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Viscosimetric and chromatographic studies of soil humic acids
Environment International, Vol. 22, No. 5, pp. 485-488, 19% Copyright 019% Elsevier Science Ltd Printed in the USA. All rights reserved 0160-4120/96 S15.00+.00
Pergamon
VISCOSIMETRIC AND CHROMATOGRAPHIC STUDIES OF SOIL HUMIC ACIDS S.S. Gonet and K. Wegner Department of Soil Chemistry, University of Technology and Agriculture, Bydgoszcz, Poland
EI 9507-3 78 M (Received I4 June 1996; accepted 23 June 1996)
The aim of this study was to obtain information on molecular parameters of humic acids (HA) extracted from soils under various fertilization treatments. HAS were extracted from a grey-brown podzolic soil and chernozem under two long-term static field experiments. The fertilization variants were control (without fertilization) and manuring. Viscosimetric and gel permeation chromatography determinations were conducted in NaOH (pH = 10.5) and NaCl (pH = 7.0) solutions. A few models were used for calculations of the shapes of the HA molecules. The results confirmed the properties of HAs typical for high-molecular polyelectrolytes. The size of their molecules depended on the pH and the concentration of HAs in solution. As pH increased, both their mean molecular weight and spheric parameters increased. Fertilization modified molecular parameters of HAS. HAS extracted from the soils under mineral fertilization had molecules of similar parameters. HAS from manured soils had lower mean molecular weight associated with a supply of fresh organic matter.
INTRODUCTION
Visser (1985), studying the viscosity phenomena, stated that variations in properties of humic acids (HAS) in solutions of varied pH and ionic strength are characteristic of ionic polymers. Some authors noted that the site and anthropogenic factors can modify their molecular weight (Dziadowiec 1992; Gonet and Wegner 1994; Kowalinski et al. 1980; Kuszelewski 1972). Results of indirect methods have been used often in studies of HAs to assess their approximate molecular weight. Among such measures, the absorbance ratio was suggested by Chen et al. (1977), who found that its value is proportional to molecular weight. The studies of the viscosity of humus substances solutions use the Fuoss-Cathers (1949) equation (Visser 1985):
Humus substances are naturally developed mixtures of organic compounds, representing various steps of transformation of plant and animal residues. The agricultural and ecological significance of humus substances results not only from their abundance in soils, but also from their properties, which are determined by their molecular structure. Many studies have placed a special emphasis on the chemical composition and structure of these molecules. At the moment, no comprehensive theory describing their structure is available. Some results indicate their globular nature, while others suggest rather a flexible model (Chen and Schnitzer 1976; Gosh and Schnitzer 1980; Kumada and Kawamura 1968; Schnitzer and Skinner 1968; Visser 1985). It is generally accepted that parameters of humus substances molecules are a function of concentration, pH, and ionic strength of the solution. Chen and Schnitzer (1976), Gosh and Schnitzer (1980), and 485
S.S. Gonet and K. Wegner
486
where, rlredis the reduced viscosity; c is the concentration of HA in the solution; and, A, B are constants, and a general equation for polymers not bearing charges (non-ionic) (McBain 1950). [rl] = lim
the intrinsic viscosity [rl] in solutions of pH 7.0 were assessed from the dependence suggested for charge-free polymers, by the extrapolation of the reduced viscosity to zero concentration (Eq. 2). The mean molecular weight of HAS was calculated from the Mark-Houwink equation (Eq. 3). The axial ratio a/b and the values of the axis a (longer) and the axis b (shorter) of HA were found from the Simha (McBain 1950) equation:
c-0
Ill1 = where, [q] is the intrinsic viscosity. On the basis of viscometric assays, Relan (1984) suggested calculation of mean viscometric molecular weight from the Mark-Houwink equation: [q] = K - M,” (3) where, K, n are constants, for HAS: K = 7.85 - 10e4,n = 0.67;
e viscometric molecular weight. MATERIALS AND METHODS HA preparations were extracted from a grey-brown podzolic soil and chemozem under two long-term static field fertilization experiments. The samples were collected from different variants of the experiment: without fertlization and with manuring. Manure was applied at the rate 30 Mg/ha for three years. Soil was sampled from the arable layer (O-20 cm) and prepared for analysis routinely according to the manual for soil science experiments. Established analytical methods were used (MAFF 1989). The HA samples of all the fertilization variants were extracted with OSM NaOH, precipitated at pH 2.0 with 0.25M HzSO,, and than purified with the mixture HE-HCI, according to the Schnitzer and Skinner (1968) method. The ash content in HA preparations was 20-50 mg/g, taken into account for analysis. Viscometric assessments were conducted in two separate systems: for the pH = 10.5 in NaOH solution, and in the presence of 0.1M NaCl at pH = 7.0 in an Ubbelohde viscometer calibrated for the flow of 3 cm3 of Hz0 per 200 s. The measurements were run in three replicates at the temperature 25°C f 0.01 “C. Concentrations of the HA solutions ranged 2.5 - 10 g/L. The HAS intrinsic viscosity [rl] in solutions of pH 10.5 was calculated from the Fuoss-Cathers formula (Eq. l), which allows the intrinsic viscosity to be estimated by plotting l/rhed against 6. The values of
