Haizhi Zhang

Ionic liquids with amino acids as cations: novel chiral ligands in chiral ligand-exchange capillary electrophoresis

Ionic liquids (ILs) with L-proline (L-Pro) as cations have been developed for the novel chiral ligands coordinated with Cu(II) in chiral ligand exchange capillary electrophoresis (CLE-CE). Four kinds of amino acid ionic liquids (AAILs), including [L-Pro][CF(3)COO], [L-Pro][NO(3)], [L-Pro][BF(4)] and [L-Pro(2)][SO(4)], were successfully synthesized. Among them, [L-Pro][CF(3)COO] was selected as the model ligand to optimize the separation conditions. The influences of AAIL concentration, pH, and methanol concentration on efficiency of chiral separation were investigated. Then it has been testified that the optimal buffer solution consisted of 25.0mM Cu(Ac)(2), 50.0 mM AAIL and 20% (v/v) methanol at pH 4.0. The interesting thing is well enantioresolution could be observed with [L-Pro][CF(3)COO] as the new chiral ligand and nine pairs of labeled D,L-AAs were successfully separated with the resolution ranging from 0.93 to 6.72. Meanwhile, the baseline separation of labeled D,L-AAs could be achieved with the other three kinds of AAILs as ligands. The results have demonstrated the good applicability of AAILs with AAs as cations for chiral separation in CLE-CE system. In addition, comparative study was also conducted for exploring the mechanism of the AAILs as new ligands in CLE-CE.

A chiral ligand exchange CE essay with zinc(II)–l-valine complex for determining enzyme kinetic constant of l-amino acid oxidase

A new strategy for the enantioseparation of D,L-amino acids employing the principle of ligand exchange capillary electrophoresis with Zn(II)-L-valine complex as a chiral selecting system in the presence of beta-cyclodextrin has been designed. Successful enantioseparation of label free and labeled amino acids have been achieved with a buffer of 100.0mM boric acid, 5.0mM ammonium acetate, 4.0mM beta-cyclodextrin, 4.0mM ZnSO(4) and 8.0mM L-valine at pH 8.1. This new method was shown to be applicable to the quantitative analysis of label free D- and L-aromatic amino acids. Furthermore, the expanding enzymatic use of L-amino acid oxidase to incubate with different L-amino acids has allowed understanding of the substrates specificity. An on-column incubation assay has been developed to study the L-amino acid oxidases catalytic efficiency. It was demonstrated that the enzyme kinetic constant could be determined by using this new method.

High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants

A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of D,L-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of D-tryptophan, with a linear range between 12.0 microM and 979.0 microM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion.

Chiral separation using capillary electromigration techniques based on ligand exchange principle

Over the last couple of decades, researchers have developed diverse chiral separation methods emerged from a few chiral separation principles. This review article is primarily focused on the application of chiral ligand-exchange (CLE) principle in capillary electromigration techniques, such as capillary electrophoresis (CE) and capillary electrochromatography (CEC). First, the most commonly used CLE-CZE separation mode by using different kinds of central ions, such as Cu(II), Zn(II), borate ion, and other metal ions, has been introduced. Meanwhile, several kinds of surfactants have been applied as the micelle-forming agents in the CLE micellar electrokinetic chromatography mode. The highlight of recent research of CLE-CEC is the exploitation of novel columns for chiral separation. Then, two kinds of capillary columns, packed capillary and monolithic capillary column, have been briefly described. Finally, the effective application of these chiral separation methods has been presented, including the application in life science and food analysis area.

Determination of sodium benzoate by chiral ligand exchange CE based on its inhibitory activity in D-amino acid oxidase mediated oxidation of D-serine.

A novel quantitative approach for the determination of sodium benzoate (SB) was proposed by the kinetic study about its competitive inhibitory efficiency to D-amino acid oxidase (DAAO) activity with a chiral ligand exchange capillary electrophoresis (CE) method, in which the Zn(II)-L-prolinamide complex was chosen as a novel chiral selector. After the optimization of buffer pH and the chiral selector concentration this chiral ligand exchange CE method was employed to determine labeled D,L-Serine with good linearity (r(2)≥0.995), efficient recovery (95.6-100.9%) and remarkable reproducibility (RSD≤1.2%). This chiral separation method was further used to observe DAAO activity through the determination of D-Serine concentration variation after being incubated with DAAO and obtain the sigmoidal inhibitory curve of SB to DAAO activity. The ascending part of this inhibitory curve was linearly fitted in a limited range for SB from 2.0 to 200 μM with an appropriate coefficient of determination (R(2)=0.990). The linearity was then validated to be a promising method for the analysis of SB with the standout merits of high selectivity and adjustable detection range. Furthermore, this proposed method was used for the pharmacokinetics study of SB.

