Journal of Chromatography A, 1250 (2012) 157171

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review

Stationary phases for packed-column supercritical uid chromatography

Colin F. Poole
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA

a r t i c l e i n f o a b s t r a c t

Article history: The properties of silica-based, chemically bonded, packed column stationary phases used in supercritical
Available online 19 December 2011 uid chromatography are described with a focus on column design and retention mechanisms. Super-
critical uid chromatography has beneted substantially from innovations in column design for liquid
Keywords: chromatography even if the separation conditions employed are generally quite different. The mobile
Supercritical uid chromatography phase composition and column operating conditions play an interactive role in modifying selectivity
Stationary phases
in supercritical uid chromatography by altering analyte solubility in the mobile phase and through
Packed columns
selective solvation of the stationary phase resulting in a wider range and intensity of intermolecular
Solvation parameter model
Column classication
interactions with the analyte. The solvation parameter model is used to identify the main parameters
that affect retention in supercritical uid chromatography using carbon dioxidemethanol as a mobile
phase and as a basis for column characterization to facilitate the identication of stationary phases with
different separation characteristics for method development. As a caution it is pointed out that these
column characterization methods are possibly a product of both the stationary phase chemistry and the
column operating conditions and are suitable for use only when columns of similar design and with
similar operating conditions are used.
2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2. Advances in column technology for liquid chromatography and its effect on supercritical uid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.1. Kinetic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.2. Retention properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
3. Effect of mobile phase composition on stationary phase properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
4. Stationary phases developed for supercritical uid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
5. Classication methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
5.1. Column screening studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.2. Carotenoid test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.3. Solvation parameter model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

1. Introduction complicated than liquid chromatography and better left to the

experts. In era that liquid chromatography has become syn-
Progress in supercritical uid chromatography has beneted onymous with reversed-phase separations, there appears to be
enormously from technological developments in liquid chro- minimal competition, since supercritical uid chromatography
matography but to a large extent is still trying to dene a is usually perceived as a potential alternative for normal-phase
role for itself in mainstream separation science in the shadow separations. Given the decline in understanding of separation
cast by the 400 lb gorilla of liquid chromatography. Super- mechanisms by those who perform the majority of routine meth-
critical uid chromatography is perceived by many as more ods and method development there is greater comfort in residing
within the single sphere of reversed-phase liquid chromatogra-
phy with its prescriptive methods and available technical support
rather than what can be, and often is, the more empirical
Correspondence address: Rm 185 Chemistry, Wayne State University, Detroit,
approaches and limited technical support for supercritical uid
MI 48202, USA. Tel.: +1 313 577 2881.
E-mail address: cfp@chem.wayne.edu chromatography.

