6 December 2003
Humic acids are precursors of petroleum and coal, produce fertile soils, interfere with industrial processes, and form carcinogens in drinking water. They were isolated more than two centuries ago, yet their structure and origin has always been disputed. In this study, humic acids were rigorously purified to study them per se, and not as part of an environmental organic “soup.” It was found that humic acids originate from polysaccharides that are common in terrestrial plants and fruits, and in sea grasses. These polysaccharides contain xylose, arabinose, and fructose, with an important source being plant xylans (hemicelluloses) and arabinans. The polysaccharides are hydrolyzed to furfural, that is then oxidized to trans- and cis-4-oxo-2-butenoic acid, that in turn readily polymerizes to humic acids. The humic acid copolymers contain a heterogeneous mixture of lactones, carboxyls, ketones (including the conjugate chelate carbonyl group), enols, gem-diols, ethers, anhydrides, methyls, methylenes (long and short chain) and methines. The lactones and carboxyls exist on the sides of an aliphatic backbone, and the carboxyls are readily decarboxylated. Both naturally derived and artificially prepared humic acids were readily decarboxylated, esterified with n-butanol and sulfuric acid, and lactonized with acetic anhydride. The derivatives were fractionated into different polarities, and had physical properties ranging from highly fluorescent, pale yellow, low viscosity oils to brittle, black solids. Solution IR and NMR spectra of some fractionated humic acid derivatives were similar to those of kerogen, bitumen, petroleum, and coal. Hopefully this research will inspire new directions in a confused field.
Humic acids are ubiquitous naturally occurring polymers, important precursors or components of kerogen, bitumen, petroleum, and coal1-3. They occur in soil and sediment4, peat5, coal6, lake water4,7, sea water8, plants and coral skeletons9,10. They increase soil fertility11, accelerate photodegradation of pesticides12, and lower the toxicity of heavy metals13. They decrease the efficiency of alumina production14, and produce carcinogens during raw water purification15. They complex to coral skeletons, giving fluorescent bands that are useful indicators of past climate16. Their structure and origin has been poorly understood and always disputed17-19.
The first record of humic acid extraction by Achard dates back to 17865, and the first major study was done by Sprengel in the 1820s. Despite such a long research history, there has been constant dispute over the structure and origin of humic acids. By the 1870s a terrestrial plant origin from the decomposition of cellulose was widely accepted. However, soon after this time it was shown that humic acid-like compounds could be obtained from simple chemicals unrelated to polysaccharides or sugars. This began a polarized, acrimonious debate that lasted for many decades. In 1920 it was claimed that humic acids originate from furfural20, a polysaccharide oxidation product, based on an earlier observation by Chardet that dry distillation of humic acids gave furfural. However, from this time on it became more commonly accepted that humic acids were derived from plant lignins due to the fact that it could be demonstrated that lignins are more persistent than polysaccharides in the natural environment. With some modifications the lignin origin idea came to predominate until the 1980s, when it was again strongly challenged17,19. The consensus then became that both polysaccharides and lignins contribute to humic acid formation, although the mechanism remained obscure. Since the 1950s it was generally believed that humic acids were formed in soils by microbial action on plant matter. However, more recently it has been demonstrated that they are widely distributed within plants themselves9. Their polymeric nature has always been accepted, although such basic properties as elemental composition, molecular weight, and type and amount of moieties that have been reported vary widely21-26. Research on minor components of humic acids has been rare1,27. A number of publications provide extensive reviews of humic acid research1,21,28-30.
A factor contributing to the poor characterization of humic acids has been their poor definition and lack of rigorous purification. Routinely humic acid preparations for research have been variable mixtures of many organic and inorganic components. In this study it was found that humic acids bind to peptides and proteins, and can bind 10 times their own weight of clay particles, even solubilizing them. No doubt this has led to many errors, such as the enormously high molecular weights that have been reported. In this study humic acids were RIGOROUSLY PURIFIED, and extracted in such a manner that excluded particulate matter and contaminant organics. Therefore the term humic acid refers to the 0.1 M NaOH soil or plant extract filtered through a 0.1µm filter, then extracted into n-butanol (BuOH) after acidification to pH 1 (humic acid precipitates at this pH). Fulvic acid refers to the acid soluble fraction of purified humic acid precipitated with concentrated HCl to pH 1. Humic acid derivatives were further purified by extracting them into organic solvents.
Novel sources of humic acids were fungi, algae, sea grasses, toasted bread, and brown banana skins. All humic acid samples contained small amounts of aliphatic dicarboxylic acids, keto acids, and long chain fatty acids, while the samples from soils, plants, and wood-rotting fungi also contained the lignin-derived vanillic and syringic acids27. By preparing novel derivatives, purifying them by silica chromatography, and using solution Fourier-Transform Infra-Red (FT-IR) and NMR analysis as the main tools, the structure and origin of humic acids has been discovered. Major discoveries reveal that polysaccharides such as plant xylans, arabinans, and pectins decompose to furfural or 5-hydroxymethyl furfural, that then oxidize to 4-oxo-2-butenoic acid, that then polymerizes to polymers and/ or copolymers consisting of ether, keto, conjugate chelate carbonyl, carboxylate, lactone, and anhydride groups. The carboxylate groups are labile, and readily leave to give hydrophobic polymers similar to long-chain hydrocarbons and fatty acids. However, they are readily distinguished from such compounds by their intense fluorescence. This study should promote a better understanding of many natural processes that humic acids are involved in, including the genesis of kerogen, bitumen, petroleum, and coal. It should also increase the understanding of soil fertility, the reduction in toxicity by humic acids of soil and water pollutants, the loss of efficiency in industrial manufacturing processes and water purification, and the formation of carcinogens during water purification. More importantly, it is hoped that this research will inspire a new direction in the confused humic acid field so that old dogmas can be put aside.
Materials and methods
Extraction of humic acids
Humic acids were extracted from soils, senescent plants, fungi, sea grasses, etc. in the state of Queensland, Australia, and prepared according to methods modified from previous reports9,31. Briefly, samples were air-dried, extracted with 0.1 M NaOH, precipitated with concentrated HCl, filtered through coarse filter paper, and dried. Sub-samples were redissolved in 0.1 M NaOH, passed through a 0.1 µm polymer filter to remove small mineral particles that are coprecipitated, acidified with concentrated HCl to pH 1, and extracted with ethyl acetate (EA) and/ or BuOH. An Aldrich (Gillingham, UK) humic acid was purified by the addition of a small amount of NH3 solution (30%), and allowed to stand for 24 hr. The soluble portion was decanted and then precipitated with concentrated HCl, and finally dried at 80oC. This process was repeated twice. Fluorescence spectra were checked for similarity in both the emission and excitation mode to monitor the consistency of sample extracts. Solvents were HPLC grade when available, otherwise analytical grade. All other chemicals were analytical grade or better, and bitumen, coal, oil and grease samples were purchased commercially.
FT-IR analysis was carried out with a Perkin-Elmer Model 1600 instrument. Solvents for underivatized humic acids were tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) and their deuterated analogues. Solvents for derivatized humic acids were THF, chloroform (CF), and dichloromethane (DCM), and their deuterated analogues, and carbon tetrachloride (CT).
