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A new fractionation of soil humic acids by adsorption chromatography
Geoderma, 47 (1990) 327-336
327
Elsevier Science Publishers B.V., Amsterdam
A new fractionation of soil humic acids by adsorption chromatography K. Yonebayashi and T. Hattori Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyoku, Kyoto 606, Japan (Received July 24, 1989; accepted after revision November 28, 1989 )
ABSTRACT Yonebayashi, K. and Hattori, T., 1990. A new fractionation of soil humic acids by adsorption chromatography. Geoderma, 47: 327-336. Macroporous, nonionic Amberlite XAD-8 resin was used for fractionation of soil humic acids in order to reduce their complexity. H+-saturated humic acid was adsorbed onto the resin at pH 3 and fractionated into four components by stepwise elution using universal buffers adjusted to pH 7, to pH 11, water, and 50% ethanol. The first component consists of a few structural subunits, assumed to be condensed aromatic rings with short al]phatic substituents and many carboxyl groups. The second component was characterized by phenolic groups, and the third by relatively long aliphatic chains. The fourth component consists of many structural subunits, assumed to be aromatic rings with long aliphatic substituents. Analysis of the distribution of these components makes possible a new classification of humic acids.
INTRODUCTION
Recently humic substances in the environment have attracted the intense attention of geochemists and environmental scientists, not to mention soil scientists and agronomists. These substances are structurally complex, polyelectrolytic, dark-colored aromatic organic acids. To reduce their complexity, they must be fractionated into a series of similar components. The following fractionation methods have been tried (Thurman and Malcolm, 1983 ): acid precipitation, metal precipitation, liquid extraction, ion-exchange, electrophoresis, and liquid chromatography. Although none of these methods is completely satisfactory, liquid chromatography separates humic substances well by such mechanisms as size exclusion, charge exclusion, hydrogen bonding on an anion exchanger, hydrophobicity, and electrostatic interaction. MacCarthy et al. (1979) and Thurman and Malcolm (1983) demonstrated that aquatic humic acids were adsorbed on the nonionic macroporous resin Amberlite XAD-8 at pH 2 and could be separated into two fractions by pHgradient elution. Curtis et al. ( 1981 ) showed that model compounds were 0016-7061/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
328
K. YONEBAYASKIAND T. HATTORI
eluted from this resin using a nearly linear pH gradient of decreasing acidity. Humic acids would be separated by the mechanism that as the pH of the eluent increases, the components having larger pKa values would be progressively ionized and desorbed (Curtis et al., 1981; Ohga et al., 1989). We attempted to improve this method so that it can be applied to soil humic acids because such acids precipitate below pH 2 prior to adsorption on hydrophobic resins. H +-humic acids treated with Amberlite IR-120 were of about pH 3 and did not precipitate when mixed with the universal buffer, an equimolar mixture of phosphoric, acetic, and boric acids, the pH of which was adjusted with sodium hydroxide. METHODS
Amberlite XAD-8 resin was pulverized and the 50-200 a m range was isolated. The sieved particles were washed with ethanol, acetonitrile, again with ethanol, and packed into a column (20 cmX 1.8 cm i.d.) which was conditioned with 0.1 M sodium hydroxide followed by the universal buffer taken to pH 3 with sodium hydroxide. Soil humic acid was extracted from Andisol, Entisol, Inceptisol, and Histosol as listed in Table I, and purified with a modified IHSS (Intern. Humic Substances Soc.) m e t h o d (Yonebayashi and Hattori, 1988): it was treated with a mixture of 0.3 M HF and 0. l M HC1, dialyzed against distilled water, passed through a column of Amberlite IR-120 resin in H+-form, and then freeze-dried. Humic acid was dissolved in 0.1 M NaOH and treated with Amberlite IR120 resin to make the H+-saturated form. Five mg of humic acid dissolved in 2 ml of aqueous solution was loaded onto the column packed with XAD-8 resin. A pH-gradient solution was prepared by titrating 200 ml of 0.02 M universal buffer, contained in an air-tight flask, with 0.1 M NaOH using a peristaltic pump, and passed through at a flow rate of 1.5 ml min-1. The pH of the column effluent was measured with a pH electrode. A water-ethanol gradient was generated by mixing 200 ml of distilled water, contained in an air-tight flask, with ethanol using a peristaltic pump. Elution was at a flow TABLEI Soil sample used Soil
Characteristics
Location
Andisol Entisol Histosol Inceptisol
Nonallophanic Andosol Gray lowland soil, paddy Buried peat soil Brown forest soil, Oak
Ahurahi, Shiga Shimogamo, Kyoto Azuchi, Shiga Kamigamo, Kyoto
All locations are in Japan.
