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Chemical and spectroscopic properties of two fractions of soil organic matter obtained by electro-ultrafiltration
The Science of the Total Environment, 114 (1992) 37-45 Elsevier Science Publishers B.V., Amsterdam
37
Chemical and spectroscopic properties of two fractions of soil organic matter obtained by electro-ultrafiltration S.A. B u f o a, A. L a t r o f a b a n d A. P a l m a c aInstituto di Chimica Agraria, Italy bDipartimento Farmaco-Chimico, Universit& degli Studi di Bari, via Amendola, 165/a, 1-70126 Bari, Italy cCRSA, Metapontam Agrobios, 1-75010 Metaponto (MT), Italy (Received November 18th, 1990; accepted December 14th, 1990) ABSTRACT A soil sample, collected from vertisol in southern Italy and containing 3.7% of organic carbon, was extracted and fractionated by electro-ultrafiltration using a relatively low electric field and negative pressure. Micropore filters (20 000 Da and 100 000 Da) were selected to obtain two fractions of soil organic matter. E4/E6 ratios and analysis data were in agreement with values of fulvic and humic acids as reported in the literature. FT-IR spectra showed high absorption values in the region of carboxylic acid groups and medium intensity peaks in the region of frequencies ascribed to aliphatic chain absorptions. IH-NMR spectra showed a resolution of signals as good as those previously reported for organic fractions that had been chemically extracted. Key words: electro-ultrafiltration; soil; organic matter; fractionation; FT-IR; NMR
INTRODUCTION In recent years a new t e c h n i q u e for the e x t r a c t i o n a n d f r a c t i o n a t i o n o f soil c o m p o n e n t s , e l e c t r o - u l t r a f i l t r a t i o n ( E U F ) , has been d e v e l o p e d [ 1 ] . T h e m e t h o d consists o f a c o m b i n a t i o n o f electrodialysis and ultrafiltration, a n d has been principally used as a n o n - e q u i l i b r i u m n o n - c h e m i c a l system to extract ions f r o m soil in o r d e r to d e t e r m i n e the e x t r a c t a b l e q u a n t i t y o f nutrients a n d also to c h a r a c t e r i z e the d y n a m i c b e h a v i o r o f soil constitutents [2-6]. W e previously utilized this t e c h n i q u e [7] to extract organic m a t t e r f r o m soil w i t h o u t using reagents which c o u l d cause chemical a n d / o r structural alterations o f native substances [ 8 - 1 3 ] . In o u r first study short e x t r a c t i o n times (5 min) a n d a m i c r o p o r e m e m b r a n e with a 20 000 D a m o l e c u l a r weight 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
38
S.A. B U F O ET AL.
cut-off were used. Most of the chemical and spectroscopic properties of the E U F extract agreed with those o f fulvic acid that had been chemically extracted from the same soil. However, chromatographic data showed that the E U F extract had a smaller molecular size distribution than corresponding chemically-extracted fractions. In the present work we analyzed two organic fractions extracted by E U F using micropore filters with two different molecular weight cut-off values in order to obtain information on organic substances with greater molecular size distributions. The extracted fractions were characterized by ~H-NMR, FT-IR and UV spectroscopy. MATERIALS AND METHODS The investigation was performed on a sample collected from a vertisol of Apenines, a calcareous sandy-clay soil from southern Italy with an organic carbon content of 3.7%. The main characteristics of this soil are reported in Table 1. A sample (5 g) o f air-dried and 1-mm sieved soil was suspended in 50 ml of water in an E U F cell [ 7]. Electrodes were covered with micropore filters
TABLE 1 Main characteristics of examined soil as averages (A) of three replicates. S.E. = standard error Unit
A
S.E.
Sand Silt Clay CaCO3 Organic Carbon N (Kjeldahl) CEC (Gillman) pH (H20)a pH (0.01 M CaCl2)a pH (l M KCI)a E.C. AR0-Ka'b
% % % % % % cmol kg-I
45.8 24.0 30.2 20.8 3.72 0.26 27.7 7.47 7.33 7.36 1.11 0.36
3.20 0.76 3.35 0.73 0.98 0.01 0.89 0.07 0.08 0.08 0.48 0.02
Clay minerals
SV+++; K+++; I-Mi+; (M)
mS cm-I
I, illite; K, kaolinite; M, montmorillonite; Mi, mica; SV, swelling vermiculite. al/2.5 (w/v) suspension. bAR0-K = aK/(aCa + aMg) 1/2 (mol l-t) I/2.
