Outline the principles of electrophoresis and review the advantages and disadvantages of
conventional electrophoresis and capillary zone electrophoresis, giving examples of their use
(March 06, Paper 1, Question 3)
Electrophoresis is the migration of charged particles in a liquid medium under the influence of
an electrical field.
Movement of the charged particles is brought about by the action of the electrical force. The
chemical environment surrounding the molecule, the electrolyte solution, must be capable of
conducting an electrical current. Therefore the choice of electrolyte is critical to achieving
optimal mobility and separation, as is the pH- effective mobility is a function of pH. For
molecules to be separated by electrophoresis they must either carry a net positive or negative
charge in their native state or be able to accept a charge.
The pH of the electrolyte solution has a critical role in determining the state of ionisation and
hence the charge of any molecule in contact with it, for example, proteins are ampholytes that
is they are either positively or negatively charged depending on the pH of the buffer. In acidic
solutions, proteins take on a positive charge and in alkaline solutions a negative charge.
Thus the rate of migration is directly proportional to the field strength and to the net charge of
the molecule.
A retarding force, which is a function of the size of the molecule and the viscosity of the
solution counteracts the forward migration induced by the electrical field. The greater the
viscosity, the slower the movement. The larger or more asymmetrical the particle, the slower
its movement.
Thus, the rate of migration is inversely proportional to the size of the particle & viscosity of
the solution
And electrophoretic mobility () can be defined as the rate of migration (cm/s) per unit field
of strength (volt/cm).
Conventional electrophoresis (zone electrophoresis)
Produces zones of proteins which are heterogeneous & physically separated from one
another- typically classified according to the type of support material used.
Applications: Serum & urine protein electrophoresis
Haemoglobin
Iso-enzymes
Nucleic acids
Advantages
 Simple, inexpensive equipment
 Cheap to run
 Commercially produced gels
 Multiple samples in parallel on the same gel
Disadvantages
 Low resolution
 High molecular weight species
 Not fully automated
 Time consuming & laborious- gel preparation, separation, staining and destaining, gel
drying
 Gel must be treated to visualise

Semi-quantitative- intensity of the stain may be poorly correlated with the amount of
protein as uptake of the stain may occur in a non-linear fashion
Capillary zone electrophoresis
The classic technique of electrophoresis is carried out in a small bore (10-100µm) fused silica
capillary column which is between 20-200cm in length.
Applications: Serum & urine protein electrophoresis
Haemoglobin
Iso-enzymes
Nucleic acids
Advantages
 The main advantage comes from efficient heat dissipation compared to traditional
electrophoresis- due to large internal surface area to volume. This enables the use of
high field strengths which deceases analysis time and minimises band diffusion.
 Separation in free solution without requirement of a casting gel
 Detection occurs as the separation progresses with resolved zones producing an
electronic signal as they migrate past a UV absorbance or fluorescence detector for
example. This configuration increases efficiency.
 No staining or destaining
 Quantitative information- results displayed as an electropherogram & integrated in
form of peak area or height
 Automated
 Separation of low molecular weight ions, proteins & other macromolecules
 Small sample volume- minimises the distortion in the applied field caused by the
presence of sample
Disadvantages
 Single sample injected- limited throughput, multiple samples are analysed serially
 Larger sample volumes required for autosampler - dead volume
 Inner diameter of the capillary tube is also the pathlength of the cuvette- limited
detection limit- lower sensitivity