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
329
rate of 1.5 ml min-1. The elution profile was determined by measuring the optical density at 400 n m after the effluent was alkalified above pH 12 by addition of l 0 M NaOH. Stepwise elution was run with universal buffer solutions adjusted to pH 7 and pH I l, distilled water, and 50% ethanol. The elution profile was determined in the same way as for the pH gradient chromatography. Each effluent was precipitated with sulfuric acid and dissolved in 0.1 M NaOH. Since the humic fraction eluted at pH 7 was not precipitated by acidification, it was adsorbed on a small XAD-8 column at pH 3 and eluted with the NaOH solution. Each of the four eluates was dialyzed against distilled water and freezedried. The amounts of carbon, nitrogen and hydrogen in the humic acids were determined by combustion in oxygen gas. The oxygen content was calculated by difference. Carboxyl and phenolic hydroxyl groups of the humic acids were analysed by the authors' nonaqueous titration method (Yonebayashi and Hattori, 1985 ). The carbonyl group was analyzed by the method based on the formation of oxime by reaction with hydroxylamine, and the total hydroxyl group content was determined by the acetylation m e t h o d (Yonebayashi and Hattori, 1988). Estimates for the content of alcoholic hydroxyl group were obtained from the difference between the amounts of total hydroxyl groups and phenolic hydroxyl group. Well-dried humic acid was dissolved in deuterium dimethylsulfoxide and a small a m o u n t of tetramethylsilane was added as an internal reference. 1HN M R spectra were obtained by Fourier transform-NMR spectroscopy on a Hitachi 90H spectrometer at 90 MHz (Yonebayashi and Hattori, 1989). Gel permeation chromatography was carried out on a column packed with Sephadex G-75, eluting with a neutral phosphate buffer containing 2 M urea to minimize the interaction between the gel and the humic acids (Yonebayashi and Hattori, 1987). Elution profiles were determined by measurement of optical density at 400 nm. RESULTS AND DISCUSSION
Humic acid treated with Amberlite IR-120 after dialysis against distilled water was recovered completely as the H+-form, was of about pH 3, and was not precipitated even if a universal buffer was added. Humic acid from Inceptisol was injected into the column packed with Amberlite XAD-8 and completely adsorbed on the resin. We first eluted the adsorbed humic acid using a pH-gradient solution (Curtis et al., 1981 ) prepared from 0.02 M universal buffer and 0.1 M sodium hydroxide (Fig. 1, a and b). Fig. 1b shows the pH profile for the effluent of the XAD-8 column. The pH gradient was not linear due to the retention of buffer components by the column (Curtis et al., 1981 ).
330
K. YONEBAYASKI AND T. HATTOR1
Fig. 1a shows the eluation pattern measured at 400 nm. The humic acid remained immobile until the pH rose to about 4. It was eluted between pH 4 and 11 in two peaks, each of which corresponded to one of the inflections in the pH-gradient curve. These embrace the pKa values of the carboxylic and phenolic groups. The fraction of humic acid eluted in the pH 5-7 region was desorbed from the hydrophobic resin because it became hydrophilic due to ionization of the carboxylic groups. The fraction eluted in the pH 8-11 region is not sufficiently hydrophilic to be desorbed from XAD resin until the phenolic groups are neutralized (Curtis et al., 1981). The results with 0.02 M, 0.05 M, and 0.1 M universal buffer were almost identical. Varying the flow rate from 0.67 to 1.5 ml min-~ had no influence on peak separation. The elution pattern was reproducible. Ravichandran et al. (1988) recently generated a linear pH gradient using a four-pump system controlled by a computer. They were not able to resolve the humic acids completely, however, no matter what linear pH gradient was used. After the pH-gradient elution, significant amounts of humic acids remained on the column. These acids were eluted with a water-ethanol gradient to resolve their hydrophobic portion (Fig. 1a) and were completely desorbed. The third peak in Fig. 1a corresponded to the fraction o f h u m i c acid desorbed from resin through the desalting of the liquid phase with water. After the aqueous elution, as the ethanol concentration in the eluent increased, the hydrophobic c o m p o n e n t was successively desorbed, yielding a broad plateau. The resolution of the peaks with pH-gradient and ethanol-gradient elutions was incomplete, however. In order to improve the resolution, the adsorbed humic acids were first eluted in two steps with buffer solutions adjusted to pH 7 and to pH 11. Next the strongly adsorbed humic acids were eluted stepwise with water and then 50% ethanol (Fig. 1c). The soil humic acid was thus separated into four components (named A, B, C and D in the order of elution) with these different solutions. Components A and B corresponded to the first and second peaks eluted by pH gradient in Fig. 1a, respectively. Components C and D corresponded to the third peak and the broad plateau eluted by water-ethanol gradient, respectively. The humic acid from Inceptisol was fractionated into these components by repeated application of the adsorption chromatography. The elemental composition and H / C and O / C atomic ratios of the four components are listed in Table II. The carbon and hydrogen content and the H / C ratio increased in the order: A < B < C < D. The O / C ratio decreased in the order; A > B > C > D. Functional group compositions were determined by nonaqueous titration methods and are also listed in Table II. The content of carboxyl and carbonyl groups decreased in the order; A > B > C > D. The functional group contents of components B and C, except for the phenolic hydroxyl group, were lower than in c o m p o n e n t A. C o m p o n e n t D was character-
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
331
b
pH 11 9 7
3
tEthanol-
IpH- g r a d i e n t
gradient
6
J
J
L
i
I
,
!
