Enantioselective liquid-liquid extraction of 2-cyclohexylmandelic acid enantiomers using chiral ionic liquids.

Obtaining optically pure compounds in an eco-friendly and cost-efficient manner plays an important role in human health and pharmaceutical industry. Racemic separation using multistage stereoselective liquid-liquid extraction has become one of the most practical and effective approach to access homochiral enantiomers. Currently, chiral ionic liquids (CILs) with structural designability have become a promising chiral additive and enable them as adjustable candidates for racemic separation. Herein, a high-effective stereoselective liquid-liquid extraction process composed of imidazolium cations and amino acid-derived anions as the chiral additive was established for racemic 2-cyclohexylmandelic acid (CHMA) separation. We have systematically investigated the choice of organic solvent, concentration of CIL, extraction temperature, and the pH of aqueous phase. For three-stage stereoselective extraction, the maximum enantiomeric excess (e.e.) for CHMA was reached up to 40.6%. Furthermore, the mechanism of steric effect and stereoselective capacity between the CILs and racemic CHMA was discussed and simulated. We envision that the work will facilitate the development of CILs in multistage liquid-liquid extraction and promote the large-scale production of optically pure enantiomers.

Lipase-based MIL-100(Fe) biocomposites as chiral stationary phase for high-efficiency capillary electrochromatographic enantioseparation.

A novel chiral porous column was fabricated by lipase immobilized MIL-100(Fe) biocomposites as chiral stationary phase through covalent coupling and applied to capillary electrochromatographic enantioseparation. MOF-based lipase biocomposites not only enhance stereoselective activities but also improve the stability and applicability of the enzyme. The functionalized porous columns were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and powder X-ray diffraction. The performance of the porous column was evaluated by enantioseparating amino acid enantiomers, affording high resolution over 2.0. Besides, the enantio-resolutions of phenylephrine, phenylsuccinic acid, chloroquine, and zopiclone were also greater than 2.0. The relative standard deviations of run-to-run, intra-, and inter-day repeatability were within 4.0% in terms of resolution and retention time, exhibiting excellent stability of the column. Conceivably, the results show that MOF-based lipase composites as chiral stationary phase offer a highly efficient means for enantioseparation in capillary electrochromatography, attributing to the enhanced enantioselective activities of lipase by highly ordered frameworks.

Growth of two-layer copolymer as the stationary phase with very high separation efficiency for separating peptides in capillary electrochromatography.

Open tubular CEC (OT-CEC) column with a very high separation efficiency was prepared for peptides separation. A pretreated silica-fused capillary was reacted with 3-(methacryloxy) propyltrimethoxysilane followed by vinylbenzyl chloride and divinylbenzene to produce first thin monolithic monolayer. The second copolymer layer was formed on thin monolithic monolayer of the capillary by reversible addition-fragmentation transfer polymerization of N-phenylacrylamide and styrene. The key parameters including buffer pH value and organic modifier were systematically evaluated to provide the optimal chromatographic condition. The resultant OT-CEC columns were validated by separating a synthetic mixture of peptides and cytochrome C tryptic digest in capillary electrochromatography. The number of theoretical plates as high as 2.4 million per column was achieved for synthetic mixture peptides. In addition, the fabricated OT-CEC column also resolved more than 18 high-efficiency digestion peptides from a mixture containing tryptic digest of cytochrome C. The column to column and inter- to intraday repeatabilities of OT-CEC column through RSD% were found better than 3.0%, exhibiting satisfactory stability and repeatability of the two-layer deposited OT-CEC column. The results reveal that the open tubular capillary column modified with two-layer copolymer shows the great prospect for the separation of proteins in capillary electrochromatography.

Hydroxypropyl-ß-cyclodextrin encapsulated stationary phase based on silica monolith particles for enantioseparation in liquid chromatography.

