Precision as an alternative to resolution

Providing retention times can be measured precisely they can be used to determine the composition of a mixture of two substances, that, although having finite differences in retention times, are eluted as a single peak on the column employed. This can be achieved, providing the Standard Deviation of the measured retention time is small compared with the difference in retention times of the two solutes. 

Consider two solutes eluted close together such that a single composite peak is produced. From the Plate Theory the concentration profile of such a peak can be described by the following equation - 

It is seen from equation (18) that when only (A) is present, the function will exhibit a maximum at t = tA, and if only solute (B) is present, it will exhibit a maximum at t = tB. It follows that the composite curve will give a range 

Thus if (D) is the voltage output from the detector, equation (18) can be put in the form - 

Equation (19) was examined by Scott and Reese (i5) employing mixtures of nitrobenzene and fully deuterated nitrobenzene as the test sample. Their retention times were 8.927 min and 9.06i min respectively giving a difference of 8.04 seconds. The separation ratio of the two solutes was 

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It follows that measurements must be made with a precision of about 0.2 second if quantitative results are to be of any value. It is seen from figure 4 that the experimental points lie very close to the line and a fairly accurate measurement of the distribution of the two isotopes can be obtained from retention time measurements. This method has very limited areas of application and is given here, more to demonstrate the effect of unresolved impurities on retention time, than to suggest it as an alternative to adequate chromatographic resolution. In some cases, however, particularly in the analysis of isotopes, it may be the only practical way to obtain a quantitative evaluation of the mixture by a liquid chromatographic method. 

The technique described here is finding increasingly widespread use for the resolution and molecular weight estimation of naturally occurring polypeptides. Its intended function is to supplement the classical exact techniques of molecular weight analysis and is not intended as an alternative procedure. Although we are aware of no instance in which invalid data have been obtained, the method can only be considered an approximate one since measurement involves calibration with secondary standards, and at present it is not suitable for precise theoretical analysis. 

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