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
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.
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.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC