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Non-Covalent Interactions in Polysaccharide Systems

Marguerite Rinaudo

CERMAV-CNRS, BP53X, 38041 Grenoble cedex 9, France

Fax: 33-476547203; E-mail: [email protected]

Received: March 8, 2006; Revised: May 14, 2006; Accepted: May 15, 2006; DOI: 10.1002/mabi.200600053

Keywords: amphiphilic polymer; hydrogen bond; hydrophobic interactions; ionic interactions; ionic selectivity; networks;

polysaccharides; physical gelation; solvation

Introduction

Knowledge of the physical chemistry of natural and modified

polysaccharides is progressing and several examples are now

available that clearly demonstrate the role of non-covalent

bonds present in aqueous solutions of polysaccharides. Be-

causeof the stereoregularity of such polymers, interactions are

often cooperative and play a role in polymer conformation and

association in solution, even in dilute regimes.

The purpose of this paper is to give examples of poly-

saccharides that have a well-characterized chemical struc-

ture in which these interactions are clearly demonstrated.

These include Coulombic interactions (polyion ion inter-

actions) and ionic selectivity, and dipolar hydrogen bonds

that affect helical structure stabilization, chain stiffness,polymerpolymer interactions, or hydration and hydro-

phobic interactions, especially in some modified polysac-

charides (block and graft copolymer types). This paper is

based mainly on our own work in which the same methods

for purification, characterization and analysis of physical

properties have been developed.

In the following examples, only aqueous solutions are

considered: the nature of the water solvent is very important

in controlling the behavior of water-soluble polysaccharides

and its high dielectric constant results in a large contribution

of electrostatic interactions. In this respect, water plays a

crucial role: i) it causes repulsion between same chargedspecies to prevent the collapse and attraction between

oppositely charged entities,and ii) it hasa role in theentropic

formation of hydrophobic interactions and also forms a

hydration shell around solute molecules because of its

dipolar character. Bulk water forms a highly cooperative

hydrogen-bonded network,in which the solute willexchange

water molecules during dissolution, which leads to a new

distribution of hydrogen bonds.[1]

It is also clear that one type of non-covalent bond is usually

not independentfrom others and the properties of the systems

result in a delicate equilibrium between the different contri-

butions, among which one dominates, depending on theactual thermodynamic conditions.

Hydrophobic Interactions

Amphiphilic molecules dispersed in aqueous media disturb

the water structure. The hydrophilic polar part of the mole-

cules interacts with water (with hydrogen-bond formation

and ion hydration) and the hydrophobic parts interact to

form a hydrophobic domain to reduce contact with water.

From this organization, interesting new properties may

Summary:This paper describes the behavior of some poly-saccharides with well-known chemical structures and in whichthe influence of cooperative secondary interactions play animportant role. The roles played by hydrophobic and ionicinteractions (including ionic selectivity) on polysaccharideconformation and gelation are discussed. Electrostatic attrac-tions are also important in the complexes formed betweensurfactants and polyelectrolytes of opposite charge. Finally,van der Waals dipolar interactions and particularly hydrogen-bond formation are examined. The role of hydrogen bonds insolubility, conformation, and especially the local stiffness ofpolysaccharides, but also in polymerpolymer complexesfrequently obtained with polysaccharides, is developed.

Repeat unit for a number polysaccharides.

Macromol. Biosci. 2006, 6, 590610 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

590 DOI: 10.1002/mabi.200600053 Feature Article


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occur. Two types of systems are examined here, based

on the chemical modification of natural polysaccharides:

methylcelluloses, which are neutral polymers, and alkyl-

ated chitosans, which are positively charged polymers

under acidic conditions. These two types of polymers have

an amphiphilic character because of the hydrophobic part

introduced by chemical modification and the hydrophilicpart composed of the polysaccharide backbone. They are

sensitive to temperature, salt concentration, and the nature

of the hydrophobic part.

Methylcelluloses

Commercial methylcelluloses are usually obtained by

methylation of semi-crystalline cellulose in the solid state;

the reaction is heterogeneous because of differences in

reactivity of the amorphous and crystalline zones. In our

work, it has been clearly demonstrated that commercial

methylcelluloses have a blockwise distribution of highlymethylated parts of thecellulosic chains. It hasbeen known for

a long time that these block copolymers behave in a very

unusual way in aqueous solution, to show a sol gel transition

at around 60 8C. This gelation induced by an increase in

temperature is directly related to the hydrophobic character-

istics of the polymer. The mechanism of gelation has been

examined on different commercial samples. Methylcelluloses

have been prepared in our laboratory under homogeneous

conditions and their behavior has been compared with

heterogeneous polymers.[25]

The first difference between the two types of methylcellu-

loses (homogeneous or heterogeneous) is that the solubility

is shown to depend on themethyl group distribution along the

chain. Homogeneous samples become water-soluble when

the degree of substitution (DS fraction of OH groupssubstituted per D-glucose unit) is around 0.9, a value that is

enough to prevent the packing of the cellulosic chains in the

dried state. With heterogeneous methylcelluloses, a DS of

1.3 is needed for solubilization in relation to the existenceof non-substituted zones (Figure 1).

In Figure 2, the rheology of a commercial methylcellu-

lose from Dow Chemicals (sample A4C) at a polymer con-centration (10 g L1) larger than the overlap concentrationC* as a functionof temperature is given. From this, it can be

observed that the situation is relatively complex: between

35 and 55 8C, a clear loose gel is formed for which G0 >G00

(at 1 Hz) followed by a strong increase of the G 0 modulus

above 60 8C. This second transition step occurs when the

polymer concentration is higher than the overlap concen-

tration (around 2 g L1 for this sample) and this gel isturbid. The gelation corresponds to a phase separation for

this low critical solution temperature polymer at a critical

temperature around 29 8C, for which the phase diagram for

this study is given in Figure 3.[6] The gelation is reversible

but with a large hysteresis as shown in Figure 4; this pheno-

menon has been observed not only by rheology but also by

DSC (differential scanning calorimetry) (Figure 5).[7]

Hysteresis is often observed in physical gelation and seems

to be related to the degree of aggregation of chains and the

Marguerite Rinaudo has a Master degree (1964) and a Ph.D. (1966) from the University of Grenoble

