UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The sodium hypochlorite oxidation of humic acids and prepared lignins Herman, William Allan 1977

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1978_A1 H47.pdf [ 3.63MB ]
Metadata
JSON: 831-1.0094514.json
JSON-LD: 831-1.0094514-ld.json
RDF/XML (Pretty): 831-1.0094514-rdf.xml
RDF/JSON: 831-1.0094514-rdf.json
Turtle: 831-1.0094514-turtle.txt
N-Triples: 831-1.0094514-rdf-ntriples.txt
Original Record: 831-1.0094514-source.json
Full Text
831-1.0094514-fulltext.txt
Citation
831-1.0094514.ris

Full Text

THE SODIUM HYPOCHLORITE OXIDATION OF HUMIC ACIDS AND PREPARED LIGNINS by WILLIAM ALLAN HERMAN B . S . A . , University of Alberta, 1972 M . S c , University of Alberta, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Soil Science University of Br i t i sh Columbia We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 (c) William Allan Herman In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date C f g j y<?77 ABSTRACT In order to investigate the relative merits of a selective oxidant for the degradation of natural polymers, humic acid extracts from three Alberta soil sites and three Bri t i sh Columbia soil s i tes , and two Kraft's prepared wood lignins were oxidized with 1.6 N NaOCl at room temperature (23°C) for 5 days. The oxidation products included highly volat i le acids and organic solvent soluble (OSS) products with the relative size of each fraction determined by carbon content. OSS products were characterized by Infrared (IR), Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) techniques and identified after methylation and separation by Gas Liquid Chromatography (GLC) methods.involving co-injecting authentic compounds and matching elution time and temperature of some of the components :with.that .of the authentic compounds. Proceeding from the known chemistry of the NaOCl reaction, the CO2 and highly volat i le acid products could only be derived from the al iphatic chain or saturated ring components of humic acids or l ignin and represented 66 to 82% of the products assuming no destruction of aromatic structure. Benzene carboxylic acid products were derived from the aromatic component of the starting materials. Estimates of the degree of aromaticity of the starting materials, using GLC and potentiometric t i trat ion data, were substantially less than those calculated from proposed model humic acid and l ignin structures i i i in the soi ls l i terature . An unidentified o i ly component was found in the oxidation products of two of the humic acid preparations. The results of this study indicated humic acid and l ignin are composed of a mixed aliphatic-aromatic compound system. The relat ively more mature humic acid preparations were found to be of greater aromaticity than the less mature samples. It was found 2 that NaOCl was not total ly selective in differentiating Sp from 3 Sp carbon hybrids; as a result the total discrimination between aliphatic and aromatic structures was not a safe assumption. It is postulated that aromatic ring opening may occur at sites of hydroxyl group substitution on the ring structures resulting in an apparent less aromaticity and the generation of al iphatic acid products from aromatic intermediates. iv TABLE OF CONTENTS PAGE ABSTRACT i i i TABLE OF CONTENTS v LIST OF TABLES v i i LIST OF FIGURES. v i i i ACKNOWLEDGEMENTS x i i INTRODUCTION 1 LITERATURE REVIEW 3 Environmental Influence.. 6 Extraction and Degradation 7 Methods for Degradation of Humic Substances 12 MATERIALS AND METHODS 24 Origin of Humic Acids and Lignins Used in the Degradation Studies 24 Extraction and Fractionation 26 Oxidation Technique 26 Highly Volati le Acids 28 Extraction of Aqueous Oxidation Products 28 Carbon Analysis. -. •. 29 Ash Content 30 Phenol Test 30 Potentiometric Titrations 32 v PAGE Spectroscopic Analyses 32 A. Infrared Analysis 32 B. Nuclear Magnetic Resonance 32 C. Mass Spectrometry 33 Chromatographic Analyses 33 A. Thin Layer Chromatography 33 B. Gas Liquid Chromatography 34 RESULTS AND DISCUSSION 37 'SUMMARY AND CONCLUSIONS 83 LITERATURE CITED 86 vi LIST OF TABLES PAGE Table 1: Origin and Chemical Characteristics of Soil Samples 25 Table 2: Structures and Elution Temperature of Standard Compounds Used to Identify GLC Peaks of Organic Solvent Soluble Oxi dati on Products . . ; . , . . . . . . . . . . . . . . . . . . . . . . 36 Table 3: Distribution of Product Carbon (C) after Oxidation of Humic Acids and Prepared Lignins with 1.6 N NaOCl 38 Table 4: Total and Relative Acid Groups in Organic Solvent Soluble Products of Humic Acids and Lignins Oxidized with 1.6 N NaOCl. 46 Table 5: Recovery of Standard Compounds Subjected to Oxidation with 1.6 N NaOCl (p-^phthalic acid I.S.) 75 Table 6: Total Weight of Aromatic Acids in Dominant GLC Peaks and Degree of Aromaticity in Organic Solvent Soluble Fraction by GLC and Titration Data 77 vi i LIST OF FIGURES PAGE Figure 1: Oxidation and Fractionation Scheme for NaOCl Degradation of Humic Acid or Lignin 31 Figure 2: Potentiometric Titrat ion Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of the Three Alberta Sites 43 Figure 3: Potentiometric Titrat ion Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of the Three B.C. Sites 44 Figure 4: Potentiometric Titrat ion Curves of the Organic Solvent Soluble Fraction of the Kraft's Hardwood and Softwood Lignins and 1, 2, 4, 5-Benzene Tetracarboxylic Acid. . . . 45 Figure 5: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Spear Grass Site after NaOCl Oxidation 48 Figure 6: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Western Porcupine Grass Site after NaOCl Oxidation 49 Figure 7: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Rough Fescue Site after NaOCl Oxidation., 50 Figure 8: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Sphagnum Moss Site after NaOCl Oxidation 51 v i i i PAGE Figure 9: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower of the Lodgepole Pine Site after NaOCl Oxidation 52 Figure 10: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Douglas-Fir Site after NaOCl Oxidation. . 53 Figure 11: IR Spectra of Kraft's Softwood Lignin (upper); and the Organic Solvent Soluble Fraction (lower) after NaOCl. Oxidation 54 Figure 12: IR Spectra of Kraft's Hardwood Lignin (upper); and the Organic Solvent Soluble Fraction (lower) after NaOCl Oxidation 55 Figure 13: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation 57 Figure 14: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation 58 Figure 15: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Rough Fescue Site after NaOCl Oxidation 59 Figure 16: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation 60 ix PAGE Figure 17: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Lodgepole Pine Site after NaOCl Oxidation 61 Figure 18: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Douglas-Fir Site after NaOCl Oxidation 62 Figure 19: NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Softwood Lignin after NaOCl Oxidation 63 Figure 20: NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Hardwood Lignin after NaOCl Oxidation 64 Figure 21: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation 66 Figure 22: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation- 67 Figure 23: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Rough Fescue Site after NaOCl Oxidation 68 Figure 24: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation 69 Figure 25: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Lodgepole Pine Site after NaOCl Oxidation 70 x PAGE Figure 26: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Douglas-Fir Site after NaOCl Oxidation 71 Figure 27: GLC Separation of the Organic Solvent Soluble Fraction of the Kraft's Softwood Lignin after NaOCl Oxidation 72 Figure 28: GLC Separation of the Organic Solvent Soluble Fraction of the Kraft's Hardwood Lignin after NaOCl Oxidation . . . . 73 Figure 29: GLC Separation of Organic Solvent Soluble Fraction of o-Phthalic Acid, 1, 2, 3-Benzene Tricarboxylic acid and 1, 2, 4, 5-Benzene Tetracarboxylic Acid Standards After NaOCl Oxidation 76 xi ACKNOWLEDGEMENTS Sincere appreciation is extended to Dr. L. E. Lowe for his guidance during the course of this study and constructive cr i t ic ism during the writing of the manuscript. I wish to thank Dr. M. Barnes and Dr. W. Barnes for their research suggestions and encouragement in my work. Appreciation is extended to Dr. S. K. Chakrabartty for our valuable discussions, materials donated to the study and assistance with the nuclear magnetic resonance data. Gratitude is expressed to Mr. G. Bohnenkamp and Mr. G. Galzy for their unselfish donation of time, experience and materials to the gas-liquid chromatography portion of this study. I also wish to thank Mr. B. von Spindler and Mr. W. Cheang for their technical assistance. I wish to thank the Department of Chemistry, U . B . C . , for assistance with the mass spectrometric data. I wish to thank the National Research Council- for the funding of this project. A sincere thank you to Mrs. J . Hollands for her patience and s k i l l f u l typing of the manuscript. I wish to thank the many people who have expressed their support and encouragement throughout my university education. F ina l ly , for the immeasurable meaning she gives to my l i f e , I dedicate my thesis to Beverley. x i i INTRODUCTION Humic substances are among the most widely distributed and important organic materials in nature. The participation and importance of humic substances in the reactions occurring in soil and water are now being recognized by earth scientists . To better understand the reaction mechanisms and environmental potential of humic substances, scientists have sought to determine their chemical structure, with much of the research concentrated on humic acid. A specific structure for humic acid has not been determined but a number of characteristics have generally been accepted. Humic acid is considered to be of three-dimensional geometry with randomly-bonded heterogeneous aromatic units or building blocks linked by several different bond types to form a dark brown to black polymer. Molecular weight estimates range from 10,000 to 200,000. It is the large size and complexity of the polymer which necessitates i ts dismemberment to allow the identif ication of the monomeric building blocks. Both the random nature of the monomeric units and the numerous types of bonds between units make the hypothesis of a representative model of the polymer from its building blocks a d i f f i c u l t task. Many of the accepted characteristics of humic acid are also characteristic of coal constituents and of l ign in . For this reason, several of the techniques for the analysis of coal and l ignin are applicable to humic acids. 1 2 In view of the limitations of past research techniques, humic acids extracted from a wide range of soils were degraded oxidatively under relatively mild reaction conditions. The primary aim of the study was to evaluate the usefulness of the NaOCl oxidation technique in elucidating differences in processes of humus formation in a variety of sites. Two prepared lignins were similarly degraded to investigate a potential l ignin component in humic acid. In accordance with reported preservation of aromatic structure in standard compounds, the degree of aromaticity was estimated for the humic acid and l ignin samples. LITERATURE REVIEW Humic substances are the major , organic accumulations of soils and waters and participate in and control many reactions that occur in these environments. Humic substances are recognized as being highly influential on the bio logical , chemical and physical properties of soi ls such as soil f e r t i l i t y , s tabi l i ty and hydrology in soi l genesis. The need for increased agricultural productivity, and more recently, the concern for ecological security of soil have fostered basic research on the nature of the chemical structures of soil components. Wallerius published observations in 1761 of humic substances forming from decomposing plants and displaying an ab i l i ty to absorb water and nutrients. Although the structure and pathways of formation remain speculative, the chemistry of humic substances has been intensely investigated in the past seventy years. The basis for the investigation of organic materials found in soils begins with recognition of the main components. Soil organic matter may be divided into three main fractions: (1) the biomass or l iv ing microorganisms and plant roots; (2) the part ia l ly decomposed plant, animal and microbial tissues; and (3) the humic substances, described as amorphous, brown or black, hydrophilic, ac id ic , polydispersed substances ranging from hundreds to tens of thousands in molecular weight (Schnitzer and Khan, 1972). The terms humic substances, humus and humic fractions are often used synonymously in the l i terature . 