Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

毛细管电色谱有机流动相双二元双台阶分离

根据毛细管电色谱中溶质保留值与有机改性剂种类和浓度之间的相互关系，首次实现了毛细管电色谱有机流动相双二元双台阶分离。利用此方法分离含１２种芳香族化合物的混合样品，各组分在六次连续进样中保留时间的ＲＳＤ（％）值均在１．６５％以内，并将样品的分离时间缩短到等度洗脱方式下的三分之一。这说明该方法分离复杂样品快速、可靠。此外还推导了双二元双台阶分离方式下溶质保留值的预测公式，并对该公式的准确性进行了检验，预测值与实测值间的相对误差在５．７３％以内。     

反相毛细管电色谱中中性化合物的保留及有机改性剂对其保留值的影响

以硫脲作为电渗流标记物测定的死时间和以苯同系物线性回归方法测定的死时间为基础，将液相色谱中溶质保留值方程应用到电色谱中，得到了容量因子与二元流动相体系中有机改性剂含量之间的关系曲线；通过实验数据说明了一些极性的电中性化合物在电场作用下也会发生迁移而引起保留值的变化，且这种变化还受有机改性剂含量的影响。     

Combination of electrophoresis,chromatography, and magnetism in a single separation technique: Part 1: a first theoretical evaluation

Magnetic nanoparticles incorporated into the layer of a (polymeric) sorbent, covering the inner surface of a fused silica capillary, can produce—upon applying an electric field across the capillary length—an electromagnetic field that would affect to some extent the separation of charged analytes. A first theoretical assessment of such phenomenon is given here with a view of developing a novel hybrid separation technique based on the principles of electrophoresis, chromatography, and magnetism. Specifically, the effect of built-in magnetic nanoparticles, varying in absolute number, on the strength of axial electric field in an open-tubular column for capillary electrochromatography (CEC) column for CEC—being expressed through the associated changes in near-wall dielectric constant—was analyzed using linearized Poisson–Boltzmann equation.     

Interplay of chromatographic and electrophoretic processes in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography and capillary zone electrophoresis, i.e., chromatographic retention and electrophoretic migration. The adsorption of the different sample components on the stationary phase can be modified by the presence of the electric field across the column. Here, we use our previously published approach to decouple chromatographic retention from electrophoretic migration that allows us to investigate the "modification" of the retention process in CEC. This paper presents a methodology for characterization of changes in the retention of neutral and charged sample components, under identical conditions of stationary and mobile phase.     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Deconvolution of electrokinetic and chromatographic contributions to solute migration in stereoselective ion-exchange capillary electrochromatography on monolithic silica capillary columns

A monolithic silica stationary phase functionalized with an enantioselective strong cation exchanger based on an aminosulfonic acid derivative was used for chiral separations of basic test solutes by nonaqueous CEC and capillary LC. The effects of the applied electric field as well as the ionic strength in the eluent on electrokinetic and chromatographic contributions to the overall separation performance in the electrically driven mode were investigated. Hence, under the utilized experimental conditions, i. e., at an electric field strength in the range of approximately 120-720 V/cm (applied voltages 4-24 kV) and an ionic strength of the counterion between 5 and 25 mM (at constant acid-to-base, i. e., co- to counterion ratio of 2:1), no deviations from the expected linearity of the EOF were observed. This led to the conclusion that an occurrence of the so-called electrokinetic effects of the second kind resulting from electric double layer overlap inside the mesopores of the monolithic stationary phase and concentration polarization phenomena were largely negligible. Additional support to this conclusion was inferred from the observed independence of CEC retention factors on the electric field strength across the investigated ionic strength range of the BGE. As a consequence, a simple framework allowing for calculation of the CEC mobilities from the individual separation contributions, viz. electroosmotic and electrophoretic mobilities as well as retention factors, could be applied to model CEC migration. There was a reasonable agreement between calculated and experimental CEC mobility data with deviations typically below 5%. The deconvolution of the individual contributions to CEC migration and separation is of particular value for the understanding of the separation processes in which electrophoretic migration of ionic sample constituents plays a significant role like in ion-exchange CEC and may aid the optimization procedure of the BGE and other experimental conditions such as the optimization of the surface chemistry of the stationary phase. In combination with the remarkable column performance evident from the low theoretical plate heights observed under CEC conditions for all test solutes (3.5-7.5 microm in the flow rate range of 0.4-1.2 mm/s, corresponding to (130,000-300,000 plates per meter), the presented framework provides an attractive tool as the basis for the assessment of chromatographic selectivities in a miniaturized CEC screening of new selectors and chiral stationary phases (CSPs), respectively, from experimental CEC data and known CE mobilities.     

