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 the resources and capacity to develop the most sophisticated chiral HPLC methods.
1. Based on the experience gained in developing chiral HPLC methods, we have developed the proprietary 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.
2. Besides poor separation, many other problems may be come across while developing a chiral HPLC separation in frequently used normal-phase mode using hexane-based mobile phases (MPs), as follows:
- poor dissolution of a polar compound used as an analyte in the MP (for example, amides, esters, schiff bases dissolve in hexane quite poorly);
- rapid degradation of an analyte caused by traces of water or amines present in the MP;
- rapid racemization of an analyte caused by traces of water or amines present in the MP, or by polar solvents used as polar mobile phase additives;
- poor robustness of a chiral normal-phase separation developed for a basic or acidic analyte.
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.
We are aware of all these possible problems and know how to handle them.
3. Many failures to achieve an appropriate chiral separation may 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.
4. In many cases a chiral substance to be separated is contained within a complex matrix. It makes the direct approach to analytes’ HPLC determination on a single column nearly impossible. 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 of the analyte under appropriate conditions.
‘Heart cutting’ 2D-HPLC is also the area of our expertise; we can provide column switching HPLC solutions for chiral separation with use of any types of chiral stationary phases (CSPs).