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Water Analysis - Trace Pollutants
of detection is also less specific than fluorescence and there is a greater possibility of chromatographic interference from other components in the sample. As with the case of phenols, the development of liquid chromatographic methods often stems from the difficulties encountered with analyses using gas chromatographic techniques. In many cases, this may be attributed to the polarities of the molecules (e.g. phenols and N-methylcarbamates), or their thermal labilities (e.g. N-methylcarbamates and phenylureas).
The techniques described so far involve the use of complex laboratory equipment and often long pretreatment stages. Ideally, an analyst would like to achieve the required sensitivity and specificity with simpler equipment and without any pretreatment being required. Field analysis would also be desirable.
Part of the solution to this problem could be the use of immunoassay but as a separate test has to be designed for each analyte, it will never be the complete answer. Field kits (necessary apparatus, reagents and calibration standards for a specific number of analyses) are available in the |xg/l range with an analysis time of 10-15 min. Laboratory kits are available for individual compounds in the ng l—1 range and are typically capable of handling 40 samples in a period of two hours. The methods commercially available include the analysis of individual pesticides (e.g. atrazine, carbofuran and paraquat), BTEX compounds (benzene-toluene-ethylbenzene-xylene(s)), total petroleum hydrocarbon (TPH), PCBs and PAHs, with the list continually expanding. Several of these methods are now approved by the US Environmental Protection Agency (EPA).
The use of these kits can be very simple, requiring little background knowledge. More thorough knowledge is necessary to understand the potential applications and limitations of the immunoassay process and the almost bewildering number of variations of the basic technique. Chemical and biological principles will both need to be understood and the techniques used sometimes seem to be more at home in a life sciences rather than a pure chemical laboratory. First of all, let us look at a simple method. Later, we will look at the background principles behind the method in an attempt to understand its particular merits.
Most field and laboratory kits use a technique known as competitive ELISA (enzyme-linked immunosorbent assay). For laboratory analyses, reactions take place in the wells of a microtitreplate (Figure 4.11). These are plastic plates which contain typically 40, 48 or 96 wells for the simultaneous analysis of the samples and standards. An automatic scanner (microtitreplate reader) measures their light absorbance at specific wavelengths. This apparatus is commonplace in biomedical laboratories.
106 Introduction to Environmental Analysis
Figure 4.11 A micropipette and microtitreplate used in immunoassay.
The wells of the plate are filled with 100 ^l sample or standards in duplicate. The reagents are then added. After a short period of time, the plate is then washed with water, further reagents are added and the plate is placed in an incubator at room temperature for a period of up to one hour. The absorption of light in each plate is then measured.
One design of field analysis kit includes individual pre-coated tubes, while another manufacturer has reagents attached to magnetic particles. The latter can be separated from the reagent and wash solutions by using a magnet, thus immobilizing the particles on the walls of the tube. Light absorbance is measured by a portable spectrometer.
The response curve is unlike any others you are likely to have come across, with a typical example being shown in Figure 4.12.
Comment on this response curve and suggest possible applications of this analytical technique.
Water Analysis - Trace Pollutants 107
log concentration ------------►-
Figure 4.12 A typical response curve found in immunoassay.
You should notice that the range over which the response can be used quantitatively is quite limited, although it can be more easily used as an indication of whether the pollutant is present or absent. Application is often as a screen for potential pollutants. This will lower the number of samples requiring more expensive GC-MS or LC-MS analysis.
So, how does the technique work? The new terms which you will need to know are summarized in Table 4.3, while Figure 4.13 shows the steps involved in the immunoassay process.
The surface of the wells of the microtitreplate have been coated with an antibody. The samples and standards are introduced into the wells of the plate, each with a fixed amount of a labelled derivative of the pollutant. The derivative molecules compete with the pollutant molecules for binding to the antibody fixed on the plate. The amount of labelled derivative binding to the surface will be determined by the relative concentrations of the pollutant and derivative, and is inversely proportional to the concentration of pollutant originally in the sample.