Tlc visualizer

All fractions were examined by a thin layer chromatography (TLC) methodology on silica gel plates (TLC Silica gel 60 F₂₅₄, Merck KGaG, 64271 Darmstadt) using (a) Chloroform-Methanol (80:20, v/v) or (b) Toulene- Ethyl Acetate-Acetic Acid (80:18:2, v/v/v) as mobile phase. The bands were visualized at white light, 254 and 366 nm wavelengths before and after derivatization with anisaldehyde using CAMAG TLC Visualizer. Eluates were then pooled into major fractions based on the TLC profiles. After evaporation of methanol, the samples were left to air-dried and residues weighed.

Grinder (GT203840, Tefal, United Kingdom), Rotamixer (Hook and Tucker Instruments Ltd., United Kingdom), Grant XB22 ultrasonic bath (Grant Instruments, United Kingdom), Centrifugator (Centrifuge 5804 R, Eppendorf, Germany), electronic balance (Sartorius CP64, Sartorius AG, Germany), Freeze dryer (ModulyoD Freeze Dryer, Thermo Fisher Scientific, United Kingdom), NMR tube (VWR international Ltd., United States), Bruker Advance 500 MHz spectrometer (Bruker, Germany), HPTLC plates silica gel 60 F 254 (Merck, Germany), Linomat 5 (CAMAG, Switzerland) coupled with a 100 μL syringe (CAMAG, Switzerland) and compressed air with 60–90 psi., Automatic Developing Chamber ADC 2 (CAMAG, Switzerland), TLC Visualizer (CAMAG, Switzerland) Microwell plate Nunclon 96 well (Thermo Scientific Nunc, United Kingdom), GalaxyB CO₂ incubator (Scientific Laboratory Supplier Ltd., United Kingdom), water bath (LAUDA Aqualine AL 12, Germany), microscope (Olympus CK40 microscope, Japan), plate shaker (MS3 basic, IKA®, Germany) microplate reader (Infinite M200, Tecan, Switzerland), Multiwave Go (Anton Paar, Graz, Austria), ICP-OES SPECTROBLUE T1 (SPECTRO Analytical Instruments, Kleve, Germany).

HPTLC for whole aqueous extracts of garlic, clove, and Indian gooseberry was performed using Alliin, ellagic acid, and gallic acid respectively as standards. Butanol+2 –Isopropanol+ Acetic acid+ Water in the ratio of 3:1:1:1 [56 (link)], Toluene+ Ethyl acetate + Methanol+ Formic acid in the ratio of 3:3:0.8:0.2 [57 (link)] and Toluene + Glacial acetic acid + Ethyl acetate + Formic acid in the ratio of 2:2:4.5:0.5 were used as mobile phases for garlic, clove, and Indian gooseberry respectively. HPTLC of fresh whole aqueous extracts were developed at 254 nm and 366 nm and derivatized by spraying Ninhydrin (1 mgmL^-1) and ferric chloride (10 mgmL^-1) to confirm the presence of alliin, ellagic acid, and gallic acid in the fresh extracts of garlic, clove, and Indian gooseberry respectively. A 10x10 cm pre-coated silica gel 60 F₂₅₄ TLC plate was used to develop the chromatogram for HPTLC. A CAMAG HPTLC system equipped with a sample applicator and linomat V, CAMAG TLC visualizer was used to capture the images.

TLC experiments were performed on 20 cm × 20 cm TLC RP-18 plates from Merck, which were cut into segments of 10 cm × 10 cm. Exactly 2 mL of the sample solutions was taken for HPLC, purged with nitrogen gas at room temperature, and concentrated to 0.2 mL. The sample solutions for TLC were spotted with 5–20 μL (∼5–20 ng of each basic colorant), and standard solutions (5 μg/mL) of the basic colorants were spotted with 1–4 μL (∼5–20 ng of each basic colorant) using a 5 μL capillary glass tube at 20 mm from the bottom of the plate.
The plates were developed up to 7 cm in a saturated developing chamber (Camag, Muttenz, Switzerland) for 10 cm × 10 cm plates. The developing solvents were 2-butanone–methanol–5 w/w% Na₂SO₄ (1:1:1, v/v/v) (solvent system A) and 2-butanone–methanol–1.6 mol/L ammonium formate (pH 2.5) (7:2:7, v/v/v) (solvent system B). After development, the plates were dried at room temperature and observed under white light for PA, AO, and RB, as well as at 254 nm for RB and 366 nm for AO and RB. The plates were documented by using a TLC visualizer (Camag).

For analytical TLC experiments, Silica 60 F₂₅₄ TLC plates (average particle size 9.5 to 11.5 μm, aluminum sheets with fluorescence indicator, Merck KGaA, Darmstadt, Germany) were used. The samples of Pinaceae exudates were dissolved in acetone to a concentration of 10 mg/mL. As reference substances for the substance class of diterpene resin acids, neoabietic acid and dehydroabietic acid were used (both dissolved in acetone, neoabietic acid to a concentration of 0.5 mg/mL, dehydroabietic acid to 1 mg/mL). Pinoresinol (as representative of lignans) was dissolved in methanol to a concentration of 1 mg/mL; ferulic acid (as representative of hydroxycinnamic acids) was also dissolved in methanol to a final concentration of 2.5 mg/mL. Five microliters of each sample solution were applied by a CAMAG Automatic TLC Sampler 4 (ATS4). Development was done in the CAMAG Automatic Developing Chamber (ADC2) with chloroform-methanol-trifluoroacetic acid (97 + 3 + 0.1) as mobile phase. The CAMAG TLC Visualizer was used to interpret and photograph the developed and dried plates under white light and at wavelengths 254 nm and 366 nm. The constituents showed improved visibility after derivatization with anisaldehyde/sulphuric acid solution, which was applied with the help of the CAMAG Chromatogram Immersion Device (III) (CAMAG, Muttenz, Switzerland).