Preliminary fractionation strategies

In water studies it is standard practice to filter the sample soon after collection, usually through a 0.45p,m membrane disc (made of cellulose acetate, cellulose nitrate or polycarbonate). This process arbitrarily divides the sample components into soluble and insoluble fractions, but as shown in Table 2.3, the average size of different chemical species varies widely, and some differentiation between species can be obtained through using filter media of different pore sizes. For example, fully dissolved compounds can be separated from finer colloidal forms by using gel filtration and dialysis, and sub-division of the total content into fractions based on particle or molecular size (see Section 2.3) has been used for speciation of elements in waters. 

With biological samples, species which cannot be determined by in situ techniques (see Table 2.2) are usually extracted, concentrated and separated by chromatographic methods, with subsequent determination of the element in the separated fractions. 

Adsorbed on mixed colloids, (inorganic/organic) Cu2+, Fe203/humic acid  

Metals adsorbed on solids M2+, MS, MC03 on clay minerals, Mniv oxides  

Metals incorporated with organic material Metals in algae  

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As shown in the purification of rat histone H2A1 53 amide synthesized using the FMOC strategy, remarkable results using a Pharmacia Biotech Mono S column were obtained for the purification of complex synthetic mixtures (Figures 3 and 4)J5H] This system has been used with similar success with several other peptides. With a complex mixture of peptide/proteins, ion-exchange chromatography was found to be an important first step for preliminary fractionation to be further purified by RP-HPLC. 

A typical isolation strategy is the preliminary purification of the n-butanol extract over dextran supports like Sephadex LH20 or Fractogel TSK, followed by further fractionation of the crude saponin mixtures [111]. A new generation of polymers has been exploited for the initial purification steps. They are highly porous polymers (Daion HP-20, MCI gel CHP-20P (both from Mitsubishi Chemical Industries, Tokyo), Amberlite XAD-2) [112], Methanol-water or acetone-water solvent gradients are used. 

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