NMR analysis was done using a Bruker Aspect AC300 instrument with TMS as internal standard. Solvents were deuterated analogues of CF, methanol (MeOH), THF, and DMSO. The DEPT spectrum distinguished between carbon connected to even or odd numbers of hydrogen.
FT-MS was done using a Bruker BioAPEX 47E instrument with electrospray injection. Solvents were MeOH/ 1% acetic acid and DCM/ 0.2% trifluoroacetic acid, and samples were aspirated at 60 µl h-1 into the system with a syringe pump. Components were detected as their mono-H+ or Na+ adducts. A series of standards was used to calibrate the system, and accuracy better than 4 ppm was achieved. Structures were derived from carbon 13/12 ratios, accurate masses, and MS-MS experiments.
GC-MS was performed with a Hewlett-Packard 5790 GC and 5970A mass-selective detector. Preliminary identification of compounds was performed with a Wiley/ BenchTop/ PBMTM mass spectral library (Palisade, New York, USA).
Fluorescence spectra were obtained with a Hitachi F-4000 spectrophotometer, and HPLC work was done with a solvent programmable Perkin-Elmer instrument, SGE silica column (25 x 0.4 cm, 5 µm particle size), and the Hitachi F-4000 fluorescence detector. Detection wavelengths were either 320/ 420 nm or 340/ 440 nm. Samples were eluted with n-hexane (HX), DCM, EA, and MeOH and/ or mixtures of these solvents at a flow rate of 1 or 2 ml/ min. Separations were done in both isocratic and gradient mode.
Preparation of derivatives
(a) Brominated humic acids (BrHA)
Aldrich humic acid was gently refluxed in MeOH/ bromine (20:1) for 1 hr, after which time the MeOH and bromine were evaporated with the aid of heat and an air stream. The resulting brown powder was washed several times with distilled water and dried at 105oC. The compound was extremely soluble in THF, but was unique in lacking fluorescence. Due to its high solubility in THF, it was ideal for investigating the dehydration of humic acids.
(b) Lactonized humic acids (LHA)
Humic acids were dissolved in a minimum of pyridine, after which a 10-fold excess of acetic anhydride was added, and the mixture was then refluxed for 2 hr. The reaction was also done with deuterated acetic anhydride instead of acetic anhydride. After refluxing, the solvent was thoroughly evaporated by heating in an air stream. DCM was added to the resulting solid, and the mixture was thoroughly shaken. Only a small fraction dissolved, so the material was filtered through coarse filter paper and the insoluble matter was discarded.
(c) Butlyated humic acids (BuHA)
Esterification with n-butanol/ conc. sulfuric acid (30:1) was by far the best method for preparing hydrophobic derivatives of humic acids. Humic acids were heated at 105oC (or refluxed) for between 4 & 24 hr in a mixture of BuOH/ conc. sulfuric acid (30:1). The reaction was terminated when no more humic acid solubilized, which was about 4 hr for Aldrich humic acid and 12-24 hr for soil extracted humic acids. Soil humic acids never totally solubilized, apparently due to decarboxylation that gave humins insoluble in sodium hydroxide solutions and organic solvents. The insoluble matter was discarded. After cooling, the solution was washed several times with an equal volume of distilled water until the aqueous layer was neutral to litmus. The BuOH was evaporated in a container of large surface area by heat and with the aid of an air stream, or under vacuum in a rotary evaporator. The resulting reddish, tar-like derivatives were very soluble in DCM, and partially soluble in HX and CT. Fractions of different polarity were extracted with variable mixtures of HX/ CT and CT/ DCM. The more hydrophobic fractions were pale yellow oils, and the less hydrophobic fractions were black, shiny solids. The reaction was also done with n-pentanol instead of BuOH.
(d) Butylated and lactonized humic acids (BuLHA)
The reddish tar from (c) was refluxed for 1 hr with acetic anhydride/ pyridine (10:1). The resulting reddish, tar-like derivatives were very soluble in DCM, and partially soluble in HX and CT. Fractions of different polarity were extracted with variable mixtures of HX/ CT and CT/ DCM. The most hydrophobic fractions were pale yellow oils, and the least hydrophobic fractions were black, shiny solids. The reaction was also done with deuterated acetic anhydride/ pyridine (10:1) to confirm that lactonization usually occurred at the expense of esterification.
(e) Decarboxylated humic acids (DHA)
Humic acids were dissolved in a concentrated NaOH solution, then calcium oxide equal to the amount of solid NaOH in the solution was added, the mixture was dried and then placed in a furnace for 48 hr at 295oC. The resulting solid was cooled, and conc. HCl was slowly added until the reaction ceased. The remaining solid was filtered, washed several times with water, and dried at 105oC. A more efficient method was to thoroughly mix BuHA or BuLHA with a mixture of equal amounts of NaOH and calcium oxide, and heated in a furnace at 295oC for 12-24 hr. Humic acids were also decarboxylated by refluxing in n-hexanol/ conc. sulfuric acid (30:1) for several hours and then evaporating the solvent under vacuum in a rotary evaporator, finally washing with distilled water. With the described methods total decarboxylation was not always obtained, perhaps because the reaction time was not long enough.
(f) Reduced, then acylated humic acids (RAcHA)
Humic acids were reduced by dissolving them in 2.5 M NaOH, adding an appropriate amount of Raney-Nickel, heating at 60oC for several hours, and decanting the solution above the Raney-Nickel. Then excess conc. HCl was slowly added to the decanted solution, and it was evaporated by heat and with the aid of an air stream, or it was evaporated under vacuum in a rotary evaporator. Sub-samples of the product were reacted with acetic anhydride/ pyridine (3:1), deuterated acetic anhydride/ pyridine (3:1) at 60oC for 1 hr, or with acetyl chloride (neat or mixed with DCM) at 25oC for 1 hr. The solvents were evaporated and the products extracted with DCM, discarding any insoluble material, and finally the DCM was evaporated.
In a second method, the BuLHA derivatives were partially dissolved in diethyl ether, which was decanted as a sub-sample, and the remaining solid was dissolved in THF. The solutions were placed in small, strong glass vials and a small amount of LiAlH4 was added to each solution. The glass vials were then capped and placed within a strong glass container (in case the vials burst) and heated at 60oC for 4-48 hr. After cooling and carefully opening the vials, excess EA was slowly added to destroy the LiAlH4, after which the solvents were evaporated by heat and with the aid of an air stream. The residue was partitioned between BuOH and 25% (v/v) aqueous HCl, and the BuOH fraction was aspirated and evaporated. Sub-samples of the product were reacted with acetic anhydride/ pyridine, deuterated acetic anhydride/ pyridine, or acetyl chloride as above. The solvents were evaporated and the products were extracted with DCM, discarding any insoluble material, and finally evaporating the DCM. Pentafluoropropionic anhydride (Sigma Chemicals, USA) was also used (without pyridine) to form the pentafluoropropionate derivative in place of the acetate derivative.
Preliminary sodium borohydride reductions with BuLHA derivatives were also investigated with ethanol as solvent, but the technique proved unsuccessful.
