Plate Theory and Extensions - Retention Measurements on Close Eluting Peaks > Page 46
Errors can also arise if two partially resolved peaks are asymmetrical (e.g., the rear half of the peak is broader the front half). It is clear that under such circumstances there can be two sources of error, which are depicted in Figure 12.
Figure 12. The Effect of Peak Asymmetry on the Apparent Composition of Closely Eluting Solutes
Several problems arise from the situation depicted in figure 12. Firstly, the retention times, as measured from the peak envelope, will be inaccurate. Secondly, because the peaks are asymmetrical (and most LC peaks are asymmetric to the extent shown in the Figure 12), the second peak appears higher. This could be interpreted as the second solute is present at a higher concentration in the mixture than the first, which, as seen, is not so. It is, therefore, important to know the specific separation ratio above which accurate measurements can still be made on the peak maxima of the individual peaks. The apparent peak separation ratio, relative to the actual peak separation ratio from columns of different efficiency, are shown in Figure 13. The data have been obtained from theoretical equations. It is demonstrated that the separation ratio must be greater than about 1.055 for a low efficiency column (2500 theoretical plates) before accurate retention measurements can be made on the composite curve.