The column fractions having cytotoxic activity (MTT assay) against A549 cells were pooled together which is labeled as pooled I (PI-with F8, F9) and pooled II (PII- with F14, F15), The PI and PII were tested against A549 cells by MTT and LDH assay to ensure their activity. Further, PI and PII was separated in precoated TLC plates (Merck, Germany) using binary mobile phase of 10% ethyl acetate in petroleum ether and 30% acetone in chloroform respectively.
Precoated tlc plates
Manufactured by Merck Group
Sourced in Germany
Precoated TLC plates are thin-layer chromatography (TLC) plates that come with a pre-applied absorbent layer. They are designed for the separation and analysis of various chemical compounds. The absorbent layer, typically made of silica gel or aluminium oxide, is pre-coated onto a rigid support, such as glass or plastic. Precoated TLC plates provide a consistent and standardized platform for TLC applications, allowing for reliable and reproducible results.
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Lab products found in correlation
400 mhz nmr spectrophotometer, by Bruker (1 mentions)
Silica gel g, by Merck Group (1 mentions)
Eca 500 spectrometer, by JEOL (1 mentions)
Hydrosphere c18 column, by YMC (1 mentions)
Inova instrument, by Agilent Technologies (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Uv lamp, by Avantor (1 mentions)
Butyric acid ba, by Merck Group (1 mentions)
N n dicyclohexylcarbodiimide dcc 4 dimethylaminopyridine dmap, by Merck Group (1 mentions)
2 4 diisopropylphenol, by Merck Group (1 mentions)
2 6 diisopropylphenol, by Merck Group (1 mentions)
2 ethyl hexanoic acid 2 eha, by Merck Group (1 mentions)
Human tumor cell lines, by American Type Culture Collection (1 mentions)
Avance 3 nanobay 400 mhz spectrometer, by Bruker (1 mentions)
10 protocols using precoated tlc plates
1
Cytotoxic Activity Screening of Extracts
2
Spectroscopic Characterization of Organic Compounds
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The reagents and solvents were purchased from commercial suppliers and used without further purification. For thin-layer chromatography (TLC), Merck pre-coated TLC plates (Silica gel 60 F254) were employed. The melting points were determined on a Thomas Hoover capillary melting point apparatus with a digital thermometer and are uncorrected. The NMR spectra were recorded in DMSO-d6 solutions at 25 °C on a Bruker 400 MHz NMR spectrophotometer. The chemical shifts expressed in ppm were relative to tetramethylsilane (TMS). The mass spectra were obtained with a VG70-70H spectrophotometer, and important fragments were given with the relative intensities in brackets.
3
Microwave-Assisted Synthesis of Coumarins
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All reagents such as l -proline, 4-hydroxy coumarin, aldehydes etc. were analytical grade and have more than 98% purity. 1H and 13C NMR spectra were recorded on BRUKER AVANCE II 500 NMR spectrometer using CDCl3 and DMSO-d6 as solvent. Purity of the compound was checked by TLC. MS 1927 microwave starter kit was used for microwave reactions. Reaction was carried out under microwave conditions at 300 W in open to air conditions. E-Merck precoated TLC plates, RANKEM silica gel G for preparative thin-layer chromatography were used. Melting points were determined in open capillary and are uncorrected.
4
Purification and Characterization of Organic Compounds
5
Solvent Removal and Characterization of Compounds
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All chemicals were purchased from Sigma-Aldrich Chemical Co. The solvent was removed at aspirator pressure using a rotary evaporator. TLC was performed with Merck precoated TLC plates, and the compounds were made visible using a fluorescent inspection lamp and iodine vapor. Gravity chromatography was done with Merck silica gel 60 (mesh size 63–200 μm). Nuclear magnetic resonance spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1H NMR and 75 MHz for 13C NMR. Chemical shifts (δ) for 1H NMR spectra are reported in ppm downfield relative to the center line of CDCl3 triplet at 7.26 ppm. Chemical shifts for 13C NMR spectra are reported in ppm downfield relative to the center line of CDCl3 triplet at 77.23 ppm. The abbreviations s, d, t, and m stand for the resonance multiplicities singlet, doublet, triplet, and multiplet, respectively. 13C spectra, are 1H decoupled, and multiciplities were determined by APT pulse sequence. The melting points were recorded on a Boëtius hot plate microscope. FT-IR spectra were recorded on FT-IR Shimadzu spectrometer (4000–400 cm−1. EI-MS and HRMS were performed with Finnigan MAT 95, EI: 70 eV, R:10000.
