Standard addition method calibration graphs using

There is one more, very important and relatively simple method to use when an interference effect is difficult to explore but its occurrence is probable and poses a threat to the reliability of analytical results. This refers to the case when an analyzed series of samples have similar chemical composition (at least in terms of the composition of interferents) and the determined component is present in all samples in a similar quantity. In this situation, the standard addition method can be used for analysis of one selected sample and the constructed calibration graph employed for interpolative determination of analyte in the remaining samples. This combined procedure is depicted in Fig. 3.16. Thus obtained results are, as a mle, more accurate than those obtained after application of the set of standards method to all the samples. In addition, the analyses are conducted faster than when all the samples are analyzed using the standard addition method. 

Although it is an elegant approach to the common problem of matrix interference effects, the method of standard additions has a number of disadvantages. The principal one is that each test sample requires its own calibration graph, in contrast to conventional calibration experiments, where one graph can provide concentration values for many test samples. The standard-additions method may also use larger quantities of sample than other methods. In statistical terms it is an extrapolation method, and in principle less precise than interpolation techniques. In practice, the loss of precision is not very serious. 

Figure 1 An illustration of the use of the standard additions method. From the aqueous standards calibration graph, the unspiked sample would appear to contain LI3 mg of determinant per litre. The spiked samples allow a calibration graph with the correct slope to be used, and give a result of 1.50 mg l 1...

Although it is possible to measure directly Mn in body fluids at normal concentrations of 18—180 nmol 1 I (1—lOpgl-1) it is difficult to achieve a precision of better than 0.10 RSD. The elimination of the considerable molecular absorption interferences requires strict control of ETA ashing temperatures and a good background correction system, and the variable condensed phase matrix interferences from the inorganic constituents necessitates the use of standard additions for calibration [64], Even this approach may not yield a viable method due to curvature of the calibration graph at very low absorbances, particularly with a diluted blood matrix [65],... 

Figure 7.17 A calibration graph using method of standard additions.

Second method ca. 0.5 g sediment was extracted with methanol/tropolone after addition of HCI. Tripropyltin was added as internal standard. Derivatiza-tion was performed by addition of pentylmagnesium bromide (2 mol L ) followed by clean-up with silica gel. Separation was by CGC (column of 25 m length, 0.2 mm internal diameter, methylphenyl silicon as stationary phase, 0.11 pm film thickness He as carrier gas at 130 mL min injector temperature at 260 °C column temperature ranging from 80 to 280 °C detector (transfer line) temperature at 280 °C). Detection was by mass spectrometry. Calibration was by calibration graph, using butyltin chloride compounds as calibrants. 

Salgado Ordonez et al. [28] used di-2-pyridylketone 2-furoyl-hydrazone as a reagent for the fluorometric determination of down to 0.2 pg aluminium in seawater. A buffer solution at pH 6.3, and 1 ml of the reagent solution were added to the samples containing between 0.25 to 2.50 pg aluminium. Fluorescence was measured at 465 nm, and the aluminium in the sample determined either from a calibration graph prepared under the same conditions or a standard addition procedure. Aluminium could be determined in the 10-100 pg/1 range. The method was satisfactorily applied to spiked and natural seawater samples. 

Either calibration graphs prepared from standards or the method of standard addition (p. 30) can be used. For the former, the standards should be as similar as possible in overall chemical composition to that of the samples so as to minimize errors caused by the reduction of other species or by variation in diffusion rates. Often, the limiting factor for quantitative work is the level of impurities present in the reagents used. 

Murillo-Pulgarin et al. used a phosphorimetry method for the determination of dipyridamole in pharmaceutical preparations [44]. Ten tablets or capsules were powdered and homogenized, and then 0.1 g was dissolved in 0.1 M sodium dodecyl sulfate. The determination of dipyridamole was carried out in 26 mM sodium dodecyl sulfate/ 15.6 mM thallium nitrate/20 mM sodium sulfite, whose pH was adjusted to 11.5 by the addition of sodium hydroxide. After 15 min at 20°C, the phosphorescence was measured at 616 nm (after excitation at 303 nm). The calibration graph was linear from 100-1600 ng/mL, with a detection limit of 16.4 ng/mL. Relative standard deviations were in the range of 0.5-7.3%, and sample recoveries were in the range of 95-97%. 