t '°°tpH 11 H20 t '°°t50%Ethanol
300
pH 7
ELUTION VOLUME (cm 3)
Fig. 1. Fractionationof soil humic acid by pH-gradient,ethanol-gradient,and stepwiseelution. a. pH-Gradient and ethanol-gradientelution curves, b. pH profile of the eluate, c. Stepwise elution with bufferadjustedto pH 7, to pH 11, water,and 50% ethanol. ized by smaller amounts of carboxyl, carbonyl, and phenolic hydroxyl groups and a larger amount of alcoholic hydroxyl group. ~H-NMR spectra of the four components are presented in Fig. 2. The chemical shifts were measured with respect to internal TMS and attributed as follows (Hatcher et al., 1980; Wilson, 1981; Wershaw, 1985; Yonebayashi and Hattori, 1989). In component A, the relatively strong absorbance in the 69.5 ppm region was attributed to aromatic protons, and the weak peaks in the 0.5-3 ppm region to aliphatic protons. In component D, the absorptions of aliphatic protons were stronger than of aromatic protons, and the extremely strong peak in the 1.0-1.9 region was attributed to protons in methyl groups fl to aromatic rings or bound to methylene or methyn carbons farther than fl to aromatic rings. The weak peaks in the 1.9-2.8 ppm region and the 0.5-1.0 ppm region were attributed to protons attached to aliphatic carbon atoms a
332
K. YONEBAYASKIAND T. HATTORI
TABLE I1 Elemental and functional group compositions of the four components of Inceptisol humic acid Component
A B C D
C (%)
49.0 50.0 53.4 58.1
H (%)
N (%)
3.8 4.5 5.1 6.6
2.9 3.4 3.4 3.5
O (%)
44.3 42.1 38.1 31.8
H/C atomic ratio
O/C atomic ratio
Functional groups ( m e q / g ) COOH
C=O
phenolic- alcoholicOH OH
0.93 1.08 1.15 1.36
0.68 0.63 0.54 0.40
4.2 4.0 3.2 2.5
6.6 5.4 3.8 1.3
0.8 1.2 1.1 0.5
4.6 4.3 1.3 5.5
Components A, B, C, and D were eluted with universal buffers adjusted to pH 7, to pH 11, water, and 50% aqueous ethanol, respectively. Humic acid was extracted from Inceptisol.
aromatic
lactone
OCHz
/ o
/ 10
i 9
i 8
i 7
6
5
4
3
2
0
ppm
Fig. 2. t H - N M R spectra of." a, c o m p o n e n t A; b, c o m p o n e n t B; c, c o m p o n e n t C; a n d d, component D. C o m p o n e n t s A to D are characterized in the legend o f Table II.
A NEW FRACTIONATIONOF SOILHUMIC ACIDS
333
to aromatic rings and attached to methyl terminal carbons y or farther from aromatic rings, respectively. The relative areas of these peaks were measured and the proton distribution percentages were calculated (Table III ). The percentage of aromatic protons decreased in the order: A > B > C > D. The ratio of aliphatic protons fl to aromatic rings to those in the o~ position increased in the order: A < B < C < D. C o m p o n e n t A had, therefore, short aliphatic chains and component D had long aliphatic chains, on the assumption that the chains had little branching (Sciacovelli et al., 1977). C o m p o n e n t A had short aliphatic chains and high content of carbon, aromatic protons, and functional groups. In contrast, c o m p o n e n t D had long aliphatic chains and low contents of carbon, aromatic protons, and functional groups. From these results it was deduced that c o m p o n e n t A had a high aromatic carbon content and component D had a low content of poorly condensed aromatic carbon. Gel permeation chromatograms of each component on Sephadex G-75 are shown in Fig. 3. C o m p o n e n t A had a small excluded fraction and a large diffused fraction; therefore, the molecular weight distribution was narrow. In contrast, c o m p o n e n t D had a large excluded fraction and a small plateau in the diffused region; therefore, the molecular weight distribution was wide with a high average molecular weight. These results indicate that component A consists of a few structural subunits assumed to be condensed aromatic rings with short aliphatic substituents and many carboxyl groups. The structural subunits of component B are similar to those of c o m p o n e n t A except that the former have relatively many phenolic groups. The many structural subunits of component D are assumed to be aromatic rings with long aliphatic substituents. The subunits of component C are similar, but the aliphatic chains are shorter. Different combinations of structural parts and functional groups in humic acids may cause different moieties to be eluted as one. However, the major TABLE III Proton distribution of the four components of Inceptisol humic acid Component
A B C D
Aromatic H
Lactone H
OCH3-H OH-H
o~-H (a-CH3
~
fl-H (fl-CH3 ~
y-H (remote~
(%)
(%)
(%)
\a-CH2,0c-CH]
\r-file,1
\CH 3
(%)
(%)
(%)
3.2 3.2 3.3 3.9
2.9 5.1 16.8 35.0
3.4 3.5 4.2 4.9
65.5 52.8 50.8 16.3
12.0 15.3 12.8 24.2
13.0 20.1 12.1 15.7
J
Components A to D are characterized in the legend to Table II. The area of each peak in the ~H-NMR spectra is given as the percentage of the total area of all the peaks in the spectrum.
