Humic substances of soils and general theory of humification [1st edition] 9781000108491, 100010849X, 9781000131222, 100013122X, 9781000151640, 1000151646, 9781003079460, 1003079466

Table of contents :
Cover......Page 1
Half Title......Page 4
Title Page......Page 6
Copyright Page......Page 7
Preface to the English Language Edition......Page 8
Table of Contents......Page 10
Introduction......Page 12
Composition of Plant Remains and Pathways of their Transformation During Humification......Page 19
Relative Stability of Organic Matter......Page 42
Problems of Separation and Nomenclature of Humic Substances......Page 51
General Remarks......Page 61
Nature of Humic Substances from their Elemental Composition......Page 73
Evaluation of Structure and Some Properties of Humic Substances from Elemental Composition......Page 79
Supplementary Data on the Elemental Composition of Humic Substances and its Change in the Course of Agricultural Exploitation of Soils......Page 91
3. Structural Units and Functional Groups of Humic Substances......Page 102
Acid Hydrolysis of Humic Substances......Page 103
Amino acids and amino sugars......Page 104
Carbohydrate Fragments......Page 111
Oxidation Products of Humic Substances......Page 113
Use of Nuclear Magnetic Resonance (NMR) Data to Describe Humic Substances......Page 122
Functional Groups and Distribution of Oxygen......Page 126
Interaction of Humic Substances with the Mineral Components of Soils......Page 137
Methods for Study of Organo-mineral, Interactions......Page 141
Composition and Properties of Compounds of Humic Substances with Metals......Page 148
Interaction of Humic Substances with Soil Minerals......Page 155
4. Electron and Molecular Absorption Spectra of Humic Substances......Page 163
Electron Absorption Spectra......Page 164
Effect of Storage and Exposure to Light on Absorption Spectra of Humic Substances......Page 180
Nature of Light Absorption by Humic Substances......Page 183
Optical Properties of Humic Substances of Various Soils......Page 188
Infra-red Spectra of Humic Substances......Page 192
5. Molecular Parameters of Humic Substances......Page 210
Minimum Molecular mass from the Data of Chemical Composition......Page 217
Mean Weighted Molecular Mass of Humic Acids from Light-scattering Data......Page 218
Molecular Mass Distribution of Particles of Humic Substances from Gel-filtration Data......Page 219
Electron Microscopic Observations......Page 224
Viscosimetric Data......Page 226
6. Hypotheses Regarding the Structure and Identification of Humic Substances......Page 228
Structural Schemes for Humic Substances......Page 229
Diagnosis and Identification of Humic Substances......Page 240
Humus Status of Soils......Page 246
Indices of Humus Status of Soils......Page 247
Humus Status of Zonal Genetic Series of Soils......Page 250
Humus in Soils of the Non-chernozem Zone of Russia......Page 252
Humus of Desert and Semi-desert Soils......Page 259
Effect of Some Ameliorants on Soil Organic Matter......Page 265
General Theory of Humification......Page 267
Hypotheses Regarding Chemical Mechanisms of Humification......Page 268
Kinetic Theory of Humification......Page 274
Biogeochemical Principles and the Laws of Humus Formation......Page 283
Literature......Page 292
Recent Publications......Page 328

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RUSSIAN TRANSLATIONS SERIES 1. K.Ya. Kondrat'ev et a,\. (editors): USSR/USA Bering Sea Experiment 2. D.V. Naiivkin: Hurricanes. Storms and Tornadoes 3. V.M. Novikov (editor): Handbook of Fishery Technology, Vol. 4. F.G. Martyshev: Pond Fisheries 5. R.N. Burukovskii: Key to Shrimps and Lobsters 6. V.M. Novikov (editor): Handbook of Fishery Technology, Vol. 4 7. V.P. Bykov (editor): Marine Fishes 8. N.N. Tsvelev: Grasses of the Soviet Union 9. L.V. Metlitskii et al.: Controlled Atmosphere Storage of Fruits 10. M.A. Glazovskaya: Soils of the World (2 volumes) 11. V.G. Kort & V.S. Samoilenko: Atlantic Hydrophysical Polygon-70 12. M.A. Mardzhanishvili: Seismic Design of Frame-panel Buildings and Their Structural Members 13. E' .A. Sokolenko (editor): Water and Salt Regimes of Soils: Modeling and Management 14. A.P. Bocharov: A Description of Devices Used in the Study of Wind Erosion of Soils 15. E.S. Artsybashev: Forest Fires and Their Control 16. R.Kh. Makasheva: The Pea 17. N.G. Kondrashova: Shipboard Refrigeration and Fish Processing Equipment 18. S.M. Uspenskii: Life in High Lotitudes 19. A.V. Rozova: Biostratigraphic Zoning and\Trilobites of the Upper Cambrian and Lower OrdOvician of the Northwestern Siberian Platform 20. N.I. Barkov: Ice Shelves of Antarctica 21. V.P. Averkiev: Shipboard Fish Scouting and Electronavigational Equipment 22. D.F. Petrov (Editor-In-Chief): Apomixis and Its Role in Evolution and Breeding 23. G.A. Mchedlidze: General Features of the Paleobiological Evolution of Cetacea 24. M.G. Ravich et a\.: Geological Structure of Mac. Robertson Lond (East Antarctica) 25. L.A. Timokhov (editor): Dynamics of Ice Cbver 26. K.\(a: Kcmdrat!ev:Changes in Global Climate 27. P.S. Nartov: Disk Soil-Working Implements 28. V.L. Kontrimavichus (Editor-in-Chief): Beringia in the Cenozoic Era 29. S.V. Nerpin & A.F. Chudnovskii: Heat and Mass Transfer in the Plant-Soil-Air System 30. T.V. Alekseeva et a\.: Highway Macbines 31. N.I. Klenin et al.: Agricultural Machines 32. V.K. Rudnev: . Digging of Soils by Earthmovers with Powered Parts 33. A.N. Zelenin et a\.: Machines for Moving the Earth 34. Systematics. Breeding and Seed Production of Potatoes 35. D.S. Orlov: Humus Acids of Soils 36. M.M. Sevemev (editor): Wear of Agricultural Machine Parts 37. Kh.A. Khachatryan: Operation of Soi/:working Implements in Hilly Regions 38. L.V. Gyachev: Theory of Surfaces of Plow Bottoms 39. S.V. Kardashevskii et al.: Tes'ting of Agricultural Technological Processes 40. M.A. Sadovskii (editor): Physics of the Earthquake Focus 41. I.M. Dolgin: Climate of Antarctica 42. V.V. Egorov et a\.: Classification and Diagnostics of Soils of the USSR . 43. V.A. Moshkin: Castor 44. E'.1. Sarukhanyan: Structure and Variability of the Antarctic CircunJpolar Current 45. V.A. Shapa (Chief Editor): Biological Plant Protection 46. A.I. Zakharova: Estimation of Seismicity Parameters Using a Computer 47. M.A. Mardzhanishvili & L.M. Mardzhanishvili: Theoretical and Experimental Analysis of Members of Eart~quake-proof Frame-panel Buildings 48. S.G. Shul'man: $eismic Pressure of Water on Hydraulic Structures 49. Yu.A. lbad-zade: Movement of Sediments in Open Channels 50. I.S. Popushoi (Chief Editor): Biological and Chemical Methods of Plant Protection 51. K.V. Novozhilov (Chief Editor): Microbiological Methods for Biological Control of Pests of Agricultural Crops 52. K.!. Rossinskii (editor): Dynamics and Thermal Regimes of Rivers 53. K.V. Gnedin: Opera'ting Conditions and Hydraulics of Horizontal Senling Tanks 54. G.A. Zakladnoi & V.F. Ratanova: Stored-grain Pests and Their Control 55. Ts.E. Mirtskhulava: Reliability of Hydro-reclamation Installations 50. 1a.S.. Ageikin: O/!-the.-road Mobility -of Au/omobiles 57. A.A. Kmito & Yu.A. Sklyarov: Pyrheliometry 58. N.S. Motsonelidze: Stability and Seismic Resistance of Buttress Dams (continued)

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of the Sea of Okhotsk and the Yellow Sea 72. N.1. Plotnikov & 1.1. R,oginets: Hydrogeology of Ore Deposits 73. A.V. Balushkin: Morphological Bases of the Systematics and Phylogeny of the Nototheniid Fishes ' 74. E.Z. Pozin et al.: Coal Cutting by Winning Machines 75. S.S. Shul'man: Myxosporidia of the USSR 76. O.N. Oogonenkov: Seismic Prospecting for Sedimentary Formations 77. I.M. Batugina & I.M. Petukhov: Geodynamic Zoning of Mineral Deposits for Planning and Exploitation of Mines 78. 1.1. Abramovich & 1.0. Klushin: Geodynamics and Metallogeny of Folded Belts 79. M.V. Mina: Microevolution of Fishes 80. K.V. Konyaev: Spectral Analysis of Physical Oceanographic Data 81. A.1. Tseitlin & A.A. Kusainov: Role of Internal Friction in Dynamic A nalysis of Structures 82. E.A. Kozlov: Migration in Seismic Prospecting 83. E.S. Bosoi et al.: Theory, Construction and Calculations of Agricultural Machines, Vol. 2 84. B.B. Kudryashov and A.M. Yakovlev: Drilling in the Permafrost 85. T.T. Klubova: Clayey Reservoirs of Oil and Gas 86. 0.1. Arnurskii et al.: Remote-sensing Methods in Studying Tectonic Fractures in Oil- and Gas-bearing Formations 87. A.V. Razvalyaev: Continental Rift Formation and Its Prehistory 88. V.A. Ivovich and L.N. Pokrovskii: Dynamic Analysis of Suspended Roof Systems 89. N.P. Kozlov (fechnical Editor): Earth's Nature from Space 90. M.M. Orachevskii and A.S. Kravchuk:- Hydrocarbon Potential of Oceanic Reefs of the World 91. K.V. Mikhailov et al.: Polymer COllCreres and TIleir Srructural Uses 92. D.S. Orlov: Soil Chemistry 93. L.S. Belousova & L.v. Denisova: Rare PlalllS of the World 94. T.I. Frolova et al.: Magmatism and Trallsformation of Active Areas of rhe Earth s Crust 95. Z.O. Ter-Martirosyan: Rlleological Parameters of Soils alld Design of Foundations 96. S.N. Alekseev et al.: Durability of Reinforced Concrete in Aggressive Media 97. EP. Olushikhin et al.: Modelling in Geomechallics 98. LA. Krupenikov: History of Soil SciellCe 99. V.A. Burkov: General Circulation of rhe World Ocean 100. B.V. Preobrazhenslo..-y: Contemporary Reefs 10 I. v.P. Petrukhin: Construction of Strul'tures on Saline Soils 102. V.v. Bulatov: Geomechanics of Deep-seated Deposits 103. A.A. Kuzmenko et al.: Seismic Effects of Blasting in Rock I~. L.M. Plotnikov: Shear Structures ill Layered Geological Bodies 105. M.!. Petrosyan: Rock Breakage by Blasting 106. O.L. Kuznetsov and E.M. Sinlkin: lransformation and Interaction of Geophysical Fields in

the Lithosphere 107. M.V. Abdulov: Phase Transformations and Rock Genesis 108. I.E. Mileikovskii and S.I. Trushin: Analysis of Thin-waIled Structures 109. Y.Sh. Barbakadze et al.: Durability of Building Stmclllres and Constmctions from

Composite Materials 110. Y.I. Kazansky: Evolutioll of Ore-bearing Precambrian Stmclllres 111. D.S. Orlov: Humic Substances of Soils and GeIleral771eory of Humification

Humic Substances of Soils

and

General Theory of Humification

Humic Substances of Soils

and

General Theory of Humification

O.S.Orlov

RUSSIAN TRANSLATIONS SERIES 111

A.A. BALKEMA/ROTTERDAM/BROOKFIELD/1995

Translation of: Gumusovye kisloti pochv i obshchaya teoriya gumifikatsii, Moscow University Press, 1990. Revised by the author in October, 1992.

©

1995 Copyright reserved

Translation Editor: Dr. V.S. Kothekar ISBN 90 6191 955 X Distributed in USA and Canada by: AA Balkema Publishers, Old Post Road, Brookfield, VT 05036, USA.

Preface to the English Language

Edition

In 1990 the publishing house of the M.V. Lomonosov Moscow State Univer­ sity (Russia) published my book Humus Acids of Soils and General Theory of Humification. In it I presented the most recent data on the structure and properties of humic and fulvic acids, their probable structure and function in the biosphere. Original ideas were presented about the process of humifi­ cation and formation of different types of humus depending on bioclimatic conditions in different natural zones. The book was popular and almost the entire print run had been sold in a matter of days. For the English language translation the text was significantly revised. Even the name of the book was changed in keeping with international nomenclature. The new title, Humic Substances of Soils and General Theory of Humification, accords with the nomenclature accepted by the International Humic Substances Society (IHSS). Considerable factual data concerning soils of the erstwhile USSR, of interest to scientists in Russia and its neigh­ bouring countries but of little interest to researchers in other regions of the world, has been deleted from the book. Corrections have been introduced relating to names of some member States of the erstwhile USSR, cities and regions in accordance with changes that occurred at the time of publication of this book in the Russian language. I have also made additions to the literature, with corresponding citations in the text, which more completely reflect studies by scientists from countries of Europe, America and Asia conducted in recent years on the subject. Finally, I have also corrected several errors which crept into the Russian text during printing. I sincerely hope that this book evokes interest not only in soil scientists, but also in a wide circle of readers in the fields of biogeochemistry, ecology and the problem of humic substances studies.

O.S.ORLOV Moscow, October 15, 1992

Contents PREFACE TO THE ENGLISH LANGUAGE EDITION INTRODUCTION

1. SOURCES OF HUMIC SUBSTANCES AND SOME PROBLEMS OF NOMENCLATURE Composition of Plant Remains and Pathways of their Transformation During Humification Relative Stability of Organic Matter Problems of Separation and Nomenclature of Humic Substances

2. ELEMENTAL COMPOSITION OF HUMIC SUBSTANCES General Remarks Nature of Humic Substances from their Elemental Compositien Evaluation of Structure and Som'e Properties of Humic Substances from Elemental Composition Supplementary Data on the Elemental CompOSition of Humic Substances and its Change in the Course of Agricultural Exploitation of Soils

3. STRUCTURAL UNITS AND FUNCTIONAL GROUPS OF HUMIC SUBSTANCES Acid Hydrolysis of Humic Substances Amino acids and amino sugars Carbohydrate Fragments Alkanes and fatty acids Oxidation Products of Humic Substances Balance of Structural Units Use of Nuclear Magnetic Resonance (NMR) Data to Describe Humic Substances Functional Groups and Distribution of Oxygen Interaction of Humic Substances with the Mineral Components of Soils Methods for Study of Organo-mineral. Interactions

v

8 8 8 31 40 50 50 62 68 80 91 92 93 100 102 102 111 1~ 1 115 126 130

viii Composition and Properties of Compounds of Humic

Substances with Metals Interaction of Humic Substances with Soil Minerals

4. ELECTRON AND MOLECULAR ABSORPTION SPECTRA OF

HUMIC SUBSTANCES Electron Absorption Spectra Effect of Storage and Exposure to Light on Absorption

Spectra of Humic Substances Nature of Light Absorption by Humic Substances Optical Properties of Humic Substances of Various Soils Infra-red Spectra of Humic Substances

5. MOLECULAR PARAMETERS OF HUMIC SUBSTANCES

137

144

152

153

169

172

177

181

199

206

Minimum Molecular mass from the Data of Chemical Composition Mean Weighted Molecular Mass of Humic Acids from Light­ 207

scattering Data Molecular Mass Distribution of Particles of Humic Substances

208

from Gel-filtration Data Electron Microscopic Observations 213

Viscosimetric Data 215

6. HYPOTHESES REGARDING THE STRUCTURE AND

IDENTIFICATION OF HUMIC SUBSTANCES Structural Schemes for Humic Substances Diagnosis and Identification of Humic Substances

7. HUMUS STATUS OF SOILS AND GENERAL THEORY OF

HUMIFICATION Humus Status of Soils Indices of Humus Status of Soils Humus Status of Zonal Genetic Series of Soils Humus in Soils of the Non-chernozem Zone of Russia Humus of Desert and Semi-desert Soils Effect of Some Ameliorants on Soil Organic Matter General Theory of Humification Hypotheses Regarding Chemical Mechanisms of Humification Kinetic Theory of Humification Biogeochemical Principles and the Laws of Humus Formation LITERATURE RECENT PUBLICATIONS

217

218

229

235

235

236

239

241

248

254

256

257

263

272

281

317

Introduction

More than 200 years ago Achard (1786) published his paper on the extrac­ tion of humic acids from peat. This is traditionally considered the beginning of studies on specific humic substances. But over 200 years of such studies notwithstanding, modern science has yet to achieve a complete understand­ ing of the structure, role and function of humic substances in the biosphere. This 'failure' is due to the complex nature of humic substances and the fact that they have been mostly investigated in various countries by small groups of scientists, predominantly engaged in soil science and the chemistry of fossil fuels. Only recently have international societies been formed and in­ dustry evinced an interest in humic substances. In 1981, the Working Group of the International Society of Soil Science passed a resolution regarding the need for establishing an international society for the study of humic sub­ stances (International Humic Substances Society-IHSS). The First Interna­ tional Conference of this society was held in 1983 in Colorado (USA). Prob­ lems pertaining to the geochemistry and separation and description of humic substances were discussed at this conference. Dr. R.L. Malcolm was the first President, Dr. R.S. Swift the first Vice-President and Dr. P. MacCarthy the first Secretary. The second conference was held in July 1984 in Birm­ ingham (England) and addressed the problems of the structure of.· humic substances, methods of investigation, the role of nitrogen, and the reactiv­ ity and ecological role of humic substances. The international symposium 'Humus et Planta' has been held regularly for over two decades in Prague (Czechoslovakia) and the aforesaid problems reviewed. The remarkable fea­ ture of the IHSS is that attention is focused on specific humic substances (humic acids, fulvic acids, humus) that form and function not only in soils, but in all the other components of the biosphere such as water, peat, coal, peloids and so on [Adhikari and Choudhury, 1989; Boyd and Sommers, 1990; Clapp et al., 1990; Deiana et al., 1990; Ghosal et al., 1991; Hernan­ dez et al., 1989; Hervas et al., 1989; Lawson and Stewart, 1989; Lobartini et al., 1991; Malcolm, 1990; Prasad and Kumar, 1989; Preston et al., 1989]. This illustrates the increasing realisation of the exceptional importance and unique role of humic substances in almost all the biogeochemical processes. If earlier attention was primarily paid to the agronomic or pedogenetic role

2

of humic substances, today no less significance is attached to their phys­ iological activity, their role in transport in the biosphere, accumulation or fixation of metals and pesticides, natural photochemical processes and so forth. It was no coincidence that one of the Dahlem workshops (Berlin, 29 March-3 April 1987) was specially devoted to humic substances and their role in the environment (Humic Substances, Dahlem Workshop, 1988). The Dahlem workshops are organised specifically to take stock of the data and prospective developments in this most important interdisciplinary problem, which once more emphasises the scientific importance of the theory of for­ mation, structure and function of humic acids. Many papers have been written on the applied aspects of the use of humic substances in industry. The Scientific Research Institute of Fertilis­ ers, Insecticides and Fungicides (NIUIF), Moscow held the first joint Soviet­ Italian symposium on humic substances in June 1987, at which firms such as Veneta Mineraria and Montedison not only presented several theoreti­ cal papers and technological schemes for obtaining humic substances, but demonstrated special preparations meant particularly for control of chloro­ sis. One of the promising directions was use of combined mineral-humic fertilisers which, according to the data presented by Italian researchers at the symposium, leads to reduction in the consumption of mineral fertilisers and an increase in crop production. Thus even the quality of the produce is changed and the adverse influence of satellite pesticide components re­ duced. Since 1957, All-Union conferences on 'Humic fertilisers-theory and practice of their application' have been held in the CIS* under the lead­ ership of LA Khristeva and conference proceedings have been published. The scientific-technical council of the CIS Ministry of Agriculture has ratified the recommendations made by Khristeva on the use of humic compounds for pretreatment of seeds, which increases the yield of wheat and corn by 3-5 qtls/ha. Sodium and ammonium humates are also produced by the Min­ istry of Petroleum Industry, the peat-processing and coal-mining industries, and several scientific research institutes. Humates perform various functions in the soil. The usual mechanisms of improvement of soil structure, optimisation of water, air and thermal regimes and increase in storage of nutrients are fairly trivial. Their protective action is manifest in combining with toxic heavy metals and some pesticides and in increasing the availability to plants of certain elements that otherwise sparingly form soluble compounds. In many soils phosphates readily form almost insoluble mineral compounds, for example hydroxy-apatite, strengite

* Commonwealth of Independent States (the former USSR)-Translator.

3 and variscite. Experimental data has shown that the use of humic com­ pounds and combined mineral fertilisers increases the availability of phos­ phorus to plants. One possible explanation for this increase is the formation of phosphate-humic compounds (through metallic element bridges such as FeOH). Such compounds protect phosphorus from being converted into in­ soluble mineral forms but readily release phosphorus when acted upon by organic complexing agents secreted by plant roots [Ahmed and Tan, 1991]. Soluble humates and many humin-like substances-oxidation products of lignin [Raskin et al., 1984], weathered coals and oil shales [Veski and Fom­ ina, 1984], synthetic metal complexes formed by decomposition of plant litter [Beskrovnyi et al., 1981] and mummies-have varying degrees of physiolog­ ical activity. The stimulatory action of humates on micro-organisms and plant and animal tissue has been established [Guminski, 1967; Khristeva, 1973; Gorovaya et al., 1977; Mulyak et al., 1983; Gorovaya and Kulik, 1980; Chen and Aviad, 1990]. The physiological effect of humic substances on plants has often been questioned since soils contain considerably more humic substances than are added to them by experimental treatments. Neverthe­ less, the beneficial effect of humic acids and their salts has been confirmed through numerous experiments on seedlings of field crops and in Khristeva's experiment with presowing treatment of wheat and corn. The mechanisms of action obviously include both the direct specific ef­ fect on plants and the protective action of humates. Some authors attach greater importance to the latter. Considerable data is available to show that the adverse effect of environment on plants can be reduced by the use of humic compounds. For instance, laboratory and field experiments have shown that the adverse effect of herbicides on cell division and DNA con­ tent in interphase nuclei of cells and apical meristems of sorghum can be reduced by presowing treatment of seeds with sodium humate at the rate of 3 ml of 2.5% solution for every kg of seed [Gorovaya, 1983; Oginova, 1985; Gorovaya et al., 1985]. Many results confirm that humic compounds increase the anti-inflammatory and antitumor activity of animal tissues [Malama et al., 1975; Ammosova et al., 1986]. Humic substances can favourably influence the functioning of mitochon­ dria and chloroplasts which facilitate respiration and photosynthesis. Re­ ports are also available regarding their capacity to influence ion selectivity of membranes and to enable a more complete realisation of genetic infor­ mation. According to Gorovaya (1983), the physiological action of humic substances becomes more clearly manifest in unfavourable reactions. The physiological action of humic substances is important and of interest not only from the agronomic or medicinal point of view; this property compels us to think of the physiological action and consequently the structure or molecu­ lar formulas of humic acids (HA) and fulvic acid (FA). The considerable and wide-ranging action of HA and FA is primarily due to the presence in them of

4 many functional groups, not only such common ones as carboxyls, phenols and alcohols, but also quinones, amines and amides, which are capable of forming electrovalent and covalent bonds and intracomplex compounds. These groups ensure regulation of the proportion of free ions to complex ions in the soil solution and in the intracellular medium. The diversity and complexity of functions are undoubtedly associated with the polychemical nature of humic substances whose molecules differ in size and functional groups and form a notable range of compounds differing in bonding ability and capacity to complex metal cations. Polydispersivity and multifunctionality ensure the high buffering capacity of humic systems in relation to acid-base, oxidation-reduction and many other reactions. Such a system must certainly actively regulate the geochemistry of met­ als and some organic compounds in the biosphere. While considering the physiological action of HA and FA we inevitably come to concepts of the similarity of animal and humus as propounded by Vernadskii, about the in­ evitable formation of a system of humic substances ensuring contemporary forms of terrestrial life. The functions of organic compounds in soils are diverse, at times even contradictory. The low-molecular-weight substances are usually readily available to micro-organisms and participate in the mobilisation of mineral constituents of soils, extracting many elements from difficultly soluble form. Humic acids play a preservative role to some extent, imparting to soils long­ term stability of important properties and functions such as humus reserve, cation exchange and buffer properties and so on. The stable reserves of humus in soils owe their origin primarily to humic acids and humins. The general principle follows from this that for accumu­ lation of humus in soils it does not suffice to add additional quantities of organic matter in the form of plant residue, manure or other organic ma­ terial. Simultaneous with such application conditions must be created that enSl)re complete humification of organic compounds, that is to say their con­ version mostly into humic acids or humin. This entails the important task of simultaneous and combined study of the structure of humus acids and the process of humification. The combined analysis makes it possible not only to understand the theoretical aspects more precisely, but also to develop effective ameliorative measures using organic manures. Consideration of only the stable, preservative properties and fractions of soil humus is obviously inadequate for solving the problems of increasing fertility and biological productivity. Soil biota need constant replenishment of .the reserves of labile organic substances. This is achieved by the addition of 'fresh' organic matter and partial mobilisation of reserves of specific humic substances. The process of mobilisation is accomplished through chemi­ cal and enzymatic hydrolysis, oxidation or reduction of humic substances and photochemical decomposition; the latter is most intense in the surface

5 layer of the upper humus-rich (or ploughed) horizon. Mobilisation of organic matter may be accompanied by, though not necessarily, the conversion of compounds into chemically and physiologically active forms. This explains in part the apparently anomalousstimulatory effect of small additions of sodium humate preparations even though the content of less active humic acids and humates in the soil may be high. It has been suggested that during extraction of humic acids from soil and preparation of sodium humates, the molecules of HA are modified and converted into active form [II'in et aI., 1973]. For ex­ ample, in an alkaline medium, under the influence of atmospheric oxygen, the degree of oxidation of HA and concentration of paramagnetic centres (free radicals) and quinonoid groups increases but the molecular weight decreases. Such changes occur very rapidly under better light, suggesting that the phenomenon is photochemical in nature. In an alkaline medium the molecules of HA acquire an 'open' configuration so that the side chains and functional groups can participate more readily in various reactions. These specific examples prove the need for understanding and codifying the most important functions of humic acids in the biosphere. In the most general form we may note five important functions: accumulation, transport, regulation, protection and physiological action. Their combination makes it possible to understand the ecological role of humic substances fairly well. Accumulative function involves accumulation in soil (and other indepen­ dent natural bodies) of organic compounds of carbon, nitrogen, phosphorus and other elements including trace elements essential for vital activity. This accumulative function should not be regarded as a passive stocking of nu­ trients since accumulation can occur even in soil solutions. Perhaps it would be expedient from this standpoint to identify another function-mobilisation. But it appears that this is overlapped by two other functions-accumulation and regulation. Transport function involves the formation of geochemical fluxes of min­ eral and organic substances, mostly in water media, due to the formation of stable but rather readily soluble (or peptisable) complexes of humic sub­ stances with metal cations, hydroxides, certain bio-organic molecules, or adsorption complexes of humic substances with complex aluminosilicates. Apparently, these are the forms in which a large part of organic and inorganic compounds migrates in the soil profile and in any landscape. Regulatory function of humic substances is complex and multifaceted. It may include: 1) formation of soil structure and hydrophysical properties of the soil [Bartoli and Philippy, 1990; Stevenson, 1985]; 2) regulation of equilibrium in ion exchange, acid-base and oxidation-reduction processes [Franzmeier et aI., 1990]; 3) regulation of conditions of mineral nutrition of plants by the influence of humic substances on the solubility of mineral components and their availability to living organisms; 4) regulation of the thermal regime of soils by influencing the spectral reflectance of the soil and

6 the heat capacity and heat conductivity of the soil mass; and 5) regulation of pro-anisotropic processes that lead to differences in chemical composition within and between horizons. Protective function involves the ability of humic substances to bind toxic elements or compounds into less mobile or difficultly dissociating compounds. It was mentioned earlier that humic substances are capable of removing the adverse effect of pesticides on crop plants. It has been demonstrated experimentally that in soils with large reserves of humic acids and humins, the permissible limiting concentration of heavy metals is much higher, so that their adverse effect on plants is manifest only at considerably higher concentrations than in Iow-humus soils. The adverse effects of excessive doses of mineral fertilisers are also eliminated. Not only heavy metals, but certain radioactive isotopes, for instance that of strontium, are converted into forms which are difficultly available to plants. The protective function involves not only the sOil-plant system, but also other components of the landscape (biocenose). It has been shown that humus-rich soils act as a geochemical barrier and obstruct the entry of many substances into ground water [Orlov and Lytkin, 1983]. The soil cover can hold a considerable amount of cations and anions and thereby maintain the quality of potable water at the desired level for a long time, despite industrial pollution. Physiological function of humic substances has been discussed above. Its manifestation is highly varied but has been very inadequately"Sludied. While discussing functions of humic substances in the biosphere no mention was made of such important aspects as their participation in the formation of composition of the atmosphere, flow of gases directed from the soil to the atmosphere with which, in part, may be associated the greenhouse effect [Orlov et al., 1987a, 1987b; Solin, 066s, Jager and others, 1986]. Mention was also not made of many other important natural mechanisms; however, a special monograph ought to be devoted to these issues. Here we must emphasise that even the partly listed functions of humic substances reflect the leading role of these compounds in many ecological links and relationships and allow us to consider that humic acids are not an incidental product of 'decay' of plant and other residues, but an essential and integral component of the soil-plant (maybe also ocean-plant and others) system formed as. a result of joint and uniform evolution of life and its surrounding medium, reflecting the integral harmony of the system. Investigations of the properties, structure and functions of humic substances are being conducted today in nearly all the developed countries. Great contributions to the understanding and resolution of these problems have been made by M. Schnitzer (Canada), K. Tan, J. Hedges, J. Martin, F. Stevenson and R. Zepp (USA), W. Flaig, W. Zeichmann, H. Seutelspacher and W.-f"ischer (West Germany); M. Hayes and

7 M. Cheshire (England), F. Swift and Goh (New Zealand), K. Kumada and K. Kyuma (Japan); N. Senesi (Italy); M. Gonsales (Spain), R. Gupta (India); D. Kleinhempel (East Germany); S. Sotakova (Czechoslovakia) and others. CIS scientists are leaders in several directions for resolving issues relat­ ing to humic substances. The CIS school is led by such eminent scientists as Academician Tyurin and Professors Kononova, Ponomareva and Alek­ sandrova. At present, groups engaged in the study of humic substances are working in Moscow (Moscow State University, Timiryazev Academy of Agricultural Sciences, Dokuchaev Soils Institute); St. Petersburg (Central Museum of Soil Science, St. Petersburg Agricultural University); Minsk (In­ stitute of Rational Utilisation of Natural Resources and Ecology); Novosibirsk (Institute of Soil Science and Arricultural Chemistry); Ashkhabad (Institute of Deserts); Dnepropetrovsk; Voronezh; Pushchino; Tyumen' and elsewhere. However, despite the length of the list, these groups have few members and are insufficiently equipped with the state-of-the-art equipment; furthermore, their activity is not properly co-ordinated. One of the main tasks in this di­ rection is the establishment of a National Committee for the Study of Humic Substances. In conclusion I would like to point out that despite the inadequacy of studies on humic substances as a whole, over the last decade or two new materials have appeared and much original data has been collected, which unveil the structure of HA and FA. This has become possible through the introduction of new methods for the study of humic substances. Such meth­ ods are improved modifications of nuclear magnetic resonance, different types of chromatography, pyrolytic methods and mass spectrometry. Our ideas have substantially changed in the field of molecular parameters of hu­ mic substances, molecular mass distribution and the formation of mineral­ organic compounds with incorporation of humic substances [Adhikari et al., 1989; Bloom and Leenheer, 1989; Chlou et al., 1990; De Nobili et al., 1990; Gonet, 1989; Leenheer and Noyes, 1989; Neto et al., 1991; Schnitzer, 1990; Wiklander and Border1, 1988]. All this requires further generalisation and interpretation of the rich, often contradictory experimental data.

