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    Chinese Journal of Chromatography
    2020, Vol. 38, No. 9
    Online: 08 September 2020

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    Volume 38,Number 9 Content
    2020, 38 (9):  0-0. 
    Abstract ( 17 )   PDF (5986KB) ( 34 )  
    Preface for the Special Issue on Capillary Electrophoresis
    2020, 38 (9):  985-985.  DOI: 10.3724/SP.J.1123.2020.07001
    Abstract ( 42 )   HTML ( 173 )   PDF (535KB) ( 40 )  
    Annual review of capillary electrophoresis technology in 2019
    ZHAO Yi, MA Yao, WEI Bo, TIAN Wenzhe, ZHAO Xinying, QU Feng
    2020, 38 (9):  986-992.  DOI: 10.3724/SP.J.1123.2020.03027
    Abstract ( 110 )   HTML ( 179 )   PDF (831KB) ( 65 )  

    This paper reviews the capillary electrophoresis (CE) in 2019. The literatures searched from ISI Web of Science published in 2019 (Jan.1st to Dec.31th) are classified and introduced based on bioanalysis, drug analysis, clinical tests and medical diagnosis, chiral separation, foods tests, other compounds and ion analysis, and the application of capillary electrophoresis-mass spectrometry (CE-MS). International and domestic conferences related to CE and the important research reports are briefly introduced.

    Single-molecule electrophoresis: renewed understanding of nanopore electrochemistry
    ZHANG Wei-Wei, YING Yi-Lun, LONG Yi-Tao
    2020, 38 (9):  993-998.  DOI: 10.3724/SP.J.1123.2020.05001
    Abstract ( 107 )   HTML ( 178 )   PDF (1977KB) ( 62 )  

    This study aims to understand nanopore technology from the standpoint of capillary electrophoresis separation. The nanopore electrochemical measurements could be regarded as "single molecule electrophoresis". Similar to the case of capillary electrophoresis, the single target molecules migrate inside a nanopore under an external electric field. The recognition ability of the nanopore mainly depends on the charge, shape, and size of the target molecules under the electric force. The confined space of an Aerolysin nanopore matches the size of single biomolecule, while the amino acid residues along the inner wall of the nanopore facilitate electrokinetic regulation inside the nanopore. Under the applied voltage, each molecule enters the nanopore, generating the characteristic migration velocity and trajectory. Therefore, statistical analysis of the current amplitude, duration, frequency, and shape of the electrochemical signals would help differentiate and identify a single analyte from the mixture. Herein, we used an Aerolysin nanopore for identifying the oligonucleotides of 5'-CAA-3' (CA2), 5'-CAAA-3' (CA3), and 5'-CAAAA-3' (CA4), which differ in length only by one nucleotide, as the model system to demonstrate single-molecule electrophoresis. The diameter of the Aerolysin nanopore is around 1 nm, and the pore length is approximately 10 nm. Under an applied voltage of 80 mV, the nanopore experiences a high electric field strength of about 80 kV/cm. The phosphate groups of the nucleotides carry negative charges in an electrolyte buffer solution of 1.0 mol/L KCl, at pH 8. Therefore, CA2, CA3, and CA4 carry 2, 3, and 4 negative charges, respectively. During nanopore sensing, CA2, CA3, and CA4 are subjected to electrophoretic forces and thus move inside the nanopore. Because the Aerolysin nanopore is anion selective, the direction of electroosmotic flow through the nanopore is consistent with the anion flow direction. Under the combined effects of the electrophoretic force and electroosmotic flow, CA2, CA3, CA4 will transverse through the Aerolysin nanopore at different migration velocities. Note that the oligonucleotide shows strong electrostatic interaction with the two sensitive regions of Aerolysin, which comprises polar amino acids around R220 and K238. The strong interaction between the sensitive region of Aerolysin and the analyte would further modulate the translocation of oligonucleotides. Therefore, each oligonucleotide follows a different migration trajectory as it individually transverses through the nanopore. The migration speed and migration trajectory are recorded as ionic blockages in nanopore electrochemistry. The scatter plots of the blockage current and blockage duration of the mixed sample of CA2, CA3, and CA4 show three characteristic distributions assigned to each type of oligonucleotide. Since the net charge increases with increasing length of the oligonucleotide, CA3 and CA4 experience a stronger electrophoretic force than does CA2 inside the nanopore, leading to higher migration velocity. Therefore, the blockage duration of CA3 and CA4 is 5 times longer than that of CA2.

    By Gaussian fitting, the fitted blockage currents of CA2, CA3, and CA4 are 20.7, 15.7, and 12.7 pA, respectively. Similar to our previous results, the blockage current increases with the chain length when the oligonucleotides comprise not more than 14 nucleotides. Therefore, nanopore-based single-molecule electrophoresis allows for the electrochemical identification of CA2, CA3, and CA4 that differ in a length by only one nucleotide. Understanding the "single-molecule electrophoresis" concept would promote the application of electrochemically confined effects in single-molecule electrophoresis separation. The combination of single-molecule electrophoresis with a microfluidic system and a nanopore array is expected to aid the separation and identification of single molecules.

