Application of the flow field flow fractionation (FFFF) to the characterization of aquatic humic colloids: evaluation and optimization of the method

The flow field flow fractionation (FFFF) is applied to determine the aquatic humic colloid size distribution. In order to determine average molecular weights of various aquatic humic acids, a calibration is carried out by using sulfonated polystyrene (PSS) reference colloids. Some interference is found to be inherent to the FFFF-method. Considerable sorption of humic substances to equipment components is observed depending on the ionic strength and pH of given carrier medium, the kind of membrane covering the accumulation wall, and the cross-flow rate. The present study shows that the sample recovery can be optimized by selecting a carrier of low ionic strength and high pH (5×10−3 mol l−1 Tris-buffer; pH=9.1) and using a regenerated cellulose membrane (cutoff: 5 kDa related to globular proteins). Under these low ionic strength conditions, the molecular weight calibration by using sulfonated polystyrene standards (PSS) is affected by ‘overloading’ effects originating from charge repulsive interaction of particles with one another and also with the accumulation wall. In general, the elution time of PSS decreases with increasing concentration. A correction for the ‘overloading’ effects is made by extrapolating the measured elution volumes to those obtained for infinitely low PSS concentrations where charge repulsion effects disappear. The molecular weight determined by this method for different aquatic humic substances is found to be in the range of 1.1–1.8 kDa for the number averaged molecular weight (Mn) and 1.8–4.1 kDa for the weight averaged molecular weight (Mw). Results are compared with the available literature data. Discrepancies can be explained to some extent by different reference colloids used in other studies for calibration.