gel G layers containing ammonium sulphate, exposing to t-butyl hypochlorite, heating, and measuring fluorescence on the plate.

E. C O P P E R ( I I ) S A L T S

A spray reagent of 3 % cupric acetate in 8 % aqueous phosphoric acid was used by Fewster et al (1969) to visualise various lipids (e.g. diglycerides, cholesterol) on silica gel H mixed with 10 % magnesium silicate; after charring by heating for 25 min at 180°C they evaluated the zones densitometrically.


Neskovic et al. (1970) also used cupric acetate-phosphoric acid to detect sulphatides, monogalactosyl diglycerides, and cerebrosides on silica gel H-Florisil ( 9 + 1 ) layers. Korolczuk and Kwasniewska (1974) chromato-graphed lipids on silica gel layers containing incorporated cupric sulphate. On heating for 1 h at 180°C they obtained dark oxide zones; 1 % of cupric salt gave zones that were too pale, and 10% influenced the Rf values of tri-glycerides.

F. P H O S P H O R I C A C I D

Phosphoric acid has been relatively little used as a charring reagent. Barrett et al. (1963) sprayed thin layers of silica gel (containing silver nitrate) with 50% phosphoric acid and heated at 340°C to char separated triglycerides for densitometric determination. Popov et al. (1967) separated glyceride types and other lipids on silica gel layers, and then charred with 10% phosphoric acid as well as with the 60% sulphuric acid previously mentioned; they heated at 130°C.

Aqueous phosphoric acid containing cupric acetate was mentioned in the preceding Section E.

4.8.2. Iod ine

Iodine is a detection reagent for many organic compounds in paper and thin layer chromatography. The usual technique is to expose the chromatogram for some minutes to iodine vapour in a closed vessel. Alternatively, the chromatogram can be sprayed with a dilute (say about 0*5%) solution of iodine in a volatile solvent such as chloroform or a lower alcohol. Excess iodine (and solvent) disappears on standing in the air afterwards, and the organic compounds on the paper or thin layer show as yellow or brown zones. An aqueous spray reagent can also be used, e.g. 0-3 g of iodine in 100 ml of 5 % potassium iodide (Merck, undated); excess iodine is then washed out with tepid water.

At first the iodine reagent was used for visualising unsaturated compounds, e.g. glycerides of unsaturated fatty acids (cf. Mangold et al., 1955). However, many other compounds yield the dark zones, although those from saturated compounds disappear more quickly than those from unsaturated compounds. This disappearance is still slow enough to permit ringing of the zones to mark their position. Probably unstable addition products are formed with the iodine, decomposing more or less slowly after the stabilising excess of iodine reagent has been removed.

Sims and Larose (1962) studied the iodine detection and stability of the coloured zone of 16 compounds, including unsaturated and saturated glycerides, glycerophosphate, lecithins, and glycerol itself. All these could be

H


detected, and the participation of the glycerol does provide justification for mentioning this detection method here. Sims and Larose preferred iodine to cone, sulphuric acid for general detection since it leaves no corrosive residues, and the colours and speeds of disappearance furnish additional information about the detected substances.

Iodine has naturally found extensive use for detecting glycerol-containing compounds, and it is clear that no more than a tiny selection of articles can be quoted. Especially in the early 1960s, glycerides were detected with iodine in reversed-phase chromatography on paper impregnated with silicone oil (e.g. Hirayama, 1961) and on thin layers impregnated with paraffin oil or silicone (e.g. Kaufmann et al, 1961; Michalec et al, 1962; Kaufmann and Das, 1962; Kwapniewski and Sliwiok, 1964; Bandi and Mangold, 1969, this last named for the glycerides of cyclopentane-carboxylic acids).

In adsorption TLC, iodine has been used for detection practically only on silica layers with, at the most, small additions of magnesium silicate, Florisil, sodium carbonate, or gypsum.

Examples of application to triglycerides are: Pokorny and Herodek (1964) on Floridin As, also for mono- and di-glycerides; Pinter et al (1964) on glass fibre paper for serum samples; Ranny (1968) for sugar glycerides; Ristrow (1968) also for higher hydrocarbons and other fatty esters, and in combination with Rhodamine B and molybdophosphate detection agents; Kundu (1969) on samples of kusum oil; Jaross and Freimuth (1970) on liver samples; Marzo et al (1971) also for mono- and di-glycerides and free fatty acids in biological samples; Vioque et al (1973) also for free fatty acids, in blood samples.

Examples of application to phosphatidyl compounds include: Skipski et al (1964) on silica gel-sodium carbonate (no gypsum); Gione and Orning (1966) for plasma; Abdel-Latif and Chang (1966) for brain phospholipids containing 3 2 P ; Neskovic (1967); Neskovic and Kostic (1968) on rat liver using silica gel H-Florisil (9 + 1); Roozemond (1969); Williams et al (1969); Nardi et al (1971) for human phospholipids; Chapman (1972); Poorthuis et al (1976) for glycerol phosphatide and derivatives.

For detecting several lipid classes (glycerides, cholesterol and esters, free fatty acids, phospholipids, etc.) iodine has been used also by many, such as Torres et al (1971) for serum lipid fractions, Kunz (1973) on plasma extracts, and Hubmann (1973).

Roughan and Batt (1968) visualised sulpholipids and galactolipids using

iodine vapour.


4.8.3. l o d i n e - a - C y c l o d e x t r i n or Starch

Iodine is used also in combination with a-cyclodextrin or starch. Thus Man-gold et al. (1955) sprayed paper chromatograms with 1% a-cyclodextrin in 30% ethanol, air-dried the chromatogram, moistened it for 1 h in a humid atmosphere (to facilitate subsequent treatment), and then exposed to iodine vapour. Lipids which occupy the dextrin as guest molecules hinder formation of the blue inclusion compound with iodine, so they appeared as white zones on a blue background. Many fatty acids, alcohols, and esters respond, and also monoglycerides.

Steiner and Bonar (1961) detected mono-unsaturated glycerides from cocoa butter in reversed-phase PC with the combined reagent but in the reverse order. After 30 min exposure to iodine vapour they sprayed the back of the paper strip with 1 % starch to obtain white zones on a blue background. Their technique was adopted by Chakrabarty et al. (1966) for triglycerides on Whatman No. 1 paper impregnated with liquid paraffin.

Chakrabarty et al. (1966) also carried out TLC of these glycerides on kieselguhr impregnated with liquid paraffin. They exposed to iodine vapour until brown zones were visible (about 5 min), then sprayed with the cyclo-dextrin reagent to obtain blue zones on white. Presumably excess iodine was allowed to evaporate from the layer before spraying, so the dextrin reacted only with the loosely held iodine of the brown zones. It served thus more as an intensifier of the iodine colour. Wessels and Rajagopal (1969) also employed this reverse order for detecting unsaturated triglycerides on kieselguhr G impregnated with paraffin.

4.8.4. M o l y b d o - and T u n g s t o - p h o s p h o r i c A c i d s

These heteropolyacids are further reagents used in chromatography for detecting and determining lipids, including the glycerol-containing com-pounds which interest us here. The molybdophosphoric acid is usually in ethanolic solution in from 1 to 20 % concentration, and yields blue or grey-blue zones on a yellowish background, probably due to reduction to "molyb-denum blue". These can be evaluated densitometrically. Tungstophosphoric acid has been much less frequently used for glycerides and phospholipids. Some examples are given in Table 4.11.

4.8.5. F luorescent De tec t ing A g e n t s

For many years fluorescent materials have been incorporated into the adsorbent used for preparing thin layers. On illuminating such layers with UV light, zones of compounds that absorb this light appear dark on the

TABLE 4.11

Visualisation of Lipids with Molybdo- and Tungsto-phosphoric Acids

Sample Layer Detection conditions References

Triglycerides, Silicic a c i d - C a S 0 4

cholesterol impregnated with esters paraffin oil

Mono, di, and Floridin AS tri-glycerides

Lipid fractions Silica g e l - C a S 0 4 (6 + 1) in blood serum

Fatty acid esters of glycerol and methanol

Silanised silica gel G impregnated with A g N Q 3

Phospholipids Whatman No. 3 paper, (also sphingo- impregnated with lipids) silicic acid

Triglycerides, Silica gel higher hydro-carbons, other fatty esters

Serum lipids Silica gel

Serum lipids Silica gel G

Serum lipids Silica gel

Lipids

Lecithin/ Silica gel sphingomyelin ratio in amniotic fluid

Acetodiacyl- Silica gel G glycerols in milk fat lipids

Lipids In PC and TLC

Mo- or W-reagent, heated 5-10 min/100°C

6 % ethanolic Mo-reagent

2 % ethanolic Mo-reagent, heated 10-15 min/100°C

Ethanolic Mo-acid, heated to 260°C (for the glycerides)

Mo-acid (also Rhodamine B) for the phospholipids (no details of reagents)

Successive treatment with Rhodamine B, iodine, and molybdo-phosphoric acid

Mo-acid, then densitometry

10% ethanolic Mo-acid, then heated 10 min/70°C

Mo-acid-ethanol-H C 1 0 4 , heated 20 min/ 85°C

5% ethanolic Mo-acid; densitometry at 720 nm

Mo-acid-ethanol-H 2 S 0 4 - H 2 0 2 , heated 1 h/140°C

Mo-acid, heated 5 min/ 180°C

20 % ethanolic W-acid, heated 20 min/70°C

Michalec et al. (1962)

Pokorny and Herodek(1964)

Baryshkov (1966)

Ord and Bamford (1967)

Mezesova (1967)

Ristrow (1968)

Wildgrube et al (1969)

Feldman and Fosslien (1971)

Chedid et al (1972)

Yamada(1972)

Verhoeven and Merkus(1974)

Parodi(1975)

Merck


fluorescent background of the layer. This method is of almost universal application and needs no further discussion here.

Many organic compounds enhance and/or modify the fluorescence of certain materials which have therefore been used as visualising agents for these. Most popular are the xanthenes, 2',7'-dichlorofluorescein, Rhodamine B (C.I. Basic Violet 10), and the ethyl ester, Rhodamine 6G (C.I. Basic Red 1).

Rhodamine 6G

Rhodamine B 2',7-Dichlorofluorescein

(one resonance form of each)

These compounds have been used with several lipid classes, including the glycerides and phosphatidylglycerol-containing compounds.

Tables 4.12-4.14 contain some examples of use in both PC and TLC of these three agents. As seen from the tables, they have been used mostly as dilute (0*001-0-2%) solutions in water or ethanol for spraying, and only rarely by incorporation into the layer (in TLC). Most work in TLC has been on silica gel, also containing borate, oxalate, silver nitrate (with 2',7'-dichloro-fluorescein and the dibromo analogue), and other metal salts, and in reversed phase. Bright yellow fluorescence is generally yielded but it depends evidently


on the nature of the lipids. Thus Dvorkin et al (1963) reported lilac fluores-cence with phosphatidyl-serine and -inositol, and orange with phosphatidyl-ethanolamines and -cholines in PC.

The nature of the reaction products does not appear to be clear. According to Schiefer and Neuhoff (1971), the basic dye Rhodamine 6G interacts with acidic phospholipids, the detection is specific for these, and neutral lipids do not interfere. Harris and Gambal (1963) also commented on the specific production of fluorescence by phospholipids (from rat tissue) with Rhodamine 6G, whereas cholesterol and neutral lipids had no effect. They used it for quantitative determination and also for monitoring phospholipids in column effluents. However, this (and the other) agents are used with the neutral lipids also. Trapp (1976) studied complex formation between Rhodamine 6G and fatty acids from C 1 2 to C 2 5 , evaluating absorbance at 510-515 nm to determine these acids in potassium fertilizer salts.

Popov and Stefanov (1968) investigated several fluorescent indicators for lipids in TLC, and also optical brighteners (Tinopals) as 0*5 % solutions in ethanol which they considered to be superior as detecting agents. Best of all were mixtures of Tinopal and Rhodamine B (in the ratio ca. 25-60:1). Klemig and Lempert (1970) also used a mixed reagent in ethanol, 0 0 5 % in Rhodamine B and 5 % in Tinopal.

TABLE 4.12

Detection of Lipids in PC and TLC with 2',7'-Dichlorofluorescein

Sample Layer and detection conditions References

Fats, oils, waxes

Lipids

Phospholipids


Nitrate esters of glycerides and glycerol ethers

Triglycerides

Phospholipids

Silicic acid; sprayed with 0*2% reagent in Mangold and ethanol; viewed in UV of 270 nm Malins (1960)

Silica gel; 002% aqueous reagent; spots Brown and scraped off and assayed radioactively Johnston

(1962)

Silicic acid-dental CaS0 4 (50 + 1); Doizaki and air-dried and sprayed with 0*2 % reagent Zieve (1962)

Fine mesh silicic acid + 10% CaS0 4 ; Rybicka (1962) 0-2% reagent

Silica gel G; evidently reagent of Malins Malins et al. and Mangold (1960) (1964)

Silica gel-AgN0 3 ; 0*1 % reagent in ethanol Jurriens (1964)

Silica gel H Parker and Peterson (1965)



Glycerides


Serum lipids

Food emulsifiers, including mono-and di-glycerides

Fat triglycerides

Plasma triglycerides

Phospholipids

Triglycerides from kusum oil

Glycerol and glycerides in "technical monoglycerides"

Triglycerides of cyclopentane acids

Triglycerides

Triglycerides

Mono- and di-glycerides in margarine

Phospholipids

Triglycerides

Neutral lipids, phospholipids

Silica gel G - A g N 0 3 ; 0-2 % reagent in ethanol

Silica gel; 0 0 2 % reagent in ethanol

Silica gel G; 0-2% reagent

Silica ge l -KH oxalate.

Silica gel G, containing 12*5 % A g N 0 3

Silica gel G

Silica gel HR

Silica gel G - A g N 0 3 ; 0-2% reagent in 95% ethanol

Silica gel; 0 1 % reagent in 95% ethanol

Kieselguhr G impregnated with 5 % paraffin in hexane; 0-2% reagent in ethanol

Silica gel G - A g N Q 3

Silica gel G - A g N 0 3 ; 001 % reagent

Silica gel-borate; 0 1 % reagent in ethanol

Silica gel G; 01 % reagent in ethanol

Silica gel G - A g N 0 3 ; ethanolic reagent

Silica gel G; 0*02% reagent; scanned at 550 nm, excitation at 365 nm

Amat et al (1966)

Ceglowska et al (1966)

Chiarioni et al (1966)

Gernert (1968)

Persmark and Toregard (1968)

Laurell(1968)

Iacono and Ishikawa (1969)

Kundu(1969)

Neissner (1969)

Bandi and Mangold (1969)

Wessels and Rajagopal (1969)

Burns et al (1969)

Kanematsu et al. (1972)

Christie (1972)

Utrilla et al (1976)

Ishida-Ichimasa et al. (1976)



Lecithin, sphingo-myelin

Silica gel; 0-25% reagent in methanol Sass et al. (1976)

Some examples of the use of the corresponding dibromofluorescein may be quoted also:

Glycerides Silica gel G - A g N 0 3 (4 + 1); 0-2% reagent in ethanol

Barrett et al. (1962)

Triglycerides Silica gel G - A g N 0 3 ; 0 2 % reagent in ethanol

Barrett et al. (1963)

Glycerides, fatty acids and their other esters

Silica gel containing 15 % C a S 0 4 ; 0 2 % reagent in ethanol

Rao and Sreenivasan (1966)

Fatty acid esters of glycerol and polyglycerols

Silica gel containing 4 % boric acid Sahasrabudhe (1967)

TABLE 4.13

Detection of Lipids in PC and TLC with Rhodamine B

Sample Detection conditions References

Glycerides, fatty acids

PC; 0-5 % aqueous reagent Hirayama (1961)


Silica gel G; Rhodamine B and 6G Skipski et al. (1962)

Serum triglycerides Silica gel G containing Rhodamine B; examined in UV of 375 nm

Krell and Hashim(1963)

Glycerides PC in reversed phase Trippel(1964)

Glycerides and sugar glycerides

Whatman No. 3 impregnated with Na silicate and NH 4 C1; 0 0 1 % reagent in 0025M K 2 H P 0 4

Ranny (1966)

Phospho- and sphingo-lipids

Whatman No. 3 impregnated with silicic acid; reagent of Rhodamine B -molybdophosphate

Mezesova (1967)

Triglycerides, also higher hydrocarbons and other esters

Silica gel; successive treatment with Rhodamine B, iodine vapour, and molybdophosphate

Ristrow(1968)

Phosphatides in sugar beet

Two-dimensional PC; visualised spots cut out and weighed

Beiss(1969)



Glycerides in cosmetic products

Lecithin and sphingomyelin ratio in amniotic fluid

Triglycerides

Paper impregnated with saturated urea or Pokorny et al. thiourea in methanol; or cellulose powder- (1973) C a S 0 4 (20 + 1); 0001 % reagent

Gelman I. TLC Type SG; reagent gave pink Blass et al. zones on blue in UV; ratio via areas (1973, 1974)

Microcrystalline cellulose containing salts of transition metals (CdCl 2 and Z n ( N 0 3 ) 2

good); 0 0 5 % reagent

Haworth et al. (1975)

TABLE 4.14

Detection of Lipids in PC and TLC with Rhodamine 6G


Phospholipids of rabbit skin



Phospholipids


Whatman No. 1 impregnated with silicic acid; 0 0 0 1 % reagent

Glass fibre paper impregnated with silicic acid; 0001 % aqueous reagent

Silica gel G; Rhodamine 6G and B

PC, using 0-001 % reagent

Schleicher and Schiill 2043b paper impregnated with silicic acid

Phosphatidyl Whatman No . 3, then Schleicher and compounds Schull 289 impregnated with silicic acid;

dipped into reagent

Serum lipids (neutral) Silica gel

Silica gel G containing 00L%reagent Triglycerides and free fatty acids

Phosphatidylcholine, Silica gel G; 0 0 5 % reagent sphingomyelin in rat tissues

Phospholipids in canine body fluids

Paper pretreated with Na silicate; 0001 % Rhodamine 6G

Schwarz et al. (196*0)

Cornatzer et al. (1962)


Dvorkin et al. (1963)

Scrignar (1964)

Letters (1964)

Whitner et al. (1965)

Fosbrooke and Tamir(1968)

Kennan et al. (1968)

Karagezyan (1969)



Phospholipids Silicic acid-impregnated paper or Whatman Chromedia S.G.81; sprayed with, not immersed in, reagent

Kilroe-Smith (1969)

Five lipid classes Silica gel G; 0-001 % reagent, 0 2 5 M in K 2 H P 0 4

Nicolosi et al. (1971)

Triglycerides and other neutral lipids

Silica gel G containing 0-005% reagent; quantitative scanning above 570 nm with excitation at 546 nm

Roch and Grossberg (1971)

Esters and ether-esters of glycerol

MgO, Anasil B, Anasil S (silica gel with 15 % MgO, with and without C a S 0 4 , resp.), silica gel

Kaufmann et al. (1971)

Six lipid classes Silica gel G; fluorimetric determination of separated zones

Hojnacki and Smith (1974)

4.8.6. Other Dyes and Ind icators

Some other dyes or indicators have been used for detection in the chromato-graphy of lipids, including those containing glycerol. It must be admitted that the glycerol moiety probably has very little to do with it but a few examples are mentioned here in passing.

