Received: 28 July 2020
Revised: 17 September 2020
Accepted: 6 October 2020
DOI: 10.1002/jssc.202000823
REVIEW ARTICLE
Two-dimensional separation techniques using supercritical
fluid chromatography
Alexander S. Kaplitz1,†
Mahmoud Elhusseiny Mostafa2,†
James L. Edwards2
James P. Grinias1
Samantha A. Calvez1
1
Department of Chemistry &
Biochemistry, Rowan University,
Glassboro, NJ, USA
High-resolution separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase resolution, multidimen-
2 Department of Chemistry, Saint Louis
University, St. Louis, MO, USA
sional chromatographic techniques have been one key solution. Supercritical
fluid chromatography provides a unique opportunity in these multidimensional
Correspondence
James P. Grinias, Department of Chemistry
& Biochemistry, Rowan University, 201
Mullica Hill Rd., Glassboro, NJ 08028,
USA.
Email: grinias@rowan.edu
James L. Edwards, Department of Chemistry, Saint Louis University, 3501 Laclede
Ave., St. Louis, MO 63103.
Email: jim.edwards@slu.edu
separations based on its potential for high solvent compatibility, rapid duty
cycles, and orthogonality to other separation modes. This review focuses on
two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and
modulation strategies used to interface supercritical fluid chromatography with
other liquid chromatography and gas chromatography techniques are described.
Particular applications of multidimensional separations using supercritical fluid
† Both of these authors contributed equally
chromatography for the analysis of oils and chiral separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for
to this work.
supercritical fluid chromatography in this field are also discussed.
Funding information
National Science Foundation,
Grant/Award Numbers: CHE-1904919,
CHE-1904454
1
KEYWORDS
chiral separations, modulation, multidimensional separations, supercritical fluid chromatography
INTRODUCTION
Supercritical fluid chromatography (SFC) has emerged as
a viable technique to achieve high separation speed and
reproducibility, while reducing solvent consumption for
the separation of complex mixtures. SFC uses supercritical
fluids, typically carbon dioxide (CO2 ), as the primary
mobile phase constituent in a chromatographic separation
[1]. SFC dates to 1962, when separations using supercritical
chlorofluoromethanes were first reported [2]. However,
developments in SFC stalled as research on HPLC began
to flourish [1]. The modern era of SFC began in the
Article Related Abbreviations: API, active pharmaceutical
ingredient; NPLC, normal phase liquid chromatography; SFC,
supercritical fluid chromatography; SFE, supercritical fluid extraction
J Sep Sci 2020;1–12.
late 2000s when more robust SFC instrumentation and
columns designed specifically for this technique became
commercially available. Other reasons for the growth in
SFC popularity have included its environmentally friendly
nature, potential for reduced operational costs, and versatility [3,4]. This versatility results from the wide variety
of conditions under which the nonpolar supercritical
CO2 can interact with mobile phase organic modifiers
and stationary phases, which provides the potential for
separation conditions that can be used for the resolution
of both polar and hydrophobic analytes [5]. Another
advantage of using supercritical CO2 as the main mobile
phase constituent is its low viscosity, ≈0.1 cP, which is
an order of magnitude lower than mobile phases that are
typically used in RP LC [3]. Low-viscosity separation conditions provide the opportunity to further increase flow
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© 2020 Wiley-VCH GmbH
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KAPLITZ et al.
rates and thus use SFC for high-throughput separations
without exceeding instrument pressure limits [6]. Analyte
diffusion increases in mobile phases with lower viscosity, which can increase mass transfer rates and reduce
C-term broadening, resulting in higher optimal velocities
compared to LC [7]. Compared to many organic solvents
used in LC, CO2 is less expensive and has lower toxicity,
providing for a “greener” analytical methodology. Several
recent reviews have focused on SFC [3,4,8–10], with the
topic of this review more specifically focused on the use of
SFC in multidimensional chromatography techniques.