1

Sources of Humic Substances and

Some Problems of Nomenclature

The question about the sources of humic substances, that is, compounds taking part in,humification and their origin, is exceedingly important since the correct answers are associated with rational concepts not only of the ways of formation of humic substances and their genesis, but also of their struc­ ture. Almost all modern investigators consider that structural units (or struc­ tural fragments) of natural biopolymers are building blocks of the molecules of humic and fulvic acids. Two extreme viewpoints differing from this con­ cept have been expressed. Williams (1947) considered humic substances as exo-enzymes of micro-organisms; in his opinion bacteria secrete humic acids into the surrounding medium while fungi produce crenic acids (fulvic acids). Troitskii (1940) held a totally opposite view. He assumed that to­ tal decay of organic matter of plant and animal remains takes place right down to formaldehyde (HCHO), which later polymerises with the incorpora­ tion of ammonium to form polymerisation products at different stages. This ultimately leads to the formation of humic acids. These views today have only historical importance. But a study of the sources (precursors) of humic substances is an important part of the general theory of humification and the chemistry of humic compounds. We must pay no less attel1tion to the question of nomenclature. In humus chemistry it is customary to present nomenclature (a list of names) as a definite hierarchic system which is often used as a classification scheme. Such schemes are convenient. Mutual understanding among researchers and reliability of the findings depend on correct arrangement, well-chosen terms and precise definitions of the concepts used. COMPOSITION OF PLANT REMAINS AND PATHWAYS OF THEIR TRANSFORMATION DURING HUMIFICATION

The chemical composition of plant remains entering the soil is very diverse. Detection of a definite set of structural units, at least kinds of carbon frame­ work, in humic substances provides direct information regarding the path­

9 ways of humification. The entire diversity of chemical compounds entering the soil from organic residues could be variously grouped for resolving the problems of interest to us. The following grouping is very basic: 1) according to the level of compounds of various classes; 2) according to the type of structure and reactive capacity; and 3) according to the biothermodynamic stability of components of plant remains. The range of levels of content of organic compounds of different classes is very large: from traces to tens of per cent. According to Aleksandrova (1980) and Grishina (1986), carbohydrates and arenes (lignin, tannins and flavonoids) are predominant in lichens, coniferous and deciduous tree species, and grass roots. The total quantity of carbohydrate reaches 40-85% (of dry, ash-free substance); only in conifer needles, leaves of tree species and legumes is the quantity of carbohydrates somewhat low. The ratio of cellulose to hemicellulose changes from group to group. The quantity of proteins, lipids and tannins is generally not high. In bacteria and fungi, the content of proteins and protein-related compounds may reach 40-70%. They often have a high content of lipids and some other compounds. The contribution of nitrogen-containing substances is high in cereals and more so in legumes. The chemical composition of different parts of tree species varies greatly: if the conifer needles and leaves contain about 8-12% protein, the woody part contains no more than 1-2%. Bacteria are distinct in their composition; they contain up to 40-70% protein and protein- , related substances. The high content of protein-related substances is also characteristic of algae. Predominance of carbohydrates and arenes has also been noted in grass roots (Table 1). Unfortunately, information about the biochemical composition of organisms has yet to be sufficiently generalised. It would be interesting to compare the biochemical composition of the roots of various plant formations with which, in the majority of cases, humus formation is possibly associated. Differentiation of the above-ground plant parts ought not to greatly influence the chemistry of humic substances, because the above­ ground plant litter is more intensively reworked by vari,ous invertebrates. Experiments conducted by Chinchaladze (1980) demonstrated a sharply dissimilar rate of decomposition and transformation of the above-ground remains and roots directly in natural surroundings. The nature of humification was also somewhat peculiar. Information about the individual substances constituting resins, waxes, pigments, etc., which could often exert a decisive influence on the course of the process, is insufficient. It has been repeatedly mentioned in the literature that there are differences in the humus formed from plant litter of different biochemical composition. Actually, in the early stages of humification in very young soils such a specificity could possibly be revealed in the litter. But for mature humus convincing data on biochemical specifics of humus are not yet available. In her exhaustive work Grishina

Vertebrates

Invertebrates

Animals

Grass roots

Legumes

Cereals

Timber

Leaves

Deciduous plants

Roots

Timber

Needles

Coniferous plants

Mosses

Lichens

Algae

5-10 8-10

5-10 0.5-1 5-7 5-10 4-6

1.5-3 0.1-0.5 1.0-1.5

2-10 5-7 20-30 1-5 3-8

Bacteria

Fungi

Ash

Object

30-50 30-40

8-12 1-2 10-12 20-25 3-4

8-10 0.5-1 2-3

40-70 35-40 10-15 2-4 8-10

compounds

containing

and nitrogen­

Protein Mono- and

6-10 8-10

9-15 2-3 10-12 22-25 2-3

10-15 2-3 2-3

3-4 3-4 3-5 3-5 10-12

oligosaccharides

0 0

15-25 45-50 30-32 20-22 30-35

28-30 40-45 34-37

0 0 0 8-10 12-25

Cellulose

Carbohydrates

10-30 8-10

20-25 20-25 30-35 25-30 20-25

22-27 20-25 25-35

20-25 25-30 50-60 50-70 30-50

carbohydrates

Other

20:"'50 30-40

3-6 4-6 8-10 3-5 3-5

10-15 3-5 4-5

15-25 20-25 1-3 1-3 7-8

Lipids

Table 1. Chemical composition of organisms (in % of dry matter) [after Grishina, 1986)

Tannins

1-2 1-2

15-20 10-12 2-4 2-3 10-12

10-15 12-15 5-8

0 0 0 1-2 15-20

and flavonoids

Arenes

0 0

20

5-6

1-2 20-22 6-10

4-6 25-30 20-25

0 0 0 8-10 0

Lignin

99

11 (1986) made an attempt to find a correlation between litter composition and humus properties. Certainly, some observable differences are readily explained by the kinetic theory of humification and need no supportive biochemical data. Hurst (1967) proposed the differentiation of humic acids of lignin, flavonoid and pigment origin but did not present convincing proof. The unique independence of composition and properties of humus from quantitative proportions of biopolymers in the composition of plant formation can be traced to several reasons. One is the mixed nature of the plant litter. Only in a few associations, for example spruce forests, do we come across plant litter composed entirely of conifer needles. However, even this contains not just needles but bark and twigs also. Root remains predominate in soil horizons. Moreover, all types of litter are subject to reworking by invertebrates, their larvae, bacteria, fungi etc. All this unifies to some extent, or at least brings closer, the composition of exudates and organic remains undergoing humification in different cenoses. The second reason is associated with the specificity of the mechanism of the humification process and owes its origin to the biothermodynamic principle of this process (see Chapter 8). The role and effect of biochemical composition of litter on humus formation are manifest most likely not through the inheritance of any chemical structures specific to individual plant species or plant formation, but through the creation of favourable, or conversely unfavourable, conditions for the activity of soil biota. Nearly all the components of plant remains are reworked and incorporated in the process of humification, which accords with the concept of plurality of the soil as a medium for the survival of micro­ organisms [Zvyagintsev, 1987]. The composition of animal remains is also specific. In this case an insignificant content of arenes, complete absence of cellulose and predominance of proteins and some types of carbohydrates are characteristic. Such composition might possibly be reflected in the mechanisms of reactions of humification. But calculations have shown that the contribution of animal remains to the components undergoing humification is not large, which naturally follows from their position in the trophic chain. Let us also note the high rate of decomposition of most animal remains because of their protein content. Only some substances, such as chitin, are relatively resistant outside the body of animals. The direct contribution of micro-organisms is not quantitatively high but - their role as transforming agents of all types of organic materials entering the soil naturally or as industrial pollutants is very high. Grouping the precursors of humic substances according to type of structure and reactive capacity, independent of their quantity in the organic remains or in soils, provides valuable information. By comparing the composition of remains and structural fragments of HA and FA we can understand the types of biopolymers mostly responsible for the formation of specific humic substances, and can also outline, at least tentatively,

12

the main types of reactions and mechanisms in their humification. At the same time, such material is suitable for understanding the reactive capacity of the formed (conditionally 'mature') humic substances. Of course, an understanding of these links is fraught with certain difficulties. In particular, the general process of transformation of organic remains during soil formation and humus formation involves simultaneous reactions of decay, decomposition and synthesis. It is difficult or even impossible to distinguish low-molecular compounds present in the soil and those participating in humification from their origin: Are these the products of partial disintegration (hydrolysis) of biopolymers and in this sense direct sources for the formation of humic substances, or have they appeared during transformation of the already formed humic acids, for instance during their partial hydrolysis or as a result of fragment renewal according to Fokin (1975)? During the process of decomposition of biopolymers partial transformational changes are unavoidable, giving monomers or oligomers. Hence for comparing the composition of initial substances and formed humic substances the carbon framework of compounds or definite classes are more convenient than specific individual substances. And finally, because of transformational changes and entirely probable intersubstitution of individual fragments of molecules of humic substances, it is expedient, in most cases, to consider groups of compounds with similar structure and not individual compounds. All this compels us during characterisation of initial substances to consider not only components of the fresh litter, but even those non-specific compounds which are present in soils in a free state, that is, not included in the composition of humic substances or humin. Several groups of biopolymers, such as those listed in Table 1, are found in the composition of plant remains. Of structural interest first of all are the linear aliphatic molecules which may be represented by carbohydrates and some of their derivatives (acids, alcohols, fats etc.) which are constituents of the lipid group. This is followed by carbohydrates· and carbohydrate-related substances, proteins and protein-related substances. The last two groups form the hydrolysable (peripheral) part of humic substances. Other forms of their incorporation in HA and FA molecules are possible in principle but these have not been studied. The fourth important group-arenes-are compounds containing in some form unsaturated six­ membered cyclic compounds with different substituents. I shall try to avoid the terms 'aromatic' and 'aromaticity' because of their considerable conceptual inaccuracies in relation to humic substances. Less studied from the point of view of humification is the five-membered group including nitrogen-containing heterocyclics and various pigments. Among these the condensed carbohydrates and their derivatives (for instance anthraquinones) whose structure can be related to some models of humic

13 acids, for instance to the scheme proposed by Kasatochkin, are quite interesting. Groupings done on the commonality of the carbon framework or reactive capacity or forms of nitrogen compounds do not match and hence all are arbitrary to some extent. The choice of grouping is dictated only by the convenience of use to solve the problem under review. Let us sequentially examine individual groups of compounds in relation to humification. Carbohydrates are the most widely distributed components of plant re­ mains. Nonetheless, they are not assigned an important role in the formation of humic substances, although in recent years NMR methods have revealed their high levels in humic substances of several soils. According to calcula­ tions done by Sadovnikova (1976), in soils with plant remains, 10-14 tonnes of carbohydrates are added per hectare every year while in humus their pro­ portion (in terms of carbon) may reach 25-30%. Using chemical methods, it has been demonstrated that in humic acids carbohydrates may account for 20 and even up to 30% of the total carbon [Singhal and Sharma, 1985]. The reason for lesser attention to carbohydrates is possibly their lower biochem­ ical stability and faster consumption by soil microflora. Furthermore, even in seasonal dynamics it is not always possible to explain the relationship between the quantity of carbohydrates, level of biological activity and num­ ber of micro-organisms. Here the reason may be that carbohydrates are not only consumed but produced by micro-organisms [Sadovnikova, 1976]. At the same time, in the zonal series of soils there is a distinct relative impov­ erishment in carbohydrates in soils with higher biological activity, especially in chernozems. This is revealed from the composition of humus as a whole and also of humic acids (Fig. 1).

% 30 20 10

7 l

3

"

S­

6 7

Soils

B

Fig. 1. Carbohydrate content in organic matter of different soils (in per cent). Soils: 1-tundra; 2-sod-podzolic; 3-grey forest; 4-chernozems; 5-chestnut; 6-serozems; 7-krasnozems and yellow soils; 8-mountain soils [after L.K. Sadovnikova).

14 Many representatives of the carbohydrate class are found in plant re­ mains and the presence of polysaccharides, oligosaccharides and monosac­ charides has been confirmed. As already noted, cellulose and hemicellu­ lose are most widespread. In some plant parts, for example in cotton fi­ bre, the cellulose content is as high as 95-98%. Cellulose has not been identified in humic substances but the products of its enzymatic and acid hydrolysis---cellobiose and glucose (a-D-glucopyranose)-are present in the soil both in free and bound state.

CH 20H

~H :~

HO

H

OH OH

a-D-glucopyranose

*'~~O~;H~H

HO

H

H

oH

H

H

H

cellobiose

During hydrolysis of hemicellulose, glucose, mannose, galactose, fruc­ tose, xylose a~d arabinose may collect in solution. Practically all types of monosaccharides forming oligosaccharides and polysaccharides are found when soils and preparations of humic substances are hydrolysed. Published data in this regard is scant and we have to depend on the indicative results obtained by Singhal and Sharma (1985). They compared the contents of monosaccharides in acid hydrolysates (72% H2 S04 ) of several forest soils and humic acids extracted from these soils. Some of their results, modified by us, are given in Table 2. It is noteworthy that in all the soils and HA inves­ tigated (20 samples), only 6 monosaccharides were found in determinable amounts. Predominance (listed in decreasing order) of glucose, galactose and mannose was reported. The most significant point about these results is the striking monotypic nature of the monosaccharide spectrum of soils as a whole and of HA. Of course, some specificity is visible but only as a ten­ dency. Soil hydrolysates of the two types of forest are somewhat different. In the soil of the mixed forest, the proportion of glucose, galactose and man­ nose is somewhat higher while that of arabinose, xylose and rhamnose is lower. Although these differences are fairly discernible, they are not so great as to practically influence the nature of humus formation and humification. And if it is possible to detect some tendency in soil hydrolysates towards a decrease in proportional content of individual monosaccharides, such dif­ ferences are totally masked in humic acid hydrolysates from different soils and horizons. The monotypic nature of monosaccharide spectra of HA can hardly be explained by selective incorporation of specific monosaccharides in HA molecules. Two other more plausible reasons are: The monotypic

105

356

168

0-27

27-64

Mixed

Mean

IV

III

Mean

"

a

26,010

150

10-29

22.7

47.9 47.6

48.7 49.1

48.0

25.4

49.5

30,000

788

0-10

22.8

25.4 26.5

46.6 47.7

49.4 48.9

26,000

47.3 47.9

41.7 40.7

30,070

18.7

48.6

25.8

26.0

19.0

18.3

19.0

22.8 22.7

22.6

22.5

22.6

21.8

22.5

40.7

23.2

47.5

40.1

18.3

48.3

40.1

350 245

31,000

34,800

35,220 31,200

b

b

a

Galactose

a

Glucose

30-50

0-30

602

0-26

26-49

Sal

b

mg/100 9

a

Total sugar

m

Profile

Depth,

Forest

type

4.1

15.1 15.2 15.4 15.6

17.5 18.7 17.9

4.4

4.0

4.7

4.6

16.6

17.1 18.4

13.2

13.1

15.1 14.6

14.0

11.4

14.1

a

13.9

15.1

14.3

b

9.3

8.9

9.3

1.7

1.6

1.6

2.0

9.5. 9.3

7.7

6.5

8.4

7.6

8.3

a

4.3

4.5

4.1

4.6

4.1

4.5

4.4

4.6

4.8

4.1

b

Xylose

9.2

9.1

9.3

9.4

9.1

b

Arabinose

14.7

14.3

14.6

13.3

16.4

a

Mannose

0.6

3.6

3.6

3.4

4.8

2.7

a

b

0.3

0.3

0.3 0.4

0.3

0.5

0.4

0.4

0.5

0.6

Rhamnose

Table 2. Content of monosaccharides in (a) soil hydrolysates and (b) HA hydrolysates, in % of total [after Singhal and Sharma, 1985]

01

16 nature of composition and similarity with soil hydrolysates could either be a consequence of incorporation of soil carbohydrates in HA preparations as an admixture, or the natural equilibrium between the free and HA-bound car­ bohydrate components. The admixture of some part of the carbohydrates cannot be ruled out. But this could hardly be the principal factor. This is indicated by some difference in the carbohydrate composition of soils and HA as also in the composition of HA extracted from soils and waters using different methods, including non-destructive ones. Presently there is no ba­ sis for refuting the possibility of incorporation of such reactive compounds as carbohydrates in the composition of HA molecules directly under soil conditions. However, two questions remain unresolved. First, do carbohy­ drates enter only into the hydrolysable part of HA molecules or are they found even in the non-hydrolysable residue? The answer to this question might be provided by such methods as NMR spectroscopy and pyrolysis­ mass spectrometry. The second question concerns the form of incorpora­ tion of carbohydrates in HA and FA: Are they represented in the molecules by fragments of polysaccharide and oligosaccharide chains or are individ­ ual monosaccharides linked to the non-hydrolysable part of the molecules of humic substances? The nature of monosaccharide spectra, however, is well explained by both a~sumptions and hence further investigations are necessary using independent methods. Also of interest is an analysis of the carbohydrate composition of soils and HA under prolonged monoculture of various plant species differing sig­ nificantly in carbohydrate composition. This would be interesting notwith­ standing the fact that soils under corn and brome grass revealed no notable differences [Baldock et al., 1987]. In view of this, it is once again neces­ sary to emphasise that soil micro-organisms could exert a unique levelling effect. Incorporation of monosaccharides by humic acids (as also attachment of amino acids) is possible through N-glucoside bonds, for example in the reaction:

H~CH20HH HO

H

~

+ H2~t -eOOH OH. R OH

H H

H~CH20H H 7 HO

H

H

HOlt

N-C-COOH

H

k

In the peripheral and (arbitrarily) in the nuclear portions of humic acid molecules there are always amino groups or other functional groups facilitat­ ing bonding or other reactions, for instance of the type of ligand exchange. According to Flaig (1988) alcoholic hydroxyls of carbohydrates are capable

17 of reacting with methylated quinones forming ester bonds. Thus the quinones are held covalent~y like polypeptides and amino acids. The need to study such reactions is obvious. But so far the reactivity of humic substances has been studied mostly in the area of binding of metal cations, some pesti­ cides and iodine, whereas the structural rearrangement of molecules has remained almost unexplored except for the simple reactions of hydrolysis and alkylation. Some years ago Shmuk (1951) and Troitskii (1940) pointed to the in­ corporation of nitrogen in humic acid molecules through an interaction of ammonia or amino groups with carbohydrates. These reactions are not re­ stricted just to monomers. It is not possible to rule out the esterification reaction as a mechanism of bonding carbohydrates in humic substances. Besides the relatively simple monosaccharides and the structural units of cellulose and hemicellulose, no small importance is attached to pectinous substances and uronic acids. Their more complex structure and richness in functional groups give a broad range of types of linking of carbohydrate components in humic compounds. The next important group of compounds comprises those with aromatic nuclei serving as the source of various phenols and quinones that are direct precursors of humic compounds. This group includes substances contained in plant remains-from microquantities to tens of per cent. Their structural formulae are quite diverse. Lignin has attracted the maximum attention, particularly in view of its stability. From the standpoint of humification, lignin is interesting not only because of its high content in plant remains and relative stability, but also because chemically it is an irregular polymer with branched macromolecules consisting of residues of substituted phenolic alcohols. This raises the possibility of recurrence of a large number. of diverse products on its decomposition-not only of low-molecular mass, but also of high molecular mass.

Ht-

H2CO\j

~fOH

I. CO

HC 11

H,cO*O.

N

Cl)

571 4 3

260 126 45 46 21 10 7 4 29 6 5 4 8

::2!

0iij

97

spectrum with considerable predominance of aspartic and glutamic acids, glycine and alanine in the amino acid composition. Table 31. Relative molar distribution of amino acids in humic substances (% of total) Amino acid

Poland, chernozems [Turski and

Japan [Tsutsuki and Kawatsuka, 1978)

Chmielewska, 1986)

India, fulvic acids [Chattopadhyay, Nayak and Varadachari,

1985) 2

2

3

4

5

6

7

HA

FA

14.6 11.5

14.3 9.59

13.4 10.6

13.3 10.5

14.9 9.5

14.6 8.9

14.8 9.7

10.2 8.9

15.6 9.6

12.8 10.6 8.36 4.16 6.32 3.76 6.23

11.6 10.9 5.86 4.66 7.14 3.87 6.51

12.3 10.4 7.63 4.86 7.87 4.08 5.86

13.0 10.8 6.3 4.8 6.9 4.2 4.6

11.3 9:5 7.5 3.8 7.2 4.1 6.0

12.4 9.9 6.3 3.7 5.8 4.0 6.4

14.9 8.9 6.1 3.1 5.7 6.2 4.2

13.8 9.2 6.2 3.2 6.0 6.2 4.7

5.28 6.60 0.00 0.89 0.00

5.81 5.36 0.71 1.40 0.00

6.09 7.18 1.14 1.77 0.00

5.54 5.57 0.81 2.17 0.00

4.9 6:5 0.9 0.5

5.5 6.19 1.8

6.4 6.4 1.4 2.0

7.0 6.4 1.1 6.6 0.5

7.1 6.0 0.8 6.8 0.6

5.89 1.67 3.31

5.57 1.55 3.48

3.80 2.66 2.82

4.16 2.68 2.27

6.1 2.4 3.7

7.0 2.5 4.5

4.5 1,9 3.5

5.6 2.1 2.4

2.2 0.6 1.4

Acidic Aspartic acid Glutamic acid Neutral Non-polar Glycine Alanine

12.6 11.9 7.76 Valine Isoleucine 4.13 Leucine 5.59 Phenylalanine 3.45 Proline 4.83

Polar Serine Threonine Methionine Tyrosine Cystine Basic Lysine Histidine Arginine

Note: Nos. 1-7 in column headings denote different soils.

The weight proportion of amino acids in FA, according to our data, ranges from 5.5 to 7.0% and that in HA from 5.8% in chernozems to 9.9% in serozems (Table 32). This means that at least 6-7% of the structural components of FA and 6:""10% of the components of HA are identical and can be used to build a structural scheme. If we consider that amino acids of FA bound to the phenol components are not subject to hydrolysis, then even for dipeptides the actual yield of amino acids will not be 50% of the theoretical value. Thus the maximum possible proportion of amino acids in FA is 12-14% and that in HA 14-20%.

98 Table 32. Weight proportions of amino acids in humic substances (%) Soils

Sod-podzolic

Land use Forest Fallow Mixed grass Legumes Rye Maize Average

Chernozem

Steppe Sainfoin Fallow Barley Average

Serozem

Steppe Mixed grass Peas Rye Cotton Maize Average

Humic

Fulvic

acids

acids

6.8 9.4 9.8 9.6 7.4 7.6 8.4

6.5 6.8 7.3 7.2 6.9 6.8 6.9

5.7 6.0 5.8 5.6 5.8

5.7 5.7 5.4 5.2 5.5

10.2 10.4 10.5 10.2 9.2 8.9 9.9

5.9 6.3 6.3 6.1 5.5 5.7 6.0

The question of the form in which amino acids are incorporated in HA and FA has not yet been resolved although models of humic substances de­ pend on whether amino acids are incorporated as individual residues or as polypeptide chains. Jenkinson and Tinsley (1960) demonstrated the pres­ ence of peptide bonds in 'Iignoprotein' based on IR spectra. Such bonds appear in the spectra of HA at 1650 and 1540 cm- 1 but either decrease in intensity or totally disappear when the HA is hydrolysed [Orlov et aI., 1972]. Experiments with enzyme hydrolysis also enable us to consider that at least a considerable part of amino acids form peptide bonds [Orlov, 1974]. A large part of the hydrolysable nitrogen is represented by the ammonium form; the average content of the NH3 groups in HA from sod-podzolic soils is 1.38%, from chernozems 0.75% and from serozems 1.34%. The ammonium nitrogen makes up a considerable fraction of the total nitrogen but its con­ tribution to the building of amino acid molecules is, on the whole, naturally low. According to Tsutsuki and Kuwatsuka (1978) the proportion of amino acid nitrogen in HA of Japanese soils is 28-57% of the total nitrogen content, that of amino sugar nitrogen 1-2% and of ammonium nitrogen

99 8-15%. Similar ranges have also been reported by other authors [Turski and Chmielewska, 1986]. The ammoniacal nitrogen content of FA is somewhat higher, from 1.60 to 1.80% by weight. Amino sugar has been reported in all fractions of humic substances. The amount expressed in terms of glucosamine (C sH11 OsN H2 , MM = 179.18) is 1.5-1.9% in HA and 2.9-4.2% in FA. These forms constitute almost the entire bulk of hydrolysable nitrogen; only a small part of it enters hydrolysates of HA in the form of FA-type compounds. This allows complete determination of the qualitative and quantitative composition of nitrogen­ containing compounds in the hydrolysable part of humic substances. The sum of nitrogen-containing compounds identified in the hydrolysable part is about 12% of the total weight of HA from sod-podzolic soils and serozems. The value for chernozems is 8.5%. In FA, for all soils it is about 11 %. Twice this value should be considered the upper limit, assuming the recovery of components to be 50%. The nitrogen content of humic substances imparts a special role to them in the nitrogen balance of the soil and nitrogen nutrition of plants. This has again and again raised the issue of mobilisation of biologically stable forms of nitrogen compounds, especially in soils rich in HA and 'humic' nitrogen. How~ ever, the conversion of nitrogen-conta1ning fragments of humic substances into forms that can be assimilated by plants is related to their decomposition. With excessive mobilisation, undesirable consequences are unavoidable: deterioration of soil structure, reduction of adsorption and buffer capacity and so on. Therefore, rational mobilisation of soil nitrogen presupposes an accelerated cycling of nitrogen compounds in the soil-plant system to pro­ vide for conservation of the optimum level of organic matter reserves. The development of such a system requires a detailed study of the forms of nitrogen compounds and their reserves in soils. In all soils the mainpart of the nitrogen reserve is represented by humins, the most stable compounds of nitrogen (Table 33). The labile components constitute only 5-10% of the total reserves in serozems and sod-podzolic soils and 2.5-3.5% of nitrogen reserves in chernozems. In absolute values the reserves of mobile nitrogen compounds in chernozems exceed those in sod-podzolic soils and serozems, but the participation of nitrogen in the biological cycle in chernozems and its utilisation are lower. A comparison of the reserves of different forms of nitrogen in different types of soils brings out the same pattern as that for the properties of HA. The more intense the humus accumulation, the higher the total nitrogen reserves in the soil and the higher the accumulation of difficultly soluble and less mobile compounds. In view of this, one of the main tasks facing proper soil management should be not just (and sometimes not so mUCh) to increase the total nutrient reserves, particularly of nitrogen, but to control the biochemical processes in soils by ensuring the optimum balance of forms of these compounds [Anderson, Sick

100 Table 33. Reserves of some organic compounds of nitrogenin the 0-20 cm layer of soil, kg/ha [after Orlov and Ovchinnikova, 1966) Nitrogen compound

Sod-podzolic

Deep

soil

chernozem

Serozem

Fulvic-acid nitrogen amine

198

273

104

ammoniacal

218

268

104

amino sugar

47

79

31

157

237

84

620

857

323

humin Total Humic-acid nitrogen amine

104

404

104

ammoniacal

83

273

83

amino sugar

19

63

9

135

980

142

341

1720

338

amine

125

336

ammoniacal

213

499

amino sugar

234

530

182

humin

806

3152

1074

humin Total Humin nitrogen

474

Total

1378

4517 .

