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.

Food Chem. 133784 (2022). Review of enzymatic protein hydrolysis, including standard and conventional techniques and their applications and insights into new strategies of detection and characterization of bioactive peptides. The paper described HPTLC methods coupled with bioassays in effect-directed analysis (EDA) to detect the bioactivities of peptides.

J Chromatogr A, 1605, 460366 (2019). Samples were standards and complex mixtures of non-ribosomally synthesized cyclic lipopeptides (CLPs) from Bacillus strains (Bacillaceae) found in soil or in manure: B. amyloliquefaciens (SS-12.6, SS-13.1, SS-27.2, SS-38.4) and B. pumilus (SS-10.7). Two extraction methods were compared: ethyl acetate extraction (Ex1), and the acidic precipitation followed by methanol extraction (Ex2). HPTLC on silica gel with chloroform – methanol – water 65:25:4. Detection under white light, UV 254 nm and 366 nm. Absorption densitometry measured at 190 nm. Derivatization for peptides, amino acids and amino derivatives, by immersion into ninhydrin – collidine reagent (ninhydrin 0.3 %, collidine 5 %, acetic acid 5 %, in ethanol), followed by heating 5 min at 110 °C. Effect-directed analysis using automated immersion: A) for free radical (DPPH•) scavengers; B) for enzymatic inhibition (acetyl-cholinesterase, α-glucosidase); C) for activity against Gram-negative (Aliivibrio fischeri bioluminescence assay) or Gram-positive bacteria (Bacillus subtilis bioassay). Active bands were eluted 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 200−2000) in positive and in negative ionization modes were recorded using heated electrospray ionization (HESI, spray voltage 3.5 kV, capillary temperature 270 °C). Active zones were assigned to be CLPs: iturin A, surfactin dimethyl-ester, and surfactin, fengycin and kurstakin homologues. Ex1 provided richer extracts compared to Ex2. Standards were seen to contain a free radical scavenging impurity.

J. Chromatogr. A 1558, 77-84 (2018). Investigation of the influence of concentration of ion-pairing additive, as well as concentration and type of organic modifier of the mobile phase on migration of peptides in a pressurized planar electrochromatography (PPEC) system with octadecyl silica-based adsorbent. Comparison of the results with those obtained by similar HPTLC and PPEC systems. Discussion of the influence of particular variables on retention, electrophoretic mobility of solutes and electroosmotic flow of the mobile phase, of the co-influence of these factors on the final selectivity of peptide separation, and of the difficulty of independent optimization of these factors due to their impact simultaneously on the retention, the electrophoretic mobility, and the electroosmotic flow. The pH of the mobile phase was the main variable for optimization of the PPEC system. Extensive tailing of peptide zones in the PPEC was observed in comparison to similar HPTLC systems.

Part VIII. Methylamides and dimethylamides of D-Leu5(-enkephalin) and D-Ala, D-Leu5(-enkephalin). Pol. J. Pharmacol. Pharm. 38, 391-402 (1986). TLC of enkephalin analogs on silica with chloroform -methanol - acetic acid 25:2:1, hexane - ethyl ether - acetic acid 11:7:1, chloroform -methanol -17 % NH3 15:1:0.1, chloroform - methanol - acetic acid 19:2:1, chloroform -methanol acetic acid 19:1:1, chloroform - methanol -25 % NH3 10:1:0.1, butanol - acetic acid - water -4:1:5 upper layer. Visualization by exposure to iodine vapor or spraying with 0.2 % ninhydrin in ethanol or with cerium-molybdenum reagent.

J. of Medicinal Chemistry 32, 391-396 (1989). TLC of vasopressin analogues on silica with 1-butanol - acetic acid - water 4:1:1 and 1-butanol - acetic acid - water - ethyl acetate 1:1:1:1. Visualizaion with starch-KI reagent.

J. of Medicinal Chemistry 32, 1366-1370 (1989). TLC of peptides on silica with butanol - acetone - water 4:1:5, butanol - acetone - water - ethyl acetate 1:1:1:1 and butanol - acetone - water - pyridine 15:3:12:10. Visualization with Pauly’s reagent.

Anal. Biochem. 217, 335-337 (1994). TLC of cyclic peptides on silica with butanol - acetic acid - water 4:1:1. Immersion of the plates in 20% trichloroacetic acid solution for 10 min, and in 0.3% aqueous Serwa Blue W solution for 5 min with gentle shaking. Detection sensitivity, less than 1 µg (0.7 nmol) peptide per spot.