HPTLC analysis was done according to Hinterdobler et al. (2019) (link) with some modifications. Metabolites were extracted from the agar by supersonication for 15 min in a mixture of 5 ml water (p.A.) and 4 ml ethyl acetate (EtOAc). Liquid-liquid extraction with 4 ml EtOAc was repeated two times. The collected organic phase was evaporated, re-collected in 140 μl methanol (MeOH) and 8 μl were applied to HPTLC separation and analysis. Samples were spotted on a silica gel plate (HPTLC silica gel 60 F254S, Merck KGaA, Darmstadt, Germany) and separated with chloroform:1 mM trifluoroacetic acid in MeOH 7:1 (v/v) as mobile phase. Pictures were taken at different wavelengths before and after derivatization with p-anisaldehyde:sulfuric acid reagent.
Hptlc silica gel 60 f254
Manufactured by Merck Group
Sourced in Germany
HPTLC Silica gel 60 F254 is a thin-layer chromatography (TLC) plate coated with silica gel. It is designed for high-performance thin-layer chromatography (HPTLC) applications. The silica gel layer contains a fluorescent indicator, F254, which allows for the detection of separated compounds under ultraviolet (UV) light.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Merck Group (2 mentions)
Perindopril, by Merck Group (2 mentions)
Ramipril, by Merck Group (2 mentions)
Hptlc diol f254, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Methanol p a, by Merck Group (1 mentions)
Twin trough glass chamber, by CAMAG (1 mentions)
Tlc scanner, by CAMAG (1 mentions)
Acetanilide, by Merck Group (1 mentions)
Theophylline, by Merck Group (1 mentions)
Acetylsalicylic acid, by Merck Group (1 mentions)
Acetaminophen, by Merck Group (1 mentions)
Lc ms grade formic acid, by Merck Group (1 mentions)
Caffeine, by Merck Group (1 mentions)
Ethyl acetate, by Avantor (1 mentions)
Cellulose tlc, by Merck Group (1 mentions)
Hlp 5, by Hydrolab (1 mentions)
Methanol, by Avantor (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Toluene, by Avantor (1 mentions)
11 protocols using hptlc silica gel 60 f254
1
HPTLC Analysis of Microbial Metabolites
2
Metabolite Profiling of Delta hda1A Mutant
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To screen for alterations in the metabolite profile of ∆hda1A compared to the WT, metabolites were extracted from the supernatants of untreated (PDB; control) and sorbitol treated liquid cultures and subjected to HPTLC. An optimized extraction method of our previously described protocol (69 (link)) was applied for metabolite extraction from liquid cultures. Liquid cultures of the WT and the ∆hda1A mutant were obtained like described above. Six milliliters of the culture supernatants were aliquoted and mixed with 1.1 mL of acetone p.a. (CarlRoth GmbH + Co KG, Karlsruhe, Germany) each. The mixture was incubated for 15 min at room temperature in the ultrasonic bath. After addition of 4.5-mL ethyl acetate p.a. (EtOAc; CarlRoth GmbH + Co KG, Karlsruhe, Germany), the samples were mixed well. Phase separation was obtained by centrifugation at 3,000 × g for 1 min, and the upper phase was transferred to a broad glass vial. The EtOAc extraction step was repeated a second time, and the EtOAc extracts were evaporated overnight. The next day, evaporated extracts were re-collected in 140 µL of methanol p.a. (Merck KGaA, Darmstadt, Germany). Chromatographic separation was done on silica gel plates (HPTLC silica gel 60 F254S, Merck KGaA, Darmstadt, Germany) via HPTLC as previously described (69 (link)).
3
HPTLC Analysis of Methanol Extracts of Kashayam
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The methanol extracts of three batches of kashayam were subjected to HPTLC analysis. The kashayam was dried in a water bath at a fixed temperature and was extracted with methanol. 4 micro litres of the extract was spotted on HPTLC silica gel 60F 254(Merck) plate as bands of length 6 mm at a distance of 10 mm. The plates were developed using Toluene: Ethyl Acetate: Formic acid (2.5: 2.0: 0.5) in the CAMAG twin-trough glass chamber, previously saturated with the solvent for 30 min. The mobile phase was chosen after testing different solvent systems of varying polarity. After development, the plates were dried in an oven at 60 °C and scanned using a CAMAG TLC Scanner in absorbance mode. Data processing was performed with winCATS planar chromatography manager software. The compounds were scanned at 254 and 366 nm.
