Tlc plate heater

The analytical standard of saccharin (>99.5%) was obtained from Aladdin (Shanghai, China). Methanol (≥99.8%), ethyl acetate (≥99.5%), acetic acid (≥99.5%), l-ascorbic acid (≥99.8%), sodium hydroxide (≥99.5%), sodium borohydride (≥96%), silver nitrate (≥99.8%), glass fiber membrane, aluminum foil and beakers were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Class backed silica gel F₂₅₄ plates (20 × 10 cm, analytical grade, serial No. 4022536067940) were supplied by Merck (Darmstadt, Germany). Prior to using, each plate was pre-washed with Methanol, followed by drying at 120 °C for 20 min on a TLC Plate Heater (CAMAG, Muttenz, Switzerland). Four beverage samples (1-iced tea, 2-cola, 3-salt soda water, 4-alcoholic drink) were purchased at local supermarkets.

The compounds in extracts were separated by using HPTLC technique. Briefly, 5 µL/band of the Centella extracts and 4 µL/band of the standards were applied on the Merck silica gel 60 F₂₅₄ glass HPTLC plates (20 × 10 cm) by a semi-automatic sampler Linomat 5 (Camag; Muttenz, Switzerland) using 8 mm bands, 11.4 mm apart, 8 mm from the lower edge, and 20 mm from the left plate edge. The plates were developed in the chamber saturation containing mobile phases. For separation of triterpenoids, the mobile phase was dichloromethane: methanol: water (14:6:1, V/V/V)¹⁶ . For separation of flavonoids, ethyl acetate: methanol: water (100:25:10, V/V/V) was used as mobile phase. The development was done with a migration distance of 70 mm using an automatic development chamber ADC2 (Camag; Muttenz, Switzerland). For observation of triterpenoids, the plate was dipped with sulfuric reagent using Chromatogram Immersion Device III (Camag; Muttenz, Switzerland) and then heated at 100 °C for 3 min on the TLC Plate Heater (Camag; Muttenz,Switzerland). The derivatized plates were visualized under white light using visualizer 2 (Camag; Muttenz, Switzerland). For detection of flavonoids, the chromatogram was heated at 110 °C for 3 min and then was dipped in NP/PEG reagent. The derivatized plates were visualized under UV light at 366 nm using visualizer 2 (Camag; Muttenz, Switzerland).

All extracts were re-dissolved in sufficient amounts of their original extraction solvents (EtOAc or MeOH) to reach a concentration of 1 mg/mL and applied on silica gel HPTLC plates (20 × 10 cm, F254) purchased from Merck (Darmstadt, Germany). A CAMAG-HPTLC system consisting of an automatic TLC sampler (ATS4), derivatizer (version 1.0 AT), TLC plate heater (version III), and TLC visualizer (CAMAG, Muttenz, Switzerland) was used. For sample application, 15 μg of EtOAc extracts were spotted in 6 mm bands. On each plate, 16 bands were applied at 10 mm from the bottom and 20 mm from the lateral edges of the plate with 8 mm distance between bands. A pool of all of samples was applied on each plate at the right edge of the plate as a quality control sample for normalization purposes. The samples were separated with a mobile phase of chloroform-acetone-formic acid (8.5:0.65:0.85, v/v/v) and developed for 75 mm from the application point. The chamber was pre-saturated for 25 min. Developed HPTLC plates were sprayed with 2 mL of anisaldehyde-H₂SO₄. Subsequently, the plates were placed on a TLC plate heater at 100 °C for 3 min. Images of the derivatized plates were recorded using a TLC visualizer at 366 nm before and after derivatization.

A CAMAG high performance thin layer chromatography (HPTLC) system equipped with an automatic TLC sampler (version 4), a derivatizer device (version 1.0 AT), a TLC plate heater (version III) and a TLC visualizer (CAMAG, Muttenz, Switzerland) was used for analysis. The separation was carried out on 20 × 10 cm F254 silica gel 60HPTLC plates (Merck, Darmstadt, Germany). Five μL of the solution were spotted in 6 mm bands with a distance of 10 mm from the bottom, 20 mm from the left and right edge, the distance between bands was 8.8 mm, allowing 18 samples per plate. Two different mobile phases were used for the analysis: ethyl acetate, formic acid, acetic acid and water (100:11:11:27, v/v/v/v) and ethyl acetate, methanol, formic acid and water (20:2.7:0.5:2, v/v/v/v) with a chamber saturation time of 20 min. The developed plates were dried with an air-stream at room temperature and scanned with the CAMAG TLC visualizer at 254, 366 nm and white light. The plates were also derivatized with 2 mL of a solution of 1g of 2-aminoethyl diphenylborinate in 100 mL of methanol (NP reagent) and after 5 min of reaction viewed at 366 nm with a CAMAG TLC visualizer.

TLC was performed on 10 × 20 TLC Si60 F254 glass-backed plates (105715) from Merck (Darmstadt, Germany). The plant extracts were applied using the Linomat 5 automatic applicator from CAMAG (Muttenz, Switzerland). TLC plates were developed with the mobile phase: F1—ethyl acetate: methanol: water, 70:20:10 (v/v), using the DS horizontal sandwich chamber from Chromdes (Lublin, Poland). The mobile phase F1 was optimized to find the best separation of both polar and nonpolar components of S.rubriflora extract. Then, the plates were documented using TLC Visualiser 2 controlled via WinCATS software from CAMAG (Muttenz, Switzerland). Next, they were subjected to chemical derivatization or bioautography using a TLC automated spraying device or TLC sprayer from Merck (Darmstadt, Germany). A TLC Plate Heater from CAMAG (Muttenz, Switzerland) was used to heat the TLC plates after derivatization, when it was necessary.