Purification of low‐concentration phenazine‐1‐carboxylic acid from fermentation broth of Pseudomonas sp. M18 via free flow electrophoresis with gratis gravity

The low‐concentration phenazine‐1‐carboxylic acid (PCA) (=0.3 mM) extracted from fermentation broth of Pseudomonas sp. M18 was selected to be purified with a newly facile free flow electrophoresis (FFE) device with gratis gravity. Three factors of pH value and concentration of background buffer, and the cooling circle of FFE device were investigated for the purification of PCA in the FFE device. It was found that the pH value and concentration of background buffer had mild influences on the separation of PCA whether with cooling circle or not. However, the cooling circle had a much greater impact on the separation of PCA. The controlling of the band zone of PCA in FFE chamber would be difficult if without cooling circle, while the controlling would become easy if with cooling circle. Under the optimal conditions (10 mM pH 5.5 phosphate as background buffer, 30 mM pH 5.5 phosphate buffer as electrode solution, 5.46 mL/min background flux, 10 min residence time of injected sample, and 500 V), PCA could be continuously prepared from its impurities with relative high purity. The flux of sample injection was 115 μL/min, viz. 7 mL sample throughput per hour, and the recovery was up to 85%. All of the experiments indicated that the FFE technique was a good alternative tool for the study on natural biological control agents.     

A simple preparative free‐flow electrophoresis joined with gratis gravity: I. Gas cushion injector and self‐balance collector instead of multiple channel pump

This paper describes a novel free‐flow electrophoresis (FFE), which is joined with gratis gravity, gas cushion injector (GCI) and self‐balance collector instead of multiple channel pump, for the purpose of preparative purification. The FFE was evaluated by systemic experiments. The results manifest that (i) even though one‐channel peristaltic pump is used for the driving of background buffer, there is still stable flow in the FFE chamber; (ii) the stable flow is induced by the gravity‐induced pressure due to the difference of buffer surfaces in the GCI and self‐balance collector; (iii) the pulse flow of background buffer induced by the peristaltic pump is greatly reduced by the GCI with good compressibility of included air; (iv) the FFE can be well used for zone electrophoretic separation of amino acids; (v) up to 20 inlets simultaneous sample injection and up to five to tenfold condensation of amino acid can be achieved by combining the FFE device with the method of moving reaction boundary. To the best of authors' knowledge, FFE has not been used for such separation and condensation of amino acids. The relevant results achieved in the paper have evident significance for the development of preparative FFE.     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

Negative‐pressure‐induced collector for a self‐balance free‐flow electrophoresis device

Uneven flow in free‐flow electrophoresis (FFE) with a gravity‐induced fraction collector caused by air bubbles in outlets and/or imbalance of the surface tension of collecting tubes would result in a poor separation. To solve these issues, this work describes a novel collector for FFE. The collector is composed of a self‐balance unit, multisoft pipe flow controller, fraction collector, and vacuum pump. A negative pressure induced continuous air flow rapidly flowed through the self‐balance unit, taking the background electrolyte and samples into the fraction collector. The developed collector has the following advantages: (i) supplying a stable and harmonious hydrodynamic environment in the separation chamber for FFE separation, (ii) effectively preventing background electrolyte and sample flow‐back at the outlet of the chamber and improving the resolution, (iii) increasing the preparative scale of the separation, and (iv) simplifying the operation. In addition, the cost of the FFE device was reduced without using a multichannel peristaltic pump for sample collection. Finally, comparative FFE experiments on dyes, proteins, and cells were carried out. It is evident that the new developed collector could overcome the problems inherent in the previous gravity‐induced self‐balance collector.     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

A simple chip free‐flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye

Here, a simple micro free‐flow electrophoresis (μFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο‐BBV) and fluorescent dye. The μFFE contained two open electrode cavities and an ion‐exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed μFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion‐exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 μm, 77 μL) avoiding the discard of μFFE due to blockage of solute precipitation in chamber. Remarkably, the μFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο‐BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10^?11 M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the μFFE.     

Isolation of terpenoids from Pimpinella anisum essential oil by high‐performance counter‐current chromatography

High‐performance counter‐current chromatography was successfully used for the isolation and purification of terpenoid compounds from the essential oil of Pimpinella anisum L. A two‐phase solvent system composed of n‐heptane/methanol/ethyl acetate/water (5:2:5:2, v/v/v/v) was suitable for the purification of linalool, terpinen‐4‐ol, α‐terpineol, p‐anisaldehyde, while n‐heptane/methanol (1:1, v/v) was used for the isolation of anethole and foeniculin. A scale‐up process from analytical to preparative was developed. Additionally, a stepwise gradient elution was applied and instead of two different runs, 40 min each, one 80 min separation was performed; although the time of separation remains the same, it was possible to repeat the efficiency even if the water‐containing mobile phase was changed to a nonaqueous system. The obtained essential oil, as well as purified compounds, was analyzed by GC. A total of 0.64 mg of linalool, 0.52 mg of terpinen‐4‐ol, 0.10 mg of α‐terpineol, 0.62 mg of p‐anisaldehyde, 15 mg of anethole, and 2.12 mg of foeniculin were obtained from 210 mg of the essential oil of P. anisum L. in a short time with purities of 99, 98, 94, 93.54, 93, and 93.6%, respectively.     

Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume

Parishins are high‐polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two‐phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two‐phase solvent system composed of butyl alcohol/acetonitrile/near‐saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high‐speed counter‐current chromatography. Fractions obtained from high‐speed counter‐current chromatography were subjected to semi‐preparative high‐performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two‐phase solvent system in high‐speed counter‐current chromatography was a suitable approach for the purification of high‐polarity compounds.     

Three‐phase hollow‐fiber liquid‐phase microextraction based on deep eutectic solvent as acceptor phase for extraction and preconcentration of main active compounds in a traditional Chinese medicinal formula

A three‐phase hollow‐fiber liquid‐phase microextraction based on deep eutectic solvent as acceptor phase was developed and coupled with high‐performance capillary electrophoresis for the simultaneous extraction, enrichment, and determination of main active compounds (hesperidin, honokiol, shikonin, magnolol, emodin, and β,β′‐dimethylacrylshikonin) in a traditional Chinese medicinal formula. In this procedure, two hollow fibers, impregnated with n‐heptanol/n‐nonanol (7:3, v/v) mixture in wall pores as the extraction phase and a combination (9:1, v/v) of methyltrioctylammonium chloride/glycerol (1:3, n/n) and methanol in lumen as the acceptor phase, were immersed in the aqueous sample phase. The target analytes in the sample solution were first extracted through the organic phase, and further back‐extracted to the acceptor phase during the stirring process. Important extraction parameters such as types and composition of extraction solvent and deep eutectic solvent, sample phase pH, stirring rate, and extraction time were investigated and optimized. Under the optimal conditions, detection limits were 0.3–0.8 ng/mL with enrichment factors of 6–114 for the analytes and linearities of 0.001–13 μg/mL (r² ≥ 0.9901). The developed method was successfully applied to the simultaneous extraction and concentration of the main active compounds in a formula of Zi‐Cao‐Cheng‐Qi decoction with the major advantages of convenience, effectiveness, and environmentally friendliness.     

Effect of aqueous and organic solutions on the performance of polyamide thin‐film‐composite nanofiltration membranes

Polyamide/polyacrylonitrile thin‐film‐composite (TFC) nanofiltration (NF) membranes for the separation of oleic acid dissolved in organic solvents (methanol and acetone) were interfacially prepared by the reaction of trimesoyl chloride in an organic phase with an aqueous phase containing piperazine and m‐phenylene diamine. The interfacial reaction was confirmed by an investigation of the attenuated total reflection infrared spectrum. The surface morphology of the polyamide TFC membranes was examined with scanning electron microscopy. The hydrophilic properties of the membrane surfaces were conjectured on the basis of the ζ potential and contact angle. The effects of the monomer concentrations of the monomer blends (aliphatic and aromatic diamines) and drying times on various aspects of membrane performance, such as the solvents (water, alcohols, ketones, and hexane), permeation rates, and organic solute [poly(ethylene glycol) 200 and oleic acid] rejection rates, were investigated. All the membranes showed good solvent resistance. The polar solvent flux for water and methanol was higher than that for a nonpolar solvent (hexane). The membranes gave good rejection rates of oleic acid dissolved in methanol and acetone. The NF membranes were compared with various commercial membranes. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 2151–2163, 2002     

A consecutive preparation method based upon accelerated solvent extraction and high‐speed counter‐current chromatography for isolation of aesculin from Cortex fraxinus

A consecutive preparation method based upon accelerated solvent extraction (ASE) coupled with high‐speed counter‐current chromatography (HSCCC) was presented and aesculin was obtained from Cortex fraxinus. The extraction condition of ASE was optimized with response surface methodology; some significant parameters such as the solvent system and its stability, the amount of loading sample in HSCCC were also investigated. The original sample was first extracted with methanol at 105°C and 104 bar for 7 min using ASE, then the extracts were consecutively introduced into the HSCCC system and separated and purified with the same ethyl acetate/n‐butanol/water (7:3:10, v/v/v) solvent system for five times without further exchange and equilibrium. About 3.1 ± 0.2 mg/g in each time and total of 15.4 mg/g aesculin with purity over 95% was isolated from Cortex fraxinus. The results demonstrated that the consecutive preparation method was time and solvent saving and high throughput, it was suitable for isolation of aesculin from Cortex fraxinus, and also has good potential on the separation and purification of effective compounds from natural product.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.     

