Purospher rp 18e column

The concentrations of the sugar esters were quantified by high-performance liquid chromatography using a Waters pump (Waters 1525) with refractive index detector (Waters 2414). A Purospher RP-18e column (5 µm×250 mm×4.0 mm², Merck) was used, and the mobile phase was a mixture of methanol and water (85∶15 v/v).

Reaction were examined by TLC on Silica gel 60 F254 aluminum sheets using chloroform: methanol: acetic acid: H₂O (75:15:5:3 v/v/v/v) as the eluting system. The compounds were colored by spraying a color agent with p-anisaldehyde–aceticacid–95% ethanol–sulfuric acid (12.5:2.5:225:12.5 v/v/v/v) and visualized by heating at 105 °C for 15 min. HPLC analysis was performed by HPLC using a Waters pump (Waters 1525, Waters, Sutton, MA, USA) with evaporative light scattering detector (ELSD, Waters 2424). A Purospher RP-18e column (5 × 250 × 4.6 mm², Merck, Darmstadt, Germany) was used, and the mobile phase was a mixture of methanol and water (90:10 v/v) at a flowrate of 1 mL min⁻¹. The yield of SML was calculated from the HPLC data.

The powdered material (0.5 g DW) of each sample was extracted with 50% methanol. After 4 h, the extract was centrifuged for 15 min at 4500 rpm (MPW 342; MPW Med. Instruments, Poland). Then, the supernatant was collected and the residue was subjected to repeated extraction. The obtained supernatants were combined and evaporated to dryness at 22 ± 2 °C (Rotavapor R-114; Büchi, Germany). The dry residue was dissolved in 2 mL of methanol with HPLC-grade. The HPLC analyses were performed using a Merck-Hitachi apparatus. The detection was set at 270 nm. A Purospher® RP-18e column (250 × 4 mm, 5 µm; Merck) was used at 35 °C. Isocratic elution was applied according to Kimura et al. (2000 (link)). The mobile phase used was composed of 0.1 M sodium dihydrogen/methanol (97:3, v/v). The flow rate was set at 1 mL/min, and the injection volume was 10 µL.