CEC columns

The plug flow profile would only be distorted in very narrow bore capillaries with a diameter smaller than the thickness of two double-layers that then overlap. To achieve an undisturbed flow, Knox suggested that the diameter should be 10-40 times larger than 6 [15]. This can easily be achieved in open capillaries. However, once the capillary is packed with a stationary phase, typically small modified silica beads that carry on their own charged functionalities, the distance between adjacent double-layers is only a fraction of the capillary diameter. However, several studies demonstrated that beads with a submicrometer size can be used safely as packings for CEC columns run in dilute buffer solutions [15,35]. 

Fig. 7. Scheme of a typical process used for packing CEC columns with beads... 

Fig. 9. Effect of pore size on the efficiency of CEC columns. (Reprinted with permission from [70]. Copyright 1997 VCH-Wiley). Conditions field strength 100-500 V/cm, capillary column 75 pm i. d., total length 30 cm, active length 25 cm, isocratic separation using 20 80 acetonitrile-100 mmol/1 phosphate buffer pH = 6.9, marker acetone... 

In order to avoid tedious procedures required to prepare packed CEC columns, some groups are studying the use of empty capillaries. Since solute-stationary phase interactions are key to the CEC process, appropriate moieties must be bound to the capillary wall. However, the wall surface available for reaction is severely limited. For example, a 100 pm i.d. capillary only has a surface area of 3xl0 4m2 per meter of length, with a density of functional sites of approximately 3.1 xlO18 sites/m2, which equals 0.5 pmol sites/m2. Moreover, surface modification cannot involve all of the accessible silanol groups, since some must remain to support the EOF. As a result, the use of bare capillaries in CEC has been less successful. 

The first approach to monolithic columns formed from beads can be assigned to Knox and Grant [15] who prepared a particle-embedded continuous-bed CEC column. They packed beads into a Pyrex glass tube of 1 - 2 mm i.d. and then drew the packed column to create a capillary. The particles were partly incorporated in the glass wall and the column was stable unless the column-to-particle diameter exceeded a value of 10. The success of this procedure was very sensitive to the presence of water in the original packing material. 

All these methods are solving the problem of column stability since the fused beads cannot move. However, these approaches often do not avoid the in situ fabrication of frits, one of the critical operations in the preparation of CEC columns. 

Although Fields already mentioned the possible preparation of monolithic silica-based CEC columns, the lack of experimental data leads to the assumption that this option has not been tested [111]. In fact, it was Tanaka et al. who demonstrated the preparation of monolithic capillary columns using a sol-gel transition within an open capillary tube [99,112]. The trick was in the starting mixture that in addition to tetramethoxysilane and acetic acid also includes poly(ethylene oxide). The gel formed at room temperature was carefully washed with a variety of solvents and heated to 330 °C. The surface was then modified with octadecyl-trichlorosilane or octadecyldimethyl-A N-dimethylaminosilane to attach the hy-... 

While only a few reports concern the in situ preparation of monolithic CEC columns from silica, much more has been done with porous polymer monoliths and a wide variety of approaches differing in both the chemistry of the monomers and the preparation technique is currently available. Obviously, free radical polymerization is easier to handle than the sol-gel transition accompanied by a large decrease in volume. 

Hoegger and Freitag modified the Hjerten s procedure and prepared a variety of monolithic acrylamide-based CEC columns [118]. Their approach allowed them to adjust both rigidity and porous properties of the monoliths and to achieve excellent separations of model compounds as well as selected pharmaceuticals. 

Zhang developed a monolithic poly(styrene-co-divinylbenzene) CEC column in which the EOF is supported by carboxyl groups of polymerized methacrylic acid [ 133]. Using benzene as a probe, column efficiencies of 90,000 -150,000 were observed within a flow velocity range of l-10cm/min (0.2-1.7 mm/s). Different families of compounds such as phenols, anilines, chlorobenzenes, phenylendi-amines, and alkylbenzenes were well separated typically in less than 5 min using 20 cm long columns. 

Since the separation process in CEC has a number of attributes similar to those of HPLC, the most important variables affecting the separation are the same for both of these techniques. However, in HPLC mobile phase, flow and separation are independent variables. Therefore, the most important operational variables are the analyte-sorbent interactions that can be modulated by the chemistry of the packing, composition of the mobile phase, and temperature. In contrast, the CEC column has a dual role as it serves as both (i) a flow driving device and (ii) separation unit at the same time. Although the set of variables typical of HPLC is also effective in CEC, their changes may affect in one way or another both column functions. Therefore, optimization of the separation process in CEC is more complex than in HPLC. 

One of the important operational variables in CEC is the analyte—sorbent interaction. In reversed-phase separations (typical in CEC) the hydrophobicity of the stationary phase determines the selectivity of the separation, and retention can be controlled by adjusting the surface chemistry of the packing, composition of the mobile phase, and temperature. In contrast to HPEC, the CEC column plays a dual role in providing a flow driving force and separation unit at the same time hence electrophoretic and chromatographic processes are operational. The stationary phase chemistry is dealt with in detail in Section III on column technology. 

