Anca-Elena Dascalu

David Speybrouck

Muriel Billamboz

David Corens

Alina Ghinet

Dr Emmanuelle Lipka
email: emmanuelle.lipka@univ-lille.fr

Analytical and preparative enantioseparations in supercritical fluid chromatography using different brands of immobilized cellulose tris (3,5-dichlorophenylcarbamate) columns: Some differences

Keywords: Cellulose tris (3,5-dichlorophenylcarbamate), Enantiomers, Loadability, Manufacturers, Supercritical fluid chromatography

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In pharmaceutical industry, the separation of enantiomers is an ever-growing field as enantiomers of the active substance can present different activities and toxicities, having a great impact on the processes of drug discovery and development. Consequently, in the last decades a strong endorsement could be observed, in the development of enantioselective syntheses and the enantioselective separation methods of new drugs.

The most important enantioseparation techniques are High-Performance Liquid Chromatography (HPLC) and Supercritical Fluid Chromatography (SFC), which has taken over the field. Chiral SFC has been providing for almost 30 years an efficient pathway to check the chiral purity of compounds, but also to provide enantiomerically pure materials for synthesis, active pharmaceutical ingredients and drugs [START_REF] Desfontaine | Supercritical fluid chromatography in pharmaceutical analysis[END_REF][START_REF] Zhao | Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry[END_REF][START_REF] Maftouh | Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery[END_REF][START_REF] Pirrone | Supercritical fluid chromatography-photodiode array detectionelectrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances[END_REF][START_REF] White | Integration of supercritical fluid chromatography into drug discovery as a routine support tool: part I: fast chiral screening and purification[END_REF].

The selection of an appropriate chiral stationary phase (CSP) represents an important step in the development of most chromatographic methods. There is a large range of CSPs, utilized for chiral resolution including polysaccharides. Polysaccharides (cellulose and amylose) derived chiral stationary phases have proved their usefulness as chiral selector in HPLC as well as in SFC, and a wide range of enantiomeric compounds have been successfully separated on these CSPs. These coated polysaccharide-based CSPs are the most popular choice firstly due to their large loadability (with saturation capacity ranged between 7.5 to more than 100 mg/g CSP) which is extremely important for preparative scale chromatography and secondly because this coated polysaccharides technology that run out of patent, has caused a revival of activity from many different manufacturers. Non-chlorinated polysaccharide-based CSPs have successfully resolved a vast majority of chiral compounds in a broad variety of structural classes [START_REF] Kalíková | Supercritical fluid chromatography as a tool for enantioselective separation; a review[END_REF][START_REF] Toribio | Applications of the Chiralpak AD and Chiralcel OD chiral columns in the enantiomeric separation of several dioxolane compounds by supercritical fluid chromatography[END_REF][START_REF] Bernal | Separation of antifungal chiral drugs by SFC and HPLC: a comparative study[END_REF], providing small and large quantities of enantiomerically pure active pharmaceutical ingredients for in-vivo and toxicological studies [START_REF] Ye | Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases[END_REF][START_REF] Wu | The impact of chiral supercritical chromatography in drug discovery: from analytical to multi gram scale[END_REF]. However, these phases have one major disadvantage: they are more or less soluble in many organic solvents (tetrahydrofuran or chlorinated solvents, amongst others) thus reducing the choice of organic modifier. However, drawbacks, such as the limited resistance of these coated phases, have been overcome by immobilizing the polysaccharide derivatives, thus allowing a greater choice of co-solvent. The immobilized versions of the existing coated phases are of the utmost importance for the preparative scale for two reasons: i) on the one hand, new co-solvents can improve resolution through improved efficiency, and ii) on the other hand, can in particular cases considerably improve the solubility of the sample, which in turn allows to increase the productivity [START_REF] Miller | Evaluation of non-traditional modifiers for analytical and preparative enantioseparations using supercritical fluid chromatography[END_REF][START_REF] Miller | Use of dichloromethane for preparative supercritical fluid chromatographic enantioseparations[END_REF][START_REF] Speybrouck | Productivity and solvent waste in supercritical fluid chromatography for preparative chiral separations: a guide for a convenient strategy[END_REF].

