# Supercritical fluid chromatography is not (only) normal-phase chromatography

Caroline West

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## To cite this version:

Caroline West. Supercritical fluid chromatography is not (only) normal-phase chromatography. Journal of Chromatography A, 2023, 1713, pp.464546. 10.1016/j.chroma.2023.464546. hal-05378651

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Supercritical fluid chromatography is not (only) normal-phase chromatography

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Caroline West

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Institute of Organic and Analytical Chemistry, University of Orleans, CNRS UMR7311, rue de

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Chartres - BP 6759, 45067 Orléans, France

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caroline.west@univ-orleans.fr

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Abstract

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Supercritical fluid chromatography (SFC), now using carbon dioxide as a major component of the mobile phase, has been known for over 60 years but still some misunderstandings remain about its capabilities. Among them, SFC is often described as a normal-phase chromatographic technique, based on different considerations: polarity of the stationary phase, elution order of the analytes, relative non-polarity of the mobile phase, non-linear retention behaviour, or adsorption retention mechanisms. All of these assumptions are true to a certain extent, and in certain circumstances. But also, all of these assumptions are wrong in different circumstances. In this paper, the criteria to categorize SFC as a normal-phase chromatographic method will be examined individually, considering all knowledge acquired from the early years of its development. Finally, it will appear that the “normal-phase” glass lens is greatly reducing the true extent of SFC’s possibilities.

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**Keywords:** elution strength; normal-phase; retention mechanisms; reversed-phase; supercritical fluid chromatography

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# 1. Introduction

Supercritical fluid chromatography (SFC) is a mature chromatographic technique, as the first experiments were presented over 60 years ago [1], and a large body of literature exists describing fundamentals [2,3] and ever-increasing applications [4]. Its potential for enantioresolution [5,6] and its interest for preparative-scale separations [7,8] were acknowledged a long time ago [9], but its capabilities for achiral and analytical-scale separations were re-discovered and more widely recognized only in the past decade [10].

This is due to the introduction of high-performance chromatographic instruments by several manufacturers, finally meeting the expectations of chromatographers in terms of repeatability [11], robustness, sensitivity, capability to transfer a method from one laboratory and instrument to another one [12,13] and hyphenation to mass spectrometry [14–16]. Although significant efforts have been made to educate chromatographers to a better use of the technique [17], some misconceptions are often expressed in published papers. The most frequent one is the supposed “normal-phase” (NP) operating mode. Indeed, very frequently, SFC is simply considered as a normal-phase technique, based on different arguments. The possible reasons expressed for this NP behaviour are based on the non-polar nature of the major mobile phase component (carbon dioxide), the observed elution order of analytes depending on their polarity, the nature of the stationary phases most employed, or finally, the supposed adsorption retention mechanism. However, considering SFC only through this angle could be deceiving, as one would expect similar chromatograms when transferring a method from NP-liquid chromatography to SFC. In addition, it would be greatly reducing the application scope of the technique. This is what we will demonstrate in this paper, considering all usual arguments to support the supposed normal-phase mode.

# 2. Experimental

All data used in this paper were previously published in [18–22]. All experimental data were determined in the author's laboratories using the same experimental protocol, mostly in isocratic mode and sometimes in gradient mode, as specified in the text and figure captions.

The compounds' descriptors of polarity were computed with MolDesc freeware (http://moldesc.icoa.fr/) using the Simplified Molecular Input Line Entry Specification (SMILES) of each molecule.

# 3. Discussion

## 3.1. Considerations related to the polarity of the stationary phase

The first reason usually expressed by the supporters of the NP theory is that SFC “is done on polar stationary phases”. There is indeed a tendency in current applications to work mostly on polar stationary phases, as observed in Figure 1 reporting the most cited columns as
reported by Plachká and co-workers [23]. It is clear that current applications are most often developed on bare silica gel, or silica bonded with polar ligands like propanediol, aminopropyl, diethylaminopropyl or pyridine ligands (most frequently ethyl-pyridine or picolylamine ligands). On the lipophilic side of the stationary phase ligands, octadecylsiloxane-bonded (ODS) stationary phases are the most cited. However, this may be somewhat confusing as ODS phases include a great variety of polarities, what with uncovered silanol groups, polar-embedded groups or hydrophilic endcapping groups. While such polar groups are not bound to drastically change the retention mechanism when ODS phases are used in reversed-phase liquid chromatographic (RPLC) mode, it was demonstrated in several occasions that they induce significant selectivity changes in SFC [24]. Other moderately polar stationary phases as phenyl- and pentafluorophenyl-type ligands have also been demonstrated to be useful in SFC in several occasions [25-32], but are clearly not the most popular. In short, all stationary phases developed for liquid chromatography can be used in SFC, but not all of them are used in practice.
There are certainly reasons for this tendency towards more polar phases. Some of them are bad reasons, like the preconception that the non-polar CO₂ can only be balanced by a polar stationary phase. Another, better reason is perhaps that many newcomers to SFC are turning to this method hoping to find one that would offer complementary selectivity to the most widespread RPLC mode, either because the RPLC separation is not satisfying, or because they wish to develop two-dimensional systems [33-37] where orthogonality must be large to improve the overall resolution. In this respect, it could seem logical to aim at polar stationary phases, which should provide different retention capabilities through different interactions with the analytes than the hydrophobic effect that dominates RPLC separations [38]. Indeed, when facing a separation problem that cannot be resolved with RPLC on ODS stationary phases, chromatographers look to diversify interactions with the analytes. When the analytes are well retained in the RPLC system but not adequately resolved, they may stick to the RPLC mode but select a different stationary phase (e.g. phenyl- or pentafluorophenyl-bonded silica), or vary the mobile phase conditions (e.g. acidic vs. basic conditions). However, when the analytes are not retained in the RPLC system, it is logical to look for a polar stationary phase to enhance the retention of polar analytes. Hydrophilic interaction liquid chromatography (HILIC), which is performed on polar stationary phases, is a possible alternative when facing such issues. But the latter is sometimes causing difficulties to non-experienced chromatographers (e.g. long column equilibration, deleterious effect of inadequate dilution solvents...). SFC is another alternative, and several examples in the literature illustrate how SFC on a polar stationary phase provided the answer to an unsuccessful RPLC analysis (for example, see [39-44]).
Another reason to choose a polar stationary phase in SFC may be that column manufacturers are mostly playing this opposite-to-RPLC-behaviour card when addressing the needs of SFC users and mostly propose polar stationary phases in their SFC catalogue pages. In other terms, the way to approach SFC practice may be influenced by manufacturers advising polar columns to SFC newcomers. This is however a rather recent practice: in older times, SFC experiments were not limited to polar stationary phases as initial screening of stationary phases often included ODS phases [45,46]. Nowadays, when ODS phases are proposed by the manufacturers for SFC use, they often contain significant elements of polarity, either through non-endcapped silanol groups or through polar embedded groups. For instance, one of the most widely used ODS phase in SFC is ACQUITY HSS C18 SB (provided by Waters Corporation), which was demonstrated to be a rather polar stationary phase, compared to well-endcapped ODS phases [47]. Its SFC retention mechanism is thus quite unexpected for an ODS phase.

