Chromatographic plates, HPTLC Si 60 F254 10 cm × 20 cm, were supplied by Merck (Darmstadt, Germany). The plates were cut into smaller pieces using a TLC plate cutter (CAMAG, Muttenz, Switzerland). The plates were washed by immersion in methanol for 1 min. Afterwards, the plates were dried in air and activated in an oven at 105‒110 °C for 15 min.

Solutions of Oil Red (Empirioskop, Warsaw, Poland), which were applied onto the chromatographic plate, were prepared as 0.02% w/V in acetone and methanol (Alfachem, Poznan, Poland).

In all experiments, the volume of solution applied to the plate was 5 μL. A steel capillary with external and internal diameter of 0.8 and 0.2 mm, respectively, was used for application, being connected to a Kwapisz Duet 20/50 syringe pump (Fabryka Aparatury Medycznej, Warsaw, Poland) to pump a solution of substance at various rates from 0.4 to 20 μL/min. The end of the steel capillary was kept in a constant position during the application of the substance solution and in contact with the adsorbent layer of the chromatography plate using a laboratory stand. The end of the steel capillary was in contact with the adsorbent layer but did not damage it. This method of sample dosing was used at atmospheric pressure, both at ambient temperature (25 °C) and at elevated temperature (40 °C and 55 °C). Samples were applied at a temperature higher than ambient temperature using a heating plate (CAMAG) on which the plates were placed. The application operation was performed after 10 min of thermal equilibration of the heating plate and the chromatography plate.

The process of applying substance solutions was also carried out under reduced pressure. For this purpose, a home-made chamber was used, the diagram of which is shown in Fig. 1. The chamber for dosing samples under reduced pressure was a plastic container with a cylindrical shape. This container was equipped with a silicone pad in its lower part, adhering to the adsorbent layer. In the upper part, there was a hole tightly fit to the steel capillary and a hole to which a vacuum pump (DryFast, Welch, Monroe, LA, USA) was connected. During the sample application process, the steel capillary touched the adsorbent layer but did not damage it.

Scheme of the sample dosing chamber under reduced pressure. 1, steel capillary; 2, tubing for connection of the chamber with the vacuum pump; 3, sealing pad; 4, chromatography plate; 5, manometer

The Oil Red spots were scanned using a TLC Scanner 4 (CAMAG) at a wavelength of λ = 520 nm. The obtained spot peaks were used to determine their width. Peak widths are presented as the arithmetic mean of eight measurements.

Photographs of the plates were taken using TLC Visualizer (CAMAG).