Solubility of Polysaccharides and Their Behavior in Solution

14 Solubility of Polysaccharides

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and Their Behavior in Solution ROY L. WHISTLER Department of Biochemistry, Purdue University, Lafayette, Ind. 47907 Polysaccharides

consisting of one type of sugar unit uni-

formly linked in linear chains are usually water insoluble even when the molecules have a low molecular weight with degrees of polymerization (DP) 20-30. Insolubility results from the fit of molecules and their preference for partial crystallization.

An exception to the rule is in (1 --> 6)-linked

homoglycans, which because of the extra degrees of freedom provided by the rotation about the C-5 to C-6 bonds gives higher solution entropy

values.

Homoglycans

with

two

types of sugar linkages or heteroglycans composed of two types of sugars are more soluble than purely homogeneous polymers.

Ionized linear homoglycans are soluble but like

all soluble linear polymers easily form gels because of segmental association which sometimes may be in a double helix formation.

As these junction zones develop

a stronger

tertiary structure, gel hardness increases.

As one examines the 300 or more natural polysaccharides that have been studied, it is seen that each polymer shows selected physical properties valuable to the biologic system which gives it eclectic origin. Besides physical service, some polysaccharides also serve as food reserves. Where service is entirely physical, it may be as a structural element, lubricant, emollient, gelation or water holding agent, complexant, or adhesive. All functionality of the polysaccharides rests to some degree on physical properties which derive from the macromolecularity of the polysaccharides and their inherent secondary molecular forces. Among the most significant of the latter are hydration characteristics which may be so extensive as to allow gelation or dissolution. Not only are water-polysaccharide relations important effectors i n biologic systems, but the physical properties of polysaccharides i n aqueous 242 Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.


14.

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Solubility of Polysaccharides

243

dispersions are widely used industrially to give desired behavior i n food and non-food products. Here, also, the useful effects are derived from the interaction of polysaccharide molecules with themselves and with molecules of their environment. Environmental molecules always include water molecules and sometimes proteins, lipids, additional polysaccha rides, and other molecules used industrially. B y interactions with these various environmental molecules, polysaccharides perform their useful practical functions which are mainly to give viscosity, solution stability, suspendability, emulsifying action, gelation without syneresis, and com patibility with proteins, other polysaccharides, or other ingredients present. Solubility of

Polysaccharides

Polysaccharides dissolve in water by continuous hydration with a transfer of interpolysaccharide binding to polysaccharide-water binding and with the action assisted to more or less degree by entropy as the molecules assume lower energy conformations. A l l polysaccharides have affinity for water; i n the dry state their affinity for initial levels of water is as great as that of phosphorus pentaoxide. A t normal humidities poly saccharides contain 8 - 1 0 % water as water of hydration. In the solid state all polysaccharides have regions where molecules or chain segments are i n a jumbled or disorganized arrangement with intermolecular forces and intermolecular hydrogen bonding only partially satisfied because of the random spatial arrangement. These amorphous regions, therefore, have numerous unsatisfied hydrogen bonding positions which can hydrate. For some polysaccharides and particularly for cellulose and starch, the relation of hydration to ambient relative humidity has been intensively examined, and hysteresis effects from moisture absorption and desorption are explained. Initial water molecules of absorption occupy hydrogen bonding positions not otherwise involved i n intermolecular bonding of the polysaccharide molecules. W h e n a soluble polysaccharide is placed in water, the abundant water molecules quickly penetrate amorphous regions and surround available polymer sites, competing for and even tually reducing to negligible numbers still other intermolecular bonds. Segments of a polysaccharide chain become fully solvated and by kinetic action move toward solution, tearing apart more interpolysaccharide bonds which are immediately solvated. Soon many sections of the poly saccharide chains are fully solvated and are solubilized while a lessening number of segments are still attached to other polysaccharide chains which are not yet completely solvated. This intermediate stage i n the dissolution of a polymer molecule represents a transient gel state and represents a universal stage i n the dissolution of all polysaccharides. As

Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.

