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There is no doubt that high-performance thin-layer chromatography (HPTLC) can be applied as a quantitative method if the technique is properly used. Densitometry is a commonly used detection mode for quantitation in HPTLC. The influence of instrumental settings on signal intensity, peak resolution, and peak positioning was rarely described in literature. Especially, quantitation of adjacent substance zones was critical when improper combinations of these settings merge. Future trends regarding ultrathin-layer chromatography and hyphenation to scanning or imaging mass spectrometry required the consideration of these delicate points. The influence of different instrumental settings on the obtained signal intensities was demonstrated for four separated parabens (each 150 ng band−1). The maximum mean signal deviations of all four compounds were 6.9% by the optical system, 16.8% by the scan slit dimension, 7.5% by the scan speed, and 1.5% by the data resolution. The influence of these settings on the quantitation of three parabens in two skin protection creams was investigated. Depending on the selected settings, deviations of the calculated substance amount of up to 5.6% were yielded, whereby determination coefficients of the polynomial calibration curves (60–300 ng band−1) varied between 0.9985 and 0.9999. The setting of integration markers between two adjacent peaks was demonstrated to be deficient if low spatial data resolution is applied; however, this challenging task will rise in interest due to the trend towards miniaturization.

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Planar chromatography is commonly used for the quality control of herbal medicines due to its many advantages. Its combination with chemometrics was proven to be a fast and reliable tool for the extraction of even more analytical information, such as similarity or dissimilarity between samples, and the identification of marker compounds. To date, depending on image processing procedures, different variables have been obtained as input data, and thus, various preprocessing procedures have been applied. In this study, we converted the chromatogram images of high-performance thin-layer chromatography to form a data matrix, by digitization of the chromatograms. Further, principal component analysis was applied on raw data and investigated after different preprocessing techniques. The proposed preprocessing techniques were successfully applied to improve the differentiation between two types of German propolis. The best multivariate models were observed in the case of warping, standard normal variate, and mean centering/autoscaling.

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Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria ( Vibrio fischeri ) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (microchemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a Vibrio fischeri -bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge Dysidea avara . This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.

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The seeds of Ocimum basilicum have traditionally been used for the prevention and treatment of a number of diseases in Xinjiang of China. In this study, six polysaccharide extracts were isolated from the seeds of O. basilicum by sequential extraction and purified. After methanolysis, the monosaccharide compositions of the six polysaccharide extracts were analyzed by high-performance thinlayer chromatography (HPTLC) on HPTLC plates silica gel 60 with a mixture of isopropyl acetate-ethyl acetate-methanol-water (5:4:1:0.1, v/v). After derivatization with the aniline diphenylamine o-phosphoric acid reagent, densitometric quantitation was performed by absorbance measurement at 370 or 630 nm. The results revealed that the polysaccharides in O. basilicum seeds consisted primarily of fructose (hR F 80), glucuronic acid (hR F 58), galacturonic acid (hR F 51), rhamnose (hR F 40), xylose (hR F 25), arabinose (hR F 18), and galactose (hR F 9). Xylose, glucuronic acid, and fructose were the three major components found and account for 45, 31, and 21%, respectively. All extracts contained uronic acids, ranged 3 to 24%. An unknown monomeric unit above glucuronic acid was characterized by mass spectrometry (MS) to be a hexuronic acid, and HPTLC-MS proved to be a well suited method for characterization of polysaccharide-based biopolymers and assignment of its monomers. The polysaccharide extracts (aqueous cold, aqueous hot, acidic, and alkaline) showed inhibitor activities of protein tyrosine phosphatase 1B in vitro with half maximal inhibitory concentration (IC50) values of 8.2, 2.2, 70.9, and 0.8 μg mL−1, respectively. For the first time, a molecular basis was provided to explain the hypoglycemic effect of the seeds of O. basilicum that has been used as antidiabetic adjuvant in traditional Chinese medicine.

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Acta Chromatographica
Authors:
Teresa Kowalska
,
Mieczysław Sajewicz
,
Danica Agbaba
,
Ivana Stanimirova-Daszykowska
,
Monika Waksmundzka-Hajnos
,
Bernd Spangenberg
, and
Gertrud Morlock
Open access