Here we discuss a basic thermodynamic description of the effect of the type and composition of the mobile phase.
In most of the HPLC separations binary eluents are employed. One of the solvents in the eluent is usually inert relative to the surface interactions.
In Reversed-phase HPLC (RP HPLC) one of the eluent components is water, which does not interact with the hydrophobic adsorbent surface. And it does not compete with the analyte for the adsorption sutes.
In normal-phase HPLC (NP HPLC) one of the eluent components is usually hexane, which also does not interact with the very polar silica surface.
Another component of any binary eluent is an active one. It usually called a "modifier" because it can interact with the adsorbent surface and compete with analyte molecules for the adsorption sites. Increasing of the concentration of the "modifier" in the eluent leads to the decreasing of the analytes retention.
As we discussed before, capacity factor is proportional to the thermodynamic equilibrium constant, and the last is an exponent of the free Gibbs energy of the system. In the formula (23) we have a difference of the free energy of analyte and eluent interactions with the surface.
For the binary eluent system we can assume that only the "modifier" can interact with the surface. For RP HPLC it will be an organic component of the eluent, and water is assumed not to interact with the surface.
As a rough approximation we can assume that the total free Gibbs energy of that adsorption system could be considered as a difference of analyte adsorption energy and a product of modifier adsorption energy and its mole fraction. Thus, component retention volume could be roughly estimated by using the equation:
where Vo is the dead volume of the column, an. is the analyte adsorption energy, x - is the mole fraction of the organic modifier in the eluent, and el. is the adsorption energy of the eluent.
Figure below is shows the experimentally measured dependencies of the retention of alkylbenzenes in reversed phase HPLC mode on C18 column with acetonitrile/water eluent at different compositions. Points are experimental values and a curves was calculated using the above equation. As we can see this simple approximation allows us to describe experimental dependencies pretty well.
Retention dependencies of alkylbenzenes vs. the eluent composition. Eluent: acetonitrile/water, column: Prodigy-C18 (150x4.6 mm)
This type of the influence of the mobile phase composition and its thermodynamic explanation are true for the chromatographic system with only hydrophobic interactions (dispersive forces). In case of the presence of any specific adsorption sites, the analyte behavior may significantly differ from that described above.
Any specific interactions of the analyte molecules with the eluent molecules also may introduce significant deviations to the analyte retention dependence. Ionizible components usually show a specific behavior.
Organic acids are easily solvated with the water molecules, which block possible interaction of the hydrophobic part of the molecule with the adsorbent surface, and lead to the very early elution of these compounds. For example, benzoic acid is eluted before the dead volume at any composition of acetonitrile/water eluent on the reversed-phase column.
Organic basis usually shows a low retention also due to solvation, but in case of presense of strong acidic accessible adsorption sites on the adsorbent they show a strong retention.
A general approach to the separation of the mixtures containing an ionisible components is to suppress their ionization. Suppression of the ionization decreases a power of the molecular solvation and exposes the hydrophobic (organic) part of the molecule to the surface interaction. Ionization suppression is usually made by the adding a buffer into the solvent, which shift a pH to the certain value.
In the absence of buffer, easy ionizible components are eluted from the column as very broad peaks. According to the Le Chatelier principle, dissolved ionizible component is present in the solution as a mixture of ions and nonionized molecules
[AB] == [A+] + [B-]
According to the above equilibrium, about 50% of all molecules are ionized in the solution . But, the chromatographic behavior of ions and neutral molecules are different. Let us assume that neutral molecules will be retained, so during the run ions will move faster, and at the first moment they will be separated from the neutral molecules. But, according to the above equilibrium, in the absence of the neutral molecules ions will tend to form them, and this new neutral molecule will also be absorbed, and so on. This process will lead to the spreading of the component along the column and causes the appearance of the broad peak.
It does not occurif the equilibrium is shifted due to the presence of the buffer
with the pH at least two units apart of pK of the component.