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CARBOHYDRATES I N SOLUTION


hydration continues, polysaccharide molecules become completely sur rounded by partially immobilized water molecules which represent the solvation layer, and molecules move into solution where they may remain monodispersed and assume preferred low energy shapes and conforma tions or where they may become involved with other polysaccharide molecules to develop various degrees of gel structure. These structures and conformations of polysaccharide molecules and their intermolecular associations give polysaccharide solutions, polysac charide dispersions, and polysaccharide gels their special properties in biologic systems and in industrial usage. The ease with which poly saccharides dissolve is related to their conformation and structure i n many the same ways as is their solution behavior. Linear polysaccharides of uniform structure can fit together for strong intermolecular binding and usually can form perfectly ordered arrays to develop crystalline regions that firmly tie molecular segments together in effective cross-links. Such molecules are difficult to dissolve and once in solution can easily segmentally recombine during crystal development. Thus, they form particles that upon continued accretion reach precipitating size. Molecules which most easily associate and form crystals have regular, extended, ribbon-like structures. Glycosidic linkages which do not form ribbon structures w i l l lead to molecules that are more easily dissolved and to solutions of greater stability. A n y structures which contain espe cially flexible units such as 1 - » 6 linkages w i l l lead to easier solubility and more stable solutions because of more favorable entropy of solution. Irregularities in the polysaccharide structure w i l l also produce greater solubility and solution stability. Branching greatly reduces the possibility of intermolecular association and usually leads to easily soluble poly saccharides which form stable solutions. Also, introducing formal ionic charges, usually anionic charges resulting from carboxyl, sulfate, or phos phate groups, develop polysaccharides that are readily soluble and which form stable solutions so long as the formal charges exist. Formal charges do not eliminate, however, the possibility of interchain association which may, occasionally, result i n a degree of cross-linking and consequent gel formation. Generally, reinforcing, cell-wall polysaccharides are least soluble while emollients, mucilaginous, and food reserve polysaccharides repre sent the most soluble group. Exceptions to the generalization that reserve food polysaccharides are easily soluble occur i n starch amylose and seed mannan. Starch amylose is readily dispersible i n most of its natural forms since it occurs mixed with easily soluble amylopectin which facili tates the dissolution of the amylose.



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Almost all low molecular weight polysaccharides—degrees of polym erization ( D P ) less than 15-20—are soluble i n water. Solubility decreases with increase in the ease with which molecules can associate and with narrowness of molecular weight distribution. Some polysaccharides classified by degree of water solubility are given i n Table I. The list includes only a few of the known polysaccha rides. Those listed have a reasonably high molecular weight, having D P of 100 to several thousand. Highly branched polysaccharides, constituting the majority of polysaccharides, are almost always very soluble in water. Table I. Solubility Group Least

Examples of Polysaccharides in Different Solubility Groups Linkage l->4

l->3 1—»3 (mainly, 1—»6 (minor

Polysaccharide

Sugar units

Cellulose Chitin

β-D-glucopyranosyl 2-acetamido-2-deoxyβ-D-glucopyranosyl β-D-xylopyranosyl Xylan a-D-glucopyranosyl Amylose Mannan (ivory nut) β - D - m a n n o p y r a n o s y l a-D-galacturonopyranosyl Pectic acid a-L-guluronopyranosyl Alginic acid β-D-mannuronopyranosyl β-D-glucopyranosyl Callose (callan) Paramylan β-D-glucopyranosyl Pachyman β-D-glucopyranosyl Laminaran β-D-glucopyranosyl

Intermediate l->6 Pustulan l->3, 1- Λ (1:1) Nigeran (1:3) Lichenan Easily Soluble l->4, l->6 (2:1) Pullulan 1—A main chain 1—>6 branches Amylopectin Glycogen Guaran l->4, l->3 (2:1) Cereal glucan (branched?) Anionic polysaccharides

β-D-glucopyranosyl a-D-glucopyranosyl β-D-glucopyranosyl a-D-glucopyranosyl

a-D-glucopyranosyl a-D-glucopyranosyl a-D-galactopyranosyl β-D-mannopyranosyl β-D-glucopyranosyl

Least Soluble Polysaccharides. As indicated, the most insoluble polysaccharides are those which are reinforcing structural polymers i n cell walls. The best example is cellulose, the universal reinforcing struc-



