Special issue: Thin layer chromatography
This is the 18th special issue of the Journal of Liquid Chromatography & Related Technologies on thin layer chromatography (TLC) that we have guest edited beginning in 1999. There is a continuing high research activity involving TLC, the primary method classified as planar chromatography, on a worldwide basis, with more than 3000 publications on that topic abstracted in Chemical Abstracts in the period November 1, 2013 to November 1, 2015, as reported in the biennial review of planar chromatography written by Sherma and published in the Journal of AOAC International in 2016 (in press). The papers in this special issue are examples of the most important current methodology and application areas of TLC as verified in that review, including chemometrics, pharmaceutical formulation analysis, phytochemical analysis, TLC-bioautography, and food analysis.
In Paper 1, Komsta reviews the multivariate analysis of retention data in TLC by chemometric techniques such as hierarchical cluster analysis and principal component analysis among others. An introduction is presented and then applications focused on retention modeling, lipophilicity determination, quantitative structure–activity relationship (QSAR)/quantitative structure-retention relationship (QSRR) models, mobile phase selection, and comparison of TLC with other separation methods. It is suggested that the use of open source software designed for multivariate data analysis can allow better understanding of results in almost all TLC researches. The use of chemometrics in all types of chromatography is growing rapidly, and this is the subject of a comprehensive book edited by Komsta, Vander Heyden, and Sherma that is now in preparation for the CRC Press/Taylor & Francis Group Chromatographic Science Series under the editorship of Dr. Nelu Grinberg.
Paper 2 by Andric et al. is a review of chromatographic methods, including TLC, used to determine soil–water partition coefficient (Koc) values. This parameter is important in the removal of toxic organics in wastewater management facilities, placing a new chemical on the market, and mobility assessment of environmentally important compounds such as pesticides in soil. The use of soil layers for direct determination is covered, and it is shown that octyldecylsily (C18) and cyano (CN) TLC are as efficient as the officially adopted CN column high performance chromatography (HPLC) method for indirect determination of Koc.
Paper 3 by Halka-Grysinska et al. describes a new semiautomatic device with horizontal developing chamber for gradient elution TLC that does not require special plate preparation; gradient TLC can give better resolution of multicomponent mixtures with large polarity differences of the components than the usual isocratic ascending mobile phase development. The device is based on two variations of the Chromdes DS-II-5 × 10 chamber. The device was tested with a 10 dye mixture, and satisfactory agreement between computer calculated and experimental elution behavior was obtained. Advantages and disadvantages of different devices for gradient elution TLC are discussed.
Papers 4–6 describe studies on the analysis of pharmaceutical drug formulations. Paper 4 by Dolowy and Pyka-Pajak compares limit of detection (LOD) and limit of quantification (LOQ) values in the determination of estradiol hemihydrate using different normal phase (NP) and reversed phase (RP) plates and densitometry at 200 nm. The best results were obtained on the plates used most widely by far for all NP TLC or high performance TLC (HPTLC) analyses, Merck silica gel 60 F containing a phosphor that emits green light when exposed to 254-nm ultraviolet (UV) radiation and allows fluorescence quenching detection of compounds that absorb at this wavelength, and on chemically bonded octylsilyl (C8) F plates for RP TLC. LODs and LOQs ranged from 0.093–3.930 ug/spot, which would be adequate not only for pharmaceutical but clinical or environmental analysis of the examined steroid as well.
Obradovic et al. reported in Paper 5 an optimized, validated method for determination of the atypical antipsychotic drug ziprasidone and its impurities in capsules. Standard and sample application with a CAMAG Linomat 5, silica gel 60 F plates, development with toluene-methanol-acetic acid (75:5:5) mobile phase in a CAMAG twin trough chamber (TTC), and densitometry at 250 and 320 nm with a CAMAG TLC Scanner 2 were used.
In paper 6, Zhang et al. applied a previously published model approach for transfer of TLC screening methods for fake and substandard pharmaceutical products to quantitative HPTLC-densitometry methods to azithromycin, imipramine HCl, and sulfadoxine + pyrimethamine tablets. The model includes use of Merck HPTLC silica gel 60 F glass Premium Purity plates, selection of mobile phases, development of calibration curves, assay of commercial formulations, determination of peak purity and identity, and validation of accuracy and precision by the standard addition method.
