Electrophoresis of podzol soil humic acids

Electrophoresis of podzol soil humic acids

EnvironmentInternational,Vol.24, No. 5/6,pp. 625--628,1998 Copyright01998 ElsevierScience Ltd Printedin the USA.All rights~ e d 0160-4120/98$19.00+.00...

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EnvironmentInternational,Vol.24, No. 5/6,pp. 625--628,1998 Copyright01998 ElsevierScience Ltd Printedin the USA.All rights~ e d 0160-4120/98$19.00+.00


PII S0160-4120(98)00049-X

ELECTROPHORESIS OF PODZOL SOIL HUMIC ACIDS Ludmilla Shirshova and Ragnar Osterberg Department of Chemistry, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden

E1 9706-163 M (Received 27 June 1997; accepted 8 March 1998)

Podzol soil humic acids prepared by a gentle procedure were studied by electrophoresis in the pH range 3 to 7 at two ionic strengths; the positive counter ion concentrations related to the negatively charged buffers were 0.035 M and 0.1 M Na +. Phosphate, acetate, and citrate buffers were used. In the pH range studied, humic acids moved to the positive pole, indicating that the particles in solution are negatively charged. The distances moved were substantially larger for 0.035 M Na+ as compared to those o f 0.1 M Na +. When these latter data, in normalized form, were plotted as a function ofpH, they compared well with proton dissociation data calculated for the pK-values 4.2 and 5.85, obtained via an acid-base titration. This indicates that the pH dependent net charge of humic acids can be essentially described by the dissociation of two groups forming negatively charged anionic groups, most likely carboxylate groups. 01998ElsevierScienceLtd

INTRODUCTION As a part of a general study on the reactions and solution structure of humic acids, previous work showed that neutron scattering from humic acids obeys a power law revealing that their structure is fractal with a fractal dimension of 2.35 (Osterberg and Mortensen 1992; Osterberg et al. 1995). These neutron scattering studies, as well as studies using atomic force microscopy (Ikai and Osterberg 1996), indicated that humic acids exist in solution in the form of discrete particles with sizes from 100 to 300 nm, dependent on source and preparation methods. Further studies, using electromotive force methods, indicated that the humic acid system is essentially a non-equilibrium system. Both the electron activity (Osterberg et al. 1995; 0sterberg and Shirshova 1997) and the proton activity (Wahlberg and Agren 1996) showed oscillations. It is possible that the proton reactions observed in the pH range 3 to 7, at least partly, may involve free radical reactions of phenolic compounds rather than carboxylic groups (Musso 1967). Also, as has been previously stated,

some of the benzene caboxylic acids isolated from humic acids might be the result of the oxidative procedure involving phenolic groups (Schulten et al. 1991). As a result, it is not known in detail whether or not "native" humic acid particles are negatively charged in the entire pH range 3 to 7 or perhaps only at neutral pHs. In this study, the net charge of humic acids was analyzed using electrophoresis; the podzol soil humic acids used were prepared by a gentle method involving special precautions to eliminate metal ions such as calcium, copper, and iron ions. Electrophoresis of humic acids has previously been studied repeatedly and the subject was reviewed by Duxbury (1989). In these earlier studies, as well as in some later studies (De Nobili and Fornasier 1994), harsher preparation methods were used, such as those involving strong alkali. Recent studies by Trubetskoj et al. (1991; 1994) involved other kinds of soil, and a detergent, as well as a denaturing agent (7 M urea), 625


were used in the electrophoretic runs. As a result, from these earlier studies, it seems difficult to draw any final conclusions regarding the pH-dependent net charge of humic acids prepared from podzol soil and, therefore, a separate study has now been done. These data are important for a detailed analysis of proton and metal ion reactions ofhumic acids.

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A1 Horizon podzol soil was taken from a suburb of Uppsala, Sweden, in late September from a meadow which had not been plowed for over a hundred years. The first three preparations (I, II, and III) were made as described previously (Osterberg et al. 1994), involving extraction with 0.05 M pyrophosphate buffer pH 7 (Buffer P), followed by precipitation of.humic acid at a low pH with HC1. The precipitate was dissolved in 0.15 M Buffer P and then the procedure of precipitation and dissolving was repeated four times. The final solution of humic acid in 0.15 M Buffer P was stored at -80°C. In order to eliminate various inorganic ions, such as iron ions, for instance, the samples were dialysed against bidistilled water followed by 0.2 M EDTA o f p H 7 and then again against bidistilled water. Spectrapor membrane tubing (Los Angeles, CA, USA), 3500 m.w. cutoff, was used. In order to remove minor impurities of silicate, Sample No III was also treated with HF. Preparation IV was prepared by using a carboxylic resin in Na*-form (Shirshova 1991) instead of the initial extraction of the soil with buffer P. The precipitation and dissolving procedures were then performed in the same way as described above for samples I-III. In all the samples studied, the humic acid concentration was estimated via analysis of C, assuming a content of 53% (by weight) C per humic acid substance (Lindqvist 1982). The C/N atomic ratios were 12.8, 16.3, 14.5, and 12.8 for preparations I, II, III, and IV, respectively. Analysis of preparation III showed _<0.1% Fe, _<0.02% Ca, and _<0.001% Cu. Electrophoresis of humic acids was done using an LKB apparatus (type 2117 Multiphor II) in 8% polyacrylamide gel. The following buffers were used: 1) Phosphate buffers of pH 6.0, 6.35, and 6.9; 2) acetate buffers o f p H 4.4, 5.0, and 5.6; and 3) citrate buffers o f p H 3.1 and 4.0. All the buffers had Na + ions as a counterion to the negatively-charged buffers. Two series were done, one involving low ionic strength with a Na + concentration of 0.035 M, and one at a higher





