Journal of Pharmaceutical and Biomedical Analysis Polysaccharide-based chiral stationary phases as efﬁcient tools for diastereo- and enantioseparation of natural and synthetic Cinchona alkaloid analogs

In this study, we present results obtained on the diastereo- and enantioseparation of some basic natural and synthetic Cinchona alkaloid analogs by applying liquid chromatographic (LC) and subcritical ﬂuid chromatographic (SFC) modalities on amylose and cellulose tris -(phenylcarbamate)-based stationary phases using n -hexane/alcohol/DEA or CO 2 /alcohol/DEA mobile phase systems. Seven chiral stationary phases in their immobilized form were employed to explore their stereoselectivity for a series of closely related group of analytes. The most important characteristics of LC and SFC systems were evaluated through the variation of the applied chromatographic conditions ( e.g ., the nature and content of the alco- hol modiﬁer, the concentration of additives, temperature). The columns Chiralpak IC and IG turned out to be the best in both LC and SFC modalities. Temperature-dependence study indicated enthalpy-controlled separation in most cases; however, separation controlled by entropy was also registered.


Introduction
Both natural and synthetic Cinchona alkaloids have a rather complex structural pattern with more than thirty representatives, among these quinine (QN), quinidine (QD), cinchonidine (CD), and cinchonine (CN) represent the main components [1]. In addition to the pharmaceutical relevance of the main alkaloids and derivatives thereof, they may also serve as catalysts in stereo-directed organic synthesis [2], and as chiral selectors in the course of the development of chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) [3]. Besides, quinine has a long tradition to be applied as a flavor component in bitter beverages. In Table 1 the chemical structures of natural and synthetic Cinchona alkaloids applied in this study are depicted. With the exception of racemic quinine, the other analyte pairs QN/QD, DHQN/DHQD, CD/CN, and epi-QN/epi-QD are diastereomers to each other, although often termed pseudo-enantiomers (Table 1). QN and QD as well as CD and CN are diastereoisomeric to each other which, in principle, eases their separation. Analytically, HPLC methods are often applied for this purpose using preferentially non-chiral stationary phases, as summarized earlier by McCalley [4]. Recently, capillary electrophoretic [5] and HPLC-based [1,6] methods have been described for the separation of the four major Cinchona alkaloids QN, QD, CD, and CN as well as dihydroquinine (DHQN) and dihydroquinidine (DHQD). Along this line, the quantitative determination of six major alkaloids implementing supercritical fluid chromatography (SFC) has recently also been reported [7].
In principle, it is not necessary to use CSPs to resolve the given four pairs of diastereoisomers (see Table 1). Nevertheless, it is still of interest to examine their stereoselective molecular recognition pattern in the context of the resolution of these analytes. However, for the resolution of enantiomeric analytes, the application of a CSP is mandatory. As we have had access to racemic quinine (rac. QN) produced through a novel, fully synthetic way [8], we had the opportunity to investigate the chromatographic resolution of rac. QN versus the diastereomeric pair QN/QD and of their epimers (9-epi-QN, 9-epi-QD). Therefore, the impact of the chiral environment of the diverse polysaccharide-type CSPs on the overall stereoselectivity parameters could be explored.  In an earlier paper Hoffmann et al. [9] studied the stereoselective resolution of QN/QD and CD/CN on a chiral cation exchanger followed by another recent investigation by Bajtai et al. [10] applying chiral zwitterionic CSPs. In terms of intermolecularly driven interaction mechanism between the chiral selector (SO) motifs of the CSPs and the chiral analytes (selectands, SAs), the ion exchangertype CSPs investigated previously and the polysaccharide-type CSPs examined in the present study (see Fig. S1) belong to structurally entirely different CSPs with respect to retention and stereoselectivity characteristics. In this study, we aimed to trace the specifics of the polysaccharide CSPs used in normal-phase liquid chromatography (NP LC) and in SFC modalities for the separation of a closely related group of Cinchona alkaloids (see Table 1).
