Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

CHAPTER 14

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography Roberta Franzini, Alessia Ciogli, Francesco Gasparrini, Omar H. Ismail, Claudio Villani Sapienza University of Rome, Rome, Italy

14.1  INTRODUCTION A supercritical fluid is defined as a “substance for which the temperature and pressure are above their critical values and which has a density close to or higher than its critical density.” Mention of the fluid density in the second part of this definition is important because some of the useful properties of the supercritical fluids, like the ability to dissolve solids or the elution ability in chromatography, are fully exploited only under those conditions where its density is not too far from the critical value (i.e. under T and P conditions near the critical values) [1]. Above the critical temperature, the vapor–liquid line in the phase diagram is no longer present, and the supercritical fluids behave as “hybrid solvents”. Their physical properties (density, viscosity, and diffusivity are chromatographically relevant) can be smoothly changed from liquid-like to gas-like by pressure or temperature variations. Table 14.1 collects some typical values for density, viscosity, and diffusivity of gases, liquids, and supercritical fluids: the hybrid nature of the latter is evident as well as the potential for fine-tuning of their properties by controlling the pressure. Supercritical fluid chromatography (SFC) was proposed for the first time in the 1960s when Klesper et al. [2] realized the first chromatographic separation of etioporphyrines using chlorofluoromethanes in supercritical state. However, among the usable supercritical fluids, carbon dioxide has become dominant as supercritical eluent in chromatography, due to its easily achievable critical parameters (Tc = 31.1 °C and Pc = 7.39 MPa). In addition, CO2 is nontoxic, chemically inert, nonflammable, environmentally safe, and relatively cheap. The principal CO2 drawback is the limited solvating and eluting power, which largely limits the applicability of pure CO2 as a chromatographic eluent, especially for polar compounds. Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00014-1 Copyright © 2018 Elsevier B.V. All rights reserved.

607

608

Roberta Franzini et al.

Table 14.1  Comparison of Physical Properties of Gases, Supercritical Fluids, and Liquids Density, ρ (kg m−3) Viscosity, η (Pa s) Diffusivity, D (m2 s−1)a

Gasb 1 Supercritical fluid 100–800c Liquid 1000

0.001 0.005–0.01c 0.05–0.1

1*10-5 1*10-7 1*10-9

Diffusion coefficient of small-molecule solutes in the given fluid. Values at room temperature. Values are pressure dependent.

a

b c

At the beginning, the advent of high-performance liquid chromatography (HPLC) and the limits of SFC instrumentation, mainly in terms of low robustness and low reproducibility, slowed down the spread of SFC. Only in late 1980s, the technique regained the attention of scientific community following the introduction of the first commercial SFC instrumentation designed for packed columns and capable to give accurate control of flow rate and mobile phase composition. Therefore, both capillary columns and pure carbon dioxide, initially employed as the preferred combination for SFC applications, were replaced by packed columns and by carbon dioxide-based eluents containing varying amounts of polar modifiers (mainly methanol or ethanol). On the one hand, the presence of organic modifier, in analogy to what happens in normal-phase HPLC, increases the solvation and elution ability and decreases retention of the solutes. On the other hand, the addition of modifiers to pure CO2 increases the critical temperature and pressure values of the mixed fluid, increases its viscosity, and decreases the diffusion of analytes. However, these changes do not obscure completely the benefits associated with using a supercritical fluid. It has to be noted that in the large majority of the chromatographic applications, a CO2/modifier mixture is used as the eluent at temperatures that are below the critical value of the mixture, and thus the eluent has to be regarded as a liquid rather than a supercritical fluid. However, the chromatographic process is generally referred as “supercritical”, even if the eluent is under “subcritical conditions”. Other definitions such as “enhanced fluidity chromatography” or “ultra-high-pressure gas chromatography” have also been proposed, both underlying the hybrid nature of the mobile phase and the associated benefits. From the late 1990s to today, SFC separations have been performed using columns packed with stationary phases that have been borrowed from HPLC applications. These stationary phases, with superficial chemistry tailored for normal phase, hydrophilic interaction

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

609

chromatography (HILIC) or reversed-phase retention mechanisms, are usually chemically and thermally stable, have broad applicability, and complementar retention behavior and selectivity. In addition, the wide choice of organic modifiers and additives in the eluents make SFC as an alternative not only to normal, but also to reversed-phase HPLC [3]. The key benefits of working under SFC conditions are mainly related to the low viscosity of the eluents and to the large solute diffusivities: given that viscosity of CO2based eluents is 3–5 times lower than in HPLC, and diffusion coefficients of solutes are 3–10 times larger than in normal liquids, one can easily run a high-speed separation (high flow rate and low back pressure) while retaining substantial efficiency values (large solute diffusivity, flat C term in the van Deemter curves). Moreover, in the post-run analysis, CO2 can be easily removed by depressurization or can be recycled, making the process green especially if developed on a large scale. With the introduction of third-generation SFC instruments (around 2010), columns packed with sub-2-micron particles or core-shell particles commonly used in ultra-high-performance liquid chromatography (UHPLC) applications were employed also in ultrafast SFC separations [3,4]. Although reversed-phase HPLC continues to have a greater diffusion (for historical reasons and for the broader application field, e.g. for strongly polar, ionizable, or high-molecular-size analytes) in the routine qualitative and quantitative analyses, SFC has a steadily growing impact in the field of chiral separations, both analytical and preparative.The first separation of enantiomers by SFC was reported in 1985 where a chiral phosphine oxide has been resolved onto the (R)-N-(3,5-dinitrobenzoyl) phenyl-glycine selector bonded to silica gel [5,6]. With the second generation of instrumentation (1990s), the interest on chiral SFC separations extended to pharmaceutical, food, agrochemical, and forensic fields, both in academic and industrial contexts. In the last case, the high productivity achievable with SFC represented and still represents a central asset in large-scale separations (easier work-up, nontoxic, nonflammable, reusable CO2, and environment-friendly system). The “speed” advantage of SFC is also extremely attractive during drug discovery and development steps, where high-throughput screening of substantial number of potentially interesting molecules is of paramount importance. Focusing in scientific production from 1992 to present, a bibliographic search carried out in Scopus for the “chiral separation by supercritical fluid” topic returns more than 400 products limited only to papers and reviews. A remarkable increase in paper production in the recent years

610

Roberta Franzini et al.

