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

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THE SODIUM HYPOCHLORITE OXIDATION OF HUMIC ACIDS AND PREPARED LIGNINS  by  WILLIAM ALLAN HERMAN B . S . A . , University of A l b e r t a , M . S c , University of A l b e r t a ,  1972 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 B r i t i s h Columbia  We accept this thesis as to  the  conforming  required standard  THE UNIVERSITY OF BRITISH COLUMBIA September,  (c)  1977  William Allan Herman  In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h 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  t h i s 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 B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  Cfgj  y<?77  ABSTRACT In order to investigate the r e l a t i v e merits of a selective oxidant for the degradation of natural polymers, humic acid extracts from three Alberta s o i l  s i t e s and three B r i t i s h Columbia soil  sites,  and two Kraft's prepared wood lignins were oxidized with 1.6 N NaOCl at room temperature (23°C) for 5 days. included  The oxidation products  highly v o l a t i l e acids and organic solvent soluble (OSS)  products with the r e l a t i v e 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 i d e n t i f i e d 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 v o l a t i l e acid products could only be derived from the a l i p h a t i c chain or saturated ring components of humic acids or l i g n i n 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 t r a t i o n data, were substantially  than those calculated from proposed model humic acid and l i g n i n  iii  less structures  in the s o i l s l i t e r a t u r e .  An unidentified o i l y component was found  in the oxidation products of two of the humic acid preparations. The results of this study indicated humic acid and l i g n i n are composed of a mixed aliphatic-aromatic compound system.  The  r e l a t i v e l y 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 t o t a l l y selective in d i f f e r e n t i a t i n g Sp  from  3  Sp  carbon hybrids; as a result the total  discrimination between  a l i p h a t i c and aromatic structures was not a safe assumption.  It  is  postulated that aromatic ring opening may occur at s i t e s of hydroxyl group substitution on the ring structures resulting in an apparent less aromaticity and the generation of a l i p h a t i c acid products from aromatic  intermediates.  iv  TABLE OF CONTENTS PAGE ABSTRACT  iii  TABLE OF CONTENTS  v  LIST OF TABLES  vii  LIST OF FIGURES.  viii  ACKNOWLEDGEMENTS  xii  INTRODUCTION  1  LITERATURE REVIEW  3  Environmental Influence..  6  Extraction and Degradation  7  Methods for Degradation of Humic Substances MATERIALS AND METHODS  12 24  Origin of Humic Acids and Lignins Used in the Degradation Studies  24  Extraction and Fractionation  26  Oxidation Technique  26  Highly V o l a t i l e Acids  28  Extraction of Aqueous Oxidation Products  28  Carbon Analysis.  -.  •.  29  Ash Content  30  Phenol Test  30  Potentiometric  Titrations  v  32  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 . . . , . . . . ;  Table 3:  ...  . . . . .  . . . .  . . . . .  36  Distribution of Product Carbon (C) after Oxidation of Humic Acids and Prepared Lignins with 1.6 N NaOCl  Table 4:  .  38  Total and Relative Acid Groups in Organic Solvent Soluble Products of Humic Acids and Lignins Oxidized with 1.6 N NaOCl.  Table 5:  46  Recovery of Standard Compounds Subjected to Oxidation with 1.6 N NaOCl (p-^phthalic acid I.S.)  Table 6:  75  Total Weight of Aromatic Acids in Dominant GLC Peaks and Degree of Aromaticity in Organic Solvent Soluble Fraction by GLC and T i t r a t i o n Data  vi i  77  LIST OF FIGURES PAGE Figure 1:  Oxidation and Fractionation Degradation  Figure 2:  Scheme for NaOCl  of Humic Acid or Lignin  31  Potentiometric T i t r a t i o n Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of  the  Three Alberta Sites Figure 3:  43  Potentiometric T i t r a t i o n Curves of the Organic Solvent Soluble Fraction of the Humic Acid Extracts of the Three B.C. Sites  Figure 4:  44  Potentiometric T i t r a t i o n Curves of the Organic Solvent Soluble Fraction of the Kraft's Hardwood and Softwood Lignins and 1, 2, 4, 5-Benzene Tetracarboxylic Acid. . . .  Figure 5:  45  IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Spear Grass Site after NaOCl Oxidation  Figure 6:  48  IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Western  Porcupine  Grass Site after NaOCl Oxidation Figure 7:  49  IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Rough Fescue Site after NaOCl Oxidation.,  Figure 8:  50  IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the Sphagnum Moss Site after NaOCl Oxidation  51  viii  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  Figure 10:  52  IR Spectra of the Humic Acid (upper); and the Organic Solvent Soluble Fraction (lower) of the  Douglas-Fir  Site after NaOCl Oxidation. . Figure 11:  53  IR Spectra of Kraft's Softwood Lignin (upper); and the Organic Solvent Soluble Fraction (lower) after NaOCl. Oxidation  Figure 12:  54  IR Spectra of Kraft's Hardwood Lignin (upper); and the Organic Solvent Soluble Fraction (lower) after NaOCl Oxidation  Figure 13:  55  NMR Spectrum of the Organic Solvent Soluble Fraction of  the  Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation Figure 14:  57  NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation  Figure 15:  58  NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Rough Fescue Site after NaOCl Oxidation  Figure 16:  59  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  Figure 18:  61  NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Douglas-Fir Site after NaOCl Oxidation  Figure 19:  62  NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Softwood Lignin after NaOCl Oxidation  Figure 20:  NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Hardwood Lignin after NaOCl Oxidation  Figure 21:  63  GLC Separation  64  of the Organic Solvent Soluble Fraction of  the Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation Figure 22:  66  GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine  Grass  S i t e after NaOCl OxidationFigure 23:  GLC Separation  67  of the Organic Solvent Soluble Fraction of  the Humic Acid Extract of the Rough Fescue Site after NaOCl Oxidation Figure 24:  68  GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation  Figure 25:  GLC Separation  69 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 Figure 27:  GLC Separation  71 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 . . . . Figure 29:  GLC Separation  72  73  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  xi  76  ACKNOWLEDGEMENTS  Sincere appreciation i s extended to Dr. L . E. Lowe for  his guidance during the course of this study and constructive  c r i t i c i s m 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 i n a l l y , for the immeasurable meaning she gives to my l i f e , I dedicate my thesis to Beverley.  xii  INTRODUCTION  Humic substances are among the most widely distributed and important organic materials in nature.  The p a r t i c i p a t i o n and  importance of humic substances in the reactions occurring in s o i l and water are now being recognized by earth s c i e n t i s t s .  To better  understand the reaction mechanisms and environmental potential  of  humic substances, s c i e n t i s t s have sought to determine their chemical structure, with much of the research concentrated on humic a c i d . A specific  structure for humic acid has not been determined  but a number of c h a r a c t e r i s t i c s  have generally been accepted.  acid is considered to be of three-dimensional  Humic  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  large size and complexity of the polymer which necessitates  the  its  dismemberment to allow the i d e n t i f i c a t i o n 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 i t s building blocks a d i f f i c u l t  task. Many of the accepted characteristics of humic acid are also c h a r a c t e r i s t i c of coal constituents and of l i g n i n . several  of the techniques  For this reason,  for the analysis of coal and l i g n i n are  applicable to humic acids.  