Polysaccharide-based PS-CSPs usually display a great variety of enantio-, diastereo-, and chemoselectivity [11][12][13][14][15][16][17]. A variety of robust CSPs with very wide spectra of applications were obtained via the immobilization of substituted amylose-and cellulose-based selectors [14,[12][13][14][15][16][17]. However, no data were found in the literature for the separation of some natural and synthetic Cinchona alkaloids and their derivatives on PS-CSPs. The main objective of the present paper is to reveal some general tendencies of a set of prominent PS-CSPs (Fig. S1) for the diastereo-and enantioseparation of the set of chiral analytes (Table 1) under NP-LC and SFC conditions. The investigation of stereoselectivity criteria of SOs and SAs was in the focus of this study. More specifically, of the three isobaric pairs of stereoisomers, QN/QD and 9-epi-QN/9-epi-QD are diastereomeric to each other, characterized by the change of only the stereogenic centers of C-8 and C-9, while the 1S, 3R, and 4S stereogenic centers of the quinuclidine ring residue remain constant. For the truly racemic QN, all five stereogenic centers are opposite to each other. For the two diastereomeric DHQN/DHQD and CD/CN pairs, the diastereomeric behavior of the analytes is the same as for QN/QD. See Table 1 for more details.
Methodological and experimental factors, such as the nature and the concentration of the different modifiers (alcohol, water, acid or base) and mobile phase compositions in NP-LC and SFC, as well as the structure of PS-CSPs and SAs and the temperature were evaluated on retention, selectivity, and resolution of stereoisomers with special attention with respect to elution sequences.

Apparatus and chromatography
Three chromatographic systems were applied in this study. The first one was a Waters Breeze apparatus consisting of a 1525 binary pump, a 487 dual-channel absorbance detector, a 717 plus autosampler, and a column thermostat. For data collection Empower 2 data manager software (Waters Corporation, Milford, MA, USA) was applied. A Lauda Alpha RA8 thermostat (Lauda Dr. R. Wobser Gmbh, Lauda-Königshofen, Germany) was used to regulate column temperature.
The second liquid chromatographic system was from Shimadzu, and it contained a low-pressure quaternary pump (LC-20AD), a photodiode array detector (SPD-M20A), a Model 7125 injector with a 20-l loop (Rheodyne, Cotati, CA, USA), and LC Solution data acquisition system (Shimadzu Corporation, Tokyo Japan). All experiments in normal-phase mode (NP) were carried out under isocratic conditions at a flow rate of 0.6 mL min -1 and at a column temperature of 25 • C (if not otherwise stated).
The third device, a Waters Acquity Ultra Performance Convergence Chromatography TM (UPC 2 , Waters Corporation) system was applied for SFC studies with a binary solvent pump, an autosampler, a backpressure regulator, a column oven, and a photodiode array detector. An Empower 2 software was used to system control and data acquisition. In every case SFC was performed in isocratic mode at a flow rate of 2.0 mL min -1 and a column temperature of 40 • C (if not otherwise stated). The outlet pressure was set at 150 bar. The mobile phases applied in SFC consisted of liquid CO 2 and MeOH, EtOH, 1-PrOH or 2-PrOH in different ratios (v/v) containing different acid and base additives.
Stock solutions of the analytes were prepared by dissolving the solid Cinchona samples in MeOH or 2-PrOH in 1.0 mg mL -1 concentration and further diluted when necessary. An injection volume of 20 L was applied in LC and 7 L in SFC. In LC the dead-time of columns (t 0 ) was determined by injection of tri-t-butylbenzene, while in SFC mode the first negative signal by injecting MeOH was used. Analytes were detected by their UV absorption at 215-230 nm.

