Page 68 - Chiral Separation Techniques

44 2 Method Development and Optimization of Enantiomeric Separations Using …

A rule of thumb has been developed after a large number of analytes were tested.
Once the selectivity was observed on the coupled column, a baseline separation can
always be achieved on a 25 cm column under optimized conditions. Since the
screening procedure already indicates the separation conditions, optimization is
straightforward and requires a minimum amount of time.
The new polar organic mode provides broad selectivity in the shortest analysis
time. Therefore, it is beneficial to start the screening process with the coupled
columns R+V+T in the new polar organic mode. The three coupled columns R+V+T
can then be directly switched to reversed phase with methanol as the organic modi-
fier. In case of high back-pressure, the coupled columns R+T could be screened and
vancomycin could be tested separately with THF as the organic modifier. In normal
phase conditions, it is advantageous to couple columns V+T and use ristocetin A as
the backup. When the column coupling method is applied, only four to five runs
(each run is within 25 min) are needed before a positive indication is reached con-
cerning the feasibility of a certain macrocyclic glycopeptide CSP and the corre-
sponding operating mode. At this point, either the separation can be optimized using
an individual CSP, or other types of CSPs can be screened.

2.4 Optimization

2.4.1 Effect of Flow Rate and Temperature on Enantiomeric
Separations

A general phenomenon observed with chiral stationary phases having hydrophobic
pockets is that a decrease of flow rate results in an increase in resolution.
This change has significant impact mostly in reversed-phase mode (see Fig. 2-10).

Fig. 2-10. The effect of flow rate on the
resolution of methylphenidate enantiomers
on vancomycin CSP (250 × 4.6 mm). The
mobile phase was methanol: 1.0 % triethyl-
ammonium acetate (95/5 v/v) pH 4.1 at
ambient temperature (23 °C).