2D chromatography is a high peak capacity separation
strategy most often used to analyze complex samples [11].
This technique provides substantial improvements in peak
capacity (nc ) compared to conventional 1D separations by
combining two orthogonal separation mechanisms into a
single method. In 2D separations, two peaks unresolved by
the first separation mechanism can ultimately be resolved
by introducing the first-dimension (1 D) co-eluting analytes
to an orthogonal chromatographic mechanism in the second dimension. As the need for analysis of complex samples in a variety of matrices continues to increase, systems
which offer improved resolution and peak capacity will be
in high demand. 2D chromatography provides the capability to analyze complicated samples in various fields [12,13],
including metabolomics [14,15], proteomics [16–18], environmental studies [19], and food applications [20].
2D separations can be undertaken using combinations
of GC, LC, CE, and/or SFC in varying configurations. The
overall peak capacity for a 2D method is the product of each
dimension’s peak capacity [21,22]:
𝑛𝑐,𝑡𝑜𝑡 = 𝑛𝑐,1𝐷 × 𝑛𝑐,2𝐷
use high-throughput 2 D separation approaches. Based on
its unique selectivity and mobile phase properties, along
with its potential for fast separations, SFC is a powerful
technique to be used in multidimensional chromatography
[30].
In this review, the variety of ways in which SFC has been
implemented in multidimensional separation techniques
is described. The use of SFC in 2D methods coupled to LC
provides an orthogonal mode to RPLC with advantages
over polar LC modes like HILIC or normal phase LC
(NPLC) in terms of solvent compatibility and throughput.
This is especially true for the use of chiral SFC as one
of the employed modes. Combined 2D-SFC techniques
are also described, in which supercritical CO2 mobile
phases are used for the separations in both dimensions.
Both comprehensive (designated with a “×” between
separation modes) and heart-cutting (designated with
a “−” between separation modes) 2D approaches will
be described. The primary focus of this review is on
manuscripts published over the last decade, as this is
when multidimensional separations using modern SFC
techniques have been achieved, although additional older
studies are also described for significant relevance and/or
historical context. For information regarding the coupling
of supercritical fluid extraction (SFE) to various analytical
approaches, readers are directed to other recent reviews
on this topic [31–33], as SFE is only mentioned in the
context of multidimensional separations using SFC here.
2
SFC AS THE FIRST DIMENSION
SEPARATION
(1)
However, to ensure that this maximum theoretical
value is obtained, the two dimensions must be completely
orthogonal [23] and a high sampling rate for 1 D fractionation into the second-dimension (2 D) separation must be
maintained [24]. Although selecting two distinct chromatography modes with high orthogonality is straightforward, the challenge for liquid-phase separations becomes
ensuring mobile phase compatibility between the two
phases [25]. In many couplings, the 1 D eluent is the strong
mobile phase solvent in the 2 D separation, leading to
poor peak shape if loaded directly onto the 2 D column.
Strategies including active solvent modulation, trapping
columns, dilution, and evaporation have all been applied
to help solve issues with solvent incompatibility between
dimensions [26]. For sampling rate, theory suggests that
the ratio of the sampling time to the standard deviation
of a 1 D peak should be no more than 2 (i.e., at least four
samples across an 8-sigma peak width) [24,27–29]. The
best approach to achieving this stringent requirement is to
Although SFC is primarily combined with various liquidphase separation modes in modern 2D techniques, there
is historical precedence of coupling 1 D SFC separations
to GC. The key challenge in this approach is minimizing
the amount of CO2 mobile phase that enters the GC column. An early study coupling SFC and GC used a restrictor tube to minimize CO2 expansion until the eluent flow
reached the injection port of the GC [34]. To ensure effective modulation between the dimensions and maintain
good peak shape, the injection port was maintained at a
lower temperature than typically used for GC. A similar
approach involves rapid temperature gradients in the 2 D
GC separation, with the initial column temperature held
low to achieve analyte focusing [35,36]. Other improvements to modulation have been achieved using thermal
desorption strategies, which allowed for controlled release
of SFC eluent onto the GC column, akin to injector actuation [37]. Strategies for coupling SFC and GC have been
applied to a variety of sample types [38,39]; significant discussion of these older studies is beyond the scope of this
KAPLITZ et al.