Bromothymol Blue is quoted for lipids by Stahl (1969) (Reagent No. 31). Urakami and Kakutani (1957) employed it to detect a- and y8-glycerol-phosphoric acids, separated in PC; it yielded the yellow acidic colour. Jatzkewicz and Mehl (1960) tested it in the TLC of lipids as a general spraying reagent, using a solution in sodium borate or very dilute sodium hydroxide. Habermann et al. (1961) visualised plasma phospholipids on silica gel with ammoniacal Bromothymol Blue, and Vacikova et al. (1962) serum lipids on alumina layers. Another example is the use to detect phosphatides by Thiele and Wober (1964). Seiffert and Alt (1969) detected glycerides on silica gel G, also impregnated with silver nitrate, using a solution of the indicator in very dilute sodium hydroxide and then exposing to ammonia. Blau (1975) obtained stable colours (blue on pale blue) from lecithin (and sphingomyelin) by exposing the zones with Bromothymol Blue (which fade rapidly) to ammonia and then spraying with 10% ethanolamine in acetone.

Sudan Black was used among other dyes by Kaufmann and Makus (1959) for glycerides on Schleicher and Schull 2040b or 2043b, and by Sliwiok and Kwapniewski (1965) to detect and determine (densitometrically) glycerides on Whatman No. 1 paper. Vereshchagin (1965) used Sudan Black B


4.8 REFERENCES 207

REFERENCES

Abdel-Latif, A. A. and Chang, F. E. (1966). / . Chromatogr. 24, 435. Abramson, D. and Blecher, M. (1964). J. Lipid Res. 5, 628 Adams, G. M. and Sallee, T. L. (1970). J. Chromatogr. 49, 552. Addison, R. F. and Ackman, R. G. (1969). Anal. Biochem. 28, 515. Aitzetmiiller, K. and Guhr, G. (1976). Fette, Seifen, Anstrichm. 78, 83. Alley, B. J. and Dykes, H. W. H. (1972a). / . Chromatogr. 71, 23. Alley, B. J. and Dykes, H. W. H. (1972b). / . Chromatogr. 72, 182. Althaus, H. H. and Neuhoff, V. (1973). Z. Physiol Chem. 354, 1073. Altmann, A., Bach, A. and Metais, P. (1967). Ann. Biol. Clin. (Paris) 25, 439. Amat, F., Marquinez, E., Utrilla, R. M. and Martin, L. (1966). Grasas Aceites (Seville)

17, 45. Amelung, D. and Bohm, P. (1954). Z. Physiol Chem. 298, 199. Amenta, J. S. (1970). Clin. Chem. 16, 339.

(C.I. Solvent Black 3) for unsaturated triglycerides and also methyl esters of fatty acids, in reversed-phase PC after removing the dodecane impregnant by heating.

Scrignar (1964) used 0 1 % Cresyl Violet solution to visualise lipids. Oil Red O was used by Kaufmann and Makus (1959) and by Castaldo

et al (1972) for triglycerides, and Bromocresol Green by Boniforti (1973) for lipids, e.g. triglycerides, of the skin surface, the first in PC and the other two in TLC. Also in TLC are applications of Amidoschwarz by Pre and Gamier (1973) for lipids in plasma and blood, on silica gel; and of Malachite Green (as 0-5% aqueous solution) by Teichman et al (1974) for lipids on silica gel G or H.

A fairly recent introduction is ANS (l-anilinonaphthalene-8-sulphonate) where enhanced fluorescence comes about probably through binding to hydrophilic regions on protein surfaces (Schiefer and Neuhoff, 1971). Gitler (1972) detected phospholipids and apolar molecules on thin layers using a 0*1 % aqueous spray reagent, then viewing in long-wave UV. Heyne-man et al (1972) used a 0-01 % solution of the magnesium salt to visualise phosphatidyl compounds derived from horse erythrocyte plasma after TLC on silica gel; they observed green-yellow spots in short-wave UV and determined the products by fluorescent scanning at 440 nm (excitation at 375 nm).

Bernard and Vercauteren (1976) comment on the probable binding of lipid amino groups to the sulphonate group in ANS, and suggest JV-phenyl-1-naphthylamine, as 0T % solution in ethanol, for detecting phospholipids and neutral lipids on thin layers (silica gel). They were thus able to detect down to 1 nmol of lecithin in short-wave UV.

208 GLYCEROL COMPOUNDS

Angelelli, L., Cavina, G., Moretti, G. and Siniscalchi, P. (1966). Farmaco, Ed. Prat. 21, 493.

Anghileri, L. J. (1964). Int. J. Appl. Radiation and Isotopes 15, 95. Anonymous (1958). Mem. Poudres 40, 217. Archer, A. W. (1975). / . Chromatogr. 108, 401. Avancini, D., Pedroni, G. and de Francesco, F. (1966). Riv. Ital. Sostanze Grasse 43,

450. Baer, D. M. (1974). Clin. Chem. 20, 502. Baldi, A. and Scuccimarra, C. (1973). Ig. Sanita Pubblica 29,304 {Chem. Abs. 82,94963). Bandi, Z. L. and Mangold, H. K. (1969). Separ. Sci. 4, 83. Bandyopadhyay, C. (1968). / . Chromatogr. 37, 123. Barnes, R. W. (1967). / . Chromatogr. 31, 606. Barr, I. G. Hamilton, R. J. and Simpson, K. (1970). Chem. Ind. (London) 988. Barrett, C. B., Dallas, M. S. J. and Padley, F. B. (1962). Chem. Ind. (London) 1050. Barrett, C. B., Dallas, M. S. J. and Padley, F. B. (1963). J. Amer. Oil Chem. Soc. 40, 580. Baryshkov, Yu. A. (1966). Lab. Delo 615. Beiss, U. (1969). Fette, Seifen, Anstrichm. 71, 363. Bernard, D. M. and Vercauteren, R. E. (1976). J. Chromatogr. 120, 211. Bezard, J. and Bugaut, M. (1969). / . Chromatogr. Sci. 7, 639. Bezard, J. and Bugaut, M. (1972). J. Chromatogr. Sci. 10, 451. Biernoth, G. (1968). Fette, Seifen, Anstrichm. 70, 402. Biezinski, J. J., Pomerance, W. and Goodman, J. (1968). J. Chromatogr. 38, 148. Blankenhorn, D. H., Rouser, G. and Weimer, T. J. (1961). J. Lipid Res. 2, 281. Blass, K. G., Thibert, R. J. and Draisey, T. F. (1973). Clin. Chem. 19,1394. Blass, K. G , Thibert, R. J. and Draisey, T. F. (1974). / . Chromatogr. 89, 197. Blau, K. (1975). Clin. Chim. Acta 64, 217. Blond, J. P., Lemarchal, P. and Le Breton, E. (1971). Biochimie 53, 1221. Boehm, O. (1966). Explosivstoffe 14, 97. Boehm, O. (1967). Explosivstoffe 15, 25. Bogaert, M. G. and Rosseel, M. T. (1972). J. Pharm. Pharmacol. 24, 737. Bombaugh, K. J., Dark, W. A. and Levangie, R. F. (1968). Z. Anal. Chem. 236, 443. Boniforti, L. (1973). Clin. Chim. Acta 47, 223. Boyne, A. W. and Duncan, W. R. H. (1970). J. Lipid Res. 11, 293. Breckenridge, W. C. and Kuksis, A. (1970) Lipids 5, 342. Brockmann, U. and Gercken, G. (1969). Clin. Chim. Acta 23, 489. Broekhuyse, R. M. (1969). Clin. Chim. Acta 23, 457. Brown, J. L. and Johnston, J. M. (1962). J. Lipid Res. 3, 480. Brown, W. H. and Stull, J. W. (1966). / . Dairy Sci. 49, 636. Buckley, J. A., Deigan, T., Muirhead, R. A. and Williams, M. J. (1970). Clin. Chim. Acta

28, 133. Bugaut, M. and Bezard, J. (1970). / . Chromatogr. Sci. 8, 380. Bugaut, M. and Bezard, J. (1973). / . Chromatogr. Sci. 11, 36. Bunn, C. R., Keele jr., B. B. and Elkan, G, H. (1969). J. Chromatogr. 45, 326. Burns, D. T., Stretton, R. J. Shepherd, G. F. and Dallas, M. S. J. (1969). J. Chromatogr.

44, 399. Camera, E. and Pravisani, D. (1964). Anal. Chem. 36, 2108. Camera, E. and Pravisani, D. (1967). Anal. Chem. 39, 1645. Camera, E., Pravisani, D. and Ohmen, V. (1965). Explosivstoffe 13, 237. Carlson, L. A. and Wadstrom, L. B. (1959). Clin. Chim. Acta 4, 197. Carracedo, C. F. and Prieto, A. (1969). Grasas Aceites (Seville) 20, 289.

REFERENCES 209

Castaldo, A., Petti, L., Colonna, G. and Merolla, R. (1972). Biochim. Appl. 18, 113. Castelli, A. H., Halik, M., Fredericks, W. E. and Pristera, F. (1954). U.S. Dept. Com.,

Office Tech. Serv. PB Rept. 157 (Chem. Abs. 58, 5444). Casu, A., Pala, V., Pantarotto, M. F. and Pecorari, D. (1974). Ital. J. Biochem. 23, 38. Cavina, G., Moretta, L., Antonini, R., Cantafora, A. and Mascagna, F. (1972). Annali

1st. Sup. Sanita 8, 122. Ceglowska, K., Schumann, K. and Szczepanska, H. (1966). Tluszcze, Srodki Piorace,

Kosmet. 10, 157 (Chem. Abs. 67, 45063). Chabard, J. L., Vedrine, F., Godeneche, D. Petit, J. and Berger, J.-A. (1976). J. Chroma-

togr. 121, 295. Chakrabarty, M. M., Bhattacharyya, D. and Gupta, A. (1966). / . Chromatogr. 22, 84. Chapman, L. R. (1972). / . Chromatogr. 66, 303. Chedid, A., Haux, P. and Natelson, S. (1972). Clin. Chem. 18, 384. Chiarioni, T., Ronchi, F., Sasso, G. F., Mancini, P. and Nardi, E. (1966). Med. Clin.

Sperm. 16, 209. Chobanov, D., Tarandjiska, R. and Chobanova, R. (1976). Amer. Oil Chem. Soc. 53,

48. Christensen, G. and Fink-Jensen, P. (1973). Farbe, Lack 79, 301. Christie, W. (1972). Analyst (London) 97, 221. Christos, T. and Spinetti, L. (1973). U.S. Bur. Mines, Rep. Invest. No . 7795 (Chem. Abs.

80, 49933). Clayton, T. A. MacMurray, T. A. and Morrison, W. R. (1970). J. Chromatogr. 47, 277. Clement, J. and Di Constanzo, G. (1963). Bull. Soc. Chim. Biol. 45, 127. Collier, R. (1962). Nature (London) 194, 771. Constantinescu, T. and Enache, S. (1974). Farmacia (Bucharest) 22, 179 (Chem. Abs.

81, 114241). Cornatzer, W. E., Sandstrom, W. A. and Reiter, J. H. (1962). Biochim. Biophys. Acta

57,568. Coupek, J., Pokorny, S., Mares, E., ztezulkova, L., Nguyen-Thien Luan and Pokorny,

J. (1976). / . Chromatogr. 120, 411. Crider, Q. E , Alaupovic, P., Hillsberry, J , Yen, C. and Bradford, R. H. (1964). J. Lipid

Res. 5, 479. Crump, G. B. (1962). Nature (London) 193, 674. Cusick, C. F. (1976). Ann. Clin. Biochem. 13, 379. Dabels, J. (1970). Z. Ges. Inn. Med. 25, 391. Davison, A. N. and Wajda, M. (1962). Biochem. J. 82, 113. Dean, F. D. (1974). Clin. Chim. Acta 50, 367. Del Campo, P. (1965). Explosivstoffe 13, 41. Del Campo, P. (1966). Inform. Quim. Anal. (Madrid) 20, 108 (Chem. Abs. 66, 30630). Delruck, H., Martens, W. and Winterfeld, M. (1968). Z. Physiol. Chem. 349, 896. De Zeeuw, R. A., Rock, R. G. and Sprinz, H. (1970). Pharm. Weekblad 105, 1072. Dieckert, J. W. and Reiser, R. (1956). J. Amer. Oil Chem. Soc. 33, 535. Dierick, W., Stockx, J. and Vandendriessche, L. (1956). Naturwissensch. 43, 82. Dittmann, J. (1966). Z. Klin. Chem. 4, 10. Dittmer, J. C. (1969). J. Chromatogr. 43, 512. Doizaki, W. M. and Zieve, L. (1962). J. Lipid Res. 3, 138. Downey, W. K., Keogh, M. K. and Murphy, R. F. (1968). Biochem. J. 110, 13P. Downing, D. T. (1968). J. Chromatogr. 38, 91. Dvorkin, V. Ya., Chetverikov, D. A. and Shmelev, A. A. (1963). Biokhimiya 28, 475

(Anal. Abs. 11,2736).


Dykes, H. W. and Alley, B. J. (1974). U.S. Patent No. 3,782,900, April 1st (Chem. Abs. 80, 66530).

Eckert, W. R. (1973). Fette, Seifen, Anstrichm. 75, 150. Edwards, L., Falkowski, C , Chileote, M. E., Hirsch, R. L. and Mather, A. (1972).

Stand. Methods Clin. Chem. 7, 69. Egge, H., Murawski, U., Mueller, J. and Zillicka, F. (1970). Z. Klin. Chem. Klin.

Biochem. 8, 488. Ellingboe, J., Nystrom, E. and Sjovall, J. (1968). Biochim. Biophys. Acta 152, 803. El-Nockrashy, A. S. and Mahfouz, M. M. (1965). Grasas Aceites (Seville) 16, 5. Erikson, J. M. and Biggs, H. G. (1973). J. Chem. Educ. 50, 631. Fedeli, E. and Camurati, F. (1969). Riv. Ital. Sostanze Grasse 46, 97. Feldman, S. A. and Fosslien, E. (1971). J. Chromatogr. 63, 63. Fewster, M. E., Burns, B. J. and Mead, J. F. (1969). J. Chromatogr. 43, 120. Folch, J., Lees, M. and Stanley, G. H. S. (1957). J. Biol. Chem. 226, 497. Fosbrooke, A. S. and Tamir, I. (1968). Clin. Chim. Acta 20, 517. Fossel, E. T. (1965). / . Gas. Chromatogr. 3, 179. Freeman, I. P. (1974). Chem. Ind. (London) 623. Freeman, C. P. and West, D. (1966). J. Lipid Res. 7, 324. Freeman, N. K , Lindgren, F. T., Ng., Y. C. and Nichols, A. V. (1957). / . Biol. Chem. 221,

449. French, J. A. and Andersen, D. W. (1973). / . Chromatogr. 80, 133. Fried, R. and Hoeflmayr, J. (1973). German Patent No. 2,139,163, Feb. 15th (Chem.

Abs. IS, 133113). Galanos, D. S., Aivazis, G. A. M. and Kapoulas, V. M. (1964). J. Lipid Res. 5, 242. Galletti, F. (1967). Clin. Chim. Acta 15, 184. Gartzke, J. and Nolte, K. D. (1973). J. Chromatogr. 84, 109. Gasbarro, L. (1972). Clin. Chim. Acta 37, 271. Gernert, F. (1968). Z. Lebensm.-Untersuch-Forsch. 138, 216. Getz, G. S., Jakovcic, S., Hey wood, J., Frank, J. and Rabinowitz, M. (1970) Biochim.

Biophys. Acta21S,44\. Giegel, J. L., Ham, A. B. and Clema, W. (1975). Clin. Chem. (Winston-Salem, N.C.) 21,

1575. Gione, E. and Orning, O. M. (1966). Scand. J. Clin. Lab. Invest. 18, 209. Gitler, C. (1972). Anal. Biochem. 50, 324. Gluck, L , Kulovich, M. V. and Brody, S. J. (1966). J. Lipid Res. 7, 750. Golborn, J. (1969). / . Amer. Oil Chem. Soc. 46, 385. Gray. G. M. (1967). Biochim. Biophys. Acta 144, 519. Guimaraes, G. (1967). Hospital (Rio de Janeiro) 72, 929 (Anal. Abs. 15, 4986). Habermann, H., Brandtlow, G. and Krusche, B. (1961). Klin. Wochschr. 39, 816. Hack, M. H. and Ferrans, V. J. (1959). Z. Physiol. Chem. 315, 157. Hack, M. H. and Leatherman, C. W. (1961). / . Chromatogr. 5, 531. Hanahan, D. H., Dittmer, J. C. and Warashina, E. (1957). / . Biol. Chem. 228, 685. Harris, R. A. and Gambal, D. (1963). Anal. Biochem. 5, 479. Haux, P. and Natelson, S. (1971). Microchem. J. 16, 68. Haworth, D. T., Kahn, D. R., Lemm, A. W. and Hoffman, N. E. (1975). Bioinorg. Chem.

4, 303 Hellwig, J. and Schoellner, R. (1973). Plaste, Kaut. 20, 216. Helman, E. Z., Blevins, E. J. and Gleason, I. O. (1971). Clin. Chem. 17, 988. Helmsing, P. J. (1967). / . Chromatogr. 28, 131. Heyneman, R. A., Bernard, D. M. and Vercauteren, R. E. (1972). J. Chromatogr. 68,285. Hirayama, O. (1961). Nippon Nogei Kagaku Kaishi 35, 367 (Chem. Abs. 60, 1941).

