Stopped-Flow FIA Measurement

The determination of ethanol in blood is the parameter most frequently measured in forensic and clinical toxicology. It is based on the following reaction scheme  

The potential of the FIA stopped-flow technique may, of course, equally well be exploited by reversing the roles of substrate and enzyme. Thus in Chapter 2.4 is shown a figure (Fig. 2.32) depicting the readout 

In Section 4.1 the potentiometric determination of urea via enzymatic degradation by urease according to the reaction  

Another interesting example of the FIA stopped-flow procedure is the. determination of sulfur dioxide in wine [71], based on the well-known West-Gaeke [4.2] method, in which a purple compound formed by the 

Inherently, the FI A stopped-flow procedure should be an ideal vehicle to determine reaction rates and rate laws, provided that an experimental approach could be designed that allows resolving the individual contributions of physical dispersion and chemical kinetics. A comprehensive treatment of this problem was recently described by Hungerford et al. [838], who pointed out that although the single-line stopped-flow system (Fig. 4.15a cf. Fig. 4.11) allows optimization of solution conditions for measurement during a selected stopped-flow time interval (fs) by choosing 

---

However, the stopped-flow FIA technique allows the fractional conversion to be measured experimentally for any FIA system by resolving the contribution of the dispersion process and of chemical kinetics. By stopping the flow (cf. Sections 2.4.3 and 4.3) any element of the dispersed sample zone can be selected and arrested in the flow cell, where the change of response with time [i.e., rf(response)/[Pg.81]

For pH measurements (e.g., in combination of FIA with hydrolytic enzyme application), glass electrodes (see subsection 2.1.1) are most frequently used. Detailed studies have been carried out on their behavior and applicability to the merging zones and stopped-flow FIA modes [348, 349]. 

Flow injection analysis (FIA) (Ruzicka and Hansen), since 1975 In continuous flow, stopped flow or with merging zones (FIA scanning or intermittent pumping) Adapted voltammetric electrodes Membranes for Partial dialysis Membrane amperometry (Clark) Differential techniques (Donnan) Computerization, including microprocessors Special measuring requirements in plant control (to avoid voltage leakage, etc., Section 5.5)... 

The hemoglobin-catalyzed oxidation of o-phenylenediamine to 2,3-diaminophenazine (100), in phosphate-citric acid buffer at pH 5.0, shown in equation 30, is the basis for a kinetic method for determination of H2O2, in a FIA system, measuring at 425 mn by the stopped-flow method. The LOD is 9.2 nM, with RSD 2.08% at 0.5 p,M and linearity in the 50 to 3500 nM range . This colorimetric method was proposed for development as a standard procedure in the Republic of China for determination of H2O2 in foodstuffs . ... 

The flow characteristics of FIA create some problems that must be addressed such as the concentration gradient, stopped flow mode, read-out/software protocol, and peak height or peak area measurements made for quantitation. 

FIA is capable of very high precision, better than 1%, as a result of very controlled sample dispersion in a continuous flow stream. Kinetic FIA measurements can be made by precisely stopping (under computer control) the flow of the analyte product in the detector cell at a position corresponding to the peak or some point past it, depending on concentrations. Then the change of signal, for example, absorbance, is recorded for a period of time to obtain the rate. The slope of this recorded signal is plotted for different concentrations to prepare a calibration curve. 

Kinetic methods. FIA, on account of Its Intrinsic features (measurements under non-equilibrium conditions), can be considered to be a fixed-time methodology. However, according to FIA jargon, a kinetic method is based on the monitoring of the evolution of the analytical signal (stopped-flow methods) or on the measurement of two or more signals at the number of times required (differential or individual kinetic determinations). 

The automation or semi-automation of a conventional manual method by FIA often results in a decrease in the number and level of interferents. Thus, in the FIA version of the determination of cyanide by the classical reaction with barbituric acid/chloramine T, nitrite and sulphide pose no Interference at concentrations ten times as high as that of the analyte, which is otherwise adversely affected by the presence of both interferents in the manual method [48], The greater tolerance to foreign species in FIA methods can be generally attributed to their kinetic character, so that undesirable side reactions scarcely have the opportunity to develop to an appreciable extent in such a short interval as the residence time. The tolerance to extransous species is even more remarkable in kinetic FIA methods based on the measurement of a reaction rate (stopped-flow). Optimization of FIA systems as regards selectivity is a relatively simple task on account of their enormous versatility. 

Except for FIA titrations, the stopped-flow method [20, 264] is, the most frequently used gradient technique. The most practical approach to reaction rate measurement is to halt the reaction mixture within the observation volume of the flowthrough detector by stopping the flow and to measure the change of the signal with time. This is readily accomplished by the FIA stopped-flow technique (Chapter 4.3), which relies on a combination of a suitable dispersion of the sample material within the reagent stream and on the subsequent reaction rate measurement during the stopped-flow period. 

Peak height at continuous flow or fixed-time reaction rate measurement at stopped flow are the most frequently used parameters of the FIA response curve for reaction-rate-based assays. 

Being suitable for conventional FIA procedures, as well as for gradient techniques and stopped-flow measurements, the hydrodynamic injection variations presented above are also ideally suited for microminiaturization. 

Multisyringe FIA is an automatic-flow method that combines the multichannel operation of FIA and the flexibility of multicommutation flow systems. In this system, the antioxidant capacity can be determined by the DPPH method for several samples using the stopped-flow condition, which enables the monitoring of the absorbance decrease with time and evaluation of the reaction kinetics (Magalhaes et al., 2006 Magalhaes et al., 2009 Kedare and Singh, 2011). The absorbance in the presence and absence of an antioxidant is measured, and the AA% or IC50 can be calculated as in the static spectro-photometric method. 

In these, the sample is introduced into the system in a continuous manner, either by keeping its speed constant and changing that of the titrant, or vice versa —alternatively, both sample and titrant can be kept at a constant speed and measurements can be made as a function of the analytical signal thus obtained. A discontinuity is occasionally introduced into the system in order to intercalate the wash solution other times (e.g. in FIA titrations, which involve injecting the sample into the titrant stream and monitoring the signal/time binomial) there is no need to stop the flow as the titrant stream itself acts as a wash solution. 

---