1730

Total nitrogen

3560

9900

3420

and Hepburn, 1989; Humic Substances in Soil and Crop Science, 1990; Stevenson and Xin Tao He, 1990]. Carbohydrate Fragments

Substances of the carbohydrate type are always present in hydrolysates of humic substances and soils as a whole. Some authors consider them an admixture in the composition of HA and FA. Yet opinions have been voiced that the carbohydrate components may be considered structural fragments of humic substances. Carbohydrates are stably bound to humic substances and there is no reason to draw a boundary between amino acids and carbo­ hydrates. It has been established that hexoses and uronic acids are present in HA of Japanese soils in amounts from small fractions of a per cent to

101 3-5% [Tsutsuki and Kuwatsuka, 1979]; in FA of Italian soils up to 63% of all carbohydrates are represented by glucose and mannose[Guidi et al., 1976]. Glucose, galactose, mannose, xylose, arabinose, ribose, rhamnose, fuc­ cose, fructose, mannitol, inositol, and glucouronic acid have been identified in the fulvic acids and hydrolysates of humic substances. Hexoses consti­ tute about 20-40% of the total sugars (glucose at times up to 40-60%), pentoses 4-9%, and uronic acids 5-20%. The latter are mostly found in the fulvic acid fraction [Reissig, 1956; Kosaka and Honda, 1957; Cheshire and Mundie, 1966; Dormaar, 1967; Havrankova, 1967; Lowe, 1969; Ogner, 1970]. According to Hayashi and Nagai (1962) hexoses in HA constitute 53%, methylpentoses 26% and pentoses 21% of all reducing sugars; in FA these values are 56, 36 and 8% respectively. Similar information is available in many other publications [Majumdar and Rao, 1978; Barriuso et al., 1985; Yadav and Jha, 1987]. The carbohydrate composition of hydrolysates of humic substances of many soils in the CIS was determined by Sadovnikova (1976). According to her results, hydrolysates of HA and FA of sod-podzolic, sod-meadow, typical deep chernozem and chestnut soils showed the presence of glucose, galactose, mannose, rhamnose, arabinose, xylose, ribose, fuccose, uronic acids, didesoxyhexoses and amino sugars. This report accords well with other published data. The content and composition of carbohydrates in hydrolysates of HA and FA are similar and, as with amino acids, do not offer an unambiguous answer to their nature: Are these the structural components of humic sub­ stances or incidental admixtures? Be that as it may, the high amounts of carbohydrates in soils and their high reactivity permit us to presume that these are constituents of HA. Carbohydrate residues have been included in the structural scheme proposed by Dragunov, who found that HA up to 10% by weight are represented by readily hydrolysable carbohydrates [Dragunov et al., 1950]. As with amino acids, once again there, is considerable similarity in carbo­ hydrate composition of various types of hydrolysates and humic substances in various soils. This permits us to consider that even in the case of carbohy­ drates there is an 'equilibrium' between humic substances and non-specific compounds, as was confirmed in Chapter 1. The structural role of carbohydrate components of humic substances is not yet fully clear. Part of the sugars, similar to amino acids, may be adsorbed by humic substances. Clark and Tan (1969) considered polysac­ charides to be fairly firmly bound to humic substances mostly through com-. plex ester bonds; polysaccharides even become the principal component in FA. Upon hydrolysis of fulvic and hymatomelanic acids, these authors isolated a fraction that was later identified from IR spectra as polysaccha­ ride. Experimental data has indicated that mono- and polysaccharides form

102 compounds with components of humic substances through complex ester bonds. It must be said that hydrolysates of humic substances contain not only amino acids and carbohydrates, but certain other compounds also. In particular, up to 5-7% of the hydrolysable substances may be represented by phenols [Tsutsuki and Kuwatsuka, 1979; Drijber and Lowe, 1991]. Some part of the hydrolysate is closer in its nature to FA [Orlov, 1974]. Thus t~e hydrolysable part of humic acids can be almost completely identified, which is reflected in the Table of structural fragments of HA and FA given later (see Table 36). Alkanes and Fatty Acids

This group of substances is not a constituent of the hydrolysable components but is extracted by organic solvents. Hence it remains uncertain whether these substances should be included under structural units of HA and FA or as admixtures. The situation is complicated by the fact that alkanes are insoluble in water but are extracted from water-soluble fractions of FA. Ac­ cording to Schnitzer and Ogner (1970), up to 0.1% FA is represented by fatty acids with a chain length of C14 to C34 and. traces of C24 hydrocarbons. The low content of fatty acids in FA would indicate that they do not have a constitutional role, albeit Schnitzer and Neyroud later (1975) reported a higher content of fatty acids and phenoxy acids in humic substances. But such data do not enable us to resolve the problem of the participation of alkanes and fatty acids in the structure of HA and FA. As shall be shown below, this is confirmed by the absence of absorption bands near 720 cm- 1 in the IR spectra of humic substances. OXIDATION PRODUCTS OF HUMIC SUBSTANCES

Oxidation as a destructive method enables us to obtain extensive and signi­ ficant information about the structural fragments of humic substances. Most commonly oxidation is done with potassium permanganate or cupric oxide in an alkaline medium. Earlier many other methods were used, including oxidation with nitric acid, hydrogen peroxide, atmospheric oxygen, alkaline nitrobenzene etc. Investigations for selecting oxidising agents were directed towards ascertaining the conditions for mild decomposition without exces­ sive destruction of the structural fragments, giving a high yield of oxidation products. On the other hand, only that method is suitable which does not produce more complex products than were present in the original substance. It is most important that aliphatic components do not turn into cyclic, which may significantly distort our ideas about the actual structure of humic sub­ stances.

103 The method and mechanisms which are most apparent and which en­ sure significant yield of destruction products and exclude secondary cyclisa­ tion are the oxidation of humic substances with alkaline solution of potassium permanganate with subsequent analysis of the resultant benzenecarboxylic acids. The yieldof the latter is fairly high and their content changes in accord­ ance with the genetic series of humic substances. I used this method over 25 years ago [Orlov and Denisova, 1962]. Its suitability was examined in detail by Chudakov and colleagues (1959). It was established that in alkaline oxi­ dation with permanganate, CO2 , acetic acid, some aliphatic acids, but mostly oxalic and benzenepolycarboxylic acids are formed. During oxidation of sim­ ple aromatic compounds more complex products are never formed. The non­ aromatic substances (cellulose) mainly produce oxalic acid, CO2 and water. Senzenepolycarboxylic acids (SPA) are formed only from compounds with a ring structure. In the presence of oxygen substituelJts, benzoid structures disintegrate and their considerable part cannot be accounted for. Nitrogen­ containing heterocyclics are more resistant than benzene rings; during oxi­ dation of quinoline (I) by potassium permanganate mostly the benzene ring is destroyed and roughly equal quantities of phthalic (Ill) and cinchomeronic (IV) acids are formed from isoquinoline (11) [Pakett, 1971].

formed of of

~

~N

n

IO,,"n04

HOOC~

HOOC~)J N

A-COOH +

'V-eaoH

m

HOOC-0

HOOC~N

l!'

The considerable content of nitrogen in the oxidation products of HA confirms this situation and allows direct estimation of the nitrogen content of heterocyclics [Orlov and Denisova, 1962]. Fears regarding the possibil­ ity of aromatisation of compounds during oxidation of humic substances by potassium permanganate may be considered exaggerated. Ovchinnikova (1965) demonstrated that the yield of SPA can be accounted for almost en c tirely by just the non-hydrolysable part of the molecule and that the aliphatic side chains do not affect the results obtained. Many authors have noted that oxidation by permanganate produces a considerable destruction of hu­ mic substances. As a result, part of the structural units is lost and it is no longer possible to arrive at a conclusion regarding the means of attachment of these units to the 'nucleus'. This was partly suggested by Maximov and

104 colleagues (1977), who remarked, that under the impact of permanganate both aliphatic and methoxy aromatic structures are destroyed. This could level the differences in humic substances of different origin observed in the experiment. A more gentle method of destruction is the use of cupric oxide. Here, a mixture of 1 g HA, 100 ml of 2N NaOH and 5 g CuO are heated in an auto­ clave for 3 hat 170°C in an atmosphere of nitrogen. This enables more com­ plete characterisation of the aliphatic oxidation products. Using this method Neyroud and Schnitzer (1974, 1977) extracted 109 mg of aliphatic com­ pounds (mostly succinic and fatty acids with a chain length of C 16 and C 1S )' about 95 mg phenolic acids, and 36 mg benzene-carboxylic acids from 1 g humic acid. In regard to the selection of method, mention must be made of one spe­ cial feature characteristic of soilS and similar materials. Harsh treatments often cause a loss of several components and some of the actual struc­ tural fragments are lost sight of by investigators. However, the use of milder methods produces other, no less undesirable, effects. Milder methods give additional 'noise' because admixtures enter into the composition of the sam­ ples and their destruction products, distorting the picture no less than the loss of individual structural fragments. Riffaldi and Schnitzer (1973) provided a good illustration in support of the foregoing. According to their results, after hydrolysis the carbon con­ tent in HA specimens from ash, peat and chernozem soils increased by 2.5-3% while the contents of hydrogen, nitrogen and sulphur decreased. This is the consequence of loss of the so-called aliphatic 'peripheral' chains which could be adsorbed by HA during its extraction from soil. The yield of oxidation products, too, changed substantially; these were identified as the respective methyl esters (Table 34). It is typical that the amount of pheno­ lic compounds in all HA increased after preliminary hydrolysis whereas the content of benzene-carboxylic acids decreased in two specimens and only in HA from chernozems did it increase. This confirms that the 'peripheral' chains (or admixture components) are linked variously with the components of the 'nucleus' in HA from various soils; the two constituent parts of HA dif­ fer sharply in HA from chernozem and are quite similar in HA from peat soil. This accords well with the earlier described concept of lesser differentiability of 'nucleus' and 'periphery' in humic acids of sod-podzolic soils compared to chernozems because of the weaker biological activity in sod-podzolic soils; this leads to more prolonged existence in sod-podzolic soils of all classes of organic compounds, both free and as an integral part 00 humic or fulvic acids [Orlov, 1974). Despite specific limitations, such observations compel us to give preference for slightly more severe methods of hydrolysis and

105 oxidation. At the present stage of investigations, it would probably be expe­ dient not to have exhaustive information but to avoid the effect of admixtures during the treatment. Aromatic compounds have long been observed in the diSintegration products of humic substances. As early as 1915, Troitskii detected terephthalic acid. In 1937, Tyurin reviewed the identified components. Attention to these compounds then waned but again picked up in the 1960s. Modern techniques of investigation enable precise quantitative estimates of aromatic compounds and their detail identification. Despite the diversity of methods employed, a fairly monotypic set of compounds is usually observed. A general survey of disintegration products was presented by me [Orlov, 19741 based on my personal and other published reports [I.V. Tyurin, A.N. Shivrina, M.D. Rydalevskaya, lA Tereshenkova, N. Atherton, L. Batistic, J. Mayadon, W. Flaig, E. Nansen, M. Schnitzer, S. Mathur, J. Dormaar, T. Hayashi, T. Nagai, F. Stevenson, and others]. The following groups of compounds are usually found among the disin­ tegration products. 1. Phenols: pyrocatechins, resorcin, fluoroglucine, nitrophenol, 2-4­ dinitrophenol, 2,4,6-trinitrophenol, trinitrodihydroxybenzene. 2. Aldehydes: n-hydroxybenzaldehyde, vanillin, syringaldehyde. 3. Acids: m- and n-hydroxybenzoic, vanillinic, syringic, veratric, protocat­ echuic, 2,4- and 3,5-dihydroxybenzoic, 3,4,5-trihydroxybenzoic, isophthalic, terephthalic, benzene-tricarboxylic, mellophanic, pyromellitic, benzene­ pentacarboxylic and mellitic acids. Depending on the methodology of the experiment these acids were ob­ tained as derivatives (mostly nitro- and methoxy). 4. Quinones: n-benzoquinone; 2,3-dimethyl-n-benzoquinone and also 2­ methyl-1 ,4-naphthaquinone, 1,4-naphthaquinone, 1,2-naphthaquinone and anthraquinone. 5. Polycyclic compounds: naphthalene, anthracene, fluoranthene, 1,2­ benzanthracene, benzofluorane, pyrene, benzopyrene, perilane, chrysene, coronene and others. 6. Nitrogen-containing heterocyclic compounds: pyrrole, indole, scatole, carbazole, acridine, benzacridine. It is hardly possible to include all the compounds in the disintegration products under structural fragments of humic substances. A rational selec­ tion of structural units must be based on the absolute yield of the products, the possibility of their secondary modification during destruction of humic substances and sources of possible incidental admixtures. The small quantities of individual aromatic compounds are practically included as admixtures of lignin or their free forms in plant microbial cells. Present-day methods have not succeeded in making the separated HA sam­ ples free of these admixtures. At the same time, one cannot overlook the fact

106 Table 34. Yield of methylated compounds after oxidation of 1 g humic acid by potassium per­ manganate (mg) [after Riffaldi and Schnitzer, 1973] Humic acids Volcanic

Chernozem Peat soil

ash soil Component Compound No. (2)

(1 )

2

2

2

2

2

2

(3)

(4)

(S)

(6)

(7)

(8)

1

Dimethylsuberate

10.6

7.2

2

Dimethyl ester of 1,2-benzene-dicarboxylic acid

7.5

8.1

Dimethyl ester of 1,3-benzene-dicarboxylic

0.4

1.1

1.4

3.4

2.2 1.6

2.9

3.2

4.5

8.0

6.5 1.7

3

5.5

5.S

4.3 2.9

11.7 21.8

12.5 6.0

acid 4

Dimethyl ester of 1,4-benzene-dicarboxylic acid

5

Trimethyl ester of 1,2,3-benzenetricarboxylic acid

37.7 24.7 47.9

55.7

18.7 6.7

6

Trimethyl ester of 1,2,4-benzenetricarboxylic acid

14.9

6.8 21.1

23.2

5.6 3.2

7

Trimethyl ester of 1,3,5-benzenetricarboxylic acid

4.4

6.1

7.2

10.9

4.5 2.5

8

Tetramethyl ester of 1,2,3,4-benzenetetracarboxylic acid

70.6 40.9 63.4

75.6

44.513.6

9

Tetramethyl ester of 1,2,4,5-benzenetetracarboxylic acid

13.9 15.6

22.5

35.8

20.0 5.7

10

Tetramethyl ester of 1,2,3,S-benzenetetracarboxylic acid

4.6

8.4

15.3

6.511.9

11

Tetramethyl ester of 5-methoxy-1 ,2,3,4benzene-tetracarboxylic acid

24.3 26.4

8.9

17.9

4.5 4.3

12

Pentamethyl ester of benzenepentacarboxylic acid

54.4 44.4 47.9

68.5

36.1 11.4

13

Pentamethyl ester of methoxy benzenepentacarboxylic acid

11.2 13.1

7.5

16.0

6.220.6

14

Not identified

4.2

5.1

4.318.1

15

Hexamethyl ester of benzenehexacarboxylic acid

27.6 70.0 11.2 31.7

8.613.4

16

-Do-

17

-Do-

5.0

4.9

2.7

2.2 13.5

1.4

7.2

1.913.0 9.4

107 (1 ) 18

(2)

(3)

Dimethyl ester of dehydrodivaratric acid

(4)

(5)

(6)

6.2 26.7

(7)

(8) 3.9

Total: benzene-carboxylic aCids (esters)

239.3225.5247.1 349.7165.477.7

phenolic substances

41.7 66.7 16.4 33.9

10.728.7

substance with two-substituted ring

10.8 12.4 17.6

33.2

21.2 9.3

substance with three-substituted ring

63.2 64.3 76.2

89.8

28.816.3

94.3 126.7 71.031.2

substance with four-substituted ring

89.5 61.1

substance with five-substituted ring

78.7 70.8 56.8

substance with six-substituted ring

38.8 83.1

86.4

40.615.7

18.7 47.7

14.834.0

Note: 1-before hydrolysis; 2-after hydrolysis.

that some structural units cannot be detected because of their decomposition during analysis. Dormaar (1969) did not detect flavonoids in the reduction products of HA when reduction was done by sodium amalgam. But specially conducted experiments showed that they break down during analysis (the pyrone ring opens up). Under the action of boiling alkali, for example, flavone disintegrates to salicylic acid, benzoic acid and acetophenone.

a

-3C -CH 2-(3C) -CH­

57 62

- OCH3 -CH 2-OH, alcohol

70; 72; 74; 76.5

HCOH, alcohol

103 112; 127; 129;132.5; 137.5; 151 162; 165; 166; 168 172; 174.5; 176.5; 179

>C (O-R)2 ·acetal of aromatic CO~-, carbonate, phenolate -COO-, COOR -CONH 2, acids, esters, amides

I

I

I

I

-C-O I

o:r:

£

-(CH)­

u""l :J:

~

0­ 0­

2n

0­

0-0­ 0­ 00­0­

0­ I

200

750

700

50

Chemical shift, ppm . J=lg.14.

13C

o

NMR spectrum of humic acids from yellow~brown soil [after Newman et aI., 1980]

115

Table 37. Distribution of C in humic acids of soils of tropical and temperate latitudes [after Lobartini and Tan, 1988] Soil

Carbon content in fragment (%) Aliphatic

Polysaccharides

Aromatic

Tropical regions Inseptisol

52.8

15.1

21.9

Ultisol

28.0

31.5

32.2 34.4

Temperate regions Entisol

32.8

23.9

Mollisol

32.2

18.8

37.2

Mollisol

26.1

20.4

40.8

Spodosol

15.6

16.2

54.9

Spodosol

29.4

25.9

33.0

Ultisol

22.7

26.9

35.5

Ultisol

22.3

25.1

38.0

The overall ranges do agree although the set of maxima and the values of the chemical shifts (in relation to tetramethylsi~ lane) vary considerably [Hatcher, Maciel and Oennis, 1981; Ogner, 1979]. Presently, it is convenient to use broad, generalised ranges. Japanese researchers have identified four ranges in the NMR spec­ tra of humic SL.)stances: 1) 0-60 ppm-aliphatic carbon, including alkyl and methoxyl; 2) 60-110 ppm---carbohydrates, primary alcohols, acetals; 3) 11~165 ppm-aromatic carbon and 4) 165-210 ppm---carbon of car­ bonyl groups [Kuwatsuka et al., 1986]. According to others [Skjemstad et al., 1983], these ranges are somewhat different: 1) ~50 ppm-alkyls; 2) 5~108 ppm---carbon of C-O; 3) 108-160 ppm-aromatic carbon and 4) 160-200 ppm-range of carboxyl carbon. The differences are not that high but in comparing published data these must be taken note of.

FUNCTIONAL GROUPS AND DISTRIBUTION OF OXYGEN Functional groups occupy a special place among the structural units of hu­ . mic substances. They not only describe in great detail the proportion of structural units, but also enable us to evaluate the actual reactive capacity of HA, HMA and FA. According to estimates the weight proportion of func­ tional oxygen alone in humic substances could be 3~35%, which could greatly affect the proposed structural formulas of HA and FA. Among the

116

/;

o

oxygen-containing groups the more important are the carboxyls (-C-OH) alcohol (R-CH 2 -OH), phenolic ©-OH, methoxyl (-O-CH 3 ), and amides (R-C-NH 2 )· 11

o

There are numerous results about the presence of quinoid, ester and keto groups although not all researchers have been able to detect them. Considerable diversity of functional groups is confirmed by many reactions, in particular alkylation. However, the quantitative indices are often conttadic­ tory, mostly because of the imperfect and incompletely developed methods of functional analysis relative to humic substances. The total number of groups reacting with cations is often judged from the absorption capacity of humic substances. However, the concept of absorp­ tion capacity has not been precisely defined for them. Many authors point to the capacity observed at different pH values, while deprotonation of carboxyl and phenol groups depends on the pH of the medium. According to Titova (1970), the exchange capacity of humic acids of the soils of arid steppes is 607-693 meq/100 g, tbat of fulvic acids 650-668 meq. For humic acids of spruce humus, values in the range of 230-300 meq have been reported [Czerney and Fiedler, 1968], that for brown and pseudogley soils 274-292 and for organic matter as a whole of these very soils 41-195 meq/100 g [Hoffmann, 1966]. The absorption capacity of humic acids from many soils of Punjab has been reported to be in the range of 390-450 and of fulvic acids 310-390 meq/100 g [Bhandari et aI., 1970]. Such a wide range of variation (twofold and more) compels us to think that either thei concept of absorption capacity is rather vague for humic substances or the substances investi­ gated were not chemically identical. The results depend on the aggregation state of the compounds. The capacity to bind with cations is not the same in a dry preparation of HA as in soluble humic substances. The absorption capacity may also be affected by blocking of that part of functional groups firmly bound to mineral particles [Krystanov, 1968]. More complete information is available regarding the content of carboxyl, methoxyl and hydroxyl groups. Selected data for some soils is presented in Table 38. The presence of carboxyl groups is confirmed by the IR spectra in which the intense absorption band at about 1720 cm- 1 is predominantly due to the non-ionised COOH group and the carboxylate ion is identified from two bands at 1690 and 1390 cm -1. The substitution of humic acids up to salts and the reverse reaction is clearly traceable from the spectra, which addi­ tionally confirms the correctness of band separation. It may be noted that

117 Table 38. Functional groups of humic acids (meq/100 g) Region, soil

COOH

OH-phenolic

(1 )

(2)

(3)

Russia, St. Petersburg district:

305

333

139-210

262-417

Podzolic

290

430-500

Podzolic

244

523

Podzolic

215-290

sod-podzolic, A 1 differing in plant residue

Source (4) Zhigunov, 1975

Russia: Natkina, 1940 Naidenova, 1968 Gemmerling, 1946

Podzolic

290

210

Grey forest

427

326

Tyurin, 1937 Simakovand Leashevich, 1971

Canada: Grey forest

230

559

Lowe, 1969

Grey, sodolised

209

537

Lowe, 1969

Grey, solonetsic

257

458

Lowe, 1969

340-390

260-380

Khan, 1970

degraded

290

210

Tyurin, 1937

typical

290

210

common

290

210

210-420

217-475

Grey forest Russia: Chernozem

Poland: Chernozem

Turski and Chmielewska, 1986

Bulgaria: Chernozem solonetsic

375

263

calcareous

408

344

light grey

297

237

390

300

Filcheva, 1976

Spain: Cherhozem

Martinez and Rodriguez, 1969

Canada: Chernozem

243

537

Lowe, 1969 Lowe, 1969

brown

243

478

brown, solonetsic

232

481

Lowe, 1969

solonetz, Ah

360

720

Khan, 1969 (Contd.)

118 Table 38. Contd. (2)

(3)

solonetz, B

400

570

s%d, Ah

380

680

Khan, 1969

s%d, B

400

550

Khan, 1969

sod s%dised Ah

sod s%dised B

410

580

Khan, 1969

420

550

Khan, 1969

(1 )

Chestnut

140-215

(4) Khan, 1969

Gemmerling, 1946

Russia: Chernozem, common, unirrigated, Ap'OU9h

480

150

A1

460

100

B1

430

140

B2

470

210

AplOugh

A1

450

180

430

190

B1

410

200

B2

460

290

A1 B1

380 420

240 240

B2 Dark chestnut, irrigated

440

200

A1

310

B1

360 370

290 320

Oko/e/ova, 1985

Chernozem, irrigated

Dark chestnut, unirrigated

B2 Light chestnut, unirrigated

290

A + B1

410

B2

340

140

BC

360

210

A + B1

370

240

B2

320

190

BC

290

220

Hydrandept

378

299

Hydrandept

409

272

Dystrandept

448

304

Dystrandept

552

147

230

Light chestnut, irrigated

Dominican Republic: Griffith and Schnitzer, 1975

119 (1 )

(2)

(3)

Dystrandept

405

218

Dystrandept

440

375

360

330

(4)

Greece: Alfisol, under oak AO

Kallianou and Yassoglou, 1985

410

420

under heath AO

A1

350

330

under grass A 1

430

330

A1

230

410

380

430

Xerochrept, shallow

Kallianou, Yassoglou and Ziechmann, 1982

Xerochrept, shallow

260

380

Chromoxerert, typical

254

426

Fluvaquent, mull

320

410

Xerofluvent, typical

350

370

235-633

56-369

Tsutsuki and

Podzol

299

96

Kumada and

Ash

555

67

Rice

304

86

Red-yellow

340

198

Mountain-meadow

299

96

Japan: Different soils

Kawatsuka, 1978 Kuwamura, 1968 Kumada and Kuwamura, 1968 Kumada and Kuwamura, 1968 Kumada and Kuwamura, 1968

the results of chemical identification of -COOH groups (comparatively sim­ ple) could be affected by the presence of S03H group, weakly acidic hy­ droxyls and others [Holtzclaw and Sposito, 1979]. Other aspects have been examined by De Nobili, Contin and Leita (1990). Still greater fluctuations are observed in the content of phenolic hydrox­ yls, for which, as for COOH, it is possible to trace some regularity only in the narrow ranges of soils. The proportion of COOH and OHphenoi groups do not remain constant. Besides phenolic hydroxyls, alcoholic OH (100-300 meq/1 00 g), was ob­ served; its presence in many cases is cpnfirmed by bands near 1100 cm- 1 in the IR spectra.

120 The amount of methoxyl groups is comparatively low and changes in an orderly manner, although the methoxyl content was used to evaluate the degree of humification. Scant data is available on the amount of different carboxyl groups. Carbonyls of the keto type have been found by many au­ thors in peat and coal humic acids in amounts up to 140-240 meq/100 g; the content of the aldehyde type was up to 35-136 meq/100 g {Ekaterinina, 1967]. Similar results have been reported by other authors although the ab­ solute levels of keto C-O differed twofold or more. Fuchs found 8.4% C=O groups in HA from brown coal while Ubaldini reported only 3.5% [Scheffer and Ulrich, 1960]. According to Flaig, the C=O content of soil HA is 2.7% [Flaig et al., 1955] whereas according to Schnitzer and Desjardins (1962), it is 2.5% for HA and 8.7% for FA. Schnitzer and Gupta later (1964) reported val­ ues of 5.0-5.3% C=O for HA and 4.8-8.4% for FA. According to the re­ sults reported even later by Schnitzer and Skinner (1966), soil HA contain 180-260 meq C=O groups per 100 g. The FA from the Bh horizon of pod­ zol contain 300-370 meq/100 g carbonyl. These authors ascribe this total amount to ketones, considering that aldehyde groups are almost absent [Schnitzer and Skinner, 1965]. Theng and Posner (1967) also reported from IR-spectral analysis not only the presence of carbonyl of COOH groups, but also of keto groups, part of which was hydrogen bonded. The possible presence of quinoid groups has not been rated uniformly. These groups could not be observed by spectroscopy in HA [Theng and Posner, 1967]. They were also not found even by NMR although Maximov and others [Glebko, Ulkina and Maximov, 1970; Maximov and Glebko, 1974] using chemical methods reported 105 meq/100 g quinoid groups in HA from forest soil and 125-340 meq/100 g in HA from peat and coal. Ekaterinina (1967) reported 75-150 meq/100 g quinoid groups for the same kind of sam­ ples. Ufimtsev (1971) found up to 570-580 meq quinoid groups in HA and 320-440 in FA per 100 g. Similar values have been reported by Mathur (1972): 350 meq/100 g in HA of chernozem and 420 meq/100 g in FA of podzol. According to Naidenova (1968), FA are richer in carboxyl (up to 275 meq/100 g) and phenolic (593 meq) groups than HA. Still higher values of carboxyl (610-910 meq) but lower values of phenolic hydroxyl (270-360 meq) have been reported by Wright and Schnitzer (1960), who in addition pointed to the presence of alcoholic hydroxyl and carboxyl groups (280-490 and 110-310 meq/100 g respectively). As a matter of fact, recent works have reported a much higher content of acidic functional groups in FA than was reported in the 1960s. It cannot be ruled out that this is attributable to improved methods of separation of fulvic

121 acids from soil and partly to the widely used method of separation of FA according to Forsyth on activated charcoal (or other sorbents). Only accord­ ing to Koryushkina is the content of carboxyls in FA 225-492 meq/100 g; according to others it lies in the range of 500-800 meq/100 g and may even reach 1120 meq (Table 39). According to most data, the content of phenolic hydroxyls is also high-up to 700-800 meq/100 g. A comparison of OHphenol and eOOH reveals a definite tendency: in many cases the increase in the number of carboxyl groups is accompanied by a decrease in the OHphenol groups. While investigating FA of soils from the Lower Volga, Okolelova showed that in more arid climatic conditions there is an enrichment of FA Table 39. Functional groups of fulvic acids (meq/100 g) Region, soil .

COOH

OH-phenolic

Source

(1 )

(2)

(3)

(4)

Russia Sod-podzolic A 1

288

763

A2

398

765

B

461

689

235

598

Chernozem, typical A B1

327

653

B2

225

853

640

630

A1

620

490

B1

590

460

B2

670

570

Chernozem, common, unirrigated, Aplough

550

450

A1

590

500

B1

600

520

B2

670

520

Chernozem, common irrigated, Aplough

Koryushkina, 1976

Okolelova, 1985

Dark chestnut, unirrigated

A1

710

710

B1

670

800

B2

680

700

630

610

B1

750

420

B2

880

610

790

660

Dark chestnut, irrigated, A 1

Light chestnut, unirrigated, A + 91 B2

620

530

BC

510

530

(Contd.)

122 Table 39. Contd. (1 ) Light chestnut, irrigated, A + B1

(2)

(3)

690

480

B2

760

490

BC

810

590

(4)

Dominican Republic: Hydrandept

735

82

Griffith and

Hydrandept

724

127

Schnitzer, 1975

Dystrandept

780

248

Dystrandept

796

34

1120

34

864

117 Kallianou and

Greece, Alfisol:

Yassoglou, 1985 under oak AO

820

410

A1

760

370

under heath AO

710

480

under grass A 1

630

450

under grass A 1

440

680

Xerochrept, shallow

820

410

Kallianou, Yassoglou

Xerochrept, shallow

658

422

and Ziechmann,

Chromoxerert, typical

710

410

1987

Fluvaquent, mull

520

740

Xerofluvent, typical

540

680

in carbonyl groups whereas FA from upper horizons of irrigated soils lose eOOH groups to some extent. A review of the available data reveals that the above-mentioned func­ tional groups (carboxyl, phenol, alcohol, quinoid, keto and methoxyl) are bound to 75-100% of the oxygen of the HA and FA molecules. Schnitzer and Gupta (1964) found that only the eOOH, OH and eo groups account for 52-74% of the total oxygen of HA and 86-100% of the oxygen of FA. Similar results have also been reported by Hansen (1966). The data on the distribution of oxygen pOints to an important fact: total absence in FA and almost total absence in HA of any other type of oxygen bonds. This rules out any significant content of simple and complex es­ ters (absorption bands of ester groups are not observed in IR spectra) and oxygen-containing heterocyclics in the structure of HA and FA molecules. The overall content of oxygen-containing functional groups in soils of different latitudinal zones according to Schnitzer is given in Table 40.

