Tlc scanner 3

Gallic acid was quantified in EP extract and fractions by HPTLC fingerprinting [30 (link)]. Briefly, test solutions of EP extract/fractions were prepared at a 2 mg/mL concentration in methanol. Gallic acid solution was prepared at a concentration of 100 μg/mL in methanol. HPTLC Silica gel 60 F254 plate (Merck Life Sciences Ltd Pvt, Mumbai) with a dimension of 20 cm x 10 cm served as the stationary phase. Mobile phase was prepared by saturating the 20 cm x 10 cm Twin trough chamber with 10 mL of Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2) for twenty minutes. Using the Camag Linomat 5 sample applicator, outfitted with liquid nitrogen tank, 10 μL per sample/standard was sprayed in the form of bands onto the HPTLC plate at a distance of 1.5 mm from the plate base. Sample solutions were loaded onto the sample applicator with a 100 μL HPTLC syringe (Camag Linomat syringe 695.0014, Hamilton Bonaduz, Schweiz). The plate was air dried and developed in the Twin trough chamber up to a distance of 80 mm from the point of application. Using Camag TLC Scanner 3, the HPTLC plate was scanned densitometrically under a scanning speed of 20 mm/s at a single wavelength of 280 nm. Subsequently, UV absorption spectrum of bands corresponding to gallic acid was examined at the same wavelength.

HPTLC was performed on 20 × 10 cm normal phase HPTLC aluminum plates (Merck, Darmstadt, Germany) precoated with 200 mm layer thickness of silica gel 60 F₂₅₄. The samples were applied at 10 mm from the lower edge of the plates, 10 mm apart, and maximum fourteen samples with bandwidth 6 mm using Linomat IV sample applicator (Camag, Switzerland) fitted with a microliter syringe. Chromatography was performed in Automatic Developing Chamber (Camag, ADC 2) previously saturated with mobile phase vapors for 15 min at 28 ± 2°C with toluene : ethyl acetate (6.5 : 3.5 v/v) as mobile phase. TLC plates were dipped in freshly prepared anisaldehyde sulphuric acid reagent and heated at 105°C for 5 min. Densitometry scanning was performed at λ 592 nm using a Camag TLC scanner 3 with winCATS software.

Lipids were extracted with chloroform/methanol (2:1, v/v) for 30 min at room temperature and then washed with 0.9% NaCl. The solvent was evaporated and lipid extracts were dissolved in an appropriate volume of chloroform/methanol (1:1, v/v). Phospholipids were analysed by loading total lipids onto HPTLC plates (60F254, Merck, Darmstadt, Germany), which were developed in methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl (25:25:25:10:9, v/v) according to Heape et al., (Heape et al., 1985 (link)). To isolate and quantify glucosylceramide (Glucer), the lipid extracts were further analysed on HPTLC plates developed with chloroform/methanol (85:15, v/v) (Hillig et al., 2003 (link)). To isolate and quantify sterols, total lipids were loaded onto HPTLC plates developed with hexane/ethylether/acetic acid (90:15:2, v/v) as described previously (Laloi et al., 2007 (link)). Lipids were identified by co-migration with known standards and quantified by densitometry analysis (Macala et al., 1983 (link)) using a TLC scanner 3 (CAMAG, Muttenz, Switzerland) as described previously (Laloi et al., 2007 (link)).

Densitometric and spectrodensitometric measurements were performed using a TLC Scanner 3 (Camag, Switzerland) densitometer. The radiation sources were deuterium and tungsten lamps. The spectrodensitometric analysis was performed in wavelength range from 200 to 800 nm. The scanning speed was 20 mm/s, the slit dimension was 12 × 0.4 mm, and the data resolution was 100 µm/step. Densitometric scanning of plates with this densitometer and winCATS 1.4.2 software was performed at the optimal wavelength of 650 nm and 550 nm, after CuSO₄×5H₂O detection and the 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively.

A high-performance thin layer chromatography (HPTLC) method was used to standardize the 70% ethanol extract of A. javanica as described elsewhere (39 (link)). The chromatography was carried out on 10×10 cm precoated silica gel F₂₅₄ RP-HPTLC plate, using rutin as the reference standard. Several mobile phases were tried to get good resolution and separation of different compounds present in the A. javanica ethanol extract. Based on our observations, we selected acetonitrile and water in the ratio of 4:6 as suitable mobile phase to carry out the standardization of the extract. The standard along with samples was applied on the HPTLC plate by CAMAG Automatic TLC Sampler-4. The plate was developed under controlled condition in CAMAG Automated Developing Chamber-2 and scanned by CAMAG TLC Scanner-3 (λ=360 nm).