See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/291385273

Metamorphosis of supercritical fluid

chromatography to SFC: An Overview

Article in TrAC Trends in Analytical Chemistry · January 2016

DOI: 10.1016/j.trac.2016.01.002

CITATIONS READS

7 202

1 author:

Abhijit Tarafder
Waters Corporation, Milford Massachusetts
48 PUBLICATIONS 694 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Scaling rules in SFC View project

All content following this page was uploaded by Abhijit Tarafder on 04 May 2016.

The user has requested enhancement of the downloaded file.

 1 Metamorphosis of Supercritical Fluid
2 Chromatography to SFC - an Overview
∗
3 Abhijit Tarafder
Waters Corporation, Milford MA 01757, USA

4 Abstract

5 SFC is expanded as supercritical fluid chromatography, but the chromatography

6 that is currently conducted under the name of SFC are not dependent on supercritical

7 conditions. An SFC practitioner is mostly neither aware of the critical temperatures

8 and pressures of the solvents, nor even require to take any measures to maintain su-

9 percriticality. Because, according to the current author, SFC has decisively shifted its

10 focus from using mobile phase in supercritical conditions to using carbon dioxide as

11 mobile phase, mixed with other co-solvents. SFC has discovered the benefits of using

12 compressed CO2 as solvent, which opened up some distinctly different and significantly

13 wide application possibilities for chromatographic analyses as a whole.

14 Here an overview will be provided on how supercritical fluid chromatography meta-

15 morphosed to SFC and why SFC may no longer be considered as a technique with

16 some special application scope, rather a complementary tool to some widely accepted

17 analytical techniques e.g. reversed-phase chromatography.

∗
Corresponding author E-mail:abhijit tarafder@waters.com

1
18 Keywords: Supercritical fluid chromatography, SFC, method, stationary phase, applica-
19 tion, metabolite, pharmaceutical, forensic, food

2
20 1 Introduction
21 Supercritical fluid chromatography was invented to extend the capability of gas chromatog-
22 raphy. Challenged with the task of analyzing heavier compounds that required high tempera-
23 tures for elution but could not withstand such temperatures without thermal decomposition,
24 Klesper et. al. [1] employed higher pressures to compensate the requirement of high tem-
25 peratures. Temperatures above the critical point was maintained, possibly to ensure that
26 the gas pressure could be continuously increased without passing through the vapor-liquid
27 biphasic conditions. This endeavor gave birth to supercritical fluid chromatography. Note
28 that the main difference between GC and SFC here is the role played by the mobile phase,
29 which was elevated from being near neutral in GC to playing an active role in compound
30 elution in SFC, by increasing the analyte solubility through increased mobile phase density.
31 Carbon dioxide, which is the principal solvent used in SFC today, was first introduced later,
32 by Sie et. al. [2]. Although many other solvents were tried, CO2 prevailed as the principal
33 solvent because of several advantageous properties that will be discussed later in this review.
34 Although the advantages of working with mobile phases at supercritical conditions drew
35 considerable attention, those were not enough to provide SFC with a strong domain of its
36 own, like LC or GC. The technique went through several phases of re-discoveries to ascertain
37 its unique capabilities and advantages - sometimes as a technique with ”liquid-like solvation
38 power and gas-like viscosity”, sometimes with ”tunable solvent” capability, sometimes ”green
39 solvents”, etc. While certainly advantageous, these features may not always look appealing
40 enough to create an extensive user domain, especially as an analytical technique. Which
41 could be a reason that SFC is perceived as a niche technique.
42 The current author believes that the most useful discovery on SFC, which came out from
43 the years of trials and errors, is the capability of compressed CO2 to mix with a wide range
44 of liquid organic solvents [3]. This capability, unconnected to being supercritical or not,
45 has empowered SFC to conduct chromatographic analysis in such a way which was never
46 witnessed before. This capability does not require SFC to look for application-scope in a
47 niche that cannot be reached by either LC or GC; rather, this makes SFC a technique that
48 is complementary to LC (also GC) deep inside their own domains.
49 The objective of this review is to present some highlights of the main transformations
50 that happened in SFC, from the time of its inception to the current state, and demonstrate
51 how this transformation affected its application range. It is hoped that this will clarify some
52 of the misconceptions shrouded around this technique and assist the non-SFC users to align
53 with the views expressed in the previous paragraph.

3
54 2 Metamorphosis of the technique
55 This section is divided in three subsections focusing on the conversion of (a) the mobile
56 phase components and composition, (b) the stationary phase and (c) the instrumentation of
57 SFC, respectively. Although not implementing a particular order, the transformation that
58 took place in the mobile phase preparation in SFC will be discussed first because of its core
59 importance in the whole process. Development of SFC-specific stationary phases and design
60 of robust instrumentations to conduct reproducible experiments will be discussed next, in
61 that order.

62 2.1 Mobile phase components and composition

63 The most significant change in SFC, from the time of early inception to the modern times,
64 has taken place in the mobile phase selection and composition. It is the condition of the
65 mobile phase (note that supercriticality is not really a separate state, rather a high pressure
66 condition of gaseous mobile phase) that earned the name supercritical fluid chromatography,
67 but the way the technique evolved, the name is currently irrelevant.

68 2.1.1 Early considerations with neat solvents

69 The earliest mobile phases were neat fluids. Apart from CO2 , compounds like ammonia (Tc =
70 405.4K, Pc = 113.33bar), nitrous oxide (Tc = 309.52K, Pc = 72.45bar), light hydrocarbons,
71 chlorofluorocarbons could be successfully used as mobile phases [4, 5]. However, CO2 emerged
72 over the other fluids as the mobile phase of choice, and the other options phased out, mainly
73 because of health & safety issues and concerns over environment and hardware damages. The
74 low critical point of CO2 (Tc = 304.13K, Pc = 73.773bar), its low toxicity and flammability,
75 and that it is carbon-neutral and widely available are the major points that made CO2 the
76 solvent of choice.
77 In the early days of SFC some unrealistic claims on the solvation capability of super-
78 ciritical CO2 were made. As noted by Berger [6, 7], initially CO2 was wrongly perceived as
79 a super-solvent based on a publication of Giddings that predicted the elutropic strength of
80 dense CO2 to be similar to isopropanol. Which implied that only through density program-
81 ming of neat CO2 one can create similar elutropic effects as with composition gradients from
82 pure hexane to pure isopropanol [6, 7].

