The Mechanism of Chromatographic Retention - The Association of Methanol with Water > Page 39

At higher methanol concentrations the amount of unassociated methanol increases rapidly thus increasing the eluting strength of the mobile phase. When using gradient elution, this rapid increase in eluting strength must be compensated by employing a convex gradient profile. The strong association of methanol with water also accounts for the fact that proteins can tolerate a significant amount of methanol in the mobile phase without them becoming denatured. This surprising tolerance to methanol is because there is very little unassociated methanol present in the mixture to cause protein denaturation, since all the methanol is associated with water and, thus, in a deactivated state.

Katz, Lochmüller and Scott (11) also showed that there was significant association between the water and acetonitrile and water and tetrahydrofuran, but not nearly to the same extent as methanol and water. At the point of maximum association in methanol-water mixtures, the solvent contained nearly 60% of the methanol/water associate. In contrast the maximum amount of THF associate that was formed was only 17%, and that for acetonitrile as little as 8%. It follows that acetonitrile/water mixtures would be expected to behave more nearly as binary mixtures than methanol/water or THF/water mixtures. The components of the mobile phase which can interact with the solute and thus control retention in a methanol-water mixture will be methanol unassociated with water, water unassociated with methanol and the methanol water associate. However, for dispersive and moderately polar materials it will be the unassociated methanol that will predominantly control solute retention. This was also demonstrated by Katz et al. (11)

Katz et al. also plotted the distribution coefficient of n-pentanol, benzonitrile and vinyl acetate against the concentration of unassociated methanol in the solvent mixture and the results are shown in Figure 16.

Figure 32. Graph of Distribution Coefficient of n-Pentanol, Benzonitrile and Vinyl Acetate against Concentration of Unassociated Methanol