334
K. YONEBAYASKI A N D T. H A T T O R I
z E) O ~t
b iii U
z
m E~
oL/3 Ca
c
d i
,oo tv t
ELUTION
VOLUME ( c m 3)
Fig. 3. Gel permeation chromatography of: a, component A; b, component B; c, component C; and d, component D.
part of the carboxylic and phenolic groups are ionized at pH 7 and 11, respectively. One therefore can safely say that components A and B are characterized by high contents of carboxyl and phenolic groups, respectively; thus we designate these components as the "carboxylic (hydrophilic)" and "phenolic (semi-hydrophilic)" component, respectively. It is well known that the aromaticity of humic acid increases with increasing carboxyl group content. Because the aromatic moieties with carboxyl groups were removed from humic acid as components A and B, residual components have hydrophobic properties with an aliphatic structure. In fact, components C and D are characterized by relatively long and even longer aliphatic substituents, respectively. We therefore designate these components as the "semi-aliphatic" and "aliphatic" component, respectively. However, these definitions are based on the frac-
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
335
tionation conditions used and may not be complete enough to apply to all humic acids. Additional separations of many kinds of humic acids are needed to confirm that in all cases each of the four components has the same structural and functional compositions. As an initial step, four types of soil humic acids were prepared from Andisol, Inceptisol, Histosol, and Entisol. Each humic acid was separated into four components by the above stepwise elution method (Fig. 4). Andisol humic acid contained abundant amounts of carboxylic components and moderate amounts of aliphatic components. This agreed with the concept that Andisol
z (3 0
~r
iii L)
Z
k
m
rv
o ~D
t pH 7
t 'oo
t
oot
p i l l 1 H20 50%Ethanol ELUTION VOLUME (cm 3)
Fig. 4. Fractionation of soil humic acids by stepwise elution: a, Andisol; b, Entisol; c, lnceptisol; d, Histosol.
336
K. YONEBAYASKI AND T. HATTORI
humic acid is highly aromatic (Tokudome and Kanno, 1968 ) and has abundant carboxyl groups (Yonebayashi and Hattori, 1988 ). Histosol humic acid contained abundant amounts of aliphatic components and little of carboxylic components. The proportions of the components of Entisol and Inceptisol humic acids were intermediate between Andisol and Histosol humic acids. These results suggest that our analysis of the distribution of these four components leads to a new way of classifying humic acids.
REFERENCES Curtis, M.A., Witt, A.F., Schram, S.B. and Rogers, L.B., 1981. Humic acid fractionation using a nearly linear pH gradient. Anal. Chem., 53:1195-1199. Hatcher, P.G., Rowan, R. and Mattingly, M.A., 1980. ~H and ~3C NMR of marine humic acids. Org. Geochem., 2: 77-85. MacCarthy, P., Peterson, M.J., Malcolm, R.L. and Thurman, E.M., 1979. Separation of humic substances by pH gradient desorption from a hydrophobic resin. Anal. Chem., 51: 20412043. Ohga, K., Aritomi, Y. and Ohtsu, H., 1989. Chromatography with pH-gradient elution of dissolved humic substances in river water. Anal. Sci., 5:215-216. Ravichandran, K., Lewis, J.J., Yin, I.-Hsiung, Koenigbauer, M., Powley, C.R., Shah, P. and Rogers, L.B., 1988. Computer-controlled linear pH gradient for high-performance liquid chromatographic fractionations of aromatic carboxylic acids and of humic and fulvic acids. J. Chromatogr., 439:213-226. Sciacovelli, O., Senesi, N., Solinas, V. and Testini, C., 1977. Spectroscopic studies on soil organic fractions, I. IR and NMR spectra. Soil Biol. Biochem., 9: 287-293. Thurman, E.M. and Malcolm, R.L., 1983. Structural study of humic substances: new approaches and methods. In: R.F. Christman and E.T. Gjessing (Editors), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Michigan, pp. 1-23. Tokudome, S. and Kanno, I., 1968. Nature of the humus of some Japanese soils. Trans. 9th. Int. Congr. Soil Sci., III: 163-173. Wershaw, R.L., 1985. Application of nuclear magnetic resonance spectroscopy for determining functionality in humic substances. In: G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Editors), Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization. Wiley, New York, N.Y., pp. 561-582. Wilson, M.A., 1981. Application of nuclear magnetic resonance spectroscopy to the study of the structure of soil organic matter. J. Soil Sci., 32:167-186. Yonebayashi, K. and Hattori, T., 1985. Nonaqueous titration of functional groups in humic acid. Org. Geochem., 8: 47-54. Yonebayashi, K. and Hattori, T., 1987. Surface active properties of soil humic acids. Sci. Total Environ., 62: 55-64. Yonebayashi, K. and Hattori, T., 1988. Chemical and biological studies on environmental humic acids, I. Composition of elemental and functional groups of humic acids. Soil Sci. Plant Nutr., 34: 571-584. Yonebayashi, K. and Hattori, T., 1989. Chemical and biological studies on environmental humic acids, II. JH-NMR and IR spectra ofhumic acids. Soil Sci. Plant Nutr., 35: 383-392.