4
 83 Although this was a misconception, SFC did provide the capability of creating varying
84 elutropic effect, albeit to much narrower extent. Through the variation of pressure and
85 temperature (density and temperature from physical point of view [8–10]) the solvation
86 power of CO2 can be considerably manipulated. Many of the early applications used density
87 gradient to achieve separation of mainly non-polar compounds [4]. This is this ”tunability”
88 of the mobile phase coupled with the use of ”carbon-neutral” CO2 as the solvent, that earned
89 the technology the ”green” tag and had attracted attentions.
90 Clarification on some mis-perceptions
91 Some misleading idea about the actual power of tunability and also about supercritical
92 conditions remain in general perception. Catchphrases like ”liquid-like dissolving power and
93 gas-like viscosity” do not explain that they cannot be achieved simultaneously, rather there is
94 a trade-off between these two properties across the supercritical space. It may not be obvious
95 that moving the experimental point either from a liquid or a gaseous state, to a supercritical
96 condition, across either the critical isobar or the critical isotherm respectively, one does not
97 create any sudden change in the physical properties of a solvent. At least not anything
98 that can clearly influence chromatographic performance. Solvent compressibility changes;
99 increasing smoothly from liquid to gaseous state, passing through the supercritical zone
100 without any sudden changes in property [11]. From chromatographic perspective, knowledge
101 of the location of the critical isotherms or isobars (cricondentherms and cricondenbars in
102 case mixtures) is useful only to locate the onset of mixed-phase conditions, not supercritical
103 conditions [12].

104 2.1.2 Inclusion of co-solvents and a great discovery

105 Although analyte solubility can be considerably modulated by varying the pressure-temperature
106 conditions, relative permittivity (r (ω)), which can be considered as an index to solvent po-
107 larity, of neat CO2 , does not vary much. The maximum achievable r (ω) of CO2 is slightly
108 more than 2.0 [11]. This is still very non-polar, which makes CO2 unsuitable for working
109 with polar analytes. To compensate for this low polarity and limited by the choice of com-
110 pressible solvents with higher polarity, researchers started using liquid polar organic solvents
111 as co-solvents. Klesper and Hartmann [13] reported one of the earlier applications of using a
112 mixture of CO2 and a co-solvent in the mobile phase. Gradually, another great property of
113 CO2 was discovered - the miscibility over a broad range of pressures and temperatures, with
114 liquid organic solvents having wide polarity ranges. This property is largely responsible for

5
115 the way SFC has evolved today. Unlike other non-polar solvents, e.g. hexane or heptane,
116 with which CO2 is often compared, the high miscibility of CO2 with polar organic solvents e.g.
117 methanol, ethanol etc. made SFC evolve as a technique with previously un-witnessed chro-
118 matographic capabilities. It made SFC versatile enough to separate a much wider range of
119 analytes than reversed-phase, especially for mixtures containing polar compounds. Not only
120 CO2 -based solvents can be used with both polar (silica, amino, cyano etc.) and non-polar
121 stationary phases (C8, C18 etc.), but one can influence the chromatography by imparting
122 solvent-gradients with a much wider choice of columns, both polar and non-polar. So the
123 unique characteristic of modern SFC is not in the state or tunability of the solvent, but in
124 the ability to separate a wider variety of compounds with one system. Chemists neither need
125 to be trained in the so-called normal-phase and reversed-phase chromatography separately
126 that now can be conducted with a single setup.

127 2.1.3 Co-solvents and additives

128 Inclusion of an organic solvent as co-solvent, and then the use of additive compounds, dra-
129 matically extended the range of solute polarity that can be analyzed in SFC [14]. Typical
130 additives used in SFC are strong acids, bases or salts. In many instances water was also used
131 as an additive to elute highly polar compounds, e.g. nucleobases and polypeptides, where
132 water introduces HILIC-like analyte partitioning [14].
133 Co-solvents influence SFC chromatography in multiple ways [15, 16] - (a) modifying the
134 polarity, hence the solvating power, of the mobile phase; this is the most important contri-
135 bution and the effect of co-solvents on solvent elution is greater than any other factors [6],
136 (b) altering the density of the mobile phase, (c) blocking active sites on the stationary phase
137 and inhibiting adsorption, (d) modifying stationary phase characteristic through sorption;
138 (e) sorbed co-solvent molecules can lead to increase the net volume of the stationary phase,
139 altering the phase ratio; and (f) selectively solvating polar compounds in the mobile phase
140 forming clusters with different distribution properties. In reference to the debate of adsorp-
141 tion versus partitioning as the retention mechanism, many of these influences indicate the
142 possibility of partition mechanism along with adsorption, e.g. which is observed in HILIC.
143 The use of additives started towards late 1980s [7], and which significantly increased
144 the range of compounds that could be analyzed with SFC. Many different classes of po-
145 lar solutes e.g. phenols, polyhydroxy, hydroxyacids, polyacids, aliphatic amines, and many
146 other drug families could be separated using additives like citric acid, trifluoroacetic acid,

6
147 isopropylamine, triethylamine, ammonium acetate, etc [7]. The suggested roles of additives
148 are diverse too [16] - (a) enhancement of solvating power of mobile phase, (b) ionization
149 suppression and ion-pairing with charged analytes, (c) modification of stationary phase sur-
150 face properties by covering active sites, changing polarity, etc. In some cases effectiveness
151 of an additive could be associated with steric considerations too. Without using additives,
152 the most polar analytes would either tail very badly or would fail to elute [14]. Note that
153 increasingly stationary phases are being designed to reduce the need for additives [16]. Wa-
154 ter as an additive needs some special mention here. Although water is sparingly soluble in
155 compressed neat CO2 [17], presence of water in SFC mobile phase can be increased as a
156 ternary mixture. This has the potential of pushing SFC boundary to more polar compounds
157 [14].