Nevertheless, as mentioned above, SFC can be practised on all sorts of stationary phases, from the most polar silica to the least polar well-endcapped ODS phases, going through a variety of bonded ligands. Typical ligands are presented in Figure 1. Elution order of the analytes will greatly vary when moving from one end of the polarity axis to the other end. As a result, it is greatly advantageous to include stationary phases with different polarities in the initial screening set when developing a new SFC method, because it maximises the chances of obtaining the adequate retention and selectivity. An example is provided in Figure 2, where the retention data issued from a set of 74 natural products, analysed with gradient elution on different stationary phases, are compared (data from ref. [22]). This set was structurally diverse, comprising acidic, neutral and basic compounds with molecular weights varying from 100 to 850 g/mol. The complete list of compounds can be found in Table S1. It appears that changing the polarity of the stationary phase from non-polar (well-endcapped ODS phase, Shim-pack GIS II) to moderately polar (pyrenyl-ethyl-bonded silica, Shim-pack PyE), and most polar (bare silica, Shim-pack Sil II) is providing different elution orders, with uncorrelated retention times when comparing the columns two by two (Figure 2a and 2b). It also appears that the least polar compounds, which were unretained on the silica phase, are differently retained on the ODS (GIS II) and aromatic (PyE) columns. For the purpose of comparison, the retention on bare silica (the most polar phase) is also compared to that on another polar phase (propanediol-bonded phase, Shim-pack Diol II), and it shows that, although elution orders will be somewhat different between these two (Figure 2c), the correlation of retention times is rather higher than in the previous cases (0.51 vs. 0.38 between silica and pyrenyl, and 0.15 between silica and ODS). Also, it is visible that those compounds that were unretained on the silica phase were also unretained on the propanediol-bonded phase, indicating that replacing one polar stationary phase by another is
134 not bound to procure an adequate retention of such compounds, although it may favour a
135 different selectivity. Note that the non-polar (ODS) and moderately-polar (aromatic) columns
136 also show some correlation of retention times between them (0.48, Figure 2d). From these
137 figures, we can conclude that limiting the exploration of SFC analyses to polar stationary
138 phases is certainly limiting the possibilities of achieving the desired retention and selectivity
139 and that most diverse stationary phases should be most useful when developing a new
140 method.

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142 **3.2. Considerations related to the elution order of the analytes**

143 A second reason usually mentioned by the supporters of the NP theory is that elution order in
144 SFC would always be according to polarity. Naturally, this is somewhat related to the
145 previous point as analytes introduced in a polar stationary phase should be retained through
146 polar interactions, thus the least polar analytes would elute first, followed by analytes of
147 increasing polarity. This point can be verified by relating retention to some polarity
148 parameter. However, which polarity parameter would be adequate to describe SFC retention
149 on any type of stationary phase is not so obvious. In RPLC, retention is usually related to
150 hydrophobicity, which can be well described by the octanol-water partition coefficient (log P).

151 Naturally, this is due to the primary effect of water in the mobile phase as the hydrophobic
152 effect is pushing hydrophobic analytes to interact with the non-polar stationary phase. In
153 SFC, as there is usually no (or very little) water in the mobile phase composition,
154 hydrophobicity cannot be a good descriptor for retention. It is reasonably good when trying to
155 relate retention of compounds with limited structural diversity [19], but when attempting to
156 plot the retention times of a diverse set of compounds depending on log P values, whatever
157 the stationary phase, only vague tendencies are visible. For instance, this can be seen in
158 Figure S1, where the retention of the above-described set of 74 natural compounds was
159 plotted against log P on the same four stationary phases as above. Another possible
160 descriptor of polarity that could be more appropriate to describe polarity, and was previously
161 observed to correlate well with SFC retention of polar analytes on polar stationary phases
162 [48], is the topological polar surface area (TPSA, expressed in Å²). This is reflecting all
163 features of the molecule that may interact through polar interactions, i.e. heteroatomic
164 functions that are bound to form hydrogen bonds or contribute to dipole-dipole interactions.

165 In Figure 2, TPSA is reflected in bubble size. However, plotting the retention times of the
166 same set of 74 natural products according to TPSA values is still not providing any clear
167 correlation, only some vague tendencies (Figure S2). These failed attempts are mostly an
168 illustration of the fact that the retention of a highly diverse set of molecules cannot be
169 described so simply as one would like, because chromatographic retention results from
170 multiple interactions and not from a single factor.
171 Another, simpler way to look at this problem is to focus on smaller sets of analytes, with
172 lower structural diversity. For instance, retaining only the 23 acidic compounds from the list of
173 74 natural products (Table S1) on the moderately polar stationary phase (Shim-pack PyE,
174 with pyrenyl ligand), plotting retention against log P values shows clearer trend lines (Figure
175 3). Indeed, parallel lines with positive slopes can be seen, where each line represents
176 compounds with similar polar surface area. The latter is better appreciated through the
177 bubble size, reflecting TPSA values (also reflected in colours in Figure 3). First, the smallest
178 bubbles, related to acidic compounds with low overall polarity (e.g. a single carboxylic acid
179 function, with no other heteroatom, where TPSA is equal to 37.3 Å²) are well aligned on the
180 bottom trend line. Second, the larger bubbles, related to acidic compounds with higher polar
181 surface area (e.g., hydroxyl groups in addition to the acidic function) are aligned on the top
182 trend lines. A single compound, with the highest polarity value in this set (glycocholic acid,
183 with TPSA value equal to 127.1 Å²) is situated above the top trend lines, where it could be
184 expected to align with other high-polarity compounds, if more of them had been included in
185 this test set. This indicates that retention on this stationary phase can be related to
186 hydrophobicity when analytes of similar capabilities for polar interactions are compared, or
187 related to topological polar surface area when compounds of similar hydrophobicity are
188 compared. This was not unexpected as aromatic phases have been demonstrated to provide
189 retention through polar interactions and dispersive interactions, thereby inducing retention of
190 non-polar as well as polar analytes [21,49,50]. On the polar stationary phases however, log P
191 was still not a good descriptor for retention trends, even with a more restricted set of
192 analytes, but TPSA was somewhat more convincing in this respect, although a perfect
193 correlation could not be observed (Figure S3).