200 60

380 200

880 220

COOH Acidic OH Weakly acidic and alcoholic OH Quinoid C-O Keto C-O OCH 3

Keto C-O OCH 3

320 240 490 230 170 40

COOH Acidic OH Weakly acidic and alcoholic OH Quinoid C-O

Functional groups

Arctic

30-40

610-850 280-570 340-460 170-310

40

30

390-450 210-250 240-320 450-560

150-570 320-570 270-350 10-180

neutral soils

acid soils

Cold, temperate

Zones

80-90

690-950 120-260

520-960 120-270

Fulvic acids

30-50

420-520. 210-250 290 80-150

Humic acids

Subtropical

90-120

30-25.0 260-520 30-150 160-270

720-1120

60-80

380-450 220-300 20-160 30-140

Tropical

30-120

260-950 120-420

520-1120 30-570

30-80

150-570 210-570 20-490 10-560

Overall range

80

820 300 610 270

60

360 390 260 290

Mean

Table 40. Content of oxygen-containing functional groups in humic substances of soils from various climatic zones (meq/100 g) [after Schnitzer, 1977]

->.

VJ

I\)

124 The data presented in Table 40 can hardly be used for calculations, quantitative estimates and strictly genetic conclusions since it includes very diverse material and is only in the nature of a review. For instance, for the available range of variations in HA, the content of weakly acidic and alcoholic groups from 20 to 490 meq/1.o0 g with a mean of 260 meq/100 g can hardly be considered as significant. The data presented confirms the monotonous nature of most functional groups in humic substances of diverse origin. The average values of the content of individual groups according to several results seem fairly close although deviations from the average are rather high. The deviations can only be explained by peculiarities of genesis or incidental analytical errors. Variation in the quantity of titratable acidic groups are most probably due to the polyelectrolyte nature of humic substances, which hinders detection of the end-point of titration. Of course, if we consider identical titration condi­ tions, different methods of finding the end-point usually give closer results. However, the large range of functional groups and their sequential involve­ ment in the reaction with an increase in pH leads to subjective interpretation of titration curves. Of great importance is the increase in number of groups dissociating in alkaline medium, which is due to dissolution of humic acids. The reaction proceeds by sequential involvement of various (including phe­ nolic, some alcoholic and other) groups during titration. It is understandable that when the absorption capacity of HA in the solid state is determined by calcium only the carboxyls react, while in the case of copper both carboxyls and a part of the hydroxyls do, which introduces significant deviations in the results. It has been repeatedly mentioned that it is not always possible to distin­ guish individual groups from the titration curves because for this distinction their dissociation constants must differ by 3-4 orders. Moreover, in view of the polyelectrolytic nature of humic acids the dissociation constants of in­ dividual groups depend on the charge of particles (degree of dissociation). All this smoothens the titration curves-a fact noted by Gamble (1970) for fulvic acids. According to his results, it is possible to distinguish titratable groups of the first type with dissociation constants of 2.5-4.7 x 10-3 and of the second type with values in the range 0.2-3.2 x 10-5 . From the ratio of constants, he considered it possible that one of the carboxyl groups is present in the ortho-position relative to the phenolic hydroxyl. Glebko (1971) examined the polyelectrolytic nature of HA for which a change in the apparent dissociation constant is quite possible with increasing substitution of protons in individual groups. It must be emphasised that such properties are inherent in the entire soil adsorption complex: the most typical ion-exchange isotherms of soil reflect the change of strength of cation bind­ ing with increasing substitution of the adsorption centres. Since the polyfunc­ tionality of humic substances and the dependence of dissociation constants

125 on the particle charge do not always facilitate separation and identification of individual groups, a more rational characteristic could be the separation of all titratable groups according to their apparent dissociation constants, as was done by Glebko in 1971 (Table 41). Such a scheme of division does not give information about the structural role of functional groups but objectively characterises the reactive capacity of humic substances. A somewhat similar evaluation was later done by Arai and Kumada (1977) who, however, separated only three ranges. The first combines very weak acidic groups of the type of phenolic hydroxyls and a small part of carboxyls reacting at pH 9.4-10.0. The second includes weakly acidic car­ boxyls of aliphatic and aromatic monomers at pH 5.0-9.4, and finally the strongly acidic components with pK close to that of phthalic and salicylic acids (pH 3.8-5.0). The average dissociation constant of humic substances usually lies in the range of 10-4 to 10-6 . For peat humic acids K1 = 3-4 x 10- 4 ; K2 = 2.5-5.6 x 10-6 ; K3 = 3.1 X 10-9 have been reported [Dragunov, 1962). Other reports [Shul'man, 1969) give pK = 3.28 - 5.42. For humic acids from solonetses, solods and sod soils pK = 5.7-6.4 [Khan, 1969]. For dr·fferent fulvic acid fractions the value of pKa was 4-6 to 9.1-9.2, which also confirms its polyfunctionality [Davis and Mott, 1981a; 1981b). The second ionisation constant for HA calculated from spectroscopic data was 3.1 x 10-9 [Goldberg et aL, 1987]. Very close values of ionisation con­ stants for HA and FA have been reported by Filcheva (1976). Samples from Bulgarian soils gave Ka values of 1.68-3.86x10-5 (pKa = 4.41-4.78) and 0.78 x 10-5 (pKa = 4.10). Water-soluble humic substances were shown to have pKa = 4.80 (from -eOOH) and pKa =8.85 (for -OH) [Bizri et al., 1984]. From photometric data pKa values for fulvic acids were found to be close to 5.5 [Orlov and Pivovarova, 1971]. The ability of functional groups to react with cations and the change in range and number of functional 9r9ups during humification are of great Table 41. Content of acidic functional groups with different pK range in humic acids [Glebko, 1971] Content of aCidic groups (meq/g) Source of HA

pK:::; 3.0

pK :::; 4.77

pK:::; 10

pK:::; 14

Soil

1.86

3.37

5.27

6.00

Peat

1.50

3.28

5.14

5.78

Brown coal

1.12

2.92

4.21

5.11

Brown coal

1.18

2.95

5.71

7.42

Weather!'ld hard coal

1.79

4.02

5.97

7.23

126 importance. Apart from investigations of the optical properties, elemental composition and molecular weights, these relationships also help reveal mechanisms of humification and some structural features of humic acids. Varying opinions have been expressed on this topic. Kumada (1956) wrote about the increase as well as decrease in the exchange capacity of HA with an increase in degree of humification. According to Scheffer and Ulrich (1960) and Dubach and Mehta (1963), the relative content of acidic groups decreased during humification. Kawaguchi and Kyuma (1959) re­ ported a rise in the adsorption capacity during the early stages of humifica­ tion. Later, however (1964), Kyuma and Kawaguchi came to the conclusion that the adsorption capacity of humic acids increased only in the initial stage of humification and gradually fell thereafter. Experiments in which ethanol was used for fractionation of preparations formed the basis for this conclu­ sion. Fractions of humic substances showed a direct correlation between the adsorption capacity and the coefficient of chromaticity whereas in the zonal soil series this correlation was inverse (the coefficient of chromaticity was taken as the measure of the degree of humification). The arbitrary nature of these estimates an,d their non-specificity are clearly due to the relativity of the coefficient of chromaticity taken as a measure of degree of humification. Given the considerable variation in the content of functional groups reported by various authors, we cannot yet express a confident preference for any of the viewpoints presented above. Tsutsuki and Kuwatsuka (1978) also conducted extensive studies into this matter. They examined in detail the functional groups of humic acids in 38 soils of Japan. The results of their study enabled them to conclude that during humification (judged from the coefficient of extinction of sodium humates) the content of carboxyl and carbonyl groups generally increased whereas the number of alcoholic and phenolic hydroxyls as well as methoxyl groups decreased. This conclusion conforms to Aleksandrova's idea of hu­ mification as a process of oxidative acid formation. INTERACTION OF HUMIC SUBSTANCES WITH THE MINERAL COMPONENTS OF SOILS

The abundance of functional groups leads to the active interaction of humic substances with a diverse range of mineral components-cations, oxides, hydroxides, silicates and aluminosilicates-which gives a wide variety of mineral-organic substances. Each interaction is governed by the laws of thermodynamics but the diversity of forms of interaction and concomitant reactions lead to a situation wherein the sum total of soil chemical reac­ tions cannot always be described by simple classical laws. Poor adsorption

127 interaction of organic and mineral components, chemisorption, fixing of or­ ganic substances in the interlayer space of clay minerals, formation of salts and complex compounds and mutual coagulation and coprecipitation are commonly observed phenomena in soils. Hence the role of organo-mineral compounds in soil formation is multifaceted and the chemical functions char­ acteristic of these compounds are quite diverse. A complete analysis of the role of all the processes and compounds formed is not possible without their rational classification and identification of the type of chemical reactions. The chemical reactions between organic substances of soils and metal cations have been almost fully investigated. The interaction between hu­ mic substances and organic compounds (herbicides, insecticides, various detergents and so on), let alone clay minerals, has been studied far less. The capacity of humic substances to' form salts or salt-like products was noted even by early investigators of humus. Berzelius [Science of Hu­ mus, 1940, pp. 114-122] separated crenic acid from the copper crenate formed and studied its derivatives in detail. These results have not lost their importance even today (Table 42). According to his data, apocrenates are similar to crenates but are blackish-dark brown and less soluble. A summary of properties of different humates was also given by Schlubler [Science of Humus, 1940, pp. 81-85]. Table 42. Properties of some crenates [after Berzeliusl Crenate

Colour

Potassium

Yellow

and sodium

Solubility

Other properties

Insoluble in absolute alcohol, slightly soluble in dilute alcohol

Ammonium

Forms dark brown mass on evaporation

Barium Calcium

Pale

Difficultly

yellow

soluble

Pale

Soluble in

Forms basic salt

yellow

water

in alkaline medium

Soluble in

Acidic salt sparingly

Magnesium

water

soluble in absolute alcohol

Alumina

Yellow

Insoluble

Soluble in excess acid

Lower oxide

Pale

Soluble in e,xcess

of manganese

yellow

acid (Contd.)

128 Table 42. Contd. Crenate Ferric oxide

Ferrous oxide Lead Cupric oxide Mercurous oxide Mercuric Silver

. Colour Dry: dirty white; wet: reddish-grey Pale yellow Light grey yellowish Light grey greenish

Solubility

Other properties

Difficultly soluble

Completely soluble in ammonia

Soluble in water Soluble in water Soluble in water

Soluble in CHa COOH and in excess crenic acid

Yellow Does not precipitate Dark purple

Soluble in nitric acid and ammonia

The interest in interaction of humic substances with metal cations owes its origin primarily to the solubility of the resultant components, on which depends,the redistribution of several mineral components in the soil profile and in a landscape. These mineral compoundsare otherwise immobile in the oxidised conditions and near-neutral soil reaction predominant in most soils. Humates of alkali metals are readily soluble although forms of com­ pounds and their quantitative characteristics of solubility have not been ad­ equately studied. Neither has proper attention been given to the solution phase. Tyurin (1937) pointed out that when humic acids are dissolved in al­ kaline solutions, true alkali humates form after the initial stage of peptisation, in the form of mono-, di-, tri- and tetra-substituted salts: Rhum NaH 3 , RhumNa2H2' Rhum Na3 H and Rhum Na4 . Antipov-Karataev also wrote about the formation of molecular solutions of sodium humates. Menkovskii and Petrovskaya (1957) examined the in­ teraction of humic acids with NaOH solution mostly as peptisation, attaching special significance to free humic acid or its salts in stabilisation of micelle of R (COOH)x' m RCOO-· (m - y) H+· Y H+. Dragunova (1954, 1957) pOinted to the complex nature of solubility of humic acids. According to her, HA molecules are bound to each other in the solid state by hydrogen bonds and adsorption forces. They then dis­ solve better in aqueous solutions of surface-active substances, which was demonstrated on examples of ethylene glycol, glycerol, acetamide, urea and

129 others. In the presence of urea, the complex formation can be schematically represented as NH2

NH2

I

HA + C= 0 + H20

I

NH2

I

--->

HA . C=O . H20.

I

NH2

Insoluble precipitates are formed when iron, aluminium, and divalents combine with humic acid. In specific pH -ranges fulvic acids, too, form in­ soluble compounds. According to Waksman, at least part of the organic substances of the acidic filtrate (designated by him as ,B-humus as distinct from humiC acid or a-humus) precipitate in the presence of iron and alu­ minium at pH 5---6. Analogous properties were reported by Tyurin and later used by him for separating fulvic acids [Ponomareva, 1964]. The conditions of formation of humates, their solubility and proportion of metals in their composition were discussed and experimentally investigated during the 1920s-1960s by many workers, including Tyurin, Harada, Kosaka and Izeki, Kukhatenko, Posner, Natkina, Gillam, Broadbent, Bradford, La­ rina and Kasatochkin. Many special studies were conducted on complex formation. There are reports regarding the formation of chelate complexes. However, there is not one comprehensive generalisation and critical anal­ ysis of the published results. The published data shows wide variation of parameters and very weak correlation with conditions of humus formation. Among the original and unique views, mention must be made of those of Troitskii, who envisaged the role of the resultant cation-humus complexes primarily in the activation, initiation or inhibition of different processes. 'Hu­ mic substances at some stage of soil formation process act as inhibitors' [Troitskii, 1949, p. 10], thereby protecting the mineral component from disin­ tegration. The mineral part may also play this protective function in relation to the organic part. According to Troitskii, the reaction of silicates with hu­ mic substances may proceed by formation of simple (or chelate) salts or by interaction between hydroxyl groups of silicates and amide groups of humic substances. In the latter case, decomposition of the organic part is inhibited since the amide and imide groups most susceptible to micro-organisms are protected. Modes of formation of complexes of metals with humic substances have been discussed by many authors. However, it must be noted that complexes in the strict chemical sense were not studied in every instance. The series of complex compounds were often considered mineral compounds of humic substances separated from soil; often the mechanical fractions of elements were termed organo-mineral complexes (silty, clayey, sandy complexes). Many investigators used (and continue to use) the terms 'complexes' and

130 'complex' in the sense of 'complicated' and 'constituent' without reference to complex compounds in their chemical sense. This has given rise to double meanings and terminological confusion. It is nigh unto impossible to refrain from using the term 'complex' in the broad sense of the word. However, it is possible to decisively avoid it whenever its use could result in ambiguous interpretation of experimental results. An example of the broadest interpre­ tation of complexes is the excellent review by Mortensen (1963), wherein he wrote: 'Soil organic matter forms complexes with metals by ion-exchange, surface adsorption, chelation and complex coagulation and peptisation reac­ tions' (p. 179). For such complexes the term 'simplex', first used by Troitskii (1949), is suggested. A stricter approach was taken by Schnitzer who used the terminology and classification of Chabereck and Martell. Schnitzer termed complexes as 'stable organo-metallic compounds in which the bonding between the metal ion and ligand can be either primarily electrostatic or mainly covalent, or intermediate between the two' [Schnitzer and Skinner, 1963; p. 87]. Most published reports suffer in general from methodological deficiencies associ­ ated with the peculiar properties of humic substances. Mention may first be made of the methods used in obtaining the compounds. Many workers ex­ tract organic substanceswith an 0.1 Nor 0.5 N NaOH solution and separate the humic acids by precipitation or adsorption; the remainder is dried and this compound considered fulvic acid. Obviously, in such a compound besides true fulvic acid the presence of other members of humic substances and even non-specific compounds is unavoidable. That such are present has been confirmed by experiments using organic solvents wherein paraffins, hydroxybenzoic acids and other compounds were extracted. The drawback is the use of the mean numerical or mean weighted molecular mass in calculations, which does not· allow us to highlight the actual role of humic substances in mobility, solubility or accumulation of inorganic substances. Other complications also arise, for instance the need to take into account not only the oxygen-containing groups, but even nitrogen fractions in the interaction of these fractions.

Methods for Study of Organo-mineral Interactions The assessment of experimental results becomes complicated not only be­ cause methods of obtaining the compounds have inherent drawbacks but also because the mechanism of reactions under experimental conditions is not always clear. For this reason, it would be appropriate to examine the important methods used in experiments on complex formation of humic substances.

131 Potentiometric titration (pH effect). Many authors have observed a some­ what significant pH effect during titration of humic compounds in the pres­ ence of metal ions. As a rule, the pH-effect is more prominently noticeable in alkaline medium, which is explained by the involvement of phenolic hydrox­ yls in the reaction. According to the magnitude of the effect, it is possible to arrange cations in a definite series, which entirely agrees withthe decreas­ ing order of solubility products of the corresponding metal hydroxides. This leads us to the assumption that hydroxides may form during titration, and their pH effect may be partial or total. The most illustrative is the following representation of the pH effect and the solubility product:

pH effect: Ca « Mn < Co < Ni < Zn < Cu < AI < Fe, Solubility products of hydroxides: 3 x 10- 5 > 4 X 10- 14 > 2 X 10- 16 >

> 9 X 10- 19 < 1.5 X 10- 17 > 6 > 2 X 10- 33 > 4 X 10- 38 .

X

10- 20 >

In this sequence only the relative position of Ni and Zn somewhat disturbs the general pattern. Khan (1969) considered the possibility of the formation of hydroxides during titration and attempted to distinguish hydroxide for­ mation from complex formation. At high metal concentrations the additional inflections in the titration curves were correlated by him with the formation of hydroxides. The effect of titration of simple salts was commensurate with the pH effect, which made interpretation of the results difficult. Using the data reported by Khan (1969), I constructed generalised titra­ tion curves for pure humic acids and a pure salt solution, assuming that titration of the mixture should consume the same amount of alkali as in sep­ arate titrations of pure components. These curves are shown by broken lines in Fig. 15. They exhibit some interesting features: In every instance the cal­ culated generalised titration curves intersect the titration curves of mixtures. The point of intersection in the case of Mn2+ corresponds to about pH 9.5, for Zn2+ about pH 8.0 and for Fe3+, pH 3.7. The concentration of metal ions in these experiments was 4 x 10-4 g.ion/l. At such concentrations all metals formed hydroxides at different pH values: Mn2+ at pH 9.0, Zn 2 + at pH 8.0 and Fe3 + at pH 3.7. Thus the points of intersection of the calculated and actual titration curves exactly correspond to the pH of hydroxide formation. Considering this feature, the pH effect could be interpreted as follows. In the acidic range, when metals exist as free ions, they react with humic acid, releasing some amount of hydrogen ions and forming compounds, including those of the type of simple salts. When an additional amount of free protons appears in the solution, the titration curve of pure salts shows lower values of pH compared to the pure HA. In this region the magnitude of the pH effect of the mixture is not high. But as a result, a considerable portion of the HA protons reacting usually at

132

Fig. 15. pH effect in titration of humic acid in the presence of metal ions. 1-humic acid; 2-humic acid + cation; 3-generalised curve constructed from results of separate titrations of humic acid and pure salt.

higher pH values, appear to have already been titrated. As the pH reaches that of hydroxide formation, simple salts change partially into basic salts: Hum (COOH)n (COONa)m_x (COOMeOH)x' Since part of the HA protons become titrated earlier and the metals are not titrated completely (up to hydroxides), the consumption of alkali for titration should be lower during titration of a mixture than the sum for pure components. This is very clearly seen in Fig. 15. In the highly alka­ line medium humates could disintegrate completely with the formation of hydroxides. In this pH range the titration curves of mixtures and calculated

133 additive curves come closer and then must overlap. Thus the pH effect is entirely satisfactorily explained by the formation initially of simple and then of basic salts and can be considered only as a conditional index of complex formation. Electrophoresis. This method, like others, is fairly dependable in the case of negative results but positive proofs in favour of complex formation are not always adequate. The entire mixture of substances remaining at the start, if it simultaneously contains organic and mineral components, is termed complex. Thus in experiments with copper with the conventionally used slightly alkaline solvent, hydroxide must form. Since the solubility prod­ uct of Cu(OHh is 5.6 x 10- 2 in a slightly alkaline medium copper must be present in the residue and exhibit low mobility. This could be taken as the index of complex formation. In an acidic medium all copper is present in solution and is readily removed from th~ system by electrophoresis. So not one of the experiments is a convincing proof. Ion-exchange equilibrium. The method of determination of stability con­ stant was first used, according to Schubert, in studies of soil organic mat­ ter by Miller and Ohlrogge (1958), Randhawa and Broadbent (1965) and Schnitzer and Skinner (1966). To calculate the stability constant Schnitzer and Skinner used the equa­ tion:

°,

log

(~

-

1) =

log K

+ n log

(FA],

where Ao is the distribution constant of the metal ion between ion exchanger and the solution in the absence of FA; A the same in the presence Of FA; K -stability constant; n-number of moles of FA reacting with one mole of the metal and [FA] the molar concentration of fulvic acid. For [FA] = 1, log

(~

-

1)

= log K, which makes it possible to readily read from the

graph the values of K and n. The method is simple to use and hence widely accepted. This equation contains two unknowns-n and K -and to solve it we must have a set of at least two equations. From two experiments with dif­ ferent [FA] it is possible to ascertain the value of

n. Denoting log

(~

-

1)

by a, we get a1 - a2 = n(log [FA]1 - log [FAh), .6.a

= n log([FA11

: [FAh) and n

[FA11

= .6.a : log [FAh·

According to the last equation, the value of n or the co-ordination number does not depend on the mode of expression of concentration and can be

134 reliably determined. However, the stability constants are directly related to the molecular mass: log K = a - n log [FA] and for a 10-fold error in the determination of the molecular mass thet value of the constant changes n-fold. The reliability of determination of molecular mass, as shall be shown in the next chapters, is not very high. When working with unfractionated compounds, interpretation of the values of the constant is all the more difficult. The method of interpretation with ionites (ion-exchange resins) is not faultless even for reasons of possible sorption of humic substances by ion­ ites, which was particularly observed by Sapek (1970). Spectrophotometry. Because of its simplicity this method has been used extensively. Incorporation of the metal ion into the molecule of an organic compound may cause change in the electron density accompanied by a bathochromatic shift. Thereby the nature of electron spectra changes and under most favourable conditions absorption bands specific for the com­ plex appear. If one of the initial components and the resultant complex are coloured, then by difference one can find out the increase in optical densi­ ties due to the reaction of complex formation. Cases of change of optical density ofHA and FA solutions with the introduction of metal ions have been described in the literature. But these effects have not been persistently con­ firmed. They are often associated with the initiation of coagulation of HA and (or) the formation of metal hydroxides. The reality of this phenomenon has been confirmed by direct turbidity measurements. It has been shown [Orlov, 1960] that even in pure humate solutions the error of measurement of optical density due to turbidity can be as much as 0.1 unit. In the initial stages of coagulation (without visible sedimentation) the turbidity of humate solutions increases sharply [Orlov and Gorshkova, 1965]. A sharp rise in turbidity with the introduction of metal ions into a solution of humic substances was clearly demonstrated by Sapek (1969). Often the changes in IR spectra are cited as proof in favour of chelate formation [De Mumbrum and Jackson, 1956; Stanchev, 1971]. However, according to manyauthors [Mortensen, 1963; Schnitzer, Shearer and Wright, 1959; Stanchev, 1971], metals only affect the carboxyl band, explained by the formation of salts (carboxylate ion). Thompson and Chesters (1970) also came to the conclusion that from a change in the bands at 1700 and 1200 cm- 1 the formation of chelates cannot be considered proved. Several other workers have reported a similar conclusion. Other methods. Karpukhin (1970) used an elegant method to determine the instability constant of iron fulvates. He used the concurrent complex formation with subsequent separation of fulvates and standard complexing agents by gel-chromatography. According to Karpukhin, the instability con­ stants of iron compounds with most fulvic acid fractions are in the range of

135 10- 16

10- 18 . This indicates very high stability of the compounds. One of· the fractions was even marked by a constant of 1.9 x. 10- 27 . This fraction -

seemed rather unusual even in other indices. The amount of iron bound with it reached 135 moles per mole of fulvic acid with mean weighted molecular mass of 11,250. Such a quantity of iron means that each of its atoms ac­ counts for FA molecular fragments with a mass equal to only 84. We shall return to this point later. Another interesting method was used by Kleist. He conducted electrolytic concentration of metals from an 8 M solution of urea in the presence of humic acids. Using emission spectral analysis he later showed that the ions of aluminium, iron, cobalt and lead in the presence of humic acids concentrated at the anode. These conclusions were confirmed by radiochromatography and electrophoresis [Kleist, 1963, 1964; Mucke and Kleist, 1965; Klocking, Kleist and Mucke, 1967]. Broadbent and associates [Broadbent, 1957; Lewis and Broadbent, 1961 a, 1961 b; Randhawa and Broadbent, 1965] proposed a unique method for studying organo-mineral interactions. They used organic matter as a solid phase in a chromatographic column and then studied the elutriation curves of the different cations. Several peaks were observed on the yield curve, the first of which was attributed to ·cations binding with carboxyl groups. Moreover, in the authors' opinion, phenol groups, possibly orthoquinones, were involved in the reactions. Compared with Ba2+ and Ca2+, copper ions react more actively and variedly, binding particularly as CuOH+. A purely chemical method was used to assess the forms of calcium bind­ ing. Organic matter of water extracts from solonetsic soils was precipitated by adding butyl alcohol. Since almost all of the calcium was precipitated with the organic matter, the authors considered it bound in the complex [Nightin­ gale and Smith, 1967]. The co-ordination bond of strontium with HA was also judged from the stability of the resultant compound against the action of 0.1 N HCI [Juo and Barber, 1969]. These methods illustrate the diversity of techniques used but do not exhaust the entire arsenal of methods. It is important to pay attention to the search for non-destructive methods allowing quantitative estimation of the cation concentration in an equilibrium solution and its content in the humate complex without destroying the latt~r. Gel-filtration is such a method. Khan (1970) used this method to distin­ guish the ionic forms of metals from those bound with organic matter. The yield curves of several cations, according to Khan, matched the yield of the low-molecular fraction of organic substances. This enabled him to suggest the presence of non-ionic forms of binding with cations for these fractions. The same method was used by Holty and Heilman (1971), who were able to demonstrate that humic acids from the B2 horizon of podzol are bound to iron: on gel-chromatograms the yield of the latter practically coincided with

136 the yields of high molecular fractions of HA. The simultaneously present cal­ cium ion, according to these results, was used up in the formation of simple salts (probably inorganic). This method was used to detect organo-mineral compounds in alkali extracts from soils and sediments [Orlov and Motok, 1974]. A considerable part of the iron and aluminium during fractionation on Sephadex G-7S and G-100 was found in the same portions of the eluate as the mean weighted molecular-mass humic substances. This confirmed the presence of stable organo-mineral compounds. However, a part of the iron and aluminium was found in the outer volume of the gel, which indicates their colloidal nature (hydroxide micelle or highly dispersed ferrialuminosilicates). Some quantity of humic substances was also found here. This fact demands greater attention. It may be assumed that the high molecular-mass fraction contains not the usual heteropolar or complex iron­ humate (or ferro-, alumino etc. fulvate) compounds but particles closer in their nature to adsorption complexes. Possibly, polymer (colloidal) particles of hydroxides of iron (aluminium) form their base. Molecules (anions) of humic substances are bound with them chemically or by adsorption. The bond may be electrovalent and/or of the donor-acceptor type. Interaction due to the hydrogen bond is also not ruled out. Organic constituents could coat or wrap the mineral particles, or the latter might act as bridges. Such a scheme makes it possible to explain the very high content of iron in the high-molecular fractions of iron fulvates observed by Karpukhin. It is felt that this scheme could explain even the disintegration of iron­ humus particles under the action of ethylenediaminetetraacetic acid (EDTA). This phenomenon has been described by Orlov and colleagues (1988). In their experiment gel-chromatograms of alkali extract from the A 1 horizon of sod-podzolic soils were obtained. Column with Sephadex G-SO was used; 0.05 M tris-(hydroxy methyl) aminomethane + 1% sodium dodecylsulphate, pH 9, was used as the eluent. The second experiment was conducted with water-soluble humic substances from the Aplough horizon of sod-podzolic soil. One or two maxima were found on the gel-chromatograms of each extract. Then EDTA was added to the extracts and chromatography done once again. In the presence of EDTA the height of the maximum for the high-molecular-mass fraction dropped to zero. One or several maxima of low-molecular-mass fractions appeared in its place. This indicated the disin­ tegration of large molecules. A possible reason could be the binding of iron with EDTA by complexation. This iron may have earlier served as a bridge between the molecules of HA or FA. The reaction of EDTA with the humic substances may be given as:

137

R1(

COO

OH

I

ooe

\Fe( \/R2+ [EDTA]4 - -­ 'COOH/ "HOOC _

-+ R1(

COO-

"COOH

+ R2 (

COO­

"COOH

+ [FeEDTA] ­

Naturally the question arises: Why does the iron-humate complex not decompose in an alkaline medium with the formation of Fe(OHh, which has a solubility product of only ~ 1O-37 ? The probable reasons could be formation of bridges by polynuclear complexes of iron of the type:

H20 H 20

H20, I ~O, I /H 20

Fe Fe H 20/ I / I "'-H 20 H 20 H 20

or [Fe OHhln.

Then the iron-humate complex could have the structure R1 - FexOy . Fen[Fe(OH)m] - R2 · Such a complex seems relatively stable in an alkaline medium but could be destroyed by the action of EDTA. In this connection it must be remem­ bered that the EPR of humic acid samples s~owed lines, presumably due to microcrystalline goethite [Babanin et aI., 1977]. This also confirms the probability of the formation of iron-humate complexes with the participation of polynuclear forms of iron. Composition and Properties of Compounds of Humic Substances with Metals Several attempts have been made to describe the composition of organo­ mineral compounds extracted directly from'soil. By and large these attempts have not been very successful because during extraction either a loss of the mineral components occurred or, conversely, an unusually high quantity of impurities was extracted. Kaolinite, hydrous mica and quartz were found in

138 the humic acids and their ash [Titova, 1969; Orlov, 1972]. The presence of quartz clearly indicates the considerable proportion of incidental impurities. Hence model experiments are more effective for studying organo-mineral compounds. According to our data [Orlov and Eroshicheva, 1967], when humic acid soil from chernozem was coagulated by the addition of metal chlorides, the following quantities (meq/100 g humic acid) of cations were bound: A1 3+

334

Fe 3+

1950

Cu2+

239

Ni 2+

86

Z2+

80

C0 2+

177

In the reaction with manganese chloride no change of concentration of Mn2+ in the solution after coagulation of HA was observed. The series in which the metals could be arranged according to the degree of their binding with humic acid conforms to the solubility product series of the hydroxides. The lower the solubility product, the more the metal binds with HA and pre­ cipitates. The data above as well as the material presented earlier confirm that the phenomena of adsorption and coagulation play a great (if not major) role in the formation of insoluble metal humus precipitates. This is confirmed by the calculations of 'equivalent weights' which, particularly in reactions with Fe3 +, are rather low. A very high iron and aluminium content (up to 800 meq/100 g) has been reported by Aleksandrova, Dorfman and Yurlova (1970); still higher values are cited by Karpukhin (1970). According to Titova (1962), the mass ratio of C : Fe in iron-humate compounds reaches 4: 1 and in iron-fulvate 2: 1, which corresponds to atomic ratios of 19: 1 and 9: 1, that is, one atom of Fe accounts for 19 or 9 atoms of carbon. At the same time, humates of Mg, Ca, Mn and Cu (according to Sapek, 1971) contain 300-330 meq metall100 g HA, humate of Fe(llI) 420 and AI 562 meq/100 g. The corresponding indices for fulvates are twice as high. Differences in es­ timates are explained by the methods used for obtaining the compounds. A higher metal content is always found in the products obtained through co­ agulation. The composition of organo-mineral compounds is determined by the amount of functional groups and pH value. At higher pH a larger number of acidic groups are involved in the reaction and the reaction products are more and more hydroxy complexes or basic salts. Therefore the proportion of metal in the composition of the compound before hydroxide formation has to have been higher.