@la)’ 15(ln2b/a-3/2)
+
3(b/a)’ 15(ln2b/l-l/2)
+_!A_ 15
(4) HAS of particular combinations in the solutions of pH 10.5 and 7.0 were fractionated on a chromatographic column filled with Fractogel TSK HW 40s (Merck). On the base of the chromatograms obtained in the system EW = f (K,,), (where E, = absorbance at 600 mu, K, = partition chromatography constant), eluates were collected for each recorded peak, corresponding to HA fractions of specific molecular weight. Optical density of the fractions was measured at 465 nm (E,,,) and 665 nm (E&. The results were compared with the corresponding values obtained for 200 mg/L HA solutions before chromatographic separation, and absorbance coefficients were calculated: E4,6(E,,:E,d for HA sofutions before chromatography, and Q4,b(ratio of absorbances at 465 nm at 665 nm - Q465:Q665)for fractions of various molecular weight. RESULTS AND DISCUSION The mean viscosic molecular weights (E, ) calculated
from the Mark-Houvink equation are given in Table 1. These values were higher in alkali solutions and lower in pH = 7.0 solutions. Higher values of z” were noted in both cases for the HA preparations for chemozem. In general, manuring of soils caused a decrease of mean molecular weight of HA in both kinds of soil. The results from Table 1 show that the absorbance ratio E4,6 is inversely proportional to HA molecular weight, both for assays made for entire HA samples (E4,&and for the fractions of various molecular weight separated chromatographically. The fractions abbreviated F I, F II, and F III indicate high molecular weight fraction (&” = 0), medium molecular weight fraction (K, = 0.3), and low molecular weight fraction (I
Viscosimetric and chromatographic studies of soil humic acids
487
Table I. Molecular parameters and optical properties of HAs. Fertilizationtreatment Parameter
Control (without fertilization) pH = 7.0
Manure
pH = 10.5
pH = 7.0.
pH = 10.5
grey-brown podzolic soil
a/b” a [nml b Inml :;Fl Q416
F I*
44,6
F 1111
0.099 f 0.002 1380 12.6 3.2 0.26 3.32 5.08
0.278 f 0.006 6370 19.3 7.0 0.36 4.36 5.10
0.096 f 0.002 1290 12.5 3.1 0.25 3.41 5.63
0.255 f 0.006 5610 18.6 6.5 0.35 4.47 5.87
3.75 4.06
5.96 7.50
3.91 3.94
5.97 7.71
chemozem
a/b” a @I b bml :;;F I Q4l6 F II 4~6 F IIII
0.102 f 0.002 1430 12.6 3.2 0.26 2.83 4.48
0.335 f 0.007 8800 21.0 8.3 0.39 3.74 3.88
0.100 f 0.002 1390 12.5 3.2 0.25 2.80 4.29
0.303 f 0.007 7240 19.9 7.5 0.38 3.78 3.85
3.06 4.32
3.87 4.29
3.20 4.52
3.60 5.40
The values of intrinsic viscosity [q] estimated from the plots were used for calculations of spheric parameters. The axial ratio a/b, as well as both separate values, were calculated for HAS. The results are given in Table 1. The axial ratio a/b values, calculated for the low range of sample concentrations in solution of pH 10.5, for HA preparations from permanently nonfertilized objects, were higher for chernozem preparations than those for grey-brown podzolic soil. Similar differences were noted for the measured parameters: a = longer axis, and b = shorter axis of the molecules. Manuring of soil caused a decrease in the values of the spheric parameters (a/b, a, and b) of HAS from both soils, but higher values were observed for the HA samples from chernozem. When neutral sa4twas present in solution (pH = 7.0), the values of the axial ratio a/b, axis a, and axis b were practically the same. This finding suggested that in neutral medium, the molecules of HAS in all combinations had similar spheric parameters. An increase in the values of qred and spheric parameters in pH = 10.5 solutions at a low sample concen-
tration reflects an expansion of HA molecules caused by a counter-action of charged groups, which dissociate during dilution. By limiting molecular interactions, the addition of salt may cause the HA molecules to fold and thus resemble neutral polymers. If the model conditions of this experiment were used for estimates made for specific soil conditions (neutral reaction), a conclusion can be drawn that, under conditions of high soil moisture (a decrease of HA concentration), better expanded HA molecules have higher specific area than spheric polyelectrolytes of drier soils. Also, HAS for chermozem and from permanently nonfertilized soil can participate more actively in the processes of ion exchange and regulation of buffering properties of soil. CONCLUSIONS
1) HAS of permanently non-fertilized soils have the highest average molecular weight. Manuring causes a decrease of the HA molecular weight. The average molecular weight is inversely proportional to the values of the E,,6 parameter determined in 0.1 M NaOH.
