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Recent trends in open-tubular capillary electrochromatography
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Recent trends in open-tubular capillary electrochromatography Kiyokatsu Jinno*
School of Materials Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan
Hirokazu Sawada
Yokogawa Analytical Systems, Inc., Musashino 180-8543, Japan Capillary electrochromatography ( CEC ), which combines the advantages of the high ef¢ciency 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 Wm particle diameter ). However, problems such as dif¢culties 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. z2000 Elsevier Science B.V. All rights reserved. Keywords: CEC; Capillary electrochromatography; Open-tubular column; Review
1. Introduction Capillary electrochromatography ( CEC ), in which the mobile phase is moved by electroosmotic £ow ( EOF ) on the surface of the packing materials or inner wall of the capillary columns instead of by a high pressure pumping system, has gained considerable attention in recent years. In 1939 Strain ¢rst examined the CEC separation of dyes by charging the voltage on both ends of the column in column chromatography [ 1 ], and then in 1974 Pretorius et al. reported the possibility of reducing band broadening by using the plug £ow *Corresponding author. Tel.: +81 (532) 47-0111; Fax: +81 (532) 47-0111. E-mail: [email protected]
pro¢le produced by EOF instead of the parabolic £ow pro¢le characteristic of pressure driven systems. They utilized both an open-tubular column and a packed column with 1 mm i.d. using nonretained solutes as a probe [ 2 ]. In the 1980's Jorgenson [ 3 ] and Knox [ 4 ] used the mobile phase £ow resulting from EOF for the separation of aromatic compounds with columns containing LC ( liquid chromatography ) reversed-phase packing materials. Since then many scientists have used the socalled CEC method for liquid phase [ 5^10 ]. CEC combines the advantages of capillary electrophoresis ( CE ) and microcolumn liquid chromatography ( micro-LC ) with regard to ef¢ciency and selectivity. The most attractive aspect of CEC is that one can use the various types of packing materials that have been developed for LC separations in order to get different selectivities and therefore it can offer high resolution, highly selective separations. Since plug £ow improves the separation ef¢ciency, one can get much higher theoretical plate numbers in CEC than those obtained by LC separations. Also, there are no effects due to pressure loss. The latter means that for the stationary phase one can use very small diameter particles, which are not useful in high pressure systems. In CEC, fused-silica capillary columns with 50^ 100 Wm i.d. have mostly been used and LC stationary phases with 1.5^5 Wm diameter particles ( generally silica-based materials ) are packed inside the capillaries. In packed-column CEC there are some major practical problems such as the dif¢culties of packing the small LC stationary phase particles in a narrow bore capillary and fabricating the frit. Fabrication of frits is an important issue in packed-column CEC and a review on this aspect has been published [ 11 ]. In addition, pressurization of both ends of the column is required to prevent bubble formation inside the packed capillary column. This
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The ¢rst report on using open-tubular CEC (OTCEC ) was by Tsuda et al. in 1982 [ 12 ], where they prepared an octadecylsilica modi¢ed inner wall of a 30 Wm i.d. soda glass capillary by a procedure involving pretreatment by 0.3 M NaOH followed by £owing octadecylethoxysilane toluene solution. They succeeded in separating some aromatic compounds by CEC with an acetonitrile and water mobile phase. Since then several different methods to prepare open-tubular columns for CEC have been developed.
enough mobile phase £ow for CEC separation. A cationic surfactant ( cetyltrimethylammonium bromide, CTAB ) was added to the buffer solution at WM concentration. Since added CTAB can adsorb on the hydrophobic surface of the polymer-coated inner wall, one can get a well-regulated osmotic £ow that is suitable for CEC. They obtained a baseline separation of several PAHs ( polycyclic aromatic hydrocarbons ) mixture using a 100 cm long polymer-coated column, 100 WM CTAB and 40% acetonitrile / citric acid buffer, at an applied voltage of 40 kV. In this case the polymer coating acts as the stationary phase, although an uncoated column did not separate the PAHs mixture. The theoretical plate number was 290 000 for the anthracene peak with a retention factor of 0.34, i.e. the HETP is 3.4 Wm. To obtain such an HETP value with a pressurized mobile phase £ow, a column inner diameter of 4.4 Wm would be required, which is almost impossible with present technology. This clearly indicated that CEC was a promising approach for achieving higher resolution and better retentivity in a shorter analysis time than open-tubular column LC separations (OTLC ). They also found that CTAB adsorbed on the inner wall of the capillary column works as an ion-exchange site and separation based on an ion-exchange mechanism in CEC was established. In addition to the existing reversed-phase separation mechanism, an ion-exchange mechanism would also be appropriate for ionic species and indicates that one can control the selectivity by changing various parameters such as salt concentration of the buffer solution, the concentration of organic modi¢ers or of CTAB in the buffer solution [ 14,15 ]. Liu et al. prepared a column with an inner wall covered by a di-molecular layer of CTAB produced by £owing 20 mM CTAB solution followed by phosphate buffer without CTAB [ 16 ]. This column ( 40 cm long, 10 Wm i.d. ) could separate ¢ve alkylbenzenes in 6 min using pH 7.2 phosphate buffer without CTAB as the mobile phase by applying 25 kV voltage.
2.1. Cationic surfactants
2.2. Porous silica layers
Yeung's group examined OTCEC using a 10 Wm i.d. polyvinylsiloxane polymer-coated capillary and cationic surfactant [ 13 ]. In this type of column the silanol groups in the inner wall surface are blocked by the polymer coating and EOF is suppressed. The reduced EOF does not produce
OTLC with a column on which the inner surface is modi¢ed by ligands cannot give enough retention due to the small phase ratio. Poppe et al. prepared a 10 Wm i.d. open-tubular column with porous silica layers on the inner wall for OTLC [ 48,49 ] and evaluated its performance in CEC [ 17 ]. A fused-silica
Fig. 1. Classi¢cation of columns for electrochromatography by the packing state: ( A ) open-tubular column; ( B ) LC stationary phase-packed column; ( C ) monolithic column.
may complicate the CEC instrument, and makes it dif¢cult to couple directly to useful detection devices such as a mass spectrometer (MS ). There are now three different types of columns available, as shown in Fig. 1. In addition to packed columns, there are open-tubular columns of which the inner surface is modi¢ed by ligands or coatings [ 12^38 ], and monolithic columns made by on-column copolymerization of various monomers which produce gels [ 39^47 ]. These latter columns do not require the tedious packing procedures and frit fabrication, and therefore seem very promising in CEC applications. It is apparent that open-tubular columns are a viable alternative to packed columns in CEC.
