All the chemicals used were of analytical reagent grade. bis(2,4,6-Trichlorophenyl)oxalate (TCPO) and H2O2 were obtained from Fluka (Buchs, Switzerland). Trimethoprim was from Sigma Aldrich (Steinheim, Germany) and sulfamethoxazole was purchased from Sigma Chemicals (St. Louis, MO). Both standards have a purity of ≥ 99.5%. trans-Resveratrol with a purity of ≥ 98% as well as methanol, ethyl acetate, n-butyl acetate, and cyclopentyl methyl ether (CPME) were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Aluminum foil silica gel 60 F254 plates (1.05554) with a fluorescent dye were from Merck (Darmstadt, Germany).

Standard solutions (St) were prepared by weighing the standards in different amounts (trimethoprim 1.608 mg) and sulfamethoxazole (11.529 mg) using an Orion Cahn® C-33 microbalance from Environmental Instruments (Beverly, MA). The standards were dissolved in 10 mL of methanol. The final standard solution (St1) was prepared by mixing 300 µL of each standard solution (St) and then adding 600 µL of methanol. The final concentrations in St1 was 40.2 ng/µL for trimethoprim and for 288.255 ng/µL for sulfamethoxazole.

Standard solutions (St) were spotted band-wise over 7 mm on predeveloped plates (predeveloped with methanol–water (8:2, V/V) using an automatic TLC Sampler (ATS 4) from CAMAG (Muttenz, Switzerland) equipped with a 25-µL syringe. Bands were spotted at a distance of 10 mm from the bottom plate edge and at a distance of 5 mm from the plate edges.

Silica gel 60 F254 plates (10 cm × 10 cm) were developed at 22 °C and 35% relative humidity in a vertical developing chamber (twin-trough glass chamber) at vapor saturation (20 min) to a distance of 50 mm (calculated from the point of application) with the solvent CPME–methanol–water (7.6:2:0.4, V/V).

Chemiluminescence can easily be induced by oxidation of diaryl ethanedioates. The compound TCPO is often used for this reaction because it is quickly oxidized by H2O2 [3,4,5,6,7,8]. In the presence of trans-resveratrol, the reaction energy can be transferred to this fluorescing compound, which emits light while relaxing from its excited state [4].

To perform chemiluminescence in HPTLC, the dry plate was dipped in a chemiluminescence solution for four seconds. This solution was prepared by dissolving 100 mg TCPO in 35 mL ethyl acetate. The amount of 1 mL H2O2 (35%) was shaken vigorously together with this solution for 1 min. Then, 14 mL of the trans-resveratrol solution (10 mg trans-resveratrol, dissolved in 100 mL of ethyl acetate), was added. The mixture is suitable for chemiluminescence measurements within 1 h.

After dipping, the wet plate was dried slightly until no more light reflection could be seen on the surface. For chemiluminescence, a sensitive CCD camera (model: Celvin® S) from Biostep company (Jahnsdorf, Germany) was used. The measurement time was 4 min with no pixel binning. The highest light intensity in the measured picture was set to J0. All other measured light intensities (J) were divided by this value. To reduce noise, 30 diodes per track were averaged as a single densitogram. The reflectance (R) was calculated using expression (1). For evaluations, the self-written program ImageTLC in PureBasic (Ver. 6.14) was used.

A TIDAS TLC S700 system from J&M (Aalen, Germany) with a reflection attachment consisting of three rows of optical fibers was used for the spectral measurements on the plate. It has a wavelength resolution of 0.8 nm and a spatial resolution on the plate of 100 µm, manufactured by the TransMIT Centre for Fibre Optics and Industrial Laser Applications (Gießen, Germany). The middle row was used for detection, and the other two rows were connected to a deuterium lamp (for ultraviolet measurements) and a 365-nm LED for fluorescence measurements. The measurement time for a single spectrum in the wavelength range from 190 to 1000 nm was 25 ms. After measuring J and J0, the wavelengths-dependant reflectance (R) was calculated according to the following equation:

$$R(\lambda )=\frac_(\lambda )}$$

(1)

J is the intensity of light reflected and/or scattered from a sample track, and J0 is the intensity of light reflected and/or scattered from a blank track.

The raw data of the measurement were evaluated using expression (2), the function of the extended Kubelka–Munk equation [1, 9, 10]:

$$KM(k,l)=\frac_-J\right)\left(k_-lJ\right)}_}=\frac$$

(2a)

$$KM(k,l)=\frac=\frac$$

(2b)

k: backscattering factor (0 ≤ k ≤ 1), a: absorption coefficient, J0: reflected light intensity measured from a neat plate part, and J: reflected light intensity measured from a sample track.

The factors k and l adjust Eq. (2) to special measurement conditions. For example, in trace analysis, not too much light is absorbed by the analyte and almost all of the illuminated light is reflected by the plate surface. This is taken into account by setting the backscattering factor k in Eq. (2) to 1, leading to Eq. (4) [1, 9, 10]:

$$KM(p=1)=\frac-1=\frac$$

(4)