The reverse-phase HPTLC analysis was performed using the CAMAG HPTLC instrument (CAMAG, Muttenz, Switzerland). The estimation of caffeine was performed on 10 × 20 cm glass plates precoated with reverse-phase silica gel 60 F254S plates (E-Merck, Darmstadt, Germany). The samples to the reverse-phase TLC plates were spotted as the 6 mm bands utilizing a CAMAG Automatic Sampler 4 (ATS4) applicator (CAMAG, Geneva, Switzerland). The sample applicator was fitted with CAMAG Microliter Syringe (Hamilton, Bonaduz, Switzerland). The application rate for caffeine estimation was constant at 150 nL s−1. The TLC plates were developed in an Automatic Developing Chamber 2 (ADC 2) (CAMAG, Muttenz, Switzerland) to a distance of 80 mm using EtOH-water (55:45, v v−1) greener mobile phase. The development chamber was saturated with vapors of EtOH-water (55:45, v v−1) for 30 min at 22 °C. Caffeine was detected using a wavelength of 275 nm at a scanning rate of 20 mm s−1 and slit size set to 4 × 0.45 mm.
Hptlc instrument
Manufactured by CAMAG
Sourced in Switzerland, Germany
The HPTLC instrument is a laboratory equipment used for high-performance thin-layer chromatography. It is designed to perform qualitative and quantitative analysis of complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Automatic sampler 4 ats4 applicator, by CAMAG (2 mentions)
Automatic developing chamber 2 adc 2, by CAMAG (2 mentions)
Tlc scanner 3, by CAMAG (2 mentions)
60 f254s plates, by Merck Group (1 mentions)
Glucuronic acid, by Merck Group (1 mentions)
Galacturonic acid, by Merck Group (1 mentions)
Arabinose, by Merck Group (1 mentions)
Xylose, by Merck Group (1 mentions)
Linomat 5, by CAMAG (1 mentions)
Silica gel g60 f254, by Merck Group (1 mentions)
Rvc2 25 cdplus, by Martin Christ (1 mentions)
Silybin, by Merck Group (1 mentions)
Ursolic acid, by Phytolab (1 mentions)
Silychristin, by ChromaDex (1 mentions)
Echinacoside, by Phytolab (1 mentions)
Tlc visualizer 2, by CAMAG (1 mentions)
Cynarin, by Phytolab (1 mentions)
Silica gel 60 f254 hptlc glass plates, by Merck Group (1 mentions)
β sitosterol, by Extrasynthese (1 mentions)
Automatic tlc sampler ats 4, by CAMAG (1 mentions)
9 protocols using hptlc instrument
1
Reverse-Phase HPTLC Quantification of Caffeine
2
HPTLC Quantification of Thymol in Natural Samples
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The “CAMAG HPTLC instrument (CAMAG, Muttenz, Switzerland)” was used to estimate thymol in its pure/bulk form, commercial formulation, essential oils of T. vulgaris and O. vulgare, TE and UBE of T. vulgaris and O. vulgare obtained from various geographical regions using normal-phase HPTLC. The analysis of thymol in normal-phase mode was carried out on “10 × 20 cm2 aluminum plates pre-coated with normal-phase silica gel 60 F254S plates (E-Merck, Darmstadt, Germany)”. Using a “CAMAG Automatic Sampler 4 (ATS4) applicator (CAMAG, Geneva, Switzerland)”, the samples to the normal-phase TLC plates were spotted as 6 mm bands. The “CAMAG microliter Syringe (Hamilton, Bonaduz, Switzerland)” was used as a sampling applicator. In normal-phase mode, the application rate for thymol analysis was set at 150 nL/s. The normal-phase TLC plates were developed at a distance of 80 mm in a “Automatic Developing Chamber 2 (ADC 2) (CAMAG, Muttenz, Switzerland)”. The greener mobile phase for thymol analysis was CY-EtOAc (85:15, v/v). For 30 min at 22 °C, the development chamber was saturated using CY-EtOAc (85:15, v/v) vapors. The slit dimensions were set at 4 × 0.45 mm2 and scanning rate was made constant at 20 mm/s.