Effects of mobile phase composition in NP-LC and in SFC
In the case of PS-CSPs, the generally accepted recognition mechanism is based on the inclusion of chiral solutes into the chiral cavities of the polysaccharide-type selector driven by additional attractive forces such as H-bonding, dipole-dipole andinteractions. In addition, the role of steric "hindrance" for the given SAs to enter deep into the chiral groves should also be considered [11][12][13][14][15][16][17]. To regulate the overall chromatographic retention, the nature and concentration of an alcohol modifier in both normal phase LC (NP LC) [19] and SFC [20] are often varied. To explore the possible effects of the alcohol modifier in NP LC and SFC, two columns were selected: the cellulose-based Chiralpak IC column (as the most effective CSP in the screening process for the investigated conformationally restricted analytes) and the amylose-based Chiralpak IE column, both possessing the same carbamate modification (tris-3,5-dichlorophenylcarbamate moiety) of the two different polysaccharide backbones.
With variation of the nature of the alcohol for the studied analytes on the 7 CSPs in NP-LC modality, a relatively similar but slight increase in k 1 was registered in the EtOH<1-PrOH<2-PrOH sequence with the exception of epi-QN/epi-QD, which was retained more significantly (Fig. S2). The best stereoselectivity performances (higher ˛ and R S ) could generally be achieved with 2-PrOH on Chi-ralpak IC. In SFC modality on the same columns, slightly higher k 1 values were obtained on mobile phases containing MeOH and 2-PrOH compared to EtOH (especially for epi-QN/epi-QD). For ˛ and R S the variations observed were quite similar to those in NP-LC; namely, Chiralpak IE exhibited much less effectiveness than Chiralpak IC. As expected, under NP-LC and SFC conditions an increase of the apolar character of the alcohol, i.e., applying alcohols with a longer or branched chain usually resulted in an enhanced analyte retention, selectivity, and resolution, especially on Chiralpak IC (Fig. S2). It should be mentioned, that opposite behaviors had also been reported in the literature [21,22]. The change in enantioand diastereoselectivity resulting from changing the alcohol modifier was previously rationalized as a result of alteration of the steric environment of the chiral cavities by different alcohol modifiers due to intra-and inter-molecular solvation effects [22]. On the basis of these results, further experiments were carried out with the application of EtOH and 2-PrOH in NP-LC as well as MeOH and 2-PrOH in SFC conditions.
For a more thorough study of the effects of alcohol concentration on chromatographic parameters in NP LC, n-hexane/EtOH/DEA and n-hexane/2-PrOH/DEA (60/40/0.1-95/5/0.1 v/v/v) mobile phases were applied. Under SFC conditions, the MeOH and 2-PrOH content in liquid CO 2 was varied from 10 to 60 v% (with 20 mM DEA in all experiments). The amylose-and cellulose-based columns applied earlier with the same selector (Chiralpak IC and Chiralpak IE, see Fig. S1) were selected for this study. Regarding the retentive characteristics, a typical NP behavior was observed for both NP LC and SFC modalities: increasing the ratio of apolar n-hexane or CO 2 resulted in an increased k 1 , especially for the epi-QN/epi-QD pair ( Fig. 1). It is noteworthy, that with the increase of the mobile phase polarity, the strength of the H-bonds between the analytes and the selector decreases and the solubility of the analytes in the mobile phase increases [23].
On Chiralpak IE the stereoselectivity exhibited only a small enhancement in both NP-LC and SFC modalities with increasing n-hexane or CO 2 content. However, on Chiralpak IC in NP-LC in n-hexane/EtOH/DEA mobile phase a slight increase, whereas in nhexane/2-PrOH/DEA mobile phase a moderate enhancement in thę value was registered. In SFC on Chiralpak IC ˛ generally slightly decreased or did not change significantly with increasing CO 2 content. The epi-QN/epi-QD pair, again, was an exception showing a slight increase on Chiralpak IC in CO 2 /MeOH. It should be noted, that slightly higher ˛ values were registered in CO 2 /2-PrOH than in CO 2 /MeOH mobile phases.
Regarding R S values, in both NP-LC and SFC, they increased significantly on Chiralpak IC and slightly on Chiralpak IE with increasing of n-hexane or CO 2 content (although an unexpected exception was found in SFC modality for DHQN/DHQD on Chiralpak IE, Fig. 1). It is worth mentioning that the change in the chromatographic performance caused by the alcohol modifier depended on the structure of the chiral selector as well; the cellulosebased selector for the investigated basic analytes outperformed the amylose-based one.
