A simple thin layer chromatography based method for the quantitative analysis of biosurfactant surfactin vis-a-vis the presence of lipid and protein impurities in the processing liquid

Abstract

Abstract The Bacillus surfactin lipopeptides, widely used as potent biosurfactants, have been increasingly researched for diverse applications, necessitating quantification during the production and purification stages. Lipopeptides are typically quantified by Reversed Phase-High- Performance Liquid Chromatography and High Performance Thin Layer Chromatography but these have inherent limitations of long processing times, high capital investment and high solvent consumption. In this paper, an economical and accessible Thin Layer Chromatography (TLC) method for the analysis of surfactin lipopeptides has been developed, its estimation capability in terms of precision and accuracy thoroughly substantiated, and its robustness under diverse solvent and sampling conditions, as well as its specificity in the presence of multiple bioproducts, comprehensively verified. A linear relationship was observed between surfactin concentration and the area formed by surfactin bands on TLC plates with surfactin concentrations in the range of 0.5 g/L and 3 g/L. The TLC analytical technique was suitable for surfactin analysis when surfactin was dissolved in different solvents and when surfactin was spotted by multiple spotting on TLC plates to accuracy and precision of up to 98% and 95% respectively. Based on inter-day error analysis, reproducible results could be obtained with high accuracy and precision (98% and ≥84% respectively) were obtained when analysis was carried out on different days (two weeks apart). While this technique has been confirmed specifically as an investigative tool for the analysis of surfactin lipopeptides, visualisation of peptides, lipids and the fengycin suggests its further application to the quantification of co-existing impurities and other lipopeptide bioproducts.