More recently introduced chlorinated polysaccharide CSPs (either coated or immobilized) can provide additional selectivity to the non-chlorinated CSPs. Chankvetadze's studies [START_REF] Chankvetadze | Chloromethylphenylcarbamate derivatives of cellulose as chiral stationary phases for high-performance liquid chromatography[END_REF][START_REF] Chankvetadze | Dimethyl-, dichloro-and chloromethylphenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography[END_REF][START_REF] Chankvetadze | High performance liquid chromatography enantioseparation of chiral pharmaceuticals using tris (chloro-methylphenylcarbamate)s of cellulose[END_REF] and Zhang's work [START_REF] Zhang | Cellulose tris (3,5-dichlorophenylcarbamate) immobilized on silica: A novel chiral stationary phase for resolution of enantiomers[END_REF] on the effect of various groups on the phenyl moiety of different carbamate CSPs, have shown a link between the structural properties and the separation performance of the CSPs. The inductive effect of the chlorine atoms, strong electron withdrawing groups (EWG), can enhance the tendency of the hydrogen atoms to form hydrogen bonds, which can modify the chemical properties of the CSP, and improve the chiral recognition. Since separation performance of a chromatographic column strongly depends on the properties of the stationary phase and therefore results from the manufacturing process, it is important to have reliable information for an adequate selection of the brand.

The aim of our study was a practical comparison of cellulose tris (3,5dichlorophenylcarbamate) columns purchased from four different manufacturers to evaluate their chromatographic performances. For this comparison, a set of different chromatographic parameters for different racemates were evaluated. In this study both commercially available and in-house synthesized compounds, containing one chiral center were used. Preparative chromatography has emerged as an asset in the synthesis of preclinical drug candidates and SFC has become a widely accepted technique for the small-scale preparation (hundreds of milligrams) of pure enantiomers of novel drug candidates needed for preclinical and early clinical studies [START_REF] Speybrouck | Preparative supercritical fluid chromatography: a powerful tool for chiral separations[END_REF]. Therefore, the loadability of each column was also studied, keeping in mind that this work is not intended to explain the differences observed because the manufacturing process is confidential and might be the same anyway, but rather to give practical information to users in order to save time in method development.

Material and methods

Chemicals

Methanol (MeOH), ethanol (EtOH), propan-2-ol (iPrOH) and acetonitrile (ACN) were of HPLC grade and were purchased from Carlo-Erba Reagents (Val-de-Reuil, France). Carbon dioxide (CO2) of 99.995% purity was purchased from Linde (Saint Priest, France).

Pharmaceutical compounds and triethylamine (TEA) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and compounds 21-29 (proprietary structure, Figure 1) were synthesized internally.

Sample solutions

For analytical screening, solutions of samples were prepared in methanol at 1 mg/mL. For the loading study, solutions of samples were prepared in methanol at 10 mg/mL (excepted 1 mg/mL for lenalidomide). The solutions were always degassed by an ultrasonic bath and filtered through a 0.45 µm PTFE syringe-filter (15 mm diameter) before use.

Supercritical Fluid Chromatography Instrument

Stationary phases

Four chiral analytical columns of cellulose tris (3,5-dichlorophenylcarbamate) from different suppliers were used for this study.

Chiralpak IC was purchased from Chiral Technologies Europe (Illkirch, France), Lux i-Cellulose-5 from Phenomenex ® (Le Pecq, France), CHIRAL ART Cellulose-SC from YMC Europe (Cluzeau Info Labo, Sainte-Foy-La-Grande, France) and the REFLECT I-Cellulose C from Regis Technologies (Thermo Fisher Scientific, Villebon-Courtaboeuf, France). All columns had dimensions of 250x4.6 mm with 5 µm particle size. The polysaccharide was immobilized on a silica-gel support.

Chromatographic system and conditions

The chromatographic system used was an SFC-PICLAB hybrid 10-20 apparatus (PIC Solution, Avignon, France) equipped with an autosampler comprising a 48-vials plate (model Alias, Emmen, Netherlands), three model 40 P pumps: two for CO2 and a third for the modifier (Knauer, Berlin, Germany), a column oven with a Valco ten-position column selection valve, and a Valco six-position solvent switching valve. The system was also composed of a Smartline 2600 diode array detector (DAD) (Knauer, Berlin, Germany).