2. Preparation of open-tubular columns in CEC
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Fig. 2. Separation of PAHs by OTCEC in two different capillaries: ( A ) capillary coated with a C8-TES /TEOS ratio of 0.2; ( B ) capillary î V50 cm ( effective length ); mobile phase, methanol / 1 mM phosphate coated with TEOS only. Conditions: column, 13 Wm i.d. A buffer ( 67:33 ); applied voltage, 30 kV; injection, 30 kV for 1 s from anode; detection, UV 220 nm. Peak identi¢cation: ( 1 ) naphthalene, ( 2 ) phenanthrene, and ( 3 ) pyrene. ( From [ 18 ], with permission from the American Chemical Society. )
capillary was treated by £owing 1 M NaOH for 2 h at room temperature, and then washed by distilled water, 0.01 M HCl, and again distilled water. The column was then maintained at 200³C for 2 h under a He gas £ow. The production of the stable porous silica layer on the inner wall was performed by precipitation of silica from polyethoxysiloxane (PES ) solution which had been prepared by the elimination of ethanol and HCl under reduced pressure after the polycondensation of tetraethoxysilane (TEOS ) by adding water containing a small amount of HCl for hydrolysis. The column was then ¢lled with pentane / monochloromethane or hexane / dichloromethane solution of PES, and the solvents were eliminated by reducing the pressure.
The remaining PES on the column's inner surface was treated by ammonia for 1 h, and then washed by distilled water for 2 h to convert it to silica, resulting in a porous silica layer. Adding 5% monochlorooctadecylsilane toluene solution to the column under 140³C for 6 h gave an open-tubular column with the inner surface coated by a porous silica layer with hydrophobic ligands. They separated eight PAHs in 7 min using a 10 Wm i.d.U49 cm length column with methanol / 50 mM phosphate buffer=50 / 50 mobile phase by laser-induced £uorescence detection. This success did not generate much popularity for this type of column as the several tedious procedures to prepare the column make the process time-consuming and the useful
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Fig. 3. SEM ( scanning electron micrograph ) picture of the inner wall of a capillary etched with ammonium hydrogen di£uoride at 300³C for 4 h. ( From [ 22 ], with permission from Elsevier Science. )
pH range is limited by the instability of the hydrophobic ligands. Colon's group applied a sol-gel method for the preparation of open-tubular columns with a porous silica-coated surface [ 18,19 ]. In order to increase the number of silanol groups on the inner surface of a 13 Wm i.d. fused-silica capillary, 1 M NaOH was passed through the column, which was then washed with distilled water and dried with nitrogen gas. The sol-gel solution was made by adding a small amount of HCl as catalyst to a mixture of TEOS, n-octyltriethoxysilane ( C8-TES ), ethanol and water. The solution was stirred for 6 h at room temperature and then it was used to ¢ll the capillary column. Excess solution was eliminated from the column under pressure and it was then dried under nitrogen gas £ow at 120³C for a night. This procedure is much easier than that of Poppe et al. [ 48,49 ].
In Fig. 2 a typical separation of PAHs with this column is demonstrated, where A shows the separation by a TEOS / C8-TES-coated column and B was obtained using a TEOS column [ 18 ]. It is clear that the octyl group included in the porous silica layer is the stationary phase in the separation of the PAHs. They compared the performance of the direct octyl modi¢ed open-tubular capillary column with this column and found that the proposed sol-gel method is an ef¢cient approach that enhances the phase ratio and increases the surface area. By changing the concentration of C8-TES in the solution when the column is prepared, one can control the concentration of octyl ligands on the inner surface. The Si^C bond between the ligand and the surface produces better stability than that of the siloxane bond. Colon et al. extended their work to produce a £uorine-containing ligand by using trideca£uoro-1,1,2,2-tetrahydrooctyl-1-triethoxysi-
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lane. It had a unique selectivity for £uorine- and halogen-containing compounds [ 20 ].
2.3. Inner wall etched columns Pesek et al. have evaluated OTCEC using etched capillary columns. In order to increase the surface area of the 50 Wm i.d. fused-silica capillary column, they used ammonium hydrogen di£uoride as the etchant [ 21^26 ]. In Fig. 3 the SEM ( scanning electron microprobe ) image of the inner surface after the etching process is shown. The increase of the surface area, about 1000 times larger than that of the non-etched surface, is clearly indicated. They then treated the surface by TES and made Si^H groups on the inner wall of the capillary. Then an ole¢ns solution with hexachloroplatinomic acid as catalyst was £ushed through the column at 100³C for 45 h ( a hydroxylation process ). As the ligand is ¢xed to the surface by a Si^C bond instead of a common siloxane bond, the column stability is superior to usual open-tubular columns. They evaluated an octadecyl bonded capillary for the separation of proteins and peptides [ 21,22,24,25 ]. They also tried to bond diol groups on the Si^H surface for peptide separation, and performed three angiotensin separations using two columns, diol modi¢ed and C18 modi¢ed etched capillaries [ 24,25 ]. The selectivity difference between the diol and the C18 modi¢ed columns is very clear and this result shows the existence of the different interactions between the solutes and two ligands bonded on the inner surface of the etched capillaries. Pesek et al. also tried to use their open-tubular capillaries for the separation of basic compounds, which gave a large peak tailing due to the interaction between silanol groups and the solutes with the packed column [ 22,23,25 ]. Since the Si^H capillary does not have so many remaining silanol groups on the surface, it gave much better peak shape for the basic solutes than the bare capillary. The bonded ligand produced the better selectivity, based on the interactions between the solutes and the ligand. Catabay et al. used the same process of the column inner surface etching and ligand bonding for cholesteryl-10undecenoate phase using 50 and 75 Wm inner diameter columns. They achieved separations based on the interaction between benzodiazepines and the cholesteryl phase with etched, modi¢ed capillary but bare and etched, unmodi¢ed capillaries did not give a good separation for those drug compounds [ 26 ]. Most recently Pesek et al. reported
the use of open-tubular capillary columns with chemically bonded liquid crystals such as 4cyano-4P-n-pentoxybiphenyl and cholesteryl-10undecenoate in CEC [ 27 ]. Separation of metabolic components of serotonin on a 50 Wm i.d. etched cyanopentoxy modi¢ed capillary is shown in Fig. 4.
2.4. Polymer-coated columns Polymer-coated open-tubular columns prepared by thermal polymerization of methacrylate monomers were reported by Tan et al. [ 28 ]. The inner surface of the column was etched with NaOH and diluted HCl, and difunctional silane reagent ( 3-( trimethoxysilyl )propyl-methacrylate ) was used for the pretreatment. The capillary was ¢lled with a toluene solution of butylmethacrylate monomers and the polymerization initiator. The polymerization process took 10 min at 120³C. At this time the inner surface was coated by the linear polymers. Due to the inef¢cient coverage of the inner surface, the EOF ( which was lower than that obtained by a bare capillary ) drove the mobile phase by the remaining silanol groups and some polymer layer contribution. The effect of the monomer concentration on the separation performance of such columns was examined and they found a critical concentration that gave the maximum retention for parabens. The best retention was obtained under the following conditions: initial monomer concentration; 45%, mobile phase; acetonitrile /phosphate buffer=20 / 80. Butylparaben gave a retention of 0.55 and non-retained acetone gave 270 000 plate numbers / m. When they prepared the column, they added cross-linking agents and investigated their effect on the separation [ 29 ]. At higher than 20% monomer concentration, the viscosity of the polymer solution was increased and therefore it was extremely dif¢cult to eliminate the remaining unbonded polymeric materials in the capillary column. The degradation of the column with very high acetonitrile concentration in the buffer mobile phase was not remarkable and the addition of the cross-linking agents reduced EOF, which clearly indicated that the cross-linked polymer covered the surface much better than the linear polymer coating. They compared the performance of such columns in OTCEC and OTLC and found that CEC gave much higher ef¢ciency, for example 200 000 plates / m for propylparaben with OTLC and 380 000 plates / m with OTCEC, respectively.
