Chiral HPLC system

Davankov, V. A., Separation of enantiomeric compounds using chiral HPLC systems. A brief review of general principles, advances, and development trends, Chromatographia, 27, 475, 1989. 

Since we used a non-chiral HPLC system, resolution of enantiomers is not possible. Consequently the racemate (structures 8 and 9, Fig. 27) must appear in peak 3 as well as the two meso forms (structures 6/7 and 10/11) which each are present in double relative molar amounts. The two pairs of racemates (structures 2-5 and 12-15), each present with a relative molar abundance of 4, belong to peaks 3 and 4, since peaks 1 and 5 must, by necessity of a relative abundance of one for each form, occupy peak 1 and 5. 

Two different chiral HPLC systems have been developed in this laboratory to evaluate the enantiomeric purity of brinzolamide. The operating conditions for these methods are also summarized in Table 9. Figure 15 shows a chiral HPLC chromatogram using the second set of conditions listed. 

In its broadest terms the discussion of HPLC detection for chiral species must include the analysis of mixtures with achiral substances as well as the quality testing of, for example, the enantiomeric purity of a chemically pure drug form. The distinction between the definitions of chemical purity versus optical purity can not be overemphasized. In an efficient chiral HPLC system the latter problem is trivial, and if retention times are significantly different then any conventional detector such as RI, electrochemical, absorption, etc., could be used. Co-elutions are a major experimental concern in separations of mixtures and at this juncture it is not only prudent but absolutely necessary to involve a chiroptical detector to preferentially identify the chiral analyte. 

The determination of the enantiomers of mefloquine in plasma and whole blood using a coupled achiral-chiral HPLC system" (38). A... 

The type of CSPs used have to fulfil the same requirements (resistance, loadabil-ity) as do classical chiral HPLC separations at preparative level [99], although different particle size silica supports are sometimes needed [10]. Again, to date the polysaccharide-derived CSPs have been the most studied in SMB systems, and a large number of racemic compounds have been successfully resolved in this way [95-98, 100-108]. Nevertheless, some applications can also be found with CSPs derived from polyacrylamides [11], Pirkle-type chiral selectors [10] and cyclodextrin derivatives [109]. A system to evaporate the collected fractions and to recover and recycle solvent is sometimes coupled to the SMB. In this context the application of the technique to gas can be advantageous in some cases because this part of the process can be omitted [109]. 

These authors further described the synthesis and resolution (by chiral HPLC) of a new C2-symmetric planar-chiral bipyridine ligand [43] (see structure 35 in Scheme 18). They obtained an X-ray crystal structure of the corresponding copper complex proving a bidentate complexation. This system led to high diastereo- (up to 94%) and enantioselectivity (up to 94%) in the... 

With this encouraging result from the model system, a gram quantity of the racemic sulfoxide 40 was prepared by oxidation of benzoxathiin 16 with mCPBA and a small amount of chiral sulfoxide (A)-40 with 94% ee was isolated by subsequent chiral HPLC separation (Scheme 5.12). When chiral sulfoxide (S)-40 was treated with borane-dimethylsulfide, a clean reduction of the olefin and the sulfoxide was observed. More surprisingly, only the desired cis-diaryl dihydrobenzoxathiin 12 was observed in high yield and unchanged 94% ee. No trans-isomer or 16 was observed. With this proof of concept in hand, an efficient... 

He, J., Shibukawa, A., Nakagawa, T., Wada, H., Fujima, H., Imai, E., Go-oh, Y. (1993). Direct injection analysis of atenolol enantiomers in plasma using an achiral/chiral coupled column HPLC system. Chem. Pharm. Bull. 41, 544—548. 

Fig. 14A-C Chromatography of the racemic monoepoxy derivatives (I—III) of Z3,Z6,Z9-18 on chiral HPLC columns A Chiralpak AD B Chiralpak AS C Chiralcel OJ-R. The solvent system for the former two normal-phase columns is 0.1% 2-propanol in n-hexane (0.45 ml/min), and that of the third column is 15% water in MeOH (0.45 ml/min). Homo-conjugated dienes, epo3,Z6,Z9-18 H (I) and Z3,Z6,epo9-18 H (III), were detected by UV (215 nm), and Z3,epo6,Z9-18 H (II) was detected by RID. The earlier eluting isomers have a 3S,4R, 6S,7R, or 9R,10S configuration...

Based on the high peak capacity of CE, the separation speed, and the availability of numerous chiral selectors and the simplicity of the systems, chiral CE is superior to chiral HPLC separations. This is as well reflected by the high number of publications on chiral CE in recent years. Chiral HPLC is suffering from low peak capacity (broad peaks), system stability (often normal phase systems), pressure sensitivity of columns (often cellulose-based column materials), and as a consequence long separation times. 