1  2  In view of the limitations of past research  techniques,  humic acids extracted from a wide range of s o i l s were degraded oxidatively under r e l a t i v e l y mild reaction conditions.  The primary  aim of the study was to evaluate the usefulness of the NaOCl oxidation technique in elucidating differences in a variety of s i t e s .  in processes of humus formation  Two prepared lignins were s i m i l a r l y  degraded to investigate a potential  l i g n i n 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 i g n i n samples.  LITERATURE REVIEW  Humic substances are the  major , organic accumulations  of s o i l s and waters and participate in and control many reactions that occur in these environments.  Humic substances are recognized  as being highly i n f l u e n t i a l on the b i o l o g i c a l , chemical and physical properties of s o i l s such as s o i l f e r t i l i t y , s t a b i l i t y and hydrology in s o i l  genesis. The need for increased a g r i c u l t u r a l productivity, and more  recently, the concern for ecological  security of s o i l have fostered  basic research on the nature of the chemical structures of s o i l components.  Wallerius published observations  in 1761 of humic  substances forming from decomposing plants and displaying an a b i l i t y to absorb water and nutrients. formation remain speculative, been intensely  investigated  Although the structure and pathways of the chemistry of humic substances has  in the past seventy years.  The basis for the investigation of organic materials found in s o i l s begins with recognition of the main components.  Soil  organic matter may be divided into three main f r a c t i o n s :  (1)  biomass or l i v i n g microorganisms and plant roots;  (2) the p a r t i a l l y  decomposed plant, animal and microbial tissues; and substances,  the  (3) the humic  described as amorphous, brown or black, hydrophilic,  a c i d i c , polydispersed substances ranging from hundreds to tens of thousands in molecular weight (Schnitzer and Khan, 1972). humic substances,  humus and humic fractions are often  synonymously in the l i t e r a t u r e . 3  used  The terms  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 c e l l  hypothesis; and  hypothesis.  (4) the microbial synthesis  autolysis  The plant a l t e r a t i o n hypothesis suggests certain plant components, p a r t i c u l a r l y l i g n i n , are resistant to microbial attack and are altered s u p e r f i c i a l l y to form humic substances. of the o r i g i n a l plant components  (cellulose,  The nature  hemicelluose,  lignin,  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 c e l l s and are released upon death and l y s i s 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 t h e i r s o l u b i l i t y in alkaline and acid media.  The f u l v i c acid  fraction is considered to be the a l k a l i - s o l u b l e component  5  of s o i l  organic matter which is also soluble in a c i d .  is also soluble  Humic acid  in alkaline but not acid media; humin is  in both a c i d i c and basic media.  insoluble  The terms f u l v i c and humic acid  have been c r i t i c i z e d 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 solubility characteristics. f u l v i c fractions  Even the boundary between the humic and  is not sharply defined.  the work of several  researchers,  Felbeck (1965) summarized  indicating that humic a c i d , f u l v i c  acid and humin d i f f e r in t h e i r elemental  composition, degree of  polymerization, molecular weight, a c i d i c functional of association  with inorganic s o i l  constituents.  groups and degree  Since much of the  is devoted to humic a c i d , this l i t e r a t u r e review w i l l  research  concentrate  on this humic f r a c t i o n . The research of several general  acceptance of several  workers has resulted in the  properties c h a r a c t e r i s t i c of humic a c i d .  Haworth (1971) defined humic acid as the d i l u t e aqueous sodium hydroxide . (NaOH) soluble fraction of s o i l precipitates  upon a c i d i f i c a t i o n .  organic matter which  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 a c i d i c nature of humic acid to a high degree of carboxyl and hydroxyl group substitution structures.  on aromatic  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 u n i t s , 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 t h e i r 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 i d e n t i c a 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  vegetative cover,  formation (type and amount of  climate, biological activity., parent  material and time) provide an almost l i m i t l e s s environmental conditions for i t s genesis.  ;  combination of  If we accept soil  to be a  continuum of b i o l o g i c a l , 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 p r a i r i e grass roots, determined that the chemistry of plant tissues varied with different plant types, establishing a potential  for differences  the chemistry of the humic products of their decomposition.  in  Dormaar  (1975a) suggested that the d i f f e r i n g chemical composition of humic substances derived fronra number of s o i l s could be used to differentiate  taxonomic s o i l groups.  the method of extraction affected  Kumada (1965) reported that  the chemistry of the humic extracts.  Shivrina et al. (1968) assumed that humic substances of different o r i g i n may vary in the composition of aromatic compounds making up t h e i r central structure or core of the molecule.  The chemical  character of the aromatic constituents may r e f l e c t the environmental influence.  EXTRACTION AND DEGRADATION The study of humic substances may be viewed as a three step process involving i s o l a t i o n of the organic materials from the mineral s o i l f r a c t i o n , degradation of the humic substances to their monomeric building blocks, and f i n a l l y a characterization of the degradation products.  The i s o l a t i o n 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 i t s chemical complexity without a l t e r i n g the chemistry of the monomers. These goals are i d e a l , 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)  relatively  drastic methods which result in high y i e l d s , but may cause structural modifications;  (2) mild methods with usually lesser  y i e l d s , which may not represent the total structurally intact.  sample but are probably  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 r e l a t i v e merits of both drastic and mild extraction techniques 1966; Posner, 1966; and Hayes etal.,  (Dubach and Mehta, 1963; Kononova, 1973).  Lindqvist (1968)  described a l l extraction methods as y i e l d i n g 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 y i e l d s with a low ash content and minimal chemical a l t e r a t i o n may be mutually exclusive.  9  Many of the extraction procedures to be discussed in this review do not subject the s o i l organic matter to an alkaline extraction followed by a c i d i f i c a t i o n .  For this reason, the  products w i l 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 c r i t i c i z e d as causing structural modifications to the humic extracts contact with a i r .  through autoxidation while in  Schnitzer and Khan (1972) cited the results of  several workers who compared d i l u t e a l k a l i to other extractants and found l i t t l e or no q u a l i t a t i v e 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 d i l u t e a l k a l i to be d i r e c t l y proportional to the length of time and temperature of extraction, within reasonable limits.  A pretreatment of soil with d i l u t e mineral a c i d , p a r t i c u l a r l y  in s o i l s 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  1966 and Schnitzer and Khan, 1972).  (Posner,  Posner (1966) maximized y i e l d s  of humic substances by pretreating a l l s o i l  samples with 0.1 N  hydrochloric acid (HC1) for 24 hours followed by extraction with 0.5N NaOH under N^ for 24 hours at room temperature.  