Chiral HPLC Method Development
Chiral methods stand head and shoulders above all other HPLC methods in terms of the amount of experience and expertise required for their development.
Chromatographic behavior of optical isomers cannot be easily predicted, since it follows the rules, which are quite different from those applicable to non-chiral compounds. Fortunately, I.B.S. has necessary experience and capacity to develop the most sophisticated chiral HPLC methods.
We have developed the state-of-the approaches for chiral separation, which are applicable in any HPLC mode, and suitable for any organic molecule. This know-how allows estimating the probability to achieve separation of a given chiral compound on a given stationary phase (CSP) on the basis of the analyte’s chemical structure.
A number of failures to achieve an appropriate chiral separation usually arise from using the normal-phase mode as the only possible approach for the chiral HPLC method development, neglecting other promising ones. By contrast, in I.B.S. we are capable of applying the variety of single and mixed LC modes to develop a chiral separation, as follows: RP, RP/CT, NP/CT, HILIC, IC, HILIC/CT. Thus, to achieve chiral separation, the full potential of liquid chromatography is being exploited.
Besides poor separation, many other problems usually are come across while developing chiral normal-phase separations, as follows:
- poor dissolution of a polar analyte in the mobile phase (this is relevant for a number of amides, esters, schiff bases);
- rapid degradation of analytes caused by traces of water or amines present in the mobile phase;
- rapid racemization of analytes caused by traces of water or amines present in the mobile phase, or by polar solvents like DMSO, DMF;
- poor normal-phase separation robustness for a basic or acidic analyte.
We are aware of all these possible problems and know how to handle them.
In some cases a chiral substance to be separated is contained within a complex matrix. It makes the direct separation on a single column nearly impossible.
Figure 1. Chiral HPLC Separation of Terizidone enantiomers developed by I.B.S.
Figure 2. Chiral HPLC Separation of Terizidone enantiomers developed by a competitor.
This calls for a more sophisticated approach named the ‘heart cutting’ column switching technique. A sample is first separated on the first HPLC column to achieve preliminary separation between the analyte and contaminants. The analyte containing aliquot is than transferred to the second chiral column in order to separate optical isomers under appropriate conditions.