139 According to Aleksandrova, iron- and aluminium-humic compounds differ substantially in properties from free humic acids. In particular, the adsorp­ tion capacity measured by calcium drops sharply; calcium does not remove iron from the products and only partially removes aluminium [Aleksandrova, 1954, 1967; Dorfman, 1969]. Decrease of the adsorption capacity is also observed in mineral-humic formations. However, a considerable increase in adsorption capacity has been reported for Cs 137 in compounds of humic acids with iron and aluminium [Sapek and Sapek, 1970]. According to Sapek (1970b, 1971), the compounds of humic substances with cations are unstable and have two regions of maximum solubility: in the acidic and alkaline ranges (Table 43). Table 43. Range 01 coagulation and solubility 01 humic compounds Irom Bh horizon [Sapek, 1971) Saturating

Coagulation,

Solubility

Coagulation

Solubility

cation

pH below

pH

pH

pH, above

Mg

1.40

> 1.40

Ca

1.50

1.55-3.75

4.10-11.0

11.0

Mn

1.40

1.45-4.30

4.80-10.4

10.4

Cu

1.50

1.55-2.80

3.00-10.4

10.4

Fe(lIl)

1.70

1.85-2.90

3.10-6.90

8.40

AI

1.45

1.50-1.95

2.10-7.85

8.60

It is characteristic that the coagulation range accounts for the most crit­ ical areas of the titration curves of humic substances. Experiments to de­ termine the stability constants of humic acid compounds with cations have been contradictory. The res!Jlts often depend on the method of determi­ nation and the values selected for molecular mass. Even the relative val~ ues of the 'constant' for various cations could play a definite role in esti­ mating the effect of humic substances on the mobility of mineral compo­ nents. The stability constants of humates and fulvates usually increase with pH. Taking into account the effect of pH and according to several publications, it is pO'ssible to indicate approximate ranges of log K for compounds with different metals. Humates HA-Fe

2.4-9.8

HA-Cu

7.0-9.7

HA-Zn

2.9-10.8

HA-Cd

6.9

HA-Pb

8.7

140

Fulvates

1.5-2.1 6.5 2.0-2.9 1.5-3.8 5.1-6.1

FA-Mg FA-AI FA-Ca FA-Mn FA-Fe

2.2-3.7 3.5-4.1 3.2-8.7 1.7-9.3 3.1-6.1

FA-Co FA-Ni FA-Gu FA-Zn FA-Pb

The lowest of the above log K values are generally seen, for an acidic medium (pH 2-4) and the highest for an alkaline medium (pH 8-10). This is partly confirmed by Schnitzer's data (Table 44). Table 44. Parameters of compounds of fulvic acids with metal cations [after Schnitzer and Skinner, 1966, 1967; Schnitzer, 1970) pH 3.5

Cation

n

pH 5.0 10gK

n

10gK

Cu(lI)

1.50

5.78

2.00

8.69

Pb(lI)

0.75

3.09

1.50

6.13 5.77

Fe(lI)

1.25

5.06

1.30

Ni(lI)

1.00

3.47

1.00

4.14

Mn(lI)

0.55

1.47

1.10

3.78

Co(lI)

0.70

2.20

1.00

3.69

Zn(lI)

0.58

1.73

0.56

2.34

Ca(lI)

0.83

2.04

0.90

2.92

Mg(lI)

0.53

1.23

0.79

2.09

AI(III)

6.45

To evaluate the effect of pH on the stability constant the data reported by Takenaga and Azo [cited from Orlov, 1979] is of great interest. In their calculations of log K, these authors used 30,000 for the molecular mass of humic acid from volcanic ash soil. According to them (Table 45), humates of Mn2y, Ca2+ and Mg2+ decomposed completely in an acidic medium. This is understandable for pH 3.0 and does not contradict the known properties of acidic soils but such a result for pH 5.0 raises some doubts. It is believed that in sod-podzolic soils with a pH of about 5.0, some part of HA is represented by calcium humates. Another fact worth noting is the decrease in stability of some components with an increase in pH. Evidently hydroxide formation, particularly of Fe3 + and Fe2+, becomes the dominant reaction. Stevenson (1976) explained the change of composition and stability of complexes with an increase in pH as successive substitution of protons of the functional groups of HA/FA and formation of hydroxo/(aqua)/complexes:

141

HA

I

/'/

~~

COO+M2+

AC~ic ~'HA MedIum

eOOH

I

/'/ ,~,

,

COO

CO

/

'M(H 0) +H+ /

2

n

~

11

o Alkaline, , Medium

HA

COO

A/

I

""A

"

co

/

OH

)M(

+H+

(H 20)n_t

11

o Besides the, pH value, the experimentally determined 'constant' is af­ fected by the ionic strength I of the solution. Stevenson (1976) found the regression correlation for a series of humates to be HA-Cu 2 +

log K = 8.9 - 4.9(1) 1/2

HA-Pb2+

log K

HA-Cd2 +

log K = 6.9 - 5.4(1)1/2.

= 8.7 - 4.7(1)1/2

The above-noted arbitrary nature of the experimentally found 'constant' is due to the complexity of the structure of organo-mineral compounds and their statistical nature. For a polydispersive compound of HA and FA, 9al­ culation of an empirical mean constant is technically feasible, but such a 'constant' describes only the given specific preparation. Replicated separa­ tions of the preparation from the same soil may lead to changes in proportion of fractions with varying molecular masses and consequently de novo deter­ mination of the constant is required. However, even compounds fractionated by salting out or gel filtration contain a large cluster of molecules with varying molecular mass. In this connection Karpukhin and Fokin (1977) presented an excellent paper. They divided FA into five fractions with molecular mass from 170 to 12,300. The observed values of instability constants of FA-Fe 3+ complexes for these fractions were 4.2 x 10- 17 , 6.9 x 10-5 , 1.8 X 10- 17 , 2.5 X 10- 18 and 4.4 x 10-26 respectively, that is, the values of the constants differed by almost 20 orders of magnitude. And if for a series of fractions the observed values agree with the published data, the complexes of other fractions seem

142 exceptionally stable. Another difficulty is associated with the unknown struc­ . ture of the resultant compounds. Organo-mineral compounds in soils could be represented by monodentate or polydentate complexes and cations could occur in the outer sphere. Chelates and polynuclear complexes are possi­ ble. Generally the forms of the compound are quite varied. The important types of possible mechanisms of interaction of HA and FA with metals have been examined by Schnitzer (1986). Among them are the following:

o 1.

0

I

I

/'\-C-O+M2+

+H20~

,~

o

,/,

OH

V

')/"

"

Y

+M2+~

"

) C-OH

U

".0

I +H+

11

"./, /M 0

0

u

c-O-

,/

"-./

C-O

"­

+MH~ ) )M+HT

,,". C-O/

»

o

0

O-Na+

O-Na+

H

C=O

C=O ... H-OM

I

I

I

,/ 4.

+H+

C

o « :3.

I

0

C-O-

2.

11

/'\-C-O-M-OH

11

'/"

OH

+Mnt (H 0)x 2

= I

n+

,/

"./,

OH

The diversity of structures and variable molar ratios make it difficult and sometimes even impossible to unambiguously interpret the results' and to use the calculated 'constants' in thermodynamic calculations. The problem becomes more acute whenever OMC are formed, as mentioned earlier, on a

143 Table 45. Log K values of compounds of humic acids with metal cations [after Takenaga and Azo, 1976] Cation

pH

3.0

5.0

7.0

9.0

11.0

8.72

9.20

FeH

11.36

8.46

6.60

CuH

6.79

12.60

12.33

NiH

5.39

7.63

9.60

FeH

5.36

6.41

4.78

Cd 2+

5.26

5.45

8.90

Zn H Mn 2+

5.05

7.15

10.34

nil

nil

5.60

Ca2+

nil

nil

6.45

7.81

8.03

Mg2+

nil

nil

5.46

6.76

8.42

colloidal base. A combination of the foregoing difficulties leads to a situation in which even a simple series of cations according to their adsorbability by humic acids hardly ever coincides with that according to their stability con­ stants. This can be seen from a comparison of the following series compiled from the results of various authors [Orlov, 1979b]. 1) According to adsorption by humic acid [Ramani and Palmer]: 3 Fe + > AI3+ » Fe2+ > Cu 2+ » Zn2+ > C02+ > Pb2+ » Ca2+ » Mn2+. 2) According to adsorption by humus [Banerjee]: Pb2+ > Cu 2+ > Mn2+ > Zn 2+ > Cd2+.

3) According to stability constants of humates from alluvial soils [Ad­ hikari]: C02+ > Cu 2+ > Zn 2+ > Ni2+. 4) According to humate formation [Pauli]:

U02 » Cu > Zn > Ni > Pb > Cd. 5) According to stability constants of fulvates [Schnitzer]: Cu2+ > Pb2+ > Fe2+ > Ni2+ > Mn2+ > Co2+ > Zn2+ > > Ca2 > Mg2+ > AI3+.

+

Thus for prognostic evaluation it is not always possible to make use of such stability series. It becomes necessary to rely on the specific values determined for preparations from specific soils [Table 46]. The problem of studying humate and tulvate complexes remains unre­ solved to date and new methodological approaches are needed for their quantitative characterisation [Khalili, 1990].

144 Table 46. Stability constants of humates and fulvates of different metals (log K) Acid, compound

Metal

Humic acids from brown soils

Cu Zn

Humic acids from brown soils solod solod solonets solo nets chestnut soil

Cu Zn Cu Zn

chestnut soil

Cu Zn

Humic acids Humic acids

Cu Zn

HA from 29 different soils HA from 5 soils Fulvic acids Fulvic acids

Zn Zn Cu Cu

Humic acids, Bulgaria Humic acids

Zn Zn

pH

5 5 5 ·5 5 5 5 5 5 3.6 5.6 7.0 7.0 6.5 3.5 3.5 5.0 3.6 5.6 7.0

Humic acids from humic gley soil Cu 2 +

Humic acids from A 1 horizon Fulvic acids, Bh horizon

PbH CdH Fe Fe Cu

Fulvic acids, Bh horizon of podzol MnH

Water-soluble substances

PbA PbA2

5 5 4.0 4.8 6.15

10gK

8.4 6.1 9.1 4.5 9.0 9.5 8.1 9.7 7.0 4.4 6.2 6.8 4.2-10.8 3.13-5.13 3.23 5.78 8.69 2.92-3.32 4.42 6.18 6.80 8.9 8.7 6.9 . 9.76 6.07 3.1-3.3 3.7-3.9 2.7

6.45 3.3 4.2 3.7

Source Rosell et aI., 1977

Filcheva, 1976 Randhawa and Broadbent, 1965

Stevenson, 1976

Fares et aI., 1982 Cressey et aI., 1983 Gamble, Schnitzer, and Skinner, 1977 Bizri et aI., 1984

Interaction of Humic Substances with Soil Minerals The interaction products of humic substances with soil minerals constitute an important part of the soil mass. The interaction reveals one of the spe­

145 cific aspects of soil formation as a unique natural process. The properties of mineral-humic formations differ from the properties of the individual com­ ponents and determine the many important features of soils. Clay minerals adsorb a wide range of organic compounds, including simple acids, alco­ hols, aldehydes, ketones, amides, peptides, various physiologically active substances (pesticides, antibiotics) and so on [Inoue et al., 1990; Kallianou and Yassoglou, 1985; Nayak et al., 1990]. Two methods were used to study the mineral-humic formations: 1) sep­ aration of 'organo-mineral colloids' from soils and subsequent study of their properties; and 2) planning experiments according to interactions of indi­ vidual minerals or clay with organic compounds. Model experiments were conducted by Khan to study structure formation. According to his results, the mineral fractions of different sizes adsorb the following quantities of humic acids (C of humic acids, g/100 g of mineral):

< 0.25 mm

< 0.2 Mm

Askanite

1.20

1.19

Gumbrin

0.96

1.14

Kaolin

0.20

0.61

Orthoclase

0.25

0.60

Muscovite

0.44

0.65

In particular, Khan showed that adsorption of humic substances is facili­ tated by pulverisation of minerals and saturation with Fe3+ and A13+ ions. The process of adsorption is largely irreversible. According to many other results, the quantity of humic substances ad~orbed by minerals depends on their concentration and, in the first approximation, could be characterised by Langmuir's adsorption isotherm. Many schemes of mineral-humus compounds have been proposed. Most are a priori in nature and assumed· the participation of various functional groups and metal cations in the adsorption of humic substances. The pro­ posed forms of interaction are quite varied. Only for humic substances did Khan distinguish physical (due to molecularand surface forces) and physico­ chemical adsorption on the mineral surfaces and adsorption on the inner surfaces for montmorillonite group minerals. According to Khan, the interaction of humic substances with minerals is possible through binding of carboxyls of humic substances with OH-groups of minerals or through the formation of mineral bridges, for instance: .

146

o

". /SI-OH + (HOOC)" R- ,11 /Si-O-C-R (COOH)n-1 ) Si.;....O-ea+ + (HOOC)n R- )Si-O-Ca-OOC-R (COOH)n-1

ooc

)Si-O-Fe (OH)! + (HOOC)n 1\ -+- ) Si-O-Fe(OOC)R (COOH)n-2 A similar scheme for soils saturated withbases was examined by Aleshin and Zhupakhina (1950):

_Si-O-Ca-OOC, /coo-Ca-ooc, /

R -Si-O-Ca-OOCI "COO-Ca-ooc l " R

In these schemes, and in many others, the possibility of participation of the most active nitrogen-containing groups of humus acids is not reflected. This fact was emphasised by Troitskii. According to Tahoun and Mortland (1966), IR spectra show that amides combine with protons in an acidic medium to be modified into cationic form and are held on the surface of montmorillonite by electrovalent forces:

o

~

[HaO]+[Montmorilloniter+ CH 3C

"

­

NH2

OH+ ... OH2]+

- [

[CH.C~

NH2

r

[.........;'"....

147

OH+ ... OH2)+

[ CH'-C~(MontmoriIonioor:;!

NH2

When dehydrated in vacuum or by heating, the amide once again changes into the molecular form and the complex disintegrates

o

~

:t; CH3C

"­NHs

+ H~Montmorillonite+ H 0. 2

In connection with this scheme it is interesting 'to remember that some exper­ iments of Scharpenseel (1968,1970) directly affirmed the possibleexistence of amino acid bridges between HA and minerals. For montmorillonite saturated with metal cations, formation of co-ordi­ nation bonds between the exchangeable cation and oxygen of the amide group has been suggested. According to many authors, the most stable com­ pounds are obtained with the participation of metal cations in the mineral-HA binding. It has been experimentally shown that fresh sesquioxide gels on the surface of particles greatly increase the adsorption of humates [Alek­ sandrova, 1~54; Schulthess and Huang, 1991]. Among metal cations iron could play a great role. Greenland (1971) considered two basic binding mechanisms: 1) through polyvalent cations interacting with the surface of mica-like minerals, and 2) through direct interaction with hydroxides of aluminium and iron. The

148 binding through cations may be direct or through hydrate bridges. In the case of hydroxides it is also possible to distinguish non-specific adsorption, which according to Greenland, may be correctly termed ligand exchange. In the latter case anions are as if an integral part of the hydroxide surface layer. Penetration of humic substances in the interlayer space of clay minerals acquires great significance. While investigating this problem major atten­ tion has been paid to minerals of the montmorillonite group since they have an expanding lattice. This problem cannot be considered finally resolved. Opinions and experimental results are marked by extreme contradictions. A review of fundamental work [Orlov et al., 1973] revealed that some authors presented data showing an increment in the interlayer spacing of montmo­ rillonite from 10 A to 30 A upon the adsorption of HA and FA while other researchers observed no lattice expansion. In our experiments, too, we observed no penetration of HA and FA into the interlayer space. Experiments were conducted in two versions. In the first version we added ammonium humate solution of pH 7.0 to samples of Mg-montmorillonite. The excess of humate was removed after two weeks of interaction, the samples were dried and diffraction patterns taken. After X- . ray diffraction the samples were moistened with glycerol and fresh diffraction patterns were recorded. The humate adsorbed on montmorillonite did not obstruct the penetration of glycerol in the interlayer space nor per se affect the interlayer spacing. In the second experiment gumbrin and askanite were used. They were saturated with Ca2+ or Na+. Interaction of these minerals with the HA solu­ tion was allowed at pH 5.0 and with the FA solution at pH 2.5. The results are presented in Table 47. Table 47. Effect of sorption of HA and FA on the X-ray diffraction patterns of gumbrin and askanite

dO.01 '

A

Before

After

After interaction

interaction

solvation with

with FA at a

interaction with

with HA or FA

glycerol

concentration, mg/ml

HA at a

Saturation Mineral

concentration

cation

Gumbrin Na+ Ca2+ Askanite Na+ Ca2+

After

13.2

19.6

3.87

13.00

14.7

14.7

15.6

19.6

15.6

13.6

19.6

14.7

16.1

18.9

16.1

of 7 mg/ml 13.2 15.6

14.7

13.6 16.1

149 Only for Na-montmorillonite was some change in the interlayer spacing (from 13.2 to 14.7 A) by adsorption of FA in an acidic medium observed. At both investigated concentrations (3.87 and 13 mg/ml) the mineral absorbed 17.6 and 52.5 mg FA per gram of mineral respectively. From this it follows that neither HA nor FA are absorbed in the interlayer space. Partial pene­ tration of some low molecular monomers into the interlayer space may be assumed, for example amino acids, which were present in the solution under the conditions of the experiment, which resulted in an expansion from 13.2 to 14.7 A. Direct experiments with amino acids confirmed this assumption. Penetration of hydrated ions of aluminium in the interlayer spaces is not ruled out. These ions could appear in the solution as a result of partial dissolution of the mineral in an acidic medium, especially in the presence of FA. The quantity of humic acids adsorbed by minerals is satisfactorily de­ scribed by the Langmuir isotherm (or Freundlich isotherm) but in many cases Table 48. Coefficients a and b of empirical equation of adsorption of HA [after Pivovarova, 1975) Soil Chernozem Adsorbent Gumbrin

pH of HA solution

a

b

Sod-podzolic a

b

5

0.29

7

0.32

0.94

1.00

9

0.14

0.80

Ca-gumbrin

7

0.15

1.00

0.25

1.00

Ca-askanite

7

1.00

5

1.00 0.83

0.18

Kaolinite

0.15 0.14

7

0.29

0.66

9

0.20

0.40 0.40

0.26

0.35

1.00

0.71

Ca-kaolinite

7

0.37

Vermiculite

7

0.48

0.47

0.70

0.49

Biotite

7

0.17

0.60

0.15

0.63 0.85

Muscovite

7

0.06

0.72

0.05

Quartz

7

0.33

0.24

0.18

0.39

Blanket loam

7

0.24

0.65

Loess-like loam

7

0.50

0.11

0.81

Sod-podzolic soil, A1

7

0.13

A2

7

0.09

0.53

B

7

0.11

0.86

Chernozem, typical, A1

7

0.10

0.74

150 a C-type (according to Giles' classification) linear adsorption isotherm has been observed. Typical HA adsorption curves on Ca-askanite and kaolinite according to Orlov and Pivovarova (1974) are presented in Fig. 16. Both the isotherms presented in the Figure obey the Freundlich equation y = ax b , similar to adsorption of HA on other minerals and soils. The parameters of the equation are presented in Table 48. The parameters of Langmuir isotherms for sorption of HA on montmorillonite are available in other works [Challa and Raman, 1985]. If in the Freundlich equation the coefficient b = 1, the isotherm is a straight line. Such isotherms are characteristic of gumbrin and askanite. The lowest values of b are typical of quartz. It may be suggested that the linearity of the isotherm is associated with the I:ligh specific surface of smectiles. Conversion of the quantity of HA absorbed to amount per unit surface (and not mass) brought the indices for montmorillonite and kaolinite closer. This enabled us to consider the linear isotherm on montmorillonite only as the initial segment of the total isotherm of the L-type (according to Giles). Linear adsorption isotherms of FA on montmorillonite were obtained by Theng (1976). According to his results the slope of the straight line or the coefficient a of the Freundlich equation depends on the type of saturating cation. This fact is well demonstrated in Fig. 17.

Fig. 16, Adsorption isotherms of humicacid

Fig. 17, Adsorption isotherms of fulvic acids

(HA) on Ca-askanite (1) and kaolinite (2) [after Orlov and Pivovarova, 1974],

on montmorilionite saturated with dif­ ferent cations [after Theng, 1976],

151 Pivovarova has also shown that low-molecular components are predom­ inantly adsorbed by minerals from the polydispersive system of humic sub­ stances. This was experimentally confirmed from the change of E-values of humic acids in an equilibrium solution and directly from the molecular mass distribution of the substance [Orlov and Pivovarova, 1974]. Tan (1976) also confirmed the greater sorption of low-molecular-mass fractions.

4

Electron and Molecular Absorption

Spectra of Humic Substances

Various interactions with electromagnetic radiation in nearly all ranges of wavelengths are characteristic of humic substances. The absorption of light in the ultraviolet, visible and infra-red ranges has .been well studied. Data is available on refractive indices and Rayleigh light scattering. Yet a reliable and precise interpretation of all types of interaction is rather difficult. The absorption spectra show a predominance of broad bands, often constituent subbands. Intense colour (stronger light absorption) and scattering of light exert considerably affect these spectra. Nevertheless, optical methods do provide exceptionally important information on the properties and structure of humic substances. However, although considerable quantities of optically active amino acids and other compounds are present in HA and FA and the presence of asymmetric residues in humic substances is entirely possible, rotation of the plane of polarisation of light in the visible range of the spectrum has not been detected in solutions of humic substances and humates. Hence it is necessary to presume that humic substances are characterised by inter­ and intramolecular compensation. The refractivity of sodium humate solution prepared from HA separated from humus-rich horizons was found to be as follows (results expressed in concentration, g/cm 3): Sod-strongly podzolised soil 0.386 0.362 Sod-moderately podzolised soil 0.426 Typical deep chernozem 0.341 Light chestnut solonetsic, ploughed Similar values have been reported by Kleist and Mucke (1966). Despite sev­ eral limitations it may be said that the values of refractivity of humic acids calculated from the Lorentz-Lorenz molar refraction equation are approxi­ mately 0.30-0.35. The refractivities thus obtained differ from the theoreti­ cally calculated (from the sum of atomic refractivities) by roughly 30%. The observed increase in values for the specific and molecular refractivities is satisfactorily explained by the high content of double bonds, carbonyl groups

153 and so on. But determination of their quantity from the specific increments has yet to be attempted.

ELECTRON ABSORPTION SPECTRA

The quantitative measurement of the colour ofhumic substances was first at­ tempted 60-70 years ago. However, spectroscopic measurements became effective in tackling problems of soils only after Kononova and Bel'chikova (1950) highlighted the changes of optical densities of HA of zonal series of soils. The absorption of light by humic substances in the visible and ultraviolet ranges of the spectrum monotonously decreases with an increase in wavelength, which, to some extent, results in featureless spectra (Fig. 18). The relative feature of humic substances is the high intensity of light absorption. This makes it possible to use spectrophotometric analysis as a sensitive diagnostic method for detection and study of humic sub­ stances. Wide use of spectrophotometry in the chemistry of humic substances presupposes knowledge of the limits of applicability of the method, especially the effect on optical properties of solution concentration, pH of the medium, presence of colloidal particles and stability of colour with time. According to Aleshin, the optical density of a solution of humic sub­ stances in the first approximation, in the 400-750 nm range, can be ex­

Fig. 18. Spectra of humic and fulvic acids from some soils. Humic acids (4 mg/100 ml): 1-southern chernozem; 2-typical chernozem; 3-common chernozem; 4-typical chernozem under forest; 5-grey forest; 6-humus carbonate; 7-solonets. Fulvic acids (15 mg/100 ml): a-typical chernozem; 9-humus-carbonate soil.

154 pressed as an exponential function of wavelength: 0). = Ke- a ).,

°

where is the optical density for wavelength '\; K and a are the constants and e is the base of natural logarithms. Putting the equation in the logarithmic form we get:

°

In 0). = In K - a'\,

that is, the value of In is a linear function of wavelength. The constant a describes the colour characteristic (coefficient of chromaticity) and can be readily found from measurements of the values of 0 1 and O2 , Since In 0 1 = In K - a'\1 and In O2

= In K - a'\2'

then

a

=

In 0 1 - In O2 '\2 - '\1

=

(In 0 1 ) O2

:

('\2 - '\1)'

In this case the coefficient a is equal to the tangent of the slope of the line In 0 - A. It follows from the equation that the value of a is constant for any wavelength only if the investigated function obeys the exponential law. To estimate the slope of the curve and correspondingly the nature of the colour of humic substances, wide use is also made of the coefficient of chromaticity Q, introduced by Springer, which is equal to the ratio of optical densities at two wavelengths, for instance, 465 and 650 nm or Q = 0465:0650' The Springer coefficient (also sometimes called the Welte coefficient) depends on which wavelengths are selected for the measurements; the wavelengths must be mentioned every time. Herein lies its relative drawback as compared to the coefficient proposed by Aleshin. The presence of non-specific pigments (see below) affects the nature of the spectra of humic substances from many soils. The quantitative spectrophotometry of solutions of humic substances is based on the applicability of the Bouguer-Lambert-Beer law to them. As shown in Fig. 19 the humate solutions of alkali metals and fulvic acids obey the Bouguer-Lambert-Beer law, that is, the optical density at any wavelength is dirfilctly proportional to the solution concentration or to the product of concentration and thickness of the absorbing layer of the solution:

0= E· C '/,

155 where C is the solution concentration, f is the layer thickness and E the proportionality coefficient. For the known molecular masses the Bouguer­ Lambert-Beer law is usually written in the form:

o=

c). .

C . f,

where c). is the molar coefficient of absorption equal to the density of the solution at a concentration of 1 M/I and f = 1 cm. For polydispersive sys­ tems such as humic substances, in place of c). we use the E-value, which is calculated similarly but with arbitrarily chosen concentration units. Vari­ ous authors have used percentages (denoted by E%) or sometimes thou­ sandths of a per cent (denoted by EO.001%). In these cases there are two basic methods for calculating the extinction coefficient, or E-value. The first method-conversion of the measured optical density of the solution of humic substances to the content of organic carbon-gives the value denoted by EC. Kononova and Bel'chikova have used this method [Kononova, 1963] and ex­ tensive data has been collected by this technique. According to Kononova, calculations were done for solutions with a C content of 136 mg/I. In the second method, optical densities are compared for equal content of the substance (denoted by EHA/FA) and not of carbon. Larina and Kasatochkin (1966) emphasised that at an equal content of carbon in the solution the optical density is a function of the ratio of the quantity of carbon in condensed aromatic groups that absorb light and the carbon of side chains transparent to light in the visible region of the spec­ trum. Such an interpretation enables estimation of the degree of aromaticity of humic substances by comparing the coefficients of extinction calculated directly. However, this suggestion is based on at least two assumptions. It

Fig. 19. Bouguer-Lambert-Beer law for sodium-humate solutions (southern chernozem).

156 is taken for granted that the side chains do not participate in the absorp­ tion of light and that the coefficients of absorption of the nuclei themselves (condensed carbon) are the same for all humic substances. As seen al­ ready in Chapter 3, the aromatic components of HA are not represented by 'pure' condensed carbon, but by a variable combination of quinoid and phe­ nolic groups. There is no justification for ignoring the conjugation between aromatic rings and aliphatic chains. Furthermore, even peripheral chains contribute some colour to humic substances, as confirmed by the colour of HA hydrolysate. Considering these facts, one can hardly estimate with confidence the ratio of nuclei and side chains from the P values. The second method-calculation of EHA-is more objective and closer to the molar absorption coefficient in concept. A distinct advantage of this method is that it describes the substances as a whole, which is very im­ portant since the optical properties of organic compounds depend on the overall molecular structure. For similar molecular mass the EHA values of humic substances of different origin could totally replace the molar coeffi­ cients of absorption. In essence, the determination of E-values is related to the absorption coefficient by the equation: EHA =

C

100 x MM'

where MM is the molecular mass; hence EHA is an average index, which may be considered an arbitrary absorption coefficient calculated in relation to the empirically obtained molecular mass. The values of EHA depend on both the molar coefficients of absorption and the molecular mass; hence the interpretation of the structure of humic substances from their optical densities should be done with extreme caution. In practice, the choice of the index-EHA or P-is often dictated by the material under investigation. For alkaline extracts in which the concen­ tration of humic substances is determined by oxidation (Tyurin's method) and converted to the carbon content, it is appropriate to use the P value. The conversion of EHA is possible but for this it is necessary to take as a basis some arbitrary content of humic substances. And this, as shown in Chapter 2, is not all that simple. Hence the conversion is not always reli­ able; but for comparison with published data one has to resort to this step. When dry preparations of HA and FA are used in preparing solutions for pho­ tometry, the result is automatically expressed in EHA values. In the following discussion, both values are used although preference is given to EHA values. One more fact requires attention in connection with spectrophotometric characteristics of humic substances. In alkali extracts from soils and some­ times even in HA preparations, various pigments are present which appre­ ciably distort the nature of the spectrum and the level of light absorption. This must be considered when discussing experimental results.

