The viscosity and molecular conformation of galactomannans in … · The galactomannan from Lotus...
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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.