Gertrud E. Morlock

Hyphenations in planar chromatography

This review is focused on planar chromatography and especially on its most important subcategory high-performance thin-layer chromatography (HPTLC). The image-giving format of the open, planar stationary phase and the post-chromatographic evaporation of the mobile phase ease the performance of various kinds of hyphenations and even super-hyphenations. Examples in the field of natural product search, food and lipid analysis are demonstrated, which point out the hyphenation with effect-directed analysis (EDA) and mass spectrometry and illustrate the efficiency gain. Depending on the task at hand, hyphenations can readily be selected as required to reach the relevant information about the sample, and at the same time, information is obtained for many samples in parallel. The flexibility and the unrivalled features through the planar format valuably assist separation scientists.

Determination of drugs and drug-like compounds in different samples with direct analysis in real time mass spectrometry.

Direct analysis in real time (DART), a relatively new ionization source for mass spectrometry, ionizes small-molecule components from different kinds of samples without any sample preparation and chromatographic separation. The current paper reviews the published data available on the determination of drugs and drug-like compounds in different matrices with DART-MS, including identification and quantitation issues. Parameters that affect ionization efficiency and mass spectra composition are also discussed.

Combined multivariate data analysis of high-performance thin-layer chromatography fingerprints and direct analysis in real time mass spectra for profiling of natural products like propolis

Sophisticated statistical tools are required to extract the full analytical power from high-performance thin-layer chromatography (HPTLC). Especially, the combination of HPTLC fingerprints (image) with chemometrics is rarely used so far. Also, the newly developed, instantaneous direct analysis in real time mass spectrometry (DART-MS) method is perspective for sample characterization and differentiation by multivariate data analysis. This is a first novel study on the differentiation of natural products using a combination of fast fingerprint techniques, like HPTLC and DART-MS, for multivariate data analysis. The results obtained by the chemometric evaluation of HPTLC and DART-MS data provided complementary information. The complexity, expense, and analysis time were significantly reduced due to the use of statistical tools for evaluation of fingerprints. The approach allowed categorizing 91 propolis samples from Germany and other locations based on their phenolic compound profile. A high level of confidence was obtained when combining orthogonal approaches (HPTLC and DART-MS) for ultrafast sample characterization. HPTLC with selective post-chromatographic derivatization provided information on polarity, functional groups and spectral properties of marker compounds, while information on possible elemental formulae of principal components (phenolic markers) was obtained by DART-MS.

Development of a planar chromatographic method for quantitation of anthocyanes in pomace, feed, juice and wine.

An efficient HPTLC method was developed, which required minimal sample preparation for quantitation of the main anthocyanes in pomace, animal feed as well as various foods. The best separation of 11 anthocyanes was achieved on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate-2-butanone-formic acid-water for anthocyanins and ethyl acetate-toluene-formic acid-water for anthocyanidins. Due to the high flexibility of the HPTLC method, both anthocyane groups could be developed in a combined 2-step method. The second development was only necessary if anthocyanidins were detected in the samples. This normal phase separation was found superior to the best separation achieved on RP-18 phases with a mixture of water-n-propanol-formic acid. Absorbance measurement was performed using the multi-wavelength scan at 505 (or 510), 520, 530 and 555 nm. The correlation coefficients of the calibrations ranged between 0.9993 and 0.9999 for the 11 anthocyanes. LOQs were all ≤90 ng/zone, most even ≤30 ng/zone and for pn-3-glc and pg-3-glc even ≤7 ng/zone. With regard to the analysis of mv-3-glc in grape seed/marc meal and supplemented animal feed samples, the mean repeatabilities were 1.4% (laboratory 1) and 1.8% (laboratory 2). The intermediate precisions within a laboratory over several months were ≤6.7%. The ruggedness of the method was ≤5.5%. The method was transferred to other sample types. Juice and wine samples, which were from the same plant source, showed a comparable anthocyanin pattern, whereas the pattern was characteristically different between plant sources. Unknown anthocyanin sample components were analyzed via HPTLC-ESI-MS by eluting the zones of interest with the TLC-MS Interface, which was helpful for further characterization of unknowns. An interesting tool was demonstrated by effect-directed analysis with regard to radical scavenging properties and general bioactivity based on detection with Vibrio fischeri bacteria.

Engineered Anisotropic Microstructures for Ultrathin-Layer Chromatography

The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 mum thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than approximately 10 mm. The limits of detection were 10 +/- 4 ng for the vertical posts and 11 +/- 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 mum. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases.

Miniaturized Planar Chromatography Using Office Peripherals

High-performance thin-layer chromatography is a separation technique commonly used to identify and quantify components in chemical mixtures. Sophisticated analytical tools are required to extract the full analytical power from this technique and especially for miniaturized planar chromatography its utility has not been harnessed. A new approach uses an elegant, simplified system assembled from ordinary consumer printers and scanners to perform separations on monolithic and nanostructured ultrathin-layer phases. This system is shown to outperform existing planar chromatographic tools for analysis on miniaturized plates. Analysis can be completed in a manner of minutes, running numerous samples in parallel at a reduced cost, with very low sample and reagent volumes, all using a familiar computer interface with common office peripherals.

ID-CUBE direct analysis in real time high-resolution mass spectrometry and its capabilities in the identification of phenolic components from the green leaves of Bergenia crassifolia L.

