The Application of C18AQ Columns In the Purification of Strong Polar Peptides

Rui Huang, Bo Xu
Application R&D Center

Introduction
A peptide is a compound composed of amino acids, each of which has unique physical and chemical properties due to the different types and order of amino acid residues constituting its sequence. With the development of solid phase chemical synthesis, the chemical synthesis of various active peptides has made great progress. However, due to the complicated composition of the peptide obtained by solid phase synthesis, the final product should be purified by reliable separation methods. The commonly used purification methods for peptides include ion exchange chromatography (IEC) and reversed-phase high performance liquid chromatography (RP-HPLC), which have the disadvantages of low sample loading capacity, high cost of separation media, complicated and costly separation equipment, etc. For the rapid purification of small molecule peptides (MW < 1 kDa), a successful application case was previously published by Santai Technologies, in which a SepaFlash RP C18 cartridge was utilized for the rapid purification of thymopentin (TP-5) and the target product meeting the requirements was obtained.

Figure 1. 20 common amino acids (reproduced from www.bachem.com).

There are 20 kinds of amino acids that are common in the composition of peptides. These amino acids can be divided into the following groups according to their polarity and acid-base property: non-polar (hydrophobic), polar (uncharged), acidic or basic (as shown in Figure 1). In a peptide sequence, if the amino acids constituting the sequence are mostly polar ones (as marked in pink color in Figure 1), such as Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, etc. then this peptide might have a strong polarity and be highly soluble in water. During the purification procedure for these strong polar peptide samples by reversed-phase chromatography, a phenomenon called hydrophobic phase collapse will occur (refer to a previously published application note by Santai Technologies: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Their Applications). Compared with the regular C18 columns, the improved C18AQ columns are most suitable for the purification of strong polar or hydrophilic samples. In this post, a strong polar peptide was utilized as the sample and purified by a C18AQ column. As a result, the target product meeting the requirements was obtained and could be used in the following research and development.