Chromatography, chiral

Chiral separations can be considered as a special subset of HPLC. The FDA suggests that for drugs developed as a single enantiomer, the stereoisomeric composition should be evaluated in terms of identity and purity [6]. The undesired enantiomer should be treated as a structurally related impurity, and its level should be assessed by an enantioselective means. The interpretation is that methods should be in place that resolve the drug substance from its enantiomer and should have the ability to quantitate the enantiomer at the 0.1% level. Chiral separations can be performed in reversed phase, normal phase, and polar organic phase modes. Chiral stationary phases (CSP) range from small bonded synthetic selectors to large biopolymers. The classes of CSP that are most commonly utilized in the pharmaceutical industry include Pirkle type, crown ether, protein, polysaccharide, and antibiotic phases [7]. 

Pirkle-type phases are amino acid derivatives possessing an aromatic entity which can undergo n-n interactions with the solute. The aromatic entity can be either a n donor or n acceptor. The CSP and the solute form a n donor/acceptor pair. This complex is then stabilized by additional interactions such as hydrogen bonding, dipole interactions, or steric repulsion [8]. The Pirkle-type phases are most commonly used in normal-phase mode in order to enhance the n-n and hydrogen bond interactions. Hexane with an alcoholic modifier, such as isopropanol, is the mobile phase of choice. These phases have 

A number of proteins are commercially available as CSPs including a-acid glycoproteins (AGP, the major plasma binding protein for basic drugs), human serum albumin (HSA, the major plasma binding protein for weakly acidic drugs), bovine serum albumin (BSA), ovomucoid (OVM), and cellobiohydro-lase (CBH) [12]. The proteins are bonded to silica and utilized in reversed-phase mode with an aqueous buffer/organic modifier eluent. Mobile-phase 

A more detailed discussion of the stationary phase types and mechanism of interaction and separation theory in relation to chiral compounds is given in Chapter 22. A large number of chiral stationary phases are currently available to meet the needs of the pharmaceutical industry for determination of the enantiomeric purity of active pharmaceutical ingredients, raw materials, and metabolites. As a consequence, there are a multitude of options in terms of columns, separation mode, and separation conditions to explore in achieving an enantioseparation. 

Derivatization is another form of sample preparation. It is utilized for the analysis of labile analytes or to enhance retention or detection with a preferred type of detector. Derivatization can be performed to enhance detection by UV/Vis, fluorescence, or electrochemical detection. Consideration must be given to the stability of the derivatize to solvolysis and thermal degradation. In our labs alendronate, a bisphosphonate with a primary amine functionality, was derivatized with FMOC to enhance detection by UV/Vis as well as to increase retention in RPLC mode [19]. An acylchloride was derivatized with 

Other packings have used esterified celluloses, chiral peptides and /3-cyclo-dextrins as enantioselective media [12]. The cyclodextrins are chiral carbohydrates formed from up to 12 glucose units. The monomers are configured such that the cyclodextrin has the shape of a hollow truncated cone or barrel-like cavity within which stereospecific guest-host interactions can occur, though other features such as steric repulsion, solvent, pH, ionic strength and temperature all affect retention. 

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of 

Initial studies on non-covalent MIPs, pioneered by Mosbach and co-workers, focused mainly on the preparation of materials selective for amino acid derivatives [13-17,19 23,30-38]. The polymers did not only possess selectivity for the amino acid used as the print molecule, but were also found to be enantioselective the 

The imprinting effects of MIPs prepared with optically active compounds as the print molecules are readily demonstrated by chromatographic evaluations. For example, when the L-enantiomer of an amino acid derivative is used as the print species, a column packed with the resulting polymer will retain the L-enantiomer longer than the o-enantiomer and vice versa when the o-enantiomer is used as the print molecule. Reference polymers prepared with the racemate or without print molecule will not be able to resolve the racemate. A steroselective memory is hence induced in the polymers by the print molecules and is in many cases very precise. 

Molecularly imprinted CSPs have been prepared for applications in HPLC, TLC (thin-layer chromatography), CE (capillary electrophoresis) and CEC (capillary electrochromatography). CSPs for HPLC are by far the most studied. 

A large number of chiral amino acids and peptides has been imprinted. Several MIPs selective for pharmaceuticals have also been described. The most widely used method has been bulk polymerisation followed by grinding, sieving and packing into HPLC columns. Alternatively, the polymers can be prepared by any of the methods discussed above. Some examples of MIP CSPs are found in Table 17.1. 

The selectivities of MIPs are in many cases comparable to those of commercially available CSPs. For example, a separation factor (a) of 17.8 was found for the separation of the two enantiomers of a dipeptide on poly(methacrylic acid-co-EDMA) imprinted with one of the enantiomers (Fig. 17.5) [43]. 

The silica gels discussed previously contain bonded alkyl chains from 8 to 18 carbon atoms. They are versatile and therefore very useful. Yet, for improving the separation of certain classes of compound the tendency is now to use one of the growing number of specific stationary phases. 

Aluminium oxide (AI2O3) or zirconium oxide (Zr02) are also used as supports of reticulated deposits based upon polymers of butadiene or styrene-divinylbenzene or hydroxymethylstyrene. Porous graphite, in the form of spheres whose surface is 100 per cent carbon and therefore completely hydrophobic, has been used in applications with compounds possessing atoms carrying lone pairs of electrons thus having high retention factors. 

