Contactless impedance sensors and their application to flow measurements.

The paper provides a critical discussion of the present state of the theory of high-frequency impedance sensors (now mostly called contactless impedance or conductivity sensors), the principal approaches employed in designing impedance flow-through cells and their operational parameters. In addition to characterization of traditional types of impedance sensors, the article is concerned with the use of less common sensors, such as cells with wire electrodes or planar cells. There is a detailed discussion of the effect of the individual operational parameters (width and shape of the electrodes, detection gap, frequency and amplitude of the input signal) on the response of the detector. The most important problems to be resolved in coupling these devices with flow-through measurements in the liquid phase are also discussed. Examples are given of cell designs for continuous flow and flow-injection analyses and of detection systems for miniaturized liquid chromatography and capillary electrophoresis. New directions for the use of these sensors in molecular biology and chemical reactors and some directions for future development are outlined.

Monitoring of arrays of amino acids in clinical samples using capillary electrophoresis with contactless conductivity detection.

Capillary electrophoresis (CE) with contactless conductivity detection (C(4)D) is readily applicable to determinations of amino acids in clinical samples. Most of these analyses employ long separation pathways. This chapter describes CE/C(4)D determinations of 28 biogenic amino acids in a short capillary with an effective length of 18 cm. All the test amino acids can be mutually separated in electrolytes of 0.5-10 mol/L acetic acid. The time of analysis does not exceed 6 min; the limits of detection vary from 0.1 to 1.7 µmol/L for all the analytes. The pretreatment of the biological material is very simple, consisting of the removal of proteins by an addition of acetonitrile and subsequent filtration. The procedure has been successfully applied to determinations of the whole amino acid spectra in blood plasma, urine, saliva, cerebrospinal, and amniotic fluid samples.

The use of a multichannel capillary for electrophoretic separations of mixtures of clinically important substances with contactless conductivity and UV photometric detection.

A fused-silica capillary with a common outer diameter, 360 µm, but containing seven internal channels, each 28 µm in diameter (a multichannel capillary), has been tested on electrophoretic separations of mixtures of dopamine, adrenaline, and noradrenaline, using a contactless conductivity and UV photometric detection. It has been demonstrated that the sensitivity of the detection of these neurotransmitters in multichannel capillary, in comparison with those obtained for a standard singlechannel capillary with similar cross-sectional area, is comparable to that for the contactless conductivity and is about 50% higher for the UV photometry. The sensitivity is increased without loss of the separation efficiency, in contrast to UV detection with bubble cell. Further possibilities of using a multichannel capillary are demonstrated on separations of mixtures of inorganic cations (K⁺, Ba²âº, Na⁺, Mg²âº, and Li⁺) and mixtures of glucose and ribose. The main advantage of multi-channel capillary in comparison with a singlechannel capillary with the same cross-sectional area becomes apparent in separations in background electrolytes of high conductivity.

Rapid determinations of saccharides in high-energy drinks by short-capillary electrophoresis with contactless conductivity detection.

The methodology for separations of saccharides in standard electrophoretic systems has been transferred to the short-capillary electrophoresis format. The laboratory-designed apparatus used employs a quartz capillary with an internal diameter of 10 µm, a total length of 10 cm, and an effective length of 4 cm, in combination with contactless conductivity detection. It has been applied to separations of neutral mono- and disaccharides. The saccharides are separated in the anionic form, in solutions of alkali hydroxides, namely, KOH, NaOH, and LiOH. The separation of a model mixture of five saccharides (sucrose, lactose, glucose, fructose, and ribose) takes less than 1 min, the LOD equaling 15, 35, 19, 17, and 24 mg L(-1) and the LOQ equaling 52, 117, 63, 53, and 79 mg L(-1) for sucrose, lactose, glucose, fructose, and ribose, respectively. The technique developed has been used to determine sucrose, glucose and fructose in high-energy drinks. The separation is finished within less than 50 s; the saccharide contents determined are identical with the declared values within the reliability interval in most cases, the RSD value being mostly less than 2%. In general, the separation system developed is very convenient for rapid analyses of large sets of similar samples, e.g., in product quality control or environmental monitoring.

Rapid monitoring of mono- and disaccharides in drinks, foodstuffs and foodstuff additives by capillary electrophoresis with contactless conductivity detection.

A capillary electrophoresis (CE) procedure with contactless conductivity detection (C(4)D) has been developed for monitoring of neutral mono- and disaccharides in drinks and foodstuffs. The separation of a mixture of seven neutral saccharides (glucose, fructose, galactose, mannose, ribose, sucrose and lactose) employed a quartz capillary, 5 µm i.d., with an effective length of 18.3 cm, and 75 mM NaOH (pH 12.8) as the background electrolyte (BGE). The limit of detection (LOD) values obtained lied within a range from 0.4 µmol L(-1) for lactose to 0.9 µmol L(-1) for ribose, with a separation time shorter than 140 s. The procedure was successfully applied to determinations of saccharides in fruit juices, Coca-Cola, milk, red and white wines, yoghurts, honey and a foodstuff additive.

Some important combinations of detection techniques for electrophoresis in capillaries and on chips with emphasis on electrochemical principles.

