Optimization Strategy for Purification of a Peptide from Complex Mixtures by Reversed‐Phase Liquid Chromatography with a Back‐Flushing Technique

Abstract

An optimization strategy for the separation and purification of a peptide from crude material by reversed‐phase liquid chromatography was developed. First, in the separation step, the tetrahydrofuran, instead of normal organic modifiers in the mobile phase, could improve the separation of the target peptide from its impurities; hence, the mass loading could be increased in each run. Furthermore, a back‐flushing technique was introduced to elute the strongly retained impurities rapidly and economically. A raw mixture of 10 mg was purified on an analytical column by an overloading operation in one run, using a mobile phase consisting of tetrahydrofuran‐water (30:70, v/v)+0.1% TFA at a flow‐rate of 1.0 mL/min and room temperature. The purity of the product was increased from 50% to 98% with recovery over 80%. The back‐flushing step was very crucial to regenerate the system for the next run in the sense of both cost and time.

Keywords:

• Peptide
• Separation
• Tetrahydrofuran
• Preparative chromatography
• Displacement effect
• Back‐flushintg