Detection wavelength was set at 254 nm. The system was controlled, and the data was acquired with the SFC PicLab Analytic Online v.3.1.2 software (PIC Solution, Avignon, France). The volume injected was 20 µL. For the loading experiment, the injected volumes were 50, 100, 250, 485 µL, respectively. During the separation optimization, the used flow rate was 4 mL/min (excepted for acebutolol (cpd 19) for which the flow rate was 6 mL/min) and the mobile phase was always CO2-modifier mixture with 15% of methanol as modifier.

The exceptions being lenalidomide, compound 25, compound 26 and compound 27 for which the modifier percentage was 30% instead of 15%. For compounds 5, 11, 12, 15, 16, 17 and 18, 5% of methanol was also tested to avoid the co-elution with the solvent peak in order to be able to evaluate the selectivity. For acebutolol (cpd 19) and 1-(1-naphthyl)ethylamine (cpd 20), 0.3% of triethylamine (TEA) was added to the co-solvent. All analyses were run in isocratic mode. The column oven temperature was 40 °C and the outlet pressure was maintained at 150 bar for all the experiments. Retention times were average values of two replicate determinations. The data was processed by SFC New Data Manager V.1.8.0 software (Pic Solution, Avignon, France).

Results and discussion

In this work, four chiral stationary phases based on immobilized tris (3,5dichlorophenylcarbamate) derivative of cellulose, coming from four different manufacturers, were evaluated and cited along the manuscript as four columns for the sake of clarity. For all of them, the polysaccharide was immobilized to the surface of a silica matrix. This work notably investigated the practical impact of the different brands on retention, selectivity, efficiency, resolution and loading capacity. Figure 1 shows the selected compounds with a broad range of lipophilicity and polar surface area. All the chromatographic parameters are summarized in Table 1. The direct comparison of the chromatographic parameters was enabled by i) the supercritical conditions used (high percentage of co-solvent and high back pressure) permitting to limit the compressibility of the mobile phase, thus limiting density variation along the column, and therefore the polarity and ii) the similar dimensions of the four columns. The inlet pressure was noticed for each column leading to a pressure drop of 37 bar on Lux i-Cellulose-5, of 25 bar on Chiralpak IC, of 26 bar on REFLECT I-Cellulose C and of 24 bar on CHIRAL ART Cellulose-SC column (these values will be discussed later in the manuscript).

Comparison of retention factors

First of all, the graph displayed in Figure 2 showed that the retention factors of the second enantiomer eluted (k2) varied a lot according to the column supplier, e.g. the retention factor of omeprazole (compound 1) ranged from 15.9 on CHIRAL ART Cellulose-SC provided by YMC to 28. Remarkably, the most retentive column was Lux i-Cellulose-5 from Phenomenex for all the selected compounds (except for compounds 18 and 28) regardless of the nature of the analyte, neutral, basic or acid. In addition, the retentiveness of the four columns in decreasing order was always the same going from Lux i-Cellulose-5 (pink plots), Chiralpak IC (orange plots), REFLECT I-Cellulose C (blue plots) to CHIRAL ART Cellulose-SC (green plots). Meaning that the retention factors and retention times as well, are higher on the former column and lower on the latter one. This behavior is exemplified for acebutolol (compound 19) in Figure S1 (in Supplementary Information). In enantioselective chromatography, retention can result from either achiral interactions, or chiral interactions, or both. The main adsorbing site of the cellulose tris (3,5-dichlorophenylcarbamate) derivative CSP is the polar carbamate group which can interact with analytes through hydrogen bonds and dipole-dipole interactions [START_REF] Enomoto | Preparation of silica gel-bonded amylose through enzyme-catalyzed polymerization and chiral recognition ability of its phenylcarbamoyl derivative in HPLC[END_REF].

