Effect of electrolyte pH on CIEF with narrow pH range ampholytes

Area of Science:

• Analytical Chemistry
• Separation Science

Background:

• Capillary isoelectric focusing (CIEF) is a powerful separation technique for amphoteric compounds.
• Sequential injection of ampholytes and sample zones offers unique analytical advantages.
• Separating compounds with isoelectric points (pIs) outside the ampholyte pH range presents challenges.

Purpose of the Study:

• To investigate the effect of anolyte and catholyte pH on CIEF selectivity and speed.
• To enhance the resolution of amphoteric compounds through optimized injection protocols.
• To explore the migration behavior of components with pIs outside the applied ampholyte pH range.

Main Methods:

• Utilized sequential injection of ampholytes and sample zones in uncoated capillaries.
• Employed three ampholyte solutions covering a two pH unit range for substituted nitrophenol dye analysis.
• Varied anolyte and catholyte pH to assess impact on isoelectric focusing performance.

Main Results:

• Anolyte and catholyte pH significantly influenced resolution and pH gradient length.
• Observed changes in the charge state of components with varying pH conditions.
• Demonstrated that components with pIs outside the ampholyte range can migrate in a charged state.

Conclusions:

• Optimized CIEF protocols, particularly anolyte/catholyte pH, enhance separation selectivity and speed.
• The migration of charged components outside the pH gradient is advantageous for hyphenation with mass spectrometry (MS).
• This approach mitigates ion suppression effects from ampholytes in CIEF-MS coupling.