弛豫理论对离子交换毛细管电色谱中峰压缩现象的研究

 在毛细管电色谱中,由于溶质在输运过程中所具有的电性质,常会产生一些特殊的现象。这些现象,如离子交换毛细管电色谱中产生超高柱效峰的现象,已经不能用一般的色谱理论加以解释。基于弛豫理论所建立的基本模型,在考虑溶质在两相中皆有可能发生正、反向迁移的情况下,得到了流出曲线一阶原点矩和二阶中心矩的理论表达式,并通过对溶质在两相中电扩散速率与电泳速率、电渗流速率关系的分析结果证实:溶质在固定相表面的电扩散行为可以使其保留变弱,出峰加快;而这种电扩散导致的超常柱效峰的出现具有不稳定性,只有在多方面因素综合影响匹配的情况下才可能出现。     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Modeling of retention behavior in capillary electrochromatography from chromatographic and electrophoretic data

A phenomenological approach was presented to describe the retention behaviors of solutes in capillary electrochromatography (CEC). Equations for calculation of the retention time and actual chromatographic retention factor for ionic solutes, weak monoprotic acid and weak monoprotic base were derived, which can be described by two general expressions regardless the charge status of the solute. The general expressions enable calculation of the retention time and retention factor in CEC from chromatographic and electrophoretic data, which were experimentally verified with a variety of compounds and a variety of CEC modes. Based on this approach, the chromatographic retention and the electrophoretic migration in the CEC systems studied were found to be two independent processes. The validity of this approach was also discussed.     

Characteristics of electroosmotic flow and migration of neutral solutes under stepwise gradient elution of capillary electrochromatography

A theoretical study on the velocity of electroosmotic flow (EOF) and the retention times of neutral solutes under multiple-step gradient of capillary electrochromatography (CEC) was carried out, focusing on that with three kinds of mobile phases. Through the model computations, the detaining time of the second kind of mobile phase in the column was proved to play an important role in affecting EOF. The variation speed of EOF was shown to be determined by the differences among dead times in different steps. In addition, the prediction of the retention times of 13 aromatic compounds under gradient mode was performed with the deduced equations. A relative error below 3.3% between the calculated and experimental values was obtained, which demonstrated the rationality of the theoretical deduction. Our study could not only improve the comprehension of stepwise gradient elution, but also be of significance for the further optimization of separation conditions in the analysis of complex samples.     

Visible diode laser induced fluorescence detection of doxorubicin in plasma using pressurized capillary electrochromatography

Pressurized capillary electrochromatography is a variant of capillary electrochromatography (CEC) in which the driving force is both electroosmotic and hydraulic. The inlet of the CEC capillary is pressurized using an HPLC pump, and an electric field is simultaneously applied. This work describes a method for the analysis of doxorubicin. Doxorubicin was reacted with Cy5.29.OSu in acetonitrile. The derivative was confirmed by RP-TLC. A CEC system equipped with a VDLIF detector was constructed and used to analyze the derivative. The reaction mixture was injected onto a capillary packed in-house with 3 microm C-18 Luna particles and separation was carried out at 25 kV using 70% acetonitrile/ 30% phosphate (10 mM, pH = 4.8) as the mobile phase. The derivatization reaction was optimized by the investigation of parameters such as reaction time, temperature and concentration of label in order to increase the yield of the derivative. The optimal conditions were determined to be 30 min, 80 degrees C and 50 nmol/mL, respectively. Doxorubicin was extracted from plasma using solid-phase extraction under alkaline conditions, derivatized and injected onto the CEC-VDLIF system. The selectivity of the assay was demonstrated by a lack of interfering peaks due to plasma constituents across the elution window of the derivative peak in blank plasma extracts (n = 6 sources). The limit of detection (LOD) of the assay in plasma calculated as 3 s(b)/m was determined to be 1.7 ng/mL. The precision of the assay determined at a concentration of 167.7 ng/mL (n = 5) was found to be within 7.04 %RSD.     