Analysis of Calix[4]arenes Using Nonaqueous Capillary Electrophoresis

In nonaqueous capillary electrophoresis (NACE), an organic solvent is used in place of an aqueous medium as the background solution to improve the solubility and selectivity for hydrophobic analytes. In this study, we employed NACE with UV detection for the analysis of eight calix[4]arenes. We examined the influence of several parameters—the buffer composition, the nonaqueous solvent‘s composition and proportion, and the concentration of the electrolyte of the nonaqueous buffer—on the efficiency of the electrophoretic separation. The separation was achieved through the analyte's different effective mobility via different degrees of deprotonation on the phenolic OH groups of the calix[4]arene. This deprotonation can further affect the analyte's ability to form a complex with the metal ion. The optimized background electrolyte (BGE), comprising a mixture of N‐methylformamide/acetonitrile (30:70, v/v) and 100 mM AcOH/20 mM NH₄OAc, provided rapid (<11 min) separation of the calix[4]arenes with good resolution. The relative standard deviations of the migration times for the eight analytes were all less than 1%. Within the calibration concentration range, the coefficients of determination (R²) were all greater than 0.9914. Thus, the present study demonstrated NACE can provide adequate separation for the analysis of calix[4]arenes.     

Separation of five oligostilbenes from Vitis amurensis by flow‐rate gradient high‐performance counter‐current chromatography

A rapid and efficient high‐performance counter‐current chromatography (HPCCC) method was developed to separate five oligostilbenes from the roots of Vitis amurensis. An n‐hexane/ethyl acetate/methanol/water system (4:8:4:10, v/v/v/v) was selected as an optimal two‐phase solvent system of which the upper phase was used as the stationary phase and the lower phase was used as the mobile one. Partition coefficient values for the target compounds under these optimized conditions were 0.28 ( 1 , ampleosin A), 7.12 ( 2 , (+)‐g‐viniferin), 2.26 ( 3 , vitisin A), 5.38 ( 4 , wilsonol C), and 11.23 ( 5 , vitisin B). Flow‐rate gradient HPCCC (4 mL/min in 0–70 min, 8 mL/min in 70–250 min) was applied to isolate the target compounds in as high purity as possible within the shortest possible run time. Under these conditions, ampelopsin A (12.1 mg), (+)‐g‐viniferin (10.4 mg), vitisin A (2.8 mg), wilsonol C (3.2 mg), and vitisin B (37 mg) were isolated with >95% purity from 150 mg of enriched oligostilbene extract. Although the K_D of the last eluted compound, vitisin B (K_D = 11.23), was relatively large, it was eluted in 115–145 min using the two‐phase solvent system. This study shows that HPCCC is an efficient tool for the isolation and purification of natural products.     

Determination of 3,5,6‐trichloro‐2‐pyridinol,phoxim and chlorpyrifos‐methyl in water samples using a new pretreatment method coupled with high‐performance liquid chromatography

A novel low‐density solvent‐based vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction with the solidification of floating organic droplet method coupled with high‐performance liquid chromatography was developed for the determination of 3,5,6‐trichloro‐2‐pyridinol, phoxim and chlorpyrifos‐methyl in water samples. In this method, the addition of a surfactant could enhance the speed of the mass transfer from the sample solution into the extraction solvent. The extraction solvent could be dispersed into the aqueous by the vortex process. The main parameters affecting the extraction efficiency were investigated and the optimum conditions were established as follows: 80 μL 1‐undecanol as extraction solvent, 0.2 mmol/L of Triton X‐114 selected as the surfactant, the vortex time was fixed at 60 s with the vortex agitator set at 3000 rpm, the concentration of acetic acid in sample solution was 0.4% v/v and 1.0 g addition of NaCl. Under the optimum conditions, the enrichment factors were from 172 to 186 for the three analytes. The linear ranges were from 0.5 to 500 μg/L with a coefficient of determination (r²) of between 0.9991 and 0.9995. Limits of detections were varied between 0.05 and 0.12 μg/L. The relative standard deviations (n = 6) ranged from 0.26 to 2.62%.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Simultaneous quantification of catecholamines in rat brain by high‐performance liquid chromatography with on‐line gold nanoparticle‐catalyzed luminol chemiluminescence detection

A new method based on high‐performance liquid chromatography (HPLC) coupled with on‐line gold nanoparticle‐catalyzed luminol chemiluminescence (CL) detection was developed for the simultaneous quantitation of catecholamines in rat brain. In the present CL system, gold nanoparticles were produced by the on‐line reaction of H₂O₂, NaHCO₃?Na₂CO₃ (buffer solution of luminol) and HAuCl_4. Norepinephrine (NE), epinephrine (EP) and dopamine (DA) could strongly enhance the CL signal of the on‐line gold nanoparticle‐catalyzed luminol system. The UV?visible absorption spectra and transmission electron microscopy studies were carried out, and the CL enhancement mechanism was proposed. Catecholamines promoted the on‐line formation of more gold nanoparticles, which better catalyzed the luminol–H₂O₂ CL reaction. The good separation of NE, EP and DA was achieved with isocratic elution using a mixture of methanol and 0.2% aqueous phosphoric acid (5:95, v/v) within 8.5 min. Under the optimized conditions, the detection limits, defined as a signal‐to‐noise ratio of 3, were in the range of 1.32–1.90 ng/mL, corresponding to 26.4?38.0 pg for 20 μL sample injection. The recoveries of catecholamines added to rat brain sample were >94.6%, with the precisions <5.5%. The validated HPLC?CL method was successfully applied to determine NE and DA in rat brain without prior sample purification. Copyright © 2014 John Wiley & Sons, Ltd.