246

CARBOHYDRATES I N SOLUTION

tural component of all higher plants. Cellulose owes its insolubility to its regular structure, consisting of β-D-glucopyranosyl units i n Reeves CI conformation joined by ( l - » 4)-linkages; the high uniformity of the ar rangement presents a ribbon-like shape. When the molecules are biosynthesized i n plant cell walls, they deposit with some amorphous regions but with many regions, constituting 5 0 % or more of the cellulose, of high crystalline order. Such regions have been extensively described by diffractionists and i n particular by Meyer and Misch ( J ) and by Her mans (2, 3 ) . The fit is so perfect, and consequently the intermolecular binding is so strong that cellulose is insoluble i n water and even i n strongly alkaline solutions. Strongly alkaline solutions, such as 18% sodium hydroxide solution, swell cellulose, perhaps i n the manner con ceived by Warwicker and Wright (4). It is believed that the cellulose chains i n ribbon-like conformation with equatorial bonds nearly i n the plane of the ribbon are laid on top of each other i n stacks which are joined i n cellulose I and II by hydrogen bonding. Alkaline solutions may cause rupture of these hydrogen bonds, allowing the stacks or sheets, which remain more or less intact to separate. Reagents which complex with cellulose molecules—e.g., cuprammonium solution—can lead to eventual dissolution of the cellulose molecules. However, the normal fit of cellulose molecules to each other is so extensive that only molecules of D P up to about 15-80 can be dissolved i n 15-17% sodium hydroxide solution, and only molecules of less than 15 D P remain soluble i n neutral aqueous solutions. A near relative of cellulose is chitin, differing only i n that the hydroxyl at C-2 of each D-glucopyranosyl unit is replaced by an acetamido group. Chitin is the reinforcing structural element i n the shells and mantles of Crustacea, such as shrimp, lobster, and crabs, and i n the exoskeleton, shells, and wings of insects. Chitin is also fairly abundant i n fungi and some green algae. L i k e cellulose it is extensively crystalline and insoluble in water and alkaline solutions. Another near relative of cellulose is xylan, differing in its lack of a hydroxymethyl group attached to C-5 but having instead only a hydrogen atom. H i g h molecular weight, linear, homogeneous xylan molecules have not been examined and may not exist i n nature. X-ray and infrared analysis suggests longer intramolecular hydrogen bonding of sugar units than occurs i n cellulose (5, 6). E v e n though almost all xylans have branched chains and the majority have other sugar units as chain substituents, most xylans are water insoluble although all are soluble i n alkaline solutions. As expected, solubility decreases when side chain substituent sugar groups are removed. Pure amylose, a linear glucan with « - D - ( 1 - » 4) linkages, is normally insoluble i n cold water but can be dissolved in hot water or i n alkaline



14.

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247

solutions. O n cooling, partial crystallization (rétrogradation) occurs as the temperature is lowered below about 65°. The precipitation rate depends also upon molecular weight (7). The glycosidic linkages cause the amylose molecule i n solution to form a statistical coil without identi fiable helical character (8). W h e n complexed with agents such as iodine and butanol, the amylose molecule forms a helical structure containing the complexing agent. In this form the regularly shaped helices associate at temperatures depending upon the nature of the complexant but usually lower than 60° and form precipitating crystals. Glucans that are /?-D-(1 - » 3) linked are insoluble i n water. Laminarabiose is readily soluble in cold water, but as the oligosaccharide series is ascended, the solubility decreases so that laminarapentaose has a solubility of less than 1%. Ivory nut mannan such as that from palm seed is the only identified water insoluble food reserve polysaccharide which occurs i n nearly pure form. It dissolves in 5 % alkaline solution, and its food reserve nature is indicated by its disappearance when the seed germinates. Perhaps its lack of solubility permits the palm seeds to withstand rather long soaking periods before germination. Most other mannans found i n other bio logical sources are branched and more readily water soluble. Thus, polysaccharide molecules with sugar units uniformly 1 - » 4 or 1 3 linked seem to produce structures which can align to form strong intermolecular associations and even crystalline regions. Such structures are insoluble in water. Even when the molecules are soluble i n alkaline solutions, neutralization leads to reassociation and precipitation. N o conclusion can be drawn for the 1 - » 2 linked β-D-glucopyranosyl polymers since the one so far known from Agrobacteria (10), though water soluble, has a D P of only 22. Glucomannans (11) are essentially linear polysaccharides with con sistent 1 4 linkages joining β-D-glucopyranosyl and β-D-mannopyranosyl units (1:2 ratio in deciduous wood) i n a rather random distribution. These diheteroglycans are soluble i n alkaline solution but are insoluble in water; thus, the chains are capable of strong association. Replacement of D-mannopyranosyl units by some D-glucopyranosyl units, both presum ably i n CI conformation, does not especially disrupt the chain regularity or its ability for intermolecular association. Alteration of the type of glycosidic linkages, such as replacement of 1 - » 4 by 1 - » 3 and especially by 1 - » 6, has a much greater effect on solubility. Polysaccharides of Intermediate Solubility. Pustulan, having a uni form 1 - » 6 linkage between ^-D-glucopyranosyl units and with one 3-0acetyl group for every ten sugar units, is insoluble i n cold water but is soluble i n hot water. It is inferred that greater solubility results from the greater freedom of rotation of sugar units provided by the low energy



