» CHROMATOGRAPHY
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[10], a screening process of candidate columns can be employed. An apparatus for automated coupling of analytical columns SFC system by Welch et al. [11] has been described for coupling of chiral column pairs. A similar setup for coupled chiral-achiral screening can likewise be employed. A suitable pre-purifi cation analysis resulting from this arrangement can lead to a single step isolation of enantiomers from achiral impurities in a coupled column preparative step.
Results and Discussion
Achiral Column Set Selection Experiment A general gradient analysis performed with an analytical SFC/ MS system on eight achiral stationary phases was used to plot the retention data of a set of 70 diverse pharmaceutical compounds on a standard test plate. The phases tested were Silica, HILIC (cross-linked Diol), Nitro, DEAP (diethylaminopropyl), Pyridyl Amide, Imidazole, 4-Ethylpyridine and 2-Ethylpyridine, all 4.6 x 100 mm, 5 micron. The linear gradient program used for this achiral column evaluation was: 5 – 60% modifi er (methanol with 20mM NH3) in 3 min., held at 60% for 0.67 min., lowered to 5% over 0.33 min., and held for re-equilibration at 5% for 1 min. The columns were installed in various combinations on a software controlled 6-channel column selector on board an external autosampler for this experiment (see Figure 1). The SFC system outlet pressure was fi xed at 100 bar, column temperature 400C, and total fl ow rate was 4.0 mL/min.
Assembling all retention times from the 70 compounds yields a frequency distribution used for comparison of the various achiral phases tested. Shown in Figure 2 is the distribution found for six of the columns studied. (Two of the other phases tested had many cases of standards not eluting, 15 in the case of Nitro, four for DEAP. These were deemed unsuitable for the application and are not represented in this fi gure.) Ideal properties for columns to be included in a general screening set are diff erences among compounds’ retention from
Figure 2. Histograms of retention times obtained from SFC gradient analysis of 70 diverse standard pharmaceutical compounds on each of six achiral columns arranged in 0.5-minute wide bins.
other columns in the set, relatively few cases of over-retention, tR 4 min. with this gradient, and a limited number under-retained, tR
> <
2 min. These properties increase the likelihood of fi nding alternative selectivity among the columns for arbitrary compounds to be encountered in the future.
In Figure 2, the histogram for the 2-ethylpyridine column “2-EP” exhibits overall shorter retention for the set shifting the retention distribution signifi cantly to the left compared with those of other phases. Although 2-ethylpyridine is one of the most widely used for achiral SFC, this data deems it a poor choice to include in an achiral screening set since it implies that two random compounds are more likely to have similar retention. The column is thus less likely to produce the added selectivity one may need in a complementary separation for this application.
exhibited the most similarity among specifi c compound retentions in the study, Pyridyl Amide was chosen to be kept in a fi nal four-column screening set (Silica, HILIC, Imidazole and Pyridyl Amide) as 4-EP added the least diversity to the group.
Coupling Diff erent Achiral Phases with One CSP
Having determined this set of achiral columns, each can be screened by coupling with the chiral column and mobile phase suitable for the racemate separation.
For example, if a good chiral separation
Figure 1. Coupled column system diagram. Flow through each of two column channels in series is possible with this fl exible setup on the SFC/MS analytical system. Bypass lines
are included in each column switching valve to allow for single column analyses as needed.
was achieved with 20% methanol modifi er on CSP #1, then achiral columns 1, 2, 3 and 4 would be individually coupled with that CSP and run sequentially with the 20% isocratic method, simply doubling the analysis time. The data from each of the four coupled methods would be compared to fi nd the most favorable separation conditions accounting for both resolution of the impurity from enantiomer peaks as well as peak shapes resulting from the two-column separation.
To illustrate the case of a chiral separation with an interfering achiral impurity, a racemate, Chlormezanone, was spiked with Chrysin and
92 | | September/October 2013 - 15TH ANNIVERSARY ISSUE
Since Pyridyl Amide and 4-ethylpyridine “4-EP”
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