Amperometric determination of underivatized amino acids at a nickel-modified gold electrode by anion-exchange chromatography

A nickel-based composite electrode obtained by anodic electrodeposition of nickel (III) oxyhydroxide film on the gold electrode substrate was characterized as an amperometric sensor and successfully applied to the determination of underivatised amino acids in flow-through systems. The electrodeposition of nickel oxyhydroxide films was obtained by cycling a gold electrode between 0.0 V and +1.0 V vs. a saturated calomel electrode in a 80 microM Ni2+ solution buffered at pH 10 with NaHCO3/Na2CO3. The resulting Au-Ni composite electrode exhibits good stability in alkaline medium and can be used as an amperometric sensor of underivatised amino acids at a fixed applied potential (+0.55 V vs. Ag/AgCl). The detection limits (S/N=3) for all investigated compounds ranged between 5 and 30 pmol injected, while the linear ranges spanned over two or three orders of magnitude. The contents of several free amino acids in two sample cheeses from different brands were evaluated by calibration graphs.     

Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection

A direct, sensitive, simple and practical method for simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection was developed. The retention behavior of amino acids and carbohydrates on the anion-exchange column and the detection of amino acids and carbohydrates at different integrated pulsed amperometric detection waveforms were investigated. The optimized gradient eluent conditions for analysis of 17 amino acids and nine carbohydrates were obtained. Separation time was 100 min. Detection limits for amino acids and carbohydrates were 5.2-207.1 nM under injection volume of 25 microl. The RSDs of peak area were 1.2-3.3%. The calibration graphs of peak area for the analytes were linear over about three orders of magnitude with a correlation coefficient of 0.9950-0.9999. The method was applied to determine amino acids and carbohydrates in a liquid condiment with satisfactory results.     

Simultaneous determination of carbohydrates and organic acids in beer and wine by ion chromatography

The simultaneous determination of mono-organic acids and carbohydrates by ion chromatography with both conductometric and pulsed amperometric detection is described. The carbohydrates, such as mannitol, arabinose, glucose, fructose, lactose, sucrose, raffinose, and maltose, as well as monoorganic acids including acetate, glycolate, formate, pyruvate, and fluoride are separated as anions by ion-exchange chromatography with 0.080 mol/L sodium hydroxide eluent at 1 mL/min within 12 min. Carbohydrates are determined by pulsed amperometric detection and mono-organic acids are determined by suppressed conductivity detection. The species in beverages are determined.     

Use of disposable gold working electrodes for cation chromatography-integrated pulsed amperometric detection of sulfur-containing amino acids

We have prepared disposable thin-film gold working electrodes on polymeric substrates. Our microfabrication process allows for inexpensive and reproducible mass production of such electrodes. We utilize this new type of electrode in flow-through electrochemical cells to replace the conventional non-disposable gold working electrodes for integrated pulsed amperometric detection (IPAD) of compounds separated by high-performance cation-exchange chromatography. Using two S-containing amino acids (homocysteine and cysteine) as test compounds, we have modified a previously reported waveform for optimum performance with disposable gold electrodes. With the help of the same two test substances we have characterized the analytical performance of disposable gold electrodes under the new conditions. Compared to non-disposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration and reproducibility. When used with a new IPAD waveform, the disposable electrodes functioned reproducibly for 3 days. At the end of the specified usage period of 3 days, the disposable electrodes are simply replaced. Reconditioning by polishing is thus no longer required.     

Analysis of oligomannuronic acids and oligoguluronic acids by high-performance anion-exchange chromatography and electrospray ionization mass spectrometry

Oligomannuronic acids and oligoguluronic acids were prepared by enzymatic hydrolysis of alginate with alginate lyases. The oligosaccharides generated up to degree of polymerization 16 were characterized by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) and electrospray ionization mass spectrometry (ESI-MS). Acetate buffer linear gradients were used as mobile phases for separations of oligosaccharides. ESI-MS and HPAEC-PAD are very effective for the analysis and characterization of anionic oligosaccharides.     

