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A new formula is presented for transforming fluorescence measurements in accordance with Kubelka-Munk theory. The fluorescence signals, the absorption signals, and data from a selected reference are combined in one expression. Only diode-array techniques can measure all the required data simultaneously to linearize fluorescence data correctly. To prove the new theory HPTLC quantification of the analgesic flupirtine was performed over the mass range 300 to 5000 ng per spot. The fluorescence calibration curve was linear over the whole range. The transformation of fluorescence measurements into linear mass-dependent data extends the technique of in-situ fluorescence analysis to the high concentration range. It also extends Kubelka-Munk theory from absorption to fluorescence analysis. The results presented also emphasize the importance of Kubelka-Munk theory for in-situ measurements in scattering media, especially in planar chromatography.
We present a videodensitometric quantification method for methadone in syrup, separated by thin-layer chromatography (TLC). The quantification is based on a derivation reaction with Dragendorf reagent. Measurements were carried out using a 16-bit flatbed scanner. The range of linearity covers two magnitudes of power using the Kubelka-Munk expression for data transformation. The separation method is inexpensive, fast, and reliable.
Vorrichtung (2) zur Analyse von Urin, umfassend: – eine Zuführ- und Abführeinrichtung (7), welche zur Zuführung einer bestimmten Urinmenge in eine wenigstens einen Analysebereich (8) aufweisende Analysekammer (9) eines Urinteststreifens (10) und zur Abführung einer bestimmten Urinmenge aus einer wenigstens einen Analysebereich (8) aufweisenden Analysekammer (9) eines Urinteststreifens (10) eingerichtet ist, wobei die Zuführ- und Abführeinrichtung (7) wenigstens ein bewegbar gelagertes Zuführ- und/oder Abführelement (28, 29) zum Zuführen einer bestimmten Urinmenge in einen Zuführbereich (33) der Analysekammer (9) des Urinteststreifens (10) und/oder zum Abführen einer bestimmten Urinmenge aus einem Abführbereich (34) der Analysekammer (9) des Urinteststreifens (10) aufweist, und – eine Erfassungseinrichtung (11), welche zur Erfassung einer zumindest abschnittsweisen Änderung wenigstens eines optisch erfassbaren Parameters, welcher sich in Abhängigkeit der Zusammensetzung einer diesen kontaktierenden Urinmenge optisch erfassbar verändert, des oder eines entsprechenden Analysebereichs (8) des oder eines entsprechenden Urinteststreifens (10) sowie zur Erzeugung einer Erfassungsinformation, welche wenigstens einen optisch erfassten Parameter des oder eines entsprechenden Analysebereichs (8) oder eine Änderung eines solchen beschreibt, eingerichtet ist.
Vorrichtung (2) zur Analyse von Urin, umfassend: – eine Zuführ- und Abführeinrichtung (7), welche zur Zuführung einer bestimmten Urinmenge in eine wenigstens einen Analysebereich (8) aufweisende Analysekammer (9) eines Urinteststreifens (10) und zur Abführung einer bestimmten Urinmenge aus einer wenigstens einen Analysebereich (8) aufweisenden Analysekammer (9) eines Urinteststreifens (10) eingerichtet ist, wobei die Zuführ- und Abführeinrichtung (7) wenigstens ein bewegbar gelagertes Zuführ- und/oder Abführelement (28, 29) zum Zuführen einer bestimmten Urinmenge in einen Zuführbereich (33) der Analysekammer (9) des Urinteststreifens (10) und/oder zum Abführen einer bestimmten Urinmenge aus einem Abführbereich (34) der Analysekammer (9) des Urinteststreifens (10) aufweist, und – eine Erfassungseinrichtung (11), welche zur Erfassung einer zumindest abschnittsweisen Änderung wenigstens eines optisch erfassbaren Parameters, welcher sich in Abhängigkeit der Zusammensetzung einer diesen kontaktierenden Urinmenge optisch erfassbar verändert, des oder eines entsprechenden Analysebereichs (8) des oder eines entsprechenden Urinteststreifens (10) sowie zur Erzeugung einer Erfassungsinformation, welche wenigstens einen optisch erfassten Parameter des oder eines entsprechenden Analysebereichs (8) oder eine Änderung eines solchen beschreibt, eingerichtet ist.
