J. Planar Chromatogr. 35, 299-311 (2022).  HPTLC of powdered herbal drugs and finished products (leaves of Mentha piperita, Olea oleuropea, Ginkgo biloba and Camellia sinensis, fruits of Styphnolobium japonicum and Piper nigrum, roots of Angelica species (A. gigas, A. sinensis, A. dahurica, A. acutiloba, and A. pubescens, Curcuma longa and poly-herbal products containing powdered extracts of Curcuma longa root and Piper nigrum fruits) on silica gel with three complementary developing solvents (CDS): low polar developing solvent (toluene - ethyl acetate 9:1); medium polar developing solvent (cyclopentyl methyl ether - tetrahydrofuran - water - formic acid 40:24:1:1); and high polar developing solvent (ethanol - dichloromethane - water - tetrahydrofuran 16:16:4:1). Detection by heating at 100 °C for 3 min, followed by spraying with NP reagent (1.0 g of 2-aminoethyl diphenylborinate in 100 mL of methanol). For Olea oleuropea and Ginkgo biloba, the derivatization with NP was followed by spraying with anisaldehyde sulfuric acid reagent and heating at 100 °C for 3 min. Analysis was performed under UV light at 254 and 366 nm. Performance of the Universal HPTLC mix (UHM) was assessed in terms of precision. The hRF values for all substances were between 20 and 80. 

J. Planar Chromatogr. 35, 313-330 (2022).  HPTLC of orange peel extract on silica gel with gradient multiple development using seven different polarity ranges: cyclohexane, cyclohexane - n-heptane 3:7, cyclohexane - methyl tert-butyl ether 43:7, cyclohexane - methyl tert-butyl ether 7:3, cyclohexane - methyl tert-butyl ether 3:7, methyl tert-butyl ether, methyl acetate - ethanol 9:1, ethyl acetate - ethanol - formic acid 44:5:1. Detection by spraying with vanillin reagent (100 mg vanillin dissolved in 9.8 mL ethanol and 0.2 mL sulfuric acid), followed by heating at 100 °C for 2 min. DPPH staining was performed with 2 mL of a DPPH solution (15 mg dissolved in 10 mL of methanol). Bioautography was performed by dipping into Aliivibrio fischeri bacteria suspension for 6 s, followed by measurement of bioluminescence within 15 min. In this sample, more than 50 compounds could be separated.

J. Planar Chromatogr. 35, 243-250 (2022). Micro TLC of three dyes (1-aminoanthraquinone, fat green and 2-nitroaniline) on silica gel with toluene at distances 1, 1.5, 2, 2.5, or 3 cm. Experiments were performed using a prototype device operated at a controlled velocity of the mobile phase, where the chromatographic plate was placed in the chamber with the adsorbent layer face-down and the mobile phase was delivered onto the adsorbent layer of the chromatographic plate by the pipette, which was driven into movement by a 3D machine controlled by a computer. Different solvents (acetone, methanol, toluene, or heptane) were used to wet and to narrow the starting zones. Detection under UV light at 286 nm. To take full advantage of the benefits of micro-planar chromatography, the size of the starting zone should be reduced as well as the processes
related to the dissolution kinetics of the starting zones of substances in the mobile phase should be optimized.
 

J Chromatogr Sci, 60(5), 472-477 (2022). Discussion of the extensive use of reversed phase chromatography in analytical and preparative applications in modern pharmaceutical industry and science, stressing a special place of the normal phase adsorption chromatography in purifying post-reaction mixtures or the separation of natural extracts, especially in wet load mode due to its simplicity and high velocity of preparation. The presented TLC gradient optimization strategy for wet load separations yields repeatable results of separations for different compounds without worrying about negative impact of wet loading on separation quality.The strategy uses an elution model of the desired compound, which is used to develop the gradient method. I also allows to standardize the separation time, because gradient methods performed by the TLC gradient optimization strategy have a similar duration time in column volumes. Therefore the method can simply be scaled using the column volume as a base unit in calculations.

