Tlc plate heater 3

A CAMAG semi‐automated system with VisionCat version 2.5 planar chromatography manager software (Camag, Muttenz, Switzerland) was used for HPTLC analysis of the sample extracts. The system comprises an automated TLC Sampler 4, TLC visualiser, automatic developing chamber ADC2, Camag derivatiser and TLC Plate Heater III. Methanol extracts were applied to the HPTLC silica gel 60 F₂₅₄ (20 cm × 10 cm) plates (Merck, Johannesburg, South Africa) as 8 mm bands. The plates were developed after optimising the mobile phase [ethyl acetate:formic acid:water (50:3:3)] for best resolution of the sample extracts. The chamber was saturated for 20 min at 33% relative humidity and 23 ± 2 °C, using 25 mL of the solvent. Prepared plates were developed by allowing the solvent (10 mL) to migrate to a distance of 70 mm from the plate edge. Once the developing solvent was optimised, three sets of plates were prepared. The first set was used for bioautography analysis to test against each of the four pathogens. The second was derivatised with 10% methanolic sulphuric acid to visualise the zones of inhibition, while plates in the third set were developed and later used to mark areas corresponding to inhibition zones on the plates for HPTLC–MS analysis.

The detection and quantification of major sugars in the honey samples was carried out as described by Islam et al. (Islam, Sostaric, Lim, Hammer and Locher, 2020a (link); 2020b (link)). In brief, aqueous methanolic honey solutions (1 mg/mL) were prepared and 2 μL of the solutions were applied as 8 mm bands at 8 mm from the lower edge of the HPTLC plate (glass plates 20 × 10 cm, silica gel 60 F254) at a rate of 50 nLs⁻¹ using a semi-automated HPTLC application device (Linomat 5, CAMAG). Standard solutions of glucose and fructose (both at 250 μg/mL) were also applied at 1 μL, 2 μL, 3 μL and 4 μL. Chromatographic separation was performed at ambient temperature in a saturated (33% relative humidity) automated development chamber (ADC2, CAMAG) to a distance of 85 mm using 1-Butanol:2-Propanol:Boric acid (5.0 mg/mL in water) in a ratio of 3:5:1 (v/v/v) as mobile phase. The plate was derivatised with 2.0 mL of Aniline-Diphenylamine-Phosphoric acid reagent (CAMAG Derivatiser) and the derivatised plate was heated for 10 min at 115 °C (CAMAG TLC Plate Heater III), then cooled to room temperature and analysed with the HPTLC imaging device under transmission white (T white) light. The chromatographic images were digitally processed and analysed using a specialised HPTLC software (visionCATS, CAMAG) which was also used to control the individual instrumentation modules.

To check the TLC profile of P. major extracts, chromatography was performed with two different solvent systems, i.e., (A) toluene: acetone: formic acid (78:22:0.15) and (B) 1,4 dioxane: xylene: propan-2-ol: 12.5% NH₃ (1:2:5:2). Samples were spotted by Camag Linomat 5 sample applicator on the HPTLC plates precoated with silica gel 60 F₂₅₄. Before development under the first solvent system (A), spotted samples were prederivatized with iodine vapor as described earlier.[5 ] Development was carried out with 10 ml of each solvent system in a Camag Twin Trough Chamber (10 cm × 10 cm) previously equilibrated with the mobile phase for 20 min at room temperature. After development, the plates were dried under warm air and subjected to derivatization by dipping in 5% sulfuric acid in methanol. TLC plates were further dried in a fume hood and then heated on a Camag TLC Plate Heater III (120°C, 3 min). The photographs were recorded by Camag Reprostar 3 under white light.

Developed plates were heated on the TLC Plate Heater III (CAMAG) at 100 °C for 3 min. After cooling, plates were dipped in the NP reagent (0.5% in ethyl acetate, immersion time 0 s, immersion speed 5 cm s⁻¹) using the Chromatogram Immersion Device). After drying, plates were immersed in the PEG solution, (5% in methylene chloride). The plates were heated at 100 °C for 5 min and chromatograms were documented at UV 366 nm.