REFERENCES 211

Hites, R. A. (1970). Anal. Chem. 42, 1736. Hoeflmayr, J. and Fried, R. (1974). Arzneimittelforsch. 24, 904. Horhammer, L., Wagner, H. and Richter, G. (1959). Biochem. Z. 331, 155. Hoffman, L. M., Fok, W. and Schneck, L. (1974). J. Lipid Res. 15, 283. Hojnacki, J. L. and Smith, S. C. (1974). J. Chromatogr. 90, 365. Holczabek, W. (1967). Fettstoffwechsel 3, 25 (Chem. Abs. 70, 112211). Hollender, A., Roos, K. and Henriksen, A. (1970). Scand. J. Clin. Lab. Invest. 25, 181. Holub, W. R. (1973). Clin. Chem. 19, 1391. Hori, S , Ikewa, Y. and Uzawa, H. (1970). Rinsho Byori 18, 588 (Chem. Abs. 74, 39036). Horrocks, L. A. (1963). / . Amer. Oil Chem. Soc. 40, 235. Hubmann, F.-H. (1973). J. Chromatogr. 86, 197. Huebner, V. R. (1961). J. Amer. Oil Chem. Soc. 38, 628. Iacono, J. M. and Ishikawa, T. T. (1969). / . Chromatogr. 40, 175. Ignatowska, H. (1964). Polskie Arch. Med. Wewnetrznej 34, 273 (Chem. Abs. 63, 3299). Ilinov, P. and Kharizanova, N. (1972). Vnitf. Bolesti 11, 48 (Anal. Abs. 26, 359). Imai, C , Watanabe, H., Haga, N. and Ii, J. (1974). / . Amer. Oil Chem. Soc. 51, 326. Ishida-Ichimasa, M., Ichimasa, Y. and Uranaka, T. (1976). Agric. Biol. Chem. 40, 1253

(Anal. Abs. 32, 2D 37). Isoda, Y. (1973). Yukagaku 22, 475 (Chem. Abs. 80, 13684). Jaross, W. and Freimuth, U. (1970). Z. Med. Labortech. 11, 322 (Anal. Abs. 22, 997). Jatzkewitz, H. and Mehl, E. (1960). Z. Physiol. Chem. 320, 251. Jones, D., Bowyer, D. E., Gresham, G. A. and Howard, A. N. (1966). J. Chromatogr. 24,

226. Jover, A. (1963). J. Lipid Res. 4, 228. Jungalwala, F. B., Evans, J. E. and McCluer, R. H. (1976). Biochem. J. 155, 55. Jurriens, G. (1964). Riv. Ital. Sostanze Grasse 41, 4. Kabara, J. J. and Chen, J. S. (1976). Anal. Chem. 48, 814. Kahovcova, J. and Odavic, R. (1969). / . Chromatogr. 40, 90. Kanematsu, H., Maruyama, T., Niiya, I., Imamura, M. and Kawakita, H. (1972). Eiyo

To Shokuryo 25, 46 (Chem. Abs. 77, 3877). Kanno, T. and Hirabayashi, T. (1969). Rinsho Byori 17, 567 (Chem. Abs. 71, 120340). Karagezyan, K. G. (1969). Lab. Delo 23. Karmen, A. (1967). Separ. Sci. 2, 387. Katyal, S. L., Eapen, S. and Venkitasubramanian, T. A. (1969). Indian J. Biochem. 6,84. Kaufmann, H. P. and Das, B. (1962). Fette, Seifen, Anstrichm. 64, 214. Kaufmann, H. P. and Grothues, B. (1961). Fette, Seifen, Anstrichm. 63, 1021. Kaufmann, H. P. and Khoe, T. H. (1962). Fette, Seifen, Anstrichm. 64, 81. Kaufmann, H. P. and Makus, Z. (1959). Fette, Seifen, Anstrichm. 61, 631. Kaufmann, H. P. and Mukherjee, K. D. (1965). Fette, Seifen, Anstrichm. 67, 183. Kaufmann, H. P. and Mukherjee, K. D. (1969). Fette, Seifen, Anstrichm. 71, 11. Kaufmann, H. P. and Schnurbusch, H. (1959). Fette, Seifen, Anstrichm. 61, 523. Kaufmann, H. P. and Wessels, H. (1964). Fette, Seifen, Anstrichm. 66, 81. Kaufmann, H. P., Makus, Z. and Das, B. (1961). Fette, Seifen, Anstrichm. 63, 807. Kaufmann, H. P., Radwan, S. S. and Ahmed, A. K. S. (1966). Fette, Seifen, Anstrichm.

68, 261. Kaufmann, H. P., Mangold, H. K. and Mukherjee, K. D. (1971). J. Lipid Res. 12, 506. Kempe, C. R. and Tannert, W. K. (1972). / . Forensic Sci. 17, 323. Kennan, R. W., Schmidt, G. and Tanaka, T. (1968). Anal. Biochem. 23, 555. Kilroe-Smith, T. A. (1969). / . Chromatogr. 41, 116. Klein, R. A. (1971). J. Lipid Res. 12, 123.


Klemig, H. and Lempert, U. (1970), J. Chromatogr. 53, 595. Kohlbeck, J. A. (1965). Anal. Chem. 37, 1282. Korolczuk, J. and Kwasniewska, I. (1974). J. Chromatogr. 88, 428. Krell, K. and Hashim, S. A. (1963). J. Lipid Res. 4, 407. Krien, G. (1963). Explosivstoffe 11, 207. Kritchevsky, D., Davidson, L. M., Kim, H. K. and Malhotra, S. (1973). Clin. Chim.

Acta 46, 63. Kuksis, A. (1967). Lipid Chromatogr. Anal. 1, 239. Kuksis, A. (1971). Fette, Seifen, Anstrichm. 73, 332. Kuksis, A. (1973). Fette, Seifen, Anstrichm. 75, 317. Kuksis, A. and Breckenridge, W. C. (1966). J. Lipid Res. 7, 576. Kuksis, A. and Ludwig, J. (1966). Lipids 1, 202. Kuksis, A. and McCarthy, M. J. (1962). Can. J. Biochem. Physiol. 40, 679. Kuksis, A., Marai, L. and Gornall, D. A. (1967). J. Lipid Res. 8, 352. Kundu, M. K. (1969). J. Chromatogr. 41, 276. Kunz, F. (1973). Biochim. Biophys. Acta 296, 331. Kunz, F. and Kosin, D. (1970). Clin. Chim. Acta 27, 185. Kwapniewski, Z. and Sliwiok, J. (1964). Mikrochim. Acta 616. Lartillot, S. and Vogel, C. (1970). Feuill. Biol 11, 39 (Chem. Abs. 74, 83874; Anal Abs.

21, 4263). Laurell, S. (1966). Scand. J. Clin. Lab. Invest. 18, 668. Laurell, S. (1968). Biochim. Biophys. Acta 152, 75. Lee, A. K. Y. and Mezei, M. (1971). Chromatographia 4, 3. Leeder, L. G. and Clark, D. A. (1967). Microchem. J. 12, 396. Lefort, D., Perron, R., Pourchez, A., Madelmont, C. and Petit, J. (1966). J. Chromatogr.

22, 266. Lemaur, R. and Le Palec, J. P. (1970). Ann. Pharm. Franc. 28, 257. Letters, R. (1964). Biochem. J. 93, 313. Lindqvist, B., Sjoegren, A. and Nordin, R. (1974). J. Lipid Res. 15, 65. Lines, J. G. (1965). Biochem. Probl. Lipids, Proc. 7th Intern. Conf. Birmingham 17

(Chem. Abs. 64, 20164). Litchfield, C. (1968). Lipids 3, 170. Litchfield, C , Harlow, R. D. and Reiser, R. (1965). / . Amer. Oil Chem. Soc. 42, 849. Lloyd, J. B. F. (1967). Forensic Sci. Soc. J. 7, 198. Loehnert, P. and Schoellner, R. (1968). Plaste, Kaut. 15, 682. Lutura, M. G and Sheltawy, A. (1972). Biochem. J. 126, 251. Macke, G F. (1968). J. Chromatogr. 38, 47. Maeda, S. (1966). Bull. Pharm. Res. Inst. (Osaka) 62, 11 (Chem. Abs. 67, 6151). Malins, D. C , Wekell, J. C. and Houle, C. R. (1964). Anal. Chem. 36, 658. Mangold, H. K. (1961). J. Amer. Oil Chem. Soc. 38, 708. Mangold, H. K. and Malins, D. C. (1960). J. Amer. Oil Chem. Soc. 37, 383. Mangold, H. K., Lamp, B. G and Schlenk, H. (1955). J. Amer. Chem. Soc. 11, 6070. Manners, M. J. Kidder, D. E. and Parsons, P. M. (1969). J. Chromatogr. 43, 276. Mano, M. L. (1965). Rev. Port. Farm. 15, 398 (Chem. Abs. 64, 11767). Mantel, M. J., Van Riel, L. H. M. and Buys Ballot, A. F. K. (1975). Clin. Chim. Acta 63,

297. Marinetti, G. V. and Stolz, E. (1956). Biochim. Biophys. Acta 21, 168. Marsh, J. B. and Weinstein, D. B. (1966). J. Lipid Res. 1, 574. Marvillet, L. (1958). Mem. Poudres 40, 273. Marzo, A., Ghirardi, P., Sardini, D. and Meroni, G. (1971). Clin. Chem. 17, 145.

REFERENCES 213

Matsui, M., Sato, K. and Ikewawa, N. (1969). Eisei Kagaku 15, 61 (Chem. Abs. 71, 79800).

Matsui, M., Watanabe, T. and Ikewawa, N. (1973). Nippon Suisan Gakkaishi 39, 367 (Chem. Abs. 79, 29175).

Matsumiya, K., Arao, M., Nakamura, M., Okishio, T. and Omori, K. (1970). Rinsho Byori 18, 383 (Chem. Abs. 73, 95313).

Medvedev, I. K. and Kalantar, I. L. (1972). Nov. Metody Modif. Biokhim. Fiziol. Issled. Zhivotnovod. No. 2,44 (Chem. Abs. 81, 60280).

Merck (Darmstadt, Germany). "Anfarbereagenzien fur Dunnschicht- und Papier-chromatographie", pp. 28-9.

Mezesova, V. (1967). Chem. Listy 61, 659. Michalec, C , Sulc, M., Mestan, J., Kolman, Z. and Jichovd, M. Cas. Lek. Cesk.

100, 909 (Anal Abs. 9,1234). Michalec, C , Sulc, M. and Meslan, J. (1962). Nature (London) 193, 63. Minnikin, D. E. and Abdolrahimzadeh, H., (1971). J. Chromatogr. 63, 452. Misra, U. K. (1968). Biochim. Biol. Sper. 7, 57. Miyashita, Y., Kodera, H. and Maeda, H. (1974). Jap. Patent No. 74 23,693, Mar. 2

(Chem. Abs. 81, 60501). Mlekusch, W., Truppe, W. and Paletta, B. (1973). Clin. Chim. Acta 49, 73. Mlekusch, W., Truppe, W. and Paletta, B. (1974). J. Chromatogr. 93, 183. Moline, C and Barron, E. J. (1969). Clin. Biochem. 2, 321. Montet, J. C , Amic, J. and Hauton, J. C. (1970). Bull. Soc. Chim. Biol. 52, 831. Morin, R. J. (1966). Clin. Chim. Acta 13, 395. Muldrey jr., J. E. Miller, N. and Hamilton, J. G. (1959). J. Lipid Res. 1, 48. Murata, T. and Takahashi, S. (1973). Anal Chem. 45, 1816. Musil, F. (1965). Cas. Lek. Cesk. 104, 965 (Chem. Abs. 65, 5841). Nardi, E , Ronchi, F., Sasso, G. F. and Mancini, L. G. M. (1971). Boll. Soc. Ital. Biol

Sper. 47, 337. Naudet, M., Pasero, J. and Biasini, S. (1965). Rev. Franc. Corps Gras 12, 515. Neissner, R. (1969).Pharm. Ind. 31, 724. Nelson, G. J. and Booth, R. A. (1967). Anal. Biochem. 20, 198. Nelson, G. J. and Freeman, N. K. (1959). J. Biol. Chem. 234, 1375. Neskovic, N. M. (1967). J. Chromatogr. 27, 488. Neskovic, N. M. and Kostic, D. M. (1968). J. Chromatogr. 35, 297. Neskovic, N. M., Nussbaum, J. L. and Mandel, P. (1970). J. Chromatogr. 49, 255. Nickell, E. C. and Privett, O. S. (1967). Sep. Sci. 2, 307. Nicolaysen, R. and Nygaard, A. P. (1963). Scand. J. Clin. Lab. Invest. 15, 79. Nicolosi, R. J., Smith, S. C. and Santerre, R. F. (1971). J. Chromatogr. 60, 111. Nishimura, T., Imai, T., Okawa, S. and Tomita, S. (1973). Rinsho Byori 21, 839 (Chem.

Abs. 80, 142625). Novitskaya, G. V. and Vereshchagin, A. G. (1969). J. Chromatogr. 40, 422. Novotny, M., Segura, R. and Zlatkis, A. (1972). Anal. Chem. 44, 9. Nutter, L. J. and Privett, O. S. (1968). J. Chromatogr. 35, 519 Ohkita, J. (1975). Japan Kokai (Patent) No. 75 72,875, June 16 (Chem. Abs. 83,168230). Ord, W. O. and Bamford, P. C. (1967). Chem. Ind. (London) 277. Ory, R. L. (1961). J. Chromatogr. 5, 153. Ovenston, T. C. J. (1949). Analyst (London) 74, 344. Owens, K. (1966). Biochem. J. 100, 354. Palmer, D. A., Kintzios, J. A. and Papadopoulos, N. M. (1972). J. Chromatogr. Sci. 10,


Parihar, D. B , Sharma, S. P. and Verma, K. K. (1967). J. Chromatogr. 31, 551. Parker, F. and Peterson, N. F. (1965). J. Lipid Res. 6, 455. Parodi, P. W. (1975). J. Chromatogr. I l l , 223. Pei, P. T. S., Henly, R. S. and Ramachandran. S. (1975). Lipids 10, 152. Persmark, U. and Toregard, B. (1968). J. Chromatogr. 37, 121. Pinter, K. G , Hamilton, J. G. and Miller, O. N. (1964). Anal. Biochem. 8, 158. Pizzoli, E. M., De Marco, O. and Notarnicola, L. (1967). Riv. Ital. Sostanze Grasse 44,

62. Pocock, D. M. E., Rafal, S. and Vost, A. (1972). J. Chromatogr. Sci. 10, 72. Pokorny, J. and Herodek, O. (1964). Sb. Vys. Sk. Chem.-Technol. v Praze, Potrav. Tech-

nol. 8, SI (Anal. Abs. 13, 1542). Pokorny, J., Kuman, M. K. and Janicek, G. (1973). J. Soc. Cosmet. Chem. 24, 753

(Chem. Abs. 80, 74256). Pollack, J. D., Clark, D. S. and Somerson, N. L. (1971). J. Lipid Res. 12, 563. Poorthuis, B. J. H. M., Yazaki, P. J. and Hosteller, K. Y. (1976). J. Lipid Res. 17, 433. Popov, A. and Steganov, K. (1968). J. Chromatogr. 37, 533. Popov, A., Chobanov, D. and Stefanov, K. (1967). Maslo-Sapunena Prom., Byul. 3, 3

(Chem. Abs. 68, 88410). Postma, T. and Stroes, J. A. P. (1970). Z. Klin. Chem. Klin. Biochem. 8, 520. Prat, Y. and Forestier, H. (1963). Mem. Poudres 45, 215. Pre, J. and Gamier, M. (1973). Pathol.-Biol. 21, 1147. Pristera, F. (1953). Anal. Chem. 25, 844. Privett, O. S. and Blank, M. L. (1962). J. Amer. Oil Chem. Soc. 39, 520. Ranny, M. (1966). Abh. Deut. Akad. Wiss., Berlin, Kl. Chem. Geol. Biol. 216. Ranny, M. (1968). Veda Vyzk. Prum. Potravin. 18, 191 (Chem. Abs. 72, 62550). Rao, P. B. and Sreenivasan, B. (1966). Chem. Ind. (London) 1376. Rastogi, S. C , Srivastava, K. C. and Tiwari, R. D. (1971). Z. Anal. Chem. 253, 208. Ripper, E. (1967). Explosivstoffe 15, 57. Ristrow, R. (1968). Deut. Lebensm.-Rundschau 64, 322. Robinson, N. and Phillips, B. M. (1963). Clin. Chim. Acta 8, 385. Roch, L. A. and Grossberg, S. E. (1971). Anal. Biochem. 41, 105. Rohleder, H , Staudacher, H. and Summermann, W. (1976). Z. Anal. Chem. 279, 152. Rojkin, M. L. Repetto, J. R. and Zaccara, F. A. (1973). Bioquim. Clin. 7, 135. Romans, J. R. and Palmer, I. S. (1972). Anal. Biochem. 49, 580. Roozemond, R. C. (1969). J. Chromatogr. 41, 270. Rosseel, M. T and Bogaert, M. G. (1972). J. Chromatogr. 64, 364. Rosseel, M. T. and Bogaert, M. G. (1973). J. Pharm. Sci. 62, 754. Roughan, P. G. and Batt, R. D. (1968). Anal. Biochem. 22, 74. Rouser, G. (1968). Biochem. Prep. 12, 73. Rouser, G , Siakotos, A. N. and Fleischer, S. (1966). Lipids 1, 85. Royer, M. E. and Ko, H. (1969). Anal. Biochem. 29, 405. Ruys, J., Crollini, C. and Hickie, J. B. (1975). Med. J. Aust. 1, 385. Ryan, W. G. and Rasho, O. M. (1967). Clin. Chem. 13, 769. Rybicka, S. M. (1962). Chem. Ind. (London) 308. Sahasrabudhe, M. R. (1967). J. Amer. Oil Chem. Soc. 44, 376. Sallee, T. L. and Adams, G. M. (1970). J. Chromatogr. 51, 545. Saracco, G. B. and Gay, M. (1971). Riv. Ital. Sostanze Grasse 48, 319. Sass, N. L., Alvarado, R. and Martin, J. P. (1976). Biochem. Med. 15, 217. Sato, K., Matsui, M. and Ikewawa, N. (1966). Bunseki Kagaku 15, 954. Sato, K., Matsui, M. and Ikewawa, N. (1967). Bunseki Kagaku 16, 1160.

REFERENCES 215

Schiefer, H. G. and Neuhoff, V. (1971). Z. Physiol Chem. 352, 913. Schlierf, G. and Wood, P. (1965). J. Lipid Res. 6, 317. Schmid, H. H. O , Mangold, H. K., Lundberg, W. O. and Baumann, W. J. (1966).

Microchem. J. 11, 306. Schroeder, W. A. (1948). Ann. N.Y. Acad. Sci. 49, 204. Schwaisch, H. and Beyrich, T. (1970). Zbl Pharm. Pharmacol, u. Lab. Diagn. 109,1161

(Anal Abs. 21, 2935). Schwartz, R. D., Mathews, R. G , Ramachandran. S., Henly, R. S. and Doyle, J. E.

(1975). J. Chromatogr. 112, 111. Schwartzman, G. (1956). J. Ass. Off. Agric. Chem. 39, 254. Schwarz, H. P., Dreisbach, L., Stambaugh, R., Kleschick, A. and Barrionuevo, M.

(1960). Arch. Biochem. Biophys. 87, 171. Scrignar, C. B. (1964). J. Chromatogr. 14, 189. Segura, R. and Gotto jr., A. M. (1974). J. Chromatogr. 99, 643. Seiffert, U. B. and Alt, A. (1969). Mikrochim. Acta 456. Shehata, A. A. Y. and De Man, J. M. (1971). Can. Inst. Food Technol. J. 4, 38. Shehata, A. A. Y., De Man, J. M. and Alexander, J. C. (1971). Can. Inst. Food Technol.

J. 4, 6. Shin, Y. S. (1963). Anal. Biochem. 5, 369. Sims, R. P. A. and Larose, J. A. G. (1962). J. Amer. Oil Chem. Soc. 39, 232. Singh, E. J. and Gershbein, L. L. (1967). J. Chromatogr. 31, 20. Sinha, S. K., Bhalla, A. K., Sahasrabudhe, S. K. and Rao, K. R. K. (1964). Paint Manuf.

