Challenges in Quantitative High-Performance Thin-Layer Chromatography — Part 1: Influence of Densitometric Settings on the Result

Summary

There is no doubt that high-performance thin-layer chromatography (HPTLC) can be applied as a quantitative method if the technique is properly used. Densitometry is a commonly used detection mode for quantitation in HPTLC. The influence of instrumental settings on signal intensity, peak resolution, and peak positioning was rarely described in literature. Especially, quantitation of adjacent substance zones was critical when improper combinations of these settings merge. Future trends regarding ultrathin-layer chromatography and hyphenation to scanning or imaging mass spectrometry required the consideration of these delicate points. The influence of different instrumental settings on the obtained signal intensities was demonstrated for four separated parabens (each 150 ng band⁻¹). The maximum mean signal deviations of all four compounds were 6.9% by the optical system, 16.8% by the scan slit dimension, 7.5% by the scan speed, and 1.5% by the data resolution. The influence of these settings on the quantitation of three parabens in two skin protection creams was investigated. Depending on the selected settings, deviations of the calculated substance amount of up to 5.6% were yielded, whereby determination coefficients of the polynomial calibration curves (60–300 ng band⁻¹) varied between 0.9985 and 0.9999. The setting of integration markers between two adjacent peaks was demonstrated to be deficient if low spatial data resolution is applied; however, this challenging task will rise in interest due to the trend towards miniaturization.