    Application, development, and challenges of capillary electrophoresis in disease prevention and control
    LIN Changying, DING Xiaojing
    2020, 38 (9):  999-1012.  DOI: 10.3724/SP.J.1123.2020.02029
    Abstract ( 105 )   HTML ( 170 )   PDF (1032KB) ( 68 )  

    Since the advent of commercial instruments in 1989, capillary electrophoresis (CE) has advanced considerably, with improvement in reproducibility and accuracy in many application fields. CE is predominantly used in research on disease prevention and control, and hygienic chemical inspection. The applications of CE range from assessment of inorganic anions and cations in drinking water to that of biological macromolecules, such as nucleic acids, in pathogenic microorganisms. Since the analytical capacity of CE ranges from inorganic ions to cell, it has become an indispensable technique in this field, particularly in public health emergency and epidemic management. Universal non-targeted analyses to detect possible pathogens, and the capability of rapid and accurate testing of large numbers of specimens are required. In the analyses of polymerase chain reaction (PCR) products, nucleic acid sequencing, mutation detection and genotyping, food-borne disease pathogens, and vaccine analyses, CE methods characterized by high through-put and sensitivity are necessary. In the public health sector, CE is essential in the analyses of food (including emergency analyses for food poisoning), cosmetics, and disinfectants. Satisfactory results of the FAPAS (Food Analysis Performance Assessment Scheme) and domestic proficiency tests indicated the accuracy of CE in quantitative analyses. Application of CE in disease prevention and control is challenged by a number of new molecular biological methods, as optimizing CE methods may not be feasible, and results are difficult to interpret. CE methods, including transformation of peaks to identification of pathogens, can be an arduous task. Thus, end-users prefer using commercialized CE systems and reagents in their routine work. Alternatively, CE methods for analysis of small molecules in product analyses, such as food safety, cosmetics and disinfectant testing, is commonly performed. A plethora of studies published within the decade, indicate that CE is still an active research area in hygienic chemical inspection. To a large extent, CE has not been used for routine analysis in the centers for disease control and prevention, accredited laboratories in China, nor regulatory agencies worldwide. This may be due to the lack of practical protocols for the standards, and the misconceptions regarding the ease of use of CE, which could have hindered its widespread application. Although CE is an environmental friendly technique with minimal usage of toxic chemicals, few standard methods of CE exist in agriculture, environmental protection, food, beverage, chemical, and pharmaceutical industries in the United States, Britain, Europe, Japan, India, Brazil, Russia, and China. Since 2002, CE was used in our laboratory to analyze a large variety of samples. We found that once the CE method has been fully verified and described in detail, it was easily standardized. It is not necessary to screen the equivalent chromatographic column, or to use a specific liquid chromatographic (LC) column. This can effectively circumvent the challenge of shifting peak orders caused by different LC column selectivity. Once combined with general, high sensitivity detectors, CE can be used in the detection of bacteria or viruses in food safety, and play a greater role in the field of disease prevention and control. In the present review, applications of CE in nucleic acid detection for viruses and bacteria, analysis of vaccines, routine testing on food, dietary supplements, medical foods, cosmetics and disinfectants, proficiency tests, and emergency analyses of food poisoning were summarized. The applications and challenges of CE in the field of disease control and prevention were analyzed, and development of this technique was prospected.

    Analysis of metabolomics and proteomics based on capillary electrophoresis-mass spectrometry
    WANG Fang, WANG Song, CONG Hailin, YU Bing
    2020, 38 (9):  1013-1021.  DOI: 10.3724/SP.J.1123.2020.02025
    Abstract ( 175 )   HTML ( 173 )   PDF (2826KB) ( 103 )  

    Capillary electrophoresis-mass spectrometry (CE-MS) has the advantages of higher sensitivity, higher efficiency, and less sample consumption. Moreover, it possesses obvious advantages during the analysis of strongly charged and highly polar samples. CE-MS has been widely applied in life sciences, medicine, and pharmacology. In the past ten years, the main factors affecting its application were system stability, reproducibility, and data accuracy. In order to solve the existing problems of CE-MS, researchers have invested significant effort in technology innovation to further expand CE-MS application. In the fields of medicine and analytical chemistry, substantial research indicates that CE-MS is superior compared to other metabolomic and proteomic approaches.

    This study aims at reviewing the latest methods and applications developed in the fields of medicine and analytical chemistry since 2015. Furthermore, it also aims at enhancing the technology development-related application value of CE-MS and serving as a reference for future development. Further development of the CE-MS technology is discussed from the aspects of coating-sample interaction, interface types, and data processing methods. Concerning the coating types, neutral coatings had been applied extensively in CE-MS and there should be no limitation to the charge of the analyte. The coating decreased sample adsorption on the inner wall by covering the surface charge, greatly reducing the electroosmotic flow (EOF). A charged capillary coating could modify such an EOF direction. The cationic coating could reduce the hydrophobic interaction between the sample and the capillary column, resulting in higher EOF. If it is applied to the sheathless interface, the resolution could be improved by extending the capillary length. Anionic coatings are predominant among the anionic compounds, shortening the separation time by reducing the interaction between the anionic compounds and the capillary. The coating type should be chosen relative to the analyte characteristics. Concerning the interface technology, all interfaces should be simple, practical, and non-dependent on sheath liquid and background electrolytes. As far as data processing methods are concerned, it is necessary to design and develop a practical method for span space data comparison and processing.

