Reference internal standard method

Because of the complex nature of the discharge conditions, GD-OES is a comparative analytical method and standard reference materials must be used to establish a unique relationship between the measured line intensities and the elemental concentration. In quantitative bulk analysis, which has been developed to very high standards, calibration is performed with a set of calibration samples of composition similar to the unknown samples. Normally, a major element is used as reference and the internal standard method is applied. This approach is not generally applicable in depth-profile analysis, because the different layers encountered in a depth profile of ten comprise widely different types of material which means that a common reference element is not available. 

Quantitative analysis using the internal standard method. The height and area of chromatographic peaks are affected not only by the amount of sample but also by fluctuations of the carrier gas flow rate, the column and detector temperatures, etc., i.e. by variations of those factors which influence the sensitivity and response of the detector. The effect of such variations can be eliminated by use of the internal standard method in which a known amount of a reference substance is added to the sample to be analysed before injection into the column. The requirements for an effective internal standard (Section 4.5) may be summarised as follows ... 

Since the peak size is directly proportional to concentration, one may think that one could prepare a series of standard solutions and obtain peak sizes to be used for a standard curve of peak size vs. concentration, a method similar to Beer s law in spectrophotometry, for example. But since peak size also varies with amount injected, there can be considerable error due to the difficulty in injecting consistent volumes, as discussed above and in Section 12.3. A method that does away with this problem is the internal standard method. In this method, all standards and samples are spiked with a constant known amount of a substance to act as what is called an internal standard. The purpose of the internal standard is to serve as a reference for the peak size measurements, so that slight variations in injection... 

Increasing standard amounts of analyte are added to the sample and the resulting peak areas, which should show an increase with concentration added, are measured. This method is not as useful in GC as it would be in atomic absorption (see Chapter 9), since the sample matrix is not an issue in GC as it is in atomic absorption, due to the fact that matrix components become separated. However, standard additions may be useful for convenience s sake, particularly when the sample to be analyzed already contains a component capable of serving as an internal standard. Thus, standard additions could be used in conjunction with the internal standard method (see Experiment 45), and the internal standard would not have to be independently added to the sample and to the series of standards — it is already present, a convenient circumstance. Area ratio would then be plotted vs. concentration added and the unknown concentration determined by extrapolation to zero area ratio. Please refer to Chapter 9 for other details of the method of standard additions. 

Note Refer to the text to refresh your memory concerning the method of standard additions and the internal standard method. Use a good fume hood when preparing the standards. All flasks and pipettes should be water-free. 

The methyl esters can be also determined by GC-FID. Using a 30 m x 0.32 mm ID x 0.25 pm (film thickness) capillary column, such as DB-1701 or equivalent, the compounds can be adequately separated and detected by FID. The recommended carrier gas (helium) flow rate is 35 cm/s, while that of the makeup gas (nitrogen) is 30 cm/min. All of the listed herbicides may be analyzed within 25 min. The oven temperature is programmed between 50 and 260°C, while the detector and injector temperatures should be 300 and 250°C, respectively. The herbicides may alternatively converted into their trimethylsilyl esters and analyzed by GC-FID under the same conditions. FID, however, gives a lower response as compared with ECD. The detection level ranges from 50 to 100 ng. For quantitation, either the external standard or the internal standard method may be applied. Any chlorinated compound stable under the above analytical conditions, which produces a sharp peak in the same RT range without coeluting with any analyte, may be used as an internal standard for GC-ECD analysis. U.S. EPA Method 8151 refers the use of 4,4,-dibromooctafluorobiphenyl and 1,4-dichlorobenzene as internal standards. The quantitation results are expressed as acid equivalent of esters. If pure chlorophenoxy acid neat compounds are esterified and used for calibration, the results would determine the actual concentrations of herbicides in the sample. Alternatively, if required, the herbicide acids can be stoichiometrically calculated as follows from the concentration of their methyl esters determined in the analysis ... 

For the internal standard method, a substance is added at the earliest possible point in the analytical scheme. This compensates for sample losses during extraction, cleanup, and final chromatographic analysis. There are two variations in the use of the internal standard technique. One involves the determination of response factors which are the ratios of the analyte peak response to the internal standard peak response. The second is referred to as response ratios which are calculated by dividing the weight of the analyte by the corresponding peak response. 

Generally, the analysis of environmental pollutants is considered as a necessary expense that is performed solely if stated by law. With less expensive screening methods and automated modern equipment to analyze suspect samples, the cost of analysis will become much lower. Hence, the attitude towards QA would most probably be more positive and the analytical work much more reliable for the customers. This also strengthens the international competitiveness of European producers. The credibility of the entire monitoring chain (screening methods, reference and standardized methods, as well as CRMs for the quality control of these methods) lies in the adequacy and integration of all three levels of the system. The adequate development and validation of methods is a prerequisite for a harmonized measurement system [80]. 