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Fig. 4. Separation of metabolic components of serotonin on a 50 Wm etched cyanopentoxy modi¢ed capillary column. Conditions: V=25 kV, mobile phase 100% aqueous pH 2.14 buffer ( 60 mM phosphoric acid and 38 mM Tris ), L=50 cm, and I=25 cm. ( From [ 27 ], with permission from the American Chemical Society. )
Another approach in CE utilizing polymer coating to counteract solutes adsorption on the inner surface, and eliminate or enhance the EOF has been examined [ 50^52 ]. In these important works Kohr and Engelhardt [ 50 ], Lee et al. [ 51 ] and Hartwick et al. [ 52 ] reported that the coating of the copolymer of anionic monomer, with a side chain containing a sulfonic acid group and acrylamide, gave a stable, constant EOF under a wide pH range. Based on such approach Sawada and Jinno prepared a 25 Wm i.d. capillary column in which the surface was coated by a copolymer of N-alkylacrylamide and anionic monomer [ 30 ]. EOF is obtained by the sulfonyl group in the linear copolymer and the hydrophobic alkyl chains in the polymer structure act as the stationary phase. Neutral ketones, parabens and PAHs, which are dif¢cult to separate by CE, could be separated by CEC. By substituting the alkyl chains with other functional groups, one can control or enhance the selectivity of chromatographic separations and the column stability can be maintained by the linear polymer coating on the inner wall. This method will be a useful technique in open-tubular CEC in the near future, but requires further enhancement of selectivity and tail-
ored phases for solving particular separation problems.
2.5. Columns for chiral isomer separations 2.5.1. Immobilized cyclodextrin columns There are many publications on chiral separations with CE such as MECC using chiral micelles, cyclodextrin modi¢ed micelles ( CD-MECC ) and immobilized CDs on capillary columns packed with polyacrylamide gel. Schurig reported CEC separation of chiral isomers using a polysiloxane polymer-coated capillary column containing permethylated-L-CD ( Chirasil-Dex column ) [ 31 ]. They immobilized the polysiloxane polymer on the 50 Wm capillary inner surface using the method that they had developed for chiral separations with supercritical £uid chromatography (SFC ) [ 53 ]. Successful results were seen in their second publication [ 32 ] where they separated antisteroid drugs with 20 mM borate / phosphate buffer ( pH 7.0 ) mobile phase. They then compared the performance of OTLC and OTCEC for pharmaceutical chiral separations and found that OTCEC gave better plate numbers and separation factors [ 33 ]. However,
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some charged drugs required longer analysis time than with OTLC due to the reduction of EOF. Armstrong et al. also reported the separation of chiral barbiturates using a derivatized CD immobilized capillary column [ 34 ]. On the other hand, Francotte et al. examined the chiral separations with a cellulose derivatives-coated capillary column [ 35 ]. Their columns were prepared by coating 3,5-dimethylphenylcarbamoyl cellulose ( DMPCC ) or pmethylbenzoylcellulose (PMBC ) on 50 Wm i.d. capillary. They also compared the performance of OTLC and OTCEC and found CEC to be better, but the column stability and its lifetime are problems in practice. The instability is due to the cellulose coatings not being immobilized on the inner wall of the capillary column.
2.5.2. Molecular imprinting polymers (MIPs ) MIPs can be prepared by the polymerization of functional monomers with cross-linking agents in the presence of template molecules. After the polymerization the template molecules are extracted from the polymeric materials in order to produce the speci¢c sites in the polymer structure. MIPs have been extensively applied to chromatographic stationary phases, particularly for selective separations, such as chiral separation. Tan et al. [ 36 ] developed a 25 Wm i.d. capillary column with its inner wall coated by MIPs and applied it in chiral separations with OTLC and OTCEC. They used Ldansylphenylalanine as the template with functional monomers of methacrylates and 2-vinylpridine, and cross-linking agents. The reaction time for polymerization is critical for better column performance. They tried to separate D- and L-dansylphenylalanines by CEC using an 85 cm effective length column with acetonitrile / 10 mM phosphate buffer at pH 7.0 and non-retained D-alanine gave 250 000 theoretical plates / m but retained L-alanine gave only 8000 plates / m, respectively. 2.5.3. Af¢nity electrochromatography Bovine serum albumin ( BSA ), K-acid glycoprotein( orosomucoid ) and ovomucoid immobilized stationary phases are very popular in LC chiral separations and they are also useful in CE as af¢nity EKC for chiral separations [ 54^56 ]. In this type of separation there were several problems such as adsorption of chiral selectors on the inner wall surface, their absorption in the UV region and low column ef¢ciency. Recently there have been attempts to apply af¢nity CEC for chiral separations with pro-
Fig. 5. Chromatograms of a ¢ve-component mixture obtained with a 65 Wm i.d. octylaminated PEEK column. Conditions: separation length 29.2 cm; detection capillary, 75 Wm i.d.U30.4 cm; no sample introduction capillary was used; mobile phase acetonitrile / 2.5 mM phosphate buffer ( pH=6.9 ) ( v / v )=50 / 50; voltage, 15 kV; current, 2 WA; detection, 215 nm. Solutes: 1, thiourea; 2, benzyl alcohol; 3, benzaldehyde; 4, benzene; 5, toluene. ( From [ 38 ], with permission from Wiley Interscience. )
tein immobilized silica stationary phase [ 57,58 ]. Schurig et al. prepared BSA bonded capillary column and examined af¢nity OTEC ( open-tubular electrokinetic chromatography ) for various dinitrophenylamino ( DNP ) acids and 3-hydroxy-1,4-benzodiazepines [ 37 ]. The preparation procedure is as follows: ¢rstly a 50 Wm i.d. column inner surface is treated with Q-glycidoxypropyltrimethoxysilane and then HCl to hydrolyze epoxides to diol groups, followed by activation by tresyl chloride, and ¢nally the capillary is ¢lled with BSA at 4³C for 24 h. Reduction of EOF was found for the BSA bonded capillary column compared to a non-processed capillary from 6 min to 20 min. They succeeded in separating DNP-alanine, glutamic acid, proline and phenylalanine in baseline separation with phosphate buffers of pH 6.0 and 8.0. Liu at al. also produced a novel column preparation method for OTCEC [ 16 ]. The adsorbed stationary phases were prepared easily by rinsing the capillary with a buffer containing cationic surfactant such as CTAB or basic protein such as lysozyme. In the latter, the adsorbed lysozyme layer was used as a chiral stationary phase for separation of enantiomers. Four
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amino acids and the drug mephenytoin were separated within 5 min with a resolution from 1.74 to 2.05. The merit of this type of CEC separations is the reduction in the required amounts of stationary phase.
