An Assessment of Progress in Studies of the Chemical Structure of Humic Substances

By David Grant BSc MSc PhD AB53 6SX UK

This document which is an updated version (portion of text in italics) of a review written many years
ago as part of an academic institute research initiative*
is also of current possible relevance to how adjustment of land cultivation practices may be achieved
so as to maximise the polymethylene contents of soil humic substances which could form the
basis of a scientifically sound method for carbon sequestration which complies with the
allowable protocols within the Kyoto agreement for the amelioration of anthropogenically
augmented atmospheric carbon dioxide levels

Contents
1 Introduction
2 Some researches on soil organic matter conducted from 1975-1981 at the University of Aberdeen
3 The five humic-substance-defining characteristics identified by MacCarthy (2001)
4 Humic substances and water activity
5 An alternative climate change amelioration strategy
6 Fractionation of Humic Matter
6a Humic substances sequester agrochemicals including inorganic nutrients as well as environmental toxins
6b Classical fractionation: aqueous alkaline solution
6b-1 Non-Aqueous vs Aqueous Solvent Extraction of Humic Acids
6c Column fractionation
7 Humic Acids : Their Chemical Structures. Some Current and Previously-Held Views
7a Molecular Weight & Elemental Composition
7a-1 Anionic Functional Groups
7b The lignin hypothesis of humic matter
7c Fungal melanins and humic acids
7d Effect of oxidants, eg alkaline KMnO4 etc on humic substances
8 Formation of aromatics during pyrolysis of aliphatics
9 Humic Acids : Resistance to Biodegradation
10 Humic Acid Particles
11 Early Attempted Use of NMR to Study Humic Acids
12 Polymethylene Chains : Key Structural/Stabilisation Features of Humic Substances
12a Polymethylene in clay humic acid complexes
13 Alkyl phthalates in soil
14 Poly maleic anhydride model of humic substances
15 Use of UV absorption spectroscopy pyrolysis GLC-MS and infrared spectroscopy for the
characterisation of humic acids
16 [15N NMR]
17 References

1 Introduction
In the decades following the conclusion made by Schnitzer and Khan in 1972 that the chemical
structure of humic acids and related substances was largely unknown, there have been many attempt to
elucidate the chemical nature of these substances including, by the use of NMR (MAS) procedures
which should, in principle, have by now finally established the constitution of humic matter, but this
task is still commonly believed not yet to have been achieved (cf eg Loh et al 2004; Pena-Mendez et al
2005).

This problem of establishing the chemical structural nature of humic substances remains of
considerable theoretical and practical importance since humic substances are probably the most
abundant class of natural carbon-based polymers on earth, being an essential part of the mechanism of
plant nutrition (many essential ‘trace’ elements being bound by them) soil classification, structure and
stability, as well as being of interest for their roles in the formation of peat (brown) coal, natural waters
and mineral oil.

2 Some researches conducted on soil organic matter from 1975-1981 at the University of Aberdeen
suggested that good quality NMR spectra of humic substances {which could be obtained following the removal of paramagnetic
ions using detergent industry polyphosphate chelation methods but without which treatment a meaningful interpretation of the
spectra had been almost impossible} clearly showed that the traditional views of the nature of humic substances were incorrect.
The most obvious (most easily identified) widely distributed chemical structures present in humic substances seemed to be
polymethylene chains (cf Grant 1977). This finding had seemed to be fully in accord with the results of previous studies which
had been conducted in industrial laboratories which demonstrated that polyethylene was uniquely the most highly stable
chemical structure when placed into a soil environment. The most abundant material which co-occurred with polymethylene in
humic substances however produced NMR spectra indicating the presence of degraded polysaccharides.

3 The five humic-substance-defining characteristics identified by MacCarthy (2001)

These should perhaps be augmented by a sixth characteristic- the binding of humic substances to
crystallisation nuclei
The abandonment by the scientific community of the traditional chemical model of humic acid and
related substances seemed by 2001 seems to have become almost complete as is suggested by the
series of review articles (Burden, Hayes & Clapp, MacCarthy, Simpson) which appeared in Soil
Science during late 2001.
It is now suggested, however, that wider attention now needs to be given to a further key fundamental
property of humic substances namely their avid adsorption onto mineral surfaces and the consequent
inhibition of the establishment of thermodynamic equilibria relating to the creation of inorganic
element buffers and the formation of sparingly soluble salts. This phenomenon apparently can control
the environmental formation of such minerals as CaCO3 (calcite), a phenomenon which may be of
critical relevance to the natural mechanism of control of the global carbon cycle; since anthropogenic
input can influence this, it therefore is of relevance to current attempts to understand how human
activities, in addition to the combustion of fossil fuel, can contribute to an increase in atmospheric CO2
contents (cf Grant, 2009).

4 Humic substances and water activity

Humic and fulvic acids may be prime examples of Haraguchi (2004) (all-element) metallomic matrices
[other examples are seawater, blood and such anionic polysaccharides as heparin/heparan sulphate cf
Grant (2010); a corollary to this hypothesis is that natural polyanionics like humic substances may
exert a key influence on water activity achievable via the modulation of the physical chemistry of water
molecule aggregation induced by ionic forces; such, as yet poorly understood, water activities per se
may be of fundamental relevance to biology].

5 An alternative climate change amelioration strategy :

formation of carbon sinks composed of stable soil organic matter
An improved knowledge of the chemical constitution of humic matter can in principle also allow for
the rational design of the kind of stable global carbon sink which is believed to be critically required to
offset the anthropogenic increase in atmospheric content of CO2 (eg cf Pr, 2008).

Strategies for reducing atmospheric CO2 which are based exclusively on restriction of the combustion
of fossil fuels might be improved in effectiveness by the inclusion of inducements for the improvement
of soils by increasing their stable soil organic matter contents; this would obviously require a soil
organic matter-chemical-analysis- methodology directed towards the regulation of agricultural
activity so as to promote long-term carbon capture by the soil organic matter.
While the dominant current belief seems to be that the correct ethical behaviour of human society
should be to focus exclusively on reducing the combustion of fossil fuels in order to prevent the
supposed potentially adverse effects of excessive global warming attendant on inappropriate
augmentation of atmospheric CO2 levels, an alternative strategy could aim to prevent such warming by
the promotion on a global scale of the formation of stable soil organic matter sinks in soils, a task
which in principle at least could be achievable in a secure scientifically-based manner.
It is now suggested that protocols and legal frameworks which focus on augmentation of the easy-to-
measure- –(CH2)n- content of soils could provide for such a simple and effective method by which
human society can offset the augmentation of atmospheric CO2 arising from the burning of fossil fuel.

(It should also be noted that the polymethylene chain structures which apparently contribute the long-
term environmental stability of humic matter could also be the origin of petroleum).

