Anal. Chem.
2010, 82, 4925–4935
Supercritical Fluid Chromatography
Larry T. Taylor
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061-0212
Review Contents
Perspective 4925
General Reviews 4926
Instrumentation 4927
SFC/MS 4928
Preparative SFC 4929
Stationary Phases 4930
Method Development 4931
Chiral SFC 4933
Literature Cited 4934
PERSPECTIVE
Supercritical fluid chromatography (SFC) has been practiced
sporadically for approximately 50 years. Early studies used packed
columns with varying degrees of success, and it would be fair to
say that SFC did not generate much interest in the separation
science community in this period. In the early 1980s, interest in
SFC exploded when it was reported that SFC could be easily
accomplished with wall coated open tubular columns similar to
the columns currently used for gas chromatography. The columns,
however, differed in two respects: (a) SFC columns had smaller
inner diameters and (b) the polymeric stationary phase that coated
the walls was more extensively cross-linked.
The emergence of SFC with a mobile phase (i.e., carbon
dioxide) that exhibited appreciable, enhanced solvating power
relative to helium and hydrogen was primarily promoted by users
of gas chromatography (GC) who saw SFC as a way to increase
the limited sample base afforded by GC. This technology flour-
ished for some time, but it had several disadvantages for wide-
scale acceptance by the analytical community. Specifically, the
technique which relied mostly on nonpolar stationary phases was
traditionally limited to relatively nonpolar analytes because (a)
pure CO2 as the mobile phase exhibited poor solvating power
similar to hexane, (b) robust sample injection was not feasible,
(c) a wide polarity range of stationary phases was not available,
(d) high quality separations required approximately 60 min,
(e) gradient CO2 pressure and flow-rate were coupled such that
flow-rate changed as mobile phase pressure changed, (f)
passive, fragile fused silica restrictors at the column outlet often
plugged and the separation had to be aborted, and (g)
multigram scale-up of a successful separation was not feasible.
One’s view of SFC today is entirely different from that of 25
years ago. Today, SFC is a separation technique similar to high
performance liquid chromatography (HPLC) using mostly the
same hardware and software as HPLC. The mobile phase is a
binary or ternary mixture with CO2 as the main component. The
separation is usually performed as a gradient elution where
composition of the mobile phase is changed versus time. Polar
stationary phases such as bare silica, cyanopropylsilica, 3-ami-
nopropylsilica, and 2-ethylpyridylsilica are routinely employed.
10.1021/ac101194x  2010 American Chemical Society
Published on Web 05/13/2010
SFC has numerous practical advantages relative to reversed
phase HPLC such as higher speed/throughput, more samples/
day, more rapid equilibration, and shorter cycle time. SFC
yields lower operating cost and lower column pressure drop,
and it is orthogonal to reversed phase HPLC. Solvent consump-
tion is low; therefore, waste generation is also minimal. Finally,
the compounds of interest can be isolated in a relatively small
amount of solvent because CO2 vaporizes away. This feature
is particularly important for preparative applications in which
elution volumes can be large.
SFC, nevertheless, will always be compared with HPLC. As a
general observation, if you can perform the normal phase HPLC
separation, you can probably carry-out the same separation or
something analogous to it by SFC. On the other hand, replacement
of reversed phase HPLC with SFC will not be totally favorable
except in limited situations. Two differences distinguish SFC from
HPLC systems. The SFC pump system must have a chilled pump
head to maintain the CO2 in the liquid state, and the whole
system for UV detection or just the packed column for MS
detection has to be under pressure.
SFC instruments come in two forms. Analytical scale instru-
ments have flow rates less than or equal to 20 mL/min. Preparative
instruments are for industrial-scale runs and can be further
subdivided. Semipreparative instruments have flow rates from 20
to 200 mL/min, whereas larger preparative systems handle flow
rates at liters per minute with columns measured in inches rather
than millimeters. Only a handful of vendors sell SFC systems.
Jasco, Inc. and Selerity Technologies (now known as Sandra
Selerity Technologies) continue to offer analytical SFC instru-
ments. TharSFC was purchased by Waters in 2009 with a complete
name change although the product line continues to be analytical
and preparative SFC instrumentation. Aurora SFC Systems, Inc.
appeared during the report period with its Fusion module that
converts a standard HPLC into an analytical SFC. One function
of the module is the precompression of CO2, and the other is
the control of the system outlet pressure. In late 2009, Aurora
and Agilent in a formal arrangement introduced this new
approach to SFC where one side of the HPLC pump is stated
to deliver compressed CO2 with the accuracy and precision of
a normal liquid.
Academic acceptance of SFC is currently not high worldwide
but even less so in the United States. Early instrumentation
involved wall coated open tubular columns which were riddled
with problems (1). Challenges to their use led to disappointment
and disillusionment. Furthermore, the early packed column
instruments were not as hardy as HPLC ones. Nowadays, SFC
hardware and software are exceedingly robust, less pure “soft-
drink” grade CO2 is readily useable, and the range of samples
Analytical Chemistry, Vol. 82, No. 12, June 15, 2010 4925
Table 1. Application Notes from Internet and Trade Publications

“Profiling the Isomerization of Biologically Relevant (E)-(Z) Isomers by SFC”, J. Cole, R. Chen, June 2009, LCGC North America (Supplement),
pp 24-25.
“Fast Separation of FFA, FAME, and Glycerol for Biodiesel Analysis by SFC”, J. Cole, J. Lefler, R. Chen, February 2008, LCGC North America
(Supplement).
“A Feasibility Study of Using SFC with UV-ELSD for Food and Beverage Analyses” J. L. Lefler, R. Chen, June 2008, LCGC North America
(Supplement).
“An Acetonitrile-Free Chromatographic Methodology for Melamine Detection and Quantification using SFC”, J. L. Lefler, R. Chen,
February 2009, LCGC North America (Supplement).
“Enhancement of UV Detection Sensitivity in SFC Using Reference Wavelength Compensation”, L. Subbarao, J. Cole, R. Chen, September
2009, LCGC North America (Supplement).
“Separation of Ionic Analytes Using Supercritical Fluid Chromatography”, L. T.
Taylor, September 2009, LCGC North America (Supplement), p 62.
“Effect of Varying Co-Solvents in SFC Method Development on a Whelk-O 1 Chiral Stationary Phase”, T. Szczerba, P. Wrezel, June 2008,
LCGC North America.
“Screening Process for Development of Enantioselective SFC Separation Methods”, G. B. Cox, February 2008, LCGC
North America (Supplement), p 15.
“Feasibility of Using ELSD to Trigger Fraction Collection in Small Scale Purification by SFC”, J. Cole, R. Chen, July/August 2009,
LCGC North America (Supplement), pp 1-2.
“Keith Bartle on SFC and his Career in Chromatography”, Chromatography Today, Feb/Mar, 2009, pp 20-22.
“A Brief Review of Interfacing SFC with MS”, R. Chen, Chromatography Today, Feb/Mar, 2009, pp 11-13.
“Recent Developments and Future Challenges in Supercritical Fluid Chromatography”, S. J. Rumbelow, Chromatography Today,
February/March 2009, pp 15-19.
“Modern Chiral Separations Using HPLC and SFC for Method Development and Prep Purification”, G. W. Yanik, Chromatography Today,
Sept. 2009, pp 44-46.
“Meeting Review: SFC 2009-3rd International Conference on Packed Column SFC”, L. T. Taylor, The Column, September 2009, p 12.
“Multicolumn Preparative SFC: An Advanced Solution to Scale-up Difficulties”, Z. Ali, J. Kocergin, V. Edwin, The Peak, March 2009, pp 16-21.
“Meeting Review: SFC for Pharmaceutical Analysis-Opportunity or Challenge?”, M. Hanna-Brown, T. Lynch, The Column.
“Separation of Ionic Analytes via SFC: Achieving the Impossible”, L. T. Taylor, Chromatography Online, June 2009.
“Packed Column SFC-Based Analysis of Lipid Modification Reactions: Overview and Applications”, D. G. Hayes, Amer. Pharm Rev.,
April/March 2009, pp 46-52.
“The Use of SFC for Chiral Pharmaceutical Analytical and Preparative Separations”, J. Van Anda, Amer. Pharm. Rev., April 2009, pp 48-53.
“SFC of Peptides-State of the Art”, L. Taylor, Amer. Pharm. Rev., May 2009, pp 48-53.
“Pushing the Limits of SFC to Resolve Chiral Molecules”, C. Kraml, Amer. Pharm. Rev., July 2008, pp 80-83.
“Preparative SFC in Drug Discovery”, L. Miller, M. Potter, Amer. Pharm. Rev., September 2008, pp 112-117.
“SFC as a Green Chromatographic Technique to Support Rapid Development of Pharmaceutical Candidates”, J. O. DaSilva, H. S. Yip,
V. Hegde, Amer. Pharm. Rev., Jan/Feb, pp 98-104.

continues to expand. In other words, the range of applicability
is getting closer and closer to the range of applicability of
reversed phased HPLC. Yet, the inertia holds and many
academicians who are accustomed to HPLC are loath to invest
time and energy to switch. In this regard, one would think that
SFC would be the first to get on the “environmentally friendly”
bandwagon. Being green is apparently fine and not a bad thing,
but most people seemingly go for SFC because of its speed
and fast method development. Experts in the field agree that
SFC has established itself as the preferred way of doing chiral
analysis on both the analytical and preparative scales. They
also say that SFC will become the norm for small-scale
purifications. Increased interest in (a) the petrochemical and
food industries, (b) the determination of environmental air
quality, (c) biodiesel quality control, (d) protein separations,
etc. can be expected in the future.
The field of SFC is solely examined in this biannual review.
The published literature in the period from January 1, 2008 to
December 31, 2009 has been considered. The document builds
upon the previous biannual review of the author published during
June 2008 in which (a) analytical scale chiral and achiral separa-
tions, (b) supercritical fluid simulated moving bed, (c) preparative
SFC, (d) SFC coupled with mass spectrometry, (e) natural product
applications, and (f) open tubular column studies were reviewed
(2). The searched database was compiled by SciFinder Scholar
which contained English and non-English journal articles, patents,
books, and abstracts of regional and National American Chemical
Society meeting presentations. The search keywords were “SFC”
4926 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
and “supercritical fluid chromatography”. Although the number
of citations fails to exceed 110 for a second two year period, the
individual contributions represent workers from all over the world.
The bulk of the peer reviewed, published investigations concerns
packed columns and pharmaceutical applications. A somewhat
disturbing trend, however, appears to be developing in that
references to articles from Internet chromatographic sites
(separationsNOW@wiley.co.uk, www.chromedia.org, chromtoday@
chromtoday-mailers.com, and www.chromatographyonline.com)
and trade publications are on the rise (see Table 1). Yet, the
customary search engines list these documents as bona fide
references. A few of these reports are highly significant and will,
thus, be covered in this biannual review. The author, however,
has serious questions regarding their peer reviewed status and
long-term availability to the chromatographic community.
GENERAL REVIEWS
The field has been well reviewed during the two year period
by a number of publications. A critical review of activity in industry
versus academia with numerous expert commentaries was pub-
lished in 2008 (3). The vendor situation in the U.S. was considered
along with the current available instrumentation for conducting
analytical and preparative separations. The separation of ionic
analytes via SFC was reviewed wherein the use of additives was
shown to dramatically extend the range of solute polarity amenable
to separation (4). Ion suppression and ion pairing were suggested
to be important mechanisms for SFC of ionic analytes. Packed
column SFC as the new modern normal phase chromatography
has been reviewed (5). Fundamental differences between packed
column and open tubular column were discussed. Divisions in
chromatography were noted to not be useful. SFC for the 21st
century appeared as a review during the biennium (6).
A field guide to new SFC instrumentation which lists informa-
tion taken from correspondences with manufacturers and their
Web sites and printed brochures appeared (7). Companies that
were polled included Chiral Technologies Inc., Jasco Inc., Modular
SFC, Novasep Process, Princeton Chromatography, Inc., Regis
Technologies, Inc., Selerity Technologies, Inc., and Thar Instru-
ments, Inc.
The enantioseparation of 123 clinically used racemic drugs by
SFC on commercial chiral stationary phases that were available
in 2006 was reviewed (8). The mobile phase compositions with
organic modifier and additives were listed. The data were
extracted and compiled from the ChirexBase database (Marseille,
France). All the drugs included are listed according to the 13
therapeutic classes of the Anatomical Therapeutic Chemical
classification. The nature of the mobile phase precluded the use
of several classes of chiral stationary phases (CSPs) such as crown
ethers, ligand exchange, and protein-based CSPs. The polysac-
charide-based CSPs were responsible for two-thirds of the enan-
tiomer separations listed. Another review highlighted the sepa-
rationsofchiralpharmaceuticalsanddrugsbyliquidchromatographic
modalities (i.e., HPLC, capillary electrochromatography, SFC,
SubSFC, and thin layer chromatography) utilizing polysaccharide
CSPs (9). Coated versus immobilized CSPs were discussed. A
comparison of CSPs commercialized by different companies was
considered.
Another review summarized a variety of novel supercritical
fluid chromatography/mass spectrometry (SFC/MS) methods for
chemical analysis that have been reported in peer-reviewed
publications (10). Efforts directed toward the establishment of a
hardware framework for SFC/MS was predicted to elevate the
technique to become the method of choice for high-throughput
chemical analysis in both academic and industrial areas. Repre-
sentative SFC/MS methods for chemical analysis were sum-
marized. Several additional reviews should also be mentioned
here. The utility of SFC in metabolomic studies was also reviewed
(11). A review of applications of SFC in cancer research, toxicol-
ogy, peptides, lipids, etc. appeared (12). Main developments and
applications of multidimensional chromatographic techniques in
food analysis have also been reviewed (13). Approaches for
efficient method development with immobilized polysaccharide-
derived chiral SP were reviewed (14). Multidimensional SFC
methodologies such as SFC × SFC, SFC × HPLC, and silver ion
SFC × RPLC were reviewed (15).