194 As a conclusion on this section, the argument of elution order according to polarity is not
195 convincing to support the NP-mode theory. Polarity can sometimes be simplified as a single
196 descriptor (e.g. log P, TPSA or number of H-bonding groups) but is most often a complex
197 feature requiring a more detailed description. Again, this is bound to cause some
198 disappointment to SFC newcomers as predicting elution order in SFC may not be so simple
199 as anticipated from the simplified NP view.

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**3.3. Considerations related to the mobile phase**

202 A third reason usually mentioned to support the NP-mode theory is that the mobile phase
203 has a low polarity, as the major component is carbon dioxide, with a polar co-solvent usually
204 introduced in smaller proportions. This mobile phase composition is usually compared to the
205 typical NP mobile phase composition comprising hexane as a major component, and a short-
206 chain alcohol as the minor component. Thus, because CO₂ was described as having a
207 polarity comparable to that of alkanes, SFC mobile phases are supposedly comparable to
208 NP mobile phases.

209 A first argument against this logic is that CO₂ is actually quite different from an alkane. As it
210 possesses local dipoles, but also π electrons and non-binding electrons, its capabilities to
211 interact with polar analytes are clearly different from those of saturated hydrocarbon chains.

212 As a result, transferring a method from NPLC to SFC sometimes causes significant changes
213 in selectivity, as demonstrated by Hirose et al. [51]. This is even worse when chiral
214 separations are compared, especially as (polymeric) chiral selectors may adopt a different
215 conformation when changing the nature of the mobile phase components [52]. In addition,
216 we may note that CO₂ has some reactivity, forming alkyloxycarbonic acids with alcohols [53],
217 thereby inducing some acidity of the CO₂-alcohol mobile phase [54], or reacting with primary
218 bases [55].

219 Another argument against this idea is that, while older experiments in SFC indeed used a
220 much larger proportion of CO₂ than the proportion of polar co-solvent, in modern SFC
221 experiments, it is not unusual to employ wide gradients of co-solvents reaching proportions
222 that are sometimes above 50%, or even reach 100% in so-called "unified chromatography"
223 experiments [56–59]. Therefore, the relative polarity of the mobile phase is not so easy to
224 define.

225 An intermediate conclusion at this stage is that comparing the CO₂-solvent mobile phase to
226 alkane-solvent mobile phase is over-simplifying and bound to cause some disillusion or, to
227 say the least, some surprises when transferring a NP method to SFC.

228 A second aspect of the mobile phase contribution is that its elution strength is supposedly
229 increasing with increasing portions of polar co-solvents. This is usually true, as increasing the
230 proportion of polar co-solvent is most often causing decreased retention. However, this is not
231 always the case, especially when non-polar compounds are analysed on non-polar stationary
232 phases. Indeed, it was demonstrated on several occasions that, for such lipophilic
233 compounds, retention first decreases when the polarity of the mobile phase increases, then
234 reaches a bottom point, and finally increases again, resulting in U-shaped retention curves.

235 Indeed, the bottom point possibly corresponds to optimal solubility of the lipophilic analyte in
236 the CO₂-solvent mobile phase. Further increases of the polar co-solvent reduce the solubility
237 of the lipophilic analyte, thereby causing increased retention. For instance, this was
demonstrated with carotenoid pigments [60], squalane [61], triacylglycerols [62,63] and
239 lipophilic antioxidants [2], or more recently with chlorophyll pigments [64].

240 While such observations are not so common, they again disagree with the NP-mode theory.

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**3.4. Considerations related to the retention mechanism**
When retention decreases with increasing proportion of polar co-solvent, the variation of retention can usually be approximated as a straight line with log k = f (log solvent %), where k is the retention factor. In the early years of SFC, when a co-solvent was introduced, this observation was often used as an argument in favour of an adsorption mechanism, such as is generally considered to dominate the retention mechanism in NP mode. However, it was later demonstrated, with the help of solvatochromic probes, that the non-linear behaviour was related to the non-linear increase of solvent strength [54,65,66], resulting from changes in polarity and density of the mobile phase. In other words, there may be an adsorption mechanism in SFC, but it cannot be deduced from the non-linear variation of retention with polar solvent proportion.

Adsorption mechanisms can be better assessed through the contribution of specific interactions with the stationary phase. A first indication of an adsorption mechanism can be found in Figure 4, where the H-bond donor character of different stationary phase ligands was plotted against the retention of H-bond acceptor analytes, as measured by quantitative structure-retention relationships in the form of the solvation parameter model or linear solvation energy relationships (LSER). Indeed, over the years, our group have characterized many chromatographic stationary phases in identical isocratic elution conditions (CO₂-methanol 90:10 v/v, 25°C, 15 MPa) [19–21]. This characterization is based on the measurement of retention data for a large set of analytes (typically over a hundred) with structural diversity, then computation of LSER system constants (see previous works for full details). In Figure 4, the LSER data obtained for 67 columns were used, including a wide diversity of stationary phases, from porous graphitic carbon or bare silica gel, to ODS phases, going through a variety of bonded ligand polarities (propanediol, ethylpyridine, phenylalkyl, pentafluorophenylalkyl, cholesteryl, etc.). The full list of columns can be found in Table S2. To each stationary phase surface or ligands, a representative compound was assigned. Some ligands are easily assigned as, e.g. 2-Ethylpyridine stationary phases can be mimicked by 2-ethylpyridine analyte. Other stationary phases are more difficult to mimic but adequate representatives can be found: for instance, porous graphitic carbon is represented by perylene, a large aromatic hydrocarbon; or a polar-embedded ODS phase containing an amide group can be represented by pentanamide. Then the basic character of this representative compound was taken as the B value of Abraham descriptors, based on literature data [67] or computed based on the chosen analyte structure [68]. This B basic character was then plotted against the basic character of the stationary phase, measured through the retention of acidic compounds with the LSER methodology (a term). A similar plot to Figure 4 was proposed in older works [19], on a much smaller set of stationary phases. The most important conclusion that can be drawn from this figure is that the retention of H-bond acceptor analytes in SFC in such conditions should be mostly related to
280 the H-bond donor character of the stationary phase, therefore related to the hydrogen bonds
281 established between the two (analyte and stationary phase). In other words, this is showing
282 the primary contribution of the stationary phase character in analyte retention, rather than
283 any form of competition with mobile phase components.

