Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

In‐column preparation of a brush‐type chiral stationary phase using click chemistry and a silica monolith

Brush‐type chiral stationary phases (CSP) have been prepared both from a silica monolith and, separately, from 10 μm porous silica beads via a process of in‐column modification including attachment of the chiral selector via copper‐catalyzed azide–alkyne cycloaddition. Azide functionalities were first introduced on the pore surface of each type of support by reaction with 3‐(azidopropyl)trimethoxysilane, followed by immobilization of a proline‐derived chiral selector containing an alkyne moiety. This functionalization reaction was carried out in dimethylformamide (DMF) in the presence of catalytic amounts of copper (I) iodide. The separation performance of these triazole linked stationary phases was demonstrated in enantioseparations of four model analytes, which afforded separation factors as high as 11.4.     

Preparation of a permethylated β‐cyclodextrin chiral stationary phase by one‐pot hydrosilylation and immobilization at the C2 position for chiral high‐performance liquid chromatography

A novel cyclodextrin intermediate, mono‐2^A‐allylcarbamido‐2^A‐deoxy‐permethylated β‐cyclodextrin, was synthesized by reacting allylamine and newly prepared mono‐2^A‐azido‐2^A‐deoxy‐permethylated β‐cyclodextrin by the Staudinger reaction and anchored onto porous silica beads by a one‐pot hydrosilylation and immobilization procedure to afford a novel chiral stationary phase. This stationary phase acts as a new member of the previous chiral stationary phase series immobilized on the cyclodextrin C2 position. This stationary phase depicted enantiomeric separation abilities toward a series of bicyclic and tricyclic racemates under reversed‐phase conditions. The resolutions for hesperetin and naringenin achieved on the current phase reached 3.91 and 1.11, respectively, much higher than the previous permethylated β‐cyclodextrin with the linkage at the C6 position.     

Effect of Chiral Additives in the Preparation of Cellulose-Based Chiral Stationary Phases in HPLC, and Effect on Enantiomer Resolution

Chiral stationary phases (CSPs) for high-performance liquid chromatographic (HPLC) have been prepared by coating silica gel with cellulose tribenzoate or cellulose trisphenylcarbamate. The effect of chiral additives on preparation of the CSPs was studied with (+)-l-mandelic acid, (−)-2-phenyl-1-propanol, (+)-1-phenyl-1,2-ethanediol and (−)-1-(1-naphthyl)ethanol as chiral additives for cellulose tribenzoate and (−)-2-phenyl-1-propanol and (+)-phenylsuccinic acid as chiral additives for cellulose trisphenylcarbamate. The results showed that chiral recognition by these stationary phases was increased in comparison with the original CSPs, especially the resolution (R _S) obtained. The method can be used to improve the efficiency of enantiomer separation by silica gel stationary phases coated with polymers.     

Graphene oxide coated capillary for chiral separation by CE

This article describes a new application of graphene oxide (GO) in CE based on the coating of fused silica capillary for chiral separation. The coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectra. The results indicated that the capillary was coated with GO. Chiral separations were carried out in the GO‐coated capillary for the ephedrine–pseudoephedrine (E‐PE) isomers and β‐methylphenethylamine (β‐Me‐PEA) isomers at the optimal buffer conditions without any chiral selector by CE. The precision and reproducibility of GO‐coated capillary were investigated, and the RSDs of migration time (n = 6) for the E‐PE isomers were 1.35–1.41%, and 0.97–3.50% for β‐Me‐PEA isomers, respectively. The LOD for E‐PE isomers and β‐Me‐PEA isomers was 3 μM and 18 μM, respectively.     

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative‐based selectors using diisocyanates

The classical method for the preparation of immobilized polysaccharide‐based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3‐aminopropyl silica gel after it was dissolved in a mixture of N,N‐dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5‐dimethylphenylcarbamate and 3,5‐dichlorophenylcarbamate of cellulose, and 3,5‐dimethylphenylcarbamate and 5‐chloro‐2‐methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6‐hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose‐based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.     

