Page 62 - Chiral Separation Techniques

38 2 Method Development and Optimization of Enantiomeric Separations Using …

2.3 Method Development with Glycopeptide CSPs

2.3.1 Method Development Protocols

The glycopeptide CSPs are multi-modal phases and can switch from one mobile
phase to another without deleterious effects. The new polar organic mobile phase
offers the advantages of broad selectivity, high efficiency, low back-pressure, short
analysis time, high capacity and excellent prospects for preparative-scale separation.
Whenever a racemic compound is targeted for separation, its structure can give a hint
as to which mobile phase should be investigated. If the compound has two or more
functional groups which are capable of interacting with the CSPs, and at least one of
these functional groups is near or on the stereogenic center, the new polar organic
phase is recommended to be tested first. In this context, functional groups include:
hydroxyl groups, halogens (I, Br, Cl, and F), nitrogen in any form (primary, sec-
ondary and tertiary), carbonyl and carboxyl groups as well as oxidized forms of sul-
fur and phosphorus.
In the new polar organic mode, the ratio of acid/base in the mobile phase affects
the selectivity and the concentration of acid and base controls the retention. It is sug-
gested to start the method development with a medium concentration (0.1 %) for
both acid and base. If retention is too long or too short, the concentration can be
increased to 1 % or reduced to 0.01 %. If no selectivity is observed in this mode,
reversed phase is recommended as the next step in the protocols.
In reversed phase, the selectivity and retention is affected by such parameters as
the type and percentage of organic modifier, the type of aqueous buffer, pH and con-
centration of buffer. Fortunately, there is an empirically optimal mobile phase com-
position for each glycopeptide phase. Vancomycin has shown its best performance
for most compounds when tetrahydrofuran (THF) is used as the organic modifier at
a concentration of 10 % with ammonium nitrate at a concentration of 20 mM and pH
5.5 (no pH adjustment is needed). Methanol seems to be the preferred organic mod-
ifier for teicoplanin and ristocetin A CSPs, and 20 % is usually a good starting com-
position. Triethylammonium acetate (TEAA) at the concentration of 0.1 % is a suit-
able buffer for both of these latter columns, although teicoplanin usually shows bet-
ter selectivity at pH 4.1 while ristocetin A has better selectivity at pH 6.8.
When analytes lack the selectivity in the new polar organic mode or reversed-
phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be
tested. Normally, 20 % ethanol will give a reasonable retention time for most ana-
lytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ris-
tocetin A CSP. The hexane/alcohol composition is favored on many occasions
(preparative scale, for example) and offers better selectivity for some less polar com-
pounds. Those compounds with a carbonyl group in the α or β position to the chiral
center have an excellent chance to be resolved in this mode. The simplified method
development protocols are illustrated in Fig. 2-6. The optimization will be discussed
in detail later in this chapter.