Electrochromatographic characterization of etched chemically-modified capillaries with small synthetic peptides

In this investigation, various capillary electrochromatographic (CEC) experiments have been employed to characterize the properties of etched, chemically-modified surfaces of open tubular capillary columns with peptides as solute probes and under conditions of variable voltage, temperature and solvent composition. The separation performance of etched capillaries with either n-octadecyl or liquid crystal moieties derived from a cholesterol phase bonded to the surface were compared. With the liquid crystal bonded species, interesting and significantly different variations in retention behavior of peptides are obtained compared to those observed with the corresponding n-octadecyl modified surfaces by changes in temperature, solvent composition and field strength. These peptide separations illustrate the usefulness of this CEC approach for practical applications, where both the retention characteristics of the charged analytes as well as the selectivity differences due to the surface properties of the etched chemically-modified surfaces of open tubular capillary columns can be rationally modulated. As in HPLC, appropriate choice of CEC experimental variables, including the chemical properties of the immobilized ligand(s), represents a powerful tool for optimizing resolution.     

“Short-end injection” rapid analysis capillary electrochromatography

Summary The analytical benefits of using “short-end injection” rapid analysis in capillary electrochromatography (CEC) have been demonstrated. These include extremely rapid analysis times, the potential for voltage gradients to be exploited, high hydrodynamic loadability and the convenience of using short detachable lengths of packed capillaries. “Short-end injection” rapid CEC has been demonstrated for the analysis of a standard neutral test mixture and three pharmaceutically active steroids. Extremely short analysis times were obtained due to the increased effective field strength experienced by the packed bed and the reduced length to the detector. In conjunction with an appropriate sampling rate, peak efficiencies in excess of 554,000 plates/metre (i.e. 41,500 plates per column) could be obtained. The repeatability of retention time and peak area compared favourably to that obtained using a conventional CEC approach.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

One‐pot synthesis of (3‐sulfopropyl methacrylate potassium)‐silica hybrid monolith via thiol‐ene click chemistry for CEC

A novel (3‐sulfopropyl methacrylate potassium)‐silica hybrid monolithic column for CEC has been prepared by a simple one‐pot approach based on efficient thiol‐ene click chemistry. In this process, the polycondensation of hydrolyzed alkoxysilanes and in situ click reaction of vinyl groups on 3‐sulfopropyl methacrylate potassium and thiol groups on the precondensed siloxanes simultaneously occurred in a pretreated capillary. Homogeneous monolithic matrix with large through‐pores tightly bonded to the inner wall of the capillary was shown by optical microscope and SEM. The minimum plate height of this hybrid monolithic column was determined as 3.9 μm for thiourea. Anilines, alkylbenzenes, and phenols were well separated on this hybrid monolithic column by CEC, which indicated typical reversed‐phase and cation‐exchange chromatographic retention mechanisms of the column.     

十二烷基键合氧化锆填充电色谱柱的研究

以十二烷基键合氧化锆(C12-ZrO2)作为固定相,制备了填充毛细管电色谱(CEC)柱,较为系统地研究了流动相条件对电渗流的影响、填充CEC柱的稳定性、碱性与中性化合物的保留与流动相pH值和有机溶剂含量的关系。C12-ZrO2固定相填充CEC柱在pH3～11．7范围内具有极好的稳定性;利用磷酸盐与氧化锆表面之间较强的相互作用,能够有效解决传统硅胶键合烷基固定相在有机溶剂含量低的流动相条件下不稳定的问题;同时吸附磷酸盐的固定相表面使得在更宽的流动相pH值范围内CEC柱有足够的电渗流,进一步拓宽CEC的应用领域。