Selective Chemometric Elimination of Co-Eluting Components in Chiral and Achiral Liquid Chromatographic Analyses

No stationary phase can address all separation problems, as such a capability is both statistically and thermodynamically implausible. Overlapping peaks in complex mixtures are unavoidable for any given eluent and stationary phase combination. Since UV spectra are broad, it is not possible to eliminate contributing signals from interfering analytes in HPLC by simply choosing different wavelengths. In the derivative space, where the differentiated pure spectrum of one component is zero, its peak profile can be selectively eliminated, yielding improved quantitative and qualitative information that was not possible from the original chromatogram. The transformed raw chromatogram is called the derivative spectrum chromatogram (DeSC). This technique is extensively useful in chiral analysis where two or more enantiomer pairs overlap in the time domain but are distinguishable in their UV–vis absorption spectra. Since differentiation degrades the signal-to-noise ratio, the improved DeSC algorithm combines time-domain Savitzky-Golay differentiation with a frequency-domain windowing technique to suppress noise in the final DeSC. The protocol can extract information from peaks with peak resolution as low as zero. Illustrative examples, including amino-acid and nicotine enantiomers, fungicides, and illicit street drugs such as tianeptine (which exhibits an overlapping impurity), were analyzed in isocratic and gradient runs with nonlinear baselines. Quantitation of extracted peaks from a real sample of street drug with overlapping impurities showed excellent calibration linearity, with an R2 value of 0.9997. For partially resolved peaks recovered from DeSC, we suggest using a generalized error-peak model for accurate area determination.