J Chromatogr A 1653, 462442 (2021). Samples were peptides obtained through tryptic hydrolysis of the 5 most abundant milk proteins: α-lactalbumin (α-LA), β-lactoglobulin (β-LG), α-, β- and κ-casein (CA). As standards, synthetic whey and pea (Pisum sativum, Fabaceae) peptides (selected based on the in silico tryptic digest of α-LA, β-LG, legumin A, and vicilin with one or zero miscleavages) were only used in the last assay for prediction of the RF values of peptides with known amino-acid (AA) sequences. Two-dimensional HPTLC on silica gel (pre-washed with methanol and activated 10 min at 100°), first with basic mobile phase sec-butanol – pyridine – ammonia – water 39:34:10:26, and (after 12h drying) in the orthogonal direction with acidic mobile phase sec-butanol – pyridine – acetic acid – water 11:8:2:5. Derivatization for peptides and proteins by immersion into fluorescamine (0.05 % in acetone); visualization under UV 254 nm and 365 nm. Computer-assisted determination of the x- and y-coordinates of the derivatized zones. Repeatability (n=8) of the 2D-HPTLC was statistically tested with the Kolmogorov-Smirnov test for normal distribution and with Dixon’s Q test for outliers. Relative standard deviation (RSD) for the RF values was 12.9 % for the first dimension (y-coordinates) and 16.5 % for the second dimension (x-coordinates). According to their higher intensity and sharpness, 15 – 20 detected zones from each protein hydrolyzate were selected, manually scraped from the derivatized layer, dissolved in formic acid solution (0.1 % in acetonitrile – water 3:2), mixed with an equal volume of matrix (dihydroxybenzoic acid 2 % in acetonitrile – water 3:7), crystallized on air on a ground steel target, before being desorbed by the laser beam of the MALDI-TOF-MS/MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry). Direct hyphenation of HPTLC to MS was not performed, to avoid zone diffusion during plate coating with the matrix and to circumvent the stronger binding of polar peptides on the layer.  The MS spectra were acquired in positive reflector mode in m/z range 340 – 4000 (10 – 2500 for fragments), using an external peptide as calibration standard. Identification of 51 from the 85 selected peptides according to AA sequences was performed, using software programs allowing m/z calculation of protein fragments and estimation of cleavage sites. Correlation of the retention behaviour of the peptides with their properties (molecular weight MW, isoelectric point IEP, charges, polarity) was tested with Student’s two-sided t-test after calculation of Pearson’s correlation coefficients. The correlation was significant with IEP, percentages of anionic AA and of non-polar AA; but not with the following properties: MW, percentages of cationic AA and of uncharged polar AA. Finally, based on the correlation results, regression formulas were found to calculate the x- and y-coordinates of any known peptide from the percentage of non-polar AA (or vice-versa). The prediction power of these formulas was verified by repeating the complete 2D-HPTLC-MS experiment with the standard peptides of whey and of peas, and measuring the absolute and relative deviations between the actual x- and y-coordinates and the predicted values. The absolute deviations were higher in the lower RF zones. The average, relative RF value deviations (range 22.1 – 25.7 %) were not different between whey and pea peptides.

