Improved ruggedness for membrane-based amperometric sensors using a pulsed amperometric method.

This paper introduces a new approach to the use of membrane-based amperometric sensors which is expected to improve the ruggedness of these sensors significantly relative to the steady-state method in common use. In this new method, the fixed-voltage source used with the conventional steady-state method is replaced by a pulsed-voltage source. Unlike the fixed-source approach, which yields steady-state currents corresponding to large differences between analyte concentrations inside and outside the isolating membrane, the pulsed-source approach permits measurement of currents corresponding to near-equilibrium conditions between solutions inside and outside the membrane. Because the measured currents correspond to near-equilibrium conditions, results are expected to be virtually independent of variables that affect rates of mass transport to and across the membrane. The new approach is evaluated using the "oxygen electrode" as a model system. Results obtained using the new method are compared with results obtained using the conventional steady-state option as well as a coulometric approach described recently. The reproducibility of the pulsed amperometric approach and the scatter of data about least-squares calibration lines are an order of magnitude or more better than for the conventional steady-state option. As expected, the pulsed amperometric method is 40-100-fold less dependent on changes in membrane thickness, stirring rate, and temperature than the conventional steady-state option.

The inseparable triad: analytical sensitivity, measurement uncertainty, and quantitative resolution.

The formal definition of sensitivity associates the term with the change in the response of a system for a small change of the stimulus causing the response, i.e., the ratio of the response of a system to the stimulus causing it. One interpretation of sensitivity associates the rate of change of the response for a small change of the stimulus as the slope of a calibration plot of response vs stimulus. An alternative interpretation associates sensitivity with the smallest value of the stimulus that can be resolved with a given degree of confidence, i.e., the detection limit. Applications of the first usage to analytical chemistry date at least to the beginning of this century; applications of the second interpretation are of more recent origin. The accompanying paper argues in favor of the second interpretation on the basis that, among other things, the "slope" interpretation conflicts with the formal definition of sensitivity and is meaningless as a descriptor of the performance of a measuring system. In this paper I offer arguments to support my belief that the slope definition of sensitivity is consistent with both formal definitions and accepted usage in analytical chemistry and, more importantly, that it is an invaluable descriptor of one of the most important characteristics of any analytical method. I include information to support my belief that proper use of the slope definition yields much more information than is available in the "detection limit" interpretation.

Measurement/data-processing method to improve the ruggedness of membrane-based sensors: Application to amperometric oxygen sensor.

This paper describes alternative measurement and data-processing approaches that can reduce effects of experimental variables on results obtained with a membrane-based sensor for oxygen. In the new approaches, the membrane-based sensor is first equilibrated with the sample solution, after which a polarizing voltage is applied and current vs. time data are recorded as the response decays toward a steady-state condition. Current vs. time data are then processed by a fixed-time option and an integration option designed to determine the charge corresponding to the total amount of oxygen inside the membrane when a polarizing voltage is applied. The current measured at a fixed time and the total charge varied linearly with oxygen concentration between 0.05 and 0.26 mmol l(-1). Pooled relative standard deviations (N = 35) for the measurement/data-processing step were near 0.4% for the new pre-equilibrium options compared to a value of 0.3% for the steady-state option. Dependencies of the pre-equilibrium options on membrane thickness and stirring rate in the most sensitive regions were at least two orders of magnitude smaller than for the steady-state option.

Unified view of kinetic-based analytical methods with emphasis on ruggedness. A review.

All analytical determinations can be grouped into two general categories, namely equilibrium-based and transient-based methods. This is an important grouping because most conventional approaches to transient-based methods are much less rugged than their equilibrium-based counterparts. As a result it is necessary to control variables within much narrower tolerances for transient-based methods than equilibrium-based methods to obtain similar degrees of reliability. This paper reviews measurement and data-processing methods developed to reduce effects of variables on transient-based methods, with emphasis on a general approach that is applicable to a wide variety of methodologies. The approach emphasized is identified as a pseudo-equilibrium method. In this method, transient data are used to compute signals that would be measured if all processes that affect the measurement could be monitored to equilibrium. Results included show that the pseudo-equilibrium method is applicable to the three most common types of responses from the transient phases of chemical and physico-chemical processes. Data included show 10- to 100-fold improvements in ruggedness relative to conventional measurement and data-processing options.

