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 substances (HS). Previous researchers have shown that HPSEC can provide values of weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity (ρ) that are comparable to values determined by other techniques such as vapor pressure osmometry and field flow fractionation. We have observed that HPSEC does not always provide reproducible results for HS—variability by 10–20% is common. Since certain applications require better precision for MW of HS, this research focuses on improving precision and reproducibility of HPSEC measurements. While the method of baseline correction or of high MW (HMW) cutoff of the HPSEC chromatogram is not critical, the choice of low MW (LMW) cutoff can greatly affect Mn and ρ. For HS, we recommend either 2% of the maximum chromatogram height or MW=50 as the LMW cutoff, whichever is the higher value, and 1% of the maximum chromatogram height as the HMW cutoff. Polystyrene sulfonate (PSS) standards are commonly used in combination with acetone; we further recommend the inclusion of salicylic acid as a charged low MW standard. Analysis of UV detection wavelengths shows that wavelengths between 230 and 280 nm give reasonable results, but that higher wavelengths can bias measurements to higher MWs. We recommend 254 nm except for low carbon concentration samples, where 230 will provide better sensitivity. Our results show that “standardization” of HPSEC procedures according to these suggestions can lead to excellent reproducibility of Mn and Mw measurements for HS (2–3% RSD).