6. Fractionation of Humic Matter

6a Humic substances sequester agrochemicals including inorganic nutrients as well as environmental toxins
Humic substances are also important to the full understanding of the physics and chemistry of the
interactions between agrochemicals and soils.
This includes the sequestration of plant nutrients such as major and trace amount required inorganic
nutrient cations, nutrient anions including silicate, borate phosphate and nitrate, pesticides and
environmental toxins including potentially carcinogenic chlorinated substances during water
treatment, long-lived combustion-produced polyaromatic substances and incineration-of -municipal-
and-industrial-waste-produced polychlorinated and polybrominated aromatic substances.
Some fractions of humic matter also seem to act in a beneficial “hormone”-like manner to stimulate the
growth of plants; the fertility of soil must therefore be related to some extent to the abundance and
distribution of these particular kinds of humic substances.
Plant growth stimulation by humic acid preparations is difficult to assess the conflicting literature data
in this topic since the chemical nature of “humic acids” studies were not adequately characterised.

6b Classical fractionation: aqueous alkaline solutions

Humic polymers were first studied by chemical methods towards the end of the eighteenth century (by
Achard (1786), who found that an aqueous alkaline solution acted as an efficient peptising agent for
humic colloids). The presently employed nomenclature of humic substances derives essentially from
18th and 19th century usage being based on fractionation techniques (cf Kononova, 1966).
‘Fulvic ‘ acid is HCl (0.1M aqueous solution) soluble, but HCl precipitates ‘humic’ salts from NaOH
solutoins of extracted humic substances. This dispersion could be fractionated into “humic acids”
which precipitated readily upon acidification.
The later use of more elaborate fractionation schemes have generated further nomenclature viz:
humic (grey and brown varieties (Springer, 1959); (Domke, 1972) crenic, apocrenic cf. Murzakov
(1969) ulmic, fulvic (having fluvanic, fulvenic, fulvinic and lignofulvonic sub-fractions, Oden (1912)
Vil’yams (1968), hymatomelanic acid and humin are also amongst the terms used for other ill-defined
humic matter fractions].

6b-1 Non-Aqueous vs Aqueous Solvent Extraction of Humic Acids

EtOH (or Et2O) extracts ‘hymatomelanic’ acids from humic acids.
Humin is the insoluble organic residue (the term is also used for that portion of the original NaOH-
soluble organic matter that cannot be redissolved after precipitation by HCl)Solvent extraction of
humic acids from the soil involves the substitution of solvent molecules for the various physical and
weak chemical links between primary particles of humic acids and replacement of the more strongly
complexed transition metal ions. Reagents which complex metal ions can be used to obtain humic
extracts in moderate yield.

NaOH solutions are however the most widely used extractant for humic acids from soil, giving the
highest yields.
The relative efficiencies of common extractants of humic acids from soils have been reported as:

NaOH (0.1M) =100 (arbitrary units) [pyrophosphate] Na4P2O7 (0.5M) = 60.5, [oxalate] Na2C2O4 36.6
and NaF (0.1M) = 34.8 (Wilk 1961).

Pyrolysis studies have suggested that aqueous Na4P2O7 solution extracts more compact humic acids
having less side chains than are present in the humic acids from aqueous NaOH solution extracts (45).

The amount and type of extract obtained form different soils by formic acid-LiBr or acetone has been
suggested to be a convenient index of soil fertility (Eloff et al 1975)

Organic solvents may also be used as extractants for humic acids but these are generally less efficient
than aqueous inorganic reagent solutions, but may cause less structural alteration to the original humic
polymer molecules.

6b Column fractionation
Al2O3 gel and charcoal columns were employed by Vil’yams (1965) to separate the several ‘fulvic’
fractions (listed above), and by Forsyth (1946) to give four fractions. Two ‘fulvic’ acid sub-fractions
were distinguished by Tyurin (1968).

The employment of dextan gel filtration, thin layer chromatography or electrophoresis is apparently
only a little more successful (Ishiwatari, 1971; Tamai, 1971; Danneberg and Schaffer, 1974;
Cameron et al. 1972; Ladd , 1969; Butler and Ladd, 1969 and Banerjee, 1974) than the above
classical fractionation techniques in producing chemically differentiated fractions form soil organic
matter solutions, although molecular size distribution curves (‘fingerprints’) can be used for
characterisation purposes.

Classical subdivision of humus and the subdivision achieved by dextran gel filtration five differently
coloured fractions, in the former case the light absorption extinction coefficients increase with
decreasing solubility (and molecular weight) whereas in the latter case the extinction coefficient was
found to decrease with increasing molecular weight (Ladd, 1969).

7 Humic Acids : Their Chemical Structures. Some Current and Previously-Held Views
A current view is that humic acids are composed of
hydrophobic polymethylene subunit stabilised aggregates
largely held together by hydrogen bonding and a range of other non-covalent linkages (Piccolo, 2001
cf., Grant 1977d) but humic substances were previously widely believed to be derived from microbial
metabolites formed (BY HUMICFICATION) I.E. FERMENTAION FOLLOWED BY FURTHER BIOTIC AND ABIOTIC
DEGRADATION OF COMPLEXES WITH INORGANIC SPECIES FROM PLANT AND ANIMAL TISSUES BY MAINLY
INITIALLY BY A DIRECT MICROBIAL DEGRADATION;
PREVIOUS [NOW DISCREDITED THEORIES cf Susic 2001, 2003 ? but also cf eg Pema-Mendez et al 2005]
HAVE HELD THAT THE FORMATON OF LIGNIN-PROTEIN complexes were of importance in
humification (Waksman, 1938, cf, tanned protein, Handely, 1954) [suitable tannins were thought to be
provided by vegetable ‘polyphenol tannins’cf. Davies et al , (1964)].

Plant as well as microbial enzymes however might play some role in the formation of humic substances
by extracellular mechanisms.

Humic acids are probably the key types of polymer structure to be solved, the other named polymers
being variants of this basic type of compound. The term ‘humic’ acid is employed in the present text in
this more general sense (in common with the usage in much of the literature on the subject) rather than
as a particular subfraction of the total humic polymers.

Other preparations which have also been labelled as humic acids are produced by the action of
concentrated mineral acids on sugars (likely as a result of the promotion of
dehydration/oxidation/polymerisation reactions).

The formation of yet another type of synthetic “humic acid”-like polymers starting from phenols (or
quinones) had stimulated postulates of the formation of soil humic acids by this mechanism to give eg,
phenol-formaldehyde resin-like products; the initial phenols are envisaged as either being leached
from green leaves, resulting from microbial metabolites or the degradation of lignin. Ring opening
polymerisation of oxidised aromatic rings could also conceivably lead to humic acid-like polymers (cf
Ludemann et al (1973).

It might have been anticipated, a priori, however, that humic acids are random heterogeneous
aggregates of carbohydrates, microbial metabolites, degraded lipids and amino acids and contain both
mixtures and complexes, inorganic soil components and various organic polymers.
This view is now re-emerging, cf Piccolo (2001); Kelleher et al (2006).

