Running the Chromatogram

The stopcock of the glass column is packed with the glass wool. The glass column is then filled with the adsorbent in dry or in wet condition. Then a thin layer of glass beads are placed on top of the packing material. On to the top of the glass beads the solvent is added so that the solvent or the mobile phase can percolate through the glass beads. Before the addition of the test sample the packing material should be saturated with the solvent (mobile phase). Solvent is filled into the column while the stopcock is open. This is to verify whether the packing material (stationary phase) is saturated with the solvent (mobile phase). When it is fully saturated the excess solvent is passed through the stopcock which is open. After knowing it’s is fully saturated the stopcock is closed. Then the sample is added onto the top as a solution. Draw the solution from the bottom while adding from the top continuously. The packing material should be saturated throughout the process thus it is essential to maintain the level of the solvent (mobile phase) always above the level of the adsorbent. Throughout the development of the chromatogram it is important to add the mobile phase at a steady rate. Collect the eluted fractions at regular intervals or in fixed volumes.