The 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 ... 

In the internal standard method the intensity of the unknown line is measured relative to that of an internal standard line. The internal line may be a weak line of the main constituent. Alternatively, it may be a strong line of an element known not to be present in the sample and furnished by adding a fixed small amount of a compound of the element in question to the sample. The ratios of the intensities of these lines — the unknown line and the internal standard line — will be unaffected by the exposure and development conditions. This method will provide lines of suitable wavelength and intensity by variations of the added element and the amount added, due regard being paid to the relative volatility of the selected internal standard element. It is important to use as internal standard pairs only those lines of which the relative intensities are insensitive to variations in excitation conditions. The line selected as standard should have a wavelength close to that of the unknown and should, if possible, have roughly the same intensity. 

Shatkay, A., Effect of Concentration on the Internal Standards Method in Gas-Liquid Chromatography, Ana/. Chem. 50, 1978, 1423-1429. 

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. 

For the example of toluene given above, the external standard method can be converted into an internal standard method by adding anisole (an appropriate internal standard) to both standard and sample. The retention time of anisole is 4.5 minutes if analyzed by the method above. To calibrate the internal standard method for toluene, toluene standards of concentration 0.3 to 1.5 mg/ml containing 0.5 mg/ml anisole were prepared. The detector response as a function of the amount of sample injected is shown in Figure 4B. 

The key step in the internal standard method is to choose an appropriate internal standard, which has polarity similar to the analyte, is inert to the conditions of extraction and processing, and elutes before or well after the peak of interest. An internal standard method is useful only for correcting for losses due to transfer or variability in dilution or injection, and it is inappropriate to use an internal standard to correct for losses due to degradation.57 This technique gives reliable, accurate, and precise results. If the internal standard is truly inert, the method is useful for determining the rate of analyte conversion in a chemical reaction. 

The improvements to the first three steps of scheme 1 were accomplished using GC as a major analytical tool. A capillary GC internal standard method, described above, was used to monitor the first three steps of scheme 1. Figure 10 is a typical chromatogram of the internal standard method for step 1 of scheme 1. To follow a reaction, a known amount of internal standard was added to the reaction vessel. Aliquots were withdrawn at intervals and analyzed on GC. A graph of yield vs. reaction time was prepared to determine the optimum time for completion of the reaction. 

Hasegawa et al. [76] measured miconazole serum concentration by a high performance liquid chromatographic method. The authors assessed whether the internal standard method produced an intra-assay error and found that the method gave more precise and more reproducible results compared to the absorption calibration curve method. With 0.5 pg/mL of miconazole, the coefficient of variation produced by that method was 3.41%, whereas that of the absorption calibration curve method was 5.20%. The concentration of absorptions calibration curve method showed higher values than the internal standard method. This indicated that the internal standard method was far more precise in measuring the miconazole serum concentrations than the absorption calibration curve method. 

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. 

Experiment 42 The Determination of Ethanol in Wine by Gas Chromatography and the Internal Standard Method... 

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. 

Compare and differentiate between the internal standard method and the standard additions method. 

Compare the internal standard method with the standard additions method in terms of ... 

Consider the quantitative gas chromatography analysis of alcohol-blended gasoline for ethyl alcohol by the internal standard method, using isopropyl alcohol as the internal standard. The peaks for these two substances are well resolved from each other and from other components. Assume there... 

In the strategy for GC, it is noted that there may be no need for weight or volume data for the sample because the sample itself may be injected directly and quantitation performed solely from the chromatographic information. It is also noted that the internal standard method is common, and the solution preparation and calibration procedure are altered accordingly. 

The internal standard method uses an internal standard substance added in a constant amount to all standards and the sample. Area ratio of analyte peak to internal standard peak is plotted vs. concentration of analyte. The standard additions method uses the addition of the analyte in increasing amounts to the sample. Peak area is plotted vs. concentration added and the line is extrapolated to zero peak area to get the sample concentration. 