J Chromatogr A 1666, 462863 (2022). Theoretical discussion on the factors determining the RF value of a given substance in a chromatographic system: A) the stationary phase (SP); B) the mobile phase (MP), the composition of which can be different from the solvent mixture prepared because of evaporation, saturation and liquid or gas adsorption effects over migration time; C) the difference of the free energies for the analyte transfer from SP to MP; D) external parameters like temperature and humidity. The universal HPTLC mixture (UHM) is a mixture of reference compounds that can be used for the system suitability test (SST) for the full RF range in all HPTLC experiments. Its composition is: thioxanthen-9-one (0.001 %), guanosine (0.05 %), phthalimide (0.2 %), 9-hydroxyfluorene, octrizole, paracetamol, sulisobenzone and thymidine (each 0.1 %), in methanol. The purpose was to study the potential of UHM to replace SST (described with specific markers in European Pharmacopoeia monographs) and to assess the quality of HPTLC results. TLC and HPTLC silica gel on different support (aluminium, glass) or with different granulometries and binders (classic, Durasil, Adamant), of the UHM, an acetonitrile extract of Abelmoschus manihot flowers (Malvaceae), a methanol extract of Sambucus canadensis flowers (Adoxaceae), and essential oils of Lavandula angustifolia, of Mentha × piperita (Lamiaceae) and of Myristica fragrans (Myristicaceae), as well as the following specific markers (standards): borneol, bornyl acetate, linalool, linalyl acetate (terpenoids), isoeugenol, isoeugenol acetate, chlorogenic acid (phenylpropanoids), gossypin (flavone), gossypetin-glucuronide, hyperoside (flavonol heterosides). Development (after 20 min plate conditioning with a saturated MgCl2 solution) with one of the following mobile phases: (MP1) toluene – ethyl acetate 19:1, especially for essential oils; (MP2) ethyl acetate – butanone – formic acid – water 5:3:1:1, especially for S. canadensis; (MP3) ethyl acetate – acetic acid – formic acid – water 100:11:11:26, especially for A. manihot. Documentation in UV 254 nm and 350 nm, and with white light (reflection + transmission), before and after derivatization. RF values were determined by scanning densitometry at 254 nm in absorption mode (for octrizole, at 366 nm in fluorescence mode with mercury lamp and optical filter K400 nm). For each HPTLC condition, intra-laboratory precision assay of UHM separation was performed (at least 5 analyses) with average RF values and 95 % prediction intervals, and calculating RF differences between pairs of UHM constituents and 95 % confidence intervals, which were max. +/-0.012 of the RF values for all UHM and markers. The sensitivity of UHM, and thus its usefulness as generic SST was demonstrated by repeating the HPTLC experiments with modifying by 10 % the quantity of one of the solvent each time. There were always significant changes in RF values of UHM components and/or in RF differences between pairs of UHM bands; it was often but no always the case with the official specific markers. UHM underwent also significant changes (although less than A. manihot extract) when several silica gel phases were compared under the same HPTLC conditions. This property is crucial to verify the right stationary phase before doing any RF correlations, and could make UHM a universal tool to identify discrepancies between different analyses. Finally, the use of UHM for a computer-supported evaluation of HPTLC results was discussed, either for zone identification and RF corrections (within confidence intervals), or for correlations of entire fingerprints as first step to implement machine learning algorithms.

Heliyon 9(2), e13469 (2023). Samples were methanolic extracts of different organs (bark, leaves, fruit pericarps, roots, twigs, seed coats and seedlings) of Dysoxylon binectariferum (= D. gotadhora = D. ficiforme, Meliaceae), as well as rohitukine (chromone piperidine alkaloid) isolated from a bark Soxhlet extract through column chromatography. TLC was used to monitor the purity of rohitukine isolation and to compare the fingerprints of the organ extracts. TLC on silica gel in 2 steps, successively with ethyl acetate – hexane 2:1, and with methanol – chloroform – dichloromethane 4:4:1. Visualization under UV 254 nm and 366 nm. Rohitukine (hRF 16) was very concentrated in bark, but present also in pericarps, leaves, twigs, seed coats and seedlings. (Editors note: Mobile phases and distribution of rohitukine were explained directly by the author (successive 2-step development, not biphasic system). The TLC figures did not show unequivocally the presence in roots, but it was confirmed by the author (and already quantified by other methods in  doi.org/10.1371/journal.pone.0158099).

Heliyon 7(2), e06116 (2021). Samples were a methanolic extract of a semi-solid ayurvedic conserve (ashwagandhadi lehyam) prepared with Withania somnifera roots (Solanaceae) and five other plants, as well as standards: withaferin A and withanolide A (= withaniol), two ergostane triterpene steroids with lactone cycle and epoxide. HPTLC on silica gel with toluene – ethyl acetate – formic acid 6:4:1. Visualization and densitometric scanning at UV 254 nm and 366 nm (deuterium lamps). Derivatization by immersion into vanillin – sulfuric acid reagent, followed by oven heating at 105 °C until optimal coloration. Documentation under white light and densitometry scanning at 540 nm (tungsten lamp). Both analytes (hRF 35 and 45 respectively) were shown at 254 nm and 540 nm (but not at 366 nm), in the standards and in the extract.