0021-9673/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2011.12.040
158 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
When commencing to write this review I was struck by the near
vacuum of technical support provided by column manufacturers
and the limited number of application notes available for super-
critical uid chromatography. While one can nd general details
of the silica morphology and the identication of different ligands
bonded to these substrates, there is almost no advice offered on
how to select a column for a particular separation, or how columns
with similar surface chemistries differ from each other. Columns
are to be purchased based on inspired guesswork, presumably, or
after a detailed literature review, in which it is hoped that someone
has already solved the problem of interest and the same column
can be taken as a suitable stating point. Column selection units
provide a practical work around for the lack of a suitable theoret-
ical framework for column selection. These units typically house
up to six columns that can be addressed in series using an auto-
mated selection valve with a constant mobile phase composition,
or by incorporation of a solvent selection valve, with different
mobile phase compositions [15]. The series of chromatograms
obtained is used to assist in selecting a suitable column. This may
not be intellectually satisfying, but can be a useful method devel-
opment tool. The process can be rather slow if a signicant number
of mobile phase compositions are simultaneously evaluated but
automation allows the operator to focus on other tasks, or the
screening sequence can be set to run overnight. The use of two
column selection valves allows for software-controlled selection of
25 different tandem column arrangements and 10 different single
column arrangements [6]. Selectivity and plate count can be opti-
mized simultaneously with this arrangement employing columns
of a single chemistry or two columns of the same or different chem-
istry connected in series. Alternatively, parallel column screening
is faster but requires as many detectors as columns included in
the screening step and is less popular [7]. These approaches are
commonly used to identify suitable conditions to separate enan-
tiomers or for compound purication in industry. Several review
articles [811] and a book [11] provide a rich source of application
information together with general descriptions of stationary phase
properties. In this review I will focus on recent developments in
column materials, methods of column characterization, and trends
and directions shaping column technology for analytical supercrit-
ical uid chromatography from the time of my previous review
[9]. Preparative scale and enantiomer separations, topics in which
supercritical uid chromatography has had considerable success,
are detailed in separate articles in this special issue and are not
explicitly covered here.
2. Advances in column technology for liquid
chromatography and its effect on supercritical uid
chromatography
From the 1990s onwards supercritical uid chromatography
has come to resemble liquid chromatography in the choice of
instrumentation and column technology. Separations are typically
performed on columns of the same dimensions and with the same
packing materials as used in liquid chromatography. In addition,
variation of the mobile phase composition and programming of
the mobile phase composition is now more commonly used than
variation of uid density or temperature to optimize separation
conditions. In many cases the separation conditions employed are
sub-critical (liquid phase) separations in which a major component
of the mobile phase is carbon dioxide to which a miscible organic
solvent is added to adjust the eluotropic strength (since carbon
dioxide is a weak solvent) and, in addition, polar additives at low
concentrations are used to improve chromatographic performance
and to control selectivity [1214]. A modern liquid chromato-
graph can be converted for supercritical uid chromatography
purposes by installing additional components to accommodate the
supply and control of compressible uids, a column temperature-
regulated oven (if not already available), a high-pressure capable
absorption detector (other detection methods including light-
scattering and mass spectrometry can be used but absorption
measurements remain the most common), and a backpressure reg-
ulator to maintain the outlet pressure (typically 100150 bar) high
enough to minimize phase separation during chromatography.
Changes in temperature can then be used to maintain a single phase
throughout the separation: (Tcol < Tcrit ) for liquid phase separations
(subcritical) and (Tcol > Tcrit ) for supercritical uid separations.
2.1. Kinetic properties
After many years in which the basic characteristics of column
packings for liquid chromatography underwent small evolutionary
changes the last decade could be considered positively revolution-
ary [15]. The driving force for change was the desire for faster
separations of mixtures of average complexity and the desire to
extend the peak capacity for the separation of complex mixtures in
a reasonable time by liquid chromatography. Monolithic columns
opened the way to faster separations because of their higher
permeability compared with particle-packed columns of similar
performance. Initially, commercially available monolithic columns
were prepared from a single block of silica with a bimodal pore size
distribution, encapsulated in a PEEK tube, and later by monoliths
prepared from porous polymers [1618]. The unique properties of
monoliths result from the larger through pores (macropores) that
provide for higher mobile phase velocities with a lower pressure
drop and smaller mesopores responsible for retention. Commer-
cially available monolithic columns have favorable mass transfer
properties but poor eddy diffusion characteristics due to radial
heterogeneity of ow streams resulting in plate heights similar
to particle-packed columns containing spherical 5 m particles
[15,19]. Typically a minimum plate height of about 815 m at
an optimum linear velocity 23 fold higher, and a column pres-
sure drop 0.50.1 times lower, than 5 m particle-packed columns
is about the best that can be obtained. Research on the optimiza-
tion of the bed structure for monoliths continues but practitioners
have shown greater interest in sub-2 m totally porous particles
operated at pressures up to about 1000 bar and sub-3 m porous
shell particles operated at pressures less than 600 bar. In practice,
a totally porous sub-2 m particle packed column can be operated
to give the same hold-up time as a silica monolithic column with a
45 fold higher column efciency and higher sample detectabil-
ity due to narrower peaks and less sample dilution (lower ow
rates and smaller diameter column format) [15,20]. Supercially
porous particles contain a solid core covered by a thin layer of sta-
tionary phase. First generation particles of 2.7 m diameter had
a fused core of 1.7 m and a 0.5 m porous shell. Compared to
totally porous sub-2 m particles the larger overall particle size
results in higher permeability (lower pressure drop) and the thin
layer of stationary phase improved kinetic performance with a min-
imum plate height equal or better than that for sub-2 m totally
porous particles [15,21,22]. For fast separations supercially porous
particles can provide similar plate counts and speed to sub-2 m
porous particles with a lower column inlet pressure, and for com-
plex samples, a larger plate count for the same separation time.
The greater intrinsic kinetic efciency of supercially porous par-
ticles arises from a smaller contribution to eddy diffusion owing
to the narrower particle size distribution and more homogeneous
bed formation and smaller contributions from axial diffusion on
account of their lower internal porosity [23]. A second genera-
tion of sub-2 m supercially porous particles is now available
and affords faster separations than totally porous particles of the
same size due to about 1.5-fold increase in optimum mobile phase
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
velocity and superior ability to minimize the deleterious effects
of the radial velocity distribution resulting from frictional heating
[24,25]. Although there is a tendency to believe that the enhanced
performance of modern liquid chromatography lies in the above
column developments coupled to the availability of instruments
with higher pressure operating limits, of equal importance is that
instrumentation for fast liquid chromatography provides lower
system volumes and faster data handling that are a large part of
the problem in adapting conventional laboratory instruments for
fast liquid chromatography when supercially porous particles are
used [26,27].
In theory faster separations should be possible using supercrit-
ical uid mobile phases compared with liquids because diffusion
coefcients are typically 310 times larger in uids and the viscos-
ity of uids is smaller by an order of magnitude or more [1113].
For identical packed columns the optimum mobile phase velocity is
shifted to higher values by about 35 fold at a pressure drop from
one-half to over an order of magnitude less. On the other hand,
supercritical uids are more compressible than typical liquids and
their properties are more dependent on operating conditions, such
as temperature, pressure, particle size, packing density, and column
length. The compressibility of uids coupled with their non-ideal
behavior results in simultaneous density, velocity, and retention
factor gradients along the column. The fact that these are not easily
handled by theory means that there is no exact form of the plate
height equation for packed column supercritical uid chromatogra-
phy suitable to direct column design [28,29]. In addition, the same
factors prevent the use of kinetic plots for a meaningful compar-
ison of the performance characteristics of liquid and supercritical
uid chromatography [15,30]. The following discussion, therefore,
built around the use of sub-5 m particle columns and monoliths
developed for fast liquid chromatography is somewhat qualitative
and descriptive, and not as denitive as the reader may wish for.
The best performance for a 25 cm 4.6 mm packed column con-
taining 5-m totally porous particles, the most common column
format for supercritical uid chromatography, is about 500 plates/s,
or about one-half of the value observed for sub-2 m particles
at 1000 bar in liquid chromatography [30]. For the fast separa-
tion of mixtures of average complexity ultra-high performance
liquid chromatography is expected to outperform supercritical
uid chromatography when conventional particle sizes and col-
umn lengths are used. However, when longer columns are required
to obtain the necessary plate count for a separation, supercriti-
cal uid chromatography can provide a higher plate count than
ultra-high performance liquid chromatography because the lower
uid viscosity facilitates the use of longer columns. The primacy
of supercritical uid chromatography to achieve high total plate
counts is well documented in the literature with many examples
of coupled-column systems providing in excess of 100,000 plates
in a reasonable time with a pressure drop <150 bar for binary uid
mixtures of relatively high density [6,12,3133]. Column coupling
may result in a signicant difference in the density drop for the
rst column in a tandem column arrangement with the result that
the prediction of the separation order from those obtained for sin-
gle column separations may not be accurate. For the same reason,
when columns containing different stationary phases are coupled
the separation obtained may depend on the order in which the
columns are coupled. The importance of column length and order
on the observed separation depends largely on the compressibility
of the selected mobile phase. Monolithic columns have signicantly
higher permeability than particle-packed columns and can be cou-
pled in series with a smaller change in uid density [34]. In this way
long column lengths can be used with near constant retention prop-
erties per column and separations are usually more predictable.
For routine applications there is little evidence for sig-
nicant use of monolithic columns in supercritical uid
159
chromatography, probably because silica and octadecylsiloxane-
bonded silica phases are not generally the rst choice stationary
phases for separations while until recently they were the only
commercially available monolithic columns available. Retention
on octadecylsiloxane-bonded silica monoliths is low compared
with comparable particle-packed columns, and their intrinsic
efciency offers no general improvement compared with 5-m
particle-packed columns, and in fact, their efciency is probably
somewhat less. Columns packed with 3-m [3537] and 1.8-m
[38,39] totally porous particles allowed shorter columns and
higher ow rates to be used with a column pressure drop <100 bar
for faster separations without loss of resolution compared with 5-
m particle-packed columns. For these conditions low-dispersion
instruments with fast electronics are obligatory, just as for ultra-
high performance liquid chromatography, even though pressure
limitations are not an issue for conventional chromatographic
instruments. A preliminary assessment of columns packed with
2.6-m supercially porous silica particles demonstrated signif-
icantly higher efciency (35,000 plates compared with 23,000)
in half the time compared with a 3-m totally porous particle
column with a column pressure drop <100 bar [36]. Theoretical
analysis of the band broadening process indicated that the higher
performance for supercially porous particles arose largely from
shorter intraparticle diffusion paths compared with totally porous
particles of a similar diameter [40]. Lesellier [41] noted an increase
in performance for a supercially porous particle column with
increasing molecular weight for members of a homologous series.
These results are different to observations in liquid chromatogra-
phy, and other parameters related to the operating conditions of
the column and system used may be involved. For those interested
in fast separations using supercritical uids, system suitability is
potentially a greater problem than column technology at present
[38]. For fast separations fronting was commonly observed for
peaks on the supercially porous particle column and could have
several origins in column or system properties. This is not unique
to supercritical uid chromatography and is also observed in
ultra-performance liquid chromatography, where it was explained
by a sample size effect [42].
Fast separations in ultra-high performance liquid chromatog-
raphy with a high column pressure drop narrow-bore columns
(2 mm internal diameter) are required for efcient distribution
of heat generated by the viscous ow of the mobile phase. Such
narrow bore columns are not essential for fast separations in super-
critical uid chromatography when using relatively high density
uid mobile phases. This can compensate to some extent for the
performance limitations resulting from extracolumn system dead
volumes. For uid phases of low relative density (e.g., conditions
close to the critical point) large pressure gradients are unavoidable
due to the compressibility of the uid. Large pressure gradients are
undesirable because the temperature of the uid phase decreases
as it expands along the column. In this case, the mobile phase will
absorb heat through the column wall forming axial and radial tem-
perature gradients inside the column. These temperature gradients
result in variations in density, retention, velocity and efciency
affecting column performance and peak shapes [15,2729,43]. In
contrast to ultra-high performance liquid chromatography tem-
perature gradients resulting from viscous uid ow cause heat to
ow from the column center to the wall, the opposite direction to
that observed in supercritical uid chromatography. In supercrit-
ical uid chromatography solute velocity is highest at the column
