ASSESSMENT OF THE EFFECT OF MOLECULAR-SIZE ON THE ELECTROPHORETIC MOBILITY OF HUMIC SUBSTANCES

The basic principles of the electrophoretic behaviour of humic substan ces (HS) still need to be systematically investigated. HS extracted fr om a Cryorthod (HS-1) and from an Haplumbrept (HS-2) were first fracti onated by ultrafiltration to obtain two series of fractions of reduced polydispersity with a nominal mean relative molecular mass (M) of 750 0, 20 000, 40 000, 75 000 and 200 000 Da. Fractions extracted from the same soil behaved like an homologous series when tested by size exclu sion chromatography, showing a linear relation of M with molecular siz e. When plotted against their mean M, the elution volumes of HS fracti ons extracted from the two different soils originated lines of differe nt slope. Absolute electrophoretic mobilities of the fractions were de termined in polyacrylamide gel slabs of increasing polyacrylamide conc entration using a single buffer system; in all gels there was a close linear relation between the electrophoretic mobility and the logarithm of the mean M of HS fractions. Extrapolation of mobilities of HS-1 at zero gel concentration gave intercepts that did not differ significan tly, showing that there was a constant mobility for all fractions in f ree solution. These results mean that charge differences have little e ffect on the electrophoretic mobilities of HS extracted from the same soil and imply the theoretical possibility of determining M distributi ons of HS by polyacrylamide gel electrophoresis in a single buffer sys tem. Mobilities of HS-2 fractions were compared with those of HS-1: th e latter fitted the regression equations of mobility against logarithm of the molecular weight obtained from HS-1 humic substances only in g els of small acrylamide concentration. Deviations were larger at small M, probably because of the increasing fulvic character of the fractio ns, and increased in gels of greater acrylamide concentration, indicat ing that charge differences may not be negligible when comparing humic substances extracted from different soils.