3
Automated TLC Characterization of Extracts
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All six extracts were air-dried and their residues were then resuspended in methanol to be ready for applying onto thin layer chromatography (TLC) plate (10 × 10 cm Merck aluminum sheet, silica gel 60, layer thickness 0.2 mm). Chromatographic bands were examined by a fully automated HPTLC instrument (CAMAG––Switzerland). The first unit is an automatic applicator (Linomate5) by which the crude extracts were loaded onto a TLC plate in a form of bands against Griseofulvin as an authentic marker. TLC plate was then transferred into an automated development chamber (ADC2 CAMAG) using toluene–ethyl acetate–formic acid 5:4:1 (v/v/v) as an elution buffer, followed by UV scanning via UV chamber unit at visible light, long UV wavelength (365 nm), short UV wavelength (254 nm), and long UV wavelength (365 nm). TLC plate was then sprayed with p-anisaldehyde 0.5% in (Conc. H2SO4–acetic acid–acetone 5:10:85) and then oven heated at (105 °C) for 10 min. Data obtained (color, Rf, and the shape of bands) were analyzed according to Paterson and Bridge (1994 ).
4
High-Performance Thin-Layer Chromatography
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HPTLC was carried out using CAMAG HPTLC instrument. A 1-mg/mL clear solution of Ethyl acetate fraction was dissolved in a solution of Methanol and Ethyl acetate (1:1, v/v) and was spotted onto aluminum silica gel plates PF254 under N2, using Linomat 5, under 0.2 μL/sec speed. Each 6-mm band contained about 30 μg of the extract. A variety of mobile phases were used for achieving the highest separation resolution. All the plates were scanned in the range of 180–800 nm, using CAMAG TLC scanner III (12 ).
5
HPTLC Analysis of n-Hexane Extract
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HPTLC was carried out using CAMAG HPTLC instrument (Muttenz, Switzerland). A 1-mg/mL clear solution of n-hexane extract was dissolved in an n-hexane and ethyl acetate (1:1, v/v) solvent mixture and was spotted onto aluminum silica gel plates PF254 under N2, using Linomat 5, under 0.2 µL/sec speed. Each 6-mm band contained about 30 µg of the extract. A variety of mobile phases were used for achieving the highest separation resolution. All the plates were scanned in the range of 180-800 nm, using CAMAG TLC scanner III (9 ).
6
HPTLC Analysis of Exopolysaccharide Composition
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HPTLC analysis was performed to deduce the monomeric composition of EPSs for which partially purified EPS was acid hydrolyzed in 2M Trifluoroacetic acid (TFA) at 100 °C for 5 h. HPTLC of hydrolysate was performed by the modified methodology of [76 (link)]. The 5µL hydrolysate was applied on 30 × 20 cm HPTLC silica gel plate (Merck, Darmstadt, Germany) along with sugar standard viz. arabinose, glucose, galactose, sucrose, fructose, maltose, glycerol, glucuronic acid, galacturonic acid, xylose (Merck, Darmstadt, Germany) via HPTLC instrument (CAMAG, Muttenz, Switzerland) of Sophisticated Instrumentation Centre for Applied Research & Testing, Sardar Patel University, India. For sugar analysis, separation was performed using butanol: ethanol: H2O (5:3:2) mobile phase, and visible bands were obtained by application of ethanolic p- anisaldehyde + ethanolic conc. H2SO4 solution used as developing agents. The plate was dried, heated at 70 °C for 15 min, and put under white light for band visualization whose image was scanned and analyzed by the CAMAG visualizer system [76 (link)].
7
Phytochemical Fingerprinting of Plant Extracts
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HPTLC instrument of CAMAG, Muttenz, Switzerland, Anchrom Enterprises (I) Pvt. Ltd, Mumbai was used in the present study. It consisting of sample applicator (Linomat 5), Twin trough chamber with lid {10×10 cm, CAMAG, Muttenz, Switzerland}, UV cabinet (Aetron, Mumbai) with dual wavelength (254/366 nm) and the HPTLC photo documentation (Aetron, Mumbai) was used for study.
HPTLC studies were carried out following the method of Harborne 9 .The prepared sample of leaf, stem and root extracts were applied separately at the concentration of 10 μL using the applicator and set at a speed of 150 nl/sec. The mobile phase was Hexane: Ethyl acetate: Toluene: Chloroform: Methanol: Formic acid (4:2.5:1.5:0.8:1:0.2) and the stationary phase was aluminum precoated sheets, Silica Gel G 60 F254 (Merck). The applicator phase was CAMAG Linomat 5. Plate was developed in a twin trough chamber. The active compounds are detected by spraying with Anisaldehyde sulfuric acid reagent and heat at 110°C for 5 minutes. The plate was scanned at 254 and 366 nm under fluorescent mode. After each observation the central points of spots appeared on chromatogram were marked with needle. The Rf values and finger print data were recorded by WIN CATS software.