    The optimized experimental conditions have effectively improved separation efficiency and data comparison analysis. Furthermore, they established a solid foundation for its application development. CE-MS analysis of complex samples in the fields of metabolomics and proteomics (e. g., of tissues, cells, body fluids, etc.) could provide a visualization method for future clinical analysis. It contributes to the development of cancer pathological analysis, drug development, disease surveillance, etc. The characteristic analysis of small molecule metabolites and protein biomarkers directly reflects on enzymatic activity in the biological systems. It could be associated with the development of various diseases/complications. Omics analysis also has an important directive to disease detection and surveillance with obvious advantages in disease diagnosis, staged treatment, drug development, and patient treatment progress. CE-MS is useful in detecting complications and promoting personalized medicine. It provides technical support for future clinical developments.

    In addition to a comprehensive review of the recent advances of CE-MS research, this paper also indicates the development directions of CE-MS. In order to avoid the problem of omics analysis and obtain the optimized analysis results, future analysis should be improved from the following three aspects:(ⅰ) The analysis conditions should be optimized concerning sample preparation methods and separation techniques. (ⅱ) The analytic techniques should be supported to adjust to capillary coating and interface technology. (ⅲ) New ideas should be developed in the fields of clinical research and statistical analysis.

    Application of poly(2-methyl-2-oxazoline) in protein separation by capillary electrophoresis
    WANG Yuchen, WANG Yanmei
    2020, 38 (9):  1022-1027.  DOI: 10.3724/SP.J.1123.2020.02028
    Abstract ( 122 )   HTML ( 7 )   PDF (1693KB) ( 57 )  

    Capillary electrophoresis (CE), a commonly used liquid-phase separation technology, has many advantages such as high analysis speed, high separation efficiency, and low sample consumption. Hence, CE has gained popularity in food analysis, medical clinical diagnosis, environmental monitoring, and biological sample separation, especially in the field of protein separation and analysis. However, the fused silica capillaries that are commonly used in CE easily adsorb proteins, resulting in unstable electroosmotic flow and poor reproducibility of the separation results. In addition, due to the short optical path of the typical ultraviolet detectors employed in commercial CE, the detection sensitivity often does not meet the requirements for the direct analysis of low-abundance proteins. Therefore, developing a coating that can prevent protein adsorption and improve detection sensitivity is one of the important challenges in CE separation and analysis of proteins. Poly(2-methyl-2-oxazoline), a peptide-like hydrophilic polymer, not only has hydrophilicity, protein-repellent ability, and biocompatibility similar to the gold standard of the anti-protein adsorption polymer (polyethylene glycol), but also shows better stability than polyethylene glycol due to its peptide-like structure. Therefore, it has been increasingly used in biomass transfer, drug carrier, and impedance protein adsorption in recent years. This article aims to review the recent applications of poly(2-methyl-2-oxazoline) in CE from two standpoints. First, poly(2-methyl-2-oxazoline) was grafted onto the capillary inner wall using polydopamine as an anchor. The resulting coated capillary successfully separated a mixture of proteins (such as lysozyme, cytochrome C, ribonuclease A, and α -pancreas chymosinogen A), in addition to preventing the non-specific adsorption of other proteins during the quantitative analysis of melamine and lactoferrin in milk powder. Thus, the detection efficiency of melamine and lactoferrin in milk powder was improved. Second, poly(2-methyl-2-oxazoline) was used to produce a binary mixed brush coating with a stimulus-responsive polymer (such as polyacrylic acid). The capillary coated with the mixed brushes could adsorb high amounts of the target protein (such as bovine serum albumin and lysozyme) under certain pH and ionic strength conditions, and most of the adsorbed proteins could be desorbed by changing the pH and ionic strength. During the release, poly(2-methyl-2-oxazoline) present on the coating would prevent the adsorption of proteins. Under the dual effects of electroosmotic flow and electrophoresis, the released protein could migrate rapidly, and the instantaneous concentration of the protein reaching the detector could be greatly increased. Therefore, the target proteins could be on-line concentrated and the detection signals could be amplified, resulting in improved detection sensitivity for the protein. Future development trends in the function of poly(2-methyl-2-oxazoline) for the separation of proteins by CE are also discussed.

    Recent advance of novel chiral separation systems in capillary electrophoresis
    ZHANG Qi
    2020, 38 (9):  1028-1037.  DOI: 10.3724/SP.J.1123.2020.02010
    Abstract ( 227 )   HTML ( 19 )   PDF (2778KB) ( 129 )  

