Calibration with internal correction

An internal standard is a known amount of a nonanalyte element or compound that is added to all samples, blanks, and standard solutions. Calibration with internal standardization is a technique that uses the signal from the internal standard element or compound to correct for interferences in an analysis. Calibration with internal standardization improves the accuracy and precision of an analysis by compensating for several sources of error. 

Calibration standards can be of two types external standards and internal standards. With external standards, multiple concentrations of the standards are injected, areas are measured, and a calibration curve is platted. Unknown samples are then injected, chromatograms run, and areas are calculated and compared with the calibration curves to determine amounts of each compound present. With internal standards, known amounts of an internal standard are added to each known concentration of standard compound and areas or peak height response factors relative to those of the internal standard are calculated. When unknowns are run, a known amount of internal standard is added to the unknown sample, response factors are calculated relative to the internal standards, and amounts of each unknown present are calculated from the standards calibration factors. Internal standards are usually used to correct for variations in injection size due to different operators and injection techniques. Internal standards can also be used to correct for extraction variation in GC/MS target compound quantitation, this standard is referred to as a surrogate standard. Generally, an internal standard is used for one purpose or the other, not both at the same time. 

Inductively coupled plasma-atomic emission spectrometry was investigated for simultaneous multielement determinations in human urine. Emission intensities of constant, added amounts of internal reference elements were used to compensate for variations in nebulization efficiency. Spectral background and stray-light contributions were measured, and their effects were eliminated with a minicomputer-con-trolled background correction scheme. Analyte concentrations were determined by the method of additions and by reference to analytical calibration curves. Internal reference and background correction techniques provided significant improvements in accuracy. However, with the simple sample preparation procedure that was used, lack of sufficient detecting power prevented quantitative determination of normal levels of many trace elements in urine. 

Analysis. Multi-element calibration standards of 0.0, 5.0 and 10.0 ppm Fe, Ca, B, Cu and A1 are prepared by dissolving 0.5 and 1.0 ml of 1000 ppm multi-element standard control stock in tetralin solvent to 100 ml with the same concentration yttrium internal standard as for samples A and B. Both samples are stirred and nebulised to determine the metal content against a standard calibration curve and corrected with an internal standard using ICP-OES. 

At pH 7, where H = OH, the voltage from the electrode is zero. This is called the isopotential point (Fig. 4). In theory, this point is temperature-independent. The International Union for Physical and Applied Chemistry (lUPAC) (1) operational pH scale is defined as fhe pH relative to a standard buffer measured using a hydrogen electrode. In practice, a pH electrode is calibrated with standard buffers of pH 7.00 and pH 4 or 9 fo defermine the isoelectric point and slope, respectively. Conventional pH meters will read accurately over a range of 2.5-11 and beyond these ranges, accuracy cannot be assured. However, recently, instruments have become available that carry out calibration to allow correction for nonideal electrode behavior allowing accurate measurements between ranges of pH 1 and 13. 

Whatever the source of radiation used, the dose delivered to the biological samples is determined by the time of exposure to radiations. Thus the dose delivered by the radiation source must be measured with precision. Dosimetry can be performed with a ferrous sulfate solution (Fricke and Morse, 1927), thermoluminescent dosimeters, bleaching of films (Hart and Fricke, 1967), Perspex dosimetry (Berry and Marshall, 1969), or calibration with standard enzymes (Beauregard et al., 1980 Beauregard and Potier, 1982 Lo et al., 1982). In many laboratories, control enzymes with known D37 are added to protein preparations as internal standards so that any variation between experiments could be corrected for. Because of the better precision of dose rate in Gammacell irradiators, this precaution is not necessary. 

Correct calibration, using calibrants with known stoichiometry, is a necessary prerequisite for assured analytical work (Quevauviller 1996). The preparation of calibrant solutions (preferably in two parallel duplicates) should be carried out according to weight and not volume, and with the use of high-purity chemicals being self-evident. New lots of calibrants must be verified, and the use of internal standards is recommended as early as possible during the ana-... 

It can be assumed that with the development and study of new methods, the ability to determine M (S), the method bias component of uncertainty, cannot be done given that it can be evaluated only relative to a true measure of analyte concentration. This can be achieved by analysis of a certified reference material, which is usually uncommon, or by comparison to a well-characterized/accepted method, which is unlikely to exist for veterinary drug residues of recent interest. Given that method bias is typically corrected using matrix-matched calibration standards, internal standard or recovery spikes, it is considered that the use of these approaches provides correction for the systematic component of method bias. The random error would be considered part of the interlaboratory derived components of uncertainty. 

The reason for the lack of RMs is the absence of reliable and accurate methods of analysis. Microanalysis is, hence, in need of at least one method that can be used as a reference tool for other techniques and to link RMs or round robin exercises to the international unit of mass. Micro-XRF can be used for this potentially, especially when used for analyzing microscopic samples, where matrix absorption effects are relatively unimportant. At present, XRF is considered to be a rather poor method for certification purposes due to intense matrix effects resulting from intense radiation absorption and enhancement by secondary fluorescence. In wavelength-dispersive XRF, reliable results can only be obtained through calibration with a set of reference samples of closely similar composition to the unknown sample. In the case of energy-dispersive XRF using monochromatic excitation, the correction for matrix effects is simpler but in this case the method suffers from a number of other drawbacks, such as spectral overlap and poor statistics in the spectra. 

Although the matrix effects are low in GDMS, results are only semiquantitative, with an accuracy of a factor 2-3 [132], A real quantitative analysis can be done either with a calibration curve or with internal standardization (i.e., relative sensitivity factors [RSFs] that correct for differences in elemental sensitivities by taking the ratio of intensity and concentration of an element x and those of a reference element). As can be observed from Figure 40.8 [155], the RSFs exhibit variations of only about one order of magnitude with only small variations of one element in different matrices, so even without matrix matching, an accuracy of 15%-20% can be achieved [132]. The RSFs are also transferable between... 

If it is not clear whether the reaction pH is correctly adjusted to around 11.5 by addition of the borate buffer, the use of an internal standard is recommended. Tlris especially holds true if hydrolysates are to be analysed. Although small amounts may be present in particulate matter, non-protein amino acids such as a-amino butyric acid or norleudne should be employed, the former having the advantage of being well separated in the HPLC system under discussion (Fig. 26-4). The internal standard should be added before the reagent. The amount added depends upon the type of analysis to be carried out and may vary from 25 to 500 pmol per injection volume. Variations in the response of the internal standard compared with a calibration run allow correction for differences in reaction pH, time and temperature. However, if these parameters have been kept constant for both calibration and sample analyses, the response should be reproducible to within about 2 %. 

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