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IV. Methods for the Analysis of Complex Polysaccharide Materials

IV. Methods for the Analysis of Complex Polysaccharide Materials

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polysaccharides from sources other than soils often occur in complex

mixtures containing other carbohydrates and a variety of impurities. The

work on glycoproteins (Neuberger and Marshall, 1966a,b; Neuberger

et al., 1966; Gibbons, 1966; Spiro, 1966), mucopolysaccharides (Jeanloz,

1963; Schmid, 1964; Brimacombe and Webber, 1964; Davidson, 1966),

plant gums and mucilages (Smith and Montgomery, 1959); and microbial

polysaccharides (Stacey and Barker, 1960; Barker, 1963; Rogers, 1966)

is most relevant to studies on soil polysaccharides.

B. ANALYTICAL

METHODS



I . Quantitative Analysis of Component Sugars

a. General. Most techniques used for the quantitative estimation of a

given sugar either in a polysaccharide or in some other carbohydratecontaining macromolecule or in a mixture of these polymers require the

liberation of the monosaccharide by hydrolysis of a glycosidic linkage.

This is due to the fact that, in general, the only groups present in the

sugar moieties of intact polysaccharides which can be recognized by

physical and chemical methods are the hydroxyl groups, and these are

common to all types of sugars. Sometimes there are carboxyl, acetamide,

phosphonic acid, sulfonic acid, and methoxyl groups, all of which can

be measured without hydrolysis of the polymer. However, the parent

sugars cannot be identified unless they are recovered intact, either as

the free monosaccharides or as suitable stable derivatives.

In certain cases use can be made of quantitative colorimetric procedures that do not require prior release of the sugars as a separate step;

color formation occurs concurrently with the liberation of the sugars, and

consequently there is no risk of destruction of the monosaccharides during hydrolysis. These procedures are of particular significance in the

measurement of acid-labile sugar components (e.g., uronic acids, by the

carbazole method of Dische, 1947) or for the routine assay of some of the

polysaccharide materials that contain only a few components (e.g.,

neutral sugars, by the orcinol method of Winzler, 1955). However, these

procedures are not very specific, and for this reason the results are very

dependent upon the purity and sugar composition of the polysaccharide

material.

b. The Problem of Hydrolysis. In order to liberate sugars from complex polysaccharides, hydrolysis by acid is employed at present almost

invariably. However, in the free state, all commonly occurring sugars are

more or less unstable in hot acid, and the glycosidic bonds joining different

sugars show different stabilities to hydrolysis by acid, some common

linkages being particularly stable.



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The acid stabilities of the various classes of monosaccharides vary

greatly, the approximate order being: hexosamines > hexoses > deoxyhexoses > pentoses > uronic acids. The particular acid used has some

bearing on this order: hexosamines are more quickly destroyed in HzS04

than in HCl of equal concentration whereas the reverse applies for the

other groups. With amino sugars the exclusion of oxygen may greatly

reduce the extent of destruction (e.g., see Walborg and Ward, I963), and

the presence of heavy metals may lead to increased destruction (Hartree,

1964).

An additional complication is the possibility of undesirable side reactions, such as “acid reversion” [the acid-catalyzed reaction of the reducing group of a liberated monosaccharide with the primary or even

secondary hydroxyl groups of another sugar molecule to give disaccharides or oligosaccharides (Pigman, 1957; Whelan, 1960; Overend er

al., 1962)], or interaction of the free sugars with amino acids (Francois

et al., 1962; Gottschalk, 1966). This last point may be particularly significant in the analysis of soil polysaccharides which have always been

found associated with polypeptide materials. Neuberger and Marshall

( 1 966a) consider that it is at present impossible to say whether these

possible sources of error affect seriously the analytical results. The reactions being bimolecular, their effect can be minimized by carrying out

the hydrolysis at low substrate concentration.

Glycosidic bonds in polysaccharides are hydrolyzed at rates that are

dependent largely on the nature of the sugar supplying the anomeric

carbon atom of the linkage (Wolfrom and Thompson, 1957; Overend er

al., 1962; Adams, 1965). Thus furanoside linkages are more labile than

pyranoside linkages (Haworth and Hirst, 1930; Shafizadeh, 1958;

Reichstein and Weiss, 1962); alpha glycosidic bonds are usually more

stable than beta (Wolfrom and Thompson, 1957; Overend et al., 1962);

with pyranoside linkages, pentoses and 2-deoxyhexoses allow easier

hydrolysis than ordinary aldohexoses; and increased resistance to hydrolysis is conferred by uronic acid groups (Smith and Montgomery,

1959) and also by amino sugars (Gottschalk and Ada, 1956; Johansen

et al., 1960).