center where temperature is the lowest (uid density is the highest)
and lowest at the column wall where the temperature is high-
est. The thermal heterogeneity inside the column is responsible
for the excessive efciency loss observed with low density uid
phases (carbon dioxide close to its critical point) [44]. Thus, oper-
ation under near critical conditions results in poor performance in
160 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
fast supercritical uid chromatography, and is avoided in practice,
although the physicochemical properties of uids would be most
favorable for high performance in this region.
Some of the speed and performance advantages of supercritical
uid chromatography could be obtained using high-temperature
liquid chromatography [4547]. At elevated temperatures liquid
viscosity decreases and analyte diffusion increases. High temper-
atures facilitate faster separations for a given particle size due to
higher optimum linear velocities, a reduction in band broadening
from resistance to mass transfer, and a more favorable use of pres-
sure due to the lower column pressure drop. These are the same
features achieved using supercritical uids, except that in the case
of uids, these advantages are obtained at lower temperatures.
High temperatures are not so easy to deploy in fast separations
because of the limited stability of many stationary phases and sam-
ples, the practical limits of instrumentation (preheating the mobile
phase and cooling it down prior to detection increases system vol-
umes and detector noise, etc.), and decrease of the separation space
due to lower retention and changes in selectivity. Most fast sepa-
rations in liquid chromatography are performed at temperatures
close to room temperature (similar in fact to supercritical and sub-
critical uid chromatography).
2.2. Retention properties
The dominant column technology for liquid and supercrit-
ical uid chromatography remains porous silica particles with
siloxane-bonded surface modications. The focus of sorbent chem-
istry directed mainly at the needs of reversed-phase liquid
chromatography has been the design of materials with improved
stability at extreme pH and materials with reduced ion-exchange
capacity arising from interactions with acidic silanol groups [48,49].
Highlights include the development of high-purity, type B, silica
substrates [16,50,51]; inorganicorganic hybrid substrates with
enhanced pH and mechanical stability [52]; chemically bonded
stationary phases with sterically protected and bidentate alkyl-
siloxane ligands [53]; chemically bonded alkylsiloxane ligands with
polar embedded functional groups [54]; chemically bonded sta-
tionary phase with high bonding densities [16]; chemically bonded
phases prepared by the hydrosilanization/hydrosilation reaction
[55]; and new endcapping reagents and procedures (but column
manufacturers simply refer to these as double or triple endcapped,
or endcapped with a hydrophilic reagent, without further expla-
nation). Columns for liquid chromatography are now considered
commodity item with most developments occurring in the manu-
facturing sector with little transfer of knowledge to the customer.
Misinformation created by marketing campaigns only adds to the
confusion. The outcome is a poor understanding of how the impact
of modern materials and reactions has contributed to improve-
ments in column characteristics.
Any column developed for liquid chromatography is avail-
able to supercritical uid chromatography but columns optimized
for reversed-phase liquid chromatography may not be optimum
for supercritical uid chromatography. The retention process in
reversed-phase liquid chromatography is controlled by the partic-
ular properties of water as a mobile phase component. These are far
removed from the general properties of typical uids used in super-
critical uid chromatography. Stability to extreme pH is not a major
concern in supercritical uid chromatography while the unde-
sirable interactions between basic compounds and acidic silanol
groups are a problem in both liquid and supercritical uid chro-
matography. What is not clear is that because the two techniques
share a common problem that they must also share a common
solution. Polar embedded alkylsiloxane ligands are supposed to
shield bases from the silica substrate silanol groups by drawing
water down into the bonded phase where they mask silanol groups
through hydrogen-bonding [54], a mechanism that would not apply
to typical supercritical uid conditions using non-aqueous mobile
phases. On the other hand, higher purity silica substrates, higher
surface coverage of bonded ligands, and improved endcapping
reactions resulting in column packings with fewer accessible acidic
silanol groups have been shown to be better suited to handling
bases in both liquid and supercritical uid chromatography.
After a signicant effort 397 silica-based, chemically bonded
stationary phases have been characterized using the hydropho-
bic subtraction model and a acetonitrile-phosphate buffer pH 2.8
(1:1) mobile phase with a standard testing protocol [5658]. The
hydrophobic-subtraction model ascribes differences in retention
to ve column characteristics: (1) hydrophobicity; (2) steric resis-
tance; (3) hydrogen-bond acidity; (4) hydrogen-bond basicity;
and (5) cation-exchange capacity. The coefcients that represent
these contributions in a linear model are standardized for an aver-
age type-B silica octadecylsiloxane-bonded column such that the
hydrophobicity (H) is 1 and the other coefcients for steric resis-
tance (S), hydrogen bond acidity (A), hydrogen-bond basicity (B)
and cation exchange capacity C-2.8 (cation exchange capacity with
the pH of the mobile phase adjusted to 2.8) are 0. Average values
for the coefcients for type-B silica columns with different bonded
phase ligands are summarized in Table 1 [57]. The dominant con-
tribution to retention on all column types is hydrophobicity of
which a major component is due to the properties of water, and
to a lesser extent, differences in the cohesion of the solvated sta-
tionary phase. The H coefcient tends to increase with the ligand
density, chain length, and extent of endcapping and decreases
with the degree of association (cohesion) of the stationary phase.
It is smaller for ligands with polar functional groups than those
with alkyl groups. For supercritical uid chromatography the water
contribution to the retention mechanism will in most cases be
absent, and consequently, contributions associated with the H
coefcient would not be expected to be as dominant as for reversed-
phase liquid chromatography. The S coefcient determines the
difculty large and bulky molecules experience in fully inserting
themselves in the solvated stationary phase. It generally tends to
reduce retention and a negative sign in Table 1 for this coefcient
is an indication that steric resistance is lower for the column in
question compared to an average silica-based, octadecylsiloxane-
bonded stationary phase. Chemically bonded phases with a phenyl
group exhibit lower steric resistance, otherwise there is little
variation for the other stationary phase types in Table 1. Steric resis-
tance is strongly dependent on the identity and volume fraction
of organic solvent in binary aqueous mobile phases, the bond-
ing density of the stationary phase, and the type of endcapping
[5861]. Steric resistance has not been observed in non-aqueous
systems and its possible role in supercritical uid chromatog-
raphy is unclear (but it should be considered). Steric resistance
is a general retention mechanism not to be mistaken for shape
selectivity exploited using predominantly polymeric chemically
bonded phases for the separation of rigid molecules by reversed-
phase liquid chromatography, although, both mechanisms may
share some features in common [58,62,63]. The hydrogen-bond
basicity of the stationary phases in Table 1 show little variation
with the stationary phase type except for alkylsiloxane-bonded
phases incorporating polar embedded functional groups in the alkyl
chain. Except as noted, it contributes little to selectivity differ-
ences for the stationary phase types. The hydrogen-bond acidity
of the stationary phases shows a different behavior with a signif-
icant variation in selectivity for different stationary phase types.
Since water is the strongest hydrogen-bond acid in the system
one possible explanation for the observed variation is the differ-
ent capabilities of the solvated stationary phase to incorporate
water into its structure, in which case, the A coefcient may not
be a good indicator of the properties of the stationary phase in
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
Table 1
Average values for the coefcients of the hydrophobic-subtraction model for type-B silica siloxane-bonded stationary phases in reversed-phase liquid chromatography with a
mobile phase of acetonitrile-phosphate buffer pH 2.8 or 7.0 (1:1). Coefcients areH: hydrophobicity; S: dipole-type; A: hydrogen-bond acidity; B: hydrogen-bond basicity;
and C-2.8 and C-7.0 are the cation exchange capacity at pH 2.8 and 7.0, respectively.
Stationary phase type Coefcients for the hydrophobic-subtraction model
H S
Butyl 0.69
Octyl 0.84
Octadecylsiloxane (ODS) 0.99
Polar end-capped ODS 0.90
Embedded polar group 0.74
Phenyl 0.63
3-Cyanopropyl 0.43
Pentauorophenyl 0.65
Fluoroalkyl 0.66
supercritical uid chromatography. The cation-exchange capacity
of the stationary phases also shows signicant variation. This inter-
action is believed to be due to the concentration of accessible acidic
silanol groups present in the stationary phase and is strongly inu-
enced by the pH of the mobile phase. For modern type-B silica
the concentration of dissociated silanol groups at pH = 2.8 should
be small but more signicant at pH = 7.0 [64]. Bases exhibit many
of the same problems in terms of excessive retention, poor peak
shape and lower efciency in supercritical uid chromatography as
observed in reversed-phase liquid chromatography. For relatively
low polarity uids the inuence of acid-base chemistry must be
different to aqueous-based systems and similar gross observations
may not translate into similar mechanistic contributions. There is
at least qualitative evidence that columns with low silanol activ-
ity in reversed-phase liquid chromatography are generally better
suited for separating bases in supercritical uid chromatography.
Marchand and Snyder have noted a considerable anion-exchange
capacity for some modern alkylsiloxane-bonded silica stationary
phases with low pH mobile phases [65]. The source of this inter-
action is unknown but might be due to the incorporation of the
catalyst used for the bonding reaction in the stationary phase.
The silica-based, alkylsiloxane-bonded stationary phases that
dominate reversed-phase liquid chromatography play only a small
role in supercritical uid chromatography. They nd applications
for the separation of compounds of low to medium polarity that
are weakly retained on polar stationary phases [8,6670]. Silica
and polar chemically bonded stationary phases are more com-
monly used facilitating separations more similar to those observed
in normal-phase or liquidsolid chromatography [16,7173]. These
or similar stationary phases are also used in hydrophilic interac-
tion chromatography, which typically employs aqueousorganic
solvent mixtures rich in organic solvent as a mobile phase [74].
In normal-phase chromatography samples are separated based on
their competition with the mobile phase for adsorption on xed or
mobility-restricted surface sites on the stationary phase. The gen-
eral elution order is opposite from that observed in reversed-phase
liquid chromatography with compounds of low polarity eluting
early and polar compounds eluting later according to the number
and type of functional groups that they contain. Internal hydro-
gen bonding between functional groups and bulky alkyl groups
adjacent to polar functional groups diminish retention. Signicant
differences in steric tting of isomers and diastereoisomers with
xed-position adsorption sites provide enhanced separations of
these otherwise difcult to separate compounds. Polar chemically
bonded stationary phases have heterogeneous surfaces with unre-
acted silanol groups and the bonded ligand as the main active sites.
The retention characteristics of 3-cyanopropylsiloxane-bonded sil-
ica resemble those of a deactivated (weak) porous silica with
accessible silanol groups as the dominant adsorption sites [73,75].
The retention properties of 3-aminopropylsiloxane-bonded silica
161
A B C-2.8 C-7.0
0.03 0.23 0.02 0.04 0.38
0 0.12 0.02 0.03 0.25
0.01 0.01 0 0 0.24
0.04 0.02 0.02 0.02 0.40
0 0.22 0.12 0.27 0.53
0.12 0.20 0.02 0.13 0.68
0.09 0.49 0 0.02 0.72
0.11 0.25 0.01 0.40 0.96
0.07 0.11 0.03 0.87 1.18
are complementary to the 3-cyanopropylsiloxane-bonded silica
phases with the amino group imparting strong hydrogen-bonding
properties to the stationary phase [76,77]. Spacer bonded propane-
diol phases are generally described as possessing properties
intermediate between those of the 3-aminopropylsiloxane-bonded
and 3-cyanopropylsiloxane-bonded stationary phases, exhibiting
lower retention for hydrogen-bonding solutes than the former
and lower retention of dipolar solutes than the latter [78,79]. A
more quantitative approach to describing selectivity differences
for these polar chemically bonded phases is provided by the sol-
vation parameter model (the features of this model are discussed
in more detail in Section 5.3). Representative data for the separation
characteristics of chemically bonded stationary phases for normal-
phase [7984] and reversed-phase [8489] liquid chromatography
is summarized in Table 2. Retention on all stationary phases for
normal-phase chromatography results from polar interactions of a
dipole-type and by hydrogen-bonding. Increasing solute size gen-
erally reduces retention and electron lone pair interactions are
weak or unimportant. Compounds with weak polar interactions are
not usually signicantly retained compared to polar compounds.
The strong inuence of selective solvation of the stationary phase
by polar components of the mobile phase is illustrated by the
change in system constants for a mobile phase containing n-hexane
and n-hexane containing 1% (v/v) methanol. These changes are too
signicant to be explained by the change in mobile phase solubil-
ity for polar compounds. The range of selectivity for normal-phase
chromatography is quite large compared with reversed-phase con-
ditions and is signicantly dependent on the composition of the
mobile phase. It is unwise, therefore, to attempt to reduce reten-
tion properties to a stationary phase effect alone. A meaningful
comparison can only be made for specic stationary and mobile
phase combinations. A striking feature of the data in Table 2 is that
the variation of selectivity for the polar chemically bonded station-
ary phases is substantially reduced when a polar solvent such as
methanol is added to the mobile phase, and although these station-
ary phases are not selectivity equivalent, they are more like each
other as a group than when n-hexane is the mobile phase. As an
example, the system constants for silica and the silica-based spacer
bonded propanediol stationary phases with n-hexane containing
1% (v/v) methanol are virtually identical.
Water is a cohesive solvent, a strong hydrogen-bond acid, and
reasonably dipolar and hydrogen-bond basic. Its control of the
separation system is illustrated by the change in sign and magni-
tude of the system constants in Table 2 compared with the results
for normal-phase liquid chromatography. The v system constant
has a positive sign indicating increasing retention with solute size
augmented by weak interactions of an electron lone pair type.
Polar interactions, especially solute hydrogen-bond basicity result
in favorable retention in the mobile phase and lower retention
than compounds of low polarity. In addition, there are signicant
162 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171

