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.     

Chiral stationary phases based on cinchona alkaloids for the HPLC resolution of racemates

Summary The employment of three chiral stationary phases (CSPs) obtained by derivatizing γ-mercaptopropyl-silanized silica gel with quinine, quinidine and N-methyl-quinium iodide, for the separation of organic racemates, is presented. They are quite useful in the resolution of alkylarylcarbinols, binaphthyl derivatives, amides and other substances of pharmaceutical interest.     

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.     

High-performance ligand-exchange chromatography of amino acids on chiral stationary phases

Three chiral stationary phases, obtained by grafting silica gel with (-)-trans-1,2-cyclohexanediamine, were studied for the resolution of α-amino acids by ligand-exchange chromatography. The packings were prepared by bonding the chiral ligand to silica gel via different hydrocarbon spacers. Separation of the optical isomers was accomplished by eluents containing a constant concentration of copper(II) acetate (0.05mM). The elution sequence of amino acids was found to be dependent on the grafting reaction selected to prepare the chiral packings.     

Some aspects of the preparative separation of enantiomers on chiral stationary phases

Summary In preparative column liquid chromatography, it is necessary to work with conditions of mass overload to obtain high throughput per unit time. The influence of sample mass and of eluent composition on the separation of R,S-1-(1-naphthylethyl)propylamide on chiral 3,5-dinitrobenzoylphenylglycine-silica (Pirkle type stationary phase) was investigated. At a sample load of 200μg of racemate per gram of stationary phase, the column becomes overloaded. At higher sample loads the peaks become triangular; therefore the first peak is always pure up to loads of 10mg g⁻¹, although resolution is low. The purity of the second peak depends on sample mass, but not on the strength (polarity) of the mobile phase. This is due to the effect that peak width, as well as resolution, decrease as the polarity of the eluent increases. In certain cases the polarimeter, used as a detector, can give more information on peak purity than the UV detector.     

Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: Effect of cyclodextrin type

Three novel chiral stationary phases (CSPs) were prepared by regioselective chemical immobilization of mono(6^A-N-allylamino-6^A-deoxy)perphenylcarbamoylated (PICD) α-, β-, and γ-cyclodextrins (CDs) onto silica support via hydrosilylation. Their enantioseparation properties in high performance liquid chromatography (HPLC) were evaluated with a large spectrum of racemates including flavanone compounds, β-adrenergic blockers, amines and non-protolytic compounds. The effect of CD's cavity size on enantioseparation abilities was studied and discussed. The results indicated that CD's surface loading at silica support played an important role in the enantioseparation on these CSPs under normal-phase conditions while inclusion phenomena contributed the major driving force under reverse-phase conditions. As expected, α-PICD demonstrated the best resolutions towards flavonone and most aromatic alcohols under normal-phase conditions with the highest surface loading; while Fujimura's competitive inclusion model can be applied to explain the better enantioseparations towards β-adrenergic blockers, amines and non-protolytic compounds with α- and β-PICD CSPs. γ-PICD CSP showed superior enantioseparation ability for sterically encumbered analytes like flavanone compounds under both normal-phase and reversed phase conditions.     

Preparation of chiral stationary phase via activated carbamate intermediate for liquid chromatographic optical resolution

Summary Chiral stationary phases (CSPs) for liquid chromatography were prepared by the way of an activated carbamate intermediate. The amino group of aminopropylsilyl silica gel was first activated by carbamylation with disuccinimido carbonate (DSC). The obtained activated carbamate silica gel (ACsil) proved useful as an intermediate for the preparation of urea-type CSPs. The reaction of ACsil with (S)- of (R)-1-(α-naphthyl)-ethylamine gave naphthylethylurea type CSPs. These CSPs were also obtained directly from aminopropylsilyl silica gel by its reaction with optically active (S)- or (R)-succinimido 1-(α-naphthyl)ethyl carbamate (SINEC). Several phenylthiohydantoin amino acid enantiomers and p-bromophenylcarbamyl amino acid enantiomers were resolved on the CSPs by elution with aqueous mobile phase.     

