Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

THE VISCOSITY AND MOJ:.,EJULAR CO:NFORMATICN

OF GALACT<lv1ANNANS IN SOLUTI<N

Wayne Robert Sharman

A thesis pr-esented in part;i.al fulfillment of the requirements far the degree of

Doctor of :Eh:ilosophy.

Chemistr,y / Biochemistry / Bi�ysics Department

Massey University April 1974

To Sharyn.

A cknowledgements

•

I wish to thank my supervisors, Professor E.L. Richards and

Pro fessor G.N. Malcolrn fer rm.1ch encouragerrent arrl assistance in this

project. I am also indebted to Mr' B.E. Jackson (Chemis try Division,

(i )

D. S.I.R., Petcne ) fer his assistance in t he part of the study concerned

with shear rate measurerrents; Dr M. J. Hardman (Massey ) for Computer

Pr ogramning and Operation; Dr R.w·. Bailey (Applied Biochemistry

Division, D.S .I.R., Palrnersto n North) for advi ce on Sugar Analyses;

Drs J . W. Lyttleton (Applied Biochemistry Division, D.S.I.R.,

Palmerstan North) , C.H. Moore (Massey) , and J . A . Lewis (Massey) far

discussions on Ultracentrifugation Results; and Mr R. Stokell far

tre translation of R.A. Schutz's (1970) paper from tre original French.

I wruld also like to thank Mrs 0. Harris and Miss M. Clark far

typing this thesis.

(ii) Abstract

Two galactomannans, from the seeds of Guar ( Cyanopsis tetragonoloba)

and Carob (Ceretonia siliqua) also !mown as Locwt Bean, have found

widespread use as industrial hydrocolloids . Many other galactomannans

hav e been isolated from legume seeds, but so far none have been widely

employed in industry.

Measurerrents of the viscosity of the solutions of some of the se

latter galactomannans (from Red Clover, Lucerne, Lotus pedunculatus ,

Sophora japonica, and Soybean ) were made along with solutions of Guar

and Carob galactomannans. A w ide range of' viscosities was found for the

various samples, with the viscosity of Guar and Carob galactomannan

solutions being t he highest.

The effects of shear rate, terrperature, and galactomannan

concentration on the solution viscosities were examined. The measured

viscosity changes correlated we ll vdth Schutz's (1970) empirical

equations explaining the viscws solutio11 behaviour of pseudopluatic

carbohydrates (including Guar galactomannan) . The effects on viscos ity

of two connnon industrial additives, sucrose and sodium chloride, were

also examined, and interpreted in terms of a dehydration mechanism.

In order to explain the viscosity results, the galactomannan

molecular weights were deter mined by measuring their sed.:irll3n tation and

diffusion coefficients in an Analytical Ultracentrifuge. Their

molecular weight distributions were obtained both from their Schlieren

sedimentation diagrams using the methcxl of Gr alen and Langermalm (1952),

and from the ratio af their upparent diffusion- coe:t:'ficicnts to their

weight-average diffUsion coefficients (Henley, 1962).

The dependence of galactomannan intrinsic viscosities,

sedimentation coefficients, and diffusion coefficients on molecular

weight (weight-weight average and sedimentation-diffusion average ) was

(iii)

examined, ani n. good linonr correlation obtained in en.ch case� with two exceptions. Lucerne gaJactomannan had a very ·wide distribution of

molecular weights. The galactomannan from Lotus peduncu latus II was

thought to differ slightly in stru cture from tte other ga lactomannans

due to its high substitution ratio of one galactose sidechain for every

mannose in the molecular backbone which changed tre conf ormation of the

mannose backbone.

The dependence of the gaJactomannan intrinsic viscosities,

sedimentation coef ficients and weight-average diffusion co efficien ts on

the weight-weight average degree of polymerisation and/or the weight­

weight average molecular weight was examined in tre light of recent

hydrodynamic t heori es. By use of tre t he orie s of Debye, Beuche and

Brinlanan; Kirkwood and. Riseman; Kuhn and Kuhn; Peter lin; and Flory,

·Fox and Mandelkern it was deduced that the galactomannan molecule

approximates to a random coil in solution. The molecule is highly

extenied, and the presence of the galactose si.dechains alters it s

draining c haracteristics, making it s solution behaviour deviate from

that expected from t he theories.

Comparison of galactomannan solution behaviour_"'With that of two

soluble ce llulose ethers, ethylhydroxyethyl celJulcse (Manley, 1956) and

hydraxyethyl cellulose (Brovm, 1962) shows many similaritie s.