The alcohol may be incorporated into the polysaccharide structure, either into the cavities or between the polymer chains, affecting the tertiary structure of the chiral polymer itself via solvation effects [24]. Under LC conditions, the main adsorbing sites are considered to be the polar carbamate residues [25] and the different involvement of the NH and CO groups in the H-bonding process were found to be responsible for the differences observed in the stereoselective and enantioselective binding process [26]. It is important to note, that in this study 10-60 v% of alcoholic modifier was employed under SFC conditions, i.e., SFC is operated under subcritical conditions, where significant deviations due to the difference of the set values and actual operational conditions cannot be expected [27]. Besides affecting the physical properties of the eluent (e.g., polarity, viscosity, and density), an alteration of the adsorption layer should also be considered under SFC conditions [27].
Besides the nature and concentration of the alcohol components, other additives (e.g., acid, base, water) are also frequently applied to modify "chiral" resolution on different types of CSPs. In SFC, beyond improvement in peak shape, additives may also have an impact on retention [28] and on the number of theoretical plates [29]. Upon increasing the concentration of the additive retention decreased in case of AcOH and slightly increased in case of water in parallel with a minor decrease of ą nd a marked reduction in R S values (Fig. 2), while the change in the efficiency was usually in the range of 10-15% (data not shown). The slight change caused by the addition of water was probably due to the less polar character of the CSP studied as observed by Armstrong et. al [29].

Effect of the structure of polysaccharide-type selectors
The stereoselectivity characteristics of the studied Cinchona alkaloids on the seven PS-CSPs were studied with different mobile phase systems, and the corresponding data are summarized in Tables 2-5. Due to the difference in linkage of glycopyranose moieties in amylose-and cellulose-based selectors, the interactions between analyte and selector may change resulting in different chromatographic behaviors, which may even affect the elution order of the resolved diastereomers and enantiomers. The contribution of the polysaccharide backbone can be evaluated by the comparison of the chromatographic data obtained on the same carbamate residue; i.e., tris-(3,5-dimethylphenylcarbamate) (Chiralpak IA vs. Chiralpak IB) and tris-(3,5-dichlorophenylcarbamate) (Chiralpak IE vs. Chiralpak IC), linked to amylose or cellulose, respectively. To facilitate the comparison between different columns, chromatographic conditions were kept constant, applying the n-hexane/EtOH/DEA and the n-hexane/2-PrOH/DEA (80/20/0.1 v/v/v) mobile phase compositions in NP-LC. In a few cases when a partial resolution occurred, the separation was further optimized. Data summarized in Table 2 show that higher retention could be obtained on amylose-than on cellulose-based CSPs in most cases. Exceptions, however, do exist especially for mobile phases containing 2-PrOH.
It should be noted, that in NP-LC, despite shorter retention times, Chiralpak IC proved to be the most effective CSP in the separation of the diastereomeric pairs of both Cinchona alkaloids and of rac. QN. In SFC modality the effect of the polysaccharide backbone was also evaluated on the same pairs of CSPs applying CO 2 /MeOH and CO 2 /2-PrOH (60/40 v/v) mobile phases all containing 20 mM DEA (Table 3). Regarding retention, the results with Chiralpak IE and IC were similar to the data obtained in NP-LC; i.e., the amylose backbone offered higher retentions. Chiralpak IA and IB, in turn, gave opposite results, with the cellulose-based CSP providing higher retentions. Among the above-mentioned four columns under SFC conditions, the best separations were achieved with Chiralpak IC, similar to those found in NP-LC.
