Chiral separation, HPLC, amino acids

Ilisz, I., Berkecz, R., and Peter, A., HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases a review, J. Sep. ScL, 29, 1305, 2006. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Macrocyclic antibiotics such as teicoplanin and vancomycin have been used in chiral stationary phases separations of amino acids, drugs, and other species using HPLC and other separations methods. These applications have been reviewed in a number of recent sources, including a 2004 monograph on chiral separations. [97, 101-107]... 

Finally, libraries aimed to chiral resolution of racemates will be covered here in particular, the use of chiral stationary phases (CSPs) has recently been reported for the identification of materials to be used for chiral separation of racemates by HPLC. The group of Frechet reported the selection of two macroporous poly methacrylate-supported 4-aryl-1,4-dihydropyrimidines (DHPs) as CSPs for the separation of amino acid, anti-inflammatory drugs, and DHP racemates from an 140-member discrete DHP library (214,215) as well as a deconvolutive approach for the identification of the best selector phase from a 36-member pool library of macroporous polymethacrylate-grafted amino acid anilides (216,217). Welch and co-workers (218,219) reported the selection of the best CSP for the separation of a racemic amino acid amide from a 50-member discrete dipeptide iV-3,5-dinitrobenzoyl amide hbrary and the follow-up, focused 71-member library (220). Wang and Li (221) reported the synthesis and the Circular Dichroism- (CD) based screening of a 16-member library of CSPs for the HPLC resolution of a leucine ester. Welch et al. recentiy reviewed the field of combinatorial libraries for the discovery of novel CSPs (222). Dyer et al. (223) reported an automated synthetic and screening procedure based on Differential Scanning Calorimetry (DSC) for the selection of chiral diastereomeric salts to resolve racemic mixtures by crystallization. Clark Still rejxrrted another example which is discussed in detail in Section 9.5.4. 

TLC using silica gel coated with an L-prolinamide-copper(II) salt mixture (Chiralplates Macherey-Nagel Co.) separates enantiomers using the ligand-exchange principle to give information on the chiral purity of amino acids and peptides. The equivalent HPLC procedure has been used for determining enantiomer ratios. 

The design and development of effective chiral separation and recognition of enantiomers is the key point of the chiral technique. Many technologies have been developed for chiral recognition and separation of amino acids and their derivatives including HPLC, CE and electrochemical sensors. However, they are expensive techniques in terms of time and reagent consumption. Accordingly, there is considerable interest in the development of simple, rapid and economical methods that will afford the analysis of enantiomeric species. 

Figure 6 Chiral HPLC separation of amino acid derivatives on imprinted stationary phases packed with Z-L-Glu-OH (a), Boc-L-phe-Gly-OEt (b), Z-L-Ala-L-Ala-Ome (c) Z-L-Ala-Gly-L-Phe-OMe (d). Mobile phase chloroform - acetic acid. Column 250 x 4.6 mm. Flow rate, 1 mL/min. Detection, UV 260 nm. Reproduced from Ref. 22, with permission.

Terminal alkynes substituted with chiral substituents have been polymerized by using a rhodium catalyst, [RhCl(NBD)]2 (NBD = norbomadiene) [6]. As shown in Scheme 3, polymerization of a chiral (carbamoyloxy)phenylacetylene 4 forms a cis-substituted polyacetylene 5. Due to the bulkiness of the substituents, these polymers show a helical conformation with no extended conjugation in the polymer chain. These materials are potentially useful as enantioselective permeable membranes to separate racemic amino acids and alcohols in water or in methanol. They can be also used as chiral stationary phase for enantioselective high-performance liquid chromatography (HPLC) analysis. 

Marchelli R, Corradini R, Galavema G et al (2006) Enantioselective separation of amino acids and hydroxy acids by ligand exchange with copptafll) cmnplexes in HPLC (chiral eluent) and fast sensing systems. In Subramanian G (ed) Chiral separation techniques. Wiley-VCH, Weinheim... 

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

The chiral reagent 122 was proposed for derivatization of enantiomeric mixtures of amino acids. Good HPLC separations were obtained for the diasteroisomer derivatives of a series of amino acids, including some unusual a-amino acids with long or bulky side chains, aryl and hetaryl groups, and -substituted /J-amino acids297. 

Since the natural target of macrocyclic antibiotics is the A-acyl-D-alanyl-D-alanine terminus (see Section 2.1), the early choice of suitable substrates for this kind of CSPs was that of amino acids [45]. However, it turned out that the macrocyclic CSPs were very successful not only in amino acids enantioresolution, but also in the separation of a wide variety of different structures. The early stages of application of macrocyclic antibiotics have been surveyed in the different fields of chromatography [1,2]. A summary of the different categories of chiral compounds separated by HPLC on glycopeptides containing CSPs is reported in Table 2.3. 

Piccinini, A.-M., Chiral separation of natural and unnatural amino acid derivatives by micro-HPLC on a ristocetin A stationary phase, J. Biochem. Biophys. Methods, 61, 11,2004. 

Lehotay, J. et al.. Chiral separation of enantiomers of amino acid derivatives by HPLC on vancomycin and teicoplanin chiral stationary phases, Pharmazie, 53, 863, 1998. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

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