284 A different way of examining retention mechanisms is to look at the retention variation in
285 homologous series. In particular, homologous series varying in carbon chain length are most
286 useful in unravelling retention mechanisms. When examining different stationary phases in
287 SFC, the difficulty in using homologous series is to find compounds that will show adequate
288 retention on all sorts of stationary phases. For instance, non-polar stationary phases can be
289 examined with alkylbenzene series, but those are not always well retained on polar
290 stationary phases. For the latter, other compound families must sometimes be found, with a
291 polar head group to offer some retention to the whole series, whatever the carbon chain
292 length. For instance, parabens or phthalates are usually adequate to serve this purpose. In
293 previous works, it was shown that increasing carbon chain length usually caused decreased
294 retention on polar stationary phases in SFC, as would be expected in a NP mode. However,
295 increasing carbon chain length on a non-polar stationary phase (like ODS phases) or
296 moderately polar stationary phases (like aromatic ligands) in SFC caused increasing
297 retention, as would be expected in reversed-phase mechanisms [21]. An example is shown
298 in Figure 5a, where the relative retention of alkylbenzene homologous series was plotted for
299 a polar stationary phase (Princeton 2-Ethylpyridine) and a non-polar stationary phase
300 (Kromasil C18 100), where the opposite trends are very clear. This is again not in
301 accordance with a universal NP mechanism in SFC.

302 In addition, the situation can be even more confusing when considering different mobile
303 phase compositions. In previous works [69], it was shown that different co-solvents employed
304 in the mobile phase induced different retention behaviour of alkylbenzene homologous series
305 analyzed on an ethyl-pyridine stationary phase (Figure 5b). Specifically, the most polar co-
306 solvent (methanol) induced typical NP retention behaviour, with increasing carbon chain
307 length causing decreasing retention, while a more lipophilic co-solvent (isopropanol) showed
308 the opposite trend, i.e. increasing carbon chain length caused increasing retention, as
309 normally observed in RP retention behaviour. A co-solvent of intermediate polarity (ethanol)
310 showed an intermediate behaviour, with all alkylbenzenes showing nearly identical retention.
311 This was an indication that the adsorbed mobile phase components could also contribute to
312 the retention mechanism, as analytes would interact with these adsorbed mobile phase
313 components. Adsorption of mobile phase components has been largely documented, notably
314 by Strubinger and co-workers [70,71]. In particular, they observed that 2% co-solvent in the
315 mobile phase could result in 40% co-solvent being present at the surface of the stationary
316 phase. Other works indicated that adsorption of the modifier should be essentially complete
after the first few percents introduced in the pressurized CO₂ mobile phase [72]. Further increases of solvent proportion should mostly result in increasing the polarity of the mobile phase, together with some variation of mobile phase density. Further works from West and Lemasson [73] and from Tarafder and Guiochon [74] were also in accordance with a multi-layer adsorption of mobile phase components on stationary phases, so it should come as no surprise that adsorbed mobile phase components could also contribute to the analyte retention. For this reason, the retention mechanism in SFC was sometimes suggested to be similar to that of hydrophilic interaction liquid chromatography (HILIC) [75].

Another concurrent observation was done earlier on porous graphitic carbon stationary phase, based on a much wider set of analytes [18]. LSER characterization had been achieved, based on the retention data measured for about 50 analytes in different isocratic elution conditions including 7 different co-solvents (methanol, ethanol, n-propanol, isopropanol, acetonitrile, tetrahydrofuran and hexane) in varying proportions (5, 10, 20, 30 and 40% in CO₂). Plotting the LSER a-term, reflecting the retention of H-bond acceptor analytes, versus the basic character of the co-solvent, expressed as Kamlet-Taft β solvatochromic parameter, resulted in trend lines. However, these trend lines changed of slope when the proportion of co-solvent increased (Figure 6): (i) the slope was positive at low percentage of co-solvent, indicating that the most basic co-solvents favoured retention of acidic analytes through their adsorption on the stationary phase; (ii) the slope became negative at high percentage of co-solvent, indicating that the most basic co-solvents now favoured elution of acidic analytes through their solvation in the mobile phase. A major conclusion of this observation is that the contribution of mobile phase components to retention mechanisms in SFC is changing depending on stationary phase but also depending on the mobile phase composition. This is also in accordance with other works from Glenne and co-workers [76,77], who observed that small proportions of co-solvent primarily adsorbed on the stationary phase (co-solvent acting as an additive), while further increases of the co-solvent proportion should mostly cause changes in the analyte elution (co-solvent acting as modifier).

Contrary to the general opinion, most of the retention mechanisms that occur in liquid chromatography can also be observed in SFC. For instance, Strubinger et al. [71] indicated that partial molar enthalpies suggested a partition-like mechanism for non-polar solutes, rather than an adsorption mechanism. Furthermore, size-exclusion mechanism [78], donor-acceptor electron mechanism [79], ion-pairing mechanism [80,81], or ion-exchange mechanism [82] were all observed in chromatographic analyses where pressurized CO₂ was present in the mobile phase, and where the exact conditions may have been supercritical, subcritical or enhanced-fluidity. Depending on stationary phase and mobile phase composition, one mechanism may dominate over others. In short, retention mechanisms in
354 SFC are diverse, take on multiple paths, and certainly cannot be summarized as a lapidary
355 "normal-phase mechanism".