39
SOIL ORGANIC MA'I'rER OBTAINED BY ELECTRO-ULTRAFILTRATION
(20 000 molecular weight cut-off), and the anode and cathode vacuum levels set at -0.6 and -0.4 bars, respectively. An electric field of 44.4 V/cm was applied to the electrodes at 150 mA and 80°C for 30 min and the first anode extract (EUF20) was collected. The micropore filters were then replaced with ones having a 100 000-Da molecular weight cut-off and electroultrafiltration was continued as before for 30 min, and a second anode extract (EUF100) was colletced. The apparatus used was equipped with a sensor to regulate the opening of an electrovalve governing the introduction of water into the E U F cell, so that the volume of the suspension remained constant during the whole extraction period. Each E U F extraction was repeated five times on different samples of the same soil in order to accumulate a large quantity of organic matter. Anode extracts obtained using the same type of micropore filter were pooled. Extracts were freeze-dried and subsequently dissolved in different ways, depending upon the test to be performed. Solubility tests were conducted by dissolving a given quantity of freezedried fraction in distilled water and adjusting the acidity to integer values of pH 4 - 9 with NaOH (0.001-0.1 N). Total acidity and carboxylic group content were determined as described by Schnitzer and Khan [ 14]; the content of phenolic hydroxy groups was calculated by difference (Table 2). Elemental carbon, hydrogen, nitrogen and sulfur were determined using specific LECO pyrolysis gas analyzers (Table 2). Metals in fractions EUF20 and EUF100 were quantified either by flameless atomic absorption or emission ICP spectrometry (Table 3). E4/E6 ratios were claculated from UV absorption spectra recorded on freeze-dried matter dissolved in 0.05 M NaHCO3. FT-IR spectra were recorded as KBr pellets with a TGS detector (16 scans, 1 cm -1 resolution). IH-NMR spectra were recorded on a Varian XR 200 spectrometer using freeze-dried matter
TABLE 2 Elemental and functional group analyses (concentrations calculated for dry matter not containing ash) C
H
O
N
S
Ha
COOH
OH
Ash
(%) (%) EUF20 EUFI00
45.16 53.88
all, total acridity.
(cmol kg -l) 6.01 5.69
42.89 34.24
3.98 4.55
1.96 1.64
10.24 11.77
4.83 5.90
5.41 5.87
3.46 9.40
40
S.A. BUFO ET AL,
TABLE 3 Main metals in the EUF anode extracts, as % (w/w) of freeze-dried material Metal
Detector
Si AI B Ca Cd Co Cr Cu Fe K Mg Mn Mo Na Ni Sn V Zn
(ICP) (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless
ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD)
EUF20
EUFI00
1.221 0.010 0.014 0.156 0.007 0.010 0.015 0.006 0.052 0.026 0.018 0.003 0.002 0.129 -0.016 0.005 0.022
3.434 0.092 0.035 0.224 0.001 0.024 0.061 0.022 0.018 0.031 0.045 0.006 0.031 0.042 0.041 0.099 0.020 0.012
dissolved in D20. H - - O D resonance was set at 4.8 ppm, partially decoupled signal as per Wilson et al. [15]. RESULTS AND DISCUSSION
The initial pH value of the suspens!on core, 7.50, remained constant during EUF extraction whereas the anode extracts EUF20 and EUF100 had pH values of 3.25 and 3.68, respectively (average of five extractions). The total quantity of freeze-dried matter obtained after five extractions was 130 mg for the EUF20 fraction and 220 mg for the EUF 100 fraction. The organic matter extracted constituted 17.8% of the initial organic matter in the soil (6.2% recovered in EUF20, 11.6% recovered in EUF100). The freeze-dried extracts were soluble over the pH range 4-9. The EriE6 ratios were 6.9 and 5.0 for EUF20 and E U F 100, respectively. Figures 1 and 2 show FT-IR spectra of the EUF extracts obtained with removal of silica absorptions. The EUF100 extract showed a stronger absorption than EUF20 in the aliphatic C - - H stretching region (3000-2920 cm-l), the aliphatic C - - H deformation region (1420-1320 cm-l), and the
41
SOIL O R G A N I C M A T T E R O B T A I N E D BY E L E C T R O . U L T R A F I L T R A T I O N
CH2 deformation region (1270-1220 cm-~). The band ascribed to the stretching of C----O in COOH (1730-1720 cm -1) is strong in both the EUF100 and EUF20 extracts, while antisymmetric stretching of C---O in COO(1650-1630 cm -1) is weak in both cases. The ~H-NMR spectra of the E U F fractions (Figs. 3 and 4), show chemical shifts which can be ascribed as follows: -
- 0 . 2 ppm, due to groups of water soluble low molecular weight organometallic complexes [ 16]; this signal is absent from the spectrum of the EUF100 fraction.