The hydrogen bonds are involved in both non-enantioselective and enantioselective interactions. For this series of compounds, the difference of retention of the second enantiomer eluted on the four different columns compared may be the consequence of a difference in achiral or chiral interactions. However, first, a similar trend was observed for the retention of the first enantiomer eluted of each racemate (k1) as for the second eluted enantiomer (k2). This means that a difference of retention for the same compound was observed between the four stationary phases whatever the enantiomers analyzed. Secondly, the graph on Figure 3, depicting the k2 versus the selectivity (α) for all the compounds, showed that there was no link between retention and selectivity. This observation allowed to conclude that the difference in retention capacity of the CSPs was more linked to achiral interactions than to chiral interactions and confirmed that retention was generally not correlated with enantio-resolution because strong interactions may occur between analytes and CSPs while these interactions do not necessarily contribute to the enantioselectivity. The difference of retentions between the four columns may be the result of the difference in manufacturing process used by the supplier. Indeed, Khater et al. [START_REF] Khater | Insights into chiral recognition mechanism in supercritical fluid chromatography IV. Chlorinated polysaccharide stationary phases[END_REF] have shown in an article dedicated to chlorinated polysaccharide stationary phases that the retention of the latter depended obviously on the nature of the polysaccharide and the type of derivatizing group attached to the glucose moieties of the polysaccharide, but also depended on the manufacturer. Indeed, two stationary phases with the same chiral selector can exhibit different retentions towards solutes. According to Khater et al., the difference linked to the manufacturers was probably due to deviations in the manufacturing procedure between the different suppliers. In our study, significant differences of retentions were observed between the four columns, while the polysaccharide and the side chains are the same for all the CSPs leading to the same conclusion about the manufacturers. One of the explanations could be linked to the process used to immobilize the polysaccharide on the support. Indeed, several methods were used to immobilize the polysaccharide derivatives on silica gel: (i) Okamoto et al. [START_REF] Okamoto | Polysaccharide derivatives for chromatographic separation of enantiomers[END_REF][START_REF] Okamoto | Chiral stationary phases for HPLC: cellulose tris (3,5-dimethylphenylcarbamate) and tris (3,5-dichlorophenylcarbamate) chemically bonded to silica gel[END_REF] developed a method using diphenyl diisocyanate as a cross linking agent and (ii) the covalent attachment of cellulose derivative via an epoxide moiety [START_REF] Chankvetadze | High-performance liquid chromatographic enantioseparations on monolithic silica columns containing a covalently attached 3,5-dimethylphenylcarbamate derivative of cellulose[END_REF]. Another possibility (iii) was the process developed by Francotte consisting in exposing the coated phases to photochemical [START_REF] Francotte | Photochemically cross-linked polysaccharide derivatives as supports for the chromatographic separation of enantiomers[END_REF] or thermal treatment. A last way (iv) which was a polymerization of a mixed 10-undecenoyl-3,5-dimethylphenylaminocarbonyl derivative of cellulose on the matrix was depicted by Minguillon et al. [START_REF] Minguillon | Bonded cellulose-derived highperformance liquid chromatography chiral stationary phases I. Influence of the degree of fixation on selectivity[END_REF]. The process of immobilization of the four columns evaluated in this work is confidential or patented. However, it is obvious that the immobilization procedure used by the different suppliers could vary with an impact on the quantity of chiral selector immobilized (carbon load percentage). In the same manner, the three-dimensional structure of the polysaccharide after immobilization can vary, resulting in some differences on the retention. In addition, as explained by Khater et al. [START_REF] Khater | Insights into chiral recognition mechanism in supercritical fluid chromatography III. Non-halogenated polysaccharide stationary phases[END_REF], the immobilization process led to the presence of residual hydroxyl groups on the polysaccharide and the percentage of hydroxyl groups may be different according to the CSPs with an impact on the retention. Finally, the nature of the silica gel may vary with a diversity of pore sizes, surface area, with the use of bare silica or aminopropyl silica or even different pre-treatment of the support leading to a diversity of physical properties of the stationary phases. All these specificities of the four columns resulted in different retention capacities. In conclusion, although the chiral selector was the same in all the four columns, a large difference in the ability to retain the compounds was observed.

Comparison of selectivity

Just like the retention factors were different on the columns for each of the tested compounds, the trend in selectivity was also diverse.

Firstly, unlike the retention factor, which varied a lot for this set of compounds, the difference of selectivity between each column towards a compound was quite low as exemplified in Figure S2 (in Supplementary Information) for compound 3 (lenalidomide) for which the selectivity values were equal to 2.44, 2.12, 2.39 and 2.31 (Table 1), respectively for Chiralpak IC, Lux i-Cellulose-5, CHIRAL ART Cellulose SC and REFLECT I-Cellulose C. Those four values of selectivity represented a relative standard deviation of 6.07% which was the highest calculated among the 29 compounds.