3 4 Felbeck (1971) l i s t s four hypotheses for the synthesis of soil humic substances: (1) the plant alteration hypothesis; (2) the chemical polymerization hypothesis; (3) the cel l autolysis hypothesis; and (4) the microbial synthesis hypothesis. The plant alteration hypothesis suggests certain plant components, particularly l i g n i n , are resistant to microbial attack and are altered superfic ial ly to form humic substances. The nature of the original plant components (cellulose, hemicelluose, l i gn in , protein) strongly influence the chemistry of the humic substances formed. The chemical polymerization hypothesis implies that plant tissues are degraded by soil-borne microorganisms which release products such as phenols and amino acids into the environment. In the s o i l , the excreted organic materials may be oxidized and polymerized to humic substances. The microbial synthesis hypothesis states that humic-like substances are synthesized within microbial cel ls and are released upon death and lysis of the microbe. It has not been established which of these four mechanisms are operative, but i t is possible that a l l four contribute to the production of humic substances. The humic substances may be further fractionated according to their so lubi l i ty in alkaline and acid media. The fulvic acid fraction is considered to be the alkali-soluble component 5 of soi l organic matter which is also soluble in acid. Humic acid is also soluble in alkaline but not acid media; humin is insoluble in both acidic and basic media. The terms fulvic and humic acid have been cr i t i c i zed as they imply discrete compounds of known structure. The humic fractions are to be viewed not as homogeneous groups, but as a series of related heteropolymers with similar so lubi l i ty characterist ics. Even the boundary between the humic and fulvic fractions is not sharply defined. Felbeck (1965) summarized the work of several researchers, indicating that humic acid, fulvic acid and humin differ in their elemental composition, degree of polymerization, molecular weight, acidic functional groups and degree of association with inorganic soil constituents. Since much of the research is devoted to humic acid , this l i terature review wil l concentrate on this humic fraction. The research of several workers has resulted in the general acceptance of several properties characteristic of humic acid. Haworth (1971) defined humic acid as the dilute aqueous sodium hydroxide . (NaOH) soluble fraction of soil organic matter which precipitates upon ac idi f icat ion. Butler and Ladd (1971) described humic acid as being dark brown to black in color with an estimated molecular weight of ranging from 10,000 to 200,000. Stevenson and Butler (1969) attributed the acidic nature of humic acid to a high degree of carboxyl and hydroxyl group substitution on aromatic structures. The organization of humic acid has been described as a 6 polymeric structure consisting of heterogeneous structural units or building blocks with heterogeneous bond types (Shivrina et al., 1968). Kononova (1968) outlined a possible humic acid structure as the random condensation product of phenolic units, amino acids and peptides. Stevenson and Goh (1971) speculated that the amino acid and peptide components were loosely attached to a more aromatic central structure or "core". The large molecular size and complexity of humic substances necessitates their degradation to smaller less complex units which can be characterized by modern techniques. Since i t is possible that no two humic acid molecules are ident ica l , the interpretation of the structural role of the degradation products, and reconstruction of a hypothetical model of the intact polymer should be approached with caution (Dubach and Mehta, 1963). ENVIRONMENTAL INFLUENCE Much of the Russian work indicates that the chemistry of humic acid is probably influenced by the environment in which i t is formed. The factors of soil formation (type and amount of vegetative cover, climate, biological activity., parent ; material and time) provide an almost l imitless combination of environmental conditions for i ts genesis. If we accept soil to be a continuum of bio logical , physical and chemical properties which vary 7 both at the time of observation and with time, i t is probable that humic substances also continuously vary in structure with time. Herman. (1974), working with freshly harvested prairie grass roots, determined that the chemistry of plant tissues varied with different plant types, establishing a potential for differences in the chemistry of the humic products of their decomposition. Dormaar (1975a) suggested that the differing chemical composition of humic substances derived fronra number of soils could be used to differentiate taxonomic soil groups. Kumada (1965) reported that the method of extraction affected the chemistry of the humic extracts. Shivrina et al. (1968) assumed that humic substances of different origin may vary in the composition of aromatic compounds making up their central structure or core of the molecule. The chemical character of the aromatic constituents may reflect the environmental influence. EXTRACTION AND DEGRADATION The study of humic substances may be viewed as a three step process involving isolation of the organic materials from the mineral soi l fract ion, degradation of the humic substances to their monomeric building blocks, and f ina l ly a characterization of the degradation products. The isolation of the humic substances aims 8 to complete the separation of organic and mineral soil components with the minimum of structural damage to the humic polymers. The subsequent degradation of the macromolecule seeks to reduce its chemical complexity without altering the chemistry of the monomers. These goals are ideal , but available techniques are less so. Recognition of humic substances as a multicomponent system including a wide range of monomers, bond types and complexes, creates problems in developing the perfect extractant. Extraction techniques f a l l into two classes: (1) relat ively drastic methods which result in high y ie lds , but may cause structural modifications; (2) mild methods with usually lesser y ie lds , which may not represent the total sample but are probably structurally intact. Both techniques are often further complicated by the presence of the extractant in the product (Kononova, 1966). The mild methods may cause more d i f f i c u l t contamination problems as they are often an organic extractant in an organic product. Several workers have compared the relative merits of both drastic and mild extraction techniques (Dubach and Mehta, 1963; Kononova, 1966; Posner, 1966; and Hayes etal., 1973). Lindqvist (1968) described a l l extraction methods as yielding polydisperse products which are d i f f i c u l t to characterize and suggested the goals of an extractant giving both high yields with a low ash content and minimal chemical alteration may be mutually exclusive. 9 Many of the extraction procedures to be discussed in this review do not subject the soil organic matter to an alkaline extraction followed by ac idi f icat ion. For this reason, the products wil l be termed humic substances rather than humic acids. Dilute aqueous NaOH remains the most commonly used and quantitatively effective extraction reagent for humic substances. Dilute aqueous NaOH has been cr i t i c i zed as causing structural modifications to the humic extracts through autoxidation while in contact with a i r . Schnitzer and Khan (1972) cited the results of several workers who compared dilute a lkal i to other extractants and found l i t t l e or no qualitative product differences. Autoxidation problems may be avoided by extracting in sealed containers under an inert atmosphere such as nitrogen. Posner (1966) and Kononova (1966) reported the amount of humic substances extracted with dilute a lkal i to be direct ly proportional to the length of time and temperature of extraction, within reasonable l imits . A pretreatment of soil with dilute mineral acid, particularly in soils high in calcium carbonate (CaCOg) and/or exchangeable calcium cation ( C a + + ) , greatly increased the yields of humic extracts by destroying C a + + bridges and reducing organo>meta,ll.i.c complexes (Posner, 1966 and Schnitzer and Khan, 1972). Posner (1966) maximized yields of humic substances by pretreating a l l soil samples with 0.1 N hydrochloric acid (HC1) for 24 hours followed by extraction with 1 0.5N NaOH under N^ for 24 hours at room temperature. Levesque and Schnitzer (1966) also found 0.4 to 0.5 N NaOH to be the most suitable concentration for extraction of humic substances with a 10 resulting low ash content. Bremner et al. (1946) suggested a standardized set of extraction parameters may be appropriate, as the various conditions applied probably lead to extraction of humics at differing states of degradation. Sodium pyrophosphate (Na^P^O )^ has gained popularity as an extractant due to i ts not requiring a decalcification step, and its extraction capability at neutral pH. Alexsandrova (1960) reported that Na^P^Oy, although not as .quantitatively effective as^dilute NaOH, extracted both humic substances and organo-metal1ic complexes. Kononova (1966) found that the efficiency of N a ^ O ^ was improved by increasing the pH of extraction from 7.0 to 9.5. A combination of 0.1 M Na^ PgOy and 0.1 M NaOH (pH 13) was recommended by Kononova and Belchikova (1970) as being an effective extractant which did not require the decalcif ication step. Organic chelating resins and water soluble chelating compounds, such as ethylene diaminetetraacetic acid (EDTA) have become popular for their extracting ab i l i t i e s without the risk of forming art i facts . Chelating resins and compounds act through the disruption of organo.-metaT.T'ic complexes in soil to form ring structures with the released metals. The resulting negatively charged humic substances, freed of their previous metallic bonds, disperse within the reaction medium. Chelating resins require a relat ively longer extraction period due to their sol id bead form requiring actual physical contact with the soil organic matter. However, the resins can be 11 easily f i l tered or centrifuged out of the medium^ thereby eliminating complexed metals from the system. Levesque and Schnitzer (1967) suggested sodium saturated chelating resins as useful agents for disrupting organo-metal1ic complexes. Ortiz de Serra and Schnitzer (1972) found close s imilarity of functional groups and infrared spectra for humic substances extracted with 0.5 N NaOH and sodium-saturated chelating resin. They concluded that the damaging effects of alkaline extraction methods, reported by other workers, might be exaggerated. Felbeck (1971) recommended a sequential extraction of soil organic matter with benzene-methanol, 0.1 M HC1, 0.1 M Na^P^Oy, 6 N HC1 at 90°C, chloroform-methanol (5:1) and f ina l ly 0.5 N NaOH to y ie ld a series of re lat ively homogeneous fractions in comparison to a s ingle,di lute a lkal i extraction. Lowe (1974) ut i l ized sequential extractions to examine differences in products from various forest floor organic matter accumulations. The use of ultrasonic vibrations, as an aid to extraction of soil with acetyl acetone, was found to be an effective method for the dispersion of humic substances, particularly organic phosphorus and sulphur compounds (Halstead et al., 1966). Felbeck (1961) successfully extracted humic substances by ultrasonic dispersion in dilute NaOH or Na^P,^ media. Hayes et al. (1975) compared several techniques and established the following order of extraction efficiencies when 12 applied to a particular organic s o i l : Na^ P-pO^ < organic chelating agents < polar aprotic solvents < pyridine < dilute NaOH with yields ranging from 13 to 63%. Successive extractions with neutral Na 4 P 2 0 7 , alkaline Na 4 P 2 0 7 , and dilute NaOH may extract humic materials in increasing order of polymerization (Anderson, 1972). The l i s t ing of extractants in order of efficiency should be considered with caution as the order may change with different soi l sites and horizon of sampling. The review of numerous extraction techniques by Schnitzer and.Khan (1972) suggests the efficiencies of a given technique may vary with changing soil types and different horizons within a given s o i l . The vast background l iterature concerning the extraction of humic substances implies some uncertainty as to the nature of the products of extraction compared to the nature of soil organic matter. Van Di j k (1963) warns that a grave error is committed in believing extractants d i f fer only in the amount of product y i e l d . The continuing