However it had previously commonly considered (cf Burges (1960)) that the various fractions
produced by solvent extraction, as outlined above in Section 6, represent size fractionation of a singly
polymeric chemical substance or closely related chemical substances and furthermore that nitrogen is
probably not present in the primary polymeric backbone structure. This statement is based primarily
on the reproducibility of the properties and the difficulty of separation of identifiable compounds from
humic substances. Amino acids, amino sugars and phenols are considered to be adsorbed at the outer
surfaces or in pores of the main humic acid “core” polymer structure.

Seasonal variation in carbon (and nitrogen) contents of humic acids extracted from soils was found to
be much less than the variability of these elements in the soil as a whole, this providing evidence for a
relatively high degree of homogeneity and chemical integrity of humic acids [Zhigunov et al (1975),
Burges (1960)].
7a Molecular Weight & Elemental Composition

An ‘average’ humic acid was described by Felbeck (1971) as a polymeric substance (ie molecular
weight in the range 103-107) having C, 54; H, 5; O, 37 and N, 4%, however, purified humic acids from
different sites showed, in general, an unacceptably wide range of elementary composition for their
structure to be regarded as that of a polymeric chemical substances having regular repeating units.

The range of composition of humic acids, e.g., from 45 soils was found (Roszk, 1963)) to be C 43-62,
H 2.0-7.3, O 29.8-47.9 and N 3.1-6.5%.
A ‘pure’ form of ‘fulvic’ acid which had C, 50.92; H, 3.34; O, 44.74; N, 0.74 and S, 0.26%, was
investigated by Barton and Schnitzer (1963). Schnitzer (1972) observed that humic acids had C, 43-60,
H, 3.3-5.9 N 0.7-8.5 and S 0.3-1.7% whilst Kononova (1966) gave C, 45-62; H, 3.3-5.8 and N, 0.9-
6.0%.
In another study, a general classification was made based on sub-division of elemental analysis values
into C-rich and C-poor types, viz. 36-39%C and 40-43%C with further subdivision according to the
‘degree of oxidation’ of the humic acid (Orlov, (1970)).
‘Fulvic’ acids end to have greater % O, perhaps further preferentially located on the peripheries of the
macromolecules present and also had greater relative numbers of COOH groups, (cf Kasatochkin, et al
1964) than the humic acids.
The % ash in humic acids was found to be correlated by Konchits and Chernikov (1977) with the % O
values.
Water-soluble humic substances have often been reported to have especially high ash contents.

Humic acids are also supposed to show characteristic UV and infrared absorptions (cf Section 16 eg
Kononova, 1966).

7a-1 Anionic Functional Groups

Classical analysis of humic acids for functional groups gives information on the amounts of COOH,
OH groups. COOH groups may occur in clusters in the humic acid structure (Posner, 1966).
(Phenolic-quinoid (Glebko et al., 1970) or otherwise activated OH groups, e.g., those adjacent to
unsaturated groups, there is however often little direct evidence for the presence of large amounts of
phenolic groups as such, a possible source of error in the estimation of phenolic OH groups by
neutralisation with Ba(OH)2 is the formation of basic salts [Lawson and Phillips (1969)]) and the
amounts of various C=O groups (Golebiowska, 1973). pH titration reveals ranges of overlapping
acidic functional groups which are generally not resolvable (Glebko et al., 1972; Tretinnik et al., 1973;
Posner, 1966 and Pommer & Breger, 1960).

Quite large variations have been reported in the amounts of the above functional groups. The presence
of COOH and salts COO-Mn+ groups is the most characteristic feature of humic substances as revealed
by conventional classical analysis (up to ca.100 mequiv/100g (Chen and Schnitzer, 1976 cf Butler and
Ladd, 1969; Hubler and Bloch 1975; Wilson and Weber, 1975); ca 1000mequiv/100g of COOH
groups have been found; 100 samples of humic acid of particle diameter <25nm had 525+
mequiv/100g COOH groups (Huber & Bloch, 1975).
In the classical work of Oden (1912) ‘fulvic ‘acid from peat was found to have an equiv. wt. of 335
(300mequiv/100g) and this suggested that it contained 3 COOH groups/molecule.

A large part of the cation exchange capacity (CEC) of soils is attributable to the functional groups of
humic acids and the stabilisation of humic colloids derives, in aqueous system, largely from the
ionisation of such groups, this providing negatively charged particles.

Examples of humic acid stoicheometry and functional group analysis are:

C75H33O17(COOH)3(OH)12(CO)2 from the Ao horizon of a podzol and C21H12 (COOH)6(OH)5(CO)2 from
a Bh horizon of a podzol (Schnitzer & Desjardins, 1962).

There is a continued debate on the existence of quinoid groups (Sevesu & Schnitzer (1977)) and the
relative amounts of aromatic structures in humic acids, one view is that the ‘core structure’ of humic
acids most characteristically is a condensed polynuclear aromatic ring system. Reductive degradation,
eg by Na amalgam yielded significant amounts of polynuclear aromatic hydrocarbons which was
thought to confirm the existence of major amounts of aromatic structures in humic acids.
These structures could, however have alternatively arisen from rearrangements of polyketide derived
linear structure of microbial metabolites, e.g. structures of a similar nature to macrolide antibiotics.
During the main treatments of soil organic matter many chemical changes may occur. Alkaline
extractants exhibit the greatest alteration effects as indicated by increased unpaired electron spins
detected by ESR (Hayesetal, Schnitzer & Desjardins, 1962). It is likely, furthermore, that the chemical
analysis methods thought to indicate the presence of aromaticity previously employed in humic
substance research, (empiricial light absorption and rate of pyrolysis data) which had also included
attempts to use X-ray diffraction to detect polyaromatic structures and their variation between soil
types (chernozems appeared to contain more of such structures than podzols; the ‘core’ held
predominantly aliphatic side chains (Kasatochkinetal, 1964) )may have greatly overestimated the
amounts of such of aromatic carbons present; eg, a specially purified fulvic acid which had been
extensively studied by Schnitzer and colleagues had been believed to contain 61% of its nuclear carbon
in aromatic structures although there was no aromatic absorptions indicated in the 1H NMR spectrum
(Schnitzer & Skinner, 1968).

7b The lignin hypothesis of humic matter

Much early literature had been devoted to the lignin hypothesis of humic matter. It should be
emphasised that there is no definite evidence of this type of structure in humic matter except from peat
etc, of low degree of humification, but even here it seems that lignin molecules do not occur in an
unaltered form.

7c Fungal melanins and humic acids

‘True’ humic acids might be derived entirely from microbial metabolites including fungi. The NMR
spectra of fungal melanins, however, showed little similarity with humic acids. Some fungal melanins
contained carbohydrates and lipid degradation products.

7d Effect of oxidant alkaline KMNO4 etc on humic substances

Small amounts (ca 3% of C) of aromatic structure seemed to be formed de novo during alkaline
KMnO4 oxidation of humic acid.
In a comparison by this KMnO4 oxidation method of humic acids from various sources it seemed that
although the products were rather similar a distinction could be evidenced between terrestial and
marine humic acids
Milder oxidants, e.g., CuO and NaOCl gave larger yelds of products than were obtained with KMnO4.
The results for NaOCl suggested an adamantane-like model of humic acid (Chakrabartty et al (1974)).

