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Effect of mobile phase amine additives on enantio selectivity for phenylalanine analogs
Abstract
The use of basic mobile phase additives enhances the chiral separation of underivatized phenylalanine analogs on a common amylosic column. These additives appear to exert their effect through differential disruption of hydrogen binding involved in the recognition process. Several examples of amine increasing retention of the second eluting enantiomer while decreasing retention of the other enantiomer were observed. This gave dramatically increased selectivity and was most commonly observed with cyclopropylamine and cyclobutylamine. The effect was attributed to steric factors involved in the elution process.
... The effect of acidic and basic mobile phase additives has been investigated intensively on coated polysaccharide-based CSPs, with significant impacts on retention and selectivity being observed [3][4][5][6][7][8][9][10][11][12][13][14]. All the published additive investigations were performed using mainly hexane, ethanol and/or isopropanol. ...
... The key roles that bulk mobile phases played and solvent effects on the enantioseparations of a broad variety of chiral compounds on CHIRALPAK IA column were reported previously [15]. Previous work on coated CSPs has shown that ethanesulfonic acid (ESA), an acidic additive, can have a significant impact on chiral amine enantioseparation using conventional normal phase solvents such as hexane with either ethanol or isopropanol [3][4][5][6][7][8][9][10]. In some cases, more than ten times improvement in selectivity improvement was observed with ESA addition [5]. ...
... ESA was selected as the acidic additive for this study based on previous reports demonstrating a significant selectivity effect on chiral separations [3][4][5][6][7][8][9][10]. The experiments were carried out with mobile phases consisting of different volume percentages of ethanol, hexane, MTBE, EtOAc, THF, acetone, 1,4-dioxane and ACN. ...
The ability to tune chiral selectivity through mobile phase modifiers is a powerful tool in chiral separations. Beyond improving efficiency and/or resolution, some mobile phase systems can even invert elution order, a highly desirable result for trace analyses or preparative scale isolations. Previous work has demonstrated that acidic modifiers, such as ethanesulfonic acid (ESA), can greatly impact separations of enantiomers. However, prior studies were primarily performed on coated chiral stationary phases (CSPs), which limited the selection of the bulk mobile phase component.
In this work, the effect of ESA modifier was studied for the enantioseparation of six pairs of amino acid esters on a CHIRALPAK® IA column, an immobilized amylose-based CSP, with different combinations of standard solvents (hexane and ethanol) as well as “non-standard” solvents, such as methyl t-butyl ether, ethyl acetate, tetrahydrofuran, acetone, or 1,4-dioxane. ESA generally improved selectivity, and multiple instances of elution order reversal were observed. A Van Deemter plot study reveals that ESA exerts its effect by pulling the enantiomer deeper into the chiral cavity of the chiral polymer to increase the interactions between the analytes and the stationary phase, which is the main reason for the increased enantioselectivity.
... The masking action of the underivatized silanols as well as of other out-of-the-stereoselective binding site functionalities usually contributes to the reduction of band broadening and peak asymmetry rather than to overall retention (Ye and Stringham, 2001a; Fulco et al., 2009). However, a decreased retention can be observed for basic analytes when chromatographed in the presence of an amine additive into the eluent (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). This behavior was explained by assuming competition for binding opportunities between the basic additive and the analyte (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). ...
... However, a decreased retention can be observed for basic analytes when chromatographed in the presence of an amine additive into the eluent (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). This behavior was explained by assuming competition for binding opportunities between the basic additive and the analyte (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). Indeed, amine additives are thought to disrupt particular H-bond contacts involved in the analyte binding (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). ...
... This behavior was explained by assuming competition for binding opportunities between the basic additive and the analyte (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). Indeed, amine additives are thought to disrupt particular H-bond contacts involved in the analyte binding (Ye and Stringham, 2001b; Ye et al., 2004a Ye et al., , 2004b). When this retention behavior goes along with a decreased or unchanged enantioselectivity, a disruption of H-bond contacts external to carbamate cleft can be realistically hypothesized ( Stringham, 2001a, 2001b). ...
Mobile phase variables have a deep influence on the chromatographic behavior with polysaccharide-based chiral stationary phases. Basic additives are generally used to minimize peak broadening arising from unwanted interactions between polar solutes and underivatized silanols. However, basic additives can improve enantioselectivity through disruption of hydrogen bonds and modification of the polymer morphology. Acidic additives are incorporated into the mobile phase during the analysis of acidic compounds as efficiency enhancers. Acidic additives can also improve enantioselectivity by minimizing within the chiral recognition site nonenantioselective retention. Peak shape without acidic additive in the eluent could be severely distorted during the analysis of salified compounds. Concentration and type of alcohol modifier can have an effect on the morphology of the polymer. The different winding of the chiral selector, caused by alcohol modifiers of different size/shape, ultimately results in different stereo environment of the chiral cavities in the polymer chain. Trace amounts of water in normal-phase eluents can affect retention time, tailing, and resolution. Deliberate addition of water to the eluent can improve peak resolution and save analysis time and solvent needs. Immobilized-type polysaccharide-derived chiral stationary phases offer new selectivity profiles and often improved enantioselectivity. Copyright © 2013 John Wiley & Sons, Ltd.
... The aromatic moieties of the chiral molecule may undergo π-π interactions with the aromatic binding sites of the polymer which are located on the exterior of the strand. 318 The discrimination is affected by the steric fit in the cavity. Changing from cellulose (1,4-β-linkage of glucose) to amylose (1,4-α-linkage of glucose) might lead to an inversion of the elution order of the enantiomers. ...
... Excellent reviews about the applicability of these CSPs have been published. [316][317][318] Cellulose and amylose derivative CSPs are mostly used, in the normal-phase mode, with n-hexane-based mobile phases containing some alcohol as modifier. Chromatographic performances, retention and selectivity, are reported to be affected by the composition of the mobile phase and the type of the alcohol present in the mobile phase. ...
... 301 Important enhancements in selectivity and resolution by addition of the additives are reported in the literature. [300][301][302][316][317][318] Inversion in the elution order of the enantiomers may occur from one column to another or when changing the type of organic modifier used. 45,319 In the reversed-phase mode, mixtures of aqueous buffer and acetonitrile are commonly used as mobile phase. ...
