毛细管电泳分析中手性化合物的定性检测

Qualitative determination of chiral compounds using capillary electrophoresis

毛细管电泳(CE)作为一种新型分离分析技术,具有分离效率高、分析速度快、样品用量少、分离模式灵活多样等众多优势,在手性物质分离等领域应用广泛。在以往的工作中,手性化合物的CE分离模式、手性拆分剂选择及提高分离度等研究已作了详尽报道,而成功分离后的手性物质定性、对映体出峰顺序确认等问题也至关重要。该文以CE手性化合物分离分析中是否依赖标准品分类,及其定性检测方法进行了总结。利用CE分离分析手性样品,若待测物有手性对映体标准品时,其定性通常通过比较标准品迁移时间或标准加入法完成。基于检测器的不同,依赖标准品的CE分析主要分为光学、质谱和电化学3类检测模式。其中光学检测又包含紫外-可见(UV)、激光诱导荧光(LIF)、化学发光(CL)等多种检测方式。不同的检测方式决定了样品前处理方式的差异,随之形成的谱图及手性化合物定性方式也大相径庭。当缺少手性对映体标准品时,可用的CE手性分离分析方法主要有酶消解和抗体添加法、计算法等。前两种方法主要依据对映体与特定消解酶或抗体间的相互作用,完成手性物质的选择定性,适用范围均较局限。相比较,借助理论计算,使CE结合圆二色光谱(CD)的计算法操作简单,定性准确且适用范围广,具有良好研究应用前景,对促进手性分子分析领域的发展意义重大。

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.