(France) on physico-chemical properties of polyelectrolytes; her work was devoted to the synthesis and

solution properties of carboxymethylcelluloses. From that time, she continued to develop her research

work on water-soluble polymers and especially on polysaccharides (cellulose, starch and chitosanderivatives, hyaluronan and other bacterial polysaccharides, seaweed polysaccharides); in her

laboratory she settled up a series of equipments and methodologies to investigate the specific behavior

of polysaccharides, which have to be considered as semi-rigid polymers. She started as full Professor

in 1968 at Joseph Fourier University (Grenoble-France); actually, she is Emeritus professor. During 12

years (19841996), she has been the director of a CNRS institute named Centre de Recherches sur

les Macromolecules Vegetales working on plant oligo- and polysaccharides. She received important

awards from the French government (Chevalier dans lOrdre National du Merite (1984) et dans

lOrdre de la Legion dHonneur (1998)); she became associate member of the Chilian academy of

sciences (2005) and of the Brazilian academy of sciences (1991). She published more than 400 original

papers and delivered more than 250 main lectures in international meetings. She also supervised more

than 50 PhD thesis and she is member of the editorial board of 5 international journals (from which

Biomacromolecules and Food Hydrocolloids).

Figure 1. Repeat unit for a number polysaccharides. Cellulose:R OH, chitin: R NHCOCH3, chitosan: R NH2 orNHCOCH3depending on the acetylation degree (DA), alkylatedchitosan: R NH2 or NHCOCH3 or NH(CH2)nCH3depending on DA and on the degree of substitution DS.

Non-Covalent Interactions in Polysaccharide Systems 591

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stability of the aggregates formed. The same type of

behavior will also be discussed later for k-carrageenan.

The progressive aggregation of methyl groups has been

observed by 13C NMR spectroscopy in the presence of

dimethyl sulfoxide (DMSO) as a reference; the mobility of

methyl substituents around 60 ppm is reflected by the ratio

of the integral of the signal of O-Me in the C-6 position and

that of the DMSO probe.[6] The results are given in Figure 6

and clearly confirm the two steps. Gelation is induced by

increasing the temperature but it also depends on the ionic

concentration (Figure 7) and on the nature of the anion and

cation that constitute the added salt. This behavior is

characteristic of hydrophobic interactions.[5] The sequence

for the second step of gelation, particularly as a function of

the nature of the ions and relative to the ability to induce

gelation at a given salt concentration (0.5 M) is as follows:

- for sodium salts:Cl

> NO3

> without salt> I

> SCN

,- for chloride salts: Na K>NH4

>Li>withoutsalt,

- for nitrate salts: Na K>NH4>without salt> Li.

The first ion cited is the one that induces gelation at the

lower temperature.

For homogeneous methylcelluloses the enthalpy obtained

by DSC remains very low and corresponds to loose inter-

actions between the modified cellulosic chains. From these

conclusions, the following mechanism is proposed: the clear

gel is attributed to the interaction of highly substituted zones

of the cellulosic chains; the second phase separation then

occurs, which involves the residual polymer, and the turbidgel is formed with a large increaseof thegel modulusoverC*.

Alkylated Chitosans

Chitosan is a modified natural polysaccharide obtained by

deacetylation of chitin, a constituent of the crustaceous

shells of crabs and shrimps.[8] Chitosan is a linear polymer

formed of partially acetylated b-(1 ! 4)-D-glucosamine(Figure 1). It is soluble in acidic aqueous media by pro-

tonation of the NH2 groups at the C-2 position; it becomes

a polycationic polymer whose charge density depends on

Figure 2. Influence of temperature on the rheological behavior of a commercialmethylcellulose in water. Sample A4C: DS 1.7, Cp 10 g L

1, G0 is thestorage modulus, and G 00 the loss modulus measured at o 1 Hz. Heating rate0.5 deg. min1.[5]

Figure 3. Phase diagram of commercial methylcellulose sampleA4C (DS 1.7) in water, with the cloud point curve (&) and thesolgel transition line (&) determined by rheology. Reprintedfrom ref. [6], Copyright 2006, with permission of Elsevier.

592 M. Rinaudo



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the degree of acetylation and on the degree of protonation,

depending on the concentration and nature of the acid used

for protonation.[9,10] It should be notedthat theintrinsic pK0is around 6 and that chitosan is soluble in acidic media of

pH < 6. In acidic conditions, chitosan, even when fully

protonated, tends to form aggregates under different condi-

tions of temperature, nature and concentration of the salt,

etc., from which it has been concluded that aggregates are

formed in solution as a result of hydrogen bonds and

hydrophobic interactions.[11a] This hydrophobic character

Figure 4. Rheological behavior of a commercial methylcellulose in water as afunction of successive heating and cooling. Sample A4M: DS 1.8,Cp 15 g L

1,G0 is the storage modulus, andG00 is the loss modulus measured at o 1 Hz.[5]

Figure 5. Thermogram obtained from a DSC experiment on heating and cooling of a solution of acommercial methylcellulose. Sample A4C: DS 1.7, Cp 30 g L

1, temperature variation rate0.5 deg. min1. Reprinted from ref.[6], Copyright 2006, with permission of Elsevier.




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is not only based on the presence ofN-acetyl groups but also

on the main polysaccharide backbone as mentioned below.

The reactive C-2 position allows specific reactions,

especially reductive alkylation with aldehydes. Aldehydes

of different chain lengths (from 3 to 14 carbon chains,

named C3 up to C14) have then been grafted onto the

chitosan backbone to give original properties in aqueous

solution.[12,13] It is recognized that the properties depend on

the degree of alkylation, the length of the alkyl chain,

temperature, pH, and on the polymer and salt concentra-

tions, as shown in Figure 8. The alkyl chains with a C12length start to give large interchain interactions, even at

concentrations lower than the overlap concentration, to

become a physical gel depending on the ionic concentra-

tion.[13] The hydrophobic interactions are counter-balanced

by electrostatic repulsions that depend on the net charge of

the chitosan (i.e., on the pH) progressively screened by the

addition of salt. Alkylated chitosan with a C12 alkyl chain

(named Chit-C12) is soluble in 0.3 Macetic acid, which is a

good solventfor unsubstituted chitosan with a characteristic

rheological solution behavior (G00 >G0 over a large frequ-ency range). The complex viscosity for 6 and 10 g L1 is

nearly Newtonian in the frequencyrangecovered(Figure 9A).In the presence of sodium acetate salt, even at concentrations

as low as 0.025 M, the complex viscosity becomes largely non-

Newtonian because of loose inter-chain interactions. Gel-like

behavior (G0 >G00) is obtained over 3 g L1 as shownearlier.[13] In Figure 9B, solution behavior with G0 G00) when some electrostatic interactions are screenedby the addition of salt (Table 1).