-
I
100
1
1
I
I
ZT 90
180
70
60
I
50
40
2U
t0
0 5250
I 4000
.l 3200
[
I
2400 2000 CM-J,
I
1
I
1600
t200
800
400
Fig. 1. FT-IR spectrum of EUF20 fraction. (Silica absorptions have been removed.)
42
S.A. BUFO ET AL.
tOO
I
1
'l
ZT 90
80
70
60
50
40
30
20
10
0 5250
Fig.
--
--
--
--
2.
I
I
4000
3EO0
I
I
2400 EO00 CM~!
I
t600
I
lEO0
800
400
FT-IR spectrum of EUFI00 fraction. (Silica absorptions have been removed.)
- 0 . 8 - 1 . 0 ppm, due to methyl, isopropyl, and tert-butyl groups of paraffinic chains. - 1.4 ppm (d, sharp) due to terminal methyl groups probably linked to - - C H O H - m o i e t i e s and coupled to a sharp quartet at - 4 . 4 ppm ( - C H - ) . These signals are most clearly seen in the spectrum of the EUF100 fraction. - 1.8-2.2 ppm, due to methylene groups bonded to free or esterified carboxylic groups. - 2 . 3 - 3 . 2 ppm, due to methyl groups bonded to sulfur and nitrogen
SOIL ORGANIC MA'I'I'ER OBTAINED BY ELECTRO-ULTRAFILTRATION
8
7
e
s
43
I
Fig. 3. IH-NMR spectrum of EUF20 fraction.
-
-
atoms and to phenyl rings and methylene and methine groups linked to nitrogen atoms. - 3.3-4.5 ppm, due to methyl and methylene groups linked to oxygen or other electron-withdrawing groups and to methines of carbons linked to oxygen.
Fig. 4. IH-NMR spectrum of EUFI00 fraction.
44
S.A. BUFO ET AL.
- - - 6.8-8.4 ppm (low broad signals, EUF100) and - 8.2 ppm (EUF20), due to protons of aromatic or heteroaromatic rings. The difference is probably due to the different size of aromatic and/or heteroaromatic units and the presence of more structured molecules [ 17,18 ] in the EUF100 fraction. The more intense signal in the aromatic proton region of EUF20 spectrum is indicative of a greater number of aromatic protons, perhaps due to less substituted aromatic rings [19]. The well defined singlet at 8.2 ppm (EUF20) may be due to the presence of highly equivalent or isochronous aromatic protons as well as to H/D exchange on aromatic ranges [ 18-22]. The decreased intensity of aromatic proton signals in the EUFI00 spectrum may be attributed to the full substitution of the aromatic rings [20]. Preliminary 13C-NMR spectra recorded on these samples (unpublished data) confirm the presence of aromatic compounds in both EUF fractions. The foregoing data indicate that electrochemical extraction in conjunction with ultrafiltration can be successfully used for size fractionation of soil organic matter with an acceptable efficiency and purity. Chemical analyses and spectroscopic data suggest that organic matter extracted by EUF is richer in aliphatic components than aromatic ones and contains a relatively high proportion of oxygen-bearing functional groups. The smaller size fraction, EUF20, appeared to contain organometallic complexes and seems to be richer in less substituted aromatic products. With five 30-min extractions, it was possible to obtain the amount of organic matter required to perform the chemical and structural characterizations reported herein but it was not possible to study the chemical reactivity of the functional groups. For this reason we are currently designing new equipment which will provide greater quantities of organic matter in a shorter time. REFERENCES 1 K. N6meth, The availability of nutrients in the soil as determined by electroultrafiltration (EUF). Adv. Agron., 31 (1979) 155-188. 