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HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows many advantages when compared to other separation techniques. The method is fast and inexpensive and does not need time-consuming pretreatments. For visualisation of the sample distribution on a HPTLC-plate we developed a new and sturdy HPTLC-scanner. The scanner allows simultaneous registrations of spectra in a range from 198 nm to 612 nm with a spectral resolution of better than 0.8 nm. The on-plate spatial resolution is better than 160 μm. The measurement of 450 spectra in one separation track does not need more than two minutes. The new diode-array scanner offers a fast survey over a TLC-separation and makes various chemometric applications possible. For compound identification a cross-correlation function is described to compare UV sample spectra with appropriate library data. The cross-correlation function herein described can also be used for purity testing. Unresolved peaks can be virtually separated by use of a least squares fit algorithm. In summary, the diode arry system delivers much more information than the commonly used TLC-scanner.
HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows a lot of advantages and options in comparison to other separation techniques. The method is fast and inexpensive and does not need time-consuming pretreatments. Using fiber-optic elements for controlled light-guiding, the TLC-method was significantly improved: the new HPTLC-system is able to measure simultaneously at different wavelengths without destroying the plate surface or the analytes on the surface. For registration of the sample distribution on a HPTLC-plate we developed a new and sturdy diode-array HPTLC- scanner which allows registration of spectra on the TLC- plates in the range of 198 nm to 610 nm with a spectral resolution better than 1.2 nm. The spatial resolution on plate is better than 160 micrometers . In the spectral mode, the new HPTLC-scanner delivers much more information than the commonly used TLC-scanner. The measurement of 450 spectra of one separation track does not need more than three minutes. However, in the fixed wavelength mode the contour plot can be measured within 15 seconds. In this case, the signal will be summarized and averaged over a spectral range having FWHM from 10 nm to 25 nm depending on the substance under test. The new diode-array HPTLC-scanner makes various chemometric applications possible. The new method can be used easily in clinical diagnostic systems easily, e.g. for blood and uring investigations. In addition, new applications are possible. For example, the rich structured PAHs were studied. Although the separation is incomplete the 16 compounds can be quantified using suitable wavelengths.
In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography (TLC). It is a simple means of quantification by measurement of the optical density of the separated spots directly on the plate. A new scanner has been developed which is capable of measuring TLC or HPTLC (high-performance thin-layer chromatography) plates simultaneously at different wavelengths without damaging the plate surface. Fiber optics and special fiber interfaces are used in combination with a diode-array detector. With this new scanner sophisticated plate evaluation is now possible, which enables use of chemometric methods in HPTLC. Different regression models have been introduced which enable appropriate evaluation of all analytical questions. Fluorescent measurements are possible without filters or special lamps and signal-to-noise ratios can be improved by wavelength bundling. Because of the richly structured spectra obtained from PAH, diode-array HPTLC enables quantification of all 16 EPA PAH on one track. Although the separation is incomplete all 16 compounds can be quantified by use of suitable wavelengths. All these aspects are enable substantial improvement of in-situ quantitative densitometric analysis.
HPTLC on amino plates, with simple heating of the plates for derivatization, has been used for quantification of glucosamine in nutritional supplements. On heating the plate glucosamine reacts to form a compound which strongly absorbs light between 305 and 330 nm, with weak fluorescence. The reaction product can be detected sensitively either by absorption of light or by fluorescence detection. The detection limit in absorption mode is approximately 25 ng per spot. In fluorescence mode a detection limit of 15 ng is achievable. A calibration plot for absorption detection is linear in the range 25 to 4000 ng glucosamine. The derivative formed from glucosamine by heating is stable for months, and the relative standard deviation is 1.64% for 600 ng glucosamine. The amounts of glucosamine found in nutritional supplements were in agreement with the label declarations.
A new diode-array scanner in combination with a computer-controlled application system meets all the demands of modern HPTLC measurement. Automatic application, simultaneous measurements at different wavelengths, and different linearization models enable appropriate evaluation of all analytical questions. The theory of error propagation recommends quantification at reflectance values smaller than 0.8; this can be verified only by use of diode-array scanning. The same theory also recommends quantification by use of peak height data, because the theory predicts best precision only for peak height evaluation. Diode-array scanning with reflectance monitoring enables appropriate validation in TLC and HPTLC analysis. All these aspects result in substantial improvement of in-situ quantitative densitometric analysis, and simultaneous recording at different wavelengths opens the way for chemometric evaluation, e.g. peak purity monitoring, which improves the accuracy and reliability of HPTLC analysis.
