Calibration of high performance size exclusion chromatography (HPSEC) for molecular weight estimation of aquatic humic substances

Three high performance size exclusion chromatography (HPSEC) columns were compared, along with mobile phase chemistry and calibration standards, for the determination of apparent molecular weight (AMW) of aquatic humic substances using a UV absorbance detector (λ = 254 nm). The addition of 10 % organic solvent did not influence peak elution times of sodium polystyrene sulfonate (PSS) standards or chromatograms of Suwannee River humic acid (SRHA) or fulvic acid (SRFA) but did result in an additional peak being detected in a treated municipal wastewater sample (WWT). Mobile phase concentration was found to have a greater effect on peak position of PSS standards and SRHA than mobile phase pH or sample injection volume. Several chemicals of known molecular weight were tested alongside PSS standards, with salicylic acid (138.1 Da) and 4-chlorobenzoic acid (156.6 Da) resulting in retention times that aligned with the PSS MW standards. Of the three columns tested, the two with comparable pore sizes (i.e., 250 and 300 Å) were found to result in similar peak elution times and highly linear calibration curves over the range of 210–17,000 Da, while the column with the smaller pore size (i.e., 100 Å) gave better peak separation in the low AMW range (i.e., < 1000 Da). Estimations of AMW of SRHA using various sets of calibration standards on one of the three columns tested ranged from 1080 to 2799 Da using 0.02 M NH4CH3CO2as eluent and 60-µL injections. AMW of SRFA ranged from 939 to 2434 Da for the same conditions.