Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC - - Chromatography Online
Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC

LCGC Europe
Volume 25, Issue 11, pp. 600-611

Figure 7: Dependence of peak efficiency and resolution degree on flow rate. Compound 11 (N-benzoyl-DL-alanine); (a) peak-2 Column: QN AX; Modifier: 35% (MeOH/FA/NH4OOCH 100/0.40/0.35 v/v/m)
From Gradient to Isocratic: The first screening of a series of acidic compounds (Figure 4) was performed by running a SFC gradient programme (Table 2) using a mixture of MeOH/FA/NH4OOCH 100/0.40/0.35 v/v/m as the modifier. Results from the gradient runs allowed easy targeting of the modifier percentage to be used in isocratic mode and compounds to be eluted in a reasonable time window.

Table 3: SFC results with and without formic acid (FA) in the modifier. Column: QN AX, Modifier: MeOH/NH4OOCH 100/0.35 v/m + FA.
Figure 5 shows the examples of straightforward method transfer from gradient to isocratic conditions. Effects of Mobile Phase Additives: In HPLC, the typical working conditions on QN AX and QD AX columns are with weakly acidic mobile phases (pH 4–7) (6). Under such conditions, the chiral selector is protonated at the quinuclidine ring (Figure 1) and the acidic analytes are dissociated. An ion-exchange mechanism is thus activated between the positively charged chiral selector and the negatively charged analyte molecules.

Table 4: Effect of the salt concentration in SFC mode. Column: QN AX; Modifier: MeOH/FA 100/0.40 v/v + NH4OOCH.
As shown in Table 3, the chiral recognition occurs without external addition of an acidic additive to the methanol/ammonium formate solution. The enantioselectivity was mainly unaffected by the absence or presence of the acidic additive and its concentration [Figure 6(b)]. For most molecules, the addition of FA did not induce notable loss in resolution degree (Table 3). The presence of FA in the mobile phase provided the benefits of reducing the analysis times to a significant extent, especially when exceeding the level of 0.20% [Figure 6(a)]. It is assumed that FA played the role of the counter ion, inducing a competitive effect and favouring the mass transfer kinetics of the chromatographic process.

Table 5: SFC enantiomer separation employing strong eluting conditions. Column: QN AX; Modifier: MeOH/FA/NH4OOCH 100/0.60/0.50 v/v/m.
The ion-exchange chromatographic process is dependent not merely on the ionization state of the chiral selector and the analyte molecules, but also on the ionic strength of the mobile phase. Addition of a certain amount of a suitable salt into the mobile phase can efficiently modulate the ionic strength of the mobile phase and regulate the adsorptiondesorption process between the analyte and the chiral selector. In practice, it is preferable to choose a salt of high solubility in the modifier or the mobile phase, high volatility for the potential LC–MS hyphenation and high UV transparency to ensure good UV detection of the analytes. NH4OOCH meets all these requirements.


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