High-performance ligand exchange chromatography on chiral diamine-bonded silica gel

Summary The resolution of racemates on chiral chemically bonded silica gel by high-performance ligand-exchange chromatography is described. The stationary phase was prepared by bonding (−) trans 1,2 cyclohexanediamine to silica gel through the coupling agent 3-glycidoxypropyl silane. The resolution of several α-amino acids and of compounds of pharmaceutical interest is reported.     

Direct separation of amino enantiomers by high performance ligand exchange chromatography on chemically bonded chiral phases

Summary Various chiral chemically bonded stationary phases for the separation of amino acid enantiomers by high performance ligand exchange chromatography are described. The phases were synthesized by treating silica gel with 3-glycidoxypropyltrimethoxysilane and bonding L-amino acids such as L-azetidine carboxylic acid, L-pipecolic acid or L-phenylalanine to the product. The best results were obtained with L-pipecolic acid as a fixed ligand. Nearly all common amino acid enantiomers could be separated.Presented at the 14th International Symposium on Chromatography London, September, 1982     

A chiral stationary phase with special hydrophobic framework for ligand-exchange chromatography

A novel chiral stationary phase (CSP1) for ligand-exchange chromatography (CLEC) was prepared by firstly using dimethyl- chlorosilane as an endcapping reagent for decreasing residual silanol group on the surface of silica gel,and then introducing L-Pro as a chiral selector and hydrophobic octyl group to the silica gel surface simultaneously.The enantioseparations of 14 DL-amino acids on CSP1 were achieved with the enantioselectivityαranging from 1.09 to 2.44 and the resolution Rs being between 0.8 and 6.3.The chromatographic performances of CSP1 with the bonded phase (CSP2) prepared using reference method were compared.The results showed that the column efficiency and resolution Rs of chiral stationary phase could be improved by using the above modifying method.     

High-performance liquid chromatographic resolution of enantiomers on chiral amine-bonded silica gel

Summary A chiral stationary phase with an immobilized, optically active diamine was prepared for the separation of enantiomers. The synthesis of the phase was carried out by bonding (–)trans-1,2-cyclohexanediamine to microparticulate silica gel through the coupling agent 3-glycidoxypropylsilane. The resolution of the racemic compounds catechin, 2,2-dihydroxy-1,1binaphthyl and trans-1,2-cyclohexandiol, is reported.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

New HPLC-chiral stationary phases for enantiomeric resolution of sulfoxides and selenoxides

Summary Enantiomers of several sulfoxides and some selenoxides can be easily separated by using a new chiral stationary phase (CSP-DACH-DNB) containing the 3,5-dinitrobenzoyl derivative of R,R-(-)1,2-diamino-cyclohexane as selector, covalently bonded to the siliceous matrix. The easy operative conditions and the high enantioselectivity values (α) allow a direct transfer of the analytical separations to a semi-preparative and preparative scale.     

High-performance liquid chromatography of nucleic acid constituents: Chromatographic examination of novel stationary phases

Summary Silica-bonded stationary phases were developed for the separation of nucleic acid constituents and their properties investigated with homologous oligoriboadenylic acids in electrostatic interaction chromatography and with alkylbenzenes in reversed-phase chromatography. Analysis of retention data confirmed the stratified molecular structure of the surface which consist of a layer of propyl chains anchoredvia siloxane bridges to the silica surface proper and of polar moieties attached to the hydrocarbonaceous functions. The polar top layer contains weak cationic and/or hydrophobic binding sites, is strongly hydrated in contact with aqueous eluents and bars the access by large biopolymers to the hydrocarbonaceous sublayer. In reversed-phase chromatography of small non polar molecules with hydro-organic eluents, however, this layer is accessible and engenders a retentive behavior typical for weak hydro-carbonaceous bonded phases. As a result the stationary phases, depending on the nature of the sample and the mobile phase, exhibit the properties of "soft" phases for the chromatography of biopolymers under mild elution conditions and those of "hard" phases for the separation of small non-polar molecules under conditions generally employed in reversed-phase chromatography. The retention of nucleic acid constituents on most of the stationary phases investigated subject to a dual mechanism as a result of the interplay of electrostatic and hydrophobic interactions between the eluites and the binding sites on the stationary phase surface. Siliceous stationary phases having surface morphology described above are suitable for the separation of nucleic acid constituents having widely ranging molecular weights up to 3 × 10⁶ Daltons provided the support has appropriate pore dimensions. This is demonstrated by the separation of mixtures arising from digesting t-RNA^pha or polyadenylic acids as well as those of ribosomal RNA’s and different forms of the plasmid pBR322 DNA. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Preparation of new hybrid organic/inorganic polymeric chiral stationary phases for ligand-exchange chromatography

