Published 1996

Chapter 35
Organic Matter Characterization
ROGER S. SWIFT, CSIRO Division of Soils, Adelaide, Australia

INTRODUCTION
It is generally accepted that the term soil organic matter refers only to the nonliving organic material in the soil, which makes up by far the major portion of the
total organic components. The living organic components, which are part of the
soil biota, comprise a minor portion of the total organic material, and they will
not be considered in this chapter. The soil organic matter can be of plant, animal
or microbial origin and may be relatively fresh or highly decomposed and transformed. It is to the characterization of this material to which this chapter will be
devoted. For in-depth reviews on this topic, the reader should consult Hayes and
Swift (1978), Stevenson (1994), Aiken et al. (1985), and Hayes et al. (1989).
In chemical terms, it is possible to identify in soil organic matter, components belonging to the main classes of naturally occurring organic compounds
found in plants and animals. Each of these compounds can be found in a wide
variety of physical environments and physicochemical associations. In addition
to these identifiable compounds, there are much larger amounts of organic matter which are not amenable to current methods of chemical characterization. To
bring some semblance of order to this diverse and complex system, it is necessary to establish and superimpose a set of classifications and definitions in order
to establish a common framework for discussion and investigation.
Given the complexity of the soil system, any attempt to rigorously categorize soil organic components is likely to be, at best, imperfect. Quite clearly the
most likely basis for classification lies in readily observable physical, chemical
and/or biochemical differences between the various components. A useful delineation based on physical characteristics is that drawn between recognizable
remains of plants (or animal) debris and the highly degraded and transformed
materials which contain no recognizable plant, animal or microbial structures.
Although this classification is essentially based on visual observation of physical
differences, in essence, it purposes to differentiate between the results of biochemical transformations. As such it is unlikely to be wholly successful. For
example, the same classes of organic compounds (e.g., carbohydrates, peptides
and amino acids) can be found in both fractions.
Copyright 1996 Soil Science Society of America and American Society of Agronomy, 677 S.
Segoe Rd., Madison, WI 53711, USA. Methods of Soil Analysis. Part 3. Chemical Methods-SSSA
Book Series no. 5.
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In an attempt to overcome this problem emphasis can be placed directly on

the chemical characteristics themselves as the basis of classification. Using this
approach, the nonliving organic matter can be divided into humic and nonhumic
substances (Hayes & Swift, 1978). In this scheme, all of the recognizable plant
debris plus all of the identifiable classes of organic compounds (such as carbohydrates and peptides) in their natural or transformed state are categorized as
nonhumic substances. The remaining amorphous, highly transformed, darkly
coloured material which cannot be identified as belonging to an established group
of organic compound are classified as humic substances.
As well as the classifications referred to above, it would be possible to
devise others based, for instance, on the distinction between free, discrete organic particles in the soil as opposed to bound organic matter which is adsorbed by
clays, oxides and other mineral surfaces in the soil. Once again, the actual arrangements in the soil are not quite so simple, as it is difficult to distinguish
absolutely between free and bound organic matter. For example, organic matter
which is apparently bound may be sorbed to the surface of a mineral particle, or
may be involved in other processes, such as occlusion, clustering or aggregate
formation of mineral particles. Once organic components are involved in sorption
or occlusion, the physical and chemical conditions in which they exist may protect them from other soil processes, such as degradation and/or complexation,
possibly for long periods of time (Theng et aI., 1992).
As can be deduced from the above discussion, the order which we may
appear to superimpose on the system by the use of definitions and classification
systems, although helpful, is, nonetheless, illusory. Soil organic matter is, and
will, remain a complex mixture of organic compounds in a wide variety of
physicochemical environments and subject to a wide raIlge of associations with
minerals, metal cations, and anthropogenic organic compounds. It is this system
which we must study either in situ in all its complexity, or we may simplify the
material to be studied by undertaking extraction, purification and fractionation
procedures.
Soil organic matter is an important pool of C in the global C budget and is
a major component of the more active fractions involved in the earth's C cycle in
terms of both total amount and annual flux. The levels of soil organic matter are
sensitive to changes in temperature, rainfall and atmospheric CO 2 concentrations
and will be both an important indicator of, and possible contributor to, global
climate change.
It has been commonly estimated that soil organic matter contains approximately 1500 x 1015 g C (Schlesinger, 1984). Although this figure is based on a
large number of measurements, it is a very difficult quantity to estimate accurately compared with, for instance, the amount of C as CO 2 in the atmosphere or
dissolved in the surface waters of the oceans. This is due to the spatial variability of soils and the paucity of data from some regions of the world. Most estimates
for soil organic C range from 1100 x 1015 to 3000 X 1015 g C (Schlesinger, 1984),
but all estimates indicate that there is more C in the soil than there is in the atmosphere or in living biomass.
Just as important as the total amount of C is the flux of CO 2 from the soil
organic matter which is estimated at 75 x 1015 g C yr-1 (Schlesinger, 1984). This

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is roughly equivalent to twice the amount of plant material entering the soil each
year. There are, of course, much larger reservoirs of C on the earth than soil organic matter (e.g., carbonate and fossil fuel deposits or CO 2 dissolved in the
oceans) but most of these are involved to a lesser extent in the annual fluxes passing through the C cycle.
As indicated above, the physically and chemically heterogeneous mixture
of materials which make up soil organic matter varies substantially in terms of
both their amount and resistance to biological decomposition. In most mineral
soils under equilibrium conditions the highly decomposed and biochemically
transformed materials are the dominant component, whereas the recognizable
plants and other biological fragments represent only a few percentage points of
the total organic matter. Of the transformed materials, the humic substances generally constitute two-thirds to three-quarters. Most of the remainder is made up
of much lesser amounts of carbohydrate and proteinaceous materials. Lesser
amounts of a wide range of other compounds can also be identified, including
lipophilic compounds such as fats and waxes.
On entering the soil, some of the more labile plant materials are decomposed very rapidly, sometimes within days or weeks, whereas the more resistant
compounds may survive for several months or even years. Similarly, the transformed materials will consist of substances with widely varying rates of decomposition. One of the characteristics of the decomposition process is that many of
the compounds which are produced are more resistant to decomposition than
their precursors. This is one of the essential features of the humification process.
Part of this stability is due to the chemical changes which result from the biological transformations which occur during decomposition and part is due to the
association of organic macromolecules with mineral surfaces and with one
another.
The overall or mean turnover time of organic matter in mineral soils is frequently several tens to a few hundred years but, due to the factors outlined
above, this is made up of turnover times of particular components from as low as
hours/days through to several thousands of years or longer for the more intractable, charcoal-like materials. Although peats and a number of other soils contain
large amounts of organic matter, most mineral soils contain just a few percentage points of organic matter (usually 1-5% C) in the surface layers. Nevertheless, this relatively small amount of organic matter has a profound effect on a
wide range of soil properties, such as: cation exchange capacity, soil structure,
the retention and cycling of nutrients, the binding of heavy metals and organic
pesticides, etc. Given the importance of these effects, it is not surprising that the
nature and properties of soil organic matter have attracted the interest of soil scientists for many years. Although considerable progress has been made in the
understanding of many of the properties of these materials, it is, perhaps, disappointing that more progress has not been made in other aspects, such as the elucidation of structure. As we shall see below, there are good reasons for this which
are inherent in the properties of the molecules which make up soil organic matter in general, and humic substances in particular.
The purpose of this chapter is to provide an outline of the experimental
methodology currently being applied to the study of organic matter in soils.

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Those methods which are commonly used, or able to be implemented with minimal cost, are reported in detail. Other methods which require expensive or specialized equipment are mentioned, but the reader is referred to more detailed reference texts for further information.
Some of the chemical methods are somewhat dated, but have not changed
in recent years. However, the instrumental methods generally have changed significantly since the last edition. The methods outlined and/or referred to in this
chapter are meant to be used as a guide to the methods which have been reported in the literature. Scientific research is a dynamic study and the researcher
should be flexible with ideas and methods, and be able to adjust these when necessary.

EXTRACTION OF SOIL ORGANIC MATTER

Although a number of techniques are now available which allow organic
matter to be studied in situ in its unaltered, natural state, most of these techniques
still require the organic matter to be removed or extracted from the soil. Among
other things, extraction results in separating the organic from the mineral soil
components, removing other inorganic interferences, increasing the organic matter concentration and rendering the organic matter soluble. The application of
many chemical procedures requires that the material being studied should be in
solution.
One criticism of extraction procedures is that, as a consequence of the
extraction process, the organic matter constituents are modified to a greater or
lesser extent. It is, of course, inevitable that associations with mineral surfaces
and other organic constituents will be disrupted, as this is a necessary part of the
extraction process. Of more concern is the possibility of chemical alteration of
the organic matter itself, resulting in artifact formation caused by the extraction
process. It is important that such chemical modification should be minimized or
avoided, if at all possible. This is more likely to be achieved through the use of
mild extraction reagents [e.g., neutral pyrophosphate (Na4PZ07)], as opposed to
strong reagents [e.g., sodium hydroxide (NaOH)]. However, this usually results
in a substantial decline in the overall yield of extracted organic matter.
Having decided to use an extraction procedure, it is as well to optimize its
usefulness and efficiency by incorporating other steps, such as purification and
fractionation into the overall procedure. Although the intention of an extraction
procedure may be to isolate a particular component, the reality is that a complex
mixture is usually obtained and, by the use of appropriate procedures, this can be
turned to advantage. Thus, it is possible to obtain samples of humic substances
and soil polysaccharides from a single extraction and to further fractionate them
both. Indeed, it is highly desirable that such procedures are employed, in order to
properly purify the particular component being studied.

Extraction and Purification of Humic Substances

The procedures used to extract, purify and fractionate humic substances
exploit the basic chemical and physical properties of these materials (Hayes,
1985). If we are to understand how the various procedures operate and whether

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they can be improved or refmed, then it is necessary to have a reasonable understanding of the composition, properties and structure of humic substances.
Humic substances are a closely related family of naturally occurring
macromolecules, with a chemically ill-defined structure. For a detailed review of
the composition and structure of humic substances the reader is referred to the
book Humic Substances II: In Search of Structure (Hayes et aI., 1989), only salient points will be noted here.
In terms of the chemical composition of humic substances, the following
statements can now be made with some degree of certainty. The presence of substituted aromatic rings in humic substances has now been independently confirmed by a range of chemical and instrumental techniques. The substituents on
the aromatic rings are predominantly carboxyl groups, hydroxyl groups, carbonyl groups, and aliphatic units. Multiple substitution on the aromatic rings is
common. Significant amounts of aliphatic C are also present with chain lengths
of 1 to 20 C atoms but with smaller chain lengths predominating. The aliphatic
units may be present as side chains or as units within the main polymer chain
attached to aromatic units at both ends. The functional groups, which are distributed throughout the length of the molecule, are certainly attached to aromatic
groups and they may also be attached to the aliphatic moieties.
The various substituted aromatic and aliphatic units described above are
assembled together in a somewhat random or, at least, a largely disordered array,
and joined mainly by strong C-C bonds and perhaps ether linkages as well. Such
assemblies make up the molecular backbone of soil humic substances. Carbohydrates and peptides can be attached to this backbone in lesser but significant
amounts.
In terms of physical properties, humic substances are variable-charge
materials in which the carboxyl and, to a lesser extent, phenolic hydroxyl functional groups dissociate progressively with increasing pH. The resultant negative
charge gives rise to cation-exchange sites and to numerous inter- and intramolecular charge interactions. Soil humic substances are extremely polydisperse and
have molecular weights ranging from a few thousand to well over a million daltons (Swift, 1989). Together with the wide range of molecular weights there will,
of course, be concomitant variation in molecular size but this will also be dependent on the molecular shape or conformation. Of the various possible models for
macromolecular conformation it has been suggested (Swift, 1989) that the random coil structure is most appropriate for humic substances. Unlike other possible conformations this model accommodates the disordered chemical structure,
the solubility and solvation properties and the charge characteristics of these
materials.
In summary, the molecules of humic substances consist of hydrophilic and
hydrophobic groupings, charged sites and counter ions, the identity and proportion of which vary from molecule to molecule. In addition, there is a substantial
molecular weight range to superimpose on the variation in chemical composition. The limitations imposed by these properties on the application and success
of extraction and fractionation procedures need to be recognized at the outset.
Soil humic substances are largely insoluble and may remain in the soil
system for long periods of time before they are degraded by relatively slow

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chemical and biological oxidative decomposition processes. Humic substances

in soil are rendered insoluble by: their own inherent chemical properties, the
composition of balancing cations, interactions with other organic molecules, and
interaction with mineral surfaces. In order to extract the humic substances, it is
necessary to displace the insolubilizing cations and to disrupt the various intraand intermolecular associations and surface interactions.
Generally, the cations responsible for the insolubility of humic substances
are a combination of di- and trivalent ions (e.g., Ca2+, Mg2+, Fe3+, and Al3+) as
well as H+. All of these ions are strongly held on the organic exchange sites so
that there is only a small amount of dissociation and, as a consequence, very little intramolecular charge repulsion. In addition, the multivalent cations simultaneously satisfy charged sites at different parts of the molecule, or between molecules, thereby forming cationic bridges. Both of these effects cause the organic
molecule to adopt a condensed conformation from which solvent (usually water)
is largely excluded. The net effect of these processes is to cause the molecules to
be insoluble.
Humic substances can form associations with other humic molecules or
with different organic macromolecules such as lignin or partially decomposed
plant materials. These associations may be via cationic bridges, polar interactions, hydrogen bonding or van der Waals forces. Whatever their nature these
interactions will tend to confer insolubility on the humic macromolecule.
Similar bonding mechanisms are also possible with mineral surfaces. In
this case, however, some of the associations may be stronger. For instance, humic
substances may bond directly with Fe or Al atoms at a clay or sesquioxide surface. The net result of the many interactions is strong attachment to a surface and
resultant insolubility. In addition to the effects listed above, the inherent chemical properties of humic substances such as the relative amounts of polar and nonpolar groups and the molecular weight will greatly affect their solubility properties.
The selection of both the extractant and the method used for the extraction
process should be made with an understanding of the chemical and physical
characteristics of the fraction(s) of humic substances required to be separated
from the soil matrix. Whitehead and Tinsley (1964) have proposed four criteria
for solvents for humic substances. In their view, an effective extractant should
have:

1. A high polarity and a high dielectric constant to assist the dispersion of

the charged molecules.
2. A small molecular size to penetrate into the humic structures.
3. The ability to disrupt the existing hydrogen bonds and to provide alternative groups to form humic-solvent hydrogen bonds.
4. The ability to remove and immobilize metallic cations.
Stevenson (1994) has listed four criteria for the effective extraction method:

1. The method leads to the isolation of unaltered materials.

2. The extracted humic substances are free of inorganic contaminants such
as clay and polyvalent cations.

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3. Extraction is complete, thereby ensuring representation of fractions

from the entire molecular weight range.
4. The method is universally applicable to all soils.
Although the four requirements for an extractant are achievable, the four
requirements for an extraction method represent more an ideal than a set of criteria that are achievable.

Extraction with Aqueous Solutions

In terms of the desirable criteria for an extractant listed above, water is a
very good solvent. It is polar, has a high dielectric constant, is able to form
hydrogen bonds and thereby disrupt other hydrogen bonds, and is small and penetrative. However, an accompanying solute or additional step is required to displace and immobilize the insolubilizing, multivalent metal ions by use of a
reagent which is capable of forming soluble complexes with these cations or of
removing them as precipitates. In this regard, solutions of pyrophosphate and
EDTA {N,W-1,2-ethanediylbis[N-(carboxymethyl)glycine]} have become popular extractants because of their ability to form stable, soluble complexes with
metal ions at near neutral pH values. Alternatively, the multivalent cations can be
removed by prior washing with dilute acid which gives a H+ -saturated soil. However, the acidic conditions are not conducive to extraction of humic substances
and the pH needs to be increased before proceeding further with the extraction
process.
Having removed the polyvalent cations from the negatively charged organic exchange sites, it is important to replace them with a counter-ion that dissociates very readily, and Na+ and K+ are the ions most commonly used for extraction. The resultant high degree of dissociation causes intramolecular charge
repulsion and leads to molecular expansion together with ingress of water which
effectively solvates polar groups and disrupts intermolecular bonds. The net
result is that the humic substances become soluble and are extracted.
One of the oldest and most frequently used methods of extracting humic
substances from soils uses sodium hydroxide as the extractant. In this procedure
many of the polyvalent cations (if not previously displaced by acid pretreatment)
are removed through the formation of insoluble hydroxides at high pH values
and are replaced by sodium. The high pH of sodium hydroxide solutions also
causes many organic functional groups to ionize resulting in a higher charge density on the molecules. The importance of this effect can be judged by the fact that
sodium hydroxide extracts far larger amounts of humic material at pH values of
12 and above than the sodium salts of complexing agents used at near neutral pH
values (Table 35-1). The problems of artifact formation by oxidation of the
humic substances which can occur at high pH values can be substantially
reduced by performing the extractions under a Nz atmosphere.
Extracts obtained by using sodium hydroxide have higher average molecular weight and lower functional group content than those extracted from the
same soil by sodium pyrophosphate (Na4PZ07) at neutral pH values. Observations such as these clearly indicate that, at any given pH value, the solubility limitations of humic substances are predominantly determined by charge density
and molecular weight considerations.

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Table 35-1. Yields and compositions of humic acid (HA) and fulvic acid (FA)t fractions extracted
with different solvents from H+ soil (adapted from Hayes et aI., 1975).
Elemental composition
Extractant

Fraction

Yield

Ash

OMF*
OMF*
Sulpholane
Sulpholane
OMSO*
OMSO*
Pyridine
Pyridine
EO~
EO~

O.5MNaOH
O.5MNaOH
1MEOTA
1MEOTA

HA
FA
HA
FA
HA
FA
HA
FA
HA
FA
HA
FA
HA
FA

16
2.0
10.0
12.0
17.0
6.0
34.0
2.0
49.0
14.0
58.0
2.0
12.5
3.8

53.4
49.9
54.0
51.8
53.5
51.5
54.7
45.3
54.7
48.2
52.0
43.9
50.8
45.7

4.5
4.0
4.8
4.3
4.1
4.1
5.0
5.1
5.7
5.4
5.9
5.9
4.0
4.0

t Excluding material lost during dialysis.

* OMSO - dimethylsulfoxide, OMF =dimethylformamide, EOA
NO =not determined.

2.6
3.1
3.2
3.2
3.2
2.1
4.3
5.8
6.2
10.5
2.8
4.2
NO
NO

1.7
1.5
2.4
1.7
1.9
1.2
NO
NO
NO
NO
NO
NO
NO
NO

1.8
4.7
0.8
3.3
2.7
6.5
2.2
3.8
3.7
5.8
2.0
2.5
2.6
5.6

=1,2-diaminoethane

International Humic Substance Society Method. A number of methods

for the extraction of humic substances from soil using sodium hydroxide solution
have been published. These methods are generally successful and yield comparable results. The following is a method which has been developed by the International Humic Substance Society (IHSS) as an acceptable method for the extraction of humic substances from soils.
It has been clearly stated by IHSS that this is not meant to be a recommended or approved method, but a method that has been found to be satisfactory
for most soil types and one which can be performed in most laboratories. It produces relatively high yields and can be used as a standard method for comparisons between and within laboratories. An important component of this method is
the use of an adsorbent resin in the purification process. This can be replaced by
dialysis if the resin is unavailable.

Materials
1.
2.
3.
4.
5.
6.
7.

