Separate and Purify

Step 4: Separate and Purify

A good method of separating substances in a mixture is called chromatography. Here are several different methods of chromatography. Which one would you pick to separate proteins?

Column Chromatography - Method 1
This type of chromatography is called "column," because you use a long glass tube to do the separations. The idea here is that if you pack a long glass tube, open at both ends, with powdered packing material of the right kind, it can adsorb the salt and let the water and proteins pass through. Then you could separate proteins of different sizes by pouring them through columns in which the packing material would only let certain-size molecules get through.

So how do you get separation of different proteins?

The idea is that certain packing materials will adsorb some substances better than others. So, for example, when you pass through the packing a water solution (loaded at the top in this picture), some molecules will stick to the packing material as it flows through the tube and out the bottom. Then, a follow-up step involves flushing the packing with an appropriate liquid that can dissolve the adsorbed chemicals and carry them out with the liquid (drained at the bottom of this picture).
This process is called "elution". Proteins come in different sizes and different sized proteins come off the column at different rates, thus achieving separation of proteins. Thus, if you collect the wash liquid for 10 seconds, for example, you may get one kind of protein, a collection of wash liquid for the next 10 seconds would get another protein, and so on.