It w as concluded tl'l:l.t galactomannans in solution can be treated as a

linear polymer series provided that the galactose : mannose ratio is not

less than 1 : 5 or mare tha n 1 1.1, and also that their molecular weight

distributions are very similar. This implies that otter gala ctomannans

could be used industrially instead of those of Guar and Carob, provided

some problems of yields and solubility could be surmounted.

It was a lso concluded that galactomannans in aqueous solution have

(�)

conformations very similar to water-soluble cellulose derivatives.

Similarities between mannans and cellulose have already been proved in

the solid state.

(v ) Preface

This work was originally intended to be a comparison of' the viscosity

of' various gaJactomannan solutions with that of some of their derivatives

(e.g. sulphates ) .

The purpose of this was to determine whether the derivatives bad

more desirable properties from an industrial viewpoint than the original

galactomannans, esp;lcially with regard to visccs ity. The idea for thi.s

came fran the large number of water-soluble cellulose deri vates that are

used industrially.

Far the above work, as simple a picture as possibJe of viscosity was

required, and this led to tre neglecting of shear rate in tre

measurerrent of the variation of galactoma.nnan viscosity with terrperatur e

and concentration.

After these first mec:.surenents had been carried out, it wr1s realised

t hat galact anannan solution viscosities were much mare corrplex than had

previous� been imagined. Accordingly, the original aim of this project

was altered, and more carrplete measurerrents of' viscosity (in:::luding shear

rate effects ) and galactom1nnan moleculDr porarreters in s olution were

made, to provide a basis for any fUture work on galnctomannan derivatives .

Contents ---

Acknowledgements

Abstract

P.re:fn.ce

Contents

List of Tables

List of Figures

Section

I Introduct ion

II Preparation of Ga.l8.ctomannans

A

B

Isolation and PUrification

(a) Materials

(b ) Extraction and Purif'ication

Chemical Analyses

(a) Hydrolysi s of Gn.lac t anannans (b ) Paper Chromatography

(c ) Quantitative Estimation of Sugars

(d) Ash and Protein Analyses

III Viscometry

A Introduction

( 1) Definition of Viscosity

(2) Preliminary Comment s

(3) Viscos ity of Galactomannan Solutions

i

ii

V

vi

xii

xiv

1.

13.

13.

13.

13.

19.

19.

19.

20.

21.

23.

23.

23.

24.

26.

(a) Observed Behaviour 26.

(b ) Interpretation of the Vis cous Behaviour of

Carbohydrate Solutions (Schutz, 1970)

(c ) Adn.ptation of Schutz's Empirical Equations

:far use wi. th Ubbelohde Viscaneters.

27.

31.

(v i)

B

( d ) Use of Schu tz's Empirical Equ�tions for Data

Ob tn:ined with Cannon-Fenske V"if3 caneters 34.

Experimental Methods

( 1) Ubbelohde Viscometers

35.

35.

(a) Construction of Ubbelohde Viscometers 35.

(b ) Construction of Wnterbat h 35.

( c ) Calibration of Viscometers 35.

( d ) Determination of Galnctomannan Solution

Viscosities at Varying Temperatures and

Concentrations

(2) Cannon-Fe nske Viscometers

38. 41.

(vii)

(a) (b ) ( c )

M odification of Constant Tempera ture Waterbath 41.

C Resul ts

Calibration of Viscometers

Measurerren t of Galactomn.nnan Solution Shear

Rates and Viscosities

( 1) Ubbelohde Viscome ters

(a) Genero..l Comments

(b ) Concent ration Dependence of Viscosity

(c ) Temperature Depen dence of Viscosity

(d ) Calculation of In trinsic Viscosity

(2) Cannon-Fenske Viscome ters

41.

47.

49.

49.

49.

53.

56.

62.

(a) General Comment s 62.

(b ) Limitations of the Apparatus 62.

(c ) Comparison of the Measured Vi scosities and

Mean Sre ar Rates of Galac tomannan Solutions

to the Schutz Equation 66.

(d) Compariso n of the Measured Viscositie s and

Mean Shear Rates of Galactomannan Solutions

with the Power Law Equati on

(e ) C alculation of the In trinsic Viscosity of

(viii )

Galactomannans c �t Mean Shenr Rate G = 1) 77.