Separation of 2-naphthol atropisomers on cyclofructan-based chiral stationary phases

A set of fifteen 2-naphthol-derived atropisomers were evaluated on three different cyclofructan-based chiral stationary phases (CSP). The cyclofructan CSPs were a dimethylphenyl-derivatized cyclofructan 7 (CF7-DMP), a isopropyl (CF6-P) and a R-naphthylethyl cyclofructan 6 (CF6-RN) derivative, all bonded to 5-µm spherical fully porous silica particles packed into three 25?cm?×?4.6?mm columns (commercially available as Larihc columns). The 15 atropisomers were analyzed in the normal-phase mode with heptane/alcohol mobile phases. The CF7-DMP column was the most effective partially or fully separating 14 of the 15 compounds (93%). All 15 compounds could be separated by at least one cyclofructan column. Five compounds could be partially or fully separated by all three CSPs. The effect of ethanol, 2-propanol and butanol as 5 and 10% v/v polar modifiers in heptane was studied. A prototype 15?cm?×?4.6?mm column packed with superficially porous 2.7?µm CF6-P bonded particles was tested with the same set of compounds and a standard HPLC system. The increased efficiency and solvent saving were significant.     

Evaluation of silica from different vendors as the solid support of anion‐exchange chiral stationary phases by means of preferential sorption and liquid chromatography

Chromatographic performance of a chiral stationary phase is significantly influenced by the employed solid support. Properties of the most commonly used support, silica particles, such as size and size distribution, and pore size are of utmost importance for both superficially porous particles and fully porous particles. In this work, we have focused on evaluation of fully porous particles from three different vendors as solid supports for a brush‐type chiral stationary phase based on 9‐O‐tert‐butylcarbamoyl quinidine. We have prepared corresponding stationary phases under identical experimental conditions and determined the parameters of the modified silica by physisorption measurements and scanning electron microscopy. Enantiorecognition properties of the chiral stationary phases have been studied using preferential sorption experiments. The same material was slurry‐packed into chromatographic columns and the chromatographic properties have been evaluated in liquid chromatography. We show that preferential sorption can provide valuable information about the influence of the pore size and total pore volume on the interaction of analytes of different size with the chirally‐modified silica surface. The data can be used to understand differences observed in chromatographic evaluation of the chiral stationary phases. The combination of preferential sorption and liquid chromatography separation can provide detailed information on new chiral stationary phases.     

Advances in enantiomeric resolution on monolithic chiral stationary phases in liquid chromatography and electrochromatography

During the last decade, chiral monolithic stationary phases have been prepared and used for rapid enantioseparations in CEC and HPLC. Various chiral selectors are used to prepare these CSPs. The preparation, properties, and applications of these CSPs are discussed in this paper. Attempts have been made to describe optimization strategies and the chiral recognition mechanisms. A comparison of chiral separations in CEC and HPLC is described. Efforts have also been made to predict the future perspectives and challenges of chiral monolithic stationary phases. The most effective chiral selectors include polysaccharides, cyclodextrins, and macrocyclic glycopeptide antibiotics. These chiral phases produced acceptable analytical enantiomeric separation of a variety of racemates. However, the development of these CSPs for preparative‐scale separations is needed.     

Application of bromoacetate‐substituted β‐CD‐bonded silica particles as chiral stationary phase for HPLC

Bromoacetate‐substituted [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy]propylsilyl‐appended silica particles (BACD‐HPS), an important and useful synthetic intermediate for preparation of novel types of macrocycles‐capped β‐CD‐bonded silica particles including crown ether/cyclam/calix[4]arene‐capped β‐CD‐bonded silica particles, have been prepared and used as chiral stationary phase for HPLC. This synthetic stationary phase is characterized by means of elemental analysis. For the first time, the chromatographic behavior of BACD‐HPS was systematically evaluated with several disubstituted benzenes and some chiral drug compounds under both normal and RP conditions in HPLC. The results show that BACD‐HPS has excellent selectivity for the separation of aromatic positional isomers and chiral isomers of some drug compounds when used as stationary phase in HPLC.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Influence of substituent position and cavity size of the regioisomers of monocarboxymethyl‐α‐, β‐, and γ‐cyclodextrins on the apparent stability constants of their complexes with both enantiomers of Tröger's base

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐α‐, β‐, and γ‐cyclodextrin and native α‐, β‐, and γ‐cyclodextrin as chiral additives at 0–12 mmol/L for β‐cyclodextrin and its derivatives and 0–50 mmol/L for α‐ and γ‐cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin–Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin–Tröger's base complexes.     