Table 2
System constants of the solvation parameter model for separations by normal-phase and reversed-phase liquid chromatography for silica-based, siloxane-bonded sorbents.
The difference of intermolecular interactions in the mobile and solvated stationary phases are indicated ase: electron loan pair; s: dipole-type; a: hydrogen-bond basicity;
b: hydrogen-bond acidity; and v: cavity formation and dispersion interactions.

Stationary phase type Mobile phase System constants

e s a b v

(i) Normal-phase liquid chromatography

Spacer bonded propanediol n-Hexane 0 1.63 2.10 3.86 1.05
3-Aminopropyl 0 1.40 1.65 3.81 0.95
3-Cyanopropyl 0 1.88 2.47 0.99 0.37
Silica n-Hexanemethanol 0 1.06 2.23 1.56 0.83
Spacer bonded propanediol (99:1) 0 1.07 2.37 1.47 0.85
3-Aminopropyl 0 0.94 2.94 1.20 0.72
3-Cyanopropyl 0 0.95 1.86 1.15 0.61
Silica n-Hexanemethyl 0.86 1.67 1.94 3.00 0
3-Cyanopropyl t-butyl ether (95:5) 0 1.43 1.10 2.80 1.20

(ii) Reversed-phase liquid chromatography

Spacer bonded propanediol Watermethanol (90:10) 0.89 0.37 0.26 0.50 0.49
3-Aminopropyl 0.37 0 0 0.52 0.38
3-Cyanopropyl 0.21 0 0.17 0.60 0.62
Octadecyl 0.02 0.56 0.45 1.97 3.35
Embedded polar group 0.28 0.47 0 2.20 3.07
Phenyl 0.28 0.35 0.58 1.49 2.36
Pentauorophenyl 0.32 0.24 0.45 1.90 3.02
Fluoroalkyl 0.49 0.49 0.48 1.74 2.38