INSTRUMENTATION
A novel method of interfacing an acoustic flame detector (AFD)
with modified SFC was presented (16). By applying resistive
heating directly to the burner region between the restrictor outlet
and the oscillating acoustic flame, infrequent severe noise, baseline
drift, and peak deformations that can occasionally be observed
with the AFD are eliminated. The approach worked equally well
for modifier levels from 0 to 100% in the HPLC mode. The
performance facilitated reliable application of the AFD as a
universal detector in modified SFC and other separation modes
that employ organic solvents in the mobile phase. The interface
was observed to reduce detector noise to near 10-25 Hz when
an appropriate temperature was achieved. The method was easy
to assemble, inexpensive to construct, and robust in its daily
operation.
Remote control of the vent/detector split flow ratio in packed
column SFC (pcSFC) with flame ionization detection (FID) was
demonstrated during the two year period by the same workers
using a dual heated restrictor method (17). Restrictors stemming
from a Tee at the separation column outlet were respectively fixed
into an FID and a vent port, and their individual temperatures
were controlled using resistively heated wires. Both system
pressure and split flow could be manipulated. Isobaric altering of
the split flow ratio was possible when opposing positive and
negative temperature gradients were applied at the two restrictors.
The method was used to establish stable detector operation in
the analysis of n-alkanes under pcSFC-FID conditions that
normally exhibit flame instability. Results indicated that this
technique could be a relatively simple and inexpensive means of
controlling system pressure and detector split flow ratios in pcSFC-
FID. Methodology was predicted to be compatible with methods
where a conventional backpressure regulator is desired for
pressure control.
An alternative means of independently controlling column
pressure in SFC by resistively heating the postcolumn restrictor
was demonstrated by Li and Thurbide (18). Compared to
conventional block heating methods, resistive heating provided
at least 4 times greater pressure programming rates and allowed
for much faster cooling times in between runs, thereby increasing
sample throughput. The methodology was found to be equally
effective for either capillary or packed column operating modes.
The technique was stated to be a simple, inexpensive, and
convenient alternative to limited passive restrictors or more costly
and complex backpressure regulators.
Four papers were published by Takahashi, et al. during the
period. A corona-charged aerosol detector (CAD) was developed
to improve the sensitivity, reproducibility, and quantitativeness
of detection as compared with evaporative light scattering detec-
tion (ELSD) using a certified reference material of poly(ethylene
glycol) (PEG) (19). The corona CAD was able to detect a 10 times
more dilute solution of uniform oligomers compared to ELSD.
Repeatability of the corona CAD was greater than ELSD. The CAD
had better signal-to-noise at very low concentrations and exhibited
a lower minimum detectable quantity. In another study, the
quantitativeness of ELSD for SFC was evaluated using an equi-
mass mixture of uniform PEG oligomers (20). All molecules have
an identical molecular mass in uniform oligomers. They are, thus,
useful for the accurate calibration of detectors. Using chromato-
grams of the equimass mixture of uniform oligomers to calibrate
SFC-ELSD, it was possible to determine exact values of not only
the average mass but also the molecular-mass distribution for a
PEG 1540 sample. The average molecular mass was shifted to a
higher value by several percentage points after calibration of the
ELSD. The ELSD 2000 was from Alltech Associates. PEGs were
separated by preparative SFC using an SFCpak SIL-5 silica gel
column (Jasco Co.) with pore size ) 6 nm and particle size ) 5
µm. Column dimensions were 250 × 4.6 mm.
Equimass mixtures of uniform PEG oligomers with various
degrees of polymerization were subjected to a precise calibration
Analytical Chemistry, Vol. 82, No. 12, June 15, 2010 4927
of SFC-ELSD by the same workers to examine the quantitativeness
of the detector in terms of the concentration and degree of
polymerization (21). The concentration dependence of the ELSD
was small, and the response of the ELSD was linear against the
injected concentration of analyte. Calibration curves for SFC-ELSD
were able to be determined for various degrees of polymerization
using data concerning the equimass mixture of uniform oligomers.
The method has been developed to permit the certification of
fractions of all the components in PEG samples that are needed
for certified reference materials issued by the National Metrology
Institute of Japan. The material is suitable for calibration against
both masses and intensities regarding matrix-assisted laser
desorption/ionization time-of-flight MS.
A condensation nucleation light scattering detector (CNLSD)
was adapted for use as a detector for SFC (22). The performance
of the CNLSD was evaluated and compared to ELSD using a well-
defined equimass mixture of poly(ethylene glycol) 1000. The
CNLSD was able to detect a 10 times less concentrated solution
of uniform oligomers compared to ELSD. The quantitativeness
of CNLSD in terms of concentration and degree of polymerization
was evaluated using an SFC system and PEG standards, and it
was high enough to obtain the molecular mass distribution of
poly(ethylene glycol) 1000 without any calibration.
On column density measurement of CO2 in packed capillary
columns using Raman microspectroscopy of the position of the
Fermi doublet has been reported (23). Correlation of the
spectrum with density was calibrated over a pressure range of
15-290 atm at 125° and 150 °C which allowed for determination
of the density gradient of fluid flowing through a packed
capillary column. Analysis was said to follow in a later
publication.
Open tubular capillary column SFC with 1-n-butyl-3-methylimi-
dazolium methyl sulfate [bmin][MeSO4] as the stationary phase
and supercritical CO2 as the carrier fluid was employed to
measure retention factors of organic solutes within 313-353
K and 8.5-23.2 MPa (24). Solute selection included 18
compounds of diverse volatilities and chemical functionalities.
At constant temperature, an increase in CO2 density produced
distinct shifts in relative retention, thus providing some pres-
sure-tunable selectivity. Analysis of the relative retention data
by regular solution theory resulted in approximate values of
the solubility parameter of CO2-expanded [bmin][MeSO4].
A new approach to SFC instrumentation, which allows a
standard HPLC system to reversibly function as an SFC system,
was reported (25). The system removes the compression require-
ments from an HPLC pump, allowing it to perform pulseless
metering. By isolating compression and metering, the detection
limits and dynamic range were improved well over an order of
magnitude. Detection limits (S/N ) 3) of 0.004% of the parent
peak were demonstrated, while both were on the same linear
calibration curve. Linearity was 0.99999 from 0.01 to 100%
(0.001-10 mg/mL racemate or 2.5 ng to 25 µg of each enantiomer
injected). Average S/N at 0.1% was 58. Retention time stability
was ±0.15% to ±0.70% over sets of five injections. Area reproduc-
ibility was ±0.27% to ±0.46% RSD when S/N was >100. It was noted
that SFC is almost never used for trace analysis. A significant
improvement in detection limits and dynamic range could allow
much more extensive use of SFC including use in regulated
4928 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
environments. The improvement in background noise, sensitivity,
and dynamic range appeared to be related to the new approach
to pumping CO2.
