What are the major parameters that need to be considered for capillary isoelectric focusing?

The major parameters that need to be considered for capillary isoelectric focusing include: voltage, polarity, temperature, solutions, chiral separations, and the capillary. Depending on the chosen mobilization strategy, electroosmotic flow needs to be minimized or eliminated. Thus, coated capillaries are often used to reduce electroosmotic flow. Increasing both the effective length and total length of the capillary reduces the electric field strength, maintaining a constant voltage. This reduction in electric field slows down the migration of analytes, resulting in increased migration time. One should use high electric fields ranging from 300 V/cm to 1,000 V/cm which are applied during focusing. For the solutions, there are several factors one should consider. One factor is that the anode buffer has a lower pH than the most acidic ampholyte, while the cathode buffer has a higher pH than the most basic ampholyte. Common choices include phosphoric acid for the anode and sodium hydroxide for the cathode. Adding a polymer like methylcellulose to the ampholyte solution increases viscosity and diminishes convective forces and electroosmotic flow. Additionally, wide pH ranges are useful for estimating the isoelectric point (pI), while narrower ranges improve accuracy. Calibration involves correlating migration time with the pI of standard protein markers; buffer additives like glycerol can prevent protein precipitation at their pI if needed. 

Temperature affects buffer viscosity, electrical conductivity, and can cause conformational changes in proteins (if increased), altering separation efficiency. Chiral selectors including cyclodextrins, polysaccharides, crown ethers and some proteins are used in chiral separations. The effectiveness of these selectors depends on their interactions with enantiomers. Testing different cyclodextrins with varied cavity sizes or modified forms with different functional groups can optimize chiral separation. Lastly, when the electrodes are set up normally (anode at the inlet, cathode at the outlet), the electroosmotic flow moves towards the cathode. But if the polarity is switched, the flow goes in the opposite direction, away from the outlet. In this case, only charged analytes that move faster than the electroosmotic flow will make it to the outlet.