Amino acids as chiral selectors in enantioresolution by liquid chromatography

Amino acids are unique in terms of their structural features and multidimensional uses. With their simple structures and the ready availability of both enantiomers, amino acids not only serve as a chiral pool for synthesis but also provide an inexpensive pool for resolution studies. There has been no attempt to review the application of amino acids as chiral selectors for chromatographic enantioresolution of pharmaceuticals and other compounds. The present paper deals with application of l-amino acids and complexes of l-amino acids with a metal ion, particularly Cu(II), as an impregnating reagent in thin-layer chromatography or as a chiral ligand exchange reagent or a chiral mobile phase additive in both thin-layer chromatography and high-performance liquid chromatography. Enantiomeric resolution of β-blockers, nonsteroidal anti-inflammatories, amino acids (and their derivatives) and certain other compounds is discussed.     

Properties of L-phenylalanine tetraamides as chiral selectors for D,L-amino acids by capillary gas chromatography

Summary A wide series of L-phenylalanine tetraamide selectors (Phe-n-O-TA) have been synthesized and used as stationary phases for chiral resolution of D,L-amino acids by capillary gas chromatography. The influence of length and polarity of the oxaalkanoyl bridge spacing the diamide chains of the selectors and the effects of steric hindrance on separating power were investigated. The characteristics of the chiral columns and the separation factors of D,L-n-butyl-N-trifluoroacetyl amino esters are reported.     

Halogen-substituted phenylalanines as enantioselective selectors for enantioselective discrimination of amino acids: effect of halogen

Halogen-substituted phenylalanines with a halogen X (X = F, Cl, Br or I) in the para position in the aromatic ring of L-phenylalanine are used as enantioselective selectors to explore the effect of the halogen substituent on the enantioselective discrimination of amino acids. Enantioselective discrimination is achieved by investigating the collision-induced dissociation spectra of the trimeric complex ion, [CuII(ref)2(A)-H]+, generated by electrospraying a solution of a mixture of D- or L-analyte amino acid (A), enantioselective reference ligand (ref) and CuCl2. The relative abundances of fragment ions resulting from the competitive loss of reference and analyte amino acids are considered for measuring the degree of enantioselective discrimination by applying the kinetic method. The enantioselectivity of the p-halogenated derivatives of L-Phe increases from fluorine to iodine for the studied amino acids (except for acidic amino acids). The validity of the present method has also been checked by cross enantioselective experiments using p-iodo-D-phenylalanine as the reference in place of p-iodo-L-phenylalanine. The enantioselectivity of fluoro-substituted L-phenylalanine is less than that obtained with L-phenylalanine. The high inductive effect of the fluorine atom decreases the strength of the pi-pi stacking interaction. The presence of halogen affects the enantioselectivity by inductive and steric effects.     

Maltodextrins as new chiral selectors in the design of potentiometric, enantioselective membrane electrodes

Maltodextrins (dextrose equivalent (DE) 4.0-7.0, 13.0-17.0, and 16.5-19.5) are proposed as novel chiral selectors for the construction of potentiometric, enantioselective membrane electrodes. The potentiometric, enantioselective membrane electrodes can be used reliably for the assay of S-captopril as raw material and in pharmaceutical formulations such as Novocaptopril tablets, by use of direct potentiometry. The best response was obtained when maltodextrin with higher DE was used for construction of the electrode. The best enantioselectivity and time-stability was achieved for the lower DE maltodextrin. L-proline was found to be the main interferent for all the proposed electrodes. The surface of the electrodes can be regenerated by simply polishing; this furnishes a fresh surface ready for use in a new assay.     

Enantiomer separation by counter-current chromatography. Optimisation and drawbacks in the use of L-proline derivatives as chiral selectors

Several L-proline and (4R)-hydroxy-L-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(+/-)-leucine and (+/-)-ketoprofen). Successful separations of DNB-(+/-)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.     

Conventional and narrow bore short capillary columns with cyclodextrin derivatives as chiral selectors to speed-up enantioselective gas chromatography and enantioselective gas chromatography-mass spectrometry analyses

