Capillary Electrochromatography Enantioseparation Research Articles

Construction of chiral crown ethers in robust covalent organic frameworks for electrochromatographic enantioseparation

ABSTRACT Capillary electrochromatography (CEC) is a rapidly emerging separation technique that merges the high separation efficiency of capillary electrophoresis with the exceptional selectivity of liquid chromatography. However, it remains a synthetic challenge to design functional chiral stationary phases (CSPs) with high chemical stability against acid and base in CEC enantioseparation. Here we demonstrate that incorporating chiral crown ethers into stable covalent organic frameworks (COFs) enables efficient and stable separations of racemates by CEC. This facilitates the crafting of two three-dimensional (3D) chiral COFs by polycondensation of a chiral 1,1'-binaphyl-20-crown-6-derived dialdehyde and tetraamines with diisopropyl substituents. Both feature an 11-fold interpenetrated diamond framework, characterized by tubular open channels decorated with chiral crown ethers serving as enantioselective recognition and binding sites. These frameworks demonstrate excellent stability in water, acid and base, thanks to the presence of bulky isopropyl groups that shield the dynamic imine linkages. Moreover, the precisely defined COF channels enhanced the accessibility of the enclosed crown ethers to the analytes while providing strong protection against harsh environments, rendering them suitable for CSPs in CEC separations. They can effectively separate some important enantiomers, including ketones, epoxides and alkaline substances, when utilized as coatings on chiral columns, particularly facilitating the chiral separation of drugs. This study advances the application of COFs in electrochromatographic separations, expanding the scope of porous materials design and engineering to create COFs with targeted enantioselective properties.

Preparation of D-histidine modified zeolitic imidazolate framework-90 coated capillary column and its application in open-tube capillary electrochromatography enantioseparation.

Metal-organic framework materials are a class of novel crystalline porous materials with regular pore structures formed by covalent bonding between metal centers and organic functional groups. Metal-organic framework materials have attracted great interest in analytical chemistry due to their unique properties such as good stability and permanent porosity. In this work, D-histidine was used to carry out chiral modification of zeolitic imidazolate framework-90 under mild conditions, and the D-histidine modified zeolitic imidazolate framework-90 coated capillary column was prepared. This chiral capillary column was used to separate epinephrine, norepinephrine, terbutaline, and tryptophan enantiomers. Under optimum conditions, baseline separations were achieved. The intra-day, inter-day, and inter-column relative standard deviations (n=3) of the four pairs of enantiomeric migration times were 0.15%-0.56%, 0.74%-2.40%, and 1.93%-3.18%, respectively. Moreover, the D-histidine modified zeolitic imidazolate framework-90 coated capillary could be reused for at least 150 runs without significant changes in the separation efficiency and migration time.

Synthesis of a novel chiral DA-TD covalent organic framework for open-tubular capillary electrochromatography enantioseparation.

Herein, a novel chiral covalent organic framework, DA-TD COF, with good chemical/thermal stability was synthesized and used as a chiral stationary phase for open-tubular capillary electrochromatography enantioseparation. The DA-TD COF coated capillary exhibited excellent enantioseparation efficiency and its separation efficiency did not show an obvious decrease over 200 runs. Furthermore, the enantioseparation mechanism was studied.

Preparation and modeling study of novel carboxymethyl-β-cyclodextrin silica hybrid monolithic column for enantioseparations in capillary electrochromatography

In this paper, an anionic carboxymethyl-β-cyclodextrin (CM-β-CD) silica hybrid monolithic column was first prepared by “one-pot” sequential reaction. The chiral stationary phase (CSP) and prepared monolithic column were demonstrated by fourier transform infrared (FT-IR), nuclear magnetic resonance (1H NMR) and scanning electron microscope (SEM). To obtain better separation efficiency, the columns’ preparation conditions (DMSO, AIBN and PEG) and separation conditions (buffer pH, ACN proportion, separation voltage and buffer solution) were optimized using model analytes. The resulting columns were finally used in chiral separations of ten racemic compounds, and satisfactory separation selectivity was obtained. The RSDs lower than 3.0% were obtained through run-to-run (n = 6), day-to-day (n = 3), and column-to-column determinations (n = 3). Furthermore, the mechanism of chiral recognition was demonstrated using molecular modeling with AutoDock. This proposed method was also well validated with good linearity (≥0.998), recovery (96.79–97.88%) and repeatability, and was successfully used for enantiomeric impurity detection of S-ofloxacin tablet. As a novel chiral stationary phase, CM-β-CD was supposed to be a promising prospect in the further analysis of chiral samples.

