Method of External Standards

Standard solutions of known concentration of analyte to provide absorbances between 0.2 and 0.8 are prepared by serial dilutions of a stock solution of an authentic standard. Each standard solution is aspirated into the flame and the observed absorbance is recorded. A calibration curve is then prepared by plotting absorbance versus concentration of analyte. As predicted by the Beer - Lambert relationship, the resulting plot wiU be linear or have a slight curvature (towards the concentration axis) at higher concentrations. The absorbance of an unknown, measured under identical operating conditions, is then related to analyte concentration in the unknown using the cahbration curve (i.e., a process of linear interpolation is used). A prerequisite to successfully using the method of external standards is that the sample and standard solutions behave identically in the flame no measurable 

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The concentration of Ca + in a water sample was determined by the method of external standards. The ionic strength of the samples and standards was maintained at a nearly constant level by making each solution 0.5 M in KNO3. The measured cell potentials for the external standards are shown in the following table. 

The purity of a sample of K3Fe(CN)6 was determined using linear-potential scan hydrodynamic voltammetry at a glassy carbon electrode using the method of external standards. The following data were obtained for a set of calibration standards. 

The concentration of Mn in steel can be determined by a neutron activation analysis using the method of external standards. A 1.000-g sample of an unknown steel sample and a 0.950-g sample of a standard steel known to contain 0.463% w/w Mn, are irradiated with neutrons in a nuclear reactor for 10 h. After a 40-min cooling period, the activities for gamma-ray emission were found to be 2542 cpm (counts per minute) for the unknown and 1984 cpm for the standard. What is the %w/w Mn in the unknown steel sample ... 

The electrode-calibration method is also referred to as the method of external standards, which is described in some detail in Section 8C-2. 

Lead was determined polarographicaUy at the dropping mercury electrode by measurements in 1 M HNO3. The limiting current on the Pb(II) wave was measured at —0.600 V versus SCE. At this potential, the residual current was 0.12 (xA. The method of external standards was used and the following results obtained ... 

For the data given in Example 31-1, compare the method of external standards to the internal standard method. Plot the analyte peak height versus percent analyte and determine the unknown with-... 

Extraction and Analysis of Alachlor. Soil was slurried with 12 mL of water and extracted twice by stirring with 50 mL of ethyl acetate for 45 minutes. The solvent was decanted after each extraction and concentrated on a steam bath. The extract was rediluted with ethyl acetate and analyzed by GLC with nitrogen-phosphorus specific detection. Residues were separated on a 90-cm x 0.2 mm i.d. glass column packed with 5% Apiezon + 0.125% DEGS maintained isothermally at 190°C. Injector and detector were held at 250°C, and gas flow rates were adjusted as needed to obtain maximum sensitivity and resolution. Residues were quantitated by the method of external standards. 

Determining the Relationship between Absorbance and Concentration 1 he method of external standards (sec Section ID-2) is most often used to establish the absorbance versus concentration relationship. After deciding on the conditions for the analysis, the calibration curve is prepared from a series of standard solutions that bracket the concentration range expected for the samples. Seldom, if ever, is it safe to assume adherence to Beer s law and use only a single standard to determine the molar ab,soq)tivity. It is never a good idea to base the results of an analysis on a literature value for the molar absorptivity. 

Kinetic methods can employ several differcnl types of reactions. Catalyzed reactions are among the most popular. With these, a catalyst is determined by its influence on the reaction rale or one of the reactants is determined. For example, iodide is a catalyst in the reaction of Cc(IV) with As(lll). Trace quantities of I can be determined by measuring the rale of this reaction as a function of the I concentration. Normally, the method of external standards is used to prepare a calibration curve of rate versus iodide concentration. More than forty inorganic cations and more than fif-icen anions have been detennined based on their catalytic effect. 

An analytical method is standardized by determining its sensitivity. There are several approaches to standardization, including the use of external standards, the method of standard addition. 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

There are three commonly encountered methods of employing these standards, namely the use of external standards, the use of standard additions and the use of internal standards. 

The determination of the relationship between detector response and the sample concentration is termed the calibration of the method. There are two types of methods in use for the quantitative analysis of a sample, i.e., the external standard and the internal standard method. An external standard method is a direct comparison of the detector response of a pure compound (standard) to a sample.2 The calibration of the method is performed by preparing standards of varying concentration and analyzing them by a developed method. Method 1 (below) was developed for toluene, and standards of varying concentration were prepared and analyzed. The results obtained are summarized in Table 2 see Figure 3. 

