Special issue: Thin layer chromatography
This is the 21th guest edited Special Issue of the Journal of Liquid Chromatography & Related Technologies (JLC&RT) published on the topic of thin layer chromatography (TLC) beginning in 1999. The issues up to 2019 have been co-guest edited by Professors Joseph Sherma and Bernard Fried of Lafayette College. This year Professor Sherma has been joined as co-guest editor by JLC&RT Editorial Board member Dr. Fred Rabel. Among his many contributions to the field of TLC, Dr. Rabel has published numerous articles and book chapters on TLC as well as other areas of chromatography, has had a critical role in commercializing TLC sorbents and plates while employed at J.T. Baker, Whatman, and EMD in the United States, and has trained over 5000 scientists on the use and optimization of TLC through training and short courses at various companies and professional meetings.
There is continuing high research activity involving TLC, the primary method classified as planar chromatography, on a worldwide basis, as documented in the biennial review of planar chromatography published in the Journal of AOAC International in 2018 (J. Sherma, Vol. 101, No. 4, pp. 905–913). The papers in this Special Issue are examples of some of the most important current methodology and application areas of TLC research covered in that review, including chemometrics, medicinal analysis, herbal analysis, effect directed analysis (EDA), TLC-mass spectrometry (MS), and food analysis.
The first three papers in the Special Issue are reviews, and the remaining eight are research papers. Paper 1 by Sobanska and Brzezinska reviews applications of micellar liquid chromatography (MLC) with focus on micellar TLC (MTLC) in drug discovery. MLC and MTLC are shown to be valuable sources of information on the physicochemical properties (mainly lipophilicity) of compounds to predict their biological activity.
Paper 2 by Segan et al. reviews TLC in medicinal chemistry. Topics covered include applications in various steps of drug discovery and development such as monitoring of synthesis, identification of bioactive substances from various natural sources and their isolation and purification by preparative TLC (PTLC), determination of lipophilicity and other physiochemical parameters, bioautography, and qualitative and quantitative analysis of drugs and their metabolites.
The final review, Paper 3 by Scrob et al., covers developed and validated HPTLC and HPTLC-MS methods for the analysis of different bioactive compounds and other derivative products found in vegetables published after the year 2000. Included is the determination of pesticide residues in vegetables, which is a health concern in other foods and the environment as well.
In Paper 4, Komsta et al. evaluate the eluotropic strength of seven water miscible solvents as well as water itself in reversed phase (RP) TLC on the medium polarity stationary phases cellulose, cyano and amino bonded silica gel, and DIOL??. A test set of 35 model compounds are studied using modern chemometric design to estimate each solvent effect accurately. Chemometrics is being increasingly applied in chromatography, and its importance is described in detail in the book published on this topic in 2018, Volume 111 in the Chromatographic Science Series edited by Dr. Nelu Grinberg for CRC Press/Taylor & Francis Group.
The next five papers are in what is probably the most active TLC application research area today, i.e., the analysis of dietary supplements and traditional medicines in herbal samples. Paper 5 is from the laboratory of the leading proponent of HPTLC to effect-directed analysis (EDA) using multiple hyphenation (e.g., with chemical derivatization detection of separated zones; biological evaluation of antioxidant, antibacterial, and cholinesterase enzyme inhibitory effects; densitometry for detection and quantification; various modes of spectrometry in situ and ex situ; and PTLC isolation and purification) for nontargeted bioprofile screening and highly targeted characterization of active compounds in plants. In the present study by Jamshidi-Aidji et al. (Paper 5), HPTLC on silica gel plates developed with ethyl acetate-toluene-formic acid-water (16:4:3:2) mobile phase was hyphenated to EDA, in situ UV/visible and fluorescence spectrometry, and high resolution (HR) MS for profiling of aqueous, fermented plant preparations from Chamomilla recutita, Allium cepa, Equisetum arvense, and Hamamelis virginiana.
The development of a harmonized HPTLC method for the determination of botanical origin of caffeine containing plant extracts, as well as detection of caffeine in cases of adulteration, is presented by Do et al. in Paper 6 from the CAMAG Laboratory, which is headed by Eike Reich. In the absence of reliable labeling information, the method can determine the caffeine content of a product, and provide a confirmatory or limit test when specific label information is given. Silica gel plates were developed with toluene-acetone-formic acid (9:9:2), and chromatographic zones were detected under 254 and 366 nm UV light and by derivatization with NP (natural product) reagent, diphenylboric acid 2-aminoethyl ester, and anisaldehyde reagent. The method was validated for robustness, specificity, precision, limit of detection (LOD), limit of quantification (LOQ), and accuracy.