SFC/MS
The summarization of a variety of novel SFC/MS methods for
chemical analysis that have been reported in peer-reviewed
publications has been published (26). Efforts toward the establish-
ment of a hardware framework for SFC/MS were predicted to
elevate the technique to become the method of choice for high-
throughput chemical analysis in both academic and industrial
areas. Another review appeared which extensively discussed
applications of SFC/tandem MS in pharmaceuticals (27).
An interesting paper appeared that describes the ionization of
samples in the absence of an applied electrospray voltage when
SFC/MS was used with some compounds showing increased
sensitivity (28). A series of test standards were analyzed with a
range of pressures and modifier percentage conditions. The
methodology was compared with three established liquid-to-gas
ionization mechanisms: thermospray, charge residue model of
electrospray ionization, and sonic spray ionization. The most
important point to note from this report was that enhanced
ionization was observed under novo-spray conditions in a SFC/
MS configuration, which in certain cases provided a lowering of
the overall limit of detection.
SFC/MS was used for the separation and analysis of integral
membrane proteins and hydrophobic peptides (29). Detergents
were rapidly and effectively separated from the proteins and
peptides, yielding them in a state suitable for direct mass
spectrometric analysis. Detergents are generally used to solubilize
membrane proteins, but they interfere with the crystallization
process which is essential to X-ray studies. They also cause severe
ion suppression effects that hinder MS analysis. Gramicidin and
bacteriorhodopsin were purified from detergents and lipids.
Photosystem II was also investigated by SFC/MS, and a total of
16 out of 26 core proteins were eluted in 15 min.
The hyphenation of SFC to atmospheric pressure chemical
ionization ion trap MS has been detailed (30). Temperature, flow
rates of gas, and mobile phase introduced in the source, position
of the restrictor, ionization additives, and conditions of autotune
were studied. The latter had to be performed with parameters as
close as possible to analytical conditions (i.e., subcritical condi-
tions). Unambiguous identification and structure elucidation of
several additives in formulated car lubricants were presented. In
another study, the separation of all six dimethylaniline isomers
by SFC without derivatization was reported (31). The combination
of SFC with ESI-MS provided selective detection in crude extracts
of spiked (40 ppb of 3,5-dimethylaniline) raw materials that are
commonly used in the manufacture of consumer hair-dye products.
An analytical system that enables the simultaneous rapid
analysis of lipids with varied structures and polarities through the
use of SFC/MS has been reported (32). Lipid mixtures included
phospholipids, glycolipids, neutral lipids, and sphingolipids. When
cyanopropylated silica gel was used for the separation, all lipids
were successfully detected and the analysis time was less than
15 min. The use of an octadecylsilylated column resulted in
separations which were dependent on differences in the unsat-
uration of the fatty acid side chains. This system is a powerful
tool for studies on lipid metabolomics because it is useful not only
as a fingerprinting method for the screening of diverse lipids but
also for the detailed profiling of individual components.
Carotenoid analysis was carried out by SFC/MS (33). The use
of an ODS packed column separated seven analytes within 15 min.
The use of a monolithic ODS column resulted in additional
improvement in both resolution and throughput. The analysis time
was reduced to 4 min by increasing the flow rate. Carotenoids in
biological samples containing complex matrixes were separated
effectively using three monolithic columns in series whose
backpressure was very low.
PREPARATIVE SFC
Exergetic life cycle analysis for the selection of chromato-
graphic separation processes in the pharmaceutical industry has
compared preparative HPLC versus preparative SFC (34). It was
concluded that, for this case, the most sustainable process as far
as integral resource consumption is Prep HPLC, unlike the general
perception that Prep-SFC outperforms Prep-HPLC. The poor score
for Prep SFC was related to the production of liquid CO2 and the
use of electricity for heating and cooling. Prep HPLC required
26.3% more resources due to its higher use of organic solvents
and 29.1% more resources quantified in exergy.
In this regard, a paper that discusses the use of preparative
HPLC and SFC to generate individual enantiomers for discovery
activities appeared (35). The usefulness of preparative chromato-
graphic resolution of racemates was demonstrated through the
presentation of numerous nonroutine, less straightforward case
studies from the laboratories of Amgen. There were some
racemates that do not scale-up as expected due to low solubility
or poor sample loading or for other unknown reasons. The
introduction of immobilized chiral stationary phase enabled
separation of racemates that would be very time-consuming and/
or impossible due to poor compound solubility.
The evaluation and implementation of a commercially available
off the shelf analytical SFC/MS and a mass directed semiprepara-
tive SFC/MS system for use in a high throughput purification
laboratory was described (36). The utilization of standard Frac-
tionLynx/AutoPurify functionalities to enable rapid incorporation
into high throughput environments was emphasized. The analyti-
cal SFC allowed for rapid screening of crude reaction products
and purity confirmation analysis of isolated fractions on a 2-ethy-
lyridine column using a 2 min gradient. The mass directed system
provided rapid separation of libraries of compounds on 10 mm
(i.d.) × 10 cm semiprep columns with no modifier for the vast
majority of the time. The expansion and controlled release of CO2
upon fractionation leaves the desired isolated product in several
milliliters of methanol, thus decreasing evaporation time from
greater than 8 h previously encountered with mass directed
reversed phase HPLC to 1 hour.
A paper that traces the economics and pressures involved in
scale-up was published (37). As a new drug moves through the
early stages of development and increasing amounts of material
are needed, the initial chromatographic method can grow in scale
with the needs of the project, often reaching the isolation of
kilogram quantities for Phase 1 clinical trials. The focus at this
point would be to find the most economical procedure for the
production of the material in time for Phase II. The cost of
chromatography for the first few grams of material will be very
different from that of the first 10 t. The cost decreases quickly
with scale and with further optimization of the separation process.
Chromatography was suggested to always be considered as one
of the options for the manufacture of pure enantiomers. A critique
of SFC, HPLC, and simulated moving bed (SMB) at the prep scale
was provided.
Several approaches for purifying difficult samples more ef-
ficiently for discovery research support were mentioned in another
paper (38). Various specialty columns were employed such as
hydrophilic interaction liquid chromatography (HILIC), hydro-
philic end-capped columns in a reversed phase purification mode,
and SFC on various chiral or bonded silica gel phases in a normal
phase purification mode. Analytical method development strate-
gies, sample preparation, scale-up from analytical to preparative,
and purification results for various discovery samples were
discussed. Difficult samples were defined as follows: (a) minimal
retention on regular reversed phase columns due to high polarity,
(b) minimal separation on reversed phase columns due to
structurally similar impurities such as diastereomers and regioi-
somers, (c) requirement for large sample amounts for numerous
injections, and (d) degradation in aqueous solutions.
Preparative batch HPLC, SFC, SMB, and steady state recycling
(SSR) were used to resolve a total of 12.2 kg of a racemic
pharmaceutical intermediate (39). The separation conditions and
results of these techniques were discussed. The productivities and
solvent costs of SFC versus HPLC and then SMB and SSR versus
HPLC were compared. Higher productivities and lower solvent
usage were observed with SMB relative to SFC, SSR, and HPLC.
