Simple, quantitative method for low molecular weight dissolved organic matter extracted from natural waters based upon high performance counter-current chromatography

A simple, high-performance counter-current chromatography method with sequential UV absorbance (254 nm) and evaporative light scattering detection (ELSD) was developed for the quantification of pre-extracted low molecular weight dissolved organic matter (DOM) extracted from natural waters. The method requires solid-phase extraction (SPE) extraction of only small volumes of water samples, here using poly(styrenedivinylbenzene)-based extraction cartridges (Varian PPL). The extracted and concentrated DOM was quantified using reversed-phase high-performance counter-current chromatography (HPCCC), with a water/methanol (5:5) mobile phase and hexane/ethyl acetate (3:7) stationary phase. The critical chromatographic parameters were optimised, applying a revolution speed of 1900 rpm and a flow-rate of 1 mL min⁻¹. Under these conditions, 50 μL of extracted DOM solution could be injected and quantified using calibration against a reference natural dissolved material (Suwannee River), based upon UV absorbance at 254 nm and ELSD detection. Both detection methods provided excellent linearity (R² > 0.995) for DOM across the concentration ranges of interest, with limits of detection of 4 μg ml⁻¹ and 7 μg ml⁻¹ for ELSD and UV absorbance, respectively. The method was validated for peak area precision (<5%), and accuracy and recovery based upon spiking seawater samples prior to extraction, together with DOM solutions post-extraction (>95% recovery). The developed method was applied to the determination of the concentration of DOM in seawater, based upon initial sample volumes as small as 20 mL.