1  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 d i f f e r i n g states of degradation. Sodium pyrophosphate (Na^P^O^) has gained popularity as an extractant due to i t s not requiring a d e c a l c i f i c a t i o n step, and i t s extraction c a p a b i l i t y 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 Kononova (1966) found that the efficiency  complexes.  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 d e c a l c i f i c a t i o n step. Organic chelating resins and water soluble  chelating  compounds, such as ethylene diaminetetraacetic acid (EDTA) have become popular for t h e i r extracting a b i l i t i e s without the r i s k of forming a r t i f a c t s .  Chelating resins and compounds act through the  disruption of organo.-metaT.T'ic complexes in s o i l to form ring structures with the released metals. substances,  The resulting negatively charged humic  freed of their previous metallic bonds, disperse within  the reaction medium.  Chelating resins require a r e l a t i v e l y longer  extraction period due to their s o l i d bead form requiring actual physical contact with the s o i l organic matter.  However, the resins can be  11  e a s i l y f i l t e r e d 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 i m i l a r i t y  of functional groups and infrared spectra for humic substances extracted with 0.5 N NaOH and sodium-saturated chelating r e s i n .  They  concluded that the damaging effects of alkaline extraction methods, reported by other workers, might be exaggerated. Felbeck (1971) recommended a sequential  extraction of  s o i l organic matter with benzene-methanol,  0.1 M HC1, 0.1 M Na^P^Oy,  6 N HC1 at 9 0 ° C , chloroform-methanol (5:1)  and f i n a l l y 0.5 N NaOH  to y i e l d a series of r e l a t i v e l y homogeneous fractions in comparison to a s i n g l e , d i l u t e  a l k a l i extraction.  extractions to examine differences  Lowe (1974) u t i l i z e d sequential  in products from various  forest  f l o o r organic matter accumulations. The use of ultrasonic v i b r a t i o n s , as an aid to extraction of s o i l with acetyl acetone, was found to be an effective method for the dispersion of humic substances,  p a r t i c u l a r l y organic phosphorus  and sulphur compounds (Halstead et al., successfully  1966).  Felbeck (1961)  extracted humic substances by ultrasonic dispersion in  d i l u t e NaOH or Na^P,^ media. Hayes et al. established  (1975) compared several techniques and  the following order of extraction e f f i c i e n c i e s  when  12  applied to a p a r t i c u l a r organic s o i l :  Na^P-pO^ < organic chelating  agents < polar aprotic solvents < pyridine < d i l u t e NaOH with y i e l d s ranging from 13 to 63%.  Successive extractions with neutral  N a P 0 , alkaline N a P 0 , and d i l u t e NaOH may extract humic materials 4  2  7  4  2  7  in increasing order of polymerization (Anderson, 1972). The l i s t i n g of extractants  in order of efficiency  should  be considered with caution as the order may change with different s o i l s i t e s and horizon of sampling.  The review of numerous extraction  techniques by Schnitzer and.Khan (1972) suggests the e f f i c i e n c i e s of a given technique may vary with changing s o i l types and different horizons within a given s o i l . The vast background l i t e r a t u r e concerning the extraction of humic substances implies some uncertainty as to the nature of the products of extraction compared to the nature of s o i l organic matter. Van Di j k (1963) warns that a grave error is committed in believing extractants d i f f e r only in the amount of product y i e l d . The continuing research with s o i l organic matter implies s o i l s c i e n t i s t s have not yet found the perfect  extractants  extraction  technique.  METHODS FOR DEGRADATION OF HUMIC SUBSTANCES Progress in deriving structural information concerning humic substances and humic acids in p a r t i c u l a r has been limited by unsatisfactory techniques for degradation (Bremner and Ho, 1962 and  13  Felbeck, 1965).  Evidence of possible s i m i l a r i t i e s  between the  chemistry of s o i l organic matter, of l i g n i n and of coal has led respective  chemists to examine numerous methods of degradation.  In  each case, the method of degradation i d e a l l y seeks to achieve high y i e l d s of chemically simpler compounds, suffering l i t t l e or no chemical alterations  compared to their status as monomers in the  o r i g i n a l 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  y i e l d s due to excessively mild degradation conditions or excessively drastic conditions destroying the humic starting materials or creating a r t i f a c t s which cannot easily be related to the parent polymer (Neyroud and Schnitzer, 1975a). Jackson et al.  (1972) recognized humic acid to be a  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 i s well  recognized in the chemical l i t e r a t u r e . 0  Interaromatic linkages probably  include - N H - , =N-, - S - , - C - , -0- and a l i p h a t i c 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 ) or consist of condensed ring systems ( [ O ^ O } )  (  K o n o n o v a  (0))  v  > 1966).  14  The wide range of bond types found in humic substances, plus probable complexing with organic and mineral constituents,  might  p a r t i a l l y explain the d i f f i c u l t y in finding the ideal degradation system. Felbeck (1965), assuming the bond energies within the aromatic monomers to be s i m i l a r to the bond energies between the monomers in a polymeric structure, reported low y i e l d s 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 s h i f t in the  efforts  of s o i l chemists from d r a s t i c to milder degradation techniques.  The  increased a v a i l a b i l i t y of gas chromatographic, mass spectrometric and computerized data processing techniques has increased the speed and s e n s i t i v i t y of research (Skinner and Schnitzer, 1975). Consideration of the merits and faults of several degradation techniques, both d r a s t i c and m i l d , may prove beneficial to the appreciation of the magnitude of the problem. The methods used to degrade humic substances chemical techniques techniques.  include  (both oxidative and reductive) and biological  Schnitzer and Khan (1972) reported the r e l a t i v e l y  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 the aromatic core of humic susbtances. sugar monomers.  information concerning  Boiling water essentially  Acid hydrolysis has been successfully  removed  used to extract  carbohydrates and nitrogenous materials prior to application of more destructive degradative methods to the nonhydrolyzable f r a c t i o n . Alkaline hydrolysis with hot NaOH or potassium hydroxide (KOH) has been c r i t i c i z e d by Cheshire et al. (1968) as creating a r t i f a c t s 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 i m i t interpretive value. Oades and Townsend (1963) found oxidation of humic substances with hydrogen peroxide structural information.  (H2O2)  to be incomplete and offering  little  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 i g n i n chemists, was u t i l i z e d by Schnitzer and Wright (1960) to i s o l a t e nitrophenolic and nitrobenzoic acid products representing only 5.3% of the o r i g i n a l humic a c i d .  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 HN0  3  to be one of the higher y i e l d i n g oxidation methods of i t s time but suspected destruction of some aromatic structures. of n i t r i t e groups ( I ^ ) -  The substitution  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 i g n i n after confirming common products in the alkaline nitrobenzene oxidation of l i g n i n and humic substances.  The apparently universal degradation products of  l i g n i n - syringic a c i d , v a n i l l i c acid and p-hydroxybenzoic acid were isolated in y i e l d s 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 y i e l d s of a l i p h a t i c acids representing 53% of the o r i g i n a l humic a c i d .  