Improving Peak Resolution when Using Capillary Electrophoresis

For research use only. Not for use in diagnostic procedures.

Answer

The overall charge within the capillary or the matrix has the greatest impact on resolution when using capillary electrophoresis (CE). With charge zonal electrophoresis (CZE) or other electrophoresis assays, the pH itself is more important because it dictates the number of charge zones (theoretical plates) within the capillary and how fast or slow the electroosmotic flow (EOF) will be if using a non-coated capillary. If using a gel polymer buffer, then more emphasis is placed on the polymers and their interaction with the analyte, as well as suppressing the EOF (unless a coating is used) to effect resolution. Capillary coatings added to the inner wall negate EOF, which then provides a more sample charge-based environment--whereby the molecule of interest's overall net charge will determine the ability for it to resolve well in an electric field.
       
Analytes that incorporate non-charged components (e.g., carbohydrates, other more hydrophobic moieties) also affect resolution and have to be considered when selecting a background electrolyte (BGE, buffer or gel polymer buffer); the BGE cannot  interact with any of the non-charged components. For example, carbohydrates can interact with SCIEX's CE-SDS gel and cause differentials in migration time (increasing them) due to the size of the molecule being different from the non-glycsoylated protein and its interaction with the polymers. Differentials in small molecules will depend on the change in charge they exhibit within a particular buffer. Proteins change with their folding properties. 

Another item to take note of is the aperture. For our CE instruments using UV detection, the smaller 200 µm aperture does have better resolution and can be used with a fixed wavelength or PDA detector.