2.6. Polymer tubings Fused-silica capillary is the most useful and popular column medium in CEC but the use of polymer tubings have also been examined [ 59^61 ]. Hydrophobic groups were bonded on the inner wall surface of PEEK ( polyether ether ketone ) tube [ 38 ]. The PEEK inner surface was sulfonated by 1,1,2,2tetrachloroethane solution of chlorosulfonic acid. This treatment increased the column surface area and thus improved the phase ratio and, at the same time, activated the surface for the subsequent reaction with alkylamines. Capillary columns prepared in this manner showed retention characteristics similar to reversed-phase LC. As seen in Fig. 5, this column works well in CEC and the results indi-
cated that potential applications of the PEEK column might be found in separations where high pH mobile phases are needed.
3. Combination with mass spectrometry Since mass spectrometry (MS ) is very useful for peak identi¢cation in GC, LC and SFC, GC^MS and LC^MS are now established and used in many different analytical areas. CE^MS is also a very promising approach and ESI ( electrospray ionization ) is the most popular ionization technique [ 62^64 ]. There are some publications on CEC^MS [ 65^73 ]. To reduce the air bubble problem in packed-column CEC, the common approach is to send the mobile phase with EOF and pressurized £ow by a typical LC pumping system, which is so-called pseudo-electrochromatography [ 65^69 ]. A CEC^ MS approach without an LC pump was also investigated by several groups [ 70^73 ]. In this pseudoCEC separation method ionic species have a differ-
Fig. 6. ( a ) Schematic of the microchip with a serpentine column geometry. ( b ) Diagram of the high voltage switching apparatus and detection / data acquisition system. ( From [ 80 ], with permission from the American Chemical Society. )
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Fig. 7. Fast open-channel electrochromatography on microchips. Channel depth, 5.2 Wm; stationary phase, octadecylsilane; mobile phase, 10 mM borate buffer ( pH 8.4 ) with a linear gradient of acetonitrile from 29% to 50% within 5 s, starting 1 s after injection; ¢eld strength, 700 V / cm; dotted line, gradient trace; analytes, coumarin dyes, ( 1 ) C440, ( 2 ) C450, ( 3 ) C460, ( 4 ) C480. ( From [ 81 ], with permission from the American Chemical Society. )
ent selectivity than with a pressurized mobile phase £ow system. As bubble formation is not a problem in OTCEC, this method combined with MS (OTCEC^MS ) would be a much easier approach than packed-column CEC^MS. The low sensitivity in UV detection due to a shorter pathlength of the narrow column diameter used in OTCEC can be solved by using the OTCEC^MS approach, especially the ESI-MS approach. Lubman's group reported the very fast separation of a peptide mixture by OTCEC^MS [ 39 ]. They prepared a 9 Wm i.d. open-tubular column using Colon's method [ 18 ] to make octyl phase on the column inner wall surface. After the preparation they treated the column with £owing
toluene solution of 5% ( v / v ) 3-aminopropyltriethoxysilane for 6 h and obtained a reversed osmotic £ow compared to the usual column. The isocratic eluent used was a mixture of 20% ( v / v ) acetonitrile and 5 mM ammonium acetate+0.05% tri£uoroacetic acid. In CEC^MS one end of the column was immersed in the buffer with negative charge, and the other end was used as the nanospray needle directly connected to the MS where the polyimide coating of the capillary column was eliminated and etched to a sharp head by HF. This edge was silver coated. They used an ESI-ion-trap / TOF ( time of £ight )-MS. The continuous ion beam produced by ESI was converted to pulsed ions in TOF-MS by using the ion-trap for the storage of
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ions. Six peptides were separated in 3 min by using a 25 cm length column at 12 kV voltage. Detection at the fmol level is possible. CEC^ESI-MS interfacings have been investigated by using quadrupole MS mainly, although this type of MS sacri¢ces the high resolution and faster separation of CE due to the relatively low scanning speed [ 63 ]. Lubman's group showed the usefulness of ion-trap /TOF-MS for fast and highly sensitive detection in CE [ 74,75 ]. They also developed a device to induce gradient elution in CEC^MS [ 39 ]. It can be concluded that CEC^MS will be a most useful analytical hyphenated technique that offers high separation power, high sensitivity and enough qualitative information.
4. Microchip technology The most recent trend in capillary separation sciences is the use of microchip technology to create the medium for the separation. In semiconductor engineering modern lithographic techniques can create patterns of electric circuits less than a micron and these techniques can be applied in the separation sciences. There are several advantages to using these miniaturized separation channels for CEC: ( 1 ) EOF can drive the £ow of the mobile phase which is very effective as it is based on the charge supply without the use of valves and pumps, ( 2 ) band broadening can be avoided as the separation by EOF is by plug £ow, ( 3 ) the ef¢ciency depends on the charge applied and is independent on the channel length in the ideal case and therefore very fast and high ef¢ciency separation can be performed using a high applied charge with the very short separation channels. Another additional but important feature of this chip type separation medium is the possibility of making a total analytical system on a chip including sample preparation, separation, reaction and detection, the so-called micro-total analysis system W-TAS. In many works published on W-TAS, CZE was the separation mode of choice, because electrophoretic mobility allows the easiest control of the separation. However, in order to extend the capability of chip type separation methods one needs to use other separation modes, such as MECC [ 76^79 ]. Ramsey's group evaluated CEC separations on a microchip in which the separation channels were coated by octadecyl chains [ 80 ]. The separation channels, 5.6 Wm deep and 66 Wm wide at half-
depth, were made by photolithography / chemical etching. Laser-induced £uorescence detection was used. The basic structure is shown in Fig. 6. The separation channels have a serpentine structure and this allows the production of very long channels on a small area ( in this case 8 mmU8 mm ). After adhering the cover glass to the chip, the coating was performed with ¢rst a £ow of 1 M NaOH and then a wash with water. Then He gas was passed through for 24 h at 125³C. After that toluene solution of 25% ( v / v ) chlorodimethyloctadecylsilane was passed through the channels by He gas for 18 h at 125³C and octadecyl chains formed on the inner surface of the channels. Using this chip they had separated three neutral dyes in 200 s and the theoretical plate height for non-retained solute was 4.1 Wm and retained solute 5.0 Wm, respectively. They also applied gradient elution [ 81 ] on the chip and Fig. 7 demonstrates the beautiful fast separation in the order of seconds. This technology appears to have a bright future for separation techniques, and even for MECC and CEC [ 82^89 ].
5. Conclusion The recent trends in open-tubular CEC (OTCEC ) have been reviewed. The many advantages of the OTCEC approach compared to packed-column CEC show a promising future for this technology in many applications. The most important point in the development of OTCEC is to produce the driving force to expand the applicability of CEC separation techniques in many ¢elds where other typical chromatographic techniques are not amenable to solving the problems.
References [ 1 ] H.H. Strain, J. Am. Chem. Soc. 61 ( 1939 ) 1292. [ 2 ] V. Pretorius, B.J. Hopkins, J.D. Schieke, J. Chromatogr. 99 ( 1974 ) 23. [ 3 ] J.W. Jorgenson, K.D. Lukacs, J. Chromatogr. 218 ( 1981 ) 209. [ 4 ] J.H. Knox, I.H. Grant, Chromatographia 24 ( 1987 ) 135. [ 5 ] M.M. Dittmann, K. Wienand, F. Bek, G.P. Rozing, LC GC 13 ( 1995 ) 800. [ 6 ] L.A. Colon, Y. Guo, A. Fermier, Anal. Chem. 69 ( 1997 ) 461A. [ 7 ] M.M. Dittmann, G.P. Rozing, J. Microcol. Sep. 9 ( 1997 ) 399. [ 8 ] M.M. Robson, M.G. Cikalo, P. Myers, M.R. Euerby, K.D. Bartle, J. Microcol. Sep. 9 ( 1997 ) 357.
