Method internal standards

7 Destructive Methods of Analysis for Metals Content in Cyanoacrylate Adhesives 

Cyanoacrylate adhesives can be prepared for metal analysis using destructive techniques e.g. dry ashing, microwave acid digestion or oxygen bomb combustion. It is a matter of 

The internal standard method is the most widely used approach in mass spectrometry quantification. The method is especially useful when the amount of the sample introduced and the instrument response may vary from run to run. In this 

The following criteria should be applied in the selection of an internal standard (1) its physical and chemical properties should exactly match those of the analyte so that any losses are accounted for accurately (2) its chromatographic characteristics are similar to those of the analyte so that the two elute close to each other (3) its molecular mass should be distinct from that of the analyte (4) its mass spectrometry behavior (i.e., ionization efficiency, fragmentation behavior, detector response, etc.) should mirror that of the analyte and (5) it should not be a constituent of the sample itself. 

Types of Internal Standards Three types of internal standards have found a niche in quantitative analysis. The first includes those compounds that have chemical and physical properties similar to those of the analyte. The only requirements are that it should not be isobaric to the analyte and it must elute from a chromatographic column at a different time. This type of internal standard is easy to find, but it does not provide the level of precision and accuracy that is 

Other analytical problems to which the direct comparison method has been applied include the determination of mixed iron oxides in the oxide scale on steel [14.10], the beta phase in titanium alloys [14.11], and mixed uranium and plutonium carbides [14.12]. 

In this method a diffraction line from the phase being determined is compared with a line from a standard substance mixed with the sample in known proportions. The internal standard method is therefore restricted to samples in powder form. 

Suppose we wish to determine the amount of phase A in a mixture of phases A, B, C. where the relative amounts of the other phases present (B, C, D.) may vary from sample to sample. With a known amount of original sample we mix a known amount of a standard substance S to form a new composite sample. Let Ca and be the volume fractions of phase A in the original and composite samples, respectively, and let be the volume fraction of S in the composite sample., If a diffraction pattern is now prepared from the composite sample, then from Eq. (14-2) the intensity of a particular line from phase A is given by 

The intensity ratio of a line from phase A and a line from the standard S is therefore a linear function of the weight fraction of A in the original sample. A calibration curve can be prepared from measurements on a set of synthetic samples, containing known concentrations of A and a constant concentration of a suitable standard. Once the calibration curve is established, the concentration of A in an unknown sample is obtained simply by measuring the ratio JJI for a composite sample containing the unknown and the same proportion of standard as was used in the calibration. 

All of these error sources, other than the minimal intrinsic error, can be reduced by using the internal standard method. The absolute measurement of the signal is replaced by the measurement of the signal ratio for the molecule that is measured and for the internal standard. The same compound can play the role of the internal standard for the quantification of various compounds within the mixture. 

For an evaluation of error sources in quantitative GC/MS determinations, the reader is referred to the paper by Claeys et al. [22], 

This method is based on a comparison of the intensities of the signal corresponding to the product that has to be quantified with the one of a reference compound called the internal standard. This method allows the elimination of various error sources other than the minimal intrinsic error due to statistical reasons. In fact, if we choose as an internal standard a molecule with chemical and physical properties as close as possible to the properties of the molecule to be measured, the latter and the internal standard undergo the same loss in the extraction steps and in the derivative or the same errors in the introduction of the sample into the mass spectrometer, when the source conditions are varied. As both 

Calibration curve for the quantitative analysis of pheno-barbital using anthracene as an internal standard. 

The method consists first of carrying out measurements on synthetic samples containing the same known quantity of the internal standard and increasing quantities of the compound to be measured. With these results a calibration curve is constructed. This allows a mathematical relationship to be obtained between the intensities of the signals corresponding to the compound to be analysed and the internal standard (/x//sti) and the quantity of compound present in the sample (Mx). As a reminder, maximum precision is obtained if the relationship corresponds to the equation of a straight line with a slope equal to one. 

This method involves the usual sourees of error sueh as those made in measuring weights and volumes. Two key potential sources of error are those occurring through poor repeatability of injection volumes and through inconsistent loss of sample in any sample preparation step. Both of these may be compensated for by the use of an internal standard 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  

Because the internal standard method eliminates some of the errors found in the external standard method it does not automatically follow that the internal standard method should always be used. The precision of many LC external standard methods is very good (e.g. 0.4% RSD for a purity determination) given that (i) the repeatability of injection volumes in modern injectors is much better than it used to be, especially if the injection is automated and (b) there are many methods for which sample 

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 

The procedure is to measure the peak sizes of both the internal standard peak and the analyte peak and then to divide the analyte peak area by the internal standard peak area. The area ratio thus determined is then plotted vs. concentration of the analyte. The result is a method in which the volume injected is not as important and, in fact, can vary substantially from one injection to the next because this ratio does not change as the volume injected changes, since both peaks are affected equally by the changes. 

Can just any substance serve as an internal standard There are certain characteristics that the internal standard should have. They are listed below  

Its peak, like the analyte s, must be completely resolved from all other peaks. 

Its retention time should be close to that of the analyte. 

