HPLC of humic substances fractionated by Flow FFF

This communication reports a study of the effect of ionic strength and electrolyte composition on fractions, separately collected by flow FFF, of a mixture of humic substances. Reverse phase HPLC analysis of three early eluting fractions suggests that the components released by the column behave as organic acids. The baseline-resolved peaks of the first two fractions, subject to higher retention in solutions of lower pH and/or higher polarity, substantiate this suggestion. The fraction with larger components, as measured by flow FFF, also appears to contain acidic species. Their retention level, however, may not be accurately modulated by varying the mobile phase properties as these species are either totally retained in acidic phases or released before the void peak at pH greater than or equal to 4.2. Besides showing the effective separation achieved in the flow FFF channel, this study reveals the pronounced difference in the physicochemical properties of some components of a humic mixture even with very close particle size.