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Recent trends in open-tubular capillary electrochromatography Kiyokatsu Jinno*
School of Materials Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan
Hirokazu Sawada
Yokogawa Analytical Systems, Inc., Musashino 180-8543, Japan Capillary electrochromatography ( CEC ), which combines the advantages of the high ef¢ciency 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 Wm particle diameter ). However, problems such as dif¢culties 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. z2000 Elsevier Science B.V. All rights reserved. Keywords: CEC; Capillary electrochromatography; Open-tubular column; Review
1. Introduction Capillary electrochromatography ( CEC ), in which the mobile phase is moved by electroosmotic £ow ( EOF ) on the surface of the packing materials or inner wall of the capillary columns instead of by a high pressure pumping system, has gained considerable attention in recent years. In 1939 Strain ¢rst examined the CEC separation of dyes by charging the voltage on both ends of the column in column chromatography [ 1 ], and then in 1974 Pretorius et al. reported the possibility of reducing band broadening by using the plug £ow *Corresponding author. Tel.: +81 (532) 47-0111; Fax: +81 (532) 47-0111. E-mail: [email protected]
pro¢le produced by EOF instead of the parabolic £ow pro¢le characteristic of pressure driven systems. They utilized both an open-tubular column and a packed column with 1 mm i.d. using nonretained solutes as a probe [ 2 ]. In the 1980's Jorgenson [ 3 ] and Knox [ 4 ] used the mobile phase £ow resulting from EOF for the separation of aromatic compounds with columns containing LC ( liquid chromatography ) reversed-phase packing materials. Since then many scientists have used the socalled CEC method for liquid phase [ 5^10 ]. CEC combines the advantages of capillary electrophoresis ( CE ) and microcolumn liquid chromatography ( micro-LC ) with regard to ef¢ciency and selectivity. The most attractive aspect of CEC is that one can use the various types of packing materials that have been developed for LC separations in order to get different selectivities and therefore it can offer high resolution, highly selective separations. Since plug £ow improves the separation ef¢ciency, one can get much higher theoretical plate numbers in CEC than those obtained by LC separations. Also, there are no effects due to pressure loss. The latter means that for the stationary phase one can use very small diameter particles, which are not useful in high pressure systems. In CEC, fused-silica capillary columns with 50^ 100 Wm i.d. have mostly been used and LC stationary phases with 1.5^5 Wm diameter particles ( generally silica-based materials ) are packed inside the capillaries. In packed-column CEC there are some major practical problems such as the dif¢culties of packing the small LC stationary phase particles in a narrow bore capillary and fabricating the frit. Fabrication of frits is an important issue in packed-column CEC and a review on this aspect has been published [ 11 ]. In addition, pressurization of both ends of the column is required to prevent bubble formation inside the packed capillary column. This
0165-9936/00/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 0 0 ) 0 0 0 5 0 - 9
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The ¢rst report on using open-tubular CEC (OTCEC ) was by Tsuda et al. in 1982 [ 12 ], where they prepared an octadecylsilica modi¢ed inner wall of a 30 Wm i.d. soda glass capillary by a procedure involving pretreatment by 0.3 M NaOH followed by £owing octadecylethoxysilane toluene solution. They succeeded in separating some aromatic compounds by CEC with an acetonitrile and water mobile phase. Since then several different methods to prepare open-tubular columns for CEC have been developed.
enough mobile phase £ow for CEC separation. A cationic surfactant ( cetyltrimethylammonium bromide, CTAB ) was added to the buffer solution at WM concentration. Since added CTAB can adsorb on the hydrophobic surface of the polymer-coated inner wall, one can get a well-regulated osmotic £ow that is suitable for CEC. They obtained a baseline separation of several PAHs ( polycyclic aromatic hydrocarbons ) mixture using a 100 cm long polymer-coated column, 100 WM CTAB and 40% acetonitrile / citric acid buffer, at an applied voltage of 40 kV. In this case the polymer coating acts as the stationary phase, although an uncoated column did not separate the PAHs mixture. The theoretical plate number was 290 000 for the anthracene peak with a retention factor of 0.34, i.e. the HETP is 3.4 Wm. To obtain such an HETP value with a pressurized mobile phase £ow, a column inner diameter of 4.4 Wm would be required, which is almost impossible with present technology. This clearly indicated that CEC was a promising approach for achieving higher resolution and better retentivity in a shorter analysis time than open-tubular column LC separations (OTLC ). They also found that CTAB adsorbed on the inner wall of the capillary column works as an ion-exchange site and separation based on an ion-exchange mechanism in CEC was established. In addition to the existing reversed-phase separation mechanism, an ion-exchange mechanism would also be appropriate for ionic species and indicates that one can control the selectivity by changing various parameters such as salt concentration of the buffer solution, the concentration of organic modi¢ers or of CTAB in the buffer solution [ 14,15 ]. Liu et al. prepared a column with an inner wall covered by a di-molecular layer of CTAB produced by £owing 20 mM CTAB solution followed by phosphate buffer without CTAB [ 16 ]. This column ( 40 cm long, 10 Wm i.d. ) could separate ¢ve alkylbenzenes in 6 min using pH 7.2 phosphate buffer without CTAB as the mobile phase by applying 25 kV voltage.
2.1. Cationic surfactants
2.2. Porous silica layers
Yeung's group examined OTCEC using a 10 Wm i.d. polyvinylsiloxane polymer-coated capillary and cationic surfactant [ 13 ]. In this type of column the silanol groups in the inner wall surface are blocked by the polymer coating and EOF is suppressed. The reduced EOF does not produce
OTLC with a column on which the inner surface is modi¢ed by ligands cannot give enough retention due to the small phase ratio. Poppe et al. prepared a 10 Wm i.d. open-tubular column with porous silica layers on the inner wall for OTLC [ 48,49 ] and evaluated its performance in CEC [ 17 ]. A fused-silica
Fig. 1. Classi¢cation of columns for electrochromatography by the packing state: ( A ) open-tubular column; ( B ) LC stationary phase-packed column; ( C ) monolithic column.
may complicate the CEC instrument, and makes it dif¢cult to couple directly to useful detection devices such as a mass spectrometer (MS ). There are now three different types of columns available, as shown in Fig. 1. In addition to packed columns, there are open-tubular columns of which the inner surface is modi¢ed by ligands or coatings [ 12^38 ], and monolithic columns made by on-column copolymerization of various monomers which produce gels [ 39^47 ]. These latter columns do not require the tedious packing procedures and frit fabrication, and therefore seem very promising in CEC applications. It is apparent that open-tubular columns are a viable alternative to packed columns in CEC.