IV The Effect of Additives (Ionic arxl Non-ionic) on the Viscosity

of Galactamannan Solutions

A

B

In trcxlu et ion

Experimental

( 1) Determin ation of the Stability of Stored

Galactomannan Solutions Containing Sucrose or

Sodium Chloride

(2) Measurerrent of the Effe cts on Galn.ctoma.nnan

Solution Visco s ity of Different Conce ntrations

Added Sucrose ar· Sodium C hloride

C Re sults

(1) Presentation

(2) Discu ssion of Results

(3) Interpretation of the Observed Effects

V Molecular Weight Determinations

A Introduction

( a) Moleculn.r -ifeight Averages

(b) Ul tro.centrifuge Methods

B Experimento.l

(a) Ma terin.J.s

(b ) Methods

(c ) Sedimenta tion Coefficient Determinations

(d) Diffusion Coefficient Determinations

84.

of

85.

87.

87.

87.

89.

93.

93.

93.

95.

99.

99.

99.

100.

103.

VI

c (e) Partial Specific Volume DeterminQtions

Results and Discussion

106.

107.

(a) Sedimentation Coef'ficients 107.

(b) Diffusion Coefficients 1 1 1.

(c) Partial Specific Volurre 1 14.

(d) Calculation of Molecular Weights 1 16.

(e) The Relationship Between Galactomannan Moleculnr

Weights, Diffusion Coefficients, rurl

Sedimentation Coefficients 1 19.

The Measurement of Galactomannan Molecular Weight

Distributions

A Measttr'errent of Sed.inentation Coefficient Distribution

121.

as an Indication of Molecular Weight Distribution 12 1.

( 1) Introduction 12 1.

(2) Measurerrent of Sedirrento.tion Coefficient

Distributions from Sedimentation Velocity

Experiments - An Outline of Available Methods 123.

(3) Determination of a Sed:i.rren tation Coefficient

Distribution Using the Ivietho::3. of Gralen and

LnngcrmaJm 125.

(4) Experirrental 126.

(a) Calculation of Results 126.

(b) Comparison of the Sedirrentation Coefficient

Distributions of the Gala.ctomannans 128.

B Diffusion Coefficients as an Index of Polyd:ispersi ty 130.

VII Conc lusions 133.·

A Tre Relati onship Between Viscosi ty and Moleculur Weight :

tre Staudinger (or Mark-Houwink) Relationship 133.

(ix)

B Galactomannan Molecular llimensions from Recent

Hydrodynamic Thear'ies

( 1) C'..enera l Bus is of the Theories (2) Outline of Recent Hydrodynamic Theories

(a) Debye, Beuche and Brinkman

(b) Kulm and Kuh.n

(c ) K irkwood md Riseman

(d) Peterlin

(e) Flory, Fox an d Mandelkern

(3 ) Calculation of Galactomannan Molecular

138.

138.

140.

140.

142.

143.

144.

145.

Conformation in Solution f':rom Solution Behaviour 147.

(a) Genera l Comments

(b) De bye, Beuche ani Brinkman

(c ) Kuhn and KuhJ1

(d) Y..irkvmod and Riseman

(e ) Peterlin

(f ) Flory, Fox and Mandelkern

(4) Comment s on the Molecular Dimensions Calculated

147.

149.

150.

152.

153.

155.

from Hydrodynamic Theories 160.

C Summary of the Solution Propert ies of Gala ctomannans 162.

Appendix I Ga lactomannans from Plant See ds 164.

Appendix II Galactomn.nnans from Yeasts , Moulds and Fungi

Appendix Ill Literature Values of Galactomannan Molecular

Weights and Viscosities

Appendix IV

Appendix V

Galactomannan Dependence on Concentration and

Temperature (Ubbe loh de Viscom etcrs ) Dependence of Galactamannan Viscosi ty on

Concentration and Mean Shear Rate at 20°0

169.

170.

172.

174.

(x)

Appendix VI-a Effect of Added Sucrose on Galactomannan

Solution Viscosity

References

-b Effect of Added Sodium Chloride on

Galactoma��an Solution Viscosity

(xi )

179.

183.

187.

Table No,

II-1

II-2

II-3

II-4

III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

List of Tables

Average Analyses of Guar and Carob Seed Endosperrns

Galactornannan Yields

Galactornruman Protein and Ash Analyses

Mannose/Galactose Analyses of Galactomannans

Viscometer Constants and Cnlibrants f or Ubbelohde

Viscorne ters

Calibration Constants of Cannon-Fenske Viscorneters

(xii)

17.

_21.

22.

38.

42.

Determination of the Mean Heads of Cannon-Fenske Viscometers 44.

