Case study 2: direct isolation
A stress degradation product was partially purified by low-pressure chromatography to remove much of the remaining main drug
peak. The degradant-enriched mixture had two significant peaks of interest (primary targets) and several of minor abundance
(secondary targets), in addition to components not of interest to the chemistry team. The HPLC chromatogram of the mixture
is shown in Figure 4. The goal for the isolation was to capture cleanly each of the primary targets for structure elucidation and to capture the
secondary target peaks if it were not "overly difficult." Preparative RP-HPLC was undesirable due to poor loading and excessive
Figure 4: Reversed-phase high-performance liquid chromatography (UV detection 295 nm, Method D) of degradant-enriched sample,
showing primary (light red) and secondary (light green) purification target peaks.
The development of an SFC method specific for a targeted minor component is often complicated by the lack of availability
of an SFC method identifying the peak of interest. Frequently, stability-indicating methods identifying minor API components
are RP-HPLC gradient-elution methods. Thus, the initial phase of method development is aimed at identifying the peak of interest
in an SFC chromatogram, followed by an optimization phase to resolve that peak from its neighbors. The final isocratic method
is scaled to a preparative column so that the peak may be accumulated by injection stacking.
Figure 5: Method development and fractionation tree for the recovery of the targets identified in Figure 4.
To facilitate the sometimes difficult process of correlating the peak in the RP-HPLC method with SFC, the authors previously
developed a co-configured HPLC/MS and SFC/MS system (6) that uses the same mass spectrometer as a detector for either chromatography.
Using this system, masses can be assigned to mixture components in both the RP-HPLC trace and the SFC chromatogram. This cross-correlation
of orthogonal reversed-phase and normal-phase peak profiles, and the subsequent development of an appropriate preparative
chromatography method to cleanly capture a target peak is part of the targeted isolation process. The standard HPLC method
is still used to confirm the successful isolation of each component (see Figure 3).
This approach to separation is straightforward, but appears cumbersome at first glance. Due to the rapidity with which isocratic
SFC methods may be developed, there is little need to spend time and effort to develop an optimal method first. A complex
sample may be sectioned into several fractions (in this case four), which are then examined using the standard HPLC method
for the presence of one or more of the targets. Once a fraction contains only a few components, the target peaks often can
be selected based on relative abundance, mass (as measured by the MS detector), or by a specific UV signature. Highly specific
methods are then developed to cleanly resolve the targets. At the end of a multicomponent isolation project, a method and
fraction tree illustrates the work flow of the complete separation (see Figure 5). Individual isolates highlighted in the figures were assayed using Method D, and deemed sufficiently pure for structure elucidation.
Table I: Supercritical fluid chromatography methods used in the target peak isolation tree, numbered according to Figure 5.
The SFC methods needed to isolate the targets are detailed in Table I. The seeming complexity of the process is belied by the speed with which the work was accomplished—in this case, less than
three weeks of effort during a period when concurrent project work also demanded laboratory resources. The rapidity derives
from the efficiency of method development, processing, and recovery from liquid fractions when using SFC; all of these facilitate
the rapid cycle time from peak to method to isolation.
The two strategies take advantage of different key attributes of SFC. The enrichment strategy leverages the speed of processing
in SFC versus HPLC, achieved through increased loading, narrow bands, and high linear solvent velocities. SFC has often been
described as 3–10 times faster than HPLC (5).
The direct isolation strategy leverages the processing speed advantage as well, but in comparison with HPLC, the speed of
method development offers a significant additional process enhancement. To achieve the same resolution in HPLC, one frequently
uses a longer column with a higher plate count. In SFC, the linear mobile phase velocity is 8–20 times that of an equivalent
HPLC method, as short columns and high flow rates are both commonly used in method development and these methods are arithmetically
scaled to larger column formats (7). Moreover, this translates into extremely fast column equilibration times, meaning that
as many as 20 different SFC methods may be evaluated for every one HPLC scouting run. In addition, it was observed that the
highly concentrated fraction volumes in SFC offer a significant advantage, i.e., the ability to quickly remove solvent and
analyze the isolate significantly speeds the cycle through processing and method development.
Finally, it was noted that the lack of oxygen and water in the SFC solvent environment limits many common degradative pathways
that would otherwise prevent the capture of certain primary degradants. Less nucleophilic alcohols, such as isopropanol and
the butanols, usually offer good selectivity and have been used when methanol reactivity is a concern. SFC may offer a greater
chance of success when the goal is to isolate primary degradants intact without secondary decomposition.