0038-075C/01/16611-810–832
Soil Science
Copyright © 2001 by Lippincott Williams & Wilkins, Inc.
November 2001
Vol. 166, No. 11
Printed in U.S.A.
THE SUPRAMOLECULAR STRUCTURE OF HUMIC SUBSTANCES
Alessandro Piccolo
The scientific understanding of the molecular size and shape of humic
substances (HS) is critically reviewed. The traditional view that HS are
polymers in soil is not substantiated by any direct evidence but is assumed only on the basis of laboratory experiments with model molecules
and unwarranted results produced by incorrectly applying either analytical procedures or mathematical treatments developed for purified and
undisputed biopolymers. A large body of evidence shows an alternative
understanding of the conformational nature of HS, which should be regarded as supramolecular associations of self-assembling heterogeneous
and relatively small molecules deriving from the degradation and decomposition of dead biological material. A major aspect of the humic
supramolecular conformation is that it is stabilized predominantly by
weak dispersive forces instead of covalent linkages. Hydrophobic (van
der Waals, -, CH-) and hydrogen bonds are responsible for the apparent large molecular size of HS, the former becoming more important
with the increase of pH. This innovative understanding of the nature of
HS implies a further development of the science and technology for the
control of the chemistry and reactivity of natural organic matter in the
soil and the environment. (Soil Science 2001; 166:810–832)
Key words: Humic substance structure, size exclusion chromatography, supramolecular associations.
substances (HS), natural organic subH
stances that are ubiquitous in water, soil, and
sediments, are of paramount importance in sus-
(including potential pollutants) in the soil environment (Tate, 1999).
Most of the difficulties encountered in chemically defining the structures and reactivities of HS
derive from their large chemical heterogeneity
and geographical variability. The substances are
undoubtedly mixtures that develop randomly
from the decay of plant tissues, from microbial
metabolism-catabolism, or from both. Thus, the
chemistry is not only highly complex, but it is also
a function of the different general properties of
the ecosystem in which it is formed, such as vegetation, climate, topography, etc. It is not surprising that, despite the efforts of many excellent scientists in the distant and recent past (Kononova,
1961; Stevenson, 1994), there is still much to be
done to achieve an appropriate awareness of humic chemistry.
The objective of this contribution is to report
on recent experimental findings leading to a new
understanding of the conformational nature of
HS and to mention the profound implications
that this may have on our understanding of soil
organic matter (SOM) functions and reactivities.
UMIC
taining plant growth and controlling both the
fate of environmental pollutants and the biogeochemistry of organic carbon (OC) in the global
ecosystem (Piccolo, 1996). Despite their role in
the sustainability of life, the basic chemical nature
and the reactivities of HS are still poorly understood. The scientific community of humic scientists has so far failed to provide a unified understanding of this field of science, and there is still,
therefore, a poor awareness of fundamental aspects of humic structures and reactivities. Nevertheless, the implications of the relevance of
awareness of HA structure should extend far beyond the interests of a few chemists; HA structures affect the ways that the soil ecosystem work,
as well as the bioavailabilty of organic substances
Dipartimento di Scienze Chimico-Agrarie, Università di Napoli “Federico II” 80055,
Portici, Italy. E-mail: [email protected].
Received June 4, 2001; accepted Aug. 21, 2001.
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SUPRAMOLECULAR STRUCTURE OF HS
Traditional Paradigms in Humus Chemistry
The amount of HS in soils is several times
greater than the amount in waters. Up to 70–80%
of the OC in mineral soils may be composed of
humic material, with recognizable plant remains
constituting a small percentage of the organic
matter (OM) of mineral soils. Even though the
abundance of OC in HS is from 2 to 3 times
greater than that in the terrestrial biomass, the latter greatly influences the OM dynamics in soils
(Insam, 1996). Biological activity rapidly decomposes labile plant materials on entering aerobic
soil environments with adequate water supplies,
but more resistant components transform slowly
in the same environment. The compositional diversities and the differences in the modes of
transformation of the components make it extremely difficult to define accurately the gross
mixtures that compose SOM, or the dissolved
(DOM), or the particulate organic matter (POM)
of waters.
Because of the heterogeneity of humic mixtures, simplification and reductionism must be
adopted. Stevenson (1994), in summarizing previous reports and definitions, stated that humus
includes a broad spectrum of organic constituents, many of which have their counterparts
in biological tissues. He distinguished between
non-HS and HS, the former of which consists of
compounds belonging to the well known classes
of organic chemistry such as amino acids, carbohydrates, lipids, lignin, and nucleic acids, whereas
the latter are unspecified, transformed, dark colored, heterogeneous, amorphous and high molecular weight (MW) materials.
The classical definitions of HS are operational only and are based on solubility properties
in the aqueous solutions used as soil extractants.
The generalized terms humic acids (HAs), fulvic
acids (FAs), and humins cover the major fractions
still used to describe HS components, but the
boundaries between these fractions have not
been yet clarified in chemical terms. Because
modern analytical techniques for organic compounds were not available during the first half of
the 20th century, the data that were available
from elemental analyses (C, H, O) and determinations of acidic functional groups suggested that
HAs and FAs from many different soils were relatively similar (Kononova, 1961). Such simple
correlations, although not based on any molecular understanding, encouraged scientists to consider humic fractions to be chemical entities having specific properties rather than complex
mixtures of nonspecific compounds.
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Many of the modern concepts of HS derive
from theories illustrated in the book of Kononova (1961). In a review of hypotheses advanced predominantly by Russian scientists,
Kononova introduced the concept of HS as a system of polymers, based on the observation that
elemental composition, optical properties, exchange acidities, electrophoretic properties, and
MW characteristics varied consistently with soil
classes. Using this concept, the various fractions
of HS obtained on the basis of solubility characteristics are imagined to be part of a heterogeneous mixture of molecules, which, in any given
soil, range in MW from as low as several hundreds
to perhaps over 300,000 Da, and exhibit a continuum of any given chemical property (Stevenson, 1994).
It must be noted that despite the existence of
data showing that the molecular dimensions (as
measured by osmometry, viscometry, and diffusion) of some HS were scarcely beyond one or
two thousand Da (Scheffer and Ulrich, 1960;
Schnitzer and Khan, 1972), more reliance was
placed on the early work of Flaig and Beutelspacher (1958) who showed, using the ultracentrifuge, that MW values were in the range of
30,000 to 50,000 Da for HAs and about 10,000
Da for FAs. One reason for such bias towards
high molecular weight (HMW) structures may
be found in the historical hypotheses that considered HS to be products of biologically-assisted
syntheses from compounds derived from degradations of lignin, polyphenols, cellulose, and
amino acids. Evidence for this polymeric assumption was researched in many classical laboratory experiments that indicated possibilities for
either abiotic or biotic condensations of simple
molecules into humic-like materials (Kononova,
1961). Some of these early laboratory studies
have been researched in more carefully defined
conditions in recent times (Haider and Martin,
1967; Martin and Haider, 1969; Flaig et al., 1975;
Flaig, 1988; Hedges, 1988). However, no direct
evidence has ever been provided for the occurrence of such polymer build-up processes in natural soil systems.
The polymeric view of HS has included the
general concept of polydispersity (Dubach and
Metha, 1963), in which the HS are made of polymers with different MW values, similar to that
which applies for other natural biological macromolecules such as proteins, polysaccharides, and
lignin. The experimental difficulties encountered
in isolating chemically homogeneous fractions of
HS was compared with those observed for bio-
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polymers with varying molecular dimensions
synthesized in the living cell. This unwarranted
similarity supported the polydisperse polymeric
concept of HS and justified the observation that
these are mixtures of compounds. The concept of
HMW and polydisperse polymers became a paradigmatic part of the descriptive definitions of
HS proposed thereafter (Schnitzer and Khan,
1972; Aiken et al., 1985; Malcolm, 1990; Stevenson, 1994).
The assumption that HS are polymers, which
has no sound molecular basis, has promulgated the
use of simple physical-chemical measurements to
characterize HS. An example is the E4/E6 index,
the ratio of the absorbance of HS at 465 nm to that
at 665 nm, introduced by Welte (1955) and reproposed by Kononova (1961). This ratio supposedly
indicates a reverse relationship with progressive
humification as well as increased condensation assumed to produce large amounts of polycondensated aromatic-ring structures. Despite its widespread and continued application, the E4/E6 ratio
has repeatedly been shown not to hold the predicted relationship with MW.At variance with the
hypotheses related to this ratio, Campbell et al.
(1967) had already indicated that humic material
with the lowest mean residence time in the soil
had the highest E4/E6 ratio, and Anderson and
Hepburn (1978), using gel permeation chromatography, showed that humic fractions with
large molecular sizes and low E4/E6 ratios were
mainly aliphatic, whereas those with molecular
size had the highest aromatic contents. Piccolo
(1988) compared the E4/E6 ratios of various HS
with their gel permeation chromatograms and
found that the results were comparable only when
the HS had been subjected to extensive purification. Summers et al. (1987) reported on the limitations of E4/E6 ratios and found that the ratio
values varied considerably with the concentrations
of ultrafiltered fractions of HS. By comparing HS
extracted from soils and soil particles before and
after long term amendments with organic wastes,
Piccolo and Mbagwu (1990) found that the
E4/E6 ratio values did not account for the increase
in molecular sizes in amended samples as observed
by gel filtration
Despite a lack of evidence, various reasons led
the scientific community to accept the polymeric
concept for HS. One of these reasons lies in the
acceptance of the description by Staudinger
(1935) of macromolecular polymers occurring in
living cells. Based on the Staudinger concept, it
seemed convenient to assume that HS were also
polymers biologically synthesized from plant tis-
SOIL SCIENCE
sues components, notwithstanding that HS arise
from cell death rather than cell biosynthesis, as occurs with other biomolecules. The rapid degradation and decomposition in soil of biopolymers
liberated from cell lysis after death is now a well
accepted process from both a biological and a
thermodynamic perspective ( Jenkinson, 1981;
Haider, 1987; Clapp and Hayes, 1999; Spaccini et
al., 2001). Surprisingly, Staudinger’s view with regard to macromolecularity is still advocated by defenders of the polymeric nature of HS (Swift,
1999).