Calculated Voll..llffis and Radii of Cannon-Fenske Viscometers

Constants (K} for the Calculation of the Mean Shear Rate

(G), and Some Sample Shear Rates

Comparison of the Experirrental Results with an Equation of

the Farm

Temperatures

log '1/ = log K i--(12)c e L e a at a Series of

Comparison of the Experimental Results with an Equation

of the Form D 1 loge L = logeK" + (-a) T Galactomannan Concentrations

Definition of Viscometry Terms

at a Series of

Maxinnlm an:l Min:i.muro Values of Visccsity and Mean Srear Rate

Obtainable with a Given Viscometer

Comparison of the Visccsi ty and Mean Sre ar Rate Data of

Galactomannans with the Schu tz Equation

Comparison of tre Viscosity and Mean Srear Rate Data of

Galactornannans wi th the POII'er Law Equation

Values of [1.]o and D in the Equation

log10 Ctl = log1{(] o ( 1-D log10G)

45.

46.

50.

54. 56.

64.

70.

so.

V-1 Values of s, s and k for Galactoii'1'.lnnans 0 s

(xiii)

109.

V-2 Calculation of Diffusion Coefficients DA and Dm

of

Galactomannans 112.

V-3

V-4

Galactomannan Partial Specific Volurres

Galactornannan Molecula r Weights

114.

117.

V-5 Comparison of Galactornannan Sed:irrentation and Diffusion

VI-1

VII-1

VII-2

VII-3

VII-4

VII-5

VII-6

VII-7

VII-8

VII-9

Coefficients 'vith Molecular ·weight Averages 120.

Values of the Ratio D -...!!: far Galactomannans 131. DA

Values of K' and "a" in the Staudinger Equation 136.

Values of K1 and " a" in � ]= K1�,w for Guaran Triacetate,

Cell ulose, and Sorr:e Cellulose Deriva tives in Var i o u s

Solvent s 136.

Galactomannan Molecular Weight s , :J)::grees of Polymerisation,

Sedimentation and Diffus ion Coefficients , and Intrinsic

Viscosi ties 14-8.

Equations RelQting Intrinsic Viscosity, Sedimentation

Coefficient, and Diffusion Coefficient to }Jioleculo.r Weigh t,

and to Degree of Polymerisation 149.

Molecular Pnrnmeters from the Kuhn and Kuhn Theory 151.

Molecular Pararr:e ters from the Kirkwood-Risemo.n Theory 154.

Molecular Ftxo.meters from the Flory-Fox-MuPdelke rn Theory 156.

(r2)t from Sedirr:entntion and Diffusion Do.t a w,w (-2).1.

r 2- from Viscosity Data w,w 158.

159.

(xiv) List of Figures

Figure No. After Page No

I-1 General Structure of Galactomannans from Legume Seeds as

I-2

I-3 a)

b)

Indicated by E..>cperimental Evidence . 4. Proposed Helical Structure of c..f-1,41 linked Manna.n Chain. 9.

Structure of EthyJl1ydroxyethyl Cellulase (Mnnley, 1 95 6 ) .

Structure of Hydroxyethyl Cellulose (Brown, 1 962 ) . 1 2 .

II- 1 Determination of Mannose and Gabctose by the Anthrone

Method - Standro-d Curves. 20.

III- 1

III-2

III-3

III-4

III-5

Definition of Viscosity.

Viscometer Types.

Typical Flow Curves of a Pseudoplastic Carbohydra.te

Solution.

Constant Temperature Waterbath .

Chonge in the Viscosity of Guo.r Galactomnnnan Solution

with Time of Refrigerated Storage .

2 3.

25.

26 .

35.

39.

III-6 Pressure Line Used for Driving Cannon-Fcnske Viscometers . 43.

III-7 a) The Dependence of Galactomannan Viscosity on

III-8

III- 9

Concentration at 20°C. 49.

b) Tre Dependence of Gnlo.ctomannnn Viscos ity on TeiJY?era.tur e. 49. Dependerc e of Gu<T Galnotoi11D.nnan Viscosity on

Concentration a.t Various Temperatures. 52.

Depende:rce of Guar Galactorrnnnan Viscosity on TeiJY?Crature

at Varying Concentrations . 5 2 .

III-10 Dependercc of Sophora japonica Galnctomannan Viscasity on

Concentration at Various Temperatures. 52.

III-1 1 Dependence of Sophora japonica Gnlactorrk�nan Viscosity on

Terrperature a.t Varying Concentrations . 52.

Figure No.

III- 1 2

III-1 3

III-14

III- 15

III- 16

III-1 7

III- 1 8

III-1 9

III-20

IV-1

(xv)

After Page No

The De pen dence of Cktlc.ctom':llma.n Int rinsic Visccsi ties on

TeiiJI:e ra illre.