J Chromatogr A, 1603, 355–360 (2019). Samples were ethyl acetate root macerates of fully flowered Tanacetum vulgare (Asteraceae). HPTLC on silica gel (classical irregular particles vs. Lichrosphere with spherical particles) previously washed with methanol, dried for 5 min at room temperature, perimeter-sealed with a polymer coat, and heated for 30 min at 100 °C. Separation with toluene or with toluene – n-hexane 7:3, in classical capillary flow or in OPLC (overpressured layer chromatography). For OPLC, off-line infusion was used (closed mobile phase (MP) outlet, automatically stopping development); external pressure 50 bar, rapid MP flush 175 and 350 µL, MP flow rate 250 and 500 µL/min, 1830 and 3475 µL MP, development time 446 s and 424 s. Derivatization by immersion into vanillin – sulfuric acid reagent, followed by 5 min heating at 110 °C; or into PABA reagent (500 mg p-aminobenzoic acid, 18 mL glacial acetic acid diluted, 20 mL water, 1 mL o-phosphoric acid, 60 mL acetone), followed by 5 min heating at 140 °C. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for activity against Gram-negative bacteria using Aliivibrio fischeri bioluminescence assay; C) for activity against Gram-positive bacteria with Bacillus subtilis bioassay. Four active polyynes were identified as hexadiynylidene-epoxy-dioxaspiro-decane (1), pontica epoxyde (nonene-triynyl-vinyl-oxirane) (2), tetradeca-triine-en-one (3) and trans-(hexadiynylidene)-dioxaspiro-decene (4), by hyphenating OPLC to quadrupole-orbitrap HRMS without eluent, using a DART interface (Direct Analysis in Real-Time, needle voltage 4kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750). Polyynes (3) and (4) were coeluting in HPTLC but not in OPLC, demonstrating that (4) is not produced by oxidation during the DART-MS procedure. Separation with OPLC compared to HPTLC was performed in a shorter time and with better resolution at the same time. Layers with spherical particles gave higher resolution; zone distortions occurring in OPLC due to dissolved air in MP were prevented by previous MP sonication.

J Chromatogr A, 1603, 355–360 (2019). Samples were ethyl acetate root macerates of fully flowered Tanacetum vulgare (Asteraceae). HPTLC on silica gel (classical irregular particles vs. Lichrosphere with spherical particles) previously washed with methanol, dried for 5 min at room temperature, perimeter-sealed with a polymer coat, and heated for 30 min at 100 °C. Separation with toluene or with toluene – n-hexane 7:3, in classical capillary flow or in OPLC (overpressured layer chromatography). For OPLC, off-line infusion was used (closed mobile phase (MP) outlet, automatically stopping development); external pressure 50 bar, rapid MP flush 175 and 350 µL, MP flow rate 250 and 500 µL/min, 1830 and 3475 µL MP, development time 446 s and 424 s. Derivatization by immersion into vanillin – sulfuric acid reagent, followed by 5 min heating at 110 °C; or into PABA reagent (500 mg p-aminobenzoic acid, 18 mL glacial acetic acid diluted, 20 mL water, 1 mL o-phosphoric acid, 60 mL acetone), followed by 5 min heating at 140 °C. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for activity against Gram-negative bacteria using Aliivibrio fischeri bioluminescence assay; C) for activity against Gram-positive bacteria with Bacillus subtilis bioassay. Four active polyynes were identified as hexadiynylidene-epoxy-dioxaspiro-decane (1), pontica epoxyde (nonene-triynyl-vinyl-oxirane) (2), tetradeca-triine-en-one (3) and trans-(hexadiynylidene)-dioxaspiro-decene (4), by hyphenating OPLC to quadrupole-orbitrap HRMS without eluent, using a DART interface (Direct Analysis in Real-Time, needle voltage 4kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750). Polyynes (3) and (4) were coeluting in HPTLC but not in OPLC, demonstrating that (4) is not produced by oxidation during the DART-MS procedure. Separation with OPLC compared to HPTLC was performed in a shorter time and with better resolution at the same time. Layers with spherical particles gave higher resolution; zone distortions occurring in OPLC due to dissolved air in MP were prevented by previous MP sonication.

