Analytical Procedures Using a Corn bined Corn bustion-Diffusion Vessel Improved Method for Combustion of Organic Compounds in Aqueous Solution JOSEPH KATZ, SAMUEL ABRAHAM, and NOME BAKER

Department of Physiology, University of California, School of Medicine, Berkeley, Calif.

OR the radioassay of carbon-14-labeled compounds in aque- F ous solutions a simple and rapid method of combustion is of great value. The use of the Folch-Van Slyke combustion mixture (11) is limited to dry and nonvolatile compounds (5), and although this method n-as adapted to the combustion of certain volatile compounds ( 1 O), the procedure and apparatus are too complex for routine analysis.

The persulfate oxidation as described by Osburn and Kerk- man (8) can be used only for water-soluble compounds, it was adopted by Weinhouse (12) and Anthony and Long (2) for carbon- 14 assay. Although this method offers obvious advantages over the use of Van Slyke and Folch mixture, it has not been used as extensively.

. A

Vacuum

FI 8

Figure 1. Combustion-Diffusion Vessel A . Evacuation of combust ion flask B . Rubber serum cap C. Transfer of alkali from combustion flask by siphon

By using a single combustion diffusion vessel similar in prin- ciple to the ones described in the previous paper (3), the authors greatly simplified the persulfate oxidation procedure.

APPARATUS AND REAGENTS

Combustion flask. Ordinary 50-ml. narrow-mouthed Erlen- meyer flasks provided with a center well for carbon dioxide ab- sorption were used. A center well similar to that used in a con- ventional Warburg flask was sealed to the bottom of the vessel. The exact dimensions of the well are not critical; a well about 12 mm. in diameter and approximately 3 em. high was found to be convenient (see Figure 1 ) . [This flask is used extensively in this laboratory as an incubation vessel for the study of tissue metabolism.

Serum bottle stoppers, sleeve type. Catalog KO. 16201, Braun, Knecht, and Heimann Co., San Francisco, Calif.; Catalog S o . 2319, A. H. Thomas Co., Philadelphia, Pa., 1950.

Siphon for carbonate sample transfer. X siphon which greatly facilitated the quantitative transfer of the carbonate solution from the center well of the combustion flask is shown in Figure 1.

Reagents. Potassium persulfate, reagent grade; 4% silver nitrate solution.

It was designed by Hans Baruch.]

PROCEDURE

About 500 to 600 mg. of solid potassium persulfate were placed in the flask so that none of the salt M+as introduced into the center well. The sample (which should yield from 10 to 80 mg. of barium carbonate) and water were added to give a volume of from 5 to 15 ml. The flask was swirled in order partially to dis- solve the persulfate. This mixture was acidified with a few drops of dilute sulfuric acid in order to liberate any carbonate present.

One milliliter of 4% silver nitrate solution ~ a a added to this mix- ture, enough standard carbon dioxidefree sodium hydroxide to absorb the carbon dioxide formed v a s ddivered into the center well, and the flask x a s stoppered with the rubber cap. Xone of the reagents need be measured accuratelv. The vessels were evacuated by inserting a 20- or 22-gage hypodermic needle which was connected to a vacuum line. The vacuum serves to remove any dissolved carbon dioxide and provides a tight seal for the flask during combustion.

brought UD to about i o " in a Deriod of 15 to 20 minutes. The flasks were placed in a vater bath a t 40" to 50" C. and

The contGnts 01 the main well darkened and gas evolution commenced. The reaction proceeded smoothly a t 70" to 75" and was usually completed n-ithin 30 minutes. At the end of this period, the dark color disappeared and the solutions became water clear.

The flasks required little attention; however, when the tem- perature was permitted to increase too fast or to exceed i 5 " , the evolution of oxygen became too vigorous and forced the cap out. The combustion may also be carried out in an oven initially set a t i s o . The reaction proceeds somewhat more slowly than in the water bath and takes about 1 to 1.5 hours; however, i t requires no attention.

After the flasks were cooled to room temperature, the vacuum was released by the insertion of a hypodermic needle through the rubber cap. The flasks were opened and the contents of the center well Tvere quantitatively transferred by means of the siphon into a suitable vessel for titration of the alkaline carbonate solution.

An analyst can burn and titrate the barium carbonate from over 20 samples in a few hours.