The analysis time for chiral HPLC separations will probably remain relatively long until CSPs with higher efficiency than the present ones become available. But monolithic columns, columns with a smaller particle size (i.e., UPLC ), and miniaturized systems would increase the efficiency and speed up the enantioseparation of existing types of CSPs. 

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... 

Lynam and Nicolas have evaluated chiral separations by HPLC versus SFC [14] The enantiomers studied were pharmaceutical synthetic precursors. Repeated injections of trans-stilbene oxide and carbobenzyloxy phe-nylalaninol were made and the chromatographic parameters Rs, N, and a were calculated daily. SFC gave superior enentiomeric resolution of peaks and there was a faster solvent equilibration. The columns were quite stable in both SFC and HPLC systems. 

Separation of enantiomers of etodolac using two different derivitization agents and three chiral stationary phases has been studied [24]. Etodolac was converted to its anilide derivative with either 1,3-dicyclohexyl-carbodiimide or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Etodolac, derivatizing agent, aniline, and dichloromethane were allowed to incubate for 30 minutes, which was followed by addition of 1 M HC1. The organic layer was removed, washed, dried, and then injected into normal phase or reverse phase HPLC. The HPLC system consisted of a 250 x 4.6 mm (5 pm particle size) column packed with chiral stationary phases, and detection was effected by the UV absorbances at 254 and 280 nm. Separation of etodolac enantiomers was achieved on only one of the stationary phases when using 20% 2-propanol in hexane as the mobile phase at a flow rate of 2.0 mL/min. 

The effect of surface polarity is even more important in separations where two or more simultaneous interactions must occur in order to achieve the desired selectivity. This is particularly true in chiral separations. Since aqueous buffer systems are almost universally used as CEC mobile phases, enantioseparations are often run under re-versed-phase conditions as opposed to the normal-phase mode typically used in chiral HPLC. Therefore, non-specific hydrophobic interactions would be highly detrimental to the discrimination process that involves subtle differences between the enantiomers. 

Riboflavin (vitamin I 2) is an important chemical species that occurs in many complex biologically related samples. The substance is chiral and strongly fluorescent and the coupling of these molecular properties into the FDCD-HPLC detection system greatly enhances both the specificity and the limit of detection capabilities of the method. In the specific case of riboflavin an HPLC system using FDCD detection where excitation is done at 325nm with a HeCd-laser a detection limit of 170 pg of riboflavin [30] is attainable. This level is about 10 times better than what is possible with a laser excitation -transmission CD-HPLC system [29]. 

The optical resolution of racemic cyclophanes can be easily achieved by chiral HPLC, and the measurement of CD spectra of chiral cyclophanes now becomes a routine. The electronic and magnetic interactions between the aromatic rings can be readily examined if the cyclophane architecture is used in the model systems. Despite this experimental and theoretical progress in relevant fields, the chiroptical properties, especially the electronic CD spectra, of chiral cyclophanes have not been systematically studied until very recently. 

A racemic mixture of three-layered [3.3]paracyclophane (45) was resolved into two enantiomers by chiral HPLC (on a Daicel OD column), and their absolute configuration was determined by a comparison of the experimental CD spectrum with the theoretical one at the TD-DFT-B3-LYP/TZVP level [55]. A simple model, composed of two p-xylenes and durene (the side chains were modeled again by methyl groups), was used to explain the origin of the chiroptical properties of the three-layered cyclophane system. Due to the flexibility of the [3.3]paracyclophanes, the solvent effects on the conformer distribution and thus on the chiroptical properties were significant (Fig. 10). 

To investigate the lipase enantiomeric selectivities, chiral HPLC was used to monitor the enantiomeric ratios of the individual ester products 27A-E. All ester products, except the disfavored product 27A, were asymmetrically resolved by the lipase transesterification process. Interestingly, the highest enantiomeric ratio of the ester products was recorded for the preferred product 27D (83% ee). This indicated that not only the lipase substrate selectivity but also the lipase enantiomeric specificity could be controlled in the dynamic cyanohydrin system. 

Separation of optically active isomers is one of the most important areas of HPLC apphcation in the pharmaceutical industry. Since most of biological systems are predominantly homochiral, different enantiomers of the same drug could have different effect and potency, and the development of enan-tioselective analytical (and preparative) separation methods is very important. Detailed description of chiral HPLC separation is given in Chapter 22 of this book here we only briefly review the specifics of distinctive types of chiral stationary phases (CSP). 

P. Borman, B. Houghtflower, K. Cattanach, K. Crane, K. Freebaim, G. Jonas, I. Mutton, A. Patel, M. Sanders, and D. Thompson, Comparative performances of selected chiral HPLC, SFC, and CE systems with a chemically diverse sample set. Chirality 15 (2003), S1-S12. 

Assuming that analyte. A, is introduced into an HPLC system with a chiral stationary phase with an R conhguration, the following equilibrium can be written ... 

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