158 2.1.4 Co-solvent mixtures and non-conventional co-solvents

159 Traditionally methanol has been the most common co-solvent used in SFC, accounting for
160 about 75% of the usage [18]. The other co-solvents generally used are ethanol, propanols,
161 acetonitrile etc. An interesting trend, however, observed in the industry, is to evaluate
162 non-conventional co-solvents, as neat or blends, in chiral separation. Organic solvents like
163 Dichloromethane, Chloroform, Methyl tert-butyl ether, Acetone, Ethyl acetate, Tetrahydro-
164 furan, Methyl tetrahydrofuran, 2,2,2-Trifluoroethanol, Cyclopentyl methyl ether, Toluene
165 and N,N-dimethylformamide were used with methanol at different proportions to prepare
166 the co-solvent blends [19] for drug candidates which are poorly soluble in traditional co-
167 solvents. Such studies were carried out and reported from leading pharmaceutical companies
168 like Amgen, Merck and Pfizer [19–21].

169 2.2 Stationary phases

170 In line with the changes that occurred in the mobile-phase of SFC, stationary phases designed
171 to be used as SFC-only have increased over the time. Some recent reviews have extensively
172 covered this subject and should be consulted for better insight [7, 16, 22]. The objective of
173 this section would be to highlight some unique nature of SFC stationary phases and some
174 major milestones.
175 Stationary phases for packed-column SFC, like in HPLC, can be broadly divided into (a)
176 chiral and (b) achiral phases. However, in SFC, the distinction between these two types are

7
177 not as clearly defined as in HPLC. In HPLC, generally the chiral separation is conducted in
178 normal-phase mode, whereas the achiral in reversed-phase or HILIC mode. Normal-phase
179 separations are mostly done in dedicated instruments and needs different kinds of expertise
180 to work with. In SFC, there is no distinction between normal-phase and reversed-phase
181 separations. Chiral chromatography can be carried out with a chiral stationary phase and
182 a mobile phase composition that could have been used with achiral stationary phases too.
183 Chiral columns can be also employed for achiral analysis in SFC, see the review by Regalado
184 and Welch [23]. Achiral and chiral columns or columns with different polarities can be
185 connected in series to create unique selectivity scope (Figure 1).
186 Chiral stationary phases used in HPLC are used in SFC as well, except few e.g. im-
187 mobilized chiral protein-based stationary phases. There is no SFC-specific chiral stationary
188 phase, but some companies have introduced targeted column dimensions for rapid screen-
189 ing by SFC, e.g. the 100mm long columns from Chiral Technologies (West Chester, PA)
190 or the Trefoil line of columns from Waters Corporation (Milford, MA). Although having
191 the same column-chemistry, some manufacturers implement different packing procedures for
192 SFC-specific columns.
193 Numbers of SFC-specific achiral columns, on the other hand, are growing noticeably.
194 Before 2000, availability of columns specific to SFC operations were rare[7], some of the ex-
195 amples being the columns used to measure aromatic content in diesel and olefins in gasoline.
196 Mostly the older normal-phase HPLC columns were being used, with little improvement [7].
197 Note that even within the scope of achiral analysis, there is no distinction between normal
198 and reversed-phase separation in SFC. Stationary phase chemistry ranging from highly polar
199 (e.g. amino-) to highly non-polar (e.g. C18) can be used in the same system using similar
200 mobile phase components and composition. Extensive reviews on the variations of retention
201 mechanism with these column-range are available [22, 24]
202 SFC-specific achiral columns were first introduced by Princeton Chromatography (Cran-
203 bury, NJ) with the 2-ethylpyridine stationary phase bonded on totally porous silica particles
204 [7]. This was followed by other column developments aiming for reduced tailing of basic
205 compounds and providing alternate selectivities in SFC. Examples being, 4-ethylpyridine,
206 pyridine amide, propyl acetamide etc from Princeton; amino phenyl, pyridyl amide etc from
207 ES Industries (West Berlin, NJ); apart from the SFC versions of more regular chemistries
208 e.g. silica, cyano and amino and diol. Waters Corporation recently introduced a set of SFC
209 specific chemistries (2-picolylamine, 1-amino-anthracene, diethylamine and diol) for achiral
210 analyses. Apart from enabling a new set of selectivities the special characteristic of these

8
211 columns is addressing the issue of retention-drift caused by silyl-ether formation (SEF) on
212 silica-based stationary phases [25]. The SEF reaction happens between alcohols on the par-
213 ticle surface and those in the mobile phase solvent, leading to shrinking surface area for
214 retention, over a period of time. This effect was mitigated with these new chemistries.
215 Regarding column geometries, for analytical studies commercial SFC columns are increas-
216 ingly being offered in sub-2µ particles, which is taking this technique significantly ahead of
217 others for high-throughput analyses. Detailed accounts of this advancement were published
218 in recent reviews and reports [5, 26]. Along with the use of sub-2µ particles, use of core-shell
219 or superficially porous particles are also increasing in SFC to achieve similar efficiency but
220 at much higher speed without higher pressure drops [27, 28].

221 2.3 Instrumentation

222 Externally, packed-column SFC instruments are not different than their HPLC counterparts,
223 except the presence of a back-pressure regulator which is often an ABPR (automated back-
224 pressure regulator), the control valve which maintains the set pressure inside the system.
225 Internally, there are differences which were mainly adapted to address the challenges posed
226 by the job of pressurizing and pressure-sustaining the compressible mobile phase without
227 affecting chromatographic performance. Comprehensive reviews on the development of SFC
228 instruments have been discussed in some of the recent publications [4, 5, 29].
229 SFC instruments have made remarkable advances in the past few years with the availabil-
230 ity of commercial instruments, which are as reliable and robust as the HPLC or UPLC/UHPLC
231 instruments. Currently available analytical SFC instruments has pushed the speed vs effi-
232 ciency envelop beyond that was achieved with the UHPLC instruments. Van-deemter plots,
233 comparing the performances of commercially available instruments with 3.5 and 1.7 µm par-
234 ticles, published by Perrenoud et. al. [30] provides a clear view of the prowess of modern
235 SFC performance vis-a-vis LC performance (see Figure 4).
236 Some major changes that took place in SFC instrument components are described below
237 -
238 Commercially available SFC pumps, which tried to mimic LC pumps, could not deliver
239 repeatable and linear results because of the compressibility of the mobile phase [31]. CO2
240 being compressible, compressing and metering with the same pump stroke can be challeng-
241 ing. It is difficult to maintain continuous flow at fixed outlet pressure, without generating
242 significant baseline noise [6]. To address this issue, some currently available commercial

9
243 instruments, e.g. Agilent Aurora SFC, Waters Acquity UPC2 , have separated the pressuriz-
244 ing and the metering task to two different stages, connected in series. Design changes have
245 taken place in CO2 handling too - e.g. for handling CO2 in a reciprocating pump, vapor from
246 the cylinder needs to be liquefied through some heat-exchanger. Traditionally chillers, sup-
247 plied with anti-freeze coolants, were employed. These are being replaced by Peltier cooling
248 elements.
249 Besides pump, changes have taken place in the choice of detectors. Detectors currently
250 used with an SFC system are UV, DAD, ELSD, CAD, MS (ESI, APCI). Remarkable changes
251 have also taken place in the design of the ABPR. E.g. Waters Acquity UPC2 uses a force
252 actuator vis-a-vis a position actuator which was earlier used for automatic control of the
253 back-pressure. The force actuator allows the needle to float to the required location to hold
254 pressure while the position actuator needs to continuously adjust when there is a change in
255 the composition or any other property of the flow [32]. Such innovations drastically improved
256 the quality of analysis in SFC.