Method

Hydrochloric acid (HCI), 1 M, 6 M

Sodium hydroxide, 1 M, 0.1 M
Potassium hydroxide (KOH), 0.1 M
Potassium chloride (KCI)
Hydrofluoric acid (HF) concentrated, 0.3 M
XAD-8 resin (Rohm & Haas Co., Philadelphia, PA)
Visking dialysis tubing (Visking Co., Chicago, IL) [MWCO (molecular
weight cut-oft)] 10 000 dalton

Remove roots and sieve the dried soil sample to pass a 2.0-mm sieve. Equilibrate the sample to a pH value between 1 to 2 with 1 M HCI at room tempera-

ORGANIC MATTER CHARACTERIZATION

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ture. Adjust the solution volume with 0.1 M HCI to provide a final concentration
that has a ratio of 10 mL liquid/1 g dry sample. Shake the suspension for 1 hand
then separate the supernatant from the residue by decantation after allowing the
solution to settle or by low speed centrifugation. Save the supernatant (FA Extract
1) for the isolation of fulvic acid using XAD-S.
Neutralize the soil residue with 1 M NaOH to pH = 7.0 then add 0.1 M
NaOH under an atmosphere of N2 to give a final extractant to soil ratio of 10:1.
Extract the suspension under N2 with intermittent shaking for a minimum of 4 h.
Allow the alkaline suspension to settle overnight and collect the supernatant by
means of decantation or centrifugation. Acidify the supernatant with 6 M HCI
with constant stirring to pH = 1.0 and then allow the suspension to stand for 12
to 16 h. Centrifuge to separate the humic acid (precipitate) and fulvic acid (supernatant - FA Extract 2) fractions.
Redissolve the humic acid fraction by adding a minimum volume of 0.1 M
KOH under N2. Add solid KCI to attain a concentration of 0.3 M [K+] and then
centrifuge at high speed to remove the suspended solids. Reprecipitate the humic
acid by adding 6 M HCI with constant stirring to pH = 1.0 and allow the suspension to stand again for 12 to 16 h. Centrifuge and discard the supernatant. Suspend the humic acid precipitate in 0.1 M HClIO.3 M HF solution in a plastic container and shake overnight at room temperature. Centrifuge and repeat the
HCI/HF treatment, if necessary, until the ash content is below 1%. Transfer the
precipitate to a Visking dialysis tube by slurrying with water and dialyze against
distilled water until the dialysis water gives a negative Cl- test with silver nitrate
AgN0 3 Freeze dry the humic acid.
Pass the supernatant designated "FA Extract I" through a column of XADS (0.15 mL of resin per gram of initial sample dry weight at a flow rate of 15 bed
volumes per h). Discard the effluent, rinse the XAD-S column containing sorbed
fulvic acid with 0.65 column volumes of distilled H20. Back elute the XAD-S
column with 1 column volume of 0.1 M NaOH, followed by 2 to 3 column volumes of distilled H20. Immediately acidify the solution with 6 M Hel to pH =
1.0. Add concentrated HF to a final concentration of 0.3 M HE The solution volume should be sufficient to maintain the fulvic acid in solution.
Pass the supernatant designated "FA Extract 2" through a column of XAD8 (1.0 mL of resin per gram of initial sample dry weight). Repeat the back elution
and acidification as for "FA Extract I" above. Combine the final eluates from
each of the fulvic acid extracts and pass this solution through XAD-8 resin in a
glass column (column volume should be one-fifth of sample volume). Rinse with
0.65 column volumes of distilled H20. Back elute with 1 column volume of 0.1
M NaOH followed by two column volumes of distilled H2 0. Pass the eluate
through W-saturated cation exchange resin [Bio-Rad AG-MP-5 (Bio-Rad, Richmond, CA) using three times the mole of Na ions in solution]. Freeze dry the eluate to recover the H+ -saturated fulvic acid.

Comments
XAD-S is a nonionic, macroporous (pore size 25 Ilm), methyl methacrylate
ester resin (see "Fractionation of Humic Substances Adsorption"). Because it is
sometimes difficult to obtain it may be necessary to use an alternative resin such

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as Polyclar, which is a cross-linked poly(vinylpyrrolidone) (PVP) (Watanabe &

Kuwatsuka 1991; De Nobili et aI., 1990a) or other equivalent resin.
Extensive purification procedures of the resins are required before use.
These methods and methods used to store the resin are detailed by Thurman &
Malcolm (1981).
If it is not possible to purify the fulvic acid using resin treatments, exhaustive dialysis against distilled H20 is an alternative but less satisfactory method of
purification. If there is a significant concentration of polyvalent cations such as
Al3+ present, these may form insoluble metal-humate complexes as the solution
is neutralized. Therefore, the dialysis should be carried out against dilute HCI initially until the concentration of any polyvalent cations has been significantly
reduced, before finally dialyzing against distilled H20. Technically, a fraction
obtained in this way should be referred to as a fulvic fraction, rather than fulvic
acid, as it is likely to contain significant amounts of unbound soil polysaccharide.
Sequential Extraction. By extracting the humic substances from the soil
at only one pH value as in the above method, variations in the physiochemical
properties of the humic substances which reflect the environment in which they
exist in the soil, may not be apparent. Sequential extraction of humic substances
at a number of different pH may be a more sensitive method for differentially
extracting the humic substances and for determining relative distribution in the
soil and their degree of interaction with soil colloidal particles. This can be done
by changing the pH alone or changing the nature of the extractant anion (Posner,
1966; Skjemstad, 1992). The sequence outlined by Posner (1966), 0.1 M pyrophosphate (PH = 7), cold 0.5 M NaOH followed by hot 0.5 M NaOH, was found
to produce fractions which were distinctly different with respect to their molecular size, functional group content and infrared spectral characteristics (Posner et
aI., 1968; Swift & Posner, 1971; Cameron et aI., 1972a). The method outlined
below is based on that of Posner (1966) and involves three different extractants
at two pH values. This methodology could be refined and developed to vary
and/or increase the number of extractants or pH values using similar procedures
as outlined by Posner (1966).

Materials
1. Sodium dihydrogen pyrophosphate/tetrapotassium pyrophosphate
(Na2H2P207~P207)' 1:1, 0.1 M, pH = 7.0.
2. Hydrochloric acid, 0.1 M, 5 M.
3. Sodium hydroxide, 0.5 M, 5 M.
4. Visking dialysis tubing.

Method
Prepare the soil by crushing and air drying before passing it through a 2.0rom sieve. Pretreat the soil with 0.1 M HCI (with stirring) for two consecutive
periods of 24 h before commencing the extraction process.
Combine this soil sample with the first extractant (pyrophosphate), in the
soil to solution ratio of 1 g/5 mL. Allow the suspension to stand for 48 h at the
desired temperature (20 or 60C) under N2. Separate the soil and extractant (Frac-

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tion 1) by centrifugation at 1200 g for 30 min. Wash the soil residue with distilled
water several times. Repeat the above extraction for each extractant (e.g., 0.5 M
NaOH) changing the temperature as required.
The humic acid components of these fractions can be obtained as follows.
Acidify the fraction to pH =1 with HCI and allow it to stand for 24 h before centrifugation. Adjust the solution volume to 600 mL with distilled water and raise
the solution to pH = 7 using a suitable alkali (e.g., 5 M NaOH). Centrifuge the
solution at 6000 g at 2 to 5C for 30 min. Repeat this precipitation and dissolution procedure a further four times, increasing the centrifugation times by 30 min
each time, to remove the clay and humins from the solution. Retain all the supernatant solutions for fulvic acid fractionation.
Finally, precipitate the humic acid at pH =1, allow the solution to stand for
24 h, centrifuge and then exhaustively dialyze the precipitated humic acid against
distilled water in Visking dialysis tubing until chloride free. Freeze dry the slurry to recover the humic acid.
Separate the fulvic acid fraction from the combined supernatants using
XAD-8, PVP or dialysis tubing, as outlined in the previous method. Freeze dry
the fulvic acid.

Extraction with Non-Aqueous Solvents

Non-aqueous solvents have been used by a number of workers to extract
humic substances from soils. The most successful solvents tried so far being pyridine and dipolar aprotic solvents such as dimethylsulfoxide (DMSO, C2H60S)
and dimethylformamide (DMF, C3H7NO) (Hayes, 1985; Hayes et aI., 1975; Piccolo, 1988; Piccolo & Mirabella, 1987; Ma'shum et aI., 1988).
These dipolar aprotic solvents have been found to work most effectively for
humic substances where the ionization of the humic molecules has been suppressed so that they will behave essentially as if they are uncharged molecules.
This is achieved by first washing the sample in dilute acid to remove the metal
cations and then maintaining the solution pH at moderately acidic levels during
the extraction with the aprotic solvent. Under these conditions the DMSO is able
to efficiently solvate the humic molecules, extracting humic substances with similar yields and molecular weight and cation-exchange capacity as those obtained
when extracting in aqueous solutions at neutral pH. By using an amphiphilic solvent consisting of a mixture of ammonium (NUt)/isopropanol Ma'shum et al.
(1988) were able to isolate the hydrophobic organic matter from a soil.
A method of supercritical gas extraction of organic matter using an organic solvent has been applied by Schnitzer et al. (1986) to extract specific organic
components from a soil matrix. The method has been further extended to sequentially extract organic matter from soils using supercritical fluid extraction, together with a range of organic solvents (Schnitzer, 1990).

Extraction and Purification of Soil Polysaccharides

As the interest in understanding the role of soil organic matter has increased
the focus has frequently been on the humic substances because they account for
a large percentage of the soil organic C. The soil polysaccharides, which are com-

1022

SWIFf

posed of a wide range of monosaccharides in both simple and complex molecular structures, form a smaller but nonetheless important part of the soil organic
matter.
Polysaccharides in the soil may occur as an original fragment of once living organic matter or may have been formed as a result of microbial activity as
part of the decomposition process. Polysaccharides exist in association with inorganic or organic colloidal components, either through sorption (van der Waals
forces, H-bonding) or through chemisorption (e.g., phenolic glycoside linkages).
Polysaccharides have a number of effects on soil properties such as the cation
exchange capacity (uronic acid group), C metabolism, biological activity and the
complexing of metals. But more importantly, it has been found that polysaccharides are involved with stability of aggregates in soils.
The percentage of the total soil polysaccharide content involved in aggregation of soil particles is probably quite low as it requires the interaction of the
polysaccharide with multiple soil components. This interaction will depend on
the type of functional groups on the polysaccharide and the conformational structure of the sugar groups, as well as the nature of the other soil components
(Williams et aI., 1967). The correlation between total polysaccharide and degree
of aggregation has been found to be poor (Oades, 1967). However, many of the
polysaccharides are not active (e.g., partly decomposed cellulose) and so do not
contribute to aggregation of the soil particles. More recent work (Haynes &
Swift, 1990) has shown that there are particularly active fractions of polysaccharide involved in the aggregation process. It has been noted (Greenland et aI.,
1962) that the aggregating effects of soil polysaccharides are more noticeable in
soils with low organic matter content.
As indicated above, one of the main reasons for studying polysaccharides
in soils is associated with their ability to bind and stabilize soil particles. Ironically, the polysaccharides involved in this process are probably those most firmly held in the soil system and, therefore, the most difficult to remove without
degrading them. Any extraction method should, ideally, be able to extract all of
the polysaccharides from the soil matrix and, if this is not possible, then be able
to extract a representative fraction of the polysaccharides. It is possible to estimate the efficiency of the extraction procedure by comparing the amount of product from the extraction with that which is obtained using hydrolysis procedures
to determine the total carbohydrate content of the soil (Cheshire, 1979). However, such a comparison does not clearly indicate whether preferential extraction of
some polysaccharides is occurring in the extraction procedure, and hence whether
the extracted fraction is representative of that in the original soil sample.
Extraction of polysaccharides from soil utilizes similar methods to those
applied to extraction of humic substances, and often is part of the procedure of
their isolation. None of the methods used are ideal and to some extent the most
suitable method will depend on the nature and origin of the soil. The most efficient methods for general application are those using 0.1 or 0.5 M sodium
hydroxide. Large amounts of other fractions of organic matter are simultaneously extracted from the soil using this method and the efficiency of separating the
polysaccharides from the bulk of the extract (especially the humic acid fraction)
is doubtful. It has been found that yields are significantly improved by pretreat-

ORGANIC MATIER CHARACTERIZATION

1023

ment of the soil with dilute HCI or HF (Swincer et aI., 1968), or sulfuric acid
(H2S04) (Barker et aI., 1967) to acidify the soil organic components. Methylation
(Cheshire et al. 1983) of the soil as a pretreatment is another alternative although
this method may be unsatisfactory as a preparative method for some analyses.
Other methods of extraction include using extractants such as hot water,
which tends to have lower yields and is thought to be degradative, and dilute mineral acids, which give lower yields but significantly reduce the amount of humic
substance being extracted, so simplifying the purification of the polysaccharide.
Organic reagents such as DMSO give a high percentage of polysaccharides in the
product. The polysaccharide can be separated from the humic substances, by
sorbing the latter on XAD-8 resin, but separating the polysaccharides from the
DMSO is difficult. Complexing agents such as EDTA give low yields and hence
a greater probability of unrepresentative fractions.
Resins such as XAD-8 or Polyclar AT [cross-linked poly(vinylpyrrolidone)] (Sanderson & Perera, 1966; Swincer et aI., 1968; Drijber & Lowe, 1990)
may be used as a means of removing humic substances from the soil extract to
obtain the polysaccharide fraction. With an appropriate eluant, the humic substances are adsorbed on the resin and the polysaccharide is eluted through the column. Purification of this polysaccharide fraction may then be achieved by dialysis to remove salts, solvents and other low molecular weight material. Adsorption
of the coloured compounds onto charcoal is another method of purification,
although it may be difficult to recover the polysaccharides from the charcoal.
Gel chromatography can be used to fractionate polysaccharides on the basis
of molecular size. Ion exchange chromatography using diethylaminoethyl-cellulose {(C6H70)..{OHhx.a[OCH2CH2N(CH3CH2)]a} (DEAE-cellulose) can then be
used to fractionate on the basis of charge density differences. In gel chromatography care must be taken to select a gel that isolates the polysaccharides as a
group without losing a specific part of the fraction; such as the smaller molecules
that bypass with the largest molecules in the case of using Sephadex G-lOO
(Pharacia, Uppsala, Sweden). The gel chromatography method given below is
based on that of Swincer et al. (1968).

Materials
1.
2.
3.
4.
5.

Hydrochloric acid, 1 M.
Sodium hydroxide, 0.5 M.
Dowex 50 (W) (Dow Co., Midland, MI).
Polyclar AT resin.
Sephadex G-25.

Method
Pretreat the soil for 16 h with 1 M HCI at 20C, then extract the soil twice
for 16 h with 0.5 M NaOH at 20C. Pass the extracts upwards through a column
containing Dowex 50 (H+) to remove the humic acids and then concentrate the
eluates in vacuo at 45C. Pass the eluates through Polyclar AT to remove the
residual coloured materials. Remove the salts and low molecular weight material by separation in a column of Sephadex G-25.

SWIFT

1024

Comments
The cation exchange resin in the H+ form was used instead of precipitating
the humic acid in acid solution, as it overcomes the problem of coprecipitation of
the polysaccharides. New resins, such as XAD-4 (polystyrene divinylbenzene)
can be used in place of Polyclar AT.

FRACTIONATION OF SOIL ORGANIC MATTER

Fractionation of Humic Substances
Because of the complexity of structure and interactions of soil humic substances, the physical and chemical properties of these natural organic mixtures
are difficult to define precisely. In order to simplify the study of humic substances, a variety of techniques have been developed to fractionate samples into
distinctive and hopefully less complex parts. Fractionating a sample of humic
substances does not result in pure homogeneous compounds but rather fractions
in which one or more of the physical or chemical properties has a narrower range
of values than the original sample.
The particular method chosen for the fractionation process will depend on
the chemical and/or physical characteristics being studied. Commonly used fractionation procedures are based on characteristics such as differences in solubility, molecular size and electrostatic charge of the molecules within the system
(Swift, 1985). Some of the techniques can be classed as preparative as they result
in fraction samples of sufficient size to be used in further study (e.g., those based
on solubility and separation according to molecular size). Other fractionation
techniques, such as some of those based on charge characteristics (e.g., electrofocusing) are more appropriate for fingerprinting. Such techniques are able to
characterize a sample for purposes of comparison with other samples chemicals,
but do not easily produce sufficient sample for further study.

Fractionation Based on Solubility

The solubility of humic substances is not only dependent on the pH of the
solution, but is also dependent on the type and concentration of the cations and
other solutes present, and on the nature of the solvent system. Theoretically, all
of these properties could be used to fractionate a humic substance sample.

Use of pH. Precipitation using changes in pH is the basis of the humic

acidlfulvic acid fractionation (Fig. 35-1). The procedure can be refined by using
smaller changes in pH to obtain more narrowly defined fractions (Flaig et aI.,
1975). Unfortunately, it is difficult to obtain fractions with limited overlap due to
significant coprecipitation which occurs in the experimental procedure. However, because of the variety of structural and functional groups within a sample of
humic substance, and of the variety of interactions between these groups, fractionation of humic substances on this basis is unlikely to yield subfractions with
substantially different chemical characteristics.

ORGANIC MATI'ER CHARACTERIZATION

1025

FRACTIONATION OF SOIL HUMIC SUBSTANCES

e.g. recognizable plant debris;
plus polysaccharides, proteins.
lignins, etc. in their natural or
transformed states.

fractionation on the

basis of jOIUbility

soluble in acid
soluble in alkali

insoluble in acid
soluble in alkali

insoluble in acid
insoluble in alkali

~
....I [ f - - - - - Decreasing molecular weight - - - - .....f - - - - - - Decreasing carbon content - - - - ......f - - - - - - Increasing oxygen content - - - - ....
I [ f - - - - - Increasing acidity and CEC - - - - I(

Decreasing nitrogen content

.....! - - - - - Decreasing resemblance to lignin - - - - -

Fig. 35-1. Diagram showing the categorisation of soil organic matter into humic and nonhumic substances, the fractionation of the humic substances and the property variations within these fractions.

A variation in using the range in solubility as a means of obtaining fractions

is to sequentially extract a soil sample using a range of solutions of increasing pH,
and ability to solubilize the humic substances present in the sample. This type of
extraction is discussed in "Sequential Extraction."
Salting Out. Increasing the salt concentration of a humic substance solution decreases the intramolecular charge repulsion and causes the polyelectrolyte
macromolecules to shrink and to exclude the solvent. Simultaneously, increasing
the salt concentration causes a decrease in the extension of the diffuse double
layer of charge, thereby decreasing intermolecular charge repulsion allowing
molecules to approach each other more closely, and hence to coagulate. It is possible to fractionate humic substances based on this behaviour (Theng et aI., 1968)
as outlined below.

Materials
1. Sodium hydroxide, 1.0 M.
2. Ammonium sulfate [(NH4)2S04], solid.
3. Sulfuric acid (H2S04), 1.0 M.

Method
Prepare 150 mL of 0.33% K+ -humate solution. Add 1.0 g of solid
(NH4)2S04, readjust the pH to seven using 1 M NaOH and shake for 5 to 6 h. Separate the precipitate by centrifugation and shake the solution overnight without

SWIFT

1026

adding more salt. Centrifuge the suspension and combine the precipitates and
retain.
Add successive increments of 1.0 g of (NH4)zS04 and repeat the procedure
until the amount of precipitate obtained after centrifugation is negligible. For
each fraction obtained from the salting out procedure, dissolve the precipitate in
distilled water or dilute alkali, adjust the pH to one using 1 M H2S04 and then
remove the excess salt by exhaustive dialysis. Freeze dry the product and weigh.
Commen~ther Precipitation

Methods

The interaction between heavy metal ions and humic substances in solution
can affect the solubility of the humic substances. Using the insolubility of the
metal-humic substances complex is one way of isolating that fraction of humic
substance which interacts with the metal concerned. MacCarthy and O'Cinneide
(1974a) used this method to study complexing of humic substances with both Cu
and Co under both acidic and alkaline conditions. Metal-humate interactions are
of interest agriculturally as they may influence the availability of trace metals in
soiVplant systems. The ability of humic substances to interact with heavy metals
is also of great interest environmentally with respect to the transport of heavy
metals through soiVwater systems and, hence, the fate of toxic metals in soils and
ground waters.
Fractionation on the basis of metal-humate solubility, and that based on
salting out, are not used regularly as they are rather tedious and the fractions are
ill-dermed due to problems of coprecipitation.
Use of Organic Solvents. Humic substances have relatively low solubility
in many conventional organic solvents which can be used as a method of fractionation (Hayes, 1985). Historically, ethanol was used to extract the hymatomelanic acid fraction from precipitated humic acid (Stevenson, 1994), but it can also
be used to fractionally precipitate alkaline solutions of humic acid (Kyuma, 1964;
Kumada & Kawamura, 1968). The method below is based on that of Kumada and
Kawamura (1968). Other water-miscible organic solvents, such as acetone
[(CH3)zCO) and methanol (CH30H), may be used in a similar way to obtain fractions of humic substances.