Analyses of very complex samples involving capillary and chip electrophoresis often require dual (multiple) detection to attain sufficient selectivity of determination. The present work reviews and critically evaluates selected combinations of electrochemical detection techniques among themselves and with absorption spectrometric, fluorescence and luminescence techniques. Amperometry, contact and contactless conductometry, UV photometry and fluorescence measurements are paid special attention. Some information is also given on combinations of spectrometric techniques with mass spectrometry. The properties of the detection systems are critically discussed, examples are illustrated in figures and some details on the characteristics of dual detectors and their applications are tabulated.

Monolithic columns based on a poly(styrene-divinylbenzene-methacrylic acid) copolymer for capillary liquid chromatography of small organic molecules.

A very simple and readily performed method is described for the preparation of poly(styrene-divinylbenzene-methacrylic acid) monolithic columns for capillary liquid chromatography. The effect of the methacrylic acid content on the morphological and chromatographic properties has been investigated. Methacrylic acid is shown to be essential for isocratic separations of small organic analytes by capillary liquid chromatography. Column efficiencies of about 28,000 theoretical plates/m have been obtained for all the test compounds. The batch-to-batch and run-to-run repeatability of the retention times is better than 1.5%.

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders.

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4)D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 µm i.d.) and the background electrolyte of 20mM 2-morpholinoethanesulfonic acid (MES)/NaOH+10% (v/v) methanol, pH 6.0 (pH is related to the 20mM MES/NaOH buffer in water). The LOD values obtained by C(4)D for the OAs which do not absorb UV radiation range from 0.6 µM (oxalic acid) to 6.8 µM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 µM (5-hydroxy-3-indoleacetic acid) to 10.2 µM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.

Differentiation among various kinds of cheese by identification of casein using HPLC-chip/MS/MS.

In proteomics, proteins can be identified by enzymatic cleavage of the target protein using an enzyme of the known specificity (primarily trypsin), sequencing the obtained specific peptides by MS and comparing the amino acid sequence of the peptides with a protein database. The sophisticated approach described above was used in this study to determine and verify the original species of cheeses. Proteins were extracted from three different cheese samples which were produced from cow, sheep and goat milks. The isolated proteins were cleaved with trypsin and the peptides obtained were sequenced and identified by a HPLC-chip/MS/MS microfluidic system. Two different extraction methods and two various chromatographic sorbents packed in plastic chips were studied. Beta-lactoglobulin and four kinds of casein were found in the cheese samples. The species of kappa-casein were identified unambiguously in all the three cheese samples and, thus, kappa-casein can be used to determine the origin of milk of the cheese. The other proteins found in the samples show very similar primary structures and cannot be recommended for identification of the cheese milk origin.

The use of capillary electrophoresis with contactless conductivity detection for monitoring of glycerol in adipose tissues during a sporting performance.

A CE procedure employing capacitively coupled contactless conductivity detection has been developed for direct determination of the glycerol and mannitol polyalcohols in biological and pharmacological samples. Both glycerol and mannitol are fully separated from the sample matrix within very short times of 3.0 and 3.9 min, respectively, when using the optimized BGE, 60 mM H3BO3+30 mM LiOH (pH 9.1). The LODs amount to 0.5 microM for glycerol and 0.3 microM for mannitol. The repeatability of the glycerol determination in real biological materials is characterized by the coefficient of variation values, 0.5 and 3.2%, for the migration time and the peak area, respectively. The procedure has been used to monitor the free glycerol concentration in adipose tissue microdialyzates. A physiological study has demonstrated that the lipolysis occurring during a sporting action can be stimulated by local application of adrenaline. The procedure has further been utilized to determine mannitol in a pharmacological preparation.

Rapid monitoring of arrays of amino acids in clinical samples using capillary electrophoresis with contactless conductivity detection.

CE with contactless conductivity detection has been used to separate 28 biogenic amino acids in a short capillary with an effective length of 18 cm. All the tested amino acids can be mutually separated in 0.5-10 mol/L acetic acid electrolytes. The time of analysis does not exceed 6 min and the LODs vary from 0.1 to 1.7 micromol/L. The CVs lie within the intervals 0.01-0.4% and 0.9-4% for the migration times and the analyte peak areas, respectively. The procedure has been successfully applied to the determinations of the whole amino acid spectra in blood plasma, urine, saliva and cerebrospinal fluid samples.

A determination of submicromolar concentrations of glycine in periaqueductal gray matter microdialyzates using capillary zone electrophoresis with contactless conductivity detection.

CE with contactless conductivity detection has been used to determine the glycine neurotransmitter in periaqueductal gray matter (PAG) of rats. The LOD for glycine has been decreased to a value of 0.2 microM by adding 75% v/v of ACN to the samples and increasing the sample zone introduced to a value of 20% of the overall capillary length. The repeatabilities of the analyte migration times and the zone areas amount to 2.1 and 2.7%, respectively. The optimized CE/contactless conductivity detection method makes it possible to determine the micromolar concentrations of glycine in PAG microdialyzates without the necessity of sample derivatization. It follows from a pharmacological study that a local inflammation initiated by an application of carrageenan increased the glycine concentration in the rat PAG seven times, compared with a control. The glycine level in PAG can be decreased and the pain suppressed by administering paracetamol.

