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.

Food Chem. 133263 (2022). HPTLC of 27 hand-filtered coffee brews of differently roasted coffee beans and 14 differently prepared and stored coffee brews on amino phase with a 3-step gradient: 1) methanol - ethyl acetate 13:7 (1), 2) ethyl acetate - toluene - formic acid - water 70:11:15:4, and stopped below the eluted alkaloids caffeine and theobromine; and 3) up to 13 mm with water - methanol 3:2. The hRF values for caffeine, theobromine, theophylline and 5-O-caffeoylquinic acid  were 95, 85, 18 and 21 in step (1), while for ferulic acid, coumaric acid, caffeic acid, nicotinic acid and gallic acid were 60, 54, 51, 35 and 10 during step (2), and for melanoids was 12 in step (3). The following effect-directed assays on the chromatogram were also performed: DPPH scavenging asay, Aliivibrio fischeri bioassay, Bacillus subtilis bioassay, acetylcholinesterase inhibition assay, α-glucosidase inhibition assay and planar yeast estrogen screen (pYES) bioassay. Further analysis by mass spectrometry using a electrospray interface. Coffee brews made by a fully automated coffee machine showed the highest antioxidative potential.

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.

Journal Chromatogr A, 1641, 461727 (2021). HPTLc of an ethanolic maceration of Solidago gigantea roots (Asteraceae) on silica gel with n-hexane – isopropyl acetate – acetone 16:3:1, or n-hexane – isopropyl acetate – acetic acid 40:9:1. With the second mobile phase, acid residues had to be eliminated by 20 min automated drying or by 2 h incubation with potassium hydroxide in the opposite twin trough (followed by 15 min cold air streaming); this latter mobile phase allowed to obtain higher hRF values, but some butyrylcholinesterase (BChE) inhibiting activities were lost. The chromatograms were documented at UV 254 nm and 365 nm and white light before and after A) derivatization with vanillin – sulfuric acid reagent; B) enzymatic reaction by immersion into acetylcholinesterase, BChE, glucosidase and amylase solutions; C) Aliivibrio fischeri and Xanthomonas euvesicatoria bioassays, to detect activity against Gram-negative bacteria; D) Bacillus subtilis bioassay to detect activity against Gram-positive bacteria; E) a new antifungal assay with Fusarium avenaceum. For this assay, the chromatograms were immersed 6 s into the isolated mycelium suspension (diluted to OD600 0.4-0.8) and incubated in a vapor chamber at 21 °C for 48-72 h. Inhibition zones were indicated by the lack of visible white fungal hyphae. An aqueous solution of iodonitrotetrazolium (INT, 1 mg/ml) was sprayed on the plate to enhance the contrast (bright zones on a purple background). Benomyl (a benzimidazole fungicide) was used as positive control. Eight clerodane diterpenes (including kingidiol, hautriwaic lactone, and solidagoic acids A and B) were identified from six multipotent zones by bioassay-guided purification through preparative flash chromatography and HPLC, followed by HRMS and NMR, as well as by HPTLC hyphenated to quadrupole-orbitrap HRMS: A) by eluting with methanol (flow 100 µL/min) the compounds from the plate through the oval elution head of an interface of heated electro-spray ionization (spray voltage 3.5 kV, capillary temperature 270 °C, nitrogen as sheath and auxiliary gas, full scan in negative and positive ionization modes in m/z range 50-750); B) without eluent with a DART interface (Direct Analysis in Real-Time, needle voltage 4 kV, grid voltage 50 V, helium as gas, temperature 500 °C, full scan in positive ionization mode in m/z range 100-750).

