Study of the Linear Range in HPLC Analyses with UV Detection: Methodology and Experimental Application to the Influence of the Analyte UV Spectrum

Abstract

Determination of the linear range is one of the main concerns in validation of an HPLC analysis method. It is particularly important since single point calibration will be then used routinely. We proposed an iterative methodology to handle this problem. The idea was, at each step, to test statistically whether the following point belonged to the same regression line. The methodology was then used to evaluate quantitatively the effect on linear range of a shift in detection wavelength or of the detector bandwidth. Although experimental results were globally in accordance with spectroscopic theory, magnitudes observed were rather large. So the linear range could vary by a factor of over 2 with changes in conditions that remained within the range of current practical values. Changes in detection wavelength were limited to about fifteen nm around λmax and the detector used was considered to be representative of modern high‐performance UV detectors. The question of how to take consequences in method validation into account was raised. The solution proposed recommended that the validation was undertaken in conditions as close as possible to those where the method would be conducted routinely. This means with the same instrumentation and on the product of interest for analyses.

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