257 3 Applications
258 There could be different ways of broadly classifying the application scopes of SFC, e.g. (a)
259 chiral and achiral, (b) analytical and preparative, (c) pharma and non-pharma, etc. In this
260 report we will classify the applications under the preparative and analytical realms. The
261 scopes and SFC-specific incentives of these two forms of operations are different in SFC and
262 will be explained below.

263 3.1 Analytical

264 Understanding the application scope of SFC for analysis is one of the most active and
265 debated area of the field. The metamorphosis of this technique in terms of solvent selection
266 (see section 2.1) is mainly driven by the continuous quest to push the application envelop.
267 Figure 2 demonstrates an overview of the current application scopes of SFC from functional
268 group point of view vis-a-vis the transformation of the mobile phase composition that has
269 taken place. It also presents a side-by-side comparison of the application ranges of different
270 techniques used in LC.
271 Very rarely now-a-days SFC is carried out with neat CO2 [33]. One of the still-existing

10
272 application scope of neat CO2 is in the analysis of petroleum fractions. Thiebaut [34, 35]
273 reviewed SFC application for simulated distillation (simdis), which is generally carried out
274 by GC, but SFC can extend the range up to C140 hydrocarbons.
275 The main incentives of current analytical applications of SFC, driven by the use of CO2
276 as a solvent, can be summarized as the following:

277 1. Application range, versatility - An interesting inference that can be drawn from Fig-
278 ure 2 is the relative generality of the application scope of modern SFC, compared to
279 different LC techniques. Note that LC does cover the widest polarity range of ana-
280 lytes in terms of selectivity, but to cover this range the technique had to be divided
281 sharply into distinct operational classes - i.e. reversed-phase, normal-phase, HILIC,
282 ion-chromatography etc. SFC, on the other hand spans over the widest application
283 range as a single technique, with the same operational characteristics. If this is com-
284 bined with the capability, that chiral analysis can be conducted in SFC using the same
285 system and similar methods as for achiral analysis, it can be confidently said that
286 modern SFC has grown as the most versatile chromatographic technique.

287 2. Orthogonality with reversed-phase - Interactions between the analytes, the mobile phase
288 and the stationary phase in CO2 -based SFC is very different compared to such inter-
289 actions in water-based reversed-phase technique. It is not surprising that mixtures
290 analyzed in these two techniques often demonstrate strongly orthogonal behavior (see
291 Figure 3). Where the analytical goal is to identify every components of a mixture,
292 importance of multi-dimensional or parallel analysis with these techniques should not
293 be oversighted.

294 3. Sample compatibility - CO2 miscibility with solvents of wide polarity range can simplify
295 the analytical task considerably. Samples can be directly injected to the system, which
296 in many other situations would need sample preparation or a derivatization step. E.g.
297 analysis of free-fatty acids is easier with SFC-MS, compared to GC or HPLC, as it does
298 not need previous sample extraction and derivatization [36]. Smaller volume of aqueous
299 solutions can be directly injected for analytical purposes however direct injection of
300 large volume of aqueous solutions should be avoided [37].

301 4. Others - SFC happens to provide unique solutions to certain problems, e.g. working
302 with analytes that degrade in water and where normal-phase cannot be used. An
303 example is the simultaneous analysis of lactones and their hydrolyzed metabolites in

11
304 biological samples. The analysis is challenging because with RPLC-MS unexpected
305 hydrolysis or reversed dehydration may occur depending on the test environments.
306 NPLC-MS may face challenges because of its relative incompatibility with several ion-
307 ization techniques. SFC-MS is found to be having a unique solution for such problems
308 [38]

309 In terms of sensitivity, modern SFC-MS systems are demonstrating more sensitivity over
310 LC-MS and/or GC-MS technique in many situations. E.g. Ishibashi et al [39] reports an
311 SFC-MS method for high throughput analysis of pesticides (72 samples per day). Their
312 work demonstrates the effectiveness of SFC-MS in pesticide analysis, over GC-MS and LC-
313 MS techniques. Novakova et al [40] compared UHPLCMS/MS and UHPSFCMS/MS, for the
314 determination of 110 doping agents in urine, in terms of sensitivity, linearity and matrix ef-
315 fects. Both the techniques had highly sensitive MS/MS detection. Overall, UHPSFCMS/MS
316 was found to be more sensitive in detecting the doping agents considered here [40]. Tao et
317 al [41] reported simultaneous determination of 7 gestagens in bovine and porcine kidney fat.
318 They found SFC-MS/MS to be more sensitive and requires less analysis time comopared to
319 their LC-MS/MS system..
320 SFC is being increasingly applied to a wide variety of applications both chiral and achiral.
321 Presenting a detailed account of these applications is not the objective here. Readers are sug-
322 gested to consult some latest and also few older reviews and other publications to understand
323 the expanding application range of SFC. Some of the application areas are (a) enantioselec-
324 tive separation (Kalikova et al [42], West [18], Klerck et al. [43]), (b) metabolite analysis
325 (Taguchi et al [44], Matsubara et al [45]), (c) food analysis (Bernal et al [46]), (d) polymer
326 analysis (Takahashi [47]), (e) analysis of peptides and ionic analytes (Taylor [48, 49]), (f)
327 clinical analysis (Abbott et al [50]), (g) analysis of non-saponifiable lipids (Kong [51]), (h)
328 analysis of fatty acids in foods [52], (h) carbohydrate analysis [53], etc.
329 From column selection perspective, because mostly polar columns are selected in SFC,
330 modern SFC is sometimes compared with the normal-phase technique. This similarity, im-
331 poses a ”niche” image even on modern SFC because of the limited practice of normal-phase
332 in LC domain. This view can be misleading. Functionally, the application space of normal-
333 phase chromatography is wider than that of reversed-phase. Reversed-phase is generally
334 preferred over normal-phase mainly because of some operational problems with the latter
335 technique, which is the reason normal-phase domain in LC is much smaller. SFC alleviates
336 majority of these problems, which empowers it to analyze compounds much beyond the cur-
337 rent application practice of normal-phase and deep into the current domain of reversed-phase