The analysis of complex real-world samples of vegetable origin requires rapid and accurate routine methods, enabling laboratories to increase sample throughput and productivity while reducing analysis costs. This study examines shortening enantioselective-GC (ES-GC) analysis time following the approaches used in fast GC. ES-GC separations are due to a weak enantiomer-CD host-guest interaction and the separation is thermodynamically driven and strongly influenced by temperature. As a consequence, fast temperature rates can interfere with enantiomeric discrimination; thus the use of short and/or narrow bore columns is a possible approach to speeding-up ES-GC analyses. The performance of ES-GC with a conventional inner diameter (I.D.) column (25 m length x 0.25 mm I.D., 0.15 microm and 0.25 microm d(f)) coated with 30% of 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-beta-cyclodextrin in PS-086 is compared to those of conventional I.D. short column (5m length x 0.25 mm I.D., 0.15 microm d(f)) and of different length narrow bore columns (1, 2, 5 and 10 m long x 0.10 mm I.D., 0.10 microm d(f)) in analysing racemate standards of pesticides and in the flavour and fragrance field and real-world-samples. Short conventional I.D. columns gave shorter analysis time and comparable or lower resolutions with the racemate standards, depending mainly on analyte volatility. Narrow-bore columns were tested under different analysis conditions; they provided shorter analysis time and resolutions comparable to those of conventional I.D. ES columns. The narrow-bore columns offering the most effective compromise between separation efficiency and analysis time are the 5 and 2m columns; in combination with mass spectrometry as detector, applied to lavender and bergamot essential oil analyses, these reduced analysis time by a factor of at least three while separation of chiral markers remained unaltered.     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

Peptide and peptidomimetic chiral selectors in liquid chromatography

The application of peptide and peptidomimetic chiral selectors in LC is reviewed in this article. In particular, methods for finding these types of selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides and peptidomimetics as general chiral selectors for LC. In terms of the library approaches, batch screening and reciprocal screening are discussed. As for general chiral selectors, one example involves the application of chiral diamide phases. Another example shows the versatility of oligoproline chiral stationary phases, which exhibit resolution for a number of racemic analytes, comparable to other well-established chiral stationary phases.     

Structural optimization of a chiral selector for use in preparative enantioselective chromatography

Utilizing the immobilized-target strategy, the structure of a proline-derived chiral stationary phase was optimized for use in the preparative chromatographic separation of the enantiomers of two chiral selectors used in commercial chiral stationary phases. In this study, various N-acylated proline anilides were prepared and chromatographed on the commercial Pirkle-1J and -Burke 2 chiral stationary phases. The analyte which displayed the greatest retention without sacrifice of enantioselectivity (the 3,5-dimethoxyanilide of N-undecenoyl proline) was chosen for incorporation into the preparative chiral stationary phase. Once prepared, this phase shows increased analyte retention and enantioselectivity comparable to that of earlier phases derived from 3,5-dimethyl anilides of proline. The increased retention allows one to use mobile phases in which the target analytes are more soluble, hence greatly facilitating an increase in the through-put of a column of a given size.     

Histamine-modified cationic beta-cyclodextrins as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids by capillary electrophoresis.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.     

Native and substituted cyclodextrins as chiral selectors for capillary electrophoresis enantioseparations: Structures,features, application,and molecular modeling

CDs are cyclic oligosaccharides consisting of α-d -glucopyranosyl units linked through 1,4-linkages, which are obtained from enzymatic degradation of starch. The coexistence of hydrophilic and hydrophobic regions in the same structure makes these macrocycles extremely versatile as complexing host with application in food, cosmetics, environmental, agriculture, textile, pharmaceutical, and chemical industries. Due to their inherent chirality, CDs have been also successfully used as chiral selectors in enantioseparation science, in particular, for CE enantioseparations. In the last decades, multidisciplinary approaches based on CE, NMR spectroscopy, X-ray crystallography, microcalorimetry, and molecular modeling have shed light on some aspects of recognition mechanisms underlying enantiodiscrimination. With the ever growing improvement of computer facilities, hardware and software, computational techniques have become a useful tool to model at molecular level the dynamics of diastereomeric associate formation to sample low-energy conformations, the binding energies between the enantiomer and the CD, and to profile noncovalent interactions contributing to the stability of CD/enantiomer association. On this basis, the aim of this review is to provide the reader with a critical overview on the applications of CDs in CE. In particular, the contemporary theory of the electrophoretic technique and the main structural features of CDs are described, with a specific focus on techniques, methods, and approaches to model CE enantioseparations promoted by native and substituted CDs. A systematic compilation of all published literature has not been attempted.     

Recent advances in peptide chiral selectors for electrophoresis and liquid chromatography

The application of peptides in chiral separations using techniques such as capillary electrophoresis (CE), electrokinetic capillary chromatography (EKC) and liquid chromatography is the focus of this review. Methods for finding peptide selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides as general chiral selectors for electrophoresis and liquid chromatography. One example shows the effectiveness of polymeric dipeptide surfactants as general chiral selectors for electrophoresis. Another example shows the versatility of oligoproline chiral stationary phases, exhibiting resolution for a number of racemic analytes comparable to other well-established chiral stationary phases.     

