Grinder (GT203840, Tefal, United Kingdom), Rotamixer (Hook and Tucker Instruments Ltd., United Kingdom), Grant XB22 ultrasonic bath (Grant Instruments, United Kingdom), Centrifugator (Centrifuge 5804 R, Eppendorf, Germany), electronic balance (Sartorius CP64, Sartorius AG, Germany), Freeze dryer (ModulyoD Freeze Dryer, Thermo Fisher Scientific, United Kingdom), NMR tube (VWR international Ltd., United States), Bruker Advance 500 MHz spectrometer (Bruker, Germany), HPTLC plates silica gel 60 F 254 (Merck, Germany), Linomat 5 (CAMAG, Switzerland) coupled with a 100 μL syringe (CAMAG, Switzerland) and compressed air with 60–90 psi., Automatic Developing Chamber ADC 2 (CAMAG, Switzerland), TLC Visualizer (CAMAG, Switzerland) Microwell plate Nunclon 96 well (Thermo Scientific Nunc, United Kingdom), GalaxyB CO2 incubator (Scientific Laboratory Supplier Ltd., United Kingdom), water bath (LAUDA Aqualine AL 12, Germany), microscope (Olympus CK40 microscope, Japan), plate shaker (MS3 basic, IKA®, Germany) microplate reader (Infinite M200, Tecan, Switzerland), Multiwave Go (Anton Paar, Graz, Austria), ICP-OES SPECTROBLUE T1 (SPECTRO Analytical Instruments, Kleve, Germany).
Automatic developing chamber adc 2
Manufactured by CAMAG
Sourced in Switzerland, Germany
The Automatic Developing Chamber ADC 2 is a laboratory equipment designed for the automated development of thin-layer chromatography (TLC) plates. It provides a controlled environment for developing TLC plates, ensuring consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Tlc visualizer, by CAMAG (2 mentions)
Automatic tlc sampler ats 4, by CAMAG (2 mentions)
Linomat 5 applicator, by CAMAG (2 mentions)
Hptlc plate, by Merck Group (2 mentions)
Automatic tlc sampler 4, by CAMAG (2 mentions)
Wincats software, by CAMAG (2 mentions)
Modulyod freeze dryer, by Thermo Fisher Scientific (1 mentions)
Hptlc plates silica gel 60 f 254, by Merck Group (1 mentions)
100 μl syringe, by CAMAG (1 mentions)
Linomat 5, by CAMAG (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
Infinite m200, by Tecan (1 mentions)
Multiwave go, by Anton Paar (1 mentions)
Silica 60 f254 tlc plates, by Merck Group (1 mentions)
Tlc visualizer 2, by CAMAG (1 mentions)
Molybdenum blue spray reagent, by Merck Group (1 mentions)
Linomat 5 sample application unit, by CAMAG (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Formononetin, by Extrasynthese (1 mentions)
60 f 254 hptlc glass plates, by Merck Group (1 mentions)
10 protocols using automatic developing chamber adc 2
1
Quantitative Chemical Analysis Protocol
2
Analytical TLC of Pinaceae Exudates
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For analytical TLC experiments, Silica 60 F254 TLC plates (average particle size 9.5 to 11.5 μm, aluminum sheets with fluorescence indicator, Merck KGaA, Darmstadt, Germany) were used. The samples of Pinaceae exudates were dissolved in acetone to a concentration of 10 mg/mL. As reference substances for the substance class of diterpene resin acids, neoabietic acid and dehydroabietic acid were used (both dissolved in acetone, neoabietic acid to a concentration of 0.5 mg/mL, dehydroabietic acid to 1 mg/mL). Pinoresinol (as representative of lignans) was dissolved in methanol to a concentration of 1 mg/mL; ferulic acid (as representative of hydroxycinnamic acids) was also dissolved in methanol to a final concentration of 2.5 mg/mL. Five microliters of each sample solution were applied by a CAMAG Automatic TLC Sampler 4 (ATS4). Development was done in the CAMAG Automatic Developing Chamber (ADC2) with chloroform-methanol-trifluoroacetic acid (97 + 3 + 0.1) as mobile phase. The CAMAG TLC Visualizer was used to interpret and photograph the developed and dried plates under white light and at wavelengths 254 nm and 366 nm. The constituents showed improved visibility after derivatization with anisaldehyde/sulphuric acid solution, which was applied with the help of the CAMAG Chromatogram Immersion Device (III) (CAMAG, Muttenz, Switzerland).