The role of pH and temperature is shown in Figure 10.

When the pH increases, the net charge of the polymer

Figure 6. Relative intensity of the signals obtained forthe OMesubstituent at the C-6 position by 13C NMR spectroscopy as afunction of temperature for a methylcellulose in D2O.Sample A4C: DS 1.7,Cp 44.6 g L

1. Reprinted from ref.[6],Copyright 2006, with permission of Elsevier.

Figure 7. Influence of salt concentration on the rheological behavior of a commercialsample as a function of temperature in aqueous medium. Sample A4M:Cp 10 g L

1,G0

is the elastic modulus, and G00 is the viscous modulus measured at o 1 Hz.[5]

594 M. Rinaudo



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(related to the degree of protonation, a) decreases, hydro-

phobic attractions dominate, and gel-like behavior is

observed with a large increase in the apparent viscosity

followed by a phase separationunder a criticalvalue ofa. At

the same time, it is shown that raising the temperature

increases the observed maximum viscosity. This behavior

provides evidence of the existing balance between electro-

static repulsion and hydrophobic attraction, with results

summarized in Figure 11.[14]

The bulk properties of these associating polymers are

very important for use as thickeners with an important non-

Newtonian character, because of the loose and reversible

inter-chain interactions. This mechanism is well demon-

strated when b-cyclodextrins (CDs) are added to C12

alkylated chitosan. Thevery high viscosity is reduced by the

progressive addition of CD because of alkyl chain/CD

complex formation, which leads to chain separation and

viscosities typical of derivatives with shorter alkyl chains

(Figure 12).[13] The micelle-like junctions form hydro-

phobic domains in a semi-dilute regime as shown by

modification of the fluorescence spectrum of pyreneadded as a probe in solution. The fluorescence spectrum

of pyrene is modified in relation to the polarity of the

environment. It has been shown that these hydrophobic

domains are able to adsorb hydrophobic substances such as

taxol or B2vitamin and behave as a reservoir for controlled

drug release.[15,16]

Another important characteristic of amphiphilic poly-

mers is their interfacial activity. It has been shown that

alkylated chitosan moderately decreases the air/water and

oil/water interfacial tension but the effect increases with

alkyl chain length, as shown previously.[13,1720] In addi-

tion, it has been shown that the mechanical properties of the

interfacial film formed are completely changed compared

to those of simple surfactants.[21]

A cooperative interaction, normal with polyelectrolytes,

is obtained between chitosan and oppositely charged

surfactants, which leads to a surfactant-polyelectrolyte

electrostatic complex (SPEC).[1921] The interfacial film isa nanostructured film stabilized by strongly interacting

surfactants and is able to encapsulate molecules.[20,22,23]

Capsules have been obtained from a chitosan gel layer

cross-linked by charged surfactant micelles and used to

encapsulate enzymes as mentioned by Babak et al.[20,22,23]

The difference between a SPEC and alkylated chitosan

having the same density of alkyl chains with the same

length per polymeric chain is very important. In fact, in the

covalently alkylated derivative, a random distribution

of the alkyl chain substituents is obtained under the

experimental conditions adopted,[12] while in a SPEC

there is a cooperative interaction between alkyl chains ofthe surfactants, which involves few polymeric chains as

soon as the distance between the ionic sites along the

polyelectrolyte is in the range of 0.5 nm, as claimed by

Osada and co-workers.[24] A critical aggregation concen-

tration (c.a.c) has been found from surface tension

measurements to be around 100 times smaller than the

critical micelle concentration (c.m.c) of the surfactant

alone.[19] The role of sulphated N-acyl chitosan (S-Cn-

Chitosan)on a lipidic membrane hasbeen compared to that

of sodium dodecyl sulfate (SDS). SDS dissociates the

membrane while the polymer increases the rigidity of the

membrane, which suggests low toxicity on bioorganisms.

In solution when the alkyl chain in S-Cn-chitosan is longer

than n 10,these polymers form more stablemicellesthanthe micelles formed with the same alkyl chain surfac-

tant.[25] An extension of this interaction comes in the field

of food application. It involves specific interactions with

phospholipids and bile acids.[26]

Ionic Interactions

A solution of charged polysaccharides has an original

set of properties as a result of electrostatic interactions

that involve all the ionic species in solution. The firsttype of interaction involves attraction between polyions

and oppositely charged ions such as counter-ions, ionic

surfactants, or polymers. The second is repulsion between

species with the same charge such as long-range electro-

static interactions between chains that cause unusual

viscosity behavior, which reaches a maximum when the

reduced viscosity is plotted as a function of polymer con-

centration at lowionic concentration or in light scattering or

neutron scattering under the same conditions.[2729] This

second type of electrostatic interaction will not be covered

in this paper.

Figure 8. Viscosity of alkylated chitosan in 0.3 M AcOH/0.1 MAcONa at zero shear rate as a function of polymerconcentration at25 8C. (&) Chitosan, (*) Chit-C6, (~) Chit-C8, (^) Chit-C10, and (&) Chit-C12. Reprinted with permission from

ref.[13]

, Copyright 2006, American Chemical Society.