Vorrichtung (2) zur Analyse von Urin, umfassend: – eine Zuführ- und Abführeinrichtung (7), welche zur Zuführung einer bestimmten Urinmenge in eine wenigstens einen Analysebereich (8) aufweisende Analysekammer (9) eines Urinteststreifens (10) und zur Abführung einer bestimmten Urinmenge aus einer wenigstens einen Analysebereich (8) aufweisenden Analysekammer (9) eines Urinteststreifens (10) eingerichtet ist, wobei die Zuführ- und Abführeinrichtung (7) wenigstens ein bewegbar gelagertes Zuführ- und/oder Abführelement (28, 29) zum Zuführen einer bestimmten Urinmenge in einen Zuführbereich (33) der Analysekammer (9) des Urinteststreifens (10) und/oder zum Abführen einer bestimmten Urinmenge aus einem Abführbereich (34) der Analysekammer (9) des Urinteststreifens (10) aufweist, und – eine Erfassungseinrichtung (11), welche zur Erfassung einer zumindest abschnittsweisen Änderung wenigstens eines optisch erfassbaren Parameters, welcher sich in Abhängigkeit der Zusammensetzung einer diesen kontaktierenden Urinmenge optisch erfassbar verändert, des oder eines entsprechenden Analysebereichs (8) des oder eines entsprechenden Urinteststreifens (10) sowie zur Erzeugung einer Erfassungsinformation, welche wenigstens einen optisch erfassten Parameter des oder eines entsprechenden Analysebereichs (8) oder eine Änderung eines solchen beschreibt, eingerichtet ist.
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.
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.
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.
A Simple and Reliable HPTLC Method for the Quantification of the Intense Sweetener Sucralose®
(2003)
This paper describes a simple and fast thin layer chromatography (TLC) method for the monitoring of the relatively new intense sweetener Sucralose® in various food matrices. The method requires little or no sample preparation to isolate or concentrate the analyte. The Sucralose® extract is separated on amino‐TLC‐plates, and the analyte is derivatized “reagent‐free” by heating the developed plate for 20 min at 190°C. Spots can be measured either in the absorption or fluorescence mode. The method allows the determination of Sucralose® at the levels of interest regarding foreseen European legislation (>50 mg/kg) with excellent repeatability (RSD = 3.4%) and recovery data (95%).
Quantification of astaxanthin in salmons by chemiluminescence and absorption after TLC separation
(2018)
Astaxanthin is a keto-carotenoid, belongs to the chemical class of terpenes and is a yellow lipid soluble compound. The compound is present in marine animals like salmons and crustacean. Its colour is due to conjugated double bonds and these double bonds are responsible for its antioxidant effect. Its antioxidant activity is ten times stronger than other carotenoids and nearly 500 fold stronger than vitamin-E. We present a new thin layer chromatography (TLC) method to measure astaxanthin on TLC-plates (Merck, 1.05554) in the visible absorption range as well as by using chemiluminescence. For separation a solvent mixture of cyclohexane and acetone (10 + 2.4, v/v) was used. The RF-value of astaxanthin is 0.14.The limit of detection in vis-absorption is 64 ng / band and the limit of quantification is 92 ng/band. In chemiluminescence the values are 90 ng / band and 115 ng/band. The method offers two independently working measurement modes on a single plate which increase the accuracy of the quantification.
Rudolf E. Kaiser
(2020)
Editorial
(2020)
Editorial
(2020)
Enzyme‐assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate
(2023)
Background
The analysis of myo‐inositol phosphates (InsPx) released by phytases during phytic acid degradation is challenging and time‐consuming, particularly in terms of sample preparation, isomer separation, and detection. However, a fast and robust analysis method is crucial when screening for phytases during protein engineering approaches, which result in a large number of samples, to ensure reliable identification of promising novel enzymes or target variants with improved characteristics, for example, pH range, thermal stability, and phosphate release kinetics.
Results
The simultaneous analysis of several InsPx (InsP1‐InsP4 and InsP5 + 6) as well as free phosphate was established on cellulose HPTLC plates using a buffered mobile phase. Inositol phosphates were subsequently stained using a novel enzyme‐assisted staining procedure. Immobilized InsPx were hydrolyzed by a phytase solution of Quantum® Blueliquid 5G followed by a molybdate reagent derivatization. Resulting blue zones were captured by DAD scan. The method shows good repeatability (intra‐day and intra‐lab) with maximum deviations of the Rf value of 0.01. The HPTLC method was applied to three commercially available phytases at two pH levels relevant to the gastrointestinal tract of poultry (pH 5.5 and pH 3.6) to observe their phytate degradation pattern and thus visualize their InsPx fingerprint.
Conclusion
This HPTLC method presents a semi‐high‐throughput analysis for the simultaneous analysis of phytic acid and the resulting lower inositol phosphates after its enzymatic hydrolysis and is also an effective tool to visualize the InsPx fingerprints and possible accumulations of inositol phosphates.
Editorial
(2022)
Editorial
(2022)
Editorial
(2020)
Editorial
(2022)
Editorial
(2021)