The Dependence of' Soybean Galactomannn.n Viscosity on Menn

Shear Rate.

The Dependence of Sophora japonica Galactomannan on Mean

Shear Rn.te.

The Dependence of' Guar Galactornruman Viscosity on Mean

Shear Rn. te.

Plot of the Logarithm of the Viscos ity at Zero Shear Rate

against Concentrati on for the Schutz Equation ,

Log-Log Plot of Viscosity against Mean Shear Rate for

Sophora jn.ponicn. GalQCt omannan.

Plot of Power Law Exponent 'a' against Galactomannan

Solution Concentration.

Logarithm of the Power Law Constant (K) Plotted against

Galact anannan Conoen tra tion.

59.

65 .

65 .

65 .

68.

73 .

75.

76 .

The Depende nce of Galo.ctomn.nnn.n Int rinsic Vis cos ities on Mean

Shear Rates. 81.

The Effect of Aging o n the Viscosity of a Guur

Galact omannan Solution Cont aining Sucr ose. 84.

IV-2 The Effect of Aging on the Viscosity of a Guor

IV-3

IV-4

Ga1actornnnnan Solution Containing Sodium Ch loride.

Effect of Vnrying Added Sucrose C oncentration on the

Visccsity of Red C lover Galactorrruman Solution.

Effect of' Varying Added Sodium C'nloride Concentrn.tion on

84.

87.

t he Viscosity of Sophora japonica GalactomannRn Solution. 87.

(xvi)

Figure No. After Page No

IV-5 Variation of the Relnti ve Visccsi ty of Galactomannnn

Solutions \>Vi th the Concentre.tion of Added Sucrose.

IV-6 Variation of the Relative Viscosity of Galactomannan

88.

Solutions with the Concentration of Added Sodium Chloride. 88.

V-1 a) Typical Schlieren Diagram Obtained wring a Sedimentation

Velocity Experiment. 102.

V-2

b) Typical Schlieren Diagram Obtained D.lring a Diffusion

Experiment ..

Calculn.tion of the Area under the Diffusion Bound-'ll'y.

V-3 Typical Galnctomannan Schlieren Diagrams Obtained During

Sedimentation Velocity Experiments.

V-4 Typical Galactomannan Schlieren Diagrams Obtained During

Diffusion Experiments.

V-5 Plots of Sedimentation Coefficient and Apparent Diffusion

Coefficient agn.inst Sedimentation-Diffusion Average

Moleculn.r iTeight.

V-6

VI-3

Plots of Sediment:.'..tion Coefficient arrl \'ieight-Average

Diffusion Coefficient against Weight-Weight Average

Molecular Weight.

Transformation and Integration of an Experimental

8ed:imerrtation Boundary.

Transforrred and Integrated Curves of Total .Area Und.er the

Sedimentation Boundary Plotted against Sedimentation

Coefficient for Sophora japonica ��lactomannan.

Extrapolation of Integrated Sed:ilnentation Boundaries to

102.

103.

107.

111.

119.

119.

125.

128.

Zero Concentration for Sophora japonica. Galactomannan. 128.

(xvii)

Figure No. After Page No

VI-4 Distribution Curves of the Limitin g Sedimen tation

Coefficient Values of the Galactomannans.

VII-1 a) The Dependence of Gal actomannan Intrinsic Viscosities on

129.

the Sedimentation-Diffusion Average Molecular Weight. 1�.

b) The Dependence of Ga1�ctomannan Intrinsic Viscosities on

the Weight-Weight Average Molecular Wei ght.

VII-2 a) The Dependence of Galactomannan Weight-Average Diffusion

Coefficients on the We ight-"Vfeight Average Degree of

Polymerisation.

b) The Dependence of Gn.lactomannan Limiting Sedirrentation

Coefficients on the Weight-Weight Average Degree of

Polyme risation . VII-3 a) The Dependence of Galactome..nnan Intrinsic Viscosities on

the Weight-1'/eight Average Degree of Polymerisation (Kuhn

and Kuhn; Petcrlin) •

b) The Dependence of Gah'lctomnnnan Intrins ic Viscosities on

148.

148.

148.

the 'Weight-Weight Aver<J..ge Degree of Polymerisation (Kirkwood

VII�

and Riseman) •

Plot - -1.

of M against M2 for Yft'!f WJW

ru Peterlin; 1950, 1952)�

148.

Ga1actomannans (after

154.