The Role of Selectivity in Practical Method Optimization

A poor separation method is mostly caused by poor peak resolution or an uneven distribution of the peaks across the chromatogram. The latter issue is always caused by the selectivity, with the cause of the former also very often by this however, there are also other parameters that can affect poor peak resolution. To obtain clarity on this and to express the characteristics of a method quantitatively, the user should first calculate the parameters plate number, N, selectivity, a, of selected peak pairs and retention factor, k, in particular for the first and the last peaks in the chromatogram. The related formulas for this calculation have been introduced previously in Section 2.2.1. Special emphasis should always be given to peak pairs, where the resolution does not meet the discussed requirements. 

We now study an example, where the resolution of a peak pair in the original method of a reversed phase separation is only =0.76. The parameter analysis yields the results shown in Table 2.1. A calculated plate number ofN = 9000 meets the expectations for a 150 mm long column packed with 5 pm particles well, and the second peak exhibits a very reasonable retention factor of kj = 2.0. The poor separation factor a = 1.05 between the two peaks is the obvious reason for the imperfect resolution and hence the method lacks selectivity to separate these two compounds. One could now reduce the organic content of the mobile phase by 10% and expect an increase in retention of factor 2. This would increase the analysis time by 67% (it is proportional to 1 + k), but the resolution would only increase to R = 0.98 and still be insufficient. Further extrapolation shows that no increase of retention without a change in plate number or selectivity could bring the resolution in the above-mentioned example R = 1.1. 

Alternatively, the plate number could be increased to Af = 15 000 by changing to a column length of L = 250 mm packed with the identical stationary phase and keeping the eluting conditions constant. Coupled with the effort and investment of organizing such a column, this would also increase the analysis time (which is proportional to the column length L) by 67%, but once again lead to insufficient resolution of R = 0.97. Could we, however, increase the selectivity from 1.05 to 1.10 and keep the retention of the second peak in the order of k2 = 2, the 

It cannot be stressed enough that selectivity is the key in liquid chromatography, but there is no simple rule available how to improve it. The following section gives us some insights into selectivity control and should help the user to address this challenge more systematically. 

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