Chromatography

There is an old joke about scientists. It was told to me many years ago by my algebra teacher in high school, Mr. Ephrimoff. It goes like this. A scientist wants to find out how jumping in frogs relates to the number of appendages they have. So he puts the frog down on the floor and yells "jump frog jump!". The frog jumps and he measures the distance and carefully records in his notebook "frog, 4 legs, 2 meters". He then cuts off one of the frog's front legs and puts the frog back down on the floor and yells "jump frog, jump!". Again he measures the distance and records "frog, 3 legs, 1 meter". He then cuts off the other front leg and repeats the experiment. This time the frog just sort of slides forward a few inches. He records "frog 2 legs, 10 centimeters". He then cuts off one of the back legs and again yells "jump frog, jump!". The frog makes a feeble attempt, rolling over in the process but progressing slightly. He writes "frog 1 leg, 2 centimeters". Finally he wacks off the last leg and yells "jump frog, jump!". Nothing happens. Again he yells "jump frog, jump!!". Still nothing. Louder still he yells, "jump frog, jump!!!". However, the frog does not move. He records in his notebook, "frog, no legs, definite lack of hearing".

However, biochemists in this day and age are considerably more sophisticated than the fellow being maligned above. We would simply put the frog in a blender and then isolate all the molecules and somehow try to understand jumping in frogs by figuring out how its molecules work. Isolating molecules however requires distinguishing between them and then finding a way to use their distinguishing characteristics to separate one from another. A number of such tricks have been devised over the years. We have talked already about centrifugation and electrophoresis, both of which use external fields to move one molecule at a different rate than another. There is another class of techniques that uses differential chemical interactions of one type or another to separate molecules. This is called chromatography.

The fundamental concept. You have all taken organic lab at some point and used a separatory funnel to separate liquids in two phases. You typically do this because the molecule you are interested in preferentially is soluble in one phase over the other. This is the basis of most chromatographic techniques. There are two phases, one moves (the mobile phase) one does not (the stationary phase) and the idea is to set things up so that the different molecules you are trying to separate spend different periods of time on average in the mobile phase. The more time the molecule spends in the mobile phase, the faster it moves. If you understand this idea, you understand chromatography. The rest is just a matter of coming up with mobile and stationary phases that work well for the molecules you like to play with. These days, there are lots of options.

When a biochemist thinks chromatography, he or she usually thinks of columns. A column is a long tube filled with some material (which either is or contains the stationary phase) and some liquid around the material (which is the mobile phase). Liquid flows through the column coming in contact with the material and the solute molecules in the liquid have some kind of interaction with the material. If they interact strongly, they will spend a lot of time associated with the material and not move down the column very fast. If the interaction is weak with the material, then the molecules will not interact strongly and the molecules will spend more time in the mobile phase.

Polarity. We could obviously try to separate molecules based on their polarity and tendency to be in hydrophobic vs. hydrophilic environments. Silica or Alumina columns work on that principle. Depending on the solvent, the solute molecules will be either more likely to stick to the Silica or Alumina or less likely.

Charge. Ion exchange columns (like you will use in lab) have charges attached to a bead of some kind normally. Thus, charged molecules are either attracted to or repulsed from the charges on the bead and thus spend more or less time there.

Size. Gel sieving columns (G-25, G-100…) are a bit different than the two above. It is not a particular chemical interaction that separates molecules. Instead, these are made of beads with holes of a particular size range in them. Small molecules can move in and out of the beads. The liquid in the beads is not moving and represents the stationary phase. The liquid outside the bead is moving and represents the mobile phase. Since small molecules go back and forth between the two phases, they are retarded. Large molecules that do not enter the beads easily, spend more time in the mobile phase and thus move faster.

Specific Interactions. It is also possible to make columns involving very specific interactions such as antibody/antigen interactions or binding between two specific proteins that naturally form a complex together. You tie one side of the interaction to the bead and then run the material of interest over the column. In principle, the only thing that sticks will be the specific molecule that has the binding site. This is called affinity chromatography. A recent and general version of this involves modifying the protein of interest at the genetic level such that it has a string of His residues (usually at least six and usually at one end or the other). It turns out that a chain of His residues in a protein will quite specifically bind to nickel. Thus, after genetically engineering the protein of interest to have the "his tag", this new expressed molecule can be pulled out of the mix using a column with exposed nickel groups.

Tricks of the trade. For chromatography to work well there are several things that need be considered. First, the molecules need to be moving forward faster than they are diffusing. On the other hand, the molecules need to have reached equilibrium between the stationary and mobile phases. This is done most completely when the column runs more slowly. The optimum speed of running a column often depends on the type of sample one is trying to purify. It is always critical to keep the column as homogeneous in structure as possible, so that no channeling occurs -- no regions of the column that run faster than others. This is most easily done by using the smallest (finest) column material size possible. However, the smaller the bead size, the slower generally is the flow rate. Finally, it is the kiss of death typically if air gets in the column. This happens when one absent mindedly lets the column run out of liquid.