Humic acids were dissolved in a slight excess of NaOH to give a solution pH of about 10. An equal volume of 100 vol. H2O2 was slowly added to this solution, after which it was shaken for 24-48 hr at 25oC. The solvent was carefully evaporated by low heat and with the aid of an air stream (evaporation under vacuum in a rotary evaporator was not possible due to the huge amount of froth that formed). When almost dry, conc. HCl was slowly added until the solution was acidic, and again the solvent was carefully evaporated by heat and with the aid of an air stream at 25oC. For GC-MS analysis the mixture was butylated by refluxing for 2 hr with BuOH/ conc. sulfuric acid (30:1), cooling, washing the solvent with distilled water until the washings were neutral to litmus, and finally evaporating the solvent by heat and with the aid of an air stream, or alternatively, under vacuum in a rotary evaporator. The product was dissolved in DCM for injection into the GC-MS system. For component identification, mass spectra were compared to those of a Wiley mass spectral database, and confirmed by preparing derivatives from authentic standards (Sigma Chemicals, USA) and comparing retention times and mass spectra.
Reactions of furfural, xylose, and glucose
Furfural was oxidized by shaking with 70% (v/v) aqueous H2O2 at 25oC for several days until the phases combined32. Residual furfural was extracted from the mixture with DCM and discarded. The resulting 4-oxo-2-butenoic acid mixture of cis and trans isomers was purified by subliming the cis form as its lactone at 60oC. This was readily converted to its Na salt by neutralizing with NaOH and evaporating to dryness. Alternatively, it was polymerized in situ by adding conc. HCl to the crude product. When polymerized in situ, the resulting precipitate (FuHA) was washed several times with distilled water, and dried. GC-MS analysis detected no 4-oxo-2-butenoic acid (as the n-butyl ester), indicating complete polymerization. 5-Hydroxymethyl furfural gave the same result as furfural. The FuHA was reacted with BuOH/ conc. sulfuric acid followed by reaction with acetic anhydride/ pyridine as described for the humic acid derivatives above. The resulting tar-like product was dissolved in DCM and filtered through glass wool to remove any insoluble material, and finally the DCM was evaporated.
Xylose and glucose were reacted at 80oC in BuOH/ conc. sulfuric acid (30:1). After several minutes xylose gave a dark brown color, but glucose took several hours to produce a color of the same intensity. The glucose products were identified by GC-MS as for the products described under Oxidation, but the xylose products were not volatile enough to be analyzed by GC-MS.
Classical Column Chromatography
Silica 60 for column chromatography (Merck, Darmstadt, Germany), particle size 50-200 µm, was used to fractionate BuHA and BuLHA derivatives. An appropriate amount of silica was placed into a beaker, excess DCM added, and the slurry was poured into a 30 x 2 cm glass chromatography column. The derivatives were dissolved in 2 ml of DCM and placed onto the column. Impurities that included phthalate esters and long-chain fatty acid esters were eluted with HX and discarded. After this step the humic acid derivates were eluted, usually beginning with 5% EA in DCM and finishing with EA or MeOH/ EA mixtures. No fractions were collected in discrete bands as there was always a continuum of material eluting. Fractions were therefore collected arbitrarily.
Summary of abbreviations
Since many abbreviations are used, a summary is provided to make it easier to follow the discussion of data.
|EA||Ethyl Acetate||IR||Infra Red|
|THF||Tetrahydrofuran||NMR||Nuclear Magnetic Resonance|
|CT||Carbon Tetrachloride||EI||Electron Ionization|
|HX||n-Hexane||UV-vis||UV/ Visible Spectroscopy|
|LiAlH4||Lithium Aluminium Hydride|
|BrHA||Brominated Humic Acids||DHA||Decarboxylated Humic Acids|
|LHA||Lactonized Humic Acids||RAcHA||Reduced and Acylated Humic Acids|
|BuHA||Butylated Humic Acids||FuHA||Furfural-derived Humic Acids|
|BuLHA||Butylated and LactonizedHumic Acids|
Results and discussion
Since environmental samples contain a plethora of organic compounds, including small organic molecules, proteins, lignins, and polysaccharides, samples in this study were rigorously purified to give solely humic acids. The lack of rigorous purification has been a major flaw in humic acid research, and studies are often conducted on some type of “soup” of organic compounds that are then falsely equated to humic acids.
When BuLHA were dissolved in DCM and analyzed by GC-MS, most of the material was not volatile enough to pass beyond the glass sleeve of the injector port, but a small amount was always volatile enough to be identified and analyzed. Small dicarboxylic acids, keto acids and aromatic carboxylic acids were identified. The highest concentrations found were for syringic acid (124 µg/ g in humic acid from a grass covered soil), vanillic acid (72 µg/ g in humic acid from a tree covered soil), and succinic acid (50 µg/ g from Aldrich humic acid). These data demonstrate that small, non-polymeric molecules are a minor component of humic acids, and it was estimated that they comprise only about 1% (w/v)27. Alkanes were not present in humic acids prior to butylation, confirmed by the inability to extract them with HX from both alkaline and acid solutions. After butylation small amounts of 17-carbon to 30-carbon n-alkanes were identified by GC-MS and NMR, far more in Aldrich humic acid than in soil or plant derived humic acids. Branched alkanes (recognized from their mass spectra) were also detected. It was also observed that in DMSO-d6 at 100oC there was a rapid increase of a methylene (CH2) peak. These data are consistent with the well known facile decarboxylation of humic acids, and suggests that some of the humic acid polymers are simply long-chain hydrocarbons with side chain carboxylate groups. Phthalic acid was present in all humic acids studied, but care had to be taken to prevent contamination by phthalic esters from plasticizers, present in even the purest solvents19. However, identification of di-n-butyl and di-n-pentyl phthalate (by GC-MS) after esterification with n-butanol and n-pentanol, respectively, confirmed the presence of phthalic acid as a minor component of humic acids.
EI-MS of BuLHA showed the presence of minute amounts of benzene carboxylic acids in this study. However, in this and other studies, mild oxidation produced very little of these acids, while severe oxidation produced much larger amounts, indicating the predominant effect of oxidation conditions33,34. Further, benzene carboxylic acids cannot be used to prove a lignin origin of humic acids because aliphatic polyketides readily produce aromatic compounds, and benzene carboxylic acids are known to be formed from both carbohydrates and lignins on oxidation35,36. No techniques in this study detected substantial amounts of aromatic compounds in unoxidized humic acids, consistent with previous studies4,17,36.
Fluorescence has been poorly utilized for humic acid research, yet in this study it was a powerful tool to demonstrate that humic acids from different and novel sources were similar, and that the derivatives prepared in this study could be linked to the parent compounds. All humic acid samples and derivatives except BrHA were fluorescent. Humic acids from different sources had similar emission and similar excitation spectra, similar to published spectra. However, in marked contrast to usual fluorescence, the range of excitation wavelengths that gave fluorescence was found to be extremely broad, and excitation wavelengths overlapped with emission wavelengths. Excitation wavelengths were varied from 230-470 nm and emission spectra collected from 30 nm above the excitation wavelength. Emission wavelengths were varied from 340-600 nm and excitation spectra collected from 30 nm below the emission wavelength. This unique property of humic acids made their identification easy. 3D-Fluorescence spectra (a plot of emission against excitation wavelengths) were also used, and proved extremely useful for comparing samples and for positively identifying humic acids and their derivatives. Emission spectra of derivatized humic acids were similar to the underivatized spectra, but excitation spectra were different. However, the phenomenon of overlaping emission with excitation spectra remained the same.