Detection of phenolic compounds in flow systems based on tyrosinase-modified reticulated vitreous carbon electrodes

The fabrication and performance of a reticulated vitreous carbon (RVC)-based tyrosinase flow-through electrode, in which the enzyme was covalently immobilized, is reported. The bioelectrode was tested as an amperometric detector for phenolic compounds. Variables affecting the construction of the enzyme flow-through electrode such as the RVC chemical pretreatment procedure, the enzyme immobilization method in the RVC matrix, the enzyme loading and the pH value of the buffer solution used, were optimized by flow-injection with amperometric detection. A good immobilization of the enzyme in the RVC matrix, in spite of the hydrodynamic conditions, was found. The same tyrosinase-RVC electrode could be used with no significant loss of the amperometric response for around 20 days, and reproducible responses could be achieved with different electrodes constructed in the same manner. Moreover, the operational stability of the bioelectrode was tested under continuous monitorization conditions. Calibration plots by flow injection with amperometric detection at -0.20 V were obtained for phenol, 2,4-dimethylphenol; 3-chlorophenol; 4-chlorophenol; 4-chloro-3-methylphenol and 2-aminophenol, with detection limits ranging from 2 mug l(-1) (4-chloro-3-methylphenol) to 2 mg l(-1).     

Flow injection analysis with amperometric detection for measurements of proton activities in solutions of strong acids and strong bases

Flow injection and reversed flow injection analysis with amperometric detection were used for the determination of the concentrations of strong acids and strong bases and the pH of the solutions. The amperometric detection was based on the reduction current of hydrogen ion or water molecules. A platinum indicator electrode cathodically polarized at a constant potential was employed, choosing the potential in the rising part of the current/potential curve. On the basis of the experimental data an equation was developed for the calculation of the concentrations of the strong acids and strong bases and the pH by a non-linear regression computer program.     

Analysis of phenolic acids in plant materials using HPLC with amperometric detection at a platinum tubular electrode

The suitability of a simple amperometric platinum tubular detector for HPLC analysis of selected phenolic acids in yacon (Smallanthus sonchifolius, Asteraceae) is reported. The detector offers good overall limits of detection for the phenolic acids of interest. Three different types of extracts from yacon leaves were analyzed and compared with respect to their content of phenolic acids. Caffeic acid was found in all yacon extracts, whereas the content of chlorogenic acid depends more on the extraction procedure used. It has been demonstrated that no complicated and sophisticated equipment is needed and HPLC with amperometric detection seems to be a very useful technique for analysis of plant extracts containing phenolic acids.     

Indirect amperometric detection of underivatized amino acids in microcolumn liquid chromatography with carbon film based ring-disk electrodes

An indirect amperometric detection of underivatized amino acids has been developed using a carbon film based ring-disk electrode (CFBRDE) in microcolumn liquid chromatography (LC). Bromide present in the mobile phase could be efficiently oxidized to bromine at the upstream (disk) electrode, and was subsequently detected at the downstream (ring) electrode. Most of the underivatized amino acids that are electroinactive under conventional amperometric conditions react rapidly with the electrogenerated bromine, the concentration of amino acids can therefore be indirectly determined by continuously monitoring the reduction current of bromine. The signal monitored at the downstream electrode was largely dependent on the bromide concentration in the mobile phase. Under optimized conditions, the response linearly increased with the concentration for most of the amino acids over a concentration range of 1-100 microM, with a correlation coefficient of 0.990-0.993. The detection limits for most of the amino acids were below 1 microM (0.2 pmol). It was demonstrated that detection with a ring-disk electrode offers the advantages of achieving a much higher collection efficiency caused by a decrease in flow rate in the microcolumn LC.     