Materials
1.
2.
3.
4.

Sodium hydroxide, 0.1 M, 0.2M.

Ethanol (C2HsOH), absolute.
Hydrochloric acid, 1:3.
Sulfuric acid, concentrated.

Method
Dissolve 150 mg of humic acid in 20 mL of 0.2 M NaOH. Add absolute
ethanol (~HsOH) to give a concentration of 10% (v/v), allow to stand overnight
and then centrifuge the suspension at 7000 rpm for 20 min. Dissolve the precipitate in 0.2 M NaOH, add ethanol to the same concentration as above and recover
the precipitate by centrifugation.

ORGANIC MAITER CHARACTERIZATION

1027

Add ethanol to the combined supernatant to give an ethanol concentration

of 20% and repeat the above procedure. Continue the process by increasing the
alcohol concentration in steps of 10% until the fmal concentration is 80% ethanol
and eight fractionated precipitates have been obtained.
Dissolve each precipitate in 0.1 M NaOH, precipitate by adding HCI (1:3)
and then wash the precipitate with ether (C4HlOO) over a glass-filter. Dry the precipitates in a desiccator containing concentrated H2S04 for 2 d and weigh the
products.
Reduce the concentration of alcohol in the 80% ethanol soluble humic fraction by distillation in a water bath, precipitate, wash and dry this fraction as
described above for the other fractions.
Comments-Use of Other Organic Solvents

Generally, extracting fractions using water-immiscible solvents gives very

low yields. However, Rice and MacCarthy (1989) have achieved some success
using a different approach with the polar water-immiscible solvent, methyl
isobutyl ketone [CH3COCH2CH(CH3)2] (MIBK). They initially separated the
aqueous fulvic acid fraction from the nonaqueous huminlhumic acid fraction at
low pH using HCI to acidify the solution. Following this the pH of the nonaqueous phase was made alkaline using NaOH allowing the separation of the humic
acid in the aqueous phase from the humin associated with the MIBK nonaqueous
phase. Rice and MacCarthy (1989) were then able to fractionate the humin residue into an acid soluble form and aqueous and nonaqueous phase products. The
study of the humin fraction of the soil is a neglected but important aspect of soil
research, and this technique provides an opportunity to study the humin fraction.
Adsorption. The desorption behaviour of humic substances sorbed to a gel
(Swincer et al., 1968), resin (Yonebayashi & Hattori, 1990), charcoal (Forsyth,
1947) or alumina (Dragunov & Murzakov, 1970), can be used as a means of fractionation. The purification of the fulvic acid fraction using resins such as XAD8 or PVP during the extraction of humic substances ("Extraction with Aqueous
Solutions") is essentially an adsorption/desorption fractionation procedure.
Materials
1. Sodium hydroxide, 0.1 M, 10 M.
2. Ethanol, 50% (v/v).
3. Universal buffer, 0.02 M.
4. Sulfuric acid,S M.
5. XAD-8 resin.
6. Amberlite IR-120 resin (Rohm & Haas Co., Philadelphia, PA).
Method

Dissolve the humic acid sample in 0.1 M NaOH and treat with Amberlite
IR-120 resin to convert it to the H+-exchanged form. Dissolve 5 mg of the H+-saturated humic acid in 2 mL of aqueous solution and load onto a column packed
with XAD-8 resin. Elute the humic acid in a stepwise fashion with universal
buffer solutions adjusted to pH =7 and pH =11, distilled water and 50% ethanol.

1028

SWIFf

Determine the elution profile by first adjusting the pH of the effluent to pH = 12

using 10 M NaOH and then measuring the optical density at 400 nm. Precipitate
the humic acid contained in each effluent using sulfuric acid and then dissolve it
in 0.1 M NaOH. If the humic acid fraction does not precipitate on acidification,
absorb it onto a small column of XAD-8 at pH = 3 and elute with NaOH solution.
Dialyze each of the eluates against distilled water and freeze dry.

Comments
It is possible to refine this method by using a greater range of buffers to separate the humic acid into a larger number of fractions. Yonebayashi and Hattori
(1990) extended the method by using both a pH gradient and a water-ethanol gradient to allow for the ability of obtaining a greater number of fractions if required.
MacCarthy et al. (1979) used a similar method to fractionate a sample of humic
substance. Instead of using buffers a pH gradient solution was generated by eluting simultaneously from two pumps, one containing 0.1 M H3P04 and the other
0.1 M NaOH. MacCarthy et al. (1979) separated out only two fractions, but it
would be possible to adapt the method to obtain more fractions, depending on the
characteristics of the humic substance and the nature of the pH gradient used.
Fractionation Based on Molecular Size
The extreme range in molecular weights associated with humic substances
extracted from soils should theoretically allow the separation of the sample into
many specific fractions. In reality the complex intermolecular associations make
it difficult to obtain fractions with insignificant overlap in molecular content.
Nevertheless, fractionation of humic substances on the basis of molecular weight
is a powerful and attractive procedure. Gel permeation chromatography and ultrafiltration are techniques which have been used successfully with proteins and carbohydrates for fractionations based on molecular size. Reasonable success has
been achieved in using these rapid and reliable techniques to purify and fractionate humic substances.
Gel Permeation Chromatography. The gels used for molecular size
chromatography have structures consisting of a system of pores, the sizes of
which are determined by the degree of cross-linking in the polymer. When a solution of humic substance is applied to the top of a gel column and eluted with solvent, large molecules which are unable to enter the pores in the beads will bypass
the beads and will be eluted first from the column. Smaller molecules which are
able to enter the pores will have their passage through the column retarded, the
extent of which will depend on the actual size and shape of the molecule. The net
result is that the solute molecules are eluted from the column in order of decreasing molecular size, and, for a given family of macromolecules, in order of
decreasing molecular weight also.
The range of molecular sizes over which the gel is able to differentiate the
molecules will depend on the type of gel chosen, but gels are available which are
able to produce fractions in the range of several thousands to millions of daltons.
Gels are selected on the basis of the exclusion limit of the gel and the molecular
weight range over which the gel is suitable. Given the extreme polydispersity of
humic substances, it is generally necessary to use a range of gels to achieve sat-

ORGANIC MATTER CHARACTERIZATION

1029

isfactory fractionation. For discussion on the calibration of gels, see "Gel Chromatography."
It is important to select gels that are inert to the solute molecules so that
there are no chemical or physical interactions between gel and solute. Otherwise
the resulting separation cannot be entirely attributed to molecular size and/or
weight differences. Swift and Posner (1971) discussed these problems fully and
show that they can be largely overcome by careful selection of the gel matrix and
by the use of appropriate buffer solutions. By successively using gels of various
exclusion limits and reapplying the excluded or included portion of a given gel to
another with a higher or lower exclusion limit it is possible to successfully separate out fractions (Schnitzer & Skinner 1968, p. 41-SS). The method outlined
below is that used by Swift et ai. (1992).

Materials
1. Sodium tetraborate, (Na2B407) 1% w/v, pH = 8.S.
2. Sephadex G-7S, G-1S0.
3. Sephrose 6B (Pharmacia, Uppsala, Sweden).

Method
Dissolve the sample of humic acid in a borate buffer (1 % w/v, pH = 8.S)
and run SO mL through Sephadex G-1S0 in a preparative (S-cm diam.) column to
fractionate the sample with respect to molecular weight. Collect the eluate as 10mL fractions and read the absorbance of these at 400 nm.
Repeat the above procedure four times and divide the eluate into four parts
corresponding to four regions of the elution curve, namely the excluded peak (A)
and three segments of the broad peak of lower molecular weights (E, C, D) (Fig.
3S-2). Dialyze and freeze dry each of the four combined eluates.
Redissolve the four fractions using borate buffer and then fractionate them
using Sepharose 6B, Sephadex G-1S0 or G-7S gels for fractions high to low molecular weight, respectively.
Refine the fractions by taking central cuts of the resulting four individual
peaks (shaded section, Fig. 3S-2) and dialyze these against distilled water
exhaustively before freeze drying.

Comments
The volume of the excluded fraction (void volume) can be determined by
using the nonadsorbing chemical, Blue Dextran 2000 (Pharmacia, Uppsala, Sweden). To determine the total effective column volume of Sephadex gels use N-2,4dinitrophenyl aspartic acid (a yellow compound), sucrose (C 12H220 11 ), or glucose
(C6H 120 6) as the low molecular weight marker. Except for gels to which it is
reversibly adsorbed (e.g., Sephadex gels) the sodium salt of 2,6-dichlorophenol
indo-phenol (a blue dye) can also be used to determine the total effective volume
of the column.
Typical elution patterns obtained by proper application of gel permeation
chromatography (e.g., Dubach et aI., 1964; Swift & Posner, 1971) should be consulted. The elution pattern shown in Fig. 3S-2, using borate or tris buffer is an
example of the type of curve that should be obtained.

SWIFf

1030
Primary fractionation (Sephadex G-150)

:, SecondarY,!ractionations".
,

Sepharose 66 : Sephadex G-150: Sephadex G-150 : Sephadex G-75

Vt Vo

Vt Va

Elution volume

Fig. 35-2. Composite diagram showing initial elution pattern (absorbance at 400 nm vs. volume eluted) for whole humic acid (upper section of diagram) and patterns for the subsequent runs of the separate fractions, FI, F2, F3 and F4 (lower section of diagram). The shaded areas of the secondary
elution patterns represent the "central cuts" taken to obtain the final secondary fractions.

Ultrafiltration. In recent years a range of polymer-based membrane filters

have been developed with pore sizes ranging from several micrometers to a few
nanometers_ The nominal molecular weight cut-off values of these range from
700 to 1 000 000 daltons and, theoretically, these membrane filters should be able
to be used to fractionate the polydisperse humic substances according to molecular weight. However, it has been observed that charge-charge interactions
between the solute and the membrane surface may interfere with the filtration
process and humic acids are highly charged. This fact together with the difficulties encountered during the manufacture of the filters and the unresolved doubts
about the relationship between molecular size and molecular weight for humic
substances, create some problems concerning the overall usefulness of the results
when studying humic substances.
Ultrafiltration is commonly used as a purification procedure in which the
membrane is used to separate the wanted fraction from the unwanted fraction. It
is also used in conjunction with other methods of fractionation, such as gel permeation and ultracentrifugation to increase the flexibility of the fractionation procedure (Cameron et aI., 1972a). The method below has been used to study the
complexing capacity of humic substances in natural waters (Smith, 1976). It is
possible to alter the method by changing the membrane types to alter the molecular size range of the fractions, and by incorporating a greater number of membranes to increase the number of fractions. The membrane selection will have to

ORGANIC MATIER CHARACTERIZATION

1031

include some with higher molecular weight cut -off values for use with soil humic
substances. It is advisable to begin the ultrafiltration using the membrane of highest cut-off value and work downwards in size in order to limit clogging of the
filters.

Materials
1. Ultrafiltration membranes, XM-l00 (Amicon, Beverly, MA), XM-50
(Amicon, Beverly, MA), PM-lO (Amicon, Beverly, MA), YM-2 (Amicon, Beverly, MA), with molecular weight cut-offs 100 x 103, 50 X 103,
10 X 103 , 1 X 103 daltons respectively.
2. Ultrafiltration cell.

Method
Flush the ultrafiltration cell and membrane with double distilled water until
the absorbance of the filtrate solution records zero magnitude [using ultraviolet
(UV)-visible spectroanalysis]. Introduce a sample of known volume (e.g., 400
mL) into the cell and pressurize the system with N2. Collect the ultrafiltrate and
retain in a preweighed flask. When the sample has been concentrated to approximately 20 mL, depressurize the system and pour the concentrate into a preweighed flask. Determine the volume of this fraction by gravimetry assuming the
specific gravity of 1.0.
Sequentially fractionate the ultrafiltrate from the above step using membranes with the next lower molecular weight cut-off until that having the smallest weight cut-off has been used. Determine the volume of the ultrafiltrate.
The above fractions could be further refined by repeating the ultrafiltration
using the membrane associated with their retention. Dialyze the fractions against
distilled water and freeze dry. Compute the mass per volume of the initial ultrafiltrate to determine the relative fraction of the original mass in the sample.
Fractionation Based on Charge Characteristics
The charge characteristics of humic substances in solution, originate predominately from the presence of carboxylic acid, phenolic and enolic functional
groups. The amount of charge relates to the degree of dissociation of these functional groups which is a function of the pH of the solution and the identity of the
counterions in the surrounding medium. By taking advantage of the differences
in charge density within a sample of humic substances it is possible to fractionate them according to their charge characteristics.
Electrophoresis. Electrophoresis is the movement of charged species in a
solution in response to an applied electrical potential. Traditionally, electrophoresis was carried out in free solution so that movement of the species was
able to be related to both charge and size of a molecule or ion. By carrying out
the process in a gel medium the movement of the charged species can be retarded according to their size and the size of the pores in the gel. In this way the fractionation of humic substances can be related to both the charge and molecular size
of the species. Experimental conditions can be modified by altering the buffer or
the characteristics of the gel, to optimize the fractionation obtained.

SWIFf

1032

Gel electrophoresis can be used as both a preparative fractionation technique, and as a means of characterizing humic substances. A preparative electrophoretic method (De Nobili et al., 1990a) which produces fractionated humic
material able to be used in other analyses has been outlined below.

Materials

1. Potassium dihydrogen phosphate buffer (KH2P04) 0.02 M pH = 7.0.

2.
3.
4.
5.

Glycerol (C3Hg03).
Polyacrylamide gel.
Sodium pyrophosphate, 0.1 M.
Poly(vinylpyrrolidone).

Method
Prepare the gel rods by casting three different overlayed gels of decreasing
acrylamide concentration (9, 7, and 5%). Prepare a sample containing 15 mglmL
organic C, mix it with glycerol, and then filter the solution through a 0.2-llm
membrane filter before application to avoid blocking the pores at the entrance to
the gel. Perform the electrophoresis in 0.02 M phosphate buffer (PH = 7.0) using
15 rnA per rod (care must be taken to ensure that the gel in the rods does not
become overheated.
Stop the run when the front of the migrating band reaches the bottom of the
9% gel segment. Immediately after the run take 2-cm long slices of the front fraction, the center fraction and the tail fraction of the migrating band of humic substances. Discard the intermediate sections. If doing the fractionation in duplicate,
mince the corresponding slices and extract them three times with 0.1 MNa4PZ07.
Concentrate the fractions on PVP columns. These fractions are suitable to be used
in the following electrofocusing analysis or can be dialyzed and freeze dried and
prepared for some other analyses.

Comments
The equipment used by De Nobili et al. (1990a) consisted of 13 x 115 mm
polyacrylamide gel rods and a Bio-Rad 155 Electrophoresis cell, powered with
an LKB 2117 power supply (LKB, Bromma, Sweden) and cooled with water circulating at 4C. Although presented as a preparative method, it should be noted
that the amounts of material obtained by this procedures are very small.
The technique of electrophoresis has been refined by incorporating a pH
gradient into the gel system. The charged species migrate until they reach the
position corresponding to their isoelectric point, a pH at which they cease to be
charged, in a process called electrofocusing. In this method, the gel has relatively large pores so that molecular size is not involved in the fractionation.
In electrofocusing t as described by De Nobili (1988), a pH gradient is set
up by the use of a mixture of compounds which are good buffers and able to act
as ampholytes in the separation medium. The range in pI (isoelectric point) values of the ampholytes in the gel must be between the pH of the electrolytes in the
anode and cathode compartments. The most acidic ampholyte (lowest pI) moves
to occupy a position closest to the anode and correspondingly the most basic ampholyte occupies the space closest to the cathode (highest pI), with intermediate

ORGANIC MATTER CHARACTERIZATION

1033

ampholytes moving to occupy spaces arranged in order of their decreasing acidity in between these two.
Electrofocusing is a potentially useful technique for characterizing humic
substances. However, a number of operational aspects indicate that any conclusions formed from using this technique should only be tentative (Duxbury, 1989).
Duxbury believes that the process is not electrofocusing but is electrophoresis in
a pH gradient. Some of the reasons for this are that humic molecules are not generally amphoteric and once near their isoelectric point tend to lose their charge
and may not even remain in solution. Those that diffuse past their pI are no longer
charged and so are not focused back to that point, i.e., true electrofocusing does
not occur.
Other problems which potentially may be associated with the process are
the interactions of humic molecules with the ampholines and the aggregation tendencies of humic substances (Duxbury, 1989).
Fractionation of Polysaccharides
The polysaccharide fraction of soil organic matter will be made up of a
mixture of components of varying molecular weight, charge density and monosaccharide composition. Because of the similarity in some of the physical and
chemical characteristics between polysaccharides and humic substances many of
the inethods used for fractionation of humic substances can be applied to the fractionation of polysaccharides, such as fractional precipitation (Parsons & Tinsley,
1961), electrophoresis (Mortensen & Schwendinger, 1963; Barker et aI., 1965),
density fractionation (Strickland & Sollins, 1987), ion-exchange chromatography
(Finch et aI., 1966; Thomas et aI., 1967) and gel filtration (Barker et aI., 1965,
1967; Swincer et aI., 1968). The last two methods are those most commonly used.
lon-Exchange Chromatography
The method below is based on that of Barker et ai. (1967). Initially, a pH
gradient elution is carried out to determine the elution characteristics of the particular polysaccharide sample. From this information, it should be possible to differentiate logical fractions within the sample and the salt concentration associated with each of these fractions. The elution is then repeated using a fresh sample
of polysaccharide, and solutions of specific salt concentrations in a buffer to separate the fractions. In this way the sample is fractionated on the basis of increasing charge density.

Materials
1. Sodium chloride, 2 M
2. Phosphate buffer (0.0371 M KH 2P0 4, 0.0043 M Na2HP04), pH = 6
3. DEAE-cellulose

Method
Prepare a column (3.4 by 45 cm) of DEAE-cellulose and carry out a gradient elution with 0 to 2 M NaCl in phosphate buffer. Monitor the elution profile by
collecting lO-mL fractions and analyzing these for sugars (Dubois et aI., 1956),

SWIFT

1034

and absorbance at 400 nm. Determine the number of fractions to be eluted and
the NaCI concentration at which their elution is maximized. For each fraction
selected, prepare 600-mL portions of buffer containing the maximum NaCl concentration associated with that fraction. This preliminary run can be carried out
using a smaller amount of polysaccharide than that used in the main preparative
experiment that follows.
Equilibrate the column with the phosphate buffer. Dissolve 200 mg of polysaccharide in 50 mL of phosphate buffer, apply it to the column and elute it successively with the 600-mL portions of buffer prepared as above, using increasing
concentrations of NaCl. Collect lO-mL fractions of the eluate and analyze as
above. Combine the polysaccharide-containing fractions for each concentration
of the salt, dialyze against distilled water and freeze dry.
Gel Filtration
This method is similar to the previous method, except that the separation is
on the basis of size, rather than charge density.
Materials

1. Sodium chloride (NaCl), 1 M.

2. Sephadex G series gels.
3. Biogel P series gels.
Method

Pretreat the gel and equilibrate the column using 1 M NaCI to eliminate ionic and adsorption effects. Load the column (54 by 3.1 cm) with 3 mL of solution
containing 200 to 300 Ilg polysaccharide/mL. Elute the column with 1 M NaCl,
collect 5-mL fractions and analyze these using absorption measurements. Determine the amount of polysaccharide (Dubois et aI., 1956) as a function of the
elution volume and separate and combine fractions as outlined in "Gel Permeation Chromatography." Suitable gels for polysaccharide analysis are the Sephadex G series (Pharmacia, Uppsala, Sweden) and Biogel P series (Calbiochem,
Los Angeles).
Density Fractionation of Soil Organic Matter
When soil organic matter becomes intimately associated with soil mineral
particles, the density of the resulting combination is greater than that of the organic matter alone. By fractionating the soil on the basis of the density of the component particles it is possible to separate the organic matter into fractions of different physical characteristics and almost certainly different chemical behavior,
based on their degree of decomposition and association with mineral particles.
Density fractionation avoids the need for solvent extraction and decreases the
possibility of artifact formation. It also is believed that this type of fractionation
may partially separate the soil organic matter according to age, with the youngest
fraction being the lightest.