Hydroxypropyl-ß-cyclodextrin-encapsulated stationary phase incorporated on silica monolith particles was prepared by physical embedding, providing a new method for the development of chiral stationary phase for enantioseparation in liquid chromatography. Ground silica monolith particles of about 2.0 µm were prepared via sol-gel reaction followed by differential sedimentation. Initially, the silica monolith particles were pretreated with 3-trimethoxysilyl propyl methacrylate to attach double-bonded ligands onto the surface, then a network structure was formed onto the surface of the particle using N-isopropyl acrylamide as functional monomer. Hydroxypropyl-ß-cyclodextrin was encapsulated inside N-isopropyl acrylamide copolymerized layer on the surface of silica monolith particles. The effect of the amount of chiral selector on the chromatographic efficiency of the chiral stationary phase was examined. The glass lined stainless steel columns (1 mm internal diameter, 300 mm length) were packed with the stationary phase for estimation of the efficiency by separation of phenylsuccinic acid, oxybutynin, equol, and naproxen enantiomers in high-performance liquid chromatography, with the resolutions of 1.54, 1.72, 2.54, and 2.31, respectively. The column to column repeatabilities through relative standard deviation were found better than 3%. The experimental results indicate that the sol-gel ground silica particles modified with ß-cyclodextrin provide a promising way for the separation of chiral enantiomers.

Fast liquid chromatography method for separation of peptides using a sub-2 µm ground silica monolith packed column.

A stationary phase based on sub-2 µm ground silica monolith particles was fabricated by in situ polymerization and applied in micro-column for separation of peptides. The sub-2 µm silica particles were obtained from monolith using sol-gel process followed by grinding and sedimentation to remove the fines. Initially, the silica monolith particles were pretreated with 3-trimethoxysilyl propyl methacrylate to attach double-bonded ligands onto the surface, then a network structure was formed onto the surface of the particle using styrene, N-isopropylacrylamide, and ethylene glycoldimethacrylate. The effect of the flow rate of the mobile phase on the separation performance was investigated. The stationary phase was characterized by field emission scanning electron microscopy, thermogravimetry, particle size distribution, and element analysis. The resultant phase was packed in glass-lined stainless steel micro-columns (2.1 mm × 50 mm) and evaluated for fast separation. An average number of theoretical plates as high as 9800 plates/column (5.10 µm plate height) was achieved for five synthetic peptides under the optimized flow rate of 0.15 mL/min. The repeatabilities of column-to-column, intraday, and interday through relative standard deviation were found better than 4%, exhibiting satisfactory repeatability of the developed micro-column for fast separation of peptides.

Open tubular capillary column immobilized with sulfobutylether-ß-cyclodextrin for chiral separation in capillary electrochromatography.

A novel chiral open tubular capillary column was fabricated with sulfobutylether-ß-cyclodextrin and glycidyl methacrylate for enantioseparation in capillary electrochromatography. First, the pretreated silica-fused capillary was treated with 3-trimethoxysilyl propyl methacrylate to attach double bond ligand onto the surface. A copolymer layer was formed on the surface of capillary using glycidyl methacrylate and ethylene dimethacrylate by in situ one-pot polymerization. Sulfobutylether-ß-cyclodextrin was encapsulated inside the copolymerized layer. The morphology of the developed column was characterized by field emission scanning electron microscopy. The effect of organic percentage and pH value of the mobile phase on electroosmotic flow and resolution was also investigated. The performance of the fabricated column was validated by separation of amlodipine besilate, 2,3-diphenylpropionic acid, tropic acid, and pantoprazole enantiomers with good resolutions of 3.67, 4.82, 3.34, and 2.61, respectively. The repeatabilities of column-to-column and day-to-day through relative standard deviation were found better than 4%, exhibiting satisfactory repeatability of the developed column. The results reveal that open tubular capillary columns modified with ß-cyclodextrin show a great prospect for enantioseparation of chiral drugs in capillary electrochromatography.

A novel one-pot strategy of preparation of N-phenylacrylamide-styrene co-polymer open tubular capillary column for peptides separation.

An open tubular capillary column (100 µm internal diameter and 120 cm length) was first fabricated with N-phenylacrylamide and styrene copolymer layer on the inner surface by in situ one-pot strategy, while ethylene dimethacrylate was used as the cross-linker. The pretreated silica-fused capillary was reacted with 3-trimethoxysilyl propyl methacrylate to attach a double-bond ligand onto the inner surface of the capillary. Further, a thick and stabile copolymer layer was generated on the inner surface of the capillary by the novel method in situ one-pot reaction of styrene-N-phenylacrylamide. The effects of reaction temperature and composition of the polymerization mixture on the morphology and permeability of the copolymer were investigated. The separation performance of the fabricated polymer columns were validated by separation of five synthetic peptides. The excellent efficiency (188 500 plates/m) of 100 µm internal diameter open tubular column with the separation media prepared by mixed one-pot copolymerization has not been achieved via the isocratic elution mode. The column to column, intraday, and interday repeatabilities evaluated from the relative standard deviation were found better than 4%, exhibiting considerable repeatability of the column.

