In the evolution of techniques used to separate molecules based on their electrophoretic mobility, capillary electrophoresis has been fine-tuned in order to obtain optimal results for varying experiments. There are six main forms of capillary electrophoresis, branching from two main subsets, one of which is further subdivided into two subdivisions.
Generally, the two main subsets of capillary electrophoresis are a continuous system capillary electrophoresis and a discontinuous system capillary electrophoresis. The discontinuous system ensures that a sample is kept in a distinct zone, which is partitioned by two dissimilar electrolytes. The only form of capillary electrophoresis that is classified in the discontinuous category is known as capillary isotachophoresis.
The continuous system has a contextual electrolyte that acts primarily as a buffering agent across the capillary. This system has two major subdivisions, namely, a steady state process capillary electrophoresis, which capillary isoelectric focusing is a member, and a kinetic process capillary electrophoresis, which is comprised of the remaining four techniques.
Capillary isotachophoresis (CITP)
Being the sole member of the discontinuous system, molecules with the CITP technique migrate in definitive zones. These zones can be measured for quantification of sample purposes. The sample is essentially added between two different buffers, one of which has a higher mobility in the separation process and is known as the leading electrolyte.
The second buffer is the slowest component in the entire system, slower than the sample molecules. The lengths of zones are proportional to the concentration of the analytes within them. CITP is particularly great in ultrasensitive mass spectrometry for the discovery of biomarkers in proteomic profiling.
Capillary isoelectric focusing (CIEF)
This form of capillary electrophoresis is commonly employed to separate peptides and proteins. The determining factors in this technique are the charge of the protein groups involved and the pH of the solutions. Since the charge is altered with altering pH, this is the basis of the mechanism for separating molecules in the mixture with this technique.
Micellar electrokinetic capillary chromatography (MEKC)
MEKC uses surfactant to form micelles (fat molecules rearranging to form a sphere when in aqueous solution). These micelles are imperative for their polar negativity, which causes a pull towards the positive pole.
The molecules that are hydrophobic (water hating) will tend to aggregate with the micelle, while those that are hydrophilic (water loving) will move fairly quickly through the solution. The key parameters for this technique are pH, surfactant concentration, any additives and the polymer coatings that are used on the capillary wall.
Capillary zone electrophoresis (CZE)
This is the most commonly used capillary electrophoresis method of capillary electrophoresis. CZE, also known as free solution capillary electrophoresis, is a separation technique that predominantly takes into account the ratio of the particle’s charge to mass, where those with large charge to mass ratio separate from the rest first; therefore, the larger the ratio, the quicker the separation.
In addition to the electrophoretic mobility of the molecules, CZE is heavily dependent on the application of constant field strength throughout the capillary and on the pH of the buffer solution. CZE is an excellent choice of technique to employ in cases where there are very small pI (isoelectric point) differences in protein isoforms.
Capillary electrochromatography (CEC)
This method resembles chromatography in many ways, as it is a combination of capillary electrophoresis and liquid chromatography. It is composed of a three parts: (i) open tubular capillary electromyography, which is a stationary phase that is immobilized, (ii) the CEC packed with some columns and (iii) the CEC with monolith.
CEC and CZE are similar in terms of having a plug-type of flow in comparison to the parabolic flow that is pumped, which increases band broadening.
Capillary gel electrophoresis (CGE)
Like CZE, CGE requires constant field strength and is dependent on the pH of the buffer solution as the particles move through the gel and separate out based on the difference of their size and shape. It is a great choice for macromolecules such as DNA and proteins. Gels are additionally advantageous in reducing solute diffusion and heat transfer.