12
338 applications. What is more interesting is the orthogonal retention behavior in SFC, which
339 can make it a complementary technique for reversed-phase. Although SFC compatibility
340 with increasing polarity of the analytes decrease and for certain analytes e.g. polypeptides
341 the performance of SFC barely matches with RPLC, but still the advantage of orthogonal-
342 ity and lesser solvent usage, especially for preparative applications, stand. It may not be
343 necessary that SFC needs to supersede RPLC in all the application domain. Both the tech-
344 niques, one water-based and the other CO2 -based, can together bring significantly stronger
345 analytical capability on a chemists benchtop.

346 3.2 Preparative

347 SFC has a much wider acceptance as a preparative separation technique, compared to analyt-
348 ical applications, because of the significant possible improvement in separation productivity
349 and product recovery time vis-a-vis traditional prep HPLC. Because of low viscosity of CO2 -
350 based mobile phases and high analyte diffusivity, separations can be performed significantly
351 faster in SFC [54]. Additionally, post separation product recovery can be done in much
352 lesser time, requiring lesser energy to isolate products. Finally, the solvent consumption in
353 SFC based purification is 2 to 10 times lower than HPLC based purifications [54]. Even for
354 applications where LC techniques could perform the same, or slightly better, the criteria of
355 low cost to procure and dispose, and significantly faster product recovery, can make SFC
356 more advantageous [55]. Additionally, CO2 is inexpensive, nontoxic and nonflammable with
357 reduced handling and storage cost. If the wide miscibility of CO2 makes the technique unique
358 for analytical tasks, its these other characteristics of CO2 that make it more attractive for
359 separations.
360 There are however, few issues that needs to be remembered, foremost are (1) difficulty
361 to measure analyte solubility with standard lab instruments leading to either (i) lower-than-
362 potential productivity, or (ii) solute precipitation inside the system leading to downtime; (2)
363 difficulty in transferring analytical methods to preparative scale [56] (3) scale of CO2 supply
364 is not always compatible for large-scale separation, which makes in necessary to opt for LC
365 if the scale is beyond 1 kg [54]. There are some hidden expenses of using CO2 at preparative
366 scale. One needs to install an elaborate CO2 supply system leading to significant capital
367 investment, although higher savings in operating cost should quickly recover this investment.

13
368 4 Conclusion
369 Although SFC is still expanded as supercritical fluid chromatography, the true potential of
370 this technique is in the use of CO2 as a mobile phase. CO2 , having high miscibility with liquid
371 organic solvents with wide polarity range, brings some very unique characteristics to SFC,
372 which can bring several complementary attributes to current chromatographic capability.
373 According to the current author, modern SFC should not be viewed as a replacement to any
374 existing technique or a technique with some ”killer” applications; rather as discussed in this
375 review, it should be viewed as a complementary technique which can significantly enhance
376 the range of analytical scope with chromatography.

14
377 5 Acknowledgment
378 Dr. Mark Baynham, Director of SFx Instruments, Waters Corporation, for helpful dis-
379 cussions. Joshua A. Shreve, Principal Mechanical Engineer, Waters Corporation, for the
380 explanation on the difference between earlier and modern back pressure regulation in SFC.

15
381 References
382 [1] E. Klesper, A. H. Corwin, D. A. Turner, High pressure gas chromatography above
383 critical temperatures, J. Org. Chem. 27 (1962) 700.

384 [2] S. Sie, W. V. Beersum, G. Rjinders, High-pressure gas chromatography and chromatog-
385 raphy with supercritical fluids. i. the effect of pressure on partition coefficients in gas-
386 liquid chromatography with carbon dioxide as a carrier gas, Sep. Sci. 1 (1966) 459.

387 [3] A. Tarafder, J. F. Hill, M. Baynham, Convergence chromatography versus sfc - whats
388 in a name?, Chromatography Today Aug/Sept (2014) 34–36.

389 [4] G. Guiochon, A. Tarafder, Fundamental challenges and opportunities for preparative
390 supercritical fluid chromatography, J. Chromatogr. A 1218 (2011) 1037–1114.

391 [5] L. Novakova, A. G.-G. Perrenoud, I. Francois, C. West, E. Lesellier, D. Guillarme,

392 Modern analytical supercritical fluid chromatography using columns packed with sub-
393 2µm particles: A tutorial, Anal. Chim. Acta 824 (2014) 18–35.

394 [6] T. Berger, Packed Column SFC, Royal Society of Chemistry, Cambridge, UK, 1995.

395 [7] R. E. Majors, T. Berger, B. Berger, A review of column developments for supercritical
396 fluid chromatography, LCGC 28.

397 [8] D. E. Martire, R. Boehm, Unified molecular theory of chromatography and its applica-
398 tion to supercritical fluid mobile phases. 1. fluid-liquid (absorption) chromatography, J.
399 Phys. Chem. 91 (1987) 2433–2446.

400 [9] M. Roth, Thermodynamics of retention in supercritical fluid chromatography: A refined

401 model, J. Phys. Chem 94 (1990) 4309.

402 [10] A. Tarafder, G. Guiochon, Use of isopycnic plots in designing operations of supercritical
403 fluid chromatography: I. the critical role of density in determining the characteristics of
404 the mobile phase in supercritical fluid chromatography, J. Chromatogr. A 1218 (2011)
405 4569–4575.

406 [11] E. W. Lemmon, M. O. McLinden, D. G. Friend, Thermophysical properties of fluid

407 systems, NIST Chemistry WebBook, NIST Standard Reference 39.

16
408 [12] A. Tarafder, G. Guiochon, Use of isopycnic plots in designing operations of supercrit-
409 ical fluid chromatography: II. the isopycnic plots and the selection of the operating
410 pressuretemperature zone in supercritical fluid chromatography, J. Chromatogr. A 1218
411 (2011) 4576–4585.