RATIONALE Bergenia crassifolia is a plant widely used in herbal medicine. Its chemical composition has been little studied, and no studies using high-resolution mass spectrometry (HRMS) have been performed. Its phenolic components are of particular interest, due to the interest in such compounds in medicine and cosmetics. The ID-CUBE, a simplified Direct Analysis in Real Time (DART) ion source, suitable for the fast MS analysis of liquids without complex sample preparation, offers a new method of studying extracts of such plant. Coupling the ID-CUBE with a high-resolution mass spectrometer can provide identification of extract components. METHODS Mass spectral conditions were optimized for model solutions of the flavonoid naringenin and used for the identification of phenolic compounds in green leaves extracts of Bergenia crassifolia. OpenSpot sample cards with a metal grid surface were used for sample introduction into the ID-CUBE ion source on an Obitrap mass spectrometer. The samples were applied as 5-μL aliquots of the extract onto the metal grid of the card. Sample ionization was stimulated in the ion source within 20 s by applying an electric current to the metal grid to thermally desorb the analytes into the gas flow of metastable helium atoms from the ID-CUBE. RESULTS Elemental compositions were assigned to abundant ions in the mass spectra of the extracts. The major phenolic components were confirmed by their [M-H](-) ions. Thirty-six other marker ions were found, and elemental compositions were suggested for 30% of them, based on a search for compounds found in herbal extracts. CONCLUSIONS The ID-CUBE-Orbitrap MS coupling allowed the rapid accurate mass determination of the phenolic components (and other compounds) in herbal extracts. Higher confidence in component identification could be provided by using additional structural elucidation methods, including tandem mass spectrometry (MS/MS), and this will be the focus of future studies.

Development of a quantitative high-performance thin-layer chromatographic method for sucralose in sewage effluent, surface water, and drinking water

Sucralose, a persistent chlorinated substance used as sweetener, can already be found in waste water, and various countries focused on the release of sucralose into the aquatic environment. A quantitative high-performance thin-layer chromatography (HPTLC) method, which is orthogonal to existing methods, was developed to analyze sucralose in water. After sample preparation, separation of up to 17 samples was performed in parallel on a HPTLC plate silica gel 60 F(254) with a mixture of isopropyl acetate, methanol and water (15:3:1, v/v/v) within 15 min. Due to the weak native UV absorption of sucralose (≤200 nm), various post-chromatographic derivatization reactions were compared to selectively detect sucralose in effluent and surface water matrices. Thereby p-aminobenzoic acid reagent was discovered as a new derivatization reagent for sucralose. Compared to the latter and to β-naphthol, derivatization with aniline diphenylamine o-phosphoric acid reagent was slightly preferred and densitometry was performed by absorbance measurement at 400 nm. The limit of quantification (LOQ) of sucralose in drinking and surface water was calculated to be 100 ng/L for a given recovery rate of 80% and the extraction of a 0.5 L water sample. The sucralose content determined in four water samples obtained during an interlaboratory trial in 2008 was in good agreement to the mean laboratory values of that trial. According to the t-test, which compares the results with the target value, the means obtained by HPTLC were not significantly different from the respective means of six laboratories, analyzed by HPLC-MS/MS or HPLC-TOF-MS with the use of mostly isotopically labeled standards. The good accuracy and high sample throughput capacity proved HPTLC as a well suited method regarding quantification of sucralose in various aqueous matrices.

Some new features of Direct Analysis in Real Time mass spectrometry utilizing the desorption at an angle option

The present study is a first step towards the unexplored capabilities of Direct Analysis in Real Time (DART) mass spectrometry (MS) arising from the possibility of the desorption at an angle: scanning analysis of surfaces, including the coupling of thin-layer chromatography (TLC) with DART-MS, and a more sensitive analysis due to the preliminary concentration of analytes dissolved in large volumes of liquids on glass surfaces. In order to select the most favorable conditions for DART-MS analysis, proper positioning of samples is important. Therefore, a simple and cheap technique for the visualization of the impact region of the DART gas stream onto a substrate was developed. A filter paper or TLC plate, previously loaded with the analyte, was immersed in a derivatization solution. On this substrate, owing to the impact of the hot DART gas, reaction of the analyte to a colored product occurred. An improved capability of detection of DART-MS for the analysis of liquids was demonstrated by applying large volumes of model solutions of coumaphos into small glass vessels and drying these solutions prior to DART-MS analysis under ambient conditions. This allowed the introduction of, by up to more than two orders of magnitude, increased quantities of analyte compared with the conventional DART-MS analysis of liquids. Through this improved detectability, the capabilities of DART-MS in trace analysis could be strengthened.

BACKGROUND MASS SIGNALS IN TLC/HPTLC–ESI-MS AND PRACTICAL ADVICES FOR USE OF THE TLC-MS INTERFACE

Among the different thin-layer and high-performance thin-layer chromatography mass spectrometry (TLC/HPTLC-MS) approaches, the elution head-based TLC-MS Interface is increasingly used due to its good detectability, reliability, and versatility in its application. The influence of different solvents, either used for prewashing or chromatography, on the mass signal background was studied. It was shown that the use of acidic solvents can cause intense background signals (acid sodium clusters) and ion suppression in the analyte mass spectra. Using a mixture of methanol–water 3:1 (v/v) for plate prewashing, background mass signals could be reduced to a minimum for both ionization modes. The TLC/HPTLC-MS background mass signals obtained from different plates with the green manganese activated zinc silicate fluorescence indicator F254, and without, did not show substantial differences. As sodium is ubiquitary in solvent, tubing, glassware, adsorbent, and so forth, the sodium adduct was often formed and observed, whereby the extent of formation was different depending on the plate type. Recommendations for plate handling for use in TLC/HPTLC-MS are provided, as well as practical advices with regard to integration of the inline filter, various sorts of pressure increase, and leakage using the TLC-MS Interface.