Enantioselective HPLC enantiomer separation

In simple experiments, particulate silica-supported CSPs having various cin-chonan carbamate selectors immobilized to the surface were employed in an enantioselective liquid-solid batch extraction process for the enantioselective enrichment of the weak binding enantiomer of amino acid derivatives in the liquid phase (methanol-0.1M ammonium acetate buffer pH 6) and the stronger binding enantiomer in the solid phase [64]. For example, when a CSP with the 6>-9-(tcrt-butylcarbamoyl)-6 -neopentoxy-cinchonidine selector was employed at an about 10-fold molar excess as related to the DNB-Leu selectand which was dissolved as a racemate in the liquid phase specified earlier, an enantiomeric excess of 89% could be measured in the supernatant after a single extraction step (i.e., a single equilibration step). This corresponds to an enantioselectivity factor of 17.7 (a-value in HPLC amounted to 31.7). Such a batch extraction method could serve as enrichment technique in hybrid processes such as in combination with, for example, crystallization. In the presented study, it was however used for screening of the enantiomer separation power of a series of CSPs. 

A special mention in the field of enantioselective HPLC separations must be made of chiro-optical detection systems, such as circular dichroism (CD) and optical rotation (OR), which can be also used to circumvent the low UV detectability of chromophore-lacking samples [40, 61]. While sensitivity of chiro-optical detection is not always sufficient to perform enantiomeric trace analysis, the stereochemical information contained in the bisignate spectropolarimetric response is useful in establishing elution order for those compounds not available as single enantiomers of known configuration. An example of application of different online detection systems (UV and CD at 254 nm) in the enantioselective separation of a racemic sulfoxide on a commercially available TAG CSP is reported in Figure 2.12, under NP conditions. 

Kleidernigg, O.P. and Kappe, C.O., Separation of enantiomers of 4-aryldihydropyrimidines by direct enantioselective HPLC. A critical comparison of chiral stationary phases. Tetrahedron Asymmetry, 8, 2057, 1997. 

Investigation on the enantiomeric composition of chiral secondary alcohols will, however, require either derivatization with an optically active reagent and separation on a conventional column or enantioselective GC using an optically active stationary phase. Today, the latter approach most frequently involves modified cyclodextrins.135 Enantioselective HPLC has also been successfully applied to separate enantiomers.136,137 Several reagents have been used in the transformation of chiral alcohols into diastereomers. Among these, acetyllactic acid138 or chlorofluoroacetic acid139 furnish volatile derivatives of pheromone... 

We then attempted purification of impure (.S )-144 by enantioselective HPLC. Fortunately, TBS derivative G was found to be separable by preparative HPLC on Chiralcel OD to give pure (S)-G. Deprotection of the TBS group of (S )-G under conventional conditions with TBAF caused partial racemization of (S)-144. However, treatment of (S )-G with dilute ethanolic hydrochloric acid at room temperature caused no appreciable racemization to give (.S )-144 (98.4% ee), [ah24 = -90.7 (acetone), in 40% yield. Similarly, (R)-144 was also synthesized by employing AD-mix-a instead of AD-mix-f) . (ft)-Cytosporone E (144 ), [a]o25 = +91.3 (acetone) could be obtained pure (>99% ee). As shown in the present case, use of preparative HPLC is becoming more and more important in the preparation of pure enantiomers. 

O.P. Kleidernigg and C. O. Kappe, Separation of Enantiomers of 4-aryldihydropyrimidines by Direct Enantioselective HPLC. A Critical Comparison of Chiral Stationary Phases, Tetrahedron Asymmetry, 8(12)(1997)2057. 

The fundamental principle of enantioselective HPLC is based on the formation of labile diastereomeric complexes of the two enantiomers with the chiral selector of the stationary phase [3], The enantiomer that forms the less stable complex will be eluted earlier, while the enantiomer that forms the more stable complex will be eluted later. The ratio of the two retention factors k determines the separation factor for the enantioselectivity [4] a (Eq. 1) of a stationary phase for two enantiomers at a certain temperatrue and for a defined solvent composition. 

The determination of retention factors has already been discussed in Chapter 1.1. In enantioselective HPLC, the void time ( dead time ) is measrued by the injection of a non-adsorbed compound, e.g., tri-tert-butylbenzene or a low molecular weight alcohol. In practice, separation factors between 1.5 and 2.5 are commonly found, yet in some cases values of a > 20 have been reported [5, 6]. A selectivity of a = 1 shows that no enantiomer separation is possible imder the conditions used values below 1 are not defined since the separation factor is always referred to the second eluting enantiomer. It is not possible to determine the a value of a racemate when using a temperature or solvent gradient. 

As discussed above, chiral recognition in HPLC (and all other kinds of enantioselective chromatography) rehes on the formation of intermediate diastereomeric complexes. For a racemic sample (identical amounts of (R)- and (S)-enantiomers), the two association constants (K and KjJ have to be different in order to observe an enantiomer separation (see Fig. 3) [3]. Both enantiomers are in solution and can interact with the selector, which is attached to the silica. If, for example, the complexation constant is higher for the (S)-enantiomer, it is bound more strongly to the chiral stationary phase and is eluted later. 

