Analytica Chimica Acta (v.404, #1)

Optical chromatography, which is a technique for the separation of microscopic particles using a radiation force, is applied to kinetic studies of an immunoreaction. Polystyrene beads coated with antibodies react with antigens to produce conjugated beads, which comprise two beads and some antigens. The resulting conjugated beads are separated from the single beads. The detection limit of the antigen is <1 ng ml−1, which is smaller than that reported in a previous paper [T. Hatano, T. Kaneta, T. Imasaka, Anal. Chem. 69 (1997) 2711], and can be achieved using 2 μm-diameter beads. The sensitivity in the present method can be further enhanced by increasing the particle size. In addition, the dissociation reaction of the conjugated beads is evaluated by using an on-column reaction technique, which allows the direct monitoring of the formation and dissociation of the conjugated beads. The rate constant for the dissociation of the conjugated beads is determined to be 4.0 × 10−3  s−1 for the case of conjugated beads combined by a single bond.
Keywords: Optical chromatography; Radiation pressure; Immunoassay; Kinetics;

Detection of pesticides from liquid matrices by ion mobility spectrometry by Kai Tuovinen; Heikki Paakkanen; Osmo Hänninen (7-17).
The detection of different pesticide compounds from liquid matrices was achieved by aspiration ion mobility spectrometry. This technique is based on ion mobility which is proportional to the molecular weight and charge. The ion mobility spectrometer is able to measure mobility changes of product ions as well as mobility changes of reactant ions. In the ion mobility spectrometer used, it is possible to measure positive and negative ion clusters at the same time in six different electrodes. Each measuring electrode detects a different portion of the ion mobility distribution formed within the cell’s radioactive source. The pattern recognition used is based on differences in the gas phase profiles of the different compounds. The results of this study reveal that the ion mobility spectrometer MGD-1 can also be used to measure pesticides even from liquid matrices. On the basis of projection calculation, the responses for 2-propanol (= background) and pesticide compounds were easily separated from each other. The greatest responses for pesticides were seen in the 2nd positive channel. Correspondingly, only minor background signals were measured in the 1st and 2nd positive channels. The detection limits of pesticides were at ng or μg levels. The sensitivity of detection of the ion mobility spectrometer for different pesticides decreased in the order: diazinon, aldicarb, dimethoate and parathion. In comparison with the traditional ion mobility spectrometric technique, the main advantages of this aspiration technique are its fast response, high sensitivity, real time vapor monitoring, straightforward maintenance and low cost. In addition, the cell tolerates high chemical concentrations and still recovers quickly.
Keywords: Ion mobility spectrometry; Pesticides; Organophosphates; Carbamate; Liquid matrices; Detection;

A valveless flow injection–capillary electrophoresis system has been developed allowing reproducible injection of samples into an electrolyte stream with the aid of programmable pumps instead of an injection valve. The electrolyte stream transports the injected samples to an interface into which a capillary has been inserted. High voltage is applied throughout the process. On reaching the capillary aperture, a small fraction of the sample plug is electrokinetically introduced. Typical relative standard deviation values in calibration tests were in the range 2–6% for small inorganic anions. The system has been assessed for on-line monitoring of the major anions present in kraft pulping liquors. The preferred electrolyte solution for this application comprised 3.5 mmol l−1 chromate and 30 μmol l−1 cetyltrimethylammonium bromide at pH 11. Samples were taken every 6 min, fully automatically. The system also allows rates of oxidation to be monitored in terms of changes in the concentrations of sulphite, sulphide and thiosulphate ions, as demonstrated by inducing and following accelerated oxidation in a test sample.
Keywords: On-line process monitoring; Flow injection analysis; Capillary electrophoresis; Pulping liquors; Sulphur anions;

We describe the rapid, facile fabrication of an immobilized protease microreactor and its characterization for peptide mapping by capillary electrophoresis (CE). A 30 cm long fused silica capillary was dry-packed with trypsin-immobilized controlled pore glass (CPG) beads. A 40 μl solution of protein standard (β-casein or insulin chain B) was perfused through the microreactor for ca. 2 h by applying low pressure at the inlet. The digest was collected at the microreactor outlet and the tryptic fragments were separated by CE and detected by UV absorbance using a diode array detector. Tryptic peptide maps obtained by solid-phase digestion in the microreactor were compared to those obtained by traditional, liquid-phase methods to evaluate the microreactor’s performance and extent of trypsin autoproteolysis (self-digestion). The effect of substrate flow rate through the packed bed of trypsin–CPG (i.e. the residence time) and effect of substrate concentration were also investigated. In general, digestion conditions in the microreactor were very reproducible, whereas CE separation conditions were found to contribute the most to migration time variability in peptide mapping. Reproducible peptide maps were obtained for 40 μl aliquots of a 2 mg ml−1 β-casein solution. The activity of the immobilized enzyme was high enough to ensure re-usability of this microreactor for at least 10 digestions.
Keywords: Peptide mapping; Capillary electrophoresis; Autoproteolysis; Immobilized enzymes;