2 H. Grimme, The use of rate equations for a quantitative description of potassium desorption in an external electric field (electro-ultrafiltration). Z. Pflanzenern/ihr. Bodenkd., 142 (1979) 57-68. 3 K. N6meth, Application of electro-ultrafiltration in agricultural production. Plant Soil, 64 (1982) 1-138. 4 A. Buondonno, D. FeUeca, S.A. Bufo, M.D.R. Pizzigallo and C. Testini, Comparison between electro-ultrafiltration and extraction methods for the determination of potassium fractions in some soils of southern Italy. Commun. Soil Sci. Plant Anal., 19 (1988) 239-258. 5 M.D.R. Pizzigailo, S.A. Bufo and A. Buondonno, Effects of fertilizer additions on
SOIL ORGANIC MATTER OBTAINED BY ELECTRO-ULTRAFILTRATION
6
7
8 9 10 11 12 13
14 15 16 17 18 19
20
21 22
45
phosphorus and potassium availability in Italian vertisol as determined by electroultrafiltration. Commun. Soil Sci. Plant Anal., 21 (1990) 1187-1198. S.A. Bufo, M.D.R. Pizzigallo and A. Buondonno, Mobility of potassium and phosphorus in soil as determined by electro-ultrafiltration. Commun. Soil Sci. Plant Anal., 21 (1990) in press. S.A. Bufo, M.D.R. Pizzigallo, F. Matteucci and L. Scrano, Preliminary characterization of soil organic matter extracted by electro-ultrafiltration. Sci. Total Environ., 81-82 (1989) 111-120. J.M. Bremner, Some observations on the oxidation of soil organic matter in the presence of alkali. J. Soil Sci., 1 (1950) 198-204. L.N. Aleksandrova, The use of sodium pyrophosphate for separating free humus substances and their organomineral compounds from soil. Pochvovednie, 2 (1960) 90-97. J.F. Dormaar, M. Metche and F. Jacquin, Extraction and purification of humic acids from a rendzina Ah and a podzol Bh horizon. Soil Biol. Biochem., 2 (1970) 285-293. R.S. Swift and A.M. Posner, Autooxidation of humic acids under alkaline conditions. J. Soil Sci., 23 (1972) 381-393. F.J. Stevenson, Humus Chemistry: Genesis, Composition, Reactions, John Wiley & Sons, New York, 1982, pp. 26-54. A. Piccolo, Characteristics of soil humic extracts obtained by some organic and inorganic solvents and purified by hydrochloric-hydrofluoric acid treatment. Soil Sci., 146 (1988) 418-426. M. Schnitzer and S.U. Khan, Humic Substances in the Environment, Marcel Dekker, New York, 1972, pp. 29-54. M.A. Wilson, P.J. Collin antl K.R. Tate, Proton nuclear magnetic resonance study of a soil humic acid. J. Soil Sci., 34 (1983) 297-304. M. Schnitzer and H. Kerndorff, Reactions of fulvic acid with metal ions. Water, Air Soil Pollut., 15 (1981) 97-108. M. Schnitzer, Aromaticity of soil fulvic acid. Nature, 316 (1985) 658. O. Sciacovelli, N. Senesi, V. Solinas and C. Testini, Spectroscopic studies on soil organic fractions. I. IR and NMR spectra. Soil Biol. Biochem., 9 (1977) 287-293. P. Ruggiero, O. Sciacovelli, C. Testini and F.S. Interesse, Spectroscopic studies on soil organic fractions. II. IR and proton NMR spectra of methylated and unmethylated fulvic acids. Geochim. Cosmochim. Acta, 42 (1978) 411-416. P. Ruggiero, F.S. Interesse and O. Sciacovelli, Proton and carbon-13 NMR studies on the importance of aromatic structures in fulvic and humic acids. Geochim. Cosmochim. Acta, 43 (1979) 1771-1775. P. Ruggiero, F.S. Interesse and O. Sciacovelli, Proton NMR evidence of exchangeable aromatic protons in fulvic and humic acids. Soil Biol. Biochem., 12 (1980) 297-299. P. Ruggiero, F.S. lnteresse, L. Cassidei and O. Sciacovelli, Proton NMR spectra of humic and fulvic acids and their peracetic oxidation products. Geochim. Cosmochim. Acta, 44 (1980) 603-609.