Paper 7 by Moricz et al. describes the evaluation of the antibacterial potential of phenols extracted from leaves of the traditional Chinese medicine Paulownia tomentosa by HPTLC hyphenated with direct bioautography (DB)using the Gram positive and Gram negative bacterial strains of Basillus subtilis and Alivibrio fischeri, respectively. Leaf Soxhlet extracts made with dichloromethane and methanol were resolved into six fractions, expectedly containing flavonoid aglycons, free phenolic acids, nonpolar flavonoid glycosides, polar flavonoid glycosides, and phenolic acids, and HPTLC-DB was performed on each fraction using development of silica gel plates with chloroform-ethyl acetate-methanol (40:6:4). HPTLC fingerprints were documented under 254 and 355 nm UV light before and after zone detection by dipping into aluminum chloride or p-aminobenzoic acid reagent.
Paper 8 by Orsini et al. reports chemical profiles of flavonoids and sugars in crude extracts from leaves and fruits of the plant caigua harvested in Slovenia and Italy. The profiles were produced on HPTLC silica gel plates (preconditioned with water) and C18 bonded plates in combination with the mobile phase - ethyl acetate-water-formic acid (17:3:2) or 5% formic acid in methanol-water (7:3), respectively. Detection of compounds was performed with NP reagent for flavonoids and diphenylamine-aniline-phosphoric acid reagent for sugars. HPTLC-MS/(MSn)analyses enabled identification of several compounds in the extracts, and HPTLC-DPPH* (2,2-diphenyl-1-picrylhydrtazyl radical) reagent assay combined with image analysis was applied for direct screening of antioxidant activity of separated zones.
Hawryl et al. in Paper 9 constructed TLC fingerprint digitized images and compared them between samples with chemometrics (similarity and distance indices with cluster analysis and principal component analysis) and evaluated antioxidant activity by TLC-DPPH* of selected lichens collected in forests near Lublin, Poland. Soxhlet dichloromethane and methanol extracts were analyzed on silica gel plates developed with toluene-ethyl acetate-formic acid (10:10:0.5). Spraying with NP reagent confirmed the presence of some phenolic compounds.
Paper 10 by Peters and Spangenberg describes research from the laboratory of Professor Bernd Spangenberg, Editor-in-Chief of the Journal of Planar Chromatography-Modern TLC. A silica gel HPTLC separation method for the phytoestrogenic active compound equol in cattle manure is presented employing the mobile phase methyl t-butyl ether-cyclohexane (1:1). After separation, the plate was scanned for the (M+!)+ signal of equol with a time of flight-direct analysis in real time (TOF-DART)-MS system. The content of equol in liquid manure was estimated to be 70 µg/g. TLC-MS is one of the most active and important current TLC instrumental research areas at this time. Most of the reported research involves hyphenation with ESI (electrospray ionization)-MS via an elution head type interface from CAMAG or Advion, but the DART-MS ambient ionization technique is less soft than ESI and results in oxidation and fragmentation products and characteristic fragment ions useful for compound analysis.
Studies of the separation and assay of synthetic drugs continues to be another currently active TLC research area as exemplified by the final two papers in this Special Issue. Obradovic et al. in Paper 11 examined the retention behavior of ivabradine, 11 of its related compounds, diltiazem, and verapamil using RP C18 bonded plates with methanol-6.25% aqueous ammonium hydroxide and tetrahydrofuram-6.25% aqueous ammonium hydroxide mobile phases, and silica gel layers with the latter mobile phase. Linear relationships were established between the retention coefficients, RMo and m, and molecular properties of the investigated drugs. Quantitative structure-retention relationship (QSRR) modeling was performed with the use of stepwise multiple linear regression to select the most important molecular properties influencing retention behavior, which were found to be lipophilicity, molecular volume, and hydrogen bond basicity of the tested compounds.
In Paper 12, Zeng et al. describe application of a previously published model process for transfer of TLC screening methods for counterfeit and substandard pharmaceutical products included in the Global Pharma Health Fund E.V (GPHF) Minilab Manual and U.S. Food and Drug Administration (FDA) Compendium to quantitative HPTLC-densitometry methods for moxfloxacin HCl, ofloxacin, and amoxicillin trihydrate. In addition, guidelines of the process were followed to develop and validate HPTLC-densitometry methods for drug products containing acetylsalicylic acid + acetaminophen + caffeine, nimesulide, irbesartan, and pantoprazole, which are not included in the GHHF-Minilab Manual or FDA Compendium. Corresponding supplemental FDA Compendium screening methods were then developed for these latter drugs and published online with open access.
We thank Dr. Nelu Grinberg, Editor-in-Chief of JLC&RT, for the opportunity to serve as guest editors of this Special Issue on TLC. We are indebted to the international experts who accepted our invitations to submit their papers. We invite readers to send us comments on this issue, as well as suggestions for topics and contributors for future issues. We also strongly encourage readers of this Special Issue to submit literature review and research papers in all areas of TLC for regular issues of JLC&RT via the ScholarOne online system.