PLoS Pathogens 19(9), e1011636 (2023). Samples were total lipid extracts from Mycobacterium tuberculosis (Mycobacteriaceae) either wild-type (1), or a mutant strain knock-out for the gene of succinyl-transferase (sucT) (2), or addback bacteria obtained through plasmide transformation of the wild-type sucT gene into the mutant (3). Development on TLC silica gel layers with chloroform – methanol – water 65:25:4. Derivatization by spraying with cupric sulphate (A) or with α-naphthol (B), followed by heating above 100°C. The following lipids were detected with both reagents (as brown spots on blue background with (A) or as purple spots on yellowish background for (B)) in all strains, without significant quantitative variations: cardiolipin, and acylated phosphatidyl–myo-inositol mannosides.

PLoS ONE 18(10), e0292514 (2023). Samples were 8 glycolipids, either sialylated or not; the two main examples were monosialotetrahexosylganglioside (GM1) and gangliotetraosylceramide (= asialo-GM1 = ASGM1). TLC on silica gel with chloroform – methanol – water – acetic acid 300:200:30:1, followed by air-drying. Visualization of glycolipids by spraying 5-methylresorcinol solution (0.2 % in 2 M sulfuric acid), followed by 5 min heating at 110°C. For immunostaining, underivatized chromatograms were immersed into a blocking gel (1% gelatin, 1% polyvinylpyrrolidone and 1mM EDTA in phosphate-buffered saline solution (PBS)), followed by immersion into a solution of monoclonal (50 ng/mL) or polyclonal (1:1,000) anti-ASGM1 antibodies purposedly produced in rabbits. After 1h reaction at room temperature, the layers were washed thrice with T-PBS (0.05 % Tween 20 in PBS) and horseradish-peroxidase-conjugated anti-rabbit IgG (1:40,000) was added. After washing thrice with T-PBS, the TLC sheets were incubated in substrate solution (tetramethylbenzidine). Blue spots indicated the glycolipids bound by the antibody. In this TLC assay, each of the five monoclonal antibodies (as well as the polyclonal serum) was specific to ASGM1 (unsialylated), whereas in ELISA (enzyme-linked immunosorbent assay) at the same concentrations three of them displayed partial cross-reactivity towards GM1, GM3 or GD1b, which are sialylated glycolipids. 

PLoS ONE 18(11), e0295012 (2023). TLC on silica gel to monitor the synthesis of 15 new camphene-based thiosemicarbazones produced by the reaction of camphene thiosemicarbazide either with benzaldehydes, or with acetophenones, or with one of the following molecules: benzophenone, cinnamic aldehyde, ethyl pyruvate, furaldehyde, menthone, pyrrole carboxaldehyde or thiophene-carboxaldehyde. Development with n-hexane – ethyl acetate 3:7 in the case of benzaldehydes, except vanillin; or 7:3 for the vanillin derivative and all others, followed by visualization of products with resublimated iodine. The aldehyde used for compound 15 is in fact vanillin.

3 Biotech 13, 32 (2023). Samples were the products of transgalactosylation operated by β-galactosidase immobilized on modified carrageenan beads in a solution of lactose. Raffinose, a trisaccharide, was used as standard. TLC on silica gel with n-propanol – water 17:3. Detection of galacto-oligosaccharides by spraying naphthol reagent (50 mg α-naphtol in 95 mL ethanol and 5 mL sulfuric acid), followed by heating. The target zones from unsprayed layers were further extracted with methanol using a TLC-MS interface into a quadrupole MS (flow rate 0.2ml/min, positive and negative electrospray ionization (ESI), m/z range 10–1200). Galactose oligomers were found, from trimers to hexamers (heptamers were observed when the reaction time was beyond 3 hours).

Marine Drugs 20(1), 2 (2022). Samples were the products of partial vs. complete hydrolysis of agar by rGaa16Bc, a recombinant form of agarase Gaa16B from Gilvimarinus agarilyticus (Cellvibrionaceae) overexpressed in Escherichia coli. D-galactose (G) and its oligomers (neoagarobiose (NA2), neoagarotetraose (NA4), neoagarohexaose (NA6)) were used as standards. TLC on silica gel with n-butanol – acetic acid – water 2:1:1. Visualization by spraying orcinol reagent (50 mg orcine monohydrate in 100 mL acetone and 8 mL sulfuric acid), followed by 10 min heating at 110° C. The observed patterns showed the apparition of NA6 and NA4 among the hydrolytic products already after 20 min reaction, whereas NA4 and NA2 were the main products after over-night complete hydrolysis.

Marine Drugs 20(4), 270 (2022). Samples were ether glycerols (EG) purified: (A) from Chimaera monstrosa liver oil (Chimaeridae); (B) from mixed liver oil of sharks Centrophorus squamosus (Centrophoridae) and Somniosus microcephalus (Somniosidae); (C) from Macaca fascicularis hearts (Cercopithecidae); (D) from tumors obtained by grafting in mice the human melanoma cell line MDA-MB-435s, and (E) from periprostatic adipose tissue of men with prostate cancer. Reduction of (phospho)ester glycerolipids into EG and fatty alcohols was part of the purification process. Octadecyl-glycerol and octadecenyl-glycerol were used as standards of alkyl- and alkenyl-glycerols, respectively. HPTLC on silica gel previously developed with chloroform – methanol 1:1, air-dried and activated for 30 min at 110° C. Application under nitrogen stream (6 bar). Development with petroleum ether – diethyl ether – acetic acid 60:140:1. After 2 h drying at room temperature under ventilation hood, visualization by 50 s immersing into sulfuric acid (7 % in ethanol), followed by 2 h drying under air-stream, and 14 min heating at 140° C. Plates were documented under white light illumination and densitometry was performed by computered scanning of the pictures. Alkyl-glycerols (mean hRF 34, LOQ 1235 ng/band) and alkenyl-glycerols (mean hRF 44, LOQ 2352 ng/band), present in all samples (except alkenyl-glycerols in shark oil), were quantified after method validation for specificity, sensitivity, accuracy, precision and repeatability. Linearity range was 1000 ng – 7000 ng for both EG types. To confirm the band identification, samples and standards were also submitted to acidic hydrolysis before HPTLC application. In this case, the bands of alkenyl glycerols did not appear, because chlorhydric acid reacted with the vinyl ether bonds to form glycerol and aldehydes.

Marine Drugs 21(1), 2 (2023). Samples were methanol extracts of cultivated marine bacteria Thalassomonas actiniarum, T. viridans and T. haliotis (Colwelliaceae),  as well as cholesterol, cholic acid, and deoxycholic acid as standards. TLC on silica gel with n-hexane – ethyl acetate – methanol – acetic acid 20:20:5:2. After drying at room temperature, visualization by spraying with phosphomolybdic acid (10 % in ethanol) and heating with a heat-gun. For isolation of cholic acid (hRF 80), present in all samples, preparative TLC on silica gel with the same mobile phase, the corresponding band was scraped off with a surgical blade and extracted with methanol overnight. The isolated cholic acid was identified by LC-MS.

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.