Figure 14.1  Milestones in SFC evolution. Growth in scientific production about chiral SFC world. Abbreviation: SFC, supercritical fluid chromatography.

is evident: 279 papers were published in the last 10 years, and 83 during the last 2 years. This vivid research activity proves that chiral SFC has emerged as a mature and consolidated methodology, exhibiting attractive features in several fields spanning from fast analysis to high-throughput screening to preparative scale separations. The reader can find recent publications concerning historical SFC development [6,7], analytical [8–13], and preparative [14–16] applications and fundamental studies [17–19] to discover the renewed world of chiral SFC. This chapter provides an overview of the progress in chiral SFC separations over the last 10 years (see Fig. 14.1). A section on chiral stationary phases (CSPs) mainly involved in supercritical separation will be presented together with some representative analytical and preparative applications. Special emphasis is placed on ultrafast separation obtained on columns packed with sub 2-micron or core shell particles, used in conjunction with third-generation SFC hardware.

14.2  CHIRAL STATIONARY PHASES FOR SFC With the introduction of packed columns, SFC methods have quickly extended to the separation of enantiomers. In addition to the common benefits, as fast runs, and easier purification of collected samples, the use of SFC as an alternative in chiral separation is also advantageous due to the improved efficiency and the short equilibration times compared to chiral separations carried out with classical LC eluents. Usually, a column packed with a given CSP produces narrower peaks when liquid mobile phases are replaced

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

611

by CO2-based eluents. However, while the favorable overall kinetic effects of supercritical fluids on the separation process are constantly observed, it is not possible to predict their impact on retention and enantioselectivity. The whole portfolio of CSPs commonly employed in liquid chromatography can be transferred to SFC methods so that now, during method development, it is a good practice to evaluate each CSP both in HPLC and SFC modalities. Most representative CSPs that are today employed in supercritical chromatography include derivatives of cellulose and amylose (coated on or immobilized to silica), brush-type (Pirkle-type) CSPs, native and derivatized cyclodextrins and cyclofructans, quinine-based and macrocyclic antibiotics-based CSPs [12]. Protein-based and ligand-exchange CSPs, due to their unique retention mechanisms and eluent requirements, have never been used in conjunction with supercritical fluids. The general structures of the most commonly used CSPs in SFC are gathered in Fig. 14.2, and representative analytical applications for each CSP class are reported in the following section.

Figure 14.2  Structures of the most commonly used chiral selectors in SFC applications. Abbreviation: SFC, supercritical fluid chromatography.

612

Roberta Franzini et al.

14.3  ANALYTICAL SEPARATIONS 14.3.1  Polysaccharide CSPs Contrary to native polysaccharides, different derivatives (esters or carbamates) of cellulose and amylose show broad chiral recognition ability when incorporated as selectors in macroporous silica-based CSPs. Introduced by Okamob in the early 1980s [20], these CSPs are nowadays commercially available by several companies. The largest chiral recognition ability of this CSP class is observed for methyl- or halogen-substituted phenylcarbamates as tris(3,5-dimethylphenylcarbamate), for both cellulose and amylose polymers. In their first version, the derivatized polysaccharides were physically coated onto inert silica particles, thus showing limited solvent compatibility and, under loosely controlled conditions, stationary phase bleeding. The second generation of CSPs was obtained by covalently immobilizing the polysaccharide derivatives to the silica matrix without compromising their resolution power compared to the coated versions.The immobilization procedure, either chemical via functional spacers, or thermal and photochemical, occurs by covalent bonding at the reactive site of the polymer chains and presumably by crosslinking of the silica-adsorbed polysaccharide chains, depending on the synthetic strategy adopted [21– 23]. While coated and immobilized CSPs show, in most cases, similar retention and selectivity under identical conditions, the immobilized versions can be used in conjunction with a variety of solvents that can improve both selectivity and sample solubility. Recent advances in the synthesis and applications of coated and immobilized polysaccharide derivatives were exhaustively reported by Chankvetadze in 2012 [23]. The CSPs based on cellulose or amylose tris(3,5-dimethylphenylcarbamate) are the two most employed materials in supercritical chromatography [24–26]. Tesarˇová and coworkers [27] focused their attention on the SFC enantioseparation of chiral, biologically active basic compounds on amylose tris(3,5-dimethylphenylcarbamate) immobilized on 3-micron silica particles. All the examined compounds were baseline resolved and the effects of cosolvent type and additives (basic, acid, or a mixture of them) were investigated: a mobile phase consisting of CO2/2-propanol with added isopropylamine/trifluoroacetic acid mixture gave the best results for a group of analytes related to amphetamine and cathinone, whereas CO2/2-propanol/methanol with added isopropylamine was the mobile phase of choice for benzofuryl and aminonaphthol derivatives (see Fig. 14.3).

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

613

Figure 14.3  Enantioseparations of chiral amines by SFC on a (150 mm × 3.0 mm i.d., 3  µm) column packed with immobilized amylose tris(3,5-dimethylphenylcarbamate); mobile phase (A): CO2/2-PrOH/TFA/IPA 90/10/0.05/0.05 (v/v/v/v); MP (B): CO2/PrOH/TFA/ IPA 95/5/0.05/0.05 (v/v/v/v); flow rate: 2.5 mL/min; Tcol: 35 °C; BP: 13.8 MPa; UV 254 nm. Reproduced with permission [27]. Abbreviation: SFC, supercritical fluid chromatography.

With the aim to understand retention and separation mechanisms of polysaccharide stationary phases, Khater et al. [28] analyzed 171 achiral probes and 97 chiral probes under supercritical fluid and normal-phase elution conditions on the two Chiralpak AD-H (amylose tris(3,5-dimethylphenylcarbamate), coated) and Chiralpak IC (cellulose tris(3,5-dichlorophenylcarbamate), immobilized) CSPs. Normal-phase LC (NPLC) eluents were converted into SFC eluents by substituting heptane with carbon dioxide, maintaining constant the nature (ethanol or isopropanol) and the amount of organic modifiers (20%). The results obtained on this large set

614

Roberta Franzini et al.

of samples were analyzed by a chemometric approach and, in some cases, the supercritical mobile phase resulted in a weaker eluent compared to the liquid mobile phase, whereas enantioselectivity for the chiral probes was basically equivalent for the two eluting systems. Differences in retention data between the two elution conditions suggest that SFC and NPLC processes are governed by different intramolecular interactions driving retention and selectivity. These and related results are not completely unexpected if one considers the molecular differences between carbon dioxide and linear alkanes: while CO2 has an overall chromatographic behavior similar to that of hexane, its elution strength is controlled mainly by the low polarizability and strong quadrupole moment. The introduction of third-generation SFC instruments, with lowered extra-column dispersion and with improved pressure-flow characteristics, suggested that faster separations with baseline resolution could be realized, leading to high-throughput screening processes involving several CSPs– solutes combinations. As reported in Fig. 14.4, during the last 20 years, run times for enantiopurity determinations by chromatography have shortened from the

Figure 14.4  Demonstration of analysis time reduction for the enantioseparation of lansoprazole, flurbiprofen and warfarin as test probes on different CSPs. Reproduced with permission [29]. Abbreviation: CSP, chiral stationary phase.

