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Die Analytik von Pflanzenkohle ist besonders im Hinblick auf einen möglichen Einsatz in der Landwirtschaft als Düngemittel Ersatz wichtig, da bei der Herstellung krebserregende polyzyklische aromatische Kohlenwasserstoffe (PAK) entstehen. Die PAK-Analytik bietet die Möglichkeit zu überprüfen und zu kontrollieren, wie viel belastetes Material auf diese Weise in die Umwelt eingeführt wird. In dieser Arbeit werden mögliche Methoden zur Extraktion und Analyse im Hinblick auf einen Schnelltest mit der bestehenden Methode bestehend aus Soxhlet-Extraktion und GC-MS Analyse verglichen. Untersucht werden die 16 EPA-PAK. Ein Schnelltest würde den Herstellern von Pflanzenkohle eine günstigere und engmaschigere Überwachung ihrer Produkte bieten.
Für die Extraktion wurden Ultraschallbad, Schüttler und Mikrowelle getestet, sowie verschiedene grüne Lösemittel als mögliche Alternativen zum momentan verwendeten Toluol. Die Extraktion mit 6 h Ultraschallbad hat mit 64% im Verhältnis zur Soxhlet am besten abgeschnitten. Bei dem Vergleich zweier Ultraschallbad-Extrakte mit den Soxhlet-Referenzen wurden jedoch 43 % und 93 % für die Summe der 16 EPA PAK gemessen. Die Methode bedarf also weiterer Untersuchungen.
Für die Analyse wurde HPTLC mit RP-18 Platten und einem Acetonitril-Wasser-Gemisch als mobile Phase getestet. Es wurden drei mögliche Methoden für die Gestaltung eines Schnelltestes getestet, ein Summenpeak mit allen 16 EPA-PAK und Naphthalin oder Benzo[a]pyren als Indikatorsubstanz. Aus diesen Versuchen geht das Standard-Additionsverfahren mit Benzo[a]pyren erfolgreich hervor, da die Ergebnisse durch die GC-MS-Analyse der gleichen Extrakte bestätigt werden konnten. Die Bestimmung von LOD und LOQ bestätigt, dass die Methode empfindlich genug ist, um den nötigen Messbereich erfassen zu können. Es wurden auch Versuche mit Coffein imprägnierten Platten durchgeführt. Diese Methode ist jedoch wegen der notwendigen Zweifachentwicklung deutlich aufwendiger und wurde daher nicht für die quantitativen Versuche gewählt.
High-performance thin-layer chromatography (HPTLC), as the modern form of TLC (thin-layer chromatography), is suitable for detecting pharmaceutically active compounds over a wide polarity range using the gradient multiple development (GMD) technique. Diode-array detection (DAD) in conjunction with HPTLC can simultaneously acquire ultraviolet‒visible (UV‒VIS) and fluorescence spectra directly from the plate. Visualization as a contour plot helps to identify separated zones. An orange peel extract is used as an example to show how GMD‒DAD‒HPTLC in seven different developments with seven different solvents can provide an overview of the entire sample. More than 50 compounds in the extract can be separated on a 6-cm HPTLC plate. Such separations take place in the biologically inert stationary phase of HPTLC, making it a suitable method for effect-directed analysis (EDA). HPTLC‒EDA can even be performed with living organism, as confirmed by the use of Aliivibrio fischeri bacteria to detect bioluminescence as a measure of toxicity. The combining of gradient multiple development planar chromatography with diode-array detection and effect-directed analysis (GMD‒DAD‒HPTLC‒EDA) in conjunction with specific staining methods and time-of-flight mass spectrometry (TOF‒MS) will be the method of choice to find new chemical structures from plant extracts that can serve as the basic structure for new pharmaceutically active compounds.
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.
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.
Two solvent mixtures for high-performance thin-layer chromatographic (HPTLC) separation of some compounds showing estrogenic activity in the yeast estrogen screen (YES) assay are presented. The new method, planar yeast estrogen screen (pYES) combines the YES assay and a chromatographic separation on silica gel HPTLC plates with the performance of the YES assay. For separation, the analytes were applied bandwise to HPTLC plates (10 × 20 cm) with fluorescent dye (Merck, Germany). The plates were developed in a vertical developing chamber after 30 min of chamber saturation over a separation distance of 70 mm, using cyclohexane‒methyl-ethyl ketone (2:1, V/V) or cyclohexane‒CPME (3:2, V/V) as solvents. Both solvents allow separation of estriol, daidzein, genistein, 17β-estradiol, 17α-ethinyl estradiol, estrone, 4-nonylphenol and bis(2-ethylhexyl) phthalate.
The research employed HPTLC Pro System and other HPTLC instruments from CAMAG® to conduct various laboratory tests, aiming to compile a database for subsequent analyses. Utilizing MATLAB, distinct codes were developed to reveal patterns within analyzed biomasses and pyrolysis oils (sewage sludge, fermentation residue, paper sludge, and wood). Through meticulous visual and numerical analysis, shared characteristics among different biomasses and their respective pyrolysis oils were revealed, showcasing close similarities within each category. Notably, minimal disparity was observed in fermentation residue and wood biomasses with a similarity coefficient of 0.22. Similarly, for pyrolysis oils, the minimal disparity was found in fermentation residues 1 and 3, with a disparity coefficient of 1.41. Despite higher disparity coefficients in certain results, specific biomasses and pyrolysis oils, such as fermentation residue and sewage sludge, exhibited close similarities, with disparity coefficients of 0.18 and 0.55, respectively. The database, derived from triplicate experimentation, now serves as a valuable resource for rapid analysis of newly acquired raw materials. Additionally, the utility of HPTLC PRO as an investigation tool, enabling simultaneous analysis of up to five samples, was emphasized, although areas for improvement in derivatization methods were identified.
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.
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%).