一种手性中间体在键合型和涂覆型纤维素手性固定相上的拆分

制备了涂覆型和键合型纤维素-(3, 5-二甲基苯基氨基甲酸酯)固定相, 分别在制备的纤维素手性固定相上成功地拆分了一种手性中间体, 通过考察流动相中的改性剂(醇、四氢呋喃、三氯甲烷)对手性拆分的影响, 优化了手性中间体在两种手性固定相上的色谱分离条件, 并比较了手性中间体在涂覆和键合型纤维素手性固定相上的拆分. 结果表明, 涂覆型和键合型手性固定相对这种手性中间体均有较好的拆分效果, 在150 mm的色谱柱上, 这两种手性固定相对这种手性中间体的拆分能力相差不大, 但键合型固定相上可选择的流动相范围更广.     

Optimization of enantiomeric separations with chiral bonded phases

Summary The preparation and properties of chiral bonded phases with amino acid groups are described. These phases were used in non-polar eluents and in aqueous systems in the presence of Cu²⁺ ions in the ligand-exchange mode. The optimization of chiral resolution is demonstrated for both cases. With non-polar eluents similarity between bonded groups and solutes is required. The elution could be accelerated by non-protonic moderators. Ligand-exchange separation is influenced by the copper content of the eluent and the stationary phase, the organic moderator concentration, the pH and ionic strength of the buffer and by the temperature. Structural requirements of both the bonded groups and of the solutes for chiral separation are discussed.     

Automated screening platform with isochronal-parallel analysis and conditioning for rapid method development of chiral separations

An automated chiral separation-screening platform was developed, allowing separation analysis on one column while another column is simultaneously equilibrated with a different mobile phase (MP). The platform can be set up from usual HPLC components at moderate cost and it considerably speeds up the screening process, allowing numerous chromatographic conditions to be tested within a short period of time and leading to a significant gain in time and productivity in preparative separations.     

Cyclodextrin carbamates as novel chiral stationary phases for capillary gas chromatography

The following carbamate derivatives of cyclodextrins (CDs) were prepared as novel chiral stationary phases for capillary gas chromatography: hexakis(2,6-di-O-pentyl)-α-cyclodextrin hexa(3-n-propyl, 3-isopropyl, and 3-phenylcarbamate), heptakis-(2,6-di-O-pentyl)-β-cyclodextrin hepta(3-n-propyl, 3-isopropyl, and 3-phenylcarbamate), and octakis(2,6-di-O-pentyl)-γ-cyclodextrin octa(3-n-propyl, 3-isopropyl, and 3-isopropyl, and 3-phenylcarbamate). Metal capillary columns coated with these stationary phases resolved many kinds of racemic mixture. In general, they were especially effective towards polar compounds such as free alcohols, amines, and epoxides. The types of sample which were effectively resolved depended on the cavity size of the CD: α-CD derivatives were specifically effective toward compounds having linear alkyl chains, and β-CD derivatives toward compounds with phenyl groups. The results indicate that chiral separation with the cyclodextrin carbamates depends on the formation of inclusion complexes and also on the hydrogen-bonding interactions between the samples and the CD carbamates.     

Enantiomer separation by gas chromatography on cyclodextrin chiral stationary phases

Fused silica capillary columns coated with several alkyl or acyl cyclodextrin derivatives, especially those of α- and β-cyclodextrins, are suitable for the enantiomer separation of a wide variety of volatile compounds of different molecular size and functionality. Positional isomers and more than 250 pairs of optical isomers have been resolved, including chiral hydrocarbons, acetals, ethers, epoxides, carbonates, lactones, esters, acids, ketones, aldehydes, alcohols, halocarbons, and also nitrogen-and sulfur-containing compounds. The physical properties of the cyclodextrin derivatives, even those obtained as viscous fluids, could be improved by dissolving them in polysiloxane liquid phases commonly used for GLC.     

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.     

Hybrid polyacrylamide chiral stationary phases for HPLC prepared by surface-initiated photopolymerization

Two hybrid polyacrylamide chiral stationary phases (CSPs) for HPLC have been synthesized by a new surface-initiated photo-induced radical polymerization approach of enantiopure N,N'-diacryloyl derivatives of (1R,2R)-diaminocyclohexane (CSP1) and (1R,2R)-diphenylethylenediamine (CSP2). This system is based on the activation of mesoporous silica microparticles by chemically bonded trichloroacetyl groups and dimanganese decacarbonyl as catalyst. UV irradiation was performed using a lab-made quartz photochemical reactor, ad hoc designed for the photo-induced polymerization process on the surface of microparticles. The two phases were evaluated and compared as chromatographic supports for the enantioselective HPLC of model chiral compounds. Their physico-chemical properties and chromatographic performances were also evaluated in comparison with those exhibited by the homologue CSPs obtained by the grafting-from thermal-induced process (CSP3 and CSP4). The new photopolymerization approach yielded higher grafting density than the thermal-induced one, especially in the case of the less reactive monomer (the diacryloyl derivative of (1R,2R)-diphenylethylenediamine), good chromatographic efficiency and a broad application field under normal phase and polar organic mode conditions.     