Fig. 1(a) shows the similarity of emission spectra between Aldrich and marine sediment humic acids obtained during HPLC separation14, 31 after stopping the flow at a peak maximum and recording the fluorescence. Fig. 1(b) shows the similarity of excitation spectra between sea water and senescent mango leaf humic acids obtained in the same way.
Figure 1. (a) Emission spectra (ex. 340 nm) of an Aldrich (A) and marine sediment (B) humic acid, obtained during HPLC separation.
Figure 1. (b) Excitation spectra (em. 420 nm) of a sea water (A) and leaf humic acid (B), obtained during HPLC separation.
IR KBr spectra of dried humic acids were similar to those published38. Bands for humic acids dissolved in organic solvents were much sharper and better resolved than those of the KBr spectra, and were therefore routinely used for identification of structures present. When humic acids were mildly oxidized with H2O2, about 34% of the carbon was lost, presumably as CO2 due to decarboxylation (the lime-water test confirmed that CO2 was produced). Other oxidation products identified by GC-MS included oxalic, malonic, ketomalonic, succinic, ketosuccinic, glutaric, 2-keto glutaric, maleic, fumaric, and malic acids. It is important to note that these compounds are similar to those reported for the oxidation of furfural (2-furaldehyde)39. When furfural was oxidized to 4-oxo-2-butenoic acid (Fig. 2 (a)) and subsequently polymerized (to form FuHA) in situ, a black, shiny, acidic, amorphous substance was produced, that gave similar UV-vis, fluorescence (excitation and emission), and IR spectra to humic acids. During column chromatography on silica the product also smeared over the entire column without forming discrete bands, the same as for humic acids. The IR spectra of Aldrich humic acid and FuHA are shown in Fig. 3. (Note that in these spectra and all subsequent IR spectra areas of solvent absorbance have been edited out.) The structural similarity is readily evident. There is a broad carboxylic acid hydroxyl band near 3100 cm-1 (partially obscured by solvent absorbance), its carbonyl band at 1731 cm-1, a lactone band at 1766 cm-1 (much larger for FuHA than Aldrich humic acid), and free H2O bands at 3566/ 3500 and a conjugate chelate carbonyl band at 1643 cm-1 (refer below for these assignments). Aldehyde bands were not observed for FuHA, indicating that the aldehyde group is readily oxidized or lactonized, or both. When xylose was reacted with BuOH/ conc. sulfuric acid (30:1), the product was similar in appearance to the BuHA and FuHA when butylated, and gave similar UV-vis, fluorescence, and IR spectra. When glucose was reacted in the same manner it gave predominantly levulinic acid (confirmed by GC-MS).
4-Oxo-2-butenoic acid occurs in the cis form (also called malealdehydic acid) and the trans form (also called fumaraldehydic acid)40-43. In the past the term ß-formyl acrylic acid was also used. The cis form readily converts to the hydroxy lactone form (Fig. 2 (b)). The trans isomer would be expected to form the aldehyde hydrate in aqueous solution (Fig. 2 (c)), although this does not appear to have been studied. When produced by the peroxide oxidation of furfural, the reported32 isomer was the cis form, although in this study substantial amounts of the trans form also occurred. 4-Oxo-2-butenoic acid readily polymerized, especially in dilute acid solution, to the same humic acid compounds that were formed by furfural without isolation of the acid. This confirmed that the 4-oxo-2-butenoic acid pathway gives the humic acid compounds.
(a) Structure of 4-oxo-2-butenoic acid.
(b) Hydroxy lactone structure of cis-4-oxo-2-butenoic acid.
(c) Hydrated aldehyde structure of trans-4-oxo-2-butenoic acid.
Figure 3. IR spectra of Aldrich (A), and furfural (B) humic acids dissolved in THF.
Broad bands between 3600 and 2600 cm-1 indicated the presence of hydroxyl groups. When Na humate was formed by neutralizing humic acid with NaOH, a carboxylate ion band at 1565 cm-1 replaced much, but not all, of the hydroxyl band. This indicated that the principal hydroxyl components are carboxylic acids. The relative intensity of the carboxylic acid band compared to the methylene bands decreased as humic acid fractions became more soluble in hydrophobic solvents, probably due to the presence of fewer carboxylic acid groups due to decarboxylation. When the concentrations of humic acid derivatives (including FuHA) in THF was decreased, small H2O bands44 began to appear near 3565/ 3495 cm-1 (doublet) and an intense band due to conjugate chelate carbonyl near 1640 cm-1 (care was taken to exclude the absorption of H2O from the air), and eventually dominated the spectrum. The 1640 cm-1 band then masked the smaller 1610 cm-1 band. Water bands are known to be characteristic of humic acids, kerogen, and coal44, and the carbonyl absorbance of the conjugate chelate carbonyl band is known to be very intense near 1640 cm-1. A concentrated solution of BrHA in THF (6 mg/ ml) showed only minor H2O bands, but during dilution (up to 500x) these bands increased in relative size. At a concentration of about 1 mg/ ml there was a sudden massive relative increase in the H2O and C=O conjugate chelate carbonyl band45,46, and at a concentration of 0.6 mg/ ml these bands dominated the spectrum. The conjugate chelate carbonyl group (refer to Fig. 8) is a special keto-enol group with aromatic-type properties due to resonance structures. Dried oxalacetic acid and its diethyl ester exhibit the same properties due to dehydration of a gem-diol group, and the mechanism is due to keto-hydrate-enol equilibria47. Fig. 4 demonstrates the difference in the bands at 3566/ 3500/ 1643 cm-1 for a concentrated and dilute solution of BrHA in THF, and for a dilute solution of oxalacetic acid in THF. The gem-diol group itself is centrosymmetric48, and therefore not observed by IR. A well-known example of this equilibrium is between methylene glycol and formaldehyde (Fig. 5). In humic acids the dehydration is generated from a gem-diol attached to adjacent carbons (Fig. 6) since there is no concomitant appearance of aldehyde IR bands.
Figure 4. IR spectra of a concentrated (A), and dilute (B) solution of BrHA (Aldrich) in THF, and of dilute oxalacetic acid (C) in THF. H2O bands and the conjugate chelate carbonyl band at 1645 cm-1 in C are marked with an asterisk.