Predictive steady-state chromatography. 1. Algorithms for leading and trailing edges of resolved and unresolved peaks in liquid chromatography.

This paper describes mathematical models and curve-fitting procedures that permit steady-state saturation signals to be computed accurately from data along leading and trailing edges of liquid chromatograms. This new approach to quantitative chromatography is called predictive steady-state chromatography (PSSC). It is shown that the computed saturation signals are virtually the same when determined from data along leading and trailing edges and they vary linearly with analyte concentration. Most importantly, the computed saturation signals for a given analyte concentration are virtually independent of experimental variables such as sample volume and flow rate. For example, for sample volumes between 25 and 45 microL, the average computed saturation signal for a 0.025 mM solution of theophylline was 0.11 V with a standard deviation of 0.00097 V (RSD = 0.8%); similar results were found for other concentrations and for changes in flow rate. Dependencies of the PSSC method on sample volume and flow rate were compared with dependencies for peak-height and peak-area methods by using relative error coefficients. Dependencies on sample volume were 0.04%/microL for the PSSC method and 3 and 4%/microL for peak-height and peak-area methods, respectively. Dependencies on flow rate were 2%/mL/min for the PSSC method and 17 and 120%/mL/min for the peak-height and peak-area methods, respectively. Thus, the predictive steady-state method is 10-100-fold more rugged than peak-height and peak-area methods.

Kinetic method for the quantitative resolution of structural isomers based on the catalytic properties of beta-cyclodextrin.

This paper describes a new approach for the quantitative resolution of mixtures of structural isomers. The method is based on the observation that rate constants for the cyclodextrin-catalyzed hydrolysis of selected structural isomers are significantly different. By using cure-fitting methods, it is possible to use these differences in rate constants to resolve kinetic responses for mixtures into the responses for the individual components. The new approach is evaluated for the ortho-, meta-, and para-isomers of nitrophenyl acetate. At pH 10, with beta-cyclodextrin as catalyst, ratios of rate constants for the three isomers differ by ratios of 1:6.7:1.6 in the order mentioned above. Results are reported for both two- and three-component mixtures. For two-component mixtures of the ortho- and para-isomers which have rate constants differing by only 1.6-fold, linear least-squares slope and intercept of determined vs prepared concentrations for the ortho-isomer were 1.00 +/- 0.02 and 2 +/- 2.2 mumol/L for three runs on each of five samples in the concentration range from 22 to 176 mumol/L. The pooled standard deviation for these 15 runs was 3.7 mumol/L, corresponding to a relative standard deviation of 3.7% for the average concentration. Similar results were obtained for other two- and three-component mixtures.

Incremento de la bioseguridad de sistemas analíticos en el laboratorio clínico / Increment of the biosecurity of analytical system in the clinic laboratory

La bioseguridad es una parte importante del conocimiento práctivo de todos los profesionales de laboratorio clínicos. Debe focalizarse la atención en la reducción del manipuleo de especímenes biológicos, en la reducción de los materiales biológicos peligrosos para el personal de laboratorio y en el mejoramiento del rotulado y envasado de los materiales biopeligrosos. En este artículo, los temas de bioseguridad se discuten en relación al diseño de sistemas analíticos, su utilización y su mantenimiento (AU)

Incremento de la bioseguridad de sistemas analíticos en el laboratorio clínico / Increment of the biosecurity of analytical system in the clinic laboratory

La bioseguridad es una parte importante del conocimiento práctivo de todos los profesionales de laboratorio clínicos. Debe focalizarse la atención en la reducción del manipuleo de especímenes biológicos, en la reducción de los materiales biológicos peligrosos para el personal de laboratorio y en el mejoramiento del rotulado y envasado de los materiales biopeligrosos. En este artículo, los temas de bioseguridad se discuten en relación al diseño de sistemas analíticos, su utilización y su mantenimiento

Systematic top-down approach to clinical chemistry.