In this paper a high-performance thin-layer chromatography (HPTLC) scanner is presented in which a special fibre arrangement is used as HPTLC plate scanning interface. Measurements are taken with a set of 50 fibres at a distance of 400 to 500 μm above the HPTLC plate. Spatial resolutions on the HPTLC plate of better than 160 μm are possible. It takes less than 2 min to scan 450 spectra simultaneously in a range of 198 to 610 nm. The basic improvement of the item is the use of highly transparent glass fibres which provide excellent transmission at 200 nm and the use of a special fibre arrangement for plate illumination and detection.
Previous studies of the hyphenation of gas chromatographic separation and spectrophotometric detection in the ultraviolet wavelength range between 168 and 330 nm showed a high potential for applications where the analysis of complex samples is required. Within this paper the development of a state-of-the-art detection system for compounds in the vapour phase is described, offering an improved behaviour compared to previous systems: Dependent on the requirements of established detection systems hyphenated with gas chromatography, the main components of the system have to be designed for optimum performance and reliability of the spectrophotometric detector: A deuterium lamp as a broadband light source has been selected for improved stability in the measurements. A new-type absorption cell based on fiber-optics has been developed considering the dynamic necessary to compete with existing techniques. In addition, the influence of the volume of the cell on the chromatogram needs to be analyzed. Tests for determining the performance of the absorption cell in terms of chemical and thermal influences have been carried out. A new spectrophotometer with adequate spectral resolution in the wavelength range, offering improved stability and dynamic for an efficient use in this application was developed. Furthermore, the influence of each component on the performance, reliability and stability of the sensor system will be discussed. An overview and outlook over the potential applications in the environmental, scientific and medical field will be given.
The identification and quantification of compounds in the gas phase becomes of increasing interest in the context of environmental protection, as well as in the analytical field. In this respect, the high extinction coefficients of vapours and gases in the ultraviolet wavelength region allow a very sensitive measurement system. In addition, the increased performance of the components necessary for setting up a measurement system, such as fibres, light sources and detectors has been improved. In particular the light sources and detectors offer improved stability, and the deep UV performance and solarisation resistance of fused silica fibres allow have been significantly optimized in the past years. Therefore a compact and reliable detection system with high measuring accuracy is developed. Within this paper possible applications of the system under development and recent results will be discussed.
In thin-layer chromatography, fiber-bundle arrays have been introduced for spectral absorption measurements in the UV-region. Using all-silica fiber bundles, the exciting light will be detected after re-emission on the plate with a fiberoptic spectrometer. In addition, fluorescence light can be detected which will be masked by the re-emitted light. Therefore, it is helpful to separate the absorption and fluorescence on the TLC-plate. A modified three-array assembly has been developed: using one array for detection, the two others are used for excitation with broadband band deuterium-light and with UV-LEDs adjusted to the substances under test. As an example, the quantification of glucosamine in nutritional supplements or spinach leaf extract will be described. Using simply heating of the amino-plate for derivation, the reaction product of Glucosamine can be detected sensitively either by light absorption or by fluorescence, using the new fiber-optic assembly. In addition, the properties of the new 3-row fiber-optic array and the commercially available UV-LEDs will be shown, in the interesting wavelength region for excitation of fluorescence, from 260 nm to 360 nm. The squint angle having an influence on coupling efficiency and spatial resolution will be measured with the inverse farfield method. Some properties of UV-LEDs for analytical applications will be described and discussed, too.
We will present the first example of a two-dimensional scanned TLC-plate, measured by use of a diode-array scanner. A spatial resolution of 250 µm was achieved on plate. The system provides real 2D fluorescence and absorption spectra in the wavelength-range from 190 to 1000 nm with a spectral resolution of greater than 1 nm. A mixture of 12 sulphonamides was separated by using a cyanopropyl-coated silica gel plate (Merck, 1.16464) with the solvent mix of methyl tert-butyl ether-methanol-dichloromethane-cyclohexane-NH3 (25%) (48:2:2:1:1, v/v) in the first and with a mixture of water-acetonitrile-dioxane-ethanol (8:2:1:1, v/v) in the second direction. Both developments were carried out over a distance of 70 mm. A separation number (spot capacity) of 259 was calculated. We discussed a new formula for its calculation in 2D-TLC separations. The drawback of this method is that measuring a 2D-TLC plate needs more than 3 h measurement time.