research with soil organic matter extractants implies soi l scientists have not yet found the perfect extraction technique. METHODS FOR DEGRADATION OF HUMIC SUBSTANCES Progress in deriving structural information concerning humic substances and humic acids in particular has been limited by unsatisfactory techniques for degradation (Bremner and Ho, 1962 and 13 Felbeck, 1965). Evidence of possible s imilari t ies between the chemistry of soil organic matter, of l ignin and of coal has led respective chemists to examine numerous methods of degradation. In each case, the method of degradation ideally seeks to achieve high yields of chemically simpler compounds, suffering l i t t l e or no chemical alterations compared to their status as monomers in the original macromolecule. The products should be of moderate chemical complexity conducive to derivation of structural information by modern analytical techniques with a view to reconstructing a model for the humic macromolecule. Deviations from the ideal include low yields due to excessively mild degradation conditions or excessively drastic conditions destroying the humic starting materials or creating artifacts which cannot easily be related to the parent polymer (Neyroud and Schnitzer, 1975a). heavily branched, three-dimensional polymer exhibiting a high bond strength between the aromatic structures of which the polymer is composed. The high bond strength of aromatic structure is well recognized in the chemical l i terature . Interaromatic linkages probably 0 include -NH-, =N-, - S - , - C - , -0- and aliphatic groups of varying carbon numbers (Kononova, 1966). Should the highly condensed core theory of humic acid structure proposed by many workers prove accurate, aromatic structures may be linked by single bonds ( ( O ) ( 0 ) ) v or consist of condensed ring systems ( [O^O } ) ( K o n o n o v a > 1966). Jackson et al. (1972) recognized humic acid to be a 14 The wide range of bond types found in humic substances, plus probable complexing with organic and mineral constituents, might part ia l ly explain the d i f f i cu l ty in finding the ideal degradation system. Felbeck (1965), assuming the bond energies within the aromatic monomers to be similar to the bond energies between the monomers in a polymeric structure, reported low yields of some degradative methods might be attributed to reaction conditions which also destroy the products after dismemberment of the polymer. Haworth (1971) stated that no successful degradations could be achieved without the use of drastic conditions. The present decade has experienced a shift in the efforts of soil chemists from drastic to milder degradation techniques. The increased ava i lab i l i ty of gas chromatographic, mass spectrometric and computerized data processing techniques has increased the speed and sensit ivity of research (Skinner and Schnitzer, 1975). Consideration of the merits and faults of several degradation techniques, both drastic and mild, may prove beneficial to the appreciation of the magnitude of the problem. The methods used to degrade humic substances include chemical techniques (both oxidative and reductive) and biological techniques. Schnitzer and Khan (1972) reported the relat ively greater success of oxidative methods might be attributed to the high oxygen content of humic substances rendering i t d i f f i c u l t to reduce. 15 Hydrolytic techniques, summarized, by Felbeck (1965), are generally considered to contribute l i t t l e information concerning the aromatic core of humic susbtances. Boiling water essentially removed sugar monomers. Acid hydrolysis has been successfully used to extract carbohydrates and nitrogenous materials prior to application of more destructive degradative methods to the nonhydrolyzable fraction. Alkaline hydrolysis with hot NaOH or potassium hydroxide (KOH) has been cr i t i c i zed by Cheshire et al. (1968) as creating artifacts which are not diagnostic of the structure of humic substances. Hydrogenation and hydrogenolysis methods subjected humic substances to conditions of 350 C and 5000 l b / i n pressure in a hydrogen atmosphere and in the presence of copper sulphate (CuSO^) catalyst. Gottlieb and Hendricks (1946) achieved high yields of a colorless o i l which they were unable to characterize; possibly a result of the drastic conditions causing rearrangements and condensation reactions which l imit interpretive value. Oades and Townsend (1963) found oxidation of humic substances with hydrogen peroxide ( H 2 O 2 ) to be incomplete and offering l i t t l e structural information. Principal products of the reaction included carbon dioxide ( C O 2 ) , water ^ 0 ) and small amounts of organic acids. The n i t r i c acid (HNOg) oxidation technique, largely developed by coal and l ignin chemists, was ut i l ized by Schnitzer and Wright (1960) to isolate nitrophenolic and nitrobenzoic acid products representing only 5.3% of the original humic acid. They 16 estimated 60% of the starting material was evolved as C0^ and detected only small amounts of benzene carboxylic acids. Hansen and Schnitzer (1967) considered 30 hours of digestion with 2 N HN03 to be one of the higher yielding oxidation methods of i ts time but suspected destruction of some aromatic structures. The substitution of n i t r i t e groups ( I ^ - ) into the aromatic products occurred at sites of humic acid aromatic-C bonds before oxidative degradation. Gottlieb and Hendricks (1946) recognized a possible connection between humic substances and l ignin after confirming common products in the alkaline nitrobenzene oxidation of l ignin and humic substances. The apparently universal degradation products of l ignin - syringic acid, van i l l i c acid and p-hydroxybenzoic acid were isolated in yields of only 0.5 to 1.0% with alkaline nitrobenzene oxidation of humic substances by Morrison (1963) and Wildung et al'.' (1970). Alkaline copper oxide oxidation has been used successfully by Neyroud and Schnitzer (1974) to achieve yields of aliphatic acids representing 53% of the original humic acid. Low product yields (0.58 to 1.33%) experienced by Greene and Steelink (1962) were improved by methylating the humic acids prior to oxidation. The alkaline CuO oxidation method is considered ineffective in degrading humic substances low in substitution by oxygen containing functional groups and C-C bonds (Neyroud and Schnitzer, 1975b). Aliphatic side chains ortho or para to phenolic hydroxyl groups on aromatic rings or interaromatic 17 T/iirika;g:es of two or more carbons are cleaved to one carbon acid (-C00H) substituents on aromatic structures (Christman, 1970). The alkaline permanganate oxidation of humic substances has been prominent in the Russian l i terature and the research of: Schnitzer and several co-workers. Randal et al. (1938), studying 60 known compounds, reported KMnO^  oxidation always yielded chemically simpler products. Benzoic acid products occurred with aromatics substituted by carbon, whereas aromatics substituted by oxygen degraded to C02> H20 and oxalic acid. Schnitzer and Desjardins (1970) fe l t low yields could be improved by methylating the humic substances prior to oxidation. Methylation prevents substituent ketonic and hydroxyl oxygen from donating electrons to the benzene rings and inhibits e lectrophil ic attack of activated rings by the oxidant. In a comparison of the oxidation products of methylated and unmethylated humic acids, Khan and Schnitzer (1971a) reported that methylation increased the y ie ld of benzene carboxylic acids about 3-fold. Maximov et al. (1971) reported underestimations in attempts to predict the amount of KMnO^  required to oxidize a measured quantity of organic substrate. They suggested structural rearrangement might be occurring during the reaction and recommended milder conditions for oxidation. Ogner (1973) suspected chemical s imilari t ies between acid nonhydrolyzable humic acid and l ignin after achieving similar products from each with KMnO,, oxidation. 18 Drastically high temperatures (500 C) employed in zinc dust d i s t i l l a t i o n techniques appeared to greatly l imit the structural information derived by several workers. Cheschire et al. (1967) concluded that the method total ly destroyed the aliphatic constituents of humic substances. Hansen and Schnitzer (1969) achieved a 0.66% yie ld of aromatic polycyclic compounds after reduction of humic acid at 530°C. Similar results achieved by Haworth (1971) suggested the products were not representative of the entire humic acid polymer but did imply a highly aromatic nature. Burges et al. (1964) subjected a series of phenolic standards to sodium amalgam reduction. The infrared spectra scanned before and after reduction implied that the technique caused significant structural alterations. Mendez and Stevenson (1966) and Tate and Goh (1973) confirmed these results . Piper and Posner (1972) fe l t the method was useful to l ignin chemists but when applied to so i l s , the products were too complex to readily provide useful structural information. Martin et al. (1974) achieved a 3 to 6% yield of extremely complex products after the reduction of several humic acids. Dormaar (1969) reported being able to dif ferentiate , humic acids from Chernozemic soil sites on the basis of their Na amalgam reduction products. Mendez and Stevenson (1966) reported that the instabi l i ty of the products of Na amalgam reduction of humic substances resulted in repolymerization reactions. 19 Guidelines differentiating drastic from mild degradation conditions have not been established in the soil science l i terature . Schnitzer and Skinner (1974b) suggested 100°C might be an upper temperature l imit above which structural alterations and formation of art i facts might be expected. Exchanging a relat ively high reaction temperature (80°C for 4 hours) for a longer peracetic acid oxidation time, Schnitzer and Skinner (1974a) reported similar yields after subjecting humic acid to 8 days oxidation with 10% aqueous peracetic acid at 40°C compared to alkaline KMnO^  oxidation of the same humic acid but after prior methylation. Additional work with 10% aqueous peracetic acid at 80°C for 4 hours, gave evidence of aromatic ring opening occurring probably in a manner similar to the electrophil ic attack of activated aromatic structures by alkaline KMnO^  (Schnitzer and Skinner, 1974a). At lower temperatures of oxidation ( 4 0 ° C ) , methylation of humic substances pr ior / to degradation was not considered necessary (Schnitzer and Skinner, 1974b). Jackson et al. (1972) proposed an approach to determine the structure of humic substances by identifying only the bond types within the polymer. Heredy and Neuworth (1962) developed a method of refluxing coal humic acids with phenol and a boron tr i f luoride (BF-j) catalyst which resulted in depolymerization through substitution of phenol for the natural aromatic structures with retention of many of the major linkage systems. When applied to humic substances, the phenol substitution technique yielded some interaromatic linkage information but contributed l i t t l e to the understanding of the structures of the aromatic monomers (Jackson et al., 1972). 