Analysis for estrogens as environmental pollutants--a review.

The approaches to the analysis for estrogen compounds as environmental pollutants are critically reviewed and evaluated on the basis of significant, recent original publications. The importance of sample pretreatment and analyte preconcentration techniques is pointed out, with an emphasis on SPE and on the use of highly selective interactions such as molecular recognition. The hyphenated systems of high-performance gas or liquid chromatography and mass spectrometric techniques are discussed as the basic methods of determination of estrogens in environmental samples. Immunochemical procedures are shown to be useful in semiquantitative screening of estrogen pollutants (e.g. ELISA kits). Classical HPLC and GC with common UV/Vis, fluorescence and electrochemical detection are useful in routine checking on higher pollutant concentrations.

Gas chromatography/mass spectrometry of oils and oil binders in paintings.

A GC/MS procedure has been developed, optimized, and applied to characterization of oil binders in paintings. The procedure involves hydrolysis of lipids to fatty acids (FAs) and derivatization of FAs to fatty acid methyl esters (FAMEs) by a solution of sodium methanolate in methanol at an elevated temperature. FAMEs are analyzed by temperature-programed GC followed by full-scan MS. Old and dried samples are subjected to extraction of nonpolymerized FAMEs into dichloromethane prior to hydrolysis. The method provides a good repeatability of results and has been applied to the characterization of common plant oils used in paintings, to commercial oil and tempera paints, to model painting samples, and to samples taken from real paintings. The fresh oils and binders can readily be identified and characterized. The ratio of the methyl esters of palmitic and stearic acids can be used to characterize oil binders in old works of art.

An evaluation of the experimental approaches to detection of small ions in CE.

This review points out some important trends in the development of the detection techniques for small ions in CE. On the basis of selected literature references it briefly discusses some general requirements on detection techniques in CE. Various optical measurements, mass spectrometric approaches and electrochemical detection techniques are dealt with. Some specific features of microchip CE separation and detection are pointed out and possibilities of dual detection are mentioned. The principal parameters of the above detection techniques are then briefly compared.

Determination of 1-methylhistidine and 3-methylhistidine by capillary and chip electrophoresis with contactless conductivity detection.

CE with capacitively coupled contactless detection (C4D) was used to determine 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH). The C4D response to 3-MH was studied in a BGE consisting of 500 mM acetic acid and ammonia at varying concentration and the results were compared with the theory. Complete separation of a model mixture of 3-MH, 1-MH, and histidine (His) was attained in two optimized BGEs, one containing 500 mM HAc, 20 mM NH4OH, and 0.1 % m/v hydroxyethylcellulose (HEC), pH 3.4 (I) and the other consisting of 100 mM morpholinoethanesulfonic acid (MES), 25 mM LiOH, and 0.1 % m/v HEC, pH 5.5 (II). These optimized BGEs were tested in CE/C4D analyses of urine. Promising results were obtained for separation and determination of 3-MH, 1-MH, and His on a silicon microchip, using aluminum strips as the C4D electrodes; the three analytes were baseline-separated within less than 30 s with a separation channel effective length of 38 mm. The LOD were satisfactory and amounted to 26.4 microM for 3-MH and 18.3 microM for 1-MH.

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins.

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.

Monolithic organic polymeric columns for capillary liquid chromatography and electrochromatography.

This review briefly summarizes the present state of the preparation and use of capillary monolithic columns for liquid chromatography (LC) and electrochromatography (EC). Most important approaches to the preparation of monolithic stationary phases based on organic polymers are outlined and the properties of the monoliths obtained are compared with those of classical particulate phases. A few selected applications of monolithic columns are shown to demonstrate the most important advantages of monolithic capillary columns. It is concluded that both the monolithic and particulate capillary columns are important and that judicious choice of the type suitable for a particular application requires careful consideration of the purpose of the separation and the properties of the solutes to be separated. Monolithic columns are substantially younger than packed ones and thus will require further theoretical and experimental study to further improve their preparation and to enable reliable prediction of their properties and applicability; nevertheless, they are very promising for the future.

Influence of the spectrophotometric detection mode on the separation performance of systems with monolithic capillary columns: on-column and external-cell detection modes.

Poly(butyl methacrylate) monolithic columns were prepared by thermally initiated radical polymerization in fused-silica capillaries of 320 microm i.d. The prepared monolithic columns were tested by capillary liquid chromatography (CLC) combined with a UV-VIS spectrophotometric detector. The influence of the detection configuration (i.e., on-column and external-cell detection modes) on the performance of the chromatographic system was investigated. In the on-column detection mode within the monolith, the detection window was located inside the column section filled with the monolith. With the on-column detection configuration after the monolith, the detection window was positioned just behind the column section containing the monolith. Using the external-cell detection mode, an additional detection capillary, provided with a detection window defining the external-cell, was connected to the monolithic capillary column. These detection modes were critically compared in terms of the principal chromatographic parameters of the system involving the prepared monolithic capillary columns.