327
Elsevier Science Publishers B.V., Amsterdam
A new fractionation of soil humic acids by adsorption chromatography K. Yonebayashi and T. Hattori Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyoku, Kyoto 606, Japan (Received July 24, 1989; accepted after revision November 28, 1989 )
ABSTRACT Yonebayashi, K. and Hattori, T., 1990. A new fractionation of soil humic acids by adsorption chromatography. Geoderma, 47: 327-336. Macroporous, nonionic Amberlite XAD-8 resin was used for fractionation of soil humic acids in order to reduce their complexity. H+-saturated humic acid was adsorbed onto the resin at pH 3 and fractionated into four components by stepwise elution using universal buffers adjusted to pH 7, to pH 11, water, and 50% ethanol. The first component consists of a few structural subunits, assumed to be condensed aromatic rings with short al]phatic substituents and many carboxyl groups. The second component was characterized by phenolic groups, and the third by relatively long aliphatic chains. The fourth component consists of many structural subunits, assumed to be aromatic rings with long aliphatic substituents. Analysis of the distribution of these components makes possible a new classification of humic acids.
INTRODUCTION
Recently humic substances in the environment have attracted the intense attention of geochemists and environmental scientists, not to mention soil scientists and agronomists. These substances are structurally complex, polyelectrolytic, dark-colored aromatic organic acids. To reduce their complexity, they must be fractionated into a series of similar components. The following fractionation methods have been tried (Thurman and Malcolm, 1983 ): acid precipitation, metal precipitation, liquid extraction, ion-exchange, electrophoresis, and liquid chromatography. Although none of these methods is completely satisfactory, liquid chromatography separates humic substances well by such mechanisms as size exclusion, charge exclusion, hydrogen bonding on an anion exchanger, hydrophobicity, and electrostatic interaction. MacCarthy et al. (1979) and Thurman and Malcolm (1983) demonstrated that aquatic humic acids were adsorbed on the nonionic macroporous resin Amberlite XAD-8 at pH 2 and could be separated into two fractions by pHgradient elution. Curtis et al. ( 1981 ) showed that model compounds were 0016-7061/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
328
K. YONEBAYASKIAND T. HATTORI
eluted from this resin using a nearly linear pH gradient of decreasing acidity. Humic acids would be separated by the mechanism that as the pH of the eluent increases, the components having larger pKa values would be progressively ionized and desorbed (Curtis et al., 1981; Ohga et al., 1989). We attempted to improve this method so that it can be applied to soil humic acids because such acids precipitate below pH 2 prior to adsorption on hydrophobic resins. H +-humic acids treated with Amberlite IR-120 were of about pH 3 and did not precipitate when mixed with the universal buffer, an equimolar mixture of phosphoric, acetic, and boric acids, the pH of which was adjusted with sodium hydroxide. METHODS
Amberlite XAD-8 resin was pulverized and the 50-200 a m range was isolated. The sieved particles were washed with ethanol, acetonitrile, again with ethanol, and packed into a column (20 cmX 1.8 cm i.d.) which was conditioned with 0.1 M sodium hydroxide followed by the universal buffer taken to pH 3 with sodium hydroxide. Soil humic acid was extracted from Andisol, Entisol, Inceptisol, and Histosol as listed in Table I, and purified with a modified IHSS (Intern. Humic Substances Soc.) m e t h o d (Yonebayashi and Hattori, 1988): it was treated with a mixture of 0.3 M HF and 0. l M HC1, dialyzed against distilled water, passed through a column of Amberlite IR-120 resin in H+-form, and then freeze-dried. Humic acid was dissolved in 0.1 M NaOH and treated with Amberlite IR120 resin to make the H+-saturated form. Five mg of humic acid dissolved in 2 ml of aqueous solution was loaded onto the column packed with XAD-8 resin. A pH-gradient solution was prepared by titrating 200 ml of 0.02 M universal buffer, contained in an air-tight flask, with 0.1 M NaOH using a peristaltic pump, and passed through at a flow rate of 1.5 ml min-1. The pH of the column effluent was measured with a pH electrode. A water-ethanol gradient was generated by mixing 200 ml of distilled water, contained in an air-tight flask, with ethanol using a peristaltic pump. Elution was at a flow TABLEI Soil sample used Soil
Characteristics
Location
Andisol Entisol Histosol Inceptisol
Nonallophanic Andosol Gray lowland soil, paddy Buried peat soil Brown forest soil, Oak
Ahurahi, Shiga Shimogamo, Kyoto Azuchi, Shiga Kamigamo, Kyoto
All locations are in Japan.