This paper introduces a systematic approach to organizing the discipline of clinical chemistry. The approach is called a top-down systems approach because it starts at the top with the most general concepts and works down through less general concepts to the most specific details and techniques. The hypothesis is that the discipline can be organized into hierarchical levels of functional processes and operational approaches to those processes. The functional processes represent what clinical scientists do; the operational approaches represent how they do it. Because functional processes change little, if at all, with time, they are used to develop a stable infrastructure or framework for the discipline. That infrastructure is then used to organize and understand operational approaches that tend to change rapidly with time in response to technological advances. This paper begins with the most general functional processes and then uses selected examples of the more general functions to illustrate lower hierarchical levels of functional processes and operational approaches.

International Federation of Clinical Chemistry (IFCC): systematic top-down approach to clinical chemistry.

This paper introduces a systematic approach to organizing the discipline of clinical chemistry. The approach is called a top-down, systems approach because it starts at the top with the most general concepts and works down through less general concepts to the most specific details and techniques. The hypothesis is that the discipline can be organized into hierarchical levels of functional processes and operational approaches to those processes. The functional processes represent what clinical scientists do; the operational approaches represent how they do it. Because functional processes change little, if at all, with time, they are use to develop a stable infrastructure or framework for the discipline. That infrastructure is then used to organize and understand operational approaches that tend to change rapidly with time in response to technological advances. This paper begins with the most general functional processes and then uses selected examples of the more general functions to illustrate lower hierarchical levels of functional processes and operational approaches.

Increasing the biosafety of analytical systems in the clinical laboratory.

Biosafety is an important part of the know-how of all clinical laboratory professionals. Biosafely must have high priority in the design and use of analytical systems. Attention should be focused on reducing the handling of biological specimens, reducing biohazards to laboratory personnel, and on improving the labelling and containment of biohazardous materials. In this paper, biosafety issues are discussed in relation to the design of analytical systems, their use and maintenance.

La expansión del rol de la robótica en el laboratorio clínico / The spansion of roll of the robotic in the clinic laboratory

Un número creciente de robots serán empleados en laboratorios químicos industriales. Muchos de ellos serán usados para reducir las tareas monótonas de preparación de muestras, para minimizar la exposición humana a entornos riesgosos o para realizar gran número de procedimientos experimentales repetitivos. Por ejemplo, buscar la condición más efectiva o sus combinaciones en síntesis química o el mejor microorganismo en un gran número de cultivos. En el laboratorio clínico la situación es ligeramente diferente y la robótica no es tan ampliamente aplicada, pero hay una tendencia definida para emplear robots o sistemas robóticos, tanto como para reducir el volumen de trabajo y la exposición del personal a posibles biopeligros y para ayudar a obtener resultados más precisos y correctos. Estas necesidades son difíciles de llenar a través delos dispositivos automáticos usuales y especialmente cuando no estan disponibles dispositivos adecuados. Aparatos especialmente diseñados deberán ser producidos para satisfacer estas demandas y la robótica jugará una parte. Finalmente necesitamos evaluar la efectividad de la introducción de la robótica en términos de economía, estrategia, bioseguridad y otros aspectos. ejemplos típicos de implementación de la robótica en el laboratorio clínico son el transporte de especímenes, la automatización de la preparación de muestras, separación, fraccionamiento en al cuotas, así como procesos seleccionados en sistemas automatizados en gran escala. Como se describió previamente, los robots que están comercialmente disponibles actualmente, no son suficientemente inteligentes para ser fácilmente manejados por personal no entrenado en robótica. Hay una necesidad de personal dedicado a robótica que se incorpore al proyecto desde el principio del plan y que pueda mantener el sistema adecuadamente. Nosotros predecimos que esta situación permanecerá de esta forma por un tiempo considerable en el futuro. Los sistemas robots o mecanismos serán gradualmente introducidos en los laboratorios clínicos. La robótica será una de las mejores formas para mantener la bioseguridad en el entorno del laboratorio clínico y en el futuro se necesitarán laboratorios más automatizados con menos personal. Una vez más nosotros queremos enfatizar que se debe establecer una interfase estandarizada y un protocolo para la comunicación entre robots, computadoras e instrumentos, antes de que la robótica sea ampliamente empleada(AU)