In practice, no perfect conditions for hydrolysis have been devised

whereby it is certain that all glycosidic linkages of polysaccharides are

cleaved and, at the same time, all the monosaccharides itre still intact at

the end of the acid treatment. However, by dividing the sugars into

groups, a set of reasonably satisfactory conditions can be derived for all

groups except the uronic acids and possibly the pentoses. The optimum

hydrolysis conditions for each group vary from one polysaccharide to

another and must be ascertained by preliminary experiments in each



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particular case. In general it can be said that HCI (4N to 8 N ) has been

most satisfactory with hexosamines; H z S 0 4( 1 N or 2 N ) with hexoses;

and more dilute acids, such as 0.1 N H2S04or HCI, with deoxyhexoses.

The uronic acids and to a lesser extent, the pentoses represent a special

problem which at present has no adequate solution. Perry and Hulyalkar

( 1965) state that even in the most favorable cases recoveries of polymerbound uronic acids rarely exceed 70 percent and, where the more acidlabile uronic acids are involved, the yields are considerably below this

figure.

Little is known about the best conditions for the release of pentoses.

Although losses are often observed, it seems that reasonable recoveries

are obtainable as long as optimum conditions are established (Saeman

el al., 1954; Ivarson and Sowden, 1962; Cheshire and Mundie, 1966).

The N-acetylamino sugars also represent a problem, although this is

not usually very difficult to solve. Under strong acid conditions the

polysaccharides containing these sugars are de-N-acetylated rapidly,

and subsequent hydrolysis of glycosidic bonds is inhibited by the resulting free amino groups which, being positively charged in acid solution,

confer some protection on the adjacent bonds (Moggridge and Neuberger,

1938; Johansen et at., 1960). However, by diluting the acid, it is possible

to reduce the rate of de-N-acetylation relative to that of glycosidic

splitting, and thus to recover a high proportion of the original N-acetylamino sugars for the purpose of identification. Once the sugars have been

identified, they can be determined quantitatively either (a) by measuring

the total N-acetyl content of the polysaccharide material, or (b) (if it is

certain that in the polymer all amino sugars are N-acetylated) by determining the free (de-N-acetylated) amino sugars following their complete release by hydrolysis with 4-8 N HCI.

A technique with important possibilities for measuring losses of sugars

during hydrolysis is the isotope dilution method employed by Frdncois

et al. ( 1 962).

c . Analysis of Polysaccharide Hydrolyzates. Methods for the separation, identification, and determination of the monosaccharides in

hydrolyzates of polysaccharides have been reviewed thoroughly elsewhere (Percival, 1963; Bishop, 1964; Davidson, 1966; Neuberger and

Marshall, 1966a; Northcote, 1966; Spiro, 1966).

Chromatographic techniques are of primary importance for the separation, detection, and preliminary identification of sugars. However,

chromatographic behavior alone does not allow unequivocal identification of an individual sugar; this requires either isolation of the pure

sugar in crystalline form or conversion of the sugar to a characteristic

crystalline derivative.



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G . D. SWINCER, J. M. OADES, AND D. J. GREENLAND



Satisfactory removal of the hydrolyzing acid prior to chromatography

is often difficult. Volatile acids are removed quite simply by evaporation,

although with HCI, particularly in the presence of heavy metals, destruction of some sugars can occur. Strong mineral acids are probably

best removed with strong anion exchange resins in the carbonale, bicarbonate, or acetate forms. However, precipitation with barium hydroxide or barium carbonate is still used regularly for the removal of

HzS04 in spite of the danger of selective adsorption of sugars by the

precipitate. An unusual approach with definite possibilities for the removal of H a S o l is selective extraction of the sulfate with an immiscible

organic liquid (Becker and Shefner, 1964).

Paper chromatography which has been the most useful routine method

for rapid preliminary characterization of sugar mixtures has not proved

completely adequate for precise quantitative work. For this reason it is

being gradually superseded by such methods as column chromatography

with ion exchange resins and gas-liquid chromatography, which offer

several distinct advantages. Ion exchange chromatography has been applied successfully not only to the charged sugars (amino sugars and uronic

acids), but also, by formation of their borate complexes, to neutral sugars.

Fully automated procedures have been developed, and the method can be

used as a preparative technique for milligram quantities of sugars. Gasliquid chromatographic analysis of sugars has been developed only very

recently, and it is almost certain that further advances will be made. This

technique offers the possibility of rapid and precise quantitative analysis

of very small samples containing a complex mixture of sugars (Oades,

1967a).

Except for gas-liquid chromatography, most of the methods used for

quantitative determination of the separated sugars are colorimetric. Because of the relatively low specificity of some of the color reactions, special

care is required to ensure that all interfering substances are taken into account. Allowance also has to be made for the variations in color yield from

sugar to sugar. Different methods are needed at least for each class of

monosaccharide (e.g., hexoses, pentoses, uronic acids, hexosamines).