8 The formation of aromatics during the pyrolysis of aliphatics

The formation of abundant amounts aromatic structures is known to occur during the incineration of
organic matter. An well-known example of the type of aliphatic-aromatic conversion process is the
conversion of polyvinyl chloride to a scrambled range of polyhalogenated aromatic substances during
the incineration of municipal and domestic waste (cf eg Choudhry and Hutzinger 1983), a phenomenon
which may also occur starting from any kind of starting organic substance incinerated in the presence
of inorganic chloride and transition metals indicates the existence of general principles where
pseudoequilibria are set up between aliphatic and aromatic moieties. [General theories of such
processes while developed many years ago in industrial laboratories are still valid; such
mathematical
models include stochasitc structural rearrangement theories of (cf eg Van Wazer (1962)) apply also to
the pseudoequilbrium situations occurring e.g. during the pyrolysis of hydrocarbons prior to the
separation of char/soot. In an analogous manner complex aromatic structures might be envisaged to
be readily formed from aliphatic humic matter during forest fires or equivalent geological processes
which give rise to the formation of coal. .

The occurrence of aromatics in common type of core structure in different humic acids sampled from a
range of soils throughout the world (including the structures which resist alkaline KMnO3 degradation
to the greatest extent) suggests that operation of a thermodynamic driven (pseudo)equilibrium process
eg catalysed by transition metals. This concept would be expected to include a series humic acid—
lignite—coal. However this idea does not easily fit in with the NMR spectra.
9 Humic Acids : Resistance to Biodegradation

Humic acids possess considerable resistance to further decomposition under soil conditions, carbon
residence times of 104 years being possible (Otsuka (1975)); such stability does not by itself give
direct evidence for the kinds of chemical structures which are present in humic matter, since many
sparingly water-soluble compounds possess a high degree of stability towards biodegradation. The
various properties which were considered to make humic acids resistant to biodegradation were listed
by Swaby (1968) as: unusual chemical bonds, steric–hindrance, high molecular weight, globular
shape, smooth topography, surface charge, disoriented polymer and copolymer structure and the
presence of cross linkages.
Graphite (from the pyrolysis of wood) was found to be degraded efficiently in the soil (Schneour
(1966)). This suggests that polycondensed aromatic ring systems such as were postulated to occur in
humic acids do not necessarily confer biological stability as might have been supposed. The presence
of disordered sp3 hybridised carbon networks (eg adamantane-like structures (cf Chakrabartty et al.,
1974) appears equally likely in humic acids of high carbon content.

Of further possible significance is the presence of structures with antibiotic properties in the humic
substances (cf the addition to soil of metal humates has been found to retard (but not prevent) the
degradation of humic anions or added glucose by microorganisms (Juste et al 1975).

10 Humic Acid Particles

Electron micrographic studies showed that humic acids exist in aqueous alkaline media as distorted
colloidal spheres (Orlov et al 1972a) of diameter several tens of nanometers.

The density of humic acids, determined by flotation, were in the range 1.4-1.7 (Orlov et al 1972b)
(this seems to be in accord with the presence of inorganic constituents within the particles).

Ultracentrifugation studies (Flaig et al 1968) and conventional viscosity studies (Flaig 1969) also
suggested that humic acids resembled aqueous hyroxyl-oxyacid (inorganic) polymer colloidal systems.
Humic acid particle densities of were suggested in one study to increase with the “degree of
humification” (Orlov et al 1965) (Bambalov 1973).

Aggregates of the primary particles of humic acid (MW = ca 103) are evidently formed in aqueous
systems, this phenomenon being pH dependent.
(‘Fulvic’ acids may, however, be less susceptible to aggregation than are the other ‘humic’ acids) (cf.
Komissarov and Loginov, (1971) and Sipos et al, (1972)).

11 Early Attempted Use of NMR to Study Humic Acids

NMR spectra of mixed solutions and colloidal dispersion from NaOH extractions of humic acids,
measured directly or after freeze drying and redissolution in D2O showed little evidence of organic
matter carbon resonances, the spectra suggested that only HO groups penetrated into the solution phase
(such groups were likely to occur at the surfaces of the colloidal particles as is found in the NMR
spectra of inorganic oxyacid polymer colloids). Progressive removal of small amounts of HO,
however, allows some organic structures to enter the solution phase, but the quality of the NMR
spectra remained were relatively poor when compared to spectra produced by initial extraction of
humic substances using organic solvents which evidently allow the colloidal size macromolecules
present to uncoil.
Early attempts to use NMR for the elucidation of humic mater structures are exemplified by the papers
by Ludemann et al, (1973) and Lentz et al (1977) who studied digested NaOD solutions of humic
substances by 100MHz 1H NMR and later the same authors used FT 100MHz NMR. The quality of
the spectra obtained were relatively poor with broad bands and poor signal to noise characteristics (this
being especially so in the first of these studies which were reported).

[Podzol and black earth humic substances showed various aliphatic and aromatic structures with the
latter apparently predominating in the podzol but the former in the black earth. However in the podzol
spectrum there was superposition of the HO and organic H-C absorption making assignments difficult.
In the 1977 work, podzols were studied further, the spectra being much improved and indicating the
predominance of aliphatic structures. The amounts of aromatic structures estimated by these authors
were almost certainly too high since they appear to have included a very broad absorption attributable
to colloidal HO or H-C-O (general environments) in their supposed aromatic absorption.
Synthetic humic acids derived from the oxidation of p-catechol appeared to be predominantly aliphatic
in nature indicting the occurrence of ring opening polymerisation during their formation].

Oka et al (1969) reported NMR evidence for carbohydrate-like and also lactone rings (attributed to the
occurrence of lignin-like structures) and well defined, but relatively minor amounts, of aromatic groups
in peat humic acids.

Ishiwatarti (1973) assigned major NMR resonances of lacustrine humic acids to alicyclic structures
[but indicative of the presence of (CH2)n chains].

Humic acids derived from river water and hymatomelanic acids from soil were reported (Reuter et al.
1972) to show the presence of polyketide structure (the alternative assignment as aromatic groups
seems less likely) with an additional association of (CH2)n chains.
Scciacovelli et al (1977) obtained high quality 1H NMR spectra from primary extraction of a Sardinian
soil with a series of ten solvents of increasing polarity followed by redissolution
in deuterated solvents. This report in agreement with agreed with previous work showed that only ca.
10% of the organic matter in the Sardinian soil was extracted in this manner; the organic matter
extracted with dioxane and EtOH apparently contained hydoquinone groups. The characteristic NMR
resonance attributable to (CH2)n groups was present in all extracts.