A second reason for acceptance of the polymeric concept is that a stable polymeric structure
would account for the refractory characteristics of
HS in soil, rather than considerations of physical
and chemical protection, as provided, for example,
by interactions with inorganic soil particles. The
OC in stable humic materials is known (Stevenson, 1994) to have extended residence times (from
250 up to 3000 years) in soil. In addition, the classical hypothesis of HS formation through condensation between amino acids and components
of degraded lignin promoted the assumption that
HS had a polymeric structure similar to that of
lignin (Waksman, 1936). Lignin is known to be
polydisperse in MW, with values ranging from
1000 to several million Da (Goring, 1971), and
its resistance to microbial degradation in soil has
been attributed repeatedly to its macromolecular
structures (Waksman, 1936; Amalfitano et al.,
1992). Similarly, polymerization processes carried
out in laboratory or in confined conditions led to
other classical hypotheses of HS formation, such
as the polyphenol theory (Flaig et al., 1975) and
the melanoidin theory based on the Maillard reaction (Maillard, 1912). Another aspect of HS
contributing to the polymeric view is their colloidal properties, which were related to those of
polyelectrolytes in aqueous media (van Dijk,
1972; Flaig et al., 1975). Most of the properties
of polyelectrolytes, such as processes of flocculation and dispersion, responses to electrolytes, and
double-layer behavior were also observed for HS,
and thus concepts of HMW properties were readily attributed to HS.
The macromolecularity of HS, despite the
wide acceptance of the concept, has never been
demonstrated unambiguously in chemical and
physical-chemical terms for soil-extracted humic
fractions. The instrumentation for organic and
physical chemistry analyses was not generally
available in the past, nor was it completely reliable. This situation has changed dramatically in
the present world of sophisticated biophysical
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
technology. Thus, the polymeric paradigm of HS
should either be proven beyond any doubt or
abandoned in favor of another description.
CONCEPTS OF CONFORMATIONAL
MODELS FOR HUMIC SUBSTANCES
Determination of the true MW of a humic
fraction is central to HS research. Conformational
aspects of structures of HS (i.e., shapes and sizes),
and ultimately their reactivities in soil and the environment, are determined by MW. It is quite evident from reviews that different methods for
measuring MW values of HS do not give the same
answers (Wershaw and Aiken, 1985; Swift, 1989;
Clapp et al., 1989; Stevenson, 1994). Generally, the
differences are not slight but can be several orders
of magnitude. The great differences in values have
been attributed either to the variability of the HS
or the intrinsic limitations of the methods when
applied to polydisperse systems. However, the
polymeric paradigm has not been questioned in
analyses of the differences, and very few attempts
have been made to decrease significantly polydispersity by classical chemical methods.
Much of the confusion with regard to MW
determinations of HS has arisen as a result of
sedimentation-velocity and diffusion methods that
became fashionable with the upsurge of biochemistry research in the 1950–1970 era. Despite the
clear indication that such semiempirical methods
are not suitable for polydisperse systems because of
the multiple diffusion coefficients and sedimentation constants for different size particles (Wershaw
and Aiken, 1985), studies were carried out with
whole HA extracts, and MW values ranging from
25,000 to more than 200,000 Da were reported
(Stevenson et al., 1953; Flaig, 1958; Piret et al.,
1960). Interestingly, Flaig and Beutelspacher
(1968), when working with polydisperse humic
solutions, found that MW values varied from
77,000 Da, when 0.2 M NaCl was added to depress repulsive negative charges, to only 2050 Da
in the absence of the background electrolyte.
Though the latter is commonly added to polyelectrolytes to reduce interferences from charge repulsion in monodisperse systems, a jump of about two
orders of magnitude in MW for the same humic
polydisperse system could suggest a molecular association rather than an extreme interference in
sedimentation-velocity of polyelectrolytes.
In an attempt to decrease the polydispersity of
HS, Cameron et al. (1972a) fractionated soilextracted HAs by ultrafiltration and by gel permeation chromatography (GPC) extensively, and
they subjected the fractions to sedimentation-
813
velocity ultracentrifugation. The fractions obtained were more homogeneous than the unfractionated solution, but these could still not be considered to be monodisperse. They reported MW
values for the fractions ranging from 2600 to a
surprising 1,360,000 Da. It is most likely that they
measured associations of smaller molecules randomly aggregated by different forces during the
fractionations procedures. Moreover, Cameron et
al. (1972a) overlooked the difficulties in assessing
sedimentation coefficients for associations of molecules such as HS. Measurement of sedimentation
coefficients of polydisperse materials that include
subunits invariably leads to erroneous values of
MW (Laue and Rodhes, 1990). Other studies using sedimentation-velocity ultracentrifuge studies
(Ritchie and Posner, 1982) and the even more
mathematically sound equilibrium centrifugation
(Posner and Creeth, 1972; Reid et al., 1990) also
showed polydispersity in HS, and, for this reason,
confirmed the ambiguity of the MW values obtained by ultracentrifuge methods.
There is more reliability in MW obtained for
FAs, or for OM dissolved in waters. There is a
general agreement that the MW of these humic
molecules is in the 400–1500 range, as determined by a variety of methods (Aiken and
Gillam, 1989; Wershaw, 1989; Stevenson, 1994).
As will be seen below, the smaller discrepancies
among MW values obtained for FAs can be ascribed to their larger hydrophilicity (small very
acidic molecules), which prevents strong molecular associations by hydrophobic forces. However, a process of molecular associations in solution can produce a polydisperse system of
apparently HMW also for FAs. In fact, FA MWs
when analyzed by ultrafiltration and gel filtration
methods, were consistently higher than when
measured with osmometry or cryoscopy (Thurman et al., 1982).
Similar contradictions (as described for the
MW values of HS) apply for the concepts of
shapes attributed to humic polymeric macromolecules. Globular shapes (Visser, 1964), flexible linear configurations (Mukherjee and Lahiri, 1959),
ellipsoidal shapes (Orlov et al., 1975), spheroid
polyelectrolytes (Ghosh and Mukherjee, 1971),
and randomly folding long chains (Cameron et al.,
1972a) have all been proposed to describe the ever
elusive polydisperse humic system. Ghosh and
Schnitzer (1980) reconciled the different views by
measuring surface pressures and viscosities of HS
at different pH values and neutral salt concentrations and adapting the results to relationships (the
Flory and Fox and the Staudinger equations) that
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had been developed for real polymers. They explained the observed behavior of HS (uncharged
matter at low pH and polyelectrolytes at high pH)
on the basis of the polymeric theory and they proposed that HS are rigid spherocolloids at high sample concentration and ionic strength and at low
pH, whereas at high pH values, low sample concentrations, and ionic strength, they behave as flexible linear polymers. This understanding had two
major flaws: it was based on studies with whole
humic extracts with full polydispersity and, despite
the lack of a direct knowledge of the real molecular structure, the data were arbitrarily used in equations specifically derived for polymers. Nevertheless, this reversible coiling model for humic
configurations soon became the most widely used
to describe HS, although it does not explain all of
the behavior of HS.
In applications of small-angle X-ray scattering to determine the particle sizes of whole HAs
in solution, or of fractions separated by adsorption chromatography using cross-linked dextran
gels (see Wershaw, 1989), it was found that HS
formed molecular aggregates in solution and
their sizes were a function of pH. It was concluded that the various fractions were different
chemically and that the differences in aggregation
behavior were a reflection of the interactions of
different bonding mechanisms. These findings,
coupled with other results that showed that humic fractions from different sources have surface
active properties (Hayase and Tsubota, 1983),
have led to a description of HS that is an alternative to the random coil polymeric structure.Wershaw (1986, 1993) proposed that HS consist of
ordered aggregates of amphiphiles, composed
mainly of relatively unaltered plant polymer segments possessing acidic functionality. In this
model, HS are aggregates held together by hydrophobic (- and charge-transfer bonds) and
H-bonding interactions, and the hydrophobic
parts of the molecules are in the interiors with
the hydrophilic parts make up the exterior surfaces. Ordered aggregates of humus in soils were
depicted to exist as bilayer membranes coating
mineral grains and as micelles in solutions.
Wershaw’s model represented a major breakthrough because it introduced the concept of aggregation of different particle sizes of humic constituents in contrast to the traditional view of
polydisperse linear humic polymers. Nevertheless, the micelle-like model did not yet solve the
issue of MWs of HS. The spontaneous aggregation of humic molecules into micellar aggregates
was advocated by other authors (Engebretson and
SOIL SCIENCE
von Wandruszka, 1994, 1997) to explain fluorescence quenching of pyrene, but the explanation
of the results was still based on the polymeric nature of the aggregating humic molecules. However, the classical concept of an ordered micelle is
hardly applicable because of the heterogeneity of
HS: the CMC (Critical Micelle Concentration)
reported in the literature for HS, in the range of
1 to 10 g L1 (Hayase and Tsubota, 1983), is
much higher than those found for surface active
compounds giving regular micellar structures
(Tanford, 1980).
