Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron(III)-loaded ion exchangers

The analytical fractionation of aquatic humic substances (HS) by means of i mmobilized metal-chelate affinity chromatography (IMAC) on metal-loaded che lating ion exchangers is described. The cellulose HYPHAN, loaded with diffe rent trivalent ions, and the chelate exchanger Chelex 100, loaded to 90% of its capacity with Fe(UI), were used. The cellulose HYPHAN, loaded with 2% Fe(III), resulted in HS distribution coefficients K-d Of up to 10(3.7) mL/g at pH 4.0 continuously decreasing down to 10(1.5) at pH 12, which were app ropriate for HS fractionation by a pH-depending chromatographic procedure. Similar distribution coefficients K-d were obtained for HS sorption onto Fe (III)-loaded Chelex 100. On the basis of Fe-loaded HYPHAN both, a low-press ure and high-pressure IMAC technique, were developed for the fractionation of dissolved HS applying a buffer-based pH gradient for their gradual eluti on between pH 4.0 and 12.0. By coupling the Chelex 100 column under high-pr essure conditions with an inductively coupled plasma mass spectrometer an o n-line characterization of HS metal species could be achieved. Using these fractionation procedures a number of reference HS were characterized. Accor dingly, the HA (humic acids) and FA (fulvic acids) studied could be discrim inated into up to 6 fractions by applying cellulose HYPHAN, significantly d iffering in their Cu(II) complexation capacity but hardly in their substruc tures assessed by conventional FTIR. In the case of using Chelex 100 exchan ger resin two major UV active HS fractions were obtained, which significant ly differ in their complexation properties for Cu(II) and Pb(LT.), respecti vely.