Specific enzyme assays have been worked out for a few monosaccharides. As further assays are developed, this approach is likely to become useful for the analysis of complex hydrolyzates since there is

normally no need for prior separation of the sugars.

2 . Physicochemical Analysis.

Most of the methods generally used on high polymers for the determination of molecular size, shape and flexibility can be used with polysaccharides. They may be listed as: (a) hydrodynamic methods (e.g.,



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sedimentation analysis by ultracentrifugation, viscometry, streaming

birefrigence measurements, determination of diffusion constants); (b)

methods based on the colligative properties of the molecules (e.g.,

osmometry, isothermal distillation); (c) methods involving measurements which depend directly on the physical size of the molecules (e.g.,

light scattering); (d) end-group determination by chemical assay; and (e)

techniques that are fundamentally separative rather than analytical in

nature (e.g., gel filtration, ultrafiltration through membranes of graded

pore diameter, electrokinetic ultrafil tration, density gradient centrifugation, free-boundary electrophoresis).

The application of these methods to polysaccharides has been reviewed

in considerable detail (Greenwood, 1952, 1956; Whistler and Smart,

1953; Whistler and Corbett, 1957; Banks and Greenwood, 1963; Horton

and Wolfrom, 1963; Gibbons, 1966). For thorough characterization of a

polysaccharide, as many methods as possible should be combined.

Many of the methods yield reliable quantitative information only with

preparations that are homogeneous (i.e., “consisting of molecules having

identical structure but not necessarily the same molecular weight” Banks and Greenwood, 1963), and satisfactory interpretation of the results often demands, in addition, a narrow distribution of molecular

weight. For example, the diffuse sedimentation boundary produced during ultracentrifugation of a very polydisperse sample allows computation only of an approximate value for the average molecular weight.

These points are of obvious significance with respect to the analysis of

soil polysaccharide preparations which have proved particularly difficult

to purify and fractionate. Clearly, it is imperative either to ensure that

homogeneous polysaccharides have been prepared before attempting

physicochemical characterization or to use only the limited range of

techniques that can be applied satisfactorily to heterogeneous preparations. Undoubtedly for the most complete and precise information

homogeneous polymers must be obtained, but the problems involved both

in isolation of the required fraction and in assessment of its homogeneity

are extremely difficult. In fact, Banks and Greenwood ( 1 963) pointed out

that “it is doubtful if any polysaccharide has been examined by sufficient

methods to prove unambiguously that it is homogeneous.”

The techniques that can be applied most satisfactorily to the characterization of polysaccharides in heterogeneous mixtures are those based on

separation methods.



C. SEPARATION

METHODS

A wide variety of methods have been used for the isolation of individual

polysaccharides from biological materials (Whistler and Smart, 1953;



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G. D. SWINCER, J. M. OADES, A N D D. J . GREENLAND



Pigman and Platt, 1957; Bouveng and Lindberg, 1960; J . E. Scott, 1960;

Banks and Greenwood, 1963; Barker, 1963; Horton and Wolfrom, 1963:

Jeanloz, 1963; Kertesz, 1963; Brimacombe and Webber, 1964; Whistler,

1965; Northcote, 1966). Initial extraction is usually the most critical step

because it is often at this point that the most drastic treatments are

needed. Separation of polysaccharides from cellular material is rarely

easy, and the problem is especially formidable with polysaccharides that

are associated intimately with an insoluble matrix as in cell walls. There

is an obvious relationship here with the problem of separating polysaccharides from the mineral matrix of soils. With almost all the procedures

sufficiently powerful to solubilize such polysaccharides, there is a definite

risk of degradation (Northcote, 1966). Any modification of the structure

of the molecules or the molecular weight distribution, or both, may invalidate many of the subsequent analyses.

Once the polysaccharides have been brought into solution, they can be

purified and fractionated by a variety of techniques. Fractional precipitation or dissolution of polysaccharides (and polysaccharide acetates or

nitrates), either by changing the solvent composition, or pH, or temperature, has been widely used. With complex mixtures this approach is only

of limited application, except for the removal of extraneous material,

because of the tendency to coprecipitation and occlusion of other polymers. Moreover, in most cases fractional precipitation merely subdivides the polymolecular system into fractions on a molecular weight

basis: each individual fraction represents a narrow molecular weight

range, but still remains a mixture of polysaccharide types.

Gel filtration is assuming a place of primary importance in the study of

heterogeneous polydisperse systems. It gives very efficient separation of

polydisperse materials into fractions covering a limited range of molecular weight and also provides a simple and effective method for removing

low-molecular-weight impurities (Granath and Flodin, 196 I ; Anderson

et al., 1965; Anderson and Stoddart, 1966; Granath and Kvist, 1967).