4
HPTLC Analysis of Pharmaceutical Compounds
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Chromatographic plates, HPTLC Silica gel 60 F254, 10 × 20 cm, were supplied by Merck (Darmstadt, Germany). Methanol, water and formic acid LC–MS grade were purchased from Merck (Darmstadt, Germany). Acetaminophen, acetanilide, aminophenazone, caffeine, acetylsalicylic acid, and theophylline were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetobutolol was purchased from Biomedicals, USA (Santa Ana, CA, USA), ciprofloxacin from Sreepathi Pharmaceutical Ldt. India (Telangana, India), Tramadol from Inogent Laboratories, India (Telengana). Bovine serum was purchased from Biomed (Lublin, Poland).
5
Development and Validation of HPTLC Methods for Antihypertensive Drugs
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Chromatographic glass plates, HPTLC silica gel 60 F254, HPTLC silica gel 60 RP-18 F254, HPTLC diol F254, HPTLC CN F254, and TLC cellulose were obtained from Merck (Darmstadt, Germany). Methanol, toluene, ethyl acetate, acetone, isopropyl, and acetonitrile of analytical grade were from Avantor (Gliwice, Poland). Deionized water was produced in the laboratory with the demineralizer HLP 5 from Hydrolab (Straszyn, Poland). The reagents used to prepare the acidic and basic mobile phases were as follows: formic acid (99.5%) and ammonia (25%) were also purchased from Avantor (Gliwice, Poland). Methanol and formic acid LC–MS grade were purchased from Merck (Darmstadt, Germany). Antihypertensive drugs (hydrochlorothiazide, indapamide, lercanidipine, nebivolol, telmisartan, valsartan) were donated from the Cardiology Clinic of the Independent Public Clinical Hospital No. 4 in Lublin (Lublin, Poland). Perindopril and ramipril were bought from Sigma–Aldrich (St. Louis, MO, USA). Albumin bovine fraction V (BSA) was from NzyTech–Genes & Enzymes (Lisbon, Portugal).
6
Bacterial Lipid Extraction and TLC Analysis
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Methods for total lipid extraction and thin‐layer chromatography (TLC) are published (Honda et al., 2019 ). In brief, isolates were cultured in Proskauer–Beck (PB) media and total lipids were extracted from bacterial pellets using 1:1 CHCl3:MeOH (chloroform:methanol). Lipid extraction was repeated three times, each time pooling the supernatants to collect the soluble lipid fractions, and evaporated using N2. For TLC, lipid fractions were resuspended in 2:1 CHCl3:MeOH and vortexed. 25 μl of each lipid fraction was spotted onto silica plates (HPTLC Silica gel 60 F254; Merck KGaA) and 65:24:4 HCCl3:MeOH:H2O (v/v/v) was used to separate the lipid species. Total lipids were visualized with CuSO4 charring spray under heat.
7
Antihypertensive Drugs Analysis by HPTLC
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Antihypertensive drugs: ramipril, lercanidipine, valsartan, hydrochlorothiazide, telmisartan, perindopril, and nebivolol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The structures, physicochemical properties and drug classes (pharmacological properties) of investigated substances are presented in Table 5 . Chromatographic glass plates, HPTLC silica gel 60 F254 and HPTLC diol F254 were supplied by Merck (Darmstadt, Germany). Acetonitrile and methanol (both MS purity) were purchased from Fisher Chemical (Waltham, MA, USA). Ethanol, toluene and formic acid (98–100%) LC-MS grades were supplied by Merck (Darmstadt, Germany). The ultra-pure water was obtained from the Millipore Direct-Q3-UV purification system (Merck, Darmstadt, Germany). The plasma samples (also a reference sample) were kindly provided by the Cardiology Clinic of the Independent Public Clinical Hospital No. 4 in Lublin (Lublin, Poland).
