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%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

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

between ethanol and n-hexane and, thus, the mixtures behave as true binary mixtures. The Association of Methanol with Water The apparent anomalous behavior of methanol-water and other water-solvent mixtures with respect to retention provoked Katz et al. (11) to investigate the association of methanol and water. This work was carried out to determine whether, in fact, such mixtures did not constitute binary mixtures but were actually ternary mixtures, the third component being the methanol-water associate. Their, arguments and subsequent experiments were as follows. An equilibrium between methanol and water can be expressed as follows,                                                                              (7) where (M) is the molar concentration of water, (W) is the molar concentration of methanol, (MW) isthemolar concentration ofmethanol/water associate, and (k) is the "

Figure 14. Graph of Refractive Index against Solvent Composition for Methanol/Water Mixtures Employing this average value for the equilibrium constant, the concentrations of the different components of a methanol water mixture were calculated for concentrations ranging from zero to 100%v/v. The curves they obtained are shown in figure 15. There are clearly three distinct ranges of methanol concentration where the solvent mixture will have very different interactive properties. At initial methanol concentrations between zero to 40%v/v, the solvent mixture will behave as though it were a binary mixture of water andmethanolassociatedwithwater. However, at methanol concentrations ranging from 40%v/v to 80%v/v, the solvent will behave as though it were a ternary mixture of water, methanol and water associated with methanol. Finally, at concentrations ranging from 80%v/v to 100%v/v of methanol the solvent will again behave as though it were again a binary mixture but this time a

strongly associating with the water and, in fact, an aqueous solution of methanol, for example, contained methanol, water and methanol associated with water. The solvent mixture was thus, a ternary system and thus the linear relation ship between the volume fraction (before mixing) and retention would not be expected to hold. The association of methanol and water was examined by Katz, Lochmüller and Scott (36) using volume change on mixing and refractive index data and established that the methanol/water solvent system was indeed a complex ternary system. They calculated both the association equilibrium constant and the distribution of the different components of a methanol water mixture form zero to 100% methanol. The curves they obtained are shown in figure 47. Figure 47. Diagram of the Ternary Solvent System for Methanol/Water Mixtures

by the more polar solvent mixtures. However, at temperatures below 25˚C, the converse applies, the least polar solvent mixtures produce the longest analysis times. It must be emphasized that although the shape of the graphs that are presented will be similar for different enantiomeric pairs, the actual values for column length and analysis times will differ widely between different chiral isomers. In addition, the results will be very different if solvents that associate (e.g. methanol/water mixtures or acetonitrile/water) are employed as the mobile phase. In order to develop the same algebraic procedure with such solvents, the association constant of the solvent pair must be known so that the ternary mixture of water–unassociated–with–methanol, methanol–unassociated–with–water and the water–methanol–associate can be calculated. The contribution of each of the three mobile phase components to solute retention must then be taken into account. It follows, that by employing