Enantioseparation of novel chiral tetrahedrane-type clusters on an amylose tris (Phenylcarbamate) chiral stationary phase

Summary Amylose tris(phenylcarbamate) (ATPC) coated on a small particle silica gel was prepared. This ATPC chiral stationary phase (ATPC-CSP) was found to be useful for the enantiomeric separation of some novel chiral tetrahedrane-type clusters. Moreover, the influence of mobile phase modifier and of the structure of chiral tetrahedrane-type clusters on the chiral separation and retention were investigated. The results suggest that not only the structure and concentration of alcohol in mobile phase, but also the subtle structural differences in racemates can have a pronounced effect on enantiomeric separation and retention.     

Correlation method for comparison of selectivity and retention on different chiral stationary phases

Summary The main chromatographic properties: selectivity and retention of two chiral stationary phases: (R)-3,5-dinitrobenzoylphenylglycine (CSP I) and (S)-3,5-dinitro-benzoylleucine (CSP II) have been compared on the basis of correlation of retention factors of derivatized esters of amino acids and derivatized aminoalcohols. The differences in retention and selectivity for the two CSPs can be easily estimated from the correlation equation. It is shown that in the case of the correlation for two sets of retention factors obtained on two different CSPs, it is mainly the intercept that decides which CSP has the better selectivity. It is demonstrated that the correlation method provides more informations on the selectivity and retention than comparison of the chromatographic data of single pairs of enantiomers. Additionally, the possible influence of the polar modifier mixed with n-hexane as diluent on the constants in the correlation equation is demonstrated.     

Direct chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using different cellulose based chiral stationary phases

Summary Two improved methods for the enantiomeric separation of racemic aminoglutethimide (±AG) and its acetylated metabolite (±AAG) have been developed. Direct liquid chromatographic resolution of the enantiomers of aminoglutethimide and its acetylated metabolite was accomplished using Chiralcel OD and Chiralcel OJ columns without any derivatization. Maximum resolution of 8.87 and 2.23 was obtained for the enantiomers of aminoglutethimide and its acetylated metabolite using a Chiralcel OD column, while maximum resolution of 10.34 and 7.01 was obtained for the enantiomers using a Chiralcel OJ column. Optimization of separation was obtained using different concentration of 2-propanol in hexane as a mobile phase.     

Influence of the structure of organic phosphonate compounds on chiral separation on a pirkle type chiral stationary phase

Summary The enantiomers of eightO,O-dialkyl-2-benzyloxycarbonyl-aminoarylmethyl-phosphonates are directly separated on anN-(3,5-dinitrobenzoyl)leucine (DNBleu) column. The influence of the mobile phase composition and the column temperature on the retention and the enantioselectivity are investigated. The influence of the length and steric hindrance of alkoxyl groups of the phosphonate ester and the nature of the substituentp-Cl and pH on the benzene ring which is attached to the chiral carbon atom on chiral separation are discussed also.     

Effect of the structure of organic phosphonate compounds on chiral separations on derivatized cellulose chiral stationary phase

Summary The enantiomers of eightO,O-dialkyl-2-benzyloxycarbonylaminoarylmethyl phosphonates have been directly separated on a tris(3,5-dimethylphenylcarbamate) cellulose column. The results are very different from those obtained by separation on anN-(3,5-dinitrobenzoyl)leucine (DNBleu) column. The effect of mobile phase composition and column temperature on retention and enantioselectivity were investigated. The effect on chiral separation of the length of, and steric hindrance by, alkoxyl groups of the phosphonate ester and of the nature of the substitutentsp-Cl andp-H on the benzene ring attached to the chiral carbon atom are also discussed.     

Assessing chiral self-recognition using chiral stationary phases

Chiral stationary phases were synthesized and their ability to separate racemic precursors from which they were derived was assessed. Taken in conjunction with homochiral recognition previously observed in the solid state, the results of this study reveal that a geometrically controlling π-π interaction has a profound influence on molecular recognition.