Effect of various size exclusion chromatography (SEC) columns on the fingerprints of extracellular polymeric substances (EPS) extracted from biological sludge

• Acquisition of fingerprints of EPS extracted from different sludges by SEC.
• Similar performances for semi-preparative and analytical columns.
• Improved selectivity for low MW by using serial-linked columns.
• Low MW (<10 kDa) mainly attributed to humic-like substances.

The fingerprints of extracellular polymeric substances (EPS), extracted from activated and anaerobic granular sludges, were obtained using size exclusion chromatography (SEC) coupled to UV (210 nm) or fluorescence (221/350 nm or 345/443 nm) detectors. Fingerprints obtained using a semi-preparative column (Amersham-Biosciences-Superdex-200-10/300-GL) or an analytical column (Agilent-Bio-SEC-300) were compared. The benefit of using two different molecular weight (MW) ranges chromatographic columns connected in series was investigated. High similarities observed on chromatograms lead to the conclusion that both analytical and semi-preparative columns can be used to obtain EPS fingerprints. As it could be expected, the low MW range column added in series to the high MW molecular separation columns only improved significantly the resolution of compounds with MW lower than 10 kDa. The combination of MW distribution and the use of three different detection conditions allowed determining several EPS main components. According to their fluorescence characteristic, low MW (<6 kDa) compounds were mainly ascribed to humic-like substances whereas higher MW (10 to >1000 kDa) were mainly recognized as proteins or probably heteroproteins.