Analyte concentration, optical measurement

This relationship provides an alternative method to determination of the concentration of the analyte of interest. Specifically, lifetime or decay time measurements can be used in fluorescence based sensors to determine the analyte concentration. These measurements provide better results than steady-state measurements. Time-domain lifetime measurements are typically performed by exciting the sensing element with a short optical pulse which is much shorter than the average fluorophor lifetime. For a single population of fluorophors, the rate at which the intensity decays over time can be expressed as ... 

OS 94][R 13][P 74]ForadmixtureofsampleswifhvaryingconcentrationsofCo(ll) and Cu(II), the respective changes in the Co(ll) chelate complex concentration as a function of contact time were optically derived [28]. Analysis was performed in the reaction/extraction area and also in the decomposition/removal area (Figure 4.102). As expected, more complex is formed in the reaction/extraction area with increasing contact time. Also, more complex results when increasing the Co(ll) concentration at constant Cu(ll) concentration. This proves that mass transfer is efficient (as high concentrations can also be handled) and that no interference from other analytes falsifies the measurement. As a result, calibration curves were derived. 

In the above-described measurement, which we call the absolute method, all pumps have equal speeds (rpm) owing to interconnection to the same drive-shaft. In order to express, if required, a deviation registered for the analyte concentration, one must calibrate with a standard by varying its rpm (B) with respect to that of the titrant (A) a B/A rpm ratio greater than unity means a proportionally lower concentration and vice versa. In general, the absolute method serves to control a sample stream with nearly constant analyte concentration as a sensor one uses not only electroanalytical but often also optical detectors. However, with considerably varying analyte concentrations the differential method is more attractive its principle is that in the set-up in Fig. 5.15 and with the sensor adjusted to a fixed and most sensitive set-point, the rpm of the sample stream (C) is varied with respect to that of the titrant (A) by a feedback control (see Fig. 5.3a) from the sensor via a regulator towards the... 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

The principle behind the test method(s) is that antibodies are made of proteins that recognize and bind with foreign substances (antigens) that invade host animals. Synthetic antibodies have been developed to complex with petroleum constituents. The antibodies are immobilized on the walls of a special ceU or filter membrane. Water samples are added directly to the cell, while soils must be extracted before analysis. A known amount of labeled analyte (typically, an enzyme with an affinity for the antibody) is added after the sample. The sample analytes compete with the enzyme-labeled analytes for sites on the antibodies. After equilibrium is established, the cell is washed to remove any um-eacted sample or labeled enzyme. Color development reagents that react with the labeled enzyme are added. A solution that stops color development is added at a specified time, and the optical density (color intensity) is measured. Because the coloring agent reacts with the labeled enzyme, samples with high optical density contain low concentrations of analytes. Concentration is inversely proportional to optical density. 

An easy calibration strategy is possible in ICP-MS (in analogy to optical emission spectroscopy with an inductively coupled plasma source, ICP-OES) because aqueous standard solutions with well known analyte concentrations can be measured in a short time with good precision. Normally, internal standardization is applied in this calibration procedure, where an internal standard element of the same concentration is added to the standard solutions, the samples and the blank solution. The analytical procedure can then be optimized using the internal standard element. The internal standard element is commonly applied in ICP-MS and LA-ICP-MS to account for plasma instabilities, changes in sample transport, short and long term drifts of separation fields of the mass analyzer and other aspects which would lead to errors during mass spectrometric measurements. 

In amperometry, we measure the electric current between a pair of electrodes that are driving an electrolysis reaction. One reactant is the intended analyte and the measured current is proportional to the concentration of analyte. The measurement of dissolved 02 with the Clark electrode in Box 17-1 is based on amperometry. Numerous biosensors also employ amperometry. Biosensors8-11 use biological components such as enzymes, antibodies, or DNA for highly selective response to one analyte. Biosensors can be based on any kind of analytical signal, but electrical and optical signals are most common. A different kind of sensor based on conductivity—the electronic nose —is described in Box 17-2 (page 360). 

Let us illustrate the benefits of higher order on a concrete analytical example measurements of concentration of Mg2+ with an ISE and with an optical sensor. After linearization of the potentiometric signal, the two experiments can be displayed as a bilinear plot (Fig. 10.2). Contained in this plot is an unusual sample point S, which clearly falls out of the linear correlation because it lies outside the statistically acceptable 3a noise level. This outlier is an indication of the presence of an interferant. Its presence is clearly identified in this bilinear plot from combined ISE and optical measurement, although it would be undetected in a first-order sensor alone. 

In contrast, a nonenzymatic optical sensor measures the analyte concentration directly and the measured signal is directly proportional to the glucose concentration. 

Fig. 5.2. (a) Evanescent field of the fundamental propagation mode in an optical waveguide, (b) Interaction of the evanescent wave with a biomolecular reaction for sensing purposes. The adsorption of the receptor layer and the recognition process produces a change of the effective refractive index of the waveguide inside the evanescent field and this change is quantitatively related with the concentration of the analyte to be measured. 

An evanescent wave biosensor was devised for determination of analytes capable of intercalation in dsDNA in a FIA system. A polyethylene lensed optical fiber is coated with a thin polymeric layer containing dsDNA which is immobilized there. The fiber is placed in a FLA system immersed in a solution of ethidium bromide (144), which undergoes intercalation in the dsDNA. The fluorescence signal of 144 is thus enhanced about a 1000-fold relative to the evanescent wave fluorescence measurement without the coating and is dependent on the concentration in solution. If an analyte is present in the same solution, it competes with 144 for intercalation in the DNA and causes fluorescence quenching, which can be measured and correlated to the analyte concentration. This method was applied to determination of various analytes, including 4, 6-diamidino-2-phenylindole dihydrochloride (145)247. 

For optical transducers, the measured signals are directly proportional to [P], so that, once again, reaction layer thickness and mass-transport kinetics determine the sensitivity of the biosensor, and signals are directly proportional to analyte concentration. For potentiometric transducers, signals are proportional to log[P], and therefore to log[S]. ... 

Measurements will be repeatable based on the presumption that the sample is inherently uniform in analyte concentration. It is also important to think about the possibility of re-absorption of the emitted fluorescence (the optical quenching... 

Fluorescence spectroscopy is used mainly as a quantitative analytical tools. The intensity of light absorbed, /abs, is related to the concentration of analyte and optical parameters of the measurement system by the Beer-Lambert law. If all of the molecules that absorbed light fluoresced, /ate would also be the intensity of fluorescence. However, the processes of internal conversion, intersystem crossing, and quenching compete with fluorescence so that the intensity of fluorescence is given by Eq. (11),... 

In flow analysis, the Schlieren effect tends to be more pronounced [28] because a perfectly mixed flowing sample is not practically achievable, and an analyte concentration gradient is always present. Undesirable concentration gradients and/or discontinuities along the monitored sample can give rise to the formation of relatively steady liquid lenses as well as a myriad of randomly distributed transient mirrors. These optical artefacts lead to fluctuations in the emergent radiation that can alter the measured signal. 

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