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
329
rate of 1.5 ml min-1. The elution profile was determined by measuring the optical density at 400 n m after the effluent was alkalified above pH 12 by addition of l 0 M NaOH. Stepwise elution was run with universal buffer solutions adjusted to pH 7 and pH I l, distilled water, and 50% ethanol. The elution profile was determined in the same way as for the pH gradient chromatography. Each effluent was precipitated with sulfuric acid and dissolved in 0.1 M NaOH. Since the humic fraction eluted at pH 7 was not precipitated by acidification, it was adsorbed on a small XAD-8 column at pH 3 and eluted with the NaOH solution. Each of the four eluates was dialyzed against distilled water and freezedried. The amounts of carbon, nitrogen and hydrogen in the humic acids were determined by combustion in oxygen gas. The oxygen content was calculated by difference. Carboxyl and phenolic hydroxyl groups of the humic acids were analysed by the authors' nonaqueous titration method (Yonebayashi and Hattori, 1985 ). The carbonyl group was analyzed by the method based on the formation of oxime by reaction with hydroxylamine, and the total hydroxyl group content was determined by the acetylation m e t h o d (Yonebayashi and Hattori, 1988). Estimates for the content of alcoholic hydroxyl group were obtained from the difference between the amounts of total hydroxyl groups and phenolic hydroxyl group. Well-dried humic acid was dissolved in deuterium dimethylsulfoxide and a small a m o u n t of tetramethylsilane was added as an internal reference. 1HN M R spectra were obtained by Fourier transform-NMR spectroscopy on a Hitachi 90H spectrometer at 90 MHz (Yonebayashi and Hattori, 1989). Gel permeation chromatography was carried out on a column packed with Sephadex G-75, eluting with a neutral phosphate buffer containing 2 M urea to minimize the interaction between the gel and the humic acids (Yonebayashi and Hattori, 1987). Elution profiles were determined by measurement of optical density at 400 nm. RESULTS AND DISCUSSION
Humic acid treated with Amberlite IR-120 after dialysis against distilled water was recovered completely as the H+-form, was of about pH 3, and was not precipitated even if a universal buffer was added. Humic acid from Inceptisol was injected into the column packed with Amberlite XAD-8 and completely adsorbed on the resin. We first eluted the adsorbed humic acid using a pH-gradient solution (Curtis et al., 1981 ) prepared from 0.02 M universal buffer and 0.1 M sodium hydroxide (Fig. 1, a and b). Fig. 1b shows the pH profile for the effluent of the XAD-8 column. The pH gradient was not linear due to the retention of buffer components by the column (Curtis et al., 1981 ).
330
K. YONEBAYASKI AND T. HATTOR1
Fig. 1a shows the eluation pattern measured at 400 nm. The humic acid remained immobile until the pH rose to about 4. It was eluted between pH 4 and 11 in two peaks, each of which corresponded to one of the inflections in the pH-gradient curve. These embrace the pKa values of the carboxylic and phenolic groups. The fraction of humic acid eluted in the pH 5-7 region was desorbed from the hydrophobic resin because it became hydrophilic due to ionization of the carboxylic groups. The fraction eluted in the pH 8-11 region is not sufficiently hydrophilic to be desorbed from XAD resin until the phenolic groups are neutralized (Curtis et al., 1981). The results with 0.02 M, 0.05 M, and 0.1 M universal buffer were almost identical. Varying the flow rate from 0.67 to 1.5 ml min-~ had no influence on peak separation. The elution pattern was reproducible. Ravichandran et al. (1988) recently generated a linear pH gradient using a four-pump system controlled by a computer. They were not able to resolve the humic acids completely, however, no matter what linear pH gradient was used. After the pH-gradient elution, significant amounts of humic acids remained on the column. These acids were eluted with a water-ethanol gradient to resolve their hydrophobic portion (Fig. 1a) and were completely desorbed. The third peak in Fig. 1a corresponded to the fraction o f h u m i c acid desorbed from resin through the desalting of the liquid phase with water. After the aqueous elution, as the ethanol concentration in the eluent increased, the hydrophobic c o m p o n e n t was successively desorbed, yielding a broad plateau. The resolution of the peaks with pH-gradient and ethanol-gradient elutions was incomplete, however. In order to improve the resolution, the adsorbed humic acids were first eluted in two steps with buffer solutions adjusted to pH 7 and to pH 11. Next the strongly adsorbed humic acids were eluted stepwise with water and then 50% ethanol (Fig. 1c). The soil humic acid was thus separated into four components (named A, B, C and D in the order of elution) with these different solutions. Components A and B corresponded to the first and second peaks eluted by pH gradient in Fig. 1a, respectively. Components C and D corresponded to the third peak and the broad plateau eluted by water-ethanol gradient, respectively. The humic acid from Inceptisol was fractionated into these components by repeated application of the adsorption chromatography. The elemental composition and H / C and O / C atomic ratios of the four components are listed in Table II. The carbon and hydrogen content and the H / C ratio increased in the order: A < B < C < D. The O / C ratio decreased in the order; A > B > C > D. Functional group compositions were determined by nonaqueous titration methods and are also listed in Table II. The content of carboxyl and carbonyl groups decreased in the order; A > B > C > D. The functional group contents of components B and C, except for the phenolic hydroxyl group, were lower than in c o m p o n e n t A. C o m p o n e n t D was character-
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
331
b
pH 11 9 7
3
tEthanol-
IpH- g r a d i e n t
gradient
6
J
J
L
i
I
,
!
