Chemiluminescence in Analytical Chemistry

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4.1 Sample Dispersion

A very important concept in understanding the theoretical background of FIA is the dispersion of the sample. Dispersion is expressed as the dispersion coefficient, D, which is defined as follows:

D C0 H0K1 A0K3

Cmax HK2 AK4

where:

C0 is the initial concentration of the sample (before the effect of dispersion takes place) while Cmax is the maximum concentration of the sample during the observation time (when the dispersion has taken place),

H0 or A0 is the height or area of the peak that would be measured by the detector if no dispersion had taken place,

H or A is the height or area of the peak during the observation time,

K1, K2, K3, and K4 are ratio factors. If the concentration of the sample is within the linear range of the detector, then the ratio factor K1 is equal to K2 and K3 is equal to K4.

When a certain volume of a sample is instantaneously introduced into the stream of the carrier solution, the sample is carried away by the carrier not as a compact plug but gradually mixes with it. Mixing of the sample with the flowing solution is achieved by the action of two different mechanisms of dispersion: axial dispersion (parallel to the direction of the flow) due to the continuous flow of the stream, and radial dispersion (in vertical direction to the flow) due to diffusion.

Measurements by FIA occur under conditions where laminar flow predominates over turbulent flow (Fig. 3, a and b) and hence a parabolic profile of the concentration of analyte solution inside the carrier stream is developed. The layers of the analyte that are adjacent to the inner surface of the transportation tube flow slowly owing to the friction forces developed between these two different

Figure 3 Schematic diagram of transportation of sample via the stream of carrier by

(a) turbulent flow, (b) laminar flow, and (c) radial diffusion of the sample in response to radial concentration gradient, within the flowing stream.

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surfaces. As the distance of the layer from the inner surface of the tube increases, the flow rate of the layer increases due to reduction of the friction forces. Hence, a concentration gradient is developed, which builds up a radial diffusion to equilibrate the different concentrations (Fig. 3c). Radial mass transfer ensures that sample and carrier are mixed and also reduces spreading out of the sample into the stream, maintaining the dilution of the sample in low level and allowing for relative short time intervals between successive sample injections.

A flow injection system can operate under low (D 1–3), intermediate (D 3–10), or high (D 10) dispersion. For example, if D 2, then the sample has been subjected to 1:1 dilution with the carrier before analytical measurement. Generally, if D n, then the sample is subjected to 1: (n 1) dilution by the carrier prior to measurement. The distribution of the sample in the flowing stream at various times after injection into the stream of the carrier is depicted in Figure 4. D increases with the distance covered by the sample zone through the transportation tube and the recorded peak becomes flat, even though the enclosed peak area usually remains constant. Thus, if peak height is used as the parameter for analytical measurement, then the sensitivity is lowered as D increases. Furthermore, sample throughput decreases with increasing D and hence flat peaks with

Figure 4 Sample dispersion (A) and signal profiles (B) at different distances within the tube (2–5) after injection of the sample into the stream (1), assuming that the detector is able to follow the sample zone as it flows.

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high risk of overlapping are recorded. Under typical operating conditions, the zone of the sample is not completely symmetrical and peaks might be tailed. Flow injection chemiluminometers usually operate under low or medium dispersion.

Dispersion depends on several experimental factors, such as volume of sample injected, length, inner diameter, and geometrical configuration to the space of the transportation tube [and the reactor(s), if there is any], and flow rate of the carrier. If all other variables are constant, then dispersion depends on the following parameters [8]:

Volume of sample: If the volume of the sample increases, then dispersion decreases but, at the same time, the width of the peak increases and hence the measurement rate is reduced.

Geometrical characteristics of flow components: If the length or the internal diameter of the transportation tube increases, then dispersion increases. Dispersion also increases if a reactor is placed between the injection point and the flow cell. However, dispersion greatly depends on the geometry of the tube and/or the reactor used. Lower dispersion is achieved by using single bed string reactors such as the one shown in Figure 5a. With this reactor, broadening of the peaks is reduced to a minimum and this feature permits the introduction of several samples with very low time interval between injections, without any risk of sample overlapping. A shortcoming of this reactor is the high ratio of area to volume, which promotes high pressures and superficial phenomena. These characteristics are desirable when immobilized enzymes or solid reagents are used online. A knitted tube (Figure 5b) has similar attributes to the former reactor, although dispersion is generally higher. A mediocre dispersion is achieved when a coiled

Figure 5 Schematic diagrams of reactors used in FIA in order of increasing dispersion:

(a) single bed string reactor, (b) knitted tube, (c) coiled tube, (d) straight tube, and (e) external mixing chamber with stirring.