Low product y i e l d s  (0.58 to 1.33%) experienced by Greene and Steelink (1962) were improved by methylating the humic acids prior to oxidation. oxidation method is considered ineffective  The alkaline CuO  in degrading humic substances  low in substitution by oxygen containing functional groups and C-C bonds (Neyroud and Schnitzer, 1975b).  A l i p h a t i c 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 t e r a t u r e 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 C0 > H 0 and oxalic a c i d . 2  2  Schnitzer and Desjardins  (1970) f e l t low y i e l d s 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 i n h i b i t s e l e c t r o p h i l i c attack of activated rings by the oxidant. In a comparison of the oxidation products of methylated and unmethylated humic a c i d s , Khan and Schnitzer (1971a) reported that methylation increased the y i e l d 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 i m i l a r i t i e s between  acid nonhydrolyzable humic acid and l i g n i n after achieving similar products from each with KMnO,, oxidation.  18  D r a s t i c a l l y 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 i m i t the structural information derived by several workers.  Cheschire et al. (1967)  concluded that the method t o t a l l y destroyed the a l i p h a t i c constituents of humic substances.  Hansen and Schnitzer (1969) achieved a 0.66%  y i e l d 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  and after reduction implied that the technique caused structural a l t e r a t i o n s .  Piper and Posner (1972) f e l t  structural  Martin et al. (1974) achieved a 3 to 6% y i e l d of  complex products after the reduction of several (1969) reported being able to d i f f e r e n t i a t e , soil  the  to l i g n i n chemists but when applied to s o i l s ,  the products were too complex to readily provide useful information.  significant  Mendez and Stevenson (1966) and Tate and Goh  (1973) confirmed these r e s u l t s . method was useful  before  humic acids.  extremely Dormaar  humic acids from Chernozemic  sites on the basis of their Na amalgam reduction products.  Mendez and Stevenson (1966) reported that the i n s t a b i l i t y of the products of Na amalgam reduction of humic substances resulted in repolymerization  reactions.  19  Guidelines d i f f e r e n t i a t i n g drastic from mild degradation conditions have not been established  in the s o i l  science l i t e r a t u r e .  Schnitzer and Skinner (1974b) suggested 100°C might be an upper temperature l i m i t above which structural alterations of a r t i f a c t s might be expected. temperature  and formation  Exchanging a r e l a t i v e l y high reaction  (80°C for 4 hours) for a longer peracetic acid oxidation  time, Schnitzer and Skinner (1974a) reported similar y i e l d s  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 attack of activated and Skinner, 1974a).  electrophilic  aromatic structures by alkaline KMnO^ (Schnitzer At lower temperatures of oxidation  (40°C),  methylation of humic substances p r i o r / 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 t r i f l u o r i d e (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. phenol substitution  When applied to humic substances, the  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 i g n i n .  Hayes et al.  (1972)  achieved a 60% y i e l d of ether-soluble materials after degrading humic acid for 2 hours at 250°C in 10% aqueous Na S. 2  The products of  degrading a series of aromatic standard compounds which broadly represented structures and linkages l i k e l y to be present in humic acid and l i g n i n indicated a p o s s i b i l i t y of humic acid being the product of modified l i g n i n and phenolic compounds (Craggs et al.,  1974).  Although not extensively applied to s o i l research, the work of Burdon et al.  (1974) and Craggs et al.  (1974) indicated Na S degradation 2  to have outstanding merit in humic acid structural  studies.  The microbiological degradation of humic substances may be the most selective or s p e c i f i c for structural determinations.  bond breakage method available  The future of biological degradation  techniques hinges on the i d e n t i f i c a t i o n or development of microorganisms capable of breaking s p e c i f i c  bonds t y p i c a l l y found in humic substances  and elucidation of t h e i r 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 i t s highest y i e l d s 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 d i f f e r e n t i a t i n g microbially  synthesized materials from humic degradation products limited the interpretive value of the method. Khan and Schnitzer (1971b) proposed a technique for examining f u l v i c acid constituents without an i n i t i a l step.  degradative  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. F u l l y methylated phenolic acids, benzoic a c i d , methyl esters and dialkylphthalate products were eluted from the column with solvents of increasing polarity.  The f u l v i c acid portion not eluted was redissolved in  benzene, dried and subjected to alkaline KMnO^ oxidation.to y i e l d products similar to the eluates but in s i g n i f i c a n t l y greater amounts. Khan and Schnftzer (1971b) suggested the eluates  represented  structures weakly l i n k e d , 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 w i l l dismantle the humic polymer in a predictable fashion y i e l d i n g stable products which, upon i d e n t i f i c a t i o n , give implications of their structural role in the polymer. further the state of knowledge,  soil  To  s c i e n t i s t s occasionally apply  22  the tested techniques of l i g n i n and coal to compare the chemical s i m i l a r i t i e s , of humic substances,  l i g n i n and c o a l .  sensitive modern instrumentation, s o i l  Aided by highly  biochemists are moving to  u t i l i z i n g r e l a t i v e l y mild degradation techniques to break the minimum number of bonds to achieve products y i e l d i n g the maximum structural information.  In short, humic substances are now being appreciated  as delicate natural polymers revealing their chemical nature under milder degradation conditions r e l a t i v e to the d r a s t i c 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 s o i l 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 i n t a c t .  Sites of oxidation on aromatic structures appeared as  carboxyl groups.in the benzene carboxylic acid., products, implying the oxidant 2 could d i f f e r e n t i a t e  Sp  3 and Sp  carbon hybrid structures.  required low reaction temperatures,as  coal is susceptible  The technique to  autoxidation at temperatures above 70°C (Chakrabartty and Berkowitz, 1976a).  23  The achievements  of s o i l  s c i e n t i s t s 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 s o i l  humic substances of varied states of maturity.  of maturation, as viewed by several  :  The concept  Russian researchers, involves a  gradual degradation of a l i p h a t i c components with concurrent in the aromatic character of the persistant materials.  increases  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 comparison of reaction products from a variety of  tool for the  soils.  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 y i e l d s might deserve  reviewing  in the l i g h t 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 p r a i r i e grassland sites previously investigated  by Herman (1974), Dormaar (1971, 1974  and 1975b) and Lutwick and Dormaar (1976) and two B r i t i s h Columbia forest  humus sites plus a sphaghum peat described in an e a r l i e r study  by Lowe (1974).  The sites were selected to include a d i v e r s i t y 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 r e l a t i v e l y undisturbed condition.  Although  the three p r a i r i e s i t e s were a l l vegetated by p r a i r i e grass species, the Ah horizons sampled included three s o i l zones and three plant communities.  distinct  The H horizons of the B.C. s i t e s were selected  where the three stages of decomposition be e a s i l y separated.  (L, F and H horizons) could  The Ah horizons sampled probably possessed a  r e l a t i v e l y more mature humic acid compared to the H horizons. The six s o i l  samples were ground to pass through a 2 mm  sieve. Commercially prepared samples of a Kraft's softwood  lignin  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. data for the l i g n i n preparations.  The manufacturer did not supply any  TABLE 1:  Origin and Chemical Characteristics of Soil Samples.  Horizon  Depth (cm)  % C  pH CaCl  H.A. % Ash  Location (Lat N/Long W)  Legal Location (Soil Zone)  4 9 ° 0 2 7 n0°26'  SE 16-2-14 W4 (Brown)  Ah  8-8  1. 32  .15  8.8  5.7  52.7  49 09'/110 5T  NE 19-2-6 W4  Ah  0-10  3. 28  .32 10.2  5.5  12.2  Rough fescue  50 06 7114 05'  SW 27-13-1 W5  Ah  0-8  11. 1  1 .10 10.1  5.8  30.9  Sphagnum species  Sphagnum moss  Fort St. B.C.  80-90 14. 4  1 .09 38.0  4.8  9.3  Pinus contorta van latifolia Engelm.  Lodgepole pine  Manning Park, B.C.  H  37.0  4.3  3.9  Pseudotsuga menziesii .(Mirb.) Franco  Douglas f i r  Campbell River, B.C.  H  1 .04 45.0  4.1  21.2  Dominant Species Vegetation  Stipa comata T r i n . & Rupr.  Common Name  Spear grass  Stipa spavtea Tri n. Western porcupine var. ouvtiseta Hitchc. Festuca Torr.  scabvella  O  O  O  0  John,  Oh  .5-2  29. 9  5-8 46. 8  % N  .81  C/N  2  r\3  26  Extraction and Fractionation The six s o i l samples were a l l subjected to identical extraction and fractionation conditions.  Each sample was i n d i v i d u a l l y 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 d i l u t e 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 b o t t l e s , 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 f u l v i c acid supernatant layer discarded.  The humic  acid centrifugate was redissolved in 0.5 N NaOH, a c i d i f i e d with 1.0 N HC1 and separated two additional times with the f u l v i c 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 i g n i n 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 autodecomposition 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) to trap CO,,. 2  The nitrogen gas flow  also served to prevent atmospheric C0 being trapped in the gas 2  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 i g n i n p a r t i c l e s adhering to the wall of the flask.  After  five days oxidation at room temperature ( 2 3 ° C ) , the resultant mixture was a c i d i f i e d with 1 N HC1 to pH 3.5; the evolved C0 was swept out 2  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 t e r e d 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 product.  2  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) gas scrubber to 2  monitor extraneous C0 entering the reaction system. 2  The reaction  was quenched and the remainder of the unreacted oxidant decomposed by further reducing the a c i d i t y 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 V o l a t i l e Acids Highly v o l a t i l e acids  in each a c i d i f i e d  aqueous reaction  mixture were removed under vacuum at 35°C and d i s t i l l e d until d i s t i l l a t e was no longer a c i d i c as tested with pH paper.  the  An aliquot  pf each d i s t i l l a t e was t i t r a t e d with 0.0505 N NaOH using phenolphthalein indicator to evaluate the highly v o l a t i l e acid products.  The  burette used for t i t r a t i o n was equipped with a trap containing KOH pellets  to prevent interference  by atmospheric CO^ (Wang et al., 1966).  The carbon content of the highly v o l a t i l e acid component was estimated  by assuming the v o l a t i l e acids for the given  included formic, acetic and  conditions  propionic acids for a probable average  of two carbon atoms per acid functional group t i t r a t e d .  Extraction of Aqueous Oxidation Products The a c i d i c aqueous reaction products removal of the highly v o l a t i l e acid fraction  remaining after 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 s o i l sample.  The material, which floated on the  surface  of the aqueous l a y e r , 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 a c i d i f i e d 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 a c i d .  The l i g n i n 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 i g n i n 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 u t i l i z i n g a vycor glass insert for the quartz enclosed graphite c r u c i b l e .  D i f f i c u l t i e s encountered in drying the  ether extracts of the humic acids extracted from the spear grass and rough fescue sites necessitated dry.  heating the samples to 100°C until  The remaining ether and methanol fractions of each s i t e 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 w i l l be  referred to as the organic-solvent-soluble fraction,abbreviated as the OSS f r a c t i o n . The oxidation and fractionation scheme for this study is summarized in Figure 1.  Ash Content Weighed samples of humic acids and l i g n i n s 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 v i o l e t colour  was considered a positive test and a yellow color for  a negative  phenolic compounds (Smith and C r y s t a l , p. 152, 1966).  test  31  Substrate (humic acid or lignin) Oxidize in 1.6 N NaOCl at pH 13 for 5 days at room temperature i Acidify to pH 3.5  Y Acidify to pH 1.0  C0  2  product  • • H i g h l y v o l a t i l e acid products  Concentrate aqueous oxidation products and extract with ether for 7 days  Dry aqueous layer and extract by shaking with hydrous methanol for 24 hours  Ether extract  Methanol extract  Carbon content  Carbon content Salt  Organic solvent soluble (OSS) extract for TLC, NMR, IR & GLC analyses  FIGURE 1:  crystals  Carbon content  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 t i t r a t e d with 0.0500 N NaOH to pH 9.0.  N HCl)and potentiometrically  A 10-ml burette equipped  with a hypodermic needle f a c i l i t a t e d dispensing of small amounts of  base.  A reference sample of 0.0600 g  of 1, 2, 4, 5-benzene  tetracarboxylic acid was t i t r a t e d 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 i n g 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, Westbury, New York).  Inc.,  The spots were chromatographed in two  dimensions with a .1:1:1 volume solvent r a t i o of 1 N acetic water and dichloromethane followed by 100:1 ratio of ether and 1 N HC1.  acid,  volume solvent  The spots were viewed under u l t r a v i o l e t  l i g h t 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 i n . (o.d.) stainless steel  columns packed with 3% SE 30 on Chromosorb w-HP, 80 to 100 mesh.  The SE 30 l i q u i d phase was selected for  its  a b i l i t y to separate both a l i p h a t i c and aromatic components on the basis of differences points.  in t h e i r molecular weights and b o i l i n g  The gas l i q u i d chromatograph was programmed from 7 5 °  to 275°C at a rate of 5°C per minute with a research grade nitrogen c a r r i e r gas flow rate regulated to 50 ml per minute.  The oxidation  products and a p-phthalic acid internal standard, added prior to d e r i v a t i z a t i o n , were methylated three times to insure maximum d e r i v a t i z a t i o n with diazomethane generated from Diazald (Schlink and Gillerman, 1960). derivatives with T r i s i l  The preparation of t r i m e t h y l i s i l y l  (Pierce,Box 117, Rockford,  Illinois)  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.  E s t e r i f i c a t i o n of the  oxidation products as methyl esters using 14% methanol in boron t r i f l u o r i d e was attempted as a t h i r d derivatization  technique  (Metcalfe, 1961); i t was rejected due to a high degree of  35  contamination in the d e r i v a t i z i n g agent.  A series of nine  aromatic and a l i p h a t i c standard compounds, selected for their a v a i l a b i l i t y and expected presence in the oxidation products according to the chemistry of the hypohalite reaction, were matched by elution time and temperature and c o - i n j e c t i n g identify some of the product peaks (Table 2).  