Page 72 - Chiral Separation Techniques

48 2 Method Development and Optimization of Enantiomeric Separations Using …

An example of the effect of acid and base concentration on the separation of pro-
pranolol is shown in Fig. 2-13. In this case, the baseline separation is achieved by
adjusting the concentration without changing the acid/base ratio.

2.4.3 Optimization of Enantiomeric Separations in Reversed Phase

In addition to temperature and flow rate, the retention and selectivity in reversed
phase are controlled by: (i) the concentration and type of organic modifier; and (ii)
the type, concentration and pH of the buffer.

2.4.3.1 Effect of Organic Modifier on Enantiomeric Separations

Various organic modifiers have been used on the glycopeptide CSPs. Methanol,
ethanol, isopropanol, acetonitrile and tetrahydrofuran are the most common solvents
which give good selectivities for various analytes. Chiral separations on glycopep-
tide CSPs arc affected by organic modifiers in two ways: (i) the percentage of mod-
ifier in the mobile phase; and (ii) the nature of the modifier. Among the chiral com-
pounds that have been resolved in reversed phase on glycopeptide CSPs, especially
on teicoplanin and ristocetin A, a large number are α and β amino acids, imino acids
and small peptides [32–35]. For these molecules, alcohols (methanol, ethanol, and
isopropanol) prove to be effective modifiers. At the same percentage, the three alco-
hols have shown somewhat different selectivity and resolution with different amino
acids (Table 2-6). Unlike traditional reversed phase operations, an increase in the
concentration of alcohols results in longer retention and thus, better resolution (Fig.
2-14). Therefore, most amino acids and small peptides are resolved at medium per-
centage (around 50 %) of alcohols as organic modifiers.

Fig. 2-14. The effect of organic modifier on retention, selectivity and resolution of methionine on
–1
teicoplanin CSP (250 × 4.6 mm). The flow rate was 1.0 mL min at ambient temperature (23 °C).