With some techniques (absolute calibration and internal standard method), the relationship mj/ms = Ajfi /Asf occurs. In such instances the calibration standard also fulfils the role of the reference compound, as... 

The reference model system method can be combined with the internal standard method. In this instance, the model system contains known amounts of the compound under determination and a calibration standard, a known amount of the standard is added to the system to be analysed and both systems are processed in the same way and under the same conditions. With this combination there obviously apply exactly the same qualifications as specified with the plain reference model system method described above. The... 

Isotope dilution mass spectrometry (IDMS) can be considered as a special case of the internal standard method the internal standard that is used is an isotopomer of the compound to be measured, for example a deuterated derivative. Note that an internal standard is necessary for every compound to be measured. This internal standard is as close as possible to perfection since the only property that distinguishes it from the compound to be measured is a slight mass difference, except for some phenomena that involve the labelled atoms, such as the isotopic effect. In that case, we have an absolute reference, that is the response coefficients of the compound and of the standard are identical. This method is often used to establish standard concentrations. The basic theory of this method rests on the analogy between the relative abundance of isotopes and their probability of occurrence [23]. 

With any form of the internal standard method, it is necessary to employ reference standards that are as near as possible to the phases in the material as regards composition, polymorphism and degree of crystallinity. This implies both knowledge of the characteristics of the particular clinker under examination and the ability to prepare the necessary specimens, or alternatively to isolate them physically from the clinker itself. One solution (G30) has been to prepare reference patterns for a wide range of specimens of each phase and to use those that appear to correspond most closely to those present in the clinker under examination. Clinkers for which results had been obtained by point counting have also been used (K11). 

Many of these difficulties can be monitored and overcome with the use of standards, either internal or external (Zevin and Kimmel 1995). For the internal standard method, a known quantity of standard material is added to an unknown mixture, and the ratio of the intensity of the standard component is compared to a previously determined calibration curve to determine the mass fraction of the unknown (e.g. one or more of the polymorphic components). In the external standard method, the entire composition of the unknown sample is determined simultaneously by comparing the measured intensities and respective calibration constants of reference intensity ratios (determined beforehand), which must all be with reference to the same reference standard. 

There are two procedures for using reference standards in the first method a weighed amount of the standard can be added directly to the sample and the area or heights of the peaks of interest compared with that of the standard. This procedure is called the internal standard method. In the second procedure a weighed amount of the standard can be made up in a known volume of solvent and chromatographed under exactly the same conditions as the unknown sample, but as a... 

Chemical interference is practically non existent as a result of the high temperature of the plasma. On the other hand, physical interference may be observed. This stems from variations in the sample atomisation speed which is usually due to changes in nebulisation efficiency caused by differences in the physical properties of the solutions. Such effects may be caused by differences in viscosity or vapour tension between the sample solutions and the standards due, for example, to differences in acidity or total salt content. The technique most commonly used to correct this physical interference is the use of internal standards. In this technique a reference element is added at an identical concentration level to all the solutions under analysis, standards, blank and samples. For each element, the ratio of simultaneous measurements of the lines of the element and the internal standard is then determined in order to compensate for any deviation in the response of the plasma. If the internal standard behaves in the same way as the element to be determined, this method can be used to improve the reliability of the result by a factor of 2 to 5. It can also, however, introduce significant errors because not all elements behave in the same way. It is thus necessary to take care when using it. Alternatives to the internal standard method include incorporating the matrix into the standards and the blank, sample dilution, and the standard addition method. 

The internal standard method is similar to the external standard method in that solutions of the reference standard are compared with solutions of the sample. The key difference is that prior to any sample pre-treatment all solutions are spiked with the same amount of a compound called the internal standard. For this method to work well, it is important to choose a suitable internal standard. Ideally an internal standard should ... 

For the precision of the HS-SPME method we can refer to the work of Vas et al. (1998) with a peaks evaluation based on percentage, and to the data of Carlin (1998) obtained more properly referring to internal standard method. Ulrich et al. (1997) also achieved precise results by using SPME analysis on fruit juices. Table 5.1, referring to the working conditions of one of the chromatograms in Figure 5.2, reports a study of repeatability for some wine compounds adsorbed in a HS-SPME 1 cm PDMS fiber and analysed by GC-MS. 

The method is based on the addition of a standard reference (internal standard) that is detected at a different wavelength from the analyte. The reference standard is added at the same concentration to samples and standards and diluted to mark in a volumetric flask. This technique uses the signal from the internal standard to correct for matrix interferences and is used with respect to precision and accuracy as well as eliminating the viscosity and matrix effects of the sample. 