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

615

standard 20–30 min to routinely obtained 15–60 s, with high-throughput as the driving force for even faster separations. Short columns packed with polysaccharide stationary phases on 3-micron silica particles have been employed to provide fast SFC analysis during method development, where speed is a key factor [29–31]. Regalado and Welch [29] introduced an approach to determine the fastest possible baseline separation (Rs > 1.5), defining the “column cutting” parameter as a speed that can be obtained through an actual reduction in column length, or by changes in mobile phase composition, flow rate, or column temperature. Using this approach, a large number of baseline enantioseparations in the sub-minute time scale were obtained on short columns packed with 3-micron CSPs based on amylose or cellulose derivatives and with 5-micron Whelk-O1 CSP, all of them using CO2 based eluents [29].

14.3.2  Pirkle-type CSPs Originally introduced and later developed by Pirkle, these CSPs are characterized by low-molecular-size selectors whose molecules form an ordered layer (brush-type) on the inert matrix surface. These selectors typically show the presence of polar sites capable of dipole–dipole, H-bonding, and aromatic–aromatic interactions with complementary sites on the analyte molecules. The widely used Whelk-O1 selector [32] bears a π–acidic 3,5-dinitrobenzamide and a π–basic naphthyl fragments close to the stereogenic center, held together in a rigid fashion and forming a cleft where aromatic portion of the analyte molecules can be accommodated while simultaneously establishing H-bond, face-to-face, and face-to-edge aromatic interactions. Spectroscopic data taken both in solution and in the solid state support this picture of enantioselective recognition.This simple 1:1 association mode and the ordered brush-type organization of the chiral selector on the silica surface have direct relevance not only on the large enantioselectivity usually observed, but also on the kinetic side of the process and ultimately on the chromatographic efficiency. The excellent kinetic performances of columns packed with CSPs incorporating brush-type selectors are peculiar of the high speed–high efficiency results obtained on the newly introduced sub-2-micron materials (see further). Working preferentially in the normal-phase mode, an easy transfer to SFC mode can be obtained with Pirkle-type CSPs. The effect of cosolvent nature was investigated by Szczerba and Wrezel [33] in chiral SFC method development on a Whelk-O1 column. Focusing the results only on the three alcohols, the authors found out that selectivity and retention decrease as polarity of alcohol decrease

616

Roberta Franzini et al.

and that isopropanol ensures highest selectivity even if compared with nonalcoholic modifier (tetrahydrofuran and acetonitrile). Byrne et al. [34] employed Whelk-O1 CSP together with polysaccharide-based CSP in the evaluation of 2,2,2-trifluoroethanol as organic modifier in CO2. Alcoholsensitive chiral compounds in addition to the other standard chiral ones were analyzed, and results were compared with those obtained with typical cosolvents (methanol, ethanol, isopropanol, and acetonitrile). It was shown that trifluoroethanol is a useful alternative to standard cosolvents and it represents a solution for separation of alcohol-sensitive compounds. Recently, in a fundamental SFC investigation, columns packed with the Whelk-O1 CSP have been included in a group of 10 chiral columns to investigate the effect of column backpressure on retention and selectivity [35].

14.3.3  Macrocyclic glycopeptide antibiotic CSPs Macrocyclic glycopeptide antibiotics are a class of medium-sized molecular size whose structure contains an aglycone cyclopeptide “basket” surrounded by carbohydrate moieties. Teicoplanin (T), teicoplanin aglycone (TAG), ristocetin (R), and vancomycin (V) are used in the preparation of a family of CSPs that have common features like a broad spectrum of application even for strongly polar and ionic compounds and the ability to work in a variety of eluting conditions including reversed-phase, polar organic, and normal-phase modes. However, only a few examples of SFC separation, in comparison to their use in liquid chromatography, have been recently reported in the literature [36,37]. Lavison and Thiébaut [36] employed a ristocetin stationary phase in SFC for the resolution of a series of structurally diverse samples. Perhaps the strong retention showed in normal elution conditions (20% of organic modifier) have restricted the development of analytical methods in SFC. However, the renewed interest about these CSPs in supercritical mode is very recent and overlaps with the introduction of a new generation of silica particles, developed for ultrafast separations (see following section).

14.3.4  Ion exchange CSPs The tert-butyl carbamates of quinine and quinidine immobilized on silica turned out to be the most useful structure variations in the design of weak-anion exchange chiral selector [38]. These selectors showed good enantioselectivity for the same compound classes of acidic chiral compounds also under SFC conditions. Pell and Lindner [39] showed in 2012

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

617

that anion-exchange, the primary interaction governing retention during HPLC elution with polar organic or aqueous mobile phases, is preserved under SFC elution with carbon dioxide containing methanol and additives. The effects of different types and amounts of additives (acids, bases, water) and of temperature on chromatographic performance were evaluated. The results indicated that retention, but not enantioselectivity, can be modulated by the amounts and types of co- and counterions (salts) in the modifier. In addition, the authors studied the effect of the “inherent acidity” of CO2–methanol mobile phases on chromatographic parameters. As counterpart of anion exchange CSPs, a strong cation exchange CSP based on the syringic acid amide derivative of trans-(R,R)-2-aminocyclohexanesulfonic acid was prepared and employed in SFC to resolve the enantiomers of chiral amines [40]. A systematic study of retention and selectivity as a function of the nature and concentration of additives (formic acid, ammonium formate, ammonia, tertiary amines) demonstrated also in this case that retention under SFC conditions is predominantly based on an ion exchange mechanism.

