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Analytical Characteristics of a Haploboroll
HA and a Spodosol FA
Element (g kgϪ1)
Functional groups (cmol kgϪ1)
all of the O in the FA is similarly distributed. The E4 /E6 ratio of the FA is almost
twice as high as that of the HA, which means that the FA has a lower particle or
molecular weight than the HA (Chen et al., 1976).
B. INFRARED (IR) AND FOURIER TRANSFORM
IR and FTIR spectra of humic substances show bands at 3400 cmϪ1 (H-bonded
OH), 2900 cmϪ1 (aliphatic C–H stretch), 1725 cmϪ1 (CuO of CO2H, CuO stretch
of ketonic CuO), 1630 cmϪ1 (COOϪ, CuO of carbonyl and quinone), 1450 cmϪ1
(aliphatic C–H), 1400 cmϪ1 (COOϪ), 1200 cmϪ1 (C–O stretch or OH deformation
of CO2H), and 1050 cmϪ1 (Si–O of silicates). The bands are usually broad because
of the extensive overlapping of individual adsorbances. IR and FTIR spectra reﬂect
the preponderance of oxygen-containing functional groups, i.e., CO2H, OH, and
CuO in humic materials. While IR and FTIR spectra provide worthwhile information on the functional groups and their participation in metal–humic interactions,
they tell us little about the chemical structure of humic materials.
Celi et al. (1997b) applied FTIR to the analysis of CO2H groups in a number of
HAs. Concentrations of CO2H groups in HAs were determined directly from FTIR
spectra by totaling adsorbances at 1720–1710 cmϪ1 (CO2H) and 1620–1600
cmϪ1 (COOϪ). Good correlations were found between total carboxyl groups determined by FTIR, a wet chemical method, and by 13C NMR. Thus, depending on
A LIFETIME PERSPECTIVE
the equipment and facilities available, soil chemists have a choice of methods that
can be used for measuring CO2H groups in HAs.
Until about the mid-1980s, when the use of liquid- and solid-state 13C NMR became more widespread, most soil chemists thought that the chemical structure of
humic substances was predominantly aromatic. 13C NMR demonstrated that
aliphatic structures in humic substances were often as important, and sometimes
even more important than aromatic structures (Schnitzer and Preston, 1986; Wilson, 1987; Norwood, 1988; Schnitzer, 1991). This was a very signiﬁcant development in our understanding of the chemistry of humic substances. Aromaticities
of HAs extracted from soils of widely differing pedological origins range from 30
to 60% (Schnitzer, 1991). A substantial portion of aliphatic carbons in HAs consists of parafﬁnic carbons. Of considerable interest are the prominent resonances
in both liquid- and solid-state 13C NMR spectra of humic substances near 130 and
132 ppm, which can be assigned to C in aromatic rings that are not substituted by
strong electron donors such as O and N but by C. Alkaylaromatics are typical structures that produce such resonances (Breitmaier and Voelter, 1978).
Of special interest in the context of this discussion is a comparison of solid-state
13C NMR spectra of a HA (extracted from the Ah horizon of a Haploboroll) and a
FA (extracted from the Bh horizon of a Spodosol). The HA spectrum in Fig. 4 shows
several distinct peaks in the aliphatic (0–105 ppm), aromatic (106–150 ppm), phe-
Figure 4 Solid-state 13C NMR spectra of HA (extracted from the Ah horizon of a Haploboroll)
and FA (extracted from the Bh horizon of a Spodosol).
nolic (155–160 ppm), and carboxyl (170–180 ppm) regions. The signals at 17, 21,
25, 27, and 31 ppm are likely due to alkyl C. The resonance at 17 ppm is characteristic of terminal CH3 groups and that at 31 ppm of (CH2)n in straight parafﬁnic
chains. The resonance at 40 ppm could also include contributions from both alkyl
and amino acid C. The broad signal at 53 ppm and the sharper one at 59 ppm may
be due to C in OCH3. Amino acid C may also contribute in this region (Breitmaier
and Voelter, 1978). Carbohydrates in HA would be expected to produce signals in
the 60 to 65, 70 to 80, and 90 to 104 ppm regions, although other types of aliphatic
C bonded to O could also do so. The aromatic region contains a relatively sharp maximum near 130 ppm due to alkyl aromatics. The peak at 155 ppm indicates the presence of O- and N-substituted aromatic C (phenolic OH and/or NH2 bonded to an
aromatic C). The broad signal near 180 ppm is due to C in CO2H groups, although
amides and esters could also contribute to this resonance.