to  To f a c i l i t a t e  precise temperature readings, a copper-constantan thermocouple was used to c a l i b r a t e the temperature programmer against an ice water  reference. Peak area c a l c u l a t i o n s , 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 e f f i c i e n c y of the oxidation and fractionation scheme followed in this study was evaluted by subjecting weighed  samples  of o-phthalic a c i d , 1, 2, 3-benzene t r i c a r b o x y l i c 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.  Compound  Fully E s t e r i f i e d Compound Elution Temperature ( C)  Structure H HOOC - C - COOH H  107  Succinnic acid  H H HOOC - C - C - COOH H H  110  p-phthalic acid  HOOC  1.  Malonic acid  2.  3.  -(O^-  125  COOH  —r-COOH  4.  1 , 2 , 3-benzene t r i c a r b o x y l i c acid  (OV C O O H  165  >—CQQQU  COOH  _ C  5.  1, 2, 4-benzene t r i c a r b o x y l i c acid  (OV V—/  COOH  1  7  0  HOOC  COOH  6.  HOOC-/O)  1, 3, 5-benzene t r i c a r b o x y l i c acid  ^—(COOH y  7.  1 , 2 , 4, 5-benzene tetracarboxyl i c acctd  174  H00C  COOH  -/O>-C00H  189  HOOC^— ,  8.  HOOC-/O/COOH  1 , 2 , 3, 4, 5-benzene pentacarboxylic acid  1,2,3,4,5, 6-benzene hexacarboxylic  196  HOOC—COOH  H 0 0 C  HOOC  acid  COOH  ^C00H  -/O>C00H  WQQZ  ^""LOOH  2  1  5  37  RESULTS AND DISCUSSION Six humic acid preparations representing a variety of soil vegetative cover and two Krafts' prepared l i g n i n s , from a softwood species and hardwood species, were oxidized at room temperature with 1.6 N NaOCl.  The eight samples studied were i d e n t i c a l l y  treated and analyzed to f a c i l i t a t e comparison of the r e l a t i v e amounts and types of oxidation products.  Comparisons were made to  evaluate  trends which might r e f l e c t an influence of the dominant plant species at each s i t e on the nature of the humic acid resulting from i t s decomposition and any possible connection between l i g n i n and humic acid formation. For the sake of b r e v i t y , the oxidation products studied are referred to by t h e i r respective dominant plant species, rather than "The NaOCl oxidation products of the humic acids extracted from the appropriate site". The r e l a t i v e amounts of carbon, expressed as a percentage of the total  humic acid or l i g n i n carbon, in ether soluble, methanol  soluble, carbon dioxide and h i g h l y - v o l a t i l e acid fractions a r i s i n g from the hypochlorite oxidation are summarized in Table 3.  The  d i s t r i b u t i o n of oxidation products is expressed in terms of the d i s t r i b u t i o n 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:  Sample Source  Distribution of Product Carbon (C) after Oxidation of Humic Acids and Prepared Lignins with 1.6 N NaOCl. {% of Total Original Carbon)  Dominant Species  Ether Soluble-C  Methanol Soluble-C  Total Organic Solvent Soluble-C  Carbon Dioxide-C  Highly Volatile 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  Rough fescue  15.03  2.02  17.05  32.12  50.27  99.44  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  Ah H  (horizon)  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 l y constituent samples.  not present in the other  six  Mass spectrometric scans of the two samples indicated,  in each case, the o i l y 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 s i t e s are r e l a t i v e l y older and probably more mature than those extracted from the H horizons of the three B r i t i s h Columbia s i t e s . proportion of the humic acid carbon appears as  A greater  ether-soluble  and carbon dioxide carbon in the more mature samples while the  carbon reverse  trend is observed in the case of the highly v o l a t i l e acid carbon obtained from the less mature samples.  A s l i g h t r i s k exists that the amounts  of highly v o l a t i l e acid products are overestimated due to HCl and H0C1 contamination during a c i d i f i c a t i o n 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 i n s i g n i f i c a n t as they contained no carbon trapped in the crystal structure. Considering the known chemistry of the sodium hypochlorite oxidation reaction, carbon dioxide and highly v o l a t i l e 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 v o l a t i l e acid products accounted for 66-82% of the humic acid or l i g n i n 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 s i t e s of previous aromatic components.  Assuming the reaction conditions t o t a l l y  preserve  aromatic r i n g s , the results of this study indicate a more aromatic character for the humic acids extracted from the spear grass and western porcupine grass s i t e s and the two l i g n i n samples compared to the other four samples.  A r e l a t i v e l y large organic solvent  soluble  (OSS) product in combination with a r e l a t i v e l y small highly v o l a t i l e acid product further implies a more aromatic and condensed structure in the two humic acids and two l i g n i n samples. The average annual p r e c i p i t a t i o n over a 30-year period for the spear grass, western porcupine grass and rough fescue sites 32, 41, and 50 cm, respectively,  is  with only the rough fescue s i t e  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 s i t e s as l i m i t i n g microbial a c t i v i t y with the resulting accumulation of l i g n i n ; possibly these conditions explain the s i m i l a r i t y in  41  oxidation product d i s t r i b u t i o n of these sites and the prepared l i g n i n s . The more moist s o i l conditions at the rough fescue s i t e apparently permit a more rapid decomposition of a l l plant constituents;  the  d i s t r i b u t i o n of the s i t e ' s humic acid oxidation products suggests a structural  s i m i l a r i t y to that of the humic acids extracted from the  three B.C. s i t e s .  Due to their close proximity, the spear grass arid  western porcupine grass sites have similar p r e c i p i t a t i o n and potential evaporation conditions, but imperfect drainage conditions at the l a t t e r s i t e 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 i g n i n , respectively,  r e l a t i v e to dry ash free  (DAF) samples.  The r e l a t i v e l y  dry s o i l conditions at the spear grass s i t e and i t s exposed condition throughout the year, probably l i m i t i n g factors for microbial a c t i v i t y , are apparently conducive to chemical and physical decomposition of plant constituents other than l i g n i n .  The l i g n i n  constituent,  which has been shown by several authors to be largely aromatic in nature (Pearl, 1967), persists while less resistant (cellulose,  plant constituents  hemicellulose and proteins) are p r e f e r e n t i a l l y degraded.  A similar situation apparently exists at the western porcupine grass site.  The i n i t i a l highest fresh root l i g n i n content at the rough  fescue s i t e and lowest OSS product of the three Alberta sites  42  indicates that the s o i l conditions at the s i t e 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 r i n g s , all  gave a negative  (yellow color)  result.  further confirmed by the i n a b i l i t y of T r i s i l  This result was to form trimethysilyl  ether derivatives of the oxidation products prior to gas l i q u i d chromatographic (GLC) analysis. An aliquot of the OSS f r a c t i o n of each sample was potentiometrically t i t r a t e d to compare the configuration of the t i t r a t i o n 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 t e , a relationship was  observed between the amount of t i t r a t a b l e 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 t e s , previously noted as having an o i l constituent  in t h e i r oxidation  products, possessed the least amount of a c i d i c 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 a l i p h a t i c groups to appear as carboxyl substituents  in the oxidation products, or possessed  r e l a t i v e l y few aromatic structures to be recovered in the OSS f r a c t i o n .  PH 10.0  0.0500  FIGURE 2:  N NaOH  (ml)  Potentiometric T i t r a t i o n 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  0 . 0 5 0 0 N NaOH  FIGURE 3:  5.0  6-0  (ml)  Potentiometric T i t r a t i o n 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 T i t r a t i o n 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.  