2. Preparation of open-tubular columns in CEC
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Fig. 2. Separation of PAHs by OTCEC in two different capillaries: ( A ) capillary coated with a C8-TES /TEOS ratio of 0.2; ( B ) capillary î V50 cm ( effective length ); mobile phase, methanol / 1 mM phosphate coated with TEOS only. Conditions: column, 13 Wm i.d. A buffer ( 67:33 ); applied voltage, 30 kV; injection, 30 kV for 1 s from anode; detection, UV 220 nm. Peak identi¢cation: ( 1 ) naphthalene, ( 2 ) phenanthrene, and ( 3 ) pyrene. ( From [ 18 ], with permission from the American Chemical Society. )
capillary was treated by £owing 1 M NaOH for 2 h at room temperature, and then washed by distilled water, 0.01 M HCl, and again distilled water. The column was then maintained at 200³C for 2 h under a He gas £ow. The production of the stable porous silica layer on the inner wall was performed by precipitation of silica from polyethoxysiloxane (PES ) solution which had been prepared by the elimination of ethanol and HCl under reduced pressure after the polycondensation of tetraethoxysilane (TEOS ) by adding water containing a small amount of HCl for hydrolysis. The column was then ¢lled with pentane / monochloromethane or hexane / dichloromethane solution of PES, and the solvents were eliminated by reducing the pressure.
The remaining PES on the column's inner surface was treated by ammonia for 1 h, and then washed by distilled water for 2 h to convert it to silica, resulting in a porous silica layer. Adding 5% monochlorooctadecylsilane toluene solution to the column under 140³C for 6 h gave an open-tubular column with the inner surface coated by a porous silica layer with hydrophobic ligands. They separated eight PAHs in 7 min using a 10 Wm i.d.U49 cm length column with methanol / 50 mM phosphate buffer=50 / 50 mobile phase by laser-induced £uorescence detection. This success did not generate much popularity for this type of column as the several tedious procedures to prepare the column make the process time-consuming and the useful
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Fig. 3. SEM ( scanning electron micrograph ) picture of the inner wall of a capillary etched with ammonium hydrogen di£uoride at 300³C for 4 h. ( From [ 22 ], with permission from Elsevier Science. )
pH range is limited by the instability of the hydrophobic ligands. Colon's group applied a sol-gel method for the preparation of open-tubular columns with a porous silica-coated surface [ 18,19 ]. In order to increase the number of silanol groups on the inner surface of a 13 Wm i.d. fused-silica capillary, 1 M NaOH was passed through the column, which was then washed with distilled water and dried with nitrogen gas. The sol-gel solution was made by adding a small amount of HCl as catalyst to a mixture of TEOS, n-octyltriethoxysilane ( C8-TES ), ethanol and water. The solution was stirred for 6 h at room temperature and then it was used to ¢ll the capillary column. Excess solution was eliminated from the column under pressure and it was then dried under nitrogen gas £ow at 120³C for a night. This procedure is much easier than that of Poppe et al. [ 48,49 ].
In Fig. 2 a typical separation of PAHs with this column is demonstrated, where A shows the separation by a TEOS / C8-TES-coated column and B was obtained using a TEOS column [ 18 ]. It is clear that the octyl group included in the porous silica layer is the stationary phase in the separation of the PAHs. They compared the performance of the direct octyl modi¢ed open-tubular capillary column with this column and found that the proposed sol-gel method is an ef¢cient approach that enhances the phase ratio and increases the surface area. By changing the concentration of C8-TES in the solution when the column is prepared, one can control the concentration of octyl ligands on the inner surface. The Si^C bond between the ligand and the surface produces better stability than that of the siloxane bond. Colon et al. extended their work to produce a £uorine-containing ligand by using trideca£uoro-1,1,2,2-tetrahydrooctyl-1-triethoxysi-
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lane. It had a unique selectivity for £uorine- and halogen-containing compounds [ 20 ].
2.3. Inner wall etched columns Pesek et al. have evaluated OTCEC using etched capillary columns. In order to increase the surface area of the 50 Wm i.d. fused-silica capillary column, they used ammonium hydrogen di£uoride as the etchant [ 21^26 ]. In Fig. 3 the SEM ( scanning electron microprobe ) image of the inner surface after the etching process is shown. The increase of the surface area, about 1000 times larger than that of the non-etched surface, is clearly indicated. They then treated the surface by TES and made Si^H groups on the inner wall of the capillary. Then an ole¢ns solution with hexachloroplatinomic acid as catalyst was £ushed through the column at 100³C for 45 h ( a hydroxylation process ). As the ligand is ¢xed to the surface by a Si^C bond instead of a common siloxane bond, the column stability is superior to usual open-tubular columns. They evaluated an octadecyl bonded capillary for the separation of proteins and peptides [ 21,22,24,25 ]. They also tried to bond diol groups on the Si^H surface for peptide separation, and performed three angiotensin separations using two columns, diol modi¢ed and C18 modi¢ed etched capillaries [ 24,25 ]. The selectivity difference between the diol and the C18 modi¢ed columns is very clear and this result shows the existence of the different interactions between the solutes and two ligands bonded on the inner surface of the etched capillaries. Pesek et al. also tried to use their open-tubular capillaries for the separation of basic compounds, which gave a large peak tailing due to the interaction between silanol groups and the solutes with the packed column [ 22,23,25 ]. Since the Si^H capillary does not have so many remaining silanol groups on the surface, it gave much better peak shape for the basic solutes than the bare capillary. The bonded ligand produced the better selectivity, based on the interactions between the solutes and the ligand. Catabay et al. used the same process of the column inner surface etching and ligand bonding for cholesteryl-10undecenoate phase using 50 and 75 Wm inner diameter columns. They achieved separations based on the interaction between benzodiazepines and the cholesteryl phase with etched, modi¢ed capillary but bare and etched, unmodi¢ed capillaries did not give a good separation for those drug compounds [ 26 ]. Most recently Pesek et al. reported
the use of open-tubular capillary columns with chemically bonded liquid crystals such as 4cyano-4P-n-pentoxybiphenyl and cholesteryl-10undecenoate in CEC [ 27 ]. Separation of metabolic components of serotonin on a 50 Wm i.d. etched cyanopentoxy modi¢ed capillary is shown in Fig. 4.
2.4. Polymer-coated columns Polymer-coated open-tubular columns prepared by thermal polymerization of methacrylate monomers were reported by Tan et al. [ 28 ]. The inner surface of the column was etched with NaOH and diluted HCl, and difunctional silane reagent ( 3-( trimethoxysilyl )propyl-methacrylate ) was used for the pretreatment. The capillary was ¢lled with a toluene solution of butylmethacrylate monomers and the polymerization initiator. The polymerization process took 10 min at 120³C. At this time the inner surface was coated by the linear polymers. Due to the inef¢cient coverage of the inner surface, the EOF ( which was lower than that obtained by a bare capillary ) drove the mobile phase by the remaining silanol groups and some polymer layer contribution. The effect of the monomer concentration on the separation performance of such columns was examined and they found a critical concentration that gave the maximum retention for parabens. The best retention was obtained under the following conditions: initial monomer concentration; 45%, mobile phase; acetonitrile /phosphate buffer=20 / 80. Butylparaben gave a retention of 0.55 and non-retained acetone gave 270 000 plate numbers / m. When they prepared the column, they added cross-linking agents and investigated their effect on the separation [ 29 ]. At higher than 20% monomer concentration, the viscosity of the polymer solution was increased and therefore it was extremely dif¢cult to eliminate the remaining unbonded polymeric materials in the capillary column. The degradation of the column with very high acetonitrile concentration in the buffer mobile phase was not remarkable and the addition of the cross-linking agents reduced EOF, which clearly indicated that the cross-linked polymer covered the surface much better than the linear polymer coating. They compared the performance of such columns in OTCEC and OTLC and found that CEC gave much higher ef¢ciency, for example 200 000 plates / m for propylparaben with OTLC and 380 000 plates / m with OTCEC, respectively.