differences in the way low polarity and polar chemically bonded
stationary phases behave in reversed-phase chromatography. This
is probably due to a combination of the characteristic properties of
the stationary phase and differences in the solvation of stationary
phases of different polarities by water. The compatible environ-
ment of the polar chemically bonded phases with water likely
facilitates the incorporation of water into the stationary phase
structure diminishing the controlling inuence of water in the
mobile phase on retention compared with low polarity station-
ary phases. The retention will generally be less on polar chemically
bonded phases for all compounds under reversed-phase conditions.
By analogy there is good qualitative support for retention on
polar chemically bonded stationary phases under typical supercrit-
ical uid conditions to be a normal-phase retention mechanism
[12,13,66,90]. Polar chemically bonded stationary phases are
widely used in supercritical uid chromatography for the sep-
aration of polar compounds with a mobile phase consisting of
carbon dioxide to which a polar organic solvent possibly containing
an additive is added [8,9,12,32,9193]. Inorganic oxide stationary
phases, mainly silica gel, are used for separating hydrocarbon-like
compounds with carbon dioxide as the mobile phase and more
polar compounds using carbon dioxide with a polar organic solvent
and possibly an additive as well as the mobile phase [9,9395]. Sil-
ica is a strong adsorbent and excessive retention and poor peak
shapes are often only avoided by deactivating the silica surface
by adsorption of polar components incorporated into the mobile
phase. Porous graphitic carbon posses a less chemically active
adsorbent surface but has strong retention properties [66,9699].
The general retention mechanism is characterized by strong dis-
persion and dipole-type interactions as well as steric selectivity
that facilitates the separation of isomers [99,100]. Because of strong
retention the molecular weight separating range for this phase is
limited to small molecules (naphthalene compounds are consid-
ered well retained). For the separation of amines it was shown
that basic additives in the mobile phase were required to mask
acidic sites on the graphite surface that give rise to a secondary
retention mechanism of low capacity [97]. Porous organic poly-
mers have been evaluated for the separation of bases to overcome
undesirable interactions with inorganic oxides that result in poor
peak shapes [9,51,90]. The retention mechanism on these sor-
bents probably involves a combination of partition and adsorption
governed by dispersion, dipole-type and polarization interactions.
They tend to exhibit strong retention and sometimes low efciency
for well retained compounds. One contributing factor to the strong
retention may be the sorption of mobile phase components by the
polymers resulting in an unfavorable phase ratio for fast separa-
tions and the separation of high molecular weight compounds.
Since these materials contain micropores additional band broad-
ening due to slow diffusion in the polymer matrix can be a problem
[101]. Immobilized ionic liquids on a porous silica substrate were
evaluated for use in supercritical uid chromatography but with
limited success owing to strong retention and poor peak shapes for
polar compounds [102,103].
3. Effect of mobile phase composition on stationary phase
properties
The elution strength of carbon dioxide is generally too weak to
elute polar compounds and to increase its solvent strength organic
solvents in amounts from about 5% to 50% (v/v) are added to it for
this purpose [8,11,12,14]. Modiers can inuence the qualities of
a separation in several ways: (1) the modier can alter the den-
sity and solvating power of the mobile phase; (2) the modier can
block active sites on the stationary phase and inhibit adsorption;
(3) sorbed modier can act as a component of the stationary phase;
(4) sorbed modier can increase the volume of the stationary phase
leading to a change in the column phase ratio; and (5) the modi-
er may selectively solvate polar compounds in the mobile phase
with the formation of clusters with different distribution properties
[104]. The combination of these factors can change retention in an
unpredictable manner while three of these factors result in a change
in stationary phase properties with a strong dependence on mobile
phase composition, column temperature, and density drop along
the column. For conditions typical of packed column supercritical
uid chromatography signicant amounts of both carbon dioxide
and modier are adsorbed by silica and chemically bonded sta-
tionary phases and function as components of the stationary phase
[12,13,56,104108]. Adsorbed modier changes the phase ratio
and alters intensity of stationary phase interactions. Contemporary
studies of stationary phase modication by adsorption of mobile
phase components are rare, although important for understanding
the retention mechanism. Adsorbed layers easily reach multilayer
thicknesses and can amend the contribution of the naked station-
ary phase to the retention mechanism. Thus, relating retention
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
properties to the chemical composition of the column packing is
far from straightforward in supercritical uid chromatography.
The use of common organic solvent modiers in supercritical
uid chromatography is not always sufcient to facilitate elution
in a reasonable time or with acceptable peak shapes for strong acids
and bases and some polar compounds [7,12,91,92,97,98]. Additives
at a concentration <1% (v/v) are used to mitigate this problem. Typ-
ical additives are strong acids (e.g., formic acid, triuoroacetic acid,
citric acid), bases (e.g., isopropylamine, triethylamine) or salts (e.g.,
ammonium acetate) [108110]. There are no general guidelines
for the choice of additives beyond that they should be a stronger
acid or base than the sample components as well as compatible
with the choice of detector. Consequently, common additives used
for mass spectrometry are frequently different to those used for
absorption detection. Neutral polar additives like formamide, 3,3,3-
triuoroethanol, and water are less commonly used today than
earlier times [9,111113]. Since additives are often used of neces-
sity rather than desire a direction of current research is the design of
stationary phases that minimize the need for additives to overcome
one or more of the following disadvantages: (1) increased noise for
low-wavelength absorbance detection; (2) erosion of silica-based
stationary phases; (3) system peaks that cause interference in the
chromatogram; (4) interference in the ionization process in mass
spectrometric detection; (5) reaction with some compounds during
chromatography; (6) and their removal presents an additional com-
plication in preparative supercritical uid chromatography [91].
Suggested roles for additives in the separation process include:
(1) enhancement of the mobile phase solvating power; (2) sup-
pression of sample ionization; (3) ion pairing with the sample;
and (4) modication of the stationary phase [111,114117]. Zheng
et al. [117] used indicator dyes to monitor the increase in acidity
of methanolcarbon dioxide mixtures with an increase in the vol-
ume fraction of carbon dioxide. This result seems counter intuitive.
The pH indicator dyes are calibrated for aqueous solutions and it is
unclear what the true meaning of a change in color for these dyes
in a uid of low dielectric constant actually represents. These color
changes are not necessarily related to proton transfer properties
and may possibly be explained by selective solvation effects.
Additives adsorb to a greater extent to the stationary phase than
modiers and the chemical identity of the naked stationary phase
may indicate little about the retention behavior of the solvated
stationary phase. The role of additives in supercritical uid chro-
matography remains speculative with few studies contributing to
a mechanistic understanding of their use while demonstrating a
practical benet in expanding the range of polar compounds that
can be analyzed. The use of additives in packed column supercritical
uid chromatography may diminish the importance of the identity
of the stationary phase for the separation of polar compounds and
requires further study.
4. Stationary phases developed for supercritical uid
chromatography
All stationary phases developed for liquid chromatography are
available to supercritical uid chromatography and remain the
largest group of stationary phases used in practice, particularly
silica-based polar chemically bonded phases. By comparison a
few stationary phases have been marketed with an eye to the
particular needs of supercritical uid chromatography, which of
course, are also available for use in liquid chromatography. His-
torically, the rst type of stationary phase to nd greater use
for supercritical uid chromatography than liquid chromatogra-
phy was the silica-based polymer-encapsulated stationary phases.
These materials consisted of porous silica particles coated with
an immobilized layer of a poly(siloxane) containing alkyl or polar
163
Table 3
Functional groups used to prepare silica-based polar chemically bonded stationary
phases marketed for supercritical uid chromatography.a
2- and 4-Ethylpyridine, Dipyridyl, Dicyanoimidazole, Morpholine
Propylacetamide, Benzamide
Methanesulfonamide, Benzenesulfonamide, 4-Fluorobenzenesulfonamide,
4-Nitrobenzenesulfonamide
Diethylaminopropyl, 3-Aminopropyl N-dinitrotoluene
Monol
a
Information from Princeton Chromatography (Cranbury, NJ, USA) and Zymor
Inc. (Wayne, NJ, USA).
substituent groups. At this time pure carbon dioxide or carbon diox-
ide with low levels of polar additives was the dominant mobile
phase and the ame ionization detector used for detection [9,16].
Density programming was generally used to change the elution
strength of the mobile phase rather than composition program-
ming widely used today. These stationary phases were somewhat
successful at masking problems associated with accessible silanol
groups on the silica substrate, but not entirely so, and are not
as widely used today. The desire to minimize undesirable silanol
interactions in the separation of polar compounds resulted in the
development of chemically bonded stationary phases with a 2-
ethylpyridine substituent [91,92,98,109,111,118]. For these phases
it is assumed that hydrogen-bonding between free silanol groups
and the nitrogen atom of the pyridine ring, or partial protonation of
the pyridine group (due to the acidity of carbon dioxidemethanol
mobile phases) would repel positively charged analytes from the
stationary phase, reducing interactions with the silanol groups
[109]. In addition, the bonded pyridine group could minimize inter-
actions with silanol groups by steric shielding. This stationary phase
is somewhat successful in the above regard and is often the rst
choice for the separation of bases with or without the use of addi-
tives. It is also a useful phase for the separation of neutral and
acidic polar compounds [118]. A number of silica-based amide,
urea, sulfonamide, and pyridine containing stationary phases are
commercially available, Table 3, and marketed specically for use
in supercritical uid chromatography. Some mixed bed column
chemistries are also available. For these columns there are virtu-
ally no application notes available or indication in trade catalogs of
what sample types they are most useful for. Beyond their availabil-
ity at the time of writing there is little that can be said about their
separation characteristics except for the observations on column
characterization in the following section.
5. Classication methods
The general lack of fundamental knowledge of the retention
mechanism combined with the relatively large number of sta-
tionary phase chemistries available has resulted in a somewhat
empirical approach to column selection and method development
in supercritical uid chromatography. For polar compounds the
diversity of mobile phase modiers is limited by the required sol-
vent strength for elution in an acceptable time. The mobile phase
also plays a key role in adjusting the stationary phase chemistry
masking or confounding the variation of stationary phase prop-
erties anticipated based on the structure of the naked stationary
phase. Empirical knowledge gathered over time is often used as
an aid to column selection and for the selection of initial separa-
tion conditions. Details of some systematic approaches to column
(and initial conditions) selection are summarized in the sections
below. The lack of clear guidelines merely reects the current state
of knowledge of the mechanisms involved and the rather complex
interrelationship between the chemistry of the stationary phase,
the mobile phase composition, and column operating parameters.
164 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
5.1. Column screening studies
If experiments can be performed quickly and efciently then
the insight obtained can be substituted for a theoretical framework
in the selection of stationary phases for method development. This
has become a common approach in the pharmaceutical industry for
the identication of suitable conditions for the purication of target
compounds produced in compound libraries [2,3,119122]. These
compounds are typically small polar molecules with a reasonable
number containing basic functional groups. From a global perspec-
tive, the results described below are somewhat biased by exclusion
of compounds not normally associated with drug discovery. Screen-
ing is usually performed on short columns with a methanol gradient
in carbon dioxide with a cycle time <10 min for neutral and weakly
basic compounds. For strong acids and bases additives are normally
required to obtain suitable retention and acceptable peak shapes.
Column selection valves, available on some instruments, facilitate
the sequential evaluation of a batch of columns for the separation of
the same sample using the same mobile phase and variation of the
stationary phase. White and Burnett [2] found a silica-based sta-
tionary phase containing siloxane-bonded 2-ethylpyridine groups
to be suitable for the separation of a wide range of mixtures of the
type described above. For strong bases 10 mM ammonium acetate
or 0.2% (v/v) isopropylamine were used as additives. Strong acids
and zwitterionic compounds could not be eluted in a reasonable
time from this column and were better handled on silica-based
3-cyanopropylsiloxane-bonded, spacer bonded propanediol, and a
mixed bed packing containing both column packings as stationary
phase. McClain et al. [120] also reported that the 2-ethylpyridine
siloxane-bonded phase was generally useful for purifying mixtures
containing neutral and weakly acidic and basic compounds. Weller
et al. [121] found four columns (ether-linked phenyl, poly(vinyl
alcohol) immobilized as a layer on porous silica, a mixed bed 3-
cyanopropylsiloxane-bonded and spacer bonded propanediol, and
2-ethylpyridine siloxane-bonded) afforded sufcient diversity as
a screen for the purication of small molecule libraries. The suc-
cess rate for isolating the target compounds was similar for the
four columns (range <10%) while providing reasonable diversity in
the separation characteristics between individual stationary phases
using a carbon dioxidemethanol gradient containing ammonium
acetate as an additive. Aurigemma and Farrell [3] described a
six column set as the basis of an open access screen to iden-
tify suitable conditions for purication of small molecule libraries
using a methanol in carbon dioxide gradient without additives.
The columns were selected initially based on prior experience
and conrmed by statistical analysis. The column chemistries
were all from Zymor Inc. and consisted of silica-based pyri-
dine and spacer bonded propanediol mixed bed, HA-dipyridine,
HA-pyridine, 4-ethylpyridine, octylsiloxane-bonded and pyridine
(endcapped) mixed bed, and a spacer bonded propanediol. Based on
observations for 1200 mixtures the column of rst choice for puri-
cation of the target compounds was the spacer bonded propanediol
(35%), 4-ethylpyridine (28%), and the pyridine and spacer bonded
propanediol mixed bed (19%). Soto-Yarrilu et al. [122] selected
eleven stationary phase chemistries for evaluation using a varied
set of standards (36 compounds) and approximately 100 mixtures
submitted for purication using a carbon dioxidemethanol gra-
dient containing 0.2% (v/v) N,N-dimethylethylamine as an additive
(except for the separation of strong acids). Five stationary phases
were selected from this group based on their success at separating
the above compounds and mixtures. The spacer bonded propane-
diol and 2-ethylpyridine stationary phases were suitable for all
compound types and a diethylaminopropylsiloxane-bonded phase
was only marginally less successful for bases. A spacer bonded
dinitrophenyl phase and a benzenesulfonamide phase provided a
useful alternatives to the rst three phases but had a lower overall
success rate for the separation of the test compounds and mixtures.
On the spacer bonded dinitrophenyl phase about half of the bases
were excessively retained. The benzenesulfonamide phase handled
bases well but acids exhibited broadened peaks. The retention on
silica and the spacer bonded propanediol phase were highly cor-
related with the spacer bonded propanediol phase being preferred
owing to its lower retention and better peak shapes. These authors
also evaluated a series of sulfonamide phases (benzenesulfon-
amide, 4-uorobenzamidesulfonamide, and methanesulfonamide)
whose retention properties were highly correlated as a group and
selected benzenesulfonamide as a suitable representative of this
group.
5.2. Carotenoid test
The carotenoid test was developed to characterize physic-
ochemical properties of silica-based, alkylsiloxane-bonded sta-
tionary phases that affect their use in reversed-phase liquid
chromatography [123126]. The conditions used for the test
are subcritical uid chromatography, however, with carbon
dioxidemethanol (85:15, v/v) as the mobile phase. The results
have some relevance for supercritical uid chromatography for
column characterization, and to a lesser extent, method develop-
ment (the diversity of the stationary phases is limited to various
types of alkylsiloxane-bonded silica stationary phases, which are
less relevant for supercritical uid chromatography compared with
reversed-phase liquid chromatography). The test consists of the
isocratic separation of three prototypical compounds: 13-cis--
carotene, all trans--carotene, and zeaxanthin. The retention factor
for -carotene provides a scale of general retention related to
the specic surface area of alkylsiloxane-bonded silica stationary
phases and their ligand density. The separation factor for 13-cis
and all trans--carotene provides a measure of shape selectiv-
ity. Since the latter depends on the type of bonding chemistry,
it can distinguish between polymer coated ( < 1), monomeric
bonded (1 < < 1.2), and polymeric ( > 1.2) alkylsiloxane-bonded
silica stationary phases. It can also be used to estimate the bond-
ing density for monomeric-bonded stationary phases and the pore
diameter for polymeric phases [126]. The separation factor for all
trans--carotene and zeaxanthin (similar structure to -carotene
but contains two terminal hydroxyl groups) provides a scale of
surface polar activity that depends on the proportion and acces-
sibility of polar sites (silanol groups, polar endcapping treatment,
polar embedded groups, etc.) The test can distinguish the major
variations in column chemistry for alkylsiloxane-bonded silica sta-
tionary phases with data for over two hundred columns available
[124126]. The scope of the test can be extended by combining the
data with the system constants of the solvation parameter model
[124].
5.3. Solvation parameter model
The solvation parameter model, based on a cavity model of sol-
vation, is a widely used tool to estimate the contribution of cavity
formation and intermolecular interactions to retention of neutral
molecules in two-phase separation systems [16,82,127130]. For
solute transfer between condensed phases the appropriate form of
the solvation parameter model is
log k = c + eE + sS + aA + bB + vV (1)
where k is the retention factor, the lower case letters are sys-
tem constants, and the capital letters solute descriptors. The
solute descriptors describe solute properties and are excess molar
refraction E, dipolarity/polarizability S, hydrogen-bond acidity A,
hydrogen-bond basicity B, and volume V (McGowans character-
istic volume scaled by division by 100). The system constants are
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171 165