3
HPTLC Analysis of Phytochemical Compounds
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High‑performance thin‑layer chromatographic (HPTLC) analyses were performed using a CAMAG-HPTLC system (CAMAG Chemie‑Erzeugnisse und Adsorptionstechnik AG, Muttenz, Switzerland) operated with winCATS software (CAMAG). Aliquots of samples (10 μL) and mixed reference compound solutions (5 μL) were applied to HPTLC glass plates coated with silica gel 60 F254 (Merck KGaA, Darmstadt, Germany) by a CAMAG Automatic TLC Sampler ATS 4. Two HPTLC separations were performed using two different mobile phase systems. In the first analysis, the application length for all the samples was set at 7 mm and a mobile phase consisting of chloroform-glacial acetic acid-methanol-water (64:32:12:8) was employed (Wagner and Bladt 1996 ). While, in the second analysis, the application length for all the samples was set at 8 mm and a mobile phase consisting of ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:26) was used (Wagner and Bladt 1996 ). HPTLC plates were developed in a CAMAG Automatic Developing Chamber ADC2 after 20 min equilibration with saturation pad and 5 min plate preconditioning to a final migration distance of 75 mm. After drying, the plates were derivatized with natural products-polyethylene glycol reagent (NP/PEG). The plates were visualized and photographed with a CAMAG TLC visualizer 2 after development and after derivatization at UV 254 and 366 nm, and at white light.
4
Lipid Extraction and Quantification Protocol
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Polar lipids were extracted from exponential-phase bacteria with chloroform-methanol (2:1 [by volume]) by the Bligh-Dyer procedure (33 (link)), vacuum dried, and dissolved in chloroform-methanol (2:1 [by volume]). For detection of Lys-PG, appropriate amounts of polar lipid extracts were spotted onto silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany) using a Linomat 5 sample application unit (Camag, Berlin, Germany) and developed with chloroform-methanol-water (65:25:4 [by volume]) in an automatic developing chamber ADC 2 (Camag, Berlin, Germany). Lys-PG content was quantified as described recently (6 (link)). Phospholipids of exponentially growing strains were selectively stained with molybdenum blue spray reagent (1.3% molybdenum oxide dissolved in 4.2 M sulfuric acid [Sigma]). Integrated lipid spot intensities of molybdenum blue-stained phospholipids were determined with ImageJ (http://rsbweb.nih.gov/ij/ )
5
HPTLC Confirmation of Crude Extract Components
6
HPTLC Analysis of Compounds
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The chromatographic stationary phase was a precoated silica gel 60 F254 HPTLC aluminum plate (20 × 10 cm, 0.1 mm thick 5–6 µm particle size; Merck, Darmstadt, Germany). The mobile phase composition was toluene: ethyl acetate: formic acid: and methanol (20:12:4:8, volume ratio). 20 µL and 2 µL of the standard solutions and samples, respectively, were spotted using ATS 4 autosampler fitted with a 100 µm Hamilton syringe. The plates were developed to a distance of 85 mm using the optimized mobile phase in CAMAG Automatic Developing Chamber (ADC 2) pre-saturated with the mobile phase. Saturation time was 20 min with 25 mL of mobile phase in a pad. Activation was done by MgCl2 (33% RH) for 10 min. The developed plates were dried for 5 min, with pre-drying, and preconditioned for 5 min. For densitometry measurements, spectra recording, and data processing, a CAMAG TLC Scanner 4 equipped with VisionCATS planar chromatography manager software was utilized to scan the dry plate. At a scan rate of 20 mm/s, the absorption/remission measuring mode was employed. Using deuterium and tungsten lamps, the standards and samples’ absorption spectra were recorded from 254 to 450 nm. The HPTLC analysis was performed at 25–26 °C and 44–46% humidity.
7
HPTLC Analysis of Crude Extract and Purified Sample
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High performance thin layer chromatography (HPTLC) experiments were performed to confirm our NMR results. Stock solutions of the crude extract (2 mg mL−1), glutamic acid (1 mg mL−1), and the purified sample (0.5 and 1 mg mL−1) were prepared. 30 µL of the crude extract, 10 µL of the glutamic acid solution and 20 µL of the purified sample were applied on a HPTLC plate (Merck, Darmstadt, Germany) using a Linomat V applicator (Camag, Muttenz, Switzerland). The bands were spotted with 10 mm width, spaced 10 mm from each other and 10 mm apart from the bottom edge of the plate. The plate was developed using the Automatic Developing Chamber ADC 2 (Camag) previously saturated with butanol:water:acetic acid (6:2:2, by vol.). Respective bands became visible by spraying the plate with 1 % ninhydrin dissolved in ethanol (cf. Fig. 5 ).