Despite its limitations, the concept of aggregation of hydrophobic parts of HS could explain:
1) results from light-scattering which showed
that addition of Cu ions to dilute soil FA increased the amount of light scattered by the
solution (Ryan and Weber, 1982);
2) the increased solubility of nonpolar compounds in humic solution because of partition/adsorption in the hydrophobic interior
of HS (Carter and Suffet, 1982); and
3) the further release of humic matter through
dialysis bags from an already extensively
dialyzed HS when this is treated with an
amphiphilic compound such as acetic acid
(AcOH) or an electrolyte (De Haan et al.,
1987; Nardi et al., 1988).
The advances that the molecular aggregation
model made for an understanding of the environmental behavior of HS (compared with the
polymeric paradigm) did not prevent recent hypotheses, based on pyrolytic analyses of HS, indicating macromolecular structures for HAs with
MW values up to about 100,000 Da (Schulten et
al., 1998). Despite the many limitations inherent
in the analytical pyrolysis of HS (Saiz-Jimenez,
1995, 1996), compounds identified by pyrolysismass spectrometry techniques were arbitrarily
linked together by covalent bonds in computer
molecular models to give graphical representations of large branched polymers. Such large
macromolecules are even being proposed as
models of humic structures and used to explain
the behavior of HS in soil (Schulten and Leinweber, 2000).
SIZE EXCLUSION CHROMATOGRAPHY
OF HUMIC SUBSTANCES:
HISTORICAL PERSPECTIVE
Gel permeation chromatography or low-pressure size exclusion chromatography on Sephadex
cross-linked dextran gel columns, a method de-
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SUPRAMOLECULAR STRUCTURE OF HS
vised to desalt and purify proteins, has been applied extensively to humic fractions to evaluate
molecular sizes and to obtain more size-homogeneous materials (see reviews of Wershaw and
Aiken, 1985; De Nobili et al., 1989). It soon became clear that separation by GPC is not a pure
size fractionation (Determann and Walter, 1968)
and that interferences may occur with HS,
namely ionic exclusion and adsorption chromatography. In the first process, electrostatic repulsion between the negative charges present on
both dissolved HS and the dextran gel enhances
the chromatographic velocity, whereas in the second process, hydrophobic interactions between
HS and the stationary phase retard the chromatographic elution of HS (Lindqvist, 1967). The
ionic strength of mobile phases should be sufficiently high to prevent electrostatic interactions
but not too high (0.5 M) to drive hydrophobic
interactions (Chicz and Regnier, 1990).
Much of the inconsistency found in HS behavior in gel permeation has been ascribed repeatedly to either ionic (electrostatic) exclusion
or to hydrophobic gel-solute interactions. Swift
and Posner (1971), by eluting a Sephadex G-100
with distilled water, showed that increasing concentrations of HS produced shifts of peaks from
high to low molecular size ranges. They disregarded the possible hydrophobic retardation and
qualitatively attributed the behavior they observed to the repulsion between HS and the negatively charged Sephadex. They also assumed that
repulsion would become stronger if a decreasing
HS concentration lowered the ionic strength. Using a classical polyelectrolytic (polymeric) view,
they claimed that “charged double layers on the
solute and the gel extend further into the solution, resulting in effectively larger solute molecules and smaller pore sizes. Charge repulsion effects therefore occur at great distances leading to
increased exclusion with decreasing sample concentration.” However, in order to corroborate
these theoretical assumptions, the authors did not
carry out measurements of actual charge density
at different sample concentrations or in the gel
bed. Notwithstanding the theoretical and experimental inconclusiveness of HS behavior in gel
permeation, buffers of high ionic strength were
repeatedly recommended as mobile phases to
suppress interferences caused by ionic exclusion
(Swift and Posner, 1971; Swift, 1989; De Nobili et
al., 1989).
High ionic-strength buffers reduce the molar
volume of HS in solution and, by thermodynamically favoring the hydrophobic associations of
815
humic molecules, invariably produce chromatograms with a bimodal distribution. However, they
also enhance hydrophobic adsorption on the gel
solid phase (Specht and Frimmel, 2000).
Yonebayashi and Hattori (1987) showed that the
addition of 2 M urea to either phosphate or borate buffers eliminates the hydrophobic adsorption observed during the GPC of HS. An explanation for the urea effect will be given below.
Adsorption of HS on gel columns has traditionally been accounted for either by salinity or by
changes in ionic strength (Lindqvist, 1967; De
Nobili et al., 1989). Anderson and Hepburn
(1978) attributed the appearance of new HS elution bands to adsorption when sodium acetate
was added to the mobile phase and regarded these
bands as unspecified artifacts created by adsorption effects. However, De Haan et al. (1987)
demonstrated for the first time that HAs in saline
solution were able to diffuse more freely across a
dialysis membrane than when in solutions of
lower ionic strength.After comparing dialysis and
gel filtration experiments, they suggested that
changes in elution profiles caused by ionicstrength variation had to be attributed to real alterations of humic conformations rather than to
any interactions between the gel and HS. This
was at variance with the classical explanations.
Further understanding came from the work
of Yonebayashi and Hattori (1987) who studied
several HS by gel filtration through Sephadex G75. The eluent was 0.1 M phosphate or borate
buffers in 2 M urea, and they thereby avoided interferences by both ionic exclusion and adsorption on the column. They first found that by increasing the pH of the buffer eluent from 4.7 to
11.2, the molecular sizes of the HS increased progressively, although the fraction excluded at high
molecular-size (Vo) was larger at pH 4.7. Interestingly, the elution pattern at pH 4.7 showed adsorption of humic material beyond the total volume (Vt) of the column. They attributed the
behavior at low pH to an aggregation process of
humic molecules but still advocated ionic repulsion as the reason for the size increase at high pH.
However, their experimental conditions (large
ionic strength and urea) should have excluded an
electrostatic effect.
Yonebayashi and Hattori (1987) also showed
that for both phosphate and borate buffers and
for all pH values from 4.7 to 11.2, the molecular
sizes of HS increased significantly with time of
standing (from 5 min to 24 h) in the buffer solution before injection into the gel column. To explain these findings, they had to invoke again a
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process of molecular association into micelle-like
aggregates. In order not to contradict the polymeric paradigm of HS, they assumed that association occurred between humic molecules of
large and small molecular sizes. However, this explanation did not account for what they further
observed when HS were treated with ethanolbenzene, 6 M HCl, 0.5 M H2SO4, or 5 M NaOH
before dilution in the buffer and separation by
GPC. After the ethanol-benzene or acid treatments, the gel chromatograms were almost the
same as those for the original HA, but the fraction excluded increased with an increase of
standing time in the buffer solution. Conversely,
the excluded fraction disappeared after the alkaline treatment, and the gel chromatogram remained unchanged regardless of the standing
time in the buffer solution before injection. Although they invoked only micellar aggregation to
explain their findings, these results were clearly a
sign of conformational changes caused by the establishment of hydrophobic interactions (see discussion below). The conformational rearrangement could take place in the buffer solution for
all pretreated samples but not in 5 M NaOH,
where a full humic dispersion (disruption of all
hydrogen bonds) and hydrophobic rearrangement had already taken place.
In an attempt to clarify the formation of micellar aggregates, Yonebayashi and Hattori (1987)
also measured the surface tension of HS. They
found that the surface tension of the whole HS
mixture in neutral phosphate buffer containing
urea decreased with an increase in sample concentration. This suggested that molecular associations increase the surface activities of humic materials. In fact, four fractions isolated by GPC
revealed that only the first HMW size fraction behaved as a surface active agent, whereas the three
smaller fractions showed low surface activity. A
cluster analysis grouping the gel behavior of several HS with their chemical and physical-chemical properties showed that HS with high surface
activities (and a large fraction excluded at Vo in
gel filtration) were rich in long aliphatic chains,
whereas HS with low surface activities (and small
excluded fractions) were composed of aromatic
rings, a high content of -COOH groups, and a
low content of -OCH3 groups. Based on the results of Yonebayashi and Hattori (1987), which
were similar to those of Anderson and Hepburn
(1978), it may be concluded that the associative
nature of HS was proven by gel filtration studies.
Fractions had different chemical compositions,
and hydrophobic attractive forces between ali-
SOIL SCIENCE
phatic components favor their association into apparently large and surface-active fractions.
The work of Ceccanti et al. (1989) has provided new evidence that HS behavior in GPC
where changes in ionic strength occurred cannot
be ascribed to simple electrostratic interactions
(ionic exclusion), as for a polyelectrolyte (a polymer holding multiple charges). They fractionated
HS by ultrafiltration and gel filtration using both
water and salt solutions as mobile phases and
found that 100% of the humic C was in the lowest molecular size fraction (less than nominal
10,000 Da) when HS were ultrafiltered in 100
mM of pyrophosphate/HCl buffer at pH 7.1.
However, no humic C was in the same fraction
when ultrafiltration was in water only, when 43%
of the C was in the highest molecular size fraction (greater than nominal 100,000 Da). Contrary to previous qualitative research, Ceccanti et
al. (1989) did measure by isoelectric focusing the
negative net charge of HS fractions isolated by
gel filtration. Despite the charge being very similar, refractionation of fractions in the Sephadex
G-50 column in water gave distinctly different
elution patterns. Moreover, fractionation by water elution from the polydextran Sephadex,
which has some residual negative charges, gave a
molecular size distribution in accordance with
that obtained on the uncharged PM-10 (polysulfone) ultrafiltration membrane. They concluded
that gel filtration occurred by a true size fractionation rather than by a charge-dependent elution. The work of Ceccanti et al. (1989) also
showed that the strength and the shape of humic
associations controlled the elution volumes of HS
fractions.