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Fig. 4. Separation of metabolic components of serotonin on a 50 Wm etched cyanopentoxy modi¢ed capillary column. Conditions: V=25 kV, mobile phase 100% aqueous pH 2.14 buffer ( 60 mM phosphoric acid and 38 mM Tris ), L=50 cm, and I=25 cm. ( From [ 27 ], with permission from the American Chemical Society. )
Another approach in CE utilizing polymer coating to counteract solutes adsorption on the inner surface, and eliminate or enhance the EOF has been examined [ 50^52 ]. In these important works Kohr and Engelhardt [ 50 ], Lee et al. [ 51 ] and Hartwick et al. [ 52 ] reported that the coating of the copolymer of anionic monomer, with a side chain containing a sulfonic acid group and acrylamide, gave a stable, constant EOF under a wide pH range. Based on such approach Sawada and Jinno prepared a 25 Wm i.d. capillary column in which the surface was coated by a copolymer of N-alkylacrylamide and anionic monomer [ 30 ]. EOF is obtained by the sulfonyl group in the linear copolymer and the hydrophobic alkyl chains in the polymer structure act as the stationary phase. Neutral ketones, parabens and PAHs, which are dif¢cult to separate by CE, could be separated by CEC. By substituting the alkyl chains with other functional groups, one can control or enhance the selectivity of chromatographic separations and the column stability can be maintained by the linear polymer coating on the inner wall. This method will be a useful technique in open-tubular CEC in the near future, but requires further enhancement of selectivity and tail-
ored phases for solving particular separation problems.
2.5. Columns for chiral isomer separations 2.5.1. Immobilized cyclodextrin columns There are many publications on chiral separations with CE such as MECC using chiral micelles, cyclodextrin modi¢ed micelles ( CD-MECC ) and immobilized CDs on capillary columns packed with polyacrylamide gel. Schurig reported CEC separation of chiral isomers using a polysiloxane polymer-coated capillary column containing permethylated-L-CD ( Chirasil-Dex column ) [ 31 ]. They immobilized the polysiloxane polymer on the 50 Wm capillary inner surface using the method that they had developed for chiral separations with supercritical £uid chromatography (SFC ) [ 53 ]. Successful results were seen in their second publication [ 32 ] where they separated antisteroid drugs with 20 mM borate / phosphate buffer ( pH 7.0 ) mobile phase. They then compared the performance of OTLC and OTCEC for pharmaceutical chiral separations and found that OTCEC gave better plate numbers and separation factors [ 33 ]. However,
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some charged drugs required longer analysis time than with OTLC due to the reduction of EOF. Armstrong et al. also reported the separation of chiral barbiturates using a derivatized CD immobilized capillary column [ 34 ]. On the other hand, Francotte et al. examined the chiral separations with a cellulose derivatives-coated capillary column [ 35 ]. Their columns were prepared by coating 3,5-dimethylphenylcarbamoyl cellulose ( DMPCC ) or pmethylbenzoylcellulose (PMBC ) on 50 Wm i.d. capillary. They also compared the performance of OTLC and OTCEC and found CEC to be better, but the column stability and its lifetime are problems in practice. The instability is due to the cellulose coatings not being immobilized on the inner wall of the capillary column.
2.5.2. Molecular imprinting polymers (MIPs ) MIPs can be prepared by the polymerization of functional monomers with cross-linking agents in the presence of template molecules. After the polymerization the template molecules are extracted from the polymeric materials in order to produce the speci¢c sites in the polymer structure. MIPs have been extensively applied to chromatographic stationary phases, particularly for selective separations, such as chiral separation. Tan et al. [ 36 ] developed a 25 Wm i.d. capillary column with its inner wall coated by MIPs and applied it in chiral separations with OTLC and OTCEC. They used Ldansylphenylalanine as the template with functional monomers of methacrylates and 2-vinylpridine, and cross-linking agents. The reaction time for polymerization is critical for better column performance. They tried to separate D- and L-dansylphenylalanines by CEC using an 85 cm effective length column with acetonitrile / 10 mM phosphate buffer at pH 7.0 and non-retained D-alanine gave 250 000 theoretical plates / m but retained L-alanine gave only 8000 plates / m, respectively. 2.5.3. Af¢nity electrochromatography Bovine serum albumin ( BSA ), K-acid glycoprotein( orosomucoid ) and ovomucoid immobilized stationary phases are very popular in LC chiral separations and they are also useful in CE as af¢nity EKC for chiral separations [ 54^56 ]. In this type of separation there were several problems such as adsorption of chiral selectors on the inner wall surface, their absorption in the UV region and low column ef¢ciency. Recently there have been attempts to apply af¢nity CEC for chiral separations with pro-
Fig. 5. Chromatograms of a ¢ve-component mixture obtained with a 65 Wm i.d. octylaminated PEEK column. Conditions: separation length 29.2 cm; detection capillary, 75 Wm i.d.U30.4 cm; no sample introduction capillary was used; mobile phase acetonitrile / 2.5 mM phosphate buffer ( pH=6.9 ) ( v / v )=50 / 50; voltage, 15 kV; current, 2 WA; detection, 215 nm. Solutes: 1, thiourea; 2, benzyl alcohol; 3, benzaldehyde; 4, benzene; 5, toluene. ( From [ 38 ], with permission from Wiley Interscience. )
tein immobilized silica stationary phase [ 57,58 ]. Schurig et al. prepared BSA bonded capillary column and examined af¢nity OTEC ( open-tubular electrokinetic chromatography ) for various dinitrophenylamino ( DNP ) acids and 3-hydroxy-1,4-benzodiazepines [ 37 ]. The preparation procedure is as follows: ¢rstly a 50 Wm i.d. column inner surface is treated with Q-glycidoxypropyltrimethoxysilane and then HCl to hydrolyze epoxides to diol groups, followed by activation by tresyl chloride, and ¢nally the capillary is ¢lled with BSA at 4³C for 24 h. Reduction of EOF was found for the BSA bonded capillary column compared to a non-processed capillary from 6 min to 20 min. They succeeded in separating DNP-alanine, glutamic acid, proline and phenylalanine in baseline separation with phosphate buffers of pH 6.0 and 8.0. Liu at al. also produced a novel column preparation method for OTCEC [ 16 ]. The adsorbed stationary phases were prepared easily by rinsing the capillary with a buffer containing cationic surfactant such as CTAB or basic protein such as lysozyme. In the latter, the adsorbed lysozyme layer was used as a chiral stationary phase for separation of enantiomers. Four
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amino acids and the drug mephenytoin were separated within 5 min with a resolution from 1.74 to 2.05. The merit of this type of CEC separations is the reduction in the required amounts of stationary phase.
