Chemical qualitative characterization of the test vegetal extracts was accomplished by high-performance thin-layer chromatography (HPTLC) and Linomat 5 instrument (CAMAG, Muttenz, Switzerland). It was used as the general method for polyphenols assessment in vegetal samples [41 ]; solvent system-ethyl acetate: acetic acid:formic acid:water, 100:12:12:26, respectively. Briefly, test vegetal extracts in volumes from 1 to 5 μL were automatically applied as 8 mm band length in the 10 × 10 cm Silica gel 60F HPTLC plate (Merck, Darmstadt, Germany) using a Hamilton syringe; test reference compounds (ref., prepared as 10−3 M solutions in 70% ethanol, v/v) and their mixtures were also applied in 0.5–2.5 μL per 8 mm band. The loaded plate was then kept in TLC twin developing chamber at 16–18 °C with the particular solvent system (mobile phase) for polyphenols assessment, up to 90 mm. The developed plate was carefully dried with a hair dryer and then immersed into the NP/PEG No. 28 identification reagents. The plate was next disposed of inside the Photo-documentation chamber of CAMAG Linomat 5 apparatus, and the image at UV-366 nm was captured. After specific chromatogram Rf measurements, polyphenols spots were assigned by comparison to literature data and particular test reference compounds used in the study.
Linomat 5
Manufactured by CAMAG
Sourced in Switzerland, Germany, India
The Linomat 5 is a laboratory instrument designed for the automated application of samples onto thin-layer chromatography (TLC) plates. It provides precise and consistent sample application, ensuring reliable and reproducible results for TLC analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tlc scanner 3, by CAMAG (18 mentions)
Tlc visualizer, by CAMAG (13 mentions)
Visioncats, by CAMAG (12 mentions)
Tlc scanner, by CAMAG (11 mentions)
Reprostar 3, by CAMAG (8 mentions)
Tlc visualizer 2, by CAMAG (8 mentions)
Tlc scanner 4, by CAMAG (7 mentions)
Silica gel 60 f254, by Merck Group (6 mentions)
Tlc plate heater 3, by CAMAG (5 mentions)
Tlc plate heater, by CAMAG (5 mentions)
Wincats software, by CAMAG (5 mentions)
Hptlc plate, by Merck Group (5 mentions)
Twin trough chamber, by CAMAG (4 mentions)
Derivatizer, by CAMAG (4 mentions)
Hptlc silica gel 60 f254 plate, by Merck Group (4 mentions)
Vision cats software, by CAMAG (3 mentions)
Microliter syringe, by CAMAG (3 mentions)
Tlc system, by CAMAG (3 mentions)
Twin trough glass chamber, by CAMAG (3 mentions)
F254 plate, by Merck Group (2 mentions)
149 protocols using linomat 5
1
HPTLC Characterization of Vegetal Extracts
2
Quantifying Histidine Levels by TLC
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The amount of His that accumulated in the medium was determined by thin-layer chromatography (TLC) using plates coated with silica gel (Merck, Germany). Samples were applied to the TLC plates using Linomat 5 (Camag, Switzerland). The plates were developed with a mobile phase consisting of propan-2-ol:acetone:25% aqueous ammonia:water = 12.5:12.5:3:2 (v/v). A solution of ninhydrin (1%) in acetone was used as the visualizing reagent; the plates were dried and then scanned at 520 nm using a Linomat 5 scanner (Camag, Switzerland).
3
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.
4
TLC Characterization of Herbal Extracts
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Ten milligrams of each extract was dissolved in one milliliter of HPLC grade methanol separately to obtain 10 mg/mL working stock solutions. Stock solutions were then filtered, and 5 μL of each extract solution was separately applied on silica gel 60 F254 precoated TLC plates, 20 × 10 cm (Merck, Germany) with the help of Camag Linomat V (Camag, Switzerland) applicator. The sample solution was applied to a 6 mm wide band using Camag Linomat V automated TLC applicator with the nitrogen flow providing a delivery speed of 120 nL/s from the syringe. Applied plates were presaturated with the mobile phase for 30 min in a Camag twin through glass tank and allowed to move the analytes in Camag horizontal developing chamber (20 × 10) at a room temperature (25°C) using solvent system toluene: ethyl acetate: formic acid (5 : 4 : 1, v/v/v). After the solvent run up to 80% of total height, the plate was air-dried. Some phenolic UV compounds showed absorption maxima at long-wave UV [13 ]. To get the optimum intensity of each compound, developed TLC plates were scanned at both 254 nm (short-wave UV) and 366 nm (long-wave UV) by a Camag TLC scanner III using the WinCATS software.
5
HPTLC Analysis of Caulerpin Compounds
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HPTLC analysis was studied using Camag Linomat V and Camag TLC-Scanner 3. TLC was performed on 20 × 10-cm plates (Merck, silica gel60 F254 TLC plates). Caulerpin samples were prepared fresh in diethyl ether and were applied to TLC by a semi-automatic sample applicator (Camag Linomat V, Muttenz, Switzerland). The twin horizontal chamber (Camag) was used for development. A Camag TLC-Scanner 3 densitometer was used to quantify the bands on the plates using a tungsten source (absorbance reflection mode). The slit dimensions were set as 6 mm and 0.3 mm in length and width, respectively. The scanning rate was set as 20-mm s−1. Diethyl ether and petroleum ether (Merck, analytical grade) were used for the extraction of the samples and used as the mobile phase. Ethyl acetate (high purity, Tekkim) was used for the maceration of samples.
