TLC scanner

High pressure xenon lamps are also employed in some TLC scanners (e.g. the scanner of Schoeffel and that of Farrand). They produce higher intensity radiation than do hydrogen or tungsten lamps. The maximum intensity of the radiation emitted lies between k = 500 and 700 nm. In addition to the continuum there are also weak emission lines below k = 495 nm (Fig. 14). The intensity of the radiation drops appreciably below k = 300 nm and the emission zone, which is stable for higher wavelengths, begins to move [43]. 

The traditional method of determining the position of an analyte spot on the plates is a visual evaluation. However, this technique is highly subjective and depends considerably on the expertise of the analytical chemist. TLC scanners, developed for exact determination not only pinpoint position but also the area, intensity and symmetry of the spot, overcome the uncertainty of the visual evaluation. Moreover, TLC scanners make possible more accurate determination of the quantity of analyte in the spot by converting spot characteristics into peak characteristics. Peak height is the distance between the peak maximum and the baseline, whereas peak area is the area of the peak between the beginning and end of the peak and the baseline. 

Cabon tetrachloride, n-hexane, chloroform, ACN, acetone, THF, pyridine, acetic acid, and their various mixtures were applied as mobile phases for adsorption TLC. Methanol, 1-propanol, ACN, acetone, THF, pyridine and dioxane served as organic modifiers for RP-TLC. Distilled water, buffers at various pH (solutions of and dipotassium hydrogen phosphate or potassium dihydrogen phosphate) and solutions of lithium chloride formed the aqueous phase. Carotenoids were extracted from a commercial paprika sample by acetone (lg paprika shaken with 3 ml of acetone for 30 min), the solution was spotted onto the plates. Development was carried out in a sandwich chamber in the dark and at ambient temperature. After development (15 cm for normal and 7cm for HPTLC plates) the plates were evaluated by a TLC scanner. The best separations were realized on impregnated diatomaceous earth stationary phases using water-acetone and water-THF-acetone mixtures as mobile phases. Some densitograms are shown in Fig.2.1. Calculations indicated that the selectivity of acetone and THF as organic modifiers in RP-TLC is different [14],... 

The ultraviolet reflectance absorbance spectrum of a benzoic acid spot on a thin-layer chromatography place was recorded on a Shimadzu CS-930 TLC scanner, and is shown in Figure 5. The spectrum was obtained on a silica-gel F254 pre-coated TLC-plate (E. Merck) after elution with 7 2 2 acetone/ammonia 25 %/isopropanol. The absorbance maximum for this system was determined to be 225 nm. 

In spite of the above advantage, the quantitation by TLC-scanner is accompanied by various difficulties. The most direct difficulty is caused by hypochromic effects in the ultraviolet spectra of copolymers, especially those containing styrene units. 

Automatic TLC-scanner (Tracemaster 20, Berthold, Bad Wildbach, Germany). Calibration... 

The method of Denda et al.14 was used to measure ceramides. After SC was stripped with adhesive cellophane tape, it was removed from the tape and washed several times with hexane, followed by drying in a vacuum desiccator. Lipids were extracted from the SC sample in a mixture of chloroform and methanol (2 1). Ceramides were separated with a silica gel column (Bond Elut SI, Analytichem International, United States) and purified for measurement by gas chromatography (GC-14A, Shimazu Manufacturing Co., Ltd., Japan). The composition of ceramides was obtained by high-performance thin layer chromatography and scanned on a recording photodensitometer (TLC Scanner CS930, Shimazu, Japan). 

Trace amounts of nimodipine could be determined using TLC on silica gel GF254 plates and a mobile phase consisting of chloroform/methanol/ methylene chloride/ra-hexane (26 12 10 5). A. TLC-scanner was used to measure the absorbance at 365 nm (Rf = 0.48). The calibration graph was linear from 5 ng to 1 pg, with a detection limit of 5 ng [12]. 

The Optical Layout of the CAMAG TLC Scanner Courtesy of CAMAG Inc. 

Densitometry equipment (e.g., Shimadzu CS-930 Dual Wavelength TLC Scanner), fitted, if possible, with a microprocessor-based data acquisition system. 

Densitometric monitoring has generally been applied to evaluate the separated bands. TLC scanners are mainly u.sed to evaluate one-dimensional (ID) separations two-dimensional (2D) scanners are mainly used to detect spots after gel electrophoresis. 

Fig. 4 Light path of TLC scanner 3. (Diagram courtesy of Camag Scientific, Inc.)...

The well-known and widely applied TLC silica gel G plates 20 x 20 cm from Merck (Darmstadt, FRG) is used. Stock solutions from the compounds given in Table 1 were prepared. After corresponding dilution, a 1-/aL sample is applied and the plate is developed for a distance of 10 cm using n-propanol-water, 8 2 (v/v), as the mobile phase. Densitometric evaluation is performed at 365 nm with a Camag TLC Scanner II in absorbance reflection mode. The plate is first air-dried and then heated in an oven for 5 min at 100°C. Immediately after drying, the plate is inserted in a tank saturated with ammonia atmosphere (ammonia solution in... 

Coupled spectroscopic methods such as TLC-UV (ultraviolet) and visible spectroscopy, TLC-mass spectrometry, and TLC-FTIR (Fourier transform infrared) have been developed to overcome this difficulty [7]. Their future application in the TLC analysis of natural pigments will markedly increase the information content of this simple and interesting separation technique. The automation of the various steps of TLC analysis (sample application, automated developing chambers, TLC scanners, etc.) greatly increased the reliability of the method, making it suitable for official control and legislative purposes [8]. 

Integration of peak areas in routine analysis is mostly achieved with electronic integrators. Two main problems arise from this evaluation technique first the definition of the baseline, and second, the determination of the time constant of the TLC-scanner and the integrator electronics. 

Table 1. Results of linearized and nonlinear regression. Substance papaverine precoated HPTLC-plate, E. Merck, Darmstadt. Solvent Acetone/toluene 20 20 (v/v) Camag TLC-scanner, reflectance at 254 nm 79)...

The visible absorbance reflectance spectrum of diosgenin on Kieselgel GF254 TLC precoated (E.Merck), eluted with CHCb/ethyl acetate (3/1) and visualized by anisaldehyde-sulphuric acid spray reagent (100°C, 10 min) was shown in Figure 3. It showed a maximum at 430 nm. The spectrum was recorded on a Shimadzu CS-930 TLC Scanner. 

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