34, 141 (Chem. Abs. 60, 11835). Sinha, S. K., Surve, R. N., Bahadur, K. and Patwardhan, W. D. (1970). Indian J. Tech-

nol 8, 33. Skipski, V. P., Peterson, R. F. and Barclay, M. (1962). J. Lipid Res. 3, 467. Skipski, V. P., Peterson, R. F., Sanders, J. and Barclay, M. (1963). J. Lipid Res. 4, 227. Skipski, V. P., Peterson, R. F. and Barclay, M. (1964). Biochem. J. 90, 374. Skipski, V. P., Good, J. J., Barclay, M. and Reggio, R. B. (1968). Biochim. Biophys.

Acta 152, 10. Sliwiok, J. and Kwapniewski, Z. (1965). Mikrochim. Acta 1. Slomiany, B. L. and Horowitz, M. I. (1970). J. Chromatogr. 49, 455. Smernoff, R. B. (1971). German Patent No. 2,113,762, Oct. 14th (Chem. Abs. 76,11795). Smith, B. G. (1974). U.S. Patent No. 3,817,706, June 18th (Chem. Abs. 81, 72350). Sobotka, J. (1971). Vnitf. Lek. 17, 600 (Chem. Abs. 76, 11726). Soeterboek, A. M. and Van Thiel, E. M. (1973). Pharm. Weekbl. 108, 854. Soloni, F. G. (1971). Clin. Chem. 17, 529. Sone, A., Shoyama, M., Ono, E. and Fukui, I. (1973). Rinsho Byori 21, 717 (Chem. Abs.

80, 24453). Spitzer, H. L. and Balint, J. A. (1963). Anal Biochem. 5, 143. Srivastava, K. C. and Rastogi, S. C. (1969). Z. Anal Chem. 244, 189. Stahl, E. (1969). "Thin-Layer Chromatography", Springer-Verlag, p. 256. Stajner, A. (1966). Vnitf. Lek. 12, 814 (Chem. Abs. 65, 20492). Steiner, E. H. and Bonar, A. R. (1961). J. Sci. Food Agr. 3, 247. Stolyhwo, A. and Privett, O. S. (1973). J. Chromatogr. Sci. 11, 20. Stone, M. C. and Thorp, J. M. (1966). Clin. Chim. Acta 14, 812. Stott, A. W. (1972). Clin. Chim. Acta 37, 535. Sundler, R. and Akesson, B. (1973). J. Chromatogr. 80, 233. Svetashev, V. I. and Vas'kovskii, V. E. (1972). J. Chromatogr. 67, 376. Swartout, J. R. and Gross, R. J. (1964). J. Amer. Oil Chem. Soc. 41, 378.


Teichman, R. J., Takei, G. H. and Cummins, J. M. (1974). J. Chromatogr. 88, 425. Thiele, O. W. and Wober, W. (1964). Z. Anal. Chem. 205, 442. Thomas III, A. E., Scharoun, J. E. and Ralston, H. (1965). J. Amer. Oil Chem. Soc. 42,

789. Thompson, J. B., Langley, R. L., Hess, D. R. and Welsh, J. D. (1969). J. Lab. Clin. Med.

73, 512. Tippett, C. F. (1963). Med. Sci. Law 3, 282. Torjescu, V., Valeanu, M. and Torjescu, A. (1968). Viata Med. 15, 1201 {Chem. Abs. 70,

103600). Torres, J. F., Jodos, D. C. de and Fregosi, E. V. de (1971). Revta Asoc. Bioquim. Argent.

36, 136 (Anal. Abs. 22, 4260). Trapp, W. (1976). Z. Chem. (Leipzig) 16, 283. Trappe, J. S., Brockington, P. B. and Vickers, M. F. (1975). Clin. Biochem. 8, 142. Trippel, A. I. (1964). Sb. Tr. Leningr. Inst. Sov. Torgovli No . 23, 151 (Chem. Abs. 63,

18497). Trowbridge, J. R., Herrick, A. B. and Bauman, R. A. (1964). J. Amer. Oil Chem. Soc. 41,

306. Trowell, J. M. (1970). Anal. Chem. 42, 1440. Truppe, W., Mlekusch, W. and Paletta, B. (1972). J. Chromatogr. 72, 405. Urakami, C. and Kakutani, Y. (1957). Bull. Chem. Soc. Japan 30, 21. Utrilla, R. M., Juarez, M. and Martinez, I. (1976) Grasas Aceitas (Seville) 27, 5 Vacikova, A., Felt, V. and Malikova, J. (1962). J. Chromatogr. 9, 301. Vandamme, D., Declercq, B., Denoc, A., Blaton, V. and Peeters, H. (1975). Anal. Bio-

chem. 69, 29. Vandercook, C. E , Guerrero, H. C. and Price, R. L. (1970). J. Agr. Food Chem. 18,

905. Van Gent, C. M. (1968). Z. Anal. Chem. 236, 344. Venkata Rao, P., Ramachandran, S. and Cornwell, D. G. (1968). Lipids 3, 187. Verder, H. and Clausen, J. (1974). Clin. Chim. Acta 51, 257. Vereshchagin, A. G. (1965). J. Chromatogr. 17, 382. Vereshchagin, A. G. (1971). Biokhim. Metody Fiziol. Rast. 208 (Chem. Abs. 11, 2430). Verhoeven, A. G. J. and Merkus, H. M. W. M. (1974). Clin. Chim. Acta 53, 229. Vioque, E , Murillo, H. and Maza, M. P. (1970). Grasas Aceites (Seville) 21, 72. Vioque, E., Maza, M. P. and Murillo, H. (1973). Grasas Aceites (Seville) 24, 94. Viswanathan, C. V. (1973). J. Chromatogr. 75, 141. Viswanathan, C. V. (1974a). J. Chromatogr. 98, 105. Viswanathan, C. V. (1974b). J. Chromatogr. 98, 129. Vogel, W. C , Diozaki, W. M. and Zieve, L. (1962). J. Lipid Res. 3, 188. Vorbeck, M. L. and Marinetti, G. V. (1965). J. Lipid Res. 6, 3. de Vries, B. (1964). J. Amer. Oil Chem. Soc. 41, 403. Walker, B. L. (1971). J. Chromatogr. 56, 320. Watts, R. and Dils, R. (1968). J. Lipid Res. 9, 40. Webb, R. A. and Mettrick, D. F. (1972). J. Chromatogr. 67, 75. Wessels, H. (1973). Fette, Seifen, Anstrichm. 75, 478. Wessels, H. and Rajagopal, N. S. (1969). Fette, Seifen, Anstrichm. 71, 543. Whitner, V. O., Grier, O. T., Mann, A. N. and Witter, R. F. (1965). J. Amer. Oil Chem.

Soc. 42, 1154. Wiklund, B. and Eliasson, R. (1972). J. Chromatogr. 68, 153. Wildgrube, H. J. and Erb, W. (1971). Verh. Deut. Ges. Inn. Med. 11, 588 (Chem. Abs. 11,

16117).

REFERENCES 217

Wildgrube, H. J, Erb, W. and Bohle, E. (1969). Z. Klin. Chem. Klin. Biochem. 7, 514. Wildgrube, H. J., Erb, W. and Bohle, E. (1973). Fette, Seifen. Anstrichm. 75, 168. Williams, A. F., Murray, W. J. and Gibb, B. H. (1966). Nature (London) 210, 816. Williams, J. H., Kuchmak, M. and Witter, R. F. (1969). Clin. Chim. Acta 25, 447. Wood, G. W. (1974). Biomed. Mass Spectrom. 1, 154. Wren, J. J. and Elliston, S. C (1961). Chem. Ind. (London) 80. Yamada, T. (1972). Bull. Pharm. Res. Inst. (Osaka) No. 96/97,1 (Chem. Abs. 78, 40008). Yamada, M. and Miyazaki, T. (1975). Rinsho Kensa 19, 197 (Chem. Abs. 83, 24612);

also Eisei Kensa 24, 391 (Chem. Abs. 83, 174985). Young, G. and Eastman, R. (1963). S. Afr. J. Lab. Clin. Med. 9, 28. Yurkiewicz, W. J. (1968). Ann. Entomol. Soc. Amer. 61, 1026. Zajac, J. (1962). Chem. Anal. (Warsaw) 7, 995. Zuckerman, J. L. and Natelson, S. (1948). J. Lab. Clin. Med. 33, 1322.

5

Analysis of Glycerol

In this chapter some words are devoted to the purity demands on glycerol as given, for example, in the various world pharmacopoeias and in specifi-cations such as those published by the British Standards Institution. Firms supplying chemicals also quote purity data for their products. The demands depend of course on the quality of the material, whether crude, technical, refined, chemically pure, or otherwise described. The discussion here is limited to the refined or chemically pure quality.

The specifications comprise tests for constituents which are undesirable or tolerable only in small amounts. The tests are devised so as to show whether these limits are exceeded or not.

5 . 1 . P H A R M A C O P O E I A TESTS

A comparison is made below of the tests of a number of pharmacopoeias, namely, those of Austria, Britain, France, the German Federal Republic (G.F.R.), the German Democratic Republic (G.D.R.), Japan, Switzerland, and the United States. Their demands and also the principles of the tests have much in common but diverge from one another in certain ways. Thus all test for acidity, chloride ion, sulphate ion, various metals, especially arsenic, and for "reducing compounds". This test for "reducing compounds" or "reducing properties" is sometimes meant more specifically for acrolein and/or glucose. In most cases, tests are carried out for ester, ammonium ion, total chlorine (i.e. organically combined chlorine as well as inorganic chloride ion), and for certain combustible products and residues (e.g. "ignition residue", "readily carbonisable material", "sulphate ash"). Some pharmacopoeias quote tests for the glycerol itself.

219

220 ANALYSIS OF GLYCEROL 5.1

Further information is presented below under the headings of the particular tests.

5 . 1 . 1 . Ch lor ide Ion

The sample in aqueous solution is treated with nitric acid and then silver nitrate; the silver chloride turbidity is compared with that from a standard reference chloride amount.

5.1.2. Su lpha te Ion

The turbidity of barium sulphate yielded by an aqueous solution of sample with a barium chloride reagent is compared with that from a standard refer-ence sulphate solution.

5.1.3. Tota l ( i n c l u d i n g Organ ic ) Ch lor ine

Organically bound chlorine is converted into chloride ion, and a silver nitrate turbidity test carried out as for free chloride ion. Slight differences in treat-ment are encountered. In the British and U.S. pharmacopoeias, 5 g of sample are refluxed with 15 ml of morpholine for 3 h before acidifying with nitric acid and adding silver nitrate. The G.F.R. procedure involves heating 5 g of sample with 1 ml of 3N sodium hydroxide and 50 mg of Raney nickel for 10 min on the boiling water bath. According to the G.D.R. pharmacopoeia, a 10 ml sample is refluxed for 2 h with a solution of 5 g of potassium hydroxide in 8 ml of water.

5.1.4. Arsen ic

Combined arsenic is reduced to arsenic hydride, AsH 3 , which is then detected. The customary reducing agent is a zinc-stannous ion reagent, and the hydride is usually detected with a paper strip saturated with a mercury(II) reagent; this yields a dark zone of mercury metal. In the U.S. procedure, the detection reagent is silver diethyldithiocarbamate, with which the arsenic hydride gives a red product.

5.1.5. Heavy Meta ls

Heavy metals are detected by treating the sample under weakly acid con-ditions (pH 3-4) with sulphide ion and observing the brown or black turbidity or precipitate of insoluble sulphides, notably of lead. Sodium or hydrogen sulphides are the usual sources of sulphide ion but the G.F.R. employ thioacetamide which gives hydrogen sulphide rapidly through hydrolysis.

5.1 PHARMACOPOEIA TESTS 221

The Austrians have a special test for iron, oxidising with bromine water to the ferric state and then adding ammonium thiocyanate which gives the characteristic red colour.

5.1.6. A c i d i t y , A l k a l i n i t y

The acid and alkali contents of the sample are tested by titration with standard alkali or acid, respectively, generally to phenolphthalein indicator. The maxi acceptable titrations are prescribed.

5.1.7. R e d u c i n g Propert ies, Mater ia ls , Impur i t ies ; A c r o l e i n ; G lucose

The test for reducing properties, etc., is usually in two stages. First, the sample is heated in alkaline solution, whereby the yellow or brown coloration must be limited. Then silver nitrate is added and the warm solution observed for a dark coloration or precipitate indicating reduction to silver metal.

The sample is generally made alkaline with ammonium hydroxide and heated for 5 min at 60°C (e.g. the French, both German, and the Japanese procedures). After adding the silver nitrate, the solution is left in the dark for some minutes (e.g. 5 min according to the British and G.F.R. and 10 min according to the French method).

The Swiss pharmacopoeia prescribes a mixed silver nitrate-sodium hydroxide reagent, warming for 15 min at 50°C .The Austrian and U.S. procedures warm only with alkali hydroxide for 5 min at 60°C and observe any yellow colour. They test at the same time for ammonium ion (see below).

5.1.8. A m m o n i u m C o m p o u n d s

All except France and the G.D.R. test for ammonium compounds. The sample is warmed with dilute sodium hydroxide; no ammonia should be detectable through smell or action on red litmus paper held in the issuing vapours. The Swiss test the solution with potassium iodobromomercurate (similar to the Nessler reagent) and the colour is compared with that from a standard.

As mentioned above, the Austrian and U.S. procedures combine this test with that for acrolein-glucose.

5.1.9. Sugars

The pharmacopoeias of Austria, Britain, and Switzerland test for reducing sugars by heating first with dilute sulphuric acid (inversion) and then with a copper sulphate-sodium hydroxide reagent. This should remain blue after


further heating and give no colour or precipitate of cuprous oxide which would indicate that reduction has occurred.

5.1.10. Esters

Ester content is evaluated by heating with a measured amount of excess alkali and back-titrating the unreacted alkali with standard acid; the back-titration must exceed a certain amount.

5 .1 .11 . Behav iour t o w a r d s S u l p h u r i c A c i d

This is sometimes termed a test for foreign organic materials or carbonisable materials. The sample is mixed with sulphuric acid and allowed to stand or heated. Any darkening which may occur must not yield a colour more intense than that of a standard, often made up from ferric, cobalt, and some-times cupric ions in acid (hydrochloric) solution. The treatments in some pharmacopoeias are given in Table 5.1.

TABLE 5.1

Behaviour towards Sulphuric Acid

Pharmacopoeia Acid Treatment

Austria ca. I N 15min/100°C G.F.R. cone. directly observed France 20% 15min/100°C Japan 95% 1 h/15-25°C U.S. cone. 1 h standing

5.1.12. Su lpha te A s h

This test is required by the pharmacopoeias of Britain, France, the G.F.R., and the G.D.R. It depends on adding sulphuric acid, charring, and finally igniting to an ash residue which may not exceed a certain limit.

5.1.13. Oxalate

The test for oxalate is required only by the Swiss authorities. It involves reaction with a calcium reagent to yield a turbidity of calcium oxalate, which must not exceed that of a standard oxalate amount.

5.1 PHARMACOPOEIA TESTS 223

5.1.14. Calcium, Magnesium, Barium Ions

The Swiss pharmacopoeia tests also for these three metals, the first two with oxalate, the last with sulphate. The tests are thus the converse of those for oxalate and sulphate (see above)

5.1.15. Oxidising Substances

This test also is to be found only in the Swiss requirements. A 10 ml sample is treated with potassium iodide and dilute sulphuric acid. After 15 min, starch is added. If blue is yielded by reaction of any liberated iodine with the starch, it must be dischargeable with 01 ml of 0*1 N thiosulphate.

5.1.16. Test for Glycerol Itself

With one exception this test depends on heating with potassium hydrogen sulphate and subsequent detection of acrolein. Table 5.2 indicates how this detection is performed.

TABLE 5.2

Detection of Acrolein yielded in Test for Glycerol

Pharmacopoeia Detection

Britain Irritating vapour G.F.R. Acrid smell; blackening of a test paper saturated with

Nessler reagent (reduction to mercury) (Ch. 1, Sect. 1.4.3)

G.D.R. Acrid smell; intense blue with paper saturated with 1 % sodium nitroprusside-piperidine (19 -1- 1) (Ch. 1, Sect. 1.4.4)

Japan Odour

The Swiss add 65 % nitric acid to the sample and then cover carefully with some drops of potassium dichromate solution. The blue ring yielded must not diffuse into the lower layer within 10 min.

A discussion of the specifications for glycerol published by standards institutions in different countries would take us too far; also, details are not given of the tests etc. carried out by firms that supply chemicals. The specifi-cations of the British Standards Institution include quantitative determina-tions as well as the limit tests. Briefly, these are: chloride ion (titration with silver nitrate); arsenic (estimation of the size of the dark zones, compared visually with those from standards); acidity and alkalinity (quantitative


titration with sodium hydroxide or hydrochloric acid, respectively, to phenolphthalein indicator); ash from ignition (gravimetric); iron (spectro-photometry using thioglycollic acid); esters (saponification with a measured amount of sodium hydroxide in excess and back-titration of the unused with sulphuric acid to phenolphthalein); propane-1,3-diol (gas chromatography); nitrogen content (Kjeldahl method); sugars (quantitative oxidation with Bertrand copper(II) reagent).

5.2. D E T E R M I N A T I O N OF W A T E R IN G L Y C E R O L

This determination merits a special section because it gives a clear intimation of the quality and value of a glycerol sample. Three general types of method can be distinguished: chemical methods, based on reaction of the water; purely physical methods; methods involving physical separation of the water.

5 .2 .1 . Chemica l M e t h o d s

Many chemical reagents have been suggested for determination of water especially in organic liquids. Unfortunately most of these enter into reaction also with organic hydroxyl groups and are therefore applicable to alcohols, such as glycerol, only when the speed of the reaction with water far exceeds that with the hydroxyl compound.

A. K A R L F I S C H E R T I T R A T I O N

The best-known method for determination of water is named after Fischer (1935) who used a reagent consisting of sulphur dioxide, iodine, pyridine, and an alcohol, usually methanol although nowadays methyl-cellosolve has gained favour. Water reacts according to the equations:

Titration can be carried out with the reagent, taking as end-point the first excess of reagent. Since the reaction products are colourless and the reagent dark coloured from the iodine, this end-point can be the first more enduring

s o 3 -

+

5.2 DETERMINATION OF WATER IN GLYCEROL 225

yellow to brown. More sensitive end-points are achieved with spectro-photometric or electrometric procedures based on indication of the electro-active iodine. The more sophisticated procedures today employ electrolyti-cally generated iodine. Burette titration with electrometric end-point determination is the procedure given in the specifications of the British Standards Institution.

B. O T H E R M E T H O D S

Glass (1970) determined water in various neutral solvents (e.g. dioxan, di-ethyl ether, and alcohols, including ethylene glycol and glycerol) using a reagent of sodium methoxide in a mixture of dry methanol and ethyl acetate. Water reacts with the alkoxide to yield sodium hydroxide which is removed by saponification reaction with the ester:

C H 3 C O O C 2 H 5 + N a O H ^ C H 3 C O O N a + + C 2 H 5 O H

This results in a loss of alkali, shown by the difference between subsequent titration of the solution to phenolphthalein with ethanolic hydrochloric acid and the titration of a control amount of alkoxide reagent without sample.

