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  • Author or Editor: Gertrud Morlock x
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A new HPTLC method has been established for separation and quantification of six given apolar, fluorescent pyrolytic heterocyclic aromatic amines (HA) of the carboline group. The mobile phase was selected by using a practical and systematic four-level optimization scheme based on the solvent classification system according to Snyder and the PRISMA model of Nyiredy et al. The amines were separated on silica gel 60 F 254 HPTLC plates by a multiple development with diethyl ether. Quantification by fluorescence measurement at λ = 366/>400 nm was performed immediately after development. Limits of detection were in the lownanogram range. The results show that modern planar chromatography is a rapid and cost-efficient alternative method for determination of HA. The method is suitable for both screening and quantification.

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High-performance thin-layer chromatography (HPTLC) proved to be a well-suited method for characterization and assignment of polysaccharide-based biopolymers in all types of plant-derived or modified thickening agents, stabilizers, or hydrocolloids on the market. The newly developed HPTLC method allows for differentiation of the polysaccharides due to its characteristic fingerprint. It is a highly effective, rapid, and cost-saving alternative or complementary method to current official methods. After a reduced sample preparation including methanolysis of the polysaccharides, up to 21 samples were separated on HPTLC silica gel plates simultaneously under identical conditions in 20 min. For determination of the methylated monomeric units in the UV-VIS range, a selective postchromatographic derivatization was performed using the aniline diphenylamine o-phosphoric acid reagent. Via spectra recording, the maximum wavelengths were obtained and absorption measurement was performed at various wavelengths showing the potential for quantification based on selected marker compounds. The limit of detection (LOD) of the methylated monomeric units was by a factor of 5 worse if compared to the non-methylated monomeric units, but this was not critical for analysis.

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Plungers with two different cutting edge geometries were compared for HPTLC-MS coupling via an extractor-based interface. Their performance was investigated on the example of the analysis of harmane and Glu-P-1, both heterocyclic aromatic amines. The plungers resulted in similar performance with regard to the delay time between switching the valve and recording the signal, the system pressure, the short negative baseline amplitude at the signal start of the elution peak, the time for complete extraction (elution peak width), and blank plate signals. The mean elution peak widths were 0.51 min for the oval plunger and 0.65 min for the circular plunger. The mean repeatability of the SIM signal for 4 ng per band harmane on three plates ( RSD , %, n = 3 plate means) was 13.3% by use of the circular plunger and 13.6% by use of the oval plunger. This was comparable with results from HPLC-MS, for which repeatability of 9.5% ( n = 6 injections) was obtained. The plunger with the oval geometry, specially designed for band-shaped zones, was better for precise positioning on the substance band of interest and for avoiding co-detection of adjacent zones. It was demonstrated that the oval plunger was more selective than the circular plunger and produced more intense signals for adjacent zones. On the other hand the circular plunger was easier to position on the zone and, for non-adjacent zones, resulted in generally higher signal intensity, because of its larger cross-section.

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Food generally enjoys public confidence; food analysis, however, is a growing and challenging sector. Analytical methods must keep pace with the continuously changing attitudes and preferences of consumers to ensure product quality. Planar chromatography, a very flexible technique for rapidly changing issues, can assist food analysts. But few users of planar chromatography (approx. 10%) use high-performance thin-layer chromatography (HPTLC). Use of appropriate instrumentation will improve the precision of the method. There is therefore, much potential to increase the efficacy and attraction of planar chromatographic applications in food analysis. Validated HPTLC methods fulfill the requirements for reliable food analysis in many fields. Planar chromatography can support food analysis by simultaneous determination of analytes with different detection principles, for example point of care analysis by digital imaging and effect-directed assays via bioactivity-based detection. Parallel analyses under identical conditions or the targeted use of mass spectrometry contributes to its cost-effectiveness. Planar chromatography is an optimum tool for national and international standards to keep analysis inexpensive — especially nowadays.

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The carbohydrate content of floral nectars of 12 Marantaceae species was analyzed by high-performance thin-layer chromatography (HPTLC). The main interest was not only to determine the sugar content of the nectar produced by the flowers but also to find potential differences within the genera and species. A total of 131 samples of this plant family, which grows in the tropical understory rainforest, were collected in the Golfo Dulce Region, Costa Rica, and analyzed. The nectar sugars found in the neotropical samples comprised melezitose, fructose, sucrose, glucose, and xylose (listed ascending in hR F values), whereof the main sugars were sucrose, glucose, and fructose. The nectars were successfully analyzed with regard to their individual sugar composition and classified according to their ratios. The overall mean sugar content of the studied species was 36%. The highest sugar contents of ≥40% were found in the nectar of the species Calathea lutea, Calathea donnell-smithii, Calathea marantifolia, Hylaeanthe hoffmannii, and Pleiostachya pruinosa. All species studied showed a ratio of sucrose to glucose and fructose between 1.3 for Calathealutea and 11.2 for Calathea donnell-smithii and, hence, were classified as sucrose dominated. HPTLC was found to be a good analytical method for nectar analysis, which took less than 1.5 h for 21 samples analyzed in parallel (ca. 4 min per sample).