6
Quantitative HPTLC Analysis of Phytochemicals
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HPTLC was performed on silica gel 60 F254 100 × 100 mm plates (Merck) with hexane: ethyl acetate (7:3 v/v) as mobile phase for the standard (β-sitosterol) solution (2–10 µL) and J.T. (0.2–0.3 µL); and hexane: ethyl acetate: 1 drop of acetic acid (6:4 v/v) as mobile phase for the standard (esculetin) solution (1–4 µL) and F.M. (1–3 µL). These were applied to the plate as 8 mm bands. Application of the sample was performed with CAMAG-Linomat 5 Automated spray on a band applicator equipped with a 100 µL syringe and operated with the settings: band length 8 mm, application rate 150 nL/s, application volume 0.20 µL, distance between track 14.4 mm, distance from the plate side edge 15.0 mm and solvent front position 70 mm. CAMAG TLC visualizes 2 and was used densitometrically to scan the bands. The scanner operating parameters were set to mode absorption/reflection at an optimized wavelength of 254, 366 nm, and in the visible range. Integration parameters were set to gauss (legacy) with sensitivity 0.1, separation 1, and threshold 0.1.
7
HPTLC Fingerprinting of Wisteria fruticosa Flowers
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TLC procedure was standardized with the solvent systems A and B which were used for the high-performance TLC (HPTLC) fingerprinting of the three samples of the W. fruticosa flowers along with the standard ellagic acid. The sequence of the tracks is given in Table 1 . HPTLC fingerprinting analysis was done using the CAMAG linomat 5 sample applicator using micro syringe (100 μl, Hamilton), CAMAG reprostar 3, Twin trough chamber, Dip tank, Win cat software - Version 1.3.3., with a development distance of 70 mm. Precoated silica gel plates 60F254 from Merck (20 cm × 10 cm) were used. Samples and standards were applied as bands on the plates in volumes 10 μl and detection was done under ultraviolet (UV) 254, UV 366, and after derivatization with 5% methanolic ferric chloride reagent.
8
TLC Identification of L-dopa in M. pruriens
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Thin layer chromatography (TLC) was used to separate and determine the presence of L-dopa in M. pruriens seed extract. The mobile phase was modified from the previous method [16 (link)]. Silica gel GF254 (Merck, Darmstadt, Germany) was used as the stationary phase and a mixture of butanol, methanol, acetic acid, and water (12.0:6.5:2.5:1.5) was used as the mobile phase. L-dopa (1 mg/mL, 5 µL) and spray-dried M. pruriens seed extract (2 mg/mL, 10 µL) were spotted on the stationary phase using Camag® Linomat 5 (Dublin, Ireland). After development, the plates were sprayed with 2% Ninhydrin solution. The plate was then photographed under visible light, 254, and 366 nm using the TLC visualizer. The original TLC images were analyzed by the winCATs TLC workstation program (Camag, Ireland).
9
Thin Layer Chromatography Analysis of Medicinal Plants
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The lyophilizates of each tissue were resuspended with distilled water to a concentration of 10 mg/mL. Then, 15 µL of these solutions were deposited in bands with a length of 8 mm each with a Camag syringe of 100 µL on aluminum support plates coated with silica gel 60F254 (20 × 10 cm) by an autosampler Camag Linomat 5.
The plates were then placed in a glass chamber previously saturated (30 min) with the mobile phase: water–methanol–ethyl acetate (10:14:76 v:v, respectively) [23 (link)]. Once the plates were dried, they were visualized in a U.V. chamber at two wavelengths of 254 and 366 nm. Additionally, T.L.C. plates were developed with specific solutions for each group of compounds: ferric chloride solution for tannin determination, trichloroacetic acid solution for glycosides, Dragendorff’s reaction for alkaloids, and aluminum chloride solution for flavonoids.
The retention factor was calculated using the following equation:
The plates were then placed in a glass chamber previously saturated (30 min) with the mobile phase: water–methanol–ethyl acetate (10:14:76 v:v, respectively) [23 (link)]. Once the plates were dried, they were visualized in a U.V. chamber at two wavelengths of 254 and 366 nm. Additionally, T.L.C. plates were developed with specific solutions for each group of compounds: ferric chloride solution for tannin determination, trichloroacetic acid solution for glycosides, Dragendorff’s reaction for alkaloids, and aluminum chloride solution for flavonoids.
The retention factor was calculated using the following equation:
10
TLC Profiling of Plant Extracts
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The comparative TLC chromatograms and fingerprint profiles were developed using pre-coated silica gel F254 plates [E. Merck (India) Ltd., alumina base, and 0.2 mm thickness]. A large number of various combinations of solvent systems were used during the present studies and the best resolution was obtained in a solvent system of toluene:ethyl acetate:acetic acid (7.4:2.4:0.2). Extracts were applied as bands using Camag Linomat 5 available in the laboratory. The running distance was kept at 8 cm and anisaldehyde sulphuric acid reagent was used as derivatizing agent followed by heating at 110 °C for 5 min or till the bands developed colour. TLC fingerprint profiles were recorded as images under UV at 254 and 366 nm before spray and under white light after derivatization on Camag Reprostar fitted with D × A 252 16 mm camera.
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