488
2) Mean viscosic molecular weights of the HAS
measured in the presence of 0.1 M NaCI, pH 7.0, were threefold lower than those measured in NaOH solutions of pH 10.5. Because of interaction of humic charges at higher pH values, the HAS create agglomerates, resulting in higher viscosity of the solutions. At the neutral reaction of solution containing salts, HAS perform as neutral polymers.
REFERENCES Chen, Y.; Schnitzer, M. Viscosity measurements on soil humic substances. Soil Sci. Sot. Am. J. 40: 866-872; 1976. Chen, Y.; Senesi, N.; Schnitzer, M. Information provided on humic substances by E, ratios. Soil Sci. Sot. Am. J. 41: 352-358; 1977. Dziadowiec, H. Ekologiczna rola pr6chnicy glebowej. (Ecological role of soil humus). Zesz. Probl. Post. Nauk Rol. 4 11: 268-282; 1992. Fuoss, R.M.; Cathers, G.I. Polyelectrolytes III. Viscosities of nbutyl bromide addition compounds of 4-vinylpyridinestyrene copolymers in nitromethane-dioxane mixtures. J. Polym. Sci. 4: 97-120; 1949. Gosh, K.; Schnitzer, M. Macromolecular structures of humic substances. Soil Sci. 129, 5: 266-276; 1980.
S.S. Gonet and K. Wegner
Gonet, S.S.; Wegner, K. The effect of mineral and organic fertilization on properties of soil humic acids. In: Senesi, N.; Miano, T., eds. Humic substances in the global environment and implications on human health. Amsterdam: Elsevier; 1994: 607612. Kowalinski, S.; Drozd, J.; Licznar, M. Sklad frakcyjny zwiazkow pr6chnicznych pod roslinnoscia uprawiana w monokulturze i zmianowaniu. (Fractional composition of humus in soils under different crop rotation systems.) Zesz. Nauk. ART Olsztyn 29: 23-3 1; 1980. Kumada, K.; Kawamura, Y. Viscosimetric characteristic of humic acid. Soil Sci. Plant Nutrition 14, 5: 190-197; 1968. Kuszelewski, L. Wplyw nawozenia organicznego i mineralnego na zawartosc i niekt6re wskazniki jakosciowe substancji prochnicowych gleby. (The effect of mineral and organic fertilization on quantity and quality of soil humic substances.) Roczn. Nauk Roln. 98, 1: 7-27; 1972. MAFF (Ministry of Agriculture, Fisheries and Food). A manual of the analytical methods used by the Agricultural Development and Advisory Service. Third edition. Ref. Book 427. London; 1989. McBain, J.W. Colloid science. Boston, MA: D.C. Health & Company; 1950: 151. Relan, P.S.; Girdhar, K.K.; Khanna, S.S. Molecular configuration of composts humic acid by viscometric studies. Plant and Soil 8 1: 203-208; 1984. Schnitzer, M.; Skinner, S.I.M. Alkali versus acid extraction of soil organic matter. Soil Sci. 105,6: 392-396; 1968. Visser, S.A. Viscosimetric studies on molecular weight fractions of fulvic and humic acids of aquatic, terrestial and microbial origin. Plant and Soil 87: 209-221; 1985.
