Normal-phase high-performance counter-current chromatography for the fractionation of dissolved organic matter from a...

Normal-phase high-performance counter-current chromatography (HPCCC) is used to obtain a preliminary fractionation of components in dissolved organic matter (DOM) from a freshwater source. The HPCCC solvent system involved a normal-phase approach with water/methanol (1:1) as the lower stationary phase and hexane/ethyl acetate (1:1) as the upper mobile phase. The critical experiment parameters were optimised: revolution speed 1800 rpm and flow rate 0.15 mL/min. Under these conditions 50 μL of a 0.50 mg/mL DOM solution was loaded. The detection wavelength was monitored at 330 nm in order to isolate the main portion of DOM, which includes substances such as carboxyl-rich alicyclic molecules. By optimising this system it was possible to isolate materials that, according to GC-MS, can be related to molecules with an analogous structural background. Where fraction analysis was not suitable for GC-MS, RP-HPLC with UV absorbance detection was used, showing unique chromatograms for each fraction at both 210 and 330 nm.