356

357 **4. Conclusions**

358 In the above, we have examined different arguments of a possible normal-phase
359 retention behaviour: polarity of the stationary phase, elution order of the analytes, relative
360 polarity and elution strength of the mobile phase, and finally retention mechanisms. A
361 general conclusion that can be drawn from all these studies is that normal-phase behaviour
362 is sometimes true, in specific conditions of stationary and mobile phases, and for specific
363 groups of analytes, but is also wrong in other conditions or for other groups of analytes. As a
364 consequence, categorizing SFC as a normal-phase method would be misleading to the
365 users, but would also limit the expected application scope of the technique. In truth, SFC
366 should be considered as a technique where all retention modes are possible. Indeed, the
367 possible retention modes in SFC are much more diverse, including not only normal-phase
368 but also reversed-phase, ion-exchange, ion-pairing, HILIC-like, mixed-mode, or even size-
369 exclusion, all depending on the choice of stationary phase and mobile phase conditions.

370

371

372 **CRediT Author Contributions**

373 **Caroline West:** Investigation, Methodology, Visualization, Writing.

374

375 **Conflicts of interest**

376 Nothing to declare.

377

378 **Acknowledgments**

379 ICOA is supported by the University of Orleans, the National Centre for Scientific Research
380 (CNRS), the projects CHemBio (FEDER-FSE 2014-2020-EX003677), Valbiocosm (FEDER-
381 FSE 2014-2020-EX003202), Techsab (FEDER-FSE 2014-2020-EX011313), QUALICHIM
382 (APR-IA-PF 2021-00149467), the RTR Motivhealth (2019-00131403) and the Labex
383 programs SynOrg (ANR-11-LABX-0029) and IRON (ANR-11-LABX-0018-01).

384

385
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547 mass spectrometry. Part II. Comparison to reversed-phase ultra-high performance liquid
548 chromatography, Journal of Chromatography A. 1663 (2022) 462771.
549 https://doi.org/10.1016/j.chroma.2021.462771.
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552 its impurities, J Pharm Biomed Anal. 125 (2016) 376–384.
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567 ionization mass spectrometry. Part I. Method development, Journal of Chromatography
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573 with phenyl and pentafluorophenyl stationary phases in supercritical fluid
574 chromatography, J Chromatogr A. 1412 (2015) 126–138.
575 https://doi.org/10.1016/j.chroma.2015.08.009.
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579 https://doi.org/10.3390/molecules24132425.
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581 supercritical fluid chromatography mobile phases for enantioselective separations on
582 polysaccharide stationary phases, J Chromatogr A. 1467 (2016) 463–472.
583 https://doi.org/10.1016/j.chroma.2016.06.060.
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585 In Situ Formation of Alkylcarbonic Acids with CO2, J. Phys. Chem. A. 105 (2001) 3947–
586 3948. https://doi.org/10.1021/jp003846y.
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588 phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the
589 mobile phase, J Chromatogr A. 1492 (2017) 136–143.
590 https://doi.org/10.1016/j.chroma.2017.02.066.
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592 Amines in Supercritical Carbon Dioxide by Proton Nuclear Magnetic Resonance
593 Spectroscopy and Implications for Supercritical Fluid Chromatography, Anal. Chem. 75
594 (2003) 622–626. https://doi.org/10.1021/ac020527p.
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596 vitamins by a novel single chromatography technique unifying supercritical fluid
597 chromatography and liquid chromatography, J Chromatogr A. 1362 (2014) 270–277.
598 https://doi.org/10.1016/j.chroma.2014.08.003.
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600 Veuthey, D. Guillarme, Applicability of Supercritical fluid chromatography–Mass
601 spectrometry to metabolomics. II–Assessment of a comprehensive library of
602 metabolites and evaluation of biological matrices, Journal of Chromatography A. 1620
603 (2020) 461021. https://doi.org/10.1016/j.chroma.2020.461021.

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605 spectrometry method to analyze free amino acids, Analytical and Bioanalytical
606 Chemistry. (2019). https://doi.org/10.1007/s00216-019-01783-5.

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608 flavonoids with unified chromatography-electrospray ionization mass spectrometry—
609 method development and application to compounds of pharmaceutical and cosmetic
610 interest, Anal. Bioanal. Chem. 412 (2020) 6595–6609. https://doi.org/10.1007/s00216-
611 020-02798-z.

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613 stationary phase and the separation of carotenes in sub-critical fluid chromatography,
614 Chromatographia. 36 (1993) 275–282. https://doi.org/10.1007/BF02263877.

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616 scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile
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619 and Chemical Structures in Subcritical Fluid Chromatography with CO2/Modifier
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624 Chromatographia. 51 (2000) 688–694. https://doi.org/10.1007/BF02505406.

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627 supercritical fluid extraction and pressurised liquid extraction, Journal of
628 Chromatography A. 1632 (2020) 461582.
629 https://doi.org/10.1016/j.chroma.2020.461582.

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689 enantioseparation mechanisms in supercritical fluid chromatography with a
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692

693
**Figure 1.** Range of polarities available for SFC stationary phases. The bar graph indicates the popularity of the stationary phase, based on the frequency of citation, as reported by Plachká *et al.* [23].

<figure><img src="image_3.png" /></figure>
**Figure 2.** Comparison of gradient-elution retention times measured on 4 different stationary phases for 74 natural products including acids, bases and neutral compounds (compound list in Table S1). Shim-pack Sil II is a bare silica stationary phase; Shim-pack Diol II is a propanediol-bonded phase; Shim-pack UC PyE has a pyrenyl-ethyl ligand; Shim-pack UC Gis II is a well-endcapped C18-bonded phase. Bubble size reflects topological polar surface area (TPSA). Based on data from [22].

709

<figure><img src="image_4.png" /></figure>

710

711

712

713
**Figure 3.** Investigation of the relation between gradient-elution retention times and log P values, on a pyrenyl-ethyl stationary phase (Shim-pack UC PyE) for 23 acidic natural products (compound list in Table S1). Bubble size and colour are related to topological polar surface area (TPSA values). Based on data from [22].