14.3.5  Cyclodextrin CSPs Cyclodextrins (CDs) are cyclic oligosaccharides consisting of several (usually 6, 7, or 8) glucose units connected by α-1,4-linkages. They possess a three-dimensional shape resembling a hollow torus. The primary 6-hydroxyl groups are located on the narrow rim of the torus, while the 2and 3-hydroxyl secondary groups are located on the wider rim, generating a hydrophilic outer surface and a hydrophobic internal cavity.The latter can accommodate nonpolar portions of the analytes and can form 1:1 inclusion complexes in water-rich media.The hydroxyl groups are amenable of selective modification, yielding a variety of derivatized cyclodextrins with extended interaction and recognition abilities.An overview about the synthesis and applications of cyclodextrins, in native or derivatized form, has been reported few years ago [41]. The use of CDs in SFC with packed columns is not widespread, probably because the low polarity of the mobile phases, even in the presence of polar modifiers, does not favor the formation of inclusion complexes with the analytes, thus limiting their interactions with the external surface of the CDs. On the other hand, cationic functionalized CDs immobilized onto vinyl silica via radical copolymerization, demonstrated good enantioselectivity in the separation of analytes containing ionizable moieties (anions) [42,43].These works highlighted the importance of

618

Roberta Franzini et al.

electrostatic forces between the ionizable group of the enantiomers and the cationic sites of CDs.

14.4  ULTRAFAST HIGH-EFFICIENT SFC SEPARATIONS The favorable fluidic properties of carbon dioxide-based eluents allow ultrafast enantiomeric separations to be performed at extremely high flow rates, with reduced drawbacks due to high column back-pressure or degraded efficiency compared to the same separations obtained with classical eluents. While some examples of fast, sub-minute SFC separations have been reported in the past [6,9,10], only in recent years the field has been deeply investigated leading to high efficient processes that are routinely completed on the seconds time scale. A number of factors have contributed to this gain in analysis speed, including improved chromatographic particle technology and dedicated instrumentation [44–46]. Particle technology is important because, to a first approximation, the column efficiency (expressed as the number of theoretical plates, N) is inversely proportional to the average size of the CSP particles that fill the column. Thus, with smaller particles, the analyte molecules can travel a shorter pathway and improve their mass transfer kinetics, which is one of the factors contributing to H, the height equivalent to a theoretical plate (H = L/N, smaller H corresponds to higher column efficiency). A compact equation accounting for the whole set of factors that contribute to H is H = Hl + He + Hm + Hf where the subscripts indicate longitudinal diffusion (l), eddy dispersion (e), liquid–solid mass transfer resistances due to slow diffusion rate across the particles (m), and frictional heating generated at elevated flow rates (f  ). Van Deemter plots correlate plate height H with mobile-phase velocity (u) according to H = A+

B + Cu u

where A, B, and C represent eddy dispersion, longitudinal diffusion, and mass transfer, respectively. Plots of experimentally measured H versus flow rate velocity are commonly used to explore the column kinetic performance and locate the optimal flow rate.

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

619

The decrease in particle size has a direct impact on the pressure required to pump the mobile phase through the column, according to the Darcy’s equation: ∆P =

Φu0 Lη d p2

where η is the viscosity of the mobile phase, L the column length, u0 the linear mobile-phase velocity (u0 = L/t0, with t0 being the hold-up time), Φ is the column resistance factor, and dp is the particle diameter. Therefore, low-viscosity mobile phases like those based on carbon dioxide are ideal when small particles (e.g. sub-2-micron fully porous particles), or long columns, are used in combination with high eluent flow rates. High speed and high efficiency enantioseparations can be realized under SFC conditions using short chiral columns packed with small particles and delivering the eluent at high flow rates: baseline resolutions that are completed in the seconds time domain have been demonstrated recently possible for a large variety of chiral solute structures. Asymmetric synthesis, enzymatic reactions, and resolution by crystallization are research areas where quick analytical methods for optical purity determinations are highly desirable. Fast enantiomeric excess measurements of a large number of chiral products can accelerate the screening of experimental conditions and the overall optimization process. SFC on CSPs is amenable to high-throughput screening of enantiomeric excess (hundreds of samples per day) and can compete with optical or sensor-based methods whose analysis cycle time is smaller than 60 s. Distinct additional advantages of high-throughput screening methods based on SFC on CSPs are the broad applicability (also for mixtures) and the inherent quantitation ability [47–49]. In a seminal paper, Gasparrini and coworkers [50] realized a proof-ofconcept study demonstrating the feasibility of high-throughput screening of large compound libraries by enantioselective ultra-high-performance SFC. Van Deemter analysis of columns packed with standard 5.0-micron and with 1.7-micron Whelk-O1 particles clearly showed the superior kinetic performance of the latter CSP when used in conjunction with carbon dioxide-based eluents (Fig. 14.5). In the high flow rate region of the plots, the efficiency loss of the columns was always less pronounced under SFC elution conditions, thus enabling short analysis times and high efficiency.

620

Roberta Franzini et al.

Figure 14.5  Comparison of van Deemter plots under LC and SFC conditions of the first eluted enantiomer of trans-stilbene oxide on 1.7-micron Whelk-O1 CSP. Adapted from [50]. Abbreviations: LC, liquid chromatography; SFC, supercritical fluid chromatography; CSP, chiral stationary phase.

A 5-cm long column packed with 1.7-micron totally porous WhelkO1 particles was used in conjunction with fast CO2–methanol gradient elution, to screen for enantioselectivity a library composed by 129 chiral compounds featuring ample structural variation. A high success rate was observed for polar neutral, and both acidic and basic compounds and transfer of the gradient conditions to unoptimized isocratic elution led to very fast, high-efficient separations with sub-minute run times as shown in Fig. 14.6.

Figure 14.6  Fast and high-efficient separations obtained on 1.7-micron Whelk-O1 CSP. Adapted from [50]. Abbreviation: CSP, chiral stationary phase.

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

621

Figure 14.7  Ultrafast, high-efficiency enantioseparations using CSPs based on 1.7-micron Whelk-O1 CSP, CO2/MeOH 80/20, 4.0 mL/min flow rate, Tcol = 35 °C, 12.4 MPa back pressure, UV detection at 220 nm. Top trace 50 × 4.6 mm column, bottom trace 10 × 4.6 mm column. Abbreviations: CSP, chiral stationary phase; UV, ultraviolet.

