Methods and materials
All instrumentation used in this work was acquired from Waters Corp. The systems included Thar AMDS analytical SFC systems,
Thar Prep80 preparative SFC systems, Waters Alliance 2795 HPLC systems, Waters 996 photodiode array detector, and Waters ZQ
single quadrupole mass spectrometry (MS) detector. All solvents were HPLC grade. Buffers and solvent additives were supplied
by Sigma-Aldrich. Column sources are indicated with the specific methods as further described.
Enrichment. Method A. The RP-HPLC method to identify and assign purity to the target impurity isolate used a Halo C18 4.6 × 150 mm 2.7-µ column
(Mac-Mod Analytical). Solvent A was water (0.1% trifluoroacetic acid [TFA]). Solvent B was acetonitrile (0.1% TFA). The solvent
gradient was 17% B over 12 min, 17–100% B over 3 min, 1-min hold, and 3-min recycle. The impurity at 9.0 min has an enhanced
absorbance relative to the main peak when detected at 260 nm.
Method B. The intermediate SFC method to collect an enriched fraction used an (S,S) Whelk-O1, 30 × 250 mm, 5-µ column (Regis Technologies). The preparative method was isocratic, 10% methanol in CO2 at 80
g/min and 120 bar pressure; 300 mg of feed were injected every 2.1 min for a productivity rate of 8.5 g/h.
Method C. For final purification of the target from the enriched sample, a new SFC method was developed using a RegisPack 4.6 × 100
mm 5-µ column (Regis Technologies). The isocratic method used 13% methanol:isopropanol (50:50) in CO2; 240 mg of enriched
fraction were processed in eight injections over 30 min.
Direct Isolation. Method D. The client's HPLC method was used to assess the stress degradation mixture, using a Waters XBridge Phenyl 4.6 × 150 mm 5-µ
column. Solvent A was 10 mM ammonium formate in water adjusted to pH 4.0. Solvent B was 10 mM ammonium formate in methanol:water
(90:10) adjusted to pH 4.0. The gradient elution at 1 mL/min used a solvent gradient 10–40% B over 30 min, 40–100% B over
5 min, and 4-min hold.
Two isolation strategies are commonly used in the authors' laboratory—enrichment and direct isolation. In the enrichment strategy,
an SFC method is developed that appears to resolve the main peak (usually the active ingredient) from neighboring peaks, and
the main peak is captured and removed to produce an enriched fraction containing all other compounds, including the desired
degradants. This approach takes advantage of the speed with which SFC can process a feedstock solution of the sample and can
be used even when the trace target peak(s) is/are not visible under preparative chromatography conditions due to limited detection
range at high loads. Using the direct-isolation strategy, a specific SFC method is developed that resolves the desired trace
peak, and that peak is collected as a purified isolate. This approach requires that the desired peak be visible with a specific
detection signature (e.g., a specific UV absorbance wavelength or a mass not shared by other compounds in the mixture). Enrichment
and direct isolation are frequently used together, and complex projects involving multiple isolates may require their sequential
application. For example, once an enriched fraction containing the desired peak is obtained, the desired peak is often an
abundant component in a fraction that becomes a small feedstock fraction for direct isolation.
In the authors' laboratories, a process termed "targeted isolation" is used to rapidly develop methods for purification of
single peaks from complex mixtures. Targeted isolation involves linking specific information, such as the UV absorbance spectrum
or mass spectral data acquired in RP-HPLC analysis, to normal phase SFC chromatograms. This approach is used to cross-correlate
peaks as analysts rapidly develop SFC methods—initially, gradient elution and finally, isocratic methods, to be scaled to
preparative chromatography. While this article does not discuss all the aspects of targeted isolation, the parts of the process
used to "close in" rapidly on the peak of interest are discussed. Case studies that illustrate these strategies and discuss
the merits and advantages of SFC technology in their application are presented.