The 13C NMR spectrum of the FA (Fig. 4) consists of a number of aliphatic resonances in the 20- to 50-ppm region, followed by signals from C in OCH3 groups,
amino acids, and carbohydrates between 50 and 85 ppm. Broad signals between
130 and 133 ppm indicate the presence of C in alkyl aromatics. The strong signal
between 170 and 180 ppm shows the presence of C in CO2H groups. In general,
fewer sharp signals are observed in the 13C NMR spectrum of the FA than in that
of the HA, possibly because of more H bonding in the FA.
13C NMR data for HA and FA are summarized in Table II in terms of the distribution of C in the different spectral regions. An examination of data in Table II
shows a similar C distribution in the two humic fractions. HA is slightly more aro-
Distribution of C (%) in a Haploboroll HA and a Spodosol FA as Determined
by 13C NMR
% of C
Chemical shift range (ppm)
Aliphatic C (0–105 ppm)
Aromatic C (106–150 ppm)
Phenolic C (151–170 ppm)
((Aromatic C ϩ phenolic C) /(Aromatic C ϩ phenolic C ϩ aliphatic C)) x 100.
A LIFETIME PERSPECTIVE
matic than FA, but FA is richer in CO2H groups, which appears to be the main difference between the two humic substances. Other differences are that HA is richer in parafﬁnic C but poorer in carbohydrate C than FA. However, on the whole,
the main structural features, as well as aromaticity and aliphaticity, are similar so
that HA and FA have similar chemical structures. These ﬁndings disagree with
those of Sprengel (1826) and other earlier workers who thought that the chemical
structures of HA and FA were quite different from each other.
Little is known about the chemical structure of humin, which is that portion of
SOM that stays behind after extraction of the soil with dilute alkali. In a more recent investigation, Preston et al. (1989) deashed the surface layer of Bainsville soil
with aqueous HCl/HF (1.16 M HCl and 2.88 M HF) at room temperature for a prolonged period of time. With progressive deashing, the humin became more soluble in 0.5 M NaOH. After extensive deashing the solid-state 13C NMR spectrum
was similar to that of HA extracted from the same soil. This suggests that humin
is essentially HA bound strongly to soil minerals.
Valuable information on the chemical structure of humic substances can be obtained by combining 13C NMR with chemical methods. In this manner, effects on
the chemical structure of humic substances of different extractants, methylation,
hydrolysis, oxidation, and reduction can be evaluated. Spectra shown in Fig. 5 illustrate this point.
Figure 5a shows the solution-state 13C NMR spectrum of HA extracted from the
Ah horizon of a Haploboroll from northern Alberta (Preston and Schnitzer, 1984).
The presence of aliphatic C (i.e., C in straight chain, branched and cyclic alkanes,
alkanoic acids, and other aliphatic compounds) is indicated by signals in the 10to 40-ppm region of the spectrum. Carbon in proteinaceous materials (amino acids,
peptides, and proteins) exhibits resonances between 40 and 60 ppm, whereas C in
OCH3 groups shows signals near 56 ppm. Signals between 61 and 105 ppm arise
from C in carbohydrates. Resonances between 106 and 150 ppm are due to aromatic C whereas those between 150 and 160 ppm arise from phenolic C. The strong
signal between 170 and 180 ppm comes mainly from C in CO2H groups, with possibly some contributions of C in esters and amides.
Figure 5b shows the solution-state 13C NMR spectrum of the same HA after hydrolysis for 24 hr with hot 6 M HCl. Most of the resonances in the 40- to 105-ppm
region arising from C in proteinaceous materials and carbohydrates are no longer
observed because of the hydrolytic removal of these materials by the hot acid.
Also, the intensity of the signal between 170 and 180 ppm, due largely to C in
CO2H groups, has been reduced because of partial decarboxylation of these groups
by the strong acid. However, intensities of the two principal HA components, i.e.,
aliphatic C (10 –60 ppm) and aromatic C (106 –150 ppm), remain undiminished.
Thus, by removing carbohydrates and proteinaceous components, acid hydrolysis
“puriﬁes” the HA and allows us to study the remaining aliphatic and aromatic components in greater detail.
Figure 5 Solution-state 13C NMR spectra of a HA extracted from the Ah horizon of a Haploboroll
before (a) and after (b) hydrolysis with hot 6 M HCl. From Schnitzer (1991), with permission of the
D. ELECTRON SPIN RESONANCE SPECTROSCOPY
Electron spin Resonance (ESR) spectroscopy measures free radicals (unpaired
electrons) in humic substances. It has been known since the early 1960s (Rex,
1960; Steelink and Tollin, 1962) that humic substances contain free radicals that
may participate in a wide variety of organic–organic and organic–inorganic interactions. The theory and a number of applications of ESR spectroscopy have been
described by Atherton (1973). The ESR spectrum of a typical HA is shown in Fig.