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Sample application is probably the most important and time-intensive step in high-performance thin-layer chromatography, owing to the fact that sample preparation can be reduced to a minimum. The modern automatic application devices offer a variety of application modes, which allow an exact application of small nanoliter volumes up to the application of the samples in the milliliter range. On the example of 4 parabens, the spotwise, bandwise, or area application was examined. In addition, the differences in the quantitative results were investigated when the same substance amount was applied via different volumes of respectively concentrated solutions. Another important factor in sample application is the dosage speed. This was examined for nine bandwise applications using dosage speeds between 50 and 1400 nL s−1 and three spotwise applications using speeds of 10, 20, and 50 nL s−1. A further point in the investigations was the band length of the application, which can decisively influence the resolution and detectability. Furthermore, it was examined whether there is a difference when the substances are applied as a mixture solution or as individual solutions via so-called overspotting, which means that the individual substance solutions are applied to exactly the same position. In order to investigate matrix effects on the application form, volume, speed, and band length, a honey sample which was spiked with 5-hydroxymethylfurfural was tested under the abovementioned conditions.

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A method was developed for effect-directed analysis (EDA) of the root extract of Pimpinella saxifraga L. High-performance thin-layer chromatography (HPTLC) was hyphenated with microchemical, biochemical, and biological assays as well as electrospray ionization– mass spectrometry (ESI–MS). This HPTLC–UV/Vis/FLD– EDA–MS method directly pointed to multi-potent compounds in the P. saxifraga L. root extract. 2,2-Diphenyl-1-picrylhydrazyl radical scavengers, acetylcholinesterase inhibitors, estrogen-effective compounds, antimicrobials against Gram-positive Bacillus subtilis bacteria, and Gram-negative Aliivibrio fischeri bacteria were discovered in the root extract. A first targeted characterization of four unknown multi-potent compounds was performed by HPTLC–ESI–MS and microchemical derivatizations. This highly streamlined effect-directed profiling is recommended for a fast and cost-efficient natural product search.

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For determination of the sweetener sucralose in the UV-visible range, derivatization is a necessity because of its weak UV absorption (≤200 nm). A new HPTLC method on silica gel plates, in which 46 chromatographic runs were performed within 13 min, has been established. Densitometry was performed either at 500 nm after post-chromatographic derivatization with 2-naphthol sulfuric acid reagent (brownish band for sucralose) or at 405 nm after post-chromatographic derivatization with aniline diphenylamine orthophosphoric acid reagent (gray-bluish band for sucralose). With the latter reagent bluish fluorescence at 366/>400 nm was also observed for sucralose; this enabled very sensitive and selective detection comparable with that on amino plates. The new methods based on silica phases were compared with the reagent-free method on amino plates with regard to limit of detection (LOD) of sucralose. LODs on silica gel measured at 366/>400 nm, 405 nm, and 500 nm and were 4, 8, and 5 ng per band, respectively. Selectivity for different matrices, for example milk-based cakes, different sorts of biscuit, chocolate, cola, milk, hard candies, and energy or sport drinks was achieved for all the detection modes used. Specificity was even obtained by use of the aniline diphenylamine orthophosphoric acid reagent because only sucralose produced a fluorescent color despite the presence of carbohydrates and other matrix constituents. Finally, the four different modes of detection were compared for analysis of sucralose in dietetic biscuits and cakes. The sucralose content measured was shown to be highly repeatable (RSD ≤ 4.4%, n = 3) and reliable with regard to the target value of the sucralose labeled on the packages. According to the target value t -test the means obtained were not significantly different from the labeled values of 45.3 mg/100 g for cakes and 24.8 mg/100 g for biscuits. The good accuracy in matrix proves HPTLC is a highly suitable method for quantification of sucralose in dietetic cakes and biscuits, and in a variety of other matrices.

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Summary

The quality of three types of beer (dark, light and non-alcoholic) was assessed using high-performance thin-layer chromatography (HPTLC) combined with high-resolution mass spectrometry and chemometrics. An HPTLC separation of the polar beer components in the ethyl acetate extract was developed. The polar components were detected either by the in situ 2,2-diphenyl-1-pic-rylhydrazyl (DPPH*) assay or by derivatization with the Neu’s reagent, followed by the PEG solution. This directly allowed the visual comparison and evaluation of the phenolic/flavonoid or radical scavenging (antioxidative) beer profile. Although the three types of beer showed a very similar chemical HPTLC pattern, the signal intensities were different. Detected by the Neu's reagent, the dark beer extracts contained a high amount of phenolic compounds, and the light beer extracts showed a moderate content, while the non-alcoholic beer extracts had the lowest phenolic content. The HPTLC-DPPH* assay confirmed the higher radical scavenging activity of dark beer extracts, if compared to light and non-alcoholic beer extracts. The most active bands with regard to the radical scavenging property were identified to be desdimethyl-octahydro-iso-cohumulone and iso-n/ad-humulone. The use of pattern recognition techniques showed a clear differentiation between dark and non-alcoholic beer extracts, while light beer extracts did overlap with both beer types. This HPTLC screening allowed the (1) direct comparison of beer samples/types via classification and pattern recognition, (2) the assessment of the beer quality with regard to its antioxidative potential, and (3) the reference to single components.

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