Three new hybrid organic/inorganic polymeric ligand-exchange chiral stationary phases were developed by radical chain transfer reaction and surface grafting on silica gel,and successfully used for the enantioseparations of DL-amino acids and DL-hydroxyl acids.The resolutions were achieved by using water containing 2.0×10~(-4) mol/L of CuAc_2 as a mobile phase,column temperature of 40℃,flow rate of 1.0mL/min and detection at UV 254 nm.The elution order of D-isomer before L-isomer was observed for all DL-amino acids resolved except DL-Pro.     

Enantioselective liquid-solid extraction for screening of structurally related chiral stationary phases

Summary An enantioselective liquid-solid batch extraction method is described for the screening of novel chiral stationary phases (CSPs) during optimization studies of chiral selectors derived form a common lead structure. Extraction enantioselectivity (α) values can be calculated from the enantiomeric excess ee-values of the selectand, which are measured in the liquid phase by enantioselective HPLC. Extraction α-values have been correlated with chromatographic α-values. The influence was studied of several experimental parameters of the assay (pH_a, buffer concentration, temperature, selector/selectand and phase ratio) on the enantiomeric excess (ee) values of the selectands and the enantioselectivity of the CSPs, respectively. The derived statistically significant model has then been implemented to predict chromatographic α-values of novel CSPs. For example, an ee of 89.3% for DNB-Leu as selectand could be achieved in batch extraction for a novel synthesized but mechanistically similarly-acting CSP derived form quinine. This corresponds to a calculated extraction α-value of 17.7. Based on this α_extraction a chromatographic α-value of 28.8 was predicted by the linear correlation model; the experimental HPLC α-value of 31.7 was in good agreement and demonstrated the validity of the proposed screening method. The method is particularly helpful in SO optimization studies.     

Enzymes as HPLC stationary phases for chiral resolutions: Initial investigations withα-chymotrypsin

Summary A new HPLC stationary phase was synthesized by the covalent immobilization of -chymotrypsin on silica. This increased the stability of the enzyme, without decreasing its activity. The initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes. The stereochemical resolutions of amino acids and amino acid derivatives are reported.     

High-performance liquid chromatographic enantioseparation of amino compounds on newly developed cyclofructan-based chiral stationary phases

Direct separation of the enantiomers of amino acid amines, amino alcohols, and diamines was performed on recently developed chiral stationary phases containing isopropyl carbamate-cyclofructan 6 (IP-CF6), (R)-naphthylethylcarbamate cyclofructan 6 (RN-CF6), or dimethylphenylcarbamate cyclofructan 7 (DMP-CF7) as chiral selectors, using n-hexane/alcohol/TFA as mobile phase. The effects of the mobile phase composition, the nature and concentrations of the alcoholic and acidic modifiers, and the structures of the analytes on the retention and resolution were investigated. In some cases, separations were carried out at constant mobile phase composition in the temperature range 5-40 °C. Thermodynamic parameters and T(iso) values were calculated from plots of lnk versus 1/T. It was found that the enantioseparations were enthalpy driven. The sequences of elution of the stereoisomers were determined but no general rule could be established.     