412 [13] E. Klesper, W. Hartmann, Parameters in supercritical fluid chromatography of styrene

413 oligomers, J. Polym. Sci Polym. Lett Ed 15 (1977) 707–712.

414 [14] L. T. Taylor, Packed column supercritical fluid chromatography of hydrophilic analytes
415 via water-rich modifiers, J. Chromatogr. A 1250 (2012) 196–204.

416 [15] J. Strubinger, H. Song, J. Parcher, High-pressure phase distribution isotherms for su-
417 percritical fluid chromatographic systems. 2. binary isotherms of carbon dioxide and
418 methanol, Anal. Chem. 63 (1991) 104.

419 [16] C. F. Poole, Stationary phases for packed-column supercritical fluid chromatography,
420 J. Chromatogr. A 1250 (2012) 157–171.

421 [17] C. R. Coan, J. A. D. King, Solubility of water in compressed carbon dioxide, nitrous
422 oxide, and ethane. evidence for hydration of carbon dioxide and nitrous oxide in the gas
423 phase, J. Am. Chem. Soc. 93 (1971) 1857.

424 [18] C. West, Enantioselective separations with supercritical fluids - review, Cur. Anal.
425 Chem. 10 (2014) 99–120.

426 [19] J. O. DaSilva, B. Coes, L. Frey, I. Mergelsberg, R. McClain, L. Nogle, C. J. Welch,

427 Evaluation of non-conventional polar modifiers on immobilized chiral stationary phases
428 for improved resolution of enantiomers by supercritical fluid chromatography, J. Chro-
429 matogr. A 1328 (2014) 98–103.

430 [20] L. Miller, Use of dichloromethane for preparative supercritical fluid chromatographic
431 enantioseparations, J. Chromatogr. A 1363 (2014) 323–330.

432 [21] J. Lee, J. Lee, W. L. Watts, J. Barendt, T. Q. Yan, Y. Huang, F. Riley, M. Hardink,
433 J. Bradow, P. Franco, On the method development of immobilized polysaccharide chiral
434 stationary phases in supercritical fluid chromatography using an extended range of
435 modifiers, J. Chromatogr. A 1374 (2014) 238–246.

436 [22] E. Lesellier, C. West, The many faces of packed column supercritical fluidchromatogra-
437 phy a critical review, J. Chromatogr. A 1382 (2015) 2–46.

17
438 [23] E. L. Regalado, C. J. Welch, Separation of achiral analytes using supercritical fluid
439 chromatography with chiral stationary phases, Tr. Anal. Chem. 67 (2015) 74–81.

440 [24] E. Lesellier, Retention mechanisms in super/subcritical fluid chromatography on packed

441 columns, J. Chromatogr. A 1216 (2009) 1881–1890.

442 [25] J. N. Fairchild, D. W. Brousmiche, J. F. Hill, M. F. Morris, C. A. Boissel, K. D.

443 Wyndham, Chromatographic evidence of silyl ether formation (sef) in supercritical fluid
444 chromatography, Anal. Chem. 87 (2015) 1735–1742.

445 [26] A. G.-G. Perrenoud, J.-L. Veuthey, D. Guillarme, The use of columns packed with sub-2
446 µm particles in supercritical fluid chromatography, Tr. Anal. Chem. 63 (2014) 44.

447 [27] E. Lesellier, Efficiency in supercritical fluid chromatography with different superficially
448 porous and fully porous particles ods bonded phases, J. Chromatogr. A 1228 (2012)
449 89–98.

450 [28] A. G.-G. Perrenoud, F. W.P., A. C.M., A. N.C., F. S., D. Guillarme, Evaluation of
451 stationary phases packed with superficially porous particles for the analysis of pharma-
452 ceutical compounds using supercritical fluid chromatography, J. Chromatogr. A 1360
453 (2014) 275–287.

454 [29] M. Saito, History of supercritical fluid chromatography: Instrumental development, J.

455 Bios. Bioeng 115 (2013) 590–599.

456 [30] A. G.-G. Perrenoud, J.-L. Veuthey, D. Guillarme, Comparison of ultra-high performance
457 supercritical fluid chromatography and ultra-high performance liquid chromatography
458 for the analysis of pharmaceutical compounds, J. Chromatogr. A 1266 (2012) 158–167.

459 [31] A. Tarafder, G. Guiochon, Accurate measurements of experimental parameters in su-

460 percritical fluid chromatography. i. extent of variations of the mass and volumetric flow
461 rates, J. Chromatogr. A 1285 (2013) 148–158.

462 [32] J. Shreve, J. Auclair, P. Keenan, E. Denecke, G. Wisser, E. Bates, E. Berthiaume,

463 M. Bower, Back pressure regulation, uS Patent App. 14/382,602 (2015).

464 [33] L. T. Taylor, Supercritical fluid chromatography for the 21st century, J. Supercrit. Fluid
465 47 (2009) 566–573.

18
466 [34] D. Thiebaut, E. Robert, Group-type separation and simulated distillation: a niche for
467 sfc, Alalusis 27 (1999) 681.

468 [35] D. Thiebaut, Separations of petroleum products involving supercritical fluid chromatog-
469 raphy, J. Chromatogr. A 1252 (2012) 177–188.

470 [36] S. Qu, Z. Du, Y. Zhang, Direct detection of free fatty acids in edible oils using super-
471 critical fluid chromatography coupled with mass spectrometry, Food Chem. 170 (2015)
472 463–469.

473 [37] Z. Wang, H. Zhang, O. Uu, B. Donovan, Implementation of achiral supercritical fluid
474 chromatography in drug development testing, Am. Pharma. Rev. 16 (2013) Apr 30.

475 [38] X.-G. Liu, L.-W. Qi, Z.-Y. Fan, X. Dong, R.-Z. Guo, F.-C. Lou, S. Fanali, P. Li,
476 H. Yang, Accurate analysis of ginkgolides and their hydrolyzed metabolites by analytical
477 supercritical fluid chromatography hybrid tandem mass spectrometry, J. Chromatogr.
478 A 1388 (2015) 251–258.

