Hptlc silica gel 60 f254 plate

Analysis of all obtained ethanolic extracts from propolis samples was performed on HPTLC Silica Gel 60 F₂₅₄ plates (20 cm × 10 cm) purchased from Merck (Darmstadt, Germany). Extracts (2 µL) were applied to the plate as 9 mm bands from the lower edge of the plate at a rate of 100 nL/s using a semi-automated HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland).
The chromatographic separation was carried out in a chromatographic tank saturated for 20 min with the mobile phase and developed to a distance 70 mm. The results obtained were documented using an HPTLC imaging device (TLC Visualizer, CAMAG) under white light, UV 254, and 366 nm. In addition, each plate was derivatized using an automated Derivatizer of TLC plates (CAMAG Derivatizer) with p-anisaldehyde reagent. After derivatization, the plates were imaged under white light and 366 nm. The obtained chromatographic images were analyzed using HPTLC software (Vision CATS, CAMAG, Muttenz, Switzerland).

The analyses were performed with the use of the CAMAG (Muttenz, Switzerland) HPTLC chromatography set, consisting of a semi-automated sample applicator (Linomat 5), an automatic developing chamber (ADC 2), a derivatizer and a visualizer. The extract samples were applied on the HPTLC plate (HPTLC Silica Gel 60 F254 plates, 20 cm × 10 cm, Merck, Darmstadt, Germany) in a volume of 5 µL, as 8 mm wide bands. Two different systems for the mobile phase and derivatization reagent were used: A–mobile phase: ethyl acetate, acetic acid, formic acid, water (10:1.1:1.1:2.6) and derivatizing agent: Natural Product Reagent/Polyethylene glycol 400 (NP/PEG); B—mobile phase: chloroform, ethyl acetate, formic acid (5:4:1) and derivatizing agent: p-anisaldehyde reagent with heating in 110 °C (10 min). After derivatization, plates were photographed in visible light and UV (366 nm). The obtained images were analyzed using HPTLC software (Vision CATS, CAMAG, Muttenz, Switzerland).

All analyses were conducted by a CAMAG TLC system (CAMAG, Switzerland) containing an automatic TLC sampler 4 with a 25 μL syringe, a TLC visualizer equipped with visionCATS (version 2.5) software, a chromatogram immersion device III, and a TLC plate heater III. The HPTLC separations were performed on 20 × 10 cm HPTLC silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). The samples and mixed standards solution (4 μL) were applied as 6 mm bands and 10 mm from the bottom edge on the HPTLC plates. The syringe was washed with methanol three times between the applications of each substance. Firstly, the applied HPTLC plate was pre-saturated with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) for 25 min in a glass twin trough chamber and developed over a path of 60 mm for the separation of high-polarity components. After the plate was dried under an airstream, the same plate was pre-equilibrated with dichloromethane-methanol (300:1, v/v) for 15 min in a twin trough chamber and developed to the distance of 90 mm to isolate the low-polarity compounds. Then, the developed plate was dried in a stream of cool air, immersed in 10% sulfuric acid in ethanol solution for one second, heated for 10 min at 105 °C on a TLC plate heater, and subsequently recorded under UV (366 nm) and white light.

TLC silica plates (aluminium HPTLC silica gel 60 F₂₅₄ plates, Merck, Darmstadt, Germany) were cut into 5 cm × 5 cm or 5 cm × 10 cm pieces according to the purpose of the experiment. To measure the minimum detectable range, 5 μL of various concentrations (50–10,000 ppm) of carbofuran solution was spotted on a TLC plate and developed in 5:5 ethyl acetate/hexane. After the TLC plate was developed, it was allowed to dry for 3–5 min, and then spots were detected under UV light at 254 nm. For the qualitative evaluation of carbofuran using TLC, the concentration of carbofuran in the sample was 5000 ppm. Two millilitres of the culture extract were volatilized using a centrifugal vacuum concentrator (HyperVac, GYROZEN, Gimpo, Korea) connected to a cooling trap device (HyperCool, GYROZEN, Gimpo, Korea) at 37 °C until the solvent was completely evaporated. The residue was dissolved in 5 mL of acetone to make a 50-fold concentrated sample. Five microlitres of concentrated extracts were spotted on the plate with 5000 ppm carbofuran in acetone as a reference.