The internal standard method can compensate for several types of errors that can be caused by sample matrix. Systematic errors due to matrix effects can sometimes be avoided. The internal standard method can also correct for fluctuations in experimental conditions amount of sample analysed, sample introduction, emission source temperature assuming that the signal analyte and internal standard are influenced to the same extent. The main advantage of the internal method over usual calibration methods is that it can provide excellent accuracy and precision and at the same time correct for variable viscosity affects. The method is limited by the availability of a suitable reference element that behaves almost as close to the analyte under test in terms of ionisation energy, solubility, low memory effects, etc. 

In the internal standard method, a known amount of a reference species is added to all the samples, standards, and blanks. The response signal is then not the analyte signal itself but the ratio of the analyte signal to the reference species signal. A calibration curve is prepared in which the y -axis is the ratio of responses and the x-axis is the analyte concentration in the standards, as usual. Figure 8-12 illustrates the use of the internal standard method for peak-shaped responses. 

The internal standard method can compensate for certain types of errors if these influence both the analyte and the reference species to the same proportional extent. For example, if temperature influences both the analyte and the reference species to the same extent, taking the ratio can compensate for variations in temperature. For compensation to occur, a reference species is chosen that has chemical and physical properties similar to those of the analyte. The use of an internal standard in flame spectrometry is illustrated in Example 8-7. 

To calibrate according to the internal standard method, the analyte components must exist as reference compounds in sufficiently high purity. To determine the correction terms, a solution is prepared that contains known amounts of the substances of interest and the internal standard. This solution is chromatographed and the correction terms are calculated based on the peak areas with Eq. (200) ... 

The three main methods of analysis differ in what is used as a reference line (1) external standard method (a line from pure a), (2) direct comparison method (a line from another phase in the mixture), and (3) internal standard method (a line from a foreign material mixed with the specimen). 

The raw GC data was converted into compositional data using the internal standard method with either N2 or Kr as the reference species. Once the species compositions were determined, values for the reactant conversion, product selecti-vities, and product yields were evaluated using the standard expressions. Errors in these parameters were calculated by closing the mass balance on the reaction. This was done by comparing the feed gas composition with the measured product gas compositions. Large errors in the mass balance (> 5%) were indicative of physical problems with the system, which were typically attributed to leaks. Smaller errors (< 5%) were caused by uncertainties in the GC molar response factors as well as fluctuations in flow rates of the MFCs on the Feed Gas Mixing Board. 

The internal standard method is a variation on the above, and is recommended for accurate quantitation. It eliminates the need for accurate injections since a reference standard is included in each sample analysed. An internal standard is selected which has a retention time such that it is eluted in a suitable gap in the chromatogram. The procedure involves analysing a test sample containing known amounts of each component plus a predetermined amount of the internal standard. Since peak area is proportional to the amount of an eluted component and the detector response factor (I>rf)... 

In the internal standardization method, a known concentration of a reference element is added to the sample being analyzed. For x-ray spectroscopy, the reference should have a characteristic radiation that will be excited and absorbed to a similar extent as the characteristic radiation of the element being determined. Therefore, the internal standard is generally an element one atomic number higher or lower than the element being determined. In some instances, it may be necessary to use an element of much higher atomic number and to make use of its L or M radiation, for example Br K (11.9 keV) and Au L i (11.4 keV). 

Within these limitations, the internal-standard method has enjoyed wide application in mineral and ore processing. It is essential that the reference element be intimately mixed with the sample on a micrometer scale. Various procedures have been used to achieve this blending, such as grinding the sample and reference with an abrasive such as silicon carbide, or by fusion using borax, carbonate, or pyrosulfate as a flux. 

The standard-addition method is similar to the internal-standard method, except that the element being determined serves as the reference standard itself. Analysis is accomplished by measuring the intensity of the characteristic spectral line before and after the addition of a known amount of the element being determined. It is assumed that a linear relationship exists between line intensity and concentration (over a limited concentration range), and that the matrix is not significantly altered by the addition. In general, the addition method is limited to the determination of trace and minor elements. All of the comments regarding sample preparation for internal standards apply directly to the standard-addition approach. 

The internal standard method is based on the use of the relative response factor of each component to be measured with respect to a marker introduced as reference. This avoids the imprecision related to the injected volumes, which is a disadvantage of the previous method. However, it requires the addition of a component to a sample dilution. In general, a calibration curve is built by ap>plying different solutions of increased concentrations of the standard analyte with a constant quantity of internal standard. When injecting such samples, we obtain the relation between the areas of the analyte and the internal standard then, it is marked in a graph according to the concentration of analyte in each solution. By means of interpolation in the graphic, we get the relation of the areas of an unknown sample, which has to contain the same quantity of internal standard. 

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