718

<figure><img src="image_5.png" /></figure>

719

720

**Figure 4.** Plot of the basic character (B Abraham descriptor) of the solute chosen as model compound for the stationary phase (Table S2) against the basic character of the stationary phase measured with the *a* coefficient of the solvation parameter model. All characterizations were done in identical chromatographic conditions (see Table S2 for conditions and column identity). Based on data published in [19–21].

721

722

723

724

725

<figure><img src="image_6.png" /></figure>

726

727
728

729

730

731

732

**Figure 5.** Variation of retention (relative log k values) when the number of carbon atoms increases in alkylbenzene carbon chains. (a) Comparison of polar (Princeton 2-Ethylpyridine) and non-polar (Kromasil C18 100) phases, with methanol co-solvent. (b) Comparison of three different co-solvents on the 2-ethylpyridine stationary phase. Other conditions: CO₂-solvent 90:10 v/v, 25°C, 15 MPa, 3 mL/min. Data from [69].

<figure><img src="image_7.png" /></figure>

733

734

735

736

737

738

739

**Figure 6.** Retention of acidic compounds on porous graphitic carbon stationary phase (Hypercarb) measured through the LSER *a* term, depending on the basic character of the co-solvent expressed as Kamlet-Taft β solvatochromic parameter. Other conditions: 25°C, 15 MPa, 3 mL/min. Data from [18].

<figure><img src="image_8.png" /></figure>

740

741
Supplementary information to:

742

743

744 Supercritical fluid chromatography is not (only) normal-phase chromatography

745

746

Caroline West

747 Institute of Organic and Analytical Chemistry, University of Orleans, CNRS UMR7311, rue de

748 Chartres - BP 6759, 45067 Orléans, France

749 caroline.west@univ-orleans.fr

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Table S1 p.23

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Table S2 p.24

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Figure S1 p.25

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Figure S2 p.26

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Figure S3 p.27

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Table S1: List of natural products used in [23] and to plot Figure 2, Figure S1 and Figure S2.
Compounds in *italics* are the acidic compounds retained in Figure 3. TPSA is the topological
polar surface area in Å, Log P is the octanol-water partition constant and MW is the exact
mass in g/mol.

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<table><thead><tr><th>Name</th><th>TPSA</th><th>Log P</th><th>MW</th><th>Name</th><th>TPSA</th><th>Log P</th><th>MW</th></tr></thead><tbody><tr><td>3-Acetylcoumarin</td><td>47.3</td><td>1.8</td><td>188.2</td><td>Fumaric acid</td><td>74.6</td><td>-0.3</td><td>116.1</td></tr><tr><td>3-Dehydroshikimic acid</td><td>94.8</td><td>-1.4</td><td>172.1</td><td>Gallic acid</td><td>98.0</td><td>0.7</td><td>170.1</td></tr><tr><td>Adonitol</td><td>101.2</td><td>-2.9</td><td>152.1</td><td>Genistein</td><td>90.9</td><td>2.7</td><td>270.2</td></tr><tr><td>α-Chaconine</td><td>220.5</td><td>1.8</td><td>852.1</td><td>Genistin</td><td>90.9</td><td>0.9</td><td>432.4</td></tr><tr><td>Amygdalin</td><td>202.3</td><td>-3.1</td><td>457.2</td><td>Glycocholic acid</td><td>127.1</td><td>2.9</td><td>465.6</td></tr><tr><td>Anabasine</td><td>24.9</td><td>1.0</td><td>162.2</td><td>Hesperetin</td><td>96.2</td><td>2.4</td><td>302.3</td></tr><tr><td>Andrographolide</td><td>87.0</td><td>2.2</td><td>350.4</td><td>Indole</td><td>15.8</td><td>2.1</td><td>117.2</td></tr><tr><td>Anthraquinone</td><td>34.1</td><td>2.5</td><td>208.1</td><td>Isovaleric acid</td><td>37.3</td><td>1.2</td><td>102.1</td></tr><tr><td>Apigenin</td><td>90.9</td><td>1.7</td><td>270.2</td><td>Kaempferol</td><td>111.1</td><td>1.9</td><td>286.2</td></tr><tr><td>Artemisinin</td><td>54.0</td><td>2.8</td><td>282.3</td><td>Laudanosine</td><td>40.2</td><td>3.7</td><td>357.4</td></tr><tr><td>Asiatic acid</td><td>98.0</td><td>5.7</td><td>488.7</td><td>Lauric acid</td><td>37.3</td><td>4.2</td><td>200.3</td></tr><tr><td>Atropine</td><td>49.8</td><td>1.8</td><td>289.4</td><td>Lupeol</td><td>20.2</td><td>9.9</td><td>426.7</td></tr><tr><td>Aucubin</td><td>149.1</td><td>-3.0</td><td>346.3</td><td>Methylcatechol</td><td>26.3</td><td>1.3</td><td>124.1</td></tr><tr><td>Baicalein</td><td>90.9</td><td>1.7</td><td>270.2</td><td>Myricetin</td><td>151.6</td><td>1.2</td><td>318.2</td></tr><tr><td>Bergamottin</td><td>52.6</td><td>5.6</td><td>338.4</td><td>Myristic acid</td><td>37.3</td><td>5.3</td><td>228.4</td></tr><tr><td>β-Carotene</td><td>0.0</td><td>13.5</td><td>536.9</td><td>Naringenin</td><td>87.0</td><td>2.4</td><td>272.3</td></tr><tr><td>Betulinic acid</td><td>57.5</td><td>8.2</td><td>456.7</td><td>Oleanic acid</td><td>57.5</td><td>7.5</td><td>456.7</td></tr><tr><td>Bilobalide</td><td>119.4</td><td>-0.3</td><td>326.3</td><td>Oleic acid</td><td>37.3</td><td>6.5</td><td>282.5</td></tr><tr><td>Boldine</td><td>62.2</td><td>2.7</td><td>327.4</td><td>Papaverine</td><td>49.8</td><td>3.9</td><td>339.4</td></tr><tr><td>Caffeic acid</td><td>77.8</td><td>1.2</td><td>180.2</td><td>Piperlongumine</td><td>65.1</td><td>2.1</td><td>317.3</td></tr><tr><td>Caproic acid</td><td>37.3</td><td>1.9</td><td>116.2</td><td>Quinine</td><td>45.6</td><td>2.9</td><td>324.4</td></tr><tr><td>Carnosic acid</td><td>77.8</td><td>4.9</td><td>332.4</td><td>Retinyl palmitate</td><td>26.3</td><td>13.6</td><td>524.9</td></tr><tr><td>Catechin</td><td>110.4</td><td>0.4</td><td>290.3</td><td>Riboflavin</td><td>161.6</td><td>-1.5</td><td>376.4</td></tr><tr><td>Catechol</td><td>40.5</td><td>0.9</td><td>110.1</td><td>Rosmarinic acid</td><td>144.5</td><td>2.4</td><td>360.3</td></tr><tr><td>Cholesterol</td><td>20.2</td><td>8.7</td><td>386.7</td><td>Rutin</td><td>269.4</td><td>-1.3</td><td>610.5</td></tr><tr><td>Chrysin</td><td>70.7</td><td>2.1</td><td>254.2</td><td>Sclareol</td><td>40.5</td><td>4.9</td><td>308.5</td></tr><tr><td>Coumaric acid</td><td>57.5</td><td>1.5</td><td>164.2</td><td>Scopolamine</td><td>62.3</td><td>0.9</td><td>303.4</td></tr><tr><td>Coumarin</td><td>30.2</td><td>1.4</td><td>146.1</td><td>Shikimic acid</td><td>98.0</td><td>-1.7</td><td>174.2</td></tr><tr><td>Daidzein</td><td>70.7</td><td>2.5</td><td>254.2</td><td>Sinapic acid</td><td>76.0</td><td>1.5</td><td>224.2</td></tr><tr><td>Daidzin</td><td>149.8</td><td>0.7</td><td>416.4</td><td>Sparteine</td><td>6.5</td><td>2.5</td><td>234.4</td></tr><tr><td>Diosmin</td><td>238.2</td><td>-0.8</td><td>608.5</td><td>Stigmasterol</td><td>20.2</td><td>8.6</td><td>412.7</td></tr><tr><td>Echinocystic acid</td><td>77.8</td><td>6.9</td><td>472.7</td><td>trans-Cinnamic acid</td><td>37.3</td><td>2.1</td><td>148.2</td></tr><tr><td>Ellipticine</td><td>28.7</td><td>4.8</td><td>246.3</td><td>Tricosanoic acid</td><td>37.3</td><td>8.3</td><td>354.3</td></tr><tr><td>Epicatechin</td><td>110.4</td><td>0.4</td><td>290.3</td><td>Trigonelline</td><td>44.0</td><td>-1.1</td><td>137.0</td></tr><tr><td>Equol</td><td>49.7</td><td>3.0</td><td>242.3</td><td>Ursolic acid</td><td>57.5</td><td>7.3</td><td>456.7</td></tr><tr><td>Esculin</td><td>149.8</td><td>-0.6</td><td>340.3</td><td>Uvaol</td><td>40.5</td><td>7.4</td><td>442.7</td></tr><tr><td>Ferulic acid</td><td>66.8</td><td>1.5</td><td>194.2</td><td>Zeaxanthin</td><td>40.5</td><td>10.9</td><td>568.9</td></tr></tbody></table>

