CO2_2016 - page 27

Chimica Oggi - Chemistry Today
- vol. 34(2) March/April 2016
more classical peak shape
is maintained for the minor
isomer. The overlap of the larger
peak with the small is reduced
relative to what it would be if a
racemate were loaded to the
same level.
This strategy works best when
the major peak is the early
eluting one. In isocratic
chromatography, the early
eluting peaks are retained
for less time on column and
undergo less diffusional band
broadening. A sharper
peak is collected at a higher
concentration in less solvent
because most of the mass of
the eluting compound is in
the center cut of the band. A
broad peak requires collection
across a broad window to
recover at high yield, and this
often means it is difficult to
limit overlapping of bands in
injection stacking.
It is frequently possible to reverse
elution order of enantiomers
by changing the selectivity of
a chiral interaction. Pirkle-type
columns capitalize on pi-bond
interactions with the phase,
and are available in both (R)
and (S) configurations from
manufacturers (7). Changing
the elution order is a matter
of changing to the opposite
phase. In our experience,
however, chiral selectivity may
often be reversed through
appropriate choice of stationary
and mobile phase. Figure
4 illustrates an elution order
change in an SFC system
mediated only by the choice
of a simple alcohol as the CO
Because enriching E1
in a feedstock offers an
advantage in processing
speed, we set out to determine whether performing
an enrichment step chromatographically prior to final
processing – in other words, a two-pass chromatography
strategy – might actually be faster than a single step. We
used warfarin (Sigma-Aldrich, St. Louis MO) as a feedstock,
which is an idealized model compound – available at high
purity as a racemate, and highly separable with good
selectivity on many chiral stationary phases.
Warfarin racemate was loaded on column (ChiralPak
in peak tailing which may
be severe enough to inhibit
resolution of enantiomers even
when the phase is selective,
The compound shown in Figure
3 is insufficiently resolved for
chromatographic resolution,
but the FMOC- and tBOC-
protected compounds are both
resolved. The Boc-protected
molecule gives and excellent
separation at high load due to
the symmetric peaks (a sign that
silanol effects are non-existent)
and good selectivity.
Protecting primary amines
with easily removed, sterically
bulky groups to improve
chromatography is part of the
essential toolbox of amino acid
analysis, developed in the early
years of proteomics research(6).
With chiral molecules, it is
important to demonstrate at a
small scale that deprotection
(generally by treatment with an
acid) does not result in inversion
at the chiral center. Therefore
we prefer to prove the strategy
in steps, first protecting the
molecule on a small scale and
develo sping an appropriate
chiral chromatography
resolution. The small sample is
resolved and each fraction’s
chiral purity measured, then
the sample is deprotected. If a
method that resolves the free
base is available, we analyze
for inversion. If not, we must re-
protect the isolates and analyze
A similar strategy may be used
with acids, generally using
esterification and saponification.
Finally, it is worth noting
that another advantage of
this approach is that a UV-
absorbent chromophore may
be introduced to a molecule
that has little UV activity,
facilitating chromatographic
A feedstock enriched in one enantiomer over the other may
usually be processed at a higher rate or efficiency. Preparative
batch chromatography is often best accomplished by
overloading the sample to a point where resolution is lost
to some degree, but selectivity is maintained. Overloading
an enriched sample generally results in a poor peak shape
(broad or tailing) for the enriched isomer, while a sharper and
Figure 3.
Comparison of “best screeing condition” chromatograms
for an FMOC- (Fluorenylmethyloxycarbonyl ) and BOC-
(t-butyloxycarbony) protected primary amine. The BOC-protected
compound was better resolved using the SFC gradient screeningx
method and was successfully scaled to a highly productive isocratic
preparative method.
Figure 4.
Example chromatograms of a racemic compound
designated AV-010163 on chiral stationary phase RegisPack
5μ (Regis Technologies, Morton Grove IL). The overlaid
chromatograms compare gradient elution of the enantiomers
using three simple alcohol co-solvents, and demonstrate a
reversal of elution order. The control of elution order is useful in
achieving higher batch processing efficiency (see text).
Table 1.
Comparison of simulated “enriched” warfarin
feedstocks and their processing and production efficiencies. The
data sets demonstrate that a) an enriched feedstock will always
be faster to process than a racemate, and b) the first eluting
enantiomer (E1) is recovered more efficiently than E2.
1...,17,18,19,20,21,22,23,24,25,26 28,29,30,31,32,33,34,35,36,37,...68
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