Secondly, the column providing the highest selectivity was not the same towards all the compounds. For the compound 20, the best separation factor was obtained within Chiralpak IC. The CHIRAL ART Cellulose-SC column provided the best selectivity for the compound 14, while the best separations of the compounds 4 and 25 were obtained on REFLECT I-Cellulose C and Lux i-Cellulose-5 respectively. Nevertheless, as illustrated in Figure S3a (in Supplementary Information), Chiralpak IC gave the best selectivity for 29.2% of the tested compounds and the highest number of compounds separated (α>1) was obtained on the CHIRAL ART Cellulose-SC and REFLECT I-Cellulose C columns with 58.7% of separation (figure S3b, in Supplementary Information). Remarkably, for eight racemates no separation was achieved on any of the four studied columns. The chiral recognition ability of the columns was different towards compounds 28 and 29. Indeed, the enantiomers of the compound 28 were separated both on CHIRAL ART Cellulose-SC and REFLECT I-Cellulose C, while no separation was observed on Chiralpak IC and Lux i-Cellulose 5 in the implemented conditions (Figure S4a, in Supplementary Information). Identically, for the compound 29, the enantiomers were not separated on Chiralpak IC while they were partially separated on the three other columns in the implemented conditions (Figure S4b, in Supplementary Information).

Regarding the fifteen compounds separated on the four columns, an average separation factor was calculated for each column to give a higher recognition ability for Chiralpak IC for those compounds. Hence, despite the same chiral selector, some differences were observed between these four different columns. Indeed, the conditions used to immobilize the chiral selector are probably different for the four columns with an impact on the chiral recognition. Like the coating conditions on silica gel significantly influence their chiral recognition ability, the process used to immobilize the polysaccharide may impact the chiral recognition too [START_REF] Okamoto | Chromatographic chiral resolution: XIV. Cellulose tribenzoate derivatives as chiral stationary phases for high-performance liquid chromatography[END_REF].

The utilization of different processes of immobilization may change the polymer configuration and/or the supramolecular structure of the polysaccharide. The high order structure of the polysaccharide is probably more or less maintained according to the manufacturer with an impact on the recognition ability. In conclusion, most of the time selectivity followed the same trend from one column to another, except for compounds 28 and 29.

Comparison of plate number and resolution

The efficiency and resolution values were reported in Table 1 (Supplementary Information).

For a fair comparison, only the 14 fully baseline resolved compounds were chosen. Mainly because efficiency can be wrongly evaluated for non-separated peak (whose peak width can be falsely increased by a beginning of separation of both enantiomers).

The plate number was slightly different between the four columns for every compound.

However, these discrepancies were not deeply studied and no conclusion was drawn from this Among the 29 compounds studied in this work, high resolutions were achieved on the four columns for compounds 1 (omeprazole); 2 (thalidomide); 4 (warfarin); 14 (tropicamide); 20

(1-(1-Naphthyl)ethylamine)) and in-house compounds 21 and 22, under 15% MeOH and for compounds 3 (lenalidomide) and in-house compound 26 under 30% MeOH, with various retention times.

Comparison of the loading capacity

The results displayed in the three previous sections showed a significant difference of retention, differences of resolution and slight difference of selectivity between the four columns for the tested compounds. In addition to retention, selectivity and resolution, loading capacity can be evaluated because it is a significant parameter in preparative chromatography. Indeed, the loading capacity of the stationary phase will have an impact on the productivity of a separation [START_REF] Speybrouck | Productivity and solvent waste in supercritical fluid chromatography for preparative chiral separations: a guide for a convenient strategy[END_REF]. Thus, the impact of increasing the amount injected on the four columns was assessed. Two methods can be used to overload a sample on a column: volume overload or mass overload. For volume overload, a feed solution was prepared by dissolving the compound with a defined concentration and then increasing volumes of the solution were injected until the peaks touched at the base (touching band) before resolution will be lost. Sometimes poor peak shape was obtained under high volume of injection. For mass overload, the sample volume was kept constant while the mass of compounds dissolved in this volume was increased progressively. Usually volume overload is used in preparative chiral separation in discovery because the method development is faster, explaining why this process was chosen in this work. Five compounds were selected, based on i) their high-resolution result under 15% MeOH as co-solvent for all of them (excepted 30% for lenalidomide) and ii) on shorter retention times (when similar resolution values were observed). Therefore omeprazole, warfarin, lenalidomide, tropicamide and the in-house compound 22 were selected.

