Reversed-phase HPLC for Mono-rhamnolipid Quantification

Reversed-phase chromatography was performed for analyzing mono-rhamnolipid concentrations based on a method developed earlier (Behrens et al., 2016 (link); Tiso et al., 2016 (link)). For sample preparation, the supernatant was mixed 1:1 with acetonitrile and stored at 4°C overnight. Subsequently, the mixture was centrifuged at 16,500 g for 2 min. All samples were filtered with Phenex RC syringe filters (0.2 μm, Ø 4 mm, Phenomenex, Torrance, USA). The HPLC system Ultimate 3000 with a Corona Veo Charged Aerosol Detector (Thermo Fisher Scientific, Waltham, MA, USA) was used. For separation, a NUCLEODUR C18 Gravity 150 × 4.6 mm column (particle size: 3 mm, Macherey-Nagel GmbH & Co. KG, Düren, Germany) was used. The flow rate was set to 1 ml min⁻¹ and the column oven temperature was set to 40°C. Acetonitrile (A) and 0.2% (v/v) formic acid in ultra-pure water (B) were used as running buffers. The method started with a ratio of 70% buffer A: 30% buffer B and a linear gradient was applied to reach a ratio of 80:20% in 8 min. The acetonitrile fraction was increased linearly from 80 to 100% between 9 and 10 min and decreased linearly to 70% between 11 and 12.5 min. The measurement was stopped after 15 min.