37
Chemical and spectroscopic properties of two fractions of soil organic matter obtained by electro-ultrafiltration S.A. B u f o a, A. L a t r o f a b a n d A. P a l m a c aInstituto di Chimica Agraria, Italy bDipartimento Farmaco-Chimico, Universit& degli Studi di Bari, via Amendola, 165/a, 1-70126 Bari, Italy cCRSA, Metapontam Agrobios, 1-75010 Metaponto (MT), Italy (Received November 18th, 1990; accepted December 14th, 1990) ABSTRACT A soil sample, collected from vertisol in southern Italy and containing 3.7% of organic carbon, was extracted and fractionated by electro-ultrafiltration using a relatively low electric field and negative pressure. Micropore filters (20 000 Da and 100 000 Da) were selected to obtain two fractions of soil organic matter. E4/E6 ratios and analysis data were in agreement with values of fulvic and humic acids as reported in the literature. FT-IR spectra showed high absorption values in the region of carboxylic acid groups and medium intensity peaks in the region of frequencies ascribed to aliphatic chain absorptions. IH-NMR spectra showed a resolution of signals as good as those previously reported for organic fractions that had been chemically extracted. Key words: electro-ultrafiltration; soil; organic matter; fractionation; FT-IR; NMR
INTRODUCTION In recent years a new t e c h n i q u e for the e x t r a c t i o n a n d f r a c t i o n a t i o n o f soil c o m p o n e n t s , e l e c t r o - u l t r a f i l t r a t i o n ( E U F ) , has been d e v e l o p e d [ 1 ] . T h e m e t h o d consists o f a c o m b i n a t i o n o f electrodialysis and ultrafiltration, a n d has been principally used as a n o n - e q u i l i b r i u m n o n - c h e m i c a l system to extract ions f r o m soil in o r d e r to d e t e r m i n e the e x t r a c t a b l e q u a n t i t y o f nutrients a n d also to c h a r a c t e r i z e the d y n a m i c b e h a v i o r o f soil constitutents [2-6]. W e previously utilized this t e c h n i q u e [7] to extract organic m a t t e r f r o m soil w i t h o u t using reagents which c o u l d cause chemical a n d / o r structural alterations o f native substances [ 8 - 1 3 ] . In o u r first study short e x t r a c t i o n times (5 min) a n d a m i c r o p o r e m e m b r a n e with a 20 000 D a m o l e c u l a r weight 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
38
S.A. B U F O ET AL.
cut-off were used. Most of the chemical and spectroscopic properties of the E U F extract agreed with those o f fulvic acid that had been chemically extracted from the same soil. However, chromatographic data showed that the E U F extract had a smaller molecular size distribution than corresponding chemically-extracted fractions. In the present work we analyzed two organic fractions extracted by E U F using micropore filters with two different molecular weight cut-off values in order to obtain information on organic substances with greater molecular size distributions. The extracted fractions were characterized by ~H-NMR, FT-IR and UV spectroscopy. MATERIALS AND METHODS The investigation was performed on a sample collected from a vertisol of Apenines, a calcareous sandy-clay soil from southern Italy with an organic carbon content of 3.7%. The main characteristics of this soil are reported in Table 1. A sample (5 g) o f air-dried and 1-mm sieved soil was suspended in 50 ml of water in an E U F cell [ 7]. Electrodes were covered with micropore filters
TABLE 1 Main characteristics of examined soil as averages (A) of three replicates. S.E. = standard error Unit
A
S.E.
Sand Silt Clay CaCO3 Organic Carbon N (Kjeldahl) CEC (Gillman) pH (H20)a pH (0.01 M CaCl2)a pH (l M KCI)a E.C. AR0-Ka'b
% % % % % % cmol kg-I
45.8 24.0 30.2 20.8 3.72 0.26 27.7 7.47 7.33 7.36 1.11 0.36
3.20 0.76 3.35 0.73 0.98 0.01 0.89 0.07 0.08 0.08 0.48 0.02
Clay minerals
SV+++; K+++; I-Mi+; (M)
mS cm-I
I, illite; K, kaolinite; M, montmorillonite; Mi, mica; SV, swelling vermiculite. al/2.5 (w/v) suspension. bAR0-K = aK/(aCa + aMg) 1/2 (mol l-t) I/2.
