The elution order of solutes in HPLC is governed by polarity. In a normal-phase separation the least polar solute spends proportionally less time in the polar station- ary phase and is the first solute to elute from the column. Retention times are con- trolled by selecting the mobile phase, with a less polar mobile phase leading to longer retention times. If, for example, a separation is poor because the solutes are eluting too quickly, switching to a less polar mobile phase leads to longer retention times and more opportunity for an acceptable separation. When two solutes are ad- equately resolved, switching to a more polar mobile phase may provide an accept- able separation with a shorter analysis time. In a reverse-phase separation the order of elution is reversed, with the most polar solute being the first to elute. Increasing the polarity of the mobile phase leads to longer retention times, whereas shorter retention times require a mobile phase of lower polarity.
Several indices have been developed to assist in selecting a mobile phase, the most useful of which is the polarity index.12 Table 12.3 provides values for the polarity index, P’, of several commonly used mobile phases, in which larger values of P’ correspond to more polar solvents. Mobile phases of intermedi- ate polarity can be fashioned by mixing together two or more of the mobile phases in Table 12.3. For example, a binary mobile phase made by combining solvents A and B has a polarity index, PA’B, of
where PA’ and PB’ are the polarity indexes for solvents A and B, and A and B are the volume fractions of the two solvents.
A useful guide when using the polarity index is that a change in its value of 2 units corresponds to an approximate tenfold change in a solute’s capacity factor. Thus, if k’ is 22 for the reverse-phase separation of a solute when using a mobile phase of water (P’ = 10.2), then switching to a 60:40 water–methanol mobile phase (P’ = 8.2) will decrease k’ to approximately 2.2. Note that the capacity factor de- creases because we are switching from a more polar to a less polar mobile phase in a reverse-phase separation.
Changing the mobile phase’s polarity index, by changing the relative amounts of two solvents, provides a means of changing a solute’s capacity factor. Such changes, however, are not very selective; thus, two solutes that significantly overlap may continue to be poorly resolved even after making a significant change in the mobile phase’s polarity.
To effect a better separation between two solutes it is often necessary to im- prove the selectivity factor, α. Two approaches are commonly used to accomplish this improvement. When a solute is a weak acid or a weak base, adjusting the pH of the aqueous mobile phase can lead to significant changes in the solute’s retention time. This is shown in Figure 12.13a for the reverse-phase separation of p- aminobenzoic acid and p-hydroxybenzoic acid on a nonpolar C18 column. At more acidic pH levels, both weak acids are present as neutral molecules. Because they par- tition favorably into the stationary phase, the retention times for the solutes are fairly long. When the pH is made more basic, the solutes, which are now present as their conjugate weak base anions, are less soluble in the stationary phase and elute more quickly. Similar effects can be achieved by taking advantage of metal–ligand complexation and other equilibrium reactions.
A second approach to changing the selectivity factor for a pair of solutes is to change one or more of the mobile-phase solvents. In a reverse-phase separation, for example, this is accomplished by changing the solvent mixed with water. Besides methanol, other common solvents for adjusting retention times are acetonitrile and tetrahydrofuran (THF). A common strategy for finding the best mobile phase is to use the solvent triangle shown in Figure 12.27. The separation is first optimized using an aqueous mobile phase of acetonitrile to produce the best separation within the desired analysis time (methanol or THF also could be chosen first). Table 12.4 is used to estimate the composition of methanol/H2O and THF/H2O mobile phases that will produce similar analysis times. These mobile phases are then adjusted, if necessary, establishing the three points of the solvent triangle. Four additional mo- bile phases are prepared using the binary and ternary mobile phases indicated in Figure 12.27. From these seven mobile phases it is possible to estimate how a change in the mobile-phase composition might affect the separation
When a separation uses a single mobile phase of fixed composition it is called an isocratic elution. It is often difficult, however, to find a single mobile-phase composition that is suitable for all solutes. Recalling the general elution problem, a mobile phase that is suitable for early eluting solutes may lead to unacceptably long retention times for later eluting solutes.
Optimizing conditions for late eluting solutes, on the other hand, may provide an inadequate sepa- ration of early eluting solutes. Changing the composition of the mobile phase with time provides a solution to this problem. For a reverse-phase separation the initial mobile-phase composition is relatively polar. As the separation progresses, the mo- bile phase’s composition is made less polar. Such separations are called gradient elutions.
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