A major problem in using size exclusion
chromatography to determine the sizes of humic
fractions is the lack of adequate standards to calibrate the gel column. A calibration of Sephadex
columns with presumably size-controlled humic
fractions differed substantially from that obtained
with globular proteins (Cameron et al., 1972b).
This indicated that the hydrodynamic behavior
of HS is different from that of globular macromolecules, and their molecular size may be more
apparent than real and could be ascribed to molecular aggregation phenomena. Hayes (1997) reported that the ability to obtain humic fractions
of homogeneous size by repetitive gel fractionation is not effective because reprocessing of
fractions results in the separation of smaller sized
components.
The difficulty of obtaining MW values of HS
by gel-filtration was noted by Reuter and Perdue
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SUPRAMOLECULAR STRUCTURE OF HS
(1981). They measured by osmometry a value of
1231 Da for the number-average MW of the HA
fraction excluded from G-50. The published
globular-protein exclusion limit for the gel is
30,000 Da. Again, the reason for the exclusion of
the humic material at such an apparent high MW
cut off may be attributable to the molecular aggregation of small molecules into apparent large
sizes. This interpretation is substantiated by the
lack of differences between the IR spectrum of
the original HA and the spectra of fractions isolated by means of gels with different MW cutoff
values (Reuter and Perdue, 1981). The association of small humic molecules into different aggregate sizes may have also been the reason why
Cornel et al. (1986) could not find sufficient similarity between the diffusion behavior of HS and
that of synthetic polymers of known compositions such as polyethylene oxides and polystyrene
sulphonates. Further questions arose when Summers et al. (1987) did not find differences in the
IR spectra of HS size fractions isolated by ultrafiltration, a separation technique that is slower
than the velocity by which humic molecules aggregate or disaggregate in solution.
EVIDENCE FOR SUPRAMOLECULAR
ASSOCIATIONS OF SMALL
HUMIC MOLECULES
Using low-pressure size exclusion chromatography (SEC), Piccolo et al. (1996a and b) reported
that the 280 nm absorption of HAs was reversibly
shifted from high to low molecular size ranges
(column total elution volume, Vt) when organic
acids were added to lower the pH of a humic solution from 9.2 to 2 before the elution in an 0.02
M alkaline borate buffer. To explain their results
they suggested that, instead of being stable polymers, HS at neutral or alkaline pH values are
supramolecular associations of relatively small
heterogeneous molecules held together by weak
dispersive forces, such as van der Waals, -,
CH-, interactions. The addition of organic
acids altered such unstable humic conformations
through the formation of energy rich hydrogen
bonds, and the subsequent chromatographic elution separated the resulting smaller subunits and
prevented the reassociation that would have occurred in static conditions.
Supporters (Swift, 1999) of the traditional
polymeric model of HS have criticized the above
results, not on the basis of an experimental replication but on theoretical and qualitative interpretations of gel-solute interactions and interferences caused by a supposed polymeric form of
817
borate. However, the results of Piccolo et al.,
(1996a and b) could not be attributed to a buffering action of the organic acid toward the alkaline
eluent because the amount of the different organic acids varied by two orders of magnitude,
but the shift to larger elution volumes remained
the same for all acids. Nor could an elution delay
caused by a solid deposition on the gel and subsequent resolubilization by the eluent have been
the cause of the shift since the treated samples remained soluble at low pH and entered the chromatographic elution immediately after deposition on the column. Nevertheless, if this had been
the case, a progressive neutralization of the acidic
buffering capacity by the alkaline eluent would
have caused a smearing out in the column of the
polydisperse humic mixture, and a diffuse chromatographic band from lower to larger elution
volumes, instead of the sharp peak at the total
volume (Vt) of the column. Furthermore, the
ionic strength effect (De Nobili et al., 1989)
could not be invoked for the reversible peak shift
because elution in an ionic strength quencher,
such as a borate buffer 10 times more concentrated, gave the same chromatographic change
upon AcOH and KOH additions.
Piccolo et al. (1996a and b) considered their
findings to be an expression of the associative nature of relatively small humic molecules that selfassemble only into apparent high-molecular size
materials. This interpretation was in accordance
with previous research, which showed that HS
behaved as molecular associations when studied
by SEC (De Haan et al., 1987; Yonebayashi and
Hattori, 1987; Ceccanti et al., 1989; Piccolo et al.,
1990a). Moreover, laboratory observations have
indicated that when AcOH is added to HS that
have been already extensively dialyzed, further
small size components are released during subsequent dialysis (Nardi et al., 1988). These low
molecular size fractions are a product of a conformational rearrangement and a chemical composition different from the bulk HS. The separated fractions were found to stimulate specific
biological properties in plants and were more biologically active than the whole humic materials
from which they were separated (Piccolo et al.,
1992).
An experimental replication of the work of
Piccolo et al. (1996a and b) that applied high performance SEC was reported by Varga et al.
(2000). They obtained the same results as Piccolo
et al., (1996a and b), but they observed that by using organic acids to lower the pH of humic solutions before injection from 9.2 to 1–3, the shift of
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PICCOLO
absorption to lower molecular size ranges may
have been caused by hydrophobic interactions on
the column upon protonation of the humic matter and the consequent change in ionic strength.
A contribution to SEC of hydrophobic interactions on the column has been long recognized in
the gel-filtration studies of HS (Lindqvist, 1967)
and of other biomolecules (Chicz and Regnier,
1990). Nevertheless, hydrophobic interactions on
size-exclusion gels have often been usefully exploited to fractionate humic aggregates into simpler molecular associations that were characterized chemically more easily than the bulk humic
material (Anderson and Hepburn, 1978; De Haan
et al., 1987; Yonebayashi and Hattori, 1987).
It was necessary to verify the innovative explanation of the conformational changes of HS using
additional experimentation, and High-Pressure
Size Exclusion Chromatography (HPSEC) was
adopted for further investigation. HPSEC is, for a
number of reasons, a more valid chromatographic
system than low-pressure GPC (Piccolo and
Conte, 2000; Piccolo et al., 2001a). The major advantages of HPSEC are:
1) high reproducibility of chromatograms (5%);
2) relatively higher rapidity of analysis (about 60
minutes per chromatogram);
3) longer column life (more than 1000 injections
per HPSEC column); and
4) higher sensitivity to chemical changes in injected samples because of a much lower loading mass.
Conte and Piccolo (1999a) have compared
two commercial HPSEC columns with respect to
their capacity to measure the molecular sizes of
HS accurately and precisely.
In the HPSEC mode, Piccolo et al. (1999) reproduced the same shift of chromatographic
peaks observed earlier by low-pressure gel filtration (Piccolo et al., 1996a and b) when the pH of
humic solutions was adjusted from 7 to 3.5 by using only small amounts (0.5 103 M) of a
number of monocarboxylic acids and, thus, without significantly changing the ionic strength of
the sample. The larger resolution and reproducibility of HPSEC columns, in comparison
with the low-pressure gel phases, allowed the authors to assess whether the alteration of the
HPSEC molecular size distribution was dependent on the number of C atoms in the organic
acids and on the hydrophilic/hydrophobic (HI/
HB) C ratio of HS, as measured by CPMAS
13C-NMR spectroscopy. It was found that the
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higher the C content of organic acids and the
lower the HI/HB ratio of humic materials, the
larger the decrease in the average molecular size
of HS.
Conte and Piccolo (1999b) conducted a second HPSEC experiment in which, instead of
lowering the pH of humic solutions before injection, they allowed the HPSEC eluent to be
slightly modified. A control eluent at pH 7 (0.05
M NaNO3, not absorbing light at 280 nm) was
modified by addition of 2.0 106 M of either
methanol, HCl, or AcOH to pH 6.97, 5.54, and
5.69, respectively, without changing the ionic
strength (I 0.0504 M). In this way the hydrophobic adsorptions, which may occur at increased ionic strengths (Lindqvist, 1967; Chicz
and Regnier, 1990; Specht and Frimmel, 2000),
were avoided. Moreover, sodium humate and fulvate solutions were previously titrated to pH 7 so
that their dissolution in the mobile phase at pH 7
would have prevented any random occurrence of
negative charges. This was to avoid any uncontrolled formation of negative charges on the
solute, ascribed to changes in the ionic strength of
humic solutions, thereby affecting the volume of
sample elution (Swift and Posner, 1971). UV-Vis
and Refractive Index (RI) detectors were used
to record the chromatograms indicating the
molecular-size distributions of the humic materials with the objective of comparing the chromatographic behavior of the chromophores (UV
at 280 nm) with that of the real humic mass (RI).
Both UV and RI detectors showed major alterations of the humic molecular-size distributions
and dramatic decreases in the weight-average
MW values in the modified eluents. The decrease
in molecular size was revealed by the shift toward
increasingly larger elution volumes for both the
UV and RI detectors and by the concomitant reductions in peak absorbance at the UV-Vis detector. In the constancy of ionic strength, the observed changes could be ascribed to the
interactions of the added chemicals with the
weakly associated molecules giving rise to an apparent macromolecular structure of the humic
matter. The slight modifications of the mobile
phases may have caused the collapse of the heterogeneous humic aggregate into molecular associations of smaller dimensions but of greater
thermodynamic stabilities than for the control
solution. This was attributed to the gain in energy
obtained by the formation of intermolecular hydrogen bonds (from 10 to 20 kJ mol1 for each
hydrogen bond) among humic molecules in the
modified mobile phases (Schwarzenbach et al.,
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
1993). The large decrease in molecular size suggests that the weak association of apparently high
molecular size, as observed for the HA in the
control solution, must, therefore, have been due
predominantly to weak intermolecular hydrophobic forces such as van der Waals, -, and
CH- (Nishio et al., 1998) bonds, which hold
small molecules together.
