Choosing an Internal Standard

It is important for a standard to possess physico-chemical proprieties as close as possible to those of the molecule to be analyzed [polarities, solubilities, sizes (steric effect) for sample preparation, volatilities for gas chromatography, ionization potentials, and basicities for mass spectrometry]. Marketed products are suitable as long as they answer all these conditions. 

An important market has developed in parallel to the success of mass spectrometry. Products marked as stable isotopes (usually deuterium or C) are sold as internal standards for GC-MS and LC-MS, particularly in the fields of environmental analyses (pesticides, dioxins, hydrocarbons, etc.) and toxicological testing (psychotropic medicines, narcotics, etc.). While these compounds are often expensive, they are used in small quantities and thus the cost may be justified. 

Along with their physico-chemical similarities to those of analytes, compounds marked with deuterium or present the advantage that they are not present in significant amounts in the environment and are not endogenous in animals or humans. 

This is an important criterion. When developing an analytical method for dosing molecnles that have no marked commercial analognes, it makes sense to choose a molecnle with a chemical structnre close to that of the analyte. It can then be wise to choose a marked prodnct to ensnre that the standard is not naturally present in the matrix to be analyzed. If the standard appeared in the matrix at a non-negligible concentration, the dosage results would be false. 

Obviously, for practical and economic reasons, one generally does not use as many standards as there are molecules to be dosed, especially if the molecules are present in large qnantities. Multi-residue methods that allow simultaneous dosing of 100 molecules or more are used frequently in environmental analysis. Such a method generally uses four to ten internal standards spread out along the chromatogram. Each molecule family is associated with the standard whose chemical structure is the closest to its own. 

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Choice of an Internal Standard. One of the difficulties in the spec-trometric trace analysis of coal ash samples, in addition to choosing a suitable comparison standard matrix, is choosing an internal standard. The first choice in both analytical methods was indium, which was used as a constant internal standard added to the graphite powder diluent-buffer. The results obtained had poor reproducibility, as previously... 

Due to the bias that can occur in the Basic Protocol, results can be as low as 1% of the correct value for the concentration of a volatile in a sample. Most results are <80% of the correct value. This bias is usually caused by differences in the recovery between the internal standard and the analytes. Choosing an internal standard thatis similar to the analyte can reduce this bias but it cannot be completely eliminated unless a separate standard is used for each analyte (see Alternate Protocol). Nevertheless, the Basic Protocol is often sufficiently accurate because the olfactory system, unlike the taste system, is compressive and insensitive to small... 

Brief mention needs to be made regarding the employment of internal standards. While it is desirable to employ an internal standard for procedures that involve significant sample preparation procedures, there is no ideal choice for an internal standard for amino acid analysis. This fact is due to the wide spectrum of chemistries exhibited by the various amino acids. If one is analyzing for a single amino acid (or class, e.g., the hydrophobic amino acids), it is possible to choose an internal standard that mimics the chemistry of that particular amino acid very well. However, for the overall amino acid profile, an internal standard will do nothing more than allow the analyst to make nonvolumetric solution transfers and correct for variability of the injection volume by the HPLC injector. Unfortunately, the employment of an internal standard can actually skew the apparent recoveries for the overall amino acid profile. 

Choosing an internal standard to correct errors due to sample preparation and injection reduced the impact of variability on the final trueness and precision of the developed method. Peak area can be corrected (peak area/migration time) to avoid the migration time drift influence, because of the temperature affecting both electro-osmosis and electrophoretic mobilities as well as buffer electrolysis, adsorption into the capillary wall and so on. 

An appropriate internal standard for MALDI must compensate not only for any crystallization irregularities but also for subsequent desorption and gas-phase effects. In choosing an internal standard, the relative polarities of the analytes and internal standard as well as their solvent solubilities should be considered (Sleno and Volmer, 2006). Structural similarities should reflect the gas-phase behavior of the involved molecules and extend to solubility. Naturally, an isotope-labeled standard is the ideal choice since its chemical behavior is nearly identical to its unlabeled counterpart (Gusev et al., 1996). Such a standard guarantees identical crystallization and gas-phase behavior of the analyte and internal standard (Kang et al., 2001). 

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. 