Use of cyclodextrin‐modified gold nanoparticles for enantioseparations of drugs and amino acids based on pseudostationary phase‐capillary electrochromatography

The application of chemical‐modified gold nanoparticles (GNPs) as chiral selector for the enantioseparation based on pseudostationary phase‐CEC (PSP‐CEC) is presented. GNPs modified by thiolated β‐CD were characterized by NMR and FT‐IR. The nanoparticle size was determined to be of 9.5 nm (+2.5 nm) by Transmission Electron Microscopy (TEM) and UV spectra. Four pairs of dinitrophenyl‐labeled amino acid enantiomers (DL‐Val, Leu, Glu and Asp) and three pairs of drug enantiomers (RS‐chlorpheniramine, zopiclone and carvedilol) were analyzed by using modified GNPs as the chiral selector in PSP‐CEC. Good theoretical plate number (up to 2.4×10⁵ per meter) and separation resolution (up to 4.7) were obtained even with low concentration of modified GNPs (0.8–1.4 mg/mL). The corresponding concentration of β‐CD in the buffer was only 0.30?0.53 mM, which was much lower than the optimum concentration of 15 mM if pure β‐CD was used as chiral selector. Our results showed that thiolated β‐CD modified GNPs have more sufficient interaction with the analytes, resulting in significant enhancement of enantioseparation. The study shed light on potential usage of chemical modified GNPs as chiral selector for enantioseparation based on PSP‐CEC.     

几种手性醇在纤维素键合的SBA-15基手性固定相上的直接拆分

在强酸性条件下, 以三嵌段聚醚P123为模板, 合成了孔径大且粒径均匀的SBA-15介孔二氧化硅微球. 将含有少量三乙氧硅丙基氨基甲酸酯残基的纤维素-三(3,5-二甲基苯基氨基甲酸酯)通过分子间缩聚作用固载到氨丙基化的SBA-15微球上, 制得手性固定相; 采用常规和非常规的流动相模式, 对一些芳香醇的消旋体进行了手性拆分. 实验结果表明, 所制备的SBA-15微球不仅分散性良好, 具有规则的二维六方孔道结构, 而且消除了微孔; 所制备的键合手性固定相不仅固载手性选择剂的量大, 而且经六甲基二硅胺烷封端后可有效改善拖尾现象, 对实验选用的手性醇具有较高的拆分能力; 与大孔硅胶为基质的同类纤维素键合手性固定相相比, 该固定相对同种手性消旋体的分离因子明显提高.     

Capillary electrochromatographic separation of enantiomers on chemically bonded type of cellulose derivative chiral stationary phases with a positively charged spacer

Positively charged chiral stationary phases (CSPs) were prepared for capillary electrochromatography (CEC) separation of enantiomers by chemically immobilizing cellulose derivatives onto diethylenetriaminopropylated silica (DEAPS) with tolylene-2,4-diisocyanate (TDI) as a spacer reagent. Anodic electroosmotic mobility was observed in both nonaqueous and aqueous mobile phases due to the positively charged amines on the surface of the prepared CSPs. For comparison, the traditionally used 3-aminopropyl silica (APS) was also adopted as the base material instead of DEAPS to prepare CSP. It was observed that the EOF on the DEAPS-based CSP was 18%-60% higher than that on the APS-based CSP under nonaqueous mobile phase conditions. Separation of enantiomers in CEC was performed on the positively charged CSPs with the nonaqueous mobile phases of pure ethanol or mixture of hexane-alcohol and the aqueous phases of acetonitrile-water or 95% ethanol. Fast separation of enantiomers was achieved on the newly prepared CSPs.     

Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g⁻¹ and 0.40 mmol g⁻¹, respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1′-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.     

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal–organic frameworks (MOFs) promising as novel stationary phases for high‐performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF‐packed columns. Herein, the fabrication of monodisperse MOF@SiO₂ core–shell microspheres as the stationary phase for HPLC to overcome the above‐mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF‐8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl‐modified silica spheres were used as the support to grow the ZIF‐8 shell. The fabricated monodisperse ZIF‐8@SiO₂ packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23 000 plates m^?1 for bisphenol A) for the HPLC separation of endocrine‐disrupting chemicals (bisphenol A, β‐estradiol, and p‐(tert‐octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01–0.39, 0.65–1.7, 0.70–1.3, and 0.17–0.91 % for retention time, peak area, peak height, and half peak width, respectively). The ZIF‐8@SiO₂ microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF‐8 crystals.     

Monolithic Silica Columns for HPLC,Micro‐HPLC,and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR‐FS) and the other prepared in a mold and clad with an engineering plastic (poly‐ether‐ether‐ketone) (SR‐PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5‐μm ODS‐silica particles and of an ODS‐silica monolith prepared in a mold and wrapped with PTFE tubing (SR‐PTFE). SR‐FS gave a lower pressure drop than a column packed with 5‐μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR‐PEEK showed higher flow‐resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8–10 μm and a lower pressure drop than popular commercial columns packed with 5‐μm particles. The evaluation of SR‐FS columns in a CEC mode showed much higher efficiency than in a pressure‐driven mode.