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.

J Chromatogr A 1638, 461830 (2021). The purpose was to find the first universal HPTLC mixture (UHM), a mixture of reference compounds that could be used for the system suitability test (SST) for the full RF range in all HPTLC experiments.
(Part 1) UHM composition: First, 56 organic molecules, detectable without derivatization, were tested on HPTLC silica gel with 20 different mobile phases (MP) belonging to different Snyder’s selectivity groups and with several polarity indices. Visualization under UV 254 nm and 366 nm. Densitometry scanning at 254 nm in absorption mode, and at 366 nm in a fluorescence mode (mercury lamp 366 nm, with wavelength filter <400 nm). For selected bands, spectra were recorded in absorbance-reflectance mode (wavelength range 190 – 450 nm, deuterium and tungsten lamp). This procedure allowed 8 molecules to be selected for their better spot resolution and for their specific RF values (at least 3 different values distributed throughout the full RF range for each MP). The final composition of UHM was: thioxanthen-9-one (0.001 %), guanosine (0.05 %), phthalimide (0.2 %), 9-hydroxyfluorene, octrizole, paracetamol, sulisobenzone and thymidine (each 0.1 %), in methanol.
(Part 2) UHM validation: Afterwards, UHM was submitted again to a panel of HPTLC assays with always two MP: (A) toluene – methanol – diethylamine 8:1:1; (B) ethyl acetate – formic acid – water 15:1:1; and for each MP, the means, standard deviation and 95 % confidence intervals of the RF values were calculated. (a) UHM was validated for intermediate intra-laboratory precision, as well as for inter-laboratory reproducibility, with ΔRF 0.045. (b) The capacity of UHM to detect small variations was demonstrated by significant changes in at least some RF values, when separation was deliberately performed at different levels of relative humidity (0 %, 33 %, 75 %, 100 %), or with smaller humidity variations (7 % compared to 0–5 %, and 49 % compared to 33 %), or when performing vs. omitting the 10min chamber pre-saturation, or when modifying the MP (+/-10% of one solvent at each time). These response characteristics (the opposite of robustness) made UHM a powerful tool for SST. (c) Finally, UHM stability was studied with UHM aliquots under several storage conditions (-78 °C, -20 °C, 4 °C, room temperature, 45 °C; or 40 °C with 75 % relative humidity) and durations (2 weeks or 2 months). The densitometric peak profiles at 254 nm were compared to those of the fresh compounds, qualitatively (RF value, UV spectrum) and quantitatively (peak area). UHM was stable at room temperature or below, for 2 months (at higher temperature, guanosine, phthalimide and paracetamol degraded).

Planta Medica 84(6/7), 465-474 (2018). The new concept “Comprehensive HPTLC Fingerprinting” was applied to define specifications for the identification and purity assessment of Angelica gigas roots, and for the quantification of its markers: the coumarins decursin and decursinol angelate. Methanolic root extracts of A. gigas (10 reference materials, 24 commercial samples), of 26 other Apiaceae species (including 10 Angelica, 9 Ligusticum, 2 Notopterygium, 4 Peucedanum, and Levisticum officinale) and of mixtures, were developed with toluene - ethyl acetate - acetic acid 90:10:1 on HPTLC silica gel (at 33% relative humidity, chamber pre-saturated for 20 min with filter paper and developing solvent) and dried for 5 min. Detection under white and UV lights before and after derivatization by dipping into 10% sulfuric acid in methanol and then heating 3 min at 100°C. Quantitative evaluation by densitometry in fluorescence mode at UV 313 nm, and luminance was also calculated from the image pixels. The study showed the presence in A. gigas of nodakenin, decursinol, 7-demethylsuberosin, imperatorin, osthole, and isoimperatorin at hRF 0, 4, 15, 33, 38 and 44 respectively. Z-ligustilide (hRF 59) was absent from A. gigas, allowing 1) to distinguish it from several other Apiaceae species; 2) to identify in mixtures with A. gigas two common adulterants (A. acutiloba, A. sinensis) even at 1% in the root powder. Minimal content of A. gigas fingerprint markers (decursin + decursinol acetate, co-eluting at hRF 27) was assessed as 3% (w/w) based on the quantified peaks from A. gigas reference materials.

J. Planar Chromatogr. 8, 349 - 356 (1995). TLC of estradiol benzoate on silica (prewashed with chloroform - methanol 1:1 with hexane - acetone - 2-propanol 45:1:1. When testing the peak purity by 2D-TLC, the mobile phase used in the second direction was chloroform - acetone 19:1. Evaluation by spectrodensitometry at 229 nm. - Determination of specificity, analyte stability, linear range, accuracy, precision (repeatability, intermediate precision), detection, and quantification limit, as well as the robustness of the method.

J. Planar Chromatogr. 17, 102-108 (2004). For sample preparation a methanolic salicylic acid solution was sprayed onto a 40x40 cm glass plate. Samples from different positions of the test plate were extracted with methanol and analyzed by HPTLC and HPLC. HPTLC of salicylic acid on silica gel with cyclohexane - isopropanol - chloroform - acetic acid 12:1:1:2. Quantitation by densitometry. Measurement uncertainty in HPTLC and HPLC is relatively small and has no effect on recovery. To come close to the true value the analytical procedure must be managed by well-considered selection of number and positions of sampling locations. TLC is an excellent analytical technique which gives reliable results.

J. Planar Chromatogr. 23, 173-179 (2010). Many manuscripts and already published articles on analytical procedures to be used in pharmaceutical quality control are characterized by several typical methodological failures and misconceptions. The autors present a collection of typical failures, misconceptions, and misleading data from articles published over the last two years in seven well-known chromatographic publications and provide at the same time a list of references describing optimum approaches to validation of specific TLC/HPTLC procedures. In particular, method specificity, linearity, accuracy, and precision very often are not determined properly and in accordance with best practise.

Planar Chromatogr. 8, 269 - 278 (1995). HPTLC of theophylline and structural related substances (i.e. theophyllidine, methylxanthine, theobromine, etophylline, caffeine) on silica with toluene - 2-propanol - acetic acid 16:2:1. Quantification by densitometry at 274 nm.

Part 3. Evaluation and calibration errors. J. Planar Chromatogr. 18, 256-263 (2005). Third part of a series discussing fundamentals of systematic quantitative errors; systematic errors caused in separation systems; evaluation and calibration errors; nonlinear separation and quantitation techniques; the ,sf4’ procedure for finding summarized systematic errors; systematic errors caused by regulation; conclusions and proposals for quantitative PLC. A correlation function is needed to obtain correct quantitative results from the raw data of a chromatogram - i. e. maximum peak height, peak area of part or or all of a PLC spot, a line or a circle (for circular chromatography): Yi = Ai + Bi x Xi + Ci x (Xi)² + Di x (Xi)³. After 1) Introduction (and example), 2) Evaluation, 3) Calibration errors (3.1 Calibration function found by polynomial interpolation, 3.2 Calibration data analysis, 3.3 Data details for polynomial interpolation, 3.4 Analysis of the ,overall data quality’, the ,data Goodnes’, 3.5 Effect of mathematical accuracy, 3.6 Positioning of the calibration sample ,i’ and the number of different concentrations/amounts to use) follows 4) A possible future of sampling and flexible precise positioning not only of the calibration substances.