20 The wood pulping industry has used aqueous sodium sulfide (Na^S) extensively to degrade l ign in . Hayes et al. (1972) achieved a 60% yie ld of ether-soluble materials after degrading humic acid for 2 hours at 250°C in 10% aqueous Na 2S. The products of degrading a series of aromatic standard compounds which broadly represented structures and linkages l ike ly to be present in humic acid and l ignin indicated a poss ibi l i ty of humic acid being the product of modified l ignin and phenolic compounds (Craggs et al., 1974). Although not extensively applied to soil research, the work of Burdon et al. (1974) and Craggs et al. (1974) indicated Na2S degradation to have outstanding merit in humic acid structural studies. The microbiological degradation of humic substances may be the most selective or specific bond breakage method available for structural determinations. The future of biological degradation techniques hinges on the identif ication or development of microorganisms capable of breaking specific bonds typical ly found in humic substances and elucidation of their biochemical pathways (Crawford and Crawford, 1976). Hurst et al. (1962) viewed fungal degradation of humic substances as a good alternative to chemical techniques. Their study achieved its highest yields under aerobic conditions confirming the degradative process as being largely oxidative. The evidence showed a reductive decarboxylation step was involved in degrading humic acid to a colorless product. Schnitzer and Khan (1972) drew attention to 21 the fact that uncertainties in differentiating microbially synthesized materials from humic degradation products limited the interpretive value of the method. Khan and Schnitzer (1971b) proposed a technique for examining fulvic acid constituents without an i n i t i a l degradative step. Fulvic acid was i n i t i a l l y methylated to dissolve in benzene and applied to an Al^Og chromatographic column. Fully methylated phenolic acids, benzoic ac id , methyl esters and dialkylphthalate products were eluted from the column with solvents of increasing polarity . The fulvic acid portion not eluted was redissolved in benzene, dried and subjected to alkaline KMnO^  oxidation.to y ie ld products similar to the eluates but in s ignif icantly greater amounts. Khan and Schnftzer (1971b) suggested the eluates represented structures weakly l inked, probably by hydrogen bonds, to a more stable polymeric structure. Methylation has been shown to reduce the strength of hydrogen bonding (Ogner and Schnitzer, 1971). The method unfortunately suffered about 50% losses of starting materials through the numerous fractionation steps. Scanning the history of research into the chemistry of humic substances reveals a series of trends. The goal of the research has been to discover a method which wil l dismantle the humic polymer in a predictable fashion yielding stable products which, upon identi f icat ion, give implications of their structural role in the polymer. To further the state of knowledge, soil scientists occasionally apply 22 the tested techniques of l ignin and coal to compare the chemical s imilarit ies , of humic substances, l ignin and coal. Aided by highly sensitive modern instrumentation, soil biochemists are moving to u t i l i z ing relat ively mild degradation techniques to break the minimum number of bonds to achieve products yielding the maximum structural information. In short, humic substances are now being appreciated as delicate natural polymers revealing their chemical nature under milder degradation conditions relative to the drastic chemistry prior to the present decade. The sodium hypochlorite (NaOCl) oxidation of organic systems has been described as being a selective oxidant devoid of any side reactions such as polymerization of products or rearrangements of reactants (Chakrabartty and Kretschmer, 1974a). Working primarily with coal humic acids and one soil humicacid sample, Chakrabartty et al.. (1974) reported NaOCl (5% active CI) reacted vigorously with aromatic systems substituted by oxygen containing functional groups; methyl, methylene and methine carbons were cleaved while leaving aromatic structures intact. Sites of oxidation on aromatic structures appeared as carboxyl groups.in the benzene carboxylic acid., products, implying the oxidant 2 3 could differentiate Sp and Sp carbon hybrid structures. The technique required low reaction temperatures,as coal is susceptible to autoxidation at temperatures above 70°C (Chakrabartty and Berkowitz, 1976a). 23 The achievements of soil scientists investigating the chemistry of humic substances include knowledge of a high degree of aromaticity and substitution of the aromatic structures with oxygen containing functional groups. It appears that structural information might be derived through application of the NaOCl oxidation technique : to soil humic substances of varied states of maturity. The concept of maturation, as viewed by several Russian researchers, involves a gradual degradation of al iphatic components with concurrent increases in the aromatic character of the persistant materials. The maturation concept includes both time and intensity factors for the accumulation condensation and further polymerization of aromatic structures. The claimed preservation of aromatic structures during oxidation with NaOCl (Chakrabartty and Kretschmer, 1974a) could be a useful tool for the comparison of reaction products from a variety of so i l s . It should be noted in concluding this review that many of the degradative techniques abandoned in the past because of the complexity of their products or small yields might deserve reviewing in the l ight of new instrumentation permitting microanalysis. MATERIALS AND METHODS  Origin of Humic Acids and Lignins Used in the Degradation Studies Six humic acid samples were used in the basic studies. They were derived from three Southern Alberta prairie grassland sites previously investigated by Herman (1974), Dormaar (1971, 1974 and 1975b) and Lutwick and Dormaar (1976) and two Brit ish Columbia forest humus sites plus a sphaghum peat described in an earl ier study by Lowe (1974). The sites were selected to include a diversity of vegetative species and to take advantage of a considerable background of accumulated information for these sites (Table 1). Each of the six sites was dominated by a single vegetative species and sampled in a relat ively undisturbed condition. Although the three prair ie sites were a l l vegetated by prairie grass species, the Ah horizons sampled included three soil zones and three dist inct plant communities. The H horizons of the B.C. sites were selected where the three stages of decomposition (L, F and H horizons) could be easily separated. The Ah horizons sampled probably possessed a relat ively more mature humic acid compared to the H horizons. The six soil samples were ground to pass through a 2 mm sieve. Commercially prepared samples of a Kraft's softwood l ignin and a Kraft's hardwoodlignin, dealer code numbers RLX 3340 : 23 and RLX 3340 : 24 respectively, were purchased from WESTVACO of Box 5207, North Charleston, South Carolina. The manufacturer did not supply any data for the l ignin preparations. TABLE 1: Origin and Chemical Characteristics of Soil Samples. H.A. Dominant Species Vegetation Common Name Location (Lat N/Long W) Legal Location (Soil Zone) Horizon Depth (cm) % C % N C/N pH CaCl 2 % Ash Stipa comata Trin . & Rupr. Spear grass 49°02 7 n0°26' SE 16-2-14 W4 (Brown) Ah 8-8 1. 32 .15 8.8 5.7 52.7 Stipa spavtea Tri n. var. ouvtiseta Hitchc. Western porcupine 49O09'/110O5T NE 19-2-6 W4 Ah 0-10 3. 28 .32 10.2 5.5 12.2 Festuca scabvella Torr. Rough fescue 50O06 7114 005' SW 27-13-1 W5 Ah 0-8 11. 1 1 .10 10.1 5.8 30.9 Sphagnum species Sphagnum moss Fort St. John, B.C. Oh 80-90 14. 4 1 .09 38.0 4.8 9.3 Pinus contorta van latifolia Engelm. Lodgepole pine Manning Park, B.C. H .5-2 29. 9 .81 37.0 4.3 3.9 Pseudotsuga menziesii .(Mirb.) Franco Douglas f i r Campbell River, B.C. H 5-8 46. 8 1 .04 45.0 4.1 21.2 r\3 26 Extraction and Fractionation The six soil samples were a l l subjected to identical extraction and fractionation conditions. Each sample was individually subjected to a pretreatment involving shaking for 24 hours with 0.1 N HC1 followed by centrifugation at 1800 rpm for 10 minutes with the supernatent solution being discarded. The humic substances were extracted from the dilute acid pretreated soil by shaking for 24 hours with 0.5 N NaOH (using a 10:1 solvent volume:soil volume ratio) under nitrogen in sealed polypropylene bottles, followed by adding 10 ml of saturated aqueous KCl solution to encourage mineral matter to flocculate. The extract was centrifuged at 2000 rpm for 30 minutes and the supernatant solution separated, adjusted to pH 1.0 with 1 N HC1 and heated in a water bath to 50°C for 2 hours to allow the humic acid to coagulate. The extract was centrifuged at 1800 rpm for 10 minutes and the fulvic acid supernatant layer discarded. The humic acid centrifugate was redissolved in 0.5 N NaOH, acidif ied with 1.0 N HC1 and separated two additional times with the fulvic acid fraction discarded each time. The humic acid preparation was lyophil'ized and stored in sealed containers. Oxidation Technique Oxidation of the humic acid and l ignin samples was achieved by dissolving approximately 0.5000 gm of substrate in 75 ml of 27 of 1.6 N NaOCl and adjusting to pH 13 with 4 N NaOH to prevent auto-decomposition of the oxidant (Chakrabartty,and Berkowitz, 1976b). Glass re-action flasks with teflon coated stoppers were used for the oxidation step. Gaseous products liberated during the reaction were swept by a continuous flow of nitrogen gas (research grade) through a gas scrubber containing 1 N Ba(0H)2 to trap CO,,. The nitrogen gas flow also served to prevent atmospheric C0 2 being trapped in the gas scrubbers and provided a mixing action as i t bubbled through the solution. The reaction flasks were occasionally swirled to rinse any humic acid or l ignin particles adhering to the wall of the flask. After five days oxidation at room temperature ( 2 3 ° C ) , the resultant mixture was acidif ied with 1 N HC1 to pH 3.5; the evolved C0 2 was swept out of the reaction flask in a flow of nitrogen to be trapped in the gas scrubber as BaCOg. The BaCOg, which was insoluble in water, was f i l tered through Whatman No. 541 f i l t e r paper in a Buchner funnel, dried at 75°C for 2 hours and weighed to evaluate the total C0 2 product. During the period that nitrogen gas was used as a carrying agent, a blank reaction flask containing 75 ml of 1.6 N NaOCl adjusted to pH 13 was maintained with a Ba(0H)2 gas scrubber to monitor extraneous C0 2 entering the reaction system. The reaction was quenched and the remainder of the unreacted oxidant decomposed by further reducing the acidity of the reaction mixture with 1 N HC1 to pH 1.5; H0C1 and HC1 fumes produced by the acid catalyzed decomposition of NaOCl were removed by exhaustion in a fume hood for 60 minutes. 28 Highly Volati le Acids Highly volati le acids in each acidif ied aqueous reaction mixture were removed under vacuum at 35°C and d i s t i l l e d until the d i s t i l l a t e was no longer acidic as tested with pH paper. An aliquot pf each d i s t i l l a t e was t i trated with 0.0505 N NaOH using phenolphthalein indicator to evaluate the highly volat i le acid products. The burette used for t i trat ion was equipped with a trap containing KOH pellets to prevent interference by atmospheric CO^ (Wang et al., 1966). The carbon content of the highly volat i le acid component was estimated by assuming the volat i le acids for the given conditions included formic, acetic and propionic acids for a probable average of two carbon atoms per acid functional group t i trated . Extraction of Aqueous Oxidation Products The acidic aqueous reaction products remaining after removal of the highly volat i le acid fraction were further concentrated under reduced pressure in a water bath at 35°C to approximately 50 ml volume and adjusted to pH 1 with 0.1 N HC1. The aqueous reaction products were transferred to a continuous ether extractor, as described by Vogel (1948), and extracted with diethyl ether for 7 days. A variable transformer insured each mantle did not heat the boiling ether over 35°C. An amorphous material insoluble in both ether and water was observed to form during the ether extraction of the oxidized humic 29 acids of each soil sample. The material, which floated on the surface of the aqueous layer, was removed from the extractor with a 100-ml pipette and subjected to a second oxidation at room temperature (23°C) with 1.6 N NaOCl at pH 13 for 3 days. The oxidation products were acidif ied to pH 1.5 with 1 N HCl and returned to their respective extractor for 5 additional days of extraction with ether. L i t t l e or no amorphous material was observed after the second oxidation of each humic acid. The l ignin samples did not require a second oxidation. The ether extract of each sample was reduced to approximately 20 ml in volume by flash evaporation at 30°C using a Buchi/Brinkman Rotary Evaporator and made up to exactly 25 ml in volume with anhydrous ether. The aqueous layer of each sample was also flash evaporated at 30°C and the residue (mostly NaCl crystals) was ground in a mortar and extracted by shaking with 50 ml of anhydrous methanol for 24 hours. The methanol extract was decanted, flash evaporated at 30°C to about 20 ml and made up to exactly 25 ml with pure anhydrous methanol. The NaCl crystals were saved for carbon analysis by tf]e Leco induction furnace method (Laboratory Equipment Corporation, Saint Joseph, Michigan). Carbon Analysis Weighed samples of the humic acid and l ignin starting materials and aliquots of the ether and methanol extraction products dried at room temperature were analyzed for total carbon content 30 by the Leco method ut i l i z ing a vycor glass insert for the quartz enclosed graphite crucible. Dif f icult ies encountered in drying the ether extracts of the humic acids extracted from the spear grass and rough fescue sites necessitated heating the samples to 100°C until dry. The remaining ether and methanol fractions of each site were reduced in volume under vacuum at room temperature, combined and made up to 25 ml with anhydrous diethyl ether. For a l l further analyses, the combined fractions wil l be referred to as the organic-solvent-soluble fraction,abbreviated as the OSS fraction. The oxidation and fractionation scheme for this study is summarized in Figure 1. Ash Content Weighed samples of humic acids and lignins were heated in crucibles on a hot plate until they smoked, followed by a pre-ashing at 250°C for 2 hours and complete ashing at 500°C for 6 hours. Phenol Test The OSS extraction products-were tested for phenolic products with a 0.1 N FeCl^ solution; a dark violet colour was considered a positive test and a yellow color a negative test for phenolic compounds (Smith and Crystal , p. 152, 1966). 