Ultrafast enantioseparations accompanied by high efficiency were achieved using a 1-cm long column, as shown in Fig. 14.7.Taking advantage of the large enantioselectivity (α = 2.5) and efficiency (N/m ∼ 250,000 on the second peak) offered by the 5 cm column packed with sub-2-micron Whelk-O1 CSP, the excess resolution available (Rs = 15) can be traded for speed of analysis by reducing the column length to 1 cm (Fig. 14.8). An extremely fast and complete separation, lasting only 10 s, was obtained on the short column (α = 2.5; N/m ∼ 205,000; Rs = 5). It should be noted that ultrafast, high-efficiency SFC enantioseparations realized on short columns, or on columns having small void volumes, pose severe hardware limitations

Figure 14.8  Preparative SFC large-scale resolution on immobilized cellulose tris(3,5-dichlorophenylcarbamate) 5-micron CSP packed on a 3 × 25 cm column, 10% methanol in CO2 as eluent delivered at 200 mL/min, T = 45 °C, 10 MPa back pressure. Abbreviations: SFC, supercritical fluid chromatography; CSP, chiral stationary phase.

622

Roberta Franzini et al.

in terms of extremely small extra-column volumes and to high detector sampling rates [49]. Ultrafast separations under SFC conditions have been reported for a variety of chiral samples on short columns packed with fully porous sub2-micron CSPs based on immobilized amylose derivative [51], Whelk-O1 [52], teicoplanin [53,54] selectors. Quinine immobilized on sub-3-micron superficially porous particle has been reported as an effective CSP capable to afford SFC separation with run times in the seconds time scale [55].

14.4.1 Preparative separations Chiral drugs are administered as single enantiomers or as racemic mixture, and the properties of every enantiomer should be studied independently as required by the regulatory authorities, so it is of high importance in pharmaceutical R&D to develop a method that allows to obtain the single isomers. Development of an asymmetric synthesis can be time consuming; on the contrary, the preparation of a racemic mixture and its resolution by chromatography shortens significantly the discovery times. Preparativescale-SFC using carbon dioxide-based eluents is nowadays extensively adopted in both academic and industrial environments to obtain enantiomers with high enantiomeric purity, bringing numerous advantages including high speed, low pressure drop, reduced solvent consumption and costs, greater safety concerning flammability and toxicity, reduced environmental impact, and fast solvent removal [56]. In chiral pSFC, the most common CSPs are those based on polysaccharide-derived selector adsorbed or immobilized on silica or Pirkle-type phases, because of the good selectivity and loadability, allowing increased amounts of racemate to be applied to a column and still achieve resolution. Other CSPs suitable for analytical SFC, like those based on cyclodextrins or on glycopeptide antibiotics, are not used on a preparative scale. 14.4.1.1  pSFC Versus pHPLC The principal difference between SFC and HPLC emerge from the properties of the mobile phase: supercritical fluid or liquid. In a preparative HPLC, hexane or heptane are used in mixture with solvents like isopropanol, methanol, acetonitrile, and methylene chloride that control both sample solubility and retention. In a preparative SFC, carbon dioxide constitutes the principal component of the mobile phase, generally above 60%, with the addition of the same polar solvents used in LC. The low viscosity and high diffusivity of the CO2-based mobile phase allows high flow rates,

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

623

thus resulting in greater chromatographic efficiencies and resolutions with shorter run times; in fact, SFC is 3–5 times faster than HPLC. Increasing the flow rates often results in higher productivity and this is of particular importance to achieve purified products for pharmaceutical testing in good yield and short times. Owing to the low viscosity of the supercritical CO2, columns packed with small-particle-size CSPs can be used, maintaining high efficiencies even at high flow rates [57].The high throughput in a preparative SFC permits the use of smaller size column compared to HPLC, thus reducing the cost of CSP material and column hardware. Another advantage of the preparative SFC versus preparative HPLC is the lower organic solvent usage. The lower solvent usage in the preparative SFC is achieved by replacing a majority of the mobile phase with CO2. Most of the mobile phase (60%–95%) is removed after chromatography by decreasing pressure, leaving only the modifier that consists usually of methanol, ethanol, or acetonitrile. The result is a reduction of the time required for post purification solvent removal and product isolation. Additionally, most of the solvents used in the preparative scale HPLC, especially in normal phase where alkanes are usually employed, are flammable. The consequence is that the preparative HPLC process must be performed in appropriate bunkers where the fire risk in minimized. Greater amounts of solvents are consumed in the preparative HPLC process compared to SFC, and usually the volume of organic solvents that can be stored in a standard laboratory are restrictive. The replacement of organic solvent with CO2 is desirable because it is nonflammable and nontoxic, so it offers less concern about safety and it is a renewable ecofriendly source, as it is generally recovered as a byproduct of manufacturing processes or condensed from the atmosphere, thus resulting in no net increase in CO2. Overall, organic solvent volumes for the preparative SFC are 2–10 times less than those used in the preparative HPLC allowing to reduce running costs considerably. However, a complete cost comparison between SFC and HPLC should consider additional aspects like initial equipment cost, maintenance time, cost of the operator, and elimination of the solvent after purification. Additionally, SFC is a new technology compared to HPLC and it still requires development because some parameters like pressure and temperature play a fundamental role in a preparative method development. Furthermore, the scale up from analytical to preparative scale that is straightforward in HPLC and is based on general rules, such as adjusting the volumetric flow and injection volume to the square of the ratio of the columns diameters, is more complicated in SFC. Indeed, an altered pressure drop along

624

Roberta Franzini et al.

the column, because of the high compressibility of the CO2-based mobile phase, will generate a density change which eventually will affect retention of the analytes and their separation. Some additional strategies, like control of the backpressure to hold up the same average density along the column, should be carried out to maintain the same chromatographic resolution and selectivity when transferring a method from the analytical to preparative scale in SFC compared to LC [58,59].

14.4.2 Application and strategy Early-stage toxicological studies require from 100 g up to few kilograms of the enantiomerically pure product, and SFC is a technique of choice for the purification in the pharmaceutical field, spacing in a range from small preparative up to larger enantioseparation scale. In the past years, successful separations of different amounts of chiral compounds have been presented in many publications. In 2008, Miller [60] reported the approach in separation of a racemate that were proprietary pharmaceutical compounds belonging to and synthesized at Amgen (Cambridge, MA, USA). In a recent publication, Leek et al. [15] reported a strategy to obtain a 5 kg separation of a compound using four CSP screening (three polysaccharide-based and Whelk-O1). When performing a large-scale separation, different aspects must be taken under consideration and one of the most important is the choice of the organic modifier in the mobile phase. The solvent is usually chosen on the basis of cost, availability, volatility, and environmental impact, but the most considerable feature is the solubility of the analyte. This is sometimes the limiting factor for preparative SFC, reducing the amount of racemate that can be injected. A method set up to afford greater separations may result unsuccessful and even causing precipitation of the compound in the column or eventually system shutdown. The solubility of the compound could change substantially in the CO2/modifier mixture compared to that in the modifier alone [16]. Eventually, the racemate must be dissolved in a solvent different from the pure modifier and depending on the injection technique, solubility issues can have undesirable effect on the chromatographic separation. Another useful strategy for increasing the productivity of the process is the “stacked injection” technique that is often applied during a preparativescale SFC [56].