248

CARBOHYDRATES IN

SOLUTION

requirement for rotation around the equatorial bond from carbon C-5 to the glycosidically bonded hydroxymethyl group (Figure 1). The low energy requirement for rotation of sugar units relative to each other should confer lower free energy of solution to the polymer. Removing the acetyl groups should lower the solubility, but the deacetylated polymer remains soluble i n hot water. Nigeran with a D P of 300-350 is also soluble i n hot water but is insoluble i n cold water. T h e irregularities i n the chain produced by com bining 1 —> 4 and 1 - » 3 linkages is expected to reduce intermolecular fit so that molecules can be more easily solvated and dissolved. Lichenan with a D P usually reported above 100 is soluble i n hot water but precipitates when its solutions are cooled.

HO Figure 1.

Rotations possible in a(l^> 6)-linked glucan

Easily Soluble Polysaccharides. Pullulan with a D P of 250 a-D-gluco pyranosyl units joined by 1 - » 4 and 1 - » 6 linkages i n a linear chain is water soluble. Again the variability i n chain linkages plus the exceptional ease of unit rotation provided by 1 - » 6 links give the polysaccharide mole cule greater numbers of low energy conformations. The poor fit of one chain to another and the low free energy of solution contribute to developing solubility solution stability. Introduction into a polysaccharide chain of substituents, w h i c h by their bulk decrease the fit of one polymer molecule to another, greatly improves water solubility and solution stability. A perfect example is the excellent solubility of guaran. This ( l - > 4)-linked chain of β-D-mannopyranosyl units has a single «-D-galactopyranosyl side chain joined by 1 6 linkages at every second unit of the main chain (12,13). The sub stituent D-galactosyl units inhibit strong intermolecular associations be tween the chains so that guaran solutions are stable; also the chains remain extended i n solution and produce high viscosity. Removal of the side chain produces insoluble mannan of the type seen i n palm seeds.


