Derivatizer

Performance of the P-YES was based on Schönborn et al. [17 (link)]. Yeast was adjusted to 1000 ± 200 FAU in fresh exposure medium. With an automated spraying chamber, Derivatizer (CAMAG) with red nozzle and spraying level six, 2 mL of yeast culture was sprayed onto HPTLC plates before or after chromatography. The HPTLC plates were incubated for 3 h at 30 °C in plastic boxes with water-saturated paper towels to maintain humidity above 80%. After incubation, the plates were dried with a hair dryer set on low heat and fan speed for 3–5 min. The indicator, 2 mL 0.5 mg/mL MUG in lacZ buffer, was then sprayed onto the plates with the Derivatizer (blue nozzle, level six) and plates were incubated at 37 °C for 20 min. The plates were dried again with a hair dryer. For cases in which fluorescent signal on a plate was uniformly less than expected, plates were exposed to NH₃ vapor, which enhanced the signal of the fluorescent product of MUG, 4-methylumbelliferone (4-MU) [13 , 30 (link)]. Images were collected with the TLC Visualizer (CAMAG) with illumination at 366 nm for 550 ms and processed for peak height and area with VisionCats v2.4 (CAMAG).

Analyses of all obtained ethanolic extracts in 70% ethanol of the drone brood samples were performed on HPTLC Silica Gel 60 F₂₅₄ plates (20 cm × 10 cm) purchased from Merck (Darmstadt, Germany). Forty microliters of each drone brood homogenate extract were applied to the plate as 11 mm bands from the lower edge of the plate at the rate of 200 nL/s using a semi-automated HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland). The chromatographic separation was performed in a chromatographic tank saturated for 20 min with the mobile phase composed of chloroform: ethyl acetate: formic acid [5:4:1 v:v:v], and developed to a distance of 85 mm. The obtained results were documented using an HPTLC imaging device (TLC Visualizer, CAMAG, Muttenz, Switzerland) under white light, 254 and 366 nm. In addition, each plate was derivatized with 0.05% DPPH reagent (in methanol) using an automated TLC Derivatizer (CAMAG Derivatizer, Muttenz, Switzerland). 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).

A 7 mM solution of ABTS was created by dissolving 38.41 mg of ABTS into 8 mL of demineralized water. A 2.45 mM solution of sodium persulfate (K₂S₂O₈) was made by dissolving 66.2 mg of K₂S₂O₈ into 10 mL of demineralized water. Then, 1 mL of 2.45 mM K₂S₂O₈ was added to 7 mM ABTS. The obtained mixture was filled up to 10 mL with water and left in the refrigerator (4 °C) for 16 h. After this time, developed and dried HPTLC plates were sprayed with the use of an automated spraying device, the Camag Derivatizer. Next, the plates were stored for 6 min in a dark place and analyzed in daylight. Bright spots of compounds with antiradical properties were observed on the blue background of the chromatograms [55 (link)].
The documentation of chromatograms and bioautograms was performed using the TLC Visualizer (Camag, Muttenz, Switzerland).

Six chromatograms were prepared in parallel. For each assay, respective positive controls were applied above the solvent front at the upper plate edge of the neutralized, dried chromatogram [6 (link)]. For example, three bands of an aqueous saccharolactone solution (100, 150 and 200 ng/band) were applied for the β-glucuronidase assay. Each plate was subjected to the respective assay solutions or suspensions by piezoelectric spraying (placed on a filter paper sheet, if not stated otherwise, yellow nozzle, level 6, Derivatizer, CAMAG). After the first spraying, the bottom side of the nozzle was manually dried with a lint-free tissue to avoid a dropping on the plate during the second spraying. For incubation (at 37 °C, if not stated otherwise), each plate was placed horizontally in a humid poly-propylene box (premoistened for 30 min at room temperature with 35 mL water spread on filter papers aligned on walls and bottom). Drying was performed in a stream of cold air (hair dryer). If not stated otherwise, documentation was performed at white light illumination in the reflectance mode (TLC Visualizer, CAMAG). Aliquoted enzyme and l-DOPA substrate solutions were stored at −18 °C, whereas other solutions were stored in the dark at 4 °C; all were stable for several months.

Analysis of polyphenolic profiles were performed on HPTLC Silica Gel 60 F254 plates (20 cm × 10 cm) purchased from Merck (Darmstadt, Germany). A 20 g aliquot of each honey sample was dissolved in 100 mL of acidified distilled water at room temperature. The solutions were then applied to a C-18 Sep Pack Cadrigde (Waters) conditioned with acidified water. Polyphenols were leached from the columns with methanol directly in a round bottom flask. A 5 µL of such prepared extract was applied to the plate as a 8 mm bands 11 mm from the lower edge, with a speed of 50 nL/s using a semi-automated HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland). The chromatographic separation was performed in a chromatographic tank saturated for 20 min with the mobile phase composed of chloroform: ethyl acetate: formic acid (50:40:10 v/v/v/v), and developed to a distance 85 mm. The results obtained were documented using a HPTLC imaging device (TLC Visualizer, CAMAG) under white light, 254 and 366 nm. In addition, each plate was derivatized with p-anisaldehyde/sulfuric acid reagent using an automated Derivatizer for TLC plates (CAMAG Derivatizer). After derivatization, the plates were heated at 110 °C for 10 min and imaged under white light and 366 nm. The obtained chromatographic images were analyzed using HPTLC software (Vision CATS, CAMAG).

Comparative analysis of polyphenolic, sugar and amino acid profiles for water extracts of the drone brood and royal jelly samples were performed on HPTLC Silica Gel 60 F₂₅₄ plates (20 × 10 cm) purchased from Merck (Darmstadt, Germany). Chosen extracts of drone brood and royal jelly showing the best results in previous analyses were applied to the plate (40 µL for polyphenolic, 5 µL for sugars and amino acids) as 10 mm bands from the lower edge of the plate at the rate of 100 nL/s using a semi-automated HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland) (Table 1).
The chromatographic separation was performed in a chromatographic tank saturated for 20 min with the mobile and developed to a distance of 70 mm. The obtained results 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). After derivatization, the plates were imaged under white light and 366 nm. The obtained chromatographic images were analyzed using the HPTLC software (Vision CATS, CAMAG).

A thin layer chromatography was performed to reveal the chemical profiles of the soy isolates extracts. The Silica gel 60 F254 plate was first documented under UV 254 nm light without chemical treatment with the TLC Visualizer 2 (CAMAG, Muttenz, Switzerland). Then, the plate was sprayed with 2 mL of a NP solution (1.0 g of 2-aminoethyl diphenylborinate in 100 mL methanol) using the Derivatizer (CAMAG) with green nozzle at level 4 and followed by a visualization at UV 366 nm. The second revelation was done with 2 mL of a PEG solution (5.0 g of PEG 400 in 100 mL ethanol) with blue nozzle at level 3. The bioautogram was documented under UV light 366 nm.