Sample collection and preparation

Despite the nature of the environmental regulations and the precautions taken by the refining industry, the accidental release of nonhazardous chemicals and hazardous chemicals into the enviromnent has occurred and, without being unduly pessimistic, will continue to occur (by aU industries—not wishing to select the refining industry as the only industry that suffers accidental release of chemicals into the environment). To paraphrase chaos theory, no matter how well one prepares, the unexpected is always inevitable. 

It is at this point that the environmental analyst has to identity the natme of the chemicals and their potential effects on the ecosystem(s) (Smith, 1999). Although petroleum itself and its various products are complex mixtures of many organic chemicals (Chapters 2 and 3), the predominance of one particular chemical or one particular class of chemicals may offer the enviromnental analyst or scientist an opportunity for the predictability of behavior of the chemical(s). 

Once the immunoassay that meets the study objectives has been identified, sample collection begins. Proper sampling is critical in order to obtain meaningful results from any type of analytical assay. An appropriate sampling scheme will support the objective of the test. For example, a plant breeder may take a single leaf punch to determine quickly whether a specific protein has been expressed in an experimental plant. A more complex sampling regime would be used to determine the expression 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

Because the protein analyte is endogenous to the plant, it can be difficult to demonstrate the efficiency of the extraction procedure. Ideally, an alternative detection method (e.g., Western blotting) is used for comparison with the immunoassay results. Another approach to addressing extraction efficiency is to demonstrate the recovery of each type of protein analyte from each type of food fraction by exhaustive extraction, i.e., repeatedly extracting the sample until no more of the protein is detected.  

Some examples are given below to illustrate extraction procedures for proteins that have been optimized for different matrices and testing strategies. 

Neomycin phosphotransferase II (NPTII) extraction from cotton leaves and cottonseed. The extraction buffer consists of 100 mM Tris, lOmM sodium borate, 5mM magnesium chloride, 0.2% ascorbate and 0.05% Tween 20 at pH 7.8. The frozen leaf sample is homogenized in cold (4 °C) buffer. An aliquot of the homogenate is transferred to a microfuge tube and centrifuged at 12 000 g for 15 min. The supernatant is diluted and assayed directly by ELISA. 

Most automated instruments available for screening procedures or high-volume analyses of samples involving a complex biological matrix, such as whole blood, serum, or plasma, are designed after the fact when it comes to sample collection and sample preparation. They assume a readily available hemolysate, a serum sample separated from its clot, or plasma separated from red blood cells although instrumentation built 

Centrifugation of samples with manual introduction and retrieval of the individual sample or groups of samples is still the mode of operation in most biochemical and clinical laboratories, and there exist a variety of automated dispensers for the introduction of these samples into continuous-flow, discrete, and parallel automated analyzers. However, the tedium of application of a quantitative amount of sample to a medium for electrophoresis has as yet no practical solution suitable for mass use. An 

FIGURE 1. Prototype system for washing a series of different erythrocyte samples in parallel, hemolyzing them, and then recovering them for electrophoretic analysis. 

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The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

For analyses in the field, the ionization chamber will usually be connected to an automated module for sample collection and preparation. 

Sample collection and preparation for the analysis of 1,2-dibromoethane in foods includes the purge-and-trap method, headspace gas analysis, liquid-liquid extraction, and steam distillation (Alleman et al. 1986 Anderson et al. 1985 Bielorai and Alumot 1965, 1966 Cairns et al. 1984 Clower et al. 1985 Pranoto-Soetardhi et al. 1986 Scudamore 1985). GC equipped with either ECD or HECD is the technique used for measuring 1,2-dibromoethane in foodstuffs at ppt levels (Clower et al. 1985 Entz and Hollifield 1982 Heikes and Hopper 1986 Page et al. 1987 Van Rillaer and Beernaert 1985). 

Standard Test Methods on the other hand, specify procedures for carrying out measurements. In the case of emissions testing, methods are usually broken down into multiple separate sections covering sampling frequency, sample collection and preparation, emissions testing and vapor analysis (see below.) Test methods may also include lists of target compounds or compounds that are commonly... 

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