Enantioselective Reversed-Phase Chromatography with Protein-based Columns

February 1, 2010

The Application Notebook

The Application Notebook, The Application Notebook-02-01-2010, Volume 0, Issue 0

Excellent reversed-phase separations using a protein-based column, CHIRAL-AGP, with volatile LC–MS compatible buffer systems have been obtained for a range of basic solutes.

Geoffrey B. Cox1, Norbert M. Maier1, Tong Zhang2, and Pilar Franco2, 1Chiral Technologies, Inc. and 2Chiral Technologies, Europe

Reversed-phase chromatography, being utilized successfully in achiral separations, can also be applied to the separation and analysis of chiral compounds. One important area of such applications is analyses of drugs and their metabolites in physiological fluids and samples of biological origin by the combination of reversed-phase chromatography and LC–MS.

This note describes use of a CHIRAL-AGP column in conjunction with a volatile mobile phase buffer appropriate for analysis and detection by LC–MS.


Agilent 1100 and 1200 HPLC systems were used for the study. Columns (150 × 4 mm) were packed with CHIRAL-AGP. Volatile mobile phases were based on 10 mM ammonium acetate buffered to a suitable pH with acetic acid and with added 2-propanol as modifier.


An important goal of the work was to simplify the development of separation methods, mainly focusing on selection of mobile phase combinations appropriate for MS detection. Mobile phases based upon ammonium acetate were found to give excellent selectivity for a wide range of solutes. Table I shows the retention and selectivity for several basic compounds, mainly beta-blockers and related molecules, using a CHIRAL-AGP column with 1% 2-propanol in 10 mM ammonium acetate at pH 5. Retention and selectivity may be adjusted by changes in pH and the concentration and nature of the modifier.

Table I: Retention, Selectivity and Resolution

The chromatogram shows the high performance and the good peak symmetry characteristic of the CHIRAL-AGP column. Further increase in performance (albeit at the expense of separation time) may be achieved by reduction in flow rate. Reduction of the flow rate by a factor of 2 increased the resolution of the four peak pairs by an average of 34%.

Figure 1


Protein-based chiral HPLC columns may be used in a wide range of reversed-phase applications. The columns in conjunction with ammonium acetate-based mobile phases have been shown to give excellent selectivity together with LC–MS compatibility. Many existing applications using other buffers can be easily modified to use ammonium acetate, thus simplifying development of analytical protocols for the protein-based chiral phases.

CHIRAL-AGP is a trademark of Chiral Technologies, Europe.

Chiral Technologies, Inc.

800 North Five Points Rd, West Chester, PA 19380

tel. 1-800-6-CHIRAL

Website: www.chiraltech.com