6
TLC-Based Chemical Peptide Detection
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The pre-purified peptide extract was chemically detected using Thin Layer Chromatography (TLC). A modified method previously described by [25 , 41 ] and also used by [24 (link)] was adopted for the TLC chemical detection. Pre-coated TLC plates (G254 MERCK, Germany) were used. The solvent system used was n-butanol: acetic acid: water (3:1:1 v/v). The solvent reconstituted aqueous peptide fraction obtained from plant samples were spotted on the TLC plate and developed in the above-mentioned solvent system. The developed plates were allowed to dry, and sprayed with freshly prepared G-250 modified stain and Ninhydrin to detect the presence of circular and linear peptides, respectively. The sprayed plates were allowed to dry and viewed under UV at 245 nm and 365 nm. For MALDI TOF MS detection, 0.5 μL each of eluted fraction obtained from the C18 pre-purification as described above was mixed well with 3 μL of CHCA (alpha cyano hydroxyl cinnamic acid) matrix, spotted on the MALDI target plate and dried away from light. Acquired spectra were processed using the 4800 Analyzer. The freeze dried fractions were reconstituted and again analysed for peptide mass peaks as described above [40 (link)].
7
Quantitative Analysis of FFH Hydrolysis
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Thin layer chromatography (TLC) was employed for the qualitative analysis of FFH hydrolysis products. The pre-coated TLC plates (E-Merck) were allowed to spot the enzyme samples; n = 2 per sample. The plates were developed in acetone: diethyl ether: water (7:3:1 by volume). The plates were sprayed thoroughly with 0.2% ninhydrin and allowed to dry at 70 °C for 20 min.14 The resulting sugars were quantitatively analyzed by a double beam HPLC (Perkins Elmer, USA). The universal column (C18, HPX-87H ion exchange, 400 mm × 118 mm) was maintained at 25 °C. The mobile phase (ethanol–butanol, 2:1) was set at a flow-rate of 0.42 mL/min. The samples were identified using a refractive index detector (Turbochron-4 software).
8
Thin-Layer Chromatography for Compound Identification
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The identification of the main compounds was performed with thin-layer chromatography (TLC) using an UV lamp VWR (Austria). For chromatographic determination, 2.5 µL of sample solutions were spotted on 20 x 20 cm Merck (Germany) pre-coated TLC plates (60 F 254 , 250 µm).The first mobile phase (1) was consisted of toluene: ethyl acetate (9.3:0.7 v/v), the second mobile phase (2) was consisted of ethyl acetate:glacial acetic acid (9.5:0.5 v/v), the third mobile phase (3) was a mixture of dichloromethane:acetone:glacial acetic acid (7.6:1.5:0.5 v/v/v), while the fourth one (4) was only toluene as an organic solvent used as a mobile phase.
9
Synthesis of Propofol-Fatty Acid Conjugates
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The raw materials needed for the synthesis of propofolfatty acid ester conjugates of selected four fatty acids, such as alpha-linolenic acid (ALA) , was extracted from eripupal oil according to a reported procedure 41) , valproic acid (VA) was purchased from Alfa-Aesar Chemicals (USA) . Butyric acid (BA) , 2-ethylhexanoic acid (2-EHA) , 2,4-diisopropylphenol, 2,6-diisopropylphenol and N,N -dicyclohexylcarbodiimide (DCC) , 4-(dimethyl amino) pyridine (DMAP) were purchased from Sigma-Aldrich Chemicals (USA) . All solvents and chemicals were of reagent grade and used directly without further purification. Silica gel (60-120 mesh) for column chromatography was purchased from Acme Synthetic Chemicals, Mumbai, India. Precoated TLC plates were purchased from Merck, Darmstadt, Germany. Human tumor cell lines were obtained from American Type Culture Collection, Manassas, VA, USA.
10
NMR Spectroscopy and Mass Spectrometry Protocol
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General Methods 1 H-NMR and 13 (link) C-NMR spectra were recorded on a Bruker Avance III Nanobay 400 MHz spectrometer (400 MHz for 1 H-NMR, 100 MHz for 13 (link) C-NMR and 376 MHz for 19 (link) F-NMR). The chemical shifts were reported in ppm on the δ scale relative to Me 4 Si (δ=0.00 for 1 H-NMR), CDCl 3 (δ=77.0 for 13 C-NMR), α,α,αtrifluorotoluene (δ=-63.72 for 19 F-NMR). Mass spectra were recorded by an electrospray ionization-time of flight (ESI-TOF) mass spectrometer (Micromass LCT). For thin layer chromatographic (TLC) analyses, Merck precoated TLC plates (silica gel 60 F 254 ) were used. Flash column chromatography was performed on neutral silica gel (40-50 µm).
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