Packed capillary columns electrokinetic packing

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

Electroosmosis refers to the movement of the liquid adjacent to a charged snrface, in contact with a polar liquid, under the influence of an electric field applied parallel to the solid-liquid interface. The bulk fluid of liquid originated by this electrokinetic process is termed electroosmotic flow. It may be prodnced either in open or in packed or in monolithic capillary columns, as well as in planar electrophoretic systems employing a variety of snpports, such as paper or hydrophilic polymers. The origin of electroosmosis is the electrical donble layer generated at the plane of share between the snrface of either the planar support or the inner wall of the capillary tube and the surronnding solntion, as a consequence of the nneven distribntion of ions within the solid/liquid interface. 

Capillary Electrochromatography. Capillary electrochromatography (CEC) is a hybrid technique that works on the basic principles of capillary electrophoresis and chromatography [41], This mode of chromatography is used on either packed or tubular capillaries/columns. The packed column approach was introduced by Pretorius et al. [60] in 1974, while open tubular CEC was presented by Tsuda et al. [61] a decade later. In 1984 Terabe et al. [62] introduced another modification in liquid chromatography, micellar electrokinetic capillary... 

An elegant study was presented by Thomas et al. who employed selective solid-phase extraction by immunoaffinity CEC (IACEC) to enhance detection limits. A model compound, fluorescein isothiocyanate biotin, was electrokinetically applied to a capillary column packed with an immunoaffinity stationary phase. The analyte was first selectively bound to the stationary phase, then eluted, migrated by zone electrophoresis, and detected by LIF [79],... 

Fig. 4.31. Separation of a test mixture on capillary columns packed by different methods (A) pressure packing, (B) by centripetal forces, using supercritical fluid, and by electrokinetic packing. Columns were 50 pm I.D., 20 cm packed (30 cm total length) mobile phase 80 20 acetonitrile-4 mmol/1 aqueous borate. Separation voltage of 20 kV. Solutes 1, thiourea 2, benzyl alcohol 3, biphenyl 4, dimethylnaphthalene 5, ethylnaphthalene 6, amylbenzene.

Fig. 10.1. Separation of polycyclic aromatic hydrocarbons (PAHs) on columns packed with Spherisorb ODS particles. Conditions (A) 35(43) cm x 50 pm i.d. fused silica capillary column packed with 3 pm Spherisorb ODS-1 particles (B) 41(53) cm x 75 pm i.d. fused-silica capillary column packed with 5 pm Spherisorb ODS-1 particles 30 kV applied voltage 5 kV, 5 s electrokinetic injection acetonitrile-50 mM Tris buffer, pH 8.1 (80 20 v/v). Peak identifications 1, benzene 2, naphthalene 3, acenaphthylene 4, fluorene 5, acenaphthene 6, phenanthrene 7, anthracene 8, fluoranthene 9, pyrene 10, benz[n]anthracene 11, chrysene 12, benzo[6]fluoranthene 13, benzo[fc]fluoranthene 14, benzo[a]pyrene 15, dibenz[n,/i]anthracene 16, indeno[7,2,3-af]pyrene 17,...

Figure 28 Capillary electrochromatography of four t-RNAs. Capillary column, packed with 2-pm nonporous ODSS stationary phase, 20.5/27 cm x 100 pm i.d. running voltage, 20 kV, electrokinetic injection, 1 kV for 2 s mobile phase in (a), hydroorganic eluent containing 1 5 mM phosphate and composed of 40% (v/v) methanol. Solutes 1, t-RNAGIU 2 t-RNAVal 3, t-RNALys 4, t-RNAphe. (Reprinted from Ref. 119, with permission.)...

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

For isocratic mode of CEC separations, standard CE instrumentation is sufficient. This applies particularly for equipment that has the provision of column pressurization. In practice this is achieved by applying a gas under a pressure of 2-12 bar to both inlet and outlet vials. Column thermostating in CEC is regarded mandatory to avoid excessive radial temperature gradients within the capillary. In such instruments, sample is typically injected electrokinetically and alternatively by applying the external gas pressure to the sample vial. Detection occurs on-column i.e. directly through a non-packed section of the capillary following immediately the end of the bed. 

Fig. 10.2. Separation of a mixture of PAHs on reversed-phase capillaries (a) without and (b) with silicate entrapment. Conditions 75 pm i.d. fused-silica capillary packed with 5 pm Nucleosil ODS particles column effective lengths 25 cm for the non-entrapped column and 17 cm for the entrapped column. Both electrochromatograms were obtained under the same conditions mobile phase, acetonitrile-0.1 M acetate buffer, pH 3.0, 80 10 (v/v) applied voltage 30 kV UV detection at 254 nm 20°C pressure 9 bar applied to both vials electrokinetic injection, 10 kV for 10s. Reproduced with permission from Chirica and Remcho [10].

Fig. 10.17. Capillary electrochromatography of PTH-amino acids with gradient elution. Column, 207 (127) mm x 50 pm i.d. packed with 3.5 pm Zorbax ODS particles, 80 A pores. Starting eluent (A), 5 mM phosphate, pH 7.55, 30% acetonitrile gradient former (B), 5 mM phosphate, pH 7.55, 60% acetonitrile flow-rate (through inlet reservoir), 0.1 ml/min gradient, 0-100% B in 20 min voltage 10 kV current, 1 pA temperature, 25°C UV detection at 210 nm electrokinetic injection, 0.5 s, 1 kV. Peaks in order of elution formamide PTH-asparagine PTH-glutamine PTH-threonine PTH-glycine PTH-alanine PTH-tyrosine PTH-valine PTH-proline PTH-tryptophan PTH-phenyialanine PTH-isoleucine PTH-leucine. The concentration of the PTH-amino acids dissolved in the mobile phase was 30-60 pg/ml. Reprinted with permission from Huber et al. [68]. Copyright 1997 American Chemical Society.

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

Stol, R., et al., Pseudo-electrokinetic packing of high efficiency columns for capillary electrochromatography, J. Chromatogr. A, 873, 293, 2000. 

Fig. 2 Electrochromatogram of naphthalene 1, fluoranthene 2, benz[a]anthracene 3, benzo[/ ]fluoranthene 4, and benzo [g/ /]pery-lene 5, using 1.5 xm non-porous ODS particles. Column dimensions 100 p.m I.D. X 6.5 cm packed length (10 cm total length). Mobile phase 70% acetonitrile in a 2 mM Tris solution. Applied voltage for separation 28 kV. Injection electrokinetic at 5 kV for 2 sec. Source From Advances in capillary electrocromotography Rapid and high-efficiancy separation of PAHs, in Anal. Chem. ...

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