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Fig. 1. Electrophoresis of humic acids at 0.1 M Na + concentration. (A) pH 3. I in citrate buffer; (B) pH 5.0 in acetate buffer; (C) pH 6.0 in phosphate buffer.

ionic strength with a Na + concentration of 0.1 M. Thymol blue was used as a marker. Before electrophoresis, humic acids were kept at 21 °C for more than 48 h at the same pH as that of the electrophoretic run

Electrophoresis of podzol soil humic acids


Table 1. Electrophoresis of humic acids at 0.1 M Na ÷ concentration. Distances moved relative to thymol blue, Rt, and normalized data Rt/f with f = 0.743) for comparison with the proton dissociation curve in Fig. 2. No





1 2

3.1 4.0

Citrate Citrate

0.44 0.23

0.59 0.31

3 4

4.4 5.0

Acetate Acetate

0.53 0.62

0.71 0.83





















(Osterberg et al. 1994). The sample volume applied was 5 ~tL ofhumic acid solution (1 to 6 mg/mL). The electrophoreses were developed by alcian blue (Castagnola et al. 1979). RESULTS AND DISCUSSION

Figure 1 gives examples of the electrophoreses obtained by fractions I, II, III, and IV of humic acids at pH 6.0, 5.0, and 3.1. As follows from Fig. 1, the present fractions yield only one major band, which in the entire pH range moves to the positive pole. Thus, the humic acid particles in solution appear to have a net negative charge in the entire pH range from 3 to 7. The different preparation procedures of the samples do not seem to have influenced the distances moved within the limits of experimental errors (Fig. 1). Except for these gentle preparation procedures involving dialysis to eliminate small molecules and ions, any polydispersity was minimized by bringing the polymerization reactions of humic acids to a steady state by keeping the samples at room temperature for 48 h before the electrophoretic runs (Osterberg and Mortensen 1992; Osterberg et al. 1994). The observation regarding the apparent homogeneity of humic acids is in agreement with previous results from neutron scattering and atomic force microscopy, indicating that an essential monodisperse population of colloids is obtained by the present preparation procedure (Osterberg et al. 1995; Ikai and Osterberg 1996). The present data also agree with the results reported by Stevenson et al. (1953). They studied organic colloids from forest soil prepared by a gentle method where small molecules were eliminated by dialysis; using free electrophoresis, they found only one single high-molecular-weight component at pH 4.8, 7.0, and 9.0. However, the data in the pre-

sent study are different from those obtained by humic acids extracted by harsher methods, such as those involving strong alkali (Duxbury 1989; De Nobili and Fornasier 1994). Their data showed some minor components in addition to one major component. Likewise, using a detergent in combination with a denaturing medium (7 M urea) yielded electrophoretic patterns with more than one component in studies of humic acids from other kinds of soil (Trubetskoj et al. 1991; 1994). Table 1 lists the distances moved for humic acids relative to those of thymol blue at the different pH studied and at the 0.1 M Na + concentration. At the lower ionic strength, 0.035 M Na+, the distances moved relative to thymol blue were substantially larger than those at the higher ionic strength. This may be due to less shielding of the negatively-charged groups on the humic acid particle by Na+ ions at the lower ionic strength. In Fig. 2, the electrophoretic data are compared with a proton dissociation curve calculated for the pK values 5.85 (pK~) and 4.2 (pK2); the pK-values were obtained from an acid-base titration. In the plot of Fig. 2, the distances moved for humic acids relative to thymol blue, Rt, were normalized by dividing them by the factor f = 0.743, so that the data for high pH would approach 2.0, corresponding to the dissociation of two protons (Table 1). The theoretical curve of Fig. 2 involves Z, the average number of protons bound per humic acids, defined by Z = (h K," + 2h 2 K," K2") / (1 + h K , " + h2K, ' Kd' ) (1) where h = [H+]. It follows from Fig. 2 that good agreement is obtained for pH a 4; however, there is a relatively large deviation for the datum at pH 3.1. It is possible that this


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1, Shirshova and R. Osterberg

REFERENCES Rt/f ..-9 .,~.'"

1 5 0" N

I 0

.•,•" •

0 5 ,.•• • •,,..."