    Chiral analysis has been an important research field in modern separation science because the enantiomers of a racemic compound often show different or even opposite bioactivities. A variety of analytical techniques have been adopted for chiral analysis over the past few decades. In comparison with conventional chromatographic methods (e. g., high-performance liquid chromatography (HPLC), gas chromatography (GC)), capillary electrophoresis (CE) has multiple advantages such as high separation efficiency, low cost, and diverse separation modes, which have made it one of the most promising analytical techniques for enantioseparation in recent years. The simplest process for CE chiral separation is the addition of a chiral selector (e. g., cyclodextrins and their derivatives, polysaccharides, antibiotics, proteins, crown ethers, chiral exchangers, chiral ionic liquids) in a running buffer to create a chiral separation environment. However, with the ever-increasing number of chiral products in the modern industrial society, satisfactory enantioseparation cannot always be achieved with conventional CE methods. Hence, scientists are endeavoring to improve CE chiral methods. The availability of various fundamental operational modes such as capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), ligand-exchange capillary electrophoresis (LECE), non-aqueous capillary electrophoresis (NACE), and capillary electrochromatography (CEC) has enabled researchers to realize flexible design of high-performance CE chiral separation systems by altering the CE operations, especially by the modification of various advanced materials. For example, ionic liquids (ILs) are a group of organic salts whose melting points are below 100℃, or more often, close to room temperature. ILs have been demonstrated to be effective modifiers in chiral CE because of their unique physical and chemical properties such as high conductivity, exceptional chemical and thermal stabilities, as well as excellent solubility in both organic and inorganic solvents. Besides, it is feasible to design and synthesize various task-specific ILs by altering their anion-cation combinations. ILs have been employed for CE enantioseparation through various modes such as achiral IL-modified conventional enantioseparation systems, chiral IL synergistic separation systems, chiral IL LECE systems, and IL-based MEKC, or by the development of novel IL chiral selectors. Nanoparticles are another class of materials that have received considerable interest for use in chiral CE. Nanoparticles have many advantages such as unique size effect, good chemical stability, significant mechanical strength, as well as ease of modification. Several studies have demonstrated that the combination of chiral selectors with nanomaterials such as gold nanoparticles, Fe3O4 magnetic nanoparticles, carbon nanotubes, and mesoporous silica nanomaterials is a promising approach to establish an EKC system or a CEC system. In this review, we summarize the current state-of-the-art of novel CE chiral separation systems, including enantioseparation systems based on achiral or chiral ILs, nanomaterials, metal-organic frameworks (MOFs), and deep eutectic solvents, as well as chiral plug-plug partial filling CE. Another important topic of research in chiral CE is the exploration of enantiorecognition mechanisms. Modern mechanistic studies focus on the applications of advanced analytical techniques such as nuclear magnetic resonance (NMR) or molecular simulations with computer technology, instead of the conventional chromatography- or CE-based thermodynamic methods. For example, nuclear Overhauser effect spectroscopy (NOESY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) have attracted attention because they provide critical information about the spatial proximity of the functional groups of chiral selectors and enantiomers. Molecular simulations have also become popular because of their powerful ability to evaluate the selector-selectand interactions, in addition to enabling visualization of the complex structures. The main objective of this paper is to provide a comprehensive review of state-of-the art of CE techniques in the field of chiral analysis, especially during the period 2015-2019. Existing problems with these techniques and future perspectives are also presented.

    Qualitative determination of chiral compounds using capillary electrophoresis
    CHEN Lixia, ZHAO Zhiyi, LIU Mingxia, LI Xiangjun
    2020, 38 (9):  1038-1045.  DOI: 10.3724/SP.J.1123.2020.03013
    Abstract ( 160 )   HTML ( 11 )   PDF (2080KB) ( 72 )  

    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.

    Research progress of molecularly imprinted polymers in separation of chiral drugs by capillary electrochromatography
    LI Zhenqun, JIA Li
    2020, 38 (9):  1046-1056.  DOI: 10.3724/SP.J.1123.2020.03018
    Abstract ( 124 )   HTML ( 17 )   PDF (924KB) ( 94 )  

    Chiral drugs exert pharmacological effects through strict matching with chiral biological macromolecules and chiral recognition. Each enantiomer has different pharmacological activities, metabolic processes and rates, as well as toxicity pharmacokinetic characteristics owing to the difference in its interactions with the chiral environment. Therefore, method development for the resolution of chiral drugs is of great significance for the synthesis of chiral drugs and for quality control during the production process. Molecularly imprinted polymers (MIPs) are prepared by using a target molecule as the template. MIPs demonstrate highly specific recognition properties toward the target molecule since they have specific spatial molecular structures and functional groups. Hence, MIPs are particularly suitable for the separation and purification of chiral drugs. Capillary electrochromatography (CEC) offers the advantages of high separation efficiency and high selectivity owing to the dual separation mechanisms including capillary electrophoresis and liquid chromatography. By using MIPs as the stationary phases for CEC, the advantages of the two technologies can be combined to achieve efficient separation of chiral drugs. MIPs were first applied to CEC for chiral resolution in 1994, and since then, there have been notable advances in this field. The four main chiral separation modes in CEC involve the use of MIPs as the stationary phases of open tubular, packed, and monolithic columns, and as the pseudostationary phase in the separation medium.

    This review summarizes the research progress of these four methods and reveals the potential of MIPs in chiral resolution by CEC. The advantages and disadvantages of these methods are commented. MIPs as the stationary phases of packed columns can allow for chiral separation. However, the preparation of packed columns in narrow capillaries is difficult. In addition, frits must be prepared at the ends of the capillaries to seal the MIPs. The frits lead to the formation of bubbles during the CEC analysis, thus resulting in poor repeatability and stability. These problems can be overcome by using MIP-based open tubular columns. Furthermore, conditioning of open tubular columns is easy and less time-consuming. However, open tubular columns have limited capacity. MIP-based monolithic columns have greater capacity than do open tubular columns, and frits are not required in this case. However, in situ preparation of MIPs monolith in narrow capillaries is still challenging. The application of MIPs to chiral CEC can also be realized by using them as pseudostationary phases (additives) in the separation medium, and this allows for ease of operation. Moreover, the amount of MIPs introduced into the capillary can be accurately controlled. Thus, the batch-to-batch reproducibility can be improved, but this has the disadvantage of increased MIP consumption.