When humic acids were diluted, aliphatic CH2 bands at 2965-2855 cm-1 disappeared with the concomitant appearance of unsaturated CCH bands at 3080-3010 cm-1, indicating the formation of enol groups. DHA (highly decarboxylated) showed a 1710 cm-1 band (resolved from the now smaller 1730 cm-1 carboxylic acid band) due to the presence of the keto carbonyl group. Fig. 7 shows the 1711 cm-1 keto carbonyl band of a highly decarboxylated and acetylated humic acid derivative dissolved in DCM, with this band readily distinguishable from the acetate band at 1737 cm-1. In polar solvents the keto band becomes smaller but does not disappear, and the enol bands become more prominent. This suggests that some, but not all, of the keto groups belong to a keto-enol system. There were broad 2600 and 1675 cm-1 bands that also varied with concentration. These bands can be assigned to hydroxyl and carbonyl groups, respectively, of conjugated carbonyl (different to conjugate chelate carbonyl) group45. Sharp bands at 2960/ 2875/ 1370 cm-1 indicate methyl groups, and sharp bands at 2930/ 2855 cm-1 indicate methylene groups. Aromatic bands were not present.
Figure 5. Dehydration of methylene glycol, a typical gem-diol, to produce formaldehyde.
Figure 6. Dehydration of a humic acid gem-diol group.
Figure 7. IR spectrum of a highly decarboxylated and acetylated soil humic acid in DCM.
Figure 8. Structure of the conjugate chelate carbonyl group.
These initial clues suggested that humic acids were derived through xylose and furfural oxidation49 and 4-oxo-2-butenoic acid polymerization. The polymers contain hydrocarbon backbones, carboxylate side chains, and other groups including the keto-enol, keto carbonyl, conjugate chelate carbonyl, and gem-diol groups. From a consideration of the 4-oxo-2-butenoic acid structure, the formation of polymers with such groups are plausible. The gem-diol group could form via the hydrated aldehyde group of trans-4-oxo-2-butenoic acid.
IR spectra of humic acid derivatives
A large number of hydrophobic humic acid derivatives were prepared so that structures of fractionated samples could be more readily investigated by solution IR and NMR. When reacted with BuOH/ conc. sulfuric acid (30:1) followed by acetic anhydride/ pyridine (10:1), FuHA gave similar IR and NMR spectra to those obtained from humic acids. In Fig. 9 the IR spectra of the CT soluble fraction of BuLHA from various sources are compared. IT IS CLEAR THAT THE STRUCTURES OF THE SOIL, ALDRICH, AND FURFURAL HUMIC ACID DERIVATIVES ARE ALMOST IDENTICAL. KBr IR spectra showed that the BuLHA are readily decarboxylated when reacted with NaOH/ calcium oxide at 290-300oC. Decarboxylation followed by reaction with acetic anhydride/ pyridine (3:1) allowed solution IR spectra to be obtained with sharp bands, and without the interference of the large ester band. Spectra were similar to the one shown in Fig. 7. EI-MS of BuHA and butylated FuHA showed a prominent CH2CO+ peak, typical of aliphatic ketones50. A fulvic acid reacted with acetic anhydride/ pyridine (3:1), but not butylated, showed major IR bands for CH2, CH3, lactone, and acetate groups, and only very minor H2O bands. The spectrum is shown in Fig. 10, highlighting the lactone and acetate bands. These data indicate that fulvic acids do no possess carboxylic acid groups to an appreciable degree, that the conjugate chelate carbonyl is minor, but that hydroxyl groups are prominent.
Figure 9. IR spectra of CT soluble BuLHA fractions from various sources dissolved in CT: soil (A), Aldrich (B), and furfural (C).
Figure 10. IR spectrum of a soil fulvic acid reacted with acetic anhydride/ pyridine and dissolved in deuterated CF.
In many BuHA and BuLHA samples the H2O bands were either present or appeared on dilution of the sample in the solvent (1605 cm-1 in CT, deuterated CF, and deuterated DCM; 1640 cm-1 in deuterated THF). These data are consistent with a gem-diol structure of the solid in which H2O is eliminated, either by dissolution or dilution, or both. Hydrated aldehydes are not the source of H2O because during butylation acetals would be formed, and they are unable to dehydrate. The gem-diol structure is in equilibrium with the keto structure, which in turn is in equilibrium with the enol structure. As noted above, the hydrated aldehyde structure of 4-oxo-2-butenoic acid would be the source of the gem-diol structure in the humic acid polymer. Fig. 11 shows the relative differences in the H2O bands of a soil BuHA dissolved in THF and CT. In THF dehydration occurs on dissolution and the H2O bands are prominent, but in CT the H2O bands are almost non-existent because the hydrate remains in the gem-diol form on dissolution, which is not detected by IR (refer above). On changing the concentration of some BuLHA fractions in deuterated DCM, enol tautomers are formed, confirmed by the appearance of bands at 2800 cm-1 (enol or conjugate chelate carbonyl OH) and 3080/ 3010 cm-1 (double bond CCH). Comparison of IR spectra of BuLHA with BuHA showed the appearance or increase of bands at 1765 cm-1 and 1185 cm-1 due to 5-ring lactone formation51. A small band at 1765 cm-1 increases in intensity when humic acids are reacted with acetic anhydride, a good reagent for promoting lactonization. An increase in the lactone band at 1765 cm-1 is always accompanied by an increase in the band at 1185 cm-1, indicating that the assignment as lactone is correct. Further, when deuterated acetic anhydride was substituted for acetic anhydride, no bands appeared for the deuterated acetate group, further confirming lactone formation at the expense of esterification. A small shoulder (at 1765 cm-1) on the large 1735 cm-1 n-butyl ester band of BuHA indicates that humic acids form some lactones even during the esterification reaction, possibly because of the presence of sulfuric acid (acids catalyze lactonization). Further, Aldrich humic acids and FuHA showed a significant lactone content prior to any reactions. The n-pentyl ester compared to the n-butyl ester of BuLHA had relatively larger CH2 bands at 2930/ 2855 cm-1, confirming that the esterification reaction proceeded as expected.
Figure 11. IR spectra of soil BuHA dissolved in THF (A), and CT (B).
In contrast to the unreduced humic acid derivatives, Raney-Nickel reduced humic acids and LiAlH4 reduced BuLHA formed acetate derivatives readily when reacted with acetic anhydride/ pyridine (3:1) or acetyl chloride. This was confirmed by similar-sized 1740/ 1235 cm-1 IR bands and 2.10/ 2.05 ppm NMR peaks. Also, they formed extremely fluorescent pentafluoropropionates with pentafluoropropionic anhydride, confirmed by the ester band at 1781 cm-1. These pentafluoropropionate derivatives give IR spectra with extremely sharp, well-resolved bands, allowing much detail to be deduced. A small band at 3067 cm-1 is assigned to the CC double bond of an enol, and a small band at 3021 cm-1 is assigned to a normal CC double bond. Prominent bands at 1781, 1300, 1222, 1151, and 1039 cm-1 are assigned to fluorine-substituted groups, indicating aliphatic and ester structures. As shown in Fig. 12 , the acetate derivatives of soil and Aldrich RAcHA give much detail in the 1750-1550 cm-1 region of the IR spectrum. The Aldrich derived sample shows a prominent keto band at 1712 cm-1, but this band is much smaller in the soil-derived sample. Other bands in the spectrum are assigned below. Comparison of the RAcHA with the BuLHA IR spectra show that the CH2 contribution in the RAcHA is much greater. Also, the 1235 cm-1 band relative to the 1735 cm-1 band is relatively larger in the RAcHA, indicating that acetate groups have replaced n-butyl groups (acetates give a relatively stronger band at 1235 cm-1 compared to the n-butyl ester). Further, the proton NMR spectrum (Fig. 13) shows prominent long-chain CH2 and acetate peaks at 1.25 and 2.12 ppm, respectively. The conjugate chelate carbonyl bands, including the CC double bond band, disappeared entirely in the IR spectrum. These data indicate that carboxylate groups are reduced to alcohols and conjugate chelate groups are reduced to alkanes. LiAlH4 is known to reduce acids and esters to alcohols without reducing CC double bonds, consistent with the carboxylate reduction. However, the conjugate chelate reduction was different to the normal mechanism.
