A concise overview (75 references) of the analytical fractionation of aquatic humic substances using sequential-stage ultrafiltration is presented. First, humic substances in aquatic environments and actual problems connected with their fractionation and analysis are briefly considered. The molecular size classification of dissolved humic substances by means of multistage ultrafiltration, with special emphasis on on-line techniques, is the focal point of the discussion. In particular, the capabilities of ultrafiltration for the size fractionation and characterization of species formed between colloidal humic substances and pollutants (e.g. metals) are stressed.

A combined analytical procedure, consisting of ligand exchange by ethylenediaminetetra-acetic acid (EDTA) off-line, size fractionation through multistage ultrafiltration (MS-UF) and metal determination using total-reflection X-ray fluorescence spectrometry (TXRF), has been developed for the speciation of heavy metals in selected aquatic humic substances (HS). Accordingly, natural HS/metal species somewhat preconcentrated by preparative ultrafiltration were reacted with equimolar amounts of dissolved EDTA as a function of time. Afterwards, a special MS-UF device was used for size fractionation of the HS under study and their metals species in the molecular mass range 1–100 kDa (six fractions), finally using TXRF (preferably with a Mo tube) for determination of the analytes (e.g. Cd, Cu, Fe, Mn, Ni, Pb and Zn) other than Al (determined by AAS). Thus, natural metal species in aquatic HS were revealed to be widely EDTA-inert, depending on the element, in contrast with those freshly formed. The TXRF measurements in HS were virtually unaffected by this matrix (up to 20 mg ml−1 HS) forming homogeneous “polymer” films on the sample carrier. Under routine TXRF conditions (10 μl sample, 300 s live time), detection limits in the lower ng ml−1 range and relative standard deviations between 4% and 7% (n=8) could be obtained in humic-rich water samples. © 1997 Elsevier Science B.V.

The molecular-size fractionation of aquatic humic substances (HS) and their metal species by means of a novel sequential-stage ultrafiltration (UF) device equipped with five appropriate ultramembranes (1, 5, 10, 50 and 100 kD) is described. First of all, the concentration dynamics of macromolecules, particulary HS, during five-stage UF and its subsequent washing step has been modelled. Based on these results, the fractionation of aquatic HS (from ground and bog water) by means of multistage UF has been optimized for an analytical scale (10 ml sample, 1 mg/ml HS, 10 ml washing solution, pH 6.0). The molecular size-distribution of selected aquatic HS (BOC 1/2 from the “DFG-Versuchsfeld Bocholt”, VM 5 from “Venner Moor”, Germany) studied by five-stage UF exhibited strong systematic influences of the procedure used for their isolation. The molecular-size distribution of HS obtained by on-line UF and gel permeation chromatography (GPC) showed a satisfactory agreement in the range 1–50 kD. Moreover, when interrupting multistage UF for > 48 h a slow transformation in the HS samples has been found as gradually additional HS fractions of < 1 kD have been formed. Besides unloaded HS molecules, the molecular-size distribution of freshly formed metal species of HS (1.0 mg metal/g HS of Al(III), Cd(II), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), each) has been characterized by multistage UF as a function of pH-value, degree of loading and complexation time. Metal determinations as carried out by flame AAS, showed that considerable metal fractions in HS especially are present in molecules > 50 kD, which seemed to be rather acid-inert. With complexation times of < 2 days a transient shift of the molecular size distribution of both HS and their metal species (e.g., Al(III), Fe(III) to higher values (> 10 kD) has been found.