8
HPTLC Analysis of Vincristine from Samples
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The HPTLC silica gel 60 F254 (Merck) plates were pre-washed with methanol by dipping them in an HPTLC chamber filled with 20 mL of methanol and letting the solvent overrun to remove any impurities, and then the plate was activated for 10 min at 120 °C in an oven. Toluene–methanol–diethylamine was used as a mobile phase in a ratio of 8.75:0.75:0.5 [16 ]. A separate chamber with its walls covered with tissue paper was allowed to mix with the mobile phase for 45 min to one hour at room temperature. The spots of 6mm band length were marked using a Linomat 5 auto-sampler and were captured under UV light. The solvent was allowed to rise over the HPTLC plate by 90% in the Camag twin-trough chamber saturated with the mobile phase. The plate was taken out and kept in an upright position for air-drying. It was then kept in the oven for 30 s at 75 °C to evaporate any traces of the mobile phase and scanned at 300 nm. The standard for vincristine was obtained from Sigma Aldrich, Bangaluru, India and a stock solution of 1 mg/mL in methanol was prepared, from which different dilutions were made. The dilutions used were 200 ng/mL, 400 ng/mL, 600 ng/mL, 800 ng/mL, and 1000 ng/mL for HPTLC analysis.
9
Quantifying Ceramide Lipid Profiles
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Ceramide contents were determined in 10-µL sample aliquots after spotting onto a (HPTLC Silica Gel 60 F254; MERCK, Darmstadt, Hessen, Germany) using a glass capillary tube (Ringcaps; Hirschman Laborgerate, Eberstadt, Postfach, Germany). Identical volumes of ceramide-2 (NS), ceramide-3 (NP), ceramide-5 (AS), and ceramide-6 (AP) were spotted as standards, and spots were developed using a mixture of chloroform:methanol: acetic acid (190:9:1). Spots were developed upward to 1 cm from the top of the HPTLC plate and were then dried and developed again. After development, 8% aqueous phosphoric acid containing 10% copper sulfate was sprayed onto the plates and the plates were then incubated at 180°C for 10 min using TLC plate heater III (Camag, Sonnenmattstrasse, Muttenz, Switzerland). Resulting spots were imaged using a Luminoimage Analyzer System (LAS-1000 Plus; Fujifilm Corporation, Tokyo, Japan), and ceramide types and contents in resulting images were quantified using a Multi Gauge (Fujifilm Corporation, Tokyo, Japan).
10
Photostability evaluation of MeO-MBM and avobenzone
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MeO-MBM and avobenzone (1%, w/w) were dissolved in ortho-xylene and applied on a 19-cm2 PMMA plate at a concentration of 1 µL/cm2. Irradiation was carried out in a suntest device (CPS+, Atlas, Chicago, IL, USA) at 250 kJ/m2, followed by extraction of the substances from the plates with 2-mL ethanol before analyzing. Equally treated samples were additionally stored in the dark without irradiation as a comparison.
HPTLC analyses were carried out by applying the sample solutions onto high-performance thin-layer chromatography (HPTLC) plates (HPTLC Silica gel 60 F254, Merck KGaA, Darmstadt, Germany) with an automatic sampler (ATS 4, CAMAG, Muttenz, Switzerland) as bands with 6.0-mm widths. The plates were developed in toluene:methanol (9:1 ratio). The chromatograms were photographed at the 254-nm wavelength (Reprostart 3, CAMAG, Muttenz, Switzerland). The retardation factor (Rf) value was obtained by using the software winCATS 1.4.8 (CAMAG, Muttenz, Switzerland).
HPTLC analyses were carried out by applying the sample solutions onto high-performance thin-layer chromatography (HPTLC) plates (HPTLC Silica gel 60 F254, Merck KGaA, Darmstadt, Germany) with an automatic sampler (ATS 4, CAMAG, Muttenz, Switzerland) as bands with 6.0-mm widths. The plates were developed in toluene:methanol (9:1 ratio). The chromatograms were photographed at the 254-nm wavelength (Reprostart 3, CAMAG, Muttenz, Switzerland). The retardation factor (Rf) value was obtained by using the software winCATS 1.4.8 (CAMAG, Muttenz, Switzerland).
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