3
F I G U R E 1 Schematic for SFC × RPLC instrument that includes trapping columns between dimensions (pictured in inset). Adapted from
[47], Copyright (2008), with permission from Wiley-VCH
text, especially as other reviews more extensively cover this
topic [40–42]. Modern applications of SFC coupled to GC
have focused on the analysis of oil and petroleum samples,
with the most advanced strategies utilizing SFC-GC × GC
[43–45] and SFC × GC × GC [46] instrumentation designed
to facilitate removal of CO2 from the 1 D fractions with minimal analyte loss prior to GC × GC analysis.
For liquid-phase 2D separations, the use of online SFC ×
RPLC was explored early in the modern SFC era as an
alternative to NPLC × RPLC [47]. NPLC × RPLC is an
appealing combination for 2D-LC due to the high orthogonality between the modes, but immiscibility between
their mobile phases can make the pairing difficult [48].
SFC coupling is much more favorable because it offers
similar selectivity to NPLC but can reduce mobile phase
incompatibility. This is because the CO2 can be eliminated
prior to injection of the eluted fractions onto the 2 D RP
column. To further improve the modulation process, C18
trapping columns were used between the dimensions to
prevent analyte loss as the CO2 dissipated (Figure 1). In
this configuration, further peak focusing was aided by
flowing aqueous mobile phases through the trap columns
before analytes were injected into the 2 D RP column. This
initial system was applied to the analysis of lemon [47]
and fish [49] oils, while a similar system was later adapted
for use in the characterization of red chili peppers [50].
Other innovations in online SFC-RPLC have included a
heart-cutting strategy with dilution-based modulation on
the trap column [51] and the use of vacuum evaporation to
remove the SFC mobile phase prior to the 2 D separation
[52]. In comparing the use of NPLC × RPLC and SFC ×
RPLC techniques for carotenoid fingerprinting in chili
pepper samples using both Q-TOF-MS and ion mobility
MS detection modes, the SFC approach had a higher
number of positive compound identifications (50 over 33)
in half the time (60 min compared to 120 min) with less
than 10% of the solvent usage (42 and 480 mL for SFC
and NPLC, respectively) [50]. These multiple advantages
demonstrate the potential for SFC × RPLC techniques as
an alternative to NPLC × RPLC methods.
Although comprehensive online methods are generally
more automated and faster than off-line 2D separations,
the use of off-line methodology can reduce some of the
challenges associated with ensuring mobile phase compatibility between two orthogonal separation modes. In
one report, SFC was coupled to a nonaqueous RP separation for the analysis of triacylglycerols in fish oil [53].
Here, 18 fractions from a 1 D SFC separation were manually collected, evaporated under nitrogen, dissolved in
a mixture of acetonitrile and isopropanol, and then analyzed with a method that used three C18 columns coupled in series. The peak capacity for this off-line strategy
was more than double the value achieved with an online
SFC × RPLC approach (7560 vs. 3000), albeit at the expense
of an approximately 20-fold increase in analysis time [53].
Similar approaches have been adapted for the analysis of
amide alkaloids from Piper longum L [54], the purification of lignan compounds from Fructus Arctii [55], and
the characterization of gangliosides in swine brain extract
[56]. In this latter study, 153 unique gangliosides were separated, with 20 detected for the first time based on the high
selectivity of this approach. In general, the high orthogonality of SFC (when used as an alternative to NPLC) and
RPLC provides a powerful combination for the analysis of
complex biological samples that contain a wide variety of
compound classes, with off-line approaches providing high
peak capacities that can increase positive analyte identifications when coupled to MS.
4
KAPLITZ et al.
