Assessment of the effect of molecular size on the electrophoretic mobility of humic substances

Abstract

SummaryThe basic principles of the electrophoretic behaviour of humic substances (HS) still need to be systematically investigated. HS extracted from a Cryorthod (HS–1) and from an Haplumbrept (HS–2) were first fractionated by ultrafiltration to obtain two series of fractions of reduced polydispersity with a nominal mean relative molecular mass (M) of 7500, 20000, 40000, 75000 and 200 000 Da. Fractions extracted from the same soil behaved like an homologous series when tested by size exclusion chromatography, showing a linear relation of M with molecular size. When plotted against their mean M, the elution volumes of HS fractions extracted from the two different soils originated lines of different slope. Absolute electrophoretic mobilities of the fractions were determined in polyacrylamide gel slabs of increasing polyacrylamide concentration 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 significantly, showing that there was a constant mobility for all fractions in free solution. These results mean that charge differences have little effect on the electrophoretic mobilities of HS extracted from the same soil and imply the theoretical possibility of determining M distributions of HS by polyacrylamide gel electrophoresis in a single buffer system. Mobilities of HS–2 fractions were compared with those of HS–1: the latter fitted the regression equations of mobility against logarithm of the molecular weight obtained from HS–1 humic substances only in gels of small acrylamide concentration. Deviations were larger at small M, probably because of the increasing fulvic character of the fractions, and increased in gels of greater acrylamide concentration, indicating that charge differences may not be negligible when comparing humic substances extracted from different soils.