Enantioselective HPLC separations

ENANTIOSELECTIVE HPLC SEPARATIONS USING THE GLYCOPEPTIDES CONTAINING 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. 

Ekborg-Ott, K.H., Liu, Y, and Armstrong, D.W., Highly enantioselective HPLC separations using the covalently bonded macrocyclic antibiotic, ristocetin A, chiral stationary phase. Chirality, 10, 434, 1998. 

K. H. Ekborg-Ott, Y. Liu and D. W. Armstrong, Highly Enantioselective HPLC Separations Using the Covalently Bonded Macrocyclic Antibiotic Ristocetin A Chiral Stationary Phase, Chirality, 10 (1998)434. 

The differences between achiral and enantioselective HPLC are smaller than is generally assumed. Enantioselective HPLC does not require special units or detectors, and all physico-chemical fundamentals of the chromatography are identical. However, it should be noted that many of the described enantioselective HPLC separations are performed imder normal-phase conditions. Applications of the popular water/mefhanol or water/acetonitrile gradients are in the minority, since many CSPs are not compatible with these gradient systems or do not show any retention. 

The fundamentals introduced in the previous sections allow some conclusions regarding optimization of enantioselective HPLC separations. 

Our group also demonstrated another combinatorial approach in which a CSP carrying a library of enantiomerically pure potential selectors was used directly to screen for enantioselectivity in the HPLC separation of target analytes [93, 94]. The best selector of the bound mixture for the desired separation was then identified in a few deconvolution steps. As a result of the parallelism advantage , the number of columns that had to be screened in this deconvolution process to identify the single most selective selector CSP was much smaller than the number of actual selectors in the library. 

Substrate 2 has also been used as a test substrate HPLC separation methods exist for 2, while ee-value determination of 1 is more difficult [6, 17]. Reflecting the general recent interest in the hydrogenation of unfunctionalized olefins, the past few years have seen the publication of a number of results for this substrate [15, 18-26]. The highest enantioselectivities were achieved using catalysts 12b [22] and 14a [26],... 

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. 

Martens-Lobenhoffer et al. [119] used chiral HPLC-atmospheric pressure photoionization tandem mass-spectrometric method for the enantio-selective quantification of omeprazole and its main metabolites in human serum. The method features solid-phase separation, normal phase chiral HPLC separation, and atmospheric pressure photoionization tandem mass spectrometry. The internal standards serve stable isotope labeled omeprazole and 5-hydroxy omeprazole. The HPLC part consists of Agilent 1100 system comprising a binary pump, an autosampler, a thermo-stated column component, and a diode array UV-VIS detector. The enantioselective chromatographic separation took place on a ReproSil Chiral-CA 5 ym 25 cm x 2 mm column, protected by a security guard system, equipped with a 4 mm x 2-mm silica filter insert. The analytes were detected by a Thermo Scientific TSQ Discovery Max triple quadrupole mass spectrometer, equipped with an APPI ion source with a... 

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 practical unavailability of (+)-sparteine means that the standard enantioselective deprotonation protocol can not be used to prepare (i ,f )-BisP diphosphines (see, however. Section 5.4.1). Some work has been performed to overcome this limitation. Imamoto and co-workers were able to prepare both enantiomers of t-Bu-BisP (BH3)2 by using both enantiomers of mrt-butyl-methylphosphine borane. Unfortunately, the preparation of the latter compounds involved a preparative HPLC separation step and therefore the strategy cannot be easily adapted for large-scale synthesis. 

Abel S, Juza M (2007) Less common applications of enantioselective HPLC using the SMB technology in the pharmaceutical industry. In Subramanian G (ed) Chiral separation techniques a practical approach, 3rd edn. Wiley-VCH, Weinheim, pp 203-273... 

Later, DeVries, Bakos, and their coworkers confirmed that the enantioselective catalyst for imine reduction is actually monosidfonated ligand 4a prepared via RP-HPLC separation of mixtures of ligands 4a-c. By using monosulfonated ligand 4a as an inseparable mixture of diastereomers (chirahty on phosphorus) under otherwise the same condition,... 

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. 

Eq. (1) implies that in order to obtain a sufficient separation factor a, a sufficient difference in the retention factors must be obtained. Therefore, gradients are rarely applied in enantioselective HPLC, wfhich would speed up isocratic separations. 

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