This technique, employing the absolute response factors, yields very reliable results with chromatographs equipped with an auto-sampler a combination of a carousel sample holder and an automatic injector. This permits numerous measurements to be made without interruption, to the condition that no change in the apparatus tuning is made between injections. 

The reference solution periodically injected affords a control that can be used to compensate an eventual baseline drift during a sequence of programmed injections. 

This method, the only one adapted to gas samples, has the added advantage that nothing needs to be added to the sample solution, unlike the method described below. 

For trace analysis it is preferable to use a method that relies on the relative response factor of each compound to be measured against a marker introduced as a reference. This means that any imprecision concerning the injected volumes, the principal constraint of the previous method, is compensated. As above, this 

The areas of the peaks to be quantified are compared with that of an internal standard (designated by IS), introduced at a known concentration within the sample solution. 

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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. 

D. of lead in brass by, 770 direct reading instruments, 775, 776 electrodes for, 763, 771 equipment for, 760, 764 excitation sources for, 763, 773, 774 general discussion of, 8, 758 internal standard method, 769 investign. of a complex inorganic mixture, 770... 

Ethanol concentration in the fermentation broth is determined by using gas chromatography (HP 5890 series II with HP Chemstation data processing software, Hewlett-Packard, Avondale, PA) with a Poropak Q Column, and a Hewlett-Packard model 3380A integrator. A flame ionisation detector (FID) is used to determine ethanol. The oven temperature is maintained at 180 °C, and the injector and detector temperature are maintained at 240 °C. The sample taken from the fermentation media has to be filtered and any internal standard must be added for analysis based on internal standard methods otherwise, the area under the peak must be compared with known standard samples for calculation based on external standard methods. 

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

HPA catalyzed liquid phase nitration was eairied out in a Teflon-lined stainless autoclave of 200 mL equipped with a magnetic stirrer. Reactants and HPA were quantitatively added to the autoclave, which was sealed and heated in an oil-bath. Products were analyzed by GC with OV-101 30 m capillary column and FID detector by using calibrated area normalization and internal standard method. All products were confirmed by GC-MASS analysis. 

This approach, called internal standard method , works even if the composition of the amorphous phase is unknown. 

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. 

Figure 4 Calibration of external and internal standard method. Chromatographic conditions — column 30 cm x 3.9 mm p-Bondapak C18 (10-pm particle size) mobile phase water acetonitrile (50 50) flow rate 1.5 ml/min column temperature ambient detector wavelength 254 nm. (A) External standard method, (B) internal standard method.

An internal standard method gives more reliable results when elaborate sample preparation is required, as in extraction of a drug substance from biological fluids, or extraction of pesticides and herbicides from soil and plant matter. The addition of internal standard (IS) to the sample and standard acts as a marker to give accurate values of the recovery of the desired compound(s). Since the determination of wt% involves the ratio of the detector responses in the two chromatograms, the injection volume is not critical as in an external standard method. 

Calibration of an internal standard method is done by preparing standard samples of varying concentration. The same amount of IS is added to each, and the standard samples are analyzed using a developed method. The detector response, area or height, of each standard is determined, and a ratio is calculated. The graph of concentration vs. area ratio is plotted. The method is considered linear if the correlation coefficient is 0.99 or better. The response factor RF is calculated as... 

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. 

Figure 10 CC Internal Standard Method for step 1, scheme 1. Chromatographic conditions were column 12 m x 0.2 mm x 0.3 mm HP-1 injector temperature 300°C detector temperature 300°C column temperature 75°C for 3 min, then +10°C/min to 280°C.

Method 3 was modified to an internal standard method into Method 5 by changing the bonded phase and the mobile phase composition. Biphenyl was used as an internal standard added into the reaction. Aliquots were withdrawn, diluted with degassed acetonitrile, and analyzed according to Method 5. This internal standard method, Method 5, was helpful in the optimization of the desired ris-1,2/1,4 product of the key step of the LANA reaction (scheme 5). 

Table 3 Product Yield (czs-1,2 /1,4 of Scheme 5) as Weight Percent Using Internal Standard Method...

Quantitative analysis using FAB is not straightforward, as with all ionisation techniques that use a direct insertion probe. While the goal of the exercise is to determine the bulk concentration of the analyte in the FAB matrix, FAB is instead measuring the concentration of the analyte in the surface of the matrix. The analyte surface concentration is not only a function of bulk analyte concentration, but is also affected by such factors as temperature, pressure, ionic strength, pH, FAB matrix, and sample matrix. With FAB and FTB/LSIMS the sample signal often dies away when the matrix, rather than the sample, is consumed therefore, one cannot be sure that the ion signal obtained represents the entire sample. External standard FAB quantitation methods are of questionable accuracy, and even simple internal standard methods can be trusted only where the analyte is found in a well-controlled sample matrix or is separated from its sample matrix prior to FAB analysis. Therefore, labelled internal standards and isotope dilution methods have become the norm for FAB quantitation. 

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. 

Precision expressed as 95% confidence intervals Spark source mass spectrometry, internal standard method From [735]... 

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... 

Select one of the quantitation procedures we have discussed (response factor method, internal standard method, or standard addition method) and describe ... 

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. 

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