    In order to further expand the potential of MIPs in chiral CEC, the following aspects must be considered. First, improvement of the preparation method. In most reported MIP-based-chiral CEC techniques, the peaks of the imprinted molecules show severe tailing, and this problem must be resolved. Improving the mass transfer rate of the prepared MIPs may be a suitable solution in this regard. Second, development of new functional monomers. A functional monomer is an indispensable component in the preparation of MIPs. New functional monomers can be prepared according to the "three-point interaction" rule. Third, selection of template molecules. A single enantiomer of chiral drugs is used as the template molecule to prepare chiral MIPs. The method is not suitable for the preparation of MIPs of chiral drugs for which a single enantiomer is difficult to obtain. Therefore, appropriate choice of the template molecules for these drugs is imperative. Fourth, discussion of chiral separation mechanism. The mechanism of interaction between the template molecules and MIPs needs to be explored further, in order to obtain theoretical guidance for the design and preparation of chiral MIPs.

    Advances in polydopamine surface modification for capillary electrochromatography
    YI Gaoyi, JI Bai'an, XIA Zhining, FU Qifeng
    2020, 38 (9):  1057-1068.  DOI: 10.3724/SP.J.1123.2020.03004
    Abstract ( 94 )   HTML ( 17 )   PDF (1708KB) ( 161 )  

    Capillary electrophoresis (CE) has a wide range of applications in analytical fields due to its advantages of low sample consumption, short separation time, and high separation efficiency. The cathodic electroosmotic flow (EOF) and single electrophoretic separation mechanism are not optimal for many CE applications. Hence, the use of an unmodified fused-silica capillary leads to insufficient separation performance that cannot meet the requirements for various complex sample systems, especially neutral and chiral compounds. Therefore, it is necessary to introduce various capillary modification strategies in CE so that its potential for practical application can be expanded. Mussel-inspired polydopamine (PDA) and PDA-derived coating materials have fascinating advantages such as simple surface coating procedures, strong surface adhesiveness, good chemical stability, latent reactivity with many functionalized molecules, and good biocompatibility. Thus, they have been widely utilized in different research fields, including catalysis, sensing, water treatment, sample pretreatment, biomedicine, chromatographic separation, and CE. The preparation of PDA coatings is simple as it involves physical adsorption, and the obtained surface adhesive coatings possess good stability similar to covalently bonded coatings. Therefore, PDA and PDA-derived coatings are well suited for the modification of fused-silica capillaries. More importantly, the PDA coating can be utilized as an intermediate reaction platform for diverse subsequent surface modification because of its strong surface adhesive property and strong latent reactivity with many functionalized molecules (such as polymers, proteins, and nanomaterials). Consequently, various chromatographic retention mechanisms can be introduced on the inner wall of the capillary, thereby contributing to the fabrication of multi-functional PDA-based stationary phases for CEC. Owing to these outstanding advantages, researchers are paying increasing attention to the great application potential of PDA and PDA-derived coatings in CEC. In this paper, recent advances in the methods for preparing PDA coatings, especially the recently developed fabrication strategies and various applications of PDA-modified silica capillary in open tubular-capillary electrochromatography (OT-CEC) and capillary electrochromatography monolithic columns, are summarized and discussed. Furthermore, the application prospects of PDA-based coating materials in CEC are prospected in this review. Although PDA and PDA-derived coating materials are seeing widespread utilization in field of CEC, researchers have still not reached a definite conclusion regarding the PDA formation and coating mechanisms, and further investigation is needed in this direction. The PDA coatings formed using existing methods are generally thin. In the early stage, many studies adopted the strategy of repeated coating to improve the coating effect of PDA in capillaries, but this method was found to be time-consuming and less efficient. In order to improve the preparation efficiency of PDA-modified CEC columns, many researchers have focused on fast deposition induced by a strong oxidant to obtain PDA-coated columns. However, the controllability of the PDA coating obtained by this method is poor. Thus, it is necessary to further explore new preparation strategies for PDA-coated CEC capillaries with better reproducibility and stronger operability. On the other hand, although a strategy for directly synthesizing functional PDA-coated CEC columns in the organic phase has been proposed, its application potential in CEC remains to be further explored. In addition, the PDA coating itself has poor porosity and a small specific surface area, which may be significantly improved by modifying the coating on the porous monolithic column surface. However, there has been limited research on the use of PDA coatings in monolithic columns, and their application potential remains to be expanded. With in-depth research into the formation mechanism and preparation methodologies of PDA coatings, PDA, which is a highly malleable biomimetic material, will play a more important role in advances in the fields of CE and CEC.

    Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry
    WANG Shuangshuang, YANG Yunhe, FAN Shanshan, LI Huihui, David D. Y. CHEN
    2020, 38 (9):  1069-1077.  DOI: 10.3724/SP.J.1123.2020.03001
    Abstract ( 64 )   HTML ( 11 )   PDF (1468KB) ( 56 )  

    The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K+, NH4+) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IRa values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×105 L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

    Comparison of two capillary electrophoresis methods for aptamer-protein affinity characterization
    MENG Qingwei, GUO Lei, XIE Jianwei
    2020, 38 (9):  1078-1084.  DOI: 10.3724/SP.J.1123.2020.04001
    Abstract ( 86 )   HTML ( 15 )   PDF (1968KB) ( 56 )  

    Affinity interaction characterization is a prerequisite for understanding the specific binding of nucleic acid aptamers to their target molecules and consequently their appropriate applications. The CE technique provides a simple and multi-mode approach to such a characterization, but different results obtained from multiple modes and systems lead to limited reliability and further applications. Thus, there is an urgent need to develop systematic comparison approaches of multi-mode applications in CE, which would allow a better investigation of the affinity between aptamers and target molecules.

    In this work, based on CE laser-induced fluorescence detection, we applied the CE frontal analysis (FA) approach for affinity evaluation and compared it with preequilibrium-capillary zone electrophoresis (PE-CZE) using thrombin and its 29-mer aptamer as a model system that specifically binds to the heparin binding site.

    The optimization conditions of the CE-FA method included 30 s injection of a mixture incubated at 37℃ for 0.5 h followed by separation at low (15℃) working temperature using short capillary (30 cm) under 15 kV in a biocompatible buffer (2×TG, pH=8.5), which provided stable plateau peaks of complex and free fluorescent-labeled 29-mer (F29-mer) aptamers. The addition of 1 g/L bovine serum albumin (BSA) enhanced the reproducibility of the plateau peak heights and migration times during the CE-FA separation.

    We then discussed in detail the results and features obtained from six fitting modes of the two methods. We applied multiple fitting modes for CE-FA and PE-CZE, including the nonlinear fitting of bound/free aptamer ratio versus free aptamer concentration, non-linear fitting of plateau peak height versus concentrations, and non-equilibrium CE of equilibrium mixtures (NECEEM) calculation. We observed that 5 of 6 fitting results showed no significant difference and all dissociation constant (Kd) values were in the range between 24 and 64 nmol/L. Three fitting modes of the CE-FA approach aligned well with each other, indicating that the association-dissociation equilibrium between the aptamer and complex could be easily maintained under the non-equilibrium CE separation conditions using the CE-FA method, and the measured Kd values were more accurate. Using the PE-CZE method, the Kd fitted from nonlinear regression through free aptamer peak height decrease versus concentration indicated a significant deviation. Moreover, we obtained accurate Kd values from the NECEEM calculation approach by choosing 0.5-2-fold thrombin against F29-mer. This approach allowed the clear observation of an exponential bridge between the two peaks in the electrophoregrams.

    The CE-FA and PE-CZE methods could be used to mutually confirm each other, improving the affinity evaluation reliability. In this work, we recommend the selection of the CE-FA evaluation method as a priority, with a fitting mode through plateau peak height derived from a series of various concentrations. It could effectively address the problem of the high voltage-affected unstable complex peaks in CE. This provides such advantages that allow wide application, robust use, and feasible and accurate fitting results.

    Application of mixed polymer brushes based on poly(2-methyl-2-oxazoline) and poly(acrylic acid) to on-line preconcentration of lysozyme by capillary electrophoresis
    ZHANG Miao, WANG Yuchen, MUHAMMAD Atif, CHEN Lijuan, WANG Yanmei
    2020, 38 (9):  1085-1094.  DOI: 10.3724/SP.J.1123.2020.02027
    Abstract ( 66 )   HTML ( 9 )   PDF (1830KB) ( 48 )  

    A capillary coated with mixed polymer brushes that shows switchability toward lysozyme adsorption was developed. This capillary was applied for the on-line preconcentration of lysozyme by capillary electrophoresis (CE) in order to enhance the detection sensitivity. First, poly(2-methyl-2-oxazoline) (PMOXA) and poly(acrylic acid) (PAA) were synthesized by cationic ring-opening polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization, respectively. Then, glycidyl methacrylate (GMA) and PMOXA were used to prepare poly(2-methyl-2-oxazoline)-random-glycidyl methacrylate (PMOXA-r-GMA) via radical copolymerization, and poly(acrylic acid)-block-poly(glycidyl methacrylate) (PAA-b-PGMA) was obtained by the RAFT polymerization of GMA and PAA. A mixed solution of PMOXA-r-GMA and PAA-b-PGMA at a certain mass ratio was then injected into the capillary. Subsequent annealing provided capillary materials coated with mixed polymer brushes based on PMOXA and PAA. X-ray photoelectron spectroscopy (XPS) analysis was performed to determine the surface composition of the capillary raw materials. When the mass concentration of the mixed solution was 20 g/L and the mass ratio of PMOXA-r-GMA and PAA-b-PGMA was 1:1, the carboxyl content in the coating increased with increasing chain length of PAA. Fluorescein isothiocyanate-labeled lysozyme (FITC-lysozyme) adsorption assay demonstrated that the coated capillary had switchable properties for lysozyme adsorption upon pH and ionic strength (I) trigger. At pH 7 (I=10-5mol/L), the capillary could adsorb a large amount of lysozyme, which could be released at pH 3 (I=10-1mol/L). Subsequently, this coated capillary was applied to the on-line preconcentration of lysozyme by CE. The sensitivity enhancement factor was 17.69 when the chain length of PAA was 2.2 times that of PMOXA. The limit of detection could reach 8.7×10-5g/L. On-line preconcentration of lysozyme was performed for five successive times on the same day and on five consecutive days. The intraday and interday relative standard deviations (RSDs) for the peak areas were 2.9% and 4.1%, respectively. The intraday and interday RSDs for the migration times were 0.9% and 2.1%, respectively. The developed method for the preparation of the coated capillary with good stability only needs one step in this work, and this research will supply a simple and effective way to analyze trace protein by CE.