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

625

With this technique, a second injection is performed before the elution of the second enantiomer of the compound is complete. The time between two subsequent injections is based on the time calculated from the beginning of the first peak and the end of the second eluted peak. Several examples of pSFC resolutions of drugs or drug-like molecules have been described by Francotte. The stacked injection technique was used to resolve a 50 g sample of chiral N-benzyl-3-methyl-piperydin-4-one on a 30 × 250 mm column packed with an adsorbed polysaccharide CSP. Using 5% ethanol in CO2 delivered at 120 mL/min and a cycle time of 1.8 min, the two enantiomers were obtained with greater than 99% enantiomeric excess, and the overall process had a productivity of 2.33 kg of racemate per day per kg of CSP [57]. Recently, an interesting paper reported a large-scale chiral SFC applications with fast run times and high loadings by using a chlorinated polysaccharide-CSP in both coated and immobilized versions [61]. In particular, the bonded version of the CSP permits the use of dichloromethane or acetonitrile as cosolvents gaining in sample solubility and loading ability for each injection and allowing the development of robust methods to resolve poorly alcohol-soluble racemates. Fig. 14.8 shows a chromatographic trace of a large-scale SFC separation of a racemate in short time (less than 6 min) and with high throughput (8.5 g/h). Additional examples highlighting the advantages of pSFC in a pharmaceutical research and development context are reported by Leek and Andersson [62]. With the availability of the immobilized version of the polysaccharides CSPs, the wider choice of solvent additives greatly improves the solute solubility in the CO2-based eluents, giving clear advantages in terms of productivity. In one example, the authors report a preparative isolation of the desired enantiomer of a chiral carboxylic acid on a 250 × 50 mm column packed with cellulose tris(3,5dichlorophenylcarbamate) immobilized on 5-micron silica particles, using 20% ethanol and 0.5% formic acid in CO2 as eluent at 12 MPa and 40 °C. Since the whole separation was completed in about 3 min, a total of 1.1 kg of the desired enantiomer was obtained in short time, with high yield and 99% enantiomeric excess. The pSFC process showed a chromatographic throughput of 5.7 kg racemate/kg CSP/day, with a solvent consumption of only 0.07 m3/kg racemate and the additional benefit of efficient solute recovery by evaporation of the mobile phase. Preparative separation of enantiomers by SFC has nowadays become the method of choice for milligrams to kilogram scale. Distinct advantages of pSFC are the high productivity,

626

Roberta Franzini et al.

the reduced consumption of organic solvent, and the easy recovery of the desired enantiomers [63].

14.5  CONCLUSIONS Enantiomer separation by SFC on packed columns is now a mature technique, thanks to the advancement in the theory behind the mechanism of retention and selectivity and to the development of improved CSPs. Parallel improvement in instrumentation quality and robustness has contributed to the acceptance of SFC as a reliable technique in the chiral separation field, complementary to HPLC and in some cases with superior performance. The unique properties of carbon dioxide-based eluents render chiral SFC the method of choice for ultrafast high-efficient separations on modern sub-2-micron CSPs and for preparative separations on a large scale.

REFERENCES [1] Darr, J. A.; Poliakoff, M. New Directions in Inorganic and Metal-Organic Coordination Chemistry in Supercritical Fluids. Chem. Rev. 1999, 99, 495–541. [2] Klesper, E.; Corwin, A. H.; Turner, D. A. High Pressure Gas Chromatography Above Critical Temperatures. J. Org. Chem. 1962, 27, 700–701. [3] Lesellier, E. Retention Mechanisms in Super/Subcritical Fluid Chromatography on Packed Columns. J. Chromatogr. A 2009, 1216, 1881–1890. [4] Perrenoud, A. G. G.; Veuthey, J. L.; Guillarme, D. Comparison of Ultra-high Performance Supercritical Fluid Chromatography and Ultra-high Performance Liquid Chromatography for the Analysis of Pharmaceutical Compounds. J. Chromatogr. A 2012, 1266, 158–167. [5] Mourier, P. A.; Eliot, E.; Caude, M. H.; Rosset, R. H.; Tambuté, A. G. Supercritical and Subcritical Fluid Chromatography on a Chiral Stationary Phase for the Resolution of Phosphine Oxide Enantiomers. Anal. Chem. 1985, 57, 2819–2823. [6] Gasparrini, F.; Misiti, D.; Villani, C. Direct Resolution in Sub- and Supercritical Fluid Chromatography on Packed Columns Containing Trans-1,2-diaminocyclohexane Derivatives as Selectors. Trends Anal. Chem. 1993, 12, 137–144. [7] Saito, M. History of Supercritical Fluid Chromatography: Instrumental Development. J. Biosci. Bioeng. 2013, 115, 590–599. [8] Taylor, L. T. Supercritical Fluid Chromatography for the 21st Century. J. Supercrit. Fluids 2009, 47, 566–573. [9] Mangelings, D.; Vander Heyden, Y. Chiral Separations in Sub- and Supercritical Fluid Chromatography. J. Sep. Sci. 2008, 31, 1252–1273. [10] Farrell, W. P.; Aurigemma, C. M.; Masters-Moore, D. F. Advances in High Throughput Supercritical Fluid Chromatography. J. Liq. Chrom. Relat. Tech. 2009, 32 (11–12), 1689–1710. [11] Lesellier, E.; West, C. The Many Faces of Packed Column Supercritical Fluid Chromatography—A Critical Review. J. Chromatogr. A 2015, 1382, 2–46. [12] Kalíková, K.; Šlechtová, T.;Vozka, J.; Tesarˇová, E. Supercritical Fluid Chromatography as a Tool for Enantioselective Separation: A Review. Anal. Chim. Acta 2014, 821, 1–33.

