Substoichiometric analysis

Combination of isotope dilution with the principle of substoichiometric analysis offers the possibility of avoiding determination either of the chemical yield of the separation procedure or of the specific activity in the isolated fraction. Two identical aliquots of the radiotracer solution are taken, both containing the tracer with mass... 

The foregoing is an example of the basic principle of substoichiometric analysis. Under... 

This substoichiometric analysis technique can be applied to most metals with a high degree of accuracy and a sensitivity of 10" to 10 ° g of X. 

The principle of the substoichiometric analysis is as follows To the element of interest (M ), one adds a known amount of its labeled radioisotope with the specific activity S = A/M, where A is the radioactivity and M the amount of carrier hence the specific activity of the mixture becomes S = A/(M -1- M). Knowing the change in specific activity from S to S, the element of interest can be sinqily determined. This is the same as the principle of isotope dilution, but in practice the accurate determination of S and S is very tedious. This is one important reason why isotope dilution analysis is not very popular in trace analysis. However, in substoichiometric analysis, equal amounts of the element (m) are isolated substoichiometrically from the radioisotope solution and the mixed solution, and subsequently the radioactivities of the separated portions (a and a ) are measured, then the amount of the element of interest can be calculated according to the equation ... 

Fractional separation of tin compounds used as stabilisers in PVC was based on substoichiometric isotope dilution analysis [446]. The tin compounds were isolated by extraction and complexed with salicylideneamino-2-thiophenol, followed by controlled addition of y-irradiated tributyl tin oxide and measurement of the y-activity. PVC containing a nominal 0.63% (Bu Sn analysed 0.614 0.016% in nine determinations. 

K. Kobayashi, T. Shigematsu, Trace determination of iron, cobalt, nickel and copper in zirconium fluoride by substoichiometric radioactivation analysis, J. Radioanal. Nucl. Chem. Articles 113 (1987) 333-341. 

Substoichiometric isotope dilution analysis was first developed by Ruzicka and Stary (1968) as another variation on the basic IDA technique. The basic idea of... 

Oxygen is so tightly bound, especially in the substoichiometric compositions, that its analysis can be difficult. Activation analysis (y, n) or the dissolution of the carbide in C-saturated Pt at 2200°C, thereby releasing the oxygen as CO, are used. Dissolved oxygen... 

Traditional methods to map posttranslational modification sites, like those of phosphorylation, have been anchored by protein digest and mass spectroscopic (MS) approaches (for a review on the classic evaluation and for MS analyses of O-glycans, see Reference (56)). Unfortunately, like many posttranslational modifications, O-GlcNAcylation occurs routinely on a protein population with substoichiometric frequency, which results in a very small detectable population of a O-GlcNAc-modified product. Also, much like O-phosphate additions, the protein-O-GlcNAc bond is labile and is detached by collision-induced dissociation (CID) during MS analysis. Often, the bond is lost before it can be detected on the peptides analyzed (57, 58). Phosphate modifications, however, can overcome this limitation by emiching the peptide mixtures... 

Two radioanalytical methods described in chapter 17 are applied preferentially in the life sciences, activation analysis and isotope dilution, the latter mainly in combination with the substoichiometric principle. 

Isotope dilution in combination with the substoichiometric principle is applied in various ways. The most important examples are radioimmunoassay for protein analysis and DNA analysis. In radioimmunoassay, radionuclides are used as tracers and immunochemical reactions for isolation. Radioimmunoassay was first described in 1959 by Yalow and Berson, and since then has found very broad application in clinical medicine, in particular for the measurement of serum proteins, hormones, enzymes, viruses, bacterial antigens, drugs and other substances in blood, other body fluids and tissues. Only one drop of blood is needed, and the analysis can be per-fonned automatically. Today more than 10 immunoassays are made annually in the United States. The most important advantages of the method are the high sensitivity and the high specificity. In favourable cases quantities down to 10 g can... 

PTMs of proteins enable many important functions in organisms (139). Proteins with PTMs can be enriched in a variety of ways antibody-based, chemical derivatization, and ionic interaction-based (89). Antibodies that are highly specific to a particular PTM site help functional assays greatly. Antibodies that preferably bind a class of PTM (e.g., proteins carrying phosphotyrosine residues) make possible the in-depth quantitative proteomic analysis of these proteins. The substoichiometric nature of PTMs yields a low concentration of modified proteins. Enrichment of these proteins allows for improved analysis. 

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS) is the central method for identifying proteins with PTMs (187,188) due to its high sensitivity, its ability to identify the site, and its ability to quantify the relative changes in PTM occupancy at distinct sites (189), among others. However, PTM analysis presents a number of analytical challenges because most modifications are low-abundance and/or substoichiometric and some are labile during MS and MS/MS. 

In contrast to other inactivators of dopamine /3-hydroxylase, p-cresol is unusual in that it does not contain a latent electrophile (Goodhart et al., 1987). Oxidation of the benzylic carbon occurs, as evidenced by production of 4-hydroxybenzyl alcohol as well as the corresponding aldehyde. Inactivation with radiolabeled p-cresol leads to substoichiometric modification of the enzyme, with the majority of the radiolabel distributed between four tryptic peptides (DeWolf et al., 1988). Analysis of two of the peptides indicates that they are of identical sequence, each containing modified tyrosine residues which differ in the structure of the p-cresol-amino acid adduct. A second pathway for inactivation is proposed which requires radical-mediated oxidation of the enzyme without incorporation of the inactivator. 

Within the series. Comprehensive Analytical Chemistry, two long chapters of particular note are those by Gibbons and Lambie (1971) treating radiochemical methods of analysis, and Stary and Ruzicka (1976) who write on substoichiometric analytical methods, some of which incorporate NAA. 

A76. Nadkarni, R.A. and B.C. Haidar Neutron activation analysis of copper by substoichiometric extraction with neocuproine Anal. Chem. 44 (1972) 1504—1506. 

Kanda, Y., Suzuki, N. (1980) Substoichiometric isotope dilution analysis of inorganic mercury and methylmercury with thionalide. Anal. Chem., 52, 1672-1675. 

Isotope dilution analysis require the determination of either the chemical yield in the separation process or of the specific activity. This can be avoided by applying the substoichiometric principle, which may also increase the sensitivity of the analytical method. 

When the substoichiometric principle is applied to isotope dilution analysis, the relationship becomes... 

Immunoassay is an application of the substoichiometric principle ( 9.3.4) developed by Yalow (Nobel laureate in 1977) for protein analysis. In the United States tens of millions radioimmunoassays are made annually in hospitals to measure hormones, enzymes, viruses, serum proteins, drugs, and so forth. Only a drop of the patient s blood is needed, reflecting the versatility and smisitivity of this technique, which can be performed automatically. Commercial RAST-kits (Radio Allergy Sorbent Tests) are used for rapid diagnosis of allergic reactions. 

Many radioanalytical methods have been introduced for trace ana is. Eadi method has its own advantages, but in comparison with ordinary instrumental nuclear m ods, these radioanalyticd methods, except for sut toichiometry, have not been applied to analysis of many actual sampla. Substoichiometric analytical methods induding substoichiometric radioactivation analysis will come to be more widely used, as a result of the many possible applications. 

Apart from the careful regulation of radioactive material handling, the main obstacle to more widespread application of these radioanalytical methods is that they involve chemical procedures for sample preparation. By examining the chemical procedures carefully, simpler methods may be found as has been demonstrated in substoichiometric radioactivation analysis and some cases of sutetoichio-metric chemical speciation. 

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