Gel-filtration column chromatography, amino acid analysis and carbohydrate determination

5 Gel-filtration column chromatography, amino acid analysis and carbohydrate determination 

The concentrated microbubble surfactant sample (0.1 ml) was applied directly to a column (1.5 cm I.D. x 35 cm) of Sephadex 

which had been prewashed with 95% ethanol over a period of 1 week and thereafter equilibrated with 95% high-purity ethanol/5% water. The column was eluted with the equilibrating solvent mixture, at a flow rate of 1 ml/4 min. The volume per fraction was 0.65 ml, and each fraction was combined with an added 0.7-ml aliquot of the same solvent mixture before being analyzed on a Beckman Model 25 spectrophotometer at 230 and 280 nm. 

Selected fractions were pooled and prepared for automated amino acid analysis by (complete) hydrolysis in 5.7 N HC1 in evacuated, sealed ampules for 24 hr at 110°C. Analyses were performed on a Beckman 120C amino acid analyzer. 

Other fractions were prepared for carbohydrate analysis by hydrolysis (of the surfactants glycosidic bonds) in 2 N HC1 in sealed ampules for 6 hr at 100°C (ref. 331). Monosaccharide analyses were performed on a programmed Tracor 985 HPLC system employing a Bio-Rad HPX-87C cation-exchange column for monosaccharide separation. 

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