8
HPTLC Analysis of Astragaloside IV and Formononetin
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Standards were diluted in methanol at a concentration of 0.1 mg/mL for formononetin (Extrasynthèse, Genay, France) and 0.51 mg/mL for astragaloside IV (European Directorate for the Quality of Medicines & HealthCare, Strasbourg, France). A. mongholicus Bunge HRE without glycerol (4 mL) was diluted in 16 mL of a mixture of ethanol and water (50/50:v/v). The resultant solution was shaken and centrifuged for 3 min at 4400 rpm. The supernatant solution was transferred into individual vials and then subjected to HPTLC analysis.
HPTLC analysis was performed on 100.0 × 100.0 mm silica gel 60 F 254 HPTLC glass plates (Merck, Germany). Standard solutions and samples were applied to the plates in bands 8.0 mm wide using a CAMAG Automatic TLC sampler (ATS 4). The plates were developed in a CAMAG Automatic developing chamber (ADC2), derivatization being accomplished using a TLC plate heater and a CAMAG Chromatogram Immersion Device. The chromatograms were recorded by a CAMAG Visualizer with WinCATS software. The specific chromatographic conditions used for the three types of compounds analyzed are presented inSupplementary Table S1 .
HPTLC analysis was performed on 100.0 × 100.0 mm silica gel 60 F 254 HPTLC glass plates (Merck, Germany). Standard solutions and samples were applied to the plates in bands 8.0 mm wide using a CAMAG Automatic TLC sampler (ATS 4). The plates were developed in a CAMAG Automatic developing chamber (ADC2), derivatization being accomplished using a TLC plate heater and a CAMAG Chromatogram Immersion Device. The chromatograms were recorded by a CAMAG Visualizer with WinCATS software. The specific chromatographic conditions used for the three types of compounds analyzed are presented in
9
HPTLC Analysis of Kochia scoparia Extract
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High-performance thin-layer chromatography (HPTLC) was carried out using the CAMAG application system (Muttenz, Switzerland) consisting of CAMAG automatic TLC sampler 4, Reprostar 3, CAMAG ADC 2 automatic developing chamber, and a digital camera. The data were processed by WinCATS (CAMAG, Muttenz, Switzerland). The 20 μL loading of each standard and sample solution was spotted in bands of width 4 mm on the HPTLC plate by CAMAG application system. The samples were separated (migration distance 7 cm) using chloroform:methanol:water (7:3:0.5, v/v/v). The spots of migrated components were detected under white light using Reprostar 3 with a digital camera (CAMAG) after spraying with 10% H2SO4, followed by heating. For the HPTLC analysis, the lyophilized K. scoparia extract (50 mg) was transferred to 50 mL volumetric flask and dissolved up to the mark with 70% methanol.
10
Lipid Class Separation and Quantification by HPTLC
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Lipid classes were determined by two different developments chromatography to separate polar and neutral classes. Lipids were quantified by a CAMAG 3 TLC scanning densitometer (CAMAG, Muttenz, Switzerland) with identification of the classes against known polar and neutral lipid standards. Lipid extracts were loaded as a spot onto 20 × 10 cm Silica gel 60 F254 HPTLC plates (Merck KGaA, Darmstadt, Germany) using an ATS 5 automatic TLC sampler (CAMAG) The HPTLC silica gel plates were developed with a mixture of solvents in an ADC2 automatic developing chamber (CAMAG). The first eluent to separate polar lipids was a mixture of methyl acetate/isopropanol/chloroform/methanol/KCl (0.25% solution) in a ratio of 25:25:25:10:9 v/v/v/v/v running to a height of 7 cm from the origin. Additionally, the second eluent was a mixture of n-hexane/diethyl ether/glacial acetic acid in a ratio of 70:30:2 v/v/v to a height of 7 cm from the origin. After being dried, the plate was dipped for 6 seconds in a reagent (10 mg of primuline, 160 mL of acetone, 40 mL of water), then scanned using a TLC Scanner 3 with WinCATs software (CAMAG). Lipid classes were identified and quantified against those of corresponding lipid standards.
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