Developing a large-volume preparative method using a handmade HPLC column to fractionate dissolved organic matter

Abstract

To purify the heterogeneous natural organic matter (NOM), various experimental methods are used (e.g., XAD resin, ultrafiltration, size exclusion chromatography (SEC)). However, these methods have few limitations such as the reproducibility, the sample recovery, and the difficulty of the experimental procedure. Preparative high-performance liquid chromatography (prep-HPLC) with a handmade column was employed as a fractionation method for NOM. The method enables accommodation of a relatively large volume (up to 5-10 mL). To optimize the efficiency of the NOM fractionation system based on the molecular size of the NOM, the preparative column was packed with polymethacrylate resin in a laboratory. Quantitative evaluation of the handmade preparative column performance was conducted for several parameters including the number of theoretical plates (N), asymmetry factor (As), height equivalent to theoretical plates, and column resolution (Rs). The N parameter increased as the packing velocity increased, and As was close to 1. The optimum flow rate was selected at the highest Rs. A greywater includes complex organic matters that generated in households without fecal contamination. To verify the separation efficiency of the handmade column, the organic matters in the greywater are fractionated. The fractions were reanalyzed by an analytical HPLC system to confirm the separation efficiency of the prep-HPLC. The polydispersity (ρ) of the fractions were around 1. A comparison of the retention time of the original and fractionated samples indicates that the prep-HPLC system with the handmade column efficiently fractionated NOM according to its molecular size.