The Use of an External Standard

Electrochemical methods covered in this chapter include poten-tiometry, coulometry, and voltammetry. Potentiometric methods are based on the measurement of an electrochemical cell s potential when only a negligible current is allowed to flow, fn principle the Nernst equation can be used to calculate the concentration of species in the electrochemical cell by measuring its potential and solving the Nernst equation the presence of liquid junction potentials, however, necessitates the use of an external standardization or the use of standard additions. 

The external standard method has the following advantages [239]. The use of an external standard permits a simpler relationship between the content of the component of interest (particular groups or structural units) in the system and the yield of the characteristic pyrolysis products to be obtained, which increases the reliability and accuracy of measurement. The introduction of an external standard with a sufficient degree of accuracy makes the selection of a standard peak no longer necessary and considerably simplifies the determination of the composition. 

A classification scheme for ranking the quality of a method has been proposed by the International Federation of Clinical Chemistry (Buttner et al., 1976). They define definitive methods as those that represent the ultimate in quality but are generally so sophisticated that they are not in common usage. Such methods for selenium are neutron activation analyses and mass spectrometry that employ isotope-dilution techniques negating the use of an external standard (Veillon and Alvarez. 1983). 

B) Externally standardized RT-PCR with online-detection using SYBR Green I technology real-time RT-PCR with SYBR Green I detection produces sensitive and reliable results. Due to the use of an external standard curve, the amplification efficiencies for the calibration curve and the analyte must be equal for accurate quantification. 

The method of standard additions can be used to check the validity of an external standardization when matrix matching is not feasible. To do this, a normal calibration curve of Sjtand versus Cs is constructed, and the value of k is determined from its slope. A standard additions calibration curve is then constructed using equation 5.6, plotting the data as shown in Figure 5.7(b). The slope of this standard additions calibration curve gives an independent determination of k. If the two values of k are identical, then any difference between the sample s matrix and that of the external standards can be ignored. When the values of k are different, a proportional determinate error is introduced if the normal calibration curve is used. 

A further critical point are the intensities correlated to spectra of the pure elements. Calculated and experimentally determined values can diverge considerably, and the best data sets for 7 measured on pure reference samples still show a scatter of up to 10%. The use of an internal standard or a simultaneously measured external standard seems to be the most successful way to reducing the inaccuracy below 10%. (Eor a more detailed discussion of background subtraction and quantification see, e.g., Seah [2.9].)... 

Under some conditions, it is difficult to incorporate an internal standard into a method. If the chromatogram is very complex, an internal standard may interfere with quantitation of a peak of interest. The development of highly precise sample transfer techniques, including modem autoinjectors, reduces the dependence of the experimentalist on the use of an internal standard to correct for effects of dilution and transfer losses. In many cases, external standardization can be used effectively. The weight percent purity is determined by comparing the area of each peak in a chromatogram with those generated by separately injected pure standards of known concentration. 

Advantages Relatively rapid test that does not rely on the use of an external force to breakdown an emulsion. Common laboratory equipment is used. Small quantities of protein are used. Disadvantages Emulsions must be prepared under standardized conditions. Some studies have found poor correlations between EAI and emulsion stabilly in food systems. 

One way around this problem is to compare cell fluorescence not with unstained cells but with the fluorescence of an external standard. This can be done by the use of fluorescent beads. In brief (this is an insiders joke you would be amazed at how much has been written about the use of beads in flow cytometry), there are commercially available polystyrene beads ( microspheres ) that have standardized... 

A selection of experimental measurements across medium Z helium-like resonances is shown in Table 3 and compared with recent theory. The most precise absolute measurement is attributed to Deslattes and co-workers [33] with a 12 ppm measurement of the w transition in argon. Our methodology is similar to that of Deslattes et al. in the use of an external X-ray calibration standard lying close to the wavelength of interest. The recoil-ion experimental method used therein also eliminates the need for Doppler corrections and uncertainties in that work as in our EBIT experiment. Argon is at the lower end of the medium Z elements where QED effects are smaller relative to the transition energies. However, this very precise measurement lies below all recent theoretical calculations shown in Table 3. 