Styrene-N-phenylacrylamide co-polymer modified silica monolith particles with an optimized mixing ratio of monomers as a new stationary phase for the separation of peptides in high performance liquid chromatography.

A stationary phase was prepared by chemical derivatization of the support particles with a layer of copolymer composed of styrene and N-phenyl acrylamide. Silica monolith particles of ca. 2.6 µm (volume-based average) have been prepared as the support particles by sol-gel reaction followed by differential sedimentation. The particles were reacted with 3-chloropropyl trimethoxysilane followed by sodium diethyldithiocarbamate to introduce an initiator moiety. Then, the copolymer layer was immobilized via reversible addition-fragmentation transfer polymerization. The resultant phase was packed in glass-lined stainless-steel micro-columns (1 x 150 mm) and evaluated for the separation of a mixture composed of five peptides (Trp-Gly, Thr-Tyr-Ser, angiotensin I, isotocin and bradykinin). The effect of monomer mixing ratio (styrene versus N-phenyl acrylamide) on the chromatographic separation efficiency of the stationary phase was examined. A number of theoretical plates (N) as high as 33 600 plates/column (224 000 plates/m, 4.46 µm plate height) was achieved using the column packed with the optimized stationary phase. The column-to-column reproducibility based on three columns packed with three different batches of stationary phase was found satisfactory in separation efficiency, retention factor, and asymmetry factor.

An optimized mixed-mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high-performance liquid chromatography.

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1-[3-(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow-bore glass-lined stainless-steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr-Tyr-Ser, Val-Ala-Pro-Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

Enantiomeric separation of oxybutynin by recycling high-speed counter-current chromatography with hydroxypropyl-ß-cyclodextrin as chiral selector.

Recycling high-speed counter-current chromatography was successfully applied to the preparative separation of oxybutynin enantiomers. The two-phase solvent system consisted of n-hexane, methyl tert-butyl ether, and 0.1 mol/L phosphate buffer solution (pH = 5.0) with the volume ratio of 6:4:10. Hydroxypropyl-ß-cyclodextrin was employed as the chiral selector. The influence of factors on the chiral separation process, including the concentration of chiral selector, the equilibrium temperature, the pH value of the aqueous phase were investigated. Under optimum separation conditions, 15 mg of oxybutynin racemate was separated with the purities of both the enantiomers over 96.5% determined by high-performance liquid chromatography. Recovery for the target compounds reached 80-82% yielding 6.00 mg of (R)-oxybutynin and 6.15 mg of (S)-oxybutynin. Technical details for recycling elution mode were discussed.

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. D-Isobutyl tartrate and hydroxypropyl-ß-cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two-phase solvent system was made up of n-hexane/methyl tert-butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.

In Vitro and In Vivo Evaluating Bioaccessibility, Bioavailability, and Antioxidant Activities of Butterfly Pea Flower Containing Bioactive Constitutes.

The antioxidant properties of butterfly pea flower (BF), which is rich in natural anthocyanins, have garnered significant attention. The impact of digestion and metabolism on BF extracts and evaluate their subsequent antioxidant activities in vivo were explored in the present study. After in vitro digestion, 42.03 ± 2.74% of total anthocyanins from BF extracts remained, indicating a negative influence of the digestion process on the bioaccessibility of phenolic compounds derived from BF. Furthermore, UPLC-LTQ-Orbitrap-MS2 analysis identified a total of four prototypes and twenty-seven metabolites in rat plasma or urine samples following the intake of BF extracts. The kinetics of key metabolites including delphinidin 3-glucoside (D3G), cyanidin-3-glucoside (C3G), and 4-hydroxybenzoic acid were subsequently determined in blood, and the Cmax values were 69.034 ± 8.05 nM and 51.65 ± 3.205 nM. These key metabolites derived from BF anthocyanins, including C3G and D3G, and flavonoid quercetin exhibited main antioxidant attributes that improved the plasmic and hepatic activities of various antioxidant enzymes and the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in a D-galactose-induced rat model. These findings provide insights into the bioaccessibility and bioavailability of bioactive constitutes derived from BF extracts, which are crucial for determining the actual efficacy of BF as well as developing functional foods based on BF.

Anthocyanin-Rich Butterfly Pea Flower Extract Ameliorating Low-Grade Inflammation in a High-Fat-Diet and Lipopolysaccharide-Induced Mouse Model.