Doubts about the value of standard reagents for water were mentioned above. Such reagents are acid halides and anhydrides, hydrides such as calcium hydride and lithium aluminium hydride, aluminium nitride, an-hydrous copper sulphate, etc. Smith and Bryant (1935) used acetyl chloride for water determination in many liquids, including alcohols. Their reagent was 1-5M in toluene and they mixed a 10 ml aliquot, cooled in ice, with 2 ml of pyridine to give the acetylpyridinium chloride:

This is much less volatile than the free acid chloride. The sample was then added and left usually for about 2 min at room temperature. They decom-posed unused reagent with absolute ethanol or methanol and then titrated with 0-5N sodium hydroxide to phenolphthalein. A control with reagent alone was carried out and the difference in titrations is a measure of the water which hydrolyses the acetylpyridinium chloride:

C O C H 3

CI


COCH3 H

N +

CI + H 2 0 CI + CH3COOH

Both titrated with alkali to phenolphthalein

COCH3 H

CI + C 2 H 5 O H — • CI + C H 3 C O O C 2 H 5

Only the pyridinium salt titrated as acid

The authors found that even an appreciable excess of many alcohols did not interfere because these react much more slowly than does the water. Un-fortunately they obtained poor results with methanol, ethylene glycol, and glycerol, probably because these react too rapidly with the reagent. This is likely to be a handicap to the application of such methods.

5.2.2. Purely Physical M e t h o d s

If the glycerol sample can be regarded as a binary mixture of glycerol and water, that is, if other materials are present in no more than trace amounts, then various physical methods of interpolation are available. This has been discussed in Chapter 1, Section 1.7, although the accent there was on glycerol determination, and in the presence not only of water. Density and refractive index measurements are easy to carry out, and the values for glycerol and water differ sufficiently to provide a fairly sensitive calibration curve relating value with water content. Viscosity is slightly more trouble-some to determine but the values for glycerol and water differ enormously and this offers a highly sensitive method. Haendel (1973) analysed water-glycerol mixtures in this way, employing a capillary viscometer.

Oehme and Ebert (1951) analysed glycerol-water mixtures with the help of dielectric constant measurements. The pure substances have values of 80 (water) and 44 (glycerol) at ca. 20°C. The authors used measuring cells of 10-15 ml capacity and set up a calibration curve relating dielectric constant to composition. The dielectric constant of water is increased by the first few % of added glycerol but thereafter falls steadily if not linearly. Since the dielectric constant values have an appreciable temperature coefficient, they must be recorded at a constant temperature. Oehme and Ebert chose

5.2 DETERMINATION OF WATER IN GLYCEROL 227

18 + 0-5°C. Gotz and Naray (1971) also used dielectric constant measure-ments to estimate the water content (in the 10-20% range) of glycerol.

Infrared spectrophotometry was used by Chapman and Nacey (1958) to determine water in glycerol. They used the water absorption band at l -93p (5180 cm" 1 ) , which is clearly sufficiently separated from the 21 p (4760 cm" 1 ) band of glycerol. A linear relation between absorbance and % water was found in the range 1-20 %.

Kameyama and Semba (1927) suggested analysing glycerol-water mixtures through the conductivity of potassium chloride solution. For a solution of 0471 mg of the salt in 100 ml of glycerol containing n ml of water, they gave the relation:

K x 10 5 = 2-24 + 0-38n + 0-0127n 2

where K is the equivalent conductivity. Other equations could be used with different amounts of potassium chloride.

5.2.3. Separation Techniques

Reference was made in Chapter 1, Section 1.8.2, to the difficulties in the way of complete removal of water from glycerol. An interesting attempt is due to Stuffins (1958) who devised a special apparatus for determining water in margarine, oils, fats, and also glycerol. It consisted of two plates, the lower of which carried the sample; this was then rubbed with the upper plate, evidently to form a film. The combined unit was weighed and placed in a vacuum oven (ca 675 mm Hg) at 58°C. The upper plate was moved away from the lower and the system left for at least 5 h (6h for glycerol). The plates were allowed to cool, brought together again, and re-weighed. The loss in weight gave the water content. The author pointed out certain advantages of the procedure, such as simplicity, easy cleaning, and not demanding particular skill in operation.

Entrainment with a suitable organic solvent has always been a favoured separation method. Horsley (1947,1949) gives over 200 compounds that form azeotropes with water, very few of which also yield azeotropes with glycerol. Many possess an unsuitably high boiling point and contain too little water for reasonably rapid removal from the sample. But several are potentially useful, and a list is given in Table 5.3. The criteria of selection are a boiling point not greater than 85°C and a water content not less than 15 %.

The hydrocarbons and halides would be especially suitable since they are immiscible with water. The distillate would thus separate into two layers and enable the water amount to be read off simply in a graduated tube.

There are, however, few examples of the application of this method to glycerol. Berth (1927) used the apparatus of Tausz and Rumm (1926) to determine water in dynamite glycerol by distilling with tetrachloroethane.


TABLE 5.3

Azeotropes of Water with Some Organic Liquids

Compound B.p. (°C) % Water in azeotrope

Nitromethane 83-6 23-6 Acetonitrile 76 15 Propionitrile 81-5 24 Isobutyronitrile 82-5 23 Ethyl nitrate 74-35 22 Propyl nitrate 84-8 20 Isobutyl formate 79-5 18-9 Aniline 75 81-8 Toluene 84-1 19-6 1,2-Dichloroethane 72 19-5 1-Chloropentane 82 32-1 Diisobutylene 81 87 But-l-yn-3-one 74 35

The water removed was forced into a graduated capillary for volume estima-tion. The author mentioned that silver nitrate had to be added to coloured samples although she offered no reason for this. Jordan and Hatch (1950) used n-butanol to remove water from glycol and glycerol (the butanol azeotrope contains 38 % water and boils at 92-4°C). They added potassium carbonate to the distillate to take up the water, and obtained the amount of water from the volume diminution that this caused. Arana Campos (1967) used xylene as entrainer for water in glycerine, and also added common soap to the sample, presumably to improve miscibility.

Gas chromatography has often been suggested for determination of water in samples but the application to glycerol appears to have been seldom. Bortnikov et al (1967) published such a determination of water in polyols (trimethylene glycol, glycerol), using a 2 m x 6 mm column at 120°C. This was packed with glass balls which had been milled and then mixed with magnesium oxide in the ratio 1:2 and subsequently kept at 80O-900°C for 4-5 h; after cooling, the product was washed with hydrochloric acid and water and dried for 2-3 h at 110-120°C before finally coating with 0-5% SE-30. Helium carrier gas and thermal conductivity detection were employed.

5.3. A S S A Y OF G L Y C E R O L IN P H A R M A C O P O E I A S

Not all the pharmacopoeias given above quote an assay for glycerol. The German Democratic Republic makes use of density values. Their pharma-ceutical product contains 84-88% glycerol, the densities being 1-219 (84%) and 1*230 (88%). Assay is by interpolation.

5.3 ASSAY OF GLYCEROL IN PHARMACOPOEIAS 229

The pharmacopoeias of Austria, Britain and the German Federal Republic

base the determination on the periodate oxidation to formic acid which

is then titrated (cf. Chapter 1, Section 1.1.14.C.1). Table 5.4 enables the

procedures to be compared.

TABLE 5.4

Assay of Glycerol

Pharmacopoeia Sample size Reaction Removal of mixture and unused duration periodate

Titration of formic acid

Austria 0 1 g + 20 ml water + 5 ml With 0-1N + 50 ml 0-05M propylene NaOH to Na periodate; glycol-water phenolphthalein 15 min (1 + 2)

Britain (for 5 ml aliquot + 150 ml water + 3 ml With 0-1N suppositories) from 8 g + Bromocresol propylene NaOH to the

sample made Blue, brought glycol; 5 min Bromocresol up to 250 ml to blue with Blue

0-IN NaOH, then + 1-6 g sodium periodate; 15 min

G.F.R. 01 g + 20 ml water + 5 ml With 0-1N + 5 0 m l 0 0 5 M ethylene N a O H to Na periodate; glycol-water phenolphthalein 15 min (1 + 2)

The procedure of the British Standards Institution can be given also for

comparison; it is stated to be technically similar to the method Ea 6-51 of the

American Oil Chemists' Society and to the procedure adopted by the Oils

and Fats Division of the Applied Chemistry section of the IUPAC (1957):

0 0 5 g (if sensibly pure glycerol)

+ 50 ml water + Bromo-thymol Blue, brought to blue with 0 0 5 N NaOH or to pH 81 + 0 1 ; + 5 0 ml 6 % N a periodate; 30 min at not above 35°C

+ 10 ml ethylene glycol; 20 min/not above 35°C

With 0-125N NaOH to pH 6-5 + 0 1 potent.; blank to pH 8-1 + 0-1

i

230 ANALYSIS OF GLYCEROL

REFERENCES

Arana Campos, J. (1967). Oleagineux 22, 33. Berth, T. (1927). Chemiker-Z. 51, 975. Bortnikov, G. N., Bortnikova, R. N. and Sdobina, R. G. (1967). Zav. Lab. 33, 160. British Standard 2621-5 (1964). "Specifications for Glycerol", British Standards

Institution. Chapman, D. and Nacey, J. F. (1958). Analyst (London) 83, 377. Fischer, K. (1935). Angew. Chem. 48, 394. Glass, R. L. (1970). Anal. Biochem. 37, 219. Gotz, M. and Naray, M. (1971). Acta Pharm. Hung. 41, 129. Haendel, M. (1973). Apotheker-prakt. Pharm.-Tech. Assistent 19, 73 (Chem. Abs. 80,

124661). Horsley, L. H. (1947). Anal. Chem. 19, 508; also (1949). ibid. 21, 831. Jordan, C. B. and Hatch, V. O. (1950). Anal. Chem. 22, 177. Kameyama, N. and Semba, T. (1927). J. Chem. Soc. Ind. (Japan) 30, 5B. Oehme, F. and Ebert, G. (1951). Pharmazie 6, 471. Pharmacopoeias:

Austria. Osterreichisches Arzneibuch, 9th Ed. (1960). p. 786; Britain. British Pharma-copoeia (1973). p. 219; France. Pharmacopee Francaise, VIII (1965). p. 530; German Democratic Republic. Arzneibuch, 2 (1975). Vol. 4; German Federal Republic. Deutsches Arzneibuch, VII (1968). p. 543; Japan. Pharmacopoeia of Japan, 8th Ed. (1973). p. 267; Switzerland. Pharmacopoeia Helvetica, 6th Ed. (1973). Vol. ii, p. 665; United States. U.S. Pharmacopoeia, XIX (1975). p. 222.

Smith, D. M. and Bryant, W. M. D. (1935). J. Amer. Chem. Soc. 57, 841. Stuffins, C. B. (1958). Analyst (London) 83, 312. Tausz, J. and Rumm, H. (1926). Z. Angew. Chem. 39, 155.

The Enzymic Determination of Glycerol

A . A . N E W M A N

The use of certain enzymes, either singly or in combination with each other, permits the determination of very small amounts of glycerol with satisfactory accuracy. Owing to their high degree of specificity these enzymes are par-ticularly valuable for the analysis of biological samples in which the multi-plicity of components would make the application of purely chemical methods difficult or even impossible.

6 . 1 . B A S I C R E A C T I O N S

In the main, four enzymic methods are available for the purpose in view. The symbols used to indicate the reactions have the following meanings:

G = sn-Glycerol GP = sn-Glycerol-3-phosphate G P T D = Glycerophosphatide ATP = Adenosine triphosphate A D P = Adenosine diphosphate N A D = Nicotinamide dinucleotide N A D H •= Nicotinamide dinucleotide (reduced form) DHA = Dihydroxyacetone D H A P = Dihydroxyacetone phosphate PEP = Phosphoenol pyruvate

6

231

232 THE ENZYMIC DETERMINATION OF GLYCEROL 6.1

GAP GTP GK PK G P D H

G D H LDH TIM GAPDH

D-Glyceraldehyde-3-phosphate Glycerate-3-phosphate Glycerol kinase (EC 2.7.1.30) Pyruvate kinase (EC 2.7.1.40) sn-Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) (In the older literature G P D H is mostly, but misleadingly, described as G D H ; the true G D H is an entirely different enzyme.) Glycerol dehydrogenase (EC 1.1.1.6) Lactate dehydrogenase (EC 1.1.1.27) Triose phosphate isomerase (EC 5.3.1.1) Glyceraldehyde phosphate dehydrogenase (EC 1.2.1.9)

6 .1 .1 . The G K - G P D H M e t h o d

Details of this, the earliest, practical enzymic method were published by Wieland in 1957. It is based on the following reactions:

G + A T P — ^ — • G P + A D P

GP + N A D + G P P H > D H A P + N A D H

The second reaction can be used for the determination of GP on its own, and there are several slightly different procedures based on it.

6.1.2. The G D H M e t h o d

Particulars of this method evolved by the Hagen group were first published in 1962. The only reaction involved is:

G + N A D — 2 ™ L + DHA + N A D H

6.1.3. The G K - G D H M e t h o d

In this method (Wieland, 1970) the basic equation is

GP + N A D G P P H > D H A P + N A D H + H +

6.1.4. The G K - P K - L D H M e t h o d

This procedure was first described in 1962 by Garland and Randle and by Eggstein and Kreutz and Kreutz. It is based on the following reaction

6.1 BASIC REACTIONS 233

sequence:

G + ATP 2 * — * GP + A D P

PK

ADP + PEP • Pyruvate + ATP LDH

Pyruvate + N A D H • Lactate + N A D

6.1.5. The GK-GPDH-TIM-GAPDH Method

Significantly increased accuracy is claimed by the developers of this method (Moller and Roomi, 1974) since it furnishes 2 molecules of N A D H per mole-cule of glycerol, whereas in the other systems so far described the ratio is 1:1. The reactions involved are:

G + ATP 2 ^ G P + A D P

GPDH

GP + N A D • D H A P + N A D H

D H A P — — — • GAP

GAP + N A D G A ? D H > GTP + N A D H

The addition of arsenic acid brings the last reaction to completion.

6.1.6. Differences Between the Various Methods

From a practical viewpoint there are important differences between these reaction groups.

In the G K - G P D H system the equilibrium of the second reaction lies strongly to the left and must be forced to the right by working at pH 9*8 and adding hydrazine. It is also a rather slow reaction, taking 30-60 min to reach completion. Also, a relatively large excess of G P D H must be used to counter-act the tendency of hydrazine to inactivate the enzyme.

Initially, the unienzymic G D H method tended to be slow, requiring 30-60 min, but later in a colorimetric form (Frings and Purdue, 1966) it has become very fast.

In contrast to the G K - G P D H system, the G K - P K - L D H system is exoenergic and requires a much shorter period, usually 1-3 min, to complete the reaction.

Whichever method is used, the determination of the glycerol is based on the stoichiometric equivalence between the amount of glycerol in the sample, and the available amount of NADH, whether formed or residual, at the end of the reaction.


6.1.7. The E n z y m i c - R a d i o m e t r i c M e t h o d

An exception to the preceding rule is Newsholme and Taylor's (1968) enzy-mic-radiometric method wherein G K only is used and the plasma glycerol is estimated by a sub-stoichiometric isotope dilution method as follows:

(i) A known amount of plasma is added to a solution containing 1 4 C -glycerol, ATP, and GK, and the ensuing reaction:

1 4 C - G + ATP 1 4 C - G P + A D P

is allowed to proceed until 20-30 % of the labelled glycerol has been converted into 1 4 C - G P .

(ii) The labelled GP thus formed is adsorbed on paper discs and the adsorbate is carefully washed with water.

(iii) The glycerol level in the plasma is then determined according to the reduction in labelled glycerol through incorporation into GP. In practice reference is made to a calibration curve prepared by incubating various amounts of unlabelled glycerol with a fixed amount of the 1 4C-glycerol.

6.2. M O D E S OF D E T E R M I N A T I O N OF N A D H

There are, so far, two practical methods available for the determination of the N A D H indicator compound: spectrophotometry and fluorimetry.

In the initial phases of development the determination of glycerol was wholly manual. Nowadays a number of automated procedures are available, permitting the serial handling of a considerable number of samples in a relatively short time. The essential details are in each case recorded in the relevant chronological literature (Section 6.7).

6.3. M E T H O D OF C A L C U L A T I O N

The formula for the calculation of glycerol, based on the photometry of NADH, and expressed in g/ml, is:

AE-V'M/e -d -v

where AE = change in optical density per minute V = total assay volume

M = molecular weight of glycerol

6.3 METHOD OF CALCULATION 235

s = extinction coefficient of N A D H d = light path (cm) v = sample volume before dilution

The value of 6 is: 6-22 cm 2 /pM at 340 nm 3-30 c m 2 / m v f a t 366 nm

To improve the accuracy of the result, AE must be corrected by taking into account the extinction behaviour of a corresponding blank. Although in practice the components of the blank (i.e. the working solution minus the glycerol) tend to be sufficiently pure to obviate any urgency to make the correction, yet it is advisable to carry out the operation at least once for every fresh batch of sample to be assayed.

Much calculation can be avoided by reading off results from a standard curve where the value of all factors other than AE can be condensed in a single quantity. Such a graphic representation of the linear relationship between AE and glycerol concentration, based on the combined results of several workers, is given in Fig. 6.1 by Pinter et a/., 1967.

0-6

- • Theoretical values

- o Results by Pinter et al.

• A Data of Garland and Randle

_L_ 0 0 4 0 0 8 012

Glycerol concentration (^m)

FIG. 6.1

016

In that particular graph the glycerol concentration was studied only between 0 and 18 pM, but if necessary the range could be extended to 0-4M (Spinella and Mager, 1966) or even 0-7M in a particular fluorimetric adapta-tion (Laurell and Tibbling, 1966).

AE

at

340

nm

o

o


In assembling the data for such graphs it must be remembered that the volume and concentration of the sample may exert a definite influence on the accuracy of the final results. Thus, Stein and Horn (1972) found that up to a glycerol concentration of 3-3 umol/ml the sample can be used directly, but with higher concentrations dilution must be applied. The importance of sample volume is well illustrated by the observations of the same two authors that the coefficient of variation for a 20 jul sample was 15 %, while the cor-responding variation for a 100 fil sample was only 3 %.

Spinella and Mager (1966) report that optimal precision could be secured when their samples contained 0-003-0*4 umol of glycerol/ml.

Pinter et al. (1967), who studied the recovery rate of added glycerol, found that whereas with 0*0088 jiM the recovery rate was 110%, with 0*14 JIM the much more satisfactory result of 98*8 % could be secured. The corresponding SDs varied from ±0*005 to ± 0 0 0 1 %.