12 Polymetheylene Chains : Key Structural/Stabilisation Features of Humic Substances

It had became apparent during the 1970s in Aberdeen University, Scotland, UK (by J. Tinsley, J. W.
Parsons and coworkers) that polymethylene was a likely prime determinant of humic substance
stability; the uniquely high stability of man-made polymethylene in soil had also been previously made
apparent in industrial laboratories (this included International Synthetic Rubber UK where the author
had researched aspects of polyolefin chemistry [cf Duck et al (1979]); Aberdeen University NMR-lab
-obtained 1H and 13C NMR soon confirmed that humic acids and related soil extracts demonstrated
the ubiquitous presence of polymethylene chains. The spectra resembled man-made polyethylene that
the 13C NMR spectra of which had been purified by a slow process of commercial polyphosphate
chelation-out of naturally occurring paramagnetic metal ions which broaden NMR resonances in such
a manner as to disallow the use of NMR of raw humic substances for structural identification
purposes. This peak broadening process which incidentally may be a key part of the humification
mechanism was slowly reversed in a systematic manner as the manganese etc ions slowly transferred
to a polyphosphate plug at the bottom of the NMR tube. This discovery was initially received with
caution [a problem seemed to be the lack of suitably qualified peer reviewers in other laboratories
engaged in humic acid research at that time but its relevance for the fuller understanding of the
chemical nature of humic substances in the global context was given some credence by the
confirmation by e.g. Ma’shum et al (1988) that abundant polymetheylene occurred in water-repellent
Australian soils.

The (CH2)n structures in humic acids may derive from (oxidative) degradation of lipids in micro-
organisms by analogy with the drying oil oxidative polymerisation.
A microbial “humic acid” obtained from Aspergillus flavus culture appeared to contain a large amount
of such material (Visser (1970).
A further source of (CH2)n structures is from aphic wax which has been found to be long-term stable
component of peat.
It should also be noted that man-made synthetic (CH2)n is very stable under soil conditions when n>30.
High density, linear polyethylene will resist degradation in soil environments over many years and
articles made from linear polyethylene will probably still be useable after many thousands of years.
(On the other hand polyolefins which contain branched hydrocarbon structures such as isotactic
polypropylene is intrinsically unstable in an aerobic environment).
Commercial articles fabricated from plastics other than linear polyethylene have lifetimes which
depends entirely on the presence of added antioxidants: a dramatic illustration of this is that highly pure
samples of test mouldings made from highly isotactic polypropylene which contained the standard
amounts of small amounts of hindered phenolic + iron chelator was unaltered over one year at 20C
(when stored in the dark under atmospheric conditions) but an identical test piece moulding of identical
resin which had been fabricated without the antioxidant mixture had become totally degraded to a
powder state under the same conditions over this time).
The above experiment is a useful if highly simplified model of how “humic material” might be
produced by a purely abiotic process by exposure to air. Whilst the intrinsically stable chemical
structures exemplified by (CH2)n where n>30 will remain largely un-degraded, most other biological
molecules are intrinsically unstable in an aerobic environment and remain intact only until the
associated antioxidant system (e.g. lignin) becomes exhausted (by a process in which the reactive
oxygen and nitrogen radicals for which it acts as a spin trap eventually degrade it, this is expected to
occur at a similar rate to those observed for man-made hindered phenolic mimics of this natural
antioxidant system).

Although the NMR spectra of humic acids from different sources reveal the presence of numerous
chemically different structures and the first NMR studies of humic substances suggested that the
spectrum was dominated by the (CH2)n –related resonances, infrared and mass spectrosocpic evidence
however also seemed to show some aromatic structures to be present.

Sephadex gel filtration of an original (methylated ) humic acid provided fractions having similar
spectra.
It is of interest to use the published elementary composition and the 1H NMR –indicated structure
analysis to obtain an estimate of any carbon-rich structure present which do not contribute to 1-H NMR
spectra). Whilst all of the fractions, on the basis of elemental analysis seemed to be aggregates of a
C15H20O9 unit, the NMR spectra suggested Cn (CH2)10CH3. The Cn might contain aromatic rings or
adamantane-like structures fully substituted by OH or COOH.
In general, 13C NMR suggested the occurrence of relatively minor amounts of genuine aromatic
structures in most humic acids and the relative scarcity of possible aromatic H-C resonances suggests
that aromatic structures are much less abundant in humic substances than was originally believed.

12a Polymethylene in clay humic acid complexes

During the formation of complexes between soil humic matter and clay minerals fulvic acids were
found to be able to penetrate the interlammelar spaces (especially at lower pH values) conferring
stability to them (Schnitzer & Kodama, 1967). Proteins may also be similarly stabilised (cf. Schnizer
and Kodima (1969); Filip 1968).
13C NMR showed that polymetlylene chain containing humic substances had penetrated the
interlammelar spaces of clays (Schnitzer et al (1988))

Alkyl phthalates in soil

An organic distillate from soil was found by Cifrulak (1969) to contain (CH2)n structure linked to
aromatic structure (reminiscent of alkyl phthalates?).

Poly maleic anhydride model of humic substances

Another model of the chemical structure of humic acid was suggested by the observation that maleic
anhydride polymers to exhibited GLC-MS patterns and IR spectra similar to humic acids (Anderson &
Russell 1976). This similarity, however, was less apparent for the NMR spectra (cf Grant, 1977).

Use of UV absorption spectroscopy for the characterisation of humic acids

UV absorption spectroscopy has been widely employed for the empirical characterisation of humic
acids. One of the alternative systems of classification of acid acids, that of Kumada et al. (1967) is
based on their light absorption characteristics. Although this method is sensitive to normal
concentrations of humic substances in solution it is insensitive to overall major chemical component
structural details. A similar criticism could be applied to the use of ESR and also to pyrolysis-GLC-
MS when applied for the elucidation of the major structural components of soil humic polymers.
On the other hand infrared spectroscopy (which has been much used historically) seem to have a
similar potential as NMR for humic matter research. A problem has been poor spectroscopic
resolution. Indicative of the complexity of the chemical structure mixtures present, which gives rise to
extensive band superposition. The present in humic matter of partially neutralised carboxylic acids
may produce hydrogen bonded arrays exhibiting very broad vibrational absorptions.

Soil organic matter and humic acids generally show some enzymic activity which indicates the
existence of protein-like fragments bound to the humic acid surfaces (such adsorption may stabilise the
adsorbed enzymes against microbial degradation).
It is possible to reduce the N content of humic acid by mineral acid (e.g. 6M HCl) hydrolysis. This
procedure was found to remove amino acids and amino sugars.

[15N NMR]
The presence of small amounts of nitrate attached to humic acid was indicated (Xu et al 2002) to be a
potentially useful indicator of the quality of forest soil; these authors also reported the presence of
small amounts of heterocyclic N in humic matter.