EXPERIMENTAL AND RESULTS

The above procedure has been used extensively in this labora- tory for the combustion of carbon-14lactate and carbon-14- acetate and to some extent for radioactive assay of carbon-14- ethyl acetoacetate, carbon-14-acetone, and carbon-14-glycerol with highly satisfactory results. However, this method is not limited to the assay of carbon-14 but can be used for the combus- tion of a large variety of water-soluble compounds including car- bohydrates, organic acids, amino acids, alcohols, and some hetero- cyclic compounds as demonstrated in Table I. It gives satis- factory results with volatile compounds such as acetaldehyde, ethyl alcohol, and the like. As can be seen from Table I, the

Table I. Carbon Recovery with Persulfate Oxidation All samples prepared by weighing analytical reagent grade compounds and dissolving them in water. Yields were determined by titration of the alkali (after addition of barium chloride) to the phenolphthaleln and the methyl orange-methylene blue end points. Each value is an average of at least two separate determinations. The duplicate titrations agreed

within 2%. Carbon Recovery Taken, of coz,

Compound M g . % Glucose Fructose Inositol Glycerol Sodium acetate Lithium lactate Succinic acid Citric acid Benzoic acid Potassium acid phthalate Ethvl acetoacetate Acetaldehyde Acetaldehyde 4- 20 mg. Glutamic acid Ethyl alcohol Histidine hydrochloride Thymine hydrochloride Adenine

NaHSOa

2 . 4 0 100 2 . 4 0 99 2 40 100 4 . 4 0 2 . 4 0 3 . 6 0 2 . 4 0 2 . 4 0 2 . 8 2 2 . 4 0 1 . 7 2 2 . 8 0 2 .80 2 . 4 0 2 . 5 2 2 . 4 0 2 . 1 0 2 . 0 5

98 99 98 98

100 95 99 98

100 100 99

102 97 96 80

1503

1504

recovery of carbonate ranged from 95 to lOOyo in all compounds tested, with the exception of adenine which gave a value of about 80%. Persulfate combustion cannot be used for water-insoluble compounds and may be found unsatisfactory for the combustion of some stable ling compounds. Also unsatisfactory results TTere obtained in the combustion of methylamine, as observed also by Anthony and Long ( 2 ) .

The results obtained n ith carbon-14-glucose and carbon-14- succinate in a comparative study using the Van Slyke and Folch combustion fluid and persulfate can be seen in Table 11. The radioactive assay of these compounds by these tvio methods was found to be identical The flask uapd here can be used in general as a diffusion flask. Although its dimensions are not as efficient for diffusion as the conventional Conway dish (j), it has the ad- vantage of easy evacuation, t h w hastening diffusion. These flasks are more convenient to use than Conw,y dishes, as they do not require greasing: and they are also much cheaper. Also, if required, reagents may be injected into the flask after it is closed.

The persulfate oxidation can also be performed in a variety of vials or vewels adapted fox this purpose (3). Thus, using an ordinary Skrip ink bottle (the top well of iThich serves as an alkali well), recoveries of 95 to lOOyo were obtained in the com- bustion of acetate, citrate, lactate, acetone, and glucose.

The special value of such flasks is that they combine the char- acteristics of a reaction and diffusion vessel. Such flasks were used by the authors in a great variety of reactions that involve the formation of carbon dioxide, such as the decarboxylation of lactate, amino acid assay with ninhydrin, and the complete degradation of carbon-14-acetate.

ACKNOWLEDGMENT

The authors wish to acknodedge the gifts of succinic acid-2- carbon-14 from B. M. Tolbert and of recrystallized adenine and thymin from Carl Emanuel.

LITERATURE CITED

(1) Abraham, S., Putman, E. W., and Hassid, W. Z., Arch. Biochem. Bzophys., 41, 61 (1952).

A N A L Y T I C A L C H E M I S T R Y

Table 11. Comparison of Van Sly-ke and Persulfate Combustion of Carbon-14 Compounds

Glucose was prepared photosynthetically (9) and was shown t o be erenly labeled ( I ) . Succinic acid used was labeled with carbon-14 in the 2-posi- tion (7) . Identical aliquots were combusted in each case. The Van Slyke combustion was carried out according to Barker (4) and all samples after the addition of barium chloride and titration of the excess alkali were mounted on filter paper according t o Entenrnan ef al. (6) and assayed

for radioactivity on a conventional end-window counter.

Persulfate Oxidation Van Slyke Oxidation Barium Specific Barium Specific

carbonate, activity, carbonate, actlvlty, mg. c./min./mg. mg. c./min./rng.

