Abstract
Spectral peak purity evaluations are often used to control inadequate selectivity issues appearing in liquid chromatography separations coupled with UV-Vis photodiode detection, despite known limitations related to (a) the lack of characteristic absorption bands in UV-Vis for target analytes and overlapping related impurities; (b) increased similarity between the spectral characteristics of co-eluting compounds; (c) large concentration differences between overlapping compounds; (d) perfect co-elution cases. We propose an alternative protocol to evaluate differences between spectra collected over a peak’s elution interval. After normalizing the acquired spectra, linear regression was applied between each pair of spectra, resulting in slope, intercepts, and correlation coefficient sets, from each comparison. The means and standard deviations of each variable are thus computed. An ellipsoid in the 3D Cartesian space illustrates these computations, having the mean coordinates as center and 2 × standard deviations of the variables as the axes. The volume of the ellipsoid should be related to the similarity between the compared spectra; a smaller volume indicates a higher spectral similarity. This approach was evaluated with respect to the (a) concentration of the analytes; (b) spectral acquisition parameters (spectral resolution, spectral frequency acquisition); (c) spectral similarity between overlapping peaks; (d) perfect co-elution situation; (e) influence of spectral processing (derivation, wavelength rationing), using test solutions of carbamazepine (a,b), acetyl cysteine and enalapril maleate (c), or nitrazepam and diazepam (d,e).
Published Version
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