This study aimed to explore the enhancive effects of butterfly pea flower (BF) extracts on metabolic and immune homeostasis in a low-grade inflammation mouse model. The BF extract was found to contain mainly anthocyanins among other flavonoids. BF supplementation alleviated metabolic endotoxemia by lowering the plasma glucose, lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α) levels and restored lipid metabolism and the balance between Treg and Th17 cells, thereby inhibiting the dysfunctional liver and abdominal white adipose tissues. BF extract increased the tight junction protein expression and reduced the expression of proinflammatory cytokines, therefore sustaining the colonic mucosa structure. Furthermore, BF extracts reshaped the gut microbiota structure characterized by significantly promoted SCFA-producing gut microbiota such as Akkermansia and Butyricicoccaceae. Additionally, BF extracts enhanced fecal primary bile acid (BA) levels and modulated bile acid signaling in the liver and ileum to facilitate BA synthesis for the restoration of lipid metabolism. In summary, anthocyanin-enriched BF extracts alleviated the profound negative dietary alterations and helped maintain the metabolic health by modulating the various aspects of the gut microenvironment and enhancing hepatic bile acid synthesis.

Enantioseparation by simultaneous biphasic recognition using mobile phase additive and chiral stationary phase in capillary electrochromatography.

Biphasic chiral recognition was first applied for the enantioseparation acidic drugs in capillary electrochromatography. The biphasic recognition system was composed of mobile phase additive (ß-cyclodextrin) and monolithic chiral stationary phase (proteins). The pretreated silica-fused capillary was treated with 3-trimethoxysilyl propyl methacrylate to attach double bond ligand onto the surface. The mixed monolithic chiral stationary phase was constructed with bovine serum albumin and pepsin using one-pot polymerization by chemical covalent coupling, while dimethyl sulfoxide and methanol were used as the progenic solvents. The effects of the type and concentration of ß-cyclodextrin additive as well as pH value of the mobile phase on the separation efficiency were optimized. The performance of the biphasic recognition system was validated by separating acidic drugs (such as ketoprofen, ibuprofen, loxoprofen, flurbiprofen and carprofen) in capillary electrochromatography, achieving outstanding separation efficiency. In terms of migration time and resolution, the run-to-run, intra-day, and inter-day repeatability through relative standards deviation were within 5.0%, exhibiting excellent stability of the biphasic recognition system. Conceivably, the experimental result reveals that biphasic chiral recognition capillary electrochromatography offers a promising prospect for enantioseparation of chiral compounds in a highly efficient manner.

Polystyrene Immobilized Sol-Gel Ground Silica Monolith Particles Using One-Pot Reaction of Enhanced Separation Efficiency.

The stationary phase based on sol-gel ground silica monolith particles has been produced by one-pot polymerization method incorporation of styrene and ethylene dimethacrylate. First, the ground silica monolith particles were prepared by a sol-gel process followed by sedimentation. The particles were then subjected to modify with styrene ligand via one-pot polymerization, whereas ethylene dimethacrylate was used as the cross-linker. The glass lined stainless steel columns (1 mm internal diameter, 150 mm length) were packed with the above phase for estimation of the chromatographic performance in high-performance liquid chromatography. An average number of theoretical plates of as high as 39,300 plates/column was obtained under the optimized elution condition. The column-to-column reproducibility was proved satisfactory in separation efficiency and retention factor. The experimental results indicate that sol-gel ground silica particles prepared by an aid of one-pot modification can provide a better way for preparation of highly efficient stationary phase.

Polystyrene bound silica monolith particles of reduced size as stationary phase of excellent separation efficiency in high performance liquid chromatograhy.

Ground silica monolith particles of quite smaller average size (2 µm) have been prepared by sol-gel process followed by soft grinding and calcination. Next a highly efficient chromatographic stationary phase has been prepared by reaction of those particles with (3-chloropropyl) trimethoxysilane followed by initiator attachment and modification of polystyrene by reversible addition-fragmentation chain transfer polymerization. The resultant phase of ca 3 µm particle size was packed in micro-columns (1.0 mm × 300 mm & 1.0 mm × 150 mm) to show the separation efficiencies as high as 67,600 and 35,500 plates/ column, respectively, for the separation of 5 small test molecules at a mobile phase flow rate of 25 µL/min. The 300 mm column shows a separation efficiency better than any of the commercially available conventional packed columns so far. Multiple shapes and high surface roughness as well as reduced particle size of the stationary phase of this study seem to contribute to such enhanced separation efficiency.