Glucose- 3 5 . 1 9 . 1 3 7 . 5 9 . 2 carbon-14 3 7 . 3 9 . 4 3 6 . 5 9 . 3

Succinic acid-2- 1 8 . 2 2 1 . 1 18 3 21.2

Blank 0 . 5 0 . 8

carbon-14 1 6 . 4 20 .5 1 8 . 0 2 0 . 7

Anthony, D. S., and Long, 31. V., Oak Ridge Xatl. Lab., Rept . 1303 (1052).

Baker, N., Feinberg, H., and Hill, R., ;ISAL. CHBM., 26, 1504 (1964).

Barker, H. A. , in “Isotopic Carbon,“ by Calvin AI . , Heidel- berger, C. , Reid, J . C., Tolbert, B. AI., and Yankwich, P. E., p. 93, New York, John Wiley &- Sons, 1949.

Conway, E. J., “Microdiffusion Analysis and Volumetric Error,” 1st ed., Sew York, D. Van Kostrand Co., 1940.

Entenman, C., Lerner, S. R., Chaikoff, I. L., and Dauben, W. G., Proc. SOC. Esptl. Bzol. M e d . , 70, 364 (1949).

Jorgenson, E. C., Bassham, J. .i., Calvin, A f . , and Tolbert, B. AI., J . Am. Chem. SOC., 74, 2418 (1952).

Osburn, 0. L., and Werkman, L. H., ISD. ENG. CHEM., ; IN~L. ED., 4, 4421 (1932).

Putman, E. W., and Hassid, IT. Z., J . Biol. Chem., 196, 749

Thorn, J. A , , and Shu, P., Can. J . Chem., 29, 558 (1952). Van Slyke, D. D., and Folch, J., J . Bzd. Chem., 136, 509

(1940). Weinhouse, S., in “Isotopic Carbon,” by Calvin, M., Heidel-

berger, C., Reid, J. C., Tolbert, B. AI., and Yankwich, P. E., p. 94, Kew York, John Wiley &- Sons, 1949.

(1952).

RECEIVED for review July 6, 1953. Accepted April 5 , 1954.

Analytical Procedures Using A Corn bined Corn bustion-Diffusion Vessel Simple Wet-Combustion Method Suitable for Routine Carbon-1 4 Analyses NOME BAKER, HAROLD FEINBERG, and ROBERT HILL

Radioisotope Unit, Veterans Administration Hospital, Cleveland, Ohio, and the Division of Physiology of the University o f California, School o f Medicine, Berkeley, Calif.

ECEKTLY Michaels et al. ( 4 ) described the use of a screw- R cap bottle for the quantitative oxidation of p-hydroxy- butyrate to acetone. By using a screw-cap bottle which has a smaller compartment within, the authors have recovered carbon dioxide from the semiquantitative oxidation of a series of organic compounds with Van Slyke-Folch combustion fluid ( 5 ) . The method, though less quantitative than the original Van Slyke- Folch procedure ( 5 ) , has proved satisfactory for the routine assay of isotopically labeled, dry, nonvolatile compounds.

APPARATUS AND REAGENTS

Screw-cap bottles of 0.5- to 2-ounce capacity with a glass well, either permanentlv attached (Figure 1, A ) or removable (Figure f,, B ) . Sheaffei’s Skrip writing fluid bottles “with the top well, 2-ounce capacity, were used successfully as combustion vessels. The enclosed well need not be in a central position for quantitative diffusion-absorption of carbon dioxide into sodium hydroxide.

Bottle caps, either metal or plastic, equipped with a full rubber

gasket (Goodrich Ameripol-D rubber, ‘/IB inch thick) previously washed in boiling soap solution. It is suggested that an extra supply of caps be kept available to replace the metal caps, which rust readily, or the lastic caps, which may crack when tightened.

Carbon dioxidegee sodium hydroxide, 31V. Van Slyke-Folch combustion mixture ( 5 ) .

PROCEDURE

Pretreatment of Apparatus. The recommended treatment of the apparatus prior to its use is similar to that commonly em- ployed in wet-combustion procedures ( 2 , 5 , 6). Bottles and wells are heated in chromic acid cleaning solution, rinsed, and oven- dried. About 5 ml. of the combustion mixture are then added to each bottle; the system is capped and heated for 30 minutes at 15 pounds pressure in either an autoclave or pressure cooker. If not treated in the above manner, new rubber gaskets, even though previously washed in boiling soap solution, may produce 8 to 9 mg. of barium carbonate blank values. After an appa- ratus has been used for an oxidation, it is rinsed with distilled water, dried a t room temperature, and stored inverted in a drawer. If the bottles and wells are not pretreated with hot