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  Section: Practical Skills in Chemistry » Instrumental techniques
 
 
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The supporting medium

 
     
 
Content
Instrumental techniques
  Basic spectroscopy
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    1H-NMR spectra
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  Mass spectrometry
    Interfacing mass spectrometry
  Chromatography ~ introduction
    The chromatogram
    Resolution
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  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 effects of convection currents (resulting from the heating effect of the applied electric field) and the diffusion of molecules within the buffer solution can be minimized by carrying out the electrophoresis in a porous supporting medium. This contains buffer electrolytes and the sample is added in a discrete location or zone. When the electric field is applied, individual sample molecules remain in sharp zones as they migrate at different rates. After separation, post-electrophoretic diffusion of selected molecules (e.g. proteins) can be avoided by 'fixing' them in position on the supporting medium, e.g. using trichloracetic acid (TCA).

The heat generated during electrophoresis is proportional to the square of the applied current and to the electrical resistance of the medium: even when a supporting medium is used, heat production will lead to zone broadening by increasing the rate of diffusion of sample components and buffer ions. Heat denaturation of certain sample types may also occur, e.g. proteins. Another problem is that heat will reduce buffer viscosity, leading to a decrease in resistance. If the electrophoresis is run at constant voltage, Ohm's law dictates that as resistance falls, the current will increase, leading to further heat production. This can be avoided by using a power pack that provides constant power. In practice, most electrophoresis equipment incorporates a cooling device; even so, distortions of an electrophoretic zone from the ideal 'sharp, linear band' can often be explained by inefficient heat dissipation.

Supporting media can be sub-divided into:
  • Inert media - these provide physical support and minimize convection; separation is based on charge density only (e.g. cellulose acetate).
  • Porous media - these introduce molecular sieving as an additional effect: their pore size is of the same order as the size of molecules being separated, restricting the movement of larger molecules relative to smaller ones. Thus, separation depends on both the charge density and the size of the molecule.
With some supporting media, e.g. cellulose acetate, a phenomenon called electro-endosmosis or electro-osmotic flow (EOF) occurs. This is due to the presence of negatively charged groups on the surface of the supporting medium, attracting cations in the electrophoresis buffer solution and creating an electrical double layer. The cations are hydrated (surrounded by water molecules) and when the electric field is applied, they are attracted towards the cathode, creating a flow of solvent that opposes the direction of migration of anionic molecules towards the anode. The EOF can be so great that weakly anionic molecules may be carried towards the cathode.

Where necessary, EOF can be avoided by using supporting media such as agarose or polyacrylamide, but it is not always a hindrance to electrophoretic separation. Indeed, the phenomenon of EOF is used in the high-resolution technique of capillary electrophoresis.

 
     
 
 
     



     
 
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