Sample Source  Total and Relative Acid Groups in Organic Solvent Soluble Products of Humic Acids and Lignins Oxidized with 1.6 N NaOCl.  Dominant Species  Total Titrated Acidic Groups in OSS Fraction (meq)  Relative Acidic ' Groups (meq/gm HA or lignin)  .76  2.87  Western porcupine grass  3.16  5.91  Rough fescue  1.45  3.26  Sphagnum moss  1.89  4.02  H  Lodgepole pine  1.83  3.55  H  Douglas f i r  1.70  4.19  Softwood 1ignin  1.61  3.77  Hardwood 1ignin  2.97  6.27  Ah horizon  Spear grass  Ah  Ah H  horizon  47  The hardwood l i g n i n OSS products were nearly twice as acidic as the softwood  l i g n i n products.  The infrared spectra of the humic acid extracts and the prepared l i g n i n samples differed s i g n i f i c a n t l y spectra of their respective 5-12).  from the  OSS oxidation products  (Figures  In a l l cases,exclusive of the sphagnum moss s i t e ,  oxidation products displayed a r e l a t i v e i n t e n s i f i c a t i o n absorption bands in the following regions: C-H stretching); 1603 and 1585 c m  1720 c m -1  and 1082,  1  (aromatic C=C stretching);  2  1052 and 970 cm  of the (aliphatic  (C-0 stretching, mainly from COOH groups);  -1  (CH and CH deformation); i285 and 1135 c m 3  2900 cm"  the  -1  -1  1465 and 1382 cm"  1  (C=0  stretching);  (aromatic C-H deformation).  The infrared  spectra provided a strong indication of both a l i p h a t i c and aromatic acids constituting the OSS oxidation products with a more pronounced a l i p h a t i c character r e l a t i v e to spectra of the unaltered humic acids. The infrared spectrum of the humic acid extract of the sphagnum moss s i t e bore l i t t l e  s i m i l a r i t y to the other five humic acids.  This  observation was probably due to the absence in moss species of vascular tissues which are p a r t i a l l y composed of l i g n i n constituents (Bold, 1970).  The oxidation products of the sphagnum moss s i t e did  display a strong 1720 c m other s o i l  sites.  -1  band but less intense r e l a t i v e to the  The infrared spectra of the two l i g n i n samples, when  compared with each other, were v i r t u a l l y identical  before and after  oxidation, indicating s i m i l a r numbers and types of bonds  existing  '  I  F  4000  3000  2000  i  1800  i  t  1  1600  1400  1200  i  1000  1  r  800  600  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  i  1—•  '  500 400 300....: .  .  I  4000  1  I  I  3000  2000  I  I  1800  1600  1  1  1  1  1400  1200  1000  800  I  600  I  I  I—  1  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  i  4000  3000  — i  2000  i  i  1800  1600  i  1400  i  1200  i  1000  i  i  800  600  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  1  500 400  f  300  I 4  0  O  !  0  3000  |  I  1  1  I  I  •  2000  1800  1600  1400  1200  1000  800  •  1  1  1  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  1  4000  3000  2000  ,  1800  1  1  1600  1400  1  1200  I  1000  I  800  1  WAVENUMBER (cm-) 1  FIGURE 12:  '  1  '  1  600 500 400 300 .  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 s o f t w o o d 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 b o r e 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  products.  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 e a c h 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  precursor.  U n f o r t u n a t e l y t h e n u c l e a r m a g n e t i c r e s o n a n c e (NMR)  spectra  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 h y d r o g e n atoms i n each sample ( F i g u r e s 13-2Q). w e l l r e s o l v e d s i g n a l s a t 6 = 1.2 of d i e t h y l ether.  and 6 = 3.6  In each s p e c t r u m ,  i n d i c a t e d the presence  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 a s 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 a n d 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 a s 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 p e a k s 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 h y d r o g e n bonded i n s t r u c t u r e s other than d i e t h y l ether.  Comparison o f each scan  with  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 type 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 b u t 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 study. The d e v e l o p m e n t o f t h e t h i n l a y e r c h r o m a t o g r a p h s o f e a c h  ^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 a n d a f a i n t t r a i l i n g shadow  ppmW  10  9  8  FIGURE 13:  7  6  5  4  3  2  N.MR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Spear Grass Site after NaOCl Oxidation.  1  0  cn  I  PPm &  10  9  8  7  FIGURE 14:  I  6  5  4  3  2  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  FIGURE 16;  6  5  4  3  2  NMR Spectrum of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Sphagnum Moss Site after NaOCl Oxidation.  I  0  o  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  ppm  W)  |  i  i  n  u  i  »  »  i  »  '  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  ppm (5)  7o"  -FIGURE 20:  NMR Spectrum of the Organic Solvent Soluble Fraction of the Kraft's Hardwood Lignin after NaOCl Oxidation.  65  as viewed under u l t r a v i o l e t l i g h t .  A p a r a l l e l run chromatographing  benzene hexacarboxylic acid and benzene pentacarboxylic a c i d , u t i l i z i n g 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 a c i d , water and dichloromethane solvent or the ether and 1 N HCl solvent and fluoresced with a mustard yellow coloration under u l t r a v i o l e t l i g h t . The gas l i q u i d 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 t h e i r 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 s i t e 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 l y substance previously observed in the spear  grass and rough fescue humic acid oxidation products apparently contributed to. the f i n a l major peak for each sample.  The chromatograms  for a l l eight samples were i n i t i a l l y produced at 4 x 10 Decreasing GLC s e n s i t i v i t y  2  by attenuating down to 64 x 10  attenuation. 2 brought  the f i n a l 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  TEMPERATURE  FIGURE 22:  200  225  250  (°C)  GLC Separation of the Organic Solvent Soluble Fraction of the Humic Acid Extract of the Western Porcupine Grass Site after NaOCl Oxidation.  275  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  TEMPERATURE  FIGURE 25:  200  225  250  (°C)  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  100  I  •  123  150  1—  175  TEMPERATURE  FIGURE 27:  I  I  200  225  I—  250  (°C)  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 e s t e r i f i c a t i o n 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 t r i c a r b o x y l i c acid trimethyl esters compared to the H horizon samples, which contained a r e l a t i v e l y greater amount of the benzene pentacarboxylic acid pentamethyl esters.  The chromatrograms of each Kraft's l i g n i n  sample closely resembled that of the lodgepole pine samples. A partial recovery of the o-phthalic a c i d , 1, 2, 4-benzene t r i c a r b o x y l i c 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 a c i d i c functions previously t i t r a t e d were in fact e n t i r e l y  ;  "  75  TABLE 5:  I n i t i a l Standard Compound Weight (gm)  Compound  o-phthalic  Recovery of Standard Compounds Subjected to Oxidation with 1.6 N NaOCl (p-phthalic acid I-S.).  Relative Derivatized Molar Ratio of I n i t i a l  Relative Peak Areas  Recovery (%)  .84  84  acid  .0125  p-phthalic acid (internal standard)  .0125  1, 2, 3 benzene t r i c a r boxylic acid  .0160  1.04  .85  88  1, 2, 4, 5 benzene t e t r a carboxylic acid  .0200  1.05  .86  90  100  p-phthalic acid o-phthalic acid  100  GURE 29:  GLC Separation of Organic Solvent Soluble Fraction of o-Phthalic A c i d , 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 T i t r a t i o n Data.  Dominant Species  Total Weight of Aromatic Acids In Dominant Peaks (gm)  Ah horizon  Spear grass  *  *  16.4  Ah  Western grass  .050  9.4  33.7  *  *  18.6  Sample Source  Degree of Aromaticity by GLC Data (%)  Minimum Degree of Aromaticity by T i t r a t i o n Data (%)  porcupine  Ah  Rough fescue  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 t r a t i o n data was  used to estimate a minimum degree of aromaticity (Table 6).  