157 In soil literature the smooth monotonous nature of the electron absorp­ tion spectra of HA was long accepted as fact. But as spectrophotometers became more available, descriptions of these spectra began to mention some maxima [Kumada, 1955; Larina and Kasatochkin, 1966; Orlov, 1966]. It was after a series of publications by Kumada and others [Kumada, 1967; Kumada and Hurst, 1967; Kumada and Sato, 1967; Kumada et al., 1967; Sato and Kumada, 1967] that attention began to be paid to these fea­ tures. From preparations of humic substances whose spectra showed a series of distinct maxima, Kumada and Sato separated by fractionation on Sephadex or cellulose a green fraction, Pg, which they called green hu­ mic acid. In the spectra of this fraction there were distinct maxima at 613, 568, 448, 430 and 281 nm (Fig. 20). The origin of the Pg-fraction was at­ tributed to sclerotia of the fungus Cenococcum graniforme (Kumada and Hurst, 1967). Chemical and spectroscopic studies led the aforesaid authors to the conclusion that the substance of the Pg-fraction is a derivative of 4,9­ dihydroxyperylene-3,10-quinone: 0

OH

OH

OH

D D D

D

'125 'ISO

500

570 500 520

nm

Fig. 20. Spectrum of sodium-humate solutions (mountain meadow alpine soil) with high content of the Pg-fraction.

1-20-25 cm; 2-6-12 cm; 3-16-20 cm.

158 Extraction of the Pg-fraction with hot ethanol yielded a pure red crys­ talline pigment which acquired an emerald-green colour in an alkaline so­ lution. The Pg-fraction extracted from soil by a mixture of alcohol and ben­ zene distinctly differs from chlorophyll by a series of absorption maxima [Ammosova, Orlov and Semenova, 1971]. Information about the structure of this green pigment is very meagre at present and warrants greater attention. The significance of the Pg-fraction is readily understood if we consider the specificity of spectra facilitating identi­ fication of substances of the fraction, probability of the condensed aromatic system, the possibility of relating the compounds constituting the fraction di­ rectly to fungal metabolites and, finally, a wider distribution in soils with clear affinity for specific conditions of soil formation. A study of the Pg-fraction holds promise in developing the theory of humification and methods of soil diagnostics. The Pg-fraction has a wide distribution in nature [Kumada, 1965; Ku­ mada and Hurst, 1967; Kumada et al., 1967; Nakabayashi and Wada, 1991; Orlov, 1985; Sato and Kumada, 1967]. It is universal in occurrence. It has been found in iron-podzols and podzolised brown soils of England, podzols and late rites of Australia, some soils of the USA, volcanic ash soils and brown forest, reddish-yellow and mountain-meadow soils of Japan. As judged from the spectra presented in several works, P-type humic acids containing varying quantity of the Pg-fraction have been found in Japan in irrigated alluvial (rice) soils, peat soils and some humus-allophane soils. Signs of the presence of the Pg-fraction have been detected in samples of podzolic soils (Poland) taken from individual plots of long-term experiments with fertilisers, in humic acids separated from German soils, in gley-podzolic soils of Rostov district formed under oak forest (wherein its maximum quan­ tity is found in the AG horizons) and in the A 1 horizon of podzolised burozem of the same district. From a study of hundreds of spectra of humic substances of diverse ori­ gin, among which were spectra with very strongly developed maxima, Orlov (1968) established the presence of the Pg-fraction in many sod-podzolic soils and sod-podzolic gley soils of Smolensk, Moscow, Irkutsk and Novosibirsk districts, in brown forest and light brown forest soils of Smolensk district, Moldova, in brown mountain-forest soils of Sverdlovsk and grey, light grey and dark grey forest soils of Sverdlovsk and Irkutsk districts; floodplain­ meadow and meadow-sod soils of many districts; in yellow soils, brown forest and mountain-meadow soils of the Caucasus and so forth. The fraction was barely traceable in spectra of HA of deep, common and southern chernozems (Kursk, Dnepropetrovsk, Crimea, Orenburg dis­ trict and Moldova) and in chestnut, serozems and many sod-calcareous and alluvial soils. The geographic distribution patterns of P-type humic acids

159 clearly show that the green pigments are confined mainly to sod-podzolic, brown forest, floodplain, mountain-meadow and other soils with at least a slight excess of moisture. The accumulation' of pigments appreciably increases even with tem­ porary gleying while ploughing and cropping of soils evidently hinder their formation. These conditions are highly conducive to the formation of fungal mycorrhiza. The link between the accumulation of Pg and conditions of moisture availability is better traced from the example of chernozems of the central Chernozem Preserve (Kursk district). The Pg-fraction was not detected in the HA in the profile of deep chernozem in the steppe on the crest. In the meadow-chernozem soil at the bottom of a nearby ravine (under steppe veg­ etation), Pg-fraction was found at 60-70 cm and under forest at 0-10 cm. In the steppe area the green pigment was found even in podzolised cher­ nozem (ravine slope, northern exposure). A comparison of the two profiles of deep chernozems on the crest yielded the same pattern: if under forest the green pigment is present in appreciable quantity, then under meadow vegetation in the glade only traces could be detected. For a quantitative estimate of the content of the Pg-fraction we proposed to calculate the percentage ratio

f:::..~

x 100, where f:::..D is the relative height

increment of the peak at 620 nm and D is the optical density of the 'normal' component [Orlov, 1968]. This relation gives the proportion of the green pig­ ment in humic acids (or ratio of Pg to the usual HA) in arbitrary units. The absolute content, also in arbitrary units, can be judged from the value of f:::..D, i.e., increment of optical density at 620 nm. For this purpose, spectra of humic substances were supplemented with a curve describing the normal components of humic acid. This curve can be superimposed or is selected according to intensity (the earlier taken spectra of usual humic acids) or a French curve used, such that it touches all areas of the ~pectrum lacking maxima (or coinciding with these areas). This method is shown in Fig. 21. After this, we found that the maximum increment of optical density f:::..D cor­ responding to this wavelength (Amax) and the increment of optical density in the first approximation could serve as a measure of content of Pg in the solution under reference. The relative content of the fraction was found by relating the value of f:::..D to the optical density of the normal component for the same wavelength and expressing it in percentage, that is,

gD

norm

x 100.

Of course, this method of calculation is somewhat arbitrary but it shows the relative contribution of Pg to the ensemble of coloured components of the alkali extract and hence does not depend on the conditions of extraction (weight of soil, volume of solution etc.).

160

Fig. 21. Analysis of absorption spectra of P-type humic acids. A-Conventional (1) and differential (2) spectra; B-Graphic determination of wavelength of maximum absorption and increment of optical density 0 by the Pg-fraction.

Of the three distinct maxima, it is most convenient to use the maximum in the region of 615-620 nm since it is marked by rather high absolute and relative intensity. The first maximum at 420 nm is on the steep slope of the spectral curve and so is difficult to measure, its intensity changes irregu­ larly, which may be due to errors of measurement. Its relative intensity, i.e. b.g420 x 100, generally does not exceed 10-12. The relative intensity for 420

maxima is fairly constant and the ratio of b.D460:b.Ds80:b.D620 is, on average, 1:0.43:1, which only for the pair b.Ds80:b.D620 is close to that for the pure Pg fraction. This is explained by the fact that for wavelengths greater than 550 nm the optical density of the usual humic acids is not high and changes little with an increase in wavelength. Hence it is possible to observe fairly stable maxima. The relative intensity maxima are the highest for 620 nm, even as high as 50-70%, whereas the relative intensity at other maxima is usually closer to 4-1 0% and rarely ever reaches 20%. Later we used the value b.:620 x 100 as a measure of the relative 620

contribution of the Pg-fraction to the content of humic acids. The results obtained by using this procedure showed that the Pg-fraction is present directly in the alkali extract of the soil as well as in the separated HA l' and HA2 fractions. In other words, Pg precipitates with HA when the extract is acidified (Table 49). It was not possible to detect Pg in fulvic acids.

161

Table 49. Relative content of Pg in soils of the Caucasian State Preserve (arbitrary units,

f1D x 100)

0

Soil

Horizon, depth in cm

Relative proportion of Pg Direct

Fraction 2

Fraction 2

8.3 25.8

19.0 43.5

27.8

27.6 22.2 21.7

46.7 3.9 40.0

22.5 17.7 16.7

22.2 41.4 31.6

37.0 45.5 60.0

23.5 11.8

A,0-5

64.9 10.4

58.5 22.9

B, 10-20

12.9

35.3

40.0

17.3 9.1 26.7 83.3 68.0

20.0 50.0 12.5 28.6

Forest litter

13.1 nil 30.0 105.0 77.8 100.4 nil 20.0 42.4 nil

62.5 Traces 35.7 35.7 Traces

37.5 nil 40.0 22.2 22.2 57.1

AOA1,3-6 A1,6-14 B, 20-30

17.7 20.0 21.1

5.8 23.8 13.0

33.3 44.4 nil

NaOH extract Mountain meadow

A1,2-5

peat alpine Mountain meadow

B1,5-10 BC, 10-50 A1,0-3 A,3-6

alpine

AB,6-12 B,12-16 BC, 16-20 C,20-25 Mountain meadow subalpine Brown forest sparse forest

B, 40-50 Forest litter A,1-5 B, 10-20 20-30 40-50

Brown forest fir forest Brown forest beech forest

Forest litter A, 2-7 AB,10-20

12.5

33.3 45.5 20.0

The same method was used to describe a large number of soils. Spec­ tra of solutions obtained when humic substances were extracted with alkali during analysis of fractional and group composition of humus were used. Soils in which, according to this data, the Pg-fraction was found in appre­ ciable quantity, are given in Table 50. We have not given the results of study of the spectra of humic substances of soils in which Pg was not detected. It was not possible to detect Pg in humate solutions of most chernozems, such as the deep chernozem of Kursk district, A1 horizon;

162 Table 50. Distribution of Pg-fraction in some soils Relative intensity of District, region

Soil

Horizon and

band at 620 nm,

depth, cm

t:. 0 620

X

Dnorm

(1 )

(2)

Smolensk district,

Sod, highly podzolised

Sychevsk region

light loam, cultivated

Sod, highly podzolised,

(3) Ap 0-29

(4) 8

A229-37

Traces

B1 37-50

44

Ap 0-26

4

light loam, cultivated, moderately eroded Sod-podzolic, gley, aggraded

Sod, highly podzolised,

B1 26-42

Traces

B242-78

Traces

ASOd 0-4 A1 4-14

10

A1 14-30

24

A2G 30-42

20

15

BG 42-62

Nil

A1 5-10

12

A230-40 A214-36

23 Traces

B1 37-57

10

medium loam Irkutsk district

Sod, highly podzolised

Moscow district,

Sod, highly podzolised, medium loam

A1 0-15

8

Sod, weakly podzolised

AO 3-8 A1 8-13

11

'Chasnikovo' station Sverdlovsk Taliisk region Smolensk district,

Sod, highly podzolised,

Shumyach and

fallow

Khislavich region

7.5

B 18-24

8

Ap 2-14

10

A216-23

10

Sod-podzolic, light loam

Ap 0-12 A220-29

10

Sod-podzolic, sandy,

B1 20-30

77

(1st terrace). weakly

A13-6

75

eroded, brown forest, contact gley, forest soil

AB 6-16

55

12

cultivated

100

163

(1 )

(2)

Ap 0-10 AB 10-17 20-30

Brown forest soil, cultivated, orchard

Ap 0-15

10 10 50 Nil

AB 20-30 Apl 0-15

40 8

AB 20-30 A230-38 AO 0-5

46 38 7

A15-9 BC 10-30 A 5-15 AB 21-31 B135-45 B255-65 AO 0-4

4

Brown mountain forest soil

Moldova

Brown forest, heavy loam soil

Sverdlovsk district, Taliisk region

Dark grey forest soil

Irkutsk district

Light grey forest soil Grey forest soil Podzolised chernozem

Smolensk district, Sychevsk region

(4)

Brown forest soil, cultivated

Light brown forest, residual highly podzolised

Sverdlovsk district, Taliisk region

(3)

Meadow-gley, floodplain (lower terrace) soil

A14-10 10-20 20-30 A1 3-14 B1 16-33 Apl 0-20 A1 0-20 AB 52-68 72-86 A,od 0-5 A1 5-30 A1 30-45 A1 45-60 A1G 60-77

Meadow peat gley soil Moderately deep, sod-meadow, deep gley, light loam (lower terrace) soil

3 10 10 10 4 3 2 3 5 2 Traces 5 3 Traces 10 11 54 62 12

Apea! 0-27 A,od 0-4

66 Nil 14

A1 4-26 B1 26-53 B2G 53-71

12 35 20

164 common chernozem Dnepropetrovsk district, AplOU9h horizon; southern cher­ nozem (Crimea); Aplough horizon, southern chernozem (Orenburg district) in the entire profile; typical deep and compact chernozems of Moldova in the entire profile. The Pg-fraction is practically absent in humic acids from the A 1 horizon of grey forest soil (Tula district), A 1 and B1 horizons of dark grey forest soil (Irkutsk district), and A 1 and B1 horizons of dark grey forest heavy-loam soils of Moldova. Maxima were also not observed on the ab­ sorption spectra for compounds separated from Asod ' A1 and B horizons of chestnut soils (Orenburg district), A 1 horizon of typical serozem (Ak-Kavkaz Experimental Station), humus horizons of compact solodised soil (Khersonsk district, Askaniya-Nova), red-coloured soils (southern coast of Crimea), sod­ calcareous, sod-leached floodplain alluvial and peat-gley forest soils (Irkutsk district). In sod-podzolic soils we often came across humic acids rich in the Pg ­ fraction although in s~veral instances, especially in the ploughed layer, we did not find it. Many sod, moderately podzolised cultivated soils of Sychevsk region, Smolensk district, sod, roughly podzolised cultivated soils of Moscow district (Lugovaya Station) and some others also did not contain this fraction. On absolutely similar soils presently UIlder forest in these regions, we did not find the Pg-fraction even though the distance between the respective profiles was not more than 40-50 m. The most intense maxima and high content of the Pg-fraction were found in the profiles of meadow-gley floodplain and sod-meadow deep gley soils of Sychevsk region (Table 50), in many brown-forest soils in the western part of Smolensk district [Glebova, 1966], and in the B horizon of sod­ podzolic soils. In the profile of sod-podzolic soils the relative content of Pg was generally very low but the amount appreciable. A definite pattern in the distribution of these components in the profile is generally not dis­ cernible although a decrease in their content in deeper layers (transition to parent material) appears probable. In the upper eluvial horizon also their content seems to be lower and the maximum accumulation is often ob­ served in the middle part of the profile, particularly if gleying is present. We have often observed a decrease in the content of the Pg-fraction or its total absence in the cultivated, less wet soils compared with their virgin counterparts. In grey forest soils maxima typical of the Pg-fraction were quite often observed but their intensity lower. Sometimes these maxima were noticed even in spectra of humic acids from chernozems. All available material leads us to conclude that the Pg-fraction or green pigments are confined mainly to soils experiencing at least relatively excess moisture; that is, sod-podzolic, brown forest and floodplair. soils, and their accumulation increases in the presence of temporary gleying. In the southern

165 region of Russia, in soils of the steppe and forest steppe zones, it was practically not possible to determine the Pg-fraction. In this respect grey forest soils occupy an intermediate position. While discussing the distribution patterns of the Pg-fraction one must bear in mind the usually higher. content of humus and humic acids in soils of the southern regions. Hence the spectra have to be recorded from relatively dilute solutions (with a wider soil: solution ratio). By this, the maxima due to Pg could become less conspicuous on the light absorption curves. However, if this fraction is present in the southern soils, their content in humus ought not to be very high. Kumada has separated many other pigments besides the green from soil organic matter. Many of them are contained in very small quantities. Carotenoids such as lutein, ,8-carotene and chlorophyll derivatives-pheophytin a, pheoforbid a and chlorophyllid a are found in appreciable quantities [Sanger, 1977]. A pink fraction with spectral maxima at 490, 520, 550, 630 and 660 nm was separated using the gel-filtration method [Lowe and Tsang, 1970]. While extracting soil with chloroform in Soxhlet's apparatus, McGrath (1970) separated a pigment which is red in concentrated H2 S04 and alkaline solution of acetone and quinone, but yellow in chloroform. Despite the low content of such pigments in soil, studies in this area could prove most promising. Even at low concentration the multinuclear systems of the oxyanthraquinone type could serve as sources for the more complex condensed systems. In the long process of accumulation of humic substances, when the most stable products are retained, the concentration of initial substances may not be of great consequence. Black, brown and other pigments are also widely known. These are pro­ duced by micro-organisms. Many of them are formed directly in cell (tissues) and accumulate in them. It was mentioned earlier that these pigments are close in composition to humic substances of soils and their electron ab­ sorption spectra entirely coincide with those of HA and FA. At the same time these pigments differ greatly from humic acids in IR spectra. Intense absorption bands with maxima at 1650, 1530, 1230-1270 and 1070 cm- 1 are prominent in their spectra. In the frequency and intensity of bands such spectra coincide with the spectra of proteins. Despite definite similarity in several features, all these pigments repre­ sent different compounds whose specificity is primarily associated with the fraction of peptides in the pigment molecules and with the nature of oxygen functions. Some of these pigments have a similarity with HA only in colour; others, differing in colour, exhibit analogous chemical properties. Some pig­ ments could be considered as a ready-made base for the formation of HA while others could act as a source of condensed structures. Some pigments probably do not participate at all in the formation of HA and FA.

166 Besides the factors listed above, many other conditions affect the spec­ tra of humic substances in the visible region. These include the presence of mineral colloids which are very difficult to eliminate, change of ionic strength of thE;! solution, photochemical oxidation, changeof pH of the medium and so on. Some of these problems have been discussed earlier [Orlov, 1974]. Here we shall discuss only the effect of the pH of the medium. Orlov (1960) investigated the quantitative increase in optical densities of fulvic acids as a function of increase of solution pH. Since fulvic acids exhibit no distinct maxima in the visible part of the spectrum, the colour tone almost does not change with pH. These spectra completely retain the general character in the pH range from 1-2 to 12-13. However, the intensity of light absorption increases in an alkaline medium (Fig. 22). In the study of a large number of preparations separated from soil samples of different types and acid-soluble fractions as a whole, we were able to observe in some cases crossing of light absorption curves and manifestation of an effect analogous to the isobestic point in the 610-620 nm region. ­ Changes of spectra can be judged from Fig. 23 in which the results of study of acid-soluble fractions are presented. Judging from the general na­ ture of the effect, we can expect a shift of the expected absorption maximum to the higher wavelength side (bathochromic shift). The colour change of FA solutions and acid-soluble fractions is re­ versible. This indicates the presence of at least two forms of fulvic acids in equilibrium in the pH range 4-8. The peculiarity of these forms and the mechanism of transitions are not yet clear. In the most general form the colour of humic substances may be explained by the presence of sys­ tems of conjugate double bonds. The presence of ionogenic electron-donor

Fig. 22. Optical density of fulvic acid solutions from grey forest soils at different pH values.

1-10.2; 2-1.0.0; 3-8.6; 4-7.4; 5-5.0; 6-2.8; 7-2.7; 8-2.6.

167 groups containing unshared pairs of electrons (-NH 2, -OH) or electrophilic .groups (N02 , C=O) affect the'colour of the substance, rather intensifying it. Dissociation of H+ ions and the formation of .anions is usually accom­ panied by a change of polarity at the ends cl the conjugate chain and shifting of the absorption maximum to the long wave region with a simul­ taneous increase in colour intensity. Such phenomena could occur during a change in colour of FA. The presence of aromatic nuclei and hydroxyl groups in their composition enables us to suggest dissociation of H+ ions from the hydroxyl (phenol) groups adjoining the conjugate chain of C=C bonds. Orlov and Zub (1963) suggested that intensification of fulvic acid colour may not be the consequence only of the formation of phenol ions, but also of keto-enol tautomerism. Stevenson (1982) also suggested that the dark colour of humic substances could be caused by quinoid structures conjugate with the ketone group. With change of pH the following transitions are pos­ sible

o

0

11 /""--CH2-C-CH2 11

~

\\

H

OH

11 I I /""--C=C-CH 2 ­

~

11

Enol form

Keto form

D

0':;0

1.2 1.2 1.2 1.2

0':;0

0.15

om:

om:

1.2

om:

1.2

1.2

om:

Fig. 23. Effect of pH on absorption spectra of acid-soluble fractions of organic matter from the A 1 horizon of sod-podzolic soil. pH values: 1-1.0; 2-3.5; 3-13.0; 4-13.5.

168 This confirms the assumption regarding the importance of keto-enol tau­ tomerism in the pH-dependent colour of humic substances. Fulvic acids of different soils similarly change their colour with pH.The average derivative curve in the t:::.D%/ t:::. pH vs pH for FA and the acid-soluble fractions isolated from samples of 15 various soils is presented in Fig. 24. It resembles the general curve of potentiometric titration. If we consider that the change in colour is directly proportional to the content of the more intensely coloured form, then from this derivative curve it is possible to approximately estimate the ionisation constant of FA. Considering that pKa = pH 1 we get pKa

= 5.5 for fulvic

2

acids. The change in colour of FA under the influence of the pH of the medium necessitates precise pH determination when spectral characteristics are studied. Sapek and Sapek (1987) have also confirmed the relationship of optical density of humic substances with pH. The optimum pH range of buffer solutions for these purposes is 11-13. Analogous changes in HA are rather difficult to observe. We observed no appreciable shift of sodium-humate spectra with a change of pH although a change in colour of the acidic medium appeared possible [Orlov, 1960] since coagulation of humic acids is accompanied by some decoloration of the solu­ tion. However, this phenomenon, too, could be the result of increased light scattering during coagulation. Kononova (1963) indicated that the optical density of humate solutions increased by 0.2 units with a rise of pH from 7.2 to 13.0. Andrzejewski (1965) also observed an increase in optical den­ sity of humates upon increasing the concentration of NaOH as a solvent. In later experiments, while obtaining the electron absorption spectra of sodium humate, we observed a tendency towards an increase in optical density in more alkaline solutions. An increase in pH from 7 to 10 was accompanied by an increase in optical density of 0.8-1 %. Such changes are more likely

Fig. 24. Mean derivative curve of dependence of optical density for acid-soluble fractions of soil organic matter on pH.

169 to be experimental errors. However, these were repeatedly observed for HA preparations from two samples of deep chernozem, one sample of common chernozem and one sample of grey forest soil. Use of the differential spectra method has confirmed a stable rise of optical density of humate solutions by 0.10-0.15 unit in the pH range from 3.0 to 13.5 [Tsutsuki and Kuwatsuka, 1979c, d]. Effect of Storage and Exposure to light on Absorption Spectra of Humic Substances This is not a new problem. Over 70 years ago, Zholtsinskii, while working in Moscow University, demonstrated the photodestruction of humic acids. How­ ever, despite the theoretical and practical importance of this phenomenon, it has been studied little. Information on the stability of humic substances during storage under usual laboratory conditions is quite meagre. In natural conditions, in summer, humic acids remain for a long time at 15-20DC and with variable, often low, moisture content. Hence the stability of HA and FA under laboratory conditions may serve as a model for their spontaneous changes in the vegetative period. The estimate of stability is essential even for standardisation of methods of obtaining important parameters of humic substances. Many workers have noted decoloration of humate solutions. An­ drzejewski (1965) reported a decrease in optical density of sodium-humate solutions after 2 and 10 days of storage. The same phenomenon was re­ ported by Nikitin (1971). Andrzejewski stressed the need to obtain spectra of humates under strictly standardised conditions (solvent, time). Menkovskii and Petrovskaya (1957) reported a decrease in optical density of alkaline solutions of humates by roughly two-thirds after storage for one year. It must be mentioned that photodestruction is being used in estimating the effect of land use on the nature of humic acids [Szczodrowska et al., 1990]. Dry preparations of HA might possibly be stored for a long time without danger of any change in properties whatsoever [Orlov, 1974]. The changes in humic substances during storage in solution can be judged from the results of experiments with HA and FA separated from the A 1 horizon of several profiles of sod-podzolic light loam soils (Moscow district). Alkaline solutions of humates as also alkali extracts were stored in the dark and portions in light. Acidic solutions containing fulvic acids were stored in a refrigerator at + 2 to + 3DC. A part of the fulvic acid solution was made alkaline with NaOH to pH 12-13 and stored at room temperature (both in light and in darkness). No significant change in spectra occurred during several months of storage. This was confirmed by th~ir external appearance, 0 465 : 0 650 ratio and relative intensity of maxima due to the Pg-fraction. However, a significant change in colour intensity did occur during storage. The optical density at 465 nm of alkali extracts of humates and fulvates of sodium decreased faster

170 in the first 3-5 months of storage. After 4-5 months a tendency towards increase in optical density was observed, which was particularly noticeable in alkali extracts. The initial alkali extracts were characterised by 0465 values from 1.4 to 2.0 which, after 15 months of storage, dropped to 36-72 of the initial value. After storage in light the average terminal optical density was 44% of the initial and after storage in darkness, 61%. Similarly, for sodium humates the average terminal optical density upon storage in light was 63% of the initial and after storage in darkness, 77%. Sodium fulvates stored in light gave an optical density of about 55% of the initial. So two general patterns emerged. In the usual room illumination the colour of all humic substances fades appreciably faster than in darkness. The probable reasons could be hydrolysis or oxidation accelerated under the influence of light (photochem­ ical destruction). The second pattern shows the relative stability of different classes of compounds. Humates appeared to be most stable in darkness. Humates in light and their alkali extracts in darkness lost their colour roughly to the same extent. Sodium fulvates appeared somewhat less stable in light than the alkali extract in darkness. Alkali extracts stored in light changed maximally. The higher stability of humates compared with fulvates is under­ standable from the general prinCiples of their structure. On this basis it is possible to expect that alkali extracts ought to occupy an intermediate place between humates and fulvates. (They might be even closer to humates be­ cause of their higher optical density.) In point of fact, alkali extracts seemed to be the least stable. This somewhat unexpected result allows us to sug­ gest the non-additive nature of some properties of HA and FA. The foregoing experiments lead to some methodological consequences. Under laboratory conditions obtaining alkali extracts of humic substances and the subsequent determination (after separation) of their optical properties usually takes from a few days to a few weeks. Although the absolute rate of fading of colour is relatively low, the results obtained require a strict stipulation of periods of measurement of the electron absorption spectra of solutions of humic substances. Significant distortions of electron absorption spectra could also arise due to heating of solutions. Hence it would be most appropriate to conduct all operations concerning the separation of humic substances from soil and their preparation for investigation only in the cold. These experiments demonstrate that one of the reasons responsible for differences in the optical properties of hUmic substances from various types of soils and their genetic horizons could be the effect of light. Photochemical processes are concentrated in the uppermost layers of soil in which, for this reason, there should be a higher content of less intensely coloured humic substances. Fading of colour of humic substances in soils of the southern regions may also be associated with increased insolation.

171

Fig. 25. Change of optical density of sodium humate from deep chernozem after ir­ radiation by mercury-quartz lamp. The initial value is taken as 100.

Fig. 26. Effect of irradiation by mercury­ quartz lamp on the molecular mass distribution of sodium humate (deep chernozem). 1-before irradiation; 2-after irradiation.