J Chromatogr A, 1602, 458–466 (2019). HPTLC of caffeine, physostigmine (alkaloids) and hydroethanolic extract of Peganum harmala seeds (Nitrariaceae, Zygophyllaceae) on silica gel prewashed twice with methanol – water 3:1, followed by 1 h drying at 120 °C. Separation, after 5 min chamber saturation, with ethyl acetate – methanol – ammonia (25%) 85:11:4 (basic mobile phase) or ethyl acetate – toluene – formic acid – water 16:4:3:2 (acidic mobile phase, requiring neutralization with phosphate-citrate buffer). Derivatization with Dragendorff’s reagent and with anisaldehyde sulfuric acid. Effect-directed analysis by spraying A) with Gram-negative bioluminescent Aliivibrio fischeri suspension for antibacterial activity (caffeine was used as standard); B) with acetyl- and butyryl-cholinesterase (AChE / BChE) solutions for enzymatic inhibition. For AChE and BChE asssays, classical immersion into the enzyme solutions was also used for comparison, and inhibition densitometry for active analytes was performed by inverse scan measurement (fluorescence without optical filter) at 546 nm using a mercury lamp; activity was expressed as physostigmine equivalents. Active bands were eluted (only after basic MP) with methanol through the oval elution head of a TLC-MS interface pump, into a quadrupole-Orbitrap mass spectrometer. Full scan mass spectra (m/z 50−750) in positive ionization mode were recorded using heated electrospray ionization (HESI, spray voltage 3.5kV, capillary temperature 270°C). By comparison to literature, AChE inhibitors (also active against A. fischeri) were assigned to be harmine, harmaline and ruine (β-carboline alkaloids), and BChE inhibitors were harmol (same class) and vasicine and deoxyvasicine (quinazoline alkaloids, also called peganine and deoxypeganine). Piezoelectric spraying had the following advantages over automated immersion: (1) it covered the whole plate surface; (2) required much lower volumes of solutions; (3) applied always fresh enzyme or reagent solutions, thus avoiding gradual inactivation; (4) avoided zone distortions, shifts or tailings occurring during immersion or withdrawal of the plate, or due to the hydrophilicity of compounds.

J Chromatogr A, 1640, 461929 (2021). Study of the impact of different strains, culture media and parameters (temperature, time, rotational speed, and glucide and amino-acid supply) on the metabolite profile of bacteria. Samples were cultivation broths of Bacillus subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens, as well as their respective supernatant liquid-liquid extracts (apolar solvents only or QuEChERS method with acetonitrile and MgSO4 – NaCl mixture 4:1). HPTLC on silica gel (normal phase and RP-18), either as bands (for small volumes of extracts) or as areas for supernatants and bigger volumes of extracts. Extract areas were focused with a three-step procedure (up to 20mm with acetone, and twice with methanol); unextracted supernatants were focused twice with methanol and once with tetrahydrofuran, but the application zone of the plate had to be cut before development, due to the high matrix load. Development with ethyl acetate – methanol – water at different ratios after activation of the plate surface with magnesium chloride (33% relative humidity), evaluation in white light and UV. Detection of antibacterial compounds with Aliivibrio fischeri bioassay. Derivatization with primuline (for lipophilic substances) and diphenylamine aniline sulfuric acid reagent (for saccharides). This method allowed a fast comparison: A) of the patterns of the different strains (presence /absence and intensity of detected or antibacterial bands); B) of cultivation parameters: the number of metabolites increased with time, rotational speed (oxygen level), and at 37°C (vs. 30°C), whereas a minimal medium allowed the detection of more metabolites, due to the lower matrix load; C) of the impact of the extraction parameters: choice of the solvents (QuEChERS method had no advantage here), solvent – supernatant ratio (1:3 showed richer patterns than 1:1); D) of the HPTLC parameters used (better separation and resolution with normal phase vs. RP18 layers).