Quantitative analysis 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 ... 

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. 

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. 

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. 

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... 

For quantitative analysis by either the external or internal standard methods, HPLC requires the use of calibration solutions that are injected under identical conditions. Thus to fully identify quantitative effects, calibration solutions plus standard solutions need to be analysed for each experiment in a ruggedness test. As duplicate determinations are required for the estimation of standard errors a single experiment can consist of up to six chromatographic experiments as shown below. 

Quantitative results were produced for each compound on the basis of internal standard method calculations. A three-point calibration curve was generated for each compound by using peak areas of a quantitation ion extracted from the mass spectrum of the compound. The ion was selected on the basis of it being a uniquely characteristic mass of the compound. The use of extracted ion quantitation produces more accurate results than total ion-current quantitation in cases in which two or more components are not completely resolved chromatographically. This situation is generally the case in complex mixture analysis. The quantitation ions selected for each of the compounds in the mix are listed in the box. 

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). 

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 ... 

A single point calibration may be used instead of a working calibration curve for quantitation by either external or internal standard method, if the response from the single point standards produces a response that deviates from the sample extract response by no more than 20%. The solvent for preparing calibration standards should preferably be the same one used to make the final sample extract. Hexane, isooctane, or methyl-feri-butylether is an appropriate solvent for the analysis of chlorinated pesticides by GC-ECD. 

Internal standard calibration is used when the changes in the analytical system are known to be frequent and substantial. To compensate for these changes, internal standards at known concentrations are added to all calibration standards, field samples, and laboratory QC samples prior to analysis. Internal standards are synthetic analogs of specific target compounds or compounds that are similar in nature to the target analytes and that are not found in environmental samples. Internal standard calibration is a requirement of GC/MS methods. Laboratories sometimes use it for GC methods as it significantly improves the accuracy of compound quantitation. 

Matisova and co-workers11 have suggested that the need for a reproducible sample volume can be eliminated by combining the standard addition method with an in situ internal standard method. In the quantitative analysis of hydrocarbons in petroleum, they chose ethyl benzene as the standard for addition, but they used an unknown neighboring peak as an internal standard to which they referenced their data. This procedure eliminated the dependency on sample size and provided better quantitation than the area normalization method they were using. 

ABA is detected and quantified by enzyme immunoassay with a monoclonal antibody.578 The detection limit of immunoassay with a monoclonal antibody is about 0.02 pmol however, enzyme immunoassay cannot exclude cross-reactivity with unknown substances, so GC- or LC-SIM analysis using the stable isotope dilution method is recommended for correct quantitation. As internal standards labeled with the stable isotope, deuterated derivatives of ABA such as [3, 5, 5, 7, 7, 7 -2H6]ABA (ABA-//6)579 and [7, 7, 7 -2H3] derivatives of phaseic and dihydrophaseic acids580 are used. 

Internal standard method is likely the most commonly used approach in a quantitative phase analysis. It is based on the following relationship ... 

The standard addition method is commonly used in quantitative analysis with ion-sensitive electrodes and in atomic absorption spectroscopy. In TLC this method was used by Klaus 92). Linear calibration with R(m=o)=o must also apply for this method. However, there is no advantage compared with the external standard method even worse there is a loss in precision by error propagation. The attainable precision is not satisfactory and only in the order of 3-5 %, compared to 0.3-0.5 % using the internal standard method 93). 

During the method development in paper II we desired to perform quantification of both the fermentation product and the substrate. Both the external and the internal standard method were evaluated, respectively. The internal standard method was chosen since it resulted in a higher precision. Initially, structurally related compounds were tested as I S. in the quantitative part described in paper II, according to the above I.S.-protocol for bioana-lytical approaches. However for biotechnological analysis no recommendations exist for the selection of internal standard. Thus in paper II an investigation was performed to see if the I.S.-protocol for bioanalytical methods could be applied also for biotechnological analysis. 

Quantitative Analysis Using Internal Standard Method [12]... 

Table 6.1 Results of analysis of cyananoacrylate adhesives with and without thickening agent (PMMA) using calibration curve, standard addition and internal standard method for quantitative analysis of Nb content...

In general, quantitative analysis by EDS in EM is similar to that of XRF. The analytical methods, however, are different for two main reasons. First, the interactions between the electron beam and specimen are different from those of primary X-ray radiation. Second, an EM specimen for chemical analysis cannot be modified as in the internal standard method. For accurate quantitative analysis of EDS in EMs, separate standard samples containing the elements in the specimen to be analyzed are necessary. The standards should be measured at identical instrumental conditions to the specimen. It means that the spectra of specimen and standard should be collected under the same conditions with regard to the following parameters ... 

Raman spectroscopy is a scattering, not an absorption technique as FTIR. Thus, the ratio method cannot be used to determine the amount of light scattered unless an internal standard method is adopted. The internal standard method requires adding a known amount of a known component to each unknown sample. This known component should be chemically stable, not interact with other components in the sample and also have a unique peak. Plotting the Raman intensity of known component peaks versus known concentration in the sample, the proportional factor of Raman intensity to concentration can be identified as the slope of the plot. For the same experimental conditions, this proportional factor is used to determine the concentration of an unknown component from its unique peak. Determining relative contents of Si and Ge in Si—Ge thin films (Figure 9.38 and Figure 9.39) is an example of quantitative analysis of a Raman spectrum. 