To understand the mechanism of fading of colour of humic substances by light, special experiments were conducted to study the effect of ultraviolet rays. Ultraviolet radiation was supplied by BUV-15 mercury-quartz lamp with maximum irradiation at 253.7 nm. As in the earlier experiments, after irradi­ ation the nature of the spectrum remained almost unchanged in the visible range but the optical density rapidly decreased with increased duration of irradiation (Fig. 25). Similarly, the colour of HA changed during prolonged storage and with partial oxidation by H2 0 2 . This allows us to consider that in all cases the change of colour is associated with the destruction of one and the same structural units, including various oxygen-containing functional groups and fragments with double bonds. The destruction of the C=C dou­ ble bond of aliphatic chains is most probable. From the data of gel filtration the HA contain a lesser quantity of high-molecular-mass components after irradiation (Fig. 26). From these premises the mechanism of destruction of humic substances could be described in the following manner. Humic acids contain comparatively stable and intensely coloured struc­ tural units of aromatic nature. These units are joined by aliphatic bridges whose double bonds are close to conjugate chains. A single system of con­ jugate double bonds of adequate length arises, which is accompanied by a sharp rise in the colour intensity. In alkaline solutions, under the influ­ ence of oxygen from the air slow oxidation occurs at double bonds, the bridges are destroyed and in place of a single large molecule the solution

172 contains two (or more) smaller molecules. Decrease of conjugation length as a result of such a rupture sharply decreases the optical density. This very slow process accelerates with the addition of strong oxidants (H 2 0 2 ), heating or illumination. Even the usual laboratory illumination promotes ap­ preciable colour fading of solutions while irradiation with a mercury-quartz lamp accelerates the process tenfold or even a hundredfold. Probably the effect of light is associated with the free radical mechanism of reaction. The suggestion about the free radical mechanism of photodestruction of humic substances is confirmed by the direct determination of paramagnetic centres (PMC). It was demonstrated that after UV-irradiation of humic substances for an hour, the concentration of PMCs increased roughly by 140% and then decreased exponentially for two days [Tichy, 1971]. This phenomenon has been confirmed in our investigations also [lI'in and Orlov, 1973; lI'in, 1975]. Nature of Light Absorption by Humic Substances

Several investigators [Kasatochkin et al., 1964; Larina and Kasatochkin, 1966] have determined the absorption spectra of humic acids in the visible range to be uniform and explained its nature by the mobility of 7r-electrons in the condensed nucleus. In their opinion, 'structural elements of aliphatic and saturated cyclic nature constituting the side radicals do not absorb light in the investigated region of the spectrum. The possible aromatic and other groups with double bonds in side radicals could be only in conjugation with aromatic rings of the nucleus' [Kasatochkin et al., 1964, p. 199]. In the ultraviolet region spectral maxima of absorption have been no­ ticed in various ranges. These appear mostly at wavelengths of 180-200 to 220-240 nm [Orlov, 1974]. It has been demonstrated that the sharp max­ imum near 200-210 nm recorded by sensitive double-beam spectrophoto­ metry should be considered as an artifact. The reason for the appearance of this maximum may be that at about 220-230 nm there is a sharp increase in absorption due to alkali. In these conditions the total light flux is attenuated and the magnitude of the signal becomes comparable to instrument 'noise'. It is characteristic that the maximum in spectra of humates described by many authors coincides precisely with a rise in the light absorption curve of 0.1 N NaOH solution. If water is used as the solvent, this maximum is not observed since the light absorption curve for water rises only gradually in the direction of decreasing wavelength. The absence of absorption maxima on the spectra of classical humic acids prompts the need to analyse their distribution and absolute inten­ sity of energy absorption and their changes during chemical transformation of humic substances. Most authors associate the nature of electron ab­ sorption spectra of humic substances with the system of conjugate double bonds and functional oxygen. In view of this, the carbon content of humic

173

E 0.8

0,6

0,'1

(1,2

3a

'10

SfJ

50

Oxygen content, % Fig. 27. Dependence of E-values on content of oxygen in the dark-coloured pigments of some fungi.

substances and the increase in degree of condensation is often cited as the direct cause for the increase in E-values. According to Kleist (1969), the darker colour of grey humic acids compared to that of brown humic acids is due to the higher content of free radicals. For HA of coals an in­ crease in optical density was reported with an increase in oxygen content in them. A direct dependence between the optical density and oxygen content (Fig. 27) was demonstrated by us for humin-like pigments [Zaprometova et aI., 1971]. This data points to the complex nature of chromaticity of humic substances. The colour intensity of humic substance is relatively high. Evidently the higher E-values typical of HA could not be due to individual radicals (R­ chromophores) for which the molar coefficients of absorption at the maxi­ mum do not exceed 2000 and are usually not below 100. Based on E-value and the possible range of molecular mass (MM) it is not difficult to calculate the probable values of E465 . From the definition 1 t: = EO,001% x MM x 100. The mean values of E2sg0 % for HA of chernozems are close to 0.1-0.2, for HA of sod-podzolic soils 0.05 and for fulvic acids 0.01. From this t:465 should be correspondingly equal to 10(MM), 4(MM) and 1(MM). If for HA of chernozem we take the range of MM from 10,000 to 100,000, then E465 values should be in the range of 105 -10 6 . In the 200-250 nm region the E-values are 5-6 times higher and then t:200 ~ 5 x 105 - 5 x 106 . These are very high values which are difficult to compare not only with t: A of individual R-bonds, but also with the total absorption of several

174 R-chromophores. For fulvic acids with mean molecular mass of the order of SOOO-1 0,000 the colour seems to be 2-S times more intense than expected for R-bonds. Such high E-values directly indicate that the colour of HA is due to conjugate systems (K-chromophores,K-bonds) with the participation of oxygen groups although the EHA values given above are high even for K-bonds. The most probable explanation may be found in the following. The system of conjugation responsible for colour is not distributed all over the HA particles with arbitrary MM = SO, 000 - 70,000 but is confined to individual isolated areas. Each molecule may contain several such areas. These areas and consequently the systems of conjugate bonds are isolated from each other and in respect of electromagnetic radiation behave independently. In this case the spectrum of HA is a sum of the absorption curves of isolated areas. Then the increase in molecular mass does not lead to a true increase in cmax due to an increase in conjugation length, but to apparent increase because of association (aggregation) of particles producing a decrease in the total number of particles in the solution. The latter is best confirmed by the effect of detergents and complex forming agents on the molecular mass distribution of humic acids (see Chapter S). If for the above-cited isolated areas we take the maximum c values of the order of 10,000-30,000, then it appears that each HA particle should comprise no less than ten such simple units with partial mass of the or­ der of SOOO-7000. Such sizes accord very well with the presumed sizes of structural units of HA [Orlov, 1974). Based only on spectroscopic data it is impossible to predict the structure of the unit. Evidently it can only be said that these must be systems with conjugate double bonds and functional oxygen. Hydrolysed HA has very high optical densities. The ~6~01 % value of HA from podzolic soils increased upon hydrolysis from 0.040-o.0S0 to 0.074-0.090 and that from chernozems from '0.10-0.11 to 0.14-o.1S. In the case of chernozems the increase in optical density was less with the E-values increasing only 1.S times, whereas in HA of podzolic soils the increase was roughly twofold. This correlates well with the hydrolysability of HA. If the non-hydrolysable residue in the chernozems HA under investigation was about 70% it was SO-SS% in HA of podzolic soils. It follows from this that the colour is almost entirely due only to the non-hydrolysable residue. The calculation of E-values of the non- hydrolysable residue from the degree of hydrolysability of HA and the initial vaiues of EHA showed good agreement with experimental results (Table S1). The combined examination of the role of 'nucleus'and peripheral part of HA in the formation of colour and the role of oxygen-containing groups allows us to explain the observed differences in E values in specific soils. The increase in extinction coefficients is largely due to a gradual loss in weakly coloured peripheral links of molecules from HA of sod-podzolic soils

175 Table 51. E-values of initial and hydrolysed humic acids

Non-hydrolysable residue, Soils

%

Sod-podzolic

55 50

Deep chernozem

Initial preparation

Non-hydrolysable residue Calculated

Observed

0.040

0.073

0.075

0.045

0.090

0.090

55

0.048

0.087

0.090

70

0.102

0.146

0.145

70

0.106

0.152

0.150

to HA of chernozems. Understandably the increase in E-values in such a situation should coincide with the increase in the carbon content of HA. This is the manner which gives rise to specific features of HA of zonal series of soils. The colour differences of the non-hydrolysable residue of HA point to another cause, namely, oxygen content. For experimental verification of the validity of the above ideas regarding the nature of the colour of humic acids, Orlov, Baranovskaya and Okolelova (1987) conducted special studies on several irrigated and unirrigated soils of the Volga region. To confirm the structural peculiarities of HA they were oxidised with potassium permanganate in an alkaline medium with sub­ sequent determination of the yield of benzene-polycarboxylic acids (BPA). To estimate the contribution of aromatic fragments in the structure of HA molecules from the yield of BPA, we used the approximation that the main part of BPA is represented by tetrabenzocarboxylic acids (TBA). We can now calculate the carbon content of aromatic fragments (C arom ) from the equation M6C

Carom = ~ xA, TBA

where M6c and MTBA are respectively the molecular masses of the ben­ zene ring and TBA (in terms of carbon) and A is the yield of TBA upon oxidation. The carbon content of aliphatic fragments (Caliph) would, in this case, be found by difference. On comparing the E-values of humic acids with CaliPh values found by the above method as well as from elemental compo­ sition, distinct inverse relationships were observed, which are given by the regression equation below. In the version of calculation where it was as­ sumed that the aromatic part is mainly made of 4-substituted benzene rings

176

and the unidentified oxygen is in the HA molecule carbonyl, the regression equation takes the form: Caliph

= 95.5 - 246.7E46S ·

From this it follows that if the HA molecules were almost entirely made of aliphatic components, that is to say up to 95.5% (carbon), then such a substance would be colourless: 95.5 = 95.5 - 2A6.7E46S ; E46S = O. For a hypothetical molecule entirely devoid of aliphatic fragments

(Caliph

= 0):

0= 95.5 - 246.7E46S ; E46S = 0.39. From this, E46S = 0.39 is the maximum possible value of optical density of humic acids with 100% benzoicity. The above relationship between the proportions of important fragments in the HA molecules and their colour agrees well with the observations of Butler and Ladd (1969) on fractionation of HA by gel filtration on Sephadex. According to these authors, with an increase in molecular mass of the separated fractions there is a distinct increase in the amino acid in the HA fractions and a decrease in the quantity of carboxyl groups and opti­ cal density both at 260 and 450 nm. There is similarly a decrease in the chromaticity coefficient (Table 52). The increase in amino acid content in­ dicates an increase in aliphatic components in high-molecular-mass frac­ tions. This, according to the above explanation, must lower the extinction coefficient. It thus follows that the more 'mature' humic acids of medium and low-molecular-mass fractions with high E-values are more developed. The benzoic part of HA according to these results is the principal carrier of carboxyl groups. Tsutsuki and Kuwatsuka (1979a, b, c) have thrown light on the nature of coloration of humic acids in their fundamental investigations. According to their results, for weakly humified substances the effect of carbonyls of keto and aldehyde groups is less on the absorption spectra, but the con­ tribution of quinones is high. When the humification process is developed the role of quinones persists but the contribution of ketones and aldehy­ des increases. They attribute relatively less to the phenolic components in the absorption of light and at the same time give preference to conjugate systems. In conclusion, fairly extensive investigations notwithstanding, the theory of the formation of colour of humic substances has not been fully devel­ oped.

177 Table 52. Some properties of humicacid fractions from red-brown soils [after Butler and Ladd,

1969) Molecular mass range Yield of Nitrogen of COOH Extinction coefficients Chromaticity of HA fractions fraction amino acids meq/g-1 for CHA = 1 mg/ml coefficient E470:E666

%

260 nm

450 nm

100,000

68.2

1.51

2.32

26.9

5.3

3.97

50,000-150,000

42.9

1.68

2.21

31.9

6.4

4.12

>150,000

31.1

1.86

2.06

20.3

4.2

3.62

OPTICAL PROPERTIES OF HUMIC SUBSTANCES OF VARIOUS SOILS

As mentioned earlier, only after the work of Kononova and Bel'chikova (1950) and Bel'chikova (1951) which demonstrated the regularities of changes of extinction coefficients of HA in the zonal-genetic series and proposed a simple method, did the measurement of optical densities of HA become one of the important techniques in genetic soil research. The sequence of extinction coefficients in the series of soils of latitudinal zonation became almost standard. Unfortunately, less attention was paid to the provincial peculiarities of humic substances and to variation of their optical properties within limits of soils of one type (contour, terrain) even though variation of extinction coefficients and the effect of local conditions might at times exert more influence on the optical properties of humic substances than the peculiarities of humification in soils of various types. We had proposed the variation of optical properties on the example of sod-podzolic deep gley. soil light loam under spruce bilberry forest (Zvenig­ orod Biological Station of Moscow State University). All samples were taken from the A horizon in a uniform territory at intervals of 15-20 m. Humic sub­ stances were extracted directly by extraction with 0.1 N NaOH. The entire acid-soluble fraction was taken as fulvic acids. The humic acids were not dried and after separation from FA were dissolved again in 0.1 N NaOH. The carbon content was determined spectrophotometrically [Orlov and Grindel', 1967]. Variations of E-values and CHA:C FA ratio reached 50% of the average value (Table 53),which is compatible with differences between types of soils. Consequently, even in regard to optical density we observe the same pat­ tern as was noticed inelemental composition. The change of E-values could

178 Table 53. Optical indices of humicsubstances and CHA:C FA ratio in A1 horizons of sodpodzolic soil Humic acids

Fulvic acids e!.OO1%C 465

e!.OO1%C 465

Sample No.

E 465 : E650

Proportion of Pg %

CHA:C FA

1

0.69

0.064

3.9

10

0.011

2

0.78

0.055

4.5

24

0.013

3

0.56

0.058

4.3

14

0.011

4

0.60

0.046

4.5

16

0.010

5

0.72

0.029

3.7

Traces

0.014 .

6

0.42

0.076

3.4

10

0.010

7

0.78

0.062

4.5

13

0.012

8

0.73

0.084

3.8

18

0.015

9

0.76

0.049

4.7

19

0.014

10

0.49

0.055

3.8

Traces

0.016

11

0.54

0.059

3.7

16

0.013

12

0.47

0.083

3.6

16

0.011

13

0.86

0.054

4.1

16

0.019

0.65

0.059

4.0

Average

0.013

be due to a change in the properties of humic acids as well as their frac­ tional composition. The method of analysis of group composition does not ensure an ideal separation of fractions and the properties of the latter are most dissimilar. Another reason could be the .non-uniform distribution of hu­ mic substances in the horizon. For most upper horizons the density of HA is due to the enrichment of these horizons with fresh organic remains and the presence of relatively 'juvenile' humic acids with developed peripheral chains. In the same samples the optical density of humic acid preparations was lower than E-values measured during analysis of group composition. This is due to the long time required for the separation of compounds, during which time humic acids could become decoloured, as described earlier. The most important reason for variation is the dissimilar conditions of separation and preparation of the compounds for investigation, although identical frac­ tions'show closer extinction coefficients. T/:1is is best confirmed by matching the E-values for HA of some soils determined by Kononova and later by Orlov (1959).

179 The extinction coefficients of HA from various types of soils also change in a wide range. These values were reported earlier [Orlov, 1974]. Later publications necessitate no change whatsoever in the already established general pattern. The large scatter of values, for reasoni cited above, permits only a probable estimation of type specificities of humic substances from the average values, The range of measured EO.00 1%HA values is fairly large. It is characteristic that the greater the number of determinations, the broader this range. However, a general pattern becomes rather evident from the average values. The average E2sgo 1%-values of humic acids of various soils are as under: Soil E-value Tundra soils 0.029 Sod-podzolic 0.049 Brown-forest 0.050 0.076 Grey forest 0.113 Chernozems 0.067 Chestnut soils 0.070 Solonetses, solods 0.093 Saline soils 0.080 Serozems 0.070 Sod, meadow etc. 0.071 Bog soils Krasnozems, red-coloured 0.073 soi.ls The maximum E-values are seen from chernozems and minimum for tundra and some high montane soils. It is not possible to establish the ab­ solute units of E-values at present. It is quite likely that for most optically dense humic acids it should not be higher than 0.25-0.40. The calculated value, as shown above, is close to 0.39. The minimum values for HA in the native state are closer to 0.02-0.03 [Orlov and Pivorarova, 1971]. Still lower values reported by some investigators should be taken as due to experi­ mental conditions. Very high E-values have been reported for some soils of Japan [Kumada, 1988] but this is predominantly for humic acids which have possibly experienced high temperatures. The spectrophotometric characteristics of fulvic acids are difficult to es­ timate. This is because for these acids the method of separation is not al­ ways mentioned and many results relate not to the true FA (as per Forsyth), but the entire ensemble of acid-soluble components of the alkali extract of soils. According to our data, the true FA and organic substances of the 1 % values of the acid-soluble fraction (including true FA) show average order of 0.013-0.015 (Table 54). For FAs investigated by us the EO.00 1%FA values are in the range of 0.007-0.018. This is a considerably narrower range than that for HA. Its upper limit overlaps minimum E-values for HA. We do not examine here the continuous series of E-values as a proof of

E2sg0

180 Table 54. E-values of fulvic acids and acid-soluble fractions Acid soluble Fulvic acid,

fractions,

Soils

EO.OO 1%FA

EO.OO1%C

Sod-podzolic

0.011-0.016

0.005-0.027 0.007-0.017

Brown forest Sandy grey

0.017

Typical chernozem

0.010-0.014

0.013-0.025 0.010-0.019

Podzolised chernozem Gommon chernozem

0.013

Southern chernozem

0.013

Red-coloured

0.011-0.017

Mountain-meadow

0.007-0.018

Humus-carbonate

0.011

Sod

0.014-0.026

Flood plain alluvial

0.015

Peat-gley

0.029 Average

0.013

0.015

similar nature of HA and FA although an analogous path of the light ab­ sorption curve points to the probable commonality of chromophore groups. The E-values of acid-solubles are in the range 0.005-0.029 (in terms of carbon) or 0.003-0.015 (in terms of FA). Besides true fulvic acids this frac­ tion includes colourless (or very light-coloured) non-specific compounds and, possibly, readily soluble representatives of humic acids, which explains the .wide range of these values. The optical density of substances of the acid-soluble fraction is 1/2-1/3 that of FA. Hence it may be considered that, on average, about half the car­ bon in them is represented by non-specific compounds. To characterise FAs determination of extinction coefficients in the acid-soluble fractions is thus not worthwhile. However, these coefficients could be useful for estimating the proportion of the non-specific constituents in these fractions. If we consider that the average EO.001%FA = 0.013, then the carbon . content is 40% and upon conversion to carbon the E-values could be taken as equal to 0.033. Considering that only fulvic acids are coloured in the acid-soluble fraction and denoting the E -value of this fraction as pOlourF, it is easy to find that part of the total carbon of the fraction which represents the true FA. It is clear that this proportion would be equal to polourF for

181 0.033. For example, for the acid-soluble fraction of flood plain alh:JVial soil (see Table 54) we get 0.015 x 0.033 = 0.45, that is, about 45% of the entire organic carbon of the fraction is due to true fulvic acids.

INFRA-RED SPECTRA OF HUMIC SUBSTANCES

Infra-red spectroscopy (IRS) provides exceptionally rich information not only about the range of important atomic groups and types of bonds, but also about the specific disposition of individual groups. The great advantage of this method lies also in the possibility of investigation of humic substances as well as of soils as a whole without taking recourse to any reaction on the object under study, except pulverisation. In other words, a non-destructive method of investigation is employed which allows us to resolve the problem of relationship between HA preparations and the native substances present in the unaltered soil [Orlov et al., 1962; Orlov, 1971]. Despite the large vol­ ume of work done in the technique of IR spectroscopy and the interpretation of spectra, there are still many unresolved problems not only in the area of precise identification of undivided absorption bands of HA and FA, but also in the comparative characteristics of substances and establishment of their identity. Complications during interpretation and use of IR spectra owe their origin to several factors. Humic substances contain a vast range of diverse atomic groups. Hence the absorption bands are usually composite and broad and owe their origin to vibrations of various groups. The position of maxima is not always precisely determined and could be intermediate between the maxima of constituent components. Mineral components contribute consid­ erable complications. Many of them give independent absorption bands in the same regions as humic substances. Interaction of mineral components with the functional groups of humic substances also significantly changes the spectra of the latter. Absorbed water contributes considerable distortion, which masks the important regions of the spectrum in which occur vibra­ tions of the OH-group at about 3400, 1600 and 1400-1300 cm- 1 . Unequal quantities of water in KBr pellets sometimes give rise to negative absorp­ tion, as a result of which pseudobands appear. In some cases, especially while investigating the 3400 cm- 1 region, the effect of water can be elim­ inated if we replace the KBr technique with preparation of samples in the form of finely pulverised powder in vaseline oil (Nujol). But then the specific absorption band of the oil in the 2920-2850, 1640 and 1380 cm- 1 regions precludes observation of the bands of humic substances in those frequency ranges.

182 The literature available on IR spectra of humic substances and then satellite components is extensive. Major attention has been paid to evaluat­ ing the structure of humic substarices whereas the investigations denoted to precise identification of individual bands are relatively few. Band designation is often done on the basis of standard tables of characteristic frequencies. Applied to humic substances such formal interpretation of spectra does not always yield reliable results. The author has been conducting a systematic study of IR spectra of hu­ mic substances since 1957. A comparative study of spectra clearly reveals that humic acids separated from various soils have monotypic IR spectra. This enables us to indicate the common motif of their structure [Orlov, et aI., 1962]. The common nature of the spectra was so well expressed as to lead to the conclusion that a special class of compounds exists whose formation is due to similar and specialised processes. The IR spectra were considered as a characteristic diagnostic feature. From the combination and intensity of bands it may be suggested that benzoid structures must play a great role in the structure of humic acids. This is confirmed by the intense absorption at 1610 cm- 1 . This band decreases but does not disappear during destruction of HA by pyrolysis and oxidation. At the same time we have shown that if the aromatic nucleus is present, then it must be represented by polysubsti­ tuted rings since in the spectra the bands of aromatic =C-H groups do not appear at 3030 cm- 1 . This is possible when four or more hydrogen atoms are substituted in the aromatic rings. According to our results, the IR spectra show the presence in HA of conjugate C=C bonds, a small content of CH 3 and CH 2 groups and the al­ most total absence of saturated hydrocarbons -(CH 2 )n - with a chain length n ;:::: 4. The small quantity of such hydrocarbons, revealed by chemical meth­ ods, appears to be a contaminant and does not significantly affect the ab­ sorption spectra. Shifting of the band of valence vibrations of -OH to the lower frequency confirms that in the solid state strong intramolecular hy­ drogen bonds develop in HA. These bonds could play a great role even in solutions facilitating association of molecules and formation of colloidal systems. In 1962, several absorption bands typical of humic acids were observed directly in soil samples using the method of differential spectrometry [Orlov et aI., 1962]. This confirmed the actual presence of important structural units in natural soils. Besides the common feature of humic substances as a special class of compounds, IR-spectra allow us to reveal some of their special fea~ tures related to structure and conditions of formation. The spectra of hu­ mic acids from sod-podzolic soils show the band of carboxyl absorption as

183 often twinned with maxima at 1685 and 1615 cm- 1 • This may be the con­ sequence of the simultaneous presence of carboxyl groups of aromatic and aliphatic groups or the higher content of ketones (quinones) albeit the ef­ fect of amides cannot be ruled out. The spectra of HA of sod-podzolic, grey forest soils and chernozems under forest, show distinct bands of CH 2 and CH 3 groups. A higher content of such groups with relatively lower role of aromatic structures in specimens separated from forest soils pOints to the varying degree of benzoicity of humic acids. The spectra of HA of steppe chernozems show distinct absorption bands typical of benzoid components. In some cases it was possible to observe that the type of vegetation affected IR spectra more noticeably than the type of soil. This was also true for fulvic acids [Orlov and Zub, 1963]. Based on published sources and our own data, we have prepared a table of absorption bands of humic substances and some satellite components (Table 55). The Table presents the maxima of absorption bands in wave numbers (cm- 1 ). For wide bands and for variation of absorption maximum the range of wave number has been indicated which is most characteristic for the given group. For the fundamental position of bands we chose that one which was most often observed and matched closely with the classical frequency for the given group. Wherever necessary, variation in the position of the absorption band is discussed below while examining these results. The last column indicates changes which the absorption bands underwent during various treatments of humic substances. The bands are given in the Table in the order of increasing wave numbers. The band pairs belonging to one group were considered jointly and their position in the Table determined by the band with lower wavelength. 3600-3300 cm- 1 region. A very wide and intense band which appears in spectra not only of humic substances, but of soil mineral components. It is due to valence vibrations of hydroxyl groups (-OH) mostly bound with intermolecular hydrogen bonds. Their presence is confirmed by methylation and acetylation. When humic substances are methylated the band intensity decreases and the maximum shifts to 3600 cm- 1 . The remaining small band in the 3650-3590 cm- 1 region is characteristic of valence vibrations of the unbound -OH groups. The formation of hydrogen bonds decreases the vibration frequency and hence the wave number decreases from 3600 cm- 1 (which is characteristic of unbound free OH groups) to 3400-3300 cm- 1 . The overall nature of the band is shown in Fig. 28. The band intensity is usually high in the spectra of HA and FA and increases upon saturation with cations, especially aluminium and iron. This effect is evidently due to absorption of hydroxyls or the formation of basic salts, as a result of which the content of hydroxyl groups increases in the sample under investigation.

184 Table 55. Important absorption bands in the IR spectra of humic substances cm- 1

/Lm

Intensity

Group and vibration

Remarks

(1 )

(2)

(3)

(4)

(5)

3600

2.8

Moderate or weak

Unbound OH, valence Appears after intense drying or methylation of humic substance, typical of clay minerals

3500-3300

2.8-3.0

Strong

OH, bound by intra­ molecular hydrogen bonds, partly NH, valence

3200

3.1

Very weak

NH, valence

2920 and 2860

3.4 and 3.5

Moderate or weak

CH 2 , (CH 3 ), valence

Increases upon methylation, decreases upon heating above 300-350°C and totally disappears above 550°C in HA and above 400°C in FA

2600

3.8

Weak

OH of carboxyls in dimers of carboxylic acids, valence

Sometimes increases on oxidation, disappears upon methylation

1745

5.7

C=O in CH 3 COO­

In acetylated fatty acids

1725-1700

5.8-5.9

Strong

Decreases upon methylation and disappears totally on heating above 40D-470°C; in interaction products of C with bivalent and trivalent cations, also typical of hydroxides and clay minerals

C=O in COOH, partly Decreases or totally disappears upon other C=O substitution giving salts, reduction by diborane and heatil)g; upon methylation shifts in the 1725 cm~l region with increase of intensity, decreases on hydrolysis of FA

185 (1 )

(2)

(3)

(4)

(5) because of partial decarboxylation

1690-1630

5.9-6.1

1650

6.0

1625-1600

C=N, valence

Appears in interaction products of C-O of FA with phenylhydrazine, hydroxylation, etc.

Variable

Amide I

Disappears upon hydrolysis with 6 N HCI (sometimes related to C-O of ketones)

6.1-6.3

Strong

Complex band due to C=C, COO­ groups, band of amide I, and partly hygroscopic water

Found in clay minerals, but of weaker intensity

1610

6--2

Moderate

C=C (aromatic), possibly participation of carbonyls, linked by hydrogen bond

Retained upon pyrolysis and oxidation

1590-1580 and 1400-1390

6.3 7.1-7.2

Variable, depending on degree of substitution

COO­

Disappears upon conversion to H­ form (acidification, dialysis with 0.5 N HCI, passage through H+-resin)

1540

6.5

Variable

Disappears upon Amide 11 (nitro group possible in HA hydrolysis oxidised by nitric acid)

1510-1500

6.6--6.7

Weak

C=C (aromatic)

1460-1440

6.8-6.9

Weak, sometimes moderate

CH in CH 2 (or CH 3 ) deformation

Increases upon methylation

1400-1390

7.1-7.2

Moderate

Complex band (CH, COO-, OH)

Increases with substitution up to salts

1375

7.3

C=CH 3

Increases in acetylated preparations (Contd.)

186

Table 55. Contd. (1 )

1325 and 780

(3)

(2)

7.5 and 12.8 -

(4)

(5)

Calcium oxalates

In oxidation products of humic acids Increases upon methylation by diazomethane, acetylation, treatment with HF; decreases upon substitution that gives salts, and. deuteration

1250-1225 8.0-8.2 (1260-1200)

Variable

Carboxyl groups (C-O, partly OH)

1150

8.7

Variable

Tertiary alcohols

1100

9.1

Variable

Secondary alcohols

1050

9.5

Variable

Primary alcohols

1080-1050

9.3-9.5

Moderate or strong

Polysaccharides

900-860

11.1-11.6

Weak, often ab­ CH (aromatic), defor­ sent mation

With one unsubstituted hydrogen atom; intensifies during pyrolysis

860-730

11.6-13.7

Weak, often ab­ CH (aromatic), defor­ sent mation

Two or more unsubstituted hydrogen atoms

750

13.3

730 and 72013.7 and 13.9 490

20.4

C-C and C-O of polysaccharides Weak, often ab­ ";'(CH 2 )n- for sent

Reported for fulvic acids, disappears upon hydrolysis with 6 N HCI

Reported for fulvic acids

n ::::: 4

Polysaccharides

According to Piccolo and Stevenson (1982), the increase in band intensity with the maximum at about 3420 cm- 1 may also be because the formed complex contains hydrated metals of the type

187

Fig. 28. Infra-red spectrum of humic acid in the range of wave numbers 3600-2600 cm- 1 .

Among other groups, NH groups may show significant absorption in this region. However, their content in humic substances is low compared to that of OH and their role in the formation of the band is probably not high. Concomitantly, the NH group may have greater significance in the prohumic substances and some pigments. Sometimes weak absorption is seen at about 3200 cm- 1 on the higher wavelength side of the principal band. This has the shape of a step or bend and corresponds to NH groups bound with hydrogen bonds. We may expect the effect of NHt groups of amino acids (or peptides) in roughly the same frequency range. 3000 cm- 1 region. In this range of frequencies a distinct band with maximum at 3030 cm- 1 serves as the diagnostic feature of =C-H groups of aromatic compounds. However, even in detailed investigations of this region with LiF prism it was not possible to detect individual bands which could have been related to aromatic =C-H groups. This seems to refute the presence of aromatic (benzoid) components in humic acids. However, such a conclusion should not be considered valid since the indicated band appears only in spectra of aromatic rings with not more than two substitutions, while with a greater number of substitutions in the ring (more than 3-4) the intensity of the 3030 cm- 1 band becomes very low. The direct determination of degree of benzoicity of humic substances done by two methods-elemental composition and yield of benzene

188 polycarboxylic acids upon oxidation of humic substances-showed that 10-50% of the entire mass' of humic acids is represented by six-member (benzoid) carbon rings. Thus the absence of a 3030 cm- 1 band is not an argument in favour of the aliphatic nature of humic acids and points to the high degree of substitution of hydrogen atoms in the six-carbon rings. The predominance of three- and four-substituted rings was also confirmed by direct chemical methods. The conclusion regarding the almost near absence of less substituted rings in HA is confirmed in other regions of the spectrum. Thus compounds with mono-, di- and trisubstituted benzene rings show a series of bands in the 2000-1800 cm- 1 region whereas humic substances show only very weak absorption in this range. Stronger bands of deformation vibrations of aromatic CH-groups also appear in the 730-900 cm- 1 region. But in spectra of humic substances this interval shows one or two.low intensity bands. 2960-2840 cm- 1 region. In spectra of humic acid preparations the higher wavelength side of the 3400 cm- 1 band shows one band near 2900, or two bands with maxima at 2920 and 2860-2850 cm- 1 . The bands are generally of moderate intensity but may be weak at times. In some preparations these bands may be practically absent. The position of the bands agrees well with the intense bands of CH 3 and CH 2 alkane groups. According to Bellami, the mean values of the absorption maxima for the methyl group CH 3 are 2962 and 2872 ± 10 cm- 1 , while for the methylene group they are 2926 and 2853 ± 10 cm- 1 • It is more probable that the bands observed for humic substances are mostly not due to the terminal methyl but due to methylene groups, which is confirmed by an almost perfect/ideal agreement of wave numbers from standard values. The predominance of methylene groups is also indicated by the nature of the band in the 1380-1480 cm- 1 region. It must be emphasised that the observed methylene groups could not be a constituent of any long paraffin chains. The -(CH2)n-chains at n ~ 4 give intense absorption at 720 cm- 1 due to vibrations of CH 2 groups; moreover, for substances in a solid state the band is twinned. Humic substances show only a weak absorption in this region and that too not in all preparations. The low intensity of the band of CH 2 and CH 3 groups agrees with the suggestions regarding the small role of alkanes in the structure of HA and FA and also indicates that their aliphatic (hydrolysable) part is almost entirely made of amino acids and carbohydrates. 2800-1750 cm- 1 region. In this region humic substances show relatively weaker absorption; distinct bands are absent. Sometimes weak absorption is observed near 2600-2500 cm- 1 which is considered as due to carboxylic acids. This band is generally weak and broad but is considered very charac­ teristic of the OH group with a strong hydrogen bond of dimers of carboxylic acids.