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Counter-Ion Interaction and Ionic Selectivity

Electrostatic interactions between a charged polymer and

counter-ions in the absence of external salt is governed by

the electrostatic potential around the polyelectrolyte as

exposed in the Katchalsky or Manning theories.[30,31] The

linear charge density imposes the activity coefficient of the

counter-ions as well as the limit over which ionic selectivity

appears related to the condensation limit, as proposed by

Manning. These theories have been compared in a previous

Figure 9. Role of salt on therheological behavior of alkylatedchitosan(Chit-C12) as a function offrequency at 10 8C:A) complex viscosity in 0.3 MAcOH (^ 6 and & 10 g L1) and in 0.3 MAcOH/0.025 MAcONa (} 6 and &10 g L1),; B) storage (G0 &) andloss(G00&) moduliin 0.3 M AcOH and G0 (*) and G00 (*) in0.3 M AcOH/0.025 MAcONa. Polymer concentration: 10 g L1.

Table 1. Influence of salt concentration on the rheology of alkylchitosan. (Concentration 10 g L1;o 1 Hz.)

Solvent G0 G00 jZ*j

Pa Pa Pa s

0.3 M AcOH 1.75 3.73 0.660.3 M AcOH/0.025 M AcONa 6.75 5.79 1.42

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paper on solution properties (activity coefficients) of a

series of carboxymethylcelluloses with different degrees of

substitution.[32] It is known that electrostatic ionpolyion

interactions become independent of the chain length as

soon as the degree of polymerization is larger than a value of

approximately 20. The different main electrostatic proper-

ties of some polysaccharides have recently been described,

based on some of our results.[29] From a general point view,

and based on these theories, it is possible to predict with

reasonable confidence the activity coefficient of counter-

ions in the absence of external salt in purified ionic poly-

saccharides as soon as the structure is known (nature of

the chain, degree of substitution, actual conformation and

distribution of the ionic sites along the polymeric chains).

With regard to these systems, it has also been shown that for

a charge parameter larger than 1, ionic selectivity occurs, as

demonstrated by different methods but the most clearly by

ultrasound absorption.[33,34] This behavior has clearly been

demonstrated for carboxymethylcelluloses obtained by

homogeneous substitution on the laboratory scale, where

the ionic selectivity is found to be:

Li >Na > K >Cs > TEA

with Li forming more ion pairs and tetraethyl ammonium,

a large hydrophobic ion used as reference, being inactive.

Data concerning different aspects of counter-ion interac-

tions are described below.

For gellan and k-carrageenan, particularly in salt free

solutions, it hasbeen demonstrated that over a critical polymer

concentration (which also meansa critical ionic concentration

Cs*), at a given temperature, a double helix forms that is

stabilized by hydrogen bonds. When the temperature is

increased, destabilization of the hydrogen bonds induces a

transition to a more flexible coiled conformation as observedby NMR spectroscopy, optical rotation, or viscometry. The

example obtained forgellan is givenin Figure 13, in which the

reduced viscosity decreases sharply around 30 8C. This

phenomenon will be discussed later but it must be pointed

out that the charge density in the helical conformation is

double that of the coiled conformation (Table 2). The electro-

static interaction with counter-ions then increases during the

coil-helix transition as demonstrated by a decrease in the

activity counter-ion.[35,36] An example is given in Figure 14

fork-carrageenan, where counter-ion activity is plotted as a

function of the polymer concentration (corresponding to an

Figure 10. Reduced viscosity of alkylated chitosan (Chit-C12)under thechlorhydrate form in water as a function of the degree ofprotonation (a) at different temperatures. Polymer concentration:0.9 g L1, DS 0.04. Reprinted with permission from ref.[14],

Copyright 2006, Wiley-VCH Publishers, Inc.

Figure 11. Phase diagram forChit-C12 deduced from the resultsof Figure 10. Reprinted with permission from ref. [14], Copyright2006, Wiley-VCH Publishers Inc.

Figure 12. Influence of the progressive addition of b-cyclo-dextrin on the viscosity of chitosan and alkylated chitosansolutions at 2 g L1 and 25 8C. (^) Chitosan, (&) Chit-C6,(~) Chit-C8, (*) Chit-C10, and (^) Chit-C12. The ratio [CD]/[Cx] is the molar ratio of CD and of alkyl chains with differentchain lengths (x). Reprinted with permission from ref.

[13],Copyright 2006, American Chemical Society.




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increase in the ionic concentration and a screening effect of

the repulsions between ionic sites on the chains). It is shown

that the activity coefficient for Na remains nearly unchanged

with ionic concentration and temperature in the domain

covered, thevalue of which corresponds to the prediction fora

single chain potential. At a lower temperature (15 8C) for K

counter-ions, a transition is observed that reduces the activity

coefficient by nearly half the initial value, which corresponds

to the formation of a dimer. The temperature for the confor-

mational transition Tm is directly related to the total ionic

concentration, as represented by a phase diagram proposed for

k-carrageenan and gellan. A linear plot is obtained when the

temperature of conformational change values (Tm) obtained

from the cooling curves by optical rotation, conductivity, or

viscosity, are represented for different polymers and external

salt concentrations (Figure 15).[3537] These phase diagrams

separate coil from double helical conformations as a function

of ionic concentration and temperature. It should be recalled

that, from this graph (see Figure 15a) over a critical ionic

concentration (Cs**) (which may be larger than Cs*) the

double helices associate to form a gel; these critical values for

gellan andk-carrageenan have been discussed previously.[36b]

An important difference between these two polymers is that

no ionic selectivity between monovalent counter-ions is

observed for gellan, which has a lower charge parameter.

With gellan, divalent counter-ions favor the helix transi-

tionasseeninFigure15b,butwith k-carrageenan, only small

differences between divalent counter-ions are observed.[35]

The phase diagram lines for divalent counter-ions (Ba, Ca,

Sr, Mg, Zn, and Co) are very close together, parallel to those

of Na and K counter-ions, and located just between their

two lines.

Figure 13. Reduced viscosity as a function of temperature for gellan under sodium andpotassium salt forms. Concentration 0.3 g L1 at 0.025 M salt concentration. Reproducedwith permission of John Wiley & Sons from ref.[36a]

Table 2. Activity coefficients of monovalent counter-ions and molecular weights of polysaccharides in relation to their conformation.

Polysaccharide lchargeparameter

gactivitycoefficientcalculated

gactivitycoefficient

experimental

Mw Ref.