The fundamental structural differences between amylose-and cellulose-based tris-(3,5-dimethylphenylcarbamate) or tris-(3,5dichlorophenylcarbamate) were found to be reflected in the stereochemical recognition patterns even for some diastereomeric analytes. Reversal of elution order between amylose-and cellulosebased CSPs containing the same substituents was registered in NP-LC containing EtOH as mobile phase for analyte CN/CD on Chiralpak IA vs. IB, and in mobile phases containing EtOH and 2-PrOH on Chiralpak IE vs. IC (Table 4). Similar behaviors were registered in SFC modality for QN/QD and CN/CD on Chiralpak IE vs. IC ( Table  2B). Examples of reversed elution orders of enantiomeric analytes on amylose-or cellulose-based columns have been described previously [19].
Applying constant chromatographic conditions in NP-LC and SFC modality, data listed in Table 2 and 3 can provide opportunity to evaluate the effect of the nature of the substituted phenylcarbamate moiety. By comparing the data obtained with Chiralpak IA vs. IE (both are amylose-based CSPs), and IB vs. IC (both are cellulose-based CSPs), higher retentions can clearly be identified for all analytes on CSPs with tris-(3,5-dichlorophenylcarbamate) moiety. The higher ˛ and R S values, observed generally in the case of CSPs with tris-(3,5-dichlorophenylcarbamate), suggest more pronounced SO-SA interactions of the studied analytes. This may be attributed to a --type interaction increment of the acidic phenylcarbamate and the -basic-type quinoline moiety of Cinchona alkaloids. However, in a few cases, in particular for epi-QN/epi-QD, lower ˛ and R S were registered on Chiralpak IE and Chiralpak IC than on Chiralpak IA and Chiralpak IB. Nevertheless, results showed that the tris-(3,5-dichlorophenylcarbamate) CSPs, in most cases, outperform other SO types. Table 2 Effect of EtOH and 2-PrOH content in n-hexane as bulk solvent for the chromatographic data, k1, ˛, R S of Cinchona alkaloids in normal phase modality.  As mentioned earlier, the structure of SO may affect the sequence of elution too. In this study, a reversal of elution sequence was registered in NP-LC modality in EtOH-containing mobile phases for analytes QN/QD using Chiralpak IA vs. IE (Table 4). The reversal of elution sequence by changing the chemical structure of substituents on the tris-(phenylcarbamate) moiety was already indicated in earlier publications [19,30,31].
The effect of the position of the substituents of the carbamate moiety of the PS-CSPs on the chromatographic performance for the given analytes was investigated by comparison of chromatographic data obtained on Chiralpak IF, IG, and ID columns in both NP-LC and SFC modalities (see Fig. 1S). In general, higher retention and better ˛ and R S values were obtained on Chiralpak IG than on IF.
Chiralpak ID seems to be as efficient as Chiralpak IG in NP-LC modality in the presence of EtOH as bulk solvent component, and less efficient in all other cases. The secondary structure of the tris-(3chloro-5-methylphenylcarbamate)-based CSP (Chiralpak IG) offers stronger retentive interactions with the analytes. Regarding elution sequences of QN/QD and rac. QN, no change was registered by altering these CSPs. In contrast, for epi-QN/epi-QD and CD/CN a reversal of elution sequence was registered in NP-LC in the presence of 2-PrOH as eluent constituent. The strong dependence of the sequence of elution as a function of the mobile phase (eluent composition and SO structure), observed in all cases, draws atten-tion to the importance of identification of each peak in the case of PS-CSPs. For chiral ion-exchangers investigated previously, this scattered tendency was not seen [10].
In the enantio-and diastereoseparation of the investigated Cinchona alkaloids in SFC modality, Chiralpak IC and IG columns performed significantly better. However, in NP-LC, the 3-chlorosubstitution in Chiralpak IC and ID promotes an increased stereodiscrimination but with lower effectiveness than in SFC modality.
The enantio-and diastereoselectivities of the seven chiral selectors for the five pairs of Cinchona alkaloid stereoisomers are summarized in Tables 4 and 5. For the separation of enantiomers, consistencies have been noticed numerous times with an elution sequence (+)QN < (-)QN, but a reversed elution sequence was registered on Chiralpak ID and IF in SFC modality applying 2-PrOH as alcohol modifier.