2.6. Polymer tubings Fused-silica capillary is the most useful and popular column medium in CEC but the use of polymer tubings have also been examined [ 59^61 ]. Hydrophobic groups were bonded on the inner wall surface of PEEK ( polyether ether ketone ) tube [ 38 ]. The PEEK inner surface was sulfonated by 1,1,2,2tetrachloroethane solution of chlorosulfonic acid. This treatment increased the column surface area and thus improved the phase ratio and, at the same time, activated the surface for the subsequent reaction with alkylamines. Capillary columns prepared in this manner showed retention characteristics similar to reversed-phase LC. As seen in Fig. 5, this column works well in CEC and the results indi-
cated that potential applications of the PEEK column might be found in separations where high pH mobile phases are needed.
3. Combination with mass spectrometry Since mass spectrometry (MS ) is very useful for peak identi¢cation in GC, LC and SFC, GC^MS and LC^MS are now established and used in many different analytical areas. CE^MS is also a very promising approach and ESI ( electrospray ionization ) is the most popular ionization technique [ 62^64 ]. There are some publications on CEC^MS [ 65^73 ]. To reduce the air bubble problem in packed-column CEC, the common approach is to send the mobile phase with EOF and pressurized £ow by a typical LC pumping system, which is so-called pseudo-electrochromatography [ 65^69 ]. A CEC^ MS approach without an LC pump was also investigated by several groups [ 70^73 ]. In this pseudoCEC separation method ionic species have a differ-
Fig. 6. ( a ) Schematic of the microchip with a serpentine column geometry. ( b ) Diagram of the high voltage switching apparatus and detection / data acquisition system. ( From [ 80 ], with permission from the American Chemical Society. )
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Fig. 7. Fast open-channel electrochromatography on microchips. Channel depth, 5.2 Wm; stationary phase, octadecylsilane; mobile phase, 10 mM borate buffer ( pH 8.4 ) with a linear gradient of acetonitrile from 29% to 50% within 5 s, starting 1 s after injection; ¢eld strength, 700 V / cm; dotted line, gradient trace; analytes, coumarin dyes, ( 1 ) C440, ( 2 ) C450, ( 3 ) C460, ( 4 ) C480. ( From [ 81 ], with permission from the American Chemical Society. )
ent selectivity than with a pressurized mobile phase £ow system. As bubble formation is not a problem in OTCEC, this method combined with MS (OTCEC^MS ) would be a much easier approach than packed-column CEC^MS. The low sensitivity in UV detection due to a shorter pathlength of the narrow column diameter used in OTCEC can be solved by using the OTCEC^MS approach, especially the ESI-MS approach. Lubman's group reported the very fast separation of a peptide mixture by OTCEC^MS [ 39 ]. They prepared a 9 Wm i.d. open-tubular column using Colon's method [ 18 ] to make octyl phase on the column inner wall surface. After the preparation they treated the column with £owing
toluene solution of 5% ( v / v ) 3-aminopropyltriethoxysilane for 6 h and obtained a reversed osmotic £ow compared to the usual column. The isocratic eluent used was a mixture of 20% ( v / v ) acetonitrile and 5 mM ammonium acetate+0.05% tri£uoroacetic acid. In CEC^MS one end of the column was immersed in the buffer with negative charge, and the other end was used as the nanospray needle directly connected to the MS where the polyimide coating of the capillary column was eliminated and etched to a sharp head by HF. This edge was silver coated. They used an ESI-ion-trap / TOF ( time of £ight )-MS. The continuous ion beam produced by ESI was converted to pulsed ions in TOF-MS by using the ion-trap for the storage of
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ions. Six peptides were separated in 3 min by using a 25 cm length column at 12 kV voltage. Detection at the fmol level is possible. CEC^ESI-MS interfacings have been investigated by using quadrupole MS mainly, although this type of MS sacri¢ces the high resolution and faster separation of CE due to the relatively low scanning speed [ 63 ]. Lubman's group showed the usefulness of ion-trap /TOF-MS for fast and highly sensitive detection in CE [ 74,75 ]. They also developed a device to induce gradient elution in CEC^MS [ 39 ]. It can be concluded that CEC^MS will be a most useful analytical hyphenated technique that offers high separation power, high sensitivity and enough qualitative information.
4. Microchip technology The most recent trend in capillary separation sciences is the use of microchip technology to create the medium for the separation. In semiconductor engineering modern lithographic techniques can create patterns of electric circuits less than a micron and these techniques can be applied in the separation sciences. There are several advantages to using these miniaturized separation channels for CEC: ( 1 ) EOF can drive the £ow of the mobile phase which is very effective as it is based on the charge supply without the use of valves and pumps, ( 2 ) band broadening can be avoided as the separation by EOF is by plug £ow, ( 3 ) the ef¢ciency depends on the charge applied and is independent on the channel length in the ideal case and therefore very fast and high ef¢ciency separation can be performed using a high applied charge with the very short separation channels. Another additional but important feature of this chip type separation medium is the possibility of making a total analytical system on a chip including sample preparation, separation, reaction and detection, the so-called micro-total analysis system W-TAS. In many works published on W-TAS, CZE was the separation mode of choice, because electrophoretic mobility allows the easiest control of the separation. However, in order to extend the capability of chip type separation methods one needs to use other separation modes, such as MECC [ 76^79 ]. Ramsey's group evaluated CEC separations on a microchip in which the separation channels were coated by octadecyl chains [ 80 ]. The separation channels, 5.6 Wm deep and 66 Wm wide at half-
depth, were made by photolithography / chemical etching. Laser-induced £uorescence detection was used. The basic structure is shown in Fig. 6. The separation channels have a serpentine structure and this allows the production of very long channels on a small area ( in this case 8 mmU8 mm ). After adhering the cover glass to the chip, the coating was performed with ¢rst a £ow of 1 M NaOH and then a wash with water. Then He gas was passed through for 24 h at 125³C. After that toluene solution of 25% ( v / v ) chlorodimethyloctadecylsilane was passed through the channels by He gas for 18 h at 125³C and octadecyl chains formed on the inner surface of the channels. Using this chip they had separated three neutral dyes in 200 s and the theoretical plate height for non-retained solute was 4.1 Wm and retained solute 5.0 Wm, respectively. They also applied gradient elution [ 81 ] on the chip and Fig. 7 demonstrates the beautiful fast separation in the order of seconds. This technology appears to have a bright future for separation techniques, and even for MECC and CEC [ 82^89 ].
5. Conclusion The recent trends in open-tubular CEC (OTCEC ) have been reviewed. The many advantages of the OTCEC approach compared to packed-column CEC show a promising future for this technology in many applications. The most important point in the development of OTCEC is to produce the driving force to expand the applicability of CEC separation techniques in many ¢elds where other typical chromatographic techniques are not amenable to solving the problems.
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