Deviations from Beer's law in electronic absorption and circular dichroism: Detection for enantiomeric excess analysis

The electronic absorption (UV) to circular dichroism (CD) signal ratio can be used for enantiomeric excess (ee) analysis within linear range. However, CD detection often requires a high sample concentration where deviations from Beer's law may occur. Individual enantiomers of four chiral compounds were separated from commercial racemates by semipreparative high-performance liquid chromatography (HPLC) with chiral columns. They were used to trace possible deviations in both UV and CD detection on achiral HPLC with a photodiode array detector and a CD detector. The CD/UV ratios for samples with the same ee value decreased by up to 7.8 to 52% when the injection volume increased, indicating that the linear standard curve of ee versus CD/UV is only valid within a narrow range. To extend the sample amount to a wider range, a data-processing method was developed based on two second-order polynomial functions, which were constructed to fit the relationship between the intensities of the UV and CD signals for two enantiomers. Moreover, a more simplified method based on a third-order polynomial function was established to calculate the ee values. The variations between the predicted and experimental ee values were within +/- 0.08 for both methods. To our knowledge, this is the first study that the deviations from Beer's law are considered in both UV and CD detection for ee analysis.