F I G U R E 2 Comparison of RPLC × SFC (left) and RPLC × RPLC (right) methods for the analysis of a bio-oil sample. The 1 D column was
a porous graphitic carbon stationary phase in both separations, with a 2-ethylpyridine stationary phase used with the 2 D SFC column and a
phenyl hexyl stationary phase used with the 2 D RPLC column. Peak capacities based exclusively on peak width measurements were 730 and
1120 for RPLC × SFC and RPLC × RPLC, respectively, but corrected to 620 and 560 when orthogonality (based on coverage of separation space)
and peak sampling frequency were considered. Adapted from [63], Copyright (2015), with permission from Elsevier
3
SFC AS THE SECOND-DIMENSION
SEPARATION
Early reports of LC coupled to capillary SFC were mainly
focused on increasing the injection volume that could be
loaded onto a 2 D SFC column to improve detection limits [57–60], rather than as a comprehensive 2D approach
that is more common now. In these modern methods,
high-throughput 2 D separations are essential for ensuring
sufficient sampling rates that maintain the 1 D chromatographic resolution [12,13,24,61]. The low viscosity mobile
phases that provide multiple advantages for SFC at high
flow rates compared to various liquid phase separation
modes make it a promising 2 D technique. Additionally,
the 1 D liquid eluent is often easier to inject into the 2 D
SFC column rather than integrating modulation strategies
needed when SFC is used in the first dimension. This technique has seen more use in the past decade, often with a
two-loop modulation strategy for online LC × SFC methods [62,63]. With NPLC × SFC (with a C18 column used
for the SFC separation to provide orthogonality to the NP
cyano column), 250 peaks were observed in a Ganoderma
lucidum sample, compared to just 17 and 34 peaks for
1 D NPLC and SFC separations using the same columns
[62]. For RPLC × SFC, higher corrected peak capacity
was observed compared to RPLC × RPLC, despite higher
observed chromatographic efficiency for the 2 D RPLC column compared to the 2 D SFC column. This was due to the
higher orthogonality between the phases, which ensured
that a higher portion of the two-dimensional separation
space was used (Figure 2) [63]. In addition to collection
loops, trap columns have been placed between dimensions
for online RPLC × SFC separations [64]. The challenge
with this approach was the balance between 1 D and 2 D
separation times: namely that higher flow rates in the 1 D
provided higher peak capacities for that separation, but
this created issues with undersampling that decreased the
overall peak capacity. Even though trace analytes were
easier to observe due to their concentration on the trap
column, the overall peak capacity loss was nearly 20%
due to this undersampling effect compared to just using
loops [64].
As with SFC × LC, off-line LC × SFC techniques
designed to reduce issues with solvent incompatibility
between dimensions have also been demonstrated, with
RPLC × SFC most commonly used due to its relatively
high degree of orthogonality. For the analysis of a complex
blackberry sage fragrant oil, 56 fractions (15 s sampling)
collected from a gradient RPLC separation were then
transferred to a 2 D SFC amino column to obtain an overall
peak capacity of 2400 in 280 min [65]. A similar approach
using a semipreparative 1 D RPLC column to increase
the volume of the fractions injected into the 2 D SFC
column (bare ethylene-bridged hybrid silica) enabled the
identification of 1033 unique peaks from two extract fractions (petroleum ether and ethyl acetate) of a traditional
Chinese medicine sample (Piper kadsura) [66]. Finally,
a method for characterizing bufadienolides in a different
traditional Chinese medicine (Venenum bufonis) was
achieved by using collecting 40 fractions from an RPLC
separation, drying them with nitrogen and reconstituting
in an acetonitrile–water mixture, and then analyzing them
with a 2 D SFC separation utilizing a C18 column [67]. 229
unique bufadienolides were found, with the method being
especially promising for the identification of structural
isomers within the complex sample.
KAPLITZ et al.