Procedure Notes Some sample types may naturally contain significant levels of yttrium. In these cases, choose a suitable alternative internal standard, or mn the test without an internal standard. Use of the internal standard is not required, but it is helpful when there are variations in the viscosity among sample types. Samples may be prepared in higher or lower concentrations as needed. Standard concentrations may be adjusted as needed. Alternative procedures should be validated before use. 

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... 

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 ... 

Consult your instructor on the proper operation of your instrument. Handle the infrared cell carefully, avoiding contact with water and the fingers. Fill the cell with pure m-xylene and obtain a spectrum on this from 2 to 15 p,m, being sure to record the last peak just before 15 pm (692 cm" )- Each time you run a sample, be sure to check 0% T by placing a card in the sample beam and adjust the pen to 0% T. Empty the cell, rinse and fill with p-xylene, and run a spectrum on this. Repeat for o-xylene. Run spectra on each of the standard mixtures. From the spectra of the pure substances, choose a peak of each isomer to measure. Using the baseline method (see Figure 16.11), measure PqIP for the peak for each compound. Prepare a calibration curve of the ratio of og PJP) J og PolP ri,o and of log(Po/P)para/ log(Po/E)ortho versus concentration for the meta and para isomers, respectively. See Chapter 20 and your CD for spreadsheet preparation using an internal standard. 

The following guidelines are used when choosing an element and wavelength to serve as an internal standard ... 

Adequate precision and accuracy are only likely to be achieved if some standardization procedure is employed and the nature of this, internal or external standards or the method of standard additions, needs to be chosen carefully. If internal standardization procedures are adopted then appropriate compound(s) must be chosen and their effect on the chromatographic and mass spectrometry methods assessed. The ideal internal standard is an isotopically labelled analogue of the analyte but, although there are a number of commercial companies who produce a range of such molecules, these are not always readily available. An analytical laboratory is then faced with the choice of carrying out the synthesis of the internal standard themselves or choosing a less appropriate alternative with implications on the accuracy and precision of the method to be developed. 

Although internal standard calibration compensates for some errors in external standard quantitation, there are several difficulties in method development. First, choosing an appropriate internal standard can often be difficult, as this compound must be available in extremely pure form and it must never appear in the samples of interest. Second, it cannot interfere in either the extraction or the chromatography of the analytes. Finally, it must be structurally similar to the analytes, so that it undergoes similar extraction and chromatography, otherwise, the compensation will be lost. 

It is well to respect what others have done but the fact that someone for whatever reason chooses not do something in a correct scientific way should not validate it as an option in an international guideline. Failure to lead by standardization via the international guideline essentially greatly diminishes its value. In its present state, it is merely a collection of worldwide practices with many conflicts. 

This comprises introducing a known element into the sample that experiences the same absorption effects as the element to be determined. A good solution is to choose a standard reference element with an atomic number close to that of the clement to be analysed it is thus possible to compare lines in the same series and the absorption coefficients are similar. A standard line from another series can be taken but, in this case, it is necessary to take account of the differences in emission conditions. In practice, the same quantity of internal standard is introduced into the samples and the standards and both the net intensity levels of the characteristic line and the intensity ratios of this line to that of the nearby line selected as the inter-... 

As mentioned in section 2.7, the internal standard (I.S.) method is useful for quantification when the matrices are complicated and sample pre-treatment is necessary. However, it is generally difficult to choose an I.S. that meets all the necessary requirements (se above in section 2.7). Thus, 4-androsten-3,17-dione (AD) was chosen as I.S. because of its great structural similarity with both substrate and product. However, it soon appeared that... 

See the Common Units and Values for Problems and Examples inside the back cover. Several problems in this section deal with perfect gases. It may be shown that for a perfect gas the enthalpy and internal energy depend on temperature alone. If a perfect gas has a constant heat capacity (which may be assumed in all the perfect-gas problems in this chapter), it is very convenient to choose an enthalpy datum that leads to h = CpT and u= CyT, where T is the absolute temperature these values may be used in the perfect-gas problems in this chapter. For Freon 12 problems, use App. A.2. For steam and COj problems, use any standard table of values. 

For bulk solid samples such as metals and alloys, the easiest way to do this is to choose an emission line of the major matrix element as the internal standard line. For example, an iron emission hne would be used in steels, a Cu line in copper alloys, and so on. If the sample is a powder, either the major element can be used or a separate element added to all standards and samples. 

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