The  degree of aromaticity calculated from the t i t r a t i o n 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 a c i d , as determined by GLC techniques,  implied a portion of the acidic functional  groups t i t r a t e d were not aromatic substituents, but possibly were short chain a l i p h a t i c acids.  This implies that the minimum degree of  aromaticity figures calculated in Table 6 from the  potentiometric  t i t r a t i o n data were inflated by the contribution of a l i p h a t i c acids which have been i n c o r r e c t l y assumed to be aromatic in nature.  An  accurate method for determining the degr.ee: of aromaticity in s o i 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 i g n i n 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 c l a s s i c a l 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 c r i t i c i s e d 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 s p r o p o r t i o n a t e  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.  work of LandoT.t (1975) confirmed the results of Ghosh Huston et al. established  (1976) fluorinated coal with elemental  al.  The (1975).  fluorine and  i t s carbon d i s t r i b u t i o n as 70% aromatic (Sp ), '20% 3  a l i p h a t i c (Sp ) and 10% substituent  carbon on aromatic rings.  work gave no support to the adamantane structure suggested as a possible  Their  (polycyclohexane)  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 recovered from the c o a l .  soluble product but no adamantane was Their results indicated adamantane, probably  did not exist in the structure of c o a l . (1976b)defended t h e i r report of 80% Sp  Chakrabartty and Berkowitz 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 suggestions for the successful  available  application of the NaOCl oxidation  technique to humic acid and l i g n i n .  The results of this study indicated  that i t was not safe to assume the technique could completely 2 discriminate between Sp the aromatic rings.  3 and Sp  carbon hybrids with no destruction of  This is i l l u s t r a t e d by the low apparent degree of  aromaticity calculated for the l i g n i n oxidation products compared to the accepted abundance of  phenyl propane building blocks constituting proposed  structures of l i g n i n .  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 a l i p h a t i c acids and CO2 as 2 by-products; as a result some previously Sp i d e n t i f i e d as Sp  hybrids.  carbon hybrids were  The research of Mayo (1975) suggests that .  hydroxyl group aromatic substituents may have transferred electrons  to  the ring followed by e l e c t r o p h i l i c attack and ring opening to y i e l d a l i p h a t i c acids.  The infrared (IR) data supported this  possibility  as both strong aromatic and a l i p h a t i c 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 l a s s i f i c a t i o n of humic acids from various sources according to their oxidation product d i s t r i b u t i o n . D i s t i n c t differences  did exist between samples taken from the Ah  horizons compared to the H horizons. related to differences of the humic acids.  These differences  may have been  in the age and possibly the r e l a t i v e maturity  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 o r i g i n a l l y 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 a l i p h a t i c 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 a l i p h a t i c carboxylic acids when a c i d i f i e d .  This reasoning is a possible  explanation  for the high benzene hexacarboxylic acid OSS product and the absence of hydroxyl groups. A connection between l i g n i n and eventual  humic acid genesis  was implied by common products of oxidation i d e n t i f i e d by GLC and the d i s t r i b u t i o n 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 l y 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 i q u i d chromatograph suggest benzene dimethyl tetracarboxylic acid as a possible structure but do not explain i t s o i l y nature. The d i f f i c u l t i e s  encountered in applying the NaOCl oxidation  technique to the investigation of s o i l organic matter further confirms the complexity of the substrate.  Successful  application of  a technique to the study of the r e l a t i v e l y simple structures of standard compounds does not assure success with natural compounds.  It seems  probable that the presence of electron donating and electronwithdrawing functional groups in a randomly bonded polymer, such as humic a c i d , can profoundly change the behavior of a degradation method in comparison to i t s performance with standard compounds.  83  SUMMARY AND CONCLUSIONS  To investigate the r e l a t i v e merits and faults of 1.6 N NaOCl as a selective oxidant for s o i l humic substances, extracts soil  humic acid  from three Alberta s o i l sites and three B r i t i s h Columbia  sites were oxidized at room temperature (23°C) for 5 days.  To  explore a possible l i g n i n component as a contributing structure in humic a c i d , 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 r e l a t i v e l y longer reaction time to l i m i t unnecessary destruction of the oxidation products. The NaOCl oxidation reaction degraded the humic acid and l i g n i n 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 a c i d .  The reaction  was a r e l a t i v e l y simple procedure to employ; neither high pressures, high temperatures nor caustic chemicals were required, other than a reaction pH of 12-13. The d i s t r i b u t i o n of the reaction products permitted classification  of the humic acid starting materials according to their  Ah mineral or H organic horizon source. humic acid extracts  If the assumption of the  from the spear grass and western porcupine grass  s i t e s being the most mature was v a l i d , 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 i g n i n component.  S i m i l a r i t i e s in the type and amounts of  oxidation products suggests a l i g n i n component in each of the humic acids studied and a p a r t i c u l a r l y strong influence on the nature of..the humic acid extracts porcupine grass  from the spear grass and western  sites.  The probable more intense microbial a c t i v i t y at the rough fescue s i t e resulted in a humic acid chemistry intermediate between that of the other two Alberta sites and three B r i t i s h Columbia sites. An unidentified o i l y component was found in the oxidation products of the spear grass and rough fescue s i t e s . The reaction products of a l l eight samples were found to be free of hydroxyl groups, despite the well documented i d e n t i f i c a t i o n of hydroxyl groups as a common functional group in humic acids and l i g n i n s .  This result and the r e l a t i v e l y low degree of aromaticity  calculated for each of the samples suggest a reasonable  possibility  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 s o l a t i o n , broken ring fragments may have been reduced to a l i p h a t i c carboxylic acids.  This result  indicates  the need for prior methylation of the humic acid or l i g n i n starting materials in future applications of the NaOCl oxidation technique. The results of this study imply the oxidation method may not have been 2 capable of t o t a l l y selecting  Sp  3 from Sp  conditions imposed and materials oxidized.  carbon hybrids under the  The extreme complexity of s o i l humic substances appears to require further modifications of the NaOCl oxidation technique for future applications/in s o i l s 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 f u l l y evaluating the potential  of the NaOCl oxidation technique in s o i l s research.  LITERATURE CITED  A c z e l , T . , M. L. Gorbaty, P. S. Maa and R. H. Schlosberg. 1975. S t a b i l i t y of adamantane to donor l i q u i f a c t i o n conditions: implications toward the structure of coal. Fuel 55: 295. Alexsandrova, L. N. 1960. 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