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<table><caption>Table S2: List of columns used in Figure 4 and mimic compounds selected to represent the ligands or surface chemistry. LSER testing conditions: CO₂-methanol 90/10 v/v, 25°C, 15 MPa. Data from [23].</caption><thead><tr><th>Column name</th><th>Mimic compound</th><th>a value</th><th>B value</th><th>Column name</th><th>Mimic compound</th><th>a value</th><th>B value</th></tr></thead><tbody><tr><td>ACQUITY UPC² HSS C18 SB</td><td>octadecane</td><td>0.48</td><td>0.00</td><td>Shim-pack CN</td><td>propionitrile</td><td>1.03</td><td>0.36</td></tr><tr><td>ACQUITY UPC² BEH</td><td>methanol</td><td>0.81</td><td>0.47</td><td>Shim-pack Diol</td><td>1-propanol</td><td>1.22</td><td>0.48</td></tr><tr><td>ACQUITY UPLC BEH Shield RP18</td><td>pentanamide</td><td>0.76</td><td>0.62</td><td>Shim-pack Py</td><td>2-ethylpyridine</td><td>1.29</td><td>0.49</td></tr><tr><td>ACQUITY UPC² BEH 2-EP</td><td>pyridine</td><td>1.39</td><td>0.49</td><td>Shim-pack HyP</td><td>phenol</td><td>0.90</td><td>0.30</td></tr><tr><td>ACQUITY UPC² CSH Fluorophenyl</td><td>pentafluorophenyl</td><td>0.37</td><td>0.10</td><td>Shim-pack NH2</td><td>propylamine</td><td>1.29</td><td>0.61</td></tr><tr><td>ACQUITY UPC² Torus 1-AA</td><td>aminoanthracene</td><td>1.32</td><td>0.63</td><td>Shim-pack Sil</td><td>methanol</td><td>0.61</td><td>0.47</td></tr><tr><td>ACQUITY UPC² Torus 2-PIC</td><td>pyridine</td><td>1.96</td><td>0.49</td><td>Shim-pack Amide</td><td>methylamide</td><td>1.12</td><td>0.52</td></tr><tr><td>ACQUITY UPC² Torus DEA</td><td>diethylamine</td><td>2.64</td><td>0.69</td><td>Shim-pack Triazole</td><td>triazole</td><td>0.78</td><td>0.62</td></tr><tr><td>ACQUITY UPC² Torus DIOL</td><td>propanediol</td><td>1.96</td><td>0.48</td><td>Hypercarb</td><td>perylene</td><td>0.78</td><td>0.42</td></tr><tr><td>Cortecs HILIC</td><td>methanol</td><td>0.94</td><td>0.47</td><td>PLRP-S</td><td>ethylbenzene</td><td>0.13</td><td>0.15</td></tr><tr><td>Ascentis Express F5</td><td>pentafluorobenzene</td><td>0.03</td><td>0.10</td><td>Pursuit Diphenyl</td><td>biphenyl</td><td>0.39</td><td>0.26</td></tr><tr><td>Nucleoshell PFP</td><td>pentafluorobenzene</td><td>-0.10</td><td>0.10</td><td>Pursuit XRs Diphenyl</td><td>biphenyl</td><td>0.25</td><td>0.26</td></tr><tr><td>Kinetex HILIC</td><td>methanol</td><td>0.94</td><td>0.47</td><td>Uptisphere PH</td><td>propylbenzene</td><td>0.26</td><td>0.15</td></tr><tr><td>Kinetex PFP</td><td>pentafluorophenyl</td><td>-0.17</td><td>0.10</td><td>Luna Phenyl-hexyl</td><td>hexylbenzene</td><td>0.01</td><td>0.15</td></tr><tr><td>Kinetex F5 (2)</td><td>pentafluorophenyl</td><td>-0.12</td><td>0.10</td><td>Gemini Phenyl-hexyl</td><td>hexylbenzene</td><td>0.07</td><td>0.15</td></tr><tr><td>Kinetex Biphenyl</td><td>biphenyl</td><td>0.12</td><td>0.26</td><td>Cosmosil 5PYE</td><td>pyrene</td><td>0.15</td><td>0.29</td></tr><tr><td>Kinetex XB C18</td><td>octadecane</td><td>-0.31</td><td>0.00</td><td>Chromegabond Fluorodecyl</td><td>trifluoroethanol</td><td>0.46</td><td>0.25</td></tr><tr><td>Synergi Polar RP</td><td>anisole</td><td>0.16</td><td>0.29</td><td>Discovery HS F5</td><td>pentafluorobenzene</td><td>0.00</td><td>0.10</td></tr><tr><td>Hypersil Gold Silica</td><td>methanol</td><td>0.87</td><td>0.47</td><td>Cosmosil 5PBB</td><td>dibromophenol</td><td>0.21</td><td>0.22</td></tr><tr><td>Hypersil Gold CN</td><td>propionitrile</td><td>0.77</td><td>0.36</td><td>Princeton Ethylpyridine</td><td>2-ethylpyridine</td><td>1.07</td><td>0.49</td></tr><tr><td>Accucore HILIC</td><td>methanol</td><td>0.72</td><td>0.47</td><td>Princeton