Electrophoretic Separations

What is Capillary Electrophoresis?

Nov 08 2014 Read 4653 Times

You don’t have to watch crime shows on television to know about DNA fingerprinting. The technique was developed by Alec Jeffries at the University of Leicester in the mid-1980s and has revolutionised crime detection. Since then thousands of crimes have been solved and many miscarriages of justice highlighted using the technique. One of the key components in DNA profiling is the separation of the DNA sample to give the classic ‘barcode’ pattern. The technique used to separate the DNA is electrophoresis.

Charged molecules

Molecules are either neutral, positively charged or negatively charged. If the number of electrons and protons in the molecule are equal, then the charge is neutral - if they are unbalanced a net positive or negative charge results.

In the absence of an electric field, the motion of charged molecules is random. But when an electric field is applied, the charged molecules begin to move in the direction of the field lines and the motion is less random. Positively charged molecules (cations) move towards the cathode (negatively charged electrode), anions move towards the anode. Electrophoresis uses the differences between charged molecules in an electric field.

Does size matter?

One of the variances utilized in electrophoresis is the mass/charge ratio (m/z) of the different molecules. With a fixed electric field, differences in mass and charge of the molecules will cause the molecules to travel towards the electrodes at differing rates. Larger molecules also face a greater resistance due to their size as they move through the separation medium, thus size also affects the separation. All these factors allow molecules to be separated by different distances — thus allowing qualitative analysis.

The medium that the electrophoresis is carried out in affects the separation; this is something an analyst can change to improve the separation of different molecules — especially by changing the pH of the separation medium.

Capillary Electrophoresis

Capillary electrophoresis is simply electrophoresis carried out in a capillary tube, which is typically a narrow bore tube containing a coating on the inside — similar to a HPLC column. The bore of the tubes used is usually in the range 20-100µm. Different coatings are used depending on the sample and the separation required. Capillary electrophoresis is preferred in certain analyses as it gives rise to a better separation than standard electrophoresis.

Capillary Electrophoresis Instrumentation

One of the reasons electrophoresis is used is due to its simple equipment. The components are simple to understand — and in capillary electrophoresis are:

  • A sample vial — containing the sample solution.
  • A source and destination vials — these contain the electrolyte chosen for the analysis.
  • A capillary column — used to carry out the separation, joins the source and destination vials.
  • Electrodes and a high-voltage power supply — used to create the electric field.
  • Detector and analysis — records and reports the analysis results.

For further information on capillary electrophoresis have a look at this article: Capillary Electrophoresis - an Attractive Technique for Chiral Separations. Then next time you are watching CSI you can impress your friends with your knowledge of DNA profiling.

Image Source: DNA in the Museum of Natural History 'Naturalis', Leiden.
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