The selenium species selenite, selenate, selenomethionine (SeMet), and trimethylselenonium iodide (TMSe+) were separated in aqueous solution by ion chromatography. The separation was performed on an Ionpac CS5 cation exchange column by elution with 10 mM oxalic acid and 20 mM potassium sulphate, pH 3. The 78 Se and 82 Se isotopes were used for the inductively coupled plasma mass spectrometry (ICP-MS) detection.Using the chromatographic system on urine diluted 1 + 1, a large shift in retention times was observed. TMSe+ could be separated from the other species, but the signal from SeMet co-eluted with the selenate signal.The calibration curve was linear in the range 5–50 μg l−1 TMSe+ determined by spiking a urine pool. The precision at the 30 μg l−1 level was 1.9%. The limit of detection and determination were 0.8 and 2.6 μg l−1, respectively. All calculation are based on the 82 Se isotope.In urine, a large interference was observed close to the retention time of TMSe+ when monitoring the 78 Se isotope. The interference was ascribed to the sodium content of the urine. Thus, the 82 Se isotope should be used for selenium speciation in urine on this chromatographic system.Urine samples from nine volunteers were analyzed. TMSe+ concentrations above the limit of detection was only found in two samples and constituted less than 10% of the total selenium content of the samples.
Keywords: Trimethylselenonium; Selenium; Speciation; Urine;

Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination by A.N. Ivanov; G.A. Evtugyn; R.E. Gyurcsányi; K. Tóth; H.C. Budnikov (55-65).
A number of potentiometric biosensors based on cholinesterases from various sources have been developed and examined for the determination of Co-Ral and Trichlorfon to establish factors affecting the analytical performance of biosensors. Acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from horse serum were immobilised on the surface of antimony electrode and on the commercial membranes (nylon and cellulose nitrate) by treatment with glutaraldehyde in vapours and aqueous solution. The biosensors developed make it possible to determine Trichlorfon and Co-Ral with detection limits 0.01 and 0.02 mg l−1, respectively.As shown, inert peptide or the polymer present in the membrane provide better sensitivity and lower detection limits of pesticides due to the hampering of the transfer of the product of enzymatic reaction between the membrane and the solution. The influence of the immobilisation procedure depends on the hydrophobicity of pesticide to be determined. The present comparative study reveals the importance of the enzyme matrix on the analytical performance of biosensors developed for inhibitor determination.
Keywords: Biosensor; Cholinesterase; Inhibition; Pesticide determination;

The lifetime and the reproducibility of novel enzyme stabilizing techniques based on polyelectrolyte agents such as diethylamimoethyl-dextran have been studied. Glucose oxidase and lactate oxidase, used as the model enzymes, are stabilized with the cationic polyelectrolyte, and the resulting enzyme–polyelectrolyte complexes are physically adsorbed into a highly porous and conductive carbon electrode for the construction of the biosensors. The amounts of diethylaminoethyl-dextran and enzyme are optimized with respect to sensor’s sensitivity and stability. Optimum results were obtained using soaking solutions of 2500 U/ml GOx and 1.0 w/v deae-dextran. Additionally, experiments with lactate oxidase sensors constructed using 200 U/ml LOx and 0.5% w/v deae-dextran solution showed improved operational, and storage stability. The sensor-to-sensor reproducibility was good, the relative standard deviation being less than 5.0%.
Keywords: Biosensor; Porous carbon electrode; Polyelectrolyte; Operational stability;

