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CHROMATOGRAPHY
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analyzed with coupled chiral and achiral columns. The recommended process for a screen to achieve a one-step chiral/achiral separation is performed by using conditions from the optimal isocratic chiral method (with acquisition time doubled from 5 to 10 minutes) applied to each of the four diff erent achiral columns in series with an Amylose chiral column (see Figure 3). The retention of enantiomers and that of the achiral compound are aff ected diff erently by the achiral column upstream of the CSP. While chiral selectivity is maintained after elution from the coupled columns, the unique selectivity of each achiral column results in a diff erent chromatographic profi le. Figure 3 data for the Nitro column shows Chlormezanone and Chrysin both retained more strongly than with other achiral columns applied, Chrysin so strongly retained it does not elute with the same isocratic method. DEAP also strongly retains Chrysin although it elutes within the 10-minute run time. With the 4-EP and Imidazole columns, Chrysin is less retained but coelutes with the second-eluting enantiomer of Chlormezanone. However with the HILIC column coupled in this example, there is good selectivity for all three peaks. With a suitable preparative scale up, enantiomers of Chlormezanone may be effi ciently isolated free from contamination with Chrysin.
Coupled Column Screen with Synthetic Racemate and Achiral Impurity
The result of a case in which the achiral/chiral column screen was performed is illustrated in Figure 4.
additive) + 80% CO2 A signifi cant achiral impurity
was present in this sample and insuffi cient separation was found in chiral screening. From the screen, the best isocratic method for chiral resolution was 20% methanol (20 mM NH3
on a
4.6 x 100 mm immobilized Amylose 3,5-dimethylphenyl carbamate column.
Using these conditions and coupling each of the four selected achiral phases with the CSP, an advantageous selectivity of
Figure 4. Comparison of 4 diff erent achiral columns coupled with immobilized Amylose 3,5-dimethylphenyl carbamate for chemistry chiral sample with large impurity using the isocratic method 20% methanol (20 mM NH3) / 80% CO2
at 4 mL/min.
enantiomers from the achiral impurity was sought. The zoomed-in chromatogram view Figure 4 reveals varying selectivities for Silica, Pyridyl Amide and HILIC, but none completely resolving the impurity from enantiomers. However the bottom chromatogram representing a coupling of the imidazole phase with immobilized Amylose reveals baseline resolution of both enantiomers from the impurity, the latest eluting peak. This is exactly the result sought from the extra screening step allowing the purifi cation of these enantiomers to be achieved in one preparative step.
Preparative SFC Comparison – Coupled vs. Two-stage Separation
To illustrate the impact of coupling columns at the preparative stage, the racemic pharmaceutical compound Diperodon was spiked or “contaminated” with 10% caff eine by weight.
The coupled column
analytical screening indicated an achiral resolution was possible at conditions which resolved the enantiomers, so a preparative scale SFC experiment with relevant columns was carried out. At the top of Figure 5 a preparative mode stacked injection sequence [12] designed to achieve a fast cycle chiral resolution for the mixture is shown. It is accepted that no achiral resolution would be achieved in this step so with favorable chiral separation conditions for an immobilized Cellulose tris (3,5)-dimethylphenyl carbamate column, the caff eine peak mostly overlaps with the front edge of the fi rst eluting enantiomer using this method (see Figure 5). The collected fraction 1 required evaporation after collection and another preparative process to resolve caff eine from enantiomer 1 isolated in the chiral separation step. On the right in the fi gure is the achiral repurifi cation trace of fraction 1 which was required following the chiral-only separation initially performed.
Figure 3. Comparison of 5 diff erent achiral columns coupled with Amylose tris [(S)-α-methylbenzylcarbamate for chiral/ achiral separation of Chlormezanone with Chrysin “impurity” using isocratic method 25% ethanol (20 mM NH3) / 75% CO2
4 mL/min. at
All the time taken for the second step is unnecessary utilizing the coupled column concept in this example. Using these same mobile phase conditions and cycle time, but with a 15 cm Pyridyl Amide column coupled with a 20 mm length of 0.020” stainless steel tubing to the 25 cm immobilized Cellulose column, the chromatographic
94 | | September/October 2013 - 15TH ANNIVERSARY ISSUE
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