Separation of r-hirudin from similar substances by capillary electrophoresis

The thrombin inhibitor r-hirudin is a peptide of 65 amino acids and with a pI of 4.4. Capillary electrophoresis (CE) was used to separate r-hirudin from seven closely related substances which may be found as by-products or from degradation. Possibly two of these substances differ only in an isoaspartyl instead of an aspartyl binding. A baseline separation was possible with an acetate buffer (pH 4.4, 60 mM) containing 0.3% (m/m) PEG 20 000 and 0.1 mM Zn²⁺. The possibilities to prevent wall adsorption are discussed.