Standard curve technique

In analytical practice, the concentration of the given analyte is, in most cases, determined by the standard curve technique. The technique is based on the determination of the relationship between the absorbance and the analyte concentration under the measuring conditions. The relationship is given in terms of the regression equation, or graphically in the form of a standard curve. For systems that obey Beer s law this curve is a straight line. 

If a series of determinations is to be made on the same type of material, it is advisable to make a standard curve by adding known amounts of methoxychlor to a stripping of the material under consideration which is known to be free of methoxychlor. Any slight variation introduced into the technique by the presence of other extracted substances will be compensated for in this way. 

The samples were analysed by injecting 25 pi aliquots into an HGA 2000 Perkin-Elmer graphite furnace attached to a Jarrell-Ash 82-800 double beam atomic absorption spectrophotometer. Graphite tubes in the furnace were replaced after 75-100 analyses. Metal concentrations were determined by comparing the peak heights of the samples to the standard curve established by the determination of at least five known standards. The detection Emits of this technique for 1% absorption were 0.9 pmol/1 (Fe), and 0.2 pmol/1 (Mn). The coefficient of variation was 11% at 6.5 pmol/1 for iron and +12% at 11.8 pmol/1 for manganese. 

The probit relationship of Equation 2-4 transforms the sigmoid shape of the normal response versus dose curve into a straight line when plotted using a linear probit scale, as shown in Figure 2-10. Standard curve-fitting techniques are used to determine the best-fitting straight line. 

Finally, there are custom two-step quantitation methods such as chromatography or ELISA that require a capture step for isolating the protein and then a quantitation step based on a standard curve of the purified target protein. The preliminary capture step may also concentrate the protein for increased sensitivity. These techniques are typically not available in a commercial kit form and may require extensive method development. They are more labor intensive and complex than the colorimetric or absorbance-based assays. In addition, recovery of the protein from and reproducibility of the capture step complicate validation. Despite these disadvantages, the custom two-step quantitation methods are essential in situations requiring protein specificity. 

One common problem of ELISAs affecting accuracy is the hook effect. This is when the signal does not increase with increasing concentration but actually decreases. Another weakness of ELISA is that, compared to other techniques, it has a limited dynamic range. Extrapolation beyond the limits of the range of the standard curve can lead to inaccuracies. 

Not all metabolites may have an internal standard, especially in techniques that measure many substrates. In this case, we can measure the ratio of the analytes of interest to a different internal standard via a standard curve and make corrections. If no unlabeled or labeled standard is available for a compound or peak of interest, then a simple ratio of masses can be calculated. Although this may be quite reproducible, it should be recognized that there is an added degree of uncertainty about the measurement and renders the analysis qualitative, or at best semiquantitative. 

Another calibration technique - standard addition - minimizes matrix effects because analytes with well defined increasing concentrations are added to a set of sample solutions to be analyzed. The measured calibration curve in the standard addition mode plots the measured ion intensities of analytes versus the concentration added to the sample solution. The concentration of analytes in the undoped sample is then determined by extrapolation of the calibration curve with the x-axis. Matrix matching is subsequently performed and the matrix effects (signal depression or interference problems) are considered. An example of the standard addition technique is described in Section 6.2.6 using solution based calibration in LA-ICP-MS. 

Gas chromatography provides a rapid analysis of citrus oil quality. This technique can be further enhanced to determine quantitative levels of individual compounds. Compounds can be measured based on the FID response. A standard curve with known concentrations is used to extrapolate an unknown concentration. 

As with any analytical technique, generation of a reproducible standard curve with minimal error is critical. An assay calibration consists of several steps during which the value of the primary standard is transferred to the calibrators used in the final assay [22]. Immunoassay optimization is usually difficult due to protein heterogeneity and matrix effects and these factors, heterogeneity and matrix effects, will also affect MIP based assays [22]. 

Oxidation of myelin surface cerebrosides by galactose oxidase. Fig. 4 shows silica HPLC of a mixture containing benzoylated-non-hydroxy and hydroxycerebroside and benzoylated derivatives of 2,4-dinitrophenylhydrazone of oxidation products from nonhydroxy- and hydroxycerebroside. Standard curves of two 6-dehydro-derivatives were shown in Fig. 5. These standard curves demonstrate that the response of the benzoylated dinitrophenylhydrazones are linear between 0.025 nmol and 0.6 nmol. Since cerebrosides containing 5 nmol can be determined without tailing to these peaks, this method should allow the determination of as little as 0.5% of the oxidation product. The fact that each curve intersects 0 point in both the abscissa and ordinate indicates that even smaller amounts of these compounds can be detected by this technique. 

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