The work of Conte and Piccolo (1999b) provided further direct evidence for the conformational model based on the reversible self-association
of small humic molecules rather than on the
macropolymeric random-coil concept. Molecular
size decreases caused by the breaking of ester and
other covalent linkages by such small amounts of
modifiers could not be proposed considering the
customary strongly alkaline and acidic conditions
to which the HS had been subjected during isolation and purification. Furthermore, using
HPSEC, it was shown that the molecular size distribution of HS must be interpreted by a combination of two factors: the elution volume, and the
molar absorptivity of the chromatographic peaks.
Earlier research had failed to address the combination of these factors either because closely similar HAs were analyzed by UV detection only and
in less sensitive low-pressure size-exclusion systems (Swift and Posner, 1971) or because weakly
UV absorbing FAs and/or (aqueous) dissolved organic matter (DOM) samples were used in studies
with HPSEC systems without the support of RI
detector systems (Becher et al., 1985; Berden and
Berggren, 1990; Chin and Gschwend, 1991; Chin
et al., 1994). More recent HPSEC investigations
which coupled UV, RI, and MALS (Multi-Angle
Light Scattering) detectors, have also indicated
different molecular size distributions for HS according to the detector employed (von Wandruszka et al., 1999).
When ionic-exclusion interferences were
carefully eliminated in the work of Conte and
Piccolo (1999b), permanent adsorption of HS
on the column was not found to occur (Conte
and Piccolo, 1999a; Piccolo et al., 2001c), in accordance with other HPSEC studies in similar
conditions (Becher et al., 1985; Mueller et al.,
2000). However, a modification of the mobile
phase by addition of methanol, HCl, and AcOH,
although in very small amounts, may have produced pore-size changes that influenced nonSEC. In order to verify that mobile phase modification did not alter the exclusion properties of
the HPSEC column, Piccolo et al. (2001b) subjected polymeric standards of known MW, such
as the negatively charged polystyrenesulphonates
819
(PSS) and neutral polysaccharides (PYR), to
SEC in the same mobile phases used by Conte
and Piccolo (1999b) for the HS studies. The
undisputed polymeric nature of the covalently
linked units of both nonionic PYR and polyelectrolytic PSS made a comparison of their
chromatographic behavior (using the same experimental conditions) with that of HS relevant.
Regardless of the charge density of the polymer,
the similar chromatographic behavior of each in
different mobile phases indicated that the slight
variations in the compositions of the mobile
phases were not sufficient to alter either the stability provided by strong covalent bonding or
their interactions with the stationary phase.
Conversely, the size-exclusion chromatograms,
based on UV or RI detector systems, of three
different HAs (HA1 from a volcanic soil, HA2
from an oxidized coal, and HA3 from a lignite)
varied dramatically (in both peak absorbance and
elution volumes) with the compositions of mobile phases.
Piccolo et al. (2001b) concluded that, whereas
slight modifications in the mobile phase did not
affect the column capacity for size exclusion, the
substantial difference between the response to
HPSEC of the polymeric standards and that of
HS in the very same chromatographic conditions
proved that humic materials have different structural assemblies. As proposed earlier, this may well
be a self-assembling association of relatively small
and heterogeneous molecules rather than a coil of
polymeric macromolecules. To verify that such
variations were not specific for the wavelength
used to record the chromatograms (280 nm), and
thereby simply accountable to shifts of peak maxima, Piccolo et al. (2001b) also recorded UV spectra of HS solutions over a range of wave lengths.
Their results showed that the three humic materials also produced different absorbance values upon
modification of their solutions over a wide range
of wavelengths. This was regarded as confirming
that the molar absorptivity of the bulk HS varied
with the composition of the solution. The absorptivity was decreased by adding chemicals
which disrupt their weakly-stabilized molecular
associations.
Piccolo et al. (2000a) also compared the chromatographic behavior of HS with that of neutral
PYR and polyelectrolytic PSS when solutions (in
0.05 M NaNO3) before injection into the
HPSEC system were titrated from pH 7 to 3.5
with either HCl or AcOH, as was done for HS in
the work outlined above (Piccolo et al., 1999).
Contrary to the work of Piccolo et al. (2001b),
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PICCOLO
the mobile phase was kept constant while the solutions under analysis were modified. It was
found that plots of retention volumes (obtained
using either the RI detector for PYR and PSS
polymers of known MW or the UV detector for
only PSS standards) versus Ln (MW) did not
show significant differences between the control
and samples added with either HCl or AcOH.
This result indicated that the capacity of the
HPSEC column in aqueous media to exclude by
size was not altered by injecting the polymer solutions brought to pH 3.5 with a very small
amount of acid. However, when HS solutions
were similarly modified by lowering pH and
chromatographed in the same mobile phase, the
elution of the acid-added humic samples differed
significantly from the control humic solution. As
in previous experiments, these results were considered to be evidence that a humic association,
stabilized only by weak dispersive forces at pH 7
(mainly hydrophobic forces), varies considerably
with respect to the arrangements of its component molecules and its size distribution when
treated with an acid. In contrast, covalently linked
polymers have more stable molecular arrangements and their HPSEC behavior is not affected
by an interaction with either a mineral or an amphiphilic acid such as AcOH.
Cozzolino et al. (2001) studied the effect of
organic acids of plant, microbial, or anthropic
origins on the molecular size distribution of dissolved HS. They used a Phenomenex Biosep
S2000 column and eluted with a 0.05 M NaN03
solution to evaluate size changes in four different
HS upon addition of hydroxy- (glycolic and
malic), keto- (glyoxylic), and sulfonic (benzenesulfonic and methanesulfonic) acids. All HS
showed a decrease in peak absorbance when humic matter was dissolved in the HPSEC mobile
phase at pH 7, and the pH of the solution was
lowered to 3.5 by acid addition before analysis.
This effect was generally accompanied by an increase in peak elution volumes. The overall decreases of the total areas of the chromatograms
compared with the control HS solutions was
again explained with a disruption of supramolecular humic aggregates into smaller sized but
more energy-rich associations brought about by
the formation of mixed intermolecular hydrogen
bonds after treatment with acid. The hydroxybicarboxylic malic acid was the most effective in
disrupting the original humic associations. This
was attributed to its greater capacity to form new
hydrogen bonds with complementary functions
of HS. Malic acid is a bicarboxylic acid (HOOC-
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CH2-CHOH-COOH) with two carboxyl
groups (pKa1 3.4; pKa2 5.11) that can either
be protonated or partially dissociated at pH 3.5,
thereby allowing a larger number of mixed hydrogen bonds to form on its oxygen-containing
functions than could be formed either by the
strong hydrochloric acid (which is a proton
donor only) or by the more weakly acidic monocarboxylic glycolic and glyoxylic acids. The extent of molecular association variation was related not only to the pKa values of the acids but
also to the chemical and stereochemical affinities
of the humic components that would allow penetration of acids into the inner humic domains,
depending on the acid structures. For example,
the strongly acidic methanesulfonic and benzenesulfonic acids showed effects that varied with
the humic properties. Methanesulfonic acid was
more effective in the disruption of the molecular
associations of HS that contained predominantly
aliphatic and alkyl moieties, and, equally, benzenesulfonic acid disrupted the distribution of
the aromatic-rich humic material because of the
probable larger - interactions with aromatic
humic components.
The indications of the supramolecular nature
of HS provided by the analytical HPSEC studies
has caused Piccolo et al. (2001c) to employ a
preparative HPSEC column to separate humic
size fractions that might be more homogeneous
chemically, and they have subjected these fractions to chemical and spectroscopic characterization. A HPSEC fractionation was carried out on
the same HS before and after adjusting the pH of
the humic solution (0.05 M ionic strength) from
7 to 3.5 with 0.5 103 M AcOH. Six fractions
were collected from the HPSEC separation of
the HA solution at pH 7.0, whereas eight fractions were obtained from the separation of the
humic solution treated with AcOH to pH 3.5
before HPSEC injections. Fractionation was run
continuously using an auto-sampler and a fraction collector, and the chromatographic pattern
monitored by means of a UV detector. Reproducibility was excellent (CV 5%) for more
than 100 runs for each fractionation series.
Moreover, elemental analyses on the sum of fractions from both untreated and AcOH-treated HS
excluded the possibility of any significant adsorption of humic matter on the column stationary phase. The molecular size distribution of the
untreated HS was significantly different from
that of the AcOH-treated HS. The latter showed
much less peak absorbance (280 nm), and several
additional peaks appeared at higher elution times
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
(120–180 min). Size-fractions were analyzed by a
Curie point (610 C) Pyrolysis-Gas Chromatography-Mass Spectroscopy (Py-GC-MS) technique and by 1H-NMR spectroscopy. Total ion
chromatograms by Py-GC-MS showed that fractions had a significantly different chemical composition after AcOH treatment, thereby confirming that AcOH had caused rearrangement of the
humic associations in solution and that preparative HPSEC is adequate for HS fractionation.
Py-GC-MS spectra showed that the AcOH addition altered the distribution of humic molecular
components in the size-fractions. The unsaturated alkyl chains were moved from size-fractions of larger molecular size into those of lower
molecular sizes. Most of the aromatic moieties,
which were found in larger molecular size fractions for the untreated HA, were spread into
fractions of lower molecular size after AcOH addition to HA. Carbohydrates, which were undetectable in any fraction of the untreated HA, appeared instead in the pyrogram of the lowest
molecular size (and the most hydrophilic fraction) after treatment with AcOH. Our results
suggested that AcOH disrupted the weakly
bound association of humic constituents and
HPSEC elution separated size-fractions of different compositions. The fractions with the largest
apparent molecular sizes were the richest in alkyl
chains, suggesting that humic molecules were
stabilized into supramolecular associations by
multiple weak interactions among apolar groups
such as alkyl chains and aromatic moieties. 1HNMR spectra of HPSEC fractions not only confirmed the findings by Py-GC-MS analyses but
also showed that the chemical compositions of
fractions were less complex after AcOH treatment. This was attributed to a less complex molecular association in the size separates, and that
allowed a larger solubility in the NMR solvent
and more favorable spin-lattice relaxation times.