The situation becomes even more complicated for the resolution of the diastereomeric pairs QN/QD, DHQN/DHQD, CD/CN, and epi-QN/epi-QD, where a clear trend cannot be seen. In both NP-LC and SFC modalities on the seven columns, the elution sequence QN < QD, DHQN < DHQD, and CD < CN and its reversal can also be observed, as a clear indication of the difficulty to interpret enantioselectivity vs diastereoselectivity (Tables 2 and 3). Unexpected reversals of the elution order of the diastereomeric (often Table 3 Comparison of the effect of MeOH and 2-PrOH content in CO2 as bulk solvent for the chromatographic data, k1, ˛, R S of Cinchona alkaloids in SFC modality. termed pseudo-enantiomeric) pairs can easily happen as a function of the composition of the mobile phase. In this context, it became particularly interesting that on Chiralpak IG applying 2-PrOH as eluent component under LC conditions the elution orders (+)DHQD < (-)DHQN and CN < CD changed in SFC modality to (-)DHQN < (+)DHQD and CD < CN (Table 4 and 5). This is another strong indication about the role of solvation of the selector and selectand moieties on the overall diastereoselectivity. Representative chromatograms for the resolution of racemic quinine and four diastereomers of Cinchona alkaloids in SFC mode are depicted in Fig. 3 and Fig. S3 on Chiralpak IC and IG applying 2-PrOH and MeOH as alcohol modifiers. Full separation and identifications of five pairs of enantiomers and diastereomers can only be achieved in two chromatographic runs.

Effect of the structure of analyte
Analytes, except DHQN/DHQD, possess an unsaturated side chain (vinyl versus ethyl group) on the quinuclidine moiety, which may influence interactions between SA and CSP, despite small differences in size and polarity. Surveying the data in Tables 2 and 3 revealed, that, in general, at a given mobile phase composition retentions in NP LC or SFC modalities differ only very slightly. In some cases, however, the difference is more significant. As a result,    Column  IA  IB  IE  IC  ID  IF  In the present case, only the overall retention but not the stereochemical differentiation of the diastereomeric analytes is affected markedly under the applied chromatographic conditions.

Effect of temperature and thermodynamic parameters
The temperature dependence of retention and enantioselectivity may provide some valuable information on the chiral recognition process [31][32][33][34][35]. Keeping in mind the limitations of the approach applied in this study [33,[32][33][34][35], the difference in the change in standard enthalpy ( H • ) and entropy ( S • ) for the enantiomers were calculated on the basis of the van't Hoff equation: where R is the universal gas constant, T is temperature in Kelvin, and ˛ is the apparent selectivity factor. In order to investigate the effects of temperature on the chromatographic parameters, a variable temperature study was carried out for five analytes in LC-NP and SFC modalities on four PS-CSPs (Chiralpak IA, IB, IE, and IC) in the temperature range 7.5-50 • C in mobile phases based on n-hexane/2-PrOH/DEA (80/20/0.1 v/v/v) and CO 2 /MeOH (90/10, 80/20 and 70/30 v/v) all containing 20 mM DEA. The experimental data are summarized in Tables S1 and Table  S2.
In both modalities on all four columns, the retention in most cases decreased with increasing temperature. The transfer of the analyte from the mobile phase to the stationary phase, in general, is an exothermic process. As a result, k and ˛ as well as R S decrease with increasing temperature. However, regarding separation factor and resolution, several exceptions were observed. On Chiralpak IA for CD/CN in the temperature range 30-50 • C and on Chiralpak IC for DHQN/DHQD and CD/CN in the temperature range 7.5-30 • C, ˛ (and R S ) increased with increasing temperature (Table  S2). Interestingly, in SFC modality for epi-QN/epi-QD on all investigated CSPs, k decreased, but ˛ and R S increased with increasing temperature in the studied temperature range (Table S2). Applying PS-CSPs, several examples were reported previously for the increase of selectivity with increasing temperature in chromatographic systems [14,19,33,35]. It should be noted, that several times in both modalities, R S increased with increasing temperature independently of the change of ˛. This phenomenon can be attributed to the enhanced kinetic effect upon increasing temperature.