6. The spectrum consists of a single line devoid of hyperﬁne splitting. From the
spectrum (by comparison with a standard), the number of free radicals per unit
weight as well as the g value (the spectroscopic splitting constant) and also the line
width can be calculated. From the magnitudes of the g values for humic materials,
with most ranging from 2.0038 to 2.0042 (Senesi and Schnitzer, 1977), it appears
that prominent free radicals in humic materials are semiquinones or substituted
A LIFETIME PERSPECTIVE
ESR spectrum of a typical HA.
semiquinones. There are two types of free radicals in these materials: (a) permanent free radicals with long lifetimes and (b) transient free radicals with relatively short lives (several hours). Transient free radicals in humic materials can be generated in relatively high concentrations by chemical reduction, irradiation, or
increase in pH (Senesi and Schnitzer, 1977). Permanent free radicals, in contrast,
appear to be stabilized by the complex chemical structures of humic materials,
which can act both as electron donors and as electron acceptors. These oxidation–
reduction reactions are reversible and can be assumed to proceed in the following
The semiquinone (shown in the center) can be produced either by the reduction
of a quinone or by the oxidation of a phenol. Under alkaline conditions, a semiquinone anion and the semiquinone dianion are formed. Except for indicating the
presence of phenols, semiquinones, and quinones in humic materials, ESR spectroscopy has so far contributed little to our understanding of the structural chemistry of these substances. The main reason for this is that it has been difﬁcult to
split the signal. However, signiﬁcant new information has been generated by ESR
on the symmetry and coordination of metals in metal–humic complexes (Cheshire
et al., 1977; Lakatos et al., 1977; Senesi et al., 1977; McBride, 1978; Boyd et al.,
1981; Cheshire and Senesi, 1998). HAs and FAs interact with paramagnetic metal ions to form a variety of metal–organic complexes. These metal ions include
Cu2+, (VO)2+, Mn2+, Mo(V) and Mo(III), Cr3+, and Fe3+. ESR parameters of complexes formed between these metal ions and HA and FA have been described in
considerable detail by Cheshire and Senesi (1998). Especially interesting is the
ESR spectrum of a Fe3+ –FA complex (Fig. 7) (Senesi et al., 1977; Cheshire and
Senesi, 1998). The signal at gϳ 2.0 (resonance C) generally arises from Fe3+ ions
in close proximity to each other. This signal is removed easily by reduction with
hydrazine. From Mössbauer studies, it is suggested that this type of Fe3+ is held
in octahedral coordination on external HA and FA surfaces.
The signal at g~ 4.3 (resonance A) is assigned to Fe3+ in an organic complex,
in which high-spin Fe3+ ions occupy sites of approximately orthorhombic sym-
ESR spectrum of a Fe3+ –FA complex.
A LIFETIME PERSPECTIVE
metry. Thus, ESR spectroscopy can provide important information on the coordination and symmetry of paramagnetic metal ions complexed by humic substances.
E. ELECTRON MICROSCOPY
Particle sizes and shapes of HAs and FAs seen under the electron microscope
are affected by pH, electrolyte concentration, and HA and FA concentrations (Chen
and Schnitzer, 1989).
Freeze dried at pH 2, FA produces a scanning electron microscopy (SEM) micrograph (Fig. 8a) that exhibits elongated ﬁbers and bundles of ﬁbers (Chen and
Schnitzer, 1976b; Chen et al., 1976). The ﬁbers are either linear or curved, ranging up to 6–7 m in length and 120–400 nm in thickness. At pH 4 (Fig. 8b), the
ﬁbers tend to become thinner and the bundles of ﬁbers more prominent. The
lengths of the ﬁbers remain unchanged, but their thicknesses are reduced to 120–
200 nm. At pH 6 (Fig. 8c), the ﬁbers diminish in numbers and thicknesses and a
greater proportion of the FA occurs in bundles of closely knit ﬁbers. A pH 7 (Fig.
8d), a ﬁne network of tightly meshed ﬁbers with parallel orientation is observed.
At pH 8 (Fig. 8e), the ﬁne network turns into a sheet-like structure of varying thicknesses. At pH 9 (Fig. 8f), the sheets tend to thicken. At pH 10 (Fig. 8e), the SEM
micrograph shows homogeneous grain-like particles.
Because of solubility constraints, the effect of pH on the structure of HA over
the pH range 6 to 10 is similar to that observed on FA (Chen and Schnitzer, 1976b).
Effects of increasing concentrations of neutral salts in aqueous solutions of HA
and FA on their SEM micrographs following freeze-drying were studied by Ghosh
and Schnitzer (1982). Fiber thicknesses increase but particle orientation gradually decreases with increases in salt concentrations. These effects are similar to those
observed when the pH is decreased from neutrality to the moderately acidic region. Thus, both pH and elevated salt concentrations affect the dissociation and
conformation of HAs and FAs in a similar manner.