HPTLC studies were carried out following the method of Harborne 9 .The prepared sample of leaf, stem and root extracts were applied separately at the concentration of 10 μL using the applicator and set at a speed of 150 nl/sec. The mobile phase was Hexane: Ethyl acetate: Toluene: Chloroform: Methanol: Formic acid (4:2.5:1.5:0.8:1:0.2) and the stationary phase was aluminum precoated sheets, Silica Gel G 60 F254 (Merck). The applicator phase was CAMAG Linomat 5. Plate was developed in a twin trough chamber. The active compounds are detected by spraying with Anisaldehyde sulfuric acid reagent and heat at 110°C for 5 minutes. The plate was scanned at 254 and 366 nm under fluorescent mode. After each observation the central points of spots appeared on chromatogram were marked with needle. The Rf values and finger print data were recorded by WIN CATS software.
8
Quantification of Lipopeptide Extracts
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Cell-free supernatants were obtained after 10 minutes centrifugation at 4700 rpm and were used for extraction of lipopeptides according to the method described by Yazgan et al. [48 (link)]. Specifically, a volume of 2 mL of the cell-free supernatant was mixed three times with 1 mL of 1-butanol 95% (v/v) by vortexing for 1 min, followed by 5 min centrifugation at 3000 rpm. The organic phases were pooled and used for evaporation of butanol phases (RVC2-25 Cdplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at 10 mbar and 60 °C. The remaining residues were dissolved in 2 mL methanol. To quantify the total amounts of lipopeptides, purified fengycin was purchased from Lipofabrik (Lesquin, France) and surfactin and iturin A standards were ordered from Sigma–Aldrich (Seelze, Germany). High-performance thin-layer chromatography (HPTLC) was performed for quantification of lipopeptides. All HPTLC instruments and chambers were from CAMAG (Muttenz, Switzerland) and instruments were controlled by winCATS Software 1.4.7 as described previously [49 (link)].
9
Analytical Characterization of Botanical Compounds
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The chemical reference standards silybin (98% pure) and caffeic acid (98% pure) were obtained from Sigma Aldrich (St. Louis, United States ), silydianin, chlorogenic acid (97% pure), caftaric acid (90% pure), and chicoric acid (97% pure) from USP (Rockville, United States), and silychristin (97.9% pure), dodec-2-ene-8,10-diynoic acid isobutylamide, and isoferulic acid (97% pure) from Chromadex (Los Angeles, United States). Taxifolin (85% pure) was purchased from Extrasynthese (Genay, France), and β-sitosterol (95% pure), ursolic acid (97% pure), echinacoside (95% pure), dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamide (93% pure), cynarin (96% pure), actein (95% pure), and cimifugin (97% pure) from Phytolab (Vestenbergsgreuth, Germany).
Solvents (≥95% pure) and reagents were purchased from Roth (Karlsruhe, Germany), Acros (Gent, Belgium), Fisher Scientific (Hampton, United States), and Merck (Darmstadt, Germany). Silica gel 60 F254 HPTLC glass plates (20 × 10 cm) were obtained from Merck (Darmstadt, Germany).
HPTLC instruments from CAMAG (Muttenz, Switzerland) were used, including Automatic TLC Sampler (ATS 4), Automatic Development Chamber (ADC 2) with humidity control, Plate Heater 3, TLC Visualizer 2, and Immersion Device 3.
Solvents (≥95% pure) and reagents were purchased from Roth (Karlsruhe, Germany), Acros (Gent, Belgium), Fisher Scientific (Hampton, United States), and Merck (Darmstadt, Germany). Silica gel 60 F254 HPTLC glass plates (20 × 10 cm) were obtained from Merck (Darmstadt, Germany).
HPTLC instruments from CAMAG (Muttenz, Switzerland) were used, including Automatic TLC Sampler (ATS 4), Automatic Development Chamber (ADC 2) with humidity control, Plate Heater 3, TLC Visualizer 2, and Immersion Device 3.
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