Piccolo et al. (2001c) showed, by combining fraction-separation by HPSEC with chemical and
spectroscopic characterization, that the fractions
with larger apparent molecular sizes were composed predominantly of a mixture of alkyl compounds of relatively low molecular sizes, whereas
fractions of lower apparent molecular sizes contained small aromatic systems and hydrophilic
compounds. These conclusions were in harmony with previous research which characterized HS fractions separated by either low-pressure GPC or ultrafiltration (Anderson and
Hepburn, 1978; Yonebayashi and Hattori, 1987;
Piccolo et al., 1990a).
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SOLUTE-GEL-ELUENT AS AN
INTERACTIVE SYSTEM IN
EXCLUSION CHROMATOGRAPHY
Although the above results by HPSEC should
be regarded as free of interferences from ionic
exclusion and adsorption (coefficient of variation
for more than five runs was consistently less than
5%, and there was a lack of adsorption of humic
matter on the columns), a further check for possible hydrophobic adsorption phenomena was
conducted. Seven molecules that might be considered monomeric constituents of HS (Stevenson, 1994) were subjected to HPSEC separately
and in a full mixture (1 g L1). Elution was with
a phosphate buffer at pH 3. The individual molecules that were size-excluded gave the elution
volumes reported in Table 1, whereas the mixture of the seven molecules produced an elution
profile with only two peaks (Fig. 1).
It must be noted that the retention volumes of
the two peaks for the mixture did not correspond
to any of the elution volumes found for the single
molecular species (Table 1). The first peak for the
mixture eluted (23.07 mL) before any of the single species, with the exception of the highly hydrophilic glucosamine, and the more intense second peak was eluted before all other monomers
except dihydroxyphenylacetic and gallic acids.
These results suggest that when the single molecular species are mixed together in solution, they
form weak mutual associations giving rise to hydrodynamic radius values larger than when they
are eluted alone. Based on the larger single elution
volumes of the more hydrophobic monomers (hydrocaffeic acid, resorcinol, and catechol), it can be
inferred that their elution is retarded by adsorption
on the stationary phase of the column. Conversely,
TABLE 1
HPSEC retention volumes (mL) of different monomers
and mixtures of the same monomers on a column
Polysep GFC-P3000 eluted with a phosphate buffer at pH 3
Monomers/Mixtures
mL
Dihydroxyphenylacetic acid
Gallic acid
Protocatechuic acid
Hydrocaffeic acid
Glucosammine (RI)
Resorcinol
Catechol
23.39
25.49
25.94
26.60
20.23
26.90
26.95
Mixture of the above (0 h)
First peak: 23.07; second
peak: 25.91
First peak: 23.01; second
peak: 25.70
Mixture of the above
(after 144 h standing)
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PICCOLO
Fig. 1. HPSEC chromatogram of a mixture (1 g.L1) of
seven monomeric precursors of HS eluted by a phosphate buffer at pH 7 through a Phenomenex column
(Polysep GFC-P3000). 1. Dihydroxyphenylacetic acid; 2.
Gallic acid;. 3:Protocatechuic acid;4. Hydrocaffeic acid;
5. Glucosammine (RI detected); 6. Resorcinol; 7. Catechol.
when the same compounds are in mixtures with
other monomers the thermodynamic drive to decrease exposure of hydrophobic components to
the aqueous medium increases the mutual attraction of monomers. This interpretation is substantiated by the observation that the retention volumes of mixture peaks decreased in value with
time of standing before injection (Table 1),thereby
indicating a further hydrophobic strengthening of
the monomer associations. A consequence is that
the hydrophobic interactions of single monomers
with the column matrix are reduced while exclusion of the monomer association is expedited.
Evidence that hydrophobic adsorption on the
column is not irreversible and affects only elution
volumes was obtained by eluting progressively
less concentrated solutions of the mixture. The
absorbance decrease of both peaks given by the
mixture gave a highly significant linear correlation with the decrease in the concentration of the
mixture, as expected by the Lambert and Beer
law (Fig. 2). This suggests that there were no
losses attributable to adsorption on the column of
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chemical species that might be regarded as representative of monomeric structures in HS.
The behavior of the monomer mixture in
HPSEC may have similarities with that of associations of humic molecules. When the apparently
large-size, weakly bound humic associations are
altered by the action of organic acids or by other
species (uncharged compounds, electrolytes, cations), smaller humic clusters are separated and
their elution is retarded by size exclusion; in addition, the hydrophobic interactions with the
column matrix will have changed.
Other evidence that humic molecules are
suprastructural associations in aqueous solutions
is given by the elution patterns of HS (in HPSEC
experiments) in 8 M urea. Concentrated urea is
employed to disrupt protein-protein interactions
and to solubilize aggregating hydrophobic proteins for their further separation (Hjelmeland,
1990). Concentrated urea has also been proposed
for low-pressure size exclusion and polyacrylamide gel electrophoresis (PAGE) separations of
HS because it is considered able to produce elution patterns similar to 0.1 M Tris-HCl buffer at
pH 9 (Trubetskoj et al., 1997).
The elution profiles of three different purified HS (0.2 g L1) dissolved in 8 M urea and
eluted in the same solution are shown in Fig. 3.
The chromatograms were detected by both UV
and RI detectors in a series. This experiment is
illustrative of the capacity of urea to separate the
hydrophobic from the hydrophilic components
of HS. The chromatograms show a net separation
of humic matter: a high molecular size fraction at
the column Vo visible only by the UV detector
Fig. 2. Relationship of peak absorbances (first and second
peak of Fig. 1) versus concentration of a mixture of seven
monomeric precursors of HS eluted by a phosphate
buffer at pH 7 through a Phenomenex column (Polysep
GFC-P3000). 1. Dihydroxyphenylacetic acid; 2. Gallic
acid;. 3. Protocatechuic acid; 4. Hydrocaffeic acid; 5. Glucosammine (RI detected); 6. Resorcinol; 7. Catechol.
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
823
and a low molecular-size fraction eluting at the
column Vt detected only by the RI detector. The
hydrophobic humic molecules are strongly associated through hydrophobic effects induced by
the highly concentrated urea. The urea interacts
more favorably than hydrophobic molecules with
the network of the water structure. Moreover,
since urea is known to form complexes with
nonionic detergents (Hjelmeland, 1990), it may
also be possible that complexes between urea and
nonionic humic hydrophobic compounds increased the hydrodynamic radius of the hydrophobic associations, causing these to elute at
the earliest exclusion volume of the column.
Differences among HS could also be noted.
The HA from an oxidized coal was the only one
to show two well separated peaks in the RI detector system (Fig. 3, I-RI), whereas only one
very intense peak (1000 mV) at the Vo column
was shown by the UV detector (Fig. 3, I-UV).
This indicates that while light-absorbing chromophores (280 nm) with high molar absorptivities were excluded rapidly from the column, their
concentrations in the humic samples were relevant and of a similar order of magnitude as the
nonabsorbing hydrophilic, probably ionized,
compounds detected by the RI detector at the
total exclusion volume of the column. The HA
from lignite gave a similar complete separation
between the large-size hydrophobic chromophore fraction (Fig. 3, II-UV) and the smallsize ionized and hydrophilic components shown
by the RI detector (Fig. 3, II-RI). However, although the molar absorptivity of the excluding
chromophores was still high (as suggested by an
absorption intensity 800 mV), the lack of corresponding peaks at the RI detector indicated
that their concentrations in the sample were
much less than those of the hydrophilic components excluded at large elution volumes. Identical
behavior was shown by the HA extracted from an
agricultural soil. Also for this sample, the chromophores association, excluded at the column
void volume (Fig. 3, III-UV), was hardly representative of the mass of humic sample since the
corresponding peak at the RI detector was almost irrelevant compared with the signal of the
Fig. 3. UV- and RI-(Refractive Index) detected HPSEC
chromatograms of humic acids dissolved and eluted in
8M urea solution. I. humic acid from an oxidized coal; II.
humic acids from a lignite; III. humic acid from a Danish
agricultural soil.
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PICCOLO
small size, nonabsorbing, hydrophilic constituents
(Fig. 3, III-RI).
The two experiments reported above suggest
that in the HPSEC analysis of associations of
molecules such as HS, the stationary and mobile
phases lose the properties associated with specific
single molecules alone. Rather, they become part
of an interactive system involving the mixture of
solutes. It is the whole solute-gel-eluent system
that produces the size-exclusion of the association under analysis. Moreover, it is the humic
conformational structure, and the degree of potential hydrophobic interaction between the associated molecules and either the stationary or
the mobile phases, that determines the elution
volume of a humic association in aqueous solution. This applies more than ionic exclusion, so
often considered in the past to explain the SEC
behavior of HS. These results also point out that
the molecular sizes of HAs are not related to the
molar absorptivity of chromophores in the humic samples, and they should be assessed by detection means that are related to the total mass of
the HAs.