A serial three-enzyme reactor system which measures hypoxanthine (Hx), inosine (HxR) and inosine 5′-monophosphate (IMP) simultaneously was prepared, characterized for its properties and applied to freshness evaluation of meats. The biosensor system was operated as a flow injection analysis mode and the reactor length combination of 15, 12 and 7 cm for Hx, HxR and IMP, respectively, was selected for a complete reaction between substrate and enzyme. The sequential enzymatic reactions by the system were performed best at 0.05 M phosphate buffer, pH 7.5 and 35°C. The effects of various possible interferants such as amino acids and sodium chloride on the system were investigated. When applied to meat freshness evaluation, the Ki - and H-values obtained by the system agreed well with those obtained by a conventional method (liquid chromatography (LC)).
Keywords: Characterization; Serial three-enzyme reactors; ATP-degradative compounds; Meat freshness;

A model for estimating the degree of cooperative ion-pairing that occurs between polyions and lipophilic ion-exchangers doped into the organic polymer membranes of polyion-sensitive electrodes has been developed. The binding of several polyanionic (e.g., heparins, carrageenans, polyphosphates, and polyglutamates) and polycationic species (e.g., protamine, polyarginine, and polylysines) with water soluble surfactants, quaternary ammonium and alkyl- and alkylbenzene sulfonates, respectively, which are structurally analogous to the ion-exchangers, is monitored in aqueous solutions with surfactant-selective electrodes. Analysis of the binding data reveals that the polyion structure, including polyion molecular weight, lipophilicity, and charge density, has a significant impact on the observed binding affinities, as does the surfactant structure. It is also shown that the measured aqueous phase binding affinities have a strong linear correlation with the equilibrium EMF changes observed with the respective polyion-sensitive electrodes, confirming the validity of using an aqueous system to model the binding behavior of ion-exchangers with polyions within the organic polymer membranes of polyion-sensitive electrodes.
Keywords: Polyion-sensitive electrodes; Ion-pairing; Surfactants; Polyions;

Mercury detection in air using a coated piezoelectric sensor by Denise P Ruys; José F Andrade; Orliney M Guimarães (95-100).
Three different methods to produce standardized vapors of mercury were tested and compared for posterior determination of metal, using a piezoelectric sensor (QCM). The generation techniques employed were: saturation, syringe dilution and permeation tube. Gold-coated piezoelectric crystals, without any substrate, were initially tested as sensor, based on the selective affinity of gold by mercury. Later, using a permeation tube as source of metal, some substances were investigated as possible substrates capable of interaction with mercury vapor. The best material found was a mixture (1 : 1 v/v) of palladium-II chloride solution (saturated in acetone) and tetrahydroxyethylethylenediamine (THEED) 50% v/v in acetone. The analytical curve is linear in the concentration range from 2.0 to 7.0 ppm of Hg. Good linearities (r  = 0.9952 and 0.9979) and sensitivities (102 and 149 Hz/ppm) were found for exposure times of 20 and 40 s, respectively. It was shown that the technique has potential application as a small, rugged, sensible and portable mercury vapor sensor.
Keywords: Mercury detection; Piezoelectric sensor; QCM;

A new type of an all-solid-state PVC-free Ca2+-selective electrode is described. The electrode membrane is prepared of soluble electrically conducting polyaniline (PANI), di(2-ethylhexyl)phosphate (H+DEHP-) and Ca2+-ionophore ETH1001. The influence of an incorporated lipophilic salt, potassium tetrakis(4-chlorophenyl)borate (KB(ClPh)4), in the electrode membrane was also studied. The non-conducting form of PANI is made electrically conductive with H+DEHP-, which is also a complexing agent for Ca2+. PANI is then dissolved in tetrahydrofuran (THF) together with the other membrane components and electrode membranes are easily prepared in a single step on a glassy carbon (GC) substrate by solution-casting. An important role of PANI is also to facilitate the charge transfer at the substrate | membrane interface.It is shown that incorporation of ETH1001 and KB(ClPh)4 improves the Ca2+-sensitivity of the electrodes studied. The best Ca2+-sensitivity, 28.6 ± 1.1 mV/log aCa (10−1–10−3  M CaCl2) in 0.1 M NaCl, was obtained for an electrode membrane containing 40% (m/m) PANI, 30% (m/m) ETH1001 and 30 % (m/m) KB(ClPh)4. No redox sensitivity was observed for a PANI electrode membrane consisting of 30% ETH1001 and 5% KB(ClPh)4 in a solution of 10 mM Fe(CN)6 3−/4− with 10−1–10−3  M CaCl2 as the ionic background. The selectivity coefficient (logK Ca,j pot) of this electrode is −1.8 ± 0.1, −1.7 ± 0.1, −1.7 ± 0.1 and approximately −2 towards j  = Na+, K+, Li+ and Mg2+, respectively. A model describing the working mechanism of the different PANI-based ion-selective electrodes is also presented.
Keywords: Polyaniline; Ion-selective electrode; Conducting polymer; Charged carrier; ETH1001; Calcium; Potentiometry;

