Peak area by triangulation

Now that we have our system set up and the column equilibrated and standardized, we are ready to carry out an HPLC separation on a real sample. We might add an internal standard (if necessary, to correct for injection variations), dilute our sample to a usable concentration, and prepare it for injection. After injection, we will record the chromatogram making sure that it stays on scale. Then, from the trace we obtain, we will calculate elution volumes either by measuring peak heights or by calculating peak areas by triangulation. 

A sample of the deuteriated polymer (0.531 g) was ozonized as a suspension in CH2CI2, and the product treated with formic acid-hydrogen peroxide as described previously in this section. Cyclization to d2-succinic anhydrides and derivatization to form the d2 N-(o-biphenyl) succinimides were also performed by previously described methods. %-NMR (acetone-d6, 200 MHz, ) resonances at 6 2.62 (s), 2.62 (d, J 4.4 Hz), 2.84 (s), 2.84 (d, J 4.4Hz), 7.48 (m). Measurement of peak areas by triangulation indicated that the d2-N-(o-biphenyl) succinimide produced in the above experiment was 55% meso-isomer and 45% d>l-isomer. 

Measurement of methylene peak areas by triangulation indicated that the d2-N-(o-biphenyl) succinimide produced in the above experiment was 48% meso-isomer and 52% d,1-isomer. 

Figure 8.4 Calculation of peak areas by triangulation and direct peak measurement.

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