In-situ and one-step preparation of protein film in capillary column for open tubular capillary electrochromatography enantioseparation

In this work, the phase-transitioned BSA (PTB) film using the mild and fast fabrication process adhered to the capillary inner wall uniformly, and the fabricated PTB film-coated capillary column was applied to realize open tubular capillary electrochromatography (OT-CEC) enantioseparation. The enantioseparation ability of PTB film-coated capillary was evaluated with eight pairs of chiral analytes including drugs and neurotransmitters, all achieving good resolution and symmetrical peak shape. For three consecutive runs, the relative standard deviations (RSD) of migration time for intra-day, inter-day, and column-to-column repeatability were in the range of 0.3%–3.5%, 0.2%–4.9% and 2.1%–7.7%, respectively. Moreover, the PTB film-coated capillary column ran continuously over 300 times with high separation efficiency. Therefore, the coating method based on BSA self-assembly supramolecular film can be extended to the preparation of other proteinaceous capillary columns.

Chiral metal-organic cages used as stationary phase for enantioseparations in capillary electrochromatography.

Since some metal-organic cages (MOCs) have been synthesized in past several years, the applications of MOCs such as drug delivery, molecular recognition, separation, catalysis, and gas storage, etc. have been witnessed with a significant increase. However, to the best of our knowledge, so far no one has used MOCs as chiral stationary phase to separate chiral compounds in CEC. In this study, three MOCs were developed as the stationary phase for CEC separation of enantiomers. The MOCs coated capillary column showed good chiral recognition ability for some chiral compounds, including amine, alcohols, ketone, etc. The influence of buffer concentration, applied voltage, pH of buffer solution on the chiral separations was also investigated. The RSDs of run-to-run, day-to-day, and column-to-column for retention time were 2.1-4.67%, 1.2-4.36%, and 3.62-6.43%, respectively. This work reveals that the chiral MOCs material is feasible for the enantioseparation in CEC.

A rapid enantioseparation system of capillary electrochromatography modified by electrostatic adsorption with transfersomes.

Transfersomes were a special kind of nanomaterials with higher deformability and flexibility. A rapid method for coated-column preparation using anionic transfersomes as a coating material by electrostatic adsorption was developed. With carboxymethyl-β-cyclodextrin added in running buffer as the chiral selector, the capillary electrochromatography enantioseparation system based on the transfersomes-coated column modified by electrostatic adsorption was established for the first time. Propranolol and metoprolol acted as model drugs to evaluate the enantioseparation performance, these two basic drugs achieved baseline separation with satisfactory resolution and selection factor in this transfersomes-electrochromatography system but only partial separation in bare column system. In order to get the optimal separation condition, concentration of chiral selector, buffer pH, and applied voltage were systematically investigated. A rapid and efficient enantioseparation electrochromatography system was established and showed that transfersomes as the stationary phase could efficiently improve chiral separation effect.

Enantioseparation of basic drugs by capillary electrochromatography using a stationary phase of transfersomes

AbstractTransfersomes, a special kind of phospholipid vesicles with higher deformability and flexibility, were applied as coating material to prepare a transfersomes‐coated capillary column. A capillary electrochromatography enantioseparation system based on the new transfersomes‐coated column with carboxymethyl‐β‐cyclodextrin added to the running buffer as a chiral selector was established for the first time. Compared with a bare column, three basic drugs achieved baseline separation with better resolution (3.17 for Propranolol, 2.38 for Metoprolol, 2.34 for Esmolol) in this transfersomes‐electrochromatography system. Several important parameters including concentration of chiral selector, buffer pH and applied voltage were systematically investigated to provide the optimal separation condition. This study constructed a flexible and efficient enantioseparation electrochromatography system with a small amount of transfersomes, and showed that transfersomes as the stationary phase could efficiently improve chiral separation effect.

In situ immobilization of sulfated-β-cyclodextrin as stationary phase for capillary electrochromatography enantioseparation

In this work, a novel sulfated-β-cyclodextrin (S-β-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-β-cyclodextrin (PDA/S-β-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-β-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%.

A novel coating method for CE capillary using carboxymethyl-β-cyclodextrin-modified magnetic microparticles as stationary for electrochromatography enantioseparation.