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Solubilization

249

Solubility of Polysaccharides of Polysaccharides by Chemical

Modification

F r o m the examples given above, it is apparent that water solubility of a polysaccharide can be instilled or improved by placing substituents on the linear structure which reduce the fit of one polysaccharide mole cule to another or by providing anionic groups which can improve hydration and present ionic charges which by coulombic repulsion aid in molecular separation. Cellulose is a good example for illustrating such effects; it has been extensively studied because of its industrial availability at low cost (14). Hydroxyalkylcellulose. Reaction of cellulose with ethylene or pro pylene oxides produces hydroxyethyl or hydroxypropyl derivatives. B y forming the hydroxyethyl derivative about the same ratio of hydrogen bonding sites to carbon atoms is provided as i n the underivatized cellu lose, but the substituent groups reduce the fit between polymer chains so that the derivative can be dissolved i n water to produce stable solu tions. T h e cellulose derivative has many of the solution properties of guaran. Methylcellulose. The placement of methyl groups on some of the hydroxyl groups of cellulose reduces the steric fit of the cellulose chains so much that they become water soluble. Certain acetylated celluloses also develop water solubility as a consequence of lowered interpolysac charide associative forces. The solubility of partially methylated cellulose shows the powerful extensive effect of disrupting or reducing intermo lecular associations. As each methyl ether grouping is formed, a strongly hydratable and effective solubilizing group is replaced by an ether linkage of equivalent hydrogen bonding capacity, and a methyl group with low hydration properties is inserted. Therefore even though some of the solubilizing hydroxyl groups are replaced by less solubilizing methyl groups, the loss i n solubility is more than made u p by the decreased association between cellulose chains, and consequently the cellulose ether dissolves. The overall solubility of the individual cellulose derivative has been reduced by the methylation. This is evident from the behavior of methylated cellulose, which, although it dissolves i n cold water, pre cipitates when the solution is heated. Thus, the molecules can suffi ciently solvate to dissolve, but they are just barely soluble, and any decrease i n the solvation—e.g., that occurring at the higher thermal en ergies of increased temperature—cause the molecules to aggregate and precipitate. Precipitation of methylcellulose with the formation of gels when its solutions are heated may also be influenced by changes i n the structure of the water surrounding the dissolved molecules. Aqueous solutions of certain types of solutes known as hydrophobic solutes cause a structuring of liquid water i n their vicinity. Such structured water



250

C A R B O H Y D R A T E S IN S O L U T I O N

would collapse when the solution is heated leaving the methylcellulose molecules free to associate to produce gels and even to form enlarged micelles which precipitate. These effects are not confined to the intro duction of methyl groups but to any groups which reduce the fit between polysaccharide molecules and simultaneously lower the solvation of all or parts of the polymer molecule. Substituents can produce kinks in the polysaccharide chains which also reduce their ease of alignment. Carboxylated Cellulose. Uniform introduction of carboxymethyl groups into cellulose to a degree of solubility ( D S ) of 0.45 or more pro duces derivatives whose alkali salts are water soluble. Solubility is a consequence of the ionic repulsion and of the disruptive effect of the substituent groups on molecular fit. If a carboxylated cellulose is pre pared by the oxidation of cellulose with dinitrogen tetraoxide and under conditions where depolymerization is minimal, a carboxyl D S of 0.8 is required to produce solubility i n the form of the sodium salt (15). Thus, introduction of ionic charge by converting the equatorial hydroxymethyl group to a carboxyl group does not extensively interfere with interchain steric fit, and absence of bulky substituents requires a greater number of coulombic repulsive forces to cause solubilization. Other sodium salts of linear polyglycuronic acids such as sodium pectate and sodium alginate are water soluble. However these, as well as soluble sodium celluronate, become insoluble and form a gel when the p H of the solution is lowered to 2.5-3 where the carboxyl group is i n the acid form and ionization is repressed. Insolubilization of alginic acid occurs regardless of its existence as a diheteroglycan i n the form of a block polymer composed of alternating stretches of D-mannuronic acids and L-guluronic acids. Cellulose Sulfates and Phosphates. Conversion of cellulose to sulfate or phosphate monoesters produces soluble derivatives. These ester groups are highly hydrated, offer steric interference to molecular fit, and are ionized at all p H levels so they continually produce coulombic repulsion. Insolubilization

of Polysaccharides

by Chemical

Modification

In reverse to solubilization, soluble polysaccharides are made less soluble by removing branches or substituents to produce a more uni formly linear polysaccharide with improved possibility of intermolecular fit, by removing formal charges, by lessening the number of strong hydra tion sites, or by completely overcoming hydration effects by introducing hydrophobic substituents. Examples of these effects are common; most xylans decrease i n solu bility when L-arabinofuranosyl side-chains are removed by mild acid