5
F I G U R E 3 Demonstration of a heart-cutting 2D LC-SFC approach to separate the active pharmaceutical ingredient from other impurities
using 1 D RPLC and then determine chiral purity with a 2 D chiral SFC separation. Adapted from [73], Copyright (2016), with permission from
Elsevier
One of the most common applications of SFC in recent
years has been in the field of chiral separations [68–70].
SFC is especially useful for high-throughput chiral separations, with nearly all analytes in a 50-compound set
of chiral drugs and synthetic intermediates obtaining
resolution of enantiomers in under 2 min, and most being
completely separated in less than 1 min, which are much
shorter analysis times compared to LC [71]. Because of
the need for short 2 D separation time in 2D methods
and the high resolution often achieved with chiral SFC
separations, the use of LC-SFC for achiral-chiral analysis
is especially promising [72]. A heart-cutting strategy using
RPLC-SFC enabled the 1 D separation of achiral impurities
from a pharmaceutical compound, with the 2 D chiral
separation used to determine the enantiomeric purity of
the same compound (Figure 3) [73]. C18 trapping columns
were used between the dimensions to focus the 1 D peaks
prior to injection onto the SFC column. This system was
then used for drug metabolism study samples, with the
1 D C18 column used to separate the active pharmaceutical
ingredient (API) and its metabolites from various matrix
interferences. Then, the 2 D chiral SFC separation was
used to monitor the enantiomeric excess of both the API
and its metabolites [74]. Without this focusing between
dimensions, the detection limits of the trace impurities
can become a potential challenge to effective achiral-chiral
analysis for APIs. When using a commercial instrument
for a multiple heart-cutting RPLC-SFC method, extracolumn dispersion between dimensions decreased the S/N
of a chiral impurity peak compared to 1D-SFC by more
than half [75]. Thus, minimizing analyte dilution during
modulation can be especially important for these critical
pharmaceutical separations of trace chiral impurities.
4
2D-SFC
SFC × SFC techniques can be promising, as the use of
supercritical CO2 in both dimensions provides higher
compatibility of the mobile phase than approaches in
which a liquid mobile phase is used in one of the dimensions [76]. SFC × SFC provides wider applicability to
nonvolatile and thermally labile compounds than GC ×
GC, and can provide higher throughput and better mobile
phase compatibility than LC × LC [77,78]. As in other
2D methods using SFC described thus far, another key
drawback is that commercial systems designed specifically
for SFC × SFC are not yet available, preventing wider
adoption of the technique. The research instruments
that have been used are generally similar in nature: a
10-port valve is used to collect eluent from the 1 D column and eventually transfer it to the 2 D column, while
6
FIGURE 4
KAPLITZ et al.
Schematic of a typical 2D-SFC instrument set-up. Adapted from [76], Copyright (2012), with permission from Elsevier
also ensuring that a BPR is connected to both columns
throughout the separation (Figure 4). Initial proof of
concept work on SFC × SFC systems used a two valve
system with a trapping mechanism to capture and degas
the eluent from the 1 D before injection into the 2 D column
[77]. In this demonstration, both dimensions used a C18
separation that limited orthogonality, but increased peak
resolution compared to 1D separations was still observed
for a number of vegetable oil and animal fat samples.
Sequential advancements on this system were implemented to improve, automate, and accelerate the analysis
of these types of samples. First, a bare silica 1 D column
was used to separate a series of fatty acid methyl esters
by the number of double bonds present in the fatty
acid chain, with a 2 D C18 column used to separate the
compounds by their chain length (Figure 5) [79]. Once
1 D eluent was passed into the open trapping tube (a
poly(dimethylsiloxane)-coated metal capillary), the tube
was depressurized which evaporated the mobile phase
and left the analytes in place to then be desorbed and
flowed to the 2 D separation. Online automation of the
valving/sampling ensured that three to four samples were
collected for each 1 D peak in accordance with multidimensional chromatography sampling theory [24]. A
subsequent advancement of the system with capillary SFC
columns in both dimensions used a stop-flow approach,
which enables sufficient sampling rates under longer 2 D
separation times [80]. Adapted from a previous GC × GC
technique [81], a synchronized pressure program enabled
the 1 D flow to be stopped while each 2 D separation
occurred, with the pressure ramp rate, sample modulation
duration, and backpressure restrictor dimensions all
affecting the quality of the separation.