The chiral separation of drug molecules and of their precursors, in the case of the synthesis of enantiomerically pure drugs, is one of the important application areas of HPLC in pharmaceutical analysis. Besides HPLC, capillary electrophoresis is another technique of choice for chiral separations. In this chapter we give an overview of the different modes (e.g., direct and indirect ones) by which it is possible to obtain a chiral separation in HPLC and CE. The direct approaches, i.e., those where the compound of interest is not derivatized prior to separation, are discussed in more detail since they are the most frequently used approaches nowadays. The latter approaches require the use of so-called chiral selectors to enable enantioselective recognition and enantiomeric separation. Many different molecules have been used as chiral selectors, both in HPLC and CE. They can be classified into three selectors, and iii) macromolecular selectors. The different classes are discussed, including their subclasses, while examples of chiral separations obtained with them are given. Finally, some practical guidelines about screening conditions to test the enantioselectivity of a given compound on a limited number of chromatographic systems, and about method optimization when an initial separation has already been obtained, are given briefly.
... Therefore, the selection of a restricted number of stationary phases showing a very broad application area is required, and the polysaccharide phases OD, AD and OJ were preferred. This selection is based on our own experience [8,9] and on many published data [29][30][31][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69]. Such selection of columns and definition of strategies also involves that for individual compounds better separations might be able on, for instance, the columns mentioned higher which were not considered. ...
... In this paper, the screening and optimisation strategies (stages 1 and 2) for the two liquid chromatography techniques, NPLC and RPLC, are described. The strategies were developed based on the results obtained from our own laboratory experiences [8,9] and on literature data [29][30][31][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69]. Finally, the strategies are illustrated with several examples. ...
... The three other polysaccharide columns (Chiralpak ® AD-H, Chiralcel ® OJ-H and Chiralcel ® OD-H) are evaluated using two modifiers. Isopropanol and ethanol are the most commonly used modifiers on the Chiralcel ® OD-H, Chiralcel ® OJ and Chiralpak ® AD columns and, according to the literature [28][29][30][31][32][33][34][56][57][58][59][60][61][62][63][64] and our own experience [8,9], allow the separation of most drug enantiomers. Two modifiers are screened because it is difficult to predict the optimal organic modifier for a given column and its effect also depends on the analyte [8,56]. ...
This paper proposes strategies in normal- and reversed-phase liquid chromatography (NP-HPLC or NPLC and RP-HPLC or RPLC), which were developed using three polysaccharide-based stationary phases. Those strategies are implemented in a knowledge-based system for the chiral separation of drug enantiomers. Each strategy includes a screening and an optimisation stage. The screening stage allows a fast evaluation of separation possibilities and enantioselectivity for many drugs in a short period of time, while the optimisation stage gives the opportunity to enhance, if needed, the initially obtained separation. Different examples demonstrate the effectiveness of the strategies for fast method development.
... Although a number of achiral stationary phases have been developed that minimize this problem, such advances have not yet been applied to CSPs. Instead, low concentrations of very polar additives [33][34][35][36][37][38][39][40][41][42][43][44] are mixed with the modifier. These additives include formic acid, acetic acid, trifluoroacetic acid, isopropyl amine, dimethylethyl amine, triethylamine (TEA), ion pairing agents, quaternary ammonium salts, sulfonic acids, etc. ...
Supercritical fluid chromatography (SFC) is becoming the technique of choice for both analytical and semi-preparative chiral separations, particularly in the pharmaceutical industry. The mobile phase consists of CO2 modified with relatively small amounts of a polar organic solvent and sometimes a very polar additive. Since the CO2 is a gas under atmospheric conditions, the intra-molecular forces are low, the viscosity is low, and the solute diffusion coefficients are high. As a result SFC is 3–5 times faster with 1/3rd to 1/5th the pressure drops in HPLC. SFC is sensitive enough for enantiomeric excess determinations. SFC has been validated for quantitation of trace contaminants in a drug formulation using sub-2 μm particle columns with gradient elution, in an intra-lab study involving 3 SFC vendors.
SFC is used for both serial and parallel chiral method development to choose the best combination of chiral stationary phase and mobile phase. Since most of the mobile phase evaporates at the end of the system, semi-preparative fractions are very small, consisting of relatively high concentrations of solute in the organic modifier. The CO2 is non-flammable, very inexpensive, has been recycled, and is considered “green”.
... Several authors have demonstrated that the addition of acidic and basic additives can also have a significant impact on peak shape [27,[33][34][35][36] . The effect of the type of additive when MeOH is used as eluent was studied replacing DEA with EDA, TEA, MEA and a disproportioned DEA/TFA mixture. ...
In this study, we describe the experimental variables influencing enantioseparation of twelve β-blockers when analyzed under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions on a column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase. Regarding polar-organic mode, two component mobile phases consisting of methanol, ethanol or acetonitrile with the addition of basic additives such as diethylamine, triethylamine, mono-ethanolamine, ethylendiamine or trifluoroacetic acid/diethylamine mixture were evaluated. Studies of retention at different temperatures were also performed. In reversed-phase mode, mixtures consisting of methanol or acetonitrile with either aqueous boric acid-borate buffer or sodium hydrogen carbonate-carbonate buffer solutions were compared aiming to study the influence of organic modifier as well as buffer type and pH on resolution. In addition, a systematic evaluation of the retention factors of β-blockers enantiomers in hydro-organic eluents containing acetonitrile in presence of diethylamine as additive was carried out by increasing progressively the water content, in order to check for retention dependencies indicative of the interplay of both hydrophilic interaction liquid chromatography and reversed-phase modes.
... Some neutral polar compounds can also be used for this purpose. Such additives improve peak shapes [183][184][185] and in SFC [76,186,187]. Tang [183] and Stringham [188] have reported on the improvement in the enantioseparation of chiral amines obtained with strong acid additives, TFA [183] and alkylsulfonic acids [188], in the mobile phase. ...
... Some neutral polar compounds can also be used for this purpose. Such additives improve peak shapes [183][184][185] and in SFC [76,186,187]. Tang [183] and Stringham [188] have reported on the improvement in the enantioseparation of chiral amines obtained with strong acid additives, TFA [183] and alkylsulfonic acids [188], in the mobile phase. ...
... When IPA was used as MP additive, prolonged retentions were observed for the majority of analytes (compared to addition of TEA or DEA). This may be due to that TEA cannot act as hydrogen bond donor and DEA is considerably weaker donor than IPA [42]. The presence of additive has also a beneficial effect on the peak shape and enantioselectivity. ...
