Variation of the eluent composition allows the chromatographer to adjust the retention times of target components in the mixture to the desired values. Usual recommendation for this adjustment is to keep the capacity factors between 1 and 10.
Typical dependencies of the retention on the eluent composition are shown in the picture below.
Retention dependencies of alkylphenones (left) and alkylbenzenes (right) vs. the eluent composition. Column: Hypersil-C8 (5µ, 150x4.6 mm), 45 °C, eluent: acetonitrile/water.
As one can see, retention dependencies on the eluent composition have an exponential shape. Reasonable separation could be conducted in the certain concentrational region of the eluent composition. If we choose k' margins between 1 and 20 as an appropriate ones, then, based on the above graphs, acetonitrile content for separation of alkylphenones will be from 40 to 70 %v/v. Same for alkylbenzenes will be 50 to 80 %v/v of acetonitrile. So, if we have to separate a mixture of all these components the best starting eluent concentration will be 60 % of acetonitrile in water.
As we mentioned, the above graphs have an exponential shape and it may be more convenient to consider a logarithmic representation of these dependencies.
Graph of the logarithm of the capacity factors on the eluent composition will reflect the changes in the excess interaction energy of the analyte molecules with the adsorbent surface.
Logarithmic dependencies of the retention vs. the eluent composition. Left: alkylphenones homologous series, right: alkylbenzenes (red - one carbon atom in alkyl chain, blue - 2, green - 3, magenta - 4)
These dependencies is not so linear as it was for homologous series retention. This effect may be explained by the excess adsorption of the eluent components. The presence of some excess amount of the acetonitrile close to the adsorbent surface will decrease the analyte interaction and also retention. This will result in a concave shape of the graph.
In general, by changing the eluent composition the chromatographer can change a component retention in very broad margins: from elution with the dead volume up to the retention in hours.
At the same time, by changing mobile phase composition we will not change the selectivity of the separation in the same order as we will change retention. Table below shows the numerical data for two points of above graph.
|Compound||Parameter||90/10 MeCN/Water||30/70 MeCN/Water|
Decreasing the acetonitrile content from 90 to 30 volumetric % changes etylbenzene retention 22 times (from 1.7 ml to 37.5). At the same time selectivity of the separation of etylbenzene and toluene changes only 1.6 times, and resolution changes 7 times.
This means that if at certain eluent composition two components of interest did not
show noticeable good selectivity then changing an eluent composition will not bring you
significant benefits without suffering an enormous retention time.