[Reader Insight] A Brief Discussion on CMPA Method for Enantiomeric Separation: Some Practical Insights

This article is submitted by expert chromatographer LI Ang. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.

The separation of enantiomers can be achieved through direct and indirect methods. The direct separation method, as the name implies, involves separating enantiomeric drugs directly without derivatization. It can be done using either chiral stationary phases (CSP) or chiral mobile phase additives (CMPA).

The CSP method involves bonding a chiral selector to a support to form a chiral stationary phase with high stability and reproducibility, making it a convenient and widely used technique.

However, if multiple types of chiral columns are not available during method development, the separation of chiral compounds may be limited. In such cases, the CMPA method can be a viable alternative for enantiomeric separation.

The CMPA method involves adding a chiral selector to the mobile phase and using a standard column to separate chiral compounds. Its advantages include the use of a standard non-chiral column, ease of operation, and the ability to optimize separation conditions by adjusting the type, concentration of CMPA, and mobile phase composition.

However, the CMPA method has drawbacks, such as lower chromatographic stability and longer equilibration times. Nonetheless, depending on the circumstances, the CMPA method is worth considering as an approach for chiral separations.

The separation mechanism can be understood as follows: due to the differing spatial configurations of enantiomers (R/S), each enantiomer interacts with the chiral selector to a different extent, leading to variations in their partitioning between phases. This causes differences in their retention factors (k), enabling separation.

Cyclodextrins are commonly used chiral additives and are primarily classified into α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and their derivatives. In chiral separation models, cyclodextrins can be viewed as “bottomless bucket” structures, with α, β, and γ types representing small, medium, and large buckets, respectively.

The inner cavity of the bucket is hydrophobic, while the top and bottom edges are hydrophilic. Hydrophobic groups of the compound enter the cavity first, while hydrophilic groups interact closely with the top and bottom edges via hydrogen bonds and other forces. During this process, the extent of interaction between cyclodextrin and each enantiomer (R/S) differs, facilitating separation.

The size of the cavity and its interaction with the nonpolar groups of the analyte are key factors for enantiomeric separation. Small groups are suitable for α-cyclodextrin-type additives, medium-sized groups (such as benzene rings and heterocycles) for β-cyclodextrin, and large groups (such as steroids) for γ-cyclodextrin additives.

In addition, there are numerous cyclodextrin derivatives available. Using these derivatives as additives not only adjusts the size of the inner cavity but also provides different hydrophilic groups, resulting in relatively distinct selectivity.

The target analyte contains nitrogen-oxygen heterocycles and amino groups. Testing revealed that these structural fragments are best separated using the β-cyclodextrin series.

The target analyte has high polarity and contains basic groups. Therefore, a C18 column with end-capping and water resistance (4.6×250 mm, 5 μm) was chosen. Various cyclodextrins and their derivatives were screened under the following chromatographic conditions:

After screening, hydroxyethyl-β-cyclodextrin proved to be the most effective chiral additive. However, the analyte’s retention factor (k) was too low. To achieve better retention, a HILIC mode was employed with the following chromatographic conditions: