Silica gel plates 60 f254

The identification and quantification of secondary metabolites was performed using the techniques reported by Cretu et al. [49 ]. Silica gel plates 60 F 254 of 10 × 20 cm (Merck^®, (Darmstadst, Germany) were used. Plates were activated by heating (TLC Plate Heater 3, CAMAG, Muttenz, Switzerland) at 100 °C for 3 min. After cooling to room temperature (22 °C ± 2), samples were applied (1 g/75 mL) with a sample applicator (ATS 4, CAMAG, Muttenz, Switzerland), and the plate was developed with 10 mL of solvent system in the automated development chamber (ADC 2, CAMAG, Muttenz, Switzerland) at 47% RH (relative humidity) (in equilibrium with a saturated solution of potassium thiocyanate KSCN) for 10 min. The plates were derivatized with a 1% natural products (NP) methanolic solution (2-aminoethyl diphenyl borate, Sigma-Aldrich^®, St. Louis, MO, USA) reagent and derivatization was performed in the immersion device (Chromatogram Immersion Device, CAMAG, Muttenz, Switzerland) at an immersion speed of 5 cm/s with an immersion time of 1 s. After derivatization, the plate was dried for 3 min at 100 °C (TLC Plate Heater 3, CAMAG, Muttenz, Switzerland). Images of each plate were documented using a TLC Visualizer (CAMAG, Muttenz, Switzerland) under visible light, at UV 254 nm and UV 366 nm.
Results were processed via the VisionCATS version 1.4.7.2018 software (CAMAG, Muttenz, Switzerland).

To analyze the hydrolytic products of beechwood xylan, 1.0% beechwood xylan in 2 ml of 0.2 M glycine-NaOH buffer was mixed with 10 μL of rXyn11A and 10 μL of rXyn11A-(His)₆, respectively. The reaction mixtures were incubated at 80°C for 24 h. The aliquots at different time intervals were withdrawn and analyzed by both thin-layer chromatography (TLC) and high-performance ion chromatography (HPIC). For analysis of TLC, 10 μL of the samples were spotted on the silica gel plates 60 F 254 (Merck, Darmstadt, Germany). A mixture containing xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose (X5) dissolved in water was used as the standard. Chromatography was developed in butanol-acetic acid-water (2:1:1, v/v) solvent system. After heating at 130°C for a couple of minutes in an oven, the plate was colorated by spraying with methanol-sulfuric acid mixture (95:5, v/v) and heated in an oven at 130°C for 5 min. The manipulations were modified from the method [45 (link)]. For analysis of HPIC, the samples at different time intervals (6 h, 12 h, 24 h) and standard xylooligosaccharides were analyzed with CarboPac^TM PA200 anion-exchange column (3 mm × 250 mm; Dionex, CA, USA), pure water as mobile phase (0.45 mL/min), and injection volumes were 10 μL. Sugar peaks were screened by ICS 5000 Electrochemical Detector (Dionex, CA, USA).

Ethanol 96%, n-hexane, and ethyl acetate (analytical grade) were obtained from Levanchimica srl (Bari, Italy). Silica gel (0.040–0.063 and 0.063–0.200 mm) and thin-layer chromatography (TLC, Silica gel plates 60 F254) were acquired from Merck (Darmstadt, Germany). TLC were visualized at 254 nm. Ultrasound-bath Branson model 3800-CPXH (Milan, Italy) was used to perform the extractions. Organic solutions were dried over MgSO₄ and evaporated on a rotary evaporator (Büchi RII) under reduced pressure. Melting points were obtained using a Gallenkamp melting point apparatus.

All reactions were carried out under a slightly positive pressure of dry argon by using standard Schlenk line techniques or in a glovebox (Braun, Labmaster SP). The oxygen and moisture concentrations in the glovebox atmosphere were monitored with an O₂/H₂O analyser to ensure both were always <0.1 p.p.m. Unless otherwise noted, all starting materials including dehydrated solvents were purchased from WAKO, KANTO, TCI or ALDRICH. Ammonium salts were prepared via reported protocols (Supplementary Methods). ArSnMe₃ were prepared through the reaction of (1) Me₃SnCl with corresponding aryl lithium or (2) Me₃SnLi with corresponding aryl bromides/iodides. Nuclear magnetic resonance (NMR) spectra were obtained on JEOL AL-300, AL-400 NMR and/or BRUKER AVANCE III HD spectrometers. Column chromatography was performed with silica gel 60 (230–400 mesh) from Merck and thin-layer chromatography was carried out on 0.25 mm Merck silica gel plates (60F-254).

Silica gel plates 60F₂₅₄ (Merck, Germany) were used for thin-layer chromatography (TLC) analysis with a solvent chloroform-methanol-water (CHC_l3-CH₃OH-H₂O) in the ratio of 65:35:10. Ginsenoside standards were used as a marker to identify GRg3-mix spots (Rg3(S), Rg3(R), Rk1, and Rg5). For the visualization of spots, 10% (v/v) H₂SO₄ was sprayed on the TLC plates, after which they were heated for 5 min at 110°C. For high-performance liquid chromatography (HPLC) of GRg3-mix, an HPLC system (Younglin Co. Ltd, Korea) with a quaternary pump, automatic injector, and single wavelength UV detector (model 730D) was used. Younglin AutoChro 3000 software was used for peak detection and integration. Prodigy ODS (2) C18 column (5 μm, 150 × 4.6 mm i.d.; Phenomenex, USA) with a guard column (Eclipse XDB C18, 5 μm, 12.5 × 4.6 mm i.d.) was used to perform the separation. The mobile phases used were B (water) and C (acetonitrile). The gradient elution started with 68% solvent B and 32% solvent C; the flow rate was kept at 1.0 mL/min. The absorbance was measured at 203 nm with an injection volume of 25μl for 28 min [35 (link)].