the complementary properties of the separation system described Table 4

System constants for silica-based chemically bonded stationary phases with carbon
as the extra polarizability associated with loosely held electron
dioxidemethanol mixtures as the mobile phase. Each column was 5 cm 4.6 mm,
pairs e, the capacity for dipole-type interactions s, the hydrogen- 5-m particle size, ow rate 3.3 ml/min, column temperature 35 C, and column
bond basicity a (because a hydrogen-bond acid interacts with a outlet pressure 100 bar [141].
hydrogen-bond base), the hydrogen-bond acidity b, and the differ-
Methanol (%, v/v) System constants
ence in cavity formation and residual dispersion interactions that
are not cancelled by transfer between phases v. The equation con- e s a b v c
stant, c term, contains a number of system and modeling properties, (i) 3-Cynanopropylsiloxane-bonded
but is usually dominated by the phase ratio for the separation sys- 5 0.13 0.31 0.88 0.13 0 0.93
tem when the dependent variable is the retention factor. The solute 10 0.10 0.24 0.54 0.13 0.06 0.65
15 0.22 0.02 0.36 0.16 0.01 0.55
descriptors are available for over 4000 compounds and are usu- 20 0.17 0.03 0.26 0.14 0.02 0.53
ally determined by calculation (V and E) and by experiment (S, A, 25 0.16 0.06 0.16 0.05 0.05 0.42
and B), as explained elsewhere [127129]. The system constants
(ii) Spacer bonded propanediol
in Eq. (1) are the parameters used to characterize the contribution 5 0.38 0.23 1.32 1.02 0.13 0.66
of the dened intermolecular interactions to the retention of neu- 10 0.34 0.13 0.84 0.63 0.10 0.51
tral compounds. They are calculated by multiple linear regression 15 0.31 0.12 0.55 0.47 0.08 0.42
analysis from the experimental retention factors determined for 20 0.31 0.16 0.43 0.46 0.06 0.45
25 0.27 0.15 0.36 0.29 0.02 0.32
a varied group of compounds with known descriptor values that
meet a set of chemical and statistical requirements for modeling (iii) 2-Ethylpyridinesiloxane-bonded
[127129,131,132]. 5 0.23 0.04 1.38 0.11 0.06 0.57
10 0.19 0.10 1.05 0.11 0.01 0.55
Early applications of the solvation parameter model to reten- 15 0.20 0.03 0.87 0.09 0.02 0.49
tion in supercritical and subcritical uid chromatography on 20 0.19 0.03 0.71 0.05 0.03 0.49
packed columns containing silica-based chemically bonded phases 25 0.16 0.04 0.52 0.06 0.02 0.34
[14,133,134] and porous polymers [135137] are reviewed in (iv) 3-Aminopropylsiloxane-bonded
[84,138]. The primary goal of these studies was to provide an under- 5 0.29 0.01 1.78 0.76 0.12 0.69
standing of the role of solvent modiers and additives on retention 10 0.21 0.08 1.19 0.54 0.07 0.47
for polar compounds with carbon dioxide as the main mobile 15 0.16 0 0.94 0.40 0.08 0.45
20 0.19 0 0.83 0.35 0.11 0.55
phase component. A limited amount of information is available for 25 0.10 0.02 0.67 0.27 0.08 0.45
supercritical uid n-heptane [136] and 1,1,1,2-tetrauoroethane
[136,137]. There are potential problems in the interpretation of
these studies due to weak retention of most compounds [133,137]
or because of the selection of test compounds with a narrow and are generally weak interactions. Other properties that depend
range of descriptor values [14,135]. The use of high temperatures signicantly on mobile phase composition become less important
and experimental conditions resulting in a relatively large density at higher mobile phase compositions. Solute size (v system con-
change along the column may confound identication of the sta- stant) and dipole-type interactions (s system constant) are weak,
tionary phase contribution and the effect of system properties on not strongly composition dependent, and result in lower retention
the retention mechanism. There is some ambiguity in the selec- for larger solutes and solutes that are more dipolar/polarizable.
tion of the appropriate form of the solvation parameter model for The dominant interactions that increase retention are the capabil-
studies at column temperatures close to the critical temperature ity of the solute for hydrogen-bonding (a and b system constants)
and low uid densities compared with column temperatures below which become less effective at higher methanol concentrations in
the critical temperature and high uid densities. The former condi- the uid phase. The contribution of electron lone pair interactions
tions are closer to gas chromatography and the V system constant is signicant at all mobile phase compositions and shows a shallow
may not be the preferred parameter for these conditions while it dependence on uid composition. The c term affects the retention
is less objectionable for the second set of conditions [138,139]. The of all solutes equally and has a signicant dependence on composi-
uncertainty in the conditions used for modeling in the above stud- tion. The major component of the c term is expected to be the phase
ies, however, does not allow a condent conclusion to be reached. ratio (although not exclusively) and its variation with composition
In the studies discussed below, the use of Eq. (1) to describe the is an indication of the increase in volume of the stationary phase
retention mechanism seems reasonable [138].
The preferred approach to demonstrate the inuence of mobile
phase composition on retention is through the use of system 1.5
maps [8489,140]. These maps illustrate the smooth change in a
system constants as a function of the composition of the mobile 1 b
System Constant

phase and should be applicable in subcritical uid chromatogra-

phy were the average density of the mobile phase is high and the
0.5
density drop along the column is small. Bui et al. [141] deter- e
mined the system constants for 3-cyanopropylsiloxane-bonded,
0 v
3-aminopropylsiloxane-bonded, 2-ethylpyridinesiloxane-bonded,
s
and spacer bonded propanediol silica stationary phases on 5 cm
-0.5
columns at 35 C with a column outlet pressure of 100 bar for car- c
bon dioxide containing from 5% to 25% (v/v) methanol, Table 4.
A system map based on this data is shown in Fig. 1 for the spacer -1
0 5 10 15 20 25
bonded propanediol stationary phase. System constants with a pos-
itive sign indicate stronger interactions in the solvated stationary Methanol (% v/v)
phase than the uid phase and vice versa. The system map shows
Fig. 1. System map for carbon dioxidemethanol mobile phase compositions on a
two types of behavior with change in methanol composition. Prop- spacer bonded propanediol stationary phase. The system constants are dened in
erties that change only slightly with mobile phase composition Section 5.3 for use in Eq. (1).
166 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171