The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC.
... Due to the intra-molecular counterion effect of the zwitteriontype SO functionalities, the simultaneous presence of an acidic and a basic additive in the eluent has been recognized to be instrumental for an improvement of the overall chromatographic performance [19,20]. This behaviour has been also observed with other types of CSPs capitalizing the underlying stereorecognition process either on ionic [41,42] or non-ionic [43][44][45] interactions. With CSP 1, acidic and basic additives regulate the strength of the SO-SA interaction without influencing the stereorecognition process, that is the stereoisomeric elution order [18][19][20]. ...
... All the eluent systems were compared by keeping constant the other experimental conditions: 1.0 mL/min flow rate, 25 • C column temperature. Moreover, 0.2% DEA (in volume) was always added as efficiency enhancer, due to its ability to mask the underivatized silanols [16][17][18][19]. ...
... Concurrently to a reduction of the enantiomeric retention time, an almost negligible improvement in terms of enantioselectivity was also recorded. The presence of TFA into the eluent did not alter the mechanism of enantiorecognition as indicated by the enantiomeric elution order inTable 2. The acidic additive can profitably interact with the residual aminopropylsilane groups on the silica surface, thus limiting the effect of unproductive secondary inter- actions13141516.Table 1 shows that CSP 2 almost generally produced the worst performance. However, a relevant improvement of the enantioresolution quality with this column was obtained with the use of ''non standard'' solvents as constituents of the mobile phase mixture (Fig. 4). ...
Three (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives (1–3) and three (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-2-ylmethyl)-9H-purine derivatives (4–6) showed relevant activity against human breast cancer cell line MCF-7, when assayed as racemates. The relevant structural analogy among these compounds stimulated us to evaluate and compare their chromatographic behaviour with five polysaccharide-based chiral stationary phases (CSPs). For a column screening purpose, four cellulose-based (Chiralcel OD-H, Chiralpak IB, Lux Cellulose-2, and Sepapak-4; CSPs 1–4, respectively) and an amylose-based (Lux Amylose-2; CSP 5) CSPs were initially assayed by employing the same “standard” eluent mixture. With CSP 2, the performance from “non standard” eluent systems was also evaluated. The different type and position of the substituents onto the carbamate moiety, the coated or immobilized nature of the modified polymer chain, and the different type of winding as well as the eluent composition were found to deeply affect the enantiorecognition mechanism. While the different winding of the polymer and the derived different morphology of the binding cleft in the 2,5-disubstituted chloromethyl phenyl carbamate amylose-based CSP 5 was found to be unsuited to get profitable enantio-discriminations of all the analyzed compounds, CSP 3 and CSP 4 produced the highest α and R
S values in the enantioselective analysis of four out of six racemates (that is 1–3 and 6). In CSP 2, the conformational change of the polysaccharide chain upon immobilization produced a profound influence on the chromatographic behaviour of compounds 1–6. A relevant improvement of the enantioresolution quality of CSP 2 was obtained when used in combination with “non standard” solvents as constituents of the mobile phase mixture.
... Here, it is important to note that the choice of these compounds did not take into account the final column selection that should be made for an updated version of the method development strategy. In this way, an extended set of compounds was selected, which simplifies the general conclusions about the utility of the optimiza- Fig. 2 is to adjust analysis time (by reducing retention factor, k) and to improve peak shapes [36][37][38][39][40]. Twenty one chiral drugs (32 optimiza-tion cases), that were baseline resolved in one or both generic (screening) mobile phases, were subjected to this optimization. ...
Earlier, a set of pharmaceuticals with different chemical structures has been used to evaluate the enantioselectivity of four recently commercialized polysaccharide-based chiral stationary phases, Lux Cellulose-1/Sepapak 1, Lux Cellulose-2/Sepapak 2, Lux Amylose-2/Sepapak 3 and Lux Cellulose-4/Sepapak 4 and of three Daicel columns, Chiralpak AD-H, Chiralcel OD-H and Chiralcel OJ-H, using the screening conditions of an existing generic separation strategy in normal-phase liquid chromatography (NPLC). In this study, the applicability of the optimization steps of the existing separation strategy was examined using 44 drugs (70 optimization cases) representing the three possible resolution situations that occur after screening. Optimizations are demonstrated by modifying parameters such as polar modifier percentages, temperatures, flow rates and additives concentration. Changing the percentage of polar modifier was found to have the largest influence on the resolution. The resolution, peak shape and the analysis time were nicely improved for 49/70 cases (70%) after the application of the original optimization steps. The introduction of some modifications to the original optimization increased this number from 49 to 62 cases, i.e. from 70% to 88.6%. Finally, an updated generic separation strategy in NPLC was proposed.
... Each mobile phase contained either DEA or TFA as additive, depending on the nature of the tested compound. It is generally accepted that basic and acidic additives enhance the separation quality, both in terms of selectivity and peak shape efficiency [53,54]. In the current study, hexane was replaced by heptane because of its lower toxicity and because retention and resolution data are very similar [48]. ...
Four recently commercialized polysaccharide-based chiral stationary phases, Sepapak(®) 1, Sepapak(®) 2, Sepapak(®) 3, and Sepapak(®) 4, now called Lux(®) Cellulose-1, Lux(®) Cellulose-2, Lux(®) Amylose-2 and Lux(®) Cellulose-4, respectively, were examined for their enantioselectivity on a set of 61 racemic compounds by applying the screening conditions of a previously developed chiral screening strategy in normal phase liquid chromatography (NPLC) [N. Matthijs et al., J. Chromatogr. A 1041 (2004) 119-133]. The enantioselectivity on these phases was compared to that on the initial set of polysaccharide-based phases, Chiralpak(®) AD-H, Chiralcel(®) OD-H, and Chiralcel(®) OJ-H, used in the earlier defined strategy. The results showed that 53 compounds out of 61 (86.9%) were resolved on the initial set of chiral stationary phases (CSPs) using two mobile phases per compound, either heptane-ethanol-diethylamine (DEA) or heptane-isopropanol-DEA for testing basic compounds and heptane-ethanol-trifluoroacetic acid (TFA) or heptane-isopropanol-TFA for acidic, bifunctional and neutral compounds. The recently commercialized set of columns gave 54 separations in total (88.5%). Our results indicated that ethanol (EtOH) as polar modifier provides a higher success rate and better resolutions than isopropanol (IPA) on both sets of stationary phases. However, the usefulness of the mobile phase with IPA as polar modifier cannot be neglected for complementarity reasons. It was found that the screening is improved by the introduction of the recently commercialized polysaccharides based CSPs since they provided enantioseparation for compounds that were not resolved by the traditional CSPs. The combination between the initial and the recently commercialized CSPs showed enantioresolution for 55 compounds out of 61 (90%), among which 47 were baseline resolved.