La expansión del rol de la robótica en el laboratorio clínico / The spansion of roll of the robotic in the clinic laboratory

Un número creciente de robots serán empleados en laboratorios químicos industriales. Muchos de ellos serán usados para reducir las tareas monótonas de preparación de muestras, para minimizar la exposición humana a entornos riesgosos o para realizar gran número de procedimientos experimentales repetitivos. Por ejemplo, buscar la condición más efectiva o sus combinaciones en síntesis química o el mejor microorganismo en un gran número de cultivos. En el laboratorio clínico la situación es ligeramente diferente y la robótica no es tan ampliamente aplicada, pero hay una tendencia definida para emplear robots o sistemas robóticos, tanto como para reducir el volumen de trabajo y la exposición del personal a posibles biopeligros y para ayudar a obtener resultados más precisos y correctos. Estas necesidades son difíciles de llenar a través delos dispositivos automáticos usuales y especialmente cuando no estan disponibles dispositivos adecuados. Aparatos especialmente diseñados deberán ser producidos para satisfacer estas demandas y la robótica jugará una parte. Finalmente necesitamos evaluar la efectividad de la introducción de la robótica en términos de economía, estrategia, bioseguridad y otros aspectos. ejemplos típicos de implementación de la robótica en el laboratorio clínico son el transporte de especímenes, la automatización de la preparación de muestras, separación, fraccionamiento en al cuotas, así como procesos seleccionados en sistemas automatizados en gran escala. Como se describió previamente, los robots que están comercialmente disponibles actualmente, no son suficientemente inteligentes para ser fácilmente manejados por personal no entrenado en robótica. Hay una necesidad de personal dedicado a robótica que se incorpore al proyecto desde el principio del plan y que pueda mantener el sistema adecuadamente. Nosotros predecimos que esta situación permanecerá de esta forma por un tiempo considerable en el futuro. Los sistemas robots o mecanismos serán gradualmente introducidos en los laboratorios clínicos. La robótica será una de las mejores formas para mantener la bioseguridad en el entorno del laboratorio clínico y en el futuro se necesitarán laboratorios más automatizados con menos personal. Una vez más nosotros queremos enfatizar que se debe establecer una interfase estandarizada y un protocolo para la comunicación entre robots, computadoras e instrumentos, antes de que la robótica sea ampliamente empleada

Analytical applications of catalytic properties of modified cyclodextrins.

This paper describes the evaluation of the catalytic properties of modified cyclodextrins for analytical applications. The beta-dimethylcyclodextrin was modified by adding one and two imidazolyl groups at carbon three positions. The modifications produced enhancements of catalytic activity for the hydrolysis of p-nitrophenyl acetate at neutral pH by factors of 1000 or more relative to the unmodified cyclodextrins. The catalytic properties of the monosubstituted cyclodextrin were evaluated for the quantification of p-nitrophenyl acetate in the concentration range of 10-90 mumol/L. Results obtained by equilibrium, initial-rate, and error-compensating predictive kinetic methods were compared. The equilibrium and predictive kinetic options yielded virtually identical results, with linear changes with concentration throughout the range studied and severalfold larger than the initial-rate option and dependencies on temperature, pH, and catalyst concentration that are 5-10-fold smaller than the initial-rate option.

Initial studies of a new approach to the design and use of enzyme-based reactor/sensor systems: amperometric system for glucose.

This paper describes the development and evaluation of a new approach to the design and use of enzyme-based reactor/sensor systems (so-called "enzyme electrodes"). In the new approach, the reactor/sensor design is such that the measured response corresponds to reaction of all substrate in a fixed volume of solution. The result is that equilibrium-based measurements can be made, which in turn should result in advantages such as extended linear ranges and reduced dependencies on experimental variables such as enzyme activity, temperature, activators, inhibitors, etc. The concept was implemented with a glucose oxidase/electron-mediator reactor system immobilized on a glassy-carbon electrode operated in an amperometric mode. The reactor/sensor system was used in a thin-layer (14 microns) cell such that the mean diffusion time of substrate (glucose) across the cell was very short (< 1 s) and the rate-limiting process was the chemical reaction at the reactor surface. In this way, it was possible to quantify the electrical charge corresponding to reaction of all the substrate in a fixed volume of solution perpendicular to the plane of the reactor system. Because the determined charge is dependent only on the total amount of substrate in the fixed volume, results exhibit linear range up to at least 2-fold the Michaelis constant and reduced dependency on pH relative to results obtained with steady-state responses from the same experimental system. A mathematical treatment is presented which yields equations that are consistent with time-dependent responses for current and charge and which provide a rational basis for several data-processing options evaluated.