Almost all enantiomer separations show a lower separation factor at higher temperatures when the temperature is increased further, enantiomer separation can be completely suppressed. This means that the entropy term in the above equation approaches the value of the enthalpy term with rising temperature, and that at a certain temperature entropy and enthalpy cancel one another out (In a = 0). For this reason, temperature increase and gradients can be neglected for the optimization of enantioselective HPLC. 

The selection of the mobile phase plays a significant role in enantioselective HPLC. Four different types of eluents can be distinguished. Besides the well-known reversed-phase and normal-phase modes, for enantiomer separations so-called polar organic and polar ionic eluents are also used (see Table 3). 

A broad range of macrocyclic compounds can be used as chiral selectors for enantioselective HPLC. Besides synthetic crown ethers, derivatized cyclodextrins and cyclic antibiotics are also used as chiral stationary phases. Enantiomer separation employing these compounds is often based on host-guest interactions [15], whereby the cyclic molecules form an inclusion compound or an association complex with the analyte. 

Another phenomenon that can only be observed in enantioselective HPLC is so-called enantiomerization, which is equivalent to a racemization of the separated enantiomers during chromatography [27]. Such enantiomerizations lead to characteristic chromatograms, as shown in Fig. 16 for compound 3 (see Fig. 15). 

The easy scalability of enantioselective HPLC can be demonstrated for the separation of omeprazole [29], one of the blockbuster drugs for the treatment of ulcers (see Fig. 17). The (S)-enantiomer, esomeprazole with the trade name Nexium, is used as an improved active pharmaceutical ingredient against inflammation and ulcers of the oesophagus (reflux oesophagitis), heartburn/ pyrosis (gastrooesophagal reflux), and ulcers of the duodenum after infection with helicobacter pylori. 

Czerwenka C, Lammerhofer M, Lindner W (2003) Structure-enantioselectivity relationships for the study of chiral recognition in peptide enantiomer separation on cinchona alkaloid-based chiral stationary phases by HPLC influence of the N-tenninal protecting group. J Sep Sci 26 1499-1508... 

The first observation of the enantioselective properties of an albumin was made in 1958 (28) when it was discovered that the affinity for L-tryptophan exceeded that of the D-enantiomer by a factor of approximately 100. This led to more studies in 1973 of the separation of DL-tryptophan [54-12-6] C22H22N2O2, on BSA immobilized to Sepharose (29). After extensive investigation of the chromatographic behavior of numerous racemic compounds under different mobile-phase conditions, a BSA-SILICA hplc column (Resolvosil-R-BSA, Macherey-Nagel GmvH, Duren, Germany) was... 

Separation of enantiomers by physical or chemical methods requires the use of a chiral material, reagent, or catalyst. Both natural materials, such as polysaccharides and proteins, and solids that have been synthetically modified to incorporate chiral structures have been developed for use in separation of enantiomers by HPLC. The use of a chiral stationary phase makes the interactions between the two enantiomers with the adsorbent nonidentical and thus establishes a different rate of elution through the column. The interactions typically include hydrogen bonding, dipolar interactions, and n-n interactions. These attractive interactions may be disturbed by steric repulsions, and frequently the basis of enantioselectivity is a better steric fit for one of the two enantiomers. ... 

A number of specialised stationary phases have been developed for the separation of chiral compounds. They are known as chiral stationary phases (CSPs) and consist of chiral molecules, usually bonded to microparticulate silica. The mechanism by which such CSPs discriminate between enantiomers (their chiral recognition, or enantioselectivity) is a matter of some debate, but it is known that a number of competing interactions can be involved. Columns packed with CSPs have recently become available commercially. They are some three to five times more expensive than conventional hplc columns, and some types can be used only with a restricted range of mobile phases. Some examples of CSPs are given below ... 

The chiral separation of cis enantiomers was improved with a decrease in temperature, whereas that of trans enantiomers was improved with an increase in temperature. The temperature dependence of enantioselectivities was studied to determine the thermodynamic parameters H°, S°, and Tiso. The thermodynamic parameters revealed that the separation of trans enantiomers was controlled by entropy in the range of temperatures examined, whereas enthalpy-controlled separation was observed for cis enantiomers. The separations of both cis and trans enantiomers, however, were controlled by enthalpy in normal phase HPLC [150],... 

HPLC with column switching and mass spectrometry was applied to the online determination and resolution of the enantiomers of donepezil HC1 in plasma [38]. This system employs two avidin columns and fast atom bombardment-mass spectrometry (FAB-MS). A plasma sample was injected directly into an avidin trapping column (10 mm x 4.0 mm i.d.). The plasma protein was washed out from the trapping column immediately while donepezil HC1 was retained. After the column-switching procedure, donepezil HC1 was separated enantioselectivity in an avidin analytical column. The separated donepezil HC1 enantiomers were specifically detected by FAB-MS without interference from metabolites of donepezil HC1 and plasma constituents. The limit of quantification for each enantiomer of donepezil HC1 in plasma was 1.0 ng/ml and the intra-and inter-assay RSDs for the method were less than 5.2%. The assay was validated for enantioselective pharmacokinetic studies in the dog. 

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