Chemical interferences methods

Interestingly, specific M +-nucleic acid interactions determined in solution for the hairpin ribozyme by spectroscopic and chemical interference methods were not later found in their X-ray crystal stmctures. Even more confusing were results of X-ray crystallography " and initial solution experiments, which both determined up to two, presumably functionally important Mg + ions in the catalytic center of the hammerhead ribozyme. However, as demonstrated recently in biochemical experiments in solution, neither of these ribozymes require either inner- or outer-sphere interactions with particular metal ions for catalysis. ... 

For a method to be useful it must provide reliable results. Unfortunately, methods are subject to a variety of chemical and physical interferences that contribute uncertainty to the analysis. When a method is relatively free from chemical interferences, it can be applied to the determination of analytes in a wide variety of sample matrices. Such methods are considered robust. 

Accuracy The accuracy of a fluorescence method is generally 1-5% when spectral and chemical interferences are insignificant. Accuracy is limited by the same types of problems affecting other spectroscopic methods. In addition, accuracy is affected by interferences influencing the fluorescent quantum yield. The accuracy of phosphorescence is somewhat greater than that for fluorescence. 

Atomic emission is used for the analysis of the same types of samples that may be analyzed by atomic absorption. The development of a quantitative atomic emission method requires several considerations, including choosing a source for atomization and excitation, selecting a wavelength and slit width, preparing the sample for analysis, minimizing spectral and chemical interferences, and selecting a method of standardization. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

What type of chemical interference in the sample necessitates the use of the method of standard additions ... 

Despite the variety of methods that had been developed, by 1960 kinetic methods were no longer in common use. The principal limitation to a broader acceptance of chemical kinetic methods was their greater susceptibility to errors from uncontrolled or poorly controlled variables, such as temperature and pH, and the presence of interferents that activate or inhibit catalytic reactions. Many of these limitations, however, were overcome during the 1960s, 1970s, and 1980s with the development of improved instrumentation and data analysis methods compensating for these errors. ... 

Selectivity The analysis of closely related compounds, as we have seen in earlier chapters, is often complicated by their tendency to interfere with one another. To overcome this problem, the analyte and interferent must first be separated. An advantage of chemical kinetic methods is that conditions can often be adjusted so that the analyte and interferent have different reaction rates. If the difference in rates is large enough, one species may react completely before the other species has a chance to react. For example, many enzymes selectively cat-... 

The advantage of the inverse calibration approach is that we do not have to know all the information on possible constituents, analytes of interest and inter-ferents alike. Nor do we need pure spectra, or enough calibration standards to determine those. The columns of C (and P) only refer to the analytes of interest. Thus, the method can work in principle when unknown chemical interferents are present. It is of utmost importance then that such interferents are present in the Ccdibration samples. A good prediction model can only be derived from calibration data that are representative for the samples to be measured in the future. 

There are a number of interferences that can occur in atomic absorption and other flame spectroscopic methods. Anything that decreases the number of neutral atoms in the flame will decrease the absorption signal. Chemical interference is the most commonly encountered example of depression of the absorption signal. Here, the element of interest reacts with an anion in solution or with a gas in the flame to produce a stable compound in the flame. For example, calcium, in the presence of phosphate, will form the stable pyrophosphate molecule. Refractory elements will combine with 0 or OH radicals in the flame to produce stable monoxides and hydroxides. Fortunately, most of these chemical interferences can be avoided by adding an appropriate reagent or by using a hotter flame. The phosphate interferences, for example, can be eliminated by adding 1 % strontium chloride or lanthanum chloride to the solution. The strontium or lanthanum preferentially combines with the phosphate to prevent its reaction with the calcium. Or, EDTA can be added to complex the calcium and prevent its combination with the phosphate. 

What is the standard additions method and why does it help with the problem of chemical interferences ... 

The standard additions method is one in which the standards prepared for the standard curve consist of the sample to which varying amounts of a standard solution have been added. It helps with the problem of chemical interferences because the sample is the matrix for the standards and the interference occurs in all measurements such that its effect is negated. 

