Limomat 5

The method was developed using a CAMAG HPTLC system (CAMAG, Muttenz, Switzerland) in the following configuration: CAMAG Limomat 5, a software-controlled applicator of CAMAG, Muttenz, Switzerland); CAMAG Automatic Developing Chamber 2 (CAMAG, Muttenz, Switzerland), and CAMAG TLC Visualizer 2 (CAMAG, Muttenz, Switzerland). The software used was “VisionCATS” (version 3, CAMAG, Muttenz, Switzerland). An ultrasonic bath (Bandelin, Berlin, Germany) was used for better dissolution of the standard solutions.

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.