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Study Notes: What’s This Thing Called ‘Electrophoresis’?

The term ‘electrophoresis’ simply means the movement of analytes by an electric force. Electrophoresis is an analytical separation technique with some similarities to chromatography but with some major differences. Most current slab gel electrophoretic techniques use a gel matrix perfused by an aqueous phase of buffer salts. This aqueous phase is not a mobile phase except in the specialised case of capillary electrophoresis.

This gel-like matrix (usually agarose or polyacrylamide) stabilises the process against solution turbulence. The primary purpose of the gel matrix is as a sieve that allows separations based on molecular size. Electrophoresis and chromatography share many characteristics. They are separation techniques used to separate complex mixtures, can be used qualitatively and quantitatively, use standards, can be used to collect bands/peaks of interest, and are relatively fast and effective methods.

On the other hand there are a number of significant differences that should be understood. These are listed in the table below.

Electrophoresis
Chromatography
Has a stationary gel but no mobile phase. Has a stationary phase and a mobile phase.
Separation is achieved by molecular sieving (based on size and charge). Separation is achieved by the relative affinity of analyte for the stationary and mobile phases (based on a number of different variables).
Movement of the sample is achieved under an electric current. Movement of the sample is achieved by movement of the mobile phase.
Is a planar technique. May be planar but usually column.
Sample volume usually about 25 µL but may be increased to mL volumes for preparative procedures. Sample volume is usually in the µL range.
Visualisation usually by staining of analyte bands. Visualisation via a detector that converts the signal to a chromatogram.
Generally used for proteins and nucleic acids. Used for a wide range of analyte types.
Equipment is relatively simple. Equipment may be simple (paper, TLC, simple columns) but is often complex and costly (HPLC, GC).

Image of vertical slab gel electrophoresis mini gel.
Vertical slab gel electrophoresis mini gel


Electrophoresis separates species of interest by moving the sample mixture through a gel of material. The sample components are moved using an electric current and hence the sample components must be charged molecules or molecules that can readily have a charge added to them.

As the molecules move through the gel they must negotiate a torturous route of small interstices (pores) through the gel. The smaller molecules move faster and hence migrate faster and the larger molecules move more slowly and hence migrate more slowly.

The electrophoresis apparatus is set up in such a way that the buffer bathing the top and bottom of the gel has a different pH (and sometimes ionic strength) to the buffer in the gel. This has the effect of concentrating like components into tight bands as they migrate down the gel in an analogous way to the banding of components in column chromatography to produce peaks.

There are many varieties of electrophoresis including gradient gel electrophoresis, disc electrophoresis, SDS electrophoresis, two-dimensional electrophoresis and isoelectric focusing.

Sodium dodecyl sulfate (SDS also called sodium lauryl sulfate) is a powerful detergent that is often used to solubilise samples prior to electrophoresis. There have been many agents used to produce the gels but the two in common use today are agarose and polyacrylamide.

Agarose is used for nucleic acid separations and polyacrylamide is used for proteins and DNA sequencing. Electrophoresis is generally used only for the separation of nucleic acids and proteins and can be used for such samples as tissue and cell extracts, serum proteins, membrane proteins and DNA restriction fragments. Intact DNA is a very large molecule and needs to be analysed in relatively small fragments.

Note: SDS-PAGE means sodium dodecyl sulfate polyacrylamide gel electrophoresis and is routinely used for the separation of proteins.

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The ionic strength of the surrounding solution and the temperature of the gel have an effect on sample mobility. Generally lower ionic strength buffers lead to faster migration but higher ionic strength buffers produce sharper bands. However, higher ionic strength buffers tend to increase current which can cause a temperature rise which in turn leads to lowered viscosity of the gel and even faster migration. The current will increase at higher temperatures leading to a spiral of even greater temperatures that can cause overheating and serious problems.

 

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