Mobile phase is the most important parameter in reversed-phase HPLC. Type of mobile phase used may have a big effect on the retention. It can promote or suppress an ionization of the analyte molecules, and it also can shield an accessible residual silanol or any other active adsorption centers on the adsorbent surface.
Proper selection of the mobile phase is the second most important step in the development of the separation method (the first one is the selection of the adsorbent type). The main requirement for the mobile phase is that it has to dissolve the analytes up to the concentration suitable for the detection.
Variation of the eluent composition provides the great flexibility of HPLC separations.
The mobile phase in reversed-phase chromatography has to be polar and it also has to provide a reasonable competition for the adsorption sites for the analyte molecules.
Table below shows the most important parameters of some common solvents often used as eluent components in RP HPLC.
| Name | MW | BP | R.I. | UV | Viscos. | µ |
| [°C] | [nm] | [cP] | [Debye] | |||
| Acetonitrile | 41 | 82 | 1.341 | 195 | .358 | 3.37 |
| Dioxane | 88 | 101 | 1.421 | 215 | 1.26 | 0.45 |
| Ethanol | 46 | 78 | 1.359 | 205 | 1.19 | 1.68 |
| Methanol | 32 | 65 | 1.326 | 205 | .584 | 1.66 |
| Isopropanol | 60 | 82 | 1.375 | 205 | 2.39 | 1.68 |
| Tetrahydrofuran | 72 | 66 | 1.404 | 215 | 2.20 | 1.70 |
| Water | 18 | 100 | 1.333 | 185 | 1.00 | 1.84 |
Note, that water has lower dipole moment (last column) than acetonitrile. But water
does not interact with the hydrophobic surface of the adsorbent in the column and
acetonitrile does. This illustrates that polarity itself is not the major factor affecting
the separation in RP HPLC.