SFC improved the separation efficiency when compared with batch
HPLC. At 10% cosolvent, the solvent usage of SFC was reduced
to half the amount compared with that of 20% cosolvent. However,
the productivity was also reduced due to the longer cycle time.
A review of SMB (i.e., continuous multicolumn chromato-
graphic process) for the separation of enantiomers appeared (40).
The review stressed design methods for robust operation, scale-
up using data obtained from analytical experiments, process
schemes, areas of design, and practical issues concerning the
operation of SMB units. Another review presented the major
developments and applications to those embarking on using SFC
for high throughput applications in other fields (41).
The semipreparative chiral separation of lansoprazole and two
related compounds (pantoprazole and rabeprazole) using SFC was
communicated (42). The volumes injected were 1, 2, and 4 mL.
The concentrations of the racemic mixtures were 3 and 6 g/L for
lansoprazole and 1.5 g/L for pantoprazole and rabeprazole. In all
cases, the recoveries, for purity higher than 99.9%, were better
for the second eluted enantiomer than for the first one. In the
case of lansoprazole, it was possible to obtain 0.025 and 0.090 mg/
min of the first and second enantiomer, respectively, with an
enantiomeric purity of 99.9%.
A comprehensive approach was described to develop a chiral
purification method for an analyte that was found to be unusually
difficult to scale-up in SFC (43). Major factors were studied such
as solubility of the analyte in the SFC mobile phase, impurity
profiles, and cycle time. Solubility in the mobile phase was
measured by a packed column technique coupled with a novel
trapping mechanism to enhance measurement precision under
SFC conditions. The SFC methods were developed to purify a
sample containing 15% of an impurity. An equal volume mixture
Analytical Chemistry, Vol. 82, No. 12, June 15, 2010 4929
of acetonitrile and ethanol was chosen for the final purification
method since this mixture demonstrated high SFC solubility
among all solvent combinations with enhanced resolution between
the analyte and the impurity and the shortest run time.
The separation of the enantiomers of flurbiprofen on an
amylase-derived chiral stationary phase, Chiralpak AD-H, by SFC
under both linear and nonlinear conditions was studied (44). The
primary objective of the work was to elucidate adsorption
isotherms in SFC and study the effect of modifier concentration
and pressure on the isotherms in order to apply this knowledge
to process optimization. At linear conditions, the isotherm was
determined directly from the chromatogram. Under overload
conditions, the elution profiles were described by competing
Langmuir and bi-Langmuir isotherms. The value of selectivity was
from 1.9 to 2.1; while the value of resolution was from 5.3 to 11.8.
The number of theoretical plates was always greater than 5000
indicating high efficiency of SFC.
STATIONARY PHASES
An overview on fluorocarbon stationary phases as alternative
reversed phases was reported (45). Solvophobicity and fluoro-
philicity of the fluorinated phases provided enhanced selectivity
for organofluorine compounds. The dual normal and reversed
phase characteristics make fluorinated phases suitable for analysis
of polar pharmaceutical and biological samples such as proteins,
peptides, nucleotides, steroids, and alkaloids. Little work was
stated to have been done in SFC.
Another review traced the development and application of
polysaccharide-based CSPs for the efficient separation of enanti-
omers (46). The chiral recognition mechanism of the polysac-
charide-based CSPs was discussed. Current applications such as
chiral separation via SFC, immobilization of polysaccharide deriva-
tives, use of monolithic silica columns, and preparative separations
were summarized.
A series of papers appeared during the period authored by
West and Lesellier. In their first paper, they evaluated specific
retention properties of 28 different packed columns based on
isocratic retention data using quantitative structure-retention
relationships (QSRR) (47). The approach gives great promise of
allowing the rapid selection of appropriate column types to
produce the desired selectivity for a particular separation. In
addition, it shows which columns would provide similar separa-
tions. The selectivity differences observed between stationary
phases (SP) were essentially due to the stationary phase itself
and not to the silica support or to differential adsorption of mobile
phase components. The oversimplified classification scheme (i.e.,
reversed phase and normal phase) exists in LC because of the
different polarities of SP and the necessary change in mobile phase
(MP) required when working with liquids. This frontier does not
exist in SFC. SFC provides analytical capability not readily
accessible with HPLC using the same MP. Thus, it is possible to
couple columns of widely differing polarity in SFC because the
MP can be identical regardless of the polarity of the SP. Naming
techniques are misleading and considering both modes as one
with the SP as variable is a more correct approach. This becomes
the basis of “unified chromatography from the SP perspective”
as a complement to Chester’s “unified chromatography” from the
MP perspective” In other words, normal phase and reversed phase
4930 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
modes are limiting behaviors, and they are bridged in SFC as
they may be bridged by emerging techniques in HPLC, such as
HILIC.
Later, it was investigated whether the number of SPs can be
reduced to an optimized set with only highly orthogonal systems
(48). The initial set of columns was defined with emphasis on
efficient SPs covering a broad selectivity range. The following
columns were picked: (a) a polar phase, 2-ethylpyridine bonded
silica, (b) an aromatic phase of intermediate polarity, phenyl-
oxypropyl bonded silica, (c) a nonpolar phase, octadecyl- and
phenylpropyl-bonded silica, and (d) two unique phases butylsi-
loxane bonded silica and pentafluorophenyl- propyl-bonded silica.
Further study (49) investigated the different chromatographic
behaviors of a variety of octadecylsiloxane-bonded SPs: (a)
classical, (b) protected against silanophilic interactions, and (c)
containing polar groups (i.e., end-capping groups or embedded
groups). Two approaches were chosen: carotenoid test and
solvation parameter model. Both tests proved to be complemen-
tary and provided precise information on the chromatographic
behavior of ODS phases. In a final paper, retention mechanisms
in super/subcritical fluid chromatography on packed columns
were reviewed (50). Whereas the retention rules of achiral
compounds are well-defined in HPLC on the basis of the nature
of the SP, some difficulties appear in SFC on packed columns.
This is mainly due to the supposed effect of volatility on retention
behaviors in SFC and to the nature of CO2 which is not polar;
thus, SFC is classified as a normal phase separation technique.
Moreover, additional effects related to density changes of the
MP or to adsorption of fluid on the SP causing a modification
of its surface are not well-known. In this paper, all these
behaviors are discussed mainly using log k-log k plots which
allow a simple comparison of SP properties.
The synthesis of mono-6-(3-methylimidazolium)-6-deoxyper-
phenylcarbamoyl-β-cyclodextrin chloride (MPCCD) and its ap-
plication in chiral SP for SFC was reported (51). The chiral
selector was coated onto the silica gel in different weight
percentages (15, 20, and 35% w/w) to obtain CSPs having different
loading content. The best enantioseparation results were obtained
using a column with 20% of MPCCD. The resolution obtained for
p-halophenylethanol analytes ranged from 3.83 to 5.65.
The first comparison (52) of a full screen-based approach using
two series of commercially available Pirkle SPs with a polysac-
charide-based SP screen (i.e., AD, OD, AS, OJ) has been reported.