K-carrageenan coil 0.68 0.72 0.37 3.4 105 [35]15 8C, K form (helix);

Cp > 102 monomol L1

helix 1.65 0.37

K-carrageenan 0.72 1.76 105 [35]45 8C, K form (coil);

Cp < 103 monomol L1

Gellan Na form coil 0.37 0.83 0.67 4.9 105 [36a]18 8C (helix) helix 0.75 0.69Gellan Na form 0.76 2.5 105 [36a]26 8C (coil)

598 M. Rinaudo



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The nature of the counter-ions and ionic selectivity is

an interesting effect based on specific interactions that

involve hydration of the ionic species. Spectroscopic techni-

ques have been used to point out the existence of ionic

selectivity that occurs when the charge parameter is larger

than a value of 1.[34] By ultrasound absorption, which is a

good technique to investigate ion pair formation based on

perturbation of the solvation shells, it has been demonstrated

fork-carrageenan that even if the charge parameter is lower

than 1, ionic selectivity in the coil conformation can be

demonstrated with K forming more ion pairs then Na.[38]

This also shows that the same selectivity plays a role to

induce the coilhelix transition and control the stability of

the double helix and that of the gel formed. The formation of

more ion pairs further reduces the net charge of the polymer,

which favors the association of two chains to form a double

helix and then association for gel formation. The ionic

selectivity observed for gellan in solution plays a role in the

ability to associate, as shown by the increase in viscosity

under the K form (Figure 13), but also on the mechanical

properties of the gel formed (Figure 16).

Anion selectivity also exists in carrageenan as demon-strated by NMR spectroscopy.[39] It has also been shown

that I has a specific interaction with k-carrageenan, to

stabilize the helical conformation and prevent the aggrega-

tion of double helices and gelation.[40,41] The mechanism of

interaction between anions and this negatively charged

polymer is still not clear but the fixation of I certainly

increases the charge density of the polymer and increases

the electrostatic repulsions between the double helices.

The same conclusions and the same model for gelation

given fork-carrageenan are also valid for gellan. The only

difference is that, because of the lower charge density of the

coiled conformation, no ionic selectivity is observed with

gellan, though it does appearwhenthe doublehelix is formed

(Figure 15b).[36,37] The ionic selectivity then appears for the

double helix conformation with the ability to induce gelation

withthe following sequenceamong monovalentcounterions:

K>Na>Li> TMA.At the same time it has been shown that the physical

properties (E, the elastic modulus and the stress at breakFmdetermined in compression experiments) of the gel are

controlled by the nature of the counter-ions (Table 3).[37]

Ionic selectivity is also recognized in alginates and pectins

in the presence of divalent counter-ions. On a poly(1,4-D-

galacturonic acid) (fully demethylated pectin), the transport

coefficient determined from conductivity (not far from the

activity coefficient) indicates a specific behavior for Mg

counter-ions compared with the other divalent counter-ions

(Table 4).[42] From these results it was concluded that under

Na and Mg salt forms, the general behavior of single chain

polyelectrolytes is confirmed, while with Ca, Ba, and Sr, a

double chain structure is stabilized.[42,43] These results are in

agreement with previous data by Kohn et al. and the egg-box

model mechanism first suggested for alginate by Rees and co-workers.[44,45] In addition, an interestingrelation is madewith

the ability to form a gel: Na and Mg forms are water soluble

while the other forms are not, while Ca, Ba, and Sr are known

to induce gelation with an ionic selectivity corresponding to

Ba> Sr> Ca for the ability to form a gel (Figure 17).[46,47]

The same sequence of selectivity and the mechanism are

involved for gelation of alginates in the presence of divalent

counterions.

Pectins with different degrees of esterification (DE) have

been prepared; demethylation is performed under alkaline

conditions (giving a random distribution of the carboxylic

Figure 14. Activity coefficient of monovalent counterions (K and Na) as a function ofpolymer concentration (Cp) at 15 and 35 8C. Reproduced with permission from ref.

[35a],Copyright 2006, American Chemical Society.




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sites) and using an enzyme (giving a blockwise distribution of

free carboxylic groups). The charge parameterof the polymers

that have the same intermediate degree of methylation

(DE 30) but different distributions does not behave in thesame way in relation to electrostatic interactions. Even if the

average charge parameter is the same, the local electrostatic

fields are not; the blockwise distribution (at least eight

carboxylic groupsper block)hasthe same chargeparameter as

the fully demethylated polymer (but not the average value

based on the degree of esterification). The gelation ability then

depends on the charge distribution as shown in Figure 18.[46] It

also plays a role in cell wall structure.[48]

Figure 15. Phase diagram giving the inverse of the temperature that corresponds to theconformational changeTmas a function of total salt concentration CT: a) for k-carrageenanunder sodium and potassium salt forms; Cs** is the critical salt concentration above whichgelation occurs. Reprinted with permission from ref.[35a], Copyright 2006, AmericanChemical Society. b) For gellan under monovalent (Na, o K, ~ TMA, and ~ Li) anddivalent counterions (& Ca and & Mg). Reprinted from ref.[36b], Copyright 2006, withpermission from Elsevier.

600 M. Rinaudo



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Polyelectrolyte Complex

The most important systems developed recently are the

electrostatic complexes that are formed when two polymers

with opposite charges are mixed. With alginate or HA in

particular, a pH-dependent complex is formed in the

presence of chitosan, whose stability depends on ionic

concentration and pH. Complexformation has been investi-

gated in dilute solution by pH-potentiometry and conductiv-

ity to determine the fraction of ion pairs (COO/NH3)

formed, depending on the experimental conditions.[49,50]

Interactions between chitosan and alginate give an electro-

staticcomplex that has been mostly used so far for biological

applications. The main applications of these electrostatic

complexes cited are antithrombogenic materials, controlled

release materials, encapsulation of drugs, immobilization of

enzymes and cells, and gene carriers. One aspect of thesecomplexes now under development is the preparation of

layer-by-layer polyelectrolyte capsules or films based on

charged biocompatible polysaccharides or chitosan/syn-

thetic polyelectrolytes.[51,52] Porous gels (sponges) can be

prepared by forming a calcium alginate gel stabilized by

complexation with galactosylated chitosan (a water-soluble

derivative).[53] Another type of complexed bead can be

obtained by the drop-wise addition of Na-alginate into a

chitosan-CaCl2 solution; the swelling of the beads being

different from that of Ca-alginate beads with a maximum

swelling at pH 9.[54]

