Internal normalization technique

Mitchell PCH (1999) Molybdenum compounds. In Ullmann s Encyclopedia of Industrial Chemistry. Volume A16. Arpe H-J (ed) John Wiley and Sons, New York, p 675-82 Moore LJ, Machlan LA, Shields WR, Gamer EL (1974) Internal normalization techniques for high accurate isotope dilution analyses. Application to molybdenum and nickel in standard reference materials. Anal Chem 46 1082-1089... 

In theory the internal normalization technique may appear ideal. But when analyzing real-life samples which may contain many components, some of which may be unresolved chromatographi-cally and of no interest to the analyst, one of the other two techniques has advantages and is generally employed. 

In the internal normalization technique a sample is injected into the chromatograph and peaks are obtained for all the sample components. Generally, area... 

In theory, the internal normalization technique may appear ideal. But in analysis of real-life samples that may contain many components, some of which may be unresolved chromatographically and of no interest to the analyst, one of the other two techniques offers more advantages and is generally employed. One analysis using this technique and performed hundreds of times each day is the component-by-component analysis of natural gas. A complete analysis is needed since the analysis is used to calculate the heating value of the sample. Thus it is natural to normalize the results. 

In this instance component A is referred to as the internal standard. All the advantages of the internal normalization technique, such as lack of knowledge regarding the exact sample size and the noncritical aspects of dilntion, carry over to this technique. The major disadvantage of internal normalization, namely, the necessity of measuring all the components of the sample, does not carry over into this technique. The cautions under internal normalization regarding system overload apply, but only to the components of interest and the internal standard, not to the entire sample. 

The obvious approach to answering this question is to remove an electron from this orbital and observe the effect on, for example, the metal-metal stretching frequency or metal-metal bond distance. Of course, removal of an electron from the delta bonding orbital creates a positive molecular ion for which determination of these properties may not be possible using normal techniques. In those cases where the ion is sufficiently stable that these properties can be measured, the meaning of the information may be clouded by changes in intermolecular interactions or other internal factors. 

The major disadvantage of this technique is that the entire mixture must be separated and detected in the chromatographic system. All peaks must be standardized via response factors whether their analysis is needed or not. Internal normalization also requires that a detector be used that responds somewhat uniformly to all components. This technique cannot be used with electron capture and flame photometric detectors, for instance. 

With care internal normalization can be used where peak size is measured by height instead of area, though this is rare. The response factor is now subject to slight variations in column temperature, injection technique, carrier flow, and the like, all mentioned under peak height measurement previously. This approach requires that the standard mix for response factors to be run as close in time to the unknown as possible and new response factors to be determined each time. Note also that response factors determined from area measurement in no way are the same for those determined from peak height. 

In the preceding example area was used to measure peak size since that was the technique used in the example for the internal normalization. The point must be made that peak height can be used as the size measure just as well as peak area. The same advantages of peak height measurement are present in this method of standardization as in any other. Likewise the same requirement for frequent standardization is present. 

Since trace analysis also includes air or gas samples, it is appropriate to point out that proper addition of an internal standard to this type of sample is difficult. This difficulty lies, not in the mechanical problem of transfer, but in the difficulty of knowing that the precisely intended volume has properly been transferred. However, the internal standard technique is still not widely used here for the same reason it is not generally used in trace analysis. This reason again is because the analyst normally has no prior knowledge of the variation in composition from sample to sample. The continual risk exists that any given sample in a series will have a component, not present in others, which elutes with the internal standard. This occurrence would introduce significant error into the quantitative calculations which result. 

Colinearities among the responses are sometimes introduced unintentionally in the course of raw data treatment. It is common to normalize chromatograms to give a sum of peak areas equal to 100 percent. If such data are used to analyze the product profile in a synthetic experiment there will be a purely artificial correlation between the relative amounts of the products in each experiment, i.e. if the amounts of one product increase, the amounts of the remaining products will decrease. Such data can lead to conclusions which are totally erroneous. To avoid such pitfalls, it is advisable to determine product distribution from chromatograms by using internal standard technique. 

The internal reflectance technique is usually called attenuated total reflection (ATR) spectroscopy. It is especially useful for studying strongly absorbing media, for example, aqueous solutions. When the infrared radiation is absorbed in the test medium, one obtains a spectrum similar to that from a transmission experiment. However, there are distortions in the ATR spectrum, especially in the region of intense bands. One reason for distortion is the fact that the depth of penetration varies with wavelength. The other effect is due to the change of the refractive index of the solution in the region of the intense band. ATR spectra should be corrected for these effects so that they may be compared to normal transmission spectra. 

Infrared spectroscopy has been used in many ways in clinical laboratories. The infrared analysis of serum and other fluids from healthy individuals and from patients with various diseases has been an aid in the diagnosis of those diseases. The adsorption of plasma proteins on the surface of polymers which are to be exposed to the bloodstream has been and is actively being investigated by means of internal reflection techniques. Normal and diseased skin surfaces have been examined by the ATR method... 

High-powered microscopes can be used to reveal the internal structure, e.g. an interpenetrating phase, which is impossible to see with the naked eye. The normal technique used to observe phase boundaries under normal light is optical microscopy. However, as some blends have very tiny components, other more powerful techniques are required, i.e. transmission electron microscopy, scanning electron microscopy and atomic force microscopy. It should be noted that in order to see the structures clearly, preliminary treatments are sometimes necessary, for example etching or staining." ... 

The technique of internal normalization is commonly apphed in both MC-ICP-MS and TIMS for the precise correction of the instrumental mass bias (see also Chapter 5) that is encountered during the analysis of radiogenic isotopic compositions [33, 34]. The ICP ion source of MC-ICP-MS, however, also features two characteristics that play an important role for isotopic analysis, where internal normalization cannot be applied. First, an ICP source operates at steady state and therefore mass fractionation is not primarily a time-dependent process, as in TIMS where the measured isotopic compositions change with time due to the progressive evaporation of a sample from the filament. The steady-state operation of an ICP ion source is beneficial for the correction of instrumental mass bias by external standardization, where the isotope ratio data obtained for a sample are referenced to the values obtained for bracketing analyses of an isotopic standard [27, 35]. Hence, this procedure is commonly termed standard-sample bracketing. 

The primary disadvantages of the double-spike technique are that (i) the preparation and calibration of a new double spike require significant effort and (ii) four interference-free isotope signals are needed for accurate data reduction, and this also rules out double-spike analysis of elements that feature only two or three isotopes. In many cases, however, these factors will be outweighed by the advantages of the method (i) it offers an instrumental mass bias correction that is similar in application and reliability to internal normalization and hence is even more robust towards matrix effects than external normalization (ii) the approach can correct for laboratory-induced mass fractionation effects, if the spike is added to the samples prior to the chemical processing and (iii) precise elemental concentration data are obtained as a byproduct of the double-spike method. Hence the double-spike method has recently found increasing popularity in MC-ICP-MS stable isotope analysis of non-traditional elements. 

Again as with internal normalization, even though the sample size is theoretically not critical, attempts should be made to use the same sample size for both standards and unknowns. This constant load on the chromatographic system gives one the best shot at the high accuracy that the technique of internal standardization is capable of producing. 

More accurate semiquantitative determinations can be made by the use of an internal standard technique. Accuracies on the order of 50% of the amount present can be achieved by this approach. A known quantity of an element that is not indigenously present in the sample being measured is added to the calibration standards before the relative sensitivity factors are determined. An internal standard element normalized relative sensitivity factor (R ) is calculated as follows ... 

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