POLYMERIZATION OF HUMIC
SUBSTANCES BY OXIDATIVE
CATALYTIC REACTIONS
To understand humus as a supramolecular association of small molecules it is necessary to
overcome the limitations imposed by the paradigmatic polymeric model. If HS are seen as
weakly bound supramolecular associations their
unstable conformations could then be stabilized
in real polymeric structures. This could be
achieved by increasing the number of intermolecular covalent bonds via an oxidative coupling reaction catalyzed by oxidative enzymes such as the
phenoloxidases. This class of enzymes has been
shown to promote, through a free-radical mechanism, oligo- and poly-merization of phenols and
anilines, and, hence, is believed to contribute to
soil detoxification from related organic contaminants (Kim et al., 1997).
Piccolo et al. (2000b) turned a loosely bound
humic superstructure into a covalently linked
polymer by treating a humic material dissolved in
0.1 M phosphate buffer at pH 7 with horseradish
peroxidase (HRP) and hydrogen peroxide (oxidant). They used HPSEC to evaluate the changes
in molecular size distribution brought about by
the oxidative reaction with HRP catalysis. Moreover, AcOH was added to the reacted humic
mixture to bring the pH to 4, and HPSEC injection was then used to assess the stability of the
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humic conformation following the polymerization reaction. The polymerized HS had HPSEC
absorptions of larger intensities, and the elution
volumes were shifted to lower values than the
control. Moreover, treatment with AcOH did not
alter significantly the chromatographic appearance of the polymerized HS, whereas it produced
disruption of the loosely bound association of the
untreated HS resulting from a significant reduction of the intensities of the peaks and their shifts
to larger elution volumes. These changes indicated a significant increase in the molecular sizes
of the humic materials resulting from oxidation
catalyzed by HRP, which was attributable to a
true polymerization of humic molecules via the
formation of C-O or C-C bonds. Furthermore,
DRIFT (Diffuse Reflectance Infrared Fourier
Transform) spectroscopy was used by Piccolo et
al. (2000b) to verify the effect of the oxidative
polymerization reaction on the molecular structure of the HS. In comparison with the control,
the DRIFT spectrum of the humic material subjected to oxidative coupling showed a substantial
change in the 1500–900 cm1 frequency interval
with the appearance of three main bands at 1247,
1097, and 947 cm1 and a decrease in the 1400
and 1227 cm1 bands. The absorption shown at
1247 and at 1097 cm1 was reasonably assigned
to bond deformation of aryl and alkyl ethers,
respectively, which were formed during freeradical coupling reactions catalyzed by HRP and,
hence, confirm the interpretation of HPSEC
measurements.
The HPSEC and DRIFT results of Piccolo et
al. (2000b) suggest that the small heterogeneous
molecules present in HS, as in weakly associated
superstructures, can be covalently bound into
true oligo- or polymers by an oxidative coupling
reaction catalyzed by a peroxidase enzyme. The
extent of covalent polymerization should be a
function of the amount of humic molecules,
mainly phenolic or benzencarboxylic acids derived from lignin and microbial biosynthesis,
which may undergo oxidative coupling reactions. However, it should be imagined that other
classes of compounds may become assimilated
into the macromolecular structures of polymerized humus.
Cozzolino and Piccolo (2001) extended the
polymerization catalysis by HRP to other HS and
studied the effects of solution pH (4.7 and 7) and
compositions of humic associations. By HPSEC
experiments they confirmed that an increase in
weight-average MW occurred invariably for all
HS subjected to oxidative polymerization. More-
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
over, a comparison of chromatograms and of MW
values obtained by treating humic solutions with
AcOH to pH 3.5 before HPSEC injection confirmed that the increase in molecular size by HRP
catalysis was stable and caused by the formation of
covalent bonds in the reacting humic molecules.
However, covalent polymerization of humic molecules was found to proceed to a greater extent at
pH 7 than at pH 4.7, despite the fact that HRP is
most active at the latter pH. The difference in reactivity was attributed to the large mobility of reacting molecules in the hydrated and relatively
smaller humic associations stabilized only by weak
dispersive (hydrophobic) forces at pH 7. Reactive
humic molecules are more mobile at neutral pH,
and the polymerization via a free-radical mechanism is more efficient. Conversely, intermolecular
hydrogen bonds formed at pH 4.7 confer a larger
size and rigidity to humic associations, whereas
the mobility as well as the reactivity of small molecules are thereby decreased.
Synthetic complexes formed with Al-(hydr)
oxide preparations of montmorillonite were made
using HAs from an oxidized coal and a lignite
(Violante et al., 1999) to model organo-mineral
complexes of soils using humic matter similar to
that of soil. In a separate experiment, these synthetic clay-humic complexes were subjected to
the oxidative reaction with HRP as a catalyst and
H2O2 as an oxidant. No changes in the OC contents were observed for the oxidative conditions
applied. Extraction of the complexes with alkaline-pyrophosphate solutions allowed the determination of the amounts of HS solubilizable before and after the treatment with HRP. Figure 4
shows that the yields extracted in the cases of
both humic-clay complexes decreased significantly after the oxidative coupling reaction, ranging from about 42 to 32% and from 40 to 29%
for the complexes made of HA from oxidized
coal and lignite, respectively. These results indicate that polymerization of humic molecules occurred also in the solid phase of the clay-humic
complexes and the increase in molecular sizes of
the humic materials was the most probable cause
for the reduction in extraction yields. General
similarities to soil HS of the humic material were
used to form the model clay-humic complexes,
and, thus, it would also seem to be possible to induce the polymerization of HS in natural soil
samples in order to control or change the properties of native SOM.
The evidence shown here that humic
supramolecular associations can be turned into
more stable covalently linked structures of larger
825
Fig. 4. Yields (%) of H. S. extracted with an alkaline-pyrophosphate solution from synthetic OH-AL-humatemontmorillonite complexes formed by using humic
acids from oxidized coal and lignite before (COX-M;
LIG-M) and after polymerization reaction catalyzed
by (HRP) horseradish peroxidase (COX-MHRP; LIGMHRP).
molecular size can be interpreted as additional
evidence that HS may be present in soils as associations of relatively small molecules.
CHEMICAL AND SPECTROSCOPIC
EVIDENCE OF SUPRAMOLECULAR
ASSOCIATIONS
The model of self-assembling supramolecular
association of HS is related to mutual affinities of
certain molecules in aqueous solutions. Molecules tend to associate by intermolecular forces
(Israelachvili, 1994), and the strength of the association depends on their molecular structures.
Particularly strong associations are formed by
apolar compounds via the hydrophobic effect
(Tanford, 1980). A humic supramolecular association in solution is thus formed by the selforganization of hydrophobic and amphiphilic
compounds. The associations are isolated progressively from the network of water structure.
Such separation results in an increase in the entropy of the system and in the overall energy stabilization as the different humic molecules form
into a superstructure.
The importance of hydrophobic humic components in phenomena of aggregation in solution
and on surfaces, as well as in controlling the reactivity of HS, is well documented (Wershaw,
1999). Investigations by fluorescence quenching
826
PICCOLO
techniques provided evidence of hydrophobic
microdomains in loose humic associations (Morra
et al., 1990; Engebretson and von Wandruszka,
1994). By following the diffusion of 1,2dichloroethane into humic matter, Aochi and
Farmer (1997) showed that there are discrete microregions of different polarities in humic structures. Chien et al., (1997) studied by 19F-NMR
spectroscopy the interactions of a trifluoromethylated atrazine with a soil HA by measuring the
NMR relaxation of atrazine in the presence of
both hydrophilic and hydrophobic paramagnetic
probes. They confirmed the existence of hydrophobic domains by showing that atrazine occupied a domain of HS accessible only to neutral
hydrophobic molecules. Similar results were obtained by Piccolo et al. (1998) who studied the
isotherms for the adsorption of atrazine by HS
extracted from two soils with four different extractants. The most hydrophobic humic extract,
isolated from soil by an acetone-HCl solution, adsorbed more and desorbed less atrazine than the
more hydrophilic humic materials extracted by
either an alkaline or an alkaline-pyrophosphate
solution. Since the acetone-HCl extract represents only a small fraction of soil HS (Piccolo et
al., 1990b), the study of Piccolo et al. (1998) inferred that humic microdomains of different polarities are also present in soil and their distributions determine their reactivities.
Direct evidence for the presence of hydrophobic domains in HS were further given by
Kohl et al. (2000), who studied the sorptive uptake
of hexafluorobenzene by two peat samples using
solid-state 19F-NMR spectroscopy. They found
that the sorption process was rapid and related to
the soil lipid content, whereas removal of the
lipids decreased significantly the sorptive capacity
of SOM. Hu et al. (2000), using solid-state NMR
and wide-angle X-ray scattering (WAXS) detected crystalline domains composed of poly(methylene) chains in several samples of SOM,
humins, and HAs from soil and coal. Their results
indicated a crystallite thickness equivalent to approximately 25 CH2 units to give hydrophobic crystalline domains. A comparable amount of
noncrystalline and more isotropically mobile poly
(methylene) chains were also found and, together
with the noncrystalline materials, larger aggregates were formed. In accordance with the
supramolecular association model described here,
Hu et al. (2000) concluded that the crystallites are
expected to be resistant to environmental attack
and, thus, inert in the soil and likely to have long
residence times, whereas amorphous regions may
SOIL SCIENCE
play a role in the sorption of nonpolar molecules
in soil.