39
SOIL ORGANIC MA'I'rER OBTAINED BY ELECTRO-ULTRAFILTRATION
(20 000 molecular weight cut-off), and the anode and cathode vacuum levels set at -0.6 and -0.4 bars, respectively. An electric field of 44.4 V/cm was applied to the electrodes at 150 mA and 80°C for 30 min and the first anode extract (EUF20) was collected. The micropore filters were then replaced with ones having a 100 000-Da molecular weight cut-off and electroultrafiltration was continued as before for 30 min, and a second anode extract (EUF100) was colletced. The apparatus used was equipped with a sensor to regulate the opening of an electrovalve governing the introduction of water into the E U F cell, so that the volume of the suspension remained constant during the whole extraction period. Each E U F extraction was repeated five times on different samples of the same soil in order to accumulate a large quantity of organic matter. Anode extracts obtained using the same type of micropore filter were pooled. Extracts were freeze-dried and subsequently dissolved in different ways, depending upon the test to be performed. Solubility tests were conducted by dissolving a given quantity of freezedried fraction in distilled water and adjusting the acidity to integer values of pH 4 - 9 with NaOH (0.001-0.1 N). Total acidity and carboxylic group content were determined as described by Schnitzer and Khan [ 14]; the content of phenolic hydroxy groups was calculated by difference (Table 2). Elemental carbon, hydrogen, nitrogen and sulfur were determined using specific LECO pyrolysis gas analyzers (Table 2). Metals in fractions EUF20 and EUF100 were quantified either by flameless atomic absorption or emission ICP spectrometry (Table 3). E4/E6 ratios were claculated from UV absorption spectra recorded on freeze-dried matter dissolved in 0.05 M NaHCO3. FT-IR spectra were recorded as KBr pellets with a TGS detector (16 scans, 1 cm -1 resolution). IH-NMR spectra were recorded on a Varian XR 200 spectrometer using freeze-dried matter
TABLE 2 Elemental and functional group analyses (concentrations calculated for dry matter not containing ash) C
H
O
N
S
Ha
COOH
OH
Ash
(%) (%) EUF20 EUFI00
45.16 53.88
all, total acridity.
(cmol kg -l) 6.01 5.69
42.89 34.24
3.98 4.55
1.96 1.64
10.24 11.77
4.83 5.90
5.41 5.87
3.46 9.40
40
S.A. BUFO ET AL,
TABLE 3 Main metals in the EUF anode extracts, as % (w/w) of freeze-dried material Metal
Detector
Si AI B Ca Cd Co Cr Cu Fe K Mg Mn Mo Na Ni Sn V Zn
(ICP) (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless (flameless
ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD) ECD)
EUF20
EUFI00
1.221 0.010 0.014 0.156 0.007 0.010 0.015 0.006 0.052 0.026 0.018 0.003 0.002 0.129 -0.016 0.005 0.022
3.434 0.092 0.035 0.224 0.001 0.024 0.061 0.022 0.018 0.031 0.045 0.006 0.031 0.042 0.041 0.099 0.020 0.012
dissolved in D20. H - - O D resonance was set at 4.8 ppm, partially decoupled signal as per Wilson et al. [15]. RESULTS AND DISCUSSION
The initial pH value of the suspens!on core, 7.50, remained constant during EUF extraction whereas the anode extracts EUF20 and EUF100 had pH values of 3.25 and 3.68, respectively (average of five extractions). The total quantity of freeze-dried matter obtained after five extractions was 130 mg for the EUF20 fraction and 220 mg for the EUF 100 fraction. The organic matter extracted constituted 17.8% of the initial organic matter in the soil (6.2% recovered in EUF20, 11.6% recovered in EUF100). The freeze-dried extracts were soluble over the pH range 4-9. The EriE6 ratios were 6.9 and 5.0 for EUF20 and E U F 100, respectively. Figures 1 and 2 show FT-IR spectra of the EUF extracts obtained with removal of silica absorptions. The EUF100 extract showed a stronger absorption than EUF20 in the aliphatic C - - H stretching region (3000-2920 cm-l), the aliphatic C - - H deformation region (1420-1320 cm-l), and the
41
SOIL O R G A N I C M A T T E R O B T A I N E D BY E L E C T R O . U L T R A F I L T R A T I O N
CH2 deformation region (1270-1220 cm-~). The band ascribed to the stretching of C----O in COOH (1730-1720 cm -1) is strong in both the EUF100 and EUF20 extracts, while antisymmetric stretching of C---O in COO(1650-1630 cm -1) is weak in both cases. The ~H-NMR spectra of the E U F fractions (Figs. 3 and 4), show chemical shifts which can be ascribed as follows: -
- 0 . 2 ppm, due to groups of water soluble low molecular weight organometallic complexes [ 16]; this signal is absent from the spectrum of the EUF100 fraction.