Figure 12. IR spectra of RAcHA for soil (A) and Aldrich (B) humic acids. The soil sample was dissolved in deuterated CF and the Aldrich sample was dissolved in DCM.
Figure 13. Proton NMR spectrum of a reduced and acetylated soil humic acid dissolved in deuterated CF.
Using well-known IR spectra, including the spectrum of oxalacetic acid as reference45,46,52,53, the following bands were assigned for BuLHA dissolved in deuterated THF:
|Wavenumber (cm-1)||Assignment||Wavenumber (cm-1)||Assignment|
|3680||Free H2O||3595 & 3475||Bonded OH|
|3000 (broad band)||OH of conjugate chelate carbonyl||2960/ 2875||CH3|
|2930/ 2855||CH2||1765/ 1185||Lactone|
|1815/ 1785||Anhydride||1735/ 1245||Butyl ester|
|1645||CO of conjugate chelate carbonyl||1630||CC double bond|
|1605||Enol CC double bond|
Many fractions smeared over the entire column during classical column chromatography and HPLC (refer below). Reactions with known reactants for carbonyl and hydroxyl groups were unsuccessful in forming derivatives that did not smear. The most probable cause for this is the high stability of the conjugate chelate carbonyl group (Fig. 8) that prevents the formation of appropriate derivatives.
NMR spectra of humic acid derivatives
Solid-state C13 NMR spectra of a soil derived and an Aldrich humic acid were obtained (not shown). Except for a long-chain methylene peak at 32 ppm in the Aldrich humic acid, only very broad bands were obtained. As in published spectra, and contrary to normal NMR studies of organic compounds, only general group types could be identified because the detail was very poor. The hydrophobic BuLHA fractions gave proton and C13 NMR spectra with sharp peaks that allowed details to be defined. However, hydrophilic fractions gave broad proton NMR bands, and C13 spectra did not register. Hence, NMR investigations were restricted to hydrophobic BuLHA fractions and RAcHA only. In Fig. 14 , proton NMR spectra of various BuLHA are shown. Peaks due to the n-butyl ester group appear at 0.9, 1.4, 2.3, and 4.1 ppm. In the Aldrich sample they are readily distinguished, but less so in the other samples where lactonization occured at the expense of esterification. All samples are similar in that they show peaks at 0.9, 1.3, and 1.7 ppm. The soil and furfural humic acids have prominent peaks at 3.9-4.3 ppm, probably due to the CH2 of a lactone. The lactone groups probably exist on the side of the main chain, like in known structures51. The proton NMR peaks are assigned thus:
|Peak (ppm)||Assignment||Peak (ppm)||Assignment|
|0.9 (multiplet)||CH3||1.3||Long-chain CH2|
|1.4||Butyl CH2||1.7||Lactone CH2 or a CH|
|2.3 (multiplet)||(a) Butyl CH2(b) CH2 next ester/ lactone||3.9-4.3 (several)||CH2 next to lactone O|
Figure 14 (a). Proton NMR spectra for HX soluble BuLHA from soil dissolved in deuterated CF. Reference was TMS. (Note: the 7.25 ppm peak is not due to aromatics, but rather to a trace of CF.)
Figure 14 (b). Proton NMR spectra for HX soluble Aldrich BuLHA dissolved in deuterated CF. Reference was TMS, and b = butyl peak.
Figure 14 (c). Proton NMR spectra for HX soluble BuLHA prepared from furfural and dissolved in deuterated CF. Reference was TMS.
Fig. 15 shows the C13 NMR DEPT spectrum of a soil BuLHA. The DEPT spectrum is extremely useful because it distinguishes between carbons that carry an odd or even number of protons. The n-butyl ester peaks occur at 13.4, 19.2, 25.7, and 65 ppm, consistent with known spectra of the n-butyl ester. Note that the aromatic region is totally devoid of peaks. Peaks are assigned as follows:
|Peak (ppm)||Assignment||Peak (ppm)||Assignment|
|10.9||Isolated CH3||13.7||CH3 connected to CH2|
|29.8||Long-chain CH2||22.9/ 23.8/ 24.5/ 29.0||Short-chain CH2|
|34.1||CH2 near O||66.9/ 68.2||CH2 next to O (lactone)|
Figure 15. C13 DEPT NMR spectrum of a soil BuLHA fraction eluted from a silica column with EA, taking the HX soluble fraction, and dissolving in deuterated CF. Reference was TMS, b = butyl peaks, and positive peaks are due to C with an odd number of protons and negative peaks are due to C with an even number of protons.
Solution NMR studies of polymers frequently show broad bands due to the large numbers of isomers that are possible. For hydrophilic fractions of humic acids this would be exacerbated by the equilibrium changes due keto-enol tautomerism and gem-diol dehydration. When the NMR relaxation time is of similar magnitude to the rate of equilibrium change, peaks are smeared over the whole spectrum and show only broad bands. This is analogous to what occurs in humic acid chromatography (refer below), and therefore limits the usefulness of NMR for elucidating humic acid structures. Solid state NMR could be more successful (it was only briefy investigated in this study), but samples must be rigorously purified if humic acids are to be measured per se.
FT-MS spectra of humic acids and derivatives
Some of the BuLHA fractions showed very strong IR bands around 1050 cm-1 that could be assigned to ether groups. This was investigated further using FT-MS, and spectra similar to those of polyethylene glycols were obtained, including the small envelope of spectra belonging to the mono-dehydrated series54. The following structures were detected:
Where R = C18H37O4 in underivatized soil humic acids and R = C18H35O4 in underivatized Aldrich humic acid
The following compounds were detected in BuLHA:
C20H40N3O5[CH2CH2O]n-H where n = 1-3
C20H40N3O5[CH2CH2CH2O]n-H where n = 1-3
C20H34N2O6 & C24H38O4
The latter two compounds are of interest because they were also found in bitumen samples. The source of the nitrogen containing compounds is unknown.
EI-MS of BuLHA gave a plethora of fragment peaks without any discernable pattern, with mass units up to about 1200 u, consistent with recently published works55,56. However, the CH2CHO+ peak (43 u), typical of polymeric ketones, was always present.