A novel all-solid-state Ca2+-selective electrode was prepared of soluble electrically conducting polyaniline (PANI), di(2-ethylhexyl)phosphate (H+DEHP) and tetraoctyl ammonium chloride (TOA+Cl). PANI is made soluble and electrically conducting in tetrahydrofuran (THF) with H+DEHP. The DEHP anion is a complexing agent of the charged carrier type for Ca2+. TOA+Cl is added to this solution and electrode membranes are then prepared by drop casting on a GC substrate. PANI membranes containing 0–40% (m/m) TOA+Cl has been studied in this work.The Ca2+-sensitivity was significantly improved by incorporation of 20–30% (m/m) TOA+Cl in the PANI electrode membrane. The best Ca2+-sensitivity, 27.0 ± 0.4 mV/log  a Ca (10−1–10−3  M CaCl2, n  = 3, LOD = 10−4  M) in 0.1 M NaCl, was obtained with an electrode membrane containing 25% TOA+Cl (PANI25). The reproducibility of the standard potential of three identical PANI25 electrodes was also very good. The selectivity coefficient (logK Ca,j pot) of this electrode towards j  = Na+, K+ and Li+ is −1.6. However, Mg2+ shows severe interference in determination of Ca2+.No redox sensitivity was observed for the PANI25 electrode in a 10 mM Fe(CN)6 3−/4− solution with 0.1 M CaCl2 as the ionic background and only a weak redox response, 5 mV/decade, could be detected with 10−3  M CaCl2 as the ionic background. The pH sensitivity of the PANI25 electrodes studied was found to be approximately 5 mV/pH within the pH range of 4.5–9.7.Furthermore, the impedance spectrum and the cyclic voltammogram of the PANI25 electrode reveal that TOA+Cl improves the ionic mobility within the PANI membrane. Finally, it is shown that the working mechanism of the PANI electrode membrane can be explained with the charge carrier model, which is usually applied to PVC-based ion-selective electrodes.
Keywords: Polyaniline; Ion-selective electrode; Conducting polymer; Charged carrier; Cationic additive; Calcium; Potentiometry;

A simple, rapid method for the spectrophotometric discrimination of monosaccharides from the oligosaccharide fraction of fruit juice, jam, syrup and honey samples is proposed. The sample, in alkaline medium, is directly introduced into a flow system and passed through an activated carbon column for its decolourization; then, a volume of 200 μl is injected into the derivatising reagent stream to start the analytical reaction, which takes place at 85°C. The two fractions are discriminated on the basis of the different colours of the derivatives formed; thus, the derivatives of the monosaccharides are yellow while those of the di- and trisaccharides are violet-carmine. The two fractions are monitored at 400 and 540 nm, respectively. Sucrose gives no reaction as it is a non-reducing sugar. The proposed method allows reducing sugars contents from 0.01% to 0.80% w/v to be determined with an average relative standard deviation of 4.5% and a sampling frequency of 10 h−1. The proposed method was validated by applying it to two milk-based and sugar candidate artificial CRMs, with good correlation. The detection limits achieved (0.01% w/v for maltose, lactose and maltotriose at 540 nm) allow adulteration of fruit juices with high fructose syrup from starch at the 4% level to be detected.
Keywords: Continuous system; Discrimination of monossacharides and oligosaccharides; Adulteration of carbohydrate-rich foods;

