Prepare your reverse phase column by equilibrating it with 10 column volumes of wash solution. This is typically known as buffer or solvent A. Buffer A is a water-based solvent with 0.1 percent of acid. The same buffer A is used, together with the organic buffer B, to elute your sample. Formic acid, trifluoroacetic acid or acetic acid are commonly used to make buffer A. Make 0.1 percent solution by adding 1 milliliter of acid into 1 liter of water.
Apply your first sample into the column. Prepare your sample by diluting it into buffer A.
Wash the column with 5 column volumes of 100 percent buffer A. This helps wash off any extra molecules in your sample that do not bind to the column, leaving only your bind sample molecule behind.
Elute your sample by gradually increasing the hydrophobicity of the elution solution. This is accomplished by gradually mixing buffer A and buffer B together. Start with 99 percent buffer A and 1 percent buffer B and gradually increase the amount of buffer B and decrease the amount of buffer A. At the end of the gradient, the elution solution is 0 percent buffer A and 100 percent buffer B.
Collect all the solvent that exits from the column after you begin the elution. This is usually done collecting few milliliter fractions into small tubes. This ensures you will not lose your sample if you do not know at what time point buffer B concentration elutes from the column matrix. Most chromatographic machines are equipped with a spectrometer that analyses each fraction for protein. Discard all fractions that contain no protein and save only the fractions that are positive for protein.
Wash your column with 10 volumes of 100 percent buffer A after you have run the entire elution gradient. After this, your column is ready for the second sample or to be regenerated and prepared for storage.