One reason is to control the pH of the solution so the compound(s) of interest interact with the column to the desired degree.
As a rule, the pH of the mobile phase buffer must be between the pI (isoelectric point) or pKa (acid dissociation constant) of the charged molecule and the pKa of the charged group on the solid support. For example, in cation exchange chromatography, using a functional group on the solid support with a pKa of 1.2, a sample molecule with a pI of 8.2 may be run in a mobile phase buffer of pH 6.0. In anion exchange chromatography a molecule with a pI of 6.8 may be run in a mobile phase buffer at pH 8.0 when the pKa of the solid support is 10.3.
Many chromatographers also use changes in pH to affect a separation. In cation exchange chromatography, raising the pH of the mobile phase buffer will cause the molecule to become less protonated and hence less positively charged.
In anion exchange chromatography, lowering the pH of the mobile phase buffer will cause the molecule to become more protonated and hence more positively (and less negatively) charged.
- Badges
- Science method