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or a straight open tube is used (Fig. 5, c and d). Because this level of dispersion is usually acceptable in CL measurements and provided that these tubes are widespread, they are very widely used. Maximum dispersion can be achieved if an external mixing chamber (Fig. 5e) is used. The peaks recorded when this reactor is used are unsymmetrical and they decline very slowly to the baseline. This characteristic reduces greatly the number of measurements per unit of time. As a conclusion, it should be pointed out that when designing a reactor to be incorporated into the flow manifold, the main requirements are maximum mixing of the sample with the reagents while the width of the peaks is maintained as small as possible.

Flow rate: If the flow rate decreases, then dispersion increases and hence the residence time of the sample within the manifold increases.

4.2 Kinetics of Chemiluminescence Reaction

The chemiluminogenic reaction is initiated as soon as the analyte and the reagent(s) mix within the flow manifold. Therefore, the emission intensity is time dependent as the reaction proceeds and the excited intermediate or product is formed. If the analyte and reagent(s) were mixed in a chamber and the emitted radiation was monitored as a function of time, then a profile similar to that in Figure 6a would be recorded. The emission intensity increases, attains a maximum value, and then declines back to the baseline (for more details, see Chapter 8). This emission profile is the result of the change in the reaction rate versus time as the analyte and the reagents are consumed. Although all CL reactions generally follow the same pattern as for the shape of this emission profile, the time required by the reaction to generate maximum intensity, which then declines to the baseline, varies extensively from less than a few seconds to several hours. Except for the nature of the analyte and CL reagents, this time interval also depends on some factors that affect the chemical environment such as temperature, solvent, ionic strength, pH, and the presence of other species. Since these factors affect greatly the rate of the CL reaction and thus the emission intensity, they should be under strict control to achieve reproducible results.

4.3 Chemiluminescence and Flow System

When maximum peak height is recorded, neither the CL reaction is complete nor the flow system has reached a physical equilibrium (particularly as for the mixing of the streams). Thus a flow system with CL detection should incorporate several features, discussed below:

Number of flow streams: The number of reagent and/or carrier streams is a very important aspect when designing a flow manifold. In contrast to other detection techniques in FIA, with CL detection there is a very important limitation

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Figure 6 CL emission profile with the time (a) after mixing of reagents, initiation of reaction, entrance and exit from the flow cell (FC) of the chemiluminescent solution (b) and typical peaks recorded after successive injections of the same analyte into the manifold

(c).

that should be encountered. This is the need for total exclusion of ambient light from the region of the detector; otherwise a high and uncontrollable noise signal will develop. Although it may seem easy to exclude ambient light from the CL detector, in many cases the transportation tubes used might act as optical fibers and lead light directly to the detector. Thus, for practical reasons and whenever it is possible, the sample is usually injected into a stream of carrier, which then merges into the stream of the CL reagent to initiate the reaction. This configuration also depends on the rate of the CL reaction. If the reaction is relatively slow, the sample can be injected directly into the stream of CL reagent, since the rate of the reaction ensures that maximum intensity will not be emitted before the analytical stream enters into the flow cell. If the reaction rate is high, the sample should be introduced into a separate stream and mix with the CL reagent near the inlet of the flow cell.

Volume of flow cell: Ideally, the solution of reactants should enter into the flow cell as soon as the radiation has such a value to generate maximum intensity while residing within the cell and start declining as soon as the solution flows

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out of the cell. On the other hand, the photosensitive area of the light detector is limited, e.g., about 2–3 cm2 for side-window PMTs. Furthermore, the construction of the flow cell also depends on glass-blowing limitations. Hence, the volume of the flow cell is usually controlled by practical limitations and cannot be easily changed according to the requirements of a single CL reaction.

Flow rate: The limitations associated with the volume of flow cell can be overcome by accurately controlling the flow rate of each stream entering into the manifold. This experimental parameter controls the residence time of the chemiluminescent solution within the cell and can be easily optimized by the operator. Flow rates are directly proportional to the rate of the CL reaction. As the rate of the reaction increases, the flow rate should be increased but, at the same time, consumption of reagents increases. The flow rate also affects the shape and the height of the peak as well as the measurement rate (number of sample or standard solutions injected per hour).

Typical flow rates in FIA vary between 0.5 and 5.0 ml/min per channel, although higher values have also been used. Most of the published work on CL with FIA is based on equal flow rates for every stream entering the manifold. Nevertheless, if different rates must be used, deterioration of repeatability and reproducibility might appear due to incomplete mixing and anomalous hydrodynamic characteristics. These problems can be avoided if the general rule that the ratio of the fastest to the slowest flow rate should not exceed the value of 3 or 4 is followed.

Volume of analyte injected: As the volume of sample and standard solution injected increases, the peak height and duration increase. Nevertheless, if the volume increases too much, dispersion of the carrier into the analyte is limited and the concentration gradient within the flow is distorted resulting in very high dispersion values at the edges and very low values in the center. As a result, double peaks are recorded that are not appropriate for analytical measurement.