... To each mobile phase, two additives are added, diethylamine (DEA) and trifluoroacetic acid (TFA). It is generally accepted that basic and acidic additives enhance the separation quality, both in terms of selectivity and peak shapes (efficiency) [23,24]. Thus the first mobile phase applied is ACN/DEA/TFA (100/0.1/0.1) ...
The screening conditions of an existing column and mobile phase selection strategy for chiral drug substances in polar organic solvent liquid chromatography (POSC) were tested for their applicability on two new chlorine-containing polysaccharide-based stationary phases. The selectors of these phases are cellulose tris(3-chloro-4-methylphenylcarbamate) and amylose tris(5-chloro-2-methylphenylcarbamate). The enantioselectivity of these phases is compared to that of the four phases (Chiralpak AD-RH, Chiralcel OD-RH, Chiralpak AS-RH and Chiralcel OJ-RH) used in the earlier defined strategy. A test set of 62 structurally diverse chiral drug substances is analyzed using the screening conditions of the strategy on the six phases. The results confirm that the acetonitrile-based mobile phase provides a higher success rate and better resolutions than the methanol-based also on the new phases. However, the importance of the methanol-based mobile phase cannot be neglected for complementarity reasons; the two mobile phases insure enantioselectivity for different compounds. A third (ethanol-based) mobile phase, not part of the strategy, was also used to screen the two new phases. The joint results led to different possibilities to upgrade the current screening strategy so that improved success rates are obtained. The chlorine-containing chiral stationary phases demonstrated to have an added value to the screening process since they provided enantioresolution for compounds not resolved by non-chlorine-containing ones.
Fentanyl analogues used in therapy and a range of highly potent non-pharmaceutical fentanyl derivatives are subject to international control, as the latter are increasingly being synthesized illicitly and sold as ‘synthetic heroin’, or mixed with heroin. A significant number of hospitalizations and deaths have been reported in the EU and USA following the use of illicitly synthesized fentanyl derivatives.
It has been unequivocally demonstrated that the enantiomers of fentanyl derivatives exhibit different pharmaco-toxicological profiles, which makes crucial to avail of suitable analytical methods enabling investigations at a “stereochemical level”.
Chromatographic methods useful to discriminate the enantioseparation of fentanyls and their derivatives are still missing in the literature. This is the first study in which the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, β-hydroxyfentanyl, and β-hydroxythiofentanyl, has been obtained under polar-ionic conditions. Indeed, the use of ACN-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively.
For the study, the two chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl.
Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems.
Nornicotine, an alkaloid constituent of tobacco, is a precursor to the carcinogen N-nitrosonornicotine that is produced during the curing and processing of tobacco. Accumulating evidence reveals that nornicotine enantiomers have different neurochemical and behavioral effects. In the present study, an accurate and rapid method was developed for the enantioseparation of (R)-(+)-nornicotine and (S)-(−)-nornicotine enantiomers in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry. Chromatographic conditions were investigated to achieve the optimal resolution of two enantiomers. Results indicated that (R)-(+)-nornicotine and (S)-(−)-nornicotine could be separated within 5 min when ammonium hydroxide was added into the co-solvent, and the best resolution (Rs = 4.76) was achieved on a immobilized cellulose tri(S)-(3,5-dichlorophenylcarbamate) chiral stationary phase. The proposed method was validated and was finally applied to analyze the compositions of (R)-(+)-nornicotine and (S)-(−)-nornicotine in three typical types of tobaccos (flue-cured, burley, and oriental). It was found that, enantiomer fraction of nornicotine (the proportion of (S)-(−)-nornicotine in the nornicotine pool) in burley tobacco samples was relatively high and constant compared with flue-cured and oriental tobaccos. The effective and rapid enantioseparation of nornicotine may help the understanding of alkaloids metabolites in different tobacco varieties and may also benefit pharmacological studies of alkaloid enantiomers.
Supercritical Fluid Chromatography is frequently used to efficiently handle separations of enantiomers. The separation of basic analytes usually requires the addition of a basic additive in the mobile phase to improve the peak shape or even to elute the compounds. The effect of increasing the concentration of 2-propylamine as additive on the elution of a series of basic compounds on a Chiralpak-AD stationary phase was studied. In this study, unusual additive concentrations ranging from 0.3% to 10% of 2-propylamine 2-propylaminein the modifier were explored and the effect on retention, peak shape, selectivity and resolution was evaluated. The addition of a large quantity of additive allowed to drastically improve the selectivity and the resolution, and even enantiomers elution order reversal was observed by changing the concentration of basic additive. The role of the ratio additive/modifier appeared a key to tune the enantioselectivity. Finally, the impact of these drastic conditions on the column material was evaluated.
We apply matched molecular pair (MMP) analysis to data from ChirBase, which contains literature reports of chromatographic enantioseparations. For the 19 chiral stationary phases we examined, we were able to identify 289 sets of pairs where there is a statistically significant and consistent difference in enantioseparation due to a small chemical change. In many cases these changes highlight enantioselectivity differences between pairs or small families of closely related molecules that have for many years been used to probe the mechanisms of chromatographic chiral recognition; for example, the comparison of N-H vs. N-Me analytes to determine the criticality of an N-H hydrogen bond in chiral molecular recognition. In other cases, statistically significant MMPs surfaced by the analysis are less familiar or somewhat puzzling, sparking a need to generate and test hypotheses to more fully understand. Consequently, mining of appropriate datasets using MMP analysis provides an important new approach for studying and understanding the process of chromatographic enantioseparation.