References

Historical Document References plus additional more recent references (in italic).
Some of the historical references which shed light on the history of humic substance research are not
specifically cited in the text

Achard FK (1786) Chemical investigation of humus (in German)

Crells Chem Ann 2, 391-403

Anderson HA (1977) Personal communication

Anderson HA Russell JD (1976)

Possible relationship between soil fulvic acid and polymaleic acid
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Anon (1972) Biodegradability lofty goal for plastics

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Arai S Kumada K Atherton NM Cranwell PA Floyd AJ Haworth RD (1967)

Humic acid – I. ESR spectra of humic acids
Tetrahedron 23 1653-1667

Azuma J Saeki H (1961) Extraction of soil organic matter with austic alkali and
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Hyogo Noka Daigaku Kenkyu Hokoku, Nogei-kakaku Hen
Chem Abs 60 2279f

Bambalov NN (1973) Viscosity of fractions of humic acids from peats of different

degrees
of decomposition (in Russian)
Vestsi Akad Navuk Belarus SSR Ser Khim Navuk (1) 92-95
Chem Abs 78 161938n

Banerjee SK (1974) Heterogeneity in soil humic acid

J Indian Soc Soil Sci 22 231-233

Barton DHR Schnitzer M (1963) A new experimental approach to the humic acid problem
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products
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Bartle KD Martin TG Williams DF (1975)

Proton decoupled pulse Fourier-transform 13C magnetic
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J Agric Sci 11 66-68

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Glycolipid character of humic acids extracted from peat (in
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CR Acad Sci Ser C 270 1030

Bracewell JM Robertson GW (1973)

Humus type discrimination using pattern recognition of the mass
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J Soil Sci 24 421-428

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Metallo-organic complexes in soil
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Burden J (2001) Are the traditional concepts of the structure of humic substances
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[It is argued that it now apparent that the traditional concepts of the
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partially degraded plant lignins and tannins plus microbial melanins and other polyketides]

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Sci Proc Roy Dublin Soc Ser A No 4 53-58

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Cameron RS Swift RS Thornton BK Posner AM (1972)

Calibration of gel permeation chromatography materials for use
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Eur J Soil Sci 23 342-349
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Chakrabartty SK Kretschmer HO Cherwonka S (1974)

Hypohalite oxidation of humic acids
Soil Sci 117 318-322

Chen Y Senesi N Schnitzer M (1977)

Information provided on humic substances by E4/E+6 ratios
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Cf Chen Y Schnitzer M (1976) ibid. 40 866-873

Cheshire MV Cranwell PA Falshas CP Floyd AJ Haworth RD (1967)

Humic acid-I, ESR spectra of humic acids
Tetrahedron 23 1653-1667

Chilom G Rice JA (2005)

Org Geochem 36 1339-1346
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Danneberg OH Schaffer K (1974) Simple colorimetric analysis of the humic system

Bodenkultur 25 366-368

Davies RI Coulson CB Lewis DA (1960)

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Domke K (1972) Charactreisation of free and fixed gray and brown humic acids
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Dormaar (1979) Alkaline cupric oxide oxidation of roots and alkaline-extractable

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Can J Soil Sci 59 27-35

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Felbeck GT (1971) Structural hypotheses of soil humic acids

Soil Sci 111 42-48

Felbeck GT (1965) Structural chemistry of soil humic substances

Adv Agron 17 327-368

Filip Z (1968) Spectroscopic study of the humus-clay complex

Rostl Vyjroba 14 697-706 Chem Abs 70 76796e

Flaig WAJ Beutelspacher H (1968)

Investigations of humic acids with the analytical ultracentriguge
Isotop Radiat Soil Org-Matter Study Proc Symp 2nd 23-30
Int At Energy Agency Vienna
Chem Abs 71, 37920s

Flaig WAJ (1969) Properties of the organic matter in soil formation

Landbauforsch Voelkenrode 19 11-20
Chem Abs 72 65932m
Flaig WAJ (1971) Physical and chemical properties of humic substances as a basis
of their characterisation
Advan Org Geochem Proc Int Meet 5th (Publ 1972) 49-67
Ed HR Von Gaertner Pergamon Oxford

Flaig W (1977) Summarizing evaluation of the international symposium “soil

organic matter studies” IAEA/FAO and AGROCHIMICA
Brauschweig Federal Republic of Germany

Forsyth WGC (1946) A fractionation of soil organic matter with special reference to
the nitrogen constituents
Ph.D. Thesis Aberdeen University

Frans JA Morry JR Campbell JA Tingey GL Pugmire RJ Grant DM (1975)

Inferences on the structure of coal: 13C and IR spectroscopy
Am C hem Soc Div Fuel Chem Prepr 20 12-18

Fuehr F Sauerbeck D (1967) Uptake of colloidal substances by plant roots as shown by

experiments with 14C labelled humus components
Isotop Plant Nutr Physiol Proc Symp Vienna 1966 317 et seq.

Glebko LI Ul’kina JU Maximov OB (1970)

A semi-micro method for the determination of quinoid groups in
humic acids
Mikrochim Acta 58 1247-1254

Glebko LI Ul’kina Zh I Kosheleva LP Vasilevskaya NA Maksimov OB (1972)

Determination of functional groups in humic acids (in Russian)
Novye Metody Issled Guminovykh Kislot 33-80
Chem Abs 79 135668a

Golebiowska D (1973) Chemiluminescence studies of humification dynamics of trifolium

leaves
Zesz Nauk Akad Roln Szczecinie No 10 119-129
Chem Abs 81 48830v

Golub MA Gargiulo RJ (1972) Thermal degradation of 1,4-polyisoprene and 1,4-polybutadiene

Polymer Letters 10 41-49

Gonzalez Vila FJ Lentz H Ludemann HD (1976)

FT-C13 nuclear magentic resosnace spectra of natural humic
substances
Biochem Biophys Res Commun 72 (3) 1063-1070

Gonzalez-Vila FJ Saiz-Jimenez C Lentz H Ludemann HD (1978)

13
C nuclear magnetic resonance spectra of fungal melanins
Z Naturforsch C Biosci 33C 291-293

Grant D (1977a) The chemical structure of humic acids

M.Sc Thesis University of Aberdeen

Grant D (1977b) Molecular composition of soil organic matter components using

proton and 13C-NMR techniques
(Abstracts of papers presented at)
International Workshop on Properties of Organic Peat
Components and Their Effect on Metabolism Braunschweig 19-
23 September 1977, No 11

Grant D (1977c) Molecular composition of soil organic matter components using

 proton and 13C-NMR techniques
(Proceedings of International Peat Society Braunschweig Sept
1977)
TELMA (Berichte der Deutschen Gesellschaft fur Moor und
Torfkunde) submitted, later discovered that publication was
delayed but not abandoned (apparently tranferred to an East
German publisher; eventual outcome of publication
details are obscure)

Grant D (1977d) Chemical structure of humic substances

Nature 270 709-710
This paper provided a brief preliminary summary of the findings of the use of NMR methods for
the elucidation of the chemical structures present in of humic substances obtained during a soil
organic matter research programme conducted by the author together with J. Tinsley and J.W.
Parsons at the Department of Soil Science King’s College University of Aberdeen.
[In this paper, while the 13C chemical shifts were reported in the normal manner by use of δ ,
the1H chemical shifts are reported in what is now an obsolete notation, as τ (= 10-δ )].
It should be noted that later workers have confirmed the general hypothesis of the nature of humic
substances which is implied by the results reported in this paper, namely that the high
environmental chemical stability of humus fractions is likely to derive at least in part from the
presence therein of polymethylene chains. It is apparent that the same (evidently
hydrophobicity-related) lack of chemical reactivity which is characteristic of polymethylene
chains in man-made polyethylene also applies to soil organic matter and indicates the unique
suitability of this type of chemical structure to act as a soil-based sink for carbon sequestration
which may be harnessed for the amelioration of atmospheric CO2 augmentation. An update of
information relating to the roles of polymethylene in humic substances is given by Hu W-G et al
loc cit and Chilom G & Rice loc cit.

