Two approaches are commonly used to accomplish this improvement. To effect a better separation between two solutes it is often necessary to im. 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. 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.Ĭhanging the mobile phase’s polarity index, by changing the relative amounts of two solvents, provides a means of changing a solute’s capacity factor. 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. Thus, if k’ is 22 for the reverse-phase separation of a solute when using a mobile Where P A ’ and P B ’ are the polarity indexes for solvents A and B, and A and B are theĪ 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. For example, a binary mobile phase made by combining solvents A and B has a polarity index, P A ’ B, of Mobile phases of intermedi- ate polarity can be fashioned by mixing together two or more of the mobile phases in Table 12.3. Larger values of P ’ correspond to more polar solvents. Values for the polarity index, P ’, of several commonly used mobile phases, in which Several indices have been developed to assist in selectingĪ mobile phase, the most useful of which is the polarity index. Increasing the polarity of the mobile phase leads to longer retention times, whereas shorter retention times require a mobile phase of lower polarity. In a reverse-phase separation the order of elution is reversed, with the most polar solute being the first to elute. When two solutes are ad-Įquately resolved, switching to a more polar mobile phase may provide an accept- able separation with a shorter analysis time. 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. Trolled by selecting the mobile phase, with a less polar mobile phase leading to longer retention times. 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. The elution order of solutes in HPLC is governed by polarity.
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