ORGANIC MA'ITER CHARACTERIZATION

1035

A number of methods have been developed to achieve this type of fractionation. These involve the separation of the soil particles on the basis of their
density in water (Turchenek & Oades 1979), and in organic liquids or salt solutions of high specific gravity (Greenland & Ford 1964; Sollins et aI., 1984;
Turchenek & Oades, 1979; Dalal & Mayer, 1986, Roth et aI., 1992; Baldock et
aI., 1990). By combining particle size fractionation (e.g., sieving, sedimentation,
and continuous flow centrifugation) with density fractionation, it is possible to
separate the organic matter in the soil into multiple fractions (Turchenek &
Oades, 1979; Oades et aI., 1987; Ducaroir et aI., 1990).
Initially, the sample is ultrasonified to break up all the aggregates and to
thoroughly disperse all the colloidal particles. The sample is then sieved, and/or
separated on the basis of settling time in water, before fractionating these resultant fractions on the basis of their density in a high specific gravity solution. The
method below is based on that of Baldock et ai. (1990).

Materials
1. Poly tungstate solution (Na3 W0 4 .9W03.H20).

Method
Add approximately 20 g of soil to 50 mL of deionized water in a 150-mL
beaker and sonicate at constant temperature and at a medium output for 5 min.
Pass the dispersed sample through a 53-J..lm sieve and then separate the sieved
fraction into >2-J..lm and ~2-J..lm fractions using gravitational separation in deionized water. Dialyze the ~2-J..lm fraction against deionized water and freeze dry.
Combine the >53-J..lm and the >2-J..lm fractions and separate these into a
light and heavy fraction by centrifugation in a sodium poly tungstate solution of
density 2.0 g cm-3. Separate the light fraction from the supernatant by filtration
using a screen with 5-llm mesh.
Resuspend the sediment in the poly tungstate solution, centrifuge and
remove and filter the supernatant as before. Repeat this process until the supernatant is clear, then wash the combined light fractions and dry at 45C. Wash the
heavy fraction five times with deionized water and then dry it at 45C.
This method gives three fractions; clay fraction (~2-J..lm diam. particles),
light fraction (>5-J..lm diam. particles with a density of ~2.0 g cm-3) and heavy
fraction (>2-J..lm diam. particles with a density >2.0 g cm-3). The method could
be extended to achieve a greater number of fractions by using different density
solutions (e.g., 1.6 g cm-3).

Comment
The earlier methods involving the use of dense organic liquids (Greenland
& Ford, 1964) are not generally used nowadays, due to the health, safety and
environmental problems associated with the use of halogenated hydrocarbons.
Freeze drying, which is unlikely to alter the composition of the fractions, may be
preferable to heat drying.

SWIFf

1036

CHARACTERIZATION OF HUMIC SUBSTANCES

Many of the chemical, physical, and spectroscopic methods already successfully used in general organic chemistry, and in the study of naturally occurring macromolecules have been applied to the study of soil humic substances to
try to determine the composition and general structure of the component macromolecules. However, because of the heterogeneous and polydisperse nature of
humic substances, and the complexity of the inter- and intramolecular reactions,
it is often difficult to interpret the results of these studies.
Because of the diverse nature of humic substances and the lack of any obvious means of referencing data, it is difficult to quantify the characteristics of these
samples. A set of standard and reference samples have been prepared by the
IHSS to assist in the comparison of humic substances within and between laboratories, and these are available for purchase from IHSS.
Characterization by Chemical Methods
The three general characteristics of a chemical compound are the elemental composition, the arrangement of these elements in the chemical structure, and
the types and locations of the functional groups in the structure.
Procedures to determine the type and relative abundance of the elements
within an organic compound are well defined and measurable experimentally.
Such procedures are not dealt with in this chapter. Because of their relatively
greater activity, it is usually far easier to determine the presence of the various
functional groups in large organic compounds than it is to determine the arrangements of the elements within the structures. However, once the elemental composition and the functional group characteristics are determined, and an estimation of the molecular weight has been made (see "Molecular Size and Shape"), it
should be possible to make an assessment of the type of structures of which
humic substances are composed.
Using general methods of organic analysis, it has been established that the
major functional groups in humic substances are the carboxylic acids (and other
groups containing the C=O functionality), phenols and alcohols, with Nand S
located in the minor functional groups. To some extent, the accuracy of these
analyses are open to question, but so long as they are reproducible they allow
comparisons to be made of humic substances from different sources.
Acidic Functional Group Concentrations
The acidic behaviour of humic substances is such that they are considered
to be a mixture of stronger (mostly carboxylic acids) and weaker organic acids
(mostly phenolic acids). The pKa of these two types of acidic groups are around
pH = 4.5 and pH = 10 respectively (Christensen et aI., 1976; Martell & Smith,
1977). Identical functional groups in a humic substance may not have identical
pKa values (Perdue, 1985). The actual pKa of an acid is not only dependent on the
nature of the acidic functional group, but also is dependent on the chemical environment in which it exists, as well as the nature of the compound to which it is
bound. There would be considerable overlap in the range of pKa values of the

ORGANIC MATIER CHARACTERIZATION

1037

acidic groups in a solution of humic substances, and the overall range of all of
these values could span the pH range from 0 to 13 (Perdue, 1985). On the basis
of pKa measurements, it would not be possible to distinguish unequivocally carboxylic acids from other types of acids in humic substances (Perdue, 1985).
A number of different types of methods for the analysis of acids have been
used to determine the acidic functional groups of humic acids (Stevensen, 1994;
Perdue, 1978; Schnitzer & Khan, 1972). These include direct and indirect titrations, as well as nonaqueous and thermometric titrations.

Total Acidity. The total acidity of a sample should include all the acidic
hydrogens present. Thus determination of total acidity would require the use of a
basic reagent at high pH so that even the weakest acids present would be accounted for. This type of method has several sources of error. First, the volume of base
required to reach equilibrium at high pH values is relatively large compared with
the amount required for reaction with the humic acid. Second, it is necessary to
carry out the titration rapidly to limit the possibility of errors arising from any
base-catalysed side reactions (Perdue, 1985). The results of the analysis are also
dependent on the conditions in solution (e.g., properties of the pH electrode, composition of the background electrolyte, etc.). Because of the foregoing considerations, the determination of the total acidity of a humic substance is a good approximation only.

Direct TItrations. There is a tendency for the pH of the solution to decrease

with time under alkaline conditions, and this effect can be considerably reduced
by carrying out the titration in the absence of O2 , This drift in pH is thought to be
due to the production of acids from side reactions associated with the presence of
the alkali (Swift & Posner, 1972). The titration can be carried either in the forward direction (i.e., against a dilute base) or in the reverse direction (i.e., against
a dilute acid). As humic substances tend to be insoluble in acid solution, it may
be difficult to achieve dissolution of the solid at moderately acidic pH values. For
this reason the reverse titration, which involves dissolving a known amount \ ,f
humic acid in KOH and titrating with dilute acid, is more rapid but the error associated with titrating in the high pH region (see above) is carried through the
whole titration.
The method below is for the forward titration (Swift & Posner, 1972) but
can easily be adapted to the reverse titration.
Materials

1. W -Dowex 50W resin.

2. Potassium chloride (KCI), 0.01 M.
3. Potassium hydroxide, 0.1 M.
Method
Prepare a solution of humic substance for titration by treating it with an
excess of H+ -Dowex 50W resin. Prepare a solution containing 5 mg of the humic
substances in 20 mL of 0.01 M KCI and titrate it against standardized 0.1 M KOH
to pH =7.0 (arbitrary end-point). Add the KOH stepwise (e.g., 50 steps) as rapidly as possible but still allowing for the solution to reach equilibrium before each

SWIFr

1038

pH reading. Repeat the procedure using a blank solution. Construct a plot of pH

vs. titre volume for each solution to determine the acidity of the humic acid as a
function of pH. All solutions should be CO2 free and the titration should be carried out under N2 at constant temperature.
Total acidity (mmolc g-l)

=(Vsample -

VblanJ X MbaselWsample(g)

where V blank and Vsample are the titre volumes (mL) associated with the blank and
sample titrations, respectively, and Wsample (g) is the mass of the humic substance
used in the experiment. (Note, mmolc designated millimole of charge.)
Comment

During the titration, the solution may not reach equilibrium in a suitably
short period of time. In this event, choose a set period of time between successive
additions of alkali or a set time over which the pH is relatively constant, as the
time at which the pH reading is taken.
Indirect titrations. The humic substance is allowed to equilibrate in an
excess of Ba(OH)2' before the unused alkali is titrated with a standard acid. The
total acidity of the humic substance is determined by calculating the difference in
titre volume with respect to pH between an analysis including the humic substance and a blank analysis under the same conditions. This method has been regularly used as a means of determining the total acidity of a humic substance and
as it involves a solution of pH > 13, it is considered that the result would be a reasonable estimation of the total acidity of the sample.
Traditionally, the method used has not specified the type of filter paper to
remove the barium salts, and it has been found (Davis, 1982) that their occurrence
in the titration solution would give an underestimation of the total acidity value.
The method below is based on that of Schnitzer and Khan (1972) with the
modifications as suggested by Perdue (1985).
Materials
1. Barium hydroxide (Ba(OH)2), 0.1 M.
2. Hydrochloric acid, 0.5 M.

Method

Place between 50 to 100 mg of humic substance in the W-form in a 125mL ground-glass stoppered Erlenmeyer flask. Add 20 mL of 0.1 M Ba(OH)2 to
the flask containing the humic substance and the same amount to a similar flask
in which there is no humic substance, to act as a blank. Displace the air in each
of the flasks with N2, stopper, and then shake for 24 h at room temperature. Filter the solutions through 0.45-Jlm membrane, then wash the residues thoroughly
with CO2 -free distilled water. Using standardized 0.5 M HCI and with the aid of

a pH meter, titrate the filtrate plus washings of both the sample and blank solutions to pH =8.4. Determine the total acidity as described above for direct titrations, using the following equation.
Total acidity (mmolc g-l)

=(Vblank - Vsample) X MaciJWsample(g)

ORGANIC MATTER CHARACTERIZATION

1039

Stronger Organic Acids (Carboxyl Groups). As there are no distinctive

equivalence points in the titration curves of humic substances, the determination
of the pH at which a specified group of acids is neutralized is somewhat arbitrary.
Because weak acids by definition must have a pKa above pH = 7.0 this value has
sometimes been used as means of differentiating the acidity associated with weak
and strong organic acids (Gamble, 1972; Burch et aI., 1978). Another more definitive method of delineation is to construct a first derivative curve of titre volume
vs. pH to determine the maximum change in pH with change in titre volume. The
volume below this point is then taken to be that associated with carboxyl groups
and that above due to the phenolic groups and other weak acids.
Direct Titrations. The method detailed below is that of Oliver et al. (1983)
and is carried out on the microscale. It is possible to scale all the apparatus upward in order to carry out the procedure using normal-sized measures and glassware. The results of this analysis give an operational estimation of the acidity
associated with strong organic acids and hence carboxylic acids (Perdue, 1985).
As for total acidity titrations, this method can be adapted to be carried out in the
reverse direction.

Materials
1. Sodium hydroxide, 0.1 M.

Method
Suspend 10 mg of humic acid (H form) in 2 mL of distilled water and
titrate the solution to pH = 7.0 with 0.1 M NaOH in 50-ilL increments. Carry out
a similar titration in the absence of humic substances.
COOH acidity (mmolc g-l) - (Vsample - Vblank)

MbasJWsample(g)

Indirect Titrations. The most common method used to determine the acidity associated with the stronger organic acids is the calcium acetate method in
which the amount of acetic acid generated from the reaction of calcium acetate
with humic substance is determined by titration with standard sodium hydroxide
solution.
There are a number of operational problems associated with this method.
Because of poor buffering in the solution, the equilibrium pH of the solution is
determined by the amount of humic acid added. Complexation of Ca 2+ with the
humic acid increases the release of protons to solution increasing the apparent
acidity (Perdue et aI., 1980). As in the barium hydroxide method above, it is also
important that the filtration step removes all solids from solution before titration.
This separation can be achieved by using fine grade filtration (Davis, 1982) or
ultrafiltration (Perdue et aI., 1980). The method below is based on that of Wright
and Schnitzer (1959) with the alteration to filtration as suggested by Perdue
(1985).

Materials
1. Calcium acetate [Ca(OAc)z], 0.5 M.
2. Sodium hydroxide, O. 1 M.

SWIFT

1040

Method
Place between 50 to 100 mg of humic substance in a 125-mL ground-glass
stoppered Erlenmeyer flask. Add 10 mL of 0.5 M Ca(OAc)2 and 40 mL of COzfree distilled water to the flask containing the humic substance and to a similar
flask in which there is no humic substance, to act as a blank. Shake the flasks for
24 h at room temperature, filter the solutions through 0.45-l1m membrane, and
then wash the residues thoroughly with CO 2-free distilled water. With the aid of
a pH meter, titrate the filtrate plus washings of the sample and blank solutions to
pH = 9.8 using standardized 0.1 M NaOH.
COOH acidity (mmole g-l) = (Vsample - Vb1ank)

MbasJWsample(g)

Comments
De Nobili et al. (1990b) has suggested an alternative method for the determination of the carboxylic acid content of humic substance based on their solubility in the presence of cetyltrimethylammonium (CfA+). The CfA+ is very efficient at quantitatively precipitating the humic substances in solutions, thus minimizing some of the problems in the calcium acetate method outlined by Perdue
(1985). The method has several other advantages including flexibility of pH for
the analysis and no apparent interference from the phenolic groups. However, the
procedure has not been widely tested and it is not possible to recommend it as a
general method at this stage.
Weaker Organic Acids (phenolic groups). The difference between the
total acidity of a humic substance and the acidity associated with the stronger
organic acids (carboxylic acids) is usually attributed to the phenolic groups, even
though other acids such as weak carboxyl groups, alcoholic groups and enols are
also probably involved. Before this assumption is accepted the structural implications of the values should be investigated for consistency (see "General Comments" below). (Note, mmolf designated millimole of functional groups.)
Phenolic-OH groups (mmolf g-l)
= total acidity (mmole g-l) - COOH acidity (mmole g-l)

Comment
It is also possible to determine the acidity characteristics of the humic substance using nonaqueous .titration methods (Stevenson, 1994) which tends to
enhance the strengths of the weak acids. A method using DMSO is detailed in
Yonebayashi and Hattori (1985) but other solvents which can also be used include
pyridine (CsHsN) and dimethyl formamide. These methods warrant more investigation than they have so far received.
Hydroxyl (OH) Groups
Total Hydroxyl (OH) Groups. The humic substances are acetylated with
acetic anhydride in pyridine. Acetyl groups are then hydrolyzed to acetic acid
(C2H40 2), which is distilled and titrated with standard base (Brooks et aI., 1958;
Schnitzer & Skinner, 1965).

ORGANIC MATTER CHARACTERIZATION

1041

Materials
1. Pyridine, 95% purity.
2. Acetic anhydride [(CH3CO)20] 97% purity.
3. Sodium hydroxide, 3 M, 0.1 M.
4. Sulfuric acid, 3 M.
5. Phenolphthalein, 0.5% in 95% ethyl alcohol.

Method
Using 5 mL of equal parts of pyridine and acetic anhydride reflux 50 to 100
mg of humic substance for 2 to 3 h under an atmosphere of N2. After cooling the
mixture, pour it into distilled water and collect the precipitate by filtration. Wash
the precipitate thoroughly with distilled water and dry under a vacuum in the
presence ofP20 s . Reflux this acetylated sample (50 mg) with 25 mL of 3 M aqueous NaOH solution for 2 h under N2. Cool the mixture, add 25 mL of 3 M H2S04
and 25 mL of distilled water and distill through a splash head.
Collect 25 mL of distillate and titrate with standardized 0.1 M NaOH using
phenolphthalein as the indicator. Add 25 mL portions of distilled water to the distillation mixture and continue the distillation. Repeat the collection of distillate,
titration and addition of 25-mL portions of the distillation mixture until the sample and reagent blanks titrate equally. Calculate the acetyl and hydroxyl contents
as follows:
Acetyl (mmolf g-l) = (Vsample - Vblank) X MbasJWsample(g)
Hydroxyl (mmolf g-l) = acetyl content/(1 - 0.042 x acetyl content)

Comments
The factor of 0.042 above is a consequence of the difference in molecular
weight of 42 between the ROH and CH3COOR. The carboxylic and phenolic OH
groups can be determined using a methylation procedure using methyl iodide as
outlined by Schnitzer and Skinner (1965)
Phenolic (OH) Groups. The phenolic content of a humic substance is
assumed to be equivalent to the concentration of weaker organic acids in the sample [see "Weaker Organic Acids (Phenolic Groups)"], i.e.,
Phenolic OH (mmolf g-l)
= total acidity (mmolc g-l) - COOH acidity (mmolc g-l)

Alcoholic (OH) Groups. The alcoholic hydroxyl content of a humic substance is assumed to be the difference between the total hydroxyl content and the
phenolic content.
Alcoholic OH (mmolf g-l)

=total hydroxyl (mmolf g-l) -

phenolic hydroxyl (mmolf g-l)

SWIFT

1042

Carbonyl (>C=O) Groups

Total Carbonyl (>C=O) Groups. The humic substances are allowed to
react with an excess of hydroxylamine hydrochloride in methanoV2-propanoi.
The unreacted hydroxylamine hydrochloride is titrated with standard HCI0 4
solution (Fritz et aI., 1959)

Materials
1. Dimethylaminoethanol [(CH3)2NCH2CH20H] 99% purity, 0.25 M (22.5
g in 2-propanol).
2. Hydroxylamine hydrochloride (NH 20H . HCI), 0.4 M (27.8 g in 300 mL
absolute methanol and dilute to 1 L with 2-propanol).
3. Perchloric acid (HCI04), 0.2 M.

Method
Place 50 mg of humic substance in a 50-mL ground-glass stoppered Erlenmeyer flask. Add 5 mL of 0.25 M 2-diethylaminoethanol solution and 6.3 mL of
0.4 M hydroxylamine hydrochloride solution to the flask containing the humic
substance and to a similar flask in which there is no humic substance, to act as a
blank. Heat the flasks on a steam bath for 15 min. Cool the solutions and back
titrate potentiometrically the excess of hydroxylamine hydrochloride with standard perchloric acid solution. Determine the end-point by plotting milliunits vs.
milliliters of acid.

Quinone Groups. In the natural environmental humic substances undergo

redox-type interactions with a number of soil components including iron (Waite
& Morel, 1984). This type of interaction can be used to determine the concentration of quinoid groups present in a sample. In the method outlined below the
humic substances are reduced in alkaline triethanolamine (TEA) solution by Fe2+.
The excess reductant is back-titrated amperometrically with standard dichromate
solution (Glebko et aI., 1970)
0= ( d = 0 + 2Fe2+-triethanolamine ~ HO-<Q>-OH + 2Fe3+

Materials
1. Sodium hydroxide, 2 M.
2. Triethanolamine (TEA) 97% purity, 2 M.
3. Ferrous ammonium sulfate hexahydrate [FeS04(NH4hS04.6H20], 0.05
M.
4. Potassium dichromate (K2Cr207), 0.004 M.