Separation techniques based on the ionic properties of the polysaccharides offer the best prospects for isolation of homogeneous materials.

These methods are amenable to all soluble polysaccharides as even those

polysaccharides with no readily ionizable groups are generally slightly

charged, particularly in alkaline solution, due to ionization of the hydroxyl

groups, while complex formation with certain ions, notably borate ions,

increases the negative charge on a polysaccharide. The techniques that

have been applied successfully include selective precipitation with metal

ions or quaternary ammonium salts, and ion exchange chromatography,

particularly with charged celluloses. These procedures can generally be



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made very sensitive to small differences in the net charge of the polymers

and they are often capable of resolving mixtures of closely related polysaccharide species. The ion exchange procedures in particular have given

some excellent separations (e.g., Jermyn, 1962; Antonopoulos et al.,

1967) and the technique offers considerable scope for further refinement.

In addition, some very good separations of mucopolysaccharides have

been achieved recently by a combination of fractional precipitation and

column chromatography (e.g., Antonopoulos et at., 1964; Pearce and

Mathieson, 1967).

Other procedures with distinct possibilities for the fractionation of

mixtures of polysaccharides are density gradient centrifugation (e.g.,

Charlwood, 1966; Franek and Dunstone, 1967) and selective precipitation with antisera (e.g., Heidelberger et af., 1955), neither of which are

based directly on differences in polymer size or charge.

V. Summary and Conclusions



Carbohydrates represent 5 to 25 percent of soil organic materials. They

consist of a wide range of monosaccharides, such as hexoses, pentoses,

deoxy- and 0-methyl sugars, uronic acids, and amino sugars. Such monosaccharides exist in polymeric molecules of various sizes and degrees of

complexity, which are associated more or less strongly with inorganic

colloids in soils.

Large proportions of carbohydrates in many soils are present in partly

decomposed plant and animal remains. Glucose, presumably in the form

of cellulose, is dominant in such materials.

Plant litter and roots, either living or dead, are the main primary source

of soil carbohydrates, but the composition of soil polysaccharides, apart

from obvious plant remains, would suggest a microbial origin, either

wholly or in part, e.g., plant materials which have been modified by the

soil flora and fauna.

Polysaccharides have been extracted from soils by many different

chemical reagents, and recently methods have been devised that enable

most of the carbohydrates to be isolated from other soil materials. The

extracted polysaccharides show a continuum of molecular sizes and contain a wide range of neutral and charged monosaccharides, amino acids,

and other unidentified nitrogenous and acid components. Carbohydrates

from different soils are similar in chemical composition suggesting that

the microbial population of different soils is qualitatively similar.

Many methods have been used to fractionate extracted soil polysaccharides usually with limited success. The most successful methods have

been based on gel filtration and chromatography on charged supports



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G . D. SWINCER, J . M. OADES, AND D. J . GREENLAND



such as cellulose. However, fractions obtained are still complex and contain a range of different components. This complexity is not surprising

in view of the wide range of substrates, organisms, and metabolic products

of organisms that are subjected to chemical extraction and fractionation

procedures. Generally the “turnover” of sugars in soil carbohydrates

appears to be rapid, but some microbial polysaccharides are resistant to

breakdown by soil organisms. The subject is complicated because of

interactions with metal cations and sorption on colloid surfaces.

The composition of soil polysaccharides suggests that in soils they may

carry charged sites and take part in exchange reactions and act as

energy sources for heterotrophic organisms. However, the main stimulus

for the study of soil polysaccharides has arisen from repeated indications

of their favorable influence on soil physical conditions. Much work has

been directed toward this aspect, and it has been shown that microbially

produced soil polysaccharides are capable of stabilizing soil aggregates

against dispersion in water. N o specific fraction has yet been definitely

identified as particularly active, but it is suggested that the larger polysaccharides produced by microorganisms in coarse pores of aggregates

are likely to be the most effective.

The mechanisms by which these polymers react with inorganic colloids is not understood, but the complex preparations obtained from soils

are sorbed from aqueous solution by clay materials, and further work

on the fractionation of carbohydrate preparations followed by studies

of the sorption of these “purer” characterized fractions will undoubtedly

prove to be worthwhile.

Methods for the isolation of polysaccharides from other soil materials

in good yield are now available and methods for the analysis of the extracted polysaccharides have been developed by carbohydrate chemists.

Combinations of these techniques in the future will enable new information about the composition, origin, and function of soil carbohydrates

to be obtained. Particularly useful information should arise from the

cooperation of chemists and microbiologists using techniques involving

isotopically labeled materials.

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