These stationary phases show a greater stability in both acidic and basic media, allowing certain columns to be rinsed with sodium hydroxide 1 M, that Si-O-C bonds would not normally resist. 

The Optical purity of an analyte defined in terms of its enantiomeric excess (e.e.), is calculated from the following expression where Ar and Aj represent the areas under the two peaks corresponding to the two enantiomers. 

The degree of interaction between the mobile phase and the stationary phase whether normal or reversed, affects the retention time of the analytes. In principle, the polarity of the stationary phase can lead to the following situations  

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Neural networks were trained on the basis of these codes to predict chiralit> -dependent properties in enantioselective reactions [42] and in chiral chromatography [43]. A detailed description of the chirality codes is given in the Tutorial in Section 8,6,... 

Analytical techniques that utilise biopolymers, ie, natural macromolecules such as proteias, nucleic acids, and polysaccharides that compose living substances, represent a rapidly expanding field. The number of appHcations is large and thus uses hereia are limited to chiral chromatography, immunology, and biosensors. 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

In recent years the solid-phase hydrosilylation reaction was successfully employed for synthesis of hydrolytically stable surface chemical compounds with Si-C bonds. Of special interest is application of this method for attachment of functional olefins, in particular of acrolein and some chiral ligands. Such matrices can be used for subsequent immobilization of a wide range of amine-containing organic reagents and in chiral chromatography. 

T. E. Beesley and R. P. W. Scott, Chiral Chromatography, John Wiley and Sons, Chichester-New York, (1998), 46. 

The screening was performed in a way similar to that of Welch, except that it involved the use of a spectropolarimeter instead of chiral chromatography to determine the selectivity. Equal amounts of the target racemate 17 were added into each of the 16 wells containing beads and the ellipticity of the supernatant liquid in each well was measured after equilibrating for 24 h at the wavelength of the maximum adsorption (260 nm). Knowing the specific ellipticity of one enantiomerically pure... 

The past two decades have seen remarkable advances in chiral chromatography, as only 20 years ago, the direct resolution of enantiomers by chromatography was still considered to be an impressive technical achievement. 

In this chapter, we will discuss the present status of CHIRBASE and describe the various ways in which two (2D) or three-dimensional (3D) chemical structure queries can be built and submitted to the searching system. In particular, the ability of this information system to locate and display neighboring compounds in which specified molecular fragments or partial structures are attached is one of the most important features because this is precisely the type of query that chemists are inclined to express and interpret the answers. Another aspect of the project has been concerned with the interdisciplinary use of CHIRBASE. We have attempted to produce a series of interactive tools that are designed to help the specialists or novices from different fields who have no particular expertise in chiral chromatography or in searching a chemical database. 

CHIRSOURCE aims to explore the use of chiral chromatography for combinatorial chemistry approaches. Combinatorial chemistry, as well as parallel synthesis. 

Utilization of intelligent systems in chiral chromatography starts with an original project called CHIRULE developed by Stauffer and Dessy [36], who combined similarity searching and an expert system application for CSP prediction. This issue has recently been reconsidered by Bryant and co-workers with the first development of an expert system for the choice of Pirkle-type CSPs [37]. 

Today, the use of CHIRBASE as a tool in aiding the chemist in the identification of appropriate CSPs has produced impressive and valuable results. Although recent developments diminish the need for domain expertise, today the user must possess a certain level of knowledge of analytical chemistry and chiral chromatography. Nevertheless, further refinements will notably reduce this required level of expertise. Part of this effort will include the design of an expert system which will provide rule sets for each CSP in a given sample search context. The expert system will also be able to query the user about the specific requisites for each sample (scale, solubility, etc.) and generate rules which will indicate a ranked list of CSPs as well their most suitable experimental conditions (mobile phase, temperature, pH, etc.). 

The Cyclobond materials are some of the most effective in separating isiomers generally and their development continues. It is likely that chiral chromatography will become increasingly important as the products from biotechnology continue to proliferate into the pharmaceutical field. 

The design of simulated moving bed chromatography and its application to the separation of cycloheptanone and cyclopentanone as test substances to validate the system for subsequent chiral chromatography has been described.27 Briefly, eight silica-packed columns were hooked up in series to form a cyclic flow path. On the first pair, preliminary separation of the components was performed, with the less-retained raffinate being directed to waste. Following the second pair of columns, eluent was added. After the... 

Volume inefficient chiral chromatography required to deliver a single enantiomer Separation at final API necessitated processing excess material through the synthesis to give the desired amount... 

Volume inefficient chiral chromatography required to deliver a single enantiomer... 

Evaluation of the above route against our initial target objectives for the synthesis of taranabant indicated a high level of success, not just for the primary objectives of removing the tin chemistry and chiral chromatography, but for a number of other process improvements (Table 9.2). Of particular note was that the three crystalline intermediates were key for purification, first the phenethylamine salt 12 for the classical resolution, secondly the HC1 salt of amine 2 allowed for upgrade of diastereomeric purity, and finally the API allowed for upgrade of enantiomeric purity via initial removal of racemic material. 

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