An open-tubular column is a capillary bonded with a wall-supported stationary phase that can be a coated polymer, bonded molecular monolayer, or a synthesized porous layer network. The inner diameters of open-tubular CEC columns should be less than 25 pm that is less than the inner diameters of packed columns. The surface area of fused silica tubing is much less than that of porous packing materials. As a result, the phase ratio and, hence, the sample capacity for open-tubular columns are much less than those for packed columns. The small sample capacity makes it difficult to detect trace analytes. 

Packed capillary columns (Figure 8) have a greater sample capacity than open-tubular columns because of the increased surface area and, hence, greater phase ratio. Greater sample capacities result in increased sensitivity and selectivity. More than 95% of the CEC columns... 

A new type of CEC column has been prepared with a charged polymer layer on the inner wall of the capillary and a neutral monolith as the bulk stationary phase (Figure 12). After silanization of the capillary wall, polyethyleneimine was covalently bound to the wall, to provide charged moieties. 

FIGURE IS Electrochromatograms obtained for the separation of basic drugs spiked in a human serum compared with a blank in a hydrophobic interaction CEC. Column 5 pm 300 A polysulfoethyl A particles, 20 cm packed length, 50 pm ID mobile phase ACN/TEAP buffer (80 20) applied voltage, 10 kV detection at 214 nm. Drugs (I) amobarbital (2) phenobarbital (3) barbital (4) caffeine (5) sulfanilamide (6) theophylline (7) 2,4-dimethylquinoline (8) propranolol. (Reproduced with permission from reference 76.)... 

The emerging of CEC and the increased scientific work on the preparation of different phases, characterization, and applications of the CEC columns have given much credence to their future potentials in microseparations. The fabrication and availability of different phases for analysis with both particle-packed and monolithic columns give the technique a great future. This is because a variety of mechanisms can be exploited in the analysis and separation of compounds that could otherwise be difficult to analyze with HPLC or CE alone. The ease of coupling CEC to sensitive detectors such as mass spectrometers for enhanced sensitivity, structural elucidation, and characterization bestows the technique with great versatility. 

CEC—MS was applied for the analysis of various pharmaceuticals using either packed-CEC, OT-CEC, or monolithic-CEC columns. 

Lord et al. analyzed a mixture of steroids by CEC-ESI/MS and interfaced externally tapered CEC columns in both sheathless and sheath-flow arrangement. Sensitivity was found 20-fold higher in the sheathless configuration. The same conclusion was drawn by Warriner et ah, who evaluated CEC-nanospray/MS vs. CEC-microspray/MS with an ion trap using five corticosteroids. Cahours et al. used CEC-ESI/MS for a drug metabolism study and obtained a simultaneous baseline separation of flunitrazepam and its major metabolites. For CEC-ESI/MS coupling, the commercially available packed-CEC column was connected... 

As discussed in previous sections, adding a chiral selector to CZE (see Section III.A.2) or MEKC (see Section III.C) buffers, either directly or indirectly using PET, is possible for analysis of CE—ESI/MS enantiomers. However, the use of such chiral selectors or additives can produce a significant enhancement of background noise. An alternative is to attach or bond the chiral selector as a chiral stationary phase (CSP) either to a packed-CEC or monolithic-CEC column, or to an OT-CEC column. ... 

Brush-type, proteins, CDs, natural molecular imprint-based polymers (MIP), and macrocyclic antibiotics have been immobilized as chiral selectors on packed-CEC columns. Zheng and Shamsi demonstrated the possibility of using chiral CEC—ESI/MS with a commercially packed column for the determination of warfarin enantiomers in human plasma using coumachlor as an internal standard (IS). Robustness of this chiral CEC capillary was recently improved by a novel procedure and applied for the simultaneous enantiosepara-tion of height /1-blockers with multimodal CSP using different combinations of vancomycin and teicoplanin, as presented in Figure 5. ... 

Kato, M., Onda, Y., Sakai-Kato, K., and Toyo oka, T. (2006). Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns. Anal. Bioanal. Chem. 386, 572-577. 

The authors prepared a monolithic MIP CEC column in 100 pm i.d. fused-silica capillary, imprinted with thiabendazole (TBZ). This compound is a commonly used post-harvest fungicide to control diseases during storage and distribution of fruits... 

Some of the more recent examples presented in this chapter show, however, that several researchers have learned how to live with this problem and succeeded in finding areas where MIP or CEC columns and MISPE cartridges or online precolumns can be used with similar or better results than competing technologies. 

There are several possible reasons for the slow development of capillary electrochromatography. The first is the difficulty involved in constructing the capillaries. However, if technology develops to the point where the capillaries can be packed easily and reproducibly, CEC columns will proba-... 

Columns employed so far in capillary electrochromatography (CEC) contain both a packed and an open segments with concomitant changes of the electric field strength and the flow velocity at the interface of these two segments in such columns. Figure 1.1 shows the schematics of a CEC column of total length L, which consists of a... 

Fig. 1.1. Schematic illustration of a CEC column consisting of a packed and an open segment. The latter is divided into pre-detection and post-detection open segments by the detector window.

---