The validity that theoretical, or quasi-theoretical, figures can be obtained under the right working conditions was also confirmed by a number of other workers (Boltralik and Noll, 1960; Chernik, 1969; Belfrage et al, 1970). The amount of care that one has to devote to preliminary preparation is also dependent on the form in which the bound glycerol is present in the sample. With the exception of glycerophosphate, which can be determined directly, all bound glycerol must be made available in a free form. Details of the required procedures are given in a later section.

6.4. N A T U R E A N D O R I G I N OF THE S A M P L E S

The forms in which glycerol may be present, either alone or as a mixture, in the samples to be assayed are:

Free glycerol (G) Glycerides (MG, DG, TG) Glycerophosphate (GP) Glycerophosphatides (GPTD)

while the origin of the samples may be roughly classified as follows:

(a) Commercial glycerol solutions or culinary fats and oils; (b) Clinical materials, such as: Blood, serum, plasma, adipose tissues, liver,

etc.; Body fluids, e.g. urine, cerebrospinal fluid; (c) Fermentation products, e.g. wort, beer, must, and wine; (d) Dairy products: milk, cheese, etc.

6.5 PRELIMINARY TREATMENT OF THE SAMPLES 237

6.5. P R E L I M I N A R Y T R E A T M E N T OF THE S A M P L E S

6 .5 .1 . A n t i c o a g u l a t i o n Treatment

This relates more particularly to blood samples wherein clotting would interfere with their subsequent processing. Sodium fluoride or heparin are the usual agents used for the purpose.

6.5.2. Depro te in isa t ion

This is routinely achieved by treatment of the sample with 3-6 % perchloric acid according as to whether one deals with blood and plasma or tissues.

Other methods of deproteinisation include the use of trichloroacetic acid, metaphosphoric acid, or zinc sulphate with barium or sodium hydroxide.

The use of perchloric acid, though widespread, cannot be unreservedly recommended since it tends to depress the activity of GK unless a large excess of the enzyme is used.

N o deproteinisation is required, of course, for commercial glycerol solutions, and the same rule applies to certain biological products such as urine or cerebrospinal fluid.

Finally, it should be mentioned that the need for the somewhat tiresome deproteinisation treatment may be altogether avoided by a newly developed procedure (Harding and Heinzel, 1969) involving the dialytic separation of glycerol.

6.5.3. L iberat ion of B o u n d Glycero l

A . FROM G L Y C E R I D E S

There are two methods available for the purpose: alkaline hydrolysis and lipase treatment.

1. Alkaline Hydrolysis This involves in most cases the use of an ethanolic solution of potassium hydroxide at 75-85°C for 30 or more minutes, although according to several workers the decomposition of the glyceride molecules can be satisfactorily achieved at temperatures no higher than 37°C coupled with a shorter period of treatment (Fleury and Le Dizet, 1954; Ukita et al, 1955; Maruo and Benson, 1959). The choice between these methods depends on whether one wishes simultaneously to hydrolyse the phosphatides that may also be present. At the lower temperature, the phosphatide molecule will remain largely unaltered. A better phosphatide-sparing action can, however, be achieved by


using a milder hydrolysing agent such as tetraethylammonium hydroxide or lithium hydroxide even at slightly higher temperatures (Chernik, 1969; Maruo and Benson* 1959).

The use of ethanol as a vehicle for the alkali is by no means mandatory; methanol (Holzl, 1967) or isopropanol (Antonis, 1967) have also been recommended.

2. Lipase Treatment In the present context, the use of lipase (EC 3.1.1.3) was demonstrated by Wiebe and his associates (Belfrage et al, 1970; Wiebe and Belfrage, 1971); cf. also Enzymatic deacylation methods, in Litchfield, "Analysis of Tri-glycerides", Academic Press, 1972.

B. FROM P H O S P H A T I D E S

This is done by means of a two-phase operation. The first of these is identical with the high-temperature hydrolysis procedure described earlier and will strip the phosphatide molecule down to glycerophosphate, while the second phase, involving the use of acid phosphatase (EC 3.1.3.2), leads to a breaking of the bond between glycerol and the phosphoric acid residue (Zollner and Warnock, 1962; Schubert, 1965). A similar effect can be obtained by using another hydrolase, namely phospholipase (EC 3.1.4.3), which has the ability to reduce the phosphatide structure to diglyceride (Horney, 1973).

When phosphatides are submitted to alkaline hydrolysis it is essential to avoid the possible alkylation of the glycerophosphate formed since this would make the resulting products unavailable for subsequent enzymic determina-tion (Letters and Markham, 1964). The danger of alkylation is particularly acute when methanol is used as solvent, whereas it can be effectively avoided with ethanol, provided that the alcoholic alkali solution contains a minimum of 2-5% water. Failure to observe this condition may not always lead to sn-glycerol-3-phosphate, the only type suited to enzymic treatment, but rather to a complex mixture of L-3, LD-3, and L-2 isomers (Schubert, 1965).

C . F R O M G L Y C E R O P H O S P H A T E

The most practical procedure for dealing with glycerophosphate would be to submit it to the second reaction indicated in the GK-GPDH method (Hohorst, 1970; Lowry and Passereau, 1972) or use the newer G D H pro-cedure of Michal and Lang (1974).

6.7 CHRONOLOGICAL LITERATURE 239

6.6. I N T E R F E R I N G S U B S T A N C E S

These can be of two kinds: those present in the enzymes and those contained in the substrate.

6 .6 .1 . In the Enzymes

Although most modern commercially available enzyme preparations are sufficiently pure to ensure reliability in the results, it is advisable to make sure that they do not contain interfering substances such as glycerol, lactate, or glyoxal.

6.6.2. In the Substrate

While it may be more difficult to avoid the presence of such interfering sub-stances as dihydroxyacetone or glyceraldehyde in some samples to be analysed, the actual danger of interference by them is less than might appear likely at first sight since, according to Garland and Randle (1962), they tend to react some 20 times more slowly than glycerol. Pinter et al (1967), who also studied the behaviour of these two compounds, found that as long as the analysis takes less than 5 min to complete there is little danger of interference. In practice this means that in all relevant cases the G K - P K - L D H system, which works satisfactorily within the stated time limit, is preferable.

6.7. C H R O N O L O G I C A L L I T E R A T U R E

This section offers a chronological list of the literature concerned with each of the enzyme systems mentioned earlier, to enable intending users to acquaint themselves with the amount of information available, and make the most suitable choice. Each reference contains the following information in a condensed form: 1. Type of material investigated. 2. Compounds analysed. 3. Whether manual or automated. 4. Special features.

6 . 7 . 1 . The G K - G P D H M e t h o d

Wieland (1957): Blood, organ extracts; G, G P ; Manual. Boltralik and Noll (1960): Prepared solutions; G, GP; Manual. Vaughan (1962): Adipose tissue; G; Manual. Zollner and Warnock (1962): Serum; GP, GPDT; Manual (Use of phos-phatase).


Mayer and Busch (1963): Must and wine; G; Manual. Spinella and Mager (1966): Plasma; G, TG; Manual (Separation by chro-matography). Laurell and Tibbling (1966): Plasma; G; Manual (Fluorimetric method). Ko and Royer (1968): Plasma; G; Automatic (Two procedures in one adapta-tion, fluorimetric). Parijs et al (1968): Plasma; G, TG; Manual. Harding and Heinzel (1969): Blood; G; Automatic (Removal of serai pro-teins by dialysis). Chernik (1969): Plasma; TG and partial glycerides; Manual (Hydrolysis with tetraethylammonium hydroxide). Belfrage et al (1970); Liver, TG, Phospholipids; Automated (Fluorimetric use of pancreatic lipase). Wiebe and Belfrage (1971): Materials and compounds as by Belfrage et al (Use of highly purified lipase from Rhizopus arrhizus).

6.7.2. The G D H M e t h o d

Hagen and Hagen (1960): Plasma; G; Manual. Hagen (1962): Plasma; G; Manual. Frings and Purdue (1966): Plasma or serum; G; Automated (Reaction rate determination). Lipparini (1967): Wine; G; Manual. Yatoron et al (1976): Serum; TG; Manual (Use of lipase). Gore (1976): Adipose tissue and other incubation media; G; Automated.

6.7.3. The G K - G D H M e t h o d

Wieland (1970): Unspecified clinical and industrial materials; G; Manual.

6.7.4. The G K - P K - L D H M e t h o d

Kreutz (1962): Plasma or serum; G; Manual. Garland and Randle (1962): Plasma, tissue extracts; Manual. Drawert (1963): Must, wine; G; Manual. Drawert and Kupfer (1963): Must, wine; G; Manual. Schubert (1965): Serum, tissues; TG and G P T D ; Manual. Eggstein and Kreutz, Pt. I (1966): Tissues or serum; G, TG; Manual. Eggstein, Pt. II (1966): (Information on reaction rates). Antonis (1967): Serum; TG; Automatic (Fluorimetric method). Holzl (1967): Tissues; TG; Manual. Pinter et al (1967); Prepared test materials; G, TG; Manual (Close study of the effect of interfering substances).

6.7 CHRONOLOGICAL LITERATURE 241

Seitz and Tarnowski (1968): Serum; G; Manual. Schmidt and von Dahl (1968): Serum, tissues; TG; Manual (Use of minimal amount of samples and reduction of reaction time). Timms et al. (1968): Serum, prepared solutions; G, TG; Manual. Mandl et al. (1968): Beer; G; Manual. Mohler and Looser (1969): Wine; G; Manual. Berner (1969a): Dairy products; G, TG; Manual. Berner (1969b): Prepared mixture of TG and partial glycerides; Manual (TLC separation). Berner and Guhr (1969): MG and D G ; G and total G; Manual. Dalton and Mallon (1970): Serum, adipose tissue; G, TG; Automated. Berner (1970): Frying fats; MG, D G ; Manual (TLC separation). Drawert et al. (1970): Wort, beer; G; Manual. Bell et al. (1970): Serum; G, TG; Automated (Fluorimetric). Sheath (1970); Maternal and foetal plasma; G; Manual (Based on a novel procedure involving two optical reading lines, one before and one after the addition of GK to cope with the determination of very small amounts of glycerol. The two lines are parallel and the drop between them corresponds to AE (see Fig. 6.2)).

0 8

0' 1 1 1 1 I I I I I I I I I

8 12 16 4 8 12 16 Time (min)

FIG. 6 .2

Mandl et al. (1971): Beer; G; Manual. Mallon and Dalton (1971): Prepared solutions, serum, fat cells; G, TG; Automated. Steiner (1972): Beer; G; Manual. Berner (1972): MDG, DG, TG, G; Manual (Improved TLC separation).

Opt

ical

den

sity


Stein and Horn (1972): Plasma; G, TG; Automated reaction rate determi-nation. Horney (1973): Serum; G, TG, G P D T ; Manual. Eggstein and Kuhlmann (1974): Blood, plasma; G, TG; Manual (Possible effects of heparin and insulin). Wahlefeld (1974): G, TG; Manual (Enzymic hydrolysis). Thiele et al (1976): Serum; TG; Manual (Enzyme kit).

6.7.5. The G K - G P D H - T I M - G A P D H M e t h o d

Mdller and Roomi (1974): Biological preparations; G; Manual (A high sensitivity method).

6.7.6. The G P D H M e t h o d for GP

Hohorst (1963): Tissues; Manual.

Lowry and Passereau (1972): Biological preparations; Manual (Two pro-cedures; in one adaptation fluorimetric). Michal and La^ig (1974): Biological preparations; Manual.

6.7.7. The E n z y m i c - R a d i o m e t r i c M e t h o d

Newsholme and Taylor (1968): Serum; G; Manual. Newsholme (1974): Biological and industrial preparations; Manual.

A C K N O W L E D G E M E N T S

The writer is grateful to all those who helped in the preparation of this survey through the donation of reprints and permission to reproduce the diagrams, and especially to Messrs Newsholme and Taylor for elucidating aspects of their radiometric procedure, and to the Bohringer Corporation of London for providing copies of articles in their library and their Test Handbook.

REFERENCES

Antonis, A. (1967). J. Amer. Oil. Chem. Soc. 44, 333-340. Belfrage, P., Wiebe, T. and Lundquist, A. (1970). Scand. J. Clin. Lab. Invest. 26, 56-30. Bell, J. B., Atkinson, S. M. and Baron, D. M. (1970). J. Clin. Path. 23, 509-513. Berner, G. (1969a). Milchwissenschaft 24, 284-289. Berner, G. (1969b). Z. Lebensm.-Untersuch.-Forsch. 141, 318-320. Berner, G (1970). Fette, Seifen, Anstrichm. 72, 735-737.

REFERENCES 243 Berner, G. (1972). J. Chromatogr. 64, 388-389. Berner, G. and Guhr. G. (1969). Fette, Seifen, Anstrichm. 71, 4 5 9 ^ 6 1 . Bohringer Corp. (London) Ltd. (J 971). "Test-Handbook" (Neutral Fat, Triglycerides). Boltralik, J. L. and Noll, H. (1960). Analyt. Biochem 1, 269-273. Chernik, S. S. (1969). Methods of Enzymology 14, 627-630. Dalton, C. and Mallon, J. P. (1970). "Advances in Automated Analysis", Vol. II, In-

dustrial Analyses, Technicon International Congr., Chicago, pp. 183-187. Drawert, F. (1963). Vitis 3, 237-239. Drawert, F. and Kupfer, G. (1963). Z. Lebensm.-Untersuch.-Forsch. 123, 211-217. Drawert, F., Hagen, W. and Barton, H. (1970). Brauwissenschaft 23, 432-438. Eggstein, M. (1966). Klin. Wochenschr. 44, 267-273. Eggstein, M. and Kreutz, F. H. (1966). Klin. Wochenschr. 44, 262-267. Eggstein, M. and Kuhlmann, E. (1974). In Bergmeyer, "Methods of Enzymatic Analysis",

2nd English Ed., Verlag. Chemie-Academic Press, Vol. Ill, pp. 1825-1831. Fleury, P. F. and Le Dizet, L. (1954). Bull. Soc. Chim. Biol. (Paris) 36, 971-981. Frings, C. S. and Purdue, H. L. (1966). Analyt. Chim. Acta 34, 225-228. Garland, P. B. and Randle, P. J. (1962). Nature 196, 987-988. Gore, M. G. (1976). Analyt. Biochem. 75, 604-610. Hagen, J. H. (1962). Biochem. J. 82, 23p-24p. Hagen, J. H. and Hagen, P. B. (1960). Can. J. Biochem. Physiol. 40, 1129-1131. Harding, U. and Heinzel, G (1969). Z. Klin. Chem. Klin. Biochem. 7, 356-360. Hohorst, H. J. (1970). In Bergmeyer, "Methoden der enzymatischen Analyse", Ver-

lag Chemie, pp. 1379-1385. Holzl, F. (1967). Fette, Seifen, Anstrichm. 69, 328-330. Horney, D. L. (1973). Clin. Chem. 19, 453-455. Ko, H. and Royer, M. E. (1968). Analyt. Biochem. 26, 18-33. Kreutz, F. H. (1962). Klin. Wochenschr. 40, 362-369, Laurell, S. and Tibbling, G (1966). Clin. Chim. Acta 13, 317-322. Letters, K. and Markham, E. (1964). Biochim. Biophys. Acta 84, (1), 91-3. Lipparini, L. (1967). Quad. Mercecol. 5(1), 69-74. Lowry, O. H. and Passereau, J. V. (1972). "A Flexible System of Enzymatic Analysis",

Academic Press, pp. 186-189. Mallon, J. D. and Dalton, C (1971). Analyt. Biochem. 40, 174-182. Mandl, B., Wullinger, F., Fischer, A. and Piendl, A. (1968). Brauwissenschaft 22, 278-

284.

Mandl, B., Wullinger, F., Schneider, K. and Piendl, A. (1971). Brauwissenschaft 2 7 , 4 3 -45.

Maruo, B. and Benson, A. A. (1959). J. Biol. Chem. 234, 254-256. Mayer, K. and Busch, I. (1963). Mttgen,. Geb. tebensm. Hyg. 54, 297-303. Michal, G. and Lang, G. (1974). In Bergmeyer, "Methods of Enzymatic Analysis", 2nd

English Ed., Verlag Chemie-Academic Press, Vol. Ill, pp. 1415-1418. Mohler, K. and Looser, S. (1969). Z. Lebensm.-Untersuch.-Forsch. 140, 149-154. Moller, F. and Roomi, M. W. (1974). Analyt. Biochem. 59(1), 248-258. Newsholme, E. A. (1974). In Bergmeyer, "Methods of Enzymatic Analysis", 2nd

English Ed., Verlag Chemie-Academic Press, pp. 1409-1414. Newsholme, E. A. and Taylor, K. (1968). Biochim. Biophys. Acta 158, 11-24; cf. also

Bergmeyer, "Methoden der enzymatischen Analyse", Verlag Chemie, Vol. II, pp. 1372-1378.

Parijs, J., Barbier, F. and Vermeire, P. (1968). Clin. Chem. Clin. Biochem. 6, 331-333.

244 REFERENCES

Pinter, J. K., Hayashi, J. A. and Watson, J. D. (1967). Arch. Biochem. Biophys. 121, 404^14 .

Schmidt, F. H. and von Dahl, K. (1968). Z. Klin. Biochem. 6, 156-159. Schubert, G E. (1965). Frankfurter Zeitschr. Pathol. 74, 460-461. Seitz, H. J. and Tarnowski, W. (1968). Z. Klin. Chem. Klin. Biochem. 6(5), 411-414. Sheath, J. B. (1970). Clin. Biochem. 3, 339-341. Spinella, C. J. and Mager, N. (1966). J. Lipid. Res. 7, 167-169. Stein, S. M. and Horn, D. B. (1972). Clin. Chim. Acta 39, 293-300. Steiner, K. (1972). Schweiz. Brauerei Rundsch. 83(1), 5-6. Thiele, O. W., Radas, A. and Bodden, A. (1976). Zentr-blatt, Veterindrmed, Reihe A

23(2), 161-166. Timms, A. R., Lawrence, A. K., Spirito, J. A. and Engstrom, R. G. (1968). J. Lipid Res.

9, 675-680. Ukita, T , Bates, N. A. and Carter, H. E. (1955). J. Biol. Chem. 216, 867-874. Vaughan, M. (1962). J. Biol Chem. 237, 3354-3358. Wahlefeld, A. W. (1974). In Bergmeyer, "Methods of Enzymatic Analysis", 2nd English

Ed., Verlag Chemie-Academic Press, Vol. IV. pp. 1831-1839. Wiebe, T. and Belfrage, P. (1971). Scand. J. Clin. Lab. Invest. 28, 453-457. Wieland, O. (1957). Biochem. Zeitschr. 329, 313; cf. Bergmeyer (1963). "Methods of

Enzymatic Analysis", Academic Press, pp. 211-214. Wieland, O. (1970). in Bergmeyer, "Methoden der enzymatischen Analyse", 2nd edn.

Verlag Chemie. Vol II, pp. 1367-1372. Yatoron, N. U., Shibata, H. and Nakegima, N. (1976). "Reagents for serum triglyceride

determination", Japan Kokai 76-68297. Zollner, N. and Warnock, S. A. (1962). Clin. Chim. Acta 7, 607-613.