Table 5 Table 6
System constants for silica-based chemically bonded stationary phases with carbon Key to abbreviations for the stationary phase types reported in Table 5 and Fig. 2.
dioxidemethanol (9:1, v/v) as the mobile phase. Each column was 25 cm 4.6 mm,
5-m particle size (mainly), ow rate 3 ml/min, column temperature 25 C, and Abbreviation Stationary phase type
column outlet pressure 150 bar [138]. C4 Butylsiloxane-bonded silica
C8 Octylsiloxane-bonded silica
Stationary phase System constants
C12 Dodecylsiloxane-bonded silica
e s a b v C18 Octadecylsiloxane-bonded silica
C18-C Octadecylsiloxane-bonded type C silica
C4 0.220 0.170 0 0.111 0.230 RPH Octadecyl(phenyl)siloxane-bonded silica
C8 0.322 0.268 0.278 0.219 0.272 MIX Octadecylsiloxane- and phenylpropylsiloxane-bonded silica
C12 0.379 0.242 0.372 0.444 0.313 PE1 Amide embedded hexadecylsiloxane-bonded silica
C18 0.587 0.459 0.457 0.479 0.439 PE2 Ether sulfonamide embedded hexadecylsiloxane-bonded silica
C18-C 0.658 0.447 0.199 0 0.276 PE3 Carbamate embedded hexadecylsiloxane-bonded silica
RPH 0.470 0.261 0.407 0.411 0.373 FD Peruorodecylsiloxane-bonded silica
MIX 0.309 0.106 0.156 0.254 0.328 SI Silica gel
PE1 0.661 0.317 1.263 0.427 0.345 DIOL Spacer bonded propanediol
PE2 0.527 0.091 0.499 0.428 0.305 PEG Poly(ethylene glycol) bonded on silica
PE3 0.577 0.214 0.795 0.462 0.308 PVA Poly(vinyl alcohol) bonded on silica
FD 0.282 0 0.457 0.961 0.501 NH2 Aminopropylsiloxane-bonded silica
SI 0.352 0.336 1.196 1.011 0.571 CN Cyanopropylsiloxane-bonded silica
DIOL 0.460 0 0.928 0.945 0.561 EP 2-Ethylpyridinesiloxane-bonded silica
PEG 0.341 0.256 1.236 0.224 0 PGC Porous graphitic carbon
PVA 0.535 0 1.189 0.878 0.477 PS Poly(styrene-divinylbenzene)
NH2 0.475 0.301 1.349 0.901 0.678 OPHE Phenyloxypropylsiloxane-bonded silica
CN 0.339 0.372 0.696 0.449 0.346 DP Diphenylpropylsiloxane-bonded silica
EP 0.588 0.564 1.053 0.790 0.692 DP-X Diphenylpropylsiloxane-bonded silica
PGC 1.552 0.391 0.593 0.307 1.145 C3P Phenylpropylsiloxane-bonded silica
PS 0.762 0 0.132 0.279 0.268 C6P-L Phenylhexylsiloxane-bonded silica
OPHE 0.346 0.099 0.185 0.101 0.192 C6P-G Phenylhexylsiloxane-bonded silica
DP 0.297 0 0.311 0.213 0.209 PYE 2-Pyrenylethylsiloxane-bonded silica
DP-X 0.311 0 0.218 0.076 0.261 PFP Pentauorophenylpropylsiloxane-bonded silica
C3P 0.280 0 0.218 0.121 0.279 PBB Pentabromobenzyloxypropylsiloxane-bonded silica
C6P-L 0.326 0 0 0.125 0.285 Chl Cholesterylundecyl-bonded silica
C6P-G 0.319 0 0.084 0.249 0.312 C6P-Z Phenylhexylsiloxane-bonded silica
C6P-Z 0.338 0 0 0.163 0.306 NAP Naphthylethylsiloxane-bonded silica
PYE 0.333 0.398 0.145 0.277 0.364 DNAP Dinitroamidopropylsiloxane-bonded silica
PFP 0.169 0.483 0.054 0 0.313 PNP p-Nitrophenylsiloxane-bonded silica
PBB 0.728 0 0.214 0 0.479 AMD Poly(amide) gel bonded on silica
Chl 0.595 0.172 0.328 0.625 0.469
NAP 0.333 0.132 0.126 0 0.307
DNAP 0.737 0.510 0.750 0.501 0.086
PNP 0.470 0.267 1.010 0.622 0.226 v = 0.69 to 1.15. The column set thus encompasses a reasonably
AMD 0.499 0.290 1.368 1.184 0.618 wide selectivity space for the separation of neutral compounds.
To identify a smaller number of columns that span the selectiv-
ity space we applied hierarchical cluster analysis with the system
at higher methanol compositions resulting in increased relative constants as variables, Fig. 2. The abbreviations used in Fig. 2
retention for all compounds. and Table 5 are the same as those suggested by West and Lesel-
System maps can be built in the same way for the other lier and are summarized in Table 6. Fig. 2 indicates that there
phases in Table 4. The main characteristic difference between are a signicant number of column chemistries with similar sep-
these four phases is the ratio of hydrogen-bond acidity to aration properties allowing a smaller number of columns to be
hydrogen-bond basicity with b/a 0.8 for the spacer bonded selected for method development. Starting from the top of the g-
diol and 0.43 for the 3-aminopropylsiloxane-bonded phases. ure; the rst cluster contains six stationary phases with similar
The 3-cyanopropylsiloxane-bonded and 2-ethylpyridinesiloxane- separation properties (C6P-L; C6P-Z; C6P-G; C4; C8; and MIX). The
bonded solvated stationary phases are only weakly hydrogen-bond average system constant and standard deviation for the station-
acidic and reasonably independent of the methanol composition ary phases in this cluster are; eav = 0.31 0.04; sav = 0.09 0.11;
resulting in b/a < 0.5 and <0.12, respectively. None of the four sol- aav = 0.06 0.13; bav = 0.19 0.06; and vav = 0.31 0.05. Reten-
vated stationary phases are signicantly dipolar/polarizable and all tion on these columns is a result of the relative difference
have similar cohesion to the uid phase so that these interactions in cohesion of the mobile and solvated stationary phases and
play a small role in adjusting selectivity [for s most signicant on favorable dispersion interactions with compounds of higher
the 3-cyanopropylsiloxane-bonded phase when methanol is 10% hydrogen-bond basicity exhibiting weaker retention. The dipolar-
(v/v) and for the spacer bonded propanediol solvated stationary ity/polarizability and hydrogen-bond acidity of compounds is not
phase for all compositions]. Changes in elution order on the four an important contributing factor to selectivity. The second cluster
phases will be most common for compounds that differ in their from the top contains ve phases of similar separation proper-
capability for hydrogen-bonding interactions and less so for com- ties (DP-X; C3P; OPHE; DP; and NAP) with PYE loosely connected
pounds with identical or limited capability for hydrogen-bonding to this group but with slightly different selectivity. The average
interactions. values for the system constants of the stationary phases in this
West and Lesellier [138,142145] have constructed a system cluster are: eav = 0.31 0.03; sav = 0.04 0.06; aav = 0.21 0.07;
constant database for 35 columns using a standardized experi- bav = 0.10 0.08; and vav = 0.25 0.05. These stationary phases are
mental protocol, Table 5, in which the mobile phase is carbon somewhat similar to those in cluster 1 except that compounds
dioxidemethanol (9:1, v/v) at 25 C. Leaving out porous graphitic of higher hydrogen-bond acidity now exhibit stronger retention
carbon because of its extreme system constant values the range compared with non-hydrogen-bonding compounds. The dipolar-
of system constants for the other stationary phases are: e = 0.46 ity/polarizability and hydrogen-bond basicity of compounds is not
to 0.56; s = 0.46 to 0.56; a = 0.46 to 1.37; b = 0.63 to 1.18; and an important contributing factor to selectivity although the weak
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171 167

Fig. 2. Cluster dendrogram for the average linkage between groups agglomeration algorithm for the 35 stationary phases identied in Table 6 with the system constants of
the solvation parameter model as variables (Table 5).
168 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
hydrogen-bond acidity of these columns favors higher retention
for strong hydrogen-bond bases. The third cluster from the top
in Fig. 2 contains three stationary phases with similar separa-
tion properties (PS; PBB; C18-C). These stationary phases are a
loosely clustered group with a wider range of system constants
than observed in cluster 1 and 2. The average values for the system
constants are: eav = 0.71 0.07; sav = 0.11 0.20; aav = 0.17 0.04;
bav = 0.09 0.08; and vav = 0.33 0.13. Compared with cluster
2 electron lone pair interactions are more important for these
phases and result in increased retention with dipolar/polarizable
compounds exhibiting weaker retention. Two stationary phases
(PE2 and Chl) are clustered together and loosely connected
with PE3. This cluster is indicated as only loosely connected
to clusters 13. The average values for the system constants
are: eav = 0.56; sav = 0.13; aav = 0.41; bav = 0.53; and vav = 0.39.
These stationary phases have different hydrogen-bonding prop-
erties to the rst three clusters and exhibit enhanced retention
for hydrogen-bond acids and reduced retention for hydrogen-
bond bases as well as strong retention for large compounds
of low polarity and compounds with polarizable electron lone
pairs. The fourth cluster from the top contains three stationary
phases (C12; RPH; C18). The average values for the system con-
stants are: eav = 0.48 0.10; sav = 0.32 0.12; aav = 0.41 0.04;
bav = 0.45 0.03; and vav = 0.38 0.06. These stationary phases
have a rather simple behavior with cavity/dispersion and electron
lone pair interactions favoring retention while interactions of a
dipole-type and hydrogen-bonding interactions result in reduced
retention. Thus non-polar compounds will be well-retained; and by
comparison; polar compounds of a similar size less well retained.
Among polar compounds the selectivity for separation is likely to
be poor unless the compounds can be distinguished principally by a
signicant difference in a single polar interaction (s; a or b). The fth
cluster of three stationary phases (CN; PNP; and DNAP) is separated
from the previous clusters by several stationary phases that show
independent behavior or form clusters of low similarity; and are
discussed later. The average values for the system constants of the
stationary phases in cluster 5 are: eav = 0.52 0.02; sav = 0.38 0.12;
aav = 0.82 0.17; bav = 0.52 0.09; and vav = 0.22 0.13. For these
stationary phases there is a signicant change in general behav-
ior to those described above. These are perceived as polar phases
with a relatively higher penalty for cavity formation resulting in a
negative v system constant and declining retention with increasing
solute size. Commensurate with more cohesive stationary phases
with a higher capacity for polar interactions; dipolar/polarizable
and hydrogen-bonding compounds exhibit strong retention. The
last cluster; cluster 6; contains two sub groups (DIOL and PVA)
and (Si; NH2; AMD); which can be distinguished from each other;
and the stationary phase (EP); which is loosely connected to
this group. The average system constant for DIOL and PVA are
eav = 0.50; sav = 0; aav = 1.06; bav = 0.91; and vav = 0.52 and for Si;
NH2; and AMD eav = 0.44 0.08; sav = 0.31 0.02; aav = 1.30 0.09;
bav = 1.03 0.14; and vav = 0.62 0.05. The main factors that dis-
tinguish the two sub groups in this cluster is their different capacity
for dipole-type interactions (signicant for Si; NH2; and AMD and
insignicant for DIOL and PVA) and the stronger hydrogen-bond
basicity of Si; NH2 and AMD. This group of stationary phases shows
strong retention of polar compounds owing to their ability to inter-
act through dipole-type and hydrogen-bonding interactions which
are offset for solutes of increasing size owing to the high cohesion
of these stationary phases. 2-Ethylpyridine fails to gain full mem-
bership of this cluster because it is more dipolar/polarizable and
signicantly less hydrogen-bond acidic and basic.
There are several stationary phases in Fig. 2 that behave
independently and thus have a combination of intermolecular
interactions that are not duplicated by the stationary phases that
can be clustered. These are PFP, PE1 and PEG (PE1 and PEG are
grouped together but with low similarity to each other and to
the other stationary phases), PGC, and FD. PFP has virtually no
hydrogen bonding interactions, is weakly dipolar/polarizable, and
moderately cohesive. It should be useful for separating small dipo-
lar molecules. PE1 and PEG selectively retain hydrogen-bond acids
while exhibiting low retention for hydrogen-bond bases (these
stationary phases have large a system constants combined with
small b system constants of opposite sign). PGC has a set of inter-
actions that are a different blend to the other stationary phases
but the large e and v system constants indicate strong retention
that limit applications to small molecules. Dipole-type interactions
are unimportant for FD and retention is governed by hydrogen-
bonding interactions (the ratio b/a 2 for this phase is unusual
among the stationary phases in Table 5). FD is also a strong cohesive
phase and increasing solute size results in lower relative reten-
tion. For method development one could choose a single stationary
phase from each cluster and any of the singleton stationary phases
with useful characteristic properties for the separation. The results
should be sound for separations that employ similar operating con-
ditions and column characteristics to those used to construct the
database. The results also demonstrate the effect of the mobile
phase in altering the separation characteristics of the columns since
many of the bonded phases lack the features in their naked struc-
tures to participate in the interactions demonstrated as important
in explaining how separations occur.
West and Lesellier [146] used spider diagrams and the angle
between system constant vectors as a column selection tool to
identify phases to span the available selectivity space with mini-
mal clustering. They used a database containing 28 of the columns
in Table 5. They recommended selection of (1) a polar phase (EP),
(2) an aromatic phase of intermediate polarity (OPHE), (3) a non-
polar phase (MIX), and (4) two unique phases C4 and PFP (although
the later is a poor choice when samples contain cationic species
due to low column performance). These recommendations are in
broad agreement with the conclusions based on cluster analysis.
West et al. [147] also demonstrated that the system constants in
Table 5 could be used to facilitate the selection of columns based
on the simulation of predicted separations in conjunction with a
multi-criteria desirability function to rank column suitability.
Mitchell et al. [148] determined system constants for eleven
columns with the same mobile phase composition employed by
West and Lesellier. West and Lesellier [132] observed that the
results of Mitchell et al. were similar to theirs for polar phases like
silica, but opposite in a number of respects for low-polarity phases
(e.g., octadecylsiloxane-bonded and cholesterylundecylsilane-
bonded silica), and quite different for some aromatic phases (e.g.,
phenylpropylsiloxane-boned, pentauorophenylsiloxane-bonded,
and naphthylethyl-siloxane bonded silica). Signicant differences
in the magnitude and sign of the v and b system constants were the
most general in the two studies. West and Lesellier accounted for
these differences by a poor choice of dead time marker compound
(signicant retention for the experimental conditions), the weak
retention of many of the compounds with an increasing likelihood
of larger errors in the retention factor values, and the inadequate
range of the V descriptor for the construction of robust models in
the study of Mitchell et al.
A comparison of the three databases described above reveals a
much wider range of the system constant values for similar station-
ary phase types with the same mobile phase compositions than is
expected viewed from similar studies of column characterization
in liquid and gas chromatography [84,127131,149]. The separa-
tion conditions employed in the three studies are summarized in
Table 7. In supercritical uid chromatography retention factors are
not independent of the column temperature, the pressure (density)
gradient along the column, the column permeability, mobile phase
ow rate, and the column outlet pressure. These parameters are
 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171 169

