High pressure size exclusion chromatography (HPSEC) of humic substances: Molecular sizes, analytical parameters, and column performance

High Pressure Size Exclusion chromatography (HPSEC) is increasingly used to evaluate molecular sizes of humic substances from different sources. Asymm etry factors (A(s)), number of theoretical plates (N), coefficient of distr ibution (k(d)), and column resolution (R-s) were determined for two differe nt HPSEC columns (TSK G3000SW and Biosep S2000) and polysaccharides of know n molecular weights were used as standards. Calibration curves were equival ent for both columns whereas analytical parameters revealed that the TSK co lumn was only slightly more efficient in separating polysaccharide standard s. M-w and M-n values for humic substances differed according to the molecu lar weight range of each column but relative standard deviation never excee ded 5% for both columns. Variations between columns were attributed to intr insic humic properties such as the stability of conformational structures. These results suggested that humic substances in solutions are loosely-boun d association of small molecules that may be consistently dispersed by diff usion through size-exclusion pores. HPSEC is confirmed to represent a highl y precise method to evaluate the relative molecular-size distribution of di ssolved humic substances. (C) 1998 Elsevier Science Ltd. All rights reserve d.