Chiral separations of multi-component natural product mixtures comprised of enantiomers, diastereomers, and unrelated molecules
are often difficult to resolve in a single isocratic method. Utilizing different chiral stationary phases can often provide
the selectivity needed for a multiple pass isolation approach.
A proprietary compound mixture recently screened consisted of multiple isomers and structurally similar components. Even though
this was not specifically an enantiomeric separation, we desired to develop chiral analytical and preparative methods under
SFC mobile phase. Upon initial screening, distinct differences between RegisPack® and Whelk-O1® were noted. Major differences
in retention times between key peaks of interest indicated a multi-pass method was needed.
Figure 1: Sample: proprietary. Column: (R,R) Whelk-O1, 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.
Under analytical SFC conditions the multi-component mixture resolved somewhat but not sufficient for a preparative separation
Figure 2: Sample: proprietary. Column: RegisPack 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Methanol 80:20. Flow rate: 4.0 mL/min.
Due to interfering peaks, isolation of the desired peaks at tr = 5.9 min and tr = 8.8 min would require a multiple chiral
stationary phase approach. Upon rescreening with the amylose based RegisPack chiral stationary phase we found enhanced resolution
and a much higher retention of the minor desired component now at tr = 14 min (Figure 2).
Figure 3: Sample: proprietary. Column: (R,R) Whelk-O1, 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.
Using a multiple pass approach, the minor desired peak was isolated in high efficiency using RegisPack with its purity confirmed
on Whelk-O1 (Figure 3). Reprocessing of the balance of the multi-component mixture using (R,R) Whelk-O1 allowed for the clean
isolation of the remaining desired major component at tr = 8.8 min.
Figure 4: Sample: proprietary. Column: (R,R) Whelk-O1 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.
Although SFC systems are optimally designed for collecting relatively pure binary mixtures, creative applications of multiple
chiral stationary phases can often allow one to isolate complex mixture components that would otherwise be isolated in poor
yield and purity. This application to a crude botanical extract is a typical example of how a comprehensive chiral screening
layout can help design an optimal preparative separation process.
Regis Technologies, Inc.
8210 Austin Ave., Morton Grove, IL 60053
tel. (847) 583-7661, fax (847) 967-1214