Method
Dissolve 20 mg of humic substance in a solution of 45 mL distilled water,
25 mL of 2 M NaOH and 25 mL of 2 M TEA in a 200-ml tall form titration cell,

ORGANIC MATIER CHARACTERIZATION

1043

fit the lid and flush with N2 continually during the analysis. Stir the solution magnetically for 30 min before adding 5 mL of 0.05 M ferrous ammonium sulfate
hexahydrate solution and leave for 30 min. Using standardized 0.004 M potassium dichromate solution, back titrate the excess reductant in solution at a constant
potential of -80mV, determined using a platinum foil (2.0 by 0.5 cm)/platinum
wire electrode system connected to a polarograph. Carry out a blank titration
under the same conditions.
Quinoid C=O (mmolf g-l) = (Vblank - Vsample)

6 x M(K2Cr207)/Wsample(g)

Ketonic C=O Groups

Ketonic C=O groups (mmolf g-l)
= total C=O groups (mmolf g-l) - quinone groups (mmolf g-l)

General Comments
Typical values of functional group content of soil humic substances from
various origins are shown in Table 35-2. Because of the complexity of humic
substances, it is not easy to assess the degree of accuracy of analytical results.
However, it is possible to determine the consistency of the data with respect to
the logical consequences of the relationships between each set of values. For a
detailed discussion of these uses the reader is referred to Perdue (1985).
With the determination of the elemental composition and the number average molecular weight, theoretical values can be determined for the amount of
Table 35-2. Distribution of oxygen-containing functional groups in humic and fulvic acids isolated
from soils of widely different climatic zones (in cmollkg)t (Stevenson, 1994).
Climatic zone

Functional group
Humic acids
Total acidity
COOH
AcidicOH
Weaky acidic +
alcoholic OH
Quinone C=O
KetonicC=O
OCH 3
Fulvic acids
Total acidity
COOH
AcidicOH
Weakly acidic +
alcoholic OH
Quinone C=O
KetonicC=O
OCH3

Cool,
Cool,
temperate temperate
Arctic acid soils neutral soils Subtropical

560
320
240
490
230
170
40
1100
880
220
380
200
200
60

t From Schnitzer, 1977.

Tropical

Range

62()"'{)60
390-450

63~770

62~750

56~90

15~570

42~520

380-450

15~570

32~570

21~250

21~250

22~300

21~570

27~350

24~320

{1~180

{45~560

40

30

57~90

Average

2~160

20-490

670
360
390
260

{8~150

{3~140

{1~560

{290

3~50

6~0

3~0

290

60

340-460

12~270

3~250

3~570

69~950

26~520

26~950

1030
820
300
610

{17~31O

{12~260

3~150

{120-420

{270

30-40

8~90

3~120

80

89~1420

64~1230

82~1030 64~1420

61~50

52~960

72~1120 52~1120

28~570

16~270
9~120

SWIFf

1044

unsaturation in the system and hence upper limits of the main functional groups
such as carboxylic acids, phenols alcohols and enols.

Characterization by Spectroscopic Methods

The use of spectroscopic methods of analysis is important in the characterization of humic substances. The individual techniques may yield only small
fragments of information but the importance of this is magnified when combined
with information from other spectroscopic techniques and/or other methods of
characterization.
The aim of this section is to outline briefly the types of spectroscopic techniques generally used in the study of humic substances, and how they have assisted in increasing the knowledge of these substances. For more detailed information about the techniques the reader should consult a recent text concerned principally with their use. For a comprehensive review of the applications of the
methods to studies on humic substances the text Humic Substances II: In Search
of Structure (Hayes et aI., 1989) is recommended.

Ultraviolet-Visible Spectroscopy
The absorption of electromagnetic radiation in the UV (200-400 nm) and
visible region (400-800 nm) is associated with the electronic transitions of the
bonding electrons. The absorption of UV-visible radiation by organic compounds
is due to the presence of specific segments or functional groups (chromophores)
which contain unbonded electrons (e.g., carbonyl groups, S, N or 0 atoms, and
conjugated C-C multiple bonds). The electronic transition within a molecular
orbital is termed local excitation and the electronic transition involving the transfer of an electron from one chromophore to another (e.g., from an aromatic ring
to an OH group) is termed electron transfer.
Generally, measurement of the absorbance of a substance is carried out by
dissolving it in a solvent and determining the difference in absorbance of that
solution from that of a solution containing only the solvent, either in sequence (in
single beam instruments) or directly (double beam instruments). The absorbance
(A) is related to the concentration of the sample in solution (c) according to the
Beer-Lambert law where
A = /c

where I is the path length of the cell and is the absorptivity

Applications of Humic Substances

The absorbance spectrum of a compound is a characteristic which can be

used in its identification. However, because the peaks of absorbance spectra are
relatively broad, it is difficult to identify a particular compound in a mixture of
even simple molecules and certainly not possible in a sample of humic substance.
The absorption spectra of humic substances are generally featureless, consisting
of a relatively smooth curve, with increasing absorption with decreasing wavelength. The curve represents the summation of the absorbances of the component
chromophores. Even though it is not known what proportion of molecules con-

ORGANIC MATIER CHARACTERIZATION

1045

tribute to the spectra, the smoothness indicates that there are a very large number
of different chromophores in the sample.
Despite the apparent lack of detailed information in the spectra, different
samples and fractions of humic substances do show slight variations which can
be measured in a number of ways to allow comparisons to be made. For example, the EJE6 value (the ratio of the absorbance at 465 nm and 665 nm, of a dilute
aqueous solution of a substance) is commonly used to characterize humic substances (Konova, 1966; Chen et aI., 1977; Stevenson, 1994) with the ratio for
humic acids being generally less than five and that for fulvic acid more than five.
Because of the known differences between humic acids and fulvic acids the ratio
would appear to be a measure of the degree of humification of a sample of humic
substance.
The magnitude of the absorbance at a given wavelength varies slightly with
pH (MacCarthy & O'Cinneide, 1974b; Baes & Bloom, 1990) which is probably
due to the ionisation of carboxylic and phenolic functional groups. This change
in absorbance with pH is dependent on the wavelength so that the EJE6 ratios of
humic acid and fulvic acid also vary with pH (Chen et aI., 1977; Ghosh &
Schnitzer, 1979). In alkaline solution the ratio decreases with increasing pH but
below pH =5.5 the EJE6 decreases with decreasing pH (Chen et aI., 1977; Ghosh
& Schnitzer, 1979).
Both absorbance and the EJE6 ratio may be affected by variations in the
salt concentration of the solution. It has therefore been suggested that the EJE6
ratio be determined in 0.05 M NaHC0 3 (pH = 8) (Chen et aI., 1977).
A more detailed study of the dependence of the absorbance on pH can be
made by referencing the absorbance reading against a spectrum of the sample at
a different pH (Tsutsuki & Kuwatsuka, 1979). These difference spectra are characterized by stronger peaks but the origin and cause of these peaks is not yet
understood with any certainty.
The UV -visible spectra of humic substances offer very little assistance in
the identification of their structure. One peculiarity is the observed absorbance at
wavelengths above 500 nm when it has been found that compounds do not normally absorb light at these wavelengths. It has been suggested that this is due to
complex unsaturated structures (Tsutsuki & Kuwatsuka, 1979) or to electron
donor-acceptor complexes (Lindqvist, 1972, 1973).

Infrared Spectroscopy
Infrared spectroscopy is the study of the molecular vibrations of bonded
atoms. The frequency of the absorption is characteristic of the atoms in the bond
and the type of motion associated with the vibration. The observed frequency can
be used to distinguish the component atoms as well as the bonding characteristics
of those atoms. Hydrogen bonding may also be apparent as it causes greater separation of the bond between H and the other atom in the covalent bond, thereby
decreasing the frequency of the absorption and broadening the bands.
Detailed interpretation of the spectra to determine the structure is possible
in simple molecules, but it is generally not so in complex molecules or mixtures
of molecules. However, useful information can be gained by comparing the spectra of different samples and noting changes in the spectra after chemically alter-

1046

SWIFT

ing a sample. Using a variety of approaches it is possible to gain information on

the functional groups such as aromatic, aliphatic and quinone groups associated
with the sample.
Infrared spectra are usually determined over the frequency range 4000 to
400 cm- l . Absorption bands in the frequency region 4000 to 1250 cm-l are relatively unaffected by the remainder of the molecule and this region is called the
"characteristic group frequency region." Absorption bands below 1250 em-I are
affected strongly by the molecular structure and so this region is called the "fingerprint region."
The spectral analysis is usually carried out on a solid sample, but as the
technique depends on the absorption of radiation, the sample is dispersed in a
medium, which is transparent to infrared radiation. The pressed pellet method of
sample preparation requires a mixture of about 1 mg of humic m~terial per 100
mg of an alkali halide (KBr) both dried and preground very finely. The mixture
is pressed (~7500 kg em-2) into a small disc about 100 mm in diameter and 1 to
2 mm thick (Stevenson, 1994). Potassium bromide is transparent over the conventional sample range 4000-400 cm-l and so does not interfere with the spectral
analysis.
Alternatively the fmely ground and dry sample can be mixed with a low
vapor pressure, medium molecular weight hydrocarbon [commonly "Nujol"
(Harry Holland & Sons, Burr Ridge, IL)]. This substance is less satisfactory as it
adsorbs radiation at 2900, 1460 and 1375 cm-l and so imposes its spectrum over
that of the sample being analyzed, and is not very suitable for the analysis of soil
humic substances.
A common problem is the incorporation of water into the sample matrix,
especially when using KBr discs. The presence of water causes bands at 3300 to
3000 cm-l and 1720 to 1500 cm-l regions. This can be reduced by heating and
evacuating the die prior to pressing or using a nonhygroscopic pelleting matrix
(Stevenson, 1994). The problems associated with water in the sample can be
avoided by using cast films or solutions in a cell (Bloom & Leenheer, 1989).

Applications to Humic Substances

The infrared (IR) spectra of humic substances are relatively simple with
only a few broad bands and no well-defmed, sharp peaks, typical of the spectra
of single compounds. As for UV-visible spectra, these undefined broad bands are
evidence that the functional groups in the sample of humic substances exist in a
wide variety of chemical environments. There are often similarities in the general appearance of spectra of different samples of humic substances. This does not
necessarily mean that the samples are composed of molecules with similar structure, but does indicate that the overall functional group content is similar.
Derivatization techniques are frequently used to study the attribution of
observed spectral bands to a particular functional group. An excellent outline of
the bands associated with the main functional groups and the type of derivatization procedures which may assist in the assignment of the bands for a particular
sample, is given in Bloom and Leenheer (1989).
In situations where the drying-out process may affect the chemical equilibrium in the sample being studied, it may be necessary to analyze the sample in

ORGANIC MATIER CHARACTERIZATION

4000

3000

1047

2000

1000

Wavenumber (cm- 1)

Fig. 35-3. Diffuse reflectance Fourier-transformed infrared spectra of four International Humic Substances Society humic acids (Neimeyer et aI., 1992). Note: the Kubelka-Munk units arise from the
application of the Kubelka-Munk transformation (Kortum, 1969) of the reflectance spectra and
result in spectra similar in appearance to absorbance spectra obtained from transmission measurements (Baes & Bloom, 1989).

Table 35-3. Fourier-transformed infrared (FTIR) bands of peat humic and fulvic acids (adapted from
Baes & Bloom, 1989.
Band

Assignment

3330-3380
3030
2930
2840
2600
1720
1610
1525
1450
1350
1270
1225
1170
1070
830

OH stretch of phenolic OH (contribution from aliphatic OH, H20 and possibly NH)
Aromatic CH stretch
Asymmetric CH stretch of -CH 2Symmetric CH stretch of -CHzOH stretch of H-bonded -COOH
-C=O stretch of -COOH
Aromatic C=C stretch and/or asymmetric -COO- stretch
Aromatic C=C stretch
-CH deformation of -CH3 and -CH bending of -CHz
Symmetric -COO' stretch and/or -CH bending of aliphatics
-C-OH stretch of phenolic OH
-C-O stretch and OH deformation of -COOH
-C-OH stretch of aliphatic OH
C-C stretch of aliphatic groups
Aromatic CH out of plane bending
Aromatic CH out of plane bending

775

1048

SWIFr

the aqueous state. This is difficult in infrared spectroscopy as the absorption

bands of water are large and centered around the major area of interest, 3400 to
1640 cm- I , and so interfere with the analysis. This may be overcome by using
D20 instead ofHzO to shift the position of the bands (MacCarthy & Mark, 1975).
More recently, Fourier Transform Infrared Spectroscopy (FTIR) and an adaptation to this method using diffuse reflectance rather than transmitted light
(DRIFT), have been applied to the study of humic substances (Baes & Bloom,
1989; Niemeyer et ai., 1992; Ristori et ai., 1992). The Fourier transform technique eliminates problems associated with the presence of water and improves
the resolution. The diffuse reflectance adaptation permits the analysis of opaque
samples so that whole soil samples can be studied (Ristori et ai., 1992; Nguyen
et ai., 1991). Reflectance methods can be used with dispersive instruments, and
KBr pellets can be used with FTIR. The combination of FTIR with diffuse
reflectance, however, seems to offer the best method for analysis of humic substances.
The small amount of sample required and the simplicity of the procedure
make IR spectroscopy one of the most commonly used methods of analysis for
humic substances. The information gained from the spectra is in many cases not
definitive as the assignment of bands to particular groups is only tentative in complex mixtures of compounds. The results should be confirmed with other methods of analysis.
Examples of typical FTIR spectra of humic acids are shown in Fig. 35-3.
The assignment of structure generally given to the absorption bands in humic
substances is well documented in Stevenson (1994) and a summary of the assignments is shown in Table 35-3.

Nuclear Magnetic Resonance

When certain nuclei with a particular type of spin are placed in a magnetic
field, they align themselves such that some have their spin magnetic moment vectors parallel to the field vector and are at slightly lower energy to others that are
antiparallel in the field.
If an oscillating magnetic field is superimposed on the steady magnetic
field with a perpendicular magnetic vector, then for a particular steady magnetic
field strength, absorption of radiation will occur at certain frequencies of oscillation, allowing transitions between different energy spin states. This resonance
condition can be met by varying the magnitude of either the steady state field or
the oscillating field. In Fourier transform nuclear magnetic resonance (NMR)
spectroscopy the sample is subjected to a pulse of radio frequency radiation comprising all frequencies.
In a molecular environment, the atoms surrounding a nucleus partially
shield it from the magnetic field so that the frequency of the oscillating field required to achieve resonance is changed. As this shielding is a function of the
chemical environment associated with the nuclei the resonance frequencies of the
atoms in the compound are able to give information about the chemical structure
of the compound. Common nuclei used for the studies with NMR are 1Hand 13e.
The NMR frequency of a given nucleus (v sample) is measured relative to
that of a standard compound {typically tetramethylsilane [(CH3)4Si] (TMS)) (v

ORGANIC MATTER CHARACTERIZATION

1049

reference). The frequency for resonance is then given as the difference, or, chemical shift (0) between these two frequencies, expressed in parts per million (ppm).

o=(Vsample -

Vreference) X

106/vreference

Common solvents used in liquid state 13C-NMR are NaOH and NaOO and
these appear to display no absorptions. The OMSO-d6 provides a sharper spectrum but the 30 to 40 ppm region is obscured unless the solvent is depleted in 13e.
In IH-NMR the aqueous H is H20, 0 20 and aqueous NaOH obscures the 3 to 5
ppm region.
There are a number of problems associated with liquid state NMR such as
the amount of sample required (100-200 mg), insolubility of some compounds in
suitable solvents, interference from water in IH-NMR and the long analysis times
required (2-12 h are common for IH-NMR and up to 1 wk for 13C NMR). In
some situations the dissolution process interferes with the analysis. The development of solid state NMR has avoided these problems as well as achieving a higher signal to noise ratio, generally giving greater sensitivity in the spectra. However, the technical problems in getting good spectra are much greater.

Applications to Humic Substances

Nuclear magnetic resonance spectroscopy is generally used to compare the
difference in concentration of the main functional groups between samples of
humic substances. Both IH-NMR and 13C-NMR are able to be used for the characterization of humic substances, although the relatively low abundance of 13C
(about 1.1%) in the latter technique places considerable demands on instrumentation and methodology. Nevertheless, these two methods provide powerful tools
for studying humic substances (Wilson et aI., 1983; Wilson & Goh, 1983).
The most common type of NMR now used in the study of humic substances
is called solid state CPMAS-NMR (cross-polarization, magic angle spinningnuclear magnetic resonance) (Wilson et aI., 1987; Krosshaven et aI., 1990). An
example of CPMAS 13C-NMR spectra of a soil humic substance is shown in Fig.

Chemical shift, S(ppm)

Fig. 35-4. Typical CPMAS 13C-NMR spectrum of a soil humic substance.

SWIFr

1050
Table 35-4. Major proton resonance of humic materialst (Wershaw, 1985).
Chemical shift, 1)
(relative to TMS as 0)

Assignment

ppm
13.0
10.0
6.0-7.5
4.0-5.5
3.7
2.6
1.3
0.9

Carboxylic acid protons

Hydroxyl protons
Aromatic protons
Lactone protons
Methoxyl protons
Aliphatic protons attached C atom IX to a benzene ring:j:
Aliphatic protons ~ to a benzene ring:j:
Aliphatic protons y to a benzene ring:j:

t Spectra measured in deuterodimethylsulfoxide [(CD3hSOj.

:j:--CaH;z-CpHrCrH2

35-4. It is now possible to compare the NMR spectra of humic substances in

whole soil samples with those of the associated extracted fractions. Using this
type of approach, Krosshaven et al. (1992) concluded that conventional humus
fractionation does not significantly change the content of the different functional
groups in a sample. Assignments of chemical shifts for IH-NMR and CPMAS
13C NMR are shown in Tables 35-4 and 35-5, respectively.
Characterization of humic substances using NMR spectroscopy is a relatively new but widespread technique, both in the number of research groups
involved and the type of research being undertaken. Nuclear magnetic resonance
spectroscopy is proving to be important, both as a source of information relating
to the structure of humic substances, as well as supporting information gained
from other types of analyses. However, there is a need to standardize the procedures and techniques used, especially in solid state NMR, to take full advantage
of this type of research with respect to humic substances.
Detailed information on the theoretical aspects of NMR spectroscopy, and
the application of this theory to the studies of humic substances, is given in Hayes
et al. (1989).

Electron Spin Resonance

When molecules containing unpaired electrons in a magnetic field are irradiated with electromagnetic radiation, the electrons can be excited to a higher
energy state and those which are already at the higher energy level can be induced
to move to the lower energy state. For a system initially in a state of thermal equilibrium there will be slightly more electrons in the lower state giving a net absorption of energy. The absorption of this energy is the basis of Electron Spin
Resonance (ESR) spectroscopy which involves the study of molecules with
unpaired electrons (free radicals).
The energy difference between the two spin states (aE) is

where h is Plank's constant, v is the frequency of the electromagnetic radiation,

~ is the Bohr magneton (a constant) and Ho
is the applied field.

g is the spectroscopic splitting factor,

ORGANIC MATIER CHARACTERIZATION

1051

Table 35-5. Chemical shift assignments in the CPMAS DC NMR spectra of fulvic and humic acids
(adapted from Malcolm, 1989).
Shift range

Possible assignments

ppm

0-50
10-20
15-50
25-50
29-33
35-50
41-42
45-46
50-95
51-61
57-65
65-85
90-110
110-160
110-120
118-122
120-140
140-160
160-230
160-190
190-230

Unsubstituted saturated aliphatic C atoms

Terminal methyl groups
Methylene groups in alkyl chains
Methine groups in alkyl chains
Methylene C (l,~, 0, E from terminal methyl group
Methylene C atoms of branched alkyl chains
(l-C in aliphatic acids
R2 NCH3
Aliphatic C singly bonded to one 0 or N atom
Aliphatic esters and ethers; methoxy, ethoxy
Carbon in CH 20H groups; C6 in polysaccharides
Carbon in CH(OH) groups; ring C atoms of polysaccharides; ether-bonded aliphatic C
Carbon singly bonded to two 0 atoms; C, anomeric in polysaccharides, acetal or ketal
Aromatic and unsaturated C
Protonated aromatic C, aryl H
Aromatic C ortho to O-substituted aromatic C
Unsubstituted and alkyl-substituted aromatic C.
Aromatic C substituted by 0 and N; aromatic ether, phenol, aromatic amines
Carbonyl, carboxyl, amide, ester C atoms
Largely carboxyl C atoms
Carbonyl C atoms

It is possible to determine the concentration of unpaired electrons (spins)

by comparing the integrated absorption with that of a standard under the same
conditions, although the accuracy of this method is believed to be quite low. Measurement of the field strength and the frequency of the electromagnetic radiation
at resonance determines the g-value, the magnitude of which is associated with
the molecular structure of the molecule. The hyperfine structure of the spectra can
be used to determine the number and types of nuclei interacting with the free
electron, and hence the structure of the molecule. The concentration of free radicals is determined by the width of the absorption line.

