Capillary zone electrophoresis analyte zones

Tsuda T, Sweedler J V, Zare R N (1990). Rectangular capillaries for capillary zone electrophoresis. Analyt. Chem. 62 2149-2152. 

Hutterer, K. M. and Jorgenson, J. W., Ultrahigh-voltage capillary zone electrophoresis. Analytical Chemistry 1999, 71, 1293-1297. 

Nielen, M. W. F. "Chiral Separations of Basic Drugs Using Cyclodextrin-Modified Capillary Zone Electrophoresis." Analytical Chemistry, 651993, 885-893. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Electrodriven Separation Techniques encompass a wide range of analytical procedures based on several distinct physical and chemical principles, usually acting together to perform the requh ed separation. Example of electrophoretic-based techniques includes capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), and capillary gel electrophoresis (CGE) (45-47). Some other electrodriven separation techniques are based not only on electrophoretic principles but rather on chromatographic principles as well. Examples of the latter are micellar... 

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

Table 1 summarizes several of the experimental methods discussed in this chapter. A need exists for new or revised methods for transport experimentation, particularly for therapeutic proteins or peptides in polymeric systems. An important criterion for the new or revised methods includes in situ sampling using micro techniques which simultaneously sample, separate, and analyze the sample. For example, capillary zone electrophoresis provides a micro technique with high separation resolution and the potential to measure the mobilities and diffusion coefficients of the diffusant in the presence of a polymer. Combining the separation and analytical components adds considerable power and versatility to the method. In addition, up-to-date separation instrumentation is computer-driven, so that methods development is optimized, data are acquired according to a predetermined program, and data analysis is facilitated. 

Kelly, J.F., Ramaley, L., Thibault, P. (1997). Capillary zone electrophoresis-electrospray mass spectrometry at submicroliter flow rates practical considerations and analytical performance. Anal. Chem. 69, 51-60. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

Boss H J., Watson D.B., and Rush R.S. (1998), Peptide capillary zone electrophoresis mass spectrometry of recombinant human erythropoietin an evaluation of the analytical method, Electrophoresis 19(15), 2654—2664. 

Dedicated applications of capillary zone electrophoresis (CZE) coupled to MS are discussed, particularly in the field of drug analysis. Development of other capillary-based electrodriven separation techniques such as non-aqueous capillary electrophoresis (NACE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) hyphenated with MS are also treated. The successful coupling of these electromigration schemes with MS detection provides an efficient and sensitive analytical tool for the separation, quantitation, and identification of numerous pharmaceutical, biological, therapeutic, and environmental compounds. 

The most important features of liquid membranes are that they olfer highly selective extraction, efficient enrichment of analytes from the matrix in only one step, and the possibility of automated interfacing to different analytical instruments such as liquid chromatography, gas chromatography, capillary zone electrophoresis, UV spectrophotometry, atomic absorption spectrometry, and mass spectrometry [82]. 

Cloud point extraction has been applied to the separation and preconcentration of analytes including metal ions, pesticides, fungicides, and proteins from different matrices prior to the determination of the analyte by techniques such as atomic absorption, gas chromatography, high performance liquid chromatography, capillary zone electrophoresis, etc. 

S Fanali, P Bocek. A practical procedure for the determination of association constants of the analyte—chiral selector equilibria by capillary zone electrophoresis. Electrophoresis 17 1912-1924, 1996. 

Hoyt and Sepaniak have used capillary zone electrophoresis to determine procaine in pharmaceuticals as a cation of benzylpenicillin [148]. A benzylpenicillin potassium tablet (250 mg) was treated with 20 mL of a 0.2% phenol solution (the internal standard), and dispersed in water. The solution was diluted to 500 mL, and samples were introduced into the fused silica capillary tube (70 cm x 50 gm) by siphoning. With 10 mM Na2HP04-6mM Na2B407 buffer as the mobile phase, the samples were subjected to electrophoresis at 30 kV (25 to 30 pA), and the emerging analytes detected at 228 nm within 10 minutes. 

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

Micellar electrokinetic capillary chromatography (MECC), in contrast to capillary electrophoresis (CE) and capillary zone electrophoresis (CZE), is useful for the separation of neutral and partially charged species [266,267]. In MECC, a surfactant, usually sodium dodecyl sulfate (SDS), is added to the buffer solution above its critical micellar concentration to form micelles. Although SDS is certainly the most popular anionic surfactant in MECC, other surfactants such as bile salts have proved to be very effective in separating nonpolar analytes that could not be resolved using SDS [268]. 

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