The analysis of pytochemicals is exemplified in the next two papers. In paper 7, Jozwiak et al. developed TLC-direct bioautography (DB) methods to examine the antimicrobial activity of selected Potentilla species. Ultrasonic extraction of plants was with dichloromethane, and extracts were separated on 0.2-mm silica gel plates developed horizontally with chloroform-diethyl ether-methanol-formic acid (30:10:1:0.2) mobile phase in a DS Teflon chamber. Samples and standards were applied to plates with a Desaga AS 30 applicator. Biological detection was performed with Bacillus subtilis DB. Preparative layer chromatography (PLC) of biologically active bands was performed on 0.5-mm silica gel layers, followed by rechromatography of the isolated fractions in various NP systems to obtain better resolution. Potentilla was found to be a rich source of medium polarity triterpenes that exhibit antibacterial activity.
In Paper 8, Skorek et al. identified flavonoids and phenolic acids contained in cosmetic raw materials such as extracts derived from wine, grape skins, pomegranate peels, juice, and green tea. Samples and standards were applied to silica gel 60 F TLC plates with micropipets, and different mobile phases and detection reagents were used to evaluate the compounds in the extracts. A Desaga CD 60 densitometer was used for the quantification of certain identified compounds.
Food analysis by different TLC approaches is described in the next four papers. Piech et al., in Paper 9, describe a TLC-DB method using Escherichia coli for screening antibiotic residues in milk. Extracts were made with acetonitrile and applied with a Linomat 5, silica gel 60 F plates with concentrating zone were developed in a DS-II-20 cm chamber, and a CAMAG TLC Immersion device for application of DB detection solutions and a CAMAG Reprostar 3 Video Camera for documentation of results were used. Compared to agar-based diffusion assays and HPLC-UV, the reported TLC-DB method was most sensitive but only semiquantitative.
In Paper 10, Kruezselyi et al. report results of the investigation of antibacterial components of button mushroom by TLC-DB and HPLC-diode array detector (DAD)-electrospray ionization (ESI)/mass spectrometry (MS). The components of methanol and ethyl acetate extracts were tested for their ability to inhibit the growth of B. subtilis using TLC-DB with microsyringe application of samples and standards, silica gel 60 F plates, and development in a TTC with hexane-ethyl acetate (7:3) mobile phase; the active components separated were also characterized by different chemical detection reagents. The major antibacterial compound was eluted from the layer and identified as linoleic acid by HPLC-DAD-ESI/MS.
In Paper 11, Hosu and Cimpoiu use HPTLC fingerprinting for authentication of 27 white wines. Wines without any pretreatment were directly applied by a Linomat 5 syringe to silica gel 60 F and C18 F plates that were developed in a TTC with ethyl acetate–formic acid–acetic acid–water (20:2:2:4) and methanol–water–formic acid (5:5:0.1) mobile phase, respectively. Developed plates were heated and dipped, in turn, into diphenylborinic acid aminoethylester and polyethylene glycol reagents in an immersion device. Images of plates under 254 and 366 nm UV light were captured as JPEG files using a CAMAG DigiStore 2 Documentation System, and images were processed using cost free ImageJ computer software. The obtained fingerprints were useful for quality control of wines and differentiation of their harvest year and vineyard.
Lotz and Spangenberg (Paper 12) present a new way to measure the polyphenolic compound trans-resveratrol (tR) in fresh and dried fruits like goldenberries. After a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction, samples and standards were applied with a CAMAG Automatic TLC Sampler (ATS 4) to silica gel 60 plates, and the separation was performed with ethyl acetate–cyclohexane–n-butanol (9:9:2) mobile phase. Chemiluminescence detection was induced by dipping the dried plate into bis(2,4,6)-trichlorophenyl)oxalate (TCPO) solution. For evaluations, the ImageTLC program written in PureBasic (version 4.50) was used. The method was specific and had an LOQ of 20.3 ng of tR/band. Goldenberries were found to be a richer source of tR than red wine.