The most accurate quantitative results in GC and LC are probably obtained by the use of an internal standard. However, it can be difficult to employ, as the procedure depends upon finding an appropriate substance that will elute in a position on the chromatogram where it will not interfere or merge with any of the natural components of the mixture. Unfortunately, for a multi-component sample, this can become difficult to the extent of being virtually impossible, under which circumstances the external standard method must be used. Having identified an appropriate reference standard, the response factors for each component of interest in the mixture to be analyzed must be determined. It should be noted that this usually does not include all the components. In many instances only certain components of the mixture need to be assayed. A synthetic mixture is then made up containing known concentrations of each of the components of interest together with the standard. 

Moreover, this method can quickly become laborious because an internal standard elution which is compatible with the analysis conditions must be found. Conditions that must be fulfilled by the internal standard are its purity must be known and it must be chemically inert toward solutes and mobile phase on the one hand, its retention time must be different from those of all the constituents present in the sample and, on the other hand, it should be as close as possible as the retention time(s) of the product(s) to be determined. It has also been demonstrated that a necessary correlation was required between chromatographic behaviors of the internal standard and the product to quantify [3]. Otherwise, the use of an internal standard can even degrade the precision of the results. A comparison of the precision of internal and external standard has also been carried out through a liquid chromatography collaborative study [4]. 

The internal standard method is less often used in liquid chromatography than in gas chromatography because injection of repeatable volumes has been made easier by the use of precise and reliable injection systems (loop valves). More generally, gradually, the internal standard method is being abandoned. The external standard method is, nowadays, the most common method and the use of an internal standard seems to be restricted to very specific applications for example, when preliminary to the chromatographic analysis, the solute of interest must be extracted by means of a complex protocol. 

Calibration of AAS methods can be performed by the use of an external calibration curve or by MSA both calibration methods were presented in Chapter 2. Internal standards are not used in AAS, because it is usually a single-element technique we cannot measure an internal standard element at the same time we measure the analyte. 

Quantitative analysis can also be performed by external calibration or by the use of an internal standard that is not a labeled analyte molecule but a compound that is not present in the sample. 

Quantitation by external calibration The most common and straightforward method of calibration in atomic absorption spectrometry is the use of an external calibration with suitable standard solutions. It is based on the assumption that the standard solution matches the composition of the sample sufficiently well. This is an assumption that must always be examined with care, since, for example, samples of different viscosity may be aspirated at different rates in flame AAS,... 

The calibration technique of standard addition is often used to minimize matrix effects in the quantification of organotins. External calibration is also used by many groups, howeveg the use of an internal standard is highly recommended with the nature of the best compound to use dependent on the measurement system involved. Tripropyltin chloride has been used by many groups as a surrogate to monitor the whole method process, and species such as tetra-butyltin and tetrapentyltin have been used as internal standards added just prior to GC analysis. 

Some conditions are required for Eq. 7 to be valid Areas and weights must be expressed in the same unit system both for analysis and calibration because precision and accuracy of this method only depend on the precision and accmacy of weighings, they depend neither on the precision and accuracy of the dilutions nor on the injected volume (unlike the external standard method). It requires no preliminary determination of the proportionality coefficients. However, if some points of the sample handling are fully corrected by the use of an internal standard, other difficulties still remain. ... 

Use of an external standard, injected separately from the sample after a predetermined delay selected so as to avoid interferences between peaks of the standard and those of the mixture components. represents an extremely useful calibration tool, and it also serves as a control for monitoring proper performance of the analytical instrument, especially in process-control analysis [58], [66], The peak area of the external, deferred standard should remain constant for as long as the chromatograph is in good working order. 

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