EFFECT OF SPECTRAL RESOLUTION, DETECTOR LINEARITY AND CHROMATOGRAPHIC RESOLUTION ON PEAK PURITY CALCULATIONS

The detection of inhomogeneity in chromatographic peaks is one of the principal benefits of photodiode array detection for HPLC. The peak homogeneity can be estimated by comparing the angles between the vector representations of the instantaneous spectrum and the peak apex spectrum, and the instantaneous spectrum and the spectral noise. When the spectral or purity angle exceeds the noise angle, the peak is not homogeneous because the differences between the instantaneous spectra and the apex spectrum cannot be explained by statistical variation of the apex spectrum. In this paper, we consider the impact of detector linearity and slit width on the purity (homogeneity) measurements of a separation of a series of compounds related to vanillin. The peak purity angle, i.e., the spectral contrast of spectra within a chromatographic peak, varies with the maximum absorbance. At small maximum absorbances (less than 0.1 AU), the variation in purity angle is dominated by system noise. At large maximum absorbances (greater than 0.5 AU), the variation in purity angle is dominated by the photometric uncertainty. The smallest measurable purity angle for a homogeneous peak is inversely proportional to the slit width and spectral bandpass of the polychrometer of the photodiode array detector.