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The mobile phase is buffered appropriately to complement the dissociation constants of the solutes. A mixture of methanol and acetonitrile is employed, the acetonitrile being used to increase the dispersive interactions in the mobile phase. The reason for the particular solvent mixture is not clear and it would appear that the separation might be achieved equally well by using a stronger solution of methanol alone or a more dilute solution acetonitrile alone. There is no particular advantage to one solvent mixture over another except for the fact that 'waste' acetonitrile produces greater solvent disposal problems than

nbsp; As the acetonitrile concentration increased, however, the concentration of adsorbed tetrabutyl ammonium salt would also be reduced and it would be eluted from the reverse phase with a resulting reduction in the ionic interactions of the solutes with the stationary phase. At higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely removed and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is

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

. The selectivity was achieved using a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent in the gradient program was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. The tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. As the acetonitrile concentration became higher, however, the tetrabutyl ammonium salt would be desorbed from the reverse phase reducing the ionic interactions of the solutes with the stationary phase. At even higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely desorbed and the interactions of the solutes with the

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.

Optimizing excitation and emission light wavelengths to obtain maximum sensitivity for a complex mixture can become quite involved as shown by the  separation of some priority pollutants depicted in figure 40. The separation was carried out on a column 25 cm long, 4.6 mm in diameter and packed with a C18 reversed phase. The mobile phase was programmed from a 93% acetonitrile, 7% water to 99% acetonitrile, 1% water over a period of 30 minutes. The gradient was linear and the flow rate was 1.3 ml/min. Courtesy of the Perkin Elmer Corporation   1 Naphthalene   9 Chrysene 2 Acenaphthene 10 Benzo(b)fluoranthene 3 Fluorene 11 Benzo(k)fluoranthene 4 Phenanthrene 12 Benzo(a)pyrene 5 Anthracene 13 Dibenz (a,h)anthracene 6 Fluoranthene 14