Nimodipine in plasma was determined by a GC method [17]. Plasma was treated with 2 M NaOH after addition of the internal standard, and then extracted with toluene. The column (10 m x 0.31 mm) consisted of cross-linked 5% phenylmethyl silicone, and the method used temperature programming from 90°C (held for 1 min) to 255°C at a heating rate of 25°C/min. Helium was used as a carrier gas, and the N-P detection mode was employed. The calibration graph was linear from 2 to 50 ng/mL, and the detection limit was 0.5 ng/mL. 

In principle there are no differences between calibration procedures for flame and ETA methods although the latter case will take longer, as has already been pointed out. Calibration standards should match the samples as nearly as possible with respect to major components otherwise standard additions must be used. Indeed the method of standard additions will have to be used at some time to check accuracy, so this procedure will usually be tried first. If the standard additions graph is parallel to the direct calibration graph then freedom from matrix interferences would be indicated. The standard additions principle as applied to ETA is now described. 

For more complex solutions and samples where it is not possible to remove the matrix during the ashing step it may be necessary to use the method of standard additions. Some workers advocate that this method should always be run initially to check for interference effects so that the best calibration procedure can be selected. If the standard additions graph and the direct calibration graph were parallel, freedom from interferences in the sample would be indicated, i.e. the element is in the same form in sample and standard immediately before atomisation, or the two forms give the same absorption response. 

Two further limitations apply to the use of the method of standard additions. Simultaneous background correction must be employed because of possibly varying amounts of matrix material present in the tube during the several firings needed to make one determination. Furthermore, all readings must be within the linear portion of the calibration graph in order... 

The method of standard additions is widely used in atomic spectroscopy (e.g. determination of Ca2+ ions in serum by atomic emission spectrophotometry) and, since several aliquots of sample are analysed to produce the calibration graph, should increase the accuracy and precision of the assay... 

Second method ca. 1 g sample was treated with 25% tetramethylammonium hydroxide followed by hexane extraction and clean-up with an alumina column. TPeT was added as internal standard. Ethylation was performed with 0.6% NaBEt4 solution in acetate buffer (pH 5). Clean-up was carried out on alumina, with hexane elution. The derivatization yield was verified by comparison between the derivatized compounds provided by SM T and un-derivatized compounds (chloride). Separation was by capillary GC (column of 30 m length, 0.25 mm internal diameter, DB-17 as stationary phase, 0.25 pm film thickness). Recoveries were assessed by spiking (standard additions) results were (66 1)% for DBT, (74 + 2)% for TBT and (55 1)% for MPhT. Calibration was by calibration graph and standard additions, using the calibrants provided by SM T. 

An SIA system for the simultaneous determination of phosphate and silicate in waste-water is proposed. The method is based on the formation of yellow vanadomolybdopho-sphate and molybdosilicate, respectively, in addition to the use of large sample volumes. The mutual interference between both analytes was eliminated by selection of the appropriate acidity and by sample segmentation with oxalic acid. The calibration graph for phosphate and silicate is linear up to 12 mg/L P and 30 mg/L Si, respectively. The detection limits are 0.2 mg/L P and 0.9 mg/L Si. The method provides a throughput of 23 samples/h with a relative standard deviation <1.4% for phosphate and <4% for silicate. The method was foimd to be suitable for the determination of these species in wastewater samples. 

A 1-ml portion of plasma was extracted twice (pH 7.4 and 12] using a 1 1 mbcture of diethyl ether and ethyl acetate after addition of methaqualone as internal standard. The extract was analyzed by GC-MS in the selective-ion monitoring (SIM) mode (m/z 185, 187, 255, 257 for lamotrigine and 235, 250 for methaqualone). A five-point calibration graph was established using spiked plasma samples [65]. The procedure was linear from 0.5 to 20 mg/L (r = 0.991) with coefficients of variation of less than 15%. The LOD was 0.1 mg/L. As we have seen, therapeutic concentrations ranged from 1 to 6 mg/L. The presented method has proved to be suitable for TDM as well as for clinical toxicology. 

The internal standard method is based on the use of the relative response factor of each component to be measured with respect to a marker introduced as reference. This avoids the imprecision related to the injected volumes, which is a disadvantage of the previous method. However, it requires the addition of a component to a sample dilution. In general, a calibration curve is built by ap>plying different solutions of increased concentrations of the standard analyte with a constant quantity of internal standard. When injecting such samples, we obtain the relation between the areas of the analyte and the internal standard then, it is marked in a graph according to the concentration of analyte in each solution. By means of interpolation in the graphic, we get the relation of the areas of an unknown sample, which has to contain the same quantity of internal standard. 

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