31 Substrate (humic acid or lignin) Oxidize in 1.6 N NaOCl at pH 13 for 5 days at room tempera-ture i Acidify to pH 3.5 Y Acidify to pH 1.0 Concentrate aqueous oxida-tion products and extract with ether for 7 days C0 2 product ••Highly volat i le acid products Dry aqueous layer and extract by shaking with hydrous methanol for 24 hours Ether extract Carbon content Methanol extract Carbon content Salt crystals Organic solvent soluble (OSS) extract for TLC, NMR, IR & GLC analyses Carbon content FIGURE 1: Oxidation and Fractionation Scheme for NaOCl Degradation of Humic Acid or Lignin. 32 Potentiometric Titrations Aliquots of the OSS fraction of the oxidation products were dried at room temperature, dissolved in 5 ml of d i s t i l l e d water (previously adjusted to pH 7.0 with 0.01 N HCl)and potentiometrically t i trated with 0.0500 N NaOH to pH 9.0. A 10-ml burette equipped with a hypodermic needle fac i l i ta ted dispensing of small amounts of base. A reference sample of 0.0600 g of 1, 2, 4, 5-benzene tetracarboxylic acid was t i trated in the same manner. A Sargent-Welch pH Meter NX equipped with a glass and reference electrode was used to evaluate pH changes. Spectroscopic Analysis A. Infrared Analysis Using a Beckman IR-20A Infrared Spectrophotometer, the infrared spectra were obtained of the humic acids and of the lignins before oxidation using 1 mg of sample mixed with 400 mg of KBr and compressed at 15,000 psi for five minutes under vacuum. Smears of the OSS oxidation products, dried at room temperature, were scanned with the same instrument u t i l i z ing AgCl windows. B. Nuclear Magnetic Resonance Aliquots of the OSS oxidation products were dried under reduced pressure in a desiccator at 33 room temperature until no solvent odor could be detected. The dry residues were dissolved in 5 ml of chloroform with a trimethylsilane internal standard and scanned with a Varian Model 360 Spectrophotometer. C. Mass Spectrometry The OSS fractions of the spear grass and rough fescue sites were scanned with a Varian Model Atlas CH4B Mass Spectrometer to determine the molecular weight of the o i l found in those two samples. Chromatographic Analyses A. Thin Layer Chromatography The OSS oxidation products of each sample were spot concentrated with a micropipette on 20 cm x 20 cm gypsum-free s i l i c a gel precoated sheets (Brinkman Instruments, Inc. , Westbury, New York). The spots were chromatographed in two dimensions with a .1:1:1 volume solvent ratio of 1 N acetic acid, water and dichloromethane followed by 100:1 volume solvent ratio of ether and 1 N HC1. The spots were viewed under ultraviolet l ight at wavelengths of 254 my and 350 my. 34 B. Gas Liquid Chromatography The OSS oxidation products were separated into their individual components with a Tracor MT220 Gas Chromatograph. This instrument was equipped with dual flame ionization detectors, dual 6 f t . x 1/8 in . (o.d.) stainless steel columns packed with 3% SE 30 on Chromosorb w-HP, 80 to 100 mesh. The SE 30 l iquid phase was selected for i ts ab i l i ty to separate both aliphatic and aromatic components on the basis of differences in their molecular weights and boiling points. The gas l iquid chromatograph was programmed from 75° to 275°C at a rate of 5°C per minute with a research grade nitrogen carrier gas flow rate regulated to 50 ml per minute. The oxidation products and a p-phthalic acid internal standard, added prior to derivatization, were methylated three times to insure maximum derivatization with diazomethane generated from Diazald (Schlink and Gillerman, 1960). The preparation of tr imethyl i s i ly l derivatives with T r i s i l (Pierce,Box 117, Rockford, I l l ino i s ) was also attempted to detect any phenolic oxidation products (Sweeley et al., 1963). The derivatizing agents and the diethyl ether solvent were checked for impurities which might be viewed as contamination peaks in the GLC work. Esterif ication of the oxidation products as methyl esters using 14% methanol in boron tr i f luoride was attempted as a third derivatization technique (Metcalfe, 1961); i t was rejected due to a high degree of 35 contamination in the derivatizing agent. A series of nine aromatic and aliphatic standard compounds, selected for their ava i lab i l i ty and expected presence in the oxidation products according to the chemistry of the hypohalite reaction, were matched by elution time and temperature and co-injecting to identify some of the product peaks (Table 2). To fac i l i ta te precise temperature readings, a copper-constantan thermocouple was used to calibrate the temperature programmer against an ice water reference. Peak area calculations, to achieve quantitative results , were determined by multiplying the height of the oxidation product peak by the width at . half the peak height and comparing the area to that of the p-phthalic acid internal standard. The efficiency of the oxidation and fractionation scheme followed in this study was evaluted by subjecting weighed samples of o-phthalic ac id , 1, 2, 3-benzene tricarboxylic acid and 1, 2, 4, 5-benzene tetracarboxylic acid to NaOCl oxidation and GLC analysis of the OSS acids recovered. Chromatographic peaks were referenced according to the numbers 1 through 9 used to denote the standard compounds in Table 2. 36 TABLE 2: Structures and Elution Temperatures of Standard Compounds Used to Identify GLC Peaks of Organic Solvent Soluble Oxidation Products. Fully Esterif ied Compound Compound Structure Elution Temperature ( C) H 1. Malonic acid HOOC - C - COOH 107 H H H 2. Succinnic acid HOOC - C - C - COOH 110 H H 3. p-phthalic acid HOOC -(O^- COOH 125 — r - C O O H 4. 1 , 2 , 3-benzene (OV C O O H 165 tricarboxylic acid > — CQQQU _ C C O O H 5. 1, 2, 4-benzene ( O V C O O H 1 7 0 tricarboxylic acid V—/ H O O C C O O H 6. 1, 3, 5-benzene H O O C - / O ) 174 tricarboxylic acid ^ — ( C O O H y COOH 7. 1 , 2 , 4, 5-benzene H 0 0 C -/O>-C00H 189 tetracarboxyl ic acctd H O O C ^ — , C O O H 8. 1 , 2 , 3, 4, 5-benzene H O O C - / O / C O O H 196 pentacarboxylic acid H O O C — C O O H 1 , 2 , 3 , 4 , 5 , H 0 0 C ^ C 0 0 H 2 1 5 6-benzene HOOC -/O>C00H hexacarboxylic acid WQQZ ^ " " L O O H 37 RESULTS AND DISCUSSION Six humic acid preparations representing a variety of soil vegetative cover and two Krafts' prepared l ignins , from a softwood species and hardwood species, were oxidized at room temperature with 1.6 N NaOCl. The eight samples studied were identical ly treated and analyzed to fac i l i ta te comparison of the relative amounts and types of oxidation products. Comparisons were made to evaluate trends which might reflect an influence of the dominant plant species at each site on the nature of the humic acid resulting from its decomposition and any possible connection between l ignin and humic acid formation. For the sake of brevity, the oxidation products studied are referred to by their respective dominant plant species, rather than "The NaOCl oxidation products of the humic acids extracted from the appropriate site". The relative amounts of carbon, expressed as a percentage of the total humic acid or l ignin carbon, in ether soluble, methanol soluble, carbon dioxide and highly-volati le acid fractions arising from the hypochlorite oxidation are summarized in Table 3. The distribution of oxidation products is expressed in terms of the distribution of the total starting carbon, rather than on a weight basis, to avoid the complications "of added oxygen and chlorine during the reaction. TABLE 3: Distribution of Product Carbon (C) after Oxidation of Humic Acids and Prepared Lignins with 1.6 N NaOCl. {% of Total Original Carbon) Total Sample Source Dominant Species Ether Soluble-C Methanol Soluble-C Organic Solvent Soluble-C Carbon Dioxide-C Highly Volat i le Acid-C Total-C . Accounted Ah (horizon) Spear grass 27.70 5.50 33.20 41.65 24.47 99.32 Ah Western porcupine grass 27.89 3.98 33.39 33.53 33.50 98.90 Ah Rough fescue 15.03 2.02 17.05 32.12 50.27 99.44 H (horizon) Sphagnum moss 13.39 4.42 17.81 7.56 74.24 99.59 H Lodgepole pine 14.97 5.35 20.32 7.07 71.85 99.24 H Douglas f i r 11.81 4.89 16.70 9.34 68.34 94.38 Softwood 1ignin 18.80 14.56 33.36 29.60 34.01 96.97 Hardwood 1ignin 29.31 6.78 36.09 19.09 36.28 91.46 CO 00 39 The ether soluble products of the spear grass and rough fescue sites presented a problem with analyses as they each appeared to contain an o i ly constituent not present in the other six samples. Mass spectrometric scans of the two samples indicated, in each case, the o i ly substance was a single compound with a molecular weight of 281. A number of interesting trends are observed i f one assumes the humic acids extracted from the Ah horizons of the three Alberta sites are relat ively older and probably more mature than those extracted from the H horizons of the three Brit ish Columbia sites. A greater proportion of the humic acid carbon appears as ether-soluble carbon and carbon dioxide carbon in the more mature samples while the reverse trend is observed in the case of the highly volat i le acid carbon obtained from the less mature samples. A sl ight risk exists that the amounts of highly volati le acid products are overestimated due to HCl and H0C1 contamination during acidif icat ion of the oxidation products in preparation for extraction with diethyl ether. The sodium chloride crystals remaining after drying the aqueous phase and extracting with anhydrous methanol were considered insignificant as they contained no carbon trapped in the crystal structure. Considering the known chemistry of the sodium hypochlorite oxidation reaction, carbon dioxide and highly volat i le acid products must be presumed to originate in saturated straight chain or 40 saturated ring structures (Chakrabartty and Kretschmer, 1974 b). Saturated straight chain structures may serve as side groups bonded to one aryl unit or as a linkage between two aromatic groups. The carbon dioxide and highly volat i le acid products accounted for 66-82% of the humic acid or l ignin oxidation product carbon. Benzene carboxylic acids can only be formed from aromatic components and are isolated in the organic solvents with the positions of carboxyl group substitution on the rings representing sites of previous aromatic components. Assuming the reaction conditions total ly preserve aromatic rings, the results of this study indicate a more aromatic character for the humic acids extracted from the spear grass and western porcupine grass sites and the two l ignin samples compared to the other four samples. A relat ively large organic solvent soluble (OSS) product in combination with a relat ively small highly volat i le acid product further implies a more aromatic and condensed structure in the two humic acids and two l ignin samples. The average annual precipitation over a 30-year period for the spear grass, western porcupine grass and rough fescue sites is 32, 41, and 50 cm, respectively, with only the rough fescue site experiencing continuous snow coverage during the winter months. Dormaar (1971) has viewed the conditions of.low precipitation and high potential evaporation at the spear grass and western porcupine grass sites as l imiting microbial act ivi ty with the resulting accumulation of l ign in; possibly these conditions explain the s imilarity in 41 oxidation product distribution of these sites and the prepared l ignins. The more moist soil conditions at the rough fescue site apparently permit a more rapid decomposition of a l l plant constituents; the distribution of the s ite's humic acid oxidation products suggests a structural s imilarity to that of the humic acids extracted from the three B.C. sites. Due to their close proximity, the spear grass arid western porcupine grass sites have similar precipitation and potential evaporation conditions, but imperfect drainage conditions at the latter site result in a decomposition process less dominated by aromatic products. In a previous study by Herman (1974) the fresh root materials sampled at the spear grass, western porcupine grass and rough fescue sites used in this study were found to be 23.7%, 29.8 % and 43.2% l ign in , respectively, relative to dry ash free (DAF) samples. The relat ively dry soil conditions at the spear grass site