A simpler approach to online SFC × SFC without requiring trapping between dimensions used neat supercritical
CO2 as a mobile phase throughout the system with a 1 D
F I G U R E 5 Two-dimensional contour chromatograms of fatty
acid methyl esters of perilla oil (top), soybean oil (middle), and butter fat (bottom) using 2D-SFC. The 1 D separation using a bare silica
column separated compounds by the degree of saturation and the 2 D
separation using a C18 column separated compounds by chain length.
Adapted with permission from [79]: Springer, Copyright (2004)
C18 column coupled to a 2 D bare silica column [76]. Here,
to avoid overloading the 2 D column with 1 D effluent, a
split-flow approach reduced the volume injected on to the
bare silica column from over 300 μL (10 s sampling time at
KAPLITZ et al.
7
F I G U R E 6 Comparison of peak width for naphthalene and anthracene without (left) and with (right) peak compression during modulation in a 2D-SFC separation. Adapted from [82], Copyright (2018), with permission from Elsevier
2 mL/min flow rate) down to 45 μL. The disadvantage of
this system is that the flow split leads to analyte loss during the transfer to the 2 D column. To mitigate this issue, an
active modulation system with a modified transfer module was developed using two ten-port valves and two independent supercritical CO2 mobile phase pumps for each
dimension to enable peak compression between dimensions [82]. By optimizing the pressure and flow in both
columns and during the analyte transfer between dimensions, peak compression at the modulator provided a fivefold decrease in the 2 D peak width without requiring a flow
split that leads to analyte loss (Figure 6).
SFC × SFC separations have also been coupled with
SFE by connecting a dual ten-port valve modulation system to an extraction chamber and trapping column [78].
Using the components in various configurations allowed
for different characteristics of polymers to be analyzed.
An SFE-SFC was used to analyze antioxidants and color
additives in a polypropylene sample by coupling multiple
extractions at increasing temperatures with an SFC separation using a C18 column. SFE-SFC × SFC was then used
for the extraction and separation of various oligomers in
polystyrene foam that were difficult to resolve using a 1D
separation.
As with LC-SFC, the use of SFC-SFC can have improvements over LC-LC for the analysis and purification of
chiral pharmaceutical compounds due to solvent compatibility issues between dimensions and low resolution
when using NPLC chiral separations in multidimensional
techniques. A preparative SFC-SFC approach for analysis
of chiral pharmaceuticals was used to separate the API
from its impurities in the first dimension and resolve
enantiomers of the API in the second dimension [83].
In this system, a dual column setup (achiral and chiral)
was operated using a single pump with four six-port
valves. 1 D effluent is split and initially monitored by
both UV absorbance and ESI-MS detectors. When the
predetermined requisite m/z value for the API is detected,
a directional valve is triggered to transfer the flow to a
trap column and subsequently to the 2 D chiral column in
a single heart-cut approach. In one example, a racemic
mixture of a pharmaceutical compound was isolated from
other reaction by-products in the 1 D achiral separation and
then separated into its two enantiomers in the 2 D chiral
separation in a single, online 10-min method (Figure 7).
5
CONCLUSIONS AND FUTURE
OUTLOOK
As demonstrated in the studies described here, the use
of SFC in multidimensional separation techniques has
proven to be a valuable strategy for the analysis of complex
mixtures, particularly for lipids and achiral-chiral mixtures. A general summary of the advantages and disadvantages of various 2D-SFC approaches based on the results
referenced in this article is shown in Table 1. The use of
SFC as a dimension in 2D separations reduces some of
the challenges that exist in 2D-LC, with the capability for
improved solvent compatibility between dimensions and
faster 2 D duty cycles. These factors proved important for
the analysis of a wide variety of food, fragrance, and natural product samples, among others. Similar to 2D-LC [84],
it is likely that the pharmaceutical industry will have a
major role in the impact of wider adoption of multidimensional separation techniques using SFC. The adoption of
industry-wide improvements in analytical method sustainability that rely heavily upon increased use of SFC [85]
and the recent call by the Enabling Technologies Consortium to instrument vendors requesting the development
8
KAPLITZ et al.
