Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography

Porous silica beads with an average particle diameter between 0.2 and 3 microm have been applied as packing material in capillary electrochromatography (CEC). The experiments were directed to investigate whether it is really feasible and as promising as expected to use such small particles. In CEC, plate heights of H approximately/= 1-2 d(p) can be achieved which is smaller than the plate heights usually attained in high-performance liquid chromatography. Using a capillary packed with 0.5 microm silica beads we achieved a plate height of H = 3 d(p) indicating the presence of dispersive effects like Joule heating. Calculations demonstrate that at a field strength of about 800 V cm(-1) one third of the plate height can be lost by Joule heating effects if the heat is not removed by a cooling system. Additionally, the H(u) curve is still descending at the maximum electroosmotic flow (EOF) velocity we generated with the modified capillary electrophoresis instrument. To fully exploit the potential of submicron size silicas higher field strengths, i.e., higher EOF velocities, must be attained. To study the influence of the kind of packing on the EOF porous as well as nonporous silicas have been applied. The experiments clearly indicate that the EOF of porous and nonporous silicas is the same. Since the EOF is more or less exclusively generated by the packing material the zeta potential of n-octyl bonded 0.5 microm silica has been determined. The dependence of the zeta potential on the pH is identical to the dependence of the EOF on the pH in a packed capillary. The point of zero charge of the silica is at pH 2-3.     

Recent developments in capillary electrophoresis and capillary electrochromatography of carbohydrate species

This review article is concerned with the recent developments in capillary electrophoresis (CE) and capillary electrochromatography (CEC) of carbohydrates. The literature shows that CE possesses impressive potential in the analysis of carbohydrates. On the other hand, CEC has just started to show promise in the analysis of carbohydrates. Advances in separation and detection approaches of derivatized and underivatized carbohydrates are discussed based on the available literature. In addition, important applications are illustrated.     

Capillary electrochromatography of peptides and proteins

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure-assisted CEC or voltage-assisted micro- high-performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.     

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.     

Recent progress in enantiomer separation by capillary electrochromatography

Enantiomer separation by electrochromatography (CEC) can be performed in three modes: (i) open-tubular capillary electrochromatography (o-CEC), in which the chiral selector is physically adsorbed coated, and thermally immobilized or covalently attached to the internal capillary wall; (ii) packed capillary electrochromatography (p-CEC), in which the capillary is either filled with chiral modified silica particles or with an achiral packing material, and a chiral selector is added to the mobile phase; and (iii) monolithic (rod)-capillary electrochromatography (rod-CEC) in which the chiral stationary phase (CSP) consists of a single piece of porous solid. We present an overview on methods and new trends in the field of electrochromatographic enantiomer separation such as CEC with either nonaqueous mobile phases or stationary phases with incorporated permanent charges, or with packing beds consisting of nonporous silica particles or particles with very small internal diameters.     

High temperature and temperature programming in capillary electrochromatography

In electrochromatography, solvent electrophoretic mobility and solute partitioning are temperature dependent processes. If temperature variations are controlled, solute selectivity and analysis times can be tailored. In this study the feasibility of temperature programming in capillary electrochromatography (CEC) was demonstrated using a reversed-phase CEC mode. The outcome of programmed separations was compared with isothermal, isocratic and isorheic (constant flow) separations. The combined effects of column temperature and mobile phase flow-rate changes during the separation run, resulted in up to a 50% reduction in the separation run time, without adversely affecting the quality of separation. For capillary electrochromatography, temperature programming may be a valuable alternative to solvent programming modes because of the great technical difficulties associated with carrying out solvent gradient elution.     

使用含离子涂层柱的毛细管电泳和毛细管电色谱的研究进展

 毛细管电色谱是近年发展起来的高效、高选择性的微分离技术。与一般的毛细管电泳和使用ODS反相填料的毛细管电色谱相比 ,含离子涂层柱的毛细管电泳和毛细管电色谱能提供较大且可控的电渗流 ,便于拓宽分离对象 ,优化分离条件。对使用含离子涂层柱的毛细管电泳和电色谱的特点、发展和应用状况进行了综述。     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

Microchip-based capillary electrochromatography using packed beds

Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.     

Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography

In the last decade, silica monolithic capillaries have focused more and more attention on miniaturized separation techniques like capillary electrochromatography (CEC), nano-liquid chromatography (nano-LC) and chip electrochromatography owing to their unique chromatographic properties and their simplified preparation compared with packed columns. They are synthesized according to a sol-gel multi-step process that includes, after a gelation step at 40 degrees C leading to the formation of the macropores network and the silica skeleton, a post-gelation step (hydrothermal treatment at 120 degrees C in basic medium) that allows to tailor the mesopores and finally a calcination or a washing step to remove remaining polymers. In order to reduce the synthesis time, the number of synthesis steps and above all the temperature synthesis, to adapt the synthesis of such silica monoliths in polymeric microsystem devices, we extensively studied the influence of the hydrothermal treatment and its duration on textural (pore size distribution) and chromatographic properties (retention, efficiency) of in situ-synthesized capillary monoliths in nano-LC and CEC. This study was performed on pure silica and octyl chains grafted silica monoliths. Untreated monoliths show small pores (<6 nm), whereas hydrothermally treated monoliths exhibit medium and large mesopores (8-17 nm). It was demonstrated that the hydrothermal treatment at 120 degrees C was not necessary for pure silica monolithic capillaries dedicated to normal phase liquid chromatography or hydrophilic interaction liquid chromatography (HILIC) and electrochromatography: the suppression of the hydrothermal treatment did not impair efficiencies in CEC and in nano-LC but contributed to increase in retention factors. Minimal plate heights of ca. 5 microm in CEC and 6 microm in nano-LC were obtained with or without hydrothermal treatment with bare silica. In the same way, the hydrothermal treatment was not necessary for grafted silica monoliths only dedicated to CEC. However, the results clearly indicate that the hydrothermal treatment becomes essential before grafting in order to preserve the efficiency of the monolithic silica capillaries dedicated to nano-LC: in this particular case, the suppression of the hydrothermal treatment leads approximately to a loss of a factor two in efficiency.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。     

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.     

以枝形大分子聚酰胺-胺(PAMAM)为键合固定相的开管毛细管电色谱柱的制备及评价

用3-氨丙基三乙氧基硅烷(KH550)作偶联剂, 在毛细管内壁上逐步合成树枝形大分子聚酰胺-胺(PAMAM), 制得了1, 2和3代PAMAM键合的开管毛细管电色谱柱, 并对其性能进行了研究. 结果表明, 随着大分子代数的增加, 毛细管电渗流(EOF)逐步下降. 利用制得的1, 2和3代PAMAM修饰的开管毛细管电色谱柱对丙氨酸和脯氨酸的分离进行对比, 结果显示, 随着大分子PAMAM代数的增加, 分离度逐步增大, 丙氨酸和脯氨酸可在3代树枝状大分子PAMAM修饰的开管毛细管电色谱柱上达到基线分离. 采用非衍生化法和3代PAMAM修饰的开管毛细管电色谱柱成功地分析了精氨酸、 丙氨酸、 脯氨酸、 甲硫氨酸和组氨酸. 结果表明, 键合毛细管柱具有良好的重现性和稳定性.     

Chiral separations in capillary high-performance liquid chromatography and nonaqueous capillary electrochromatography using helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) as chiral stationary phase.

Enantioseparations in nonaqueous capillary electrochromatography (CEC) are reported in this study for the first time, using wide-pore aminopropyl silica gel coated with helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) (PDPM) as chiral stationary phase (CSP). The anodic electroosmotic flow (EOF) in a methanolic solution of ammonium acetate was used for the migration of neutral analytes through the packed bed in the capillaries. Four different techniques, high-performance liquid chromatography (HPLC) in common-size columns, capillary HPLC, pressure-assisted CEC and CEC were compared from the viewpoint of separation parameters. The latter three were performed with the same experimental setup, varying the relative contribution of the pressure-driven and the electrically driven flow to the overall mobility of the analyte. Capillary HPLC offers clear advantages compared to enantioseparations in common-size columns. However, for a given particle size of the packing material, CEC was not obviously advantageous compared to pressure-driven separations.     

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Use of nanoparticles in capillary and microchip electrochromatography

Applications of nanoparticles are of rising interest in separation science, due to their favorable surface-to-volume ratio as well as their applicability in miniaturization. A stationary phase with large surface area in combination with an electroosmotic flow-driven system has great potential in a highly efficient separation system. This review covers the use of various nanoparticles as stationary or pseudostationary phase in capillary and microchip electrochromatography. The use of nanoparticles in pseudostationary phase capillary electrochromatography and open-tubular capillary electrochromatography are thoroughly discussed. The stationary and pseudostationary phases that are described include polymer nanoparticles, gold nanoparticles, silica nanoparticles, fullerenes and carbon nanotubes.