Index In this index, substituted organic compounds are indexed under the names of their parent compounds.

Acenaphthene-5-carboxylic acid, fluorescence reagent, 52

Acetic acid esters boiling points, 113 fractional extraction, 114 gas chromatography, 39, 76, 104-105,

144-145 Acetic acid, trifluoro-, esters

gas chromatography, 39, 42, 104, 105 N M R studies, 35

"Acetin" method, 35-36 Acetoin, liquid chromatography, 77 Acetone, acetyl-, ammoniacal, reagent

for formaldehyde, 24-25, 137-141, 150

Acetone, dihydroxy-, 1 determination, 9 paper chromatography, 72, 73

Acidity, determination, 221, 223 Acrolein

detection and determination, 46-53, 143-144, 223

detection in glycerol, 221 Acrylaldehyde, see Acrolein Adipic acid esters

solvent extraction, 159 thin layer chromatography, 117

Adipose tissue analysis of triglycerides in, 164, 165,

173 determination of glycerol in, 41, 239,

240

determination of glycerol and triglycerides in, 241

Alcohols, analysis of Q - C 3 , 68 Aldoses, analysis of reduction products,

45 Alimentary pastes, analysis, 115, 122 Alkalinity determination, 221, 223 Alkoxides, transesterifying agents, 140 Alkyd resins, 97

analysis, 110, 146,188 analysis of aminolysis products, 40,

41,44, 147-148 solvent extraction, 159

Allyl alcohol, reaction with peracetic acid, 18

Allyl esters, gas chromatography, 105 Amidoschwarz, visualisation reagent, 207 Ammonium compounds, detection, 221 Ammonium sulphate, charring reagent,

196 Amniotic fluid

analysis, 182 analysis of glycerol ethers derived

from, 124, 149 analysis of lipids in, 118, 177, 192, 200 sphingomyelin/lecithin ratio in, 181,

182, 189, 205 Anhydroribitol, trimethylsilylated

gas chromatography, 43 thin layer chromatography, 45

/?-Anisidine, visualisation reagent, 14 ANS, visualisation reagent, 207

245

246 INDEX

Anthrone, colour reagent, 50-51 Antifreeze materials, analysis, 47 Arachis oil, analysis, 157, 165, 172 Arsenic, detection, 220, 223 Arsenite complexes, 60 Ash, determination, 222, 224

Bacterial cell walls, analysis, 59, 73 Bacterial lipids, analysis, 180 Beer, analysis, 44, 241 Benzaldehyde, /?-dimethylamino-, colour

reagent, 53 Benzaldehyde, /7-hydroxy-, colour

reagent, 53 Benzidine, visualisation reagent, 13 Benzoic acid esters, 34 Benzoic acid, 3,5-dinitro-, esters, 34-35 Benzoic acid, 4-nitro-, esters, 34 Benzothiazolin-2-one, 3-methyl-,

hydrazone, see MBTH Beverages, see also Beer, Liqueurs,

Wines analysis, 3,11

Bisulphite compound, of formaldehyde,

22-23 Blood

analysis, 178, 185 analysis of lipids in, 179, 195, 207 anticoagulation treatment, 237 determination of free fatty acids in,

198 determination of free fatty acids and

triglycerides in, 174 determination of glycerides in, 141 determination of glycerol in, 15, 20,

240 determination of glycerol and

glycerophosphate in, 239 determination of glycerol and sorbitol

in, 21 determination of glycerol and

triglycerides in, 242 determination of nitrate esters in, 163 solvent extraction, 159, 160, 161 sphingomyelin/lecithin ratio in, 189

Blood plasma analysis, 174, 179 analysis of lipids in, 177, 181, 198,

203, 207

analysis of phospholipids in, 176 deproteinisation, 237 detection of glucose and glycerol in,

32, 73 determination of cholesterol and

triglycerides in, 139 determination of free fatty acids and

triglycerides in, 172 determination of glycerides in, 135 determination of glycerol in, 234,

240, 241,242 determination of nitrate esters in,

164, 165 determination of triglycerides in,

135, 137, 139, 156, 172, 241, 242 solvent extraction, 159, 160, 162

Blood serum analysis, 175, 176, 179, 181, 185 analysis of lipids in, 116, 118, 119,

120, 121, 125, 127, 169, 171, 173, 176, 177, 178, 180, 184, 186, 187, 189, 191, 192, 193, 194, 196, 198, 200, 203, 205, 206

analysis of phospholipids in, 191, 196 determination of cholesterol and

triglycerides in, 139 determination of glycerides in, 135,

136-137 determination of glycerol in, 40, 240,

241,242 determination of glycerophosphate in,

239 determination of glycerophosphatides

in, 239, 242 determination of triglycerides in, 50,

135, 137, 139-140, 144, 155-156, 169, 171, 174, 198, 204, 241,242

solvent extraction, 159, 160, 161 Borate complexes with polyols, 59-60,

103-104 Boronates

gas chromatography, 104 paper electrophoresis, 14

Brain tissue analysis of lipids in, 178, 185, 186 analysis of phospholipids in, 175,

176, 198 sphingomyelin/lecithin ratio in, 189

Brake fluids, analysis, 63, 78, 126 British Standards Institution

INDEX 247

specifications, 223-224, 225, 229 Bromine, oxidising agent, 1-3, 142 Bromocresol Green, visualisation

reagent, 207 Bromothymol Blue, visualisation

reagent, 206 Butane-2,3-diol

detection, 11 determination, 20 ion exchange, 80 liquid chromatography, 77, 79 thin layer chromatography, 76

Butterfat, analysis, 110, 163, 188 Butyrin, determination, 101

Calcium, detection, 223 Carbazide, 1,5-diphenyl-, colour

reagent, 8 Carbazole-sulphuric acid, colour

reagent, 3 Carbohydrates, see also Polysaccharides,

Sugars analysis, 44 analysis of hydrogenation products, 41 detection, 9, 10, 31, 32 paper chromatography, 71, 72, 73 separation of borate complexes, 59,

60 Carbon dioxide, determination, 6 -7 Cellophane, analysis, 62, 63 Cephalins, 96

analysis, 2 Cerebrosides, visualisation reagent for,

197 Cerebrospinal fluid, analysis, 185, 237 Cerium(IV), oxidising agent, 3 -5 Charring reagents, 190-197 Cheese, analysis, 163 Chloramine T, oxidising agent, 5 -6 Chloride, determination, 220, 223 Chlorine, determination of total, 220 Cholesterol

colorimetric determination, 139 solvent extraction, 159, 161 thin layer chromatography, 116,

119, 120, 121, 122, 173 visualisation reagents for, 192, 193,

195, 196, 198

Cholesterol esters gas chromatography, 165, 166 liquid chromatography, 187 paper chromatography, 124 thin layer chromatography, 116,119,

120, 121, 122, 170, 171, 173 visualisation reagents for, 190, 192,

193, 195, 196, 198 Chromatography, see names of specific

techniques Chromotropic acid, reagent for

formaldehyde, 19-20, 28,101, 135-137

Cigarette smoke, see Tobacco smoke Citronella oil, analysis, 134 Citrus juice, analysis, 188 Cocoa butter, analysis, 163, 199 Coconut, analysis of desiccated, 18, 67 Coconut oil

analysis, 121, 164, 165, 185 glycerolysis, 112

Codeine, colour reagent, 2, 51 Column chromatography, see Liquid

chromatography Conductivity determination, 227 Copper(II), reagent for glycerol, 55-58 Copper(III), oxidising agent, 8, 60 Copper(II) salts, charring reagents,

196-197 Cosmetics, see also Skin lotions,

Vanishing creams analysis, 70, 71 analysis of glycerides in, 205 analysis of polyols in, 31 detection of glycerol in, 46, 51, 52 determination of alcohols in, 79 determination of glycerol in, 10,17,

68, 72 determination of polyols in, 72 solvent extraction, 65

Cotton, analysis of sized, 7, 35 Cottonseed oil

analysis, i65, 172 glycerolysis, 124

Cresyl Violet, visualisation reagent, 207 Critical solution temperature, 62, 156 Crotonaldehyde, detection, 50 Cyclohexane-1,3-dione, 5,5-dimethyl-,

see Dimedone Cyclopentanecarboxylic acid glycerides,

248 INDEX

thin layer chromatography, 173, 198, 203

Dairy products, see also Butterfat, Cheese analysis, 164, 241

Dehydration of glycerol, 46-53, 143-144, 221

Density determination, 62, 226, 228 Dentifrice humectants, analysis, 43 0-Dianisidine, reagent for acrolein, 47,

143 Dichromate, oxidising agent, 4, 6-8 ,

10, 142 Dichromate-sulphuric acid, charring

reagent, 194-195 Dielectric constant determination,

226-227 Diethylene glycol

detection, 10 gas chromatography, 68, 70 separation, 59 thin layer chromatography, 76

Diethylene glycol acetates, detection, 10

Differential thermal analysis, 157 Diglycerides, 95, 96

characterisation of 1-alkoxy-, 156 detection and determination

by enzymic methods, 241 by periodate oxidation, 134 in linseed oil, 106

esterification, 104, 106 gas chromatography, 106-109, 128 IR spectra, 110 liquid chromatography, 125-128 N M R analysis, 111 paper chromatography, 123-124 solvent extraction, 159 sulphoquinovosyl, see Sulpholipids thin layer chromatography, 103,

103, 115-123 urea complexes, 112 visualisation reagents for, 191, 193,

194, 196, 197, 198, 200, 202, 203 Diglycerol, determination, 40 Dimedone, reagent for formaldehyde,

23, 101 Dioleins, determination, 107 Diols, see Glycols

Dioxan, analysis, 225 Dipalmitins

gas chromatography of trimethylsilylated, 107, 108

liquid chromatography, 127 Distearins

gas chromatography of trimethylsilylated, 107, 108

IR analysis, 110 liquid chromatography, 127

Distillation, 66-67, 113-114, 227-228 Drugs, analysis, 7, 46 Dulcitol, detection, 51 Dyes, analysis of suspensions, 65 Dynamite, analysis of residues, 184

Eggs and egg products, analysis, 18, 47, 158,175

Emulsifiers, analysis, 101, 118, 126, 128, 134, 203

Enzymic analysis, 231-242 Epidermal lipids, analysis, 180 Epihydrinaldehyde, detection, 46 Erythritol

detection, 24 ion exchange, 81 thin layer chromatography, 76

Erythrocyte plasma, analysis, 180, 207 Erythrocytes, analysis, 178 Esterification, 34-42, 104-106, 144-145 Esters, determination, 222, 224 Ethanol

determination, 4 ion exchange, 81

Ethanolamines, ion exchange, 81 Ether, diethyl, analysis, 225 Ether formation, 42-45, 106-109, 145 Ethyl centralite, determination, 157 Ethylene glycol

analysis, 225, 226 detection, 8, 22, 51, 52 determination, 4, 5, 21, 30 gas chromatography, 68, 69, 70, 71 ion exchange, 80 liquid chromatography, 77, 79 refractive index of solution, 62 separation, 59 thin layer chromatography, 74

Ethylene glycol dinitrate

INDEX 249

gas chromatography, 163 solvent extraction, 161

Ethylene glycol esters liquid chromatography, 125 visualisation reagent for, 195

Explosives, see also Propellants analysis, 122, 158, 168, 183-184 solvent extraction, 161

Faeces analysis of lipids in, 179, 195 analysis of triglycerides in, 173, 195 determination of glycerol in, 2 solvent extraction, 159

Fast Green FCF, reagent for cerium(IV), 5

Fats and oils, 95; see also Frying fats, Vegetable oils, and names of specific fats and oils

analysis of digestion products, 119 analysis of glycerides in, 141-142, 172 analysis of glycerolysis products, 169 analysis of phosphatides in, 177 analysis of triglycerides in, 164, 168,

171, 203 characterisation, 170 detection, 143, 191,202 detection of glycerides in, 144 detection of partial glycerides in, 193 detection of split, 31 determination of glycerides in, 2, 45,

145 determination of glycerol ethers in,

101 determination of monoglycerides in,

99, 101, 104, 109 differential thermal analysis, 157 extraction of monoglycerides from,

114 fractionation, 111 gas chromatography, 163 "monethanolamine indices", 148

Fatty acids liquid chromatography, 187, 189 paper chromatography, 124 thin layer chromatography, 116, 119,

120, 171, 172, 173, 174

visualisation reagents for, 190, 191, 192, 193, 195, 198, 199, 202, 204, 205, 207

Fermentation media analysis of polyols in, 69 detection of glycerol in, 31 determination of glycerol in, 3, 18,

20, 22, 73, 78 Fertilisers, analysis, 202 Fish oils, analysis, 165 Flavours, analysis, 70, 79 Flour, analysis, 185, 192 Fluorescein, dibromo-, visualisation

reagent, 201, 204 Fluorescein, dichloro-, visualisation

reagent, 11, 103, 105, 201, 2 0 2 -204

Fluorescent visualisation reagents, 199, 201-207

Foods analysis of polyols in, 71 detection of glycerol in, 46, 51 determination of glycerol and

glycerides in, 44 determination of propylene glycol in,

18 solvent extraction, 71

Formaldehyde, detection and determination, 8, 12, 19-25, 28, 30, 52, 101, 134-141, 150

Formic acid, detection and determination, 8, 17-19, 229

Fruit pastilles, analysis, 18, 78 Frying fats, analysis, 189, 195, 241

Galactolipids liquid chromatography, 187 solvent extraction, 159 thin layer chromatography, 176 visualisation reagent for, 198

Gallic acid, colour reagent, 51 Gas chromatography

of acylation products, 39-42, 104-105, 105, 144-145

of allyl esters, 105 of isopropylidene derivatives, 45,

102-103 of methylation products, 45

250 INDEX

of trimethylsilyl ethers, 42-45 ,104 of underivatised compounds, 67-71,

128, 147, 162-166 of water in glycerol, 228

Germanate complexes, 60, 61 Gladstone constant, 62 Glucose

analysis of hydrogenolysis products, 14, 73, 80

detection, 27, 32, 221 determination, 9 paper chromatography, 73 thin layer chromatography, 75

Glyceraldehyde, 1 Glyceraldehyde phosphate

dehydrogenase, 233, 242 Glycerides, see also Diglycerides,

Monoglycerides, Triglycerides determination, 142-143, 144-145 glycerol liberation from, 237-238 paper chromatography, 167, 168, 169 separation of hydrolysis products,

146-147 solvent extraction, 158-161 thin layer chromatography, 170-174 visualisation reagents for, 10, 190-

207 Glycerol

analysis, 219-228 British Standard specification, 223 detection and determination

by enzymic methods, 231-242 by esterification methods, 8, 35-42 by oxidation methods, 1-33 by physical methods, 61-64 pharmacopoeia tests, 223, 228-229 via dehydration, 46-53 via ether formation, 42-45 via hydriodic acid reaction, 53-55 via reaction with inorganic ions,

55-61 identification, 34-35, 38-39 separation from other compounds

by column chromatography, 77-79

by distillation, 66-67 by gas chromatography, 67-71 by ion exchange, 79—81 by paper chromatography, 71-73,

167

by paper electrophoresis, 79,146 by solvent extraction, 64-66 by thin layer chromatography,

74-77, 146 visualisation reagents for, 193, 197,

203 Glycerol acetates

boiling points, 113 fractional extraction, 114 gas chromatography, 39-42, 104,

144-145 thin layer chromatography, 10, 76,

121 Glycerol compounds, 95-97; see also

Glycerides detection and determination

methods based on participation of complete molecule, 155-189

methods based on residual hydroxyl groups, 97-111

via aminolysis, 147-149 via hydrolysis, 133-147 via reduction, 149 via thermal cleavage, 150

separation, 111-128, 158-189 as urea complexes, 112 by distillation, 113-114 by gas chromatography, 128,

162-166 by liquid chromatography, 124-

128, 184-189 by paper chromatography, 123-124,

166-169 by solvent extraction, 114-115,

158-162 by thermal diffusion, 111 by thin layer chromatography,

115-123, 170-184 Glycerol dehydrogenase, 232, 233, 238,

240 Glycerol dinitrate, chloro-, gas

chromatography, 163 Glycerol ethers, 95

boiling points, 113 critical solution temperatures, 156 esterification, 104-106 gas chromatography of methylated,

109 gas chromatography of isopropylidene

derivatives, 102

INDEX 251

gas chromatography of trimethylsilylated, 107

oxidation, 101 paper chromatography, 124, 149 separation, 102-103 thin layer chromatography, 118,

122,149 visualisation reagent for, 202

Glycerol formates, boiling points, 113 Glycerol kinase, 232, 233, 234, 239-40,

242 Glycerol lactates, gas chromatography

of trimethylsilylated, 108 Glycerol nitrates, see also Glycerol

trinitrate gas chromatography, 128, 164 gas chromatography of trimethyl-

silylated, 108 liquid chromatography, 127 thin layer chromatography, 118, 119,

183-184 visualisation reagent for, 202

Glycerol phosphates, paper chromatography, 100, 123

Glycerol stearate, analysis of technical-grade, 105, 127

determination, 100 Glycerol trifluoroacetates, gas

chromatography, 39, 105 Glycerol trinitrate, 97

gas chromatography, 163, 164, 165 IR spectrophotometry, 157-158 liquid chromatography, 184, 188 solvent extraction, 161-162 thin layer chromatography, 122,

183-184 sn-Glycerol-3-phosphate dehydrogenase,

232, 233, 238, 239-240, 242 Glycerophosphates, 97

detection, 143 determination by enzymic methods,

232, 239, 242 determination by oxidation methods,

98, 102, 111 isomerisation, 100 paper chromatography, 167 visualisation reagents for, 197, 206

Glycerophosphatides, 96 detection, 101 enzymic determination, 239, 240, 242

gas chromatography, 166 gas chromatography of

trimethylsilylated, 108 mass spectrometry, 157 reduction, 149 thermal cleavage, 150 thin layer chromatography, 182

Glycerose, see Acetone, dihydroxy-Glycolipids

gas chromatography, 166 thin layer chromatography, 179

Glycollic acid, ion exchange, 81 Glycols

analysis of mixtures, 12 detection, 50 determination, 9, 11, 13, 29, 55 gas chromatography, 68-70 gas chromatography of

trimethylsilylated, 43 ion exchange, 80-81 N M R study, 35 paper chromatography, 72, 73 separation, 103 thin layer chromatography, 58, 74,

76 Glycopeptides, analysis of reduction

products, 44 Glyoxal, methyl-, 1, 32 "Glyptals", 97 Gold(III), oxidising agent, 9 Greases, analysis, 7, 142 Griess reagents, 60 Groundnut oil, see Arachis oil Guaiacol, colour reagent, 3, 51 Guaiacol RL, colour reagent, 52

Hair, analysis, 176 Heat of combustion, 13 Heavy metals, detection, 220 Hexoses, analysis of oxidation products,