When running corresponding chromatography conditions, often-
times Pirkle SPs can provide resolution opportunities similar to
the polysaccharide phases prevalent in the pharmaceutical indus-
try. Literature suggested an 80% success rate for polysaccharide
phases, while in this study a 70% success rate was observed for
Pirkle phases. An advantage of the additional capabilities and
solvent choices (i.e., 1,2-dichloroethane, methylene chloride,
chloroform, and THF) of Pirkle SPs was noted. Two commercially
available Pirkle chiral phase platforms were chosen for compari-
son: Chirex Pirkle columns from Phenomenex and Pirkle columns
from Regis.
Poly(trans-1,2-cyclohexanediyl-bis-acrylamide) (P-CAP) with
SFC was investigated via the analysis of 40 commercial and 100
proprietary compounds using a 12 min gradient with methanol
as a modifier (53). P-CAP demonstrated separation of 25% of the
140 compounds, while each of the polysaccharide phases (AD-H,
AS-H, OD-H, OJ-H) separated 46%. A key advantage of P-CAP is
the fact that it is available in both enantiomeric forms, allowing
manipulation of the elution order of enantiomers which is helpful
for preparative applications. P-CAP, however, showed less chiral
discrimination power compared to the derivatized polysaccharide-
base CSPs.
Four cationic β-cyclodextrin derivatives have been synthesized
and physically coated onto porous spherical silica gel to obtain a
novel chiral SP (54). The performance of these columns was
studied using 18 racemic aryl alcohols as test analytes. Mono-6-
(3-octylimidazolium)-6-deoxyperphenylcarbamoyl-B-cyclodextrin
chloride (OPCCD) showed the best separation results for all the
analytes via both HPLC and SFC. Chromatographic studies
revealed that CSPs consisting of an n-octyl group on the imida-
zolium moiety and phenylcarbamoyl groups on the CD ring
provided enhancement of analyte-chiral substrate interactions
over CSPs bearing a methyl group on the imidazolium moiety
and 3,5-dimethylphenylcarbamoyl groups on the CD ring.
The applicability of monolithic silica columns versus conven-
tional columns packed with spherical particles for the analysis of
complex hydrophobic metabolites was reported (55). The struc-
tural isomers of carotenoids were separated (monolithic ODS
column (100 × 4.6 mm) for 10 min, seven carotenoids, methanol
1-5% in 8 min, 0.1% ammonium formate, inlet pressure as low as
15 mPa, outlet pressure 10 mPa, and flow rate of 3 mL/min). A
method for profiling biological samples containing complex
matrixes was also developed.
Two new synthetic polymeric chiral stationary phases based
on trans-(1S,2S)-cyclohexanedicarboxylic acid bis-4-vinylphenyla-
mide and trans-N,N-(1R,2R)-cyclohexanediyl-bis-4-ethenylbenza-
mide monomers were prepared and evaluated by HPLC and SFC
(56). A variety of chiral compounds were separated on these two
CSPs. The different orientation of the amide groups in the two
CSPs resulted in a striking difference in the enantioselective
properties. Resolutions generally were higher with HPLC than
with SFC; while SFC provided faster separations. Another study
concerned the synthesis of monosubstituted positively charged
cyclodextrins and their application to SFC in chiral separations
(57).
An ionic liquid (IL)-functionalized SP based on 1-octyl-3-
propylimidazolium chloride covalently bonded to silica gel was
communicated (58). A 3.2 mm × 250 mm column for the
simultaneous separation of acidic, basic, and neutral compounds
using CO2 was considered. The effects of pressure, temperature,
cosolvent, and additive on the retention behavior of the analytes
were studied. Simultaneous separation of acidic, basic, and
neutral compounds via SFC was successful at a cosolvent
content of 20% methanol, a pressure of 110 bar, and a column
temperature of 35 °C. RSDs of the retention times and peak
areas at 50 ppm were all less than 4% and 8% (n ) 6),
respectively.
METHOD DEVELOPMENT
The separation of phenacyl esters of the fatty acids originating
from a fish oil extract by means of a comprehensive analysis using
silver ion (SI) SFC and reversed phase liquid chromatography in
the first and second dimensions, respectively, is described (59).
The construction of the SI-SFC × RP-LC interface consisted of
two 2-position/ten port switching valves, of which one was
equipped with two loops packed with ODS particles. In the SFC
dimension, high efficiency and loadability were obtained by
coupling two wide-bore columns (4.6 mm id) in series. Evaporative
light scattering detection (ELSD) and UV detection with standard
and high pressure flow cells were evaluated in terms of data
acquisition speed and suppression of signal interferences origi-
nating from supercritical CO2 expansion.
A quantitative structure retention relationship model to predict
SFC retention of some organic compounds in various percents of
organic modifiers in the MP using linear and nonlinear feature
mapping techniques was published (60). The data set contained
retention information for 35 various organic compounds in a MP
which contained 0, 2, 4, and 6% methanol.
Prediction of SFC retention based upon the solvation parameter
model was attempted, given only the knowledge of the solute
structures without any prior experiment (61). The Derringer
desirability function proved to be useful for ranking chromato-
graphic systems according to their separation performance. Even
if retention factors were not exactly matched, elution order and
separation factors were correctly predicted within 10%. The
capacity of the method to determine the column with the largest
probability to succeed in a given separation was suggested to have
been proven. Chlorotriazine pesticides were separated.
The effects of particle size and thermal insulation on retention
and efficiency in packed column SFC with large pressure drops
was described for the separation of a series of model n-alkanes
(62). The three principle causes of band broadening were stated
to be (a) the normal dispersion processes described by the van
Deemter equation, (b) changes in the retention factor due to axial
density gradient, and (c) radial temperature gradients associated
with expansion of the MP. Positive effects were attributed to the
elimination of radial temperature gradients and the concurrent
enhancement of the axial temperature gradient. Thermal insulation
had no significant effect on chromatographic performance at the
higher density.
The possibility for achieving high resolution separations
concurrently with HPLC equivalent analysis times through serial
coupling of five columns was presented (100 000 plates) (63). A
highly effective screening method for a set of 17 structurally and
physico-chemically diverse pharmaceutical standards was obtained
in less than 20 min with optimum results obtained on five serially
connectedcyanopropylsilicacolumnswith(a)acetonitrile-methanol
(1:3) modifier, (b) diisopropylamine and trifluoroacetic acid
additives (0.5% each), and (c) 100 bar outlet pressure. The effect
of temperature on both efficiency and selectivity was significant
and even amplified when a long column setup was used.
The effect of increased concentrations of ammonium acetate
(AA) in SFC was studied on silica, 2-ethylpyridine, and end-capped
2-EP phases (64). The study involved the addition of increasing
concentrations of AA either in the MP modifier or in the sample
solvent. Compounds that exhibited satisfactory chromatographic
behavior in the absence of the additive were virtually unaffected
by its presence in the MP or sample solvent. Compounds that
exhibited late elution and strong peak tailing when pure methanol
was used showed dramatically improved chromatographic behav-
ior in the presence of the additive.
