Absolute quantitation

Both the ICAT (Jenkins 2006) and ITRAQ methodologies (Ross 2004) can be adapted to enable absolute rather than relative quantitation of proteins, essentially by tagging a (natural isotope) synthetic version of the peptide chosen as the surrogate analyte with one of the labeled reagents. There is no question that such approaches can work well but they suffer from the same disadvantages as when used for relative quantitation and, moreover, are more laborious than some more recent methods described below. 

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It is extremely difficult to know values for all of these parameters precisely. Therefore, absolute quantitation is almost never attempted. The determination of relative atomic ratios is an inherently more tractable approach, however. This method is best illustrated by consideration of a binary material composed exclusively of atoms A and B that is perfectiy homogeneous up to the surface. In this case, independent equations can be developed relating the number of atoms sampled to the xps intensity for each atom as follows ... 

The method of Carius for the determination of chlorine in organic compounds is, of course, absolutely quantitative, but is very tedious, and is scarcely suitable for the detection of very small traces of chlorine, as the weight of oil taken in a Carius determination never exceeds 0 5 grams. 

In a different example, traceability in the amount-of-substance analysis of natural potassium, thorium, and uranium by the method of passive gamma-ray spectrometry was demonstrated by Nir-El (1997). For an absolute quantitative determination, accurate values of two parameters were required (i) the emission probability of a gamma-ray in the decay of the respective indicator radionuclides, and (2) the detection efficiency of that gamma-ray. This work employed a number of CRMs in the critical calibration of the detection efficiency of the gamma-ray spectrometer and the establishment of precise emission probabilities. The latter results compared well with literature values and provided smaller uncertainties for several gamma-rays that were critical for the traceabUity claim. The amount-of-substance analytical results of the long lived naturally occurring radionucHdes K, Th, and... 

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

Solid samples can also be measured in transmission, although reflection or transflection measurements are more common. Open arrangements with the source on one side of the sample and the spectral analyser on the other side are prevalently used, e.g. in industrial process control. For absolute quantitative analysis the thickness of the object must either be constant or be measured. 

Almeida, P., Ribeiro, M. J., Bottlaender, M. et al. Absolute quantitation of iodine-123 epidepride kinetics using single-photon emission tomography comparison with carbon-11 epidepride and positron emission tomography. Eur. J. Nucl. Med. 26 1580-1588,1999. 

It is interesting to observe here that the above reaction is absolutely quantitative under experimental parameters. Therefore, it forms the basis for the estimation of pharmaceutical substances essentially containing a free primary amino function as already illustrated earlier. 

Grayson DR, Bovolin P, Santi M-R. 1993. Absolute quantitation of y-aminobutyric acid A receptor subunit mRNAs by competitive polymerase chain reaction. Methods in neuroscience, vol 12. Academic Press San Diego pp. 191-208. 

In conventional electrochemistry in solution, quantitation of analytes can be obtained by using several techniques. Thus, exhaustive electrolysis provides an absolute quantitation of an electroactive component in the sample. Voltammetric measurements (linear potential scan, cyclic, pulse, and square-wave techniques) can be used for determination of analytes in solution via calibration because peak currents (and peak areas) are usually proportional to the analyte concentration under fixed electrochemical conditions. 

In spite of its evident limitations, solid-state electrochemical methods can also be used for quantifying electroactive components in sparingly soluble solids. As discussed in this chapter, relative quantitative methods and absolute quantitations are available from voltammetric data. 

Absolute quantitation of individual components in the FID chromatograms was achieved using the method described by Larter and Senftle (44) in which a known amount of polymer standard... 

Targeted analysis refers to metabolome analysis that targets one, or a few metabolites, and typically uses an internal standard for quantitation. The most common method is isotope dilution mass spectrometry (IDMS) [34], which relies on the use of stable isotope internal standards to enable the absolute quantitation of metabolites. This method has proven highly effective and has been successfully used in numerous studies. 

Sensitivity 200 ppm Absolute quantitation is not a straightforward procedure, appropriate standards and some approximations of correction factors are always involved in the practical application... 

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