In packed columns, a stationary phase is chemically bonded to a support particle that is then packed into a fused-silica tube of dimensions similar to those used in CE. Most of the reported workrelating to CEC separa-tions has been focused on the use of reversed phases [73±80]. However, materials which are suitable for HPLC may not in all cases be suitable for CEC [66]. In liquid chromatography the remaining silanol functions are not favorable for separating basic compounds due to unde-sired ionic and/or hydrophobic interactions, hence they are usually endcapped by trimethylsilylation. However, such end capping is undesirable in CEC, since the decrease of the density of the silanol group causes reduc-tion of the velocity of EOF due to the decrease of negativ-ity on the particle surface. Some manufacturers (Waters, Hewlett-Packard, Supelco and others) are now providing chromatographic material that has been specifically designed for CEC. Several groups have reported separa-tions on ion-exchange column packing material [80±93], on size-exclusion stationary phases [94], and on chiral stationary phases [95±97]. Djordjevic et al. [90] prepared mixed bed stationary phases by blending bare silica and reversed phase. They also studied the retention behavior of neutral compounds in CEC.
Mixed packing CEC with the stationary phase comprising a mixture of strong cation exchanger (SCX) and octade-cylsilane (ODS) phase was developed by Zhang et al.
[91]. With the existence of a sulfonic acid group on the surface of SCX, not only could the 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 C18on the surface of ODS, both the retention and the res-olution of samples were improved. The columns were used for the analysis of strong polar solutes as well as for the high-speed separation of acidic, basic and neutral compounds in a single run. Recently, Ye et al. [92] re-ported the preparation of a column packed with an SCX packing material and dynamically modified with cetyltri-methylammonium bromide (CTAB), which was added to the mobile phase. CTAB was adsorbed onto the surface of the SCX packing material, and the resulting hydropho-bic layer on this packing was used as the stationary phase for the separation of neutral solutes. A mixture con-taining the acidic, basic, and neutral compounds was also separated in this mode with a low-pH mobile phase; how-ever, peaktailing for basic compounds was observed.
Hoffman and Dovletoglou [98] reported the transition metal-mediated separation of isomeric pneumocandins by CEC using capillaries packed or coated with ODS
par-ticles (C18) or with glycerol bound to silica through a car-bon chain linker. They found that the separation achieved with the glyerol-coated capillary was much better than the separation achieved with the C18 coated or C18 packed capillaries. Suzuki et al. [99] prepared chemically bonded silica gels by pumping an ethanolic solution of a silylating reagent, such as octadecyltrimethoxysilane, 3-aminopro-pyltrimethoxysilane and dimethyloctadecyltrimethoxysilyl-propylammonium chloride into a heated capillary packed with bare silica particles. The silylation reactions were completed in a short time and columns so prepared showed high column efficiency and high reproducibility.
Examples are shown for the separation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldopentoses on a 3-aminopropylated silica column (Fig. 4) and ben-zoate homologues as well as PMP derivatives of the com-ponent monosaccharides of glycoproteins on an octade-cylammonium column (Fig. 5). The advantage of in-column derivatization is simultaneous introduction of ionic groups to both packing materials and capillary inner wall.
In this paper, the authors fixed the bed of modified silica gel particles to the capillary inner wall by a cross-linking technique to solve the drawbackof frit making. The sepa-ration of dimethylphthalate and thiourea was rather good as compared to that on a column with frits.
Figure 4. Comparison of the separation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldopentose isomer on various columns. Columns prepared (a) by in-column 3-aminopropylation of Nucleosil silica gel; (b) by packing a commercial sample of amino silica, Develosil NH2, in an APTMS-treated capillary; (c) by packing Develosil-NH2 in an uncoated capillary. Eluent, (25 mM HEPES-NaOH, pH 6.0)-acetonitrile (2:1 v/v); sample concentration, 50 nmol in 100 mL of eluent; injection,
±2 kV for 3 s (from the cathodic end); applied voltage, ±20 kV; detection, UV absorption at 245 nm.
Peaks: Ara,D-arabinose; Xyl,D-xylose; Rib,D-ribose; Lyx,D-lyxose, all as PMP derivatives. Reprinted from [99], with permission.
Silica particles coated with a mixture of sulfonic acid groups or amino groups and alkyl chain moieties were also reported [85, 100]. The alkyl chains with sulfonic acid groups at low pH will give cathodal EOF, while those with amino groups at low pH will give anodal EOF. Huang et al. [100] used a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized CEC (PCEC) with an ion trap storage/reflection time-of-flight mass spectrometer detector. PCEC is a novel analytical method in which both pressure and electric field are applied to a packed capillary to achieve separation of analytes [101]. PCEC combines various aspects of CEC and LC. In PCEC, an EOF caused by the voltage is superimposed on a pressure-induced hydrodynamic flow.
Therefore, the separation efficiency is intermediate be-tween that of pure CEC and LC. The major advantages over pure CEC include the stability of the mobile phase flow due to bubble suppression, the increased speed of separation, and the enhanced selectivity for charged par-ticles. The macrocyclic antibiotics vancomycin and teico-planin, widely used as chiral selectors in HPLC, have also been employed in packed CEC [102±104].
3.1.2 Packing methods
Several protocols have been reported for the fabrication of packed capillary columns for CEC. Colon et al. [105]
reported that one can still consider column fabrication in CEC as an art. As in HPLC, variables affecting column packing include quality of packing material, slurry compo-sition, packing procedure, velocity at which particles
arrive to the accumulating bed, and the characteristics of the tube to be packed. A reliable and reproducible column performance depends on the column fabrication. The fab-rication of a typical packed column for CEC is schemati-cally presented in Fig. 6 [105].
Columns that have been packed poorly can lead to low efficiency, poor resolution and asymmetric peakshapes.
Packing CEC columns is really a skill that requires experi-ence. Five different packing methods are discussed by Colon and co-workers [105] to deliver packing material into the capillary column: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using cen-tripetal forces, and packing by gravity. Entrapment of par-ticulate material by sintering and sol-gel technology is also mentioned. The efficiency of columns packed by the different procedures mentioned above varies considera-bly. Colon et al. [105] reported that electrokinetic packing is the simplest and easiest method to implement and use.
In general, the preference of which method to use seems to depend on the familiarity of a particular procedure in a given laboratory.
The performance of bed-retention frits in CEC is likely the most important experimental parameter influencing bub-ble formation. A variety of approaches to frit preparation have been reported [67, 73, 106, 107]. However, good quality frits can be difficult to achieve, especially when small particles (i.e.
<
3 mm particle size) are used.Recently, Cassidy and co-workers [106] reported the preparation of porous silica frits via spot-heating of a sili-Figure 5. Comparison of the separation of alkylbenzoate homologues on (a) an octade-cylammonium column and (b) an ODS column. The columns were prepared by in-column derivatization of DaisoGel silica (30 nm, 3 mm) with dimethyl- octadecyltrimethoxysilylpropy-lammonium chloride. Reprin-ted from [99], with permission.
cate solution, and the effects of several experimental parameters on their performance. Zare and co-workers [107] reported that macroporous polymer frits can be fab-ricated in fused-silica capillaries by the UV photopolymeri-zation of a solution of glycidyl methacrylate and trimethyl-olpropane trimethacrylate. This in situ preparation is a simple, rapid, and reproducible process.