    Preparation of a two-dimensional azine-linked covalent organic framework-coated capillary and its application to the separation of nitrophenol environmental endocrine disruptors by open-tubular capillary electrochromatography
    ZHAO Lingyi, LÜ Wenjuan, NIU Xiaoying, PAN Congjie, CHEN Hongli, CHEN Xingguo
    2020, 38 (9):  1095-1101.  DOI: 10.3724/SP.J.1123.2020.02031
    Abstract ( 110 )   HTML ( 11 )   PDF (1471KB) ( 62 )  

    As a class of new porous crystalline materials, covalent organic frameworks (COFs) are attracting the attention of a large number of scientists. Because of their large specific surface area, low density, high stability, and tunable pore size, COFs have been widely applied in many fields, including analytical chemistry. Open-tubular capillary electrochromatography (OT-CEC) is a mode of capillary electrochromatography. In recent years, a variety of materials such as porous organic frameworks have been used as the stationary phase for OT-CEC to overcome the disadvantages of low phase ratio and column capacity, thereby improving the separation efficiency. However, there are a few reports on the use of COFs as the stationary phase to improve the separation efficiency of OT-CEC. Environmental endocrine disruptors (EEDs) are a large class of exogenous chemicals that can disturb the effect of the normal endocrine substances and adversely affect the endocrine and reproductive systems of human beings. Considering the widespread existence of EEDs and the disadvantages of existing detection methods (e. g., gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry), such as complicated operation and large sample consumption, it is necessary to develop new methods for the separation and determination of EEDs in complex samples. OT-CEC is a good choice in this regard because of its low sample dosage, simple operation, and high analytical speed. Accordingly, a two-dimensional azine-linked covalent organic framework (ACOF-1) with a large surface area and small pore size was synthesized according to the reference method. Then, an ACOF-1-coated capillary was fabricated using ACOF-1 as the stationary phase, via covalent bonding, and used as the separation channel to establish a new OT-CEC method for the separation and detection of nitrophenol EEDs. X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) were used to characterize the synthesized ACOF-1 and the ACOF-1-coated capillary. The peak pattern in the XRD spectrum confirmed the successful synthesis of ACOF-1. The absorption peaks in the FT-IR spectrum and the morphology of the inner wall seen in the SEM images also demonstrated that the ACOF-1-coated capillary was fabricated successfully. A series of experiments were carried out to investigate the effects of the organic additive (methanol) content, pH, and concentration of the borate buffer on the resolution, migration time, and peak shape. Based on the results, the optimal separation conditions for the four nitrophenol analytes were 15 mmol/L borate (pH 9.20) with 10% (v/v) methanol. Under the optimum conditions, 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), 2, 4-dinitrophenol (DNP), and 2, 4, 6-trinitrophenol (TNP) could be baseline separated within 20 min by the established OT-CEC method. The linear range for 2-NP and 4-NP was 10-500 mg/L, while that for DNP and TNP was 20-1000 mg/L. The determination coefficients (R2) were greater than 0.99. For the four analytes, the limits of detection and limits of quantitation were in the ranges of 0.13-0.23 mg/L and 0.45-0.60 mg/L, respectively. The intraday, interday, and column-to-column relative standard deviations (RSDs) of the migration time and peak area were less than 9.4%. These results revealed that the established method has good repeatability and high stability, thus being suitable for the separation and detection of nitrophenol EEDs. The mechanism studies revealed that the pore size of ACOF-1 was the main factor influencing the separation behavior of each analyte. This work demonstrated the feasibility of using capillary electrochromatography with COFs as the stationary phase for the separation and detection of EEDs. Future research will continue to focus on the preparation of COF-coated capillaries and their application to OT-CEC separation and determination of EEDs.