Application to Humic Substances

The ESR spectra of humic substances are generally only a single line which
limits the amount of information that can be gained from the analysis. The hyperfine structure in ESR spectra, typical of simple systems, is absent although a few
instances have been reported with splitting (Atherton et aI., 1967; Senesi et aI.,
1977). An example of an ESR spectrum of a humic substance is shown in Fig.
35-5 with some typical values of the measurable parameters for soil humic substances in Table 35-6. These values are only meant to be indicative of those commonly found as the magnitude is dependent on solution pH, metal ion content and
solvent effects.
Results obtained from this type of research have led to the realization that
the free radicals in humic substances are remarkably stable with respect to time
and chemical attack. This stability has been attributed to the stabilization of an
unpaired electron over an aromatic system (Theng & Posner, 1967) suggesting
that humic substances contain free radicals of the semi-quinone type (Steelink,

SWIFT

1052

A
B

-5

+5

Fig. 35-5.ESR spectra of a podzol FA in NaOD-D20: (A) immediately after mixing fulvic acid with
NaOH; (B) 48 h after mixing. g =2.0040; line width =5.0 x 10-4 T; frequency =9.451 GHz; field
center =3342.0 (Steelink et aI., 1983).

1964). Basic solutions of humic substances contain exceptionally long-lived free

radicals. It has also been found that the spin content generally increases markedly with increasing pH.
The concentration of free radicals in humic substances is relatively small
(Schnitzer & Skinner, 1969), and as the concentration varies with the type of solvent used in the extraction procedure it may even be an artifact of extraction technique used (Hayes et aI., 1975). Other techniques such as NMR and infrared (IR)
are more useful than ESR in characterizing the functionality of soil humic substances. However, ESR is a source of additional information and should be
included in the general research procedure. If the technique was to become more
reliable and informative the potential of its use would increase as it is concerned
with one of the more reactive fractions of humic substances, the free radical content.

Pyrolysis-Mass Spectroscopy
Pyrolysis (Py) is the degradation of a substance through the action of heat.
This process is usually carried out in a vacuum, or in a rapid stream of inert gas
to restrict the formation of secondary products. The absorption of thermal energy
causes excitation of the bond vibrational modes resulting in the cleavage of the
weaker bonds. The number and variety of the products formed by using this techTable 35-6. Electron spin resonance parameters for various humic and fulvic acids (Steelink et aI.,
1983).
Sample

State

Free-radical
concentration
spinsg-1x
10- 17

Soil humic
acid
Soil fulvic
acid
Soil humic
acid
Soil fulvic
acid

Line width
T

Spectroscopic
splitting factor Reference
(g value)

Solid

5-10

4.8 x 10-4-5.2 x 10-4 2.0032-2.0047 Chen et al. (1978)

Solid

1-2

4.5 x 10-4-7.5 x 10-4 2.0037-2.0050 Chen et al. (1978)

Aqueous
solution
Aqueous
solution

2.1

2.5 x 10-4

2.0037

1.3

2.5 x 10-4

2.0038

Varadachari et al.
(1983)
Varadachari et al.
(1983)

ORGANIC MAlTER CHARACTERIZATION

1053

nique to study humic substances is very large. It is therefore essential to incorporate mass spectrometry (Py-MS) or gas chromatography (Py-GC) into the system,
to separate and identify the reaction products. A further, and highly desirable,
improvement on the technique is to use a combination of both gas chromatography and mass spp.ctrometry (Py-GC-MS). This system allows the volatile reaction
products to be separated prior to the analysis using the mass spectrometer. Alternatively, soft-ionization techniques such as field ionization (FI) and field-desorption combined with pyrolysis mass spectrometry, (Py-FIMS, Py-FDMS) extend
the range in size of molecular ions which can be observed by Py-MS by including the small and larger molecular ions, respectively.
The data gained from each analysis can be used as ajingerprint of that sample under the particular pyrolysis conditions used. This fingerprint can then be
compared with the results of analysis on other samples of humic substances or on
known chemical compounds. Because of the large amount of data collected using
these analytical techniques, multivariate analysis with the aid of computers is
used to collate the results (Howarth & Sinding-Larsen, 1983). The theoretical and
technical aspects of these pyrolysis techniques and the methods of treatment of
the accumulated data are described by Bracewell et ai. (1989).

Applications to Humic Substances

Pyrolysis techniques are used to study humic substances in an attempt to
correlate samples of humic substances with their parent biopolymers (Meuzelaar
et aI., 1982). Evidence from the studies using pyrolysis indicate that the humic
macromolecules have polysaccharides, polypeptides and lignins incorporated
into their structures (Greenland & Oades, 1975; Halma et aI., 1978; Bracewell et
aI., 1980). Pyrolysis techniques have also been used to determine differences
between the various fractions of humic substances (Saiz-Himinez et aI., 1978,
1979). The studies carried out so far indicate that there is very little variation in
the pyrolysates of humic acids from different soils and different soil types, whereas the pyrolysates of a fulvic acid fraction are dependent on the origin of the soil
and the method of extraction (Bracewell et aI., 1989).
One of the most important aspects of pyrolysis with respect to applications
to humic substances is the ability of this technique to analyze whole soil samples.
This permits a relatively rapid analysis of soil organic matter in general and
hence, comparison of organic matter associated with soils from different origins
(Baldock et aI., 1991) or as a result of different management practices (Schulten
& Hempfling, 1992). Figure 35-6 shows Py-FI mass spectra [intensity of the ion
vs. the mass of m/z (mass to charge ratio of the ion)] of the pyrolysates obtained
from a soil as a result of different intensities of crop rotation and manure treatment. These two soil samples exhibited almost identical total concentrations of C
and N. Schulten and Hempfling determined that there was little difference
between the pyrolysates of the soil samples in the mass range m/z 50 to 200, but
in the higher molecular weight products of the pyrolysis the intensities were
greater for the soil that had less intense cropping and had been treated with
manure.
Comparison of Py-FIMS spectra of whole soil samples with those of the
extracted humic fractions permits the verification of consistency of results when

SWIFf

1054
5.0

96

r--xl1f -

4.0 58
3.0

i
l1.0

2.0

82

110

67

208

,!;' 0

Cf)
100
50
c:
~ 5.058-_96

c:
. 4.0
i
~ 3.0

(a)

150

200

250

300

350

400
(b)

110

2.0
1.0
0
50

340

100

150

200

250

m/z-

300

350

400

Fig. 35-6. Averaged Py-FI mass spectra of soil samples from the same site and with the same Nand
C content, but under different management systems and (a) with manure addition, (b) without
manure addition.

characterizing the fractions (Hempfling & Schulten, 1991). It has been found that
humic acid extracts from soils are enriched in polypeptides relative to the whole
soil pyrolysate, and the fulvic acid extracts are deficient in polypeptides but
enriched in polysaccharides or pseudo-polysaccharides (Haider et aI., 1977). The
"humin" extract had a similar fingerprint to that of the humic acid indicating that
the nonextracted material of a soil humic substance has similar chemical content
to that of the extracted material. The effects of different methods of extraction of
fractions of humic substances have also been observed using Py-FIMS by Haider
& Schulten (1985).
Pyrolysis techniques also provide supporting data for the results obtained
when using other types of chemical analysis and instrumental analysis. Zech et
al. (1990) used chemical analysis combined with IR, 13C-NMR and Py-FIMS
instrumental methods to characterize and compare the organic matter in soils
from different soil horizons. Beyer et al. (1992) concluded that the Py-FIMS
results confirmed and extended the results obtained from wet chemistry methods
and NMR methods.
Pyrolysis techniques used to study whole soil samples allow the characterization of the organic matter without going through the potentially destructive
and time-consuming processes of extraction and purification of the humic substances. It is for both of these reasons that this approach to organic matter characterization is receiving increasing interest and holds considerable promise.

Fluorescence Spectroscopy
The UV-visible radiation absorbed by a molecule may be dissipated as heat
and/or electromagnetic radiation at a longer wavelength than the incident radiation. This emitted radiation is known as fluorescence and it is a characteristic of
humic substances.
There are three types of spectra generally associated with this technique.
Excitation spectra are obtained by scanning the incident radiation and determin-

ORGANIC MAITER CHARACTERIZATION

1055

ing the intensity of radiation emitted at a fIxed wavelength. Emission spectra are
obtained by fixing the incident radiation and determining the intensity of radiation emitted over a wavelength range. Synchronous excitation spectra are
obtained by setting the difference between the emitted and absorbed radiation to
be a constant (LlA) and determining the intensity of the emitted radiation for a
range of wavelengths.

Application to Humic Substances

In general, only a small fraction of molecules are known to fluoresce, and
so the proportion of molecules in a sample of humic substance contributing to the
observed fluorescence is relatively small. Additionally, this observed fluorescence is not the total fluorescence as humic substances absorb radiation over a
wide range of wavelengths and some of the fluorescence radiation will be reabsorbed by the sample.
Fluorescence spectroscopy has been used to characterize samples of humic
substances but for a number of reasons it has not gained general acceptance (Senesi, 1990). Because of the reabsorbance of the fluoresced radiation the relative
intensity of the observed radiation at a particular wavelength is a function of the
concentration of humic substances in solution (Spark & Swift, 1994). When
using UV -visible absorbance spectroscopy increasing the solution concentration
of dilute solutions increases the absorbance in a regular manner. In fluorescence
spectroscopy increasing the concentration of humic substances in dilute solution
does initially increase the observed intensity of fluorescence, but eventually this
reaches a maximum and then begins to decrease. The concentration at which the
fluorescence reaches a maximum is dependent on the wavelength of excitation
and emission as well as on the nature of the humic substance. As yet it has not
been possible to correlate the detail in the observed fluorescence spectra of humic
substances with known characteristics of these substances so the usefulness of
this technique is limited.

Measurements of Physical Properties

The physical properties generally determined for humic substances are
molecular size, shape and weight, and charge characteristics. The principles of
these determinations are theoretically soundly based, but in practice the results
are somewhat limited in value by the large variation in range of the properties in
even the most highly fractionated samples.

Molecular Size and Shape

Molecular size and shape of humic molecules are two of the most elusive
properties of humic substances. Considerable research on humic substances has
been directed towards the determination of these properties, but as knowledge of
humic substances has increased, so has the realization of the complexities associated with assigning values to the size and shape of humic molecules. It is probably because of these problems, as well as the rapid development of spectroscopic instrumentation, that this area of humic substance research has made little
progress in recent years with respect to other areas.

1056

SWIFT

The techniques generally used to study the molecular size and shape of
humic substances are gel permeation chromatography and viscosimetry. A variety of other techniques such as light scattering (see "Light Scattering"), Flow
Field Flow Fractionation (FFFF) (Becket et aI., 1987), ultracentifugation (Posner
& Creeth, 1972; Ritchie & Posner, 1982), and colligative data calculations
(Reuter & Perdue, 1981) have also been used.

Gel Chromatography. The general principles relating to gel chromatography have previously been discussed with respect to the fractionation of humic
substances (see "Ion-Exchange Chromatography"). For molecules of the same
shape and structure it is possible to calibrate the gel column with respect to elution volume vs. molecular weight using known compounds, and hence determine
the molecular weight of an unknown compound. As elution time is dependent on
the molecular shape and structure, the elution time for two molecules of the same
molecular weight, one of which is spherical and the other a long chain molecule,
may be quite different.
The elution volume for a given solute is dependent on the geometry of the
column, the length of the gel bed and differences in the packing density, and so
cannot be used to characterize a solute. The distribution coefficient (Kd) can be
used to compare the migration rates of different solutes in different experiments.

where Vc is the elution volume, Vo is the void (excluded) volume Vi is the inner
volume (i.e., the solvent volume inside the gel beads). Because of the difficulty
in measuring the magnitude of Vi> the total volume of the column (VI) is used to
determine an approximation of Kav according to the equation

Applications to Humic Substances

A variety of media are now used in this type of work including dextran
(C 6H lO OS)m polyacrylamide {[-CH2 C(CONH2)O-]}, agarose gels and porous
glass beads. The elution characteristics that can affect the elution time are interactions between the gel and solute, ionic strength and pH of the solution (Tsutsuki & Kuwatsuki, 1984), concentration of the sample and the type of buffer used
(Swift & Posner, 1971). Swift and Posner (1971) found that two buffer systems
tris (2-hydroxy-2-aminopropane-1,3-diol) and borate buffers at pH 9, are particularly well suited for use with humic substances.
Calibrations of the gels are usually made using polysaccharides or proteins
of known molecular weights. It has been found (Cameron et al., 1972b) that there
is a better correlation between humic substances with polysaccharides than with
proteins. This may be because the more diffuse or open molecular configuration
of polysaccharides is closer to that of the humic substances in solution, than are
the condensed protein configurations.
Calibration of the column is carried out by determining the elution characteristics of humic substances of which the molecular weight has been estimated

ORGANIC MATI'ER CHARACTERIZATION

1057

using some other technique, such as ultracentifugation (Cameron et aI., 1972b).

Dawson et al. (1981) had some success calibrating the column using chemicals
thought to be similar to those of humic substances.

Ultracentrifugation. Ultracentrifugation is the centrifugation of solutions

at high speed (100 0()(}-600 000 g) under a vacuum to cause sedimentation of
solute species. The velocity of sedimentation can be used to separate molecules
and to determine molecular shape and size.
Sedimentation of a macromolecule requires its movement through solution
which may be hindered by a number of factors including frictional resistance
from the solvent, molecular enlargement due to solvation, and interactions with
other sedimenting molecules and/or solute components. In addition to these factors if the sedimenting molecule is charged its sedimentation velocity will be
decreased due to the accumulation of small counter ions behind it. The sedimentation velocity may also be affected by the sedimentation characteristics of the
buffer or background electrolyte.
A number of different techniques are used in ultracentrifugation. Sedimentation velocity involves the use of high rotor speed and requires the development
of well-formed boundaries, which is not possible in polydisperse systems. Sedimentation equilibrium uses lower rotor speeds for long periods of time, until the
macromolecules in solution are in equilibrium. The number average (Mn),
weight-average (Mw) and z-average (Mz) molecular weights of the sample can be
determined using this method for samples with a limited amount of poydispersity without the need for any other measurements.
Both of the above techniques rely on the use of analytical centrifuges which
have optical detection systems attached. When preparative centrifuges, which do
not have associated optical systems, are used, it is necessary to carry out the separation using the density gradient technique to overcome problems of convection
and mechanical disturbance. The density gradient is formed in the centrifuge tube
by using increasing concentrations of a solute such as sucrose, an inorganic salt
or mixtures of H20 and D20.
Applications of Humic Substances
There are a number of problems associated with applying this technique to
the study of humic substances. The color of humic substances restricts the transmission of light and hence limits the accuracy of the refractive index determination. The uncertainty of the molecular structure and conformation of humic substances makes it difficult to nominate suitable compounds to be used as internal
standards. However, the method is still valuable in being able to determine relative values of molecular weight between different fractions, or changes in the
molecular weight of a fraction associated with certain conditions or treatments.
It is necessary to use a relatively high concentration of background electrolyte
concentration (0.1-0.2 M) when analyzing humic substances to limit interferences associated with the highly charged nature of the molecules in these samples. This concentration of electrolyte will also influence the configuration and
degree of expansion of the humic substance in solution.
The three types of techniques, sedimentation velocity (Stevenson et aI.,
1953; Cameron et aI., 1972b), equilibrium ultracentrifugation (Posner & Creeth,

SWIFf

lOS8

1972) and density gradient methods (Ritchie & Posner, 1982) have been applied
to samples of humic substances. In the latter two techniques, carried out using
fractionated samples of humic substances, the large variation in the values of
(Mn), (Mw) and (MJ are strong evidence for the polydispersity of humic substances (Swift, 1989). Because of the highly polydisperse nature of humic substances the density gradient technique is potentially the most applicable, particularly for preparative procedures.
Light Scattering. The scattering of light by particles in solution can be
used to determine their molecular weight, a technique commonly used for the
study of polymers. The molecular weight (M) of the particles in a monodisperse
system at a density p can be determined by the using the relationship M =PV. The
volume of the particle (V) is determined using the Raleigh equation where the
ratio of the intensity of the scattered light [/(9)] to the intensity of the incident
light (/0) (Hunter, 1989) is given by

1(9)
10

= 91t2W(n2 -

1)2(1 + cosZS)
2,.z~..4(n2 + 2)

where N, and r are the number and radius of the of particles respectively, n is the
refractive index of the light scattering particles relative to that of solvent, A. is the
wavelength of the light and 9 is the angle between the incident beam and the scattered beam. It is assumed that the particles are uniform in size and spherical and
that the particle size is small relative to the wavelength of the scattered light.

Applications to Humic Substances

Very little use has been made of this technique to study humic substances.
Theoretically, light scattering techniques may be extended to study the changes
in solution in response to change in pH (Wershaw, 1989) and to study the complexation of humic particles in the presence of heavy metal ions (MacCarthy &
Mark, 1976; Ryan & Weber, 1982).
In practice problems arise from the absorption and fluorescence of light by
humic substances which would significantly interfere with the analysis at wavelengths below 500 nm (Wershaw, 1989). Reid et aI., (1991) used laser light at 633
nm to study the aggregation characteristics of humic molecules in solution. Using
laser lights with the new technique of quasi-elastic light scattering is also a possibility (Wershaw, 1989).
Viscosity. Information about the size and shape of humic and fulvic acids,
as well as their weight, polyelectrolytic characteristics and interaction behavior
with other macromolecules can be determined from measurements of viscosity.
The equipment required for this type of analysis is relatively inexpensive and the
experimental procedures are easier to conduct than other methods available for
determining the characteristics of humic substances. However, it must be noted
that the values obtained from the measurements tend to be relative values only.
Both the theory behind this technique, as well as the experimental information,
are well documented by Clapp et al. (1989).

ORGANIC MATIER CHARACTERIZATION

1059

Application to Humic Substances

The viscosity of humic substances in solution has been found to depend on
both the pH and the salt concentration of the solution (Chen & Schnitzer, 1976;
Stevenson, 1994). The results obtained from studies using different concentrations of salts (Ghosh & Schnitzer, 1980) support the theory that humic macromolecules in solution are polyelectrolytic in character with the degree of coiling
dependent on the concentration of the salt. It has been found that for a given molecular weight, humic substances from different sources exhibit varying degrees
of expansion (Visser, 1985).
Besides being used simply as a method of characterizing a humic substance
(Tomar et aI., 1992), viscosity measurements have also been used to study the
interactions between contaminants and humic substances (Chen et aI., 1992) and
between soil minerals and humic substances (Zhang et aI., 1991).

Other Techniques
A number of other techniques are used to characterize humic substances.
Two of these are chemical degradation studies and electron microscopy. Neither
of these techniques are discussed in detail, and the reader is referred to other
sources of information.

Chemical Degradation
The macromolecular size, the diverse arrangement of functional groups and
the multitude of interactions possible between these groups make the task of determining the structure of humic substances extremely difficult. The aim of
chemical degradation studies of humic substances is usually to break down the
large macromolecules into smaller, more recognizable units. From these units it
may then be theoretically possible to determine the origin of the parent molecules
and, hence, build up a picture of the structure of humic substances.
The process of rebuilding the molecular structure of humic substances from
the component molecules is complex in itself, but this work is made much more
difficult by the inability to control the degradative process to achieve a satisfactory set of simpler molecules. The ideal method should yield high amounts of
degradative products of moderate molecular complexity (Stevenson, 1994). If the
degradation producer is too mild, the total amount of each type of recognizable
molecules is too small to be determined analytically. And, if the degradation procedure is too extreme, the types of recognizable molecules produced are so simple that little information is gained about the nature of the parent molecule. Some
of those problems may be overcome by using a combination of different procedures and a range of severity (Stevenson, 1994; Hayes et aI., 1989).
The types of chemical procedures used in degradative studies include oxidation, reduction, hydrolysis and depolymerization.
The compounds produced when applying these procedures to humic substances are numerous, including complex phenolic and benzenecarboxylic acids,
hydroxycarboxylic acids, nitrophenols and polycyclic ring compounds (Stevenson, 1994). The text Humic Substances II. In Search of Structure (Hayes et aI.,
1989) provides a detailed reference for this type of research.