Humic acid structure and origin
From the data and evidence obtained in this study, it is concluded that humic acids are aliphatic polymers containing primarily carboxylate, keto (and keto-enol), conjugate chelate carbonyl, and lactone groups, with a lesser amount of anhydride and ether groups. Minor amounts of syringic and vanillic acids are considered to be lignin derived contaminants, and minor amounts of dicarboxylic and keto acids are considered to be furfural or humic acid oxidation products. The source of phthalic acid is unknown. The molecular weights of the major components appear to be around a maximum of 1200 u. This equates to the polymerization of 12 monomeric 4-oxo-2-butenoic acid units, and no doubt there are polymers with less than this number of monomers. Carboxylates are attached as side chains and decarboxylation readily occurs to give hydrocarbons and hydrocarbons with some keto groups, structures that are prominent in bitumen and petroleum. From the well-known chemistry of xylans, furfural, and xylose39, the genesis of humic acids can be deduced. Cellulose degradation can be discounted because cellulose hydrolyzes very slowly to furfural via glucose, but the pathway to levulinic acid (via glucose) is more rapid. In the natural environment microorganisms would metabolize most of the cellulose before it could degrade chemically to levulinic acid. In contrast, xylose, arabinose, and other furanose sugars rapidly form furfural, and fructose readily forms 5-hydroxymethyl furfural39,57. Although fructose is normally thought of as a pyranose sugar like glucose, it can exist in the furanose form that gives 5-hydroxymethyl furfural. Furfural and 5-hydroxymethyl furfural readily oxidize to 4-oxo-2-butenoic acid, that readily polymerizes to humic acids. Furfural and 5-hydroxymethyl furfural are commonly found in plant matter, for example in coffee oil and orange powder58-60. Unlike the slow pyranose pathway to levulinic acid, the furanose pathway to furfural is rapid, therefore not allowing microorganisms sufficient time to metabolize furanose sugars. Subsequent to this, humic acids are extremely resistant to microbial attack, which accounts for their persistence in the natural environment to eventually form the huge deposits of bitumen, petroleum, and coal, and to comprise the bulk of organic matter in many environmental samples. For example, data gathered from mangrove pore waters (unpublished results) show that 98+/-5% of the organic matter is due to humic acids.
The furfural origin of humic acids and coal was first proposed in 192020, so the idea is not new. However, at that time the route via 4-oxo-2-butenoic acid was unknown. We can now be confident that the principal route of humic acid genesis is via furfural and 4-oxo-2-butenoic acid from living things and decaying matter that possess xylose, arabinose, and fructose in their structures, such as plant xylans (hemicelluloses), arabinans, and fruit polysaccharides. Xylans are extremely abundant in nature, rivalling the amounts of cellulose and lignin. Xylans occur in practically all land plants and are present in some marine algae. For example, woody plant tissues are known to contain 34-58% cellulose, 14-34% lignin, and 7-30% pentosan (xylan)61. Some plants (e.g. switch grass and corn cobs) actually contain a greater amount of xylan than lignin. Arabinose is widely distributed in plants such as in pectins, and also occurs in mycobacteria. Ribose is a furanose but can be discounted in nature as a major source of humic acids because of its rarity. Fructose is an abundant sugar in fruits. In this study humic acids were also found in abundant quantities in sea grasses. Their origin was not investigated, although it can be assumed that they are derived from furanose sugars in sea grasses. No doubt sea grasses are a major source of marine humic acids, for which the aliphatic structure has never been disputed. However, in contrast to this study, an origin from unsaturated lipids has previously been proposed62.
In this study the polymerization of 4-oxo-2-butenoic acid and the in situ polymerization of furfural both yielded mainly aliphatic long-chain humic acids with lactone and carboxylate side chains. Furfural can oxidize to both the cis and trans isomers of 4-oxo-2-butenoic acid, but the cis form (which readily converts to the hydroxy lactone) was the predominant isomer in this study. From theoretical considerations 4-oxo-2-butenoic acid could copolymerize with itself at different sites to form diketo groups separated by two methylenes, conjugate chelate carbonyl groups, and ether groups. The hydrated aldehyde form would be the precursor of the gem-diol group in humic acids. The aldehyde structure originating from 4-oxo-2-butenoic acid was not preserved in humic acids since no band at 2720 cm-1 due to aldehyde hydrogen was ever observed. The conjugated aldehyde band of 4-oxo-2-butenoic acid at 1684 cm-1 was also never observed in humic acids. Therefore, the band observed at 1710 cm-1 was due to keto carbonyl. However, the lactone structure was always observed. It was a major structure in fulvic acids, FuHA, and Aldrich humic acid. 4-Oxo-2-butenoic acid readily converts to the hydroxy lactone form, and this compound could readily polymerize through carbons 2 and 3 (Fig. 2(b)), similar to the polymerization of acrylic acid and its derivatives. Since hydroxyl groups were not present together with the lactone group in any substantial amounts, the hydroxyl group is probably lost due to hydrolysis, as is known to occur in similar polymers containing poly (gamma-crotonolactone)51.
Even though humic acid molecular weights up to about 1200 u were detected, amounts above about 800 u were small, so the number of repeating units is probably =<10. Possible head-to-tail structures of lactone polymers containing identical repeating units are shown in Fig. 16 (a) & (b), where n = 0 – 8. Head-to-head structures could also be possible51, but are not shown. In addition to these structures there could be numerous other structures where the lactones are randomly oriented to each other. These structures would be derived from cis-4-oxo-2-butenoic acid in the lactone form. The carboxylic acid group is also prominent in humic acids, and to have carboxylic acid groups without aldehyde groups present suggests that the aldehyde groups are readily oxidized to carboxylic acids. The polycarboxylate structure could be derived from cis-4-oxo-2-butenoic acid in the open form to give polymaleic acid (Fig. 17 (a)) or from trans-4-oxo-2-butenoic acid to give polyfumaric acid (Fig. 17 (b)), where n= 0 – 8. There could be numerous other structures where the carboxylates are randomly orientated. It is significant to note that because of its properties polymaleic acid has been proposed as a model for fulvic acids37. Vicinal carboxyl groups readily dehydrate to yield anhydride groups, which were observed in low amounts in some humic acid fractions.
Figure 16. Proposed head-to-tail humic acid polymers containing the lactone group.
Figure 17. Proposed humic acid polymers containing the carboxyl group.
Except for the most hydrophobic fractions, BuHA fractions eluted from a classical silica column all showed the same type of moieties when examined by solution IR, but the relative ratios of these moieties varied. The more hydrophobic fractions (pale yellow, highly fluorescent oils) contained more methyl, methylene and lactone groups, whereas the more hydrophilic fractions (shiny black, brittle, low fluorescent solids) contained more conjugate chelate carbonyl and gem-diol groups. It is therefore proposed that humic acids consist of repeating units of identical moieties (polymers) as well as different moieties (copolymers) . This is due to the existence of both trans- and cis-4-oxo-2-butenoic acid and different sites within each molecule where polymerization could occur. The exact process would depend on environmental conditions such as pH, temperature, and the surrounding matrix. Obviously this would result in a huge number of different polymeric structures, consitent with the enormous complexity of humic acids.