Microchip capillary electrophoresis with electrochemical detector for fast measurements of aromatic amino acids

A method based on microchip capillary electrophoresis with amperometric detection was developed for the rapid separation and direct detection of oxidizable aromatic amino acids (without prior derivatization). The working electrode was a thick-film carbon strip electrode positioned opposite the outlet of the separation channel. Factors influencing the separation and detection processes were examined and optimized. The five aromatic amino acids, tyrosine, 5-hydroxytryptophan, tryptophan, p-aminobenzoic acid, and m-aminobenzoic acid, can be well separated within 5 min using a separation voltage of 2000 V and a 25 mM phosphate buffer (pH 7.0) run buffer containing 50 mM sodium dodecylsulfate. Most favorable amperometric detection was obtained at +0.95 V. Linear calibration plots are observed for micromolar concentrations of the oxidizable amino acids. The new protocol offers good stability and for reproducibility, with relative S.D. of less than 5% for both migration times and peak currents (n=8). It should be useful for the analysis of aromatic amino acids, as desired for life sciences.     

Potentiometric detection of organic acids in ion-exclusion chromatography using different types of liquid-membrane electrodes

Four different liquid-membrane electrodes were tested in a potentiometric flow-cell, in combination with an LC ion chromatography system (Aminex HPX-87H column). This setup was used for the determination of weak organic acids. The flow-through detector was of the wall-jet type. Conditions were established to achieve the best separation and detection characteristics. The sensitivity, selectivity and response time of the different electrodes were compared. Calibration curves and detection limits were measured for several organic acids, and compared with conductometric, and with low-wavelength UV detection. The detection limits were improved by inserting a post-column ion-suppressor system between the column and the detector. Several biological samples were analyzed to demonstrate the possibilities of the potentiometric detector.     

Flow injection analysis with amperometric detection for measurements of proton activities in solutions of strong acids and strong bases

 Flow injection and reversed flow injection analysis with amperometric detection were used for the determination of the concentrations of strong acids and strong bases and the pH of the solutions. The amperometric detection was based on the reduction current of hydrogen ion or water molecules. A platinum indicator electrode cathodically polarized at a constant potential was employed, choosing the potential in the rising part of the current/potential curve. On the basis of the experimental data an equation was developed for the calculation of the concentrations of the strong acids and strong bases and the pH by a non-linear regression computer program. Received: 26 October 1995/Revised: 3 April 1996/Accepted: 14 April 1996     

土壤中氨基酸分析方法的研究进展

本文对土壤中氨基酸的分析方法做了较详细的评述,并针对土壤氨基酸的特点,就土壤样品的前处理(主要包括水解和纯化)及目前常用的色谱分离分析方法进行了总结(主要包括柱后衍生阳离子交换色谱、气相色谱、柱前衍生反相高效液相色谱、阴离子交换色谱-积分脉冲安培检测等方法)。     

Quantification of sugars and organic acids in hygroscopic pharmaceutical herbal dry extracts

Three chromatographic methods have been employed for the determination of hydrophilic compounds, namely carbohydrates and organic acids in herbal dry extracts of Eschscholtzia californica Cham. The hydrophilic compounds were separated from the other components of the dry extracts by solid-phase extraction methods, which were optimised with respect to recovery rates. Carbohydrates were quantified using high-performance anion-exchange chromatography with pulsed amperometric detection. Organic acids were analysed by ion-exclusion chromatography with evaporative light scattering detection and gas chromatography-mass spectrometry. Using the latter method, large amounts of glyceric acid were separated from the extracts of Eschscholtzia californica Cham. This substance together with sugars may be responsible for the increased hygroscopicity and the poor processing behaviour of the extracts.     

Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase

The flow-through amperometric biosensor is presented for determination of carboxylic acids. It is based on two sensor layers that are deposited on a platinum electrode. The inner layer serves to eliminate interferences by limiting diffusion of electrochemically active substances such as ascorbic acid. This layer is electro-polymerized using an equimolar mixture of o-phenylenediamine and resorcinol. The outer layer is prepared by cross-linking the enzyme sarcosine oxidase and bovine serum albumin using glutaraldehyde. The formation of enzymatically produced hydrogen peroxide is monitored at 600 mV vs. an Ag/AgCl reference electrode. The addition of carboxylic acids causes competitive inhibition of the enzyme and a decrease in signal. The assay was optimized for determination of carboxylic acids in wine samples. Following 10-fold dilution, most samples contain 1–10 mM individual carboxylic acids and thus a 5 mM concentration of sarcosine was chosen as being optimal for competition. In case of real samples, the biosensor measures the sum of all carboxylic acids, which serves as a parameter describing the quality of wines. Results from testing several wine samples are reported.     

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.     

A nickel oxide amperometric detector in the chromatographic separation of amino acids

Reverse-phase ion-pair chromatography with a nickel oxide amperometric detector is evaluated for separation and detection of several amino acids. A simple T-junction allows post-column mixing of detector electrolyte. Mixer and detector dimensions are shown to contribute negligibly to band spreading. Picomole amounts of some amino acids can be detected. The system is evaluated for determination of amino acids in hydrolyzed bovine A-chain insulin and urine samples.     

Nickel-titanium alloy electrodes for stable amperometric detection of underivatized amino acids in anion-exchange chromatography

Nickel-titanium (Ni-Ti) alloy electrode was used as an electrochemical detector for the analysis of underivatized amino acids in flow systems. In strong alkaline solution, an oxide film on the Ni-Ti alloy electrode surface exhibited a high catalytic activity toward the oxidation of amino acids. Cyclic voltammetry experiments confirmed that electrogenerated Ni(III)O(OH) functioned as the key redox mediator associated with the oxidation of the amine group in amino acids. The electrochemical behavior of the Ni-Ti electrode in alkaline medium was very similar to the Ni electrode. However, the oxide film was found to be much stable on Ni-Ti than on Ni. Consequently, the Ni-Ti alloy electrode exhibited an excellent stability for constant-potential amperometric detection of amino acids in flow systems. For example, the relative standard deviation (R.S.D.) for the repetitive 100 injections of 50 muM (1.2 nmol) glycine over 10 h was less than 1%. It was postulated that the presence of Ti in the alloy stabilizes the microstructure of oxide layer on the electrode surface. The sensitivities of amino acids at the electrode were different, depending on their chemical structures. The detection limits obtained in a range from 0.9 pmol for arginine to 90.2 pmol for leucine and isoleucine. The Ni-Ti alloy electrodes have been demonstrated to be very suitable for the amperometric detection of underivatized amino acids in anion-exchange chromatography.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Some factors affecting separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Some factors influencing the separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection were investigated. These factors include eluent concentration, column temperature, and detection waveform. The selectivity changes in weakly retained amino acids are slight with changing sodium hydroxide eluent concentration. When sodium acetate eluent concentration is changed, the selectivity variations between strongly retained amino acids containing two carboxyl groups and containing only one carboxyl group are obviously different. Significant but slight selectivity changes in weakly retained amino acids can be achieved through changing the column temperature. Sodium hydroxide and sodium acetate eluent concentration affect the detection of amino acids. Detection sensitivity of amino acids can be improved by increasing the concentration of sodium hydroxide and sodium acetate in a certain concentration range. The detections of amino acids at two different detection waveforms were compared. The hydroxyl amino acids can be selectively detected by choosing a modified detection waveform. The optimized gradient elution condition and column temperature for analyzing 19 amino acids were obtained. The time for the gradient elution program was 60 min. The column temperature was 35 degrees C. Under the optimized conditions, detection limits for 19 amino acids were 0.15-4.52 pmol. The calibration graphs of peak area for all the analytes were linear for about three orders of magnitude. The RSDs (n=5) of peak area were 0.6-5.6%. The determination of trace amino acid impurities in valine product is shown as an application example.