and i ts exposed condition throughout the year, probably l imiting factors for microbial ac t iv i ty , are apparently conducive to chemical and physical decomposition of plant constituents other than l ignin . The l ignin constituent, which has been shown by several authors to be largely aromatic in nature (Pearl, 1967), persists while less resistant plant constituents (cellulose, hemicellulose and proteins) are preferentially degraded. A similar situation apparently exists at the western porcupine grass s i te . The i n i t i a l highest fresh root l ignin content at the rough fescue site and lowest OSS product of the three Alberta sites 42 indicates that the soil conditions at the site are conducive to the decomposition of l i g n i n , the most resistant of plant constituents. The OSS fractions of a l l eight samples, when tested with 0.1 N FeCl^ for hydroxyl group substitution on aromatic rings, a l l gave a negative (yellow color) result. This result was further confirmed by the inabi l i ty of T r i s i l to form trimethysilyl ether derivatives of the oxidation products prior to gas l iquid chromatographic (GLC) analysis. An aliquot of the OSS fraction of each sample was potentiometrically t i trated to compare the configuration of the t i t ra t ion curves of the eight samples to that of 1, 2, 4, 5-benzene tetracarboxylic acid. The curves of a l l eight samples were sigmoid in shape, similar to that of the standard compound (Figures 2-4). Excluding the spear grass s i te , a relationship was observed between the amount of t i tratable acidic functional groups and the amount of ether-soluble product carbon (Tables 3 and 4). The humic acids extracted from the spear grass and rough fescue s i tes , previously noted as having an o i l constituent in their oxidation products, possessed the least amount of acidic funtional groups. According to the known chemistry of the hypohalite reaction, this result indicates that the humic acid extracts of these two samples were either highly condensed,with few aliphatic groups to appear as carboxyl substituents in the oxidation products, or possessed relat ively few aromatic structures to be recovered in the OSS fraction. PH 10.0 0 . 0 5 0 0 N NaOH (ml) FIGURE 2: Potentiometric Titrat ion Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of the Three Alberta Sites. pH 10.0. 0 1.0 2.0 3.0 4.0 5.0 6-0 0 . 0 5 0 0 N NaOH (ml) FIGURE 3: Potentiometric Titrat ion Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of the Three B.C. Sites. PH 11.0-0 1.0 2.0 3.0 4.0 5.0 6.0 0.0500 N NaOH (ml) FIGURE 4: Potentiometric Titration Curves of the Organic Solvent Soluble Fraction of the Kraft's Hardwood and Softwood Lignins and 1, 2, 4, 5-Benzene Tetracarboxylic • Acid. 46 TABLE 4. Total and Relative Acid Groups in Organic Solvent Soluble Products of Humic Acids and Lignins Oxidized with 1.6 N NaOCl. Sample Source Total Titrated Dominant Acidic Groups in Species OSS Fraction (meq) Relative Acidic ' Groups (meq/gm HA or lignin) Ah horizon Ah Ah H horizon H H Softwood 1ignin Hardwood 1ignin Spear grass Western porcupine grass Rough fescue Sphagnum moss Lodgepole pine Douglas f i r .76 3.16 1.45 1.89 1.83 1.70 1.61 2.97 2.87 5.91 3.26 4.02 3.55 4.19 3.77 6.27 47 The hardwood l ignin OSS products were nearly twice as acidic as the softwood l ignin products. The infrared spectra of the humic acid extracts and the prepared l ignin samples differed signif icantly from the spectra of their respective OSS oxidation products (Figures 5-12). In a l l cases,exclusive of the sphagnum moss s i te , the oxidation products displayed a relative intensification of the absorption bands in the following regions: 2900 cm"1 (aliphatic C-H stretching); 1720 cm - 1 (C-0 stretching, mainly from COOH groups); 1603 and 1585 cm - 1 (aromatic C=C stretching); 1465 and 1382 cm"1 (CH3 and CH2 deformation); i285 and 1135 cm - 1 (C=0 stretching); and 1082, 1052 and 970 cm - 1 (aromatic C-H deformation). The infrared spectra provided a strong indication of both aliphatic and aromatic acids constituting the OSS oxidation products with a more pronounced al iphatic character relative to spectra of the unaltered humic acids. The infrared spectrum of the humic acid extract of the sphagnum moss site bore l i t t l e s imilarity to the other five humic acids. This observation was probably due to the absence in moss species of vascular tissues which are part ia l ly composed of l ignin constituents (Bold, 1970). The oxidation products of the sphagnum moss site did display a strong 1720 cm - 1 band but less intense relative to the other soil s ites. The infrared spectra of the two l ignin samples, when compared with each other, were v ir tual ly identical before and after oxidation, indicating similar numbers and types of bonds existing ' I F i i t 1 i 1 r I i 1—• ' 4000 3000 2000 1800 1600 1400 1200 1000 800 600 500 400 300....: WAVENUMBER (cm->) . . FIGURE 5: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Spear Grass Site after NaOCl Oxidation. I 1 I I I I 1 1 1 1 I I I I — 1 4000 3000 2000 1800 1600 1400 1200 1000 800 600 500 400 300 WAVENUMBER (cm-i) FIGURE 6: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Western Porcupine Grass Site after NaOCl Oxidation. • -c* o 4000 i 3000 — i i i i i i i i i 1 f 2000 1800 1600 1400 1200 1000 800 600 500 400 300 WAVENUMBER (crrrO FIGURE 8: IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Sphagnum Moss Site after NaOCl Oxidation. I ! | I 1 1 I I • • 1 1 1  4 0 O 0 3000 2000 1800 1600 1400 1200 1000 800 600 500 400 300 WAVENUMBER (cm-') FIGURE 9: IR Spectra of the Humic Acid (upper); and the Organic Solvent ' Soluble Fraction (lower) of the Lodgepole Pine Site after NaOCl Oxidation. (Tl ro 4000 3000 1 , 1 1 1 I I 1 ' 1 ' 1 2000 1800 1600 1400 1200 1000 800 600 500 400 300 . WAVENUMBER (cm-1) FIGURE 12: IR Spectra of Kraft's Hardwood Lignin (upper); and the Organic Solvent Soluble Fraction (lower) after NaOCl Oxidation. 56 w i t h i n t h e s t r u c t u r e o f hardwood and softwood l i g n i n . The i n f r a r e d s p e c t r a o f t h e two l i g n i n samples a f t e r o x i d a t i o n bore a c l o s e s i m i l a r i t y t o t h e s p e c t r a o f t h e D o u g l a s - f i r and l o d g e p o l e p i n e o x i d a t i o n p r o d u c t s . In g e n e r a l , t h e o r g a n i c s o l v e n t s o l u b l e f r a c t i o n o f each sample d i s p l a y e d both a s t r o n g e r a c i d i c and a l i p h a t i c c h a r a c t e r compared t o i t s r e s p e c t i v e humic a c i d p o l y m e r i c p r e c u r s o r . U n f o r t u n a t e l y t he n u c l e a r magnetic r e s o n a n c e (NMR) s p e c t r a o f t h e OSS f r a c t i o n s y i e l d e d l i t t l e i n f o r m a t i o n r e g a r d i n g t h e n a t u r e o f t h e hydrogen atoms i n each sample ( F i g u r e s 13-2Q). In each spectrum, w e l l r e s o l v e d s i g n a l s a t 6 = 1.2 and 6 = 3.6 i n d i c a t e d t h e p r e s e n c e o f d i e t h y l e t h e r . T h i s r e s u l t was d i f f i c u l t t o e x p l a i n as t h e samples were e x h a u s t i v e l y d r i e d t o f r e e them o f t h e i r e t h e r s o l v e n t p r i o r t o a n a l y s i s and then r e d i s s o l v e d i n c h l o r o f o r m so as t o g i v e a w e l l r e s o l v e d s i g n a l a t 5 = 7.3. M i n o r peaks t h r o u g h o u t each s p e c t r u m i n d i c a t e d s m a l l amounts o f sample hydrogen bonded i n s t r u c t u r e s o t h e r t h a n d i e t h y l e t h e r . Comparison o f each scan w i t h r t h e s c a n s o f s t a n d a r d compounds d i d n o t match t h e s i g n a l s w i t h a n o t h e r compound e x c e p t d i e t h y l e t h e r . A s i m i l a r t y p e o f spectrum e x i s t e d f o r p r o p i o n i c a c i d but w i t h s i g n a l s a t 6 = 1.2 and 6 = 2.7. I f t h e w e l l r e s o l v e d peaks were i n f a c t d i e t h y l e t h e r , t h e r e a p p e a r e d <to-be i n s u f f i c i e n t o x i d a t i o n p r o d u c t s t o make NMR a u s e f u l a n a l y t i c a l -tool f o r t h i s s t u d y . The development o f t h e t h i n l a y e r chromatographs o f each ^sample p r o d u c e d o n l y a s i n g l e d i s t i n c t s p o t and a f a i n t t r a i l i n g shadow ppmW 10 9 8 7 6 5 4 3 2 1 0 FIGURE 13: N.MR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation. cn I I PPm & 10 9 8 7 6 5 4 3 2 FIGURE 14: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation. f 1 I ppm (5) FIGURE 15: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Rough Fescue Site after NaOCl Oxidation. I ppm(S) 10 9 8 7 6 5 4 3 2 I 0 NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation. o FIGURE 16; NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Lodgepole Pine Site after NaOCl Oxidation. I FIGURE 18: NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Douglas-Fir Site after NaOCl Oxidation. ro ! i I | i i n u i » » i » ' ppm W) 10 9 8 7 6 5 4 3 2 I 0 FIGURE 19; NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Softwood Lignin after NaOCl Oxidation. CO 7o" ppm (5) -FIGURE 20: NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Hardwood Lignin after NaOCl Oxidation. 65 as viewed under ultraviolet l ight . A parallel run chromatographing benzene hexacarboxylic acid and benzene pentacarboxylic ac id ,ut i l i z ing the same conditions,suggested the single sample spot could have been either or both of the standard compounds. A major portion of the OSS fraction spot of the spear grass and rough fescue sites did not migrate in either the 1 N acetic acid, water and dichloromethane solvent or the ether and 1 N HCl solvent and fluoresced with a mustard yellow coloration under ultraviolet l ight . The gas l iquid chromatographs (Figures; 21--28)-of each "sample supported the results of the thin layer chromatography. The chromatographed species were methylated before analysis and are referred to as their respective methyl ester derivatives. The dominant peak in each sample was the benzene hexacarboxylic acid hexamethyl ester except in the case of the Douglas-fir site which also yielded a benzene pentacarboxylic acid pentamethyl ester as a major peak. Each sample yielded a strong peak, merged with the solvent peak, probably representing acetic acid methyl ester or oxalic acid dimethyl ester. The o i ly substance previously observed in the spear grass and rough fescue humic acid oxidation products apparently contributed to. the final major peak for each sample. The chromatograms 2 for a l l eight samples were i n i t i a l l y produced at 4 x 10 attenuation. 2 Decreasing GLC sensit ivity by attenuating down to 64 x 10 brought the final major peak in both the spear grass and rough fescue samples on to the recorder scale, but only a single peak was observed under 66 FIGURE 21: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Spear Grass Site after NaOCL Oxidation. 67 o 100 125 150 175 2 0 0 2 2 5 2 5 0 2 7 5 T E M P E R A T U R E ( ° C ) FIGURE 22: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation. FIGURE 23: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Rough Fescue Site after NaOCl. Oxidation. FIGURE 24; GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation. 100 125 150 175 2 0 0 2 2 5 2 5 0 T E M P E R A T U R E ( ° C ) FIGURE 25: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Lodgepole Pine Site after NaOCl Oxidation. GURE 26: GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Douglas-Fir Site after NaOCl Oxidation. — I I • 1— I I I — 100 123 150 175 2 0 0 2 2 5 2 5 0 T E M P E R A T U R E ( ° C ) FIGURE 27: GLC Separation of the Organic Solvent Soluble Fraction of the Kraft's Softwood Lignin after NaOCl Oxidation. FIGURE 28: .. GLC Separation of the Organic Solvent Soluble. Fraction of the Kraft's Hardwood Lignin after NaOcl Oxidation. 