It is most likely that the conjugate chelate carbonyl group causes the precipitation of humic acids in acidic solutions due to an equilibrium change to the diketo tautomer. The presence of the conjugate chelate carbonyl, keto-enol, and carboxylate groups would give humic acids great chelating power, a fact that is observed even for sodium and ammonium ions31. Tautomerism of the various types of keto groups in equilibrium with their respective enol groups together with equilibrium between the gem-diol and its dehydrated form gives humic acids a very dynamic nature. This is consistent with recent discoveries63 that humic acid macromolecular structures are different under different solution chemical conditions.
Structure of individual humic acid components
HPLC as well as classical column chromatography separations are hampered because humic acids undergo keto-enol tautomerism (from isolated carbonyls and conjugate chelate carbonyls) and gem-diol dehydration during separation that smears components over the column. During HPLC analysis (silica column) of hydrophobic derivatives, the column has to be flushed numerous times with MeOH injections after each sample injection or the enol tautomers adhering to the column cause subsequent injections to be almost totally adsorbed onto the column. As noted above, the tautomerism could only be eliminated by derivative formation after reduction. However, some of the most hydrophobic fractions did not show tautomerism and could be chromatographed without smearing. These fractions also gave sharp NMR spectra as described above.
HPLC analysis confirmed that individual humic acid components are extremely numerous, similar to the situation for petroleum components. In this study small fractions of derivatized humic acids were collected by column chromatography and then separated by HPLC. Fig. 18 shows typical HPLC separations for a soil BuHA. The large end peak is due to a MeOH flush that removes all the enol tautomers from the column. The HPLC traces occasionally showed well resolved individual peaks (Fig. 19), but generally the peaks were part of an envelope of peaks and resolution was poor. It is estimated that there are thousands of individual components, consistent with the complex polymerization of 4-oxo-2-butenoic acid described above.
An interesting observation was that fluorescence emission maxima correlated closely with the polarity of humic acid derivatives, although not many fractions were examined. Eluates from the HPLC experiment described in Fig. 18A were collected and their excitation and emission spectra obtained, and compared to the HX eluate from a classical silica column. Excitation spectra (em. 440 nm) were reasonably similar with no discernable pattern, but emission spectra maxima ranged from 340 nm for the HX eluate to 440 nm for the MeOH HPLC column flush (enol tautomers). These spectra are shown in Fig. 20.
Figure 18. HPLC of a soil BuHA eluted with DCM (A), and EA (B). Elution was at 2 ml/ min from a silica column with fluorescence detection at 340/ 440 nm. Prior to HPLC analysis the samples were eluted from a classical silica column with DCM and from which a HX soluble fraction was then obtained.
Figure 19. HPLC of an Aldrich BuHA eluted with DCM/ 10% HX. Elution was at 2 ml/ min from a silica column with fluorescence detection at 320/ 420 nm. Prior to HPLC analysis the sample was eluted from a classical silica column with DCM.
Figure 20. Fluorescence emission spectra (ex. 280 nm) for a HX eluate from a classical silica column (1), HPLC eluates collected in sequence each 5 min (2-4), and the HPLC MeOH flush (5). Prior to HPLC analysis the sample was eluted from a classical silica column with EA .
Much progress has been made to identify individual petroleum components by using GC-MS and FT-MS, but there is no equivalent technology currently available for humic acids because the components are not labile enough for GC-MS work, except for perhaps the reduced pentafluoropropionate derivatives, which were not investigated by GC-MS in this study. FT-MS may show some promise for elucidating the structures of individual humic acid components. Unfortunately, in this study only the minor structures containing ether and nitrogen groups could be ionized by electrospray techniques, even though many solvent systems were investigated. Other drawbacks of this technology is the interference between compounds of higher molecular weight56, and its limited availability due to extremely high costs.
Comparison of humic acids with kerogen, bitumen, petroleum and coal
It is well known that humic acids are precursors of kerogen, bitumen, petroleum, and coal. Published KBr IR spectra of humic acids and kerogens were similar to spectra of humic acids obtained in this study. Also, in this study fluorescence spectra of humic acids, BuLHA, FuHA, bitumen, commercial vehicle oil and grease, and DMSO soluble coal extracts were similar in both emission and excitation modes, consistent with a polymeric keto structure45. Solution IR spectra showed that bitumen has lactone, keto, enol, methyl and methylene groups. When a bitumen solution in THF is diluted, a relatively large H2O band is present, indicating that a gem-diol group dehydrates to a keto group. The keto band was abnormally wide, suggesting that it was unresolved from the nearby conjugate chelate carbonyl band. When bitumen was reduced with LiAlH4, the keto group was readily reduced to a hydroxyl group. This was confirmed by the acetate band that occurred after acetylation that was readily achieved with acetic anhydride and pyridine. When the keto band was compared to the methyl and methylene bands of a commercial vehicle oil, and then compared to the same bands in bitumen, it was clear that the commercial vehicle oil had more aliphatic structure and less keto structure than bitumen. Further, the commercial oil had a pronounced band due to CC double bonds, and lacked the conjugate chelate carbonyl band. A DMSO soluble coal IR spectrum showed anhydride doublet bands, a small keto band, a small conjugate chelate carbonyl band, a large H2O band, minor unsaturated aliphatic bands, methyl and methylene bands, and a major ether band (1050 cm-1).
Keto groups were present in all of the materials, but only humic acids and kerogen showed carboxylate groups. The conjugate chelate carbonyl group was even more restricted in its occurence, only being present in substantial amounts in hydrophilic humic acid fractions. Decarboxylation alters the humic acid structures during diagenesis but leaves both hydrophobic and hydrophilic fractions. In the natural environment the most hydrophilic fractions will gather as kerogens, the most hydrophobic fractions as petroleum, and intermediate fractions as bitumen. Because of its high ether content, coal probably has a different formation pathway, perhaps via the trans 4-oxo-2-butenoic acid route. This is feasible because the trans and cis isomers of 4-oxo-2-butenoic acid have quite different chemistries41.
1. To study humic acids per se, environmental samples must be rigorously purified. This includes filtering an alkaline solution through a 0.1 µm filter to remove minerals, extracting an acidified solution into an organic solvent such as ethyl acetate and/ or n-butanol, and preparing a derivative such as the n-butyl ester and extracting it into an organic solvent such as dichloromethane.
2. Care must be taken with investigative methods to ensure that humic acids per se are being measured, and not contaminants such as lignin degradation products. NMR and FT-MS techniques may not detect humic acids, and chromatography can result in a smearing of components over the entire column. Solution IR and fluorescence techniques have been found to be the most useful. The most promising techniques for elucidating structures of individual humic acids appear to be column chromatography separations of derivatized humic acids followed by HPLC-MS.
3. Humic acids originate from polysaccharides containing xylose, arabinose, and fructose. Xylose and arabinose oxidize and polymerize via the furfural and 4-oxo-2-butenoic acid pathway, and fructose oxidizes and polymerizes via the 5-hydroxymethyl furfural and 4-oxo-2-butenoic acid pathway.
4. Humic acids are a complex mixture of polymers and copolymers containing the following groups (how these groups are arranged is not currently known, and end groups are not shown):
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This manuscript was written in January 2001 and edited with some changes and additions on 6 December 2003. I thank a large number of colleagues and reviewers for expert technical and editorial help.
I dedicate this work to my father, Louie Susic, who passed away on 7 April 2003.