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Figure 12.13 shows such a separation, using a 40 cm, 75 mm uncoated fused silica capillary operated at 30 kV. A solution of ampholytes (5%) and sample (1 mg/mL of each protein) in the running buffer are loaded throughout the capillary. The application of the electric eld results in slow, continuous separation and detection due to the slow electroosmotic ow. With this method, no salt mobilization is required. SUGGESTED REFERENCES
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C. Horvath and J. F. Nikelly, Eds., Analytical Biotechnology: Capillary Electrophoresis and Chromatography, American Chemical Society, Washington, DC, 1990. O. W. Reif, R. Lausch, and R. Freitag, in Advances in Chromatography, Vol. 34, P. R. Brown and E. Grushka, Eds., Marcel Dekker, New York, 1994, pp. 1 56. (See also 4, by Z. El Rassi, pp. 177 250). D. Schmalzing, S. Buonocore, and C. Piggee, Capillary Electrophoresis-Based Immunoassays, Electrophoresis 21, 2000, 3919 3930.
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1. A. G. Ewing, R. A. Wallingford, and T. M. Ole rowicz, Anal. Chem. 61, 1989, 300A 303A. 2. R. A. Wallingford and A. G. Ewing, in Advances in Chromatography, Vol. 29, J. C. Giddings, E. Grushka, and P. R. Brown, Eds., Marcel Dekker, New York, 1989, p. 22. 3. G. J. M. Bruin, Electrophoresis 21, 2000, 3931 3951. 4. Y.-F. Cheng and N. J. Dovichi, Science 242, 1988, 562 564. 5. J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, and C. M. Whitehouse, Science 246, 1989, 64 71. 6. R. D. Smith, J. A. Olivares, N. T. Nguyen, and H. R. Udseth, Anal. Chem. 60, 1988, 436 441. 7. R. A. Wallingford and A. G. Ewing, Anal. Chem. 60, 1988, 258 263. 8. R. A. Wallingford and A. G. Ewing, Anal. Chem. 60, 1988, 1972 1975. 9. S. L. Pentoney, Jr., R. N. Zare, and J. F. Quint, in Analytical Biotechnology: Capillary Electrophoresis and Chromatography, C. Horvath and J. F. Nikelly, Eds., American Chemical Society, Washington, DC, 1990. 10. H. Swerdlow, J. Z. Zhang, D. Y. Chen, H. R. Harke, R. Grey, S. Wu, N. J. Dovichi, and C. Fuller, Anal. Chem. 63, 1991, 2835 2841. 11. J. R. Mazzeo and I. S. Krull, Anal. Chem. 63, 1991, 2852 2857.
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PROBLEMS 1. A CZE experiment was performed using an open 50-mm diameter silica capillary, 50 cm long. A 5-mM carbonate buffer at pH 10 was used, with a separation voltage of 25 kV, following a 15 s, 1-kV electrokinetic injection of an FITC-derivatized amino acid mixture. Detection using laser-induced uorescence showed that the mixture contained three main components, with elution times of 4.5, 6.3, and 10.2 min. How would these elution times be expected to
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change if (a) The length of the capillary was increased to 1 m, (b) the separation voltage was increased to 50 kV, and (c) a length of 1 m and a voltage of 50 kV were used 2. Untreated silica capillaries show a cathodic drift of buffer, that is a ow of buffer from the anodic to the cathodic end, allowing an on-column detector to be placed near the cathodic end of the capillary. At which end of the capillary should a detector be placed if the inner silanol surface of the capillary has been covalently modi ed with aminopropyltri(ethoxy) silane, APTES (cf. 4) 3. Under acidic conditions, an analyte protein of unknown molecular weight is known to possess multiple positive charge, between 5 and 8. A mixture containing this protein and others were subjected to capillary electrophoresis in 10-mM tri uoroacetic acid, and the eluate from the capillary was fed directly into the ionization source of an electrospray mass spectrometer. As the protein eluted from the capillary, the m/z range of the mass spectrometric detector was scanned, and peaks were observed at m/z values of 938, 1071, 1250, and 1500. What is the molecular weight of the protein 4. Detection limits obtainable with most CE detectors increase (worsen) as the diameter of the capillary decreases. However, ultramicroelectrode-based amperometric detectors behave in the opposite manner, with improved detection limits for smaller capillary diameters, allowing smaller quantities of sample to be used. Suggest a precolumn derivatization reaction that could be used to allow the electrochemical detection of amino acids following a CZE separation. 5. What is the main advantage of using a coincidence radiochemical CE detector over one that uses a single photomultiplier
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