Analytical Chemistry, Vol. 82, No. 12, June 15, 2010 4931
 The retention behaviors of pharmaceutical and druglike
compounds in SFC with polar SPs were evaluated using linear
solvation energy relationships (LSER) (65). More than 200
compounds were used. The dominant contribution to positive
retention was the hydrogen bond donor acidity of the solutes
particularly for pyridine and amino columns. Another significant
contributor to retention behavior was the hydrogen bond acceptor
basicity of the solutes made particularly for diol and amino
columns. The LSER results showed that the SFC retention
behavior of compounds using CO2-MeOH with polar SP was
close to that reported for normal phase HPLC using hexane-
methanol. A greater knowledge of the nature of solute/SP and
solute/MP interaction allows a more rapid and efficient choice
in high throughput screening of compound libraries.
In a related study, the connection between the observable
output in column chromatography (retention time, retention
volume, retention factor, separation factor, etc.) and system
properties (hold-up volume, pressure, temperature, isotherm
behavior, etc.) was discussed from a practical and mechanistic
perspective for SFC and other chromatographies (66). The
unifying feature of these techniques was that retention can be
described by a partition model, although not always exclusively.
Applications and prospects of two phase tunable solvent
systems composed of ionic liquids and supercritical fluids with
an emphasis on supercritical CO2 were reviewed (67). SFC has
emerged as a powerful technique to study the interphase
distribution of highly dilute solutes. A major issue to be
resolved in the future is the reliability of SFC data, namely the
role of spurious retention mechanisms and the ensuing
systematic errors in the resultant partition coefficients. An
overview of methods for predictive thermodynamic modeling
of binary (IL-CO2) and ternary (solute-IL-CO2) systems was
included.
Several papers discussed very unusual experimentation. Enan-
tiomeric resolution by chiral SFC of a series of neutral iridium
complexes with greater than 95% enantiomeric purity was com-
municated (68). Water has been used as a polar modifier, and
µ-Porasil has been used as a saturator column (69). The µ-Porasil
column was inserted between the pump outlet and the injection
valve. During the passage of the supercritical fluid MP through
the silica column, water could be dissolved in the pressurized fluid.
Under these conditions, PEG was separated via SFC. More peaks
were separated with the saturator column than with pure CO2.
These results agreed well with those reported in the case of
PDMS. A Nucleosil packed diol column was used (i.e., 100 ×
2 mm id).
The application of SFC to the analysis of naturally occurring
polyprenols and lipid mixtures has been documented as an
example for the separation of hydrophobic metabolites (70). Under
optimized SFC conditions, individual homologues with 10-100
mers were separated. When a cyanopropylated silica gel packed
column was used, the separation of 14 lipids was successfully
detected, and the time required for analysis was less than 15 min.
2-Propanol and methanol were compared as alcohol/water
modifiers of supercritical CO2 in packed column SFC (71).
2-Propanol was found to allow 5 times more MP water content
in SFC than methanol. Of note, a 1:1 2-propanol/water ratio
was attainable relative to a maximum 9:1 ratio realized for
4932 Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
methanol/water. In test separations of polar analytes, the
methanol system easily eluted compounds from a nonpolar
PRP-1 column but failed to elute the most polar analytes from
a polar silica gel or diol column. By comparison, 2-propanol
systems could elute all of the analytes from each of these
columns. Thus, 2-propanol can assist in increasing the water
capacity of supercritical CO2 and potentially facilitate the
analysis of various polar hydrophilic analytes.
Isolation, fractionation, and identification of sucrose esters from
aged oriental tobacco employing supercritical fluids have been
completed (72). In addition to supercritical fluid extraction,
semipreparative SFC provided for an additional purification of the
sucrose ester enriched fraction after column optimization. Struc-
tural assignments of the SFC fractions were facilitated using GC/
MS accompanied by N,O-bis(trimethylsilyl)trifluoroacetamide-
dimethylformamide derivatization of the free hydroxyl groups and
HPLC-MS.
The path forward for validation in analytical SFC was discussed
(73). Few SFC instruments can be found in drug development
laboratories where current Good Manufacturing Practice (cGMP)
is followed. Drug discovery group emphasis is on isolating the
individual chiral compound in the enantiomeric mixture. Drug
development group emphasis is on quantifying the enantiomeric
impurity in the chiral active. Method validation, method transfer,
and instrument qualification, etc. must be addressed. Certain
improvements concerning instrument sensitivity, chromatographic
data system and system qualification were touted to be necessary
for SFC to fulfill the analytical needs in drug development
laboratories.
Separation of furocoumarins has become a great interest for
the cosmetic industry and human health. Due to the variety of
compounds and their subtle structural differences, their separation
requires high performance methods. Isocratic conditions were
established to obtain a satisfactory separation in 10 min (74). A
two-dimensional analysis was also investigated by performing first
a class fractionation of compounds on a 2-ethyl pyridine phase,
then by separating each class on a pentafluorophenyl phase with
the selected isocratic MP. These approaches allowed the structure
of the compound to be related to retention behavior which was
unusual due to the selected pentafluorophenyl SP.
A study aimed to develop a generic gradient method and
validate it, for a pharmaceutically relevant application was the
goal of this publication (75). A secondary goal was the
determination of thiourea in a pharmaceutical intermediate at
low level (0.01% w/w). The applied method validation ac-
ceptance criteria were consistent with those used for Active
Pharmaceutical Ingredient late stage development and regula-
tory submission. This suggested that with some further
research and instrumental developments, SFC could potentially
reach the same maturity as GC and LC. Generic conditions
were CO2/MeOH containing 20 mM ammonium acetate
(AA). The modifier was programmed from 5%, held for 1
min, to 40% at 2%/min. The pressure was 150 bar; the
temperature was 40 °C, and the flow rate was 2.0 mL/min.
The advantage of AA as an additive was that this is the
preferred volatile salt for SFC/MS. System suitability and
precision based on six injections of a 7.5 µg/mL thiourea
(0.05% w/w) solution showed an RSD of 3.13% for peak area
and of 0.09% for retention time. Late stage development and
regulatory submission performance criteria were met.
An achiral 22-component mix, representing wide diversity and
previously found to be extremely difficult to resolve, was chromato-
graphed between 20° and 70 °C in 10 °C increments using a fixed,
doubling gradient (76). Between 17 and 22 maxima were found
depending upon the temperature although baseline resolution of 22
peaks was not achieved in the 12 min of a fixed gradient. Overlapped
trace peaks, representing <0.1% of the largest component peak, with
S/N < 30 were resolved using changes in temperature only. Noise
at 70 °C remained below 0.05 mAU peak to peak. The results
suggested that small temperature changes can be effective in
resolving difficult pairs in methods that can be validated.
