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.
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.
Authors:Petar M. Ristivojević and Gertrud E. Morlock
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.
Authors:Petar M. Ristivojević and Gertrud E. Morlock
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.
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.