2.3. Plate Prewashing and Remarks

A set of HPTLC plates was developed with methanol–water, 4:1 (V/V) up to the upper plate edge in the Simultan Separating Chamber (biostep, Burkhardtsdorf, Germany), dried in an oven at 110 °C for 20 min, covered by a clean counter glass plate (placed on top of the stacked plates) and wrapped in aluminum foil for storage in a desiccator. As standards of high purity are expensive, the application parameters were selected so that less microliter volume was lost for the automated syringe operation (filling vacuum time 1 s; rinsing vacuum time 6 s; rinsing/filling cycles 1; return unused sample into vial; Automatic TLC Sampler ATS4, CAMAG, Muttenz, Switzerland). Plate drying was always performed in a stream of cold air (hair dryer or Automatic Developing Chamber 2, ADC 2, CAMAG), immediately after application (0.5 min) and development (5 min). The relative humidity of the ambient air was ca. 40 ± 5% during the developments. If required, this humidity of the plate can be adjusted using a saturated sodium acetate solution. All instrument operation and acquired data were processed with the software visionCATS (version 3.0, CAMAG).