To illustrate this loading capacity linked to the baseline return (or touching band), the following representations were chosen: i) overloaded chromatograms and ii) the plot of the separation criterion (S) in function of the amount injected.

Firstly, considering the overloaded chromatograms, they were represented for the in-house compound 22 (Figure 5) and for omeprazole (Figure 6). As shown in Figures 5 and6, increasing volume of feed solution (10 mg/mL) impacted the peak shape. This modification of the peak shape may lead to a reduction of the resolution with, for some compounds, the observation of one broad peak instead of two for the larger injected volume. For the in-house compound 22, a loss of baseline separation was observed for 2.50 mg injected (250µL of feed solution) on the four columns. Remarkably, for omeprazole, the retention times at the peak ends were constant whatever the injection volume, and the retention times at the beginning of the peak decreased in proportion to the injection volume. For the analysis of omeprazole (Figure 6), a baseline resolution was observed for 4.85 mg injected (485µL) on Chiralpak IC while a slight overlap of the peaks was observed for the three additional columns. However, the phenomenon was more the consequence of a lower selectivity on the three columns compared to Chiralpak IC and a lower retention on the CHIRAL ART Cellulose-SC column than a question of loadability since any significant difference in the peak shapes was observed.

As the resolution is difficult to determine for broad and asymmetric peak shape, the measurement of the separation criterion (S) was also used to monitor the quality of the separation of two successive peaks according to the injected amount [START_REF] Noireau | Purification of drug degradation products supported by analytical and preparative supercritical fluid chromatography[END_REF]. S is defined as the difference between the retention time at the beginning of the second peak (tB2) and the retention time at the end of the first peak (tE1); peaks are baseline resolved if S is positive: S= tB2-tE1. The graphs for omeprazole and in-house compound 22 on Figure 7 (and for lenalidomide, tropicamide and warfarin on Figure S5, in Supplementary Information) showed the same trend for all the compounds regardless column brand. The increase in injected volume caused a reduction of the separation criterion with no significant difference observed between the four columns.

The loading capacity of a stationary phase towards a compound strongly depends on the selectivity (α) but is also impacted by the efficiency of the column and the retention factor. In other words, it also depends on the resolution. In addition, the loadability of a stationary phase is a function of the number of accessible interaction sites per mass unit of phase, so is dependent of the density of chiral selectors or carbon load [START_REF] Francotte | Chiral stationary phases for preparative enantioselective chromatography[END_REF]. The behavior of the four columns towards increasing injected amount didn't differ substantially, leading to the conclusion that the four columns have quite similar density of chiral selectors.

Conclusion

The comparison of the different immobilized cellulose tris (3,5-dichlorophenylcarbamate) columns i.e. Chiralpak IC, Lux i-Cellulose 5, CHIRAL ART Cellulose-SC and REFLECT I-Cellulose C, by use of 29 representative racemic mixtures showed some differences. Indeed, the retention factors were significantly different with a higher retention on the Lux i-Cellulose 5 and with the lowest retention on Chiral ART Cellulose-SC for all compounds (excepted compounds 18 and 28, see Figures 2 andS4a). The best selectivity of each of the four columns depended on the nature of the compounds but differences between selectivity values were minor. Besides, the Chiralpak IC column showed the highest rate of separation. Concerning the resolution parameter, once again, the best resolution of each of the four columns depended on the compounds but differences between resolution were quite significant for compounds 1 (omeprazole), 2 (thalidomide), 3 (lenalidomide) and in-house 27 with standard relative deviation comprised between 11% and 20%. Finally, for the loading capacity, no strong difference was registered between the different columns, thus for smallscale separation each of them can be chosen. This study showed that despite the same chiral selector, the four stationary phases from different manufacturers were not identical probably due to differences in the manufacturing process such as the immobilisation of the polysaccharide that we do not intend to further explain here, as these processes are confidential. From a practical point of view, if one needs high retention capacity from its column, then choose a Lux i-Cellulose-5, on the contrary if your compounds elute too late, one should opt for CHIRAL ART SC-cellulose. If one has an important amount of racemates to separate then the Chiralpak IC seems the most appropriate column and will certainly lead to a high success rate in terms of selectivity.

