Mechanism of Separation in Open Column Chromatography

Mobile phase carries the test sample through the stationary phase. Solvent and solute molecules within the sample compete for the active sites on the solid adsorbent. Active sites are polar sites. Since the molecules are chosen by the stationary phase from the degree of their polarity, more polar molecules will be able to adhere on to the solid adsorbent than the less polar ones. Strongly adsorbed molecules take more time to elute out. Weakly adsorbed molecules will be eluted out with the solvent earlier. Separation is based on the polarity of the test sample that is carried by the mobile phase.

If the test sample is a complex mixture then a sequential elution should be carried out. In sequential elution separation process starts from a less polar solvent and then the polarity of the solvent is gradually increased. So that elution of the molecules will take place in a polarity range which means least polar will come very first and most polar will be the last. In here several mobile phases are used considering their polarity differences. Also in sequential elution the user can control the rate of flow using the stopcock by taking compounds regularly at the bottom end.

Sequential elution