From the chromatographic data van't Hoff plots were constructed. As a general trend, ln ˛ vs. 1/T curves gave clearly linear van't Hoff plots, as indicated by the correlation coefficients listed in Table 6. The differences in the changes in standard enthalpy and entropy in NP-LC modality ranged between -4.5 to +2.3 kJ mol −1 and -12.3 to +8.1 J mol -1 K -1 , while in SFC modality between -5.4 to +5.5 kJ mol -1 and -14.1 to +18.8 J mol -1 K -1 . If k and ˛ decrease with increasing temperature, negative ( H • ) values for a pair of enantiomer or diastereomers accompanied by a negative ( S • ) provide information on the relative ease of transfer of analytes from the mobile to the stationary phase. A negative ( S • ) reflects an increase in the order/or loss in the degrees of freedom in the course of interaction between the stereoisomers and CSP, and the number of solvent molecules released from the chiral SO and the analyte when the molecule is associated with the CSP. If k decreased but į ncreased with increasing temperature, ( H • ) and ( S • ) were positive. In this case, the change in the adsorption enthalpy with increasing temperature has a positive effect on enantioselectivity. On the other hand, the positive ( S • ) compensated the positive ( H • ) and resulted in negative ( G • ). An exceptional behavior in NP-LC on Chiralpak IC for DHQN/DHQD and CD/CN pairs was registered (Fig. S4); namely, in the temperature range 7.5-30 • C, ˛ increased with increasing temperature, while above 30 • C d ecreased with increasing temperature. That is, the separation is governed in the lower temperature range by entropy, while in the higher temperature range by enthalpy.
To estimate the enthalpy/entropy contribution to the free energy, Q values [Q = ( H • )/[298 × ( S • )] were calculated. According to the data in Table 6, the discrimination process was enthalpically or entropically driven depending on both the nature of analyte and the chiral selector. Under SFC conditions on all investigated CSPs, epi-QN/epi-QD in SFC modality exhibited entropy-controlled separation, but no other general trend could be observed.

Conclusions
In this comprehensive study we have investigated the performance of a set of chiral polysaccharide-based stationary phases for the separation of some closely related natural and synthetic Cinchona alkaloid analogs. As evidenced by chromatographic data summarized in Tables 2-4 and, several characteristic features can be extracted. Consequently, it was of interest to investigate the impact of seven different polysaccharide-type CSPs for their chromatographic resolution. Specific conclusions are as follows: a) with respect to the effect of the nature of alcohol in NP-LC and SFC, the use of 2-PrOH and in some cases EtOH in NP-LC and MeOH in SFC were favored for this class of compounds; b) the "fitting" of the studied analytes to the amylose-or cellulosebased polymeric chain-type selectors characterized by the shape and size of the chiral groves may markedly depend on the different solvation effects of the alcohol components of the mobile phase; c) results showed that the tris-(3,5-dichlorophenylcarbamate)based CSPs (Chiralpak IC and Chiralpak IE) always outperform the tris-(3,5-dimethylphenylcarbamate)-based ones (Chiralpak IA and Chiralpak IB); d) the extremely high retentions of 9-epi-QN and 9-epi-QD compared to those of QN/QD observed in a few cases can be attributed to the change of the configuration of the C-9 atoms for the (-)epi-QN from (9R) to (9S) and for the (+)-epi-QD from (9S) to (9R) resulting in the full change in the overall steric configuration of the analytes; whereas it results in a stronger interaction with the selector sites of the CSP, it is not necessarily accompanied with an increased stereodifferentiation; e) overall, the enantio-and diastereoselectivity of these chiral columns for the stereochemically rather restricted Cinchona alkaloids was moderate, which hints to a restricted adoptive conformation (induced fit phenomenon) of the analytes towards the chiral selectors and vice versa; it should be mentioned that a further screening of alternative mobile phase compositions may reveal some additional effects.

Declaration of Competing Interest
The authors report no declarations of interest.