74 either conditions of temperature programming or an isothermal run at 200°C for 2 hours. Dissolving the two nonmigratory thin layer chromatography spots in diethyl ether, followed by esterif ication with diazomethane, and analysis:under the same GLC conditions produced a single major peak at 215°C in both cases. The chromatograms of the Ah horizon samples generally displayed more benzene tricarboxylic acid trimethyl esters compared to the H horizon samples, which contained a relat ively greater amount of the benzene pentacarboxylic acid pentamethyl esters. The chromatrograms of each Kraft's l ignin sample closely resembled that of the lodgepole pine samples. A partial recovery of the o-phthalic acid, 1, 2, 4-benzene tricarboxylic acid and 1, 2, 4, 5-benzene tetracarboxylic acid standards indicated a potential for a 10% to 20% loss of oxidation products through handling and/or destruction of benzene carboxylic acids during oxidation (Table 5 and Figure 29). The overwhelming dominance of the benzene hexacarboxylic acid hexamethyl ester and, in one case the penta-substitute, made i t reasonable to consider only their peak area in the quantitative aromatic product calculations (Table 6). Quantitative determinations, on the basis of GLC peak areas, were not done with the spear grass and rough fescue samples since an unidentified component co-chromatographed with the esters of the aromatic acids. Assuming the reaction conditions did not destroy any aromatic rings and that the OSS acidic functions previously t i trated were in fact entirely ; " 75 TABLE 5: Recovery of Standard Compounds Subjected to Oxidation with 1.6 N NaOCl (p-phthalic acid I-S.) . Compound Ini t ia l Standard Relative Compound Weight Derivatized Molar Relative Recovery (gm) Ratio of In i t ia l Peak Areas (%) o-phthalic acid .0125 .84 84 p-phthalic acid (internal standard) .0125 100 1, 2, 3 benzene tr icar-boxylic acid .0160 1.04 .85 88 1, 2, 4, 5 benzene tetra-carboxylic acid .0200 1.05 .86 90 p-phthalic acid o-phthalic acid 1 0 0 GURE 29: GLC Separation of Organic Solvent Soluble Fraction of o-Phthalic Acid, 1, 2, 3-Benzene Tricarboxylic acid and 1, 2,4, 5-Benzene Tetracarboxylic Acid Standards After, NaOCl Oxidation. TABLE 6: Total Weight of Aromatic Acids in Dominant GLC Peaks and Degree of Aromaticity in Organic Solvent Soluble Fraction by GLC and Titrat ion Data. Total Weight of Aromatic Acids Degree of Minimum Degree of Sample Dominant In Dominant Aromaticity by Aromaticity by Source Species Peaks (gm) GLC Data (%) Titrat ion Data (%) Ah horizon Spear grass * * 16.4 Ah Western porcupine grass .050 9.4 33.7 Ah Rough fescue * * 18.6 H horizon Sphagnum moss .045 9.6 22.9 H Lodgepole pine .043 8.3 20.3 H Douglas f i r .060 14.8 23.9 Softwood lignin .055 12.9 21.5 Hardwood lignin .110 23.3 35.8 *could not be calculated. 78 benzene hexacarboxylic acid molecules, the t i trat ion data was used to estimate a minimum degree of aromaticity (Table 6). The degree of aromaticity calculated from the t i t rat ion data exceeded that calculated by GLC quantitative evaluation,indicating the acidic groups were not exclusively aromatic acids. The dominance of the OSS fractions by benzene hexacarboxylic acid, as determined by GLC techniques, implied a portion of the acidic functional groups t i trated were not aromatic substituents, but possibly were short chain aliphatic acids. This implies that the minimum degree of aromaticity figures calculated in Table 6 from the potentiometric t i t ra t ion data were inflated by the contribution of al iphatic acids which have been incorrectly assumed to be aromatic in nature. An accurate method for determining the degr.ee: of aromaticity in soi l organic matter fractions has not been developed, but several estimated structures for humic acid and l i g n i n , cited by Kononova (Chapter 3, 2 1966), attributed a minimum of 40% of the molecular carbon to Sp hybrids in aromatic structures. Pearl (1967) cited several estimations of the structure of l ignin in which 55% to 50% of the molecular carbon were S.p hybrids in aromatic structures. The results of this study indicated that the humic acids and lignins studied were much less aromatic than estimated by classical methods; however, the hypochlorite oxidation apparently did destroy some aromatic structures, thus giving the impression of a lesser aromaticity. 79 The recent research by several workers has cr i t i c i s ed the techniques of Chakrabartty and Kretschmer (1972, 1974a and 1974b). Mayo (1975) reported 2-naphthol and 2-naphthoic acid reacted vigorously with ring opening at the sites of substitution when reacted at 65°C for 1 to 3 hours. He also reported a rapid d i spropor t ionate of NaOCl to NaCl and NaClOg at acid pH which quenches the oxidation reaction. Ghosh et al. (1975) oxidized a naphthol-formaldehyde 3 polymer and fluorescein, containing 9% and 10% Sp carbon, respectively, with 1.6 N NaOCl and obtained as by-products.32% and 50% of their respective carbon as C02« Their work indicated that the oxidant did 2 3 not always discriminate between Sp . and Sp carbon hybrids. The work of LandoT.t (1975) confirmed the results of Ghosh al. (1975). Huston et al. (1976) fluorinated coal with elemental fluorine and established i ts carbon distribution as 70% aromatic (Sp ), '20% 3 aliphatic (Sp ) and 10% substituent carbon on aromatic rings. Their work gave no support to the adamantane structure (polycyclohexane) suggested as a possible structure for coal by Chakrabartty and Berkowitz (1974b). Aczel et al. (1975) subjected coal samples and adamantane to coal liquefaction conditions using tetralin:substrate, 2:1, at 400°C for 130 minutes; 96% of the adamantane was recovered unchanged as a cyclohexane soluble product but no adamantane was recovered from the coal. Their results indicated adamantane, probably did not exist in the structure of coal . Chakrabartty and Berkowitz (1976b)defended their report of 80% Sp carbon in coal structures by claiming that the selective NaOCl oxidation reaction was maintained 80 at room temperature, at high pH (11-12) to preserve the anionic form of the products and in an excess of 1.6 N NaOCl to insure complete oxidation of the substrate. The present research project incorporated a l l available suggestions for the successful application of the NaOCl oxidation technique to humic acid and l ignin . The results of this study indicated that i t was not safe to assume the technique could completely 2 3 discriminate between Sp and Sp carbon hybrids with no destruction of the aromatic rings. This is i l lustrated by the low apparent degree of aromaticity calculated for the l ignin oxidation products compared to the accepted abundance of phenyl propane building blocks constituting proposed structures of l ignin . Although intermediates from the oxidation reaction could not be characterized, i t seems reasonable to assume that some of the aromatic rings were broken producing aliphatic acids and CO2 as 2 by-products; as a result some previously Sp carbon hybrids were identif ied as Sp hybrids. The research of Mayo (1975) suggests that . hydroxyl group aromatic substituents may have transferred electrons to the ring followed by electrophil ic attack and ring opening to y ie ld aliphatic acids. The infrared (IR) data supported this poss ibi l i ty as both strong aromatic and al iphatic peaks were observed. Conversion of hydroxyl groups to methyl ethers with diazomethane prior to oxidation with NaOCl may have preserved some aromatic structures; as was the case with the work Schnitzer and Desjardins (1970) and Schnitzer and Khan (1972). 81 The goal of this study was to examine the potential of the NaOCl oxidation technique for the c lass i f icat ion of humic acids from various sources according to their oxidation product distribution. Distinct differences did exist between samples taken from the Ah horizons compared to the H horizons. These differences may have been related to differences in the age and possibly the relative maturity of the humic acids. Further distinctions among the three Ah horizons or the three H horizons were d i f f i c u l t to deduce as probably destruction of some aromatic rings altered the character of the products. Although humic acid structural determinations were not original ly intended in the goals of this study, the results do infer possible structures. The dominance of the GLC data by benzene hexacarboxylic acid suggests that the core of the humic acid may consist of a network of aromatic structures highly substituted by aliphatic linkages at a l l aromatic carbon sites which are oxidized to carboxyl groups during the NaOCl reaction. Moving to the exterior of the humic acid polymer, many of the aromatic rings may be substituted by hydroxyl groups, in addition to other functional groups, and are destroyed during the NaOCl reaction and reduced to al iphatic carboxylic acids when acidi f ied. This reasoning is a possible explanation for the high benzene hexacarboxylic acid OSS product and the absence of hydroxyl groups. A connection between l ignin and eventual humic acid genesis was implied by common products of oxidation identified by GLC and the distribution of oxidation product carbon approximately 82 intermediate in amounts between the humic acids extracted from the Ah horizons and the H horizon. No explanation as to the source or chemical nature of the o i ly substance in the oxidation products of the spear grass and rough fescue sites could be deduced. The molecular weight of 281 and the elution temperature from the gas l iquid chromatograph suggest benzene dimethyl tetracarboxylic acid as a possible structure but do not explain its o i ly nature. The d i f f i cu l t i e s encountered in applying the NaOCl oxidation technique to the investigation of soi l organic matter further confirms the complexity of the substrate. Successful application of a technique to the study of the relatively simple structures of standard compounds does not assure success with natural compounds. It seems probable that the presence of electron donating and electron-withdrawing functional groups in a randomly bonded polymer, such as humic acid, can profoundly change the behavior of a degradation method in comparison to i ts performance with standard compounds. 83 SUMMARY AND CONCLUSIONS To investigate the relative merits and faults of 1.6 N NaOCl as a selective oxidant for soi l humic substances, humic acid extracts from three Alberta soil sites and three Brit ish Columbia soil sites were oxidized at room temperature (23°C) for 5 days. To explore a possible l ignin component as a contributing structure in humic acid , two Krafts' prepared lignins were also oxidized under the same conditions. The study attempted to incorporate the benefits of a mild reaction temperature and a relat ively longer reaction time to l imit unnecessary destruction of the oxidation products. The NaOCl oxidation reaction degraded the humic acid and l ignin starting materials to chemically simpler structures of C O 2 and carboxylic acid products. The reaction yielded no products of molecular weight greater than benzene hexacarboxylic acid. The reaction was a relat ively simple procedure to employ; neither high pressures, high temperatures nor caustic chemicals were required, other than a reaction pH of 12-13. The distribution of the reaction products permitted c lass i f icat ion of the humic acid starting materials according to their Ah mineral or H organic horizon source. If the assumption of the humic acid extracts from the spear grass and western porcupine grass sites being the most mature was va l id , the results of this study indicate the maturation process tends toward a more aromatic chemistry in the humic acid with possibly a greater dominance by the plant 84 l ignin component. Similarit ies in the type and amounts of oxidation products suggests a l ignin component in each of the humic acids studied and a particularly strong influence on the nature of..the humic acid extracts from the spear grass and western porcupine grass sites. The probable more intense microbial act iv i ty at the rough fescue site resulted in a humic acid chemistry intermediate between that of the other two Alberta sites and three Brit i sh Columbia sites. An unidentified o i ly component was found in the oxidation products of the spear grass and rough fescue sites. The reaction products of a l l eight samples were found to be free of hydroxyl groups, despite the well documented identif ication of hydroxyl groups as a common functional group in humic acids and l ignins. This result and the relat ively low degree of aromaticity calculated for each of the samples suggest a reasonable poss ibi l i ty of ring opening at sites of hydroxyl group substitution on aromatic structures.with the result of an apparent lesser aromaticity and no hydroxyl group substitution in the products. Under the eventual acid conditions of product i so lat ion, broken ring fragments may have been reduced to aliphatic carboxylic acids. This result indicates the need for prior methylation of the humic acid or l ignin starting materials in future applications of the NaOCl oxidation technique. The results of this study imply the oxidation method may not have been 2 3 capable of total ly selecting Sp from Sp carbon hybrids under the conditions imposed and materials oxidized. The extreme complexity of soil humic substances appears to require further modifications of the NaOCl oxidation technique for future applications/in soi ls research. Application of the technique to a large number of standard compounds suspected to be representative of structures occurring in humic substances and lignins may prove most beneficial to fu l ly evaluating the potential of the NaOCl oxidation technique in soils research. LITERATURE CITED Aczel , T . , M. L. Gorbaty, P. S. Maa and R. H. Schlosberg. 