SWIFf

1060

Electron Microscopy
Electron microscopy has been used to study the physical appearance of
humic substances. This technique requires that the sample is dried which,
undoubtedly, alters some of the physical characteristics of the humic substances;
It is known that factors, such as pH, ionic strength, metal complexation and concentration, affect the size and shape of the dried particles of humic substances
(Chen & Schnitzer, 1989). The method of drying the sample also affects the
resultant appearance of the particle.
It is because of these types of problems that the results from electron
microscopy are thought to accentuate or distort the characteristics of humic substances in the natural environment, but do provide an insight into the possible
physical appearance of those substances. Recent advances in electron microscopy
allow samples to be studied under moist conditions, and it is important that this
technique be applied to the study of humic substances.

CHARACTERISTICS OF SOIL POLYSACCHARIDES

Many of the chemical and physical methods for characterizing humic substances can similarly be used to characterize soil polysaccharides (Cheshire,
1979; Cheshire & Hayes, 1990). This section will concentrate on those methods
peculiar to polysaccharides.

Determination of Monosaccharide Composition

Characterization of a sample of soil polysaccharides has similar problems
to humic substances with respect to the complexity of the sample hindering most
attempts at detailed analysis. However, because of the polymeric nature of this
soil fraction it is theoretically possible to convert the larger macromolecules
using hydrolysis procedures, into smaller and perhaps more recognizable units.
Hydrolysis of polysaccharides usually involves the reaction with hot acid
to the sample to break the glycosidic bonds joining the different sugars. This
method is generally used to carry out an estimation of total sugars. Because of the
different natures of the monosaccharide units, the nature of the hydrolyzing acid
and the analytical conditions needed to effect link breakages without destruction
of the monosaccharide units depends on the type of sugar units present. Sidereactions such as acid-reversion [Acid-catalyzed disaccharide or oligosaccharide
formation (Overend et aI., 1962)] are possible.
Another approach to the characterization of polysaccharides is the methylation technique (Cheshire et aI., 1979, 1983). By methylating the polysaccharides
prior to hydrolysis, it is theoretically possible to identify the monosaccharide
linkages (unmethylated sites on the monosaccharide products) within the polysaccharide macromolecule. Complete methylation is difficult even when using
extracted samples which may be due to steric hindrances arising from the association of hydroxyl groups with nonsugar components (Cheshire & Hayes, 1990).
No ideal conditions exist whereby the hydrolysis conditions are optimal for
all the general groups of monosaccharides. Therefore a variety of analysis are
usually performed for each of the main groups of monosaccharides. These

ORGANIC MAITER CHARACTERIZATION

1061

include hexosamines, hexoses, deoxyhexoses, pentoses and uronic acids. The

conditions generally used are: hexosamines, HCI (4-8 M); hexoses, H2S04 (0.5
or 1 M); deoxyhexoses, HCI or H2S04 (0.1 M) (Swincer et aI., 1969). It is more
difficult to achieve a satisfactory degree of recovery for the uronic acids and the
pentoses.

Hydrolysis of Polysaccharides
The method below based on that of Oades et ai. (1970), uses 2.5 M H2S04
to extract one fraction of polysaccharides prior to a more vigorous extraction procedure with 0.5 and 12 M H2S04,

Materials
1. Sulfuric acid, 12 M (72% w/w, sp. gr = 1.634), 2.5 M
2. Sodium hydroxide, concentrated.

Method
Combine 2 g of finely ground air dried soil with 25 mL of 2.5 M H2S04 in
a 50-mL round bottom flask and reflux the suspension for 20 min. Filter the mixture through a sintered glass filter, wash the residue with distilled water and dry
over P20S' Retain the filtrate and washings for analysis or to be combined with
the hydrolysate from the next part of the method.
Soak the residue in 12 M H2S04 for 16 h and then dilute the suspension to
0.5 M H2S04, Stopper the flask with a capillary air leak and heat at 100C for 5
h. Cool the mixture and filter through a sintered glass filter (porosity x 3). Combine the filtrate with that from the above procedure, neutralize this resulting solution to pH = 7 using NaOH, make the volume up to 100 mL and then filter again
(Whatman no. 1 paper). This solution is then ready for analysis of hydrolysates.

Analysis of Hydrolysates
The analysis of the sugar mixture released by the hydrolysis procedure is
often carried out using an alkaline ferricyanide and anthrone method (Cheshire,
1979). An alternative to this involves reduction and acetylation of the sugars followed by analysis of the solution by gas-liquid chromatography (glc) or high performance liquid chromatography (hplc). For a simple monosaccharide mixture
hplc is probably the most suitable technique. However, monosaccharide mixtures
derived from samples of humic substances are relatively complex. It has been recommended that glc is a more suitable method for quantitative and qualitative
analysis of these samples (Chaplin & Kennedy, 1986).
The method below, based on that used by Oades et ai. (1970), is designed
for analysis of the sugars using Gc.

Materials
1.
2.
3.
4.

Sodium borohydride (NaBH4)

Acetic anhydride (CH3CO)zO
Chloroform (CHCI3)
Methylene chloride (CH 2Cl 2)

SWIFf

1062

Method
Place a solution (water or methanol solvent) of the sugars (1-10 mg),
including internal standards, in a 12.4 by 1.6-cm screw top test tube, add 1 mg of
solid sodium borohydride and leave to stand overnight. Place this reduced sugar
solution in a warm bath at 60C and blow to dryness using a stream of air.
Add 1 to 2 mL of methanol containing 10% (v/v) glacial acetic acid to each
tube in the 60C water bath and blow it to dryness again using a jet of air. Repeat
this procedure four times. Add 1 to 2 mL of acetic anhydride, then seal the tubes
using screw caps fitted with Teflon inserts and heat at 130 to 135C for 2 h. Dry
the reaction mixture using the water bath and stream of air as before.
Extract the alditol acetates using chloroform and filter the solution under
pressure through a small column (5 by 10 mm) of Silica Gel G (Bio-Rad, Richmond, CA) using chloroform as the eluant to remove the fine black solids present
when analysing soil hydrolysates. Evaporate the chloroform filtrates to dryness
then dissolve the alditol acetates in methylene chloride before injection into a gas
chromatograph.

Comment
Two chemicals suitable for use as an internal standard for the above method
are myoinositol (C6H 120 6) and quebrachitol (C7H 140 6) (Oades et aI., 1970). A
suitable internal standard to be used for plant-derived carbohydrate analysis is
pentaerythritol (CSH 120 4) (Chaplin & Kennedy, 1986).

FUTURE DEVELOPMENTS
It must be obvious to the reader that there is still much to learn about the
nature of soil organic matter. As each new technique is applied to this area of scientific research it becomes more apparent that, in the short term, the chances of
a major breakthrough' are unlikely. The amount of information gained from each
individual study or from the application of a single technique is usually small, but
the significance of that information may then be magnified when combined with
that from other studies or results. In this context, it is important that as many techniques as possible are used to study the same material and the information obtained integrated to maximize our knowledge of this material. The standard and
reference samples prepared by the International Humic Substances Society will
prove to be invaluable for this purpose.
It is anticipated that the type of analysis associated with soil organic matter
research will tend towards the desirable situation in which the analytical procedures are capable of studying organic matter in situ in the soil matrix. This is likely to involve instrumentation of the complexity and costliness of the type of solidstate NMR, with other nondestructive instrumental techniques also playing a
major role. In the long term the future directions of soil organic matter research
will be governed by the requirements of our society. As the needs for increased
agricultural production and decreased environmental pollution grow, so will the
need for a better understanding of the chemical and physical characteristics of
soil chemistry and, hence, soil organic matter.

ORGANIC MATIER CHARACTERIZATION

1063

ACKNOWLEDGMENT

The author is very grateful to Dr. Kaye Spark for invaluable assistance in
the preparation of this chapter.
REFERENCES
Aiken, O.R, D.M. McKnight, R.L. Wershaw, and P. MacCarthy. 1985. Humic substances in soil, sediment and water. Geochemistry, isolation and characterization. Wiley-Intersci., New York.
Atherton, N.M., PA Cranwell, AJ. Floyd, and RD. Haworth. 1967. Humic acid I. ESR spectra of
humic acids. Tetrahedron 23:1653-1667.
Baes, AV., and P.R. Bloom. 1989. Diffuse reflectance and transmission fourier transform infrared
(DRIFI) spectroscopy of humic and fulvic acids. Soil Sci. Soc. Am. J. 53:695-700.
Baes, A.V., and P.R Bloom. 1990. Fulvic acid ultraviolet-visible spectra: Influence of solvent and
pH. Soil Sci. Soc. Am. 1. 54:1248-1254.
Baldock, JA, 1.M. Oades, A.M. Vassallo, and MA Wilson. 1990. Solid state CP/MAS 13C N.M.R.
Analysis of particle size and density fractions of a soil incubated with uniformly labelled 13C_
glucose. Aust. J. Soil Res. 28:193-212.
Baldock, I.A., G.J. Currie, and J.M. Oades. 1991. Organic matter as seen by solid state 13C NMR and
pyrolysis tandem mass spectrometry. p. 45-{i0. In W.S. Wilson (ed.) Advances in soil organic
matter research: The impact on agriculture and the environment. Proc. Jt. Symp., Univ. Essex,
3-4 Sept. 1990. R. Soc. Chern., Cambridge, England.
Barker, S.A, P. Finch, M.H.B. Hayes, R.G. Simmonds, and M. Stacey. 1965. Isolation and preliminary characterization of soil polysaccharides. Nature (London) 205:68-{i9.
Barker, S.A, M.H.B. Hayes, R.G. Simmonds, and M. Stacey. 1967. Studies on soil polysaccharides.
I. Carbohyd. Res. 5:13-24.
Becket, R, Z. Jue, and J.C. Giddings. 1987. Determination of molecular weight distributions of fulvic and humic acids using flow field-flow fractionation. Environ. Sci. Technol. 21:289-295.
Beyer, L., H.-R. Schulten, and R. Friind. 1992. Properties and composition of soil organic matter in
forest and arable soils of Schleswig-Holstein: 1. Comparison of morphology and results of wet
chemistry, CPMAS-13C-NMR spectroscopy and pyrolysis-field ionisation mass spectrometry.
Z. Pflanzenerniihr. Bobenk. 155:345-354.
Bloom, P.R., and J.A Leenheer. 1989. Vibrational, electronic, and high-energy spectroscopic methods for characterizing humic substances. p. 409-446./n M.H.B. Hayes et al. (ed.) Humic substances II. In search of structure. Wiley-Intersci., Chichester, England.
Bracewell, I.M., K. Haider, S.R. Larter, and H.-R. Schulten. 1989. p. 180-222. Thermal degradation
relevant to structural studies of humic substances. In M.H.B. Hayes et al. (ed.) Humic substances II. In search of structure. Wiley-Intersci., Chichester, England.
Bracewell, 1.M., G.W. Robertson, and B.L. Williams. 1980. Pyrolysis-mass spectrometry studies of
humification in a peat and a peaty podzol. J. Anal. Appl. Pyrol. 2:239-248.
Brooks, J.D., R.A Durie, and S. Sternhell. 1958. Chemistry of brown coals. Aust. J. Appl. Sci.
9:303-320.
Burch, R.D., C.H. Langford, and D.S. Gamble. 1978. Methods for the comparison of fulvic acid samples: The effects of origin and concentration on acidic properties. Can. J. Chern.
56:11%-1201.
Cameron, R.S., B.K. Thornton, R.S. Swift, and AM. Posner. 1972a. Molecular weight and shape of
humic acid from sedimentation and diffusion measurements on fractionated extracts. J. Soil
Sci. 23:394-408.
Cameron, RS., R.S. Swift, B.K. Thornton, and A.M. Posner. 1972b. Calibration of gel permeation
chromatography materials for use with humic acid. 1. Soil Sci. 23:342-349.
Chaplin, M.E, and I.E Kennedy. 1986. Carbohydrate analysis. IRL Press, Oxford.
Chen, S., w.P. Inskeep, S.A. Williams, and P.R Callis. 1992. Complexation of I-Naphthol by humic
and fulvic acids. Soil Sci. Soc. Am. J. 56:67-73.
Chen, Y., and M. Schnitzer. 1976. Viscosity measurements on soil humic substances. Soil Sci. Soc.
Am. J. 40:866-872.
Chen, Y., and M. Schnitzer. 1989. Sizes and shapes of humic substances by electron microscopy. p.
62140. In M.H.B. Hayes et al. (ed.) Humic substances II. In search of structure. Wiley-Intersci., Chichester, England.

1064

SWIFT

Chen, Y., N. Senesi, and M. Schnitzer. 1977. Information provided on humic substances by EJE6
ratios. Soil Sci. Soc. Am. J. 41:352-358.
Chen, Y., N. Senesi, and M. Schnitzer. 1978. Chemical and physical characteristics of humic and fulvic acid extracted from soils of the mediterranean region. Geoderma 20:87-104.
Cheshire, M.V. 1979. Nature and origin of carbohydrates in soils. Acad. Press, London.
Cheshire, M.V., and Hayes, M.H.B. 1990. Composition, origin, structures, and reactivities of soil
polysaccharides. p. 307-336. In M.E De Boodt et al. (ed.) Soil colloids and their association
in aggregates. Plenum Press, New York.
Cheshire, M.V., J.M. Bracewell, e.M. Mundie, G.w. Robertson, J.D. Russell, and AR. Fraser. 1979.
Structural studies on soil polysaccharides. J. Soil Sci. 30:315-326.
Cheshire, M.V., e.M. Mundie, J.M. Bracewell, G.W. Robertson, J.D. Russell, and AR. Fraser. 1983.
The extraction and characterization of soil polysaccharide by whole soil methylation. J. Soil
Sci. 34:539--554.
Christensen, J.J., L.D. Hansen, and R.M. Izatt. 1976. Handbook of proton ionization heats and related thermodynamic quantities. J. Wiley & Sons, Inc., New York.
Clapp, C.E., W.W. Emerson, and AE. Olness. 1989. Sizes and shapes of humic substances by viscosity measurements. p. 497-514. In M.H.B. Hayes et al. (ed.) Humic substances II. In search
of structure. Wiley-Intersci., Chichester, England.
Dalal, R.e., and R.J. Mayer. 1986. Long-term trends in fertility of soils under continuous cultivation
and cereal cropping in southern Queensland. IV. Loss of organic carbon from different density fractions. Aust. J. Soil Res. 24:293-300.
Davis, J.A 1982. Adsorption of natural dissolved organic matter at the oxide/water interface.
Geochim. Cosmochim. Acta 46:2381-2393.
Dawson, H.J., B.E Hrutfiord, R.J. Zasoski, and Ee. Ugolini. 1981. The molecular weight and origin
of yellow organic acids. Soil Sci. 132:191-199.
De Nobili, M. 1988. Electrophoretic evidence of the integrity of humic substances separated by means
of electrofocusing. J. Soil Sci. 39:437-445.
De Nobili, M., G. Bragato, J.M. Alcaniz, A Puigbo, and L. Comellas. 199Oa. Characterization of
electrophoretic fractions of humic substances with different electrofocusing behaviour. Soil
Sci. 150:763-770.
De Nobili, M., M. Contin, and L. Leita. 1990b. Alternative method for carboxyl group determination
in humic substances. Can. J. Soil Sci. 70:531-536.
Dragunov, S.S., and B.G. Murzakov. 1970. Heterogeneity of fulvic acids of a chernozem. Prochvovedenie 3:115-121.
Drijber, R.A, and L.E. Lowe. 1990. Nature of humus in andosols under differing vegetation in the
Sierra Nevada, Mexico. Geoderma 47:221-231.
Dubach, P., N.e. Mehta, T. Jakab, E Martin, and N. Roulet. 1964. Chemical investigations on soil
humic substances. Geochim. Cosmochim. Acta 28:1567-1578.
Dubois, M., KA Gilles, J.K Hamilton, P.A Rebers, and E Smith. 1956. Colorimetric method for
determination of sugars and related substances. Anal. Chern. 28:350--356.
Ducaroir, J., P. Cambier, J.-P. Leydecker, and R. Prost. 1990. Application of soil fractionation methods to the study of the distribution of pollutant metals. Z. Pflanzenerniihr. Bodenk.
153:349--358.
Duxbury, J.M. 1989. Studies of the molecular size and charge of humic substances by electrophoresis. p. 593-620. In M.H.B. Hayes et al. (ed.) Humic substances II. In search in structure.
Wiley-Intersci., Chichester, England.
Finch, P., M.H.B. Hayes, and M. Stacey. 1966. p. 19--32. In G.v. Jacks (ed.) Soil chemistry and fertility. Trans. Jt. Meet. Comm. 2, 4, Int. Soc. Soil Sci. 1966. Univ. Press, Aberdeen, Scotland.
Flaig, w., H. Beutelspacher, and E. Rietz. 1975. Chemical composition and physical properties of
humic substances. p. 28. In J.E. Gieseking (ed.) Soil components. Vol. 1. Organic components.
Springer-Verlag, New York.
Forsyth, W.G.e. 1947. Studies on the more soluble complexes of soil organic matter. I. A method of
fractionation. Biochem. J. 41:176--181.
Fritz, J.S., S.S. Yamamura, and E.C. Bradford. 1959. Determination of carbonyl compounds. Anal.
Chern. 31:260--263.
Gamble, D.S. 1972. Potentiometric titration of fulvic acid: Equivalence point calculations and acidic
functional groups. Can. J. Chern. 50:2680--2690.
Ghosh, K, and M. Schnitzer. 1979. UV and visible absorption spectroscopic investigations in relation
to macromolecular characteristics of humic substances. J. Soil Sci. 30:735-745.
Ghosh, K, and M. Schnitzer. 1980. Macromolecular structures of humic substances. Soil Sci.
129:266--276.