Martelanc et al. in Paper 13 determine common triterpenoids and polysterols in dichloromethane extracts of cuticular waxes of 12 vegetables waxes by HPTLC on silica gel 60 and C18 plates with n-hexane-ethyl acetate (5:1) and ethyl acetate-acetonitrile (3:2) mobile phases, respectively. Postchromatographic derivatization with anisaldehyde detection reagent enables densitometry at 535 nm (TLC Scanner 3) and image analysis at 366 nm and in white light (DigiStore 2 and CAMAG VideoScan software). It was confirmed that the studied vegetables contained up to 1.5 mg total triterpenols/100 g, making them an important source of these compounds in the human diet. A comparison between unhydrolyzed and hydrolyzed cuticular waxes suggested the presence of conjugated triterpenoids and phytosterols in the vegetable waxes.
Thin layer chromatography is a valuable method for analysis of a wide range of biological samples. This is shown in Paper 14 by Armour et al. where Analtech HPTLC silica gel plates with channels and a preadsorbent zone were used to study the effects of 4- to 20-day starvation on the neutral and polar lipid contents of the medically important snail Biomphalaria glabrata. The mobile phase petroleum ether–diethyl ether–glacial acetic acid (80:20:1), phosphomolybdic acid detection reagent, and densitometry with a TLC Scanner 3 at 610 nm were used for neutral lipids, and chloroform–methanol–water (65:25:4.5), cupric sulfate–phosphoric acid detection reagent, and densitometry at 370 nm for polar lipids. Significant differences were observed in the triacylglycerol and steryl acid levels in the starved versus the control snails. Preadsorbent laned plates are optimal for HPTLC-densitometry with manual sample and standard application because preferred band-shaped zones are automatically formed at the preadsorbent-silica gel interface, and the scored lanes facilitate setting of the densitometer slit in the correct location.
We thank the Editor-in-Chief, Dr. Nelu Grinberg, for the opportunity to serve as guest editors of this yearly special issue, and the preeminent international experts who accepted our invitations to submit their research papers. We will begin to solicit papers in August, 2016, for our 2017 co-guest edited special TLC issue of this journal. We invite readers to send us comments on our present and past special issues, as well as suggestions for topics and contributors for the next issue. We are especially interested in review papers on active research areas of TLC in our future special issues. In addition, as pointed out by Spangenberg in an editorial in Volume 26, Issue 5 of his Journal of Planar Chromatography-Modern TLC, TLC is the only separation method that detects in a dry stationary phase, thereby making biological systems possible as detectors (e.g., Papers 7, 9, and 10 in this issue) and allowing detection based on a particular biological activity, i.e., toxicity (determined against Aliivibrio fischeri), free radical scavenger activity [2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) reagent], estrogenic activity [planar yeast estrogen, screen (pYES)], antibiotic activity (Bacillus subtilis), or genotoxicity (Salmonella typhimurium). He suggested advances in TLC would be more in this field of activity analysis (effect directed analysis, EDA) than in classic detection. (A film by Morlock and Klingelhoefer demonstrating the EDA of estrogen-effective compounds in beer using state-of-the-art HPTLC-pYES-MS techniques and instrumentation is available as a prime teaching tool at <https://youtube.be/Q7AGuljcFvQ>.) Therefore, we will welcome in our 2017 special TLC issue research papers on EDA involving hyphenation of TLC with biological detection (bioautography), and MS. Papers describing research on all types of online couplings of TLC with MS (a book on techniques, instrumentation, and applications of planar chromatography-MS edited by Kowalska, Sajewicz, and Sherma and just published as Volume 110 of the Chromatographic Science Series will benefit all who wish to include confirmation of identity of analytes by online MS in their TLC analyses) are also welcome. Finally, we strongly encourage the submission of literature review and research papers in all areas of TLC for regular issues of the Journal of Liquid Chromatography & Related Technologies.
Dr. Joseph Sherma
Dr. Bernard Fried
Lafayette College
Easton, Pennsylvania
February, 2016