4.4Optimization of Flow Injection Chemiluminescence Measurements

The target for optimization in FIA with CL detection is to adjust all experimental factors in such a way so that the detector views as much radiation as possible while the chemiluminescent solution flows through the cell. Hence the kinetics of the flow and detector system should be monitored to match the kinetics of the reaction and generate maximum intensity inside the cell. The effect of experimental variables on the CL signal cannot be exactly predicted in advance and there is not enough theoretical background to support any suggestion.

Some empirical observations can reduce the number of experimental factors that must be investigated. CL reactions require low or medium dispersion to achieve high detection sensitivity. Hence since the predominant feature is the

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time interval between mixing the sample with the reagent(s) and production of maximum emission intensity, optimization should be focused on this parameter. This time interval depends on the flow rate of each stream of the manifold and the length and diameter of the transportation tube between the mixing point and the flow cell. Subsequently, the detector sensitivity can be maximized by controlling these parameters so that the observation time window is near the peak of the CL emission intensity (Fig. 6a). If the volume of the flow cell and the geometrical characteristics of the tube from mixing to the cell are constant, then a change in the flow rate of solutions will alter the position and the width of the observation window. In addition, an extensive study of the effect of concentration of each reagent used will probably help to increase the sensitivity even more.

In some cases, the use of the stopped-flow technique might improve sensitivity. BL reactions or CL reactions with very low rate might benefit from this technique. By stopping the pump when the sample zone has reached the flow cell, it is possible to extend the time allowed for the analyte to react with the immobilized enzyme or with the reagents within the cell. In this way, sample dispersion is kept within low levels and sensitivity remains high. Implementation of this technique presupposes that the propulsion system has the ability to stop instantaneously and reproducibly to entrap always the same portion of sample zone within the flow cell. For very slow CL reactions, stopping of the flow can be done before the sample zone reaches the flow cell and for a time interval enough to generate maximum radiation and then the flow is reestablished for analytical measurement. Flow injection systems that operate under computer control of both the injection system and the propulsion system can be used for this purpose.

5. INSTRUMENTATION

The instrumentation used for FIA with CL detection is usually simple and is composed of the components depicted in Figure 2. These components are readily assembled to form the analytical manifold, although there are also commercially available flow injection systems with CL detection. Spectrophotometric or fluorimetric flow injection systems can often be used for CL measurements after some modifications.

The components required for a dedicated flow injection chemiluminometer are briefly discussed.

5.1 Propulsion Units

Ideally, the flow rate should be constant and perfectly reproducible to achieve reproducible residence time and maintain constant dispersion throughout the sys-

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Figure 7 Schematic diagram of the drum of a peristaltic pump showing the rollers, which squeeze the tubes. The arrows show the direction of the flow.

tem. For this purpose, peristaltic pumps are commonly used. These types of pumps are subjected to pulsation, which depends on the diameter of the roller used to squeeze the tubes (Fig. 7) and the distance between them. Pumps that operate with high-frequency, low-amplitude pulses and/or with pulse dumber are satisfactory. The number of channel (streams) used varies from one to 16, but usually is four.

The flow rate can be altered either by changing the diameter of tubes or by changing the rotation speed of the roller, provided that all other parameters are kept constant. Modern pumps can be controlled by microcomputer and different flow rates can be established during one analytical measurement. The pump is usually located before the injection port but sometimes it can be placed after the detection cell to reduce the effect of pulsation.

Alternatively, but not commonly, propulsion of the solutions can be achieved by gravity-based units, which rely on the difference in height between the solution(s) reservoir(s) and the flow lines of the manifold. Also, gas-pressure units, which rely on the action of pressure by an inert gas inside the vessels, which contain the solutions, can be used. Both these uncommon propulsion units yield pulse-free flow but require periodic refilling of solution reservoirs and adjustment of the desired flow rate is very difficult.

5.2Flow Lines, Connectors, and Intermediate Reaction Systems

Tubes made by polyvinylchloride (PVC), Teflon, silicone, or any other similar material (with internal diameter between 0.1 and 2.5 mm) are usually used to transfer analytical streams along the manifold. A shortcoming of most tubes is that they are not suitable to transfer all kinds of organic solvents. They also

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require periodic replacement depending on the time and conditions of operation. Long-time operation alters the initial length and diameter of the tube and hence the flow rate of the tube changes. This problem is more intense when the propulsion system operates at high rotation speed. A gradual change in the flow rate would reduce sensitivity, which is not easily noticeable and may lead to erroneous results.