The separation of endogenous 17- or 18-hydroxylated corticosteroids of the 21-hydroxylated 4-pregnen series was obtained by capillary electrophoresis of their charged borate chelate complexes (323). Aldosterone, 18-hydroxycorti-costerone, 18-hydroxy-deoxycorticosterone, cortisone, cortisol and 11-deoxycor-tisol are separated and resolved with 400 mM borate buffer at pH 9.0. The corticosteroid/borate chelation complex as indicated by CE data correlated well with 11 B-NMR. The separation of corticosteroids and benzothiazin analogs were studied by MEKC and a comparison with CZE was made (324). Bile salts, which have a similar carbon skeleton to the corticosteroids, were used for the separation of these steroids. A short analysis time, 15 min, and a high number of theoretical plates (150,000-350,000) were obtained. Sodium cholate was found to be very effective. The MEKC method was applied to the determination of the drug substance in tablets and cream formulations. An internal standard method was used for quantitation. The purity of the drug substance was also determined. 

The GC-FID analysis is conducted by injection of 1 to 2 fil of FI or F3 into a gas chromatograph equipped with a high resolution capillary column (operated in sphtless injection mode). The injector and detector temperatures are set at 290 and 300°C, respectively. The GC temperature program is selected to achieve near-baseline separation of all of the saturated hydrocarbons. Quantitation of the individual components is performed by the internal standard method. The relative response factor (RRF) for each component is calculated relative to the internal standard. The TPH is also quantified by the internal standard method using the baseline corrected total area of the chromatogram and the average hydrocarbon response factor determined over the entire analytical range. ... 

The GC-MS analysis is conducted by injection of 1 /xL of FI orF2 into a gas chromatograph-mass spectrometer. The MS detector is operated in the scan mode to obtain spectral data for identification of components and in the selected ion mode (SIM) for quantitation of target compounds. An appropriate temperature program is selected to achieve near-baseline separation of all of the target components. Quantitation of the alkalized PAH homologues, other EPA priority PAHs and biomarker compounds are performed by the internal standard method with the RRFs for each compound determined during the instrument calibration. The ions monitored for alkylated PAH and biomarker analyses are listed in Table 27.4 and Table 27.5, respectively. 

The peak height or peak area of an eluatc from a GC column has been widely used for quantitative and semi-quantitative analyses. An accuracy of I % relative is attainable under carefully controlled conditions using either the external or the internal standard method. As with most analytical tools, reliability is directly related to the control of variables the nature of the sample also plays a part in determining the potential accuracy. The general discussion of quantitative chromatographic analysis given in Section 26F-2 applies to GC as well as to other types therefore, no further consideration of this topic is given here. 

Emission spectroscopy is widely used for both qualitative and quantitative analysis. The high sensitivity and the possible simultaneous excitation of as many as 72 elements, notably metals and metalloids, makes emission spectroscopy especially suited for rapid survey analysis of the elemental content in small samples at the level of 10 /ug/g or less. With control over excitation conditions to maintain constant and reliable atomization and excitation, the spectral line intensities can be used for quantitatively determining concentrations. An analytical curve must be constructed with known standards, and often the ratio of analyte intensity to the intensity of a second element contained in, or added to, the sample (the internal-standard method) is used to improve the precision of quantitative analyses. Preparation of standards for arc and spark techniques requires considerable care to match chemical and physical forms to the sample this is not commonly required for ICP discharge. 

Like all classical quantitative analysis methods, NMR spectroscopy needs calibration, calibration standards and a validation procedure. The standard techniques are used for calibration external calibration, the standard addition method and the internal standard method. A fourth is a special NMR calibration method, the tube-in-tube technique. A small glass tube (capillary) containing a defined amount of standard is put into the normal, larger NMR tube filled with the sample for analysis. In most cases, there are slight differences in the chemical shift of corresponding signals of the same molecule in the inner... 

Values allow to get a rapid orientation regarding the ratios of the components of the sample since their values express relative step lengths proper of the equimolar mixture of the analyzed substances. They additionally permit the internal standard method to be used, thus enabling to perform the quantitative evaluation with the aid of the only analysis without knowing the injected volumes. Volume Vs at molarity m of a suitable standard substance S is added to a defined volume of sample V at molarity which is to be determined. Then, a suitable amount of the mixture thus obtained is injected and the step lengths, and L, are evaluated. Molarity m, which is determined, is thra given by the relationship... 

In order to obtain a more quantitative picture of the mechanochemical reaction, we performed a quantitative XRD phase analysis using the Rietveld refinement method. In addition to the amount of the crystalline phases, i.e., Na2CC, Nb20s and NaNbCh, we determined also the contribution from the XRD background, which we denoted as "XRD-amorphous" phase. This was done using an internal standard method details of the method can be found in Kuscer et al. (2006) and Rojac et al. (2008b). 

---