Grant D (2009)
Humic substances inhibit calcite crystallization
http://www.scribd.com/doc/23967637/Humic-Substances-
Inhibit-Calcite-Crystallizn-II

Grant (2010) Notes on the inorganic biochemistry of heparin/heparan sulphate

http://www.scribd.com/doc/26994439/Publication-2-Web /
[Similarities, e.g. in the inorganic ion binding characteristics between algal
and animal anionic polysaccharides and humic substances are apparent which could suggest that the
former have evolved to mimic the latter in vivo]

Haworth RD (1971) The chemical nature of humic acid

Soil Sci 111 71-79

Handley WRC (1954) Mull and mor formation in relation to forest soils
Bull For Comm 23 (HMSO London)

Haraguchi H (2004) Metallomics as integrated biometal science

J Anal At Spectrom 19 5-14

Hatcher PG Dria KJ Sunghwan K Fraizer

Modern Analytical Studies of Humic Substances
Soil Sci 166 770-794

Hayes MHV Clapp CE Humic Substances: Consideration of Composition, Aspects of

Structure and Environmental Influences
Soil Sci 166 723-737

Hayes MHV Swift RS Wardle RE Brown JK (1975)

Humic materials from organic soil: a comparison of extractants
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Geoderma 13 231-245
Hu W-G Mao J Xing B Rohr KS (2000)
Poly(methylene) crystallites in humic substances detected by
nuclear magnetic resonance
Environ Sci Technol 34 530-534

Hubler JC Block JM (1975) Electrochemical determination of a sodium humate in ethylene

glycol (in French)
CR Hebd Seances Acad Sci Ser C 281 677-679

Ishiwatari R (1971) Molecular weight distribution of humic acids from lake and
marine sediments
Geochem J 5 121-132

Ishiwatari R (1973) Chemical characterization of fractionated humic acids from lake

and marine sediments
Chem Geol 12 113-126

Juste C Derlas J Langon M (1975) Comparison of the biological stability of different metal humates
(in French)
CR Hebd Seances Acad Sci Ser D 281 1685-1688

Kastochkin VI Kononova MM Larina NK Egorova OI (1964)

Theses on “Special and X-ray investigation of chemical
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Proc 8th Int Congr Soil Sci Bucharest III 81-86

Kelleher BP Simpson AJ (2006) Humic substances in soils: are they really chemically distinct
Environ Sci Technol 40 4605-4611

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Geoderma 4 47-55

(1970b) Pyrolysis gas chromatography of soil organic matter. 2.

The effect of extractant and soil history on the yields of products
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Geoderma 4, 57-71

Komissarov ID Loginov LF (1971) Structural scheme and modelling of macromolecular humic acids
Nauch Tr Tyumen Se-Khoz Inst 1970 (Publ 1971) No 14 141-142

Kononova MM (1966) Soil organic matter, its nature, its role in soil formation and soil
fertility
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Kumada K Sato O Ohsumi Y Ohta S (1967)

Humus composition of mountain soils in Central Japan with
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Soil Sci Plant Nutr 13 151-158

Lawson GJ Phillips CV (1969) Acid equivalent of some humic acids derived from fossil fuels
Fuel 48 398-400

Ladd JN (1969) The extinction coefficients of soil humic acids fractionated by

Sephadex gel filtration
Soil Sci 107 303-306
Lentz H Ludemann H-D Ziechmann W (1977)
Proton resonance spectra of humic acids from the solum of a
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Geoderma 18 325-328

Lindbeck MR Young JL (1966) Polarography and coulometry in dimetylsulfoxide, of nitric acid

oxidation products from soil humic acid
Soil Sci 101 366-372

Lindqvist I Bergman B (1966) Differential spectrosscopy of lignin and humic acids at 100MHz
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Seawater dissolved organic matter (DOM) is the largest reservoir
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Cf Repele DJ Aluwihare LI (2006)

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Cf Susic A (2001) loc cit.

Ludemann H-D Lenz H Zeichmann W (1973)

Proton resonance spectroscopy of lignin and humic acid at 100
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Erdol Kohle-Erdgas-Petrochem-Brennstoff-Chem 26 506-509

MacCarthy P (2001) The principles of humic substances

Soil Sci 166 738-775
[A: Humic substances (HS) are devoid of a regular recurring extended
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molecular structure or set of structures that fully describe the connectivity with molecules of a humic substance]

Mclaren AD (1975) Soil as a system of humus and clay immobilized enzymes

Chimica Scripta 8 97-99

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Pochvovedenie 79-85
(Soviet Soil Science 1102-1107)

Ma’shum M Tate ME Jones GP Oades JM (1988)

Extraction of characterization of water-repellent materials from
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J Soil Sci 39 99-110

Martin F Saiz-Jiminez C Cert A (1977)

Pyrolysis-gas chromatogrphy-mass spectrometry of soil humic
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Soil Sci Soc Am J 41 1114-1118

Mato MC Fabregas R Mendez J (1971)

 Inhibitory effect of soil humic acids on indolacetic acid-oxidase
Soil Biol Biochem 3 285-288

Maximon OB Shvets TV Elkin Yu N (1977)

On permanganate oxidation of humic acids
Geoderma 19 63-79

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Zh Prikl Khim (Leningrad) 42 1373-1376
Chem Abs 71 90257g

Oden S (1912) Contribution to the understanding of humic acids from sphagnum

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Chem Ber 454 651-660

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Geoderma 19 237-245

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Canad J Chem 49 1053-1063

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Acta Chem Scand 27 1601-1612

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Studies on the structure of peat humic acid (1)- study on the
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Journal of the Fuel Society of Japan (Nenryo Kyokai-shi) 48
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O’Reilly WF Murrman RP (1974) Identification of soil organics using a gas

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USNTIS AD Rep No 782991/4GA 17pp
Chem Abs 73 3014a

Orlov DS Gorshkova EJ (1965) Size and shape of humic acid particles from chernozem and soddy-
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Nauchn Dokl Vysshei Shkoly, Biol Nauki (1) 207-212

Orlov DS (1970) Elementary composition and the degree of oxidation of

humic acids (in Russian)
Biol Nauki 5-20
Chem Abs 73 3014a

Orlov DS Ammosova Ya M Glebova GI Gorshkova ET Ilin NP Kolesnikov MP (1971)

Molecular weights, sizes and configuration of humic-acid
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Pochvovedenie 43-56
 (Soviet Soil Sci 637-687)

Orlov DS Glebova GI (1972a) Electron-microscopy investigation of humic acids (in

Russian)
Agrokhimiya 131-136
(Soviet Soil Sci 445-452)

(1972b) Density and structure of humic acids (in Russian)

Biol Nauki 15 119-124
Chem Abs 77 138885p

Orlov DS Min’ko OI Demin VV Sal’nikov VG Izmailova NB (1988)

Form of metal humus complex: characterisation and mechanism
Pochvovedenie (9) 43-52 Chem Abs 109 210098h
[EDTA can dis-aggregate the humic metal ion complexes]

Otska H (1975) Volcanic soil at Ohnobaru, Tarumi, Kagoshima Prefecture V.