Effects of varying the concentrations of HA and FA in solution prior to freezedrying on the sizes and shapes of the micromolecules under transmission electron
microscopy were examined by Stevenson and Schnitzer (1982). Single drops of
dilute aqueous HA and FA solutions, adjusted to different pH values, were spread
uniformly on strips of freshly cleaned mica. The solution-bearing strips were tilted and then frozen rapidly in Freon. This allowed the surface ﬁlm to run toward
the lower edges of the mica and form a concentration gradient. The smallest individual particles that appear in the dilute areas are spheroidal with diameters ranging from 9 to 27 nm. Spheroids tend to coalesce to form round-shaped aggregates
or linear chain-like structures. At higher concentrations, the spheroids and chainlike structures form ﬂat, elongated, multibranched ﬁbers (Fig. 9), similar to those
observed earlier by Chen and Schnitzer (1976b). The width of the ﬁbers (or ﬁlaments) ranges from 20 to 100 nm. At higher concentrations, parallel arrays of ﬁbers
Figure 8 Scanning electron micrograph of a freeze-dried FA at various pH values. From Chen and Schnitzer (1976b), with permission of the publisher.
A LIFETIME PERSPECTIVE
Figure 9 Transmission electron micrograph of a 0.01% HA solution. From Schnitzer (1991), with
permission of the publisher.
tend to coalesce to form sheet-like structures. Spheroids observed at low concentrations are larger and range in diameters from 12 to 50 nm. With increasing concentrations of HA and FA, spheroids coalesce into aggregates of spheroids and then
into chain-like structures, followed by ﬁbers and ﬂattened sheets.
An interesting conclusion drawn by Chen and Schnitzer (1989) is that humic
substances in aqueous solutions act like ﬂexible, linear polymers rather than spheroids. When sample concentrations are high and the pH of the solution is low or
when appreciable amounts of neutral electrolytes are present, humic particles may
assume a spheroidal shape. At low concentrations and neutral to basic pH, the particles stretch, forming slightly coiled ﬁbers. The latter consist of a large number of
oriented molecules. At low solute concentration, low ionic strength, and high pH,
the coils stretch further, the ﬁlamentous structure disintegrates, and complete dispersion takes place. If, under similar conditions, the concentration of humic substances is high, sheet-like structures may be formed due to coagulation.
IV. CHEMICAL STRUCTURE
OF HUMIC SUBSTANCES
A. OXIDATIVE DEGRADATION
One of the most useful methods for obtaining information on the chemical structure of complex organic substances is oxidative degradation. Over the course of
20 years, my co-workers and I have investigated the oxidative degradation of HAs,
FAs, humins, and whole soils using a variety of methods. These included the oxidation of methylated and unmethylated humic substances with an alkaline KMnO4
solution (Schnitzer and Khan, 1978; Grifﬁth and Schnitzer, 1989). The somewhat
milder oxidation with alkaline CuO, as well as the sequential oxidation with CuONaOH ϩ KMnO4 and with CuO-NaOH ϩ KMnO4 ϩ H2O2 solutions, has also
been employed (Schnitzer and Khan, 1978; Schnitzer, 1978). Humic substances
have also been degraded under acidic conditions with peracetic acid and nitric acid
(Schnitzer, 1978). Other oxidants used include alkaline nitrobenzene, sodium
hypochlorite, and H2O2 solutions (Schnitzer and Khan, 1978). Degradations with
Na2S and phenol have also been carried out (Hayes and O’Callaghan, 1989).
Major compounds produced by the oxidation of methylated and unmethylated
humic substances from widely differing pedological and geographical origins under alkaline as well as under acidic conditions are aliphatic carboxylic, phenolic,
and benzenecarboxylic acids (Schnitzer and Khan, 1978; Schnitzer, 1978; Grifﬁth
and Schnitzer, 1989).
Among aliphatic oxidation products are mono-, di-, tri-, and tetracarboxylic
acids. Major aromatic oxidation products are benzenecarboxylic acids such as the
tri, tetra, penta, and hexa forms (Fig. 10), whereas phenolic acids include compounds containing between one and three OH groups and between one and ﬁve
CO2H groups per aromatic ring (Fig. 11).
From the oxidation products identiﬁed and from 13C NMR spectra of humic
substances, it appears that aromatic rings are cross-linked by parafﬁnic chains (Fig.
12). On oxidation, the aliphatic carbons closest to the rings become the C of CO2H
groups and remain bonded to the rings whereas the other carbons in the aliphatic
chains are oxidized to either aliphatic acids or CO2. The formation of CO2 from
Major benzenecarboxylic oxidation products.