A novel amphipathic block copolymer coating forming micelle-like aggregates for separation of steroids in open tubular capillary electrochromatography

A new amphipathic block copolymer, poly(tert-butyl acrylate)(127)-block-poly(glycidyl methacrylate)(86), was developed for the coating in open tubular capillary electrochromatography. The self-assembly characters of the coating, which could form micelle-like aggregates under proper conditions, were observed by atomic force microscopy. Compared with bare capillary, this coating could act as surfactant and lead to improve the separation of steroids. In addition, the influence of pH, buffer concentration and organic solvents on the separation was investigated. The best separation of the three model steroid analytes could be achieved using 20.0mM borate buffer at pH 10.5. For covalent bonding, the coating showed good repeatability and stability with RSD of u(EOF) less than 3.3%. Then, this proposed method was well validated with good linearity (≥ 0.999), recovery (91.0-94.0%) and repeatability, and was successfully used for separation of steroids in spiked serum samples, which indicated that this new OT-CEC method could provide a potential tool to determine steroids in real biological system without interference.

Influence of ionic liquids as electrolyte additives on chiral separation of dansylated amino acids by using Zn(II) complex mediated chiral ligand exchange CE

In this work, investigation of the comparative influence of diverse ionic liquids (ILs) as electrolyte additives on the chiral separation of dansylated amino acids by using Zn(II)-L-arginine complex mediated chiral ligand exchange CE (CLE-CE) was conducted. It has been found that not only the varied substituted group number, but also the alkyl chain length of the substituted group on imidazole ring in the structure of ILs show different influence on chiral separation of the analytes in the CLE-CE system, which could be understood by their direct influence on EOF. Meanwhile, the variation of anion in the structure of ILs displayed remarkably changed performance and the ILs with Cl(-) showed the most obvious promoting effect on the chiral separation performance. Among the investigated seven ILs, 1-butyl-3-methylimidazolium chloride was validated to be the proper electrolyte additive in the CLE-CE system. Moreover, it has been observed that 1-butyl-3-methylimidazolium chloride also has obvious promotive effect on the labeling performance. The results have demonstrated that the ILs with different structures have important relation to their performance in CLE-CE and to their labeling efficiency in dansylation of the analytes.

l‐Lysine‐derived ionic liquids as chiral ligands of Zn(II) complexes used in ligand‐exchange CE

Amino acid ionic liquids (AAILs) with l‐lysine (l‐Lys) as anion were synthesized and applied as new chiral ligands in Zn(II) complexes for chiral ligand‐exchange CE. After effective optimization, baseline enantioseparation of seven pairs of dansylated amino acids was achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [C6mim][l‐Lys] at pH 8.2. To validate the unique behavior of AAILs, a comparative study between the performance of Zn(II)‐l‐Lys and Zn(II)‐[C6mim][l‐Lys] systems was conducted. In Zn(II)‐[C6mim][l‐Lys] system, it has been found that the improved chiral resolution could be obtained and the migration times of the three test samples were markedly prolonged. Then the separation mechanism was further discussed. The role of [C6mim][l‐Lys] indicated clearly that the synthesized AAILs could be used as chiral ligands and would have potential utilization in separation science in future.

Study on alanine aminotransferase kinetics by microchip electrophoresis

Alanine aminotransferase (ALT), which catalyzes the reversible conversion between L-glutamic acid (L-Glu) and L-alanine (L-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (L-Glu and L-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of L-Glu and L-Ala was conducted and limits of detection (signal/noise=3) for L-Glu and L-Ala were 4.0 × 10⁻⁷ and 2.0 × 10⁻⁷ M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (L-Ala and L-Glu), and the K(m) and V(max) values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.

Development of a new open‐tubular capillary electrochromatography method for in vitro monitoring of toxic aromatic amines distribution in rat blood

Exposure to aromatic amines from different industrial and agricultural activities entails a substantial risk of deleterious somatic effects, genetic damage and cancer development. Thus, a new and simple method for separation and analysis of aromatic amines has been developed by open-tubular capillary electrochromatography with a novel amphipathic block copolymer (poly(tert-butyl acrylate)(127)-block-poly(glycidyl methacrylate)(86)) coating based on its self-assembled property. Key factors affecting the separation efficiency of the test analytes, such as pH, buffer concentration and selective solvent, were studied in detail. Meanwhile, method validation was well evaluated by linearity (≥0.998), detection limit and recovery. Application of this developed protocol on in vitro monitoring of the target aromatic amines distribution in rat blood demonstrated its potential usage for separation and determination of aromatic amines in biological samples. Additionally, for assimilating more polymeric materials into analysis of aromatic amines, the effect of morphology changes of the amphipathic block copolymer coating on open-tubular capillary electrochromatography separation was also studied, and the result revealed that the block copolymer coating could play the same role as surfactant.