Refine
Year of publication
Document Type
- Article (reviewed) (43) (remove)
Language
- English (43)
Is part of the Bibliography
- yes (43) (remove)
Keywords
- Dünnschichtchromatographie (16)
- HPTLC (5)
- Chromatographie (4)
- Faseroptik (3)
- Belastung (2)
- Densitometrie (2)
- Diode-array detection (2)
- Fluorescence enhancement (2)
- High-performance thin-layer chromatography (2)
- Kubelka-Munk theory (2)
Institute
Open Access
- Closed Access (22)
- Open Access (7)
- Closed (4)
- Hybrid (2)
- Gold (1)
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.
The use of a TLC scanner can be regarded as a key step in high performance thin layer chromatography (HPTLC). Densitometric measurements transform the substance distribution on a TLC plate into digital computer data. Systems that allow quantitative measurements have been available for many years for either fluorescence or ultraviolet absorption measurements, while lately the reflection analysis mode for both types is the most common application. New scanning approaches are designed to aid the analyst who has common demands for TLC-densitometry without using special data, such as scanned images. Two examples that have been developed lately in the laboratories of the authors are described in this paper. These approaches were developed on the basis of current needs for analysts who employ TLC as a tool in research, as well as in routine analysis. One approach is aimed to support analysts in economically disadvantaged areas, where cost intensive apparatus is unsuitable but trace analysis by simple means is required. The other system, allows the spectral determination of chromatographic spots on TLC plates covering the ultraviolet and visible range, thus, revealing highly desired information for the analyst.
A systematic toxicological analysis procedure using high-performance thin layer chromatography in combination with fibre optical scanning densitometry for identification of drugs in biological samples is presented. Two examples illustrate the practicability of the technique. First, the identification of a multiple intake of analgesics: codeine, propyphenazone, tramadol, flupirtine and lidocaine, and second, the detection of the sedative diphenhydramine. In both cases, authentic urine specimens were used. The identifications were carried out by an automatic measurement and computer-based comparison of in situ UV spectra with data from a compiled library of reference spectra using the cross-correlation function. The technique allowed a parallel recording of chromatograms and in situ UV spectra in the range of 197–612 nm. Unlike the conventional densitometry, a dependency of UV spectra by concentration of substance in a range of 250–1000 ng/spot was not observed.
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.
An algorithm is presented that has successfully been utilized in practice for several years. It improves data analysis in chromatography. The program runs in an extremely reliable way and evaluates chromatographic raw data with an acceptable error. The algorithm requires a minimum of preliminaries and integrates even unsmoothed noisy data correctly.
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.
A Validated Quantification of Sudan Red Dyes in Spicery using TLC and a 16-bit Flatbed Scanner
(2018)
We present a video-densitometric quantification method for Sudan red dyes in spices and spice mixtures, separated by TLC. Application was done band-wise in small dots using a 5 μL glass pipette. For separation, the RP-18 plates (20 × 20 cm with fluorescent dye; Merck, Germany, 1.05559) were developed in a vertical developing chamber without vapor saturation from the starting point to a distance of 70 mm by using acetonitrile, methanol, and aqueous ammonia solution (25%; 8 + 1.8 + 0.2, v/v) as mobile phase. The quantification is based on direct measurements using an inexpensive 16-bit flatbed scanner for color measurements (in red, green, and blue). Evaluation of only the green channel makes the measurements very specific. For linearization, an extended Kubelka-Munk expression for data transformation was used. The range of linearity covers more than two magnitudes and lies between 20 and 500 ng. The extraction from a 2 g sample with acetonitrile, evaporation, and reconstitution to 200 μL with methanol and the band-wise application (7 mm) of a 10 μL sample allows a statistically defined LOD of less than 500 ppb of Sudan red dyes. To perform the analysis, a separation chamber, RP-18 plates, 5 μL glass pipettes, and a 16-bit flatbed scanner for 105 € are needed; therefore, the separation method is inexpensive, fast, and reliable.
We present a planar chromatographic separation method for the phytoestrogenic active compound equol, separated on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic cattle manure extract contains this phytoestrogenic active compound to a larger amount. As solvents for the mobile phase, hexane, ethyl acetate, and acetone (45:15:10, v/v); acetone and water (15:10, v/v); and n-hexane, CH2Cl2, ethyl acetate, methanol, and formic acid (40:40:20:5:1, v/v) have been used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505 containing an estrogen receptor. Its activation by equol induces the reporter gene lacZ which encodes the enzyme β-galactosidase. The enzyme activity is measured directly on the TLC plate by using the substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside) or the substrate X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside). β-Galactosidase cleaves MUG into a fluorescing compound. X-β- Gal is also hydrolyzed and then oxidized by oxygen forming the deep-blue dye 5,5′-dibromo-4,4′-dichloro-indigo. Both reactions in combination with a thin-layer chromatography (TLC) separation allow very specific detecting of equol in cattle manure, although that is a very challenging matrix. Preliminary results show that the average content of equol in liquid manure is roughly 60 μg g−1. The value for urine is 50 μg mL−1.