Dipolar Interactions and Hydrogen-BondNetwork Formation

Hydrogen bonds are loose dipolar interactions that involve

hydrogen atoms interacting with highly electronegative

atoms such as oxygen or nitrogen. Hydrogen-bond inter-

action analysis is very important to understand the

physicochemical properties of polysaccharides in aqueous

solution. These polymers are very rich in OH groups or

C O. At low temperatures, hydrogen bonds can be formed

to give rise to intrachain or interchain networks. Interaction

with the solvent (water) is also favored in relation tosolvation and dissolution. Usually, the existence of ionic

sites along the chains prevents interchain interactions,

which exist for neutral polysaccharides. The situation of

galactomannans is clear in this respect and has been

discussed previously;[55] in this case loose interactions

exist, which lead to loose gel-like behavior in solution.

Solubility

From a general point of view, the greater theinteraction of a

solute with solvent molecules, the greater the solubility. In

Table 3. Physical properties of gellan in the gel form underdifferent ionic forms. Gellan concentration Cp 10 g L

1; saltconcentration [XCl] 0.1 M. Reprinted from ref. [36b], Copyright2006, with permission from Elsevier.

X E(104) Fm

Pa N

Li 0.44 Na 11.9 13.3K 31.4 23.1

Figure 16. Elastic modulusE for purified gellan under potas-sium () and sodium (*) forms as a function of polymerconcentration in salt excess. Salt concentration 0.1 M, temperature25 8C. Reprinted from ref.[36b], Copyright 2006, with permissionfrom Elsevier.

Table 4. Transport coefficient obtained by conductivity fordifferent counterions in a poly-D-galacturonate solution.Reprinted from ref. [42], with permission of John Wiley & Sons.

Counter-ion Transport coefficient of polygalacturonatea)

Na 0.62Ba 0.095Ca 0.11Sr 0.11Mg 0.28

a)lcharge parameter for a single chain 1.58 and for a doublechain 3.16.




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fact, solubilization of polysaccharides is a difficult problem

because of the existence of a cooperative network formed

by interchain hydrogen bonds. The difficulty in solubilizing

such a system in water comes from too high a temperature

and total dehydration when purified polysaccharides are

first isolated making OH groups no longer accessible to

interact with water molecules. The existence of charged

groups such as SO3 or COO favors the dissociation of

Figure 17. Influence of the progressive addition of divalent counter-ions on thecritical gel point for polygalacturonic sodium salt form followed by light scattering.Reproduced with permission from ref.[46], Copyright Society of Chemical Industry.Permission granted by John Wiley & Sons Ltd on behalf of the SCI.

Figure 18. Influence of the distribution of carboxylicgroups along the polygalactur-onic chain for DE 30 pectins obtained by enzyme (PE: blockwise distribution) and byalkaline treatment (PH: random distribution) on gelation in the presence of calciumcounter-ions. Reproduced with permission from ref.[46], Copyright Society ofChemical Industry. Permission granted by JohnWiley & Sons Ltdon behalfof theSCI.

602 M. Rinaudo



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aggregates by electrostatic repulsion and increases the

ability to fix water because of the presence of the ionic sites

and their counter-ions.[56] The progressive hydration of HA

has recently been studied by infrared spectroscopy.[5759]

This shows why heterogeneous methylcellulose with a

DS 1 is not water soluble while homogeneous samples are

soluble, the dimension of the methyl substituents regularlydispersed along the chain prevent the packing of poly-

hydroxylated molecules.

In the majority of oligo- and polysaccharides, the hydro-

philic character as a result of the OH groups is counter-

balanced by the hydrophobic contribution of the CH entity

of the glycosidic cycle and the ether-like O-4 and O-5 oxygen

atoms.[60] Such hydrogen-bond formation and the additional

hydrophobic character of polysaccharides have been demon-

strated for chitosan under acidic conditions.[11a] With

hyaluronan, the self association of segments in 0.15 M NaCl

has also been discussed.[11b] These associations are related to

the local stiffness of the polysaccharides which promotescooperative interactions.

The interaction of water molecules with a model molecule,

maltose, has been approached by molecular modeling.[61]

The location of water molecules has been described (albeit by

a dynamic process) and the results have been used to predict,

with good agreement, the hydration of amorphous amylose

(on average, 3.8 water molecules are firmly interacting with

one D-glucose moiety). Experiments on amorphous amylose

confirm the prediction and have been extended to acetylated

starch in relation to the influence of the relative humidity on

the mechanical properties of films.[62,63]

Hydrogen bonds play an important role in solubility, as

has recently been investigated for cyclodextrins.[64] The

role of hydrogen bonds has been displayed by comparing

their behavior in H2O a n d in D2O . In D2O, stronger

hydrogen bonds are formed than in H2O and the solubility

of CD decreased in D2O. We have also shown that when the

helical conformation of polysaccharides is stabilized

by intrachain hydrogen bonds, the conformation is more

stable in D2O, as has been found with xanthan and other

stereoregular polysaccharides. The example obtained for

gellan in H2O and D2O is shown in Figure 19, where the

optical rotation is plotted as a function of temperature,[36]

and the double helix coil transition temperature increases

by around 6 8C in D2O under the experimental conditionsused. Another interesting behavior in relation to hydrogen-

bond formation has been obtained regarding hyaluronan

viscosity (Figure 20).[65] When the pH decreases, the net

charge of the polymer decreases as well as the electro-

static repulsions between polysaccharide chains, which

favors the stabilization of aggregates to give a gel-like

behavior. In this situation, pH 2.4 may also represent apseudo isoelectric point as a result of an equilibrium

between a few anionic groups from the uronic unit and the

amino groups from the N-acetyl-D-glucosamine unit (see

Figure 21).