Pergamon
VISCOSIMETRIC AND CHROMATOGRAPHIC STUDIES OF SOIL HUMIC ACIDS S.S. Gonet and K. Wegner Department of Soil Chemistry, University of Technology and Agriculture, Bydgoszcz, Poland
EI 9507-3 78 M (Received I4 June 1996; accepted 23 June 1996)
The aim of this study was to obtain information on molecular parameters of humic acids (HA) extracted from soils under various fertilization treatments. HAS were extracted from a grey-brown podzolic soil and chernozem under two long-term static field experiments. The fertilization variants were control (without fertilization) and manuring. Viscosimetric and gel permeation chromatography determinations were conducted in NaOH (pH = 10.5) and NaCl (pH = 7.0) solutions. A few models were used for calculations of the shapes of the HA molecules. The results confirmed the properties of HAs typical for high-molecular polyelectrolytes. The size of their molecules depended on the pH and the concentration of HAs in solution. As pH increased, both their mean molecular weight and spheric parameters increased. Fertilization modified molecular parameters of HAS. HAS extracted from the soils under mineral fertilization had molecules of similar parameters. HAS from manured soils had lower mean molecular weight associated with a supply of fresh organic matter.
INTRODUCTION
Visser (1985), studying the viscosity phenomena, stated that variations in properties of humic acids (HAS) in solutions of varied pH and ionic strength are characteristic of ionic polymers. Some authors noted that the site and anthropogenic factors can modify their molecular weight (Dziadowiec 1992; Gonet and Wegner 1994; Kowalinski et al. 1980; Kuszelewski 1972). Results of indirect methods have been used often in studies of HAs to assess their approximate molecular weight. Among such measures, the absorbance ratio was suggested by Chen et al. (1977), who found that its value is proportional to molecular weight. The studies of the viscosity of humus substances solutions use the Fuoss-Cathers (1949) equation (Visser 1985):
Humus substances are naturally developed mixtures of organic compounds, representing various steps of transformation of plant and animal residues. The agricultural and ecological significance of humus substances results not only from their abundance in soils, but also from their properties, which are determined by their molecular structure. Many studies have placed a special emphasis on the chemical composition and structure of these molecules. At the moment, no comprehensive theory describing their structure is available. Some results indicate their globular nature, while others suggest rather a flexible model (Chen and Schnitzer 1976; Gosh and Schnitzer 1980; Kumada and Kawamura 1968; Schnitzer and Skinner 1968; Visser 1985). It is generally accepted that parameters of humus substances molecules are a function of concentration, pH, and ionic strength of the solution. Chen and Schnitzer (1976), Gosh and Schnitzer (1980), and 485
S.S. Gonet and K. Wegner
486
where, rlredis the reduced viscosity; c is the concentration of HA in the solution; and, A, B are constants, and a general equation for polymers not bearing charges (non-ionic) (McBain 1950). [rl] = lim
the intrinsic viscosity [rl] in solutions of pH 7.0 were assessed from the dependence suggested for charge-free polymers, by the extrapolation of the reduced viscosity to zero concentration (Eq. 2). The mean molecular weight of HAS was calculated from the Mark-Houwink equation (Eq. 3). The axial ratio a/b and the values of the axis a (longer) and the axis b (shorter) of HA were found from the Simha (McBain 1950) equation:
c-0
Ill1 = where, [q] is the intrinsic viscosity. On the basis of viscometric assays, Relan (1984) suggested calculation of mean viscometric molecular weight from the Mark-Houwink equation: [q] = K - M,” (3) where, K, n are constants, for HAS: K = 7.85 - 10e4,n = 0.67;
e viscometric molecular weight. MATERIALS AND METHODS HA preparations were extracted from a grey-brown podzolic soil and chemozem under two long-term static field fertilization experiments. The samples were collected from different variants of the experiment: without fertlization and with manuring. Manure was applied at the rate 30 Mg/ha for three years. Soil was sampled from the arable layer (O-20 cm) and prepared for analysis routinely according to the manual for soil science experiments. Established analytical methods were used (MAFF 1989). The HA samples of all the fertilization variants were extracted with OSM NaOH, precipitated at pH 2.0 with 0.25M HzSO,, and than purified with the mixture HE-HCI, according to the Schnitzer and Skinner (1968) method. The ash content in HA preparations was 20-50 mg/g, taken into account for analysis. Viscometric assessments were conducted in two separate systems: for the pH = 10.5 in NaOH solution, and in the presence of 0.1M NaCl at pH = 7.0 in an Ubbelohde viscometer calibrated for the flow of 3 cm3 of Hz0 per 200 s. The measurements were run in three replicates at the temperature 25°C f 0.01 “C. Concentrations of the HA solutions ranged 2.5 - 10 g/L. The HAS intrinsic viscosity [rl] in solutions of pH 10.5 was calculated from the Fuoss-Cathers formula (Eq. l), which allows the intrinsic viscosity to be estimated by plotting l/rhed against 6. The values of