Supercritical fluid chromatography (SFC) is a separation technique closely related to high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UPLC or UHPLC), except most of the mobile phase is replaced by dense carbon dioxide (CO2). The physical characteristics of CO2 allow much higher speed separation, compared to HPLC or UPLC, on the same-sized particles, while at the same time causing much lower pressure drops across the column. The solvation characteristics of mobile phases consisting of CO2 mixed with one or more organic modifiers makes it ideal for the separation of small drug-like molecules. SFC is increasingly used at both the analytical and semi-preparative scale for chiral separations, due to its high speed, low cost, easy dry-down and "green" aspects. For semi-preparative scale chromatography, the higher inherent flow required in SFC translates directly into much higher throughput, compared to HPLC. SFC has the major advantage that most of the mobile phase flash vaporizes when the pressure is dropped to near atmospheric, leaving much smaller organic fractions to dry down.An SFC is essentially an HPLC with several critical modifications/additions, such as a back pressure regulator, to keep the entire the system at elevated pressure. This chapter attempts to elucidate the differences in SFC instrumentation compared to HPLC/UPLC, and discusses differences in infrastructure requirements. The chapter also describes the main uses of SFC, at both the analytical and semi-preparative scale.
This paper proposes strategies in normal- and reversed-phase liquid chromatography (NP-HPLC or NPLC and RP-HPLC or RPLC), which were developed using three polysaccharide-based stationary phases. Those strategies are implemented in a knowledge-based system for the chiral separation of drug enantiomers. Each strategy includes a screening and an optimisation stage. The screening stage allows a fast evaluation of separation possibilities and enantioselectivity for many drugs in a short period of time, while the optimisation stage gives the opportunity to enhance, if needed, the initially obtained separation. Different examples demonstrate the effectiveness of the strategies for fast method development.
The separation of enantiomers of 16 basic drugs was studied using polysaccharide-based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate-based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several β-blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase. Chirality 00:000-000, 2015. © 2015 Wiley Periodicals, Inc.
© 2015 Wiley Periodicals, Inc.
A L18 (21 × 37) OAD was employed to investigate the effects of SFC parameters on the asymmetric factor of a basic compound on C18 columns. The effect of active sites of silanol group on SFC could be much more significant than that of HPLC due to the low viscosity and high diffusivity of the supercritical fluid. Three Waters C18 columns with different silanol activities were employed to examine the effect of the SFC parameters on the asymmetric factor of 5-(α-phenylethyl)-semioxamazide. The OAD data were evaluated by ANOVA to determine significant parameters of SFC on the asymmetric factor. The results of ANOVA indicate that the silanol activity is the most dominant factor affecting the asymmetric factor. The influence of other parameters on the asymmetric factor follows the order of: modifier type > modifier concentration > additive type > pressure > temperature ∼ additive concentration > injected sample concentration.
An extensive series of free amino acids and analogues were directly resolved into enantiomers (and stereoisomers where appropriate) by HPLC on zwitterionic chiral stationary phases (Chiralpak ZWIX(+) and Chiralpak ZWIX(–)). The interaction and chiral recognition mechanisms were based on the synergistic double ion-paring process between the analyte and the chiral selectors. The chiral separation and elution order were found to be predictable for primary α-amino acids with apolar aliphatic side chains. A systematic investigation was undertaken to gain an insight into the influence of the structural features on the enantiorecognition. The presence of polar and/or aromatic groups in the analyte structure is believed to tune the double ion-paring equilibrium by the involvement of the secondary interaction forces such as hydrogen bonding, Van der Waals forces and π–π stacking in concert with steric parameters. The ZWIX chiral columns were able to separate enantiomers and stereoisomers of various amphoteric compounds with no need for pre-column derivatisation. Column switching between ZWIX(+) and ZWIX(–) is believed to be an instrumental tool to reverse or control the enantiomers elution order, due to the complementarity of the applied chiral selectors.This article is protected by copyright. All rights reserved
To the best of our knowledge enantioselective chromatographic protocols on β-amino acids with polysaccharide-based chiral stationary phases (CSPs) have not yet appeared in the literature. Therefore, the primary objective of this work was the development of chromatographic methods based on the use of an amylose derivative CSP (Lux Amylose-2), enabling the direct normal-phase (NP) enantioresolution of four fully constrained β-amino acids. Also, the results obtained with the glycopeptide-type Chirobiotic T column employed in the usual polar-ionic (PI) mode of elution are compared with those achieved with the polysaccharide-based phase. The Lux Amylose-2 column, in combination with alkyl sulfonic acid containing NP eluent systems, prevailed over the Chirobiotic T one, when used under the PI mode of elution, and hence can be considered as the elective choice for the enantioseparation of this class of rigid β-amino acids. Moreover, the extraordinarily high α (up to 4.60) and R
S (up to 10.60) values provided by the polysaccharidic polymer, especially when used with camphor sulfonic acid containing eluent systems, make it also suitable for preparative-scale enantioisolations.
A strategy aimed at developing faster chiral screening approaches is proposed in this paper by mixing samples and simultaneously screening the resulting mixture of racemates. The data matrix of the mixture obtained by diode array detector or mass spectrometry is deconvoluted into resolved chromatograms and spectra through a multivariate curve resolution–alternating least squares algorithm. The individual racemates are then identified through the resolved UV spectra, and enantiomeric excess ratios can be measured via the resolved chromatograms. Two representative experiments were carried out to verify the feasibility of the strategy. A mixture containing five pairs of racemic solutes was successfully screened on Chiralcel OD column in one-fifth of the conventional analysis time. Another mixture containing 10 racemates gained nine-tenths of the original screening time on three CSPs with a predictive accuracy above 90 %.
In this study, amylose and cellulose derivatized with 3,5-dimethylphenylcarbamate (DMPC) are compared using polar-organic eluents. Investigation of different mobile phase modifiers and acid/base additives showed that these stationary phases vary greatly in their retention and chiral selectivity for coumarin-based solutes. The DMPC-cellulose phase was found to have excellent resolution for coumatetralyl and adequate resolution for coumachlor enantiomers. However, no selectivity was shown for warfarin and coumafuryl enantiomers. In contrast, the DMPC-amylose phase was found to be the most promising for the separation of these solutes. The enantiomers of warfarin, coumachlor, and coumatetralyl have high resolution in this stationary phase. The type and concentration of organic modifiers affect the thermodynamic and kinetic aspects of the separation in these phases. Methanol and acetone decrease the selectivity in both phases, but tetrahydrofuran increases the selectivity of coumafuryl and coumatetralyl enantiomers on the DMPC-amylose phase. Interesting behavior was observed when the kinetic aspects of the separation were compared by using coumatetralyl as a probe and tetrahydrofuran as a modifier. As the concentration of tetrahydrofuran increased, the rate constants of sorption and desorption for the first-eluted enantiomer increased in both stationary phases. In contrast, the rate constants for the second-eluted enantiomer decreased on DMPC-amylose, but increased on DMPC-cellulose. Ultimately, when 10% THF was used on DMPC-amylose, the first-eluted enantiomer underwent rapid kinetics with a desorption rate constant of 63 s, whereas the second-eluted enantiomer showed sluggish kinetics with a desorption rate constant of 0.08 s.