479 [39] M. Ishibashi, Y. Izumi, M. Sakai, T. Ando, E. Fukusaki, T. Bamba, High-throughput

480 simultaneous analysis of pesticides by supercritical fluid chromatography coupled with
481 high-resolution mass spectrometry, J. Agric. Food Chem. 63 (2015) 4457–4463.

482 [40] L. Novakova, M. Rentsch, A. G.-G. Perrenoud, R. Nicoli, M. Saugy, J.-L. Veuthey,
483 D. Guillarme, Ultra high performance supercritical fluid chromatography coupled with
484 tandem mass spectrometry for screening of doping agents. ii: Analysis of biological
485 samples, Anal. Chim. Acta 853 (2015) 647–659.

486 [41] Y. Tao, R. Paula, A. Stolker, D. Chen, Z. Yuan, Simultaneous determination of seven
487 gestagens in kidney fats by ultra performance convergence chromatography tandem
488 mass spectrometry, J. Chromatogr. B 988 (2015) 143–148.

489 [42] K. Kalkov, T. Slechtov, J. Vozka, E. Tesarov, Supercritical fluid chromatography as a

490 tool for enantioselective separation; a review, Anal. Chim. Acta 821 (2014) 1–33.

491 [43] K. D. Klerck, D. Mangelings, Y. V. Heyden, Supercritical fluid chromatography for the
492 enantioseparation of pharmaceuticals, J. Pharm. Biomed. Anal. 69 (2012) 77.

493 [44] K. Taguchi, E. Fukusaki, T. Bamba, Supercritical fluid chromatography/mass spec-

494 trometry in metabolite analysis, Bioanal. 6 (2014) 1679–1689.

19
495 [45] A. Matsubara, E. Fukusaki, T. Bamba, Metabolite analysis by supercritical fluid chro-
496 matography, Bioanal. 2 (2010) 27.

497 [46] J. Bernal, M. Martin, L. Toribio, Supercritical fluid chromatography in food analysis,
498 J. Chromatogr. A 1313.

499 [47] K. Takahashi, Polymer analysis by supercritical fluid chromatography, J. Biosci. Bioeng
500 116 (2013) 133.

501 [48] L. T. Taylor, Supercritical fluid chromatography of peptides - state of the art, Am.
502 Pharm. Rev. 12 (2009) 48.

503 [49] L. T. Taylor, Separation of ionic analytes using supercritical fluid chromatography,
504 LCGC Eur. 22.

505 [50] E. Abbott, T. Veenstra, H. Issaq, Clinical and pharmaceutical applications of packed-
506 column supercritical fluid chromatography, J. Sep. Sci. 31.

507 [51] L. Kong, X. Li, H. Zou, H. Wang, X. Mao, Q. Zhang, J. Ni, Analysis of non-saponifiable
508 lipids by super-/subcritical-fluid chromatography, J. Chromatogr. A 936.

509 [52] F. Senorans, E. Ibanez, Analysis of fatty acids in foods by supercritical fluid chromatog-
510 raphy, Anal. Chim. Acta 465.

511 [53] M. Lafosse, B. Herbreteau, L. Morin-Allory, Supercritical fluid chromatography of car-

512 bohydrates, J. Chromatogr. A 720.

513 [54] L. Miller, Preparative enantioseparations using supercritical fluid chromatography, J.

514 Chromatogr. A 1250 (2012) 250–255.

515 [55] E. Francotte, Why sfc should be the first choice technology for the purification of phar-
516 maceuticals, 30th International Symposium on Chromatography Salzburg, Austria.

517 [56] A. Tarafder, C. Hudalla, P. Iraneta, K. J. Fountain, A scaling rule in supercritical fluid
518 chromatography. i. theory for isocratic systems, J. Chromatogra. A 1362 (2014) 278–293.

519 [57] K. W. Phinney, L. C. Sander, S. A. Wise, Coupled achiral/chiral column techniques in

520 subcritical fluid chromatography for the separation of chiral and nonchiral compounds,
521 Anal. Chem. 70 (1998) 2331–2335.

20
522 [58] C. H. Arnaud, Supercritical fluid chromatography seeks new users, Chem. Eng. News
523 92 (2014) 10–13.

524 [59] Z. Wang, H. Zhang, O. Liu, B. Donovan, Development of an orthogonal methodfor

525 mometasone furoate impurity analysis using supercritical fluid chro-matography, J.
526 Chromatogr. A 1218 (2011) 2311–2319.

21
527 Figure captions
528 1 Separation of β-blockers on (a) a Chiralcel OD column, (b) an achiral cyano
529 bonded phase, and (c) coupled cyano/Chiralcel OD system. Chromatographic
530 conditions: 20% methanol (containing 0.5% isopropylamine) in carbon diox-
531 ide, 2.0 mL/min, 15 MPa, 30 o C, λ = 280 nm. See ref. [57] for details and
532 more examples. 23
533 2 An overview of the application range of SFC with co-solvents and additives
534 (from [58]). Note that, functionally SFC has a wider application domain
535 compared to RPLC, although the method development becomes difficult with
536 analytes of high polarity. 24
537 3 Demonstration of orthogonal selectivity between SFC on a polar stationary
538 phase and RPLC technique. SFC conditions - 100 bar, 30 o C, 4.0 mL/min,
539 co-solvent gradient was methanol 515% in 15 min, on a silica column. RPLC
540 conditions - 25 o C, 1.5 mL/min, varying water/acetonitrile (58:42, v/v) to
541 water/acetonitrile (48:52, v/v) in 60 min, Ultrasphere ODS column was used.
542 See ref [59] for details. 25
543 4 Comparison of kinetic performance and normalized pressure drops, as a func-
544 tion of linear velocity, for 1.7 and 3.5 µm particles between UHPLC and
545 UHPSFC. (A) Van Deemter curves for butylparaben on 2 systems equipped
546 with 1.7 or 3.5 µm particles columns. XTerra RP18 50 mm 4.6 mm, 3.5 µm
547 (blue dots), Acquity Shield RPC18 50 mm 2.1 mm,1.7 µm (red diamonds)
548 both columns tested in LC conditions: H2 O/ACN (60/40, v/v), 30 o C, 1 µL
549 injected, 254 nm. Acquity UPC2 BEH 2-EP 100 mm 3.0 mm, 3.5 µm (pur-
550 plesquares) and 100 mm 3.0 mm, 1.7 µm (green triangles) columns tested in
551 SFC conditions: CO2/MeOH (96/4, v/v), 40 o C, 150 bar ABPR pressure, 1
552 µL injected, 254 nm. (B)Corresponding generated column pressure drop nor-
553 malized to 1 m of column, to avoid influence of column geometry variations.
554 For details see [30] 26