Accumulated state of humus in the soil profile and sugar, uronic acid and amino acid
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Nippon Dojo-Hiryogaku Zasshi 46 180-184
Chem Abs 83 177210h Geoderma 14 139-145

Pena-Mendez EM Havel J Patocka J (2005)

Humic substances-compounds of still unknown structure:
applications in agriculture, industry, environment, and
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J Appl Biomed 3 13-24

Piccolo A (2001) The supramolecular structure of humic substances

Soil Sci 166 810-832
Cf Spaccini R Piccolo A loc cit “innovative soil management
practices employing hydrophobic humic substances may increase the biological
stability of soil organic matter and thus contribute to significant mitigation of CO2
emission from agricultural soils”

Pr (2008) Proposal for carbon sequestration

(“Why Use Humic Substances for Carbon Sequestration”)
http://ihccs.org/humic.htm
While this International Humic Consortium for Carbon
Sequestration (IHCCS) document notes that the detailed
molecular structures of humic acids are unknown at present the
refs cited include a proposal by Davies G et al (1997)for a humic
acid building molecule which contains a hollow interior which
can permit facile water retention

Cf also Gorrison A deVisser AJC (2004) Modelling carbon sequestration

Note 304 Plant Research International Wageningen
http://library.wur.nl/wasp/bestanden/LUWPUBRD_00333153_A502_001.pdf
Note that “since it is the slow SOM (sol organic matter) pool that determines
C-sequestration, it is important to experimentally identify this pool”

Smith P (2008) loc cit also draws attention to the important role of tropical
soil organic matter degradation for future augmentation of atmospheric CO2

Pommer AM Breger IA (1960) Potentiometric titration and equivalent weight of humic acid
Geochim Cosmochim Acta 20 30-44

Posner AM (1966) The humic acids extracted by various reagents from a soil. Part 1,
 yield, inorganic components, and titration curves
J Soil Sci 17 65-78

(1964) Titration curves of humic acid

Proc 8th Intern Congress of Soil Science, Bucharest, Romania
(1964)
Vol III 161-174

Potts JE Clendinning RA Ackart WA (1973)

The effect of chemical structure on the biodegradability of plastics
Proc Symp Degradability of Polymers and Plastics 27-28 Nov
1973, Plastics Institute, London, 12, 1-12, 10

Reuter JH Perdue EM (1972) Chemical characterization of dissolved organic mater and its
influence on the chemistry of river water
US Natn Tech Inform Serv Rep PB210 714

Rifaldi R Schnitzer M (1972) Electron spin resonance spectrometry of humic substances

Soil Sci Soc Aner Proc 36, 301-305

Roszyk E (1963) Elementary composition of humic acids from some types of soils
Zeszyty Nauk, Wyzszej Szkoly Rolniczej Wroclawiu Rolnictwo
No 16 (49) 20-36
Chem Abs 62 12396b

Schneour EA (1966) Oxidation of graphitic carbon in certain soils

Science 1151 991-992

Sciacovelli O Senesi N Solinas V Testini C (1977)

Spectroscopic studies on soil organic fractions I, IR and NMR
Spectra
Soil Biol Biochem 9 287-293

Senesi N Schnitzer M (1977) Effect of pH, reaction time, chemical reduction and irradiation on
ESR spectra of fulvic acid
Soil Sci 123 224-234

Schnitzer M Desjardins JG (1962) Molecular and equivalent weights of the organic matter of a
podzol
Soil Sci Soc Am Proc 26 362-365

Schnitzer M Desjardins JG (1970) Alkaline permanganate oxidation of methylated and

unmethylated fulvic acid
Soil Sci Soc Amer Proc 34 77-79

Schnitzer M Kodama H (1967) Reactions between a podzol fulvic acid and Na-montmorillonite
Soil Sci Soc Amer Proc 31 632-636

Schnitzer M Skinner SIM (1968) Gel filtration of fulvic acid, a soil humic compound
Isotop Radiat Soil Org-Matter Stud Proc Symp 2nd 41-55
Int At Energy Agency Vienna

Schnitzer M Khan SU (1972) Humic Substances in the Environment

Dekker New York

Schnitzer M Ortiz de Serra MI (1973) The chemical degradation of a humic acid

 Can J Chem 51 1554-1566

Schnitzer M Skinner SIM (1974) The low temperature oxidation of humic substances
Can J Chem 52 1072-1080

Schnitzer M Neyroud JA (1975) Further investigations on the chemistry of fungal “humic acids”
Soil Biol Biochem 7 365-371

Schnitzer N Ripmeester JA Kodema H (1988)

Characterization of the organic matter associated with a soil clay
Soil Sci 145 (6) 448-454
[IR and NMR spectra of the initial clay-organic complex showed the
prominence of long-chain aliphatics {i.e. polymethylene}; n-hexane and CHCl3 extracted n-alkanes C16-35, long
chain alcohols and more complex aliphatics; these authors were of the opinion that the polymethylene structures
had been transported into the clay spaces by supposed ‘traditional’ humic poylmers]

Cf also Shulten HR Schnitzer M (1990) loc cit

Sciacovelli O Senesi N Solinas V Testini C (1977)

Spectroscopic studies on soil organic fractions I, IR and NMR
spectra
Soil Biol Biochem 9 287-293

Sinha A Shukla RN (1966) Degradative products of humic substances with alkaline

potassium permanganate and their identification by paper
chromatograpy
Technol (Sindri) 3 17-21
Chem Abs 66 45816z

Simpson A (2001) Multidimensional solution state NMR of humic substances, a

practical guide and a review
Soil Sci 166 795-805

Smith P (2008) Land use change and soil organic carbon dynamics
Nutrient Cycling in Agrosystems 81 169-178

Schulten HR Schnitzer M (1990) Aliphatics in Soil Organic Matter in Fine-Clay Fraction

Soil Sci Soc Am J 54 98-105
[Fine clay fractions were separated from 3 agricultural soils and extracted with n-
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*The original document was produced in conjunction with the soil organic matter research programme
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