COMBINATION OF GEL CHROMATOGRAPHY AND PAA G ELECTROPHORESIS FOR PREPARATIVE ACCUMULATION OF HUMIC-ACID FRACTIONS

Humic acids (HA) from chernozem were fractionated by gel filtration on Sephadex G-75 in the presence of Tris-HCl or 7 M urea. Several absorb ance peaks with shoulders between them were resolved in the elution pr ofiles of HA samples in both cases. Seven pools from each experiment w ere collected, dialyzed, lyophilized and assayed by 10% polyacrylamide gel electrophoresis in the presence of denaturing agents (PAGE). The distribution of HA fractions in Sephadex gel matrix has shown that ure a is more effective as eluent. HA from three different soils were frac tionated by gel filtration on Sephadex G-75 in the presence of 7 M ure a without any absorption on the gel matrix. All added HA were eluted f rom the gel within the total column volume and independently from samp le concentration and flow rate. The elution profiles of all samples we re similar. Two absorbance peaks with a shoulder between them were res olved. Several fractions from each experiment were collected, dialyzed , lyophilized and assayed by PAGE. The unfractionated HA from all sour ces were used as reference. Each chromatographic fraction produced onl y one electrophoretic zone corresponding closely to the band of the re ference sample with some admixture of preceding or following fractions . The results indicate that PAGE fractionation of HA is based on molec ular weight differences and this technique can be successfully used fo r checking the purity of HA fractions obtained by gel filtration.