Error-compensating kinetic method for enzymatic determination of DNAs.

We describe the adaptation and evaluation of an error-compensating method for kinetic determinations of deoxyribonucleic acids (DNAs). The DNA is first reacted with ethidium bromide to produce a fluorescent intercalation complex. Subsequent treatment of the complex with DNase catalyzes hydrolysis of the DNA, causing a time-dependent decrease in fluorescence, which is monitored. A model for two-component parallel first-order processes is fit to the decay curve to predict the total change in fluorescence expected if the process were monitored to equilibrium. The predicted change in fluorescence response varies linearly with DNA concentration with an intercept corresponding to 0.13 mg/L DNA. Results by the predictive method are 47-, 58-, and 250-fold less dependent on DNase activity, temperature, and ethidium bromide concentration, respectively, than are results for an initial-rate method utilizing the same data. Moreover, the predictive method yields a significantly wider linear range than the initial-rate method, and is much less affected by blank fluorescence and RNA interference than is an equilibrium method based on the reaction of DNA with ethidium bromide alone.

Development and evaluation of an error-compensating predictive data-processing method for liquid chromatography.

This paper describes an alternative data-processing approach for liquid chromatographic responses. Transient data from the leading edges of chromatographic peaks for nonsaturating amounts of sample are used with a suitable mathematical model and curve-fitting program to predict the steady-state response that would be measured if sufficient sample were used to saturate the system. Results obtained by this approach are compared with peak-height and peak-area options by using aspirin as an analyte. For aspirin concentrations from 0.6 to 5.0mmol/L, each data-processing option yields linear calibration plots for each of several sample volumes from 50 to 100 microL and flow rates from 1.0 to 3.0 mL/min. As expected, the predictive option yielded lower dependency on sample volume (20-30-fold improvement) and flow rate (10-fold improvement) than the peak-height and peak-area options. However, the peak-height option provided slightly better (approximately 2-fold) calibration statistics.

Improved data-processing method for atomic absorption spectroscopy with electrothermal atomization.

A new approach is described for processing transient data from electrothermal atomizers used in atomic absorption spectroscopy. The transient responses are first integrated and then a pseudo-first-order model is fit to the time-dependent integrals in order to predict the response that would be measured if the atomization process were monitored to completion. The principal advantage expected and observed for the new approach is its ability to reduce effects of variables such as atomization temperature. For all elements studied (Cr, Mn, K, Yb, Fe), the new predictive approach is shown to be virtually independent of temperature in the range from 2200 to 2600 degrees C. The predictive approach exhibited lower temperature coefficients than either the peak-height or peak-area options for all elements examined. For the more volatile elements (Mn, K, Yb, Fe), the improvement ratio at 2400 degrees C of the predictive approach relative to the others ranged from 1.4 to 8.2. For chromium at 2400 degrees C, the temperature coefficient of the predictive method was approximately 10- and 30-fold lower than those for the peak-area and peak-height options, respectively.

Kinetic study of the reaction of acetoacetate with glycine and sodium nitroprusside.

This paper describes an extensive kinetic study of the reactions involved in the determination of acetoacetate in body fluids. It is concluded that acetoacetate reacts with glycine to produce an imine intermediate that tautomerizes to an enamine. It is also concluded that nitroprusside reacts with the imine intermediate to produce an unstable product with an absorption maximum near 540 nm. This product decays slowly to produce a stable product with an absorption maximum near 393 nm. A proposed reaction pathway is used to develop kinetic equations, rate constants, equilibrium constants, and molar absorptivity of the unstable product that permit quantitative prediction of the kinetic behavior for a wide range of reactant concentrations.