Cyano</td><td>propionitrile</td><td>0.65</td><td>0.36</td></tr><tr><td>Accucore 150-Amide-HILIC</td><td>methylamide</td><td>1.70</td><td>0.52</td><td>Uptisphere RPH</td><td>octadecylbenzene</td><td>-0.41</td><td>0.08</td></tr><tr><td>Accucore Urea-HILIC</td><td>methylurea</td><td>1.68</td><td>0.64</td><td>Nucleodur Sphinx RP</td><td>octadecylbenzene</td><td>-0.16</td><td>0.08</td></tr><tr><td>Accucore Phenyl-X</td><td>benzene</td><td>0.58</td><td>0.15</td><td>Supelcosil ABZ+</td><td>pentanamide</td><td>1.29</td><td>0.62</td></tr><tr><td>Accucore Phenyl-hexyl</td><td>benzene</td><td>-0.10</td><td>0.15</td><td>Uptisphere C4</td><td>butane</td><td>-0.02</td><td>0.00</td></tr><tr><td>Accucore PFP</td><td>pentafluorobenzene</td><td>-0.14</td><td>0.10</td><td>Uptisphere C8</td><td>octane</td><td>-0.26</td><td>0.00</td></tr><tr><td>Speedcore Diphenyl</td><td>biphenyl</td><td>0.38</td><td>0.26</td><td>Synergi Max RP</td><td>dodecane</td><td>-0.37</td><td>0.00</td></tr><tr><td>COSMOCORE Cholester</td><td>cholestane</td><td>0.48</td><td>0.00</td><td>Kromasil C18 100</td><td>octadecane</td><td>-0.44</td><td>0.00</td></tr><tr><td>Shim-pack PBr</td><td>dibromophenol</td><td>0.14</td><td>0.22</td><td>Kromasil Sil 100</td><td>methanol</td><td>0.96</td><td>0.47</td></tr><tr><td>Shim-pack PyE</td><td>pyrene</td><td>0.09</td><td>0.29</td><td>Discovery HS PEG</td><td>dioxane</td><td>1.24</td><td>0.64</td></tr><tr><td>Shim-pack Phenyl</td><td>propylbenzene</td><td>0.32</td><td>0.15</td><td>Princeton Amino</td><td>propylamine</td><td>1.44</td><td>0.61</td></tr><tr><td>Shim-pack RP</td><td>pentanamide</td><td>0.71</td><td>0.62</td><td>Princeton Diol</td><td>1-propanol</td><td>0.98</td><td>0.48</td></tr><tr><td>Shim-pack Choles</td><td>cholestane</td><td>0.19</td><td>0.56</td><td>YMC-Pack PVA-Sil</td><td>1-propanol</td><td>1.03</td><td>0.48</td></tr><tr><td>Shim-pack GIS II</td><td>octadecane</td><td>-0.23</td><td>0.00</td><td></td><td></td><td></td><td></td></tr></tbody></table>
**Figure S1:** Investigation of the relation between gradient-elution retention times and log P values, on 4 different stationary phases for 74 natural products including acids, bases and neutral compounds (compound list in Table S1). Shim-pack Sil II is a bare silica stationary phase; Shim-pack Diol II is a propanediol-bonded phase; Shim-pack UC PyE has a pyrenyl-ethyl ligand; Shim-pack UC Gis II is a well-endcapped C18-bonded phase. Data from [23].

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<figure><img src="image_9.png" /></figure>

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**Figure S2:** Investigation of the relation between gradient-elution retention times and TPSA values, on 4 different stationary phases for 74 natural products including acids, bases and neutral compounds (compound list in Table S1). Shim-pack Sil II is a bare silica stationary phase; Shim-pack Diol II is a propanediol-bonded phase; Shim-pack UC PyE has a pyrenyl-ethyl ligand; Shim-pack UC Gis II is a well-endcapped C18-bonded phase. Data from [23].

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<figure><img src="image_10.png" /></figure>

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<figure><img src="image_11.png" /><figcaption>**Figure S3:** Investigation of the relation between gradient-elution retention times and topological polar surface area (TPSA values), on a silica stationary phase (Shim-pack UC Sil II) and a propane-diol bonded stationary phase (Shim-pack UC Diol II), for 23 acidic natural products (see compound list in Table S1). Bubble size is related to log P values. Data from [23].</figcaption></figure>

<figure><img src="image_12.png" /></figure>