In some applications, additional components acting as reactors for specific chemical pretreatment are incorporated within the flow manifold. Typical examples are ion-exchange microcolumns for preconcentration of the analyte or removal of interferences and redox reactors, which are used either to convert the analyte into a more suitable oxidation state or to produce online an unstable reagent. Typical examples of online pretreatment are given in Table 2. Apart from these sophisticated reactors, a simple and frequently used reactor is a delay coil (see also Fig. 4), which may be formed by knitting a segment of the transfer line. This coil allows slow CL reactions to proceed extensively and enter into the flow cell at the time required for maximum radiation. The position of the reactors within the manifold is either before or after the injection port depending on the application.

The connectors used in FIA to join the tubes when the analytical system uses more than one stream usually have the shape of those in Figure 8. These connectors provide adequate mixing of the several analytical streams, while dispersion remains in relatively low levels, to achieve high measured values. The connector in Figure 8d is normally used for very fast CL reactions and allows mixing of the reagents with the analyte in front of the light detector. The choice of the connector depends mainly on the CL reaction rate (Table 3).

5.3 Sample Introduction Unit

The purpose of the injection unit is to insert accurately and reproducibly a very small volume of analyte into the flowing stream. This volume is usually within the range of 25–250 L and very rarely less or more. Injection of the solution into the flowing stream of carrier or reagent should not cause any disturbance to flow of the solution and should be done fast enough to allow a high sampling rate. These requirements are fulfilled by volume-based injection units, such as the typical six-port rotary valve shown in Figure 9, which are based on the entrapment of a certain volume of the sample inside a loop. This is the most commonly used insertion unit and can be controlled either manually or by microcomputer via a pneumatic actuator for instance. By changing the loop with a lager or a smaller one, the volume of the solution injected can be altered. When operating the sample valve, it is essential to avoid the presence of air bubbles as they can modify the flow pattern and affect the mixing process with a subsequent change of the analytical signal.

Table 2

Typical Examples of Online Pretreatment in Flow Injection CL

Analyte

Comments

Anions

Column packed with anionic luminol derivative bound to a strong anion-exchange

resin, displaced derivative–H

O

-[Fe(CN)

]

3

CL

2

2

6

Catecholamines

Online electrogeneration of ClO-, inhibition of luminol CL

Citrate

Photochemical reaction, luminol CL, Pharmaceuticals, soft drinks

Cobalt

8-Quinolinol column, gallic acid–H

O

2

CL, seawater

2

Chromium

Anion exchange column, luminol-H

O

2

CL, speciation

2

Cyanide

Resin with immobilized luminol and Cu

2

,

Tap and waste water

Dopa

Online electrogeneration of ClO-, inhibition of luminol CL

Etamsylate

Online electrogeneration of ClO-, inhibition of luminol CL

Glucose

Immobilization of glucose oxidase, peroxyoxalate-H

O

2

CL

2

Glucose, cholesterol

Immobilized glucose oxidase or cholesterol oxidase to H

O

, luminol CL

2

2

Glutamate, glutamine

Coimmobilized glutamate oxidase

glutaminase, Luminol-H

O

2

CL

2

Hydrazine

Ion-exchange columns to remove interfering metal ions, hydrazine-NaClO CL

Hydrogen peroxide

Removal of interferences by anion and cation resin, luminol CL, rainwater

Inorganic anions

Anion-exchange column, KOH-HNO

CL, water

3

Iron

8-Hydroxyquinoline column, luminol-H O

2

CL, seawater,

on board analysis

2

Iron

8-Hydroxyquinoline column, luminol CL, seawater

Iron

Preconcentration on 8-hydroxyquinoline-5-sulfonic acid column, luminol-H

O

2

CL

2

Isoniazid

Online electrogeneration of BrO

sensitization of luminol CL

,

Nitrate

Photochemical activation, luminol CL, natural waters

Persatin

Online electrogeneration of BrO

inhibition of luminol CL

,

Pesticides

Immobilized choline oxidase/peroxidase, luminol-H

O

2

CL

2

Vanadium

Potassium dichromate-KI reactor, luminol CL

Vanadium

Jones reductor, lucigenin CL, tapwater, river water, industrial wastewater

Xanthine hypoxanthine

Immobilization of peroxidase or xanthine oxidase, luminol-H

O

2

CL

2

L.o.D.
1.0 M SO	4	2	–5.0	M
Br
0.6–1 nM

0.2M 10 ng/L

0.05g/L Cr(III)

0.1g/L Cr(VI) 2 ng/mL

0.8ng/mL

0.6ng/mL

0.05M Not reported

0.1M glutamate, 1 M glutamine

0.5nM 35 nM

70–100 ng/mL 0.021 nM

40 pM

2 pg/mL

7 ng/mL

70 nM

4 g/L

4 g/L aldicarb

0.75g/L paraoxon

0.7nM

0.3ng/mL

2 and 5 M

Ref.

34

35 36 37 38

39 40 41 42 43 44

45 46 47 48 49 50 51, 52 53 54 55

56 57 58

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