Direct separation of 2-hydroxy acid enantiomers by high-performance liquid chromatography on chemically bonded chiral phases

Summary The resolution of 2-hydroxy acid enantiomers by the principle of ligand exchange chromatography on chemically modified silica gel is described. The phases were synthezised by fixing L-amino acids via 2-glycidoxypropyl-trimethoxysilane to silica gel. The highest enantioselectivity for 2-hydroxy acids was obtained by using L-hydroxyproline as fixed ligand and Cu(II) as complexing agent. A number of aromatic as well as aliphatic 2-hydroxy acids could be resolved on this sorbent. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

High-performance liquid chromatographic investigation of the enantioselectivity and mechanism of chiral recognition of new cholic acid-based stationary phases

Summary To elucidate the mechanism of chiral recognition of cholic acid-based stationary phases, four new cholic acid derivatives, with differently substituted carbamate or three acetoxy groups, were bonded to a hydrosilyl-modified silica gel. Their capacity to discriminate between enantiomers was evaluated in normal-phase high-performance liquid chromatography. The results were compared with those from equivalent separations on trihydroxy- and 3α-phenylcarbamate-substituted cholic acid-based bonded phases. The influence of mobile phase composition of the separation of the enantiomers of amino alcohols was shown. Different mechanisms of chiral discrimination are discussed, highlighting the influence of the nature of the carbamate on enantioselectivity.     

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 μmol/m²) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids. Chromatographic separation of methionine enantiomers using a dinaphtyl neamine-based ligand-exchange chiral stationary phase     

The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography

Summary As an alternative to the known three-point interaction model describing recognition of optical isomers by a chiral resolving agent, a new concept has been developed stating that two interaction points between the resolving agent and the enantiomers are also sufficient for achieving chiral recognition of the latter, provided that the diastereomeric adducts formed by the resolving agent with the enantiomers additionally interact with a non-chiral chromatographic sorbent. This concept is based on the results of ligand-exchange chromatography of -amino acid enantiomers with copper(II) complexes of chiral bifunctional ligands as the resolving agents in chromatographic systems.     

Selectivity studies on branched and linear alkyl bonded stationary phases in reversed-phase liquid chromatography

Summary Reversed-phase liquid chromatographic retention characteristics for the sixteen acyclic C₁−C₅ N-alkylbenzamide congeners were measured on various branched and linear, alkyl bonded hydrocarbon stationary phases. Retention factors, k′, were determined in acetonitrile-water mobile phase compositions on ethyl, n-octyl, n-dodecyl, n-octadecyl, 1-ethyladamantyl, 4-butyloctyl, and 2,4,4-trimethylpentyl stationary phases. Statistical analysis of the two main effects investigated — type of stationary phase and percentage of organic modifier (acetonitrile) in the mobile phase — described greater than ninety percent of the variability in the data for most of the comparisons. Selectivity effects due to variation in the mobile phase dominated the results.     

Enantiomeric separation of precursors of rho-kinase inhibitors by HPLC on polysaccharide-based chiral stationary phases

Summary The separation of enantiomers of substituted cyclohexanecarboxamides, benzamides and chemical precursors of Rho-kinase inhibitors was achieved using derivatized polysaccharide-based chiral stationary phases. Separations were by normal phase HPLC with a mobile phase ofn-hexane-alcohol (methanol, ethanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), tris-methylbenzoate (Chiralcel OJ), a silica-based amylose tris-(S)-1-phenylethylcarbamate (Chiralpak AS), or tris-3,5-dimethylphenylcarbamate (Chiralpak AD). The effects of cencentration of various aliphatic alcohols in the mobile phase were investigated. The effect of structural features on the discrimination between the enantiomers was examined. The isolation of milligram amounts of enantiomers of two derivatives was performed on an analytical column by multiple repetitive injections under overload conditions.     

Separation of the enantiomers of pyrethroic acids and their esters by high-performance liquid chromatography on a polysaccharide-derived chiral stationary phase

Summary The liquid-chromatographic separation of the enantiomers of pyrethroic acids and their esters has been investigated on a polysaccharide-derived chiral stationary phase (CSP), Chiralpak AS. Good separation of the enantiomers of underivatized pyrethroic acids was achieved on the column, and the enantiomers of pyrethroic acid methyl and ethyl ester derivatives were also resolved.     

Chromatographic properties in normal-mode HPLC of chiral stationary phases based on substituted β-cyclodextrins

Summary Substituted β-cyclodextrin chiral stationary phases having different types of phenyl carbamate substituents have been prepared and evaluated (retention, selectivity, resolution) for the liquid chromatographic separation of several series of enantiomers. The influence on separations of the degree of substitution and the structure of the substituent are discussed. Different mechanisms are suggested to explain separations in normal mode conditions.