@la)’ 15(ln2b/a-3/2)
+
3(b/a)’ 15(ln2b/l-l/2)
+_!A_ 15
(4) HAS of particular combinations in the solutions of pH 10.5 and 7.0 were fractionated on a chromatographic column filled with Fractogel TSK HW 40s (Merck). On the base of the chromatograms obtained in the system EW = f (K,,), (where E, = absorbance at 600 mu, K, = partition chromatography constant), eluates were collected for each recorded peak, corresponding to HA fractions of specific molecular weight. Optical density of the fractions was measured at 465 nm (E,,,) and 665 nm (E&. The results were compared with the corresponding values obtained for 200 mg/L HA solutions before chromatographic separation, and absorbance coefficients were calculated: E4,6(E,,:E,d for HA sofutions before chromatography, and Q4,b(ratio of absorbances at 465 nm at 665 nm - Q465:Q665)for fractions of various molecular weight. RESULTS AND DISCUSION The mean viscosic molecular weights (E, ) calculated
from the Mark-Houvink equation are given in Table 1. These values were higher in alkali solutions and lower in pH = 7.0 solutions. Higher values of z” were noted in both cases for the HA preparations for chemozem. In general, manuring of soils caused a decrease of mean molecular weight of HA in both kinds of soil. The results from Table 1 show that the absorbance ratio E4,6 is inversely proportional to HA molecular weight, both for assays made for entire HA samples (E4,&and for the fractions of various molecular weight separated chromatographically. The fractions abbreviated F I, F II, and F III indicate high molecular weight fraction (&” = 0), medium molecular weight fraction (K, = 0.3), and low molecular weight fraction (I
Viscosimetric and chromatographic studies of soil humic acids
487
Table I. Molecular parameters and optical properties of HAs. Fertilizationtreatment Parameter
Control (without fertilization) pH = 7.0
Manure
pH = 10.5
pH = 7.0.
pH = 10.5
grey-brown podzolic soil
a/b” a [nml b Inml :;Fl Q416
F I*
44,6
F 1111
0.099 f 0.002 1380 12.6 3.2 0.26 3.32 5.08
0.278 f 0.006 6370 19.3 7.0 0.36 4.36 5.10
0.096 f 0.002 1290 12.5 3.1 0.25 3.41 5.63
0.255 f 0.006 5610 18.6 6.5 0.35 4.47 5.87
3.75 4.06
5.96 7.50
3.91 3.94
5.97 7.71
chemozem
a/b” a @I b bml :;;F I Q4l6 F II 4~6 F IIII
0.102 f 0.002 1430 12.6 3.2 0.26 2.83 4.48
0.335 f 0.007 8800 21.0 8.3 0.39 3.74 3.88
0.100 f 0.002 1390 12.5 3.2 0.25 2.80 4.29
0.303 f 0.007 7240 19.9 7.5 0.38 3.78 3.85
3.06 4.32
3.87 4.29
3.20 4.52
3.60 5.40
The values of intrinsic viscosity [q] estimated from the plots were used for calculations of spheric parameters. The axial ratio a/b, as well as both separate values, were calculated for HAS. The results are given in Table 1. The axial ratio a/b values, calculated for the low range of sample concentrations in solution of pH 10.5, for HA preparations from permanently nonfertilized objects, were higher for chernozem preparations than those for grey-brown podzolic soil. Similar differences were noted for the measured parameters: a = longer axis, and b = shorter axis of the molecules. Manuring of soil caused a decrease in the values of the spheric parameters (a/b, a, and b) of HAS from both soils, but higher values were observed for the HA samples from chernozem. When neutral sa4twas present in solution (pH = 7.0), the values of the axial ratio a/b, axis a, and axis b were practically the same. This finding suggested that in neutral medium, the molecules of HAS in all combinations had similar spheric parameters. An increase in the values of qred and spheric parameters in pH = 10.5 solutions at a low sample concen-
tration reflects an expansion of HA molecules caused by a counter-action of charged groups, which dissociate during dilution. By limiting molecular interactions, the addition of salt may cause the HA molecules to fold and thus resemble neutral polymers. If the model conditions of this experiment were used for estimates made for specific soil conditions (neutral reaction), a conclusion can be drawn that, under conditions of high soil moisture (a decrease of HA concentration), better expanded HA molecules have higher specific area than spheric polyelectrolytes of drier soils. Also, HAS for chermozem and from permanently nonfertilized soil can participate more actively in the processes of ion exchange and regulation of buffering properties of soil. CONCLUSIONS
1) HAS of permanently non-fertilized soils have the highest average molecular weight. Manuring causes a decrease of the HA molecular weight. The average molecular weight is inversely proportional to the values of the E,,6 parameter determined in 0.1 M NaOH.
