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Excluding additives in chiral preparative separations

Mobile phase additives are commonly used in analytical chiral separations without trouble. But in preparative scale, additives make solvent recycling tricky and can cause re-racemization. By using Kromasil AmyCoat or CelluCoat it is possible to exclude additives in chiral preparative separations.

Background

Peak performance in analytical …

The use of acidic or basic additives in mobile phases used for the screening of chiral separation is very common. While the presence of an additive is no burden for analytical HPLC, additives make the solvent recycling a much more troublesome and can lead to re-racemization during the solvent evaporation.

Necessity of an additive for analytical separations Figure 1 illustrates the impact presence of 0.1% diethylamine (DEA) has on the peak shape of metoprolol.

chromatogram of analytical injection of metoprolol on Kromasil CelluCoat with and without additive

Figure 1: Separation of metoprolol on Kromasil CelluCoat 5 μm (4.6 × 250 mm). Mobile phase: heptane/2-propanol (90/10) left chromatogram (a) with 0.1% DEA, right chromatogram (b) without DEA. Flow rate: 1 ml/min. Detection: UV 223 nm.

The chromatograms above clearly confirm the necessarily to have a basic additive present for the successful analytical separation.

Scale-up

… may be weak performance in preparative

Often, the conditions that led to a successful analytical separation are brought along to the scale-up if a preparative separation the racemate is required. Figure 2 shows the separation of metoprolol under preparative conditions, with and without 0.1% DEA in the mobile phase.

chromatogram of preparative injection of metoprolol on Kromasil CelluCoat with and without additive

Figure 2: Separation of 40 mg metoprolol on Kromasil CelluCoat 10 μm (4.6 × 250 mm). Mobile phase: heptane/2-propanol (90/10) left chromatogram (a) with 0.1% DEA, right chromatogram (b) without DEA. Flow rate: 1 ml/min. Detection: UV 223 nm.

The detector signal shows almost no difference. These findings were confirmed by collecting fractions along the elution profile which were subsequently analyzed in order to allow calculating purity and yields based on the reconstructed elution profile displayed in Figure 3.

elution profiles from fraction anaysis for preparative injection of metoprolol on Kromasil CelluCoat with and without additive
RacematePurity [%]Yield [%]
110097.9
299.788.1
RacematePurity [%]Yield [%]
110099.5
299.597.4

Figure 3: Reconstructed elution profile for the preparative chiral separations of metaprolol on Kromasil CelluCoat 10 μm, with (0.1%) and without DEA as mobile phase additive

Conclusion

The study shows that there is a big potential to exclude additives in preparative purifications using Kromasil. The need for additive differs between manufacturers of CSP even though the phases are similar in theory (same selector supported on a silica matrix).

Overloaded injections are needed early in method development in order to decide if mobile phase additive is needed or not.

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