Tlc silica gel 60 w f254s

Thin-layer chromatography (TLC) profile of the most active extract was carried out using pre-coated TLC plates (Merck TLC Silica Gel 60 W F254s–normal phase) and methanol–dichloromethane (1:13) as solvent system through one-way ascending technique. The developed chromatogram was visualized under UV light at 365 and 254 nm and retention factor (R_f) values of each band were calculated (Jayashree et al., 2014 (link)). Direct bioautography of the chromatogram was further performed by covering the surface of the TLC plate with a soft agar (MHA 0.8% agar) seeded with 1 × 10⁶ CFU/mL of S. aureus ATCC BAA-44 and incubated overnight. The zone of inhibition of antibacterial components was visualized by detecting mitochondrial reductase enzyme activity of viable cells using a resazurin reduction-based reaction. The metabolically active viable cells convert resazurin (blue colored compound) to its reduced form resorufin (pink colored compound). Thus, the zone of inhibition appears as blue spot over a pink background (Choma and Grzelak, 2010 (link); Choma and Jesionek, 2015 (link); Jesionek et al., 2015 (link)).

Metabolic profiles of the test compound in fillet and liver/hepatopancreatic tissue of trout and carp were determined. Aliquots of acetonitrile extracts (approximately 5 Bq) were applied to a normal‐phase silica gel 60 thin‐layer chromatography (TLC) plate with fluorescence indicator (Merck TLC Silicagel 60WF254s, 20 × 20), along with non‐labelled parent reference compound and two reference metabolites (each approximately 20 µg). The TLC plates were developed in ethyl acetate/2‐propanol/water (65:25:15 by volume) and then dried and analysed by a BAS‐1000 Bio‐Imaging Analyser (Fujifilm, Tokyo, Japan) and AIDA software (Raytest, Straubenhardt, Germany). Non‐labelled standards were visualised by UV light.