Chromatogram subtraction

Figure 13.10 LC-LC chromatogram of a surface water sample spiked at 2 p.g 1 with ati azine, and its metabolites (registered at 220 nm). Conditions volume of sample injected, 2 ml clean-up time, 2.60 min ti ansfer time, 4.2 min The blank was subtracted. Peak identification is as follows 1, DIA 2, HA 3, DEA 4, atrazine. Reprinted from Journal of Chromatography, A 778, F. Hernandez et al, New method for the rapid detemiination of triazine herbicides and some of thek main metabolites in water by using coupled-column liquid cliromatography and large volume injection , pp. 171-181, copyright 1997, with permission from Elsevier Science.

Figure 3.20 Reconstructed ion chromatograms for the ions observed in the background-subtracted mass spectrum obtained from the component eluting after 5.05 min in the LC-MS analysis of a pesticide mixture. From applications literature published by Micromass UK Ltd, Manchester, UK, and reproduced with permission.

To characterize the difference between two chromatograms, an overall mismatch index and a list of the areas, helglits, and positions of the main peaks in the difference chromatogram are provided. The difference chromatogram is the result of subtracting the standard chromatogram for that class of specimen from the chromatogram of the specimen. 

In the mass balance approach, all impurities are quantified and subtracted from the absolute value of 100%. This approach will result in a purity value that, if all impurities are accounted for, is more accurate than the external or internal standard methods. However, the ability to identify all impurities in a given drug substance may require the use of hyphenated detection techniques and could be extremely costly to complete on a regular basis. Therefore, a related approach, called Area Normalization, is often used where the majority of the impurities can be identified and quantified in a single chromatogram. In the simplest case, all of the impurities would be assumed to have the same relative response... 

FIGURE 22 (a) HPLC/UV chromatogram, (b) ESI LC/MS total ion chromatogram, (c) extracted mass spectrum for RT = 25.35 min peak without background subtraction, (d) Extracted mass spectrum for the same peak with background subtraction. 

This volume, designated by Fs, does not appear on the chromatogram. It can be determined by subtracting the volume of the mobile phase from the total volume inside the column. 

The stochastic model applies to processes involving the stationary phase. To analyze the chromatogram, we need to subtract contributions to peak broadening from dispersion in the mobile phase and extra-column effects such as finite injection width and finite detector volume. These effects account for the width of the unretained peak. To subtract the unwanted effects, we write... 

Mass spectra recorded using LC/MS software may be displayed individually, signal averaged, and background subtracted. Furthermore, these data may be used to plot computer-reconstructed selected ion or total ion chromatograms. 

If it suspected that the enantiomers of interest are coeluting, a correction to the areas of the affected peaks may be applied. The correction can be determined from a knowledge of the peak areas on a nonpolar and polar achiral column. If the shape of the peak and the areas are the same on both columns, then no coelution is occurring. If the areas are different, then a correction factor can be applied by comparing retention times of the separated enantiomers, and subtracting the areas of those peaks that are coeluting, which were determined from the chromatograms obtained from the achiral columns. It should be emphasized that this is not an appropriate correction to make if accurate quantitative information is required. 

Gas chromatograms were obtained on a twin Hewlett-Packard model 5750 Research Chromatograph. The dual columns were 6-m lengths of copper tubing 3 mm in diameter, packed with 3% OV-1 on Chromosorb G-HP (methylsilicone on calcined diatomaceous earth). Samples were introduced as about 10% solutions in carbon bisulfide. Detection was by hydrogen-flame ionization, the non-sample contribution from the idle column being subtracted from the total contribution of the active column to provide a sample chromatograph corrected for extraneous ionization. 

Internal standard areas and acceptance limits Raw data for each sample, blank, spike, duplicate, and standard (quantitation reports, reconstructed ion chromatograms) Raw and background subtracted mass spectra for each target analyte found Tentatively identified compounds mass spectra with library spectra of 5 best-fit matches Sample preparation bench sheets Gel permeation chromatography clean-up logs S S S S S S S ... 

The standard curves obtained from the CP-80,794 assay are shown in Figure 6.32. Background subtraction routines are applied to obtain the best linear regression analyses and smallest y-intercept. The accuracy and precision of this assay are highlighted in Table 6.17, and representative HPLC chromatograms are shown in Figure 6.33. 

The center peak (28.986 minutes) is clearly a mixture of two compounds, as indicated by the shoulder in the total ion chromatogram, by the strong 85 and 91 ions in the mass spectrum, and the presence of two carbonyl bands in the IR. The individual compounds are most easily identified by spectrum subtraction in the IR, yielding resolved spectra of the overlapping components. The first is gamma-hexalactone and the second phenylacetaldehyde (Figure 7). 

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