Tham khảo tài liệu 'hplc a praactical user's guide part 2', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | OTHER WAYS TO MAKE MY SEPARATION 9 The key to changing the separation is to change the difference in polarity between the column packing and the mobile phase. Making the solvent polarity more like the column polarity lets compounds elute more rapidly. Increasing the difference in polarities between column and mobile phase makes compounds stick tighter and come off later. The effects are more dramatic with compounds that have polarities similar to the column. On a nonpolar column running in acetonitrile we could switch to a more polar mobile phase such as methanol to make compounds retain longer and have more time to separate. We can achieve much the same effect by adding a known percentage of water which is very polar to our starting acetonitrile mobile phase step gradient . We could also start with a mobile phase containing a large percentage of water to make nonpolar compounds stick tightly to the top of the column and then gradually increase the amount of acetonitrile to wash them off solvent gradient . By changing either the initial amount of acetonitrile the final amount of acetonitrile or the rate of change of acetonitrile addition we can modify the separation achieved. Separation of very complex mixtures can be carried out using solvent gradients. There are however penalties to be paid in using gradients. More costly equipment is required solvent changes need to be done slowly enough to be reproducible and the column must be re-equilibrated before making the next injection. Isocratic separations made with constant solvent compositions can generally be run in 5-15 min. True analytical gradients require run times of around 1 hr with about a 15-min re-equilibration. But some separations can only be made with a gradient. We will discuss gradient development in a later section. Ranges of Compounds Almost any compound that can be retained by a column can be separated by a column. HPLC separations have been achieved based on differences in polarity size shape
