by comparison with literature data for lipids analysis in the extracellular polymeric substances the methods proposed by smolders et al 1994 were used with modification pure fatty acids sigma aldrich were used as external standard freeze dried extracellular polymeric substance samples and fatty acid standards were weighed using an analytical balance and transferred into tubes with screw caps one milligramme of c15 fatty acid in 1 propanol was used as internal standard 1 5 ml of a mixture of concentrated hcl and 1 propanol 1 4 and 1 5 ml of dichloroethane were added into the tubes and heated for 2 h at 100 u00b0 c after cooling free acids were extracted from the organic phase with 3 ml water one millilitre of the organic phase was filtered over water free sodium sulphate into gc vials the lipids in the organic phase were analyzed by gas chromatography model 6890n agilent usa equipped with a fid on an hp innowax column size exclusion chromatography was performed with a superdex 75 10 300 gl column akta purifier system ge healthcare elution was carried out at room temperature using pbs at constant 0 4 ml min flow rate and detection was monitored by following the absorbance of the eluted molecules at 210 nm superdex 75 10 300 gl ge healthcare column separates molecules of 1 000 to 150 000 daltons da with a total exclusion volume of 7 9 ml measurement of the elution volume of dextran standards 1000 da 5000 da 12000 da 25 000 da and 50 000 da led to the calibration equation mw molecular weight of the molecule in dalton da ve elution volume in ml assayed at the top of the peak chromatogram profiles were recorded with unicorn 5 1 software ge healthcare peak retention.