Normalising peak areas

The area of each peak is obtained from a series of replicate (5+) injections of a mixture containing equal (or known) amounts of all the components. Acceptable precision is essential to obtain satisfactory data. One component is chosen as the reference and the relative responses of the other components are determined by dividing the peak areas by that of the reference component. The detector response factors (Z rf) may then be used to calculate corrected peak areas (. 4correct) for other analyses involving these components and hence their percentage ratios in the mixture may be 

Area for reference. 4ref Corrected area for a component in a sample is correct — -Drf x chrom 

The internal standard method is a variation on the above, and is recommended for accurate quantitation. It eliminates the need for accurate injections since a reference standard is included in each sample analysed. An internal standard is selected which has a retention time such that it is eluted in a suitable gap in the chromatogram. The procedure involves analysing a test sample containing known amounts of each component plus a predetermined amount of the internal standard. Since peak area is proportional to the amount of an eluted component and the detector response factor (I rf) 

Response factors for all components are calculated in the same way. Analysis of an unknown mixture is achieved by adding an accurately known amount of internal standard and then carrying out the chromatography. The concentration of each component calculated using the equation above rearranged to give 

The precision of the analysis is not dependent on injection of an accurately known amount of sample, but does depend on accurate measurement of peak areas. This is not a problem with electronic integrators and an overall precision or covariance of 4% should be readily obtained. 

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Nbasba eqnalling 1. All normalised peak areas were then used to calculate the number-average sequence length for both styrene and -butyl acrylate (Table 7.7). 

Procedure. Inject 1 fiL of the sample solution and obtain a chromatogram. Under the above conditions the compounds are separated in about 3 minutes, the elution sequence being (1) aspirin (2) phenacetin (3) caffeine. Measure peak areas with an integrator and normalise the peak area for each compound (i.e. express each peak area as a percentage of the total peak area). Compare these results with the known composition of the mixture discrepancies arise because of different detector response to the same amount of each substance. 

Correct the peak areas initially obtained by dividing by the appropriate response factor and normalise the corrected values. Compare this result with the known composition of the mixture. 

To allow for this, before the peak areas are normalised we must first correct each area so as to get the area we would have obtained had the detector response been the same for each of the three compounds. We will now use the results from our mixture to determine calibration factors (relative response factors) for the detector, and then use these for the analysis of a commercial tablet. 

Once response factors have been obtained, using data from a standard mixture, we can then use them to correct peak areas for an unknown mixture of the same compounds, and determine the mixture by normalising the corrected areas. 

Altria, K. D. (1993). Essential peak area normalisation for quantitative impurity content... 

Note aTemperature is 380 °C bRate is MHSV in hr"l cResults are normalised on propane free basis Raw integrated peak areas in arbitrary units shown in parentheses Conversion of methanol is 100 X in all cases. 

Detector response factors need to be accurately determined for good reproducible quantitative analyses [60]. They may be conveniently obtained by the constant volume method, that is, approximately 10 repeatable volumes of a sample containing an equal amount of the analytes are injected and the mean determined. Drf values are calculated hy normalising each peak area to that of the peak to be used as the reference. 

Normalised area% is obtained by first correcting the peak area from the chromatogram, /4chrom. of each component for the relative response of the detector to that component. Detector response factor (Drp) is calculated with reference to a specified reference standard or internal standard see Section 2.7. The corrected area, y4correct of each peak is then used in the equation above to obtain the normalised area%... 

Calibrations could be made on the basis of the decomposition of solid standards, injections of volatile liquids, standard gas mixtures, injections of pulses of gas, or steady injection rates of gases into the purge gas stream. With proper normalisation procedures, all methods gave equivalent results.A good linear relationship between EGA peak area and amount of product was found for between a few and a few hundred microgrammes of gas when using a jet separator. 

Figure 1.27 Normalised DSC scans showing the change in the exothermic peak area as a function of % azo. (a) sample B, (b) sample c, (c) sample E and (d) sample F Reproduced with permission from A. Prasad and M. Shanker, Cellular Polymers, 1999, 18,1, 35. Rapra Technology [95]From Prasad and Shankar, Cellular Polymers [95]...

Figure 1.27 shows the relationships between the degree of hydrogenation determined by the iodine value method and the peak area of Cy-MN(A) and Cu-MN(A) normalised... 

Figure 7.15 shows the pyrogram of the trimer area for five different mole compositions of STY/n-BA. The number-average sequence lengths were calculated for all five polymers. The peak areas were normalised on the basis of the summation of and... 

To obtain an accurate quantitative analysis of the composition of a mixture, a knowledge of the response of the detector to each component is required. If the detector response is not the same for each component, then the areas under the peaks clearly cannot be used as a direct measure of the proportion of the components in the mixture. The experiment described illustrates the use of an internal normalisation method for the quantitative analysis of a mixture of the following three components ethyl acetate (ethanoate), octane, and ethyl n-propyl ketone (hexan-3-one). 

Figure 4.11—Standard chromatogram producedhy a recording integrator showing retention time (min), area of the peak (arbitrary units) and corresponding % of area. The % area of the peaks must not be confused with the % quantity of the sample constituents since the response factors are not identical for all compounds (non-normalised report).

The total fallout from the low-yield trials in Nevada and Utah (outside the NTS boundary) was estimated by Hardy at 26 TBq (700 Ci), compared with about 9 TBq of global 239+240pu falling in the same area. The peak measured fallout, outside the N.T.S., was 600 Bq m-2 at Eureka, NV. This compares with mean global fallout of 60 Bq m-2 in the USA (Hardy, 1976) and 50 Bq m-2 (normalised to 1000 mm a-1 rainfall) in the UK (Peirson et al., 1982). Within the N.T.S., the deposition of Pu was much greater, ranging from 4 x 104 to 2 x 108 Bq m-2 (Martin Bloom, 1980). 

When an analysis has been completed, the results can be expressed directly as weight percentages of the fatty acids for presentation in tabular form. On the other hand, it is often necessary to calculate the molar amounts of each acid as, for example, in most lipid structural studies (positional distributions and molecular species proportions). This is performed simply by multiplying the area of each peak by an arithmetic factor, obtained by dividing the weight of a selected standard ester (say 16 0) by the molecular weight of the component, followed by re-normalising to 100%. For convenience, these factors are also listed in Table 5.4. It should be noted that if fatty acid... 

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