F I G U R E 7 Achiral-chiral 2D-SFC-MS analysis for the separation of a three-component pharmaceutical mixture. (A) 1 D achiral SFCMS total ion current (TIC) chromatogram showing separation of a chiral pharmaceutical compound from two impurities. (B) 2D-SFC-UV
chromatogram with heart-cut fraction transfer to 2 D chiral separation. The dotted line shows the mobile phase gradient profile in both panels.
Adapted from [83], Copyright (2011), with permission from Elsevier
TA B L E 1
Comparison of SFC × LC and LC × SFC
SFC × LC
LC × SFC
Advantages
∙ Improved solvent compatibility compared to
NPLC × RPLC [48]
∙ Improved MS-based analyte identification [50]
∙ Fast
∙ Reduced solvent consumption
∙
∙
∙
∙
∙
Limitations
∙ May require off-line sample collection and
re-injection [53–55], which can lead to 20-fold
increase in analysis time
∙ Potential undersampling at higher LC flow rates [64]
∙ May require off-line sample collection and re-injection [67]
∙ Extra-column band broadening decreases S/N [75]
of commercially viable LC × SFC technology [86] indicate that this may occur sooner than later. In the pharmaceutical industry, it is likely that combined achiral-chiral
methods for impurity monitoring and purification will be
a primary motivation for implementing 2D-LC-SFC and
2D-SFC [73,74,83].
While most iterations of 2D-SFC have used RPLC and
NPLC 1 D columns, the use of 1 D IEX and SEC separations
coupled to 2 D SFC columns is inevitably on the horizon.
Although the use of ion-exchange columns and ion-pairing
reagents has been demonstrated with SFC for the separation of ionic compounds [87–90], the use of hydrophobic,
high proton affinity tags to derivatize polar analytes
Higher sampling rates [61]
High SFC flow rates
Elution/sampling compatibility
High nc [63]
Well-suited for achiral-chiral separations [73–75]
and membrane proteins offers unique opportunities to
leverage multiple aspects of analyte preconcentration in
IEX with high-speed SFC separations in a combined 2D
method. Additionally, polymer analysis was undertaken
with SFC × SFC [78], but the use of SEC may offer a more
orthogonal approach for improved resolution of complex
polymers based on previous SEC-capillary SFC methods
[57,58]. Although not the primary focus here, the use of
MS detection in the analysis of very complex samples was
common to most of the studies described here, and further
enhancements to SFC-MS will only increase this trend
[91]. In terms of instrument design, higher efficiency 2 D
SFC separations are currently limited by extra-column
KAPLITZ et al.
9
effects that limit the ability to reduce column dimensions
and increase overall throughput [92,93], although the
capability to minimize these effects is starting to emerge
[94,95]. 2D-SFC has made substantial advancements in
terms of overcoming solvent compatibility and increasing
throughput, but there is still exciting work in the future
with continued needs for improved sensitivity, expanded
analyte coverage, and integrated commercial solutions.
AC K N OW L E D G E M E N T S
This work is supported by funding from the National Science Foundation to J.L.E. (CHE-1904919) and to J.P.G.
(CHE-1904454).
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
James P. Grinias
https://orcid.org/0000-0001-9872-9630
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How to cite this article: Kaplitz AS, Mostafa ME,
Calvez SA, Edwards JL, Grinias JP.
Two-dimensional separation techniques using
supercritical fluid chromatography. J Sep Sci.
2020;1-12. https://doi.org/10.1002/jssc.202000823