In this study, 6-azido-2,3-di(p-chlorophenylcarbamoylated) cellulose was synthesized and bonded onto aminized silica gel to obtain a new chiral stationary phase. Enantioselectivity of the chiral stationary phase and Chiralcel OF suggested promising chiral separation ability of the new cellulose chiral stationary phase. In addition, the effect of trifluoroacetic acid, diethylamine on enantioselectivity and retention factors on the chiral stationary phase in high performance liquid chromatography was investigated. Experimental results revealed that resolution increased as the trifluoroacetic acid concentration increased to 0.3% while resolution declined as the diethylamine concentration increased. Therefore, the optimal concentrations of trifluoroacetic acid and diethylamine were determined to be 0.3 and 0.1%, respectively. In most cases, trifluoroacetic acid shortened the retention of the first eluted enantiomer while it increased the retention of the other. For acidic compounds, with the existence of diethylamine in the mobile phase, the retention of both enantiomers decreased. But for basic compounds, the retention of both enantiomers increased.
A sensitive and specific high performance liquid chromatography method for the separation and determination of tapentadol enantiomers has been developed and validated. Ten different chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 2.5 for all enantiomers was achieved on Chiralpak AD-H using mixture of heptane-propan-2-ol-diethylamine (980:20:1, v/v/v). The detection was carried out using fluorescence detector at excitation wavelength of 295nm and emission wavelength of 273nm. The influence of mobile phase composition, namely organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including linearity, limit of detection, limit of quantification, precision, accuracy and selectivity was performed according to the International Conference on Harmonization guidelines. The advantage of the method is a good enantioseparation, short analysis time (less than 20min) and therefore this method is suitable for routine determination of chiral purity of (R,R)-tapentadol in enantiopure active pharmaceutical ingredient.
Introduction Structure Characterization for Polysaccharide-Based CSPs Chiral Discrimination Mechanism Study by Polysaccharide-Based CSPs Conclusions References
Because of the large number of commercially available chiral selectors both for electrophoretic and chromatographic techniques, the experimental possibilities to separate enantiomers are numerous. As a result, the development of a proper separation method for a chiral molecule is challenging. Generic separation strategies can present a useful approach for chiral method development. This paper overviews some strategies that have been developed earlier for different electrophoretic and chromatographic techniques. These strategies can be used either for impurity determination of enantiomers or for the enantioseparation of racemic mixtures. They are not only generic, i.e. applicable on diverse molecules, but are also fast, i.e. requiring only a limited number of experiments to reach a decision.
This chapter discusses chiral separation of active pharmaceutical ingredients (APIs) and their related compounds by various methods. Particular emphases have been put on chiral separation by HPLC on chiral stationary phases (CSPs). Different types of CSPs, including polysaccharide derivatives, macrocyclic glycopeptides (antibiotics), cyclodextrins, Pirkletype, proteins, ligand exchange, crown ethers, synthetic polymers/molecularly imprinted polymers (MIPs), are discussed. Separation mechanisms and method development for chiral molecules using these phases are also discussed.
The enantioselectivities of β-blockers (propranolol, metoprolol, atenolol and pindolol) on four different types of chiral stationary phases (CSPs): Chiralpak AD-H, Lux Cellulose-1, Chirobiotic T and Sumichiral OA-4900 were compared using polar organic (PO) elution mode and normal phase (NP) elution mode. Method optimizations were demonstrated by modifying parameters such as organic modifier composition (ethanol, 2-propanol and acetonitrile) and basic mobile phase additives (triethylamine, diethylamine, ethanolamine, and buthylamine). In normal phase elution mode with Lux Cellulose-1, the four pairs of enantiomers can be separated in the same run in gradient elution mode. Additionally, a simple chiral HPLC-DAD method using a newly commercialized polysaccharide-based CSP by Phenomenex (Lux Cellulose-1) in NP elution mode for enantioselective determination of propranolol in water samples by highly selective molecularly imprinted polymers extraction was validated. The optimized conditions were a mobile phase composed by n-hexane/ethanol/DEA (70/30/0.3, v/v/v) at a flow rate of 1.0 mL min(-1) and 25 °C. The method is selective, precise and accurate and was found to be linear in the range of 0.125-50 μg mL(-1) (R(2)>0.999) with a method detection limit (MLD) of 0.4 μg mL(-1) for both enantiomers. Recoveries achieved with both enantiomers ranged from 97 to 109%.
This study describes using 0.1% of a 28-30% ammonium hydroxide solution as an additive to alcohol modifiers in SFC to improve chromatographic peak shapes for basic molecules. Ammonium hydroxide's high volatility leaves no residual additive in the purified sample unlike classical additives in preparative chromatography such as diethylamine and triethylamine. We demonstrate that the silica support is stable despite having ammonium hydroxide in the modifier by running a durability study for over 350 h (105 L of solvent, 105,000 column volumes) on an analytical Chiralcel OJ column and a second study for 30 h (7.2 L, 14,400 column volumes) on an analytical Lux Cellulose-1 column. The peak shape of small, basic molecules is greatly improved with the use of ammonium hydroxide and this improvement is very similar to those having 0.1% diethylamine as a mobile phase additive. Electrospray ionization is also enhanced with the presence of ammonium hydroxide compared with that of diethylamine. We have found that the age of the 28-30% bottle of ammonium hydroxide solution can have significant effects on the chromatography and we describe how this can be overcome. Finally, we analyzed 23 racemic and basic compounds on six different chiral stationary phases and found there to be very little chiral selectivity difference between ammonium hydroxide and diethylamine, triethylamine, ethanolamine and isopropylamine.
