Size exclusion chromatography of humic substances: Limits, perspectives and prospectives

The fractionation range of chromatographic media used in size exclusion chromatography (SEC) depends strongly on the kind of substances eluted, Although the fractionation range reported by the manufacturer for globular proteins on Sephadex G-200 is 5000 to 250,000 daltons and that for dextrans 1000 to 150,000 daltons, phenolcarboxylic acids (pH = 8, 0.1 mmol/L NaOH) are eluted much earlier than would be predicted based on their molecular weight (MW). The actual MWs for this group of compounds ranges from 200 to 500 daltons, whereas their MWs determined by SEC calibrated with proteins is much higher (7200-180,000 daltons). Ion exclusion, which is the basis for this behavior, originates from residual ionized groups and limits the possibility of applying SEC to the study of conformational changes of humic substances (HS). A correct selection of experimental conditions enables the use of SEC for various applications such as the study of metal-binding equilibria and fractionation according to molecular size. To determine the MW distribution of HS, columns should be calibrated with standard humic and fulvic acids with known MWs determined by colligative properties such as vapor pressure osmometry. It should be noted, however, that SEC studies on fractions of HS originating from various soils, result in a series of significantly differing calibration curves. The Linearity of the relationship between the distribution coefficient (K-av) values and the logarithm of the MWs implies the constancy of partial specific volumes. It also suggests that HS extracted from the same soil constitute a homologous series in which the structure of the larger components is formed by the repetition of structurally similar units and is, therefore, more regular than previously believed. The experimental results obtained by SEC also suggest that in regard to the sensitivity of the technique, charge density differences are small within a certain series compared with size differences.