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.

Proc. Intern. Symposium on TLC with special Emphasis on OPLC, Szeged, 28 (1984). OPLC (OPTLC) of leu-, gly-, glu-, his-, ser-, tyr-, met on silica with 1) chloroform - ethanol - acetic acid 90.10:2, followed by a second run with dichloromethane-ethyl acetate 9:1. Detection by UV. Fluorescence scanning (excitation at 275 nm). Detection limit 50 ng. Sequencing of some polypeptides from the N-terminus.

Polish Journal of Pharmacology and Pharmacy 39, 303-307 (1987). TLC of two new 1-ß-ß-disubstituted analogues of oxytocin polypeptide hormones on silica with chloroform - methanol 7:3 or 1-butanol - acetic acid - water - pyridine 15:3:3:10 or 1-butanol - acetic acid - water 4:1:5 or ethanol - 0.1 mol/L aqueous pyridine - 0.1 mol/L aqueous acetic acid 4:1:1 or benzene - methanol - acetic acid 2:15:1 or n-hexane - ethyl ether - acetic acid 11:7:1 or benzene - methanol - acetic acid 15:2:1. Visualization with ninhydrin or iodine.

Part XI. 2- (2-phenyl-1, 3-indandionyl)- amides of enkephalin analogs. Synthesis and analgesic activity. Polish J. of Pharmacology and Pharmacy 40, 303-311 (1988). TLC on silica with butanol - methanol - water 4:1:1, ethyl acetate - petrol ether 1:1, chloroform - methanol 10:1 chloroform - methanol - acetone 19:1:1, dichloromethane - ethyl acetate 9:1, chloroform - methanol - acetone 19:3:1, butanol - acetone water 4:1:1, benzene - ethyl acetate 1:4, ethyl acetate - petrol ether 1:4, butanol - acetone - water 4:1:5 (upper phase), chloroform - methanol 5:1.

J. Chromatogr. Sci. 27, 540-544 (1989). Reversed-phase TLC of 22 peptides on paraffin-impregnates silica with methanol, ethanol or propanol. Study of the dependence of the silanophyl effect on the chemical structure of peptides and on the type of organic mobile phase.

Dünnschicht-Chromatographie InCom Sonderband 1996, 85-87. TLC of MCD-peptide (mast cell degranulating peptide) on silica with butanol-pyridine-acetic acid-water 15:10:3:12 resp. 15:10:6:12. Detection with ninhydrin.