Tlc silica gel 60 f 254 aluminum plates

Thin-layer chromatography (TLC) was applied to examine the components of the plant extract, and retention factor (Rf) values were determined. To this aim, the components of P. granatum ethanol extract were examined. TLC silica gel 60 F 254 aluminum plates (Merck, Frankfurt, Germany) were cut into 10 × 10 cm sizes. A 1 cm gap was left from the edges and bottom of the plates. In the study, chloroform/methanol solvents were used in a ratio of 7:3, respectively. The sampling was made with capillaries in the form of 5 spots and the spots were dried by a dryer. The plate was placed in the solvent tank obliquely so that 0.5 cm from the bottom remained in the solvent mixture. The run was continued for about 15 min and then the plates were left to dry at room temperature, after which, the Rf values were determined [51 (link),52 (link)]. Rf values were calculated using the formula Rf = x1/x0 (x1 = distance traveled by the solute, x0 = distance traveled by the solvent).

The high-performance thin-layer chromatography (HPTLC) system (Camag, Muttenz, Switzerland) consisted of a Limomat 5 connected to compressed air, an Automatic Developing Chamber 2 (ADC 2), and a TLC Visualizer 2 supported with visionCATS software. An analytical grade of ferulic acid (Merck, Germany) was used to prepare 400 µg ml⁻¹ in methanol as a calibration standard against dry leaves’ extracts of micropropagated plants. TLC silica gel 60 F₂₅₄ aluminum plates (10 × 20 cm, Merck, Darmstadt, Germany) were used for the TLC analysis. Standard and samples were applied to plates as 8 mm bands, 8 mm from the bottom edge of the layer, using Linomat 5. A ferulic acid standard solution of 400 µg ml⁻¹ of a volume of 2–9 µL was applied against 2, 4, 6, 8, 10, 12, and 14 µL of dry leaves’ extract. A mixture of ethyl acetate/methanol/water (100:13.5:10, v/v/v) was used as the mobile phase. Plates were developed at room temperature and 60% humidity in an ADC2 automated development chamber. The migration distance of the mobile phase was 70 mm with a development time of 9 min. After development, the chromatogram was visualized and photographed by Visualizer 2 at 254 and 366 nm. The ferulic acid content in the samples was expressed as mg g⁻¹ DW.

Chromatographic separations were carried out on 4 × 2 cm TLC Silica gel 60 F₂₅₄ aluminum plates (Merck). The complex lipid mixture was applied on the edge of the plates (at the sample spotting line) using a glass capillary. The samples were allowed to run for 4 cm in a 8 × 4 cm glass chamber, previously saturated with the mobile phase. Two distinct mobile phases were used - cyclohexane:ethyl acetate (4:1) and chloroform:methanol:water (75:25:2.5) - which respectively allow for the separation of neutral lipids and the separation of phospholipids by head group polarity. The plates were then left exposed to air to allow for evaporation of the mobile phase residual. For differential detection of neutral lipids and phosphorous-containing lipids, iodine vapor and a modified Dittmer–Lester reagent13 (link) were employed for dual staining. The plates were scanned immediately after drying.