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hydrolysis. Guaran decreases i n solubility as its D-galactopyranosyl single unit side chains are hydrolyzed off; amylopectin develops an insoluble fraction when its «-D-( 1 - » 6) branch points are hydrolyzed by pululanase. As indicated above, polyglycuronates become insoluble as the molecular charge is eliminated by repressing ionization of the carboxyl group. Here the lower hydration of a carboxyl group compared with the carboxylic anion is important and may facilitate insolubility. Working with cellulose and starch sulfates ( 16), we have shown that progressive introduction of 3,6-anhydro rings, through alkali induced dis placement of the sulfate group at carbon C-6 by attack of the oxygen at C-3, gradually leads to lower solubility and eventually to insolubility. Insolubility results from excessive loss of hydration capacity because of the sulfate groups and of the hydroxyls at C-3 positions and their replace ment by a single ether linkage. Introduction of hydrophobic groups decrease the water solubility of a polysaccharide. W i t h large hydrophobic groups, such as benzyl or stearoyl, insolubility is produced at low levels of derivatization. One might properly conclude that neutral polysaccharides, when soluble are often just barely so and that withdrawal of a small degree of hydration can lead to insolubilization. Solution Behavior of

Polysaccharides

Depending upon chemical structure and the conformations that are possible, polysaccharides in solution may develop secondary structures such as helices, tertiary structures formed from junction zones or by double helix or triple helix unions and even quaternary structures from the cross linking of tertiary structures. Polysaccharides thus mimic pro teins and nucleic acids, which are specific types of sugar-phosphoric acid copolymers. Methods used to obtain conformational information and establish secondary, tertiary, and quaternary structures involve electron micros copy, x-ray diffraction, refractive index, nuclear magnetic resonance, infrared radiation, optical rotation, and anisotropy, as well as a variety of rheological procedures and molecular weight measurements. Extra polation of solid state conformations to likely solution conformations has also helped. The general principles of macromolecules i n solution has been reviewed by Morawetz (17), and investigative methods are dis cussed by Bovey (18). Several workers have recently reexamined the conformations of the backbone chain of xylans (19, 20, 21). Evidence seems to favor a left-handed chain chirality with the chains entwined perhaps i n a two fold screw axis. Solution conformations are more dis ordered than those in crystallites (22). However, even with the disorder-


252

C A R B O H Y D R A T E S IN

SOLUTION


ing effects of branching and fewer intermolecular hydrogen bonds per sugar unit i n xylan, compared with a glycohexan, the polymers associate i n solution. Thus, Blake, M u r p h y , and Richards (23,24) show that upon cooling a heated aqueous dispersion of hemicellulose, changes occur i n optical rotation, viscosity, and light transmission which indicate molecular aggregation.

H

Figure 2. κ-Carrageenan Rees et al (25) have extensively examined the solution conformation and interactions of a number of polysaccharides, especially carrageenan fractions. Kappa-carrageenan is unusual as it forms gels when a solution of its potassium salt is cooled. Rees conceives that double helix formation occurs i n solution and, interlocking helices develop i n the gel state. It is possible that gel formation is abetted by insolubihty factors within the polysaccharide make up. Kappa-carrageenan is thought to be a barely soluble polysaccharide. It contains enough 3,6-anhydro rings to insolubilize a neutral polysaccharide (Figure 2 ) . Solubility is main tained by the presence of the 4-O-sulfate groups. Yet it appears that

3001-

200

lOOl-

0.2

0.4

0.6

0.8

1.0

Figure 3. Viscosity of sodium and potassium salts of waxy corn starch sulfate (DS 0.5)



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replacement of the counter ion sodium with potassium is enough to i n solubilize the structure. The insolubilization is influenced b y the much weaker electronic fields surrounding potassium ions as compared with sodium ions and their consequent reduced structuring effect on adjacent water molecules. F o r example, the large difference between the potassium and sodium salts of a waxy corn starch sulfate are evident in the viscosities of their solutions (26) (Figure 3 ) . Rees and coworkers (27, 28) have suggested that optical rotatory changes during solution cooling of *- and i-carrageenans can be attributed to the formation of helices. W i t h kappa-carrageenan they obtain optical rotatory changes on cooling the solution. W e have obtained similar results as shown in Figure 4. Because of the value of gelling agents i n various applications we are examining specifically modified starch and cellulose

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Solubility of Polysaccharides and Their Behavior in Solution

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