Figure 13. Curves Relating Apparent Separation Ratio Relative to Actual Separation Ratio for Two Closely Eluting Peaks
In contrast, the separation ratio need only be in excess of about 1.035 on the high efficiency columns (10,000 theoretical plates), before accurate retention measurements can be made on the composite curve. It follows that,:
Considerable care must be taken when accessing closely eluting peaks. If the resolution is inadequate, measurements must be taken on the individual solutes, chromatographed separately.
Quantitative Analysis from Retention Measurements
A consequence of the above discussion on composite peak envelopes is that if the retention times of a pair of solutes are accurately known, then the measured retention time of the composite peak will be related to the relative quantities of each solute present. It follows that an assay of the two components could be obtained from accurate retention measurements only. This method of analysis was shown to be feasible and practical by Scott and Reese (12).
Consider two solutes that are eluted so close together that a single composite peak is produced. Employing the Gaussian form of the elution equation, the concentration profile of such a peak can be described by the following equation: