Influence of the structural fragments of the analyte molecules on the retention in reversed-phase HPLC could more clearly seen in the example of the homologous series retention. The picture below demonstrates the dependencies of retention of three different homologous series on the number of carbon atoms in their alkyl chain.
Dependencies of retention of alkylbenzenes (1), alkylphenones (2), and alkylparabenes (3) plotted against the number of carbon atoms in alkyl chain. Column: Hypersil-C18 (5 Ám, 150x4.6 mm); eluent: MeCN/Water 60/40, 1 ml/min
Based on this picture we can see that alkylbenzenes expose much stronger retention than phenones. Presence of carbonyl group in phenones molecules makes it more polar and thus reduces the strength of dispersive interactions with hydrophobic surface.
Retention of alkylparabenes is the smallest in all three series. Parabenes is an esters of p-hydroxybenzoic acid. Nuclei of this homologous series are very polar and do not show any significant interaction with the surface.
More information about the energy of the surface interactions can give us a plot of the logarithm of capacity factor against the number of carbon atoms.
Dependencies of ln(k') on the number of carbon atoms
In the picture above all three homologous series show linear dependencies which are parallel to each other. This means that each -CH2- group introduces the same energetic increment in the interaction of the analyte molecule with the surface. This increment is independent of the position of -CH2- group in the chain and it also independent of the type of the nucleus of homologous series.
This means, that if we know the retention of at least two members of homologous series then we can calculate the retention of all others.
The difference between the lines represents the influence of the nucleus on the molecular interactions with the adsorbent surface.
We have to emphasize that to be able to estimate the values of the interaction energy of -CH2- groups with the surface we have to measure the temperature dependencies of the retention.
Because of the competitive nature of HPLC retention, calculated values of interaction
energy will not represent an absolute value but the difference of the analyte and eluent