Reverse Phase Chromatography

   In reverse phase chromatography, the polarities of the mobile and stationary phases are opposite to what they were when performing normal phase chromatography. Instead of choosing a non-polar mobile phase solvent, a polar solvent wil be chosen. Or, if the experiment requires a solvent polarity gradient, the gradient must be carried out with the most polar solvent first and the least polar solvent last (reverse order of normal phase chromatography). Common polar solvents mixtures of solvents include water, methanol, and acetonitrile. It is slightly more difficult and expensive to obtain a column where the stationary phase is non polar, as all solid adsorbents are polar by nature. The non polar stationary phase can be prepared by coating silanized silica gel with a non polar liquid. Silanizing the silica gel reduces the silica gel's ability to adsorb polar molecules. Common non polar liquid phases include silicone and various hydrocarbons. An alternative to this type of column is used in HPLC, in which a bonded liquid phase is used as the stationary phase. The less polar liquid is chemically bonded to the polar silica gel in the column. So using reverse phase, the most polar compounds in the sample solution will be eluted first, with the components following having decreasing polarities.