Liquid Chromatography - Aqueous Solvent Mixtures 2
It is seen from figure 47 that there are three distinct ranges of methanol concentration where the solvent will behave very differently. From zero to 40%v/v of methanol in the original mixture, the solvent will largely behave as though it were a binary mixture of water and methanol associated with water. From 40%v/v to 80%v/v of methanol in the original mixture, the solvent will predominantly behave as though it were a ternary mixture of water, methanol and water associated with methanol. From 80%v/v to 100%v/v of methanol in the original mixture, the solvent will again behave as though it were again a binary mixture but this time a mixture of methanol and water associated with methanol.
The curves shown in figure 47 explain some of the unique characteristics of mobile phases consisting of methanol water mixtures when used in reversed phase LC. From figure 47 it is seen that when the original mixture contains 50%v/v of methanol there is little free methanol available in the mobile phase to elute the solutes as it is mostly associated with water. Subsequently, however, the amount of methanol unassociated with water increases rapidly in the solvent mixture and this rapid increase must be accommodated by the use of a convex gradient profile when employing gradient elution. The convex gradient will compensate for the strongly concave form of the unassociated methanol concentration profile shown in figure 47 which will be the strongest eluting component of the mobile phase. The strong association of methanol with water could also account for the fact that proteins can tolerate a significant amount of methanol in the mobile phase before they become denatured. It is clear that this is because there is virtually no unassociated methanol present in the mixture which could cause protein denaturation since all the methanol is in a deactivated state by association with water.
Katz, Lochmüller and Scott also examined acetonitrile/water, and tetrahydrofuran(THF)/water mixtures in the same way and showed that there was significant association between the water and both solvents but not to the same extent as methanol/water. At the point of maximum association for methanol, the solvent mixture contained nearly sixty percent of the methanol/water associate. In contrast the maximum amount of THF associate that was formed amounted to only about 17% and for acetonitrile the maximum amount of associate that was formed was 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.