Table 7
Operating conditions for the column characterization studies summarized in Section 5.3 with carbon-dioxidemethanol (9:1, v/v) as the mobile phase.

Parameter Bui et al. [141] West and Lesselier [138] Mitchell et al. [148]

Column temperature ( C) 35 25 35
Flow rate (ml/min) 3.3 3 3
Column outlet pressure (bar) 100 150 100
Column length (cm) 5 25 15
Column internal diameter (mm) 4.6 4.6 3 or 4.6
Particle size (m) 5 5 (mainly) 2.75

also interdependent and in absolute terms it is difcult to speculate
with condence how these differences affect the variation in the
system constants. It is likely that the temperature range (2535 C)
has only a small affect on relative retention but differences in the
outlet pressure (100150 bar), column length (525 cm), and par-
ticle size (2.75 m), could affect relative retention signicantly
through variation in the average column density and changes to
the composition of the solvated stationary phase. These effects
have to be superimposed on the expected differences between col-
umn chemistries dened only by the nominal identication of the
stationary phase by its general chemical type. All in all, the differ-
ences in the system constants might be less alarming if they are
considered to represent quite different experimental domains. The
column inlet pressure is not indicated in the three studies but is
an important parameter as an indication of the average column
density as a reference point for columns of a similar type oper-
ated with similar experimental conditions. In addition, rather than
a single mobile phase composition system maps for a range of uid
compositions would be more useful for method development and
to establish chemical and statistical problems in model building.
The system constants for similar stationary phases employed
in the above studies are summarized in Table 8 [138,141,148].
Columns from the same manufacturer were studied by Bui et al.
[141] and West and Lesellier [138] with the main difference being
the column length and the column outlet pressure (the 10 C
temperature difference was assumed to be less important), see
Table 7. It is expected that the density drop along the column
would be larger in the study of West and Lesellier and the average
column density would be higher. The largest variation for the com-
pared columns is observed for the v system constant with smaller,
but statistically signicant differences, observed for the s, a and b
system constants. There is greater disparity in the system proper-
ties for similar stationary phases in the studies of Mitchell et al.
[148] and West and Lesellier [138]. Mitchell et al. used shorter
columns with a lower permeability per unit length (in most cases
a smaller particle size) as well as a higher temperature and a lower
column outlet pressure, Table 7. The column density drop and
average column density is anticipated to be higher in the study
of Mitchell et al., although in the absence of experimental data
this is speculation. However, it may explain part of the signi-
cant difference in the system constants between the two studies
as well as statistical differences arising from the different number
and descriptor range of the test solutes and weak retention, as dis-
cussed in [132]. For similar stationary phases studied by Mitchell
et al. [148] that differ in column permeability (supercially porous
particles and totally porous particles of different particle diameters
and/or column internal diameter at a constant mobile phase ow
rate), Table 8, the system constants are signicantly different, with
differences in the v system constant being the largest. There is at
least qualitative evidence that the variation in the system constants

Table 8
Comparison of system constants for nominally similar stationary phases.

Source System constants Reference

e s a b v

Kromasil SIL-100 0.35 0.34 1.20 1.01 0.57 [138]

Ascentis silica 0.22 0.61 0.73 1.22 0.50 [148]
Ascentis Express Silica 0.15 0.37 0.68 1.31 0.70 [148]

Uptisphere PH 0.28 0 0.22 0.12 0.28 [138]

Ascentis phenyl 0.38 0.03 0.01 0.09 0.23 [148]

Cosmosil -NAP 0.33 0.14 0.13 0 0.31 [138]

Cosmosil -NAP 0.35 0.25 0.08 0.46 0.14 [148]

Discovery HS F5 0.17 0.48 0.05 0 0.31 [138]

Discovery HS F5 0.24 0.92 0.30 0.19 0.38 [148]
Kinetex PFP 0.55 0.58 0.05 0.04 0.86 [148]

Kromasil C!8 100 0.59 0.46 0.46 0.48 0.44 [138]

Zorbax XDB-C18 0.65 0.22 0.12 0.86 0.07 [148]

Cogent Bidentate C18 0.66 0.45 0.20 0 0.28 [138]

Cogent Bidentate C18 0.63 0.10 0.60 1.29 0.62 [148]

Cosmosil Cholester 0.60 0.17 0.33 0.63 0.47 [138]

Cogent UDC cholesterol 0.63 0.50 0.21 1.32 0.53 [148]

Princeton Amino 0.48 0.30 1.35 0.90 0.68 [138]

Princeton Amino 0.29 0.01 1.78 0.76 0.12 [141]

Princeton Diol 0.46 0 0.93 0.95 0.56 [138]

Princeton Diol 0.38 0.23 1.32 1.02 0.13 [141]

Princeton CN 0.34 0.37 0.70 0.45 0.35 [138]

Princeton CN 0.13 0.31 0.88 0.13 0 [141]

Princeton ethylpyridine 0.59 0.56 1.05 0.79 0.69 [138]

Princeton ethylpyridine 0.23 0.04 1.38 0.11 0.06 [141]
170 C.F. Poole / J. Chromatogr. A 1250 (2012) 157171
in the above studies is determined in part by differences in system
properties (column design and operating conditions). The system
constant databases described here may only be useful in describing
the retention properties of columns of similar design and employ-
ing similar operating conditions, as well as for the same or similar
stationary phases.
6. Conclusions
Supercritical uid chromatography by age could be considered
a mature separation technique but by knowledge of the separa-
tion process it lags well behind gas and liquid chromatography.
Both kinetic optimization and retention mechanisms seam more
complex and interdependent on the system operating conditions
resulting in the lack of a comprehensive model for column design.
Most of what is known about the factors that are responsible for
separations is descriptive and at best semi-empirical in super-
critical uid chromatography and in need of further study. In the
absence of a general framework it will continue to have doubters
who stay away from the technique because it seems difcult and
unpredictable. These features make it difcult to introduce into
routine laboratories if less of a burden in a research environment
where a lack of understanding can be a reason for a engagement.
The contents of this review reect these concerns and highlight
the lack of focus on fundamental studies on column design and
method development. Supercritical uid chromatography is a high
performance technique so mixtures of moderate complexity fre-
quently get separated under conditions far from optimum since
the idea of what is optimum and what is easily achieved are not
so easy to differentiate without a general metric of what to expect.
Hopefully, when the next review of column design and station-
ary phase chemistry is written it will be against a background of a
deeper understanding, control, and predictive capability of the sep-
aration process similar to the other main stream chromatographic
methods.
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