Isolation of high‐purity peptide Val‐Val‐Tyr‐Pro from Globin Peptide using MCI gel column combined with high‐speed counter‐current chromatography

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high‐speed countercurrent chromatography was developed for the separation of high‐purity peptide Val‐Val‐Tyr‐Pro from Globin Peptide. First, the fraction of Val‐Val‐Tyr‐Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high‐purity Val‐Val‐Tyr‐Pro was separated by high‐speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso‐propyl alcohol/(NH 4 ) 2 SO 4 Saturated solution /H 2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high‐speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78 mg of Val‐Val‐Tyr‐Pro was successfully purified with the purities of 98.80% from 30 g crude Globin Peptide. The amino acid sequence of the Val‐Val‐Tyr‐Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large‐scale separation of pure peptide Val‐Val‐Tyr‐Pro from Globin Peptide, and provides a reference method for obtaining high‐purity peptide from other polypeptide mixtures.