Tlc scanner

The stationary phase was 10 × 10 cm silica gel (Merck KGaA, Darmstadt, Germany). For the mobile phases, chloroform (Merck KGaA, Darmstadt, Germany), methanol (Merck KGaA, Darmstadt, Germany), ammonia 32% (VWR Chemicals, Radnor, PA, USA), and water (Carl Roth GmbH, Karlsruhe, Germany) solution was used in the ratio 161:75:5:10; detection with copper-II-sulphate 10% (Merck KGaA, Darmstadt, Germany), phosphoric acid 8% (Carl Roth GmbH, Karlsruhe, Germany), methanol 5% in water, and baking at 120 °C for 60 min [36 (link), 37 (link)]. The plate was scanned in a TLC scanner (CAMAG, Wilmington, NC, USA) and Rf value (retardation factor) and intensity (in arbitrary units (AU)) were compared to standards using the VisionCats program 2.4 (CAMAG, Wilmington, NC, USA). Digital data were processed with CorelDRAW 2017 (Corel Corporation, Ottawa, Canada).

HPTLC was performed on aluminium backed HPTLC plates (10 × 10 cm) coated with 0.2 mm layers of silica gel 60 F254 (E. Merck, Germany). Samples were applied on the plate with band width 6 mm employing Linomat IV sample applicator (Camag, Switzerland) fitted with a microlitre syringe. Linear ascending development of the plates to a distance of 80 mm was performed with mobile phase hexane: ethyl acetate: acetic acid (6.2: 2.8: 1.0 v/v/v) in a twin trough glass chamber previously saturated with mobile phase vapour for 10 min at 25 °C. The dried plate was scanned at the wavelength of 272 nm (λmax of galangin) using a Camag TLC scanner 3 with CATS 4 software.
For the calibration curve, a standard stock solution (1 mg/mL) was prepared by dissolving 1 mg accurately weighed galangin in methanol and diluting it to 10 mL in the volumetric flask. Working standard galangin solutions 3, 6, 9, 12 and 15 μg/mL of different concentrations; 50, 100, 150, 200 and 250 ng, respectively were prepared by diluting the stock solution. Each solution (10 μL) was applied on the plate and the plate was developed under predetermined conditions described above. The procedure was repeated thrice to plot a graph of response (peak area) and amount of galangin.

PQS was extracted from 10 mL of culture supernatant of P. aeruginosa using 10 mL acidified ethyl acetate (0.01% acetic acid) (57 (link)). The organic phase was transferred to a new tube, and the solvent was evaporated via a nitrogen stream. A normal-phase silica 60 F₂₅₄ high-performance thin-layer chromatography (HPTLC) plate was soaked in KH₂PO₄ (5%) and activated at 100°C for 1 h. The extracted PQS was dissolved in methanol and spotted on the HPTLC plates. The plate was placed in the HPTLC chamber presaturated with the mobile phase (2-propanol/ethyl acetate [4:6]). The commercial PQS (2-heptyl-3-hydroxy-4-quinolone; Merck) was used as a positive control and for quantifying the PQS produced by P. aeruginosa. The densitometry of the applied PQS spots was performed on a Camag TLC scanner at 254 nm (58 (link)).