5 pH

Fig. 2. Normalized electrophoretic data of humic acids, P~/f (Table 1), plotted against pH (filled circles); R, is the distance moved relative to thymol blue at 0.1 M Na+ concentration; f is a normalizing factor (= 0.743)• The dotted curve, (2-Z) vs. pH, was calculated from pK~ = 5.85 and pK 2 = 4.2 using Eq. 1; the pKvalues were obtained from an acid-base titration of the same podzol soil humic acids.

d e v i a t i o n h a s to d o w i t h the i n f l u e n c e o f e l e c t r o e n d o s m o t i c f l o w . A t this pH, the c o n c e n t r a t i o n o f H + ions is substantial, a n d t h e y are p r e s e n t in e x c e s s o f the c o n s t a n t N a + c o n c e n t r a t i o n . A l s o , the H + ion has f a s t e r m o b i l i t y t h a n a n y o t h e r p o s i t i v e ion. A t this pH, the d i s t a n c e m o v e d is s m a l l and, t h e r e f o r e , even a s m a l l contribution from electroendosmosis would influence the distance recorded.

CONCLUSIONS T h e d a t a i n d i c a t e t h a t the n e t c h a r g e o f p o d z o l soil h u m i c a c i d s is n e g a t i v e in the e n t i r e p H r a n g e s t u d i e d , p H 3.1 to p H 7. T h e p H d e p e n d e n c e o f the net n e g a t i v e c h a r g e c o m p a r e s w i t h the r e s u l t o f an a c i d - b a s e titration, i n d i c a t i n g that this net c h a r g e can be d e s c r i b e d b y the n e g a t i v e l y c h a r g e d g r o u p s s u c c e s s i v e l y r e v e a l e d b y p r o t o n d i s s o c i a t i o n . T h i s r e s u l t a l s o s u p p o r t s the i d e a that c a r b o x y l i c g r o u p s e x i s t in n a t i v e p o d z o l soil h u m i c a c i d s a n d , thus, n o t o n l y as a r e s u l t o f a d e g r a d a t i o n procedure. Acknowledgment--We are indebted to The Swedish Agricultural

Sciences Research Council for financial support.

Castagnola, M.; Nigro, C.; Marini Bettolo, (i.B.: Milan& A; 1Iotas, R.G Characterization of soil humic acid by combined polyacu'lamide disc electrophoresis and chromatic reactions .I Chromatogr. 177: 130-134, 1979. De Nobili, M. Fornasier, F. Polyacrylamide gel electrophorcsis ol humic substances fractionated by ultrafiltration. In: Senesi, N : Miano, T.M., eds. flumic substances in the global environment and implications on human health. Amsterdam: Elsevier: 1994: 269-272, Duxbuo,', J.M Studies of the molecular size and charge of humic substances by electrophoresis. In: Hayes, M.H.B.; McCarthy', t' • Malcolm, R.L.: Swirl, P.S., eds. ttumic substances II - In search of structure. Chicester: Wiley; 1989: 593-620. lkai, A.: ()sterberg, R. Atomic torce microscopy of humic acids Scanning Microsc. 10: 994-997: 1996. Lindqvist, 1. Charge-transfer interaction of humic acids with donor molecules in aqueous solutions. Swed. J. Agric. Res. 12: 105t 09; 1982. Musso, tl. Phenol coupling. In: Iaylor, W.I.; Batterby, H.I,L, eds Oxidative coupling of phenols. New York, NY: Marcel Dekker: 1967: 1-94. ()sterberg, R.: Mortensen, K. Fractal dimension of humic acids, .,k small-angle neutron scattering study• Eur. Biophys. J. 21: 163167: 1992. Osterberg, R.; Szajdak, L.; Mortensen, K. lemperature-dependent restructuring of fractal humic acids: A proton dependent process Environ. Int. 20: 77-80; 1994. ()sterberg, R.: Mortensen, K.; lkai, A. Direct observation ofhumic acid clusters• A non-equilibrium system with fractal structure. Naturwissenschaften 82: 137-139: 1995. ()sterberg. R. Shirshova, L. Oscillating, non-equilibrium redox properties of humic acids• Geochim, Cosmochim. Acta 61: 45994603: 1997. Schuhcn, It.R.: Plagc, 17,.; Schnitzer, M. A chemical structure to~ humic substances• Naturwissenschaften 78:311-312:1991 Shirshova, I,. Polydispersity of soil humic substances. Moscow: Nauka Pub.; 1991 (in Russian). Stevenson, F.J,: Van Winkle, G.: Martin, W.P. Physicochemical investigations of clay-adsorbed organic colloids• Soil Sci. Amer Proc. 17: 31-34; 1953. Trubetsk@ O.A.: Kuduavceva, I,Y.: Shirshova, L. Charactcrb zation of soil humic matter by polyacrylamide gel electrophoresis in the presence of denaturing agents• Soil Biol. Biochem. 23: 1179-1181: 1991. Trubetskqi, O.A. I'rubctskaya, O.E.: Markova, L.F.; Muranova. T.A. Comparison of amino acid composition and E4/E~ ratios of soil and water humic substances fractions obtained by polyacrylamide gel electrophoresis. Environ. Int. 20: 387-389: 1994. Wahlberg, 04 Agren, S. Kinetic studies of fulvic and humic acids in 0.1 M NaCI aqueous solution at 25 ° C. Characterization of the steady states• Acta Chem. Scand. 50: 564-566; 1996.