22
mine

per-
05A
and
d at
tem
ters
the
med
Data
were
per-

the
ific,
eld,
NH2
OD
icle
lcel
mns
r to
aph.

ons
OD
ame
.27,28
the
only Figure 4. Separation of β-blockers on the Chiralcel OD CSP (a),
hiral the achiral cyano1:bonded
Figure phase (b),ofand
Separation the coupled on
β-blockers cyano/Chiralcel
(a) a Chiralcel OD column, (b) an achiral cyano
n of OD system (c). Chromatographic conditions: 20% methanol (contain-
bonded phase, and (c) coupled cyano/Chiralcel OD system. Chromatographic conditions:
ing 0.5% isopropylamine) in carbon dioxide, 2.0 mL/min, 15 MPa, 30
sary
°C, λ )20% methanol (containing 0.5% isopropylamine) in carbon dioxide, 2.0 mL/min, 15 MPa,
280 nm.
hiral
30 o C, λ = 280 nm. See ref. [57] for details and more examples.
ytes separation of all the components. Different achiral stationary
hiral phases were evaluated to improve the separation. Very little
the retention of the β-blockers was observed on a C18 bonded phase
dual under the mobile phase conditions used for the chiral column,
and coupling of the C18 phase with the chiral column did not
are improve the separation. Use of a diol column did not 23 yield
diac separation of alprenolol from oxprenolol. The achiral separation
mine of the four β-blockers on a cyano bonded phase is shown in Figure
e 1. 4b. Serial coupling of the cyano and Chiralcel OD columns
es a produced the separation shown in Figure 4c. No changes in
  

Figure 2: An overview of the application range of SFC with co-solvents and additives (from
[58]). Note that, functionally SFC has a wider application domain compared to RPLC,
although the method development becomes difficult with analytes of high polarity.

24
 Author's personal copy

Trends in Analytical Chemistry, Vol. 37, 2012 Trends

Figure 1. (a) The orthogonal selectivity of SFC method vs. RP-HPLC method. SFC conditions: 100 bar, 30C, 4.0 mL/min, modifier: methanol
5–15% in 15 min, silica column. RP-HPLC conditions: 25C, 1.5 mL/min, water/acetonitrile (58:42, v/v) to water/acetonitrile (48:52, v/v) in
Figure 3: Demonstration of orthogonal selectivity between SFC on a polar stationary phase
60 min, Ultrasphere ODS column. (b) Comparison of retention factor (kÕ) in SFC method and in RP-HPLC method [16].
and RPLC technique. SFC conditions - 100 bar, 30 o C, 4.0 mL/min, co-solvent gradient was
methanol 515% in 15 min, on a silica column. RPLC conditions - 25 o C, 1.5 mL/min, varying
water/acetonitrile (58:42, v/v) to water/acetonitrile 25 (48:52, v/v) in 60http://www.elsevier.com/locate/trac
min, Ultrasphere ODS85
column was used. See ref [59] for details.
 No. of Pages 16 ARTICLE IN PRESS
V. Desfontaine et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2015) xxx–xxx

tic performance and normalized generated pressure drop as a function of linear velocity for 1.7 and 3.5 ␮m particles in UHPLC and UHPSFC. (A) Van Deem
aben on 2 systemsFigure
equipped 4: with
Comparison of kinetic
1.7 or 3.5 ␮m particlesperformance
columns. XTerra and normalized
RP18 50 mm × 4.6pressure
mm, 3.5 ␮m drops,
(blueas a function
dots), of RPC18 50 mm
Acquity Shield
linear velocity,
diamonds) both columns for
tested in LC 1.7 and H
conditions: 3.5 µm particles
2 O/ACN between
(60/40, v/v), ◦ UHPLC
30 C, 1 ␮L injected,and UHPSFC.
254 nm. Acquity UPC (A) Van
2 Deemter
BEH 2-EP 100 mm × 3.0 mm, 3.5 ␮
◦
d 100 mm × 3.0 mm, 1.7 ␮m (green triangles) columns tested in SFC
curves for butylparaben on 2 systems equipped with 1.7 conditions: CO 2 or 3.5 µm particles columns. XTerra 1 ␮L injected, 2
/MeOH (96/4, v/v), 40 C, 150 bar backpressure,
ng generated column pressure drop normalized to 1 m of column, to avoid influence of column geometry variations. (For interpretation of the referenc
RP18 50 mm 4.6 mm, 3.5 µm (blue dots), Acquity Shield RPC18 50 mm 2.1 mm,1.7 µm
e legend, the reader is referred to the web version of this article.) o
(red diamonds) both columns tested in LC conditions: H2 O/ACN (60/40, v/v), 30 C, 1 µL
m [24] with permission.
injected, 254 nm. Acquity UPC2 BEH 2-EP 100 mm 3.0 mm, 3.5 µm (purplesquares) and
100 mm 3.0 mm, 1.7 µm (green triangles) columns tested in SFC conditions: CO2/MeOH
o
ns, the UHPSFC(96/4, v/v), 40with
can compete C, UHPLC,
150 barfrom
ABPR pressure, elsewhere
a kinetic 1 µL injected, 254 nm.
[26–28], (B)Corresponding
also for pharmaceutical compounds
generated column pressure drop normalized
iew, as shown in Fig. 2. In Fig. 2A, the kinetic performance to 1
Indeed, columns packed with SPP of
m of column, to avoid influence in column
the range 2–3 ␮m ha
mter curves) geometry variations.
of conventional HPLCForand
details
SFCsee [30]
columns 26 parable efficiency to the one achieved on UHPLC column
ith 3.5 ␮m particles and the “modern” versions of these porous particles of sub-2 ␮m), at much lower pressure. T
es, UHPLC and UHPSFC columns packed with 1.7 ␮m parti- interest of using SPP is linked to the too restrictive pressure
compared. For both separation modes, the minimal plate UHPSFC systems. Several studies have already shown grea
min ) when using small particles was greatly decreased tial for this column technology in UHPSFC, with an impres
d to the conventional particles. Moreover, Hmin for UHPSFC
View publication stats increase in efficiency compared with 3 ␮m fully porous p