Chemiluminescence in Analytical Chemistry

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1. INTRODUCTION

Chemiluminescence (CL) is usually generated by fast reactions and hence the phenomenon can be followed only when the chemical reaction is initiated in front of the light detector. A plethora of analytical research on CL was carried out with modified fluorimeters [1] that allowed mixing of reagents and injection of the analyte through a syringe maintaining the lighttightness of the unit. The airsegmented continuous-flow system is not appropriate for CL measurements since the time between mixing of the reagents and passing the final solution through the debubbler before measurement is sometimes longer than the time required for maximum CL intensity. Nevertheless, a plethora of analytes have been determined by nonsegmented continuous-flow manifolds [2] and some of them are summarized in Table 1.

The great boost of analytical CL appeared soon after the discovery of flow injection analysis (FIA) by Ruzicka and Hansen [3]. The speed with which the solutions of reagents can be supplied to the detector proved to be the best for CL reactions. Various mixing coils were investigated and this was the beginning of an avalanche of research on CL [4].

In this chapter the basic principles and various designs of flow injection chemiluminometers will be presented.

2. BASIC PRINCIPLES OF FLOW INJECTION ANALYSIS

FIA [5–7] is a version of continuous-flow analysis based on a nonsegmented flowing stream into which highly reproducible volumes of sample are injected, carried through the manifold, and subjected to one or more chemical or biochemical reactions and/or separation processes. Finally, as the stream transports the final solution, it passes through a flow cell where a detector is used to monitor a property of the solution that is related to the concentration of the analyte as a

function of time. A typical recording has the shape of a peak (Fig. 1). The height of the peak (H) is related to the concentration of the analyte. The area (A) under this peak or the width (Wi) of the peak at a fixed height can also used for correlation with the concentration of the analyte. The time interval between sample injection and recording of the maximum value of the peak is called residence time (T). During this time interval, all necessary physical and chemical processes occur to generate the analytical signal. Wash time (tw) is defined as the time interval between the maximum value of the peak height until the removal of the sample zone from the detection area. The dispersion or dilution of the analyte or its reaction product can be controlled through the geometrical and hydrodynamic features of the flow system. Neither physical equilibrium (homogeneous flow) nor chemical equilibrium (completion of reaction) is reached by the time of signal detection. In a well-defined flow injection system the time required for completion of a single measurement ranges between 20 and 60 s.

Analytical measurement by CL is very sensitive to a variety of experimental factors and even slight variations of these factors affect extensively the emitted radiation. Thus, analytical CL measurements require highly reproducible mixing of the reagents necessary for the chemiluminescent reaction and in such a way to ensure repetition of the whole procedure at different times. A suitable observation cell (flow cell) that allows detection of the CL emission at an appropriate time after mixing of analyte with reagents and initiation of the reaction is also needed. Therefore, combining FIA with CL is a powerful tool to exploit the advantages of both these techniques.

Figure 1 Schematic diagram of a typical FIA peak. S, time of sample introduction; T, residence time; H, peak height; A, area under peak; Wi, peak width at fixed height; tw, wash time.