-
I
100
1
1
I
I
ZT 90
180
70
60
I
50
40
2U
t0
0 5250
I 4000
.l 3200
[
I
2400 2000 CM-J,
I
1
I
1600
t200
800
400
Fig. 1. FT-IR spectrum of EUF20 fraction. (Silica absorptions have been removed.)
42
S.A. BUFO ET AL.
tOO
I
1
'l
ZT 90
80
70
60
50
40
30
20
10
0 5250
Fig.
--
--
--
--
2.
I
I
4000
3EO0
I
I
2400 EO00 CM~!
I
t600
I
lEO0
800
400
FT-IR spectrum of EUFI00 fraction. (Silica absorptions have been removed.)
- 0 . 8 - 1 . 0 ppm, due to methyl, isopropyl, and tert-butyl groups of paraffinic chains. - 1.4 ppm (d, sharp) due to terminal methyl groups probably linked to - - C H O H - m o i e t i e s and coupled to a sharp quartet at - 4 . 4 ppm ( - C H - ) . These signals are most clearly seen in the spectrum of the EUF100 fraction. - 1.8-2.2 ppm, due to methylene groups bonded to free or esterified carboxylic groups. - 2 . 3 - 3 . 2 ppm, due to methyl groups bonded to sulfur and nitrogen
SOIL ORGANIC MA'I'I'ER OBTAINED BY ELECTRO-ULTRAFILTRATION
8
7
e
s
43
I
Fig. 3. IH-NMR spectrum of EUF20 fraction.
-
-
atoms and to phenyl rings and methylene and methine groups linked to nitrogen atoms. - 3.3-4.5 ppm, due to methyl and methylene groups linked to oxygen or other electron-withdrawing groups and to methines of carbons linked to oxygen.
Fig. 4. IH-NMR spectrum of EUFI00 fraction.
44
S.A. BUFO ET AL.
- - - 6.8-8.4 ppm (low broad signals, EUF100) and - 8.2 ppm (EUF20), due to protons of aromatic or heteroaromatic rings. The difference is probably due to the different size of aromatic and/or heteroaromatic units and the presence of more structured molecules [ 17,18 ] in the EUF100 fraction. The more intense signal in the aromatic proton region of EUF20 spectrum is indicative of a greater number of aromatic protons, perhaps due to less substituted aromatic rings [19]. The well defined singlet at 8.2 ppm (EUF20) may be due to the presence of highly equivalent or isochronous aromatic protons as well as to H/D exchange on aromatic ranges [ 18-22]. The decreased intensity of aromatic proton signals in the EUFI00 spectrum may be attributed to the full substitution of the aromatic rings [20]. Preliminary 13C-NMR spectra recorded on these samples (unpublished data) confirm the presence of aromatic compounds in both EUF fractions. The foregoing data indicate that electrochemical extraction in conjunction with ultrafiltration can be successfully used for size fractionation of soil organic matter with an acceptable efficiency and purity. Chemical analyses and spectroscopic data suggest that organic matter extracted by EUF is richer in aliphatic components than aromatic ones and contains a relatively high proportion of oxygen-bearing functional groups. The smaller size fraction, EUF20, appeared to contain organometallic complexes and seems to be richer in less substituted aromatic products. With five 30-min extractions, it was possible to obtain the amount of organic matter required to perform the chemical and structural characterizations reported herein but it was not possible to study the chemical reactivity of the functional groups. For this reason we are currently designing new equipment which will provide greater quantities of organic matter in a shorter time. REFERENCES 1 K. N6meth, The availability of nutrients in the soil as determined by electroultrafiltration (EUF). Adv. Agron., 31 (1979) 155-188. 2 H. Grimme, The use of rate equations for a quantitative description of potassium desorption in an external electric field (electro-ultrafiltration). Z. Pflanzenern/ihr. Bodenkd., 142 (1979) 57-68. 3 K. N6meth, Application of electro-ultrafiltration in agricultural production. Plant Soil, 64 (1982) 1-138. 4 A. Buondonno, D. FeUeca, S.A. Bufo, M.D.R. Pizzigallo and C. Testini, Comparison between electro-ultrafiltration and extraction methods for the determination of potassium fractions in some soils of southern Italy. Commun. Soil Sci. Plant Anal., 19 (1988) 239-258. 5 M.D.R. Pizzigailo, S.A. Bufo and A. Buondonno, Effects of fertilizer additions on
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