J. Planar Chromatogr. 34, 503-512 (2021). HPTLC of β-acetoxyisovalerylalkannin (1), acetylshikonin (2) and β,β’-dimethylacrylalkannin (3) in Arnebia guttata on silica gel with petroleum ether (60-90°C) - xylene - ethyl acetate - formic acid 16:6:1:2. Quantitative determination by absorbance measurement at 522 nm. The hRF values for (1) to (3) were 31, 46 and 64. Linearity was between 200 and 1200 ng/zone for (1), 76 and 760 ng/zone for (2) and 201 and 1206 ng/zone for (3). Interday and intra-day precisions were below 5 % (n=6). The LOD and LOQ were 38 and 125 ng/zone for (1) and (3) and 50 and 167 ng/zone for (2). Average recovery was 100.7 % for (1), 98.9 % for (2) and 100.1 % for (3). 

J. Planar Chromatogr. 34, 361-366 (2021). HPTLC of 19 iridoids, including ten iridoid glycosides (catalpol, aucubin, ajugol, hastatoside, loganin, geniposide, harpagoside, verbenalin, agnuside, nuzhenide), six secoiridoid glycosides (harpagide, sweroside, swertiamarin, gentiopicroside, oleuropein, amarogentin) and three nonglycosylated iridoids (loganic acid, genipin, valtrate) in samples of Gentiana lutea, Verbena officinalis, Olea europaea and Harpagophytum procumbens on silica gel with nine different mobile phases. Detection by spraying with anisaldehyde reagent, vanillin reagent, sulfuric acid reagent, respectively, followed by heating at 100 °C for 3 min. After derivatizing the plate with Ehrlich’s reagent, the plate was heated at 100 °C for 5 min. Digital images were recorded under UV light at 254 nm and 366 nm. The data is part of a HPTLC database under development for different families of phytochemicals.

Phytochem. Anal. 3078 (2021). HPTLC of thymoquinone on silica gel with cyclohexane - ethyl acetate 9:1 (1) and on RP with ethanol - water 4:1 (2). Quantitative determination by absorbance measurement at 259 nm. The hRF value of thymoquinone was 42 for system 1 and 51 for system 2. Linearity was between 25 and 1000 ng/zone for (1) and 50 and 600 ng/zone for (2). The intermediate precision was below 1 % (n=6) for (1) and (2). The LOD and LOQ were 8 and 25 ng/zone for (1) and 17 and 50 ng/zone for (2), respectively. Recovery rate was between 99.0 % and 100.9 % for (1) and 98.4 % and 101.2 % for (2). Analytical GREEnness (AGREE) scores for the systems were predicted using the AGREE software according to the 12 principles of green analytical chemistry.

Food Chem. 357, 129135 (2021). HPTLC of cinnamon on silica gel with toluene - ethyl acetate - methanol 6:5:3. Nine detection modes were used: 1) white light illumination, 2) UV 366 nm, 3) UV 254 nm, and six different derivatization reagents applied by immersion: 4) primuline reagent (100 mg primuline, 20 mL water and 80 mL acetone), 5) p-anisaldehyde sulfuric acid reagent (1 mL methoxy benzaldehyde, 140 mL methanol, 16 mL acetic acid and 8 mL sulfuric acid), 6) vanillin sulfuric acid reagent (1 g vanillin, 80 mL ethanol and 0.8 mL sulfuric acid), 7) diphenylamine aniline o-phosphoric reagent (2 % each of diphenylamine and aniline in 100 mL isopropanol plus 20 mL o-phosphoric acid), 8) Fast Blue B salt reagent (100 mg Fast Blue B salt in 100 mL ethanol, 70 %) and 9) natural product reagent (1 g 2-aminoethyl diphenyl borate in 100 mL ethanol), followed by heating at 110 °C (5), 120 °C (4, 6) or 140 °C (7, 8) for 3-5 min. Effect-directed profiling was performed through eight different assays: HPTLC–Aliivibrio fischeri bioassay, HPTLC–Bacillus subtilis bioassay, HPTLC–tyrosinase inhibition assay and densitometric evaluation, HPTLC–α–glucosidase and β–glucosidase inhibition assays, HPTLC–AChE and BChE inhibition assays, HPTLC–DPPH assay. Compounds were further characterized by heated electrospray ionization high–resolution mass spectrometry (HESI–HRMS).