t '°°tpH 11 H20 t '°°t50%Ethanol
300
pH 7
ELUTION VOLUME (cm 3)
Fig. 1. Fractionationof soil humic acid by pH-gradient,ethanol-gradient,and stepwiseelution. a. pH-Gradient and ethanol-gradientelution curves, b. pH profile of the eluate, c. Stepwise elution with bufferadjustedto pH 7, to pH 11, water,and 50% ethanol. ized by smaller amounts of carboxyl, carbonyl, and phenolic hydroxyl groups and a larger amount of alcoholic hydroxyl group. ~H-NMR spectra of the four components are presented in Fig. 2. The chemical shifts were measured with respect to internal TMS and attributed as follows (Hatcher et al., 1980; Wilson, 1981; Wershaw, 1985; Yonebayashi and Hattori, 1989). In component A, the relatively strong absorbance in the 69.5 ppm region was attributed to aromatic protons, and the weak peaks in the 0.5-3 ppm region to aliphatic protons. In component D, the absorptions of aliphatic protons were stronger than of aromatic protons, and the extremely strong peak in the 1.0-1.9 region was attributed to protons in methyl groups fl to aromatic rings or bound to methylene or methyn carbons farther than fl to aromatic rings. The weak peaks in the 1.9-2.8 ppm region and the 0.5-1.0 ppm region were attributed to protons attached to aliphatic carbon atoms a
332
K. YONEBAYASKIAND T. HATTORI
TABLE I1 Elemental and functional group compositions of the four components of Inceptisol humic acid Component
A B C D
C (%)
49.0 50.0 53.4 58.1
H (%)
N (%)
3.8 4.5 5.1 6.6
2.9 3.4 3.4 3.5
O (%)
44.3 42.1 38.1 31.8
H/C atomic ratio
O/C atomic ratio
Functional groups ( m e q / g ) COOH
C=O
phenolic- alcoholicOH OH
0.93 1.08 1.15 1.36
0.68 0.63 0.54 0.40
4.2 4.0 3.2 2.5
6.6 5.4 3.8 1.3
0.8 1.2 1.1 0.5
4.6 4.3 1.3 5.5
Components A, B, C, and D were eluted with universal buffers adjusted to pH 7, to pH 11, water, and 50% aqueous ethanol, respectively. Humic acid was extracted from Inceptisol.
aromatic
lactone
OCHz
/ o
/ 10
i 9
i 8
i 7
6
5
4
3
2
0
ppm
Fig. 2. t H - N M R spectra of." a, c o m p o n e n t A; b, c o m p o n e n t B; c, c o m p o n e n t C; a n d d, component D. C o m p o n e n t s A to D are characterized in the legend o f Table II.
A NEW FRACTIONATIONOF SOILHUMIC ACIDS
333
to aromatic rings and attached to methyl terminal carbons y or farther from aromatic rings, respectively. The relative areas of these peaks were measured and the proton distribution percentages were calculated (Table III ). The percentage of aromatic protons decreased in the order: A > B > C > D. The ratio of aliphatic protons fl to aromatic rings to those in the o~ position increased in the order: A < B < C < D. C o m p o n e n t A had, therefore, short aliphatic chains and component D had long aliphatic chains, on the assumption that the chains had little branching (Sciacovelli et al., 1977). C o m p o n e n t A had short aliphatic chains and high content of carbon, aromatic protons, and functional groups. In contrast, c o m p o n e n t D had long aliphatic chains and low contents of carbon, aromatic protons, and functional groups. From these results it was deduced that c o m p o n e n t A had a high aromatic carbon content and component D had a low content of poorly condensed aromatic carbon. Gel permeation chromatograms of each component on Sephadex G-75 are shown in Fig. 3. C o m p o n e n t A had a small excluded fraction and a large diffused fraction; therefore, the molecular weight distribution was narrow. In contrast, c o m p o n e n t D had a large excluded fraction and a small plateau in the diffused region; therefore, the molecular weight distribution was wide with a high average molecular weight. These results indicate that component A consists of a few structural subunits assumed to be condensed aromatic rings with short aliphatic substituents and many carboxyl groups. The structural subunits of component B are similar to those of c o m p o n e n t A except that the former have relatively many phenolic groups. The many structural subunits of component D are assumed to be aromatic rings with long aliphatic substituents. The subunits of component C are similar, but the aliphatic chains are shorter. Different combinations of structural parts and functional groups in humic acids may cause different moieties to be eluted as one. However, the major TABLE III Proton distribution of the four components of Inceptisol humic acid Component
A B C D
Aromatic H
Lactone H
OCH3-H OH-H
o~-H (a-CH3
~
fl-H (fl-CH3 ~
y-H (remote~
(%)
(%)
(%)
\a-CH2,0c-CH]
\r-file,1
\CH 3
(%)
(%)
(%)
3.2 3.2 3.3 3.9
2.9 5.1 16.8 35.0
3.4 3.5 4.2 4.9
65.5 52.8 50.8 16.3
12.0 15.3 12.8 24.2
13.0 20.1 12.1 15.7
J
Components A to D are characterized in the legend to Table II. The area of each peak in the ~H-NMR spectra is given as the percentage of the total area of all the peaks in the spectrum.
