Considerations in the use of high-pressure size exclusion chromatography (HPSEC) for determining molecular weights of aquatic humic substances

High-pressure size exclusion chromatography (HPSEC) is a powerful technique for determining molecular weight (MW) distributions of aquatic humic subst ances (HS). Previous researchers have shown that HPSEC can provide Values o f weight average molecular weight (M-w), number average molecular weight (M -n), and polydispersity (rho) that are comparable to values determined by o ther techniques such as vapor pressure osmometry and held flow fractionatio n. We have observed that HPSEC does not always provide reproducible results for HGS-variability by 10-20% is common. Since certain applications requir e better precision for MW of HS, this research focuses on improving precisi on and reproducibility of HPSEC measurements. While the method of baseline correction or of high MW (HMW) cutoff of the H PSEC chromatogram is not critical, the choice of low MW (LMW) cutoff can gr eatly affect M-n and rho . For HS, we recommend either 2% of the maximum ch romatogram height or MW = 50 as the LMW cutoff, whichever is the higher val ue, and 1% of the maximum chromatogram height as the HMW cutoff. Polystyren e sulfonate (PSS) standards are commonly used in combination with acetone; we further recommend the inclusion of salicylic acid as a charged low MW st andard. Analysis of UV detection wavelengths shows that wavelengths between 230 and 280 nm give reasonable results, but that higher wavelengths can bi as measurements to higher MWs. We recommend 254 nm except for low carbon co ncentration samples, where 230 will provide better sensitivity. Our results show that "standardization" of HPSEC procedures according to these suggest ions can lead to excellent reproducibility of M-n and M-w measurements for HS (2-3% RSD). (C) 2000 Elsevier Science Ltd. All rights reserved.