A drying step in the protocol to pack capillary columns by centripetal forces for capillary electrochromatography.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.     

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．     

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 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.     

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.     

Influence of the unpacked section on the chromatographic performance of duplex strong anion-exchange columns in capillary electrochromatography

This work describes initial investigations of strong anion-exchange (SAX) packing materials for capillary electrochromatography (CEC). The use of SAX phases in CEC is theoretically appealing for the analysis of negatively charged species. The reversed direction of the electroosmotic flow (EOF) generated by SAX phases (in comparison to reversed phases and strong cation-exchange phases) means that negative species can migrate with the EOF, not against it, hence the analysis times, of such species should be decreased and efficiencies improved. Duplex CEC columns (the standard for instruments using UV detection) consist of a packed and an unpacked section. Using common reversed-phase packing materials the direction of the EOF in both sections is co-linear, however when normal fused-silica capillaries are packed with SAX material the direction of the EOF in the two sections oppose one another. It has been shown, using conventional duplex CEC columns and fully packed CEC-MS columns that the opposing direction of EOF causes a massive degradation in column performance. Consequentially, it is demonstrated that if the EOF in the open section of the duplex SAX column can be controlled via pH or capillary derivatisation then good, reproducible CEC can be performed on anionic species using SAX packed CEC columns.     

The Development of Reversed‐Phase Capillary Electrochromatography for the Separation of Steroids

This paper describes the preparation and optimization of packed capillary columns for reversed‐phase separation of steroids with CEC. The fabrication of on‐column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused‐silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100‐μ end‐frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5‐μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column‐to‐column, day‐to‐day, and run‐to‐run are less than 7%, 2%, and 1% for four steroids, respectively.     

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.     

Capillary electrochromatography with segmented capillaries for controlling electroosmotic flow

Capillaries consisting of two segments each packed with a different stationary phase were introduced for the control and manipulation of the electroosmotic flow (EOF) in capillary electrochromatography (CEC). This kind of column configuration was called segmented capillary where one segment was packed with octadecyl silica (ODS) and served as the separation segment while the other segment was packed with bare silica and functioned as the EOF accelerator segment. The average flow in the segmented capillary increased linearly with increasing fractional length of the EOF accelerator segment, and consequently the analysis time was reduced. Under a given set of conditions, the average flow can be varied over a certain range that extends from the EOF in the individual ODS capillary at the lower end to the EOF in the individual bare silica capillary at the higher end. The pore size of the bare silica in the EOF accelerator segment influenced the average flow in the segmented capillary. Because of the difference in the EOF of the individual segments, the average flow across the segmented capillary is partially degenerated from EOF to viscous flow. Furthermore, the retaining frits in CEC columns are restrictive points which slow down the average flow, thus furthering the degeneration of the flow from EOF to viscous flow. In other words, in CEC columns containing retaining frits, the flow of the mobile phase is not only based on electroosmosis but is contaminated by a viscous component.     

Preparation of fritless capillary using avidin immobilized magnetic particles for electrochromatographic chiral separation

In capillary electrochromatography (CEC), magnetic particles (MPs) were packed in a fused silica capillary by using the magnetic field to be retained without frits. For a chiral CEC separation, avidin was immobilized onto the surface of the MPs (AVI-MPs) as a stationary phase by using the physical adsorption technique. The injected AVI-MPs into the capillary were stably captured with the magnet (surface magnetic flux density, 250 mT) under the separation voltage of 10 kV (190 V/cm). By employing the fritless AVI-MPs packed capillary, the chiral separation of ketoprofen was successfully attained with the packing length of only 5 cm. Effects of the modification condition of avidin, pH of background solution, and the packing length on the enantioseparation were also investigated. Under the optimal condition, furthermore, the repeatability for the retention time of ketoprofen was better than 1.5% in the relative standard deviation and the capillary-to-capillary reproducibility was also acceptable in the prepared fritless capillaries.     

Capillary electrochromatography without external pressure assistance. Use of packed columns with a monolithic inlet frit

A fused silica capillary column was packed with RP(18) silica stationary phase entrapping the particles between two frits obtained by two different procedures. The inlet frit consisted of a short organic polymer made via a thermopolymerization process while the outlet frit was prepared by sintering the octadecylsilica (ODS) material. The packed column was employed in capillary electrochromatography (CEC) experiments for the separation of three selected test compounds. Retention time and separation efficiency were evaluated. Results were compared with those ones obtained with a packed capillary containing the same stationary phase entrapped between two sinterized frits. The novel packed column exhibited comparable separation efficiency and resolution with the traditional one. However, it allowed experiments without pressure support during the runs with no bubble formation.     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

Packing capilllary electrochromatography columns using vacuum--a preliminary study

This paper introducesa novel method for packing Capillary Electrochromatography Columns (CEC). Using vacuum packing methodology, silica particles as small as 1 microm were successfully packed into the capillary columns with 75 microm inner diameter. The columns are verystable and show no noticeable loss in efficiency after 200 sample injections. The performance of these vacuum packed capillary columns was evaluated with a mixture of aromatic and non-aromatic compounds. A 24 cm long capillary column can produce peak efficiencies of around 45,000 plates for benzene.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

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.     

Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 μm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2–4% whereas the plate heights of an unretained marker increased by 30–50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-μm stationary phases up to 10–15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.     

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.     

Microcolumns with self-assembled particle frits for proteomics

LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.     

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.     

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.