Structural Biochemistry/Proteins/Purification/Capillary Electrophoresis

Capillary Electrophoresis
Capillary Electrophoresis is a family of techniques that use narrow-bore capillaries to perform high efficiency separations of both large and small molecules. Using a high voltage power supply, the solution travels from the anode to the cathode through the capillary. By doing so, the solution passes through the detector and based on the flow of the molecules, the integrator computes the separation of the molecules from the original solution. There are five modes of capillary electrophoresis which include capillary zone electrophoresis, isoelectric focusing, capillary gel electrophoresis, isotachophoresis, and micellar electrokinetic capillary chromatography.

Capillary Zone Electrophoresis
Capillary zone electrophoresis is a separation mechanisms that is based on the differences in the charge-to-mass ratio of the molecules. The homogeneity of the buffer solution as well as the constant filed strength are fundamental to the capillary zone electrophoresis process. It can be used to separate both large (DNA) and small (drugs) molecules. Capillary Zone Electrophoresis is the simplest form of capillary electrophoresis.

Isoelectric Focusing
Isoelectric focusing is when the solution tested is run through a pH gradient where the pH is low at the anode and high at the cathode. Therefore, when a voltage is applied, the ampholyte mixture separates in the capillary.

Capillary Gel Electrophoresis
Capillary gel electrophoresis is conducted in an anticonvective medium, oftentimes such as polyacrylamide or agarose gel. The composition of the media thus serves as a molecular sieve for size separations.

Isotachophoresis
In isotachophoresis, there is zero electroosmotic flow with the heterogeneous buffer. In fact, the capillary is filled with a leading electrolyte with a higher mobility than any of the sample components as well as a terminating electrolyte where the ionic mobility of the electrolyte is lower than any of the sample components. As a result, the solution is separated based on the leading and terminating electrolytes.

Micellar Electrokinetic Capillary Chromatography
In Micellar Electrokinetic Capillary Chromatography (MECC or MEKC), the use of micelle-forming surfactant solutions can give rise to separations that resemble reverse-phase liquid chromatography. Based on the hydrophobic and electrostatic interactions, the analytes are organized at the molecular level.

Electroosmotic Flow
In comparison to HPLC which uses hydrodynamic flow, capillary electrophoresis is based on electroosmotic flow (EOF). The factors that influence the rate of electroosmotic flow are pH, voltage, temperature and the concentration of the buffer.

pH
At neutral to alkaline pH, the electroosmotic flow is sufficiently stronger than the electrophoretic migration such that all species are swept towards the negative electrode. At high pH, the electroosmotic flow is large and the peptide is negatively charged; despite the peptide’s electrophoretic migration towards positive electrode (anode), the EOF is overwhelming and the peptide migrates towards negative electrode (cathode). At low pH, peptide is positively charged and EOF is very small, thus resulting in peptide electrophoretic migration and EOF towards the negative electrode. However, most solutes migrate towards negative electrode regardless of charge when buffer pH is above 7.0. Oftentimes, the pH selected is at least two units above or below pKa of the analyte in order to ensure complete ionization.

Voltage
High voltages provide for greatest efficiency by decreasing the separation time.

Temperature
At high temperatures, the viscosity of the solution is lower and the electroosmotic flow increases as a result. However, some buffers are known to be pH-sensitive with temperature.

Buffer Concentration
When the buffer concentration is reduced, the peak efficiency of the results is reduced by decreasing the focusing effect.