ORGANIC MATTER CHARACTERIZATION

1065

Glebko, L.I., J.U. U1kina, and O.B. Maximov. 1970. A semi-micro-method for the determination of
quinoid groups in humic acids. Mikrochim. Acta. 1247-1254.
Greenland, 0.1., and G.w. Ford. 1964. Separation of partially humified organic materials from soils
by ultrasonic dispersion. p. 137-148. In Trans. 8th Int. Congr. Soil Sci. 1964. Acad. Socialist
Rep. Romania, Bucharest, Romania.
Greenland, OJ., G.P. Lindstrom, and J.P. Quirk. 1962. Organic materials which stabalise natural
aggregates. Soil Sci. Soc. Am. Proc. 26:366-371.
Greenland, O.G., and J.M. Oades. 1975. Sacharrides. p. 213-261. In 1.E. Gieseking (ed.) Soil components. Vol. 1. Organic compounds. Springer, New York.
Haider, K, and H.-R. Schuten. 1985. Pyrolysis-field ionisation mass spectrometry of lignins, soil
humic compounds and whole soil. 1. Anal. Appl. Pyro .. 8:317-331.
Haider, K, B.R. Nagar, H.L.e. Meuzelaar, e. Saiz-Jimenez, and J.P. Martin. 1977. Studies on soil
humic compounds, fungal melanins and model polymers by pyrolysis-mass spectrometry. p.
213-220. In Soil organic matter studies. Proc. Symp. Soil Organic Matter Studies, Braunschueis, Germany. 6-10 Sept. 1976. Int. At. Energy Agency, Vienna.
Halma, G., M.A. Posthumus, R. Miedema, W. van de Westeringh, and H.L.C. Meuzelaar. 1978. Characteriation of soil types by pyrolysis-mass spectrometry. Agrochimica 22:372-381.
Hayes, M.H.B. 1985. Extraction of humic substances from soil. p. 329-361. In G.R. Aiken et al. (ed.)
Humic substances in soil, sediment and water. Geochemistry, isolation and characterization.
Wiley Intersci., New York.
Hayes, M.H.B., and R.S. Swift. 1978. The chemistry of soil organic colloids. p. 179-320. In OJ.
Greenland and M.H.B. Hayes (ed.) The chemistry of soil constituents. Wiley-Intersci., New
York.
Hayes, M.H.B., R.S. Swift, R.E. Wardle, and J.K Brown. 1975. Humic materials from an organic
soil: A comparison of extractants and of properties of extracts. Geoderma 13:231-245.
Hayes, M.H.B., P. MacCarthy, R. Malcolm, and RS. Swift. 1989. Humic substances II. In search of
structure. Wiley-Intersci., Chichester, England.
Haynes, RJ., and R.S .Swift. 1990. Stability of soil aggregates in relation to organic constituents and
soil water content. J. Soil Sci. 41:73-83.
Hempfling, R., and H-R. Schulten. 1991. Pyrolysis-(gas chromatography!) mass spectrometry of agricultural soils and their humic fractions. Z. Pflanzenernahr. Bodenk. 154:425-430.
Howarth, RJ., and R Sinding-Larsen. 1983. Multivariate analysis. p. 207-289. In R.J. Howarth (ed.)
Statistics and data analysis in geochemical prospecting. Elsevier, Amsterdam.
Hunter, R.J. 1989. Foundations of colloid science. Vol. 1. Oxford Press, Oxford, England.
Konova, M.M. 1966. Soil organic matter. Permagon, Elmsford, New York.
Kortum, G. 1969. Reflectance spectroscopy. Springer-Verlag, New York.
Krosshaven, M., J.O. Bj0rgum, J. Krane, and E. Steinnes. 1990. Chemical structure of terrestial
humus materials formed from different vegetation characterized by solid-state \3C NMR with
CP-MAS techniques. 1. Soil Sci. 41:371-377.
Krosshaven, M., I. Kiigel-Knabner, T.E. Southon, and E. Steinnes. 1992. The influence of humuc fractionation on the chemical composition of soil organic matter studied by solid-state \3C NMR
J. Soil Sci. 43:473-483.
Kumada, K, and Y. Kawamura. 1968. On the fractionation of humic acids by a fractional precipitation technique. Soil Sci. Plant Nutr. 14:198-200.
Kyuma, K 1964. A fractional precipitation technique applied to soil humic substances. Soil Sci. Plant
Nutr. 10:33-35.
Lindqvist, I. 1972. Charge transfer interaction of humic acids with donor molecules in aqueous solutions. Swed. J. Agric. Res. 12:105-109.
Lindqvist, I. 1973. Partial reduction of a humic acid. Swed. J. Agric. Res. 13:69-73.
MacCarthy, P., and H.B. Mark, Jr. 1975. Infrared studies on humic acid in deuterium oxide. I. Evaluation and potentialities of the technique. Soil Sci. Soc. Am. Proc. 39:663-668.
MacCarthy, P., and H.B. Mark, Jr. 1976. An evaluation of Job's method of continuous variations as
applied to soil organic matter-metal ion interactions. Soil Sci. Soc. Am. J. 40:267-276.
MacCarthy, P., and S. O'Cinneide. 1974a. Fulvic acid. II. Interaction with metal ions. J. Soil Sci.
25:429-437.
MacCarthy, P., and S. O'Cinneide. 1974b. Fulvic acid. I. Partial fractionation. J. Soil Sci. 25:420-428.
MacCarthy, P., M.J. Peterson, R.L. Malcolm, and E.M. Thurman. 1979. Separation of humic substances by pH gradient desorption from a hydrophobic resin. Anal. Chern. 51:2041-2043.
Malcolm, R.L. 1989. Application of solid-state 13C-NMR spectroscopy to geochemical studies of
humic substances. p. 339-371. In M.H.B. Hayes et al. (ed.) Humic substances. Vol. 2. Wiley
Intersci., Chichester, England.

SWIFf

1066

Martell, A.E.; and RM. Smith. 1977. Critical stability constants. Vol. 3. Other organic ligands.
Plenum, New York.
Ma'shum, M.E., M.E. Tate, G.P. Jones, and J.M. Oades. 1988. Extraction and characterization of
water-repellent materials from Australian soils. J. Soil Sci. 39:99-109.
Meuzelaar, H.L.C., J. Haverkamp, and F.D. Hileman. 1982. Pyrolysis-mass spectrometry of recent
and fossil biomaterial~ompendium and atlas. Elsevier, Amsterdam.
Mortensen, J.L., and R.B. Schwendinger. 1963. Electrophoretic and spectroscopic characterization of
high molecular weight components of soil organic matter. Geochim. Cosmochim. Acta
27:201-208.
Niemeyer, 1., Y. Chen, and J.-M. Bollag. 1992. Characterization of humic acids, composts, and peat
by diffuse reflectance Fourier Transform Infrared Spectroscopy. Soil Sci. Soc. Am. J.
56:135-140.
Nguyen, T.T., L.J. Janik, and M. Raupach. 1991. Diffuse reflectance infrared fourier transform
(DRIFI) spectroscopy in soil studies. Aust. J. Soil Res. 29:49-67.
Oades,1.M. 1967. Carbohydrates in some Australian soils. Aust. J. Soil Res. 5:103-115.
Oades, 1.M., MA Kirkman, and G.H. Wagner. 1970. The use of gas-liquid chromatography for the
determination of sugars extracted from soils by sulfuric acid. Soil Sci. Soc. Am. Proc.
34:230-235.
Oades, 1.M., A.M. Vassallo, A.G. Waters, and M.A. Wilson. 1987. Characterization of orf,anic matter in particle size and density fractions from a red-brown earth by solid-state 1 C N.M.R.
Aust. J. Soil Res. 25:71-82.
Oliver, B.G., E.M. Thurman, and RL. Malcolm. 1983. The contribution of humic substances to the
acidity of colored waters. Geochim. Cosmochim. Acta 47:2031-2035.
Overend, w.G., C.W. Rees, and J.S. Sequira. 1962. No. 675 Reactions at position 1 of carbohydrates.
Part III. The acid-catalyzes hydrolysis of glycosides. J. Chern. Soc. 3:3429-3440.
Parsons, J. W., and 1. Tinsley. 1961. Chemical studies of polysaccharide material in soil composts
based on extraction with anhydrous formic acid. Soil Sci. 92:46-53.
Perdue, E.M. 1978. Solution thermochemistry of humic substances-I. Acid-base equilibria of humic
acid. Geochim. Cosmochim. Acta 42:1351-1358.
Perdue, E.M. 1985. Acidic functional groups of humic substances. p. 493-526. In G.R Aiken et al.
(ed.) Humic substances in soil, sediment and water. Geochemistry, isolation and characetrization. Wiley-Intersci., New York.
Perdue, E.M., J.H. Reuter, and M. Ghosal. 1980. The operational nature of acidic functional group
analyses and its impact on mathematical descriptions of acid-base equilibria in humic substances. Geochim. Cosmochim. Acta 44:1841-1851.
Piccolo, A. 1988. Characteristics of soil humic extracts obtained by some organic and inorganic solvents and purified by HCI-HF treatment. Soil Sci. 146:41~26.
Piccolo, A., and Mirabella, A. 1987. Molecular weight distribution of peat humic substances extracted with different inorganic and organic solutions. Sci. Total Environ. 62:39-46.
Posner, A.M. 1966. The humic acids extracted by various reagents from a soil. Part I. Yield, inorganic components, and titration curves. J. Soil Sci. 17:65-78.
Posner, A.M., and 1.M. Creeth. 1972. A study of humic acid by equilibrium ultracentrifugation. 1. Soil
Sci. 23:333-341.
Posner, A.M., B.K.G. Theng, and 1.RH. Wake. 1968. The extraction of soil organic matter in relation
to humification. p. 153-162. In 1.W. Holmes (ed.) Trans. 9th Int. Congr. Soil Sci., 3. 1968.
August Robertson, Sydney, Australia.
Reid, P.M., A.E. Wilkinson, E. Tipping, and M.N. Jones. 1991. Aggregation of humic substances in
aqueous media as determined by light-scattering methods. 1. Soil Sci. 259-270.
Reuter, J.H., and E.M. Perdue. 1981. Calculation of molecular weights of humic substances from colligative data: Application to aquatic humus and its molecular size fractions. Geochim. Cosmochim. Acta 45:2017-2022.
Rice, 1., and P. MacCarthy. 1989. Isolation of humin by liquid-liquid partitioning. Sci. Total Environ.
81/82:61~9.

Ristori, G.G., E. Sparvoli, M. De Nobili, and L.P. D' Acqui. 1992. Characterization of organic matter
in particle-size fractions of vertisols. Geoderma 54:295-305.
Ritchie, G.S.P., and A.M. Posner. 1982. The effect of pH and metal binding on the transport properties of humic acids. 1. Soil Sci.33:233-247.
Roth, C.H., W. Wilczynski, and C. de Castro Filho. 1992. Effect oftiIIage and liming on organic matter composition in a Rhodic Ferralsol from Southern Brazil. Z. Pflanzenerniihr. Bodenk.
155:175-179.

ORGANIC MATIER CHARACTERIZATION

1067

Ryan, D.K., and 1.H. Weber. 1982. Fluorescence quenching titration for determination of complexing
capacities and stability constants of fulvic acid. Anal. Chern. 54:986--990.
Saiz-limenez, e., K. Haider, and H.L.e. Meuzelaar. 1979. Comparisons of soil organic matter and its
fractions by pyrolysis-mass spectrometry. Geoderma 22:25-37.
Saiz-limenez, C., e. Martin, K. Haider, and H.L.e. Meuzelaar. 1978. Comparison of humic and fulvic acids from different soils by pyrolysis-mass spectrometry. Agrochimica 22:353-359.
Sanderson, G.W., and B.P.M. Perera. 1966. Removal of poly phenolic compounds interfering with carbohydrate determinations in plant extracts with an insoluble polyphenol adsorbent. Analyst
91:335-336.
Schlesinger, W.H. 1984. Soil organic matter: A source of atmospheric CO2, p. 111-127. In G.M.
Woodwell (ed.) The role of terrestrial vegetation in the global carbon cycle: Measurement by
remote sensing. SCOPE, Wiley, Chichester, England.
Schnitzer, M. 1977. Recent findings on the characterisation of humic substances extracted from soils
from widely differing climatic zones. p. 117-131. In Proc. Symp. Soil Organic Matter Studies, Braunschweig, Germany. 6--10 Sept. 1976. Int. At. Energy Agency, Vienna.
Schnitzer, M. 1990. Selected methods for characterization of soil humic substances. p. 65-90. In P.
MacCarthy et al. (ed.) Humic substances in soil and crop sciences: Selected readings. ASA
and SSSA, Madison, WI.
Schnitzer, M., e.A. Hindle, and M. Megliz. 1986. Supercritical gas extraction of alkanes and alkonoic
acids from soils and humic materials. Soil Sci. Soc. Am. 1. 50:913-919.
Schnitzer, M., and S.U. Khan. 1972. Humic substances in the environment. Marcel Dekker Inc., New
York.
Schnitzer, M., and S.I.M. Skinner. 1965. Organo-metallic interactions in soils: 4. carboxyl and
hydroxyl groups in organic matter and metal retention. Soil Sci. 99:278-284.
Schnitzer, M., and S.I.M. Skinner. 1968. Gel filtration of fulvic acid, a soil humic compound. Isotopes
and radiation in soil organic matter studies. Int. At. Energy Agency, Vienna.
Schnitzer, M., and S.I.M. Skinner. 1969. Free radicals in soil humic compounds. Soil Sci.
108:383-390.
Schulten, H.-R., and R. Hempfling. 1992. Influence of agricultural and management on humus compositon and dynamics: Classical and modem analytical techniques. Plant Soil 142:259-271.
Senesi, N. 1990. Molecular and quantitative aspects of the chemistry of fulvic acid and its interactions
with metal ions and organic chemicals. Part II. The fluorescence spectroscopy approach. Anal.
Chim. Acta 232:77-106.
Senesi, N., Y. Chen, and M. Scnitzer. 1977. Hyperfine splitting in electron spin resonance spectra of
fulvic acid. Soil BioI. Biochem. 9:371-372.
Skjemstad,1. 1992. Genesis of podzols on coastal dunes in southern Queensland. III. The role of aluminum-organic complexes in profile development. Aust. J. Soil Res. 30:645-{)65.
Smith, R.G., Jr. 1976. Evaluation of combined applications of ultrafiltration and complexation capacity techniques to natural waters. Anal. Chern. 48:74-76.
Soli ins, P., G. Sprycher, and e.A. Glassman. 1984. Net nitrogen mineralization from light- and heavyfraction forest soil organic matter. BioI. Biochem. 16:31-37.
Spark, K.M., and R.S. Swift. 1994. Investigation of some of the parameters affecting the fluorescence
spectra of humic substances. p. 153-160. In N. Senesi and T.M. Miano (ed.) Humic substances
in the global environment and implications on human health. Elsevier, Amsterdam.
Steelink, e.A 1964. Free radical studies of lignin, lignin degradation products, and soil humic acid.
Geochim. Cosmochim. Acta 28:1615-1622.
Steelink, C.A., M.A Mikita, and K.A Thorn. 1983. Magnetic studies of humates and related model
compounds. p. 83-105. In R.E Christman and E.J. Gjessinger (ed.) Aquatic and terrestrial
humic materials. Ann Arbor, Sci., Ann Arbor, MI.
Stevenson, FJ. 1994. Humus chemistry. Genesis, composition, reactions. 2nd ed. John Wiley & Sons,
New York.
Stevenson, FJ., Q. van Winkle, and w.P. Martin. 1953. Physiochemical investigations of clayadsorbed organic colloids II. Soil Sci. Soc. Am. Proc. 17:31-34.
Strickland, T.e., and P. Sollins. 1987. Improved method for separating light- and heavy-fraction
organic material from soil. Soil Sci. Soc. Am. I. 51:139(}"1393.
Swift, R.S. 1985. Fractionation of soil humic substances. p. 387-408. In G.R. Aiken et al. (ed.) Humic
substances in soils, sediment, and water. Wiley-Intersci., New York.
Swift, R.S. 1989. Molecular weight, size, shape, and charge characteristics of humic substances: some
basic considerations. p. 449-466. In M.H.B. Hayes et al. (ed.) Humic substances II. In search
of structure. Wiley-Intersci., Chichester, England.
Swift, R.S., and AM. Posner. 1971. Gel chromatography of humic acid. J. Soil Sci. 22:237-249.

1068

SWIFT

Swift, R.S., and A.M. Posner. 1972. Autoxidation of humic acid under alkaline conditions. J. Soil Sci.
23:381-393.
Swift, R.S., R.L. Leonard, R.H. Newman, and B.K.G. Theng. 1992. Changes in humic acid composition with molecular weight as detected by \3C-nuclear magnetic resonance spectroscopy. Sci.
Total Environ. 117/118:53-61.
Swincer, G.D., I.M. Oades, and D.J. Greenland. 1968. Studies on soil polysaccharides. I. The isolation of polysaccharides from soil. Aust. 1. Soil Res. 6:211-224.
Swincer, G.D., I.M. Oades, and OJ. Greenland. 1969. The extraction, characterization, and significance of soil poysaccharides. Adv. Agron. 21:195-235.
Theng, B.K.G., and A.M. Posner. 1967. Nature of the carbonyl groups in soil humic acid. Soil Sci.
104:191-201.
Theng, B.K.G., K.R. Tate, and P. Becker-Heidmann. 1992. Towards establishing the age, location, and
identity of the inert soil organic matter of a spodosol. Z. Pflanzenerniihr. Bodenk.
155:181-184.
Theng, B.K.G., 1.R.H. Wake, and A.M. Posner. 1968. The fractional precipitation of soil humic acid
by ammonium sulphate. Plant Soil 29:305-316.
Thomas, R.L., J.L. Mortenson, and EL. Himes. 1967. Fractionation and characterization of a soil
polysaccharide extract. Soil Sci. Soc. Am. Proc. 31:568-570.
Thurman, E.M., and R.L. Malcolm. 1981. Preparative isolation of aquatic humic substances. Environ.
Sci. Technol. 15:463-466.
Tomar, N.K., R.P. Yadav, and P.S. Relan. 1992. Characterisation of humic and fulvic acids extracted
with NaOH and NaOH-Na-pyrophosphate mixture from soils of arid and subhumid regions 1.
Analytical characteristics. Arid Soil Res. Rehab. 6:177-185.
Tsutsuki, K., and S. Kuwatsuka. 1979. Chemical studies on soil humic acids. VII. pH dependent
nature of the ultra-violet and visible absorption spectra of humic acids. Soil Sci. Plant. Nutr.
25:373-384.
Tsutsuki, K., and S. Kuwatsuka. 1984. Molecular size distribution of humic acid as affected by the
ionic strength and degree of humification. Soil Sci. Plant Nutr. 30:151-162.
Turchenek, L.W., and I.M. Oades. 1979. Fractionation of organo-mineral complexes by sedimentation and density techniques. Geoderma 21:311-343.
Varadachari, C., A.K. Barman, and K. Ghosh. 1983. Electron spin resonance and scanning
microscopy of humic sbustances. J. Indian Soc. Soil Sci. 31:28-30.
Visser, S.A. 1985. Viscosimetric studies on molecular weight fractions of fulvic and humic acids of
aquatic, terrestrial and microbial origin. Plant Soil 87:209-221.
Waite, T.D., and M.M. Morel. 1984. Coulometric study of the redox dynamics of iron in seawater.
Anal. Chern. 56:787-792.
Watanabe, A., and S. Kuwatsuka. 1991. Fractionation of soil fulvic acids using polyvinyl-pyrrolidone
and their ionization difference spectra. Soil Sci. Plant Nutr. 37:611-617.
Wershaw, R.L. 1985. Application of nuclear magnetic resonance spectroscopy for determining functionality in humic substances. p .561-584. In G.R. Aiken et al. (ed.) Humic substances in soils,
sediment and water. Wiley-Intersci., New York.
Wershaw, R.L. 1989. Sizes and shapes of humic substances by scattering techniques. p. 545-559. In
M.H.B. Hayes et al. (ed.) Humic substances II. In search of structure. Wiley-Intersci., Chichester, England.
Whitehead, D.C., and J. TInsley. 1964. Extraction of soil organic matter with dimethylformamide.
Soil Sci. 97:34--42.
Williams, B.G., OJ. Greenland, and J.P. Quirk. 1967. The effect of polyvinyl alcohol on the nitrogen
surface area and pore structure of soils. Aust. J. Soil Res. 5:77-92.
Wilson, M.A., and K.M. Goh. 1983. NMR spectroscopy of soils. Structure of organic material in sodium deuteroxide extracts from Patua Loam, New Zealand. J. Soil Sci. 34:305-313.
Wilson, M.A., A.H. Gillam, and PJ. Collins. 1983. Analysis of the structure of dissolved humic substances and their phytoplankton precursors by lH and \3C nuclear magnetic resonance. Chern.
Geol. 40:187-201.
Wilson, M.A., A.M. Vassallo, E.M. Perdue, and I.H. Reuter. 1987. Compositional and solid-state
nuclear magnetic resonance study of humic and fulvic acid fractions of soil organic matter.
Anal. Chern. 59:551-558.
Wright, I.R., and M. Schnitzer. 1959. Oxygen-containing functional groups in the organic matter of a
podzol soil. Nature (London) 184:1462-1463.
Yonebayashi, K., and T. Hattori. 1985. Non-aqueous titration of functional groups in humic acid. Org.
Geochem. 8:47-54.

ORGANIC MATTER CHARACTERIZATION

1069

Yonebayashi, K., and T. Hattori. 1990. A new fractionation of soil humic acids by adsorption chromatography. Geoderma 47:327-336.
Zech, W., R. Hempfling, L. Haumaier, H.-R. Schulten, and K. Haider. 1990. Humification in subalpine rendzinas: Chemical analyses, IR and 13C NMR spectroscopy and pyrolysis-field ionisation mass spectometry. Geoderma 47:123-138.
Zhang, T., H. Gan, and P.F. Low. 1991. Effect of sodium-humate on the rheological characteristics of
montmorillonite suspensions. Soil Sci. Soc. Am. I. 55:989-993.