Chemiluminescence in Analytical Chemistry

Figure 8 Schematic diagram of connectors used to join different streams in FIA: (a) Y-shaped, (b, c) T-shaped, and (d) concentric tubes. The arrows show the direction of the flow.

A disadvantage of injection via rotary valves is that while the volume of the sample that is injected is very small, the procedure for filling the loop of the valve requires a high volume of sample to flow through. This may reduce application of this technique in cases where there is only a limited quantity of sample available.

5.4 Flow Cell

The flow cell is the most important component of a flow injection manifold for CL measurements since maximum radiation should be generated while the solution is flowing in front of the detector. Other attributes of the flow cell are the small ‘‘dead volume’’ of the cell to allow fast and effective washing between injections

Table 3 Typical Examples of Connectors Used in Flow Injection with CL Detection

Figure 9 Schematic diagram of typical six-port rotary injection valve at (a) filling and

(b) emptying position. (1) Sample-loop inlet, (2) carrier inlet, (3) outlet to the flow cell,

(4) sample-loop outlet, (5) outlet to waste, and (6) sample inlet.

as well as accurate and reproducible control of dispersion and/or mixing of the reagents within the construction. A variety of flow cells used in FIA with CL detection are shown in Figure 10.

The first design of flow cell was based on mixing of reagents by blowing air into the solution but the presence of bubbles disturbed severely the CL radiation [9] (Fig. 10a). A modification of this design allows mixing by introducing the reagents from opposite sides of the cell [4] (Fig. 10b) and it was the first used in FIA with CL detection. Another design of flow cell incorporates a movable back plate to adjust the volume of the cell [10] (Fig. 10c). Nevertheless, the most widely used flow cell is the flat spiral of glass tube (Fig. 10d), introduced by Burguera et al. in 1980 [11]. The design is very simple and can be constructed very easily. It can be placed very close to the light-sensitive area of the detector and, therefore, maximize light intensity. A shortcoming of the cylindrical shape of this flow cell is that only a very thin layer of solution emits light opposite to the detector while light is also emitted to other directions, which are not viewed by the detector.

A recent development of the ‘‘technology’’ of the flow cells is the fountain cell [12] (Fig. 10e). An advantage of this cell for CL is that it has a relatively large surface, where the CL reaction takes place, and more light is emitted, but it must be placed horizontally; otherwise the gravity will influence mixing and dispersion of the flowing solution.

Figure 10 Schematic diagrams of flow cells: (a) flow cell using air bubbles for better stirring (reprinted with permission from Ref. 9), (b) flow cell using counterbalancing inlet flows, (c) flow cell with changeable volume (reprinted with permission from Ref. 10), (d) coiled flow cell (reprinted with permission from Ref. 11), and (e) fountain cell (reprinted with permission from Ref. 12). The arrows show the direction of the flow.

Flow cells may also act as reactors. In BL, enzymes may be immobilized inside the cell either by chemical bonding on the inner surface or by entrapping the enzyme as a heterogeneous system by mechanical ways. This approach has the advantage of low consumption of expensive reagents and enhancement of their stability, which is usually low. Many bioluminescent reactions have utilized the benefit of this process. The flow cell is also used as a reactor in the case of electrogenerated chemiluminescence (ECL) when used with FI manifolds. Some of these applications are included in Table 4.

5.5 Detector

Any device that can monitor radiation can be used as detector. In most cases, end-on and side-on photomultiplier tubes (PMT) are used. The end-on PMT has the benefit of circular photocathode, which views more effectively the radiation emitted from the circular flow cells. Nonetheless, the side-on PMTs are more commonly used due to their lower cost. The signal from the PMT is current and, hence, in most applications a current-to-voltage converter is required to convert the current to voltage, which is then monitored.

Light detection can also be achieved by semiconductor photodiodes or by photodiode array detectors. Their sensitivity, so far, is lower than that of PMTs but they possess the great advantages of much smaller dimensions and lower demand on power supply. These features make them attractive, especially for the construction of portable chemiluminometers. The sensitivity of these detectors

Table 4 Applications of Electrogenerated Chemiluminescence FIA

is expected to improve considerably within the next few years and probably in the near future chemiluminometers will depend solely on them.

Apart from the obvious dependence of the output of a given detector to the concentration of the chemiluminescent species, several other factors also affect the output [13]:

The area of the photosensitive surface of the detector, A.

The distance of the flow cell from the photosensitive area of the detector, d. The three-dimensional shape of the cell.

The use of mirrors. The use of lenses.

The first two factors are very important and it has been found that the detected radiation, L, is related to the intensity of emission, I, by the expression

L I A/d2

Therefore, the sensitivity of the measurement is greatly improved by decreasing the distance of the flow cell from the detector. Practically, the flow cell should be positioned as close as possible to the photosensitive area of the detector.

The effect of the other factors is less significant but sensitivity can still be improved if, for example, mirrors are used to focus the radiation on the detector.

5.6 Data Acquisition System

In the early years of FIA, the signal was followed by recorders and the height of the recorded peak was used as the analytical parameter. Although today recorders are still in use, the progress in computer technology has led to their wide use in handling the analytical signal produced by the detector. They can also be used to control the whole system of the flow injection manifold, including control of the injection and propulsion system. Thus, apart from the height, the area of the recorded signal is now used in the interpretation of the received data, which in some cases extends the linear range of the measurement. Integrators may also be used to record the analytical signal.

