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  Section: Practical Skills in Chemistry » Instrumental techniques
 
 
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Capillary electrophoresis

 
     
 
Content
Instrumental techniques
  Basic spectroscopy
    Introduction to spectroscopy
    UV Ivisible spectrophotometry
    Fluorescence
    Fluorescence spectrophotometry
    Phosphorescence and luminescence
    Atomic spectroscopy
  Atomic spectroscopy
    Atomic Absorption Spectroscopy
    Atomic Emission Spectroscopy
    Inductively coupled plasma
    Decomposition techniques for solid inorganic samples
  Infrared spectroscopy
  Nuclear magnetic resonance spectrometry
    1H-NMR spectra
    13C-NMR spectra
  Mass spectrometry
    Interfacing mass spectrometry
  Chromatography ~ introduction
    The chromatogram
    Resolution
    Detectors
  Gas and liquid chromatography
    Gas chromatography
    Liquid chromatography
    High-performance liquid chromatography
    Interpreting chromatograms
    Optimizing chromatographic separations
    Quantitative analysis
  Electrophoresis
    The supporting medium
    Capillary electrophoresis
    Capillary zone electrophoresis (CZE)
    Micellar electrokinetic chromatography (MEKC)
  Electroanalytical techniques
    Potentiometry and ion-selective electrodes
    Voltammetric methods
    Oxygen electrodes
    Coulometric methods
    Cyclic voltammetry
  Radioactive isotopes and their uses
    Radioactive decay
    Measuring radioactivity
    Chemical applications for radioactive isotopes
    Working practices when using radioactive isotopes
  Thermal analysis
    Thermogravimetry
    Applications

The technique of capillary electrophoresis (CE) combines the high resolving power of electrophoresis with the speed and versatility of HPLC. The technique largely overcomes the major problem of carrying out electrophoresis without a supporting medium, i.e. poor resolution due to convection currents and diffusion. A capillary tube has a high surface-area-to-volume ratio, and consequently the heat generated as a result of the applied electric current is rapidly dissipated. A further advantage is that very small sample volumes (5 - 10 nL) can be analysed. The versatility of CE is demonstrated by its use in the separation of a range of molecules, e.g. amino acids, proteins, nucleic acids, drugs, vitamins, organic acids and inorganic ions; CE can even separate neutral species, e.g. steroids, aromatic hydrocarbons.

The components of a typical CE apparatus are shown in Fig. 33.3. The capillary is made of fused silica and externally coated with a polymer for mechanical strength. The internal diameter is usually 25-50,µm, a compromise between efficient heat dissipation and the need for a light path that is not too short for detection using UV/visible spectrophotometry. A gap in the polymer coating provides a window for detection purposes. Samples are injected into the capillary by a variety of means, e.g. electrophoretic loading or displacement. In the former, the inlet end of the capillary is immersed in the sample and a pulse of high voltage is applied. The displacement method involves forcing the sample into the capillary, either by applying pressure in the sample vial using an inert gas, or by introducing a vacuum at the outlet. The detectors used in CE are similar to those used in chromatography, e.g. UV/visible spectrophotometric systems. Fluorescence detection is more sensitive, but this may require sample derivitization. Electrochemical and conductivity detection are also used in some applications, e.g. conductivity detection of inorganic cations such as Na+ , K+.

EOF, described above, is essential to the most commonly used types of CE. The existence of EOF in the capillary is the result of the net negative charge on the fused silica surface at pH values over 3.0. The resulting solvent flow towards the cathode is greater than the
 
Components of a capillary
Fig 33.3 Components of a capillary
electrophoresis system.
attraction of anions towards the anode, so they will flow towards the cathode (note that the detector is situated at the cathodic end of the capillary). The greater the net negative charge on an anion, the greater is its resistance to the EOF and the lower its mobility. Separated components migrate towards the cathode in the order: (1) cations, (2) neutral species, (3) anions.


 
     
 
 
     



     
 
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