1975. Stabi l i ty of adamantane to donor l iquifact ion conditions: implications toward the structure of coal. Fuel 55: 295. Alexsandrova, L. N. 1960. The use of sodium pyrophosphate for separating free humic substances and their organic-mineral, compounds from the s o i l . Soviet Soil Sc i . 1961(2): 190-197. Anderson, D. 1972. The characteristics of the organic matter of grassland, transitional and forest so i l s . Ph.D. Thesis, Univ. of Sask., Saskatoon, Sask. 138 pp. Bold, H. C. 1970. The plant kingdom. 3 r d ed. Prentice-Hall , Inc. Bremner, J . M . , S. G. Heintzl , P. G. J . Mann, and H. Lees. 1946. Metallo-organic complexes in s o i l . Nature 158: 790-791. Bremner, J . M. and C. L. Ho. 1962. Use of Dowex A- l chelating resin for extraction of soil organic matter. Prepublication Report, Dept. Agron., Iowa State Univ. , Ames Iowa. 5 pp. Burdon, 0. , J . D. Craggs, M. H. Hayes, and M. Stacey. 1974. Reactions of sodium sulphide. I. With compounds containing hydroxyl groups. Tetrahedron 30: 2729-2733. Burges, N. A . , H. M. Hurst, and S. B. Walkden. 1964. The phenolic constituents of humic acid and their relation to l ignin of the plant cover. Geochim. Cosmochim. Acta 28: 1547-1564. Butler, J . H. A. and J . N. Ladd. 1971. Importance of the molecula weight of humic and fulvic acids in determining their effect on protease act iv i ty . Soil B io l . Biochem. 3: 249-257. Chakrabartty, S. K. and H. 0. Kretschmer. 1972. Studies on the structure of coal: Part 1. The nature of al iphatic groups. Fuel 51: 160-163. Chakrabartty, S. K. and H. 0. Kretschmer. 1974a. Studies on the structure of coals. Part 2. The valence state of carbon in coal. Fuel 53: 132-135. Chakrabartty, S. K. and H. 0. Kretschmer. 1974b. Sodium hypochlorite as a selective oxidant for organic compounds. J . Chem. Soc. Perkin Transactions I: 223-228. Chakrabartty, S. K . , H. 0. Kretschmer, and S. Cherwonka. 1974. Hypohalite oxidation of humic acids. Soil Sc i . 117: 318-322. Chakrabartty, S. K. and N. Berkowitz. 1976a. Aromatic structures in coal. Nature 261: 76-77. Chakrabartty, S. K. and N. Berkowitz. 1976b. Non-aromatic skeletal structures in coal. Fuel 55: 362-364. Cheshire, M. V . , P. A. Cranwell, C. P. Falshaw, A. J . Floyd, and R. D. Haworth. 1967. Humic acid-II . Structure of humic acids. Tetrahedron 23: 1669-1682. Christman, R. F. 1970. Chemical structures of color producing organic substances in water. In Organic matter in natural waters. D. H. Wood, ed. Institute of Marine Science, Univ. Alaska, pp 181-194. Craggs, J . D. M . , H. B. Hayes, and M. Stacey. 1974. Sodium sulphide reactions with humic acid and model compounds. 10th I .S.S.S. Trans. II: 318-324. Crawford, D. L. and R. L. Crawford. 1976. Microbial degradation of 1ignocellulose: the l ignin components. Appl. Env. Microbiol. 31: 714-717. Dormaar, J . F. 1969. Reductive cleavage of humic acids of Chernozemic so i l s . Plant and Soil 31: 182-184. Dormaar, J . F. 1971. Total and carboxyl acidity and methoxyl groups of humic acid preparations of Chernozemic so i l s . Soil Sc i . Plant Anal. 2: 345-351. Dormaar, J . F. 1974. Comparison of several methods for extracting organic matter from Chernozemic and transformed Chernozemic Ah horizons. Can. J . Soil Sci . 54: 241-244. Dormaar, J . F. 1975a. Susceptibil ity of organic matter of Chernozemic Ah horizons to biological decomposition. Can. J . Soil Sc i . 55: 473-480. Dormaar, J . F. 1975b. Effects of humic substances from Chernozemic Ah horizons on.nutrient uptake by Phaseolus vulgaris and Festuca scabrella. Can. J . Soil Sci . 55: 111-118. Dubach, P. and N. C. Mehta. 1963. The chemistry of soi l humic substances. Soils Fert. 26: 293-300. 88 Felbeck, G. T. 1965. Structural chemistry of soi l humic substances. Adv. Agron. 17: 328-368. Felbeck, G. T. 1971. Chemical and biological characterization of humic matter. In Soil biochemistry, Vol . 2, A.D. McLaren and J . Skujins, eds. Marcel Dekker, Inc. , New York. p. 36-59. Ghosh, G . , A. Banerjee and B. K. Mazumdar. 1975. Skeletal structure of coal. Fuel 54: 294-295. Gottl ieb, S. and S. B. Hendricks. 1945. Soil organic matter as related to newer concepts of l ignin chemistry. S .S .S .A.P. 10: 117-125. Greene, G. and C. Steelink. 1962. Structure of soi l humic acid. II. Some copper oxidation products. J . Org. Chem. 27: 170-174. Halstead, R. L . , G. Anderson, and N. M. Scott. 1966. Extraction of organic matter from soils by means of ultrasonic dispersion in aqueous acetylacetone. Nature 211: 1430-1431. Hansen, E.H. and M. Schnitzer. 1967. Nitr ic acid oxidation of Danish i l l u v i a l organic matter. S.S.S.AP.. 31: 79-85. Hansen, E. H. and M. Schnitzer. 1969. Zinc dust d i s t i l l a t i o n and fusion of a soi l humic and fulvic acid. S .S .S .A.P. 33: 29-36. Haworth, R. D. 1971. The chemical nature of humic acid. Soil Sc i . I l l : 71-79. Hayes, M. H. B . , M. Stacey and R. S. Swift. 1972. Degradation of humic acid in a sodium sulphide solution. Fuel 51: 211-213.. Hayes, M. H. B . , M. Stacey and J . Standley. 1973. Studies on the humification of plant tissue. 9th Int. S.S.S. Trans. I l l : 247-255. Hayes, M. H. B . , R. S. Swift, R. E. Wardle, and J . K. Brown. 1975. Humic materials from an organic s o i l : a comparison of extractants and of properties of extracts. Geoderma 13: 231-245. Hereby, L. A. and M. B. Neuworth. 1962. Low temperature depolymerization of bituminous coal. Fuel 41: 221-231. Herman, W. A. 1974. Chemical composition of roots and decomposing root residues from three grass species. M.Sc. Thesis, Univ. Alberta, Edmonton, Al ta . 100 pp. Hurst, H. M . , A. Burges, and P. Latter. 1962. Some aspects of the biochemistry of humic acid decomposition by fungi. Phytochemistry 1: 227-231. 89 Huston, J . L . , R. G. Scott and M. H. Studier. 1976. Reaction of fluorine gas with coal and the aromaticity of coal. Fuel 55: 281-286. Jackson, M. P . , R. S. Swift, A. M. Posner, and J . R. Knox. 1972. Phenolic degradation of humic acid. Soil Sc i . 114: 75-78. Khan, S. U. and M. Schnitzer. 1971a. The potassium permanganate oxidation of methylated and unmethylated humic acids extracted from Solonetz, Solod and Chernozem Ah horizons. Israel J . Chem. 9: 667-677. Khan, S. U. and M. Schnitzer. 1971b. Further investigations on the chemistry of fulvic acid, a soil humic fraction. Can. J . Chem. 49: 2302-2309. Kononova, M. M. 1966. Soil organic matter. 2nd ed. , Pergamon Press, New York, N.Y. , U.S.A. 554 pp, Kononova, M. M. 1968. Transformations of organic matter and their relation to soi l f e r t i l i t y . Sov. Soil Sc i . 7: 894-900. Kononova, M. M. and N. P. Bel'chikova. 1970. Use of sodium pyrophosphate to separate and characterize organo-iron and organo-aluminum compounds in s o i l . Soviet Soil Sc i . 351-364. Kukharenko, T. A . , V. I. Bekikova, and L. V. Motovilova. 1971. Oxidation of humic acid fractions and derivatives by potassium permanganate in an alkaline medium. Sov. Soil Sc i . 304-313. Kumada, K. 1965. Studies on the color of humic acids. Part 1 on the concepts of humic substances and humification. Soil Sc i . and Plant Nut. 11: 11-16. Landolt, R. G. 1975. Oxidation of coal models. Reaction of aromatic compounds with sodium hypochlorite. Fuel 54: 299. Levesque, M. and M. Schnitzer. 1967. The extraction of soi l organic matter by base and chelating resin. Can. J . Soil Sc i . 47: 76-78. Levesque, M. and M. Schnitzer. 1966. Effects of NaOH concentration on the extraction of organic matter and of major inorganic constituents from a s o i l . Can. J . Soil Sc i . 46: 7-12. Lindqvist, I. 1968. On the extraction and fractionation of humic acids. Lantbrukshogsk. Ann. 34: 377-389. Lowe, L. E. 1974. A sequential extraction procedure for studying the distribution of organic fractions in forest humus layers. C. J . For. Res. 4: 446-454. Lutwick, L. E. and J . F. Dormaar. 1976. Relationships between the nature of soi l organic matter and root lignins of grasses in a zonal sequence of Chernozemic so i l s . Can. J . Soil Sc i . 56: 363-371. Martin, J . P . , K. Haider, and C. Saiz-Jimenez. 1974. Sodium amalgam reductive degradation of fungal and model phenolic polymers, soi l humic acids, and simple phenolic compounds. S .S.S.A.P. 38: 760-765. Maximov, 0. B . , V. E. Schapovalov, and T. V. Shvets. 1972. Alkaline permanganate oxidation of methylated humic acids. Fuel 51: 185-189. Mayo, F. R. 1975. Application of sodium hypochlorite oxidations to the structure of coal. Fuel 54: 273-275. Mendez, J . and F. J . Stevenson. 1966. Reductive cleavage of humic acids with sodium amalgam. Soil Sc i . 102: 85-93. Metcalfe, L. D. and A. A. Schmitz. 1961. Rapid preparation of fatty acid esters for gas chromatography analysis. Anal. Chem. 33: 363-364. Morrison, R. I. 1963. Products of the alkaline nitrobenzene oxidation of soi l organic matter. J .S .S . 14: 201-216. Neyroud, J . A. and M. Schnitzer. 1974. The exhaustive alkaline cupric oxide oxidation of humic acid and fulvic acid. S.S.S.A 38: 907-913. Neyroud, J . A. and M. Schnitzer. 1975a. The alkaline hydrolysis of humic substances. Geoderma 13: 171-188. Neyroud, J . A. and M. Schnitzer. 1975b. The mild degradation of humic substances. Agrochimica 19: 116-126. Oades, J . M. and W. N. Townsend. 1963. The influence of iron on the s tabi l i ty of soi l organic matter during peroxidation. J . S . S . 14: 134-143. Ogner, G. and M. Schnitzer. 1971. Chemistry of fulvic acid, a soi l humic fract ion, and i ts relation to l ign in . Can. J . Chem 49: 1053-1063. Ogner, G. 1973. Oxidation of nonhydrolyzable humic residue, and its relation to l ign in . Soil Sc i . 116: 93-99. 91 Ortiz de Serra, M. and M. Schnitzer. 197^ 2. Extraction of humic acid by a lka l i and chelating resin. Can. J . Soil Sci 52: 365-374. Pearl , I. A. 1967. The chemistry of l ign in . Marcel Dekker, Inc. New York, N. Y. 339 pp. Piper, T. J . and A. M. Posner. 1972. Sodium amalgam reduction of humic acid. Soil B io l . Biochem. 4: 513-531. Posner, A. M. 1966. The humic acids extracted by various reagents from a s o i l . Part 1. Yie ld , inorganic components, and t i t ra t ion curves. J . S . S . 17: 65-78. Randall, R. B . , M. Benger, and C. Groocock. 1938. The alkaline permanganate oxidation of organic substances selected for thei bearing upon the chemical constitution of coal. Proc. Roy. Soc. A. 165: 432-452. Schlink, H. and J . L. Gillerman. 1960. Esteri f icat ion of fatty acids with diazomethane on a small scale. Anal. Chem. 32: 1412-1414. Schnitzer, M. and J . R. Wright. 1960. Ni tr ic acid oxidation of the organic matter of a podzol. S .S .S .A.P. 24: 273-276. Schnitzer, M. and J . G. Desjardins. 1970. Alkaline permanganate oxidation of methylated and unmethylated fulvic acid. S .S .S .A.P . 34: 77-79. Schnitzer, M. and S. U. Khan. 1972. Humic substances in the environment. Marcel Dekker, Inc. New York, N . Y . , U.S.A. 327 Schnitzer, M. and S. I. M. Skinner. 1974a. The low temperature oxidation of humic substances. Can. J . Chem. 52: 1072-1080. Schnitzer, M. and S. I. M. Skinner. 1974b. The peracetic acid oxidation of humic substances. Soil Sc i . 118: 322-331. Skinner, S. I. M. and M. Schnitzer. 1975. Rapid identif ication by gas chromatography-mass spectrometry - computer of organic compounds resulting from the degradation of humic substances. Analytica Chimica Acta 75: 207-211. Shivrina, A. N . , M. D. Rydalevskaya, and I. A. Tereshenkova. 1968. Aromatic compounds as components of humic acids. Sov. Soil Sc i . 62-67. 92 Smith, L. 0. and J . S. Cr i s to l . 1966. Organic chemistry. Reinhold Publishing Corp. New York, N .Y . , U.S.A. 966 pp. Stevenson, F. J . and J . H. A. Butler. 1969. Chemistry of humic acids and related pigments, in Organic geochemistry: methods and results. G. Eglington and Sister M. T. J . Murphy, eds. Springer, New York. pp. 534-557. Stevenson, F. J . and K. M. Goh. 1971. Infrared spectra of humic acids and related substances. Geochim. Cosmochim. Acta 35: 471-483. Sweeley, C. C . , R. Bentley, M. Makita, and W. W. Wells. 1963. Gas-liquid chromatography of trimethylsi lyl derivatives of sugars and related substances. J . A . C . S . 85: 2495-2507. Tate, K. R. and K. M. Goh. 1973. Reductive degradation of humic acids from three New Zealand so i l s . N . Z . J . Sci . 16: 59-69. van Dijk, H. 1963. Some physico-chemical aspects of the investigation of humus. Report of the FAO/IAEA Technical Meeting, pp. 129-141. Vogel, I. A. 1948. Practical organic chemistry. Longmans, Green and Co. New York, N .Y. , U.S.A. p. 151. Wang, T. S. C , S. Cheng, and H. Tung. 1967. Extraction and analysis of soi l organic acids. Soil Sc i . 103: 360-366. Wildung, R. E . , Chesters, G . , and D. E. Behmer. 1970. Alkaline nitrobenzene oxidation of plant lignins and soil humic col loids . Plant and Soil 32: 221-237. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094514/manifest

Comment

Related Items