A normal-phase enantioselective high-performance liquid chromatographic method is developed for the separation of the undesired enantiomer from T-3811ME and quantitaion of the undesired enantiomer at low levels using a Chiralpak AD-H column (4.6 x 150 mm) packed with modified amylose stationary phase. The 2% water-modified 2-propanol is used for the method development activities, including exploration of various organic modifiers, optimization of additive acid concentration, column screening, and column temperature optimization. The final optimized method separated the undesired enantiomer from T-3811ME and is proven to be robust, sensitive, linear, accurate, and precise.
A simple and rapid HPLC method using a polysaccharide-based chiral stationary phase (Chiralpak AD-H) in polar-organic phase mode has been developed for direct resolution of glycidyl nitrobenzoate (GNB) and 2-methyl glycidyl nitrobenzoate (MGNB) enantiomers. ACN and methanol were used as mobile phase and the effects of the addition of ethanol and 2-propanol as organic modifier in the mobile phase, flow rate and the column temperature were tested. The optimized conditions were: methanol/ethanol (80:20) at a flow rate of 0.9 mL/min and 40 degrees C. Analysis time was < or = 13 min and the chiral resolution was > or = 2. The method was validated and resulted to be selective, precise and accurate. The method was found to be linear in 2-300 microg/mL range (R(2) >0.999) with an LOD nearly 0.5 microg/mL for four enantiomers. GNB and MGNB enantiomers were obtained by asymmetric epoxidation of allyl alcohol and 2-methyl allyl alcohol, respectively, using chiral titanium-tartrate complexes as catalyst and dichloromethane as solvent after in situ derivatization of the intermediate glycidols derivatives. The quite simple and rapid validated method was applied successfully for direct determination of the enantiomeric excess (> or = 90%) and yield obtained in real samples of asymmetric epoxidation of allylic alcohols without further purification, workup or solvent removal. The method provides a useful and value-added tool for controlling the enantiomeric purity of the synthesized epoxides.
A polysaccharide-based chiral stationary phase (Sepapak-4), with cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector, has been investigated in liquid chromatography (LC). Its enantioresolution power was evaluated towards 13 basic amino-drugs with widely different structures and polarities, using polar organic mobile phases. After preliminary experiments, acetonitrile was selected as the main mobile phase component, to which a low concentration of diethylamine (0.1%) was systematically added in order to obtain efficient and symmetrical peaks. Different organic solvents were first added in small proportions (5-10%) to acetonitrile to modulate analyte retention. Polar organic modifiers were found to decrease retention and enantioresolution while hexane had the opposite effect, indicating normal-phase behaviour under these conditions. The addition of an organic acid (formic, acetic or trifluoroacetic acid) was found to strongly influence the retention of the basic amino drugs in these nonaqueous systems. The nature and proportion of the acidic additive in the mobile phase had also deep impact on enantioresolution. Therefore, the studied compounds could be subdivided in three groups in respect to the acidic additive used. All analytes could be enantioseparated in relatively short analysis times (10-20 min) using these LC conditions.
We packed an ionic liquid (IL)-functionalized stationary phase--based on 1-octyl-3-propylimidazolium chloride covalently bounded to silica gel--into a 3.2mmx250mm column for the simultaneous separation of acidic, basic, and neutral compounds using carbon dioxide subcritical/supercritical fluid chromatography (SFC), and examined the effects of the pressure, temperature, co-solvents, and additives on the retention behavior of the analytes. The model compounds tested for SFC separation are acetaminophen, metoprolol, fenoprofen, ibuprofen, naphthalene, and testosterone. The data indicate that hydrogen-bonding and hydrophobic interactions between the analytes and the IL-modified stationary phase seem to involve in the separation process. Simultaneous separation of acidic, basic, and neutral compounds via SFC was successful at a co-solvent content of 20% MeOH, a pressure of 110 bar, and a column temperature of 35 degrees C. The relative standard deviations of the retention times and peak areas at 50 ppm were all less than 4 and 8% (n=6), respectively.
Sulfonic acids have been shown to be more effective than the commonly used trifluoroacetic acid (TFA) in the chiral resolution of underivatized aromatic amino acids on an amylosic column. Sulfonic acid additives give a more UV transparent mobile phase, possibly allowing the detection of non-aromatic analytes. Work presented demonstrates that through the combination of sulfonic acid mobile phase additives, amine mobile phase additives and solvent modifier variations, the enantiomers of 20 of 25 probe amino acids are fully resolved, four are partially resolved with only one failing to be separated on a common amylosic column.
Using chiral probes shown to be sensitive to the presence of mobile phase additives, a memory effect for these additives by an amylosic column was demonstrated. Exposure to these additives gave prolonged chromatographic performance changes even after their removal from the mobile phase. This finding is consistent with strong binding of the additives to the stationary phase. A procedure to remove bound additives was developed.
A strategy for rapid screening for the separation of chiral molecules of pharmaceutical interest by normal-phase liquid chromatography using three cellulose/amylose stationary phases is proposed. In a first step, the most important parameters for the separations were determined and studied for their effects by means of experimental designs. Results showed that the cellulose tris-(3,5-dimethylphenylcarbamate), the amylose tris-(3,5-dimethylphenylcarbamate) and the cellulose tris-(4-methylbenzoate) stationary phases have very broad and complementary enantiorecognition properties. The type of organic modifier used in the mobile phase appeared to have a dramatic influence on the quality of the separation. Based on the results of the preliminary study, a screening strategy was developed and successfully applied to a set of 36 diverse drugs. Enantiomeric separation was observed in 89% of cases and the analysis times were usually shorter than 20 min.
Two different automated generic liquid chromatography screens for the separation of chiral compounds of pharmaceutical interest have been evaluated. The test set comprised 53 chemically diverse chiral compounds involving 55 enantiomeric pairs from the pharmaceutical industry (i.e. starting materials, synthetic intermediates and drug substances). The first screen utilised four polysaccharide-based columns with five mobile phases and showed enantioselectivity for 87% of the test compounds. The second screen employed three macrocyclic glycopeptide columns with two mobile phases and showed enantioselectivity for 65% of the test compounds. Merging of the two screening procedures resulted in an enantioselectivity for 96% of the chiral compounds. It is anticipated that the systematic use of the automated chiral HPLC screens described in this report will substantially reduce the necessary time for method development of pharmaceutically related chiral analytical methods.
A thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) (i.e., Chiralpak AD) chiral stationary phase (CSP) in the enantioseparation of dihydropyrimidinone (DHP) acid racemate was studied for the first time by quasi-equilibrated liquid chromatography with cyclic van't Hoff and step temperature programs and solid-state ((13)C CPMAS and (19)F MAS) NMR using ethanol and trifluoroacetic acid (TFA)-modified n-hexane as the mobile phase. The conformational transition was controlled by a single kinetically driven process, as evidenced by the chromatographic studies. Solid-state NMR was used to study the effect of the temperature on the conformational change of the solvated phase (with or without the DHP acid enantiomers and TFA) and provided some viable structural information about the CSP and the enantiomers.
Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.
The effects of ethanesulfonic acid (ESA) and n-butylamine as additives were studied for a wide variety of chiral compounds using the polysaccharide chiral stationary phase (CSP), Chiralpak AD. The mobile phase consisted of hexane-ethanol (90:10, v/v). The additives typically had small effects, with one exception: the acidic additive had an enormous effect on the chiral selectivity of amino acid esters. The improved chiral selectivity was largely due to the longer retention of the later eluting enantiomer. Retention behavior of amines indicated that the higher selectivity for amino acid esters owes to increased hydrogen-bonding donation by the amine group of the analyte. Computation establishes the feasibility of a planar complex between the analyte and the cliral stationary phase, involving a pair of complementary hydrogen-bonding groups on each species, enabled by protonation of the analyte. Retention behaviors for a range of structures point to steric hindrance as the third interaction to comprise the requisite three interactions in chiral recognition.
The ability ammonium trifluoroacetate as an additive for elution of acids and bases from derivatized polysaccharide chiral stationary phases was first observed in the process of developing normal-phase chiral HPLC methods in our lead-generation programs. To demonstrate this ability on a broader scale, chiral HPLC methods containing this additive in the mobile phases were developed to resolve selected acidic, basic, and neutral racemates, which are considered standards in the pharmaceutical industry and for which published methods exist. The mobile phases of these published methods contain acidic and/or basic additives (e.g., trifluoroacetic acid or diethylamine). This article demonstrates the versatility of ammonium trifluoroacetate additive in resolving the enantiomers of acidic and basic racemates on the same derivatized polysaccharide chiral columns. This resolution is achieved without changing the mobile phase between the analysis of acidic and basic racemates and also without observation of stationary-phase "memory effect." This chiral method development strategy can result in significant savings of cost and time.
Molecular chirality is a fundamental consideration in drug discovery, one necessary to understand and describe biological targets as well as to design effective pharmaceutical agents. Enantioselective chromatography has played an increasing role not only as an analytical tool for chiral analyses, but also as a preparative technique to obtain pure enantiomers from racemates quickly from a wide diversity of chemical structures. Different enantioselective chromatography techniques are reviewed here, with particular emphasis on the most widespread high performance liquid chromatography (HPLC) and the rapidly emerging supercritical fluid chromatography (SFC) techniques. This review focuses on the dramatic advances in the chiral stationary phases (CSPs) that have made HPLC and SFC indispensable techniques for drug discovery today. In addition, screening strategies for rapid method development and considerations for laboratory-scale preparative separation are discussed and recent achievements are highlighted.
The chiral separation of basic compounds by subcritical fluid chromatography (SFC) is often unsuccessful, due possibly to multiple interactions of the analyte with the mobile and stationary phase. Incorporation of a strong acid, ethanesulfonic acid (ESA), into the sample diluent and mobile phase modifier gives a dramatic improvement in these separations. Screening with ethanol containing 0.1% ESA on CHIRALPAK AD-H gave separation of 36 of 45 basic compounds previously not separated in SFC. The mechanism appears to involve the separation of an intact salt pair formed between the basic compound and ESA. Other modifiers, other acids and one additional stationary phase were examined and found to yield additional separations.
Cyclohexylcarbamates of cellulose and amylose were prepared and their resolving abilities for enantiomers were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The CSPs showed high resolving abilities, which are comparable to those of popular CSPs, tris(3,5-dimethylphenylcarbamate)s of cellulose and amylose. The cycloalkylcarbamates could also be used as CSPs for thin-layer chromatography because of the absence of a phenyl group, which causes the difficulty of detection by UV radiation. In addition, these two derivatives were soluble in chloroform and exhibited chiral discrimination to some chiral compounds in 1H NMR spectroscopy as well as in HPLC.
Chiral recognition of many enantiomeric solutes by a chiral amide stationary phase is based mainly on hydrogen bonding. A
chiral-recognition-factor CHI is proposed, given by the difference of the enthalpy change in the enantiomer discrimination,
standardized with respect to the specific interaction of the solutes with the diamide core of the stationary phase. The röle
of the entropy part is also discussed. By extrapolation of the retention behaviour to elevated temperature, peak inversion
of enantiomers is predicted.
A series of 28 chiral α-alkyl arylcarboxylic acids were chromatographed on an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (AD-CSP). The retention data were correlated to a series of molecular descriptors to produce quantitative structure-enantioselective retention relationships (QSERR) incorporating the hydrogen bonding ability and aromaticity of the solutes.The QSERR equations were used to guide molecular modelling experiments designed to investigate the chiral recognition mechanism responsible for the observed enantioselective separations. The results of the study indicate that unlike the standard “three-point interaction” model of chiral recognition, enantioselectivity was due to a “conformationally driven” chiral recognition process.
An empirical relationship was derived which relates properties of the mobile phase modifier to the chiral selectivity factor for a given analyte/chiral selector combination. Using carbon dioxide and heptane-based mobile phases, the effect of various mobile phase modifiers on Pirkle-type stationary phases may be accurately modeled using a two-parameter equation. Similar results are obtained using cellulosic stationary phases with carbon dioxide-based mobile phases. Modeling separations performed using heptane-based mobile phases with cellulosic stationary phases were not successful. The predictive ability of this modeling approach was demonstrated using novel modifiers and chiral analytes.
Recent developments of polysaccharide-based chiral stationary phases (CSPs) for the direct separation of enantiomers in high-performance liquid chromatography (HPLC) are mainly reviewed together with the results on mechanistic studies by means of chromatography, NMR and mass spectroscopies, and computational methods. Miscellaneous applications of polysaccharide derivatives to the newly developed, chiral dynamic high-performance liquid chromatography (DHPLC) for obtaining a nonracemic compound are also described.
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