The assay of stressed samples will usually require the use of some type of external standard. The external standard could be an established reference standard, however, the preferred method is to use the same material/lot as is being stressed. This is easily accomplished by weighing additional samples (that will not be stressed) for use as standards at the same time as the stress test samples are weighed. The standards should then be stored under conditions that will assure that no degradation will occur (e.g., freezer). At the time of analysis, the stressed samples are simply assayed vs. the freshly prepared unstressed standards and the results calculated as percent initial. 

Integration of a peak is simply the first step in data manipulation for the determination of component concentrations in a sample. Peak integration is performed in order to convert the detector signal into numerical data. There are four principal techniques for determining relative composition information about the sample, all of which rely on the construction of calibration curves. These methods are normalization, the internal standard method, the external standard method, and the method of standard additions. 

Rieger and Ballschmiter [43] developed a low resolution method for PCA analyses, based on GC/ECNI-MS in the SIM mode. Their method of quantifying PCAs in environmental samples was based on a triangulation method previously developed in their laboratory for the analysis of toxaphene [56], For PCAs, typically four ions,known to be prominent in the external standard, were monitored in separate injections of a known amount of standard and in the sample, and the areas of the broad PCA peak were then compared. The choice of external standard used was based on pattern matching, i.e., visually comparing the elution time and signal structure of the sample to those of a number of in-house standards [57],... 

Linearity can be established by visual evaluation of a plot of the area as a function of the analyte concentration. The correlation coefficient, y intercept, slope, and RSD for all the generated response ratios (= area/concentration) should be calculated. The y intercept should statistically not differ from 0. The slopes of these curves (Fig. 7) are divided by the slope of the active compound curve (Fig. 5) to determine the RRFs these are recorded in the method procedure if the method does not prescribe the use of external standards for related compounds. 

Quantitation was made through the use of external standards. The calibration curve, constructed from measurement of the total area under the chromatogram versus known weights of standard LAS, was linear from 0 to 3 ug LAS injected. The sensitivity for total LAS was 10 ppb for aqueous samples and 100 ppb for solid samples when using the suggested sample volumes. The reproducibility of the entire method expressed as a relative standard deviation, was 4 % for aqueous samples and 10 % for solid samples. Recovery of standard additions of LAS was 94% minimum. 

Internal standard calibration similarly requires the preparation of external standard solutions, but in addition a constant concentration of a second compound is added to each sample. The sample concentration is directly proportional to the ratio of the analyte to internal standard. This method is used for the analysis of biological samples and other more difficult analyses. 

Make a series of 0.5 pi injections of solvent to be analysed and optimise resolution for the components in the sample by adjusting column temperature and mobile phase flow-rate. Under the optimum conditions determined make at least three 0.5 pi injections of the standard solutions and of all sample solutions on both columns. Check the reproducibility of the area data. Repeat injections if greater than 5% deviation from mean of analyte peak. Check the linearity of detector response for both systems and by the method of external standardisation determine the concentration of water in the sample. Compare the order of elution of the analytes on the two columns and account for any differences (Figures 9.13 and 9.14). The experiment can be extended by using the technique of standard addition to determine the water content of methanol. A series of standard water solutions are prepared (e.g. 0.5, 1.0, 1.5 and 2.0% (v/v) water together with an accurate volume of methanol) these standards and the sample prepared in an analogous fashion are analysed as above. The water content in the solvent can be obtained by extrapolation of the calibration line the intercept with the x-axis giving the concentration of water in the sample. 

Four techniques are commonly used to convert peak heights or areas into relative composition data for the sample. These are the normalization method, the external standard method, the internal standard method and the method of standard additions [264,284]. In the normalization method the area of all peaks in the chromatogram are summed and then each analyte is expressed as a percentage of the summed areas. All sample components must elute from the column and their responses must fall within the linear operating range of the detector. This method will always lead to totals representing 100%. If the detector response is not the same for all compounds then response factors are required to adjust the peak areas to a common scale. Response factors are usually determined as the slope of the calibration curve and converted to relative response factors since these tend to be more stable than absolute values. 

The most straigbiforward method foi quantitative chromatographic analyses involves the preparation ( 1 a series of external-standard solutions that approxi-... 

Calibration methods include the use of external standards, and may include the use of internal standards to improve precision. The internal standard may be an added element or elements not present in the sample, but this is a very time-consuming endeavor, both in making the standards and samples and in measuring all the lines. Frequently, the scattered radiation from the X-ray tube is used as an internal standard this is a reasonable approach because the scattered radiation (background) is very dependent on the sample matrix. The ratio of analyte line to scattered radiation results in better precision than the measurement of the analyte line alone. 

It is important to remark that the use of automatic injectors increases the reproducibility in the injection, which has allowed for the spread in the use of external standards because of its simplicity. However, in the case of manual injection, the internal standard method turns out to be simple and adaptable to everyone. 

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