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

627

[13] Płotka, J. M.; Biziuk, M.; Morrison, C.; Namieśnik, J. Pharmaceutical and Forensic Drug Applications of Chiral Supercritical Fluid Chromatography. Trends Anal. Chem. 2014, 56, 74–89. [14] Desfontaine,V.; Guillarme, D.; Francotte, E.; Novákovác, L. Supercritical Fluid Chromatography in Pharmaceutical Analysis. J. Pharm. Biomed. Anal. 2015, 113, 56–71. [15] Leek, H.; Thunberg, L.; Jonson, A. C.; Öhleń, K.; Klarqvist, M. Strategy for Large-Scale Isolation of Enantiomers in Drug Discovery. Drug Discov.Today 2017, 22 (1), 133–139. [16] Miller, L. Preparative Enantioseparations Using Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 250–255. [17] Guiochon, G.; Tarafder, A. Fundamental Challenges and opportunities for preparative supercritical fluid chromatography. J. Chromatogr. A 2011, 1218, 1037–1114. [18] Rajendran, A. Design of Preparative–Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 227–249. [19] Vajda, P.; Guiochon, G. The Modeling of Overloaded Elution Band Profiles in Supercritical Fluid Chromatography. J. Chromatogr. A 2014, 1333, 116–123. [20] Shibala, T.; Mori, K.; Okamob, Y. Polysaccharide Phases. In Chiral Separation by HPLC; Krstulovic, A. M., Ed.; Ellis Horwood: New York, 1989; pp. 336–398. [21] Francotte, E. R. Enantioselective Chromatography as a Powerful Alternative for the Preparation of Drug Enantiomers. J. Chromatogr. A 2001, 906, 379–397. [22] Francotte, E.; Huynh, D. Immobilized Halogenophenylcarbamate Derivatives of Cellulose as Novel Stationary Phases for Enantioselective Drug Analysis. J. Pharm. Biomed. Anal. 2002, 27, 421–429. [23] Chankvetadze, B. Recent Developments on Polysaccharide-based Chiral Stationary Phases for Liquid-phase Separation of Enantiomers. J. Chromatogr. A 2012, 1269, 26–51. [24] West, C.; Guenegou, G.; Zhang, Y.; Morin-Allory, L. Insights into Chiral Recognition Mechanisms in Supercritical Fluid Chromatography. II. Factors Contributing to Enantiomer Separation on Tris-(3,5-dimethylphenylcarbamate) of Amylose and Cellulose Stationary Phases. J. Chromatogr. A 2011, 1218, 2033–2057. [25] Felix, G.; Berthod, A.; Piras, P.; Roussel, C. Part III Supercritical Fluid Separations. Sep. Purif. Rev. 2008, 37, 229–301. [26] West, C.; Zhang,Y.; Morin-Allory, L. Insights into Chiral Recognition Mechanisms in Supercritical Fluid Chromatography. I. Non-enantiospecific Interactions Contributing to the Retention on Tris-(3,5-dimethylphenylcarbamate) Amylose and Cellulose Stationary Phases. J. Chromatogr. A 2011, 1218, 2019–2032. [27] Geryk, R.; Kalíkova, K.; Schmid, M. G.; Tesarˇová, E. Enantioselective Separation of Biologically Active Basic Compounds in Ultra-performance Supercritical Fluid Chromatography. Anal. Chim. Acta 2016, 932, 98–105. [28] Khater, S.; Lozac’h, M. -A.; Adam, I.; Francotte, E.; West, C. Comparison of Liquid and Supercritical Fluid Chromatography Mobile Phases for Enantioselective Separations on Polysaccharide Stationary Phases. J. Chromatogr. A 2016, 1467, 463–472. [29] Regalado, E. L.; Welch, C. J. Pushing the Speed Limit in Enantioselective Supercritical Fluid Chromatography. J. Sep. Sci. 2015, 38, 2826–2832. [30] Regalado, E. L.; Helmy, R.; Green, M. D.; Welch, C. J. Chromatographic Resolution of Closely Related Species: Drug Metabolites and Analogs. J. Sep. Sci. 2014, 37, 1094–1102. [31] Schafer, W.; Chandrasekaran, T.; Pirzada, Z.; Zhang, C.; Gong, X.; Biba, M.; Regalado, E. L.;Welch, C. J. Improved Chiral SFC Screening for Analytical Method Development. Chirality 2013, 25, 799–804. [32] Pirkle, W. H.; Welch, C. J. An Improved Chiral Stationary Phase for the Chromatographic Separation of Underivatized Naproxen Enantiomers. J. Liq. Chromatogr. 1992, 15, 1947–1955. [33] Szczerba, T.; Wrezel, P. Effect of Varying Co-solvents in SFC Method Development on a Whelk-O1 Chiral Stationary phase. LCGC N. Am. 2008, 21.

628

Roberta Franzini et al.