The comparison of the chromatographic parameters obtained on the four immobilized columns with those obtained on the coated version of this chiral selector would have been interesting to determine the impact of the immobilization. But to be realistic, the coated and the immobilized columns should be provided by the same manufacturer. In addition, as explained by S. Carradori, this coated CSP (named as Sepapak-5 from Sepaserve GmbH affiliated to Phenomenex) is no longer commercially available, due to high solubility of this polysaccharide in mobile phase consisted of n-hexane/iPrOH mixture [START_REF] Carradori | A chromatographic study on the exceptional chiral recognition of 2-(benzylsulfinyl)benzamide by an immobilized-type chiral stationary phase based on cellulose tris(3,5-dichlorophenylcarbamate)[END_REF].

Figure Captions

  3 on Lux i-Cellulose-5 provided by Phenomenex with intermediate values for both Chiralpak IC and REFLECT I-Cellulose C from Daicel and Regis, respectively.

Figure 1 :

 1 Figure 1: Molecular structures, names and numbers of the analyzed compounds within the SFC study.

Figure 2 :

 2 Figure 2: Retention factor for the 29 compounds with the four CSPs. • acidic compounds, neutral compounds, ▲ basic compounds.

  489

Figure 3 :

 3 Figure 3: Selectivity vs retention factor for the 29 compounds on the four stationary phases.

Figure 4 :

 4 Figure 4: Resolution for the 29 compounds with the four CSPs. • acidic compounds, neutral compounds, ▲ basic compounds.

Figure 5 :

 5 Figure 5: Chromatograms of the analysis of the in-house compound 22 on the four columns (Chiralpak IC, Lux i-Cellulose 5, CHIRAL ART Cellulose-SC and REFLECT I-Cellulose C). Conditions: mobile phase: CO2/MeOH 85/15; flow rate: 4 mL/min; temperature: 40 °C; outlet pressure: 150 bar; sample concentration: 10 mg/mL; sample volume: from 20 µL to 250 µL; λ= 254 nm.

Figure 6 :

 6 Figure 6: Chromatograms of the analysis of omeprazole on the four columns (Chiralpak IC, Lux i-Cellulose 5, CHIRAL ART Cellulose-SC and REFLECT I-Cellulose C). Conditions: mobile phase: CO2/MeOH 85/15; flow rate: 4 mL/min; temperature: 40 °C; outlet pressure: 150 bars; sample concentration: 10 mg/mL; volume injected: from 20 µL to 485 µL; λ= 254 nm.

Figure 7 :

 7 Figure 7: Separation criterion (S) vs injected amount on the four CSPs (Chiralpak IC, Lux i-Cellulose 5, CHIRAL ART Cellulose SC and REFLECT I-Cellulose C) for omeprazole (10 mg/mL from 20 µL to 485 µL) and in-house compound 22 (10 mg/mL from 20 µL to 250 µL).

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 4 Figure 5

 45 Figure 4

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

observation because the inlet pressure measured for every column was not the same (as depicted before) resulting in a difference of diffusion coefficient in the mobile phase with a potential impact on the efficiency.

Like the selectivity, the difference of resolution between each column towards a compound depended on the compound itself as explained by Figure 4. Some discrepancies were observed as the relative standard deviation were calculated for the four resolution values obtained on the four columns for each compound. For instance, the difference between the four columns was equal to 6.24% for compound 7 (indoprofen) which was quite low, meaning that the four results obtained on different manufacturer columns were similar. But for some compound this difference reached 17.56% for compound 1 (omeprazole) with resolution values equal to 10.27; 6.64; 9.01 and 9.39 and even 19.78% for compound 2 (thalidomide) with values equal to 4.39; 2.82; 4.52; 3.89 which was significant. For each compound, the four columns were classified either first or second position (out of four) depending on the resolution obtained. Among the fourteen compounds, the Chiralpak IC appeared seven times in first position and four times in second position, allowing the best resolution for eleven compounds out of fourteen. This column was followed by the CHIRAL ART Cellulose SC column with four first positions and three second positions. But with watching the Figure 4 and Table 1, there were no drastic differences between columns, at two exceptions. For compound 28 a full resolution occurred for two columns (CHIRAL ART Cellulose SC and REFLECT I-Cellulose C) while no enantiomeric separation was observed nor on Lux i-Cellulose-5 neither on Chiralpak IC in the implemented conditions (Figure S4, in Supplementary Information). And for compound 29 for which the enantiomers were not separated on Chiralpak IC while they were partially separated on the three other columns in the implemented conditions (Figure S4b, in Supplementary Information).