Advantage The major plus points of the standard addition method is that it tends to compensate for variations caused by physical and chemical interferences in the sample solution. 

Ideally, the standards should be made up in a solution containing the same normally expected levels of matrix elements as occur in the sample solution. It should be borne in mind that even if they exert no chemical interference, they could possibly exert a viscosity effect on a nebulized solution (especially with high concentrations of phosphoric or sulphuric acids). If it is not possible to determine the matrix components or prepare standards in a matrix solution, and unless experiments have shown matrix interference to be insignificant, then the method of standard additions, or spiking, should be carried out. This is where known amounts of the analyte are added to the sample or sample solution before determination by, e.g. AAS or colorimetry. 

Flame atomic absorption spectrometry can be used to determine trace levels of analyte in a wide range of sample types, with the proviso that the sample is first brought into solution. The methods described in Section 1.6 are all applicable to FAAS. Chemical interferences and ionization suppression cause the greatest problems, and steps must be taken to reduce these (e.g. the analysis of sea-water, refractory geological samples or metals). The analysis of oils and organic solvents is relatively easy since these samples actually provide fuel for the flame however, build-up of carbon in the burner slot must be avoided. Most biological samples can be analysed with ease provided that an appropriate digestion method is used which avoids analyte losses. 

The distillation method (see Alternate Protocol 1) involves recovering malonaldehyde from an acidified food product. It is similar to that of the direct heating approach (see below), except that TBA is reacted only with an aliquot of the distillate. Consequently, physical and chemical interference by extraneous food constituents in the reaction with TBA is minimized because the food is never directly in contact with TBA. Unfortunately, direct heating of a food under acidic conditions enhances the degradation of existing lipid hydroperoxides as malonaldehyde precursors, and generates additional reactive radicals and scission products other than malonaldehyde that can react with TBA (Raharjo and Sofos, 1993). More malonaldehyde or reactive substances will be gener-... 

If an analytic method lacks sufficient specificity, chemical interferences will result in an erroneously high reported concentration. If a measured chemical is introduced as an artifactual contaminant during sample collection or analysis, reported concentrations will also be overestimated. For example, credibly estimating human exposure to phthalates was hindered by the difficulties involved in avoiding specimen contamination with these ubiquitous chemicals the problem was resolved by focus on the much less prevalent metabolic product, the phthalate half-ester (Silva et al. 2004). Alternatively, a chemical measured as a marker of exogenous exposure may be identical with a chemical formed by an unrelated endogenous metabolic pathway. In each of those cases, a rigorous laboratory-method validation should detect the problem before data are reported. More subtly, the measured biomarker of exposure may be chemically identical with a dietary... 

Analytical methods for mycotoxins have historically utilised chemical detection methods which are easily quantifiable, highly sensitive, less subject to interference by co-extractives but can be quite time demanding. Most quantitative methods use a final chromatographic separator followed by a suitable detection... 

Metals can be conveniently determined by emission spectroscopy using inductively coupled plasma (ICP). A great advantage of ICP emission spectroscopy as applied to environmental analysis is that several metals can be determined simultaneously by this method. Thus, multielement analysis of unknown samples can be performed rapidly by this technique. Another advantage is that, unlike atomic absorption spectroscopy, the chemical interference in this method is very low. Chemical interferences are generally attributed to the formation of molecular compounds (from the atoms) as well as to ionization and thermochemical effects. The principle of the ICP method is described below. 

To help determine the presence of chemical interference from the sample matrix, laboratories use the serial dilution technique, described in Chapter 4.4.4.5. Once the presence of chemical interferences from the sample matrix has been determined, the matrix can be modified to remove interferences or to stabilize the analyte. The sample can be also analyzed using the method of standard additions (MSA), described in Chapter 4.4.4.5. 

If the difference between the two results exceeds the method criterion, physical and chemical interferences may be present, and laboratories should perform a post digestion spike addition to verify their presence. 

---