Magnetic microparticles (MMPs) have been extensively studied and aroused considerable interest in separation science owing to their superior characteristics. In this paper, a novel coated capillary with carboxymethyl-β-cyclodextrin-functionalized magnetic microparticles (CD-MMPs) as stationary phase was constructed and then applied to establish an open-tubular capillary electrochromatography enantioseparation system. The preparation of the CD-MMP-coated open-tubular column was very convenient because the coating of the magnetic microparticles onto the capillary column could be easily manipulated by an external magnetic field. The preparation conditions of the coated capillary such as magnetic field intensity and coating time are discussed in detail. The new constructed CD-MMP capillary system was applied to separate enantiomers of several racemic drugs. Compared to the uncoated capillary system, obviously preferable separations of tested enantiomers were obtained. Several important parameters affecting the enantioseparation, such as CM-β-CD concentration, running buffer pH, organic solvent, and applied voltage, were systematically optimized. Furthermore, satisfactory repeatability and chemical stability of this new CD-MMP capillary system were achieved in the experiment. Graphical abstract ᅟ.

The preparation of poly-levodopa coated capillary column for capillary electrochromatography enantioseparation

Levodopa (L-DOPA) is promising as chiral stationary phase for open tubular capillary electrochromatography (OT-CEC) enantioseparation owing to its self-polymerization adhesive property and chiral recognition potential. In this work, CuSO4/H2O2 was used as a trigger agent to accelerate the polymerization process of L-DOPA and the poly-levodopa (poly-(L-DOPA)) coated column was successfully prepared for the first time by depositing it on the inner wall of fused silica capillary via the rapid and in-situ approach at room temperature. The performance of the poly-(L-DOPA) coated capillary was validated by the separation of different chiral analytes, including chiral amine drugs, neurotransmitters and amino acids, and the good enantioseparation efficiencies were achieved. For five consecutive runs, the relative standard deviations (RSDs) for the migration time of the analytes for intra-day, inter-day and column-to-column were in the range of 0.19–1.33%, 0.96–4.47%, and 2.21–7.79%, respectively. Additionally, the poly-(L-DOPA) coated capillary column could be successively used over 250 runs without observable change in the separation efficiency.

Nano-amylose-2,3-bis(3,5-dimethylphenylcarbamate)-silica hybrid sol immobilized on open tubular capillary column for capillary electrochromatography enantioseparation.

The chiral organic-inorganic hybrid materials can exhibit a high loading, and the chiral selector nanoparticles can create efficient stationary phases for open-tubular capillary electrochromatography (OT-CEC). Hence, a novel protocol for the preparation of an OT column coated with nano-amylose-2,3-bis(3,5-dimethylphenylcarbamate) (nano-ABDMPC)-silica hybrid sol through in situ layer-by-layer self-assembly method was developed for CEC enantioseparation. By controlling the assembly cycle number of nano-ABDMPC-silica hybrid sol, a homogeneous, dense and stable coating was successfully prepared, which was confirmed by SEM and elemental analysis. As the main parameter influencing the chiral separating effect, the nano-ABDMPC bearing 3-(triethoxysilyl)propyl residues concentration was investigated. The experimental results showed that 10.0 mg/mL nano-ABDMPC bearing 3-(triethoxysilyl)propyl residues coated OT capillary column possessed chiral recognition ability toward the six enantiomers (phenylalanine, tyrosine, tryptophan, phenethyl alcohol, 1-phenyl-2-propanol, and Tröger's base) at some of the different conditions tested. Additionally, the coated OT column revealed adequate repeatability concerning run-to-run, day-to-day and column-to-column. These results demonstrated the promising applicability of nano-ABDMPC-silica hybrid sol coated OT column in CEC enantioseparations.

Homochiral zeolite-like metal-organic framework with DNA like double-helicity structure as stationary phase for capillary electrochromatography enantioseparation

In recent years, artificial materials with double helix structure have attracted widespread attention due to their unique properties such as the DNA like double-helicity, intrinsic chirality and diverse functional groups. Developing novel chiral stationary phases (CSPs) for capillary electrochromatography enantioseparation is of intriguing interest. Herein, a novel homochiral zeolite-like metal-organic framework (ZMOF) JLU-Liu23 with unique DNA like double-helicity structure was firstly utilized as the CSP in open tubular capillary electrochromatography (OT-CEC) for enantioseparation of chiral monoamine neurotransmitters and analogues. Owing to the unique DNA like double-helicity structure of the homochiral ZMOF JLU-Liu23, the good enantioseparation of four monoamine neurotransmitters and analogues was achieved on the prepared homochiral ZMOF JLU-Liu23 coated capillary column. The relative standard deviations (RSDs) of the analytes migration time for intra-day, inter-day and column-to-column were in the range of 0.3–0.6%, 0.8–2.2% and 3.5–6.5%, respectively.

Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation

Bacteria, the microorganism with intrinsic chirality, have numerous fascinating chiral phenomena such as various chirality-triggered biological processes and behaviors. Herein, bacteria were firstly explored as novel chiral stationary phases in open-tubular capillary electrochromatography (OT-CEC) for enantioseparation of fluoroquinolone enantiomers and simultaneous separation of six fluoroquinolone antibiotics. The model strain, i.e. non-pathogenic Escherichia coli (E. coli) DH5α, was adhered onto the inner surface of positively charged polyethyleneimine (PEI) modified capillaries based on the bacterial adhesion characteristics and strong electrostatic interaction. The morphology and thickness of the bacteria adhesive coatings in the capillary were characterized by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). Baseline separation of ofloxacin and partial separation of lomefloxacin enantiomers could be achieved by the E. coli coated columns. The preparation parameters including the coating time and concentration of bacteria that affecting the chiral resolution were intensively investigated. The electrophoretic parameters, including pH, buffer concentration and applied voltage, were also optimized. The developed method was validated (linearity, LOD, LOQ, intra-day, inter-day and column-to-column repeatability and recovery) and successfully utilized for the quantitative analysis of ofloxacin enantiomers in the ofloxacin tablets. Moreover, only a slight decrease in the separation efficiency was observed after 90 consecutive runs on the E. coli@capillary. These results demonstrated that bacteria are promising stationary phases for chiral separation in CEC.

One-step synthesis of mussel-inspired molecularly imprinted magnetic polymer as stationary phase for chip-based open tubular capillary electrochromatography enantioseparation

A facile approach for preparation of molecularly imprinted polymers was developed and successfully used as chiral stationary phase for rapid enantioseparation by open tubular capillary electrochromatography (OT-CEC). In this work, molecularly imprinted polymers were one-step prepared employing Fe3O4 nanoparticles (NPs) as the supporting substrate and dopamine as the functional monomer. By simply mixing Fe3O4 NPs with template molecules in a weak alkaline solution of dopamine, a thin adherent polydopamine (PDA) film imprinted with template molecules was formed by the self-polymerization of dopamine on the surface of Fe3O4 NPs. After extracting the embedded template molecules, the produced imprinted Fe3O4@PDA NPs are of three dimensional shape of template molecules favoring high binding capacity and magnetism property for easy manipulation. The imprinted Fe3O4@PDA NPs prepared with l-tryptophan, l-tyrosine, Gly-l-Phe or s-ofloxacin as template molecules were packed in the PDMS microchannel via magnetic field as novel stationary phase for the successful enantioseparation of corresponding target analysts. In addition, the imprinted Fe3O4@PDA NPs-based OT-CEC system exhibited excellent reproducibility, stability and repeatability, which provides a powerful protocol for separation enantiomers within a short analytical time and opens up a promising avenue for high-throughput screening of chiral compounds.

Facile preparation of protein stationary phase based on polydopamine/graphene oxide platform for chip-based open tubular capillary electrochromatography enantioseparation

A novel chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC) was developed employing bovine serum albumin (BSA) conjugated polydopamine–graphene oxide (PDA/GO) nanocomposites (PDA/GO/BSA) as stationary phase. After the poly(dimethylsiloxane) (PDMS) microfluidic chip was filled with a freshly prepared solution containing dopamine and graphene oxide, PDA/GO nanocomposites were formed and deposited on the inner wall of microchannel as permanent coating via the oxidation of dopamine by the oxygen dissolved in the solution. The PDA/GO-coated PDMS microchips not only have the adhesion of PDA that make them easily immobilized in the microchannel, but also have the larger surface and excellent biocompatibility of graphene which can incorporate much more biomolecules and well maintain their biological activity. In addition, incorporation of GO in PDA film can make surface morphology more rough, which is beneficial for enhancing the loading capacity of proteins in the microchannels and increasing sample capacity of OT-CEC columns. BSA was stably immobilized in the PDMS microchannel to fabricate a protein-stationary phase. Compared with the native PDMS microchannels, the modified surfaces exhibited much better wettability, more stable electroosmotic mobility, and less nonspecific adsorption. The efficient separation of chiral amino acids (tryptophan and threonine) and chiral dipeptide demonstrate that the constructed OT-CEC columns own ideal enantioselectivity. The presented strategy using PDA/GO coating as a versatile platform for facile conjugation of proteins may offer new processing strategies to prepare a functional surface designed on microfluidic chips.