488
2) Mean viscosic molecular weights of the HAS
measured in the presence of 0.1 M NaCI, pH 7.0, were threefold lower than those measured in NaOH solutions of pH 10.5. Because of interaction of humic charges at higher pH values, the HAS create agglomerates, resulting in higher viscosity of the solutions. At the neutral reaction of solution containing salts, HAS perform as neutral polymers.
REFERENCES Chen, Y.; Schnitzer, M. Viscosity measurements on soil humic substances. Soil Sci. Sot. Am. J. 40: 866-872; 1976. Chen, Y.; Senesi, N.; Schnitzer, M. Information provided on humic substances by E, ratios. Soil Sci. Sot. Am. J. 41: 352-358; 1977. Dziadowiec, H. Ekologiczna rola pr6chnicy glebowej. (Ecological role of soil humus). Zesz. Probl. Post. Nauk Rol. 4 11: 268-282; 1992. Fuoss, R.M.; Cathers, G.I. Polyelectrolytes III. Viscosities of nbutyl bromide addition compounds of 4-vinylpyridinestyrene copolymers in nitromethane-dioxane mixtures. J. Polym. Sci. 4: 97-120; 1949. Gosh, K.; Schnitzer, M. Macromolecular structures of humic substances. Soil Sci. 129, 5: 266-276; 1980.
S.S. Gonet and K. Wegner
Gonet, S.S.; Wegner, K. The effect of mineral and organic fertilization on properties of soil humic acids. In: Senesi, N.; Miano, T., eds. Humic substances in the global environment and implications on human health. Amsterdam: Elsevier; 1994: 607612. Kowalinski, S.; Drozd, J.; Licznar, M. Sklad frakcyjny zwiazkow pr6chnicznych pod roslinnoscia uprawiana w monokulturze i zmianowaniu. (Fractional composition of humus in soils under different crop rotation systems.) Zesz. Nauk. ART Olsztyn 29: 23-3 1; 1980. Kumada, K.; Kawamura, Y. Viscosimetric characteristic of humic acid. Soil Sci. Plant Nutrition 14, 5: 190-197; 1968. Kuszelewski, L. Wplyw nawozenia organicznego i mineralnego na zawartosc i niekt6re wskazniki jakosciowe substancji prochnicowych gleby. (The effect of mineral and organic fertilization on quantity and quality of soil humic substances.) Roczn. Nauk Roln. 98, 1: 7-27; 1972. MAFF (Ministry of Agriculture, Fisheries and Food). A manual of the analytical methods used by the Agricultural Development and Advisory Service. Third edition. Ref. Book 427. London; 1989. McBain, J.W. Colloid science. Boston, MA: D.C. Health & Company; 1950: 151. Relan, P.S.; Girdhar, K.K.; Khanna, S.S. Molecular configuration of composts humic acid by viscometric studies. Plant and Soil 8 1: 203-208; 1984. Schnitzer, M.; Skinner, S.I.M. Alkali versus acid extraction of soil organic matter. Soil Sci. 105,6: 392-396; 1968. Visser, S.A. Viscosimetric studies on molecular weight fractions of fulvic and humic acids of aquatic, terrestial and microbial origin. Plant and Soil 87: 209-221; 1985.