189 Bands of anhydrides appear in this region. These are absent in the orig­ inal preparations but appear in some reactions, particularly during pyrolysis. Cyclic anhydrides could be formed when the COOH groups lie in pairs in the aromatic ring in orthoposition-this gives rise to five-member anhydride (I). When COOH groups are located on adjacent diphenyl rings in orthoposition,

a seven-member anhydride is formed (11). In soil HA such a disposition has been proposed for roughly 30% of all carboxyl groups [Wagner and Stevensen, 1965]. 1700 cm- 1 region. Humic substances have strong absorption in this re­ gion. However, according to various results the band maximum may shift from 1725 to 1700 cm- 1 . For humic acids the most characteristic position of the absorption maximum is at 1720 cm- 1 (Fig. 29). This band is typical

Fig. 29. Infra-red spectra of humic acids in the range of wave numbers 2000-700 cm- 1 • 1--grey forest soil; 2-typical chernozem.

190 for the carbonyl group, which may be represented by ketones, aldehydes and carboxylic acids. The position of the band depends greatly on conju­ gation, substituents and the presence of hydrogen bonds. In humic acids this band is mainly due to carboxyl groups; during the formation of different salts of humic acids (Na+, Ca2+, Ba2+, C02+, Ni 2+, Zn 2+ and others) this band disappears, though not always completely, and in its place appear two characteristic bands of carboxylate ion at 1590 and 1390 cm- 1 • The effect of substitution is reversible and upon treatment of humates or fulvates with acid or after passing them through cationic resin in H-form the band near 1700 cm- 1 reappears. However, even with the most complete substitution of hydrogen of carboxyls to salts, the spectra of humates (and partly ful­ vates) still show residual absorption, generally evidenced by a small rise on the low wavelength side of the 1650-1610 cm- 1 band. The residual band may be attributed to ketones, partly quinones. Some authors also attribute it to aldehydes and complex ester bonds, although the latter usually show absorption at higher frequencies (1730-1800 cm -1). During the formation of humate complexes of various metals it is quite probable that .substitution of the COOH group is incomplete. This may be because complex formation leads to a near-neutral reaction of the medium at which not all carboxyl groups are capable of ionisation. A detailed analysis of the 1700, 1600-1400 and 3600-330 cm -1 regions enabled Piccolo and Stevenson (1982) to conclude that besides the carboxyl groups other groups too, especially conjugated ketones, are involved in complex formation:

The spectra of hymatomelanic acids too change similarly [Glebova,

1985] during the formation of hymatomelanates. Distinct transformation of hymatomelanates into hymatomelanic acid and back to sodium hymatome­ lanate was observed by Glebova when she treated the original prepara­ tion of hymatomelanic acid first with hydrochloric acid and then with NaOH (Fig. 30, A). The original HMA preparation was evidently represented by a mixture of hymatomelanic acid and some of its salts. The salts may have been hymatomelanates of iron, and calcium in the natural soil, or of sodium formed during extraction of HMA from the soil. The usual method of sep­ arating humic substances does not ensure 100% conversion into H-form. After special treatment with hydrochloric acid it was possible to remove all metal cations and the HMA transformed to the H-form. Correspondingly, the

191 band intensity at 1720 cm- 1 rose sharply but there was decreased absorp­ tion in the 1600 and 1400 cm- 1 regions. This pure HMA was treated with NaOH to convert it into sodium hymatomelanate. As a result the 1720 cm- 1 band disappeared completely but the absorption band of carboxylate ion appeared. The reaction with fulvic acid proceeded similarly. The original prepa­ ration of fulvic acid was characterised by a very weak band at 1700 cm- 1 (Fig. 30, B). After passing the fulvic acid through cationic resin in the H-form, the picture changed drastically. An intense absorption band appeared with a maximum at 1700 cm- 1 which was attributed to the carboxyl group. Con­ comitantly there was an almost complete absence of the band at 1400 cm- 1 . The band with maximum at 1595 cm- 1 shifted to 1610 cm- 1 . The latter happened because the band at 1595 cm- 1 is a complex band and includes absorption due to C=C groups. The maximum of such a band occupies some average position at about 1595 cm- 1 . After the disappearance of the band of COO- at 1590 cm- 1 , the maximum of the C=C band occupied its true position near 1610 cm- 1 . This experiment illustrates the need for careful interpretation of spectra. Thus the low intensity of the COOH band in the original preparations of FA and HA does' not in any way indicate the low carboxyl content in them. The above examples confirm not only the need for a very careful inter­ pretation of spectra but also indicate the optimum conditions for obtaining

Fig. 30. Infra-red spectra of hymatomelanic acid (A) and fulvic acid (8). 1-hymatomelanic acid in H-form (after treatment with Hel); 2-same, as sodium hymatomelanate (after treatment with NaOH); 3-fulvic acid before passing through cationic resin; 4-same, after passing through resin.

192 them. In particular, while studying spectra in the band intervals of carboxyl groups and carboxylate ions, more reliable results are obtained only when the sample is in 100% acid form (one of the indices being low ash content). The spectra of preparations with low ash content enable us to obtain true representation of the most important functional groups of humic substances, i.e., COOH groups. Moreover, the effect of ionised groups on the spectra is eliminated. These groups show absorption at 1590 and 1390 cm- 1 and thus distort the representation of C=C and CH groups in humic substances. It unambiguously follows from the above discussion that in order to char­ acterise absorption due to carbonyl it is necessary to work with highly stan­ dardised preparations of humic substances [Orlov and Pivovarova, 1971; Orlov, 1972]. 1690-1630 cm- 1 region. This range is not represented very clearly in spectra of humic substances. In many cases this band, due to absorption by carbonyls, directly merges with the absorption band at 1620 -1610 cm- 1 • It is then not possible to identify any additional bands against the background of an overall strong absorption. Under favourable conditions the amide I band can be identified at about 1650 cm- 1 . Band of anhydride groups. In the spectra of many preparations of humic substances, especially when the nitrogen content is high, individual bands at 1650-1640 and 1550-1540 cm- 1 which are typical of amide groups appear. The first band may be considered as due to carbonyl of the amide group or the OCN group. The second band is due either to deformation vibrations of NH or mixed vibrations of OCN and NH. Its position in the spectra of HA usually corresponds to secondary amides which allows us to relate the en­ tire- range of amide bands to polypeptides. Such affinity correlates well with the intensity of the bands established for spectra of many proteins. Here it is important to emphasise that for several dark brown and red pigments produced by certain micro-organisms we observed a spectrum which was very similar to that of proteins, in which the main outline was due to three amide bands with gradually decreasing intensities [Orlov, 1974]. Sometimes the bands at 1650 and 1540 cm- 1 could not be observed in the spectra of HA; this does not mean that humic acids were totally devoid of polypeptide components. The fact is that absorption by peptide groups may be super­ imposed on stronger absorption by carbonyls or C=C double bonds. As a result, an independent band does not arise but the maximum of the com­ posite band shifts towards· 1650 cm- 1 . This is one of the reasons explaining the unstable position of the absorption band in the interval 1625-1600 cm- 1 • . Upon hydrolYSis with 6N HCI the first amide band disappears (Fig. 31). 1625-1610 cm- 1 region. In this region of the spectra of humic sub­ stances is found the third intense absorption band. Among the groups re­ sponsible for absorption near 1600 cm- 1 could be aromatic C=C, carbonyl C=O linked by hydrogen bonds and quinones also bound to OH groups

193

Fig. 31. Infra-red spectra of humic acid from sod-podzolic soil. 1---before hydrolysis; 2-after hydrolysis with 6 N Hel.

by hydrogen bonds. The presence of conjugate C=C and C=O groups or the effect of polar substituents is usually emphasised in the literature. This wide range in interpretation is due to the complex and often contradictory behaviour of this band during various reactions of humic substances. The band intensity also decreases on heating above 250-300°C whereas the formation of salts may lead to its increase. We were able to observe that on the action of different oxidants (KMn04 , H2 0 2 ) on humic acids and on heating in air the 1625-1610 cm- 1 band considerably narrows and its inten­ sity decreases. At the same times its· maximum occupies a highly specific position at 1610 cm- 1 and presists even after completion of the reaction. This led us to suggest that the aromatic C=C are responsible for this band; their maximum lies at 1610 cm- 1 . Broadening of the band and its shift to­ wards the short wave side could be due to superimposition of the amide band; a shift towards the long wave side is due to the effect of carboxylate ion. Moreover, near 1625 cm- 1 absorption due to water appears since its content in HA is rather difficult to control. Band of carboxylate-ion. In humates and fulvates the ionised carboxyl group shows stable absorption near 1590 and 1400 cm -1. But it does not always appear as individual bands. The band at 1590 cm- 1 merges with the 1610 cm- 1 band, shifting its maximum to 1600 cm- 1 ; the band at 1400 cm- 1 is superimposed on the region of absorption due to deformation vibrations of CH 2 and CH 3 groups. Weak bands of carboxylate have been reported in the literature even for free HA. However, this phenomenon is more likely explained by partial formation of potassium humates during pressing of humic acid with KBr. 1510-1500 cm- 1 band. This band in association with the absorption near 1610 cm- 1 indicates the presence of aromatic components. The inten­ sity of absorption at 1510 cm- 1 in spectra of humic substances is low even though in ~pectra of aromatiC compounds the 1500 cm- 1 band is usually more intense than the 1600 cm- 1 band. This fact once again highlights the

194 complex nature of the 1600 cm- 1 band and suggests that the contribution of benzoid rings to the structure of HA and FA is not as high as repeatedly reported in the literature. 1470-1370 cm- 1 region. In this region the spectra of all humic substances have two, sometimes more bands. The maximum number of bands is observed at high resolution of the instrument. The important bands of humic substances in this range are due to deformation vibrations of C-H although near 1390 cm- 1 the deforma~ion vibration ofO-H and vibrations of the C-O group could also appear .. The first group of bands in the 1460-1440 cm- 1 region almost coincides with absorption bands due to the antisymmetric vibrations of C-CH3 group and -CH 2 - group. It is almost impossible to distinguish these groups just from the position of the bands. However, since in the region of valence vibrations preference ought to be given to the methylene group, it would be appropriate to relate the deformation vibration band at 1460-1440 cm- 1 to CH 2 . The band near 1370 cm- 1 could be attributed to the symmetric deformation vibration of C-CH3. The intensities of these bands are low and. correlate poorly with each other. In this range superimposition of the carboxylate band is possible, as also the deformation vibrations of -CH 2 group, OH group (and possibly C-O) of certain alcohols and phenols. The complex nature of absorption and strong dependence on the state of the substance compels us to give only limited importance to this region. 1260-1200 cm- 1 region. In this region humic substances show vari­ able intensity of absorption, changing from weak to moderate .. The posi­ tion of the maximum too is not stable and at high resolution several bands become apparent (1260-1250, 1220, 1200 cm- 1 ). The band intensity de­ creases sharply on the formation of humates and fulvates, which indicates that the band is mainly related to the carboxyl group of carboxylic acids. Gen­ erally a concomitant indication of C-O and O-H vibrations is also manifest. The participation of bands of aromatic groups CO-Me has been reported. Without rejecting the complex nature of absorption, it should nonetheless be attributed to deformation vibrations of atoms of the carboxyl group. 1100-1000 cm- 1 region. This region and that of long wavelengths have been inadequately studied. These regions are influenced by the numerous mineral components present in humic substances as admixtures or as their compounds. Very intense and broad bands are given by all silicates (clay minerals) with the maximum at 1050-100 cm- 1 while inorganic orthophos­ phates show absorption at 1050 cm- 1 • After various treatments of humic substances in the presence of sulphate ions, an absorption band by 8=0 groups with the maximum at 1040 cm- 1 may be observed. Characteris­ tic bands appear here for several organic substances which could either be constituents of humic substances or simply incidental impurities. Thus in the region 1180-950 cm- 1 bands appear due to absorption by sugars

195

and glucoside bonds. For identification of polysaccharides, use is made of absorption at 1080 cm- 1 • For low ash preparations of humic substances from soils absorption near 1000 cm- 1 is attributed to vibrations of the alcohol groups. The entire 1100-1000 cm- 1 region in the spectra on HA might be attributed to COH of alcohols albeit the type of vibrations have yet to be assertained. In spectra of ash-free preparations of humic substances near 1100 cm- 1 it is possible to observe two, sometimes three bands. The most intense absorption is at about 1050 cm- 1 . This may be tentatively explained by the presence of primary alcohol groups. Bands were observed in the spectra of fractions extracted from soils using organic solvents at 1080, 1050, 970 and 900 cm- 1 . This may be considered the result of planar and extraplanar deformation vibrations of aromatic OH groups. Of course, these are not masked by other bands. Re~ion of wave numbers less than 1000 cm- 1 • Humic and fulvic acids exhibit no intense absorption whatsoever in the interval from 1000 to 600 cm- 1 . It is possible to observe only, relatively weak and diffused bands-but not in every sample! According to the literature and our results two or three bands appear between 900 ana 750 cm- 1 , possibly due to extraplanar deformation vibrations of C-H in the aromatic rings. With one unsubstituted hydrogen atom in the benzene ring a band of moderate intensity usually appears near 900-860 cm- 1 • This band is often observed in spectra of humic substances, but has low intensity. At lower frequencies vibrations of the CH group or aromatic rings appear, Which have two or more unsubstituted hydrogen atoms. Thus the nature of the spectra in the 900-700 cm- 1 region confirms earlier Conclusions drawn regarding the relatively lower content of aromatic components in humic substances and the absence of rings with more than two unsubstituted hydrogen atoms. Organic (non-specific) admixtures exert some influence on the IR spectra of humic substances. Thus in spectra of lipids (wax-resin fraction, 'bitumens') intense bands in the 1380-1480 cm- 1 region are attributed to CH 2 groups and CH 3 of saturated and unsaturated hydrocarbons. The presence of unsaturated hydrocarbons is confirmed by a band of moderate intenSity at 1820 cm- 1 , which correlates in the first approximation with the iodine number and the band at 880 cm- 1 attributed to terminal RC=CH 2 groups. Carbonyl absorption is weakly expressed in the 1710-1780 cm- 1 interval. Very characteristic intense bands of alcohol groups appear in the 1040-1080 cm- 1 interval. Aromatic rings give only weak bands at 1580-1600 cm- 1 . Many other bands are also observed, which give on the whole a specific character to the spectrum [Ammosova, Orlov and Semenova, 1971]. In the spectra of peat waxes distinct absorption bands have been no­ ticed at 2960-2920,2860,1740,1715,1640,1610,1520,1470, 1420, 1380,

196 1270, 1175 and 730-720 cm -1 . Among them those at 1740 and 1175 cm- 1 relate to saturated complex esters; 1715, 1640, 1430 and 1270 cm- 1 to un­ saturated complex esters; 2960-2860, 1470, and 730-720 to long chains [Yurkevich, 1971]. Among substances extracted from soils by a mixture of benzene-methonol-acetone, phthalic esters and phthalates have been iden­ tified besides paraffins, alcohols and their esters. For these a doublet at 1603-1585 cm- 1 is characteristic; it is related to the esters of phthalic acid. The bands at 1285 and 1135 cm- 1 are due to C-O group of phthalates and benzoates. Deformation vibrations or aromatic CH have been observed at 1082, 1050,970 and 700 cm- 1 . Another group of more probable admixtures is represented by polysac­ charides which may sometimes predominate in some fractions of humic sub­ stances. For IR spectra of the polysaccharide fraction of soil, Lowe (1968) gave a list of bands (Table 56) which coincide closely with bands of hu­ Table 56. Absorption bands of polysaccharide fraction [Lowe, 1968] Absorption maximum, cm-

Intensity

Affinity

1

3400

Strong

O-H

2920

Strong

Aliphatic C - H

1730

Strong

C=O of carboxyls or esters

Strong

C=O of aldehydes or ketones

Weak

COO­

1630-1650 1550 1360-1390

Moderate

C - H of methyl

1220-1250

Moderate

C-O of esters and others

Weak

C-O-C, C-OH or aliphatic C-C

Strong

O-H or C-O-C in ring

1140 1030-1070 890

Weak

C-H

810

Weak

Not determined

mic substances. The relative intensity of bands in the spectra of HA and of polysaccharides differs. Inorganic admixtures could introduce considerable distortions in the spectra, more so because preparations of humic substances with a low ash content are rather difficult to obtain. All silica-containing compounds, . phosphates, sulphates, carbonates, as well as some oxides and hydroxides may very strongly influence the spectra. Figs. 32 and 33 present typical

197

Fig. 32. IR spectra of mineral components of soils.

Fig. 33. IR spectra of alluvial soils and their clay fraction [Orlov and Osipova, 1988].

1-amorphous silica; 2-quartz; 3-mont­ morillonite; 4-kaolinite.

A-3800-3200 cm- 1 region: 1-soil as a whole, 2--clay fraction of the same soil; 8-1300-400 cm- 1 region: 1-soil as a whole, 2--clay fraction of the same soil.

spectra of soils, clay fractions and characteristic minerals. Table 57 gives the most intense bands of possible mineral admixtures. A similar analysis of the influence of inorganic components was given earlier [Orlov and Osipova, 1988]. Table 57. Wave numbers of moist intense bands of characteristic mineral admixtures Compound

Wave number, cm- 1

(1 )

(2)

Carbonates Na2C03

1450, 1375, 880

MgCO:j

1520, 1420,855

CaC03

1420, 870,710

Dolomite

1450, 880, 730

Siderite

1430,1170,895,795 Sulphates

Na2S04, K2S04

1130,615

CaS04

1150,670

Gypsum

1630,1150,1115,670;605

Anhydrite

1060, 680, 615 (Contd.)

198 Table 57. Contd. (2)

(1 )

Phosphates

CaHP04 ·2H2 0 Apatite Vivianite

1645, 11~, 1060,985,870

1095, 1045

1045, 1005,960,945

Nitrates

Quartz Quartz Quartz

1370, 890, 820

1365, 835, 1270

Oxides and hydroxides

Quartz

1170,1090,795 and 770 (doublet), 470

Opal

1080, 470

8i02 , amorphous

1100

1160,680,460

a-A1 2 0 3

'"Y-AI 2 0 3

790-610

Gibbsite

3600-3400, 1020, 800, 750

a-FeOOH Muscovite Biotite Vermiculite Chlorite Kaolinite lIIite Montmorillonite

3200, 900, 800, 400

Aluminosilicates

3640,1070-970,520,470

3600, 1615, 1020-960

3410, 1000, 450

3570, 985-955, 650, 440

3700,1090,1035,1015,915,695,545,470

3630, 1030, 470

3450-3420, 1030, 1015,520,470

5

Molecular Parameters of Humic

Substances

Molecular parameters of humic substances include such fundamental char­ acteristics as the mean molecular mass, shape and size of molecules, de­ gree of polydispersion and the nature of molecular mas.s distribution. Molecular parameters should be considered the most important funda­ mental characteristics necessary in solving problems of the structure of hu­ mic and fulvic acids as well as in evaluating their pedogeochemical role. The stability, capacity to migrate in a porous medium, sorption by soil minerals and potential assimilation by micro-organisms and higher plants depend on the size and configuration of particles of humic substances. Any structural model of humic substances should be based on the shape and size of its molecules. Determination of molecular mass and shapes of particles of specific hu­ mic substances is a complex task. This is because humic and fulvic acids are insoluble in the usual organic solvents and humic acids are almost insoluble in water. Solutions of humic substances are strongly coloured, which de­ creases the precision of many measurements. Samples of humic substances isolated from soils usually contain mineral elements and low-molecular-mass organic compounds as contaminants. However, the greatest difficulties arise because of polydispersion and heterogeneity of humic substances. In the early period of investigation, humic substances were considered individual substances and simple formulae were proposed and molecular weights were calculated for them as for simple organic compounds. After their polydispersion and heterogeneity was proven many investigators gen­ erally refrained from attempts to estimate the molecular mass of humic sub­ stances. Development of the chemistry of high-molecular-mass compounds again generated interest in the problem of the molecular mass of humic substances. Prof. Gemmerling of Moscow University was the first to pay attention to the polydispersion of humic substances and the dependence of intensity of colour of HA on their particle size. According to the scheme proposed by him in 1921, humic substances, based on their colloid-chemical prop­ erties, could be subdivided into coarsely dispersed (represented by humic

200 coals), colloidal systems (humic and fulvic acids) and molecular dispersed. Using this scheme, Gemmerling clearly explained the dissimilar coloration of several soils with equal humus content and some other properties. Polydispersion of humic and fulvic acids has been proven by experi­ ments and conclusively established by many methods of ultracentrifugation, adsorption chromatography [Yonebayashi and Hattori, 1990] and gel filtra­ tion. Simultaneously it became known that it is necessary to distinguish two types of polydispersion of humic acids. True polydispersion is due to the simultaneous presence of molecules of different size in the composition of the substance while secondary poly(jispersion is due to the capability of hu­ mic acids to form associations of molecules because of hydrogen bonds or intermolecular interactions. The former does not depend on the state of mat­ ter nor composition and concentration of solutions, whereas the degree of formation of associations of molecules is affected by the ionic strength of the solution, pH, presence of polyvalent cations and concentration of the solu­ tion. It is not always possible to experimentally differentiate the two types of polydispersion, although this is most important for estimating the properties and behaviour of humic substances. Some ways of solving this problem are discussed in this chapter. Published data on the size and shape of particles of humic substances is quite varied and at times contradictory. The values of molecular masses reported by several authors can be conveniently placed in two groups: first group-from a few hundreds to 10,000-20,000 daltons; and the second group-from 20,000-30,000 to 100,000-200,000 daltons (higher values have also been reported). In explaining such sharp variation Aleksandrova wrote: 'Obviously it is necessary to differentiate molecular and micellar (particulate) weight of humic acids' [Aleksandrova, 1962; p. 83]. Aleksandrova's contention was absolutely justified when results obtained by comparative methods were compared. At the same time, it was shown that the grouping of molecular mass is largely associated with the methods of its determination [Orlov and Grishkova, 1965). For HA the calculated molecular masses and those determined by chemical methods, are usually in the range of 1300-13,000. Osmometry, cryoscopy, ebullioscopy, dialysis, diffusion and viscosimetry give values of the order of 700-26,000 (for FA the lower limit drops to 200-300). Ultracentrifugation and light scattering methods give values of the order of 30,000-80,000. In recent years accurate results based on various methods confirming and enlarging the earlier published data have been obtained [Aiken and Gillam, 1989; Chen and Schnitzer, 1989; Clapp et aI., 1989; De Nobili et aI., 1989; Duxbury, 1989; Hayes et aI., 1989; Orlov, 1992; Swift, 1989; Wershaw, 1989]. Methods based on estimates of the total number of particles in solu­ tion give the lowest values of molecular mass. Higher values are obtained when methods which measure molecular mass as a function of particle size

201 (volume) are used. The average values for the polydispersed system differ depending on the method used. Unfortunately, in soil literature molecular mass values are often reported without mentioning the method of determi­ nation and hence the method of averaging. Hence, according to Lansing and Kremer, depending on the method of averaging results, we may distinguish the mean numerical molecular mass M n' mean weighted molecular mass M wand the mean molecular mass M z' The mean numerical molecular mass is equal to the sum of the mass of all molecules in a mixture divided by their total number:

M = L,MxNx n L,Nx ' where Nx is the total number of molecules of x type; M x is their molecular mass. This is the simplest method of averaging. The mean numerical mass is found by methods which enable us to calculate the total number of particles in a solution. These are the methods of determining terminal groups, cryoscopy, ebullioscopy and osmometry. These methods give very low values of molecular mass of HA and FA. The mean weighted molecular mass takes into account the weight (mass) fractions of molecules of each size and its value is essentially analogous to the weighted mean value

M

= w

L,M;Nx L,MxNx'

The mean weighted molecular mass is found by light scattering, diffusion and gel filtration methods. Mz, the mean molecular mass, is determined by the equilibrium sedi­ mentation method. Its value is related to the molecular mass of individual components by the equation:

M =L,M;Nx z L,M1Nx ' Moreover, use is also made of the so-called mean viscous molecular mass. It is found from the results of viscosimetric measurements using the relation:

[1]]

= K·

M~,

where [1]] is the characteristic viscosity, M b the mean viscous molecular mass and 0: and K are constants. The mean viscous molecular mass is close to the mean weighted mass in many cases but may also significantly differ from it. The more the de­ pendence between viscosity of the solution and molecular mass deviates from the linear, the greater the divergence of values. In other words, if 0: = 1

202 then M b = Mw. The characteristic viscosity can be used to calculate the molecular mass if the constant K is known, which is unique for each series of polymer homologues. In monodispersed systems all methods of averaging lead to the same value of the molecular mass: Mn=Mb=Mw=Mz­

In the polydispersed system the mean molecular mass found by various methods could differ by several orders of magnitude. Moreover Mz>Mw>M n·

The coincidence of values of M z' Mw and M n is proof of the monodis­ persed system, while the M w/ M n is used as an index of degree of polydis­ persion. Since the properties of HA are dictated by the fraction predominant by mass, the mean weighted molecular mass should more or less be well cor­ related with the properties of the preparations. However, this is also possible by other methods of estimation and calculation. It is advantageous to cal­ culate the mean weighted molecular mass based on simplest molecular for­ mulae as these give an idea of the size of the simplest elementary units and thereby describe the possible values of mean numerical molecular mass. In the chemistry of humic substances there is as yet no commonly ac­ cepted terminology for molecular parameters. For this purpose we think it fit to use the following concepts. Minimum molecular mass. Its value corresponds to the simplest formula (and elementary unit) of humic substance calculated from the elemental composition. This value denotes the lowest limit of the possible molecular masses. Structural fragment. This is that part of the molecule of a humic· sub­ stance separated during decomposition and having a relatively simple struc­ ture. Changes in structural fragments are possible during decomposition. Structural cell. This is that part of the humic substance which contains all the important (essential) structural units in minimal yet proportional quantity. Its size is determined from the yield of the destruction products of benzene­ polycarboxylic acids or by multiplying the minimum molecular mass by the number of the principal functions of the element present in minimum quantity. Generally this is nitrogen. Molecule of humic substance. An individual particle in which the bonds between atoms are solely due to the forces of the principal valence electrons and which include one or several structural cells. Simple molecular associations (dimers, etc.). These are formed due to the accessory valence electrons and intermolecular interactions and are the result of the first stage of formation of supramolecular structures.

203 Complex associations (micelles or aggregates). These are formed as a result of intermolecular forces when the forces of mutual repulsion of molecules are eliminated under the influence of pH of the medium, elec­ trolytes and so on. As applied to the simple and complex associations of humic substances, we use the term 'particles', and 'particulate mass'. Such terms should be used for dispersed formations of any nature and size. Simple and complex associations could obviously produce diverse forms of supramolecular structures of higher orders: chains, clusters and so on. Dif­ ficulties arising during analysis of the molecular mass of humic substances are often due to the variable degree of polydispersion. Different instances of non-homogeneity are possible in the system of hu­ mic substances when: 1) the molecules differ only by size (length of chain); 2) molecules close in size differ in composition; 3) the size and composition of molecules change concomitantly and 4) homogeneous or heterogeneous molecules interact because of mineral bridges or hydrogen bonds. The first three cases are related to true molecular polydispersion. The last case char­ acterizes the formation of associations if hydrogen bonds are present. The particle associations formed due to the forces of accessory valence electrons belong to supramolecular structures [Rafikov et aI., 1963]. We shall include dimers and associates formed due to hydrogen bonds, intermolecular inter­ actions and bond bridges under supramolecular structures. The extent of their formation depends on the ionic strength, pH of the medium, ion-salt composition and equilibrium solutions. Whichever way we look at the data published on molecular parame­ ters, we have to unambiguously accept that the properties of a polydis­ persed substance depend predominantly on that fraction which occurs in maximum quantity (predominance by mass). Hence the mean weighted molecular mass should most completely describe such a substance. Ac­ cording to numerous published results, the molecular mass of soil humic substances varies over a wide range. Chemical methods give values from 1300 to 13,000; osmometry, cryoscopy, ebullioscopy and dialysis give from 700 to 26,000. For fulvic acids, the lower limit of molecular mass drops to 200-300. The methods of ultracentrifugation and light scattering give values of the order of 30,000-80,000. Attempts to use non-standard methods lead to reports of very high molecular mass. Thus the molecular mass calculated from the concentration of paramagnetic centres reach a figure of hundreds of thousands or several million dalton. X-ray observations serve as the ba­ sis for a discussion regarding values of the order of 210,000 and 1,000,000. Electron microscopic observations show particles with size corresponding to relative mass of the order of 100,000 to several million. All these differences are clearly associated with the above-mentioned methods of averaging the values. The group of relatively low values of molecular mass (from 200-300 to 10,000-20,000) observed by methods of osmometry, ebullioscopy etc.

204 characterises mean numerical molecular mass. Such results greatly depend on low-molecular-mass admixtures including water and simple salts. Several published values defy logic. Reportedly, the experimentally ob­ served values of M n for several FAs were equal to 262-300 [Solominskaya, 1969], whereas based on the nitrogen content (4.03 and 4.97%) the mini­ mum molecular mass should be 696 and 564 if it is considered that an FA molecule contains at least two atoms of nitrogen. Such results (unfortunately published quite often) force us to conclude that either the determination of molecular mass has not been don~ at the required level (use of a method incompatible with the substance) or the fraction under reference cannot be considered as an individual substance but a mixture of diverse molecules. Very low values have been obtained by cryoscopy, isothermal distillation and other methods. As demonstrated by Schnitzer, large errors may crop up in determination of molecular mass because of the dissociation of humic substances. In his first experiments the M n values of fulvic acids were of the order of 460-670 [Schnitzer and Desjardins, 1962, 1969; Barton and Schnitzer, 1963]; later, after introducing corrections for dissociation and taking recourse to fraction­ ation, these values increased to 920-970 and in some fractions even up to 3570 [Hansen and Schnitzer, 1969; Schnitzer, 1968], which accord well with elemental composition and structural units. Some results from Schnitzer's work are reproduced in Table 58. To estimate the effect of dissociation on the results of determination of molecular mass we can use the equation:

C(1 - a)

+ nCa + Ca =

C(1

+ na),

Table 58. Effect of dissociation on values of molecular mass of fulvic acids and their fractions [after Schnitzer and Skinner. 1968] Yield.

M n in osmometry

9 Fraction

Experi­

Corrected for

mental

dissociation

444

688

1500

3570

0.232

710

1179

0.141

493

754

0.265

276

337

Unfractionated fulvic acid

A 8

0.164

C

0