334
K. YONEBAYASKI A N D T. H A T T O R I
z E) O ~t
b iii U
z
m E~
oL/3 Ca
c
d i
,oo tv t
ELUTION
VOLUME ( c m 3)
Fig. 3. Gel permeation chromatography of: a, component A; b, component B; c, component C; and d, component D.
part of the carboxylic and phenolic groups are ionized at pH 7 and 11, respectively. One therefore can safely say that components A and B are characterized by high contents of carboxyl and phenolic groups, respectively; thus we designate these components as the "carboxylic (hydrophilic)" and "phenolic (semi-hydrophilic)" component, respectively. It is well known that the aromaticity of humic acid increases with increasing carboxyl group content. Because the aromatic moieties with carboxyl groups were removed from humic acid as components A and B, residual components have hydrophobic properties with an aliphatic structure. In fact, components C and D are characterized by relatively long and even longer aliphatic substituents, respectively. We therefore designate these components as the "semi-aliphatic" and "aliphatic" component, respectively. However, these definitions are based on the frac-
A NEW FRACTIONATION OF SOIL HUMIC ACIDS
335
tionation conditions used and may not be complete enough to apply to all humic acids. Additional separations of many kinds of humic acids are needed to confirm that in all cases each of the four components has the same structural and functional compositions. As an initial step, four types of soil humic acids were prepared from Andisol, Inceptisol, Histosol, and Entisol. Each humic acid was separated into four components by the above stepwise elution method (Fig. 4). Andisol humic acid contained abundant amounts of carboxylic components and moderate amounts of aliphatic components. This agreed with the concept that Andisol
z (3 0
~r
iii L)
Z
k
m
rv
o ~D
t pH 7
t 'oo
t
oot
p i l l 1 H20 50%Ethanol ELUTION VOLUME (cm 3)
Fig. 4. Fractionation of soil humic acids by stepwise elution: a, Andisol; b, Entisol; c, lnceptisol; d, Histosol.
336
K. YONEBAYASKI AND T. HATTORI
humic acid is highly aromatic (Tokudome and Kanno, 1968 ) and has abundant carboxyl groups (Yonebayashi and Hattori, 1988 ). Histosol humic acid contained abundant amounts of aliphatic components and little of carboxylic components. The proportions of the components of Entisol and Inceptisol humic acids were intermediate between Andisol and Histosol humic acids. These results suggest that our analysis of the distribution of these four components leads to a new way of classifying humic acids.
REFERENCES Curtis, M.A., Witt, A.F., Schram, S.B. and Rogers, L.B., 1981. Humic acid fractionation using a nearly linear pH gradient. Anal. Chem., 53:1195-1199. Hatcher, P.G., Rowan, R. and Mattingly, M.A., 1980. ~H and ~3C NMR of marine humic acids. Org. Geochem., 2: 77-85. MacCarthy, P., Peterson, M.J., Malcolm, R.L. and Thurman, E.M., 1979. Separation of humic substances by pH gradient desorption from a hydrophobic resin. Anal. Chem., 51: 20412043. Ohga, K., Aritomi, Y. and Ohtsu, H., 1989. Chromatography with pH-gradient elution of dissolved humic substances in river water. Anal. Sci., 5:215-216. Ravichandran, K., Lewis, J.J., Yin, I.-Hsiung, Koenigbauer, M., Powley, C.R., Shah, P. and Rogers, L.B., 1988. Computer-controlled linear pH gradient for high-performance liquid chromatographic fractionations of aromatic carboxylic acids and of humic and fulvic acids. J. Chromatogr., 439:213-226. Sciacovelli, O., Senesi, N., Solinas, V. and Testini, C., 1977. Spectroscopic studies on soil organic fractions, I. IR and NMR spectra. Soil Biol. Biochem., 9: 287-293. Thurman, E.M. and Malcolm, R.L., 1983. Structural study of humic substances: new approaches and methods. In: R.F. Christman and E.T. Gjessing (Editors), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Michigan, pp. 1-23. Tokudome, S. and Kanno, I., 1968. Nature of the humus of some Japanese soils. Trans. 9th. Int. Congr. Soil Sci., III: 163-173. Wershaw, R.L., 1985. Application of nuclear magnetic resonance spectroscopy for determining functionality in humic substances. In: G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Editors), Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization. Wiley, New York, N.Y., pp. 561-582. Wilson, M.A., 1981. Application of nuclear magnetic resonance spectroscopy to the study of the structure of soil organic matter. J. Soil Sci., 32:167-186. Yonebayashi, K. and Hattori, T., 1985. Nonaqueous titration of functional groups in humic acid. Org. Geochem., 8: 47-54. Yonebayashi, K. and Hattori, T., 1987. Surface active properties of soil humic acids. Sci. Total Environ., 62: 55-64. Yonebayashi, K. and Hattori, T., 1988. Chemical and biological studies on environmental humic acids, I. Composition of elemental and functional groups of humic acids. Soil Sci. Plant Nutr., 34: 571-584. Yonebayashi, K. and Hattori, T., 1989. Chemical and biological studies on environmental humic acids, II. JH-NMR and IR spectra ofhumic acids. Soil Sci. Plant Nutr., 35: 383-392.