Quantification of the separated amino acids is usually performed by using external calibration or the internal standard method. Due to the large differences in chemical structure exhibited by the various amino acids, there is not a single ideal standard for the overall amino acid profile. Nevertheless, a suitable internal standard must be stable to hydrolysis and offer chromatographic resolution. The most popular choices comprise norleucine, norvaline, and a-amino-n-butanoic acid (AABA) [196]. 

Amine quantification is usually accomplished using the internal standard method. Different amines have been used for this purpose 1,7-diaminoheptane [282], 1,3-diaminopropane [287], and benzylamine [311]. 

Calculation of the Hey concentration is performed using the internal standard method ... 

Quantification of faecal BAs is carried out in SIM mode by using the internal standard method, and peak areas are obtained from the chromatograms generated by data handling. Component identification is based on fragmentation and comparison of the retention times with those of standards. 

This method is more precise if several injections of the standard and the samples are carried out. The internal standard method is of general use and is reproducible but relies on the proper choice of compound for the internal standard, which should have the following characteristics ... 

To measure serotonin (5-hydroxytryptamine), by the internal standard method, a 1 ml aliquot of the unknown solution is added to 1 ml of a solution containing 30 ng of N-methyl-serotonin. This mixture is then treated to remove all other compounds which could interfere with the experiment. The operation performed was an extraction in the solid phase to isolate the serotonin and its methyl derivative, diluted in a suitable medium. 

With attention to the purity of the standards and to the lack of interference of any solvent impurities, the precision of the internal standard method is controlled by the ability to quantitate peak size. That certainly qualifies this technique as the most precise method of quantitative analysis by GC, and where precision is paramount, the internal standard technique should be applied. Its advantages far outweigh the slight increase in effort required for standard and sample preparation. An excellent, detailed, how-to approach for the internal standardization technique as applied to a practical problem has been detailed by Barbato, Umbreit, and Leibrand (35). 

All of the above points about liquid injection should be considered even when using a standard technique that does not require an accurate volume to be known. Selective evaporation cannot be tolerated even with the internal standard method. The size measurement errors obvious from the above discussion certainly point to the substantial advantage of the internal standard technique for accurate analysis. 

Analysis of extracts was performed on a gas chromatograph (GC) (5880A, Hewlett-Packard) equipped with an electron-capture detector (ECD) and a 30-m fused silica capillary column with an outer diameter of 0.25 mm and a film thickness of 0.25 xm (Durabond DB-5, J W Scientific). The internal standard method developed by Dunnivant and Elzerman (15) was used, except that that only one internal standard was used (Aldrin) to minimize run time on the gas chromatograph. Daily working standards were composed of 80% Aroclor 1016 and 20% Aroclor 1254. This ratio was chosen because it matches the Aroclor distribution found in the sediments by Polansky (13). Quantification and collation of data were done on microcomputers with a spreadsheet program (SuperCalc 4, Computer Associates International). 

The international standard method given in ISO 8147 uses a disc test piece 3 0.1 mm thick and between 35 and 40 mm in diameter, bonded to metal plates which are approximately 0.1 mm less in diameter than the rubber. The slightly smaller size of the metals is intended to prevent the rubber tearing from the edges of the metals during test. 

The internal standard method is more reliable than the external standard method. Equal amounts of one or more internal standards are added onto equal volumes of sample extracts and the calibration standards. The response factor (RF) is then calculated as follows ... 

It may be noted that for calibration each analyte is needed to be spiked with its labeled analog. This enhances the cost of the analysis. High cost and often unavailability of labeled analogs are the major drawbacks of isotope dilution method as compared with external and internal standard calibration methods. The isotope dilution method should be, theoretically, more accurate than the internal standard method, as the chromatograpic response and the retention times of the labeled analogs are closest to the compounds. 

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 ... 

The quantitation is performed by the internal standard method. The SIM response for the isomers at their primary characteristic m/z are compared against the internal standard(s). A detection limit in the range of 2 to 5 ppt (0.002 to 0.005 pg/L) can be achieved for aqueous samples concentrated as above and analyzed using low resolution mass spectrometry method. A lower detection limit in 0.01 to 1 ppt range may be achieved by HRMS technique. 

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