Conformation and Stiffness

Hydrogen bonds play an important role in the local

conformation of stereoregular polysaccharides as many of

them adopt a helical conformation in the solid state, as has

been established for gellan,[66] k-carrageenan,[67] hyaluro-

nan,[68]

chitin,[69]

and xanthan.[70]

In aqueous solution, undergiven thermodynamic conditions, they adopt an ordered

conformation assumed to be helical, as displayed by circular

dichroism, optical rotation, or NMR spectroscopy, while the

conformational transition is induced by increasing the

temperature or changing the ionic concentration.[3537,71,72]

The transition results from a decrease in the attraction by

hydrogen bonds when the temperature increases and the

effect of electrostatic repulsions between ionic groups on the

chain.

The intrachain hydrogen bonds in such an ordered

conformation are very important because they control the

local stiffness of the polymer, reflected by its intrinsic

persistence length (Lp), as seen, for example, for hyaluronan

in Figure 21. SomeLp valuesare given in Table5. In relation

to this rigidity, the worm-like chain model was introduced a

few years ago to analyze the behavior of polysaccharides,

the local stiffness of the polymer being characterized by an

intrinsic persistence lengthLp.[73,74] For a charged polymer,

it is necessary to introduce an electrostatic contributionLe(the total persistence length being Lt Lp Le at a givenionic concentration) following the Odijk development.[75]

The local stiffness of the polysaccharides allows the high

viscosity obtained to be interpreted for a given molecular

weight compared to a flexible polymer. The Lp value,

obtained in a large excess of salt, is characteristic of thelocal structure of the polysaccharides and can be predicted

from molecular modeling, as recently shown.[7678]

The experimental values of intrinsic viscosity and radius

of gyration can, therefore, be interpreted as a function of the

molecular weights of the polysaccharides. Theoretically,

the persistence length Lp of a chain and the molecular

weight directly control the radius of gyration Rg and the

intrinsic viscosity [Z] on the basis of the worm-like chain

model under y conditions, following the Benoit Doty

equation:[79]

R2gL Lp=3L2p 2L

3p=L2L

4p=L

21expL=Lp

1

in which L is the contour length calculated from the

chemical structure and the molecular weight (M). For high

molecular weights, one can limit the analysis to the first

right-hand term of Equation (1). The intrinsic viscosity is

given by the formula proposed by Yamakawa and Fujii:[80]

Z FM;Lp; dML=2Lp3=2

M1=2 2

withd, the hydrodynamic diameter, andML, the mass per

unit length of the chain, which is known from the chemical

structure.




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Experimentally, the intrinsic viscosity [Z] (expressed in

mL g1) can be obtained directly from steric exclusionchromatography (equipped with a refractometer, viscom-

eter, and light scattering detectors on line) at the same time

as the molecular weight but, more usually, it is determined

in a separate experiment. It is obtained by extrapolation to

zero polymer concentration of the reduced viscosity (Zred)

of the polymeric solution plotted as a function of the

polymer concentration (Cin g mL1) in accordance withthe Huggins relation:

Zred ZZ0=Z0C Z k0Z2C 3

in which Z is the viscosity of the polymer solution, Z0 the

viscosity of the solvent, andk0 the Huggins constant.

Figure 19. Determination of theconformational transition on gellanby optical rotationasa function of temperature in H2O () Tm 24 8C and in D2O (*) Tm 31 8C. Polymerconcentration 6 g L1. Reprinted with permission of John Wiley & Sons from ref.[36a]

Figure 20. Complex viscosity of hyaluronan as a function of the pH at 25 8C.Polymer concentration 10 g L1, frequency 1 rad s1. Reprinted with permis-sion from ref.[65], Copyright 2006, American Chemical Society.

604 M. Rinaudo



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In dilute and semi-dilute solutions, the most important

characteristic for applications of water-soluble polysac-

charides is the thickening character of the solution, which

depends on the intrinsic viscosity, molecular weight, and

concentration of the polymerat a given temperature and in a

given solvent.

In this case, the viscosity of a polymer solution at zero

shear rate is directly described by the following equation:[81]

Zsp CZ1k1CZ k2CZ2 k3CZ

3

4

in which k1 0.4, k2 k1/2!; k3 k1/3!. This relation isvery important, as it shows that the overlap parameter

(C[Z]) controls the viscosity of the solution at zero shear

rate and allows this behavior to be predicted. For a given

polymer, under given thermodynamic conditions, all the

viscosity values for different polymer concentrations and

molecular weights appear on the same curve when plotted

as a function ofC[Z]. Thek1value represents the Huggins

constant and for many perfectly water-soluble polysac-

charides it equals 0.4. All deviations from this reference

curve indicate interchain interaction or aggregation, such as

that observed with galactomannan.[55]

The intrinsic viscosity is related to the molecular weightof the polymer, following the empirical MarkHouwink

relationship:

Z KMav 5

in whichMv is the viscometric-average molecular weight,

and Kand a are two parameters that depend on thepolymer,

solvent, and temperature. The important thing is that poly-

saccharides usually deviate from the behavior of synthetic

polymers, which are flexible, and it is demonstrated that

even under y conditions, for polysaccharides, the a

exponent of the Mark-Houwink relationship is never equal

Figure 21. Hyaluronan: A) repeat unit, B) local conformation of a segment of hyaluronan,representation of intrachain hydrogen bonds.

Table 5. Intrinsic persistence length for different polysaccharidesobtained by molecular modeling and experiments at 25 8C.

Polysaccharide Lp Ref.

nm

HAa) 8 [82]HAb) 7.5 [77]Xanthan Coila) 5 [73b]Xanthan Helixa) 40 [73b]Gellan Coila) 5 [36]

Gellan Helixa) 35 [36]Chitinb) 12.5 [78a]Chitosan DA 0a) 10 [78b]Chitosan DA 0b) 9 [78a]Mannanb) 14.5 [76b]Galactomannan M/G 1:1b) 9.6 [76b]Galactomannan M/G 1:1a) 9.3 [76a]Alginate M/G 0.28a) 9 [73b]M/G 1.92a) 4 [73b]Succinoglycan Helixa) 35 [73b]Succinoglycan Coila) 5 [73b]

a) Determined experimentally.b) Determined by molecular modeling.




17/21

to 0.5, excluding, for example, the direct use of the Flory-

Fox relation.