Disruption of humic supramolecular associations as a result of the formation of hydrogen
bonds stronger than the hydrophobic forces stabilizing the original conformation was shown by
Miano et al. (1992). They used fluorescence and
infrared spectroscopy to investigate the effects of
the addition of glyphosate [N-(phosphonomethyl)
glycine] herbicide to a purified humic solution to
bring the pH from 9 to progressively lower values. The excitation spectra revealed increasing
fluorescence quenching effects with increasing
glyphosate contents at high wavelengths. Synchronous spectra showed a decrease in the main
peak intensity with increasing additions of herbicide. These results were interpreted in terms of a
disaggregation of the humic supramolecular association as the result of the formation of multiple
hydrogen bonds between glyphosate and the
small humic molecules. This implied that a decrease in electron delocalization, attributable to
the apparent large molecular size of the HS, was
responsible for the bands at high wavelengths.
The molecular and conformational structures of
HS were confirmed as determinants of the adsorption of glyphosate on different humic materials (Piccolo et al., 1996c). The high content of
aliphatic components and the large and flexible
molecular size increased adsorption of the herbicide on HS, possibly because of the involvement
of dispersive hydrophobic interactions, together
with hydrogen bonds.
Conte et al. (1997) showed that the formation
of tightly bound hydrophobic domains in solutions of HS could alter the quantitative evaluation
of humic C distribution in 13C-NMR spectra
recorded in the liquid state. A comparison between liquid-state NMR spectra and solid-state
CPMAS-NMR spectra of several HS showed that
the amounts of alkyl groups measured by the former technique were invariably lower than for the
latter method. The authors attributed this result to
the separated phase that the humic hydrophobic
components create to decrease the total solvation
energy in solution and to the consequent limited
spin-lattice relaxation time of hydrophobic carbons.Although liquid-state spectra may, therefore,
show lower signals in the alkyl-C region, this does
not occur in the solid-state mode, where signal
recording occurs by polarization transfer from
protons near the carbons measured.
In another approach, Kenworthy and Hayes
(1997) used the fluorescence quenching of
pyrene by bromide to investigate the nature of
VOL. 166 ~ NO. 11
SUPRAMOLECULAR STRUCTURE OF HS
humic associations. They found that pyrene in a
HS solution was protected from bromide
quenching. This protection was lost, however,
when AcOH, followed by a base, was added to
the medium. The authors considered that hydrophobic associations of the humic molecules
protected the pyrene from the bromide. Treatment of the humic solution with AcOH, as proposed by Piccolo et al. (1996a and b), removed
that protection because of disaggregation of the
loose humic superstructure. This suggested that
the HS in solution were associations of low MW
masses held together by hydrophobic bonding
and in which pyrene was enveloped.
Supramolecular associations of HS were
found by Haider et al. (2000) and Ricca et al.
(2000) to be disrupted into smaller components
by the action of derivatizing reagents (trimethyl
silyl or halogenated alkyl compounds) to silylate
or methylate acidic oxygenated functions. Such
simple derivatizations, which could not break
ether and ester linkages, disaggregated the weakly
assembled humic supramolecular structures into
smaller entities that readily dissolve in organic
solvents, elute in the low molecular size ranges by
HPSEC (Haider et al., 2000), and produce better
resolved NMR and FTIR spectra (Ricca et al.,
2000). Wanner et al. (2000) used the concept of
disaggregation of humic supramolecular associations to explain the shift in gel permeation to
larger elution volumes of the radioactivity of a
14C-labeled dithianon fungicide bound to HS
extracted from soil after 64 days of incubation together with straw. The same material extracted
from soil without incubation with straw indicated that the radioactivity was mainly in the
large molecular size fraction of the HS. The authors reasoned that organic acids or other amphiphilic compounds produced during microbial
degradation of maize would have disrupted the
association of humic molecules as proposed by
Piccolo et al. (1996a and b).
Data from Simpson et al. (2000), who used
Diffusion Ordered Spectroscopy (DOSY) NMR,
which has enhanced sensitivity because the probe
circuitry is kept at 22 oK, support the supramolecular model proposed by Piccolo and coworkers.
In their preliminary study, the authors observed
that the Nuclear Overhauser Effect (NOE) of a
humic material was explained more by an association of small molecules rather than by a macromolecular structure.
Buurman et al. (2001) studied the thermal stabilities of soil humic extracts exchanged with H,
Na, Ca, or Al before and after wetting with organic
827
solvents such as methanol, formic acid,and AcOH.
The thermal stabilities of Na-humates, upon addition of the organic solvents, shifted to high temperatures, whereas hardly any effect was observed
for H-, Ca-, and Al-humates. This was explained
by the mobilities of relatively small humic molecules that were forced by the organic solvent to
enhance their intermolecular hydrophobic interactions and strengthen their Na-humate supramolecular structures. More energetic bonds, such
as hydrogen bonds in H-humates, and electrostatic
bridges with divalent and trivalent cations in Caand Al-humates, provided an increased humic
conformational rigidity and prevented a molecular
rearrangement in the organic solvents.
CONCEPTS OF SUPRAMOLECULAR
ASSOCIATIONS OF HUMIC
SUBSTANCES
The results of the experiments described that
used either analytical or preparative SEC cannot
be explained by analytical interferences or by the
traditional polymeric model of HS. Rather, they
can be interpreted by the concept of loosely
bound humic supramolecular associations. In this
concept, one can imagine HS to be relatively
small and heterogeneous molecules of various
origins that self-organize in supramolecular conformations. Humic superstructures of relatively
small molecules are not associated by covalent
bonds but are stabilized only by weak forces such
as dispersive hydrophobic interactions (van der
Waals, -, and CH- bondings) and hydrogen
bonds, the latter being progressively more important at low pH values. Hydrophilic and hydrophobic domains of humic molecules can be
contiguous to or contained in each other and, in
hydration water, form apparently large molecular
size associations. In humic supramolecular organizations, the intermolecular forces determine
the conformational structure of HS, and the
complexities of the multiple noncovalent interactions control their environmental reactivity.
The definition given by Lehn (1995) may
well be applied to HS:“Supramolecular assemblies
(are) molecular entities that result from the spontaneous association of a large undefined number
of components into a specific phase having more
or less well-defined microscopic organization and
macroscopic characteristics depending on its nature (such as films, layers, membranes, vesicles, micelles, mesomorphic phases, solid state structures,
etc.).”
On consideration of the concept of supramolecular associations, the classical definitions of
828
PICCOLO
HAs and of FAs should be reconsidered. The FAs
may be regarded as associations of small hydrophilic molecules in which there are enough
acidic functional groups to keep the fulvic clusters dispersed in solution at any pH. The HAs are
composed by associations of predominantly hydrophobic compounds (polymethylenic chains,
fatty acids, steroid compounds) that are stabilized
at neutral pH by hydrophobic dispersive forces
(van der Waals, -, and CH- bondings). Their
conformations grow progressively in size when
intermolecular hydrogen bonds are increasingly
formed at lower pH values until they flocculate.
FUTURE PERSPECTIVES IN
RESEARCH AND TECHNOLOGY
A clarification of the aggregate structures of
HS represents a major innovation in humus
chemistry. The concepts presented here emphasize that HS are not macromolecular polymers, as
they have been described for so long, but are
rather superstructures of apparent large size only
and are self-assembled from relatively small heterogeneous molecules held together mainly by
hydrophobic dispersive forces. These concepts
provide a new opportunity to enlarge knowledge
of both the detailed chemistry and the management of HAs in soil and in the environment.
Chromatographic methods of separation such
as HPSEC were found to produce reproducible
and more homogenous fractions of humic superstructures.Awareness of the weak forces that cause
the self-assembling of humic molecules allows
methods to be devised based on interactions with
chemical species, such as amphiphilic organic
acids, urea, mono- and polyvalent cations, that can
disrupt the apparently large humic associations
and obtain fractions that are chemically simpler
and more homogeneous.
The combination of chromatographic methods with spectroscopic techniques such as NMR,
IR, and ESR spectroscopy, together with the different modern variations of mass-spectrometry,
has increased enormously the potential to derive
a complete picture of the secondary molecular
structure of HS. The time is near when there will
be good grasp of the molecular structures and associations of HS, based on chemical methods
rather than on computer models. Our ability to
obtain a full molecular structure of any humic
material or natural organic matter (NOM) molecule from any environment and ecosystem will
then be limited by only the advances in analytical
automation. When we consider the rapidity with
which certain biological research fields can ad-
SOIL SCIENCE
vance, this limitation could easily be overcome,
provided there is the necessary public and private
interest to pursue knowledge in a field that is vital for the well-being of our planet and the biological life on it.
The novel understanding of HS as supramolecular associations as described here has great implications in soil and environmental management. One example is the possibility of turning
the loose humic superstructures into real covalently linked polymers by catalytic technology
that can ensure polymerization of humic molecules in both water and soil environments. Such
technology can improve our capacity to control
SOM management, reduce the risk of erosion,
and limit soil desertification with the obvious improvement of soil productivity. Another example
of the potential of the polymerization of soil
humic molecules in situ is the possibility of controlling CO2 emission from agricultural soils by
sequestering the organic C in more stable polymerized humus. Finally, a large number of observations indicate that humic molecules released
from large supramolecular associations can influence nutrient uptake by plants and increase crop
yields significantly. Combinations of refined
chemical analyses of humic molecules with physiological studies on their effect on plants may
clarify the mechanism(s) by which soil HS increase crop yields and, possibly, revive the former
interests in humus based fertility. The use of humic molecules, either native or exogenous, in
combination with inorganic fertilizers, to maximize plant nutrient uptake and final yields, may
also have a tremendous impact on increasing the
economic efficiency of fertilizers and protecting
the environment from the pollution caused by
excess uses of fertilizers.
ACKNOWLEDGMENTS
The author thanks his former doctoral students, Drs. P. Conte and R. Spaccini, for most of
the original research presented here and Ms. A.
Cozzolino, who is now completing her doctorate.
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