A versatile polydopamine platform for facile preparation of protein stationary phase for chip-based open tubular capillary electrochromatography enantioseparation

A novel, simple, and economical method for the preparation of chiral stationary phases for chip-based enantioselective open tubular capillary electrochromatography (OT-CEC) using polydopamine (PDA) coating as an adhesive layer was reported for the first time. After the poly(dimethylsiloxane) (PDMS) microfluidic chip was filled with dopamine (DA) solution, PDA film was gradually formed and deposited on the inner wall of microchannel as permanent coating via the oxidation of DA by the oxygen dissolved in the solution. Due to possessing plentiful catechol and amine functional groups, PDA coating can serve as a versatile multifunctional platform for further secondary reactions, leading to tailoring of the coatings for protein bioconjugation by the thiols and amines via Michael addition or Schiff base reactions. Bovine serum albumin (BSA), acting as a target protein, was then stably and homogeneously immobilized in the PDA-coated PDMS microchannel to fabricate a novel protein stationary phase. Compared with the native PDMS microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and electroosmotic flow of PDA/BSA-coated PDMS substrate were measured to be 44° and 2.83×10−4cm2V−1s−1, compared to those of 112° and 2.10×10−4cm2V−1s−1 from the untreated one, respectively. Under a mild condition, d- and l-tryptophan were efficiently separated with a resolution of 1.68 within 130s utilizing a separation length of 37mm coupled with in-column amperometric detection on the PDA/BSA-coated PDMS microchips. This present versatile platform, facile conjugation of biomolecules onto microchip surfaces via mussel adhesive protein inspired coatings, may offer new processing strategies to prepare a biomimetic surface design on microfluidic chips, which is promising in high-throughput and complex biological analysis.

Hybrid monolithic columns coated with cellulose tris(3,5-dimethylphenyl-carbamate) for enantioseparations in capillary electrochromatography and capillary liquid chromatography

A hybrid monolithic capillary column synthesized with (3-chloropropyl)-trimethoxysilane (CPTMS) and tetramethoxysilane (TMOS) via sol–gel chemistry was in situ coated with cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) for enantioseparations in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC). Prior to coating, the prepared CP-silica hybrid monolith was straightforwardly modified with diethylenetriamine (DETA) to introduce NH2 functionalities via the nucleophilic substitution reaction, which generate the stronger EOF for CEC. The coating condition was optimized to obtain a stable and reproducible chiral stationary phase for enantioseparation. The results indicated that racemic benzoin was baseline separated on the resulting hybrid monolith coated with 30mg/mL CDMPC in CEC, while several racemates were successfully enantioseparated on the resulting CP-silica hybrid monolithic column coated with 60mg/mL CDMPC in CLC with RP and NP modes.

Preparation of a positively charged cellulose derivative chiral stationary phase with copolymerization reaction for capillary electrochromatographic separation of enantiomers.

A positively charged chiral stationary phase (CSP) was prepared by chemically immobilizing cellulose 3,5-dimethylphenylcarbamate onto methacryloyldiethylenetriaminopropylated silica (MCDEAPS) via a radical copolymerization reaction. The prepared CSP was evaluated for enantiomer separation in nonaqueous capillary electrochromatography (CEC). Electroosmotic flow (EOF) generated on the prepared CSP could be significantly improved with introduction of positive charges into the CSP, and separation of enantiomers in CEC has been achieved with mobile phases of ethanol and hexane-ethanol, respectively. In addition, we investigated the solvent versatility of the immobilized CSP on enantioseparations in CEC and capillary liquid chromatography (CLC) due to the elimination of dissolution of chiral selector in a number of solvents. Chiral resolution of some enantiomers was improved by adopting tetrahydrofuran (THF) and chloroform as mobile-phase modifiers, respectively.

Enantioseparations in capillary liquid chromatography and capillary electrochromatography using amylose tris(3,5-dimethylphenylcarbamate) in combination with aqueous organic mobile phase

In this study the potential of amylose tris(3,5-Dimethylphenylcarbamate) (ADMPC) was evaluated for enantioseparations in capillary liquid chromatography (CLC) and capillary electrochromatography (CEC). This material was previously used for enantioseparations in CEC with nonaqueous alcohols and the first study in aqueous-organic mobile phase indicates its potential for CEC enantioseparations. The effect of various experimental parameters on the separation characteristics was investigated in both CLC and CEC mode. Under optimized conditions the plate numbers in CEC mode reached 500 000 plates/m for thiourea and exceeded 240 000 plates/m for the enantiomers of some chiral compounds investigated. Comparative enantioseparations performed in CLC and CEC mode revealed significant advantages of CEC.