A flow-through solid phase UV spectrophotometric biparameter sensor for the sequential determination of ascorbic acid and paracetamol by A Ruiz-Medina; M.L Fernández-de Córdova; M.J Ayora-Cañada; M.I Pascual-Reguera; A Molina-Dı́az (131-139).
For the first time, a continuous flow system with solid phase UV spectrophotometric detection (an optosensor) is described for the sequential determination of two analytes based on the alternate use of two carrier/self-eluting agents. The selective and sequential sorption of both on an active solid support (an anion exchanger gel placed in the detection zone into an appropriate quartz flow cell) is performed and their respective UV intrinsic absorbances monitored. Each carrier itself elutes the respective analyte from the solid support, so regenerating the sensing zone.Ascorbic acid and paracetamol in concentrations ranging from 0.3 to 20 μg ml−1 and from 0.4 to 25 μg ml−1, respectively, could be determined with this UV flow-through optosensor using sodium acetate/acetic acid (pH 5.6) and 0.05 M NaCl (pH 12.5), respectively as carrier/self-eluting solutions and Sephadex QAE A-25 anion exchanger gel as solid phase placed in the inner of an 1 mm optical path length quartz flow cell. The RSDs % (n  = 10) were lower than 1.3 (for ascorbic acid) and than 1.5 (for paracetamol). Detection limits (criterion 3σ) as low as 0.02 μg ml−1 were achieved in both cases.Application to the analysis of pharmaceutical samples (in addition to synthetic ones) testifies the utility of this sequential sensor, which tolerates amounts of the species usually accompanying the analytes much higher than those ones found in these samples.
Keywords: Flow-through sensor; Solid phase spectroscopy; Ascorbic acid; Paracetamol;

Flow injection biamperometric determination of chloramphenicol and related nitro compounds by on-line chemical photodegradation by J.A.Garcı́a Bautista; J.V.Garcı́a Mateo; J.Martı́nez Calatayud (141-150).
An unsegmented continuous-flow assembly for the determination of chloramphenicol is proposed. The determination is based on the on-line photodegradation of the drug in an NH4 +/NH3 buffer at pH 10.4 by using a photoreactor consisting of a 697 cm long × 0.8 mm ID piece of PTFE tubing coiled around an 8 W low-pressure mercury lamp. Photodegraded chloramphenicol is detected by photolytic cleavage of nitrite from the parent compound as well as by organic oxidizing photofragments, and their subsequent reaction with iodide ion, which is monitored biamperometrically. Triiodide thus formed is detected in excess iodide solution by polarizing two platinum electrodes at 100 mV. The calibration graph is linear up to 8 mg l–1 chloramphenicol; the limit of detection is 0.05 mg l–1, the relative standard deviation is 0.4% (for 25 replicates of 10 mg l–1 drug) and the throughput 68 samples h–1. The proposed method was used to determine chloramphenicol in commercially available pharmaceutical formulations and human urine.
Keywords: Chloramphenicol; Flow injection analysis; Pharmaceutical and clinical analyses; Biamperometry; Photodegradation;

Uric acid (UA) and ascorbic acid (AA) present in urine were rapidly determined by the amperometric method in association with flow injection analysis. An array of gold microelectrodes modified by electrochemical deposition of palladium was employed as the working electrode. Uric and ascorbic acids were quantified in urine using amperometric differential measurements at +0.75 and +0.55 V, respectively. This method is based on three steps involving the flow injection of: (1) the sample spiked with a standard solution, (2) the pure sample, and (3) the enzymatically treated sample. The enzymatic treatment was carried out with ascorbate oxidase, uricase, and peroxidase at pH 7. The calibration curves for freshly prepared ascorbic and uric acid standards were very linear in the concentration ranges of 0.44–2.64 mg l−1 (AA) and of 0.34–1.68 mg l−1 (UA) with a relative standard deviation (RSD) <1 %. For uric acid, the real sample analyses where compared with the classical spectrophotometric method, showing deviations between 3.1 and 8.6% (n  = 9).
Keywords: Ascorbic acid; Uric acid; Flow injection analysis; Urine; Modified microelectrodes;

Voltammetric study of uranyl–selenium interactions by R. Djogić; I. Pižeta; M. Zelić (159-166).
Uranyl–selenium interactions were studied at two different ionic strengths (I  = 0.1 and 3.0 mol/l) using square wave voltammetry. The uranyl(+6) reduction signal is only slightly affected by selenium(+4) reduction to HgSe, which appears in virtually the same potential range. On the other hand, the height and shape of the selenium stripping peak (which corresponds to dissolution of accumulated HgSe) can be significantly changed by the uranyl(+6) concentration in the solution. The type and magnitude of such effects are highly dependent on the ionic strength. As a result of uranyl(+6) interaction with selenium(+6), a coordination species UO2SeO4 is formed, whose stability constant at I  = 3 mol/l (log  β 1  = 1.57 ± 0.01) is in very good agreement with the literature value based on spectrophotometric data. The presence of higher complexes could not be confirmed unambiguously.
Keywords: Uranyl(+6); Selenite; Selenate; Voltammetry; Complexation; Stability constants;