Type 60 f254

FTIR and SEM were applied to examine the new adsorbent (a Hitachi S-4160 scanning electron microscope). The concentration of metal ions was determined via inductively coupled plasma (ICP-AES) (Varian liberty 150 XL).
The Perkin-Elmer Frontier was applied to take FT-IR spectra. Dimethyl sulfoxide (DMSO) or chloroform (CDCl₃) were applied as solvent materials for routine spectra of NMR at ambient temp. on an Avance TM 400 spectrometer. It is important to note that all chemical modifications are stated in ppm δ relative to the trace resonance of protonated chloroform, CDCl₃ dimethyl sulfoxide, DMSO, and external 85% aqueous H₃PO_4, as (δ 7.25 ppm), (δ 77.0 ppm), or (δ 2.50 ppm), (δ 39.51 ppm) and (δ 0.0 ppm), respectively.
A mass spectrum was examined by using a GC Finnigan MAT SSQ-7000 type of mass spectrometer. The progress of the reactions and examination of the compound’s purity was completed. The latter was done via thin Layer Chromatography (TLC) on silica gel-precoated aluminium sheets (Type 60, F 254, Merck, Darmstadt, Germany) with an eluent of petroleum ether (60–80 °C)/ethyl acetate, and the spots were identified by exposure to UV light at a lamp at λ₂₅₄ nm for several seconds. The chemical names of the synthesized chemicals are designated using the IUPAC nomenclature. Conventional drying and purification processes were utilized.

All chemicals were from Sigma-Aldrich or Acros (USA) and were used without further purification. Compounds BB-A and BB-B were provided by prof. Preobrazhenskaya from previous collaborations2 (link) and ee of BB-A was accepted as provided. Analytical TLC for checking the homogeneity of the compounds was made using TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) with chloroform-methanol as a mobile phase and the spots were detected by exposure to a UV-lamp at 254 nm. The structures of all synthesized compounds were confirmed by 400 MHz ¹³C-NMR spectra (Varian VXR-400) and high-resolution ESI mass-spectrometry (microTOF-Q II, Bruker Daltonics GmbH). ¹³C-NMR spectra were recorded in DMSO-d₆ or in CDCl3. Purity was checked by HPLC (column Kromasil C-18, 250 × 4.5 mm, PDA, mobile phase 0.03% HCOOH (pH = 3) or 0.03% NH4COOH pH = 7.8, gradient with acetonitrile: from 10 to 95 w/w%. Final products were purified chromatographically. On the first stage they were purified by column chromatography on silica gel Merck 60 (using ISCO instrument with detector at 254 nM). About 10 or 20 mL of sorbent was used for 100–250 mg of reaction mixture. The gradient CHCl3–MeOH–NH4OH (0.1%) was used for elution to give compounds of 60–80% purity. For further purification, pTLC method on Merck 40F 254 plates was used with mobile phase from CHCl3–MeOH–NH4OH (0.1%).

The CtCel7 hydrolysis product profile was qualitatively determined by thin layer chromatography (TLC) after incubation for various time periods (10, 30, 60, 120, and 180 min) with glucan, xylan, PASC, and filter paper as the substrates (Cheng et al., 2012 (link)). The hydrolysis products at various periods and the oligosaccharide mixture standard (Gentaur, Kampenhout, Belgium) were individually spotted on a silica plate (type 60 F254; Merck, Germany), and the plate was subsequently developed with an ethyl acetate/methanol/water/acetic acid (4:2:1:0.5, v/v/v/v) solvent system in a covered chamber (Hua et al., 2018 (link)). The results were visualized by dipping into a solution containing 2% (w/v) N-phenylaniline, 2% (v/v) phenylamine, and 85% (v/v) phosphoric acid in acetone, followed by heating at 85°C for 15 min (Li W. et al., 2018 (link)). Commercial TlCBHI and TlXyn were used as controls under their individual optimal reaction conditions for 180 min. The relative proportions of different oligosaccharide products were evaluated through gray-value analysis of the TLC results using ImageJ software.

Melting points (°C) were determined with a Kofler hot bench and were uncorrected. Analytical thin-layer chromatography (TLC) on silica-gel-precoated aluminum sheets (Type 60 F254, 0.25-mm thickness; from Merck, Darmstadt, Germany) was employed to follow the progress of the reactions and to check the purity and homogeneity of the synthesized products. Nuclear-magnetic-resonance spectra (NMR) were recorded on a Brucker DRX-400 Avance spectrometer (at 400 MHz for ¹H and 100 MHz for ¹³C), using dimethylsulfoxide (DMSO-d₆) as the solvent and tetramethylsilane (TMS) as internal standard. The chemical shifts are expressed in parts per million (ppm) and the multiplicities of ¹H NMR signals were designated as follows: s: singlet; d: doublet; t: triplet; q: quartet; and m: multiplet. Coupling constants were expressed in hertz (Hz). High-resolution mass spectra (HRMS) were carried out by using a Bruker micrOTOF-Q II spectrometer (Bruker Daltonics) in positive electrospray ionization time-of-flight at UCA Clermont Ferrand, France.

All thin layer chromatography (TLC) analyses were performed on type 60 F 254 silica gel n silica-gel-precoated aluminum sheets (Type60 F254, 0.25-mm thickness; from Merck, Darmstadt, Germany) with detection using a UV lamp. Melting points were determined on the Kofler and Buchner bench and were not corrected. Infrared (IR) spectra were recorded using a Perkin Elmer device whose range of precision is from 4000 to 400 cm⁻¹ in powders (dispersed in a KBr pellet). All NMR spectra were recorded using a Brucker AV400 Avance spectrometer (at 400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts are expressed in parts per million (ppm) using TMS as an internal standard in CDCl₃.
5-Amino-1-phenyl-1H-1,2,4-triazole 1a–b was prepared according to the literature [41 (link)] by gently refluxing in methanol.
All other reagents and solvents used were analytical grade.