Recent trends in open-tubular capillary electrochromatography

Capillary electrochromatography (CEC), which combines the advantages of the high efficiency of capillary electrophoresis (CE) and the high selectivity of liquid chromatography (LC), has recently received considerable attention. Most CEC experiments have been performed with capillary columns packed with small LC packing materials (1.5–5 μm particle diameter). However, problems such as difficulties in packing the small LC packing materials and fabricating the frits still exist in preparing the CEC column. The use of open-tubular columns in CEC is therefore an alternative approach that can eliminate the problems encountered in packed-column CEC. So far, several types of open-tubular columns have been proposed for CEC separations and in this article recent progress in this area is reviewed.     

Packing structure and self-heating in capillary electrochromatography

The origin of bubble formation during operation of capillary electrochromatography (CEC) has been an issue of debate. Ohmic heating resulted from current passed through a packed column was proposed as the primary cause. However, this explanation has been questioned on the ground that the current measured in CEC is much lower than that measured with open-tubular separation systems where no bubble formation occurs. To resolve this issue, we carried out a theoretical study correlating self-heating of the electrolyte with packing structure of the column. We used a bundle of capillary tubes, a bundle of two types of capillary tubes and two bundles of capillary tubes connected serially to model, respectively, the flow channels in the column of non-porous particles, in the column of porous particles and in the column of various packing densities. The results from this study indicate that, for columns of homogeneous packing density, the heat output is indeed smaller than that in open-tubular columns of the same dimensions. In this case, the self-heating cannot be a key factor responsible for the bubble formation in CEC. However, for columns of heterogeneous packing density, a large excess of heat release may be produced in column sections of high packing density and, in turn, over-heating in such sections may become the primary cause for the formation of bubbles. It follows from this study that preparation of columns of homogeneous packing structure is essential to obtain reproducible and bubble-free CEC systems.     

Packing columns for capillary electrochromatography

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.     

磁场辅助毛细管电色谱

磁场辅助毛细管电色谱是液相色谱研究领域中出现的新技术．它利用外加磁场的引力将置于毛细管内的具有磁响应性的硅胶微球或四氧化三铁微球固定在管内任意位置．磁场固定微球聚集体既可用作填充柱,直接用于电色谱分离;也可用作柱筛,用于填装由商品色谱填料组成的色谱柱．这一技术的优势在于制备简便易行,柱管可以再生使用,适合于微流控芯片上柱筛或柱床的制作．本文简要评述磁场辅助毛细管电色谱的进展,包括磁性色谱填料的制备,磁场固定柱床电色谱,磁性柱筛电色谱及毛细管柱内柱结构参数的测定等方面．     

Towards high peak capacity separations in normal pressure nanoflow liquid chromatography using meter long packed capillary columns

Single shot proteomics is a promising approach to high throughput proteomics analysis. In this strategy, long capillary columns are needed to perform long and shallow gradients to achieve high peak capacity and good peak width for informative mass spectrometric detection. Herein, we report that meter long capillary columns, packed with 5 μm particulate material, can be facilely fabricated based on single particle fritting technology. The long columns could reliably generate high peak capacities of 800 in 10 h long gradients for protein digest separations. The operation was within the pressure range (40 MPa) of the most widely used normal pressure nanoLC systems. Due to the excellent life time (>100 injections) and inter-column performance consistency, the meter long capillary columns reported here should be of practical usefulness in single shot proteomics without the need for ultra-high pressure instrumentation.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Recent progress in polar stationary phases for CEC

This review summarizes most of the recent developments in the preparation and application of polar stationary phases for CEC covering the literature published since the year 2004. These polar stationary phases have been adopted for separation of analytes by the modes of packing column CEC, open-tubular CEC (o-CEC) and monolithic column CEC. Currently, development of o-CEC using biomolecules, such as protein and DNA, as the immobilized ligands is highlighted partly due to the simplicity of preparation. Furthermore, monolithic columns have been extended quickly, particularly inorganic materials-based monoliths, such as silica, zirconia, hafnium, etc., as an alternative to packed columns have been developed quickly.     

Towards the column bed stabilization of columns in capillary electroendosmotic chromatography. Immobilization of microparticulate silica columns to a continuous bed

This article discusses a novel method generating a continuous bed inside the CEC column. The column bed composed of microparticulate reversed-phase silica is completely immobilized by a hydrothermal treatment using water for the immobilization process. This process eliminates the manufacture of frits of both ends of the column and all problems associated with their preparation. Fundamental studies on operational parameters will be presented such as the dependence of the immobilization on the column temperature, the type of stationary phase and the column back pressure. The immobilized CEC columns show the same high column efficiency as packed columns with frits.     

Monolithic Molecularly Imprinted Columns for Chromatographic Separation

Monolithic molecularly imprinted columns are a new class of column that emerged in the early 1990s. These monolithic materials are typically prepared directly inside stainless steel columns or capillary columns without the tedious procedures of grinding, sieving, and column packing. Furthermore, the preparation of this type of MIP is more cost-efficient, because the amount of template molecules required is much lower. In recent years monolithic supports as stationary phases in high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) have attracted significant interest because of their ease of preparation, high reproducibility, versatile surface chemistry, and rapid mass transport.     

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.     

功能化聚合物基质整体色谱柱的研究进展

新药研发、蛋白质组学分析、中药有效成分分析等前沿领域的快速发展,对分析检测方法的灵敏度、样品通量等提出了更高的要求,色谱分析技术面临着新的严峻挑战。作为色谱技术的核心,高效快速、高选择性的新型色谱柱开发是当前研究的热点之一。有机聚合物整体色谱柱因具有制备简单、通透性好、耐酸碱性能强、特别适合复杂样品的快速分离分析等优点,有望克服传统色谱分离介质的不足而备受关注。近年来分析对象越来越复杂,基于整体材料制备简单的特点,开发特殊功能化的新型整体色谱材料,成为分析科学及材料科学等领域研究者高度重视的方向之一。该文系统介绍了整体柱的发展史,并主要综述了有机聚合物整体色谱柱的发展及其功能化研究。     

Properties and Applications of Mixed Packing Capillary Electrochromatography

Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C₁₈ on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.     