6.NEW CHEMILUMINESCENCE REACTIONS AND FLOW INJECTION ANALYSIS

When a reaction is under investigation to establish possible chemiluminogenic properties, a batch chemiluminometer is preferable to be used (Fig. 11). This system can reveal the emission profile of the reaction and provide useful information about the kinetics of the reaction. It is suitable for reactions of all rates even

Figure 11 Schematic diagram of a batch chemiluminometer. H.V., high-voltage power supply; I/V, current-to-voltage converter.

though in cases of extremely fast reactions the rising part of the profile would not be representative of the reaction rate. By using the optimization data from batch systems, it is possible to predict with good accuracy, many of the experimental parameters for establishing the same reaction within a flow system, saving valuable time and reagents [14]. In general, if both systems are available, the batch system should be used for understanding the reaction and the flow system for applying the reaction.

7.FLOW INJECTION ANALYSIS VERSUS SEGMENTED FLOW ANALYSIS AND SEPARATION TECHNIQUES

Two independent groups of scientists developed FIA in the middle of seventies: Ruzicka and Hansen in Denmark [3] and Stewart et al. in the United States [15] and since then it has been developed rapidly. The first group developed the method using primarily instrumentation normally associated with segmented flow analyzers (SFA). In contrast, the second group based their initial work on HPLC components. These two different origins of FIA are responsible for several features of the technique related to SFA and /or HPLC that may characterize FIA as a hybrid of SFA and HPLC.

The main differences between SFA and FIA lie in the absence of air bubbles, which are used in SFA to prevent carryover. This shortcoming is not associated with FIA because the hydrodynamic conditions and the geometry of the system provide sufficient mixing of the solutions without the risk of sample over-

lapping. The absence of air bubbles makes the measurement procedure less sophisticated in the instrumentation used and allows for better sensitivity, reproducibility, and precision. In addition, it reduces significantly the interval between mixing of the solutions and detection of the signal, which is very important for the usually fast-rated CL reactions. Thus, an extremely wide variety of organic analytes have been measured by CL utilizing flow injection manifolds (Table 5).

In relation to separation techniques (mainly HPLC), FIA has the disadvantage that it is not able to separate the sample to its constituents and if the sample contains more than one analyte, then unless the kinetics of the reactions are totally different, it is not possible to measure them. Nonetheless, by placing a separator (e.g., an ion-exchange column) before the flow cell, it might be possible to eliminate these problems. Nevertheless, the scope of each technique is different. Separation techniques are aimed at separating and measuring the constituents of a mixture, while FIA is oriented toward the rapid determination of one or two species in a large number of samples. Also, the FIA instrumentation is much simpler than the corresponding for a liquid chromatographic technique.

8. RECENT FLOW INJECTION ANALYSIS VERSIONS

Progress in developing new flow injection techniques has led to sequential injection analysis (SIA). This technique is relatively new [16] and utilizes simpler instrumentation without reducing the precision comparing to classical flow systems. SIA, contrary to FIA, does not push the analytical stream continuously in the same direction, but the direction and the rate of the flow are altered according to the progress of the analytical procedure. SIA utilizes only one flowing stream, which can move in both directions. Initially the propulsion system pumps a washing solution, followed by the sample and finally the solution of the reagent. All these solutions are introduced via a special injection valve into the same tube, which serves as a medium for the reaction, the incubation, and finally for the detection of the analytical signal. This valve ensures that this successive introduction of different solutions inside the tube is synchronized with the rate of the pump. After the reaction process has been completed, the pump alters the direction of the flow and thus the solutions are flown out of the tube and then the system is ready for the next measurement sequence. Mixing of the reagent(s) with the sample is done by diffusion of one into the other. The magnitude and the time of mixing depend on the residence time of solutions at different sections of the tube, as well as by the direction of the flow and the flow rate. The aim is to obtain the maximum signal possible when the solutions exit the tube and pass through the detector, in the final stage of the analytical procedure. SIA has already been used with CL detection [17] (Table 6).

9. CONCLUSIONS

Combination of FIA with CL detection offers several advantages for CL measurements. Some of them are, briefly:

Small analysis time: The typical time needed for a complete sequence of a measurement in FIA is usually less than 60 s. This feature makes FIA very attractive when a large number of samples have to be measured.

Automation: CL measurements can be performed very rapidly and with minimal human participation if the flow system is carefully designed. Several reagents can be added to the sample if multiple flow lines are used and at predetermined times to fulfill the best condition for maximum CL sensitivity. Introduction of special devices into the flow line, which allow procedures otherwise timeconsuming such as solvent extraction or ion exchange, improve substantially the sensitivity and selectivity of the technique.

Precision: FIA measurements typically show low relative standard deviations (RSD) on replicate measurements, mainly due to the definite and reproducible way of sample introduction. This is a very important feature especially for CL, which is very sensitive to several environmental factors and sensitivity relies greatly on the rate of the reaction.

Versatility: Another benefit that derives from the fact that CL reagents are continuously mixed in front of the detector, regardless of the presence of the analyte, is implementation of the analytical procedure even in cases of reaction where the reagents produce a low background emission. This happens because this background emission can be regarded as the baseline since it is constant with the time and, hence, it will not interfere with the analytical signal produced by the analyte.

On the other hand, there are a few shortcomings in the use of FIA with CL detection:

Kinetics of CL reaction: CL reactions with complex kinetics are not always easily handled. The correlation of peak height with concentration of analyte is

not always linear since the characteristics and limitations in the design of the flow cell allow only a fraction of the emitted radiation to be viewed by the detector.

Physical properties of solutions: Hydrodynamic problems and disturbance of the flow profile might appear when solutions with different properties, such as density or viscosity, are introduced into the manifold.

Fundamental research: Flow systems are not suitable for fundamental CL research, as they do not allow observation of the whole emission profile of a reaction. In addition, in cases of very fast or very slow CL reactions a flow system might lead to wrong conclusions. Therefore, a fast or a slow chemiluminogenic reaction might be missed if the flow system is not well established.

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