Heliyon 8(8), e10103 (2022). Samples were a methanolic extract of Cymbopogon giganteus leaves (= C. caesius subsp. giganteus, Poaceae), as well as flavones as standards: isorhamnetin, luteolin and orientin (=luteolin 8-C-glucoside). HPTLC on silica gel with ethyl acetate – acetic acid – formic acid – water 100:11:11:26. Derivatization for flavones with Neu’s reagent (ethanolamine diphenylborate – PEG). Visualization under UV 365 nm. The standards (hRF 75, 70-72 and 96, respectively) were not detected in the extract. Some analytes detected by the reagent were scraped from the underivatized plate into a tube, and injected through a TLC-MS interface into a double-quadrupole – time-of-flight MS (electrospray ionization). Full mass scan spectra were recorded in positive and negative ionization modes in m/z range 150–550. For 3 of the compounds, isolated through MPLC columns, the HPTLC-MS results, combined to the NMR and HPLC-MS analyses, allowed the identification as epicatechin (hRF 86, a flavanol, not coloured by Neu’s reagent) and as luteolin 8-C- and 6-C-glucosides (hRF 67-70).

Plant Med 88(2), 163-178 (2022). TLC was used to verify the purity of acetoxychavicol acetate (a phenylpropanoid) isolated through column chromatography from a hexane extract of Alpinia galanga rhizomes (Zingiberaceae). TLC on silica gel with n-hexane – ethyl acetate 17:3, evaluation under UV 254 nm.

Front. Pharmacol. 13, 925298 (2022). HPTLC of milk thistle on silica gel with toluene - ethyl formate - formic acid 8:10:1. Detection by dipping in NP reagent and subsequently in PEG reagent, followed by heating at 100 °C for 5 min. Qualitative analysis under UV light at 254 and 366 nm. HPTLC of alkylamides in coneflower on silica gel with ethyl acetate - ethyl methyl ketone - water - formic acid 5:3:1. Detection by dipping in NP reagent and subsequently in PEG reagent, followed by heating at 100 °C for 5 min. Qualitative analysis under UV light at 254 and 366 nm. HPTLC of black cohosh on silica gel with toluene - ethyl formate - formic acid 5:3:2. Detection by dipping into sulfuric acid reagent (20 mL of sulfuric acid in 180 mL of methanol), followed by heating at 100 °C for 5 min. 

Chinese Medicine 15, 76 (2020). This review compared the 2020 editions of Chinese (ChP) and European Pharmacopoeas (EuP) in different aspects of quality control of traditional Chinese medicinal plants (73 of which drugs were common to both, but with differences in species or organs for 17 of them). Discussed points included history, identification, plant origin and processing, sample preparation, marker selection, tests and assays, as well as advanced analytical techniques for quality control and for the establishment of comprehensive quality standard. TLC was discussed in relation to its following aspects: purposes, markers/references, techniques and result description.
(A) The main uses of TLC and HPTLC were (1) chemical-based identification of the plant in a more accurate and precise method than by macroscopic and microscopic observation only, and in a more direct and easily interpretation than HPLC, and allowing the simultaneous analysis of multiple samples in parallel; (2) control of possible adulterants; (3) quantification of active compounds. Both uses (1) and (2) were combined in some EuP monographs: as example were given the roots of Angelica dahurica, A. pubescens, A. sinensis, using TLC for identification of the species and of adulterants from other species (Angelica, Levisticum and Ligusticum).
(B) In ChP, identification through TLC was in most cases achieved by fingerprint comparison to an official reference extract or herb (herbal reference substance). At the opposite, EuP often indicated analytical markers, irrespective of any pharmacological activity, but chosen only for analytical purposes in TCM identification and quantification. Examples were: aescin and arbutin as analytical markers for TLC identification of Anemarrhena asphodeloides rhizome and Panax notoginseng root.
For the TLC system suitability assessment tests, ChP used the same intensity markers or active markers that were chosen for the identification or assay; whereas EuP often used other specific references, e.g. isoeugenol and methyleugenol in the case of Ophiopogon japonicus roots.
(C) For the techniques, conventional separations and chemical derivatizations were used. Hyphenations of TLC to other analytical methods (e.g. MS) were absent. Only one monograph applied an effect-directed analysis directly on TLC chromatogram (free DPPH• radical scavenging assay for TLC identification of Rehmannia glutinosa root, in ChP).
Sometimes, the TLC methods were different between both reference books for the same species. Example was given for Belamcanda chinensis (=Iris domestica) rhizome: in EuP, development on silica gel with cyclohexane – ethyl acetate – acetic acid 20:80:1, detection under UV 254 nm, comparison to standards coumarin and irisflorentin; whereas in ChP, development on polyamide layer with chloroform – butanone – methanol (3:1:1), detection under UV 365nm after derivatization with aluminium chloride, comparison to a reference rhizome powder.
 (D) Finally, the results in ChP were described as a text stating the similarity of sample profile with the profile of the chosen reference, whereas the results in EuP were described with a schematic box indicating the positions of bands of interest.