    Determination of gastrodin activity inhibition on acetylcholinesterase by capillary electrophoresis
    ZHANG Jian, ZHANG Bo, HE Maofang, HAN Lu, GAO Dongyu, LIU Chunye
    2020, 38 (9):  1102-1106.  DOI: 10.3724/SP.J.1123.2020.02015
    Abstract ( 152 )   HTML ( 10 )   PDF (1290KB) ( 49 )  

    Alzheimer's disease (AD) is the most common cause of dementia in elderly individuals. Currently, acetylcholinesterase inhibitors (AChEI) are the most effective clinical treatment for AD. AChEIs in natural products may have therapeutic potential and should be screened for use in AD treatment. The authors describe a simple and reliable method for AChEI screening by capillary electrophoresis (CE). A hexadimethrine bromide (HDB) solution was pushed into a capillary (0.015 MPa×10 s) and incubated for 5 min. The capillary was flushed with deionized water for 5 min to remove free HDB, followed by plugging with an acetylcholinesterase (AChE) solution. After a 5 min incubation, the AChE was immobilized on the positively charged coating by ion binding, and the micro-reactor was created. The substrate solution, acetylthiocholine iodide (AThC), was injected into the capillary and incubated in the micro-reactor for 1 min. The unreacted substrate and the enzymolysis product were separated by CE. Gastrodin, an important component of Gastrodia elata, can inhibit AChE activity. After a certain amount of gastrodin was spiked into the substance solution, the peak area of the product decreased. Greater peak area reduction indicated stronger inhibition of AChEI. We observed good reproducibility of the product peak, with relative standard deviation (RSD) values less than 5.3%. The micro-reactor can be reused up to 300 times, which greatly improves efficiency. When the concentration of gastrodin was 5.24 μmol/L, the inhibition rate of AChE reached 64.8%. The IC50 of gastrodin was (2.26±0.14) μmol/L (R2=0.9983), which was consistent with the result of traditional UV method (2.09±0.18 μmol/L). If the function of the micro-reactor deteriorates, it can be conveniently renewed by flushing the column to remove the enzyme and repeating the AChE immobilization protocol. The proposed method is simple, efficient, and low cost, and can be used to screen AChEI from natural products, thus contributing to the improvement of AD treatment.

    Analysis of three antipyretic analgesic drugs by open-tubular capillary electrochromatography
    LIU Lili, QIAO Juan, ZHANG Hongyi, QI Li
    2020, 38 (9):  1107-1114.  DOI: 10.3724/SP.J.1123.2020.01006
    Abstract ( 99 )   HTML ( 8 )   PDF (3443KB) ( 39 )  

    The advantages of capillary electrophoresis, such as small sample consumption, high separation efficiency, and multiple separation modes, have been known for decades. However, exploring unique capillary electrophoresis techniques for the analysis of fluid drugs in living bio-systems remains an important and urgent task. Owing to the similar structures and mass-to-charge ratios of antipyretic analgesic drugs, efficient baseline separation of these analytes by capillary zone electrophoresis method cannot be easily achieved. Micellar electrokinetic chromatography can improve the baseline separation of these drugs, but the substantial amounts of non-volatile surfactants (such as sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium deoxycholate and cetylammonium bromide) in running buffer solutions would pollute the ion source during mass spectrometric analysis. For this reason, it is difficult to analyze unknown drugs by capillary electrophoresis-electrospray ionization-mass spectrometry. To overcome these drawbacks, much attention has been paid to capillary electrochromatography (CEC) because combines the high separation efficiency of capillary electrophoresis with the high selectivity of high performance liquid chromatography (HPLC). Recent challenges encountered in open-tubular capillary electrochromatography (OT-CEC) expanding the range of suitable functional polymer monomers and improvement of the separation efficiency by tuning the characteristics of the polymer coatings without using any organic solvent additives. In this study, a protocol based on OT-CEC using a block co-polymer coating is proposed for the analysis of three test antipyretic analgesic drugs (4-aminoantipyrine, antipyrine and phenacetin), without adding organic solvents and surfactants in the running buffer solutions. First, an amphiphilic block co-poly(styrene-co-glycidyl methacrylate) (P(St-GMA)), was synthesized by reversible addition-fragmentation chain transfer polymerization under mild conditions. Then, P(St-GMA) was coated onto the capillary surface, and an OT-CEC analysis was performed. Next, the effect of some key factors, including the polymerization time for obtaining P(St-GMA) with different molecular weights, coating concentrations of the block copolymer, the species of the running buffer solutions, pH and concentrations of the running buffer solutions, and organic solvent additives, on the OT-CEC separation efficiency were investigated. Under the optimized conditions with 50.0 mmol/L NaAc-HAc as the running buffer solution at pH 5.7, the three test antipyretic analgesic drugs were base-line separated by the constructed OT-CEC system. Good linear relationships between peak area and concentration of the test analytes in the range of 8.0-2.5×103 μmol/L were obtained (R2 ≥ 0.995). The limits of detection (LODs) were 1.0-2.5 μmol/L. Furthermore, the reason for the OT-CEC separation efficiency was clarified based on the decreased electro-osmotic flow in the coated capillary compared with that in the uncoated capillary. Finally, the proposed OT-CEC assay without using any organic solvents and surfactants as additives was applied for analysis of the three test antipyretic analgesic drugs in rat serum samples. Importantly, it was found that despite peak tailing, the OT-CEC separation efficiency of the drugs was dramatically enhanced because the block co-polymer could self-assemble in the solution and form pseudo-micelles, which further increased the interactions between the P(St-GMA) and these drugs. Our results not only reveal the great potential of block co-polymer coatings in OT-CEC for the analysis of drugs in real biological samples, but also serve asa platform for the preparation of diverse block co-polymers to be used in OT-CEC analysis. We believe that in the near future, the peak tailing problem in OT-CEC analysis can be resolved by using the designed unique block co-polymers, which possess a greater number of functional sites, as coatings and by appropriately tuning the interactions between the analytes and the coatings.