We present a video-densitometric quantification method in combination with diode-array quantification for the methyl-, ethyl-, propyl-, and butylparaben in cosmetics. These parabens were separated on cyanopropyl bonded plates using water-acetonitrile-dioxane-ethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. The quantification is based on UV-measurements at 255 nm and a bioeffectively-linked analysis using Vibrio fischeri bacteria. Within 5 min, a Tidas S 700 diode-array scanner (J&M, Aalen, Germany) scans 8 tracks and thus measures in total 5600 spectra in the wavelengths range from 190 to 1000 nm. The quantification range for all these parabens is from 20 to 400 ng per band, measured at 255 nm. In the V. fischeri assay a CCD-camera registers the white light of the light-emitting bacteria within 10 min. All parabens effectively suppress the bacterial light emission which can be used for quantifying within a linear range from 100 to 400 ng. Measurements were carried out using a 16-bit MicroChemi chemiluminescence system (biostep GmbH, Jahnsdorf, Germany), using a CCD camera with 4.19 megapixels. The range of linearity is achieved because the extended Kubelka-Munk expression was used for data transformation. The separation method is inexpensive, fast, and reliable.
A 2D-separation of 16 polyaromatic hydrocarbons (PAHs) according to the Environmental Protecting Agency (EPA) standard was introduced. Separation took place on a TLC RP-18 plate (Merck, 1.05559). In the first direction, the plate was developed twice using n-pentane at −20°C as the mobile phase. The mixture acetonitrile-methanol-acetone-water (12:8:3:3, v/v) was used for developing the plate in the second direction. Both developments were carried out over a distance of 43 mm. Further on in this publication, a specific and very sensitive indication method for benzo[a]pyrene and perylene was presented. The method can detect these hazardous compounds even in complicated PAH mixtures. These compounds can be quantified by a simple chemiluminescent reaction with a limit of detection (LOD) of 48 pg per band for perylene and 95 pg per band for benzo[a]pyrene. Although these compounds were separated from all other PAHs in the standard, a separation of both compounds was not possible from one another. The method is suitable for tracing benzo[a]pyrene and/or perylene. The proposed chemiluminescence screening test on PAHs is extremely sensitive but may indicate a false positive result for benzo[a]pyrene.
We present a two-dimensional (2D) planar chromatographic separation method for phytoestrogenic active compounds on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic extract of liquorice (Glycyrrhiza glabra) roots contains four phytoestrogenic active compounds. As solvent, in the first direction, the mix of hexane, ethyl acetate, and acetone (45:15:10, v/v) was used, and, in the second direction, that of acetone and water (15:10, v/v) was used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside). The enzyme can also hydrolyse X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranosid) into β-galactose and 5-bromo-4-chloro-3-indoxyl. The indoxyl compound is oxidized by oxygen forming the deep-blue dye 5,5β-dibromo-4,4β-dichloro-indigo which allows to detect phytoestrogenic activity more specific in the presence of native fluorescing compounds.
We present a two-dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 W (Merck, 1.14296) phase. A mixture of 8 substances was separated using a solvent mix consisting of hexane, ethyl acetate, acetone (55:15:10, v/v) in the first direction and of acetone and water (15:10, v/v) in the second direction. Separation was performed on an RP-18 W plate over a distance of 70 mm. This 2D-separation method can be used to quantify 17α-ethinylestradiol (EE2) in an effect-directed analysis, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside).
We present a two dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 (Merck, 1.05559) and silica gel (Merck, 1.05721) phase. A mixture of 13 substances was separated using a solvent mix consisting of methanol–acetonitrile–water (2 + 2 + 1, v/v/v) on RP-18 phase in the first direction and cyclohexane–butylacetate–methanol (8 + 6 + 1, v/v/v) in the second direction on silica gel plate. Both developments were carried out over a distance of 70 mm. We used the grafted method to combine both plates in a 2D-separation. This 2D-separation method can be used to quantify 17α-ethinylestradiol (EE2) in an effect-directed analysis using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducting the reporter gene lacZ that encodes the enzyme ß-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl ß-D-galactopyranoside).