Recent progress in enantiomeric separation by capillary electrochromatography

Recent progress in enantiomeric separations by capillary electrochromatography (CEC) is reviewed. The development of simple and robust CEC column technologies plays an important role for popularization of CEC. During the last several years, various approaches for the preparation of enantioselective columns have been reported. Currently, the monolithic column technology (continuous beds) represents the most advanced approach for the preparation of CEC columns. The development of new chiral stationary phase used for CEC is another important issue in this field. Fundamental investigations on electrochromatographic behaviors of various CSPs are necessary in order to understand the separation mechanism and thus improve the separation performance. Some chiral stationary phases performed better under nonaqueous CEC conditions than reversed-phase conditions. Coupling CEC with mass spectrometry (MS) provides a powerful tool for enantiomeric separation. Finally, some applications of enantiomeric separation by CEC are summarized.     

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography.

Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.     

Selection of two reliable parameters to evaluate the impact of the mobile-phase composition on capillary electrochromatography performance with monolithic and particle-packed capillary columns

Different models have been described in the literature to evaluate the total porosity of CEC columns: gravimetric, flow as well as conductivity-based methods. In this study, these models have been compared for two kinds of CEC columns: two mixed-mode silica particle stationary phases and different monolithic columns (acrylate or polystyrene divinylbenzene-based). The total porosities measured from the conductivity-based methods were lower than the total column porosities obtained by gravimetric or flow methods for all the investigated columns while the wide distribution of observed values shows that conductivity-based methods discriminate columns more efficiently with very different properties. We propose a conductivity-based method taking into account the actual length proposed by Horvath, to evaluate what we call an "actual electrokinetic" porosity (AEP). This parameter, based on electrokinetic theory only, affords the most consistent evaluation of porosity under experimental CEC conditions for the packed- and acrylate-based monolithic columns. To illustrate the potential of AEP and actual EOF for the estimation of the performances of a CEC system (stationary and mobile phases) we studied the influence of the mobile-phase composition on these parameters for CEC separations with an ammonium embedded packed stationary phase. The AEP and the actual electroosmotic mobility should allow a better understanding of the perfusive EOF and stationary-phase wettability. For neutral compounds (substituted phenols), AEP evaluation allowed us to predict the mobile-phase conditions able to enhance the efficiency while both AEP and actual EOF had to be considered in the case of peptide analysis.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Comparison of sub-2-microm particle columns for fast metabolite ID

The use of sub-2-microm particle columns for fast high throughput metabolite ID applications was investigated. Three LC-MS methods based on different sub-2-microm particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 microm particle column coupled to an MS, methods B and C utilized a 1.7 microm particle column (BEH 50 x 2.1 mm2 id) and 1.8 microm particle column coupled to a Q-TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N=10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O-demethylverapamil (ODMVP) in human-liver microsomal incubates. Results show that 1.8 microm particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MS(E) mode of the Q-TOF instrument compared to 1.7 microm particle columns. Additionally, the study also confirmed that sub-2-microm particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384-well plate format of up to 48 compounds in incubates of human-liver microsomes was discussed.     

Acrylic-based high internal phase emulsion polymeric monolith for capillary electrochromatography

The use of high internal phase emulsion polymers (polyHIPEs) for CEC applications has remained relatively unexplored. A few reports exist in the literature for the preparation of similar structures. In this study, polyHIPEs having high porosity, and interconnected open-cell structure, were introduced and evaluated as stationary phase for CEC. The polyHIPE monolithic columns were prepared by the in situ polymerization of isodecylacrylate (IDA) and divinylbenzene (DVB) in the continuous phase of a high internal phase emulsion (HIPE). Due to its well-defined polyHIPE structure with interconnected micron size spherical voids, the columns synthesized with different initiator concentrations were successfully used for the separation of alkylbenzenes. Furthermore, the columns indicated a strong electroosmotic flow (EOF) without any additional EOF generating monomer probably due to the presence of ionizable sulfate groups coming from the water-soluble initiator used in the preparation of polyHIPE matrix. The best chromatographic performance in the separation of alkylbenzenes was achieved by using 70% ACN in the mobile phase with high column efficiency (up to 200 000 plates/m).     

Use of a fritless dual tapered column and a low flow interface for capillary electrochromatography-mass spectrometry

To avoid problems associated with the use of sintered frits to retain packing material, tapered columns were investigated for use with capillary electrochromatography-mass spectrometry (CEC-MS) analysis. Taking the advantage that negatively charged stationary phase particles have a net velocity directed towards the buffer reservoir (inlet) over a wide range in pH, a fritless CEC column with a single taper tip was prepared for CEC-MS analysis. During CEC-MS analysis, the tapered end was immersed in the buffer reservoir and the unmodified end was pointed toward the ionization source. For better sensitivity, this single tapered CEC column was coupled to ESI/MS using a low flow sheath liquid interface. With this setup, occasional blockage of the ESI sprayer by stationary phase particles was observed. In addition, significant dead volume was observed because the unmodified tip could not be inserted into the very end of the sprayer of the low flow sheath liquid interface. To circumvent these problems, a dual tapered CEC column was prepared. This fritless dual tapered column CEC-MS approach alleviated the problems of frit, sprayer blockage and extensive dead volume.     

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.