Qualitative determination of chiral compounds using capillary electrophoresis

As a well-established analytical separation technique, capillary electrophoresis (CE) is widely used in the separation of chiral substances because of its numerous advantages such as high separation efficiency, short analysis time, small sample dosage, and flexible separation modes. In the previous studies, the CE separation mode, selection of the chiral dispersant and improvement of the separation degree of chiral compounds have been reported in detail. Moreover, it is important to determine the quality of chiral substances and confirm the order of enantiomer peaks after successful separation. This paper summarizes the qualitative detection methods for CE chiral compounds, based on whether the separation analysis of chiral compounds depends on the classification of standard materials.
There are two common methods for the qualitative determination of chiral substances in CE. One method compares the difference in the peak migration time, while the other compares the change in peak area before and after the addition of a single enantiomer standard. Both methods are accurate and simple to operate. A diverse range of detectors are used in CE. Based on the type of detector, CE analysis based on standard products is divided into optical, mass spectrometric and electrochemical detection modes. Optical detection involves the use of ultraviolet-visible (UV-vis), laser-induced fluorescence (LIF), chemiluminescence (CL) and other detectors. Among these, the UV detector has the advantages of stable performance, simple structure, and high cost performance and it is most widely used in CE detection as one of the fixed commercial detectors. The LIF detector is a kind of fluorescence detector with a laser as an excitation source. It has the advantages of low background and high signal-to-noise ratio, which make it the most sensitive CE detector. However, this detector is expensive and the sample must be able to provide a fluorescence signal. Otherwise, it is necessary to use a derivatization reagent for sample pretreatment, which renders the analysis complicated. The type of detector leads to a difference in the sample pretreatment, and the resulting spectra and qualitative methods for chiral compounds are also widely different. Therefore, we must choose the most suitable detector to be combined with CE depending on the sample properties and experimental requirements.
The above mentioned methods require at least one chiral molecular standard of one configuration. However, for many chiral enantiomers, especially some novel chiral drugs, there is no single commercially available standard enantiomer, and even if there is one, it is very expensive. In this case, the available CE chiral separation methods include enzyme digestion, addition of antibodies, and computational methods. Enzyme digestion entails the addition of refers to adding the corresponding degrading enzyme or oxidase into the tested sample, so that qualitative analysis is possible by observing the change in peak area. In the method based on antibody addition, it is necessary to find antibodies that respond to a single enantiomer. Nevertheless, it is difficult to acquire the corresponding digestion enzymes or specific antibodies for most chiral molecules. Hence, the application scope of these two methods is limited. On the other hand, the computational method is a chiral qualitative method that is rapid, economical, and simple to operate, with a wide range of applications. This technique combines the circular dichroism (CD) spectroscopy with theoretical calculations within the premise of CE separation. The underlying principle of this method covers two aspects. On the one hand, all chiral compounds can give CD signals, and a racemic mixture of isomers gives equal signals but with opposite signs. However, in the case of a non-racemic chiral isomer mixture, only the CD signal of the dominant isomers observed. Using the time-dependent density functional theory (TDDFT), the theoretical CD spectrum of a single isomer can be obtained. By comparing the theoretical and experimental spectra, the configuration of the dominant enantiomer is determined. On the other hand, the peak area in electrophoresis is linearly related to the enantiomeric excess in the CE spectrum. By combining CE with CD, accurate separation and qualitative detection of chiral substances can be easily achieved. The method of calculation, independent of the standard, will greatly promote the development of chiral molecular analysis.