The persistence length of polysaccharides depends mainly

on the intrachain hydrogenbonds.For example, it reducesthe

mobility of the proton as determined by NMR spectroscopy

and the signals obtained depend directly on the temperature,

as has been described before and confirmed by molecular

modeling for hyaluronan and chitosan.[82a,82c] A stronger

effect is obtained when the helical conformation is stabi-

lized; the mobility is reduced so much in a solution of

polysaccharides in the helical conformation that all the

characteristic broad signals disappear. When the temperature

Figure 23. Storage modulus (&G0) and loss modulus (&G0) for a LBG/Xanthan (50/50) mixture in wateras a function of temperature on heating. Frequency 1 Hz.

Figure 22. DSC thermograms obtained on heating (0.2 deg. min1) for xanthan/galactomannan mixture (4:2 g L1): a) in water, b) in 5 103 M NaCl, and c) in102 M NaCl. Tgel is the temperature for melting of the mixed gel and Tm is thetemperature for the conformational transition of xanthan. Reproduced withpermission from ref.[86], Copyright 2006, Wiley-VCH Publishers Inc.

606 M. Rinaudo



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increases under given conditions (salt concentration, nature

of counter-ions, and polymer concentration), a conforma-

tional transition is observed that corresponds to the appear-

ance of the characteristic signals for protons, such as the

results obtained for succinoglycan and gellan.[36b,71] This

helix-coil conformational transition is related to the

equilibrium between electrostatic repulsions and hydrogen-

bond attractions.

Polymer/Polymer Complex

Usually, when two different polymers in solutionare mixed,

a phase separation occurs, while in many polysaccharide

systems in aqueous solutions cooperative interactions are

established, which increases the viscosity up to physical

gelation. This phenomenon is well known when xanthan is

mixed with galactomannans or glucomannans.[8388] When

deacetylated xanthan and galactomannan are mixed, it has

been shown that two types of gel exist:

- A low temperature gel that meltsat around 258C, which

involves disordered xanthan (coil conformation) andgalactomannan with different mannose/galactose ratios at

a low ionic concentration. These form an ordered complex

stabilized by the hydrogen bond network, which is demon-

strated by circular dichroism and differential scanning

calorimetry (Figure 22).[8387]

- A second type of gel formed in water or in the presence

of salts especially with locust bean gum (LBG) galacto-

mannan with a relatively high mannose/galactose ratio,

with a melting point of around 60 8C as shown in Figure 23.

The melting temperature is estimated from the crossing

point ofG0 andG00 (Table 6).[88] The interaction is clearly

demonstrated in Figure 24, where the storage modulus is

plotted for the galactomannan and xanthan solutions

(10 g L1 in water) as a function of frequency. From thesedata, it can be seen that a loose interaction exists for the

xanthan/guar mixture as the modulus is larger than that of

the corresponding additivity; a larger increase of the

modulus is obtained for the mixture with LBG. In

Figure 25, the change in behavior for LBG, from a

viscoelastic solution to a gel (G0 > G00 in all the frequencyranges covered) is shown. The largest modulus is obtained

after first heating above 70 8C and then cooling. It also

shows that the modulus decreases in the presence of

external salt but that the melting temperature increases

(data not shown). The modeling of the interactions between

the two polymers (xanthan/galactomannan) has been

investigated by Chandrasekaran and Radha[89] and different

hydrogen-bonding interactions may involve side-chain/

side-chain interactions, main-chain/main-chain interac-

tions, etc.

Figure 24. Storage modulus as a function of the frequency for the different systems tested at 25 8C and10 g L1 in water.

Table 6. Rheology of galactomannans, xanthan, and their 50:50mixtures. (Concentration 10 g L1; solvent H2O;o 1 Hz.)

Sample G0 G00 j j TF(G0 G00)

Pa Pa Pa s 8C

Guar M/G 1.7 5.05 6.39 1.30LBG M/G 2.44 4.46 9.79 1.72Xanthan 18.06 5.52 3.02Xanthan/LBG 148.6 11.33 23.72 56.5Xanthan/Guar 16.13 7.87 2.87 50.9




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Conclusion

This paper covers the secondary interactions that exist in

aqueous solution for a few representative polysaccharides.

This includes the role of hydrophobic interactions in amphi-

philic derivatives such as heterogeneous methylcelluloses,

which areknown to form gels when the temperature increases.

The mechanism of gelation and the influence of the different

thermodynamic parameters are discussed. The role of

heterogeneity of methyl group substitution (blockwise distri-

bution) is clearly demonstrated. Grafted alkylated chitosans

have original bulk properties and give rise to large increases in

solution viscosity up to gelation, depending on the solvent

composition as a result of the aggregation of grafted alkyl

chains that form hydrophobic domains which act as points of

contact. These derivatives also have interesting interfacial

properties with the formation of stiff films able to stabilize

emulsions.

Ionic interactions are also of great interest. Charged

polysaccharides are characterized by an electrostatic poten-

tial, which imposes the counter-ion distribution dependingon the conformation. The activity coefficient of counter-

ions may be predicted when confirmation and structure of

the linear chain are known. For higher charge densities,

ionic selectivity is shown to play an important role in some

stereoregular polysaccharides. Ionic selectivity based on

ion pair formation controls the stability of the conforma-

tion, the more ion pairs that are formed, the more stable is

the helical conformation that results in a balance between

electrostatic repulsions and attractive hydrogen bonds. As a

consequence of this, ionic selectivity is related to the ability

of gelation and the mechanical properties of the gels.

Finally, the importance of the hydrogen bond in these

systems is discussed. They play a role in the local stiffness

and persistence length and also rheological properties in

solution. The cooperative hydrogen bonds allow the

stabilization of the helical conformation whose stability

depends on the temperature, salt concentration, and nature

of the counter-ions. Hydrogen bonds are alsoinvolvedin the

hydration and dissolution of polysaccharides. Interchain

hydrogen bonds allow the formation of original cooperative

polymer A/polymer B complexes, against the usual rule of

polymer incompatibility.

In all these secondary interactions, the microstructure of

the polymers, their chemical structure, and the thermody-

namic conditions (temperature, solvent composition, pH,

salt nature) are all important for controlling the conforma-

tion, interchain interaction, and stiffness of the chain, as

well as the solution and gel properties.

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608 M. Rinaudo



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610 M. Rinaudo

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