Corrected peak area

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. 

Since CE is an on-column detection technique, analytes migrate with different velocities through the detection window. Thus, slower migrating compounds will have the same peak height but a larger peak area than faster migrating compounds. Therefore, it is common to work with corrected peak areas, i.e., peak area divided by migration time. The work on a racemic mixture of enantiomers demonstrates the importance of this correction. 

In the Quantification section, the necessity for using corrected peak areas, i.e., peak areas divided hy the corresponding migration time, is emphasized. Corrected peak areas are used in order to... 

Integration of the electropherogram for an antibody to quantify main protein species percent light chain and heavy chain (%LC and %HC) and minor species such as percent non-main and percent high molecular weight species (%non-main species and %HMW) is shown in Figure 5. Corrected peak areas were used for quantification. The % corrected peak area is defined as ... 

Figure 7 shows the results for the corrected peak area plotted against injection time for the HC, LC, and non-HC and LC species called non-main species. The corrected peak area values are used because they compensate for velocity differences between species of different molecular size as they pass through the detector. These results demonstrate that the reduced CE-SDS method has a wide injection linearity range (5—40 s at —5kV) for the HC, LC, and non-main species. 

To further confirm the ability of the reduced CE-SDS method to be used for determination of purity, percent corrected peak area (%HC, %LC, and %non-main) was calculated for each component with respect to the total corrected peak area. Eigure 8 presents the relationship between %HC, %LC, %non-main species, and injection time. 

FIGURE 7 Linear relationship between corrected peak area and injection time. 

FIGURE 8 % Corrected peak areas for HC, LC, and non-main versus injection time. Panel A ... 

TABLE 2 % Corrected Peak Areas Obtained from the Injection Time Linearity Study... 

The % corrected peak area values are shown in Table 2. When an injection time of 20 to 40 sec is used, the assay generated reasonable precision. The overall percent relative standard deviation (%RSD) are 0.7, 1.6, and 7.6 for the HC, LC, and non-main species, respectively. The %RSD of HMW species is relatively high due to poor resolution between the HMW and HC peaks. In this case, we recommend that the HMW species not be integrated separately from the HC component unless there is a clear graphic valley point for integration. 

The corrected peak area was plotted against antibody concentration for each component, and the results are shown in Figure 10. The % corrected peak areas for each component (HC, LC, and non-main) are linear over a wide protein concentration range, with coefficient of determination (R ) values greater than 0.99. 

The % corrected peak areas for HC, LC, and non-main species were plotted against protein sample concentrations and are presented in Figure 11. The % corrected peak area values are shown in Table 3. These results show that the assay is linear for sample concentrations above 0.5mg/mL (Table 3), and that the assay is capable of quantitating all three components with overall %RSDs of 0.8, 1.0, and 11.7% for the HC, LC, and non-main species, respectively. The recommended final protein concentration is 1 mg/mL. 

The purpose of sample acceptance criteria is to confirm that the run for a test article is suitable for quantification. The corrected peak area of the sample should be within linear range of the assay the baseline should be suitable for integration the corrected peak percentage should be in a reasonable range. Again, for a purity assay, migration time is usually not a critical parameter. 

At this time the peak areas in the unknown can be corrected for individual variation in detector response by dividing each area by the response factor for that component. The result is then a corrected peak area. The weight percent is then determined by dividing the corrected area by the total corrected area and multiplying by 100. 

Conversion of corrected peak area, (now in weight °/o) to moles. Corrected peak area, ... 

Conversion of mole-corrected peak area, to mole °/o of FAME,. 

The denominator for Equation E6.2 is calculated by performing steps 1 and 2 for all of the FAMEs present in a single triacylglycerol sample. The mole-corrected peak areas are added together to obtain the total of all mole-corrected areas. If we assume that the extent of conversion of free fatty acids to FAMEs is essentially quantitative, or at least equal for all fatty acids in our experiment, the above calculation leads directly to the combined mole percent composition of fatty acids in all positions of the triacylglycerol. 

As previously described (9), the St content is measured from the peak of the phenyl proton at 2.7-n3.1 t, the DOL content from the corrected peak area at 5.3 t and 6.4 t, and the BCMO content from the peak area of 6.5 r. 

As has been discussed, several requirements must be met to predict the explosiveness of self-reactive substances on the basis of the results of measurements by SC-DSC. First, a leak-free sealed cell must be used that is sufficient pressure-proof. Such cells are available from Nippon Kayaku Co. and Seiko Instruments Inc. Next, correct peak areas must be measured. Determination of correct exothermic peaks is one requisite for this purpose, and it is necessary to acquire some experience in this task. 

Quantitation of a PCR product is possible through the generation of a standard curve showing linearity between amount of DNA template present prior to amplification and the corrected peak area of resulting PCR product. To develop this standard curve, a serial dilution of template is performed. Alternatively, a ratio of the unknown template s peak area relative to that of an internal standard can be determined (e.g., for competitive PCR). A linear polyacrylamide sieving buffer was used to determine viral burden. The buffer, which contained the intercalating dye EnhanCE, permitted visualization by LIF and quantitation of the products of reverse transcriptase (RT) PCR... 

Figure 7.10 Plot of corrected peak area versus RNA template added for RT-PCR. Peak area for the 53 bp Sabin 3 product were corrected for transit time through the detector and plotted as a function of the amount of RNA used in the RT-PCR. A linear relationship is observed up to 2.0 fiL RNA added. Inclusion of the 5.0 fiL RNA point demonstrates PCR plateauing.

Corrected peak area using photoelectric cross-sections relative to C Is. 

To measure detector linearity, increasing concentrations of a sample are injected and analyzed using a preconditioned capillary prepared as described in Section 12.3.2. The linearity of tlie detector is measured over tlie concentration range of 0.1 to 5.0 inM. Prior to each injection, tlie preconditioned capillary is rinsed with buffer at high pressure (100 kPa). Four sample concentrations that can be used are 0.1, 0.5, 1.0, and 5.0 inM. After tlie 3-s 5-kPa injection, a constant voltage is applied and tlie detector set to collect data at an appropriate wavelengtli for tlie sample. The temperature is set to a value between 20 and 25°C, and tlie data rate is set to a moderate value (5 Hz). A calibration curve of corrected peak area versus sample concentration is constructed and a correlation coefficient calculated. A linear response should be observed and tlie value of should be greater tlian 0.99 to pass tlie test. 

In principle, one could calculate an absolute Raman cross section from the response of an instrument calibrated with a standard radiometric source. This approach is difficult but has been used to provide the cross sections in Table 2.2. If the relative response function is calibrated accurately, however, it is much simpler to determine cross sections by comparison to standards. Provided the sample positioning and optics permit quantitative Raman signal reproducibility, cross sections of liquids may be determined by comparing the response-corrected peak area to a band with known absolute cross section, such as the benzene 992 cm band. For response-corrected spectra, the ratio of the peak areas under identical experimental conditions equals the ratio of the absolute cross sections. 

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