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IV. Methods for the Analysis of Complex Polysaccharide Materials
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.
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
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.
G. D. SWINCER, J . M . OADES, A N D D. J . GREENLAND
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,
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
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
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
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,
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.,
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
A wide variety of methods have been used for the isolation of individual
polysaccharides from biological materials (Whistler and Smart, 1953;
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
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
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
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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