44 Humectants, analysis, 11, 40, 43, 44,

65, 6 8 , 7 1 , 7 4 Hydrazine, phenyl-, reagent for

formaldehyde, 21 Hydrazines, reagents for acrolein,

47-48 , 102, 134-135, 141 Hydriodic acid, reagent for glycerol,

53-55, 145

252 INDEX

Hydroxyl groups, determination, 36 Hypohalites, oxidising agents, 9 Hypophosphite, determination, 9

Ice cream, analysis, 101 Infrared spectrophotometry, 63

of alkyd resins, 110 of diols, 55 of explosives and propellants, 157-158 of glycerides, 110 of lecithin and triglycerides, 158 of polyols in polyesters, 63, 145-146 of water in glycerol, 227

Insect phospholipids, analysis, 177 Iodate

detection and determination, 25-26 oxidising agent, 10

Iodine, visualisation reagent, 10, 197-199

Iodoform reaction, 3 Ion exchange, 79-81 Iron, detection, 221, 224 Isomannide nitrate, gas chromatography,

128, 164 Isoprene, analysis of synthesis products,

58 Isopropylidene derivative formation,

45, 102-103, 141-142 Isopropyl iodide, detection and

determination, 53-55, 145 Isosorbide nitrates, gas chromatography,

128, 164, 165 Isotope dilution analysis, 234, 242

J-acid, reagent for formaldehyde, 21 Jet fuel, analysis of additives, 63 Juices, see also Citrus juice

analysis, 20

Karl Fischer titration, 224-225 Kusum oil

analysis, 173, 198, 203 solvent extraction, 159

Lacquers, analysis of extracts, 61, 64

Lactate dehdrogenase, 232-233, 239, 240 Lactic acid, determination, 65 Lard, analysis, 7, 157,163 Laurin, determination, 101 Laurins, separation, 125 Lead, detection, 220 Lead(II), reagent for glycerol, 58 Lead(IV), oxidising agent, 10-12, 102 Leather, analysis, 47, 57, 65 Lecithins, 96

analysis, 2, 148 IR spectrophotometry, 158 liquid chromatography, 189 mass spectrometry, 157 thin layer chromatography, 180, 181,

182 visualisation reagents for, 197, 200,

204, 205, 206, 207 Light scattering, see Nephelometry Linseed oil

analysis, 106, 148, 172, 185 glycerolysis, 112, 124

Lipase hydrolysis of glycerides, 238 Lipids, see also Galactolipids, Glycerides,

Glycolipids, Phospholipids gas chromatography, 166 IR spectra, 110 liquid chromatography, 184-189 solvent extraction, 158—161 thin layer chromatography, 174-182 visualisation reagents for, 189-207

Liqueurs, analysis, 32, 65, 76 Liquid chromatography

of glycerol esters and ethers, 124-128 of glycerol-containing compounds,

184-189 of polyols, 77-79,81

Liver tissue analysis of lipids in, 178, 181 analysis of phospholipids in, 175, 176,

177, 191, 198, 240 analysis of triglycerides in, 140, 173,

195, 240 solvent extraction, 159-160 sphingomyelin/lecithin ratio in, 189

Lung tissue analysis of glycerides and

phospholipids in, 186 analysis of lipids in, 191

Lyes, analysis, 7, 8, 80

INDEX 253

Lymph, analysis, 142-143 Lysolecithin

paper chromatography, 167 thin layer chromatography, 182

Magnesium, detection, 223 Maize oil, analysis, 157 Malachite Green, visualisation reagent,

207 Malarial parasites, analysis, 179 Malonaldehyde, detection, 50 Maltitol, detection, 24 Maltose

detection, 27 thin layer chromatography, 75

Manganate, oxidising agent, 12 Manganese dioxide, determination, 30 Mannitol

detection, 27, 51 determination, 4, 16, 21, 30 thin layer chromatography, 59, 75

Margarine, analysis, 103, 120,163,188, 203, 227

Mass spectrometry, 157 MBTH, reagent for formaldehyde,

23-24, 141 Methanol

analysis, 226 determination, 4, 8

Methyl ether formation, 109 Milk

analysis of lipids in, 105-106, 119, 200 analysis of triglycerides in, 188 determination of monoglycerides in,

100, 101 Milk products, see Dairy products Molybdate complexes, 61 Molybdophosphoric acid, visualisation

reagent, 198, 199, 200 Monoglycerides, see also Emulsifiers

characterisation of 1,2-dialkoxy-, 156 detection and determination

by enzymic methods, 241 by oxidation methods, 98-102, 134 in linseed oil, 106 via borate complex formation, 103

esterification, 104-106 gas chromatography of

trimethylsilylated, 106-109 IR spectra, 110 isomerisation, 100-101 liquid chromatography, 125-128 paper chromatography, 123-124, 169 thermogravimetry, 111 thin layer chromatography, 103,

115-123 urea complexes, 112 visualisation reagents for, 191, 193,

194, 196, 198, 200, 202, 203 Monosaccharides, see also Hexoses,

Pentoses analysis of hydrogenation products,

14, 69, 70 analysis of reduction products, 40 gas chromatography of

trimethylsilylated, 44 ion exchange, 81 visualisation reagent for, 31

Muscle tissue, analysis, 118 Must, analysis, 20, 49, 240 Mustard-seed oil, analysis, 172 Myristin, gas chromatography of

trimethylsilylated, 107 liquid chromatography, 127

Naphthalene-2,7-diol, colour reagent, 3, 52, 143

Naphthalene-3,6-disulphonic acid, 1,8-dihydroxy-, see Chromotropic acid

Naphthalene-3,6-disulphonic acid, 2-hydroxy-, colour reagent, 3

Naphthalene-8-sulphonate, 1 -anilino-, see ANS

Naphthalene-3-sulphonic acid, 6-amino-l -hydroxy-, see J-acid

a-Naphthol, colour reagent, 51 j5-Naphthol, colour reagent, 2, 51 1-Naphthylamine, JV-phenyl-,

visualisation reagent, 207 Neatsfoot oil, analysis, 187 Nephelometry, 155-156 Nessler reagent, 12, 47 Nicotinamide dinucleotide (reduced),

determination, 234-236 Nitric acid esters, see also Glycerol

nitrates

254 INDEX

gas chromatography, 162, 163, 164, 165

thin layer chromatography, 105, 183-184

visualisation reagent for, 202 Nitrite, determination, 9 Nitrocellulose, liquid chromatography,

184 Nitrogen, determination, 224 Nitroglycerine, see Glycerol trinitrate Nitroprusside-piperidine (or

morpholine), colour reagent, 47, 143

Nuclear magnetic resonance studies, 35, 64, 111

Nuts, analysis, 20

Oil Red O, visualisation reagent, 207 Ointments, analysis, 18, 76, 118 Oleic acid methyl ester, analysis, 18 Olein

determination, 101 gas chromatography of

trimethylsilylated, 107 Oleins, separation, 111, 126 Olive oil, analysis, 157, 171 Optical brighteners, visualisation

reagents, 202 Orcinol, colour reagent, 3, 9 Oxalate, detection, 222 Oxalic acid, determination, 29-30 Oxidising substances, detection, 223 Oxine, colour reagent, 3

Paint, analysis, 147 Palm oil

analysis, 121, 136,164,185 analysis of hydrogenated, 18

Palmitic acid liquid chromatography, 127 thin layer chromatography, 120

Palmitin determination, 101 gas chromatography of

trimethylsilylated, 107 Palmitins, separation, 120, 122, 126, 127 Paper

analysis, 117

analysis of parchment, 50, 64 Paper chromatography

of glycerol esters or ethers, 123-124 of glycerol-containing compounds,

166-169 of polyols, 71-73, 146 visualisation reagents, 190-207

Paper electrophoresis, 59, 79 Pentaerythritol, paper chromatography,

146 Pentaerythritol nitrate, thin layer

chromatography, 183 Pentoses, determination, 3 - 4 Peracetic acid, reaction with allyl

alcohol, 18 Periodate, oxidising agent, 4, 9 ,13-26 ,

28, 98-102,134-141, 150, 229 Permanganate

oxidising agent, 4, 26-30, 142-143 reagent for formaldehyde, 25

Pharmaceutical preparations, see also Drugs, Ointments, Suppositories

detection of glycerol in, 31, 51, 52, 73 determination of glycerol in, 12,15,

18, 29-30, 33, 62, 68 determination of glycerol trinitrate in,

165, 188 determination of propylene glycol in,

18 extraction of glycerol from, 65 extraction of glycerol trinitrate from,

161,162 Pharmacopoeia tests

for glycerol analysis, 219-228 for glycerol determination, 228-229

p-Phenylenediamine, JV^-dimethyl-, visualisation reagent, 27

Phloroglucinol colour reagent, 32 reagent for acrolein, 46 reagent for formaldehyde, 24

Phosphatase breakdown of phosphatides, 238

Phosphatides, see also Lecithins determination of glycerol in, 137,145 glycerol release from, 238 liquid chromatography, 185, 186 paper chromatography, 166,167,

168, 169 thin layer chromatography, 116, 175,

INDEX 255

176,177 visualisation reagents for, 190,191,

196, 198, 201-202, 204, 205 Phosphoglycerides, see

Glycerophosphatides Phospholipase breakdown of

phosphatides, 238 Phospholipids, see also Phosphatides,

Sphingolipids determination of glycerol in, 21, 137,

144 enzymic determination, 240 liquid chromatography, 184,186,

187, 188, 189 mass spectrometry, 157 paper chromatography, 124, 167,

168, 169 solvent extraction, 159, 161 thin layer chromatography, 120,121,

175, 176, 177, 178, 179,180, 181,182

visualisation reagents for, 190, 191, 192, 193,194, 195, 196, 198, 200, 202, 203, 204, 205, 206, 207

Phosphoric acid, charring reagent, 197

Placental phosphatides, analysis, 177 Plant lipids

liquid chromatography, 187 solvent extraction, 159 thin layer chromatography, 176

Plasmalogens, 95-96 paper chromatography, 167 solvent extraction, 159 visualisation reagent for, 191

Polyesters, 97 analysis, 144-146, 148 analysis of hydrolysates, 63

Polyethylene glycol esters, liquid chromatography, 125

Polyglycerols analysis of mixtures, 13, 31, 35 detection, 27 gas chromatography of

trimethylsilylated, 107 monitoring of formation of, 62 paper chromatography, 72, 73 thin layer chromatography, 74, 75, 146

Polysaccharides, analysis of oxidation products, 43

Polyurethanes, analysis, 41,44,144-145 Potable spirits, analysis, 42 Preservatives, analysis, 2 Propane, trimethylol-, gas

chromatography, 70,147 Propane- 1,2-diol

detection, 52 determination, 21 determination in nuts, 20 determination of glycerol in, 17-18 gas chromatography, 68, 69, 70, 71 gas chromatography of

trimethylsilylated, 43 ion exchange, 81 liquid chromatography, 79 separation, 59 visualisation reagent for, 22

Propane-1,2-diol esters, thin layer chromatography, 103, 120

Propane-1,3-diol, gas chromatography, 67, 69, 70, 224

Propellants, analysis, 108,127, 157-158,164,165,

183-184 solvent extraction, 161

Propylene glycol, see Propane-1,2-diol Pyridine, 3-methyl-, detection, 48 Pyrocatechol, colour reagent, 2, 52 Pyrogallol, colour reagent, 51 Pyruvate kinase, 232-233, 239, 240

Quinoline, 5,7-dichloro-8-hydroxy-2-methyl-, colour reagent, 33

Quinoline, 8-hydroxy-, see Oxine Quinoline derivatives, detection and

determination, 49-50

Radiometric analysis, 234, 242 Rayon, analysis of sized, 47, 142 Reducing properties, detection, 219, 221 Reducing sugars, detection, 221-222 Refractive index, determination, 61-62,

226 Resorcinol

colour reagent, 2, 32, 51,142 thin layer chromatography, 183

Resorcinol, 4-hexyl-, colour reagent, 52, 144

256 INDEX

Rhodamine B, visualisation reagent, 198, 201, 202, 204-205

Rhodamine 6G, visualisation reagent, 201, 205-206

Ribitol gas chromatography of

trimethylsilylated, 43 thin layer chromatography, 75 thin layer chromatography of

trimethylsilylated, 45 visualisation reagent for, 53

Rosaniline, visualisation reagent, 11, 22 ,60

Safflower oil, analysis, 172 Salicylic acid, colour reagent, 2, 51 Sardines, analysis, 175 Schiff reagents

reagent for acrolein, 47, 143, 144 reagent for formaldehyde, 28, 30 reagent for polyols, 22

Sea water, analysis, 81 Seed oils, analysis of hydrolysed, 107 Sesame-seed oil

analysis, 172 glycerolysis, 112

Shortening, analysis, 103, 120, 126 Silver reagents, 11, 24, 26, 31-32, 47,

143 Skin lipids, see also Epidermal lipids

analysis, 184, 205 Skin lotions, analysis, 18 Skraup reaction, 49-50, 144 Soap

analysis, 4, 7, 34 analysis of waste liquors, 61

Solvent extraction, 64-66, 114-115, 158-162

Sorbitol analysis of hydrogenation products, 80 detection, 51, 61 determination, 16, 21, 42 thin layer chromatography, 59, 74, 76

Soy sauce, analysis, 66 Soya bean oil, analysis, 119, 148, 167 Soya beans, analysis, 158 Sphingolipids

gas chromatography, 166 visualisation reagent for, 204

Sphingomyelin liquid chromatography, 189 thin layer chromatography, 181,182 visualisation reagents for, 200, 204,

205, 206 Spinach extracts, analysis, 186 Stannate complexes, 60, 61 Stearin, see also Glycerol stearate

determination, 99 gas chromatography of

trimethylsilylated, 107 liquid chromatography, 127

Stearins, separation, 112,125,127 Sterols, see also Cholesterol

thin layer chromatography, 117,119 visualisation reagents for, 191, 196

Sucroglycerols, analysis, 18 Sucrose

liquid chromatography, 78 refractive index of solution, 62

Sucrose esters paper chromatography, 124 thin layer chromatography, 172

Sudan Black, visualisation reagent, 206-207

Sugar beet, analysis, 169, 204 Sugar esters, thin layer chromatography,

116 Sugars, see also Aldoses,

Monosaccharides, Reducing sugars

analysis of 1 4C-labelled, 43 analysis of reduction products, 41, 69,

80 detection, 61 determination, 224 ion exchange, 80, 81 paper chromatography, 61, 72, 73 separation, 59 thin layer chromatography, 74, 75, 76 visualisation reagents for, 13, 32

Sulphate, determination, 220 Sulphate ash, determination, 222 Sulphatides, visualisation reagent for,

197 Sulpholipids (sulphoquinovosyl

diglycerides), 96 liquid chromatography, 187 solvent extraction, 159 thin layer chromatography, 176

INDEX 257

visualisation reagent for, 198 Sulphur dioxide, solvent for lipids, 115 Sulphuric acid, charring reagent, 190-

193 Sulphuric acid test, 222 Sulphuryl chloride, charring reagent,

195-196 Suppositories, analysis, 18, 118 Surface tension, 62 Surfactants, non-ionic,

analysis, 78 analysis of hydrolysis products, 32, 146

Syrups, analysis, 32, 65, 76

Tallow, analysis, 163 Tartaric acid, detection, 50, 52 Terephthalic acid esters, 146, 147 Terpenes, tri-, visualisation reagent for,

196

Tetrabase, visualisation reagent, 14 Tetrahymena pyriformis lipids, analysis,

181 Textile sizing agents, analysis, 2, 47, 142 Thermal diffusion, 111 Thermogravimetry, 111 Thin layer chromatography

of glycerol acetates, 105 of glycerol esters and ethers, 115-123 of neutral lipids, 170-174 of phospholipids, 174-182 ofpolyol nitrates, 183-184 of polyols, 74-77, 146-147 visualisation reagents for, 189-207

Threonine, determination, 16 Thymol, colour reagent, 2, 32, 51 Tinopals, visualisation reagents, 202 Tissues, see also specific tissues

analysis, 177, 180, 185 enzymic methods for, 239, 240, 241,

242 analysis of extracts of, 175 analysis of lipids in, 177, 186 analysis of metabolites in, 43 analysis of phospholipids in, 188, 205 deproteinisation, 237 determination of triglycerides in, 139,

140 solvent extraction, 159

Tobacco determination of glycerol in, 15, 72, 78 determination of glycerol and glycols

in, 69 identification of glycerol in, 34 solvent extraction, 64, 65

Tobacco additives, analysis, 31, 59, 65, 73

Tobacco humectants, analysis, 11, 40, 44, 65, 68, 71, 74

Tobacco smoke, analysis, 44, 68, 70 Triacetin

gas chromatography, 144 IR spectrophotometry, 157-158 thin layer chromatography, 183

1,2,4-Triazole, 4-amino-3-hydrazino-5-mercapto-, reagent for formaldehyde, 141

Triethylene glycol detection, 52 gas chromatography, 68 ion exchange, 80

Triglycerides detection and determination, 144, 145 by enzymic methods, 240, 241, 242 by IR spectrophotometry, 158 by nephelometry, 156 by oxidation methods, 134-142 differential thermal analysis, 157 gas chromatography, 128,162-166 liquid chromatography, 125-128,

166, 185, 186, 187, 188,189 mass spectrometry, 157 paper chromatography, 123-124,168,

169 solvent extraction, 159,160, 161 thin layer chromatography, 115-123,

170-174 visualisation reagents for, 190-207

Trimethylene glycol, analysis, 228 Trimethylsilylation, 42-45, 104, 106-109 Triolein

determination, 141, 145 liquid chromatography, 127 paper chromatography, 169

Triols gas chromatography of

trimethylsilylated, 43 thin layer chromatography, 58-59

Triose phosphate isomerase, 233, 242

258 INDEX

Tristearin determination, 145 liquid chromatography, 127

Tungstate complexes, 61 Tungstophosphoric acid, visualisation

reagent, 199, 200

Urea complexes, 112 Urethanes, 34 Urine analysis, 43, 176, 237

Vanadate, oxidising agent, 32-33 Vanilla extracts, analysis, 18 Vanillin,

colour reagent, 51, 52 gas chromatography, 71

Vanillin, ethyl-, gas chromatography, 71 Vanishing creams, analysis, 16 Vegetable oils, see also Frying fats,

Seed oils, and names of specific oils

analysis, 43,145 Veratrole, colour reagent, 2 Vinegar, analysis, 16, 71, 72, 77 Viscosimetry, 63, 226

Water, see also Sea water, Waste water determination, 224-228

Waxes analysis, 202 liquid chromatography, 187

Whale oil, analysis, 163, 187 Wheat flour, analysis, 185, 192 Wine

analysis, 71 determination of diols and glycerol in,

70, 79, 80 determination of ethanol, sugars and

glycerol in, 81 determination of glycerol in, 2, 7,

15, 20, 24, 30 ,47 ,49 ,240 ,241 separation of glycerol from, 66 separation of glycerol and lactic acid

from, 65 Wort, analysis, 241

Xylitol, ion exchange, 80, 81

Yeast, analysis, 179

Zone electrophoresis, 59

Waste water, analysis, 127,189

Analytical Methods for Glycerol

Documents

Transcript of Analytical Methods for Glycerol