[34] Byrne, N.; Hayes-Larson, E.; Liao, W. W.; Kraml, C. M. Analysis and Purification of Alcohol-Sensitive Chiral Compounds Using 2,2,2-Trifluoro Ethanol as a Modifier in Supercritical Fluid Chromatography. J. Chromatogr. B 2008, 875, 237–242. [35] Wang, C. L.; Zhang,Y. R. Effects of Column Backpressure on Supercritical Fluid Chromatography Separations of Enantiomers Using Binary Mobile Phases on 10 Chiral Stationary Phases. J. Chromatogr. A 2013, 1281, 127–134. [36] Lavison, G.; Thiébaut, D. Evaluation of a Ristocetin Bonded Stationary Phase for Subcritical Fluid Chromatography of Enantiomers. Chirality 2003, 15, 630–636. [37] Liu, Y.; Berthod, A.; Mitchell, C. R.; Xiao, T. L.; Zhang, B.; Armstrong, D. W. Super/ Subcritical Fluid Chromatography Chiral Separations with Macrocyclic Glycopeptide Stationary Phases. J. Chromatogr. A 2002, 978, 185–204. [38] Maier, N. M.; Nicoletti, L.; Lämmerhofer, M.; Lindner, W. Enantioselective Anion Exchangers Based on Cinchona Alkaloid-Derived Carbamates: Influence of C8/C9 Stereochemistry on Chiral Recognition. Chirality 1999, 11, 522–528. [39] Pell, R.; Lindner, W. Potential of Chiral Anion-Exchangers Operated in Various Subcritical Fluid Chromatography Modes for Resolution of Chiral Acids. J. Chromatogr. A 2012, 1245, 175–182. [40] Wolrab, D.; Kohout, M.; Boras, M.; Lindner, W. Strong Cation Exchange-type Chiral Stationary Phase for Enantioseparation of Chiral Amines in Subcritical Fluid Chromatography. J. Chromatogr. A 2013, 1289, 94–104. [41] Xiao,Y.; Ng, S.; Tan, T. T.Y.; Wang,Y. Recent Development of Cyclodextrin Chiral Stationary Phases and Their Applications in Chromatography. J. Chromatogr. A 2012, 1269, 52–69. [42] Wang, R. Q.; Ong, T. T.; Ng, S. C. Chemically Bonded Cationic β-Cyclodextrin Derivatives as Chiral Stationary Phases for Enantioseparation Applications. Tetrahedron Lett. 2012, 53, 2312–2315. [43] Wang, R. Q.; Ong, T. T.; Ng, S. C. Chemically Bonded Cationic (–)Cyclodextrin Derivatives and Their Application in Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1224, 97–103. [44] Guillarme, D.; Bonvin, G.; Badoud, F.; Schappler, J.; Rudaz, S.;Veuthey, J. -L. Fast Chiral Separation of Drugs Using Columns Packed with Sub-2 µm Particles and Ultra-high Pressure. Chirality 2010, 22, 320–330. [45] Kotoni, D.; Ciogli, A.; D’Acquarica, I.; Kocergin, J.; Szczerba, T.; Ritchie, H.; Villani, C.; Gasparrini, F. Enantioselective Ultra-high Performance Liquid Chromatography: A Comparative Study of Columns Based on the Whelk-O1 Selector. J. Chromatogr. A 2012, 1269, 226–241. [46] Grand-Guillaume Perrenoud, A.; Veuthey, J. -L.; Guillarme, D. The Use of Columns Packed with Sub-2 µm Particles in Supercritical Fluid Chromatography. Trends Anal. Chem. 2014, 63, 44–54. [47] Hamman, C.;Wong, M.; Aliagas, I.; Ortwine, D. F.; Pease, J.; Schmidt, D. E., Jr.;Victorino, J. The Evaluation of 25 Chiral Stationary Phases and the Utilization of Sub-2.0 µm Coated Polysaccharide Chiral Stationary Phases via Supercritical Fluid Chromatography. J. Chromatogr. A 2013, 1305, 310–319. [48] Hamman, C.; Wong, M.; Hayes, M.; Gibbons, P. A High Throughput Approach to Purifying Chiral Molecules Using 3-µm Analytical Chiral Stationary Phases via Supercritical Fluid Chromatography. J. Chromatogr. A 2011, 1218, 3529–3536. [49] Barhate, C. L.; Joyce, L. A.; Makarov, A. A.; Zawatzky, K.; Bernardoni, F.; Schafer, W. A.; Armstrong, D. W.; Welch, C. J.; Regalado, E. L. Ultrafast Chiral Separations for High Throughput Enantiopurity Analysis. Chem. Commun. 2017, 53, 509–512. [50] Sciascera, L.; Ismail, O.; Ciogli, A.; Kotoni, D.; Cavazzini, A.; Botta, L.; Szczerba, T.; Kocergin, J.;Villani, C.; Gasparrini, F. Expanding the Potential of Chiral Chromatography for High-throughput Screening of Large Compound Libraries by Means of Sub-2 µm

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

629

Whelk-O1 Stationary Phase in Supercritical Fluid Conditions. J. Chromatogr. A 2015, 1383, 160–168. [51] Berger, T. A. Preliminary Kinetic Evaluation of an Immobilized Polysaccharide Sub2µm Column Using a Low Dispersion Supercritical Fluid Chromatograph. J. Chromatogr. A 2017, 1510, 82–88. [52] Berger, T. A. Kinetic Performance of a 50 mm Long 1.8 µm Chiral Column in Supercritical Fluid Chromatography. J. Chromatogr. A 2016, 1459, 136–144. [53] Barhate, C. L.; Farooq Wahab, M.;Tognarelli, D. J.; Berger,T. A.; Armstrong, D.W. Instrumental Idiosyncrasies Affecting the Performance of Ultrafast Chiral and Achiral Sub/ Supercritical Fluid Chromatography. Anal. Chem. 2016, 88, 8664–8672. [54] Ismail, O. H.; Ciogli, A.;Villani, C.; De Martino, M.; Pierini, M.; Cavazzini, A.; Bell, D. S.; Gasparrini, F. Ultra-fast High-efficiency Enantioseparations by Means of a Teicoplaninbased Chiral Stationary Phase Made on Sub-2 µm Totally Porous Silica Particles of Narrow Size Distribution. J. Chromatogr. A 2016, 1427, 55–68. [55] Patel, D. C.; Breitbach, Z. S.;Yu, J. J.; Nguyen, K. A.; Armstrong, D. W. Quinine Bonded to Superficially Porous Particles for High-efficiency and Ultrafast Liquid and Supercritical Fluid Chromatography. Anal. Chim. Acta 2017, 963, 164–174. [56] Speybrouck, D. Preparative Supercritical Fluid Chromatography: A Powerful Tool for Chiral Separations. J. Chromatogr. A 2016, 1467, 33–55. [57] Francotte, E. Practical Advances in SFC for the Purification of Pharmaceutical Molecules. LC–GC Europe April 2016. [58] Tarafder, A. A Scaling Rule in Supercritical Fluid Chromatography. I. Theory for Isocratic Systems. J. Chromatogr. A 2014, 1362, 278–293. [59] Åsberg, D.; Enmark, M.; Samuelsson, J.; Fornstedt, T. Evaluation of Co-solvent Fraction, Pressure and Temperature Effects in Analytical and Preparative Supercritical Fluid Chromatography. J. Chromatogr. A 2014, 1374, 254–260. [60] Miller, L. Preparative Enantioseparations Using Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 250–255. [61] Wu, D. -R.; Yip, S. H.; Li, P.; Sun, D.; Mathur, A. From Analytical Methods to Largescale Chiral Supercritical Fluid Chromatography Using Chlorinated Chiral Stationary Phases. J. Chromatogr. A 2016, 1432, 122–131. [62] Leek, H.; Andersson, S. Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development. Molecules 2017, 22, 158–166. [63] Welch, C. J. The Use of Preparative Chiral Chromatography for Accessing Enantiopurity in Pharmaceutical Discovery and Development. Comprehensive Org. Synth. 2014, 9, 143–159 Second Edition.