The effect of pressure drop on the performance of SFC using
a modified MP (CO2 + ethanol) was studied (77). Experiments
were performed on a Lichrosphere RP-18 column with phenan-
threne as a solute. Experiments yielding both small and large
pressure drops were performed. A good match between the
experimentally measured and calculated values of pressure
drop, retention time, and column efficiency was observed. At
low back pressure and modifier composition, significant loss
of column efficiency was observed. The column efficiency was
described using a model that takes into account axial disper-
sion, fluid film mass transfer, and pore diffusion. Good
comparisons were obtained between experimental and calcu-
lated HETP curves.
How fluid compressibility affects efficiency in supercritical fluid
separations has been studied (78). On column detection revealed
spatial focusing of the analyte as it moved down the column
density gradient. It was speculated that spatial band focusing
arising from the band velocity gradient could be utilized to improve
the peak capacity of supercritical fluid separations.
Retention characteristics of sulfonamides were calculated (79).
Literature studies of quantitative structure-retention relationships
showed that these characteristics can be correlated with simple
molecular descriptors to derive equations predicting the retention
behavior of new compounds. Measured retention characteristics
were found to correlate with total dipole moment, molecular
surface area, and the electronic charge on the most negatively
charged atom. The correlation of chromatographic measurements
with calculated molecular descriptors may allow the prediction
of retention behavior for an unknown compound provided its
properties are known.
CHIRAL SFC
A SFC chiral assay was implemented to determine the
possibility of interconversion of the desired R and less active S
isomers of a drug candidate (80). The interconversion study was
performed in a classical off-line stationary system and monitored
by an enantioselective method. The rate constants of racemization,
the half-life of racemization, and the enantiomerization energy
barrier were determined for the R to S and S to R conversions.
The method was selective and sensitive enough to detect less that
1% interconversion occurring under the conditions studied.
Enantioselective chromatography was reviewed (81). It has
proven to be a powerful tool for the discrimination of biotic and
abiotic transformation processes for chiral environmental pollut-
ants. Another review of chiral SFC covering the literature from
2000 on also appeared (82). 2,2,2-Trifluoroethanol (TFE) was
evaluated as an alternative modifier for the analysis and purifica-
tion of alcohol sensitive compounds using SFC (83). Four chiral
compounds selected for their sensitivity to alcohols were analyzed
using TFE with polysaccharide and Pirkle-type SPs to produce
selectivity and resolution as high as 1.4 and 7.2.
The enantioseparation of trans-3-ethoxycarbonyl-4(4’-fluorophe-
nyl)-1-methylpiperidine-2,6-dione which is one of the important
racemic precursors of trans-(-)-paroxetine has been investigated
using SFC on a Chiralpak AD column (84). Using a van’t Hoff
plot, the isoenantioselective temperature was calculated to be 410
K. The enantioseparation process was “enthalpically driven” under
the experimental conditions. The retention factors were satisfac-
torily correlated by a simplified lattice-fluid model with average
absolute deviation of both enantiomers smaller than 1.76%.
Many 3-substituted-4-arylquinolinones containing an ortho
substituent on the aryl ring are known as a class of compounds
with maxi-K opening activity. These quinolinones exhibit atropi-
somerism. To explore the potential of SFC to separate the
atropisomers, six 3-substituted-4-arylquinolinones with various
hydrophilic and hydrophobic substituents in various positions
were screened using three alcoholic modifiers (methanol, ethanol,
2-propanol) with four polysaccharide-based chiral SPs (Chiralpak
AD-H and AS-H, Chiralcel OD-H and OJ-H) (85). Results showed
that all six compounds studied were successfully resolved under
multiple SFC conditions within 10 minutes regardless of structural
differences and polarity. The Chiralpak AD-H column appeared
to be superior to the other three chiral columns, and methanol
and ethanol showed a higher success rate than 2-propanol.
Simulated moving bed, preparative HPLC, and SFC have been
illustrated by three case studies in a published paper (86). The
different methods were compared, and their strengths and
limitations were discussed. The most powerful separation technol-
ogy was suggested to be SMB which is reflected by some
commercial drugs where the manufacturing process involves
SMB. Investment costs are highest, and SMB is restricted to
binary separations. SFC is expected to become dominant for
enantiomer separation. SFC is superior to HPLC, but HPLC
systems are omnipresent. The biggest progress in chiral chro-
matography will be made in the field of stationary phases, but it
is important to note that some patents of the most successful CSP’s
have expired.
Analysts perform over 90% of their routine work using reversed
phase systems. RP chiral methods are stated in this paper to be
preferred by analysts, for they do not require the complex system
rinses needed when changing a reversed phase system to a normal
phase setup. Polysaccharide, macrolide, and Pirkle-based SPs in
the RP mode have been evaluated and compared to traditional
normal phase screening capabilities (87). Depending on the screen
and compounds applied, a RP screen achieved chiral recognition
60-95% of the time, making it a viable alternative to traditional
NP chiral screens.
Enantiomeric excess was evaluated for two internally synthe-
sized compound libraries using a high-throughput automated
intelligent four channel parallel SFC/MS system equipped with a
multiplexed ion source interface (SFC/MS-MUX) (88). The
system analyzed each sample simultaneously against four chiral
columns using up to six organic modifiers. A reversal of elution
order was observed for several samples across multiple CSPs and
Analytical Chemistry, Vol. 82, No. 12, June 15, 2010 4933
modifiers. The relationship between elution order and percent
enantiomeric excess (%ee) accuracy was presented for compounds
exhibiting high, middle, and low %ee values. Despite incidences
in which the minor enantiomer eluted prior to the major enanti-
omer with less baseline resolution, the overall %ee was in
agreement with separations in which full baseline resolution was
achieved.
A report appeared that noted improved selectivity was being
achieved using SFC relative to HPLC (89). It was not clear
whether it was the nature of the mobile phase or the modifiers
used in each study that were influencing the difference in
enantiomeric selectivity. Because SFC and HPLC both operate in
a normal phase mode and with the same columns, a significant
difference in chromatography to a point where only one mode
shows selectivity to the enantiomeric pair was not expected. The
goal was to see if the apparent discrepancy was repeatable when
corresponding chromatographic conditions were applied. The
results presented illustrated that the difference resulted from the
modifiers used rather than from the chromatographic mode used.
Proline derivatives can serve as important building blocks and
intermediates. The separation of prolines on Chiralpak AD-H was
reported and compared to that of HPLC (90). Selectivity was
impacted less by change of ethanol content in the MP under SFC
conditions than with HPLC. With SFC, the contribution from
hydrogen bonding was most likely a dominant interaction for
retention rather than chiral recognition. This was supported by
limited thermodynamic data and the elution order change for Boc-
proline between SFC and HPLC.
ACKNOWLEDGMENT
Thanks are extended to Negin Nazem for her generous
assistance concerning the preparation of this review.
Larry T. Taylor holds B. S. and Ph. D. degrees from Clemson University.
He joined the Virginia Tech chemistry faculty in 1965. From 1998 to
2004, he served as Chair of the Department of Chemistry. He currently
holds the title of Emeritus Professor of Chemistry. He has authored over
400 peer reviewed publications. He currently coteaches short courses on
supercritical fluid chromatography with applications directed toward the
pharmaceutical industry.
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