Macrocyclic antibiotics as chiral selectors in the design of enantioselective, potentiometric membrane electrodes for the determination of S-flurbiprofen

Construction of three novel enantioselective, potentiometric membrane electrodes based on carbon paste impregnated with different macrocyclic antibiotics vancomycin and teicoplanin as chiral selectors are described. The solutions for the construction of electrodes were prepared in phosphate buffer pH 4 for the vancomycin-based electrode (VCM), pH 6 and pH 6/40% acetonitrile solutions for teicoplanin-based electrodes, TCP I and II, respectively. The proposed electrodes were applied in the assay of S-flurbiprofen raw material and its pharmaceutical formulation by use of direct potentiometry, VCM electrode exhibiting the best enantioselectivity. The surfaces of the electrodes are easily renewable by simply polishing on an alumina paper.     

A (4R)-hydroxy-L-proline-derived chiral scaffold and its oligomers as chiral selectors in liquid chromatography chiral stationary phases for enantioseparation

The chromatographic behaviour of a poly-L-proline-derived chiral stationary phase (CSP) is compared to the corresponding single proline-derived CSP. Structurally diverse racemic test compounds and mobile phases, including normal- and RP conditions, were used. Although the application domain of the poly-L-proline-derived CSP (CSP-3) was considerably restricted, this CSP showed a higher retention and a slightly broader application domain than the monomeric analogue (CSP-1) when heptane/2-PrOH was used as mobile phase. The presence of an alcohol in the mobile phase was essential for enantioseparation in the poly-L-proline-derived CSP when normal-phase conditions were applied.     

Novel cinchona alkaloid carbamate C9-dimers as chiral anion-exchange type selectors for high-performance liquid chromatography

Nine new quinine (QN) carbamate C9-dimers (QN-X-QN), with different aliphatic and cyclic spacers (X), have been synthesized and immobilized onto porous silica gel for HPLC. The chiral discriminating behavior of these "dimeric" anion-exchange type chiral stationary phases (CSPs) has been investigated in detail, to elucidate the role of the presence of a second QN subunit on the chiral selector (SO), as well as the influence of the structure and length of the spacer, on the overall chiral recognition of a set of N-derivatized amino acids and other acidic drugs. The bulkiness of the intermediate spacer tuned the chiral recognition abilities of these SOs, with the 1,3-adamantylen-derived CSP being the one that led to the best separations. Shorter spacers reduced the chiral discrimination abilities of the "dimeric" selectors, with the n-hexylen bridge being the most favorable distance to allow a nearly independent interaction of the two QN subunits with the racemic analytes. The comparison to five "monomeric" CSPs showed that the "dimeric" ones usually retain the chiral analytes more strongly, though the enantioseparation is not improved. Nevertheless, the exceptional resolution abilities of dimeric SOs with a trans- 1,2-diaminocyclohexylen-bridge for the separation of DNP-derivatives of amino acids and certain acidic drugs of therapeutical interest (e.g., profens) seemed to be superior to most of the other CSPs.     

Macrocyclic antibiotics as chiral selectors in the design of enantioselective, potentiometric membrane electrodes for the determination of l- and d-enantiomers of methotrexate

Three enantioselective, potentiometric membrane electrodes (EPMEs) based on macrocyclic glycopeptide antibiotics—vancomycin and teicoplanin (modified or not with acetonitrile)—were proposed for the determination of l- and d-enantiomers of methotrexate (Mtx). The linear concentration ranges for the proposed enantioselective membrane electrodes were between 10⁻⁶ and 10⁻³ mol l⁻¹ for l- and d- methotrexate. The slopes of the electrodes were 58.00 mV/pl-Mtx for vancomycin-based electrode; 57.60 mV/pd-Mtx for teicoplanin-based electrode and 55.40 mV/pd-Mtx for teicoplanin modified with acetonitrile-based electrode. The detection limits of the proposed electrodes were of 10⁻⁸ mol l⁻¹ magnitude order. The surfaces of the electrodes are stable and easily renewable by polishing on alumina paper. All proposed electrodes proved to be successful for the determination of the enantiopurity of Mtx as raw material and of its pharmaceutical formulations (tablets and injections).     

Gas chromatography of bile acids as their hexafluoroisopropyl ester-trifluoroacetyl derivatives.

Bile acids, such as cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids, were allowed to react with hexafluoroisopropanol and tri-fluoracetic anhydride at 37 for 30 min. The resulting derivatives were gas chromatographed on QF-1, with flame ionization detection, and were identified by gas chromatography-mass spectrometry. Separation was good. By using this method, these acids were detected in samples of human duodenal fluid; the ratios of each were 24.4, 41.5, 24.9, 2.3 and 6.9%, respectively.