Structural Biochemistry/Proteins/Purification/Ion-Exchanged chromatography
Ion Exchange Chromatography (IEC) is a purification method aimed at separating proteins based on charge. A column is packed with a resin (usually cellulose or agarose) with a charged group bonded to it. For a positively charged resin, a protonated diethylaminoethyl (DEAE) group is used. For negatively charged resin, a deprotonated carboxymethyl (CM) group is used. Ion exchange chromatography consists of cation exchange chromatography and anion exchange chromatography. Whatever one wants to purify is known as the sample and the parts that are separated are known as the analytes. The sample is added to the top of the column and a buffered solution is used to elute it.
In cation exchange chromatography, a sample consisting of a certain protein that bears a net positive charge at a certain pH is a added to a column. In anion exchange chromatography, a sample with a protein that bears a net negative charge at a certain pH is added to a column. (Anet charge is the sum of partial charges for each amino acid's particular R group at a given pH). The columns have resin that consists of cellulose (or agarose) beads, which have a function group covalently bonded to it. For cation exchange a carboxylate group is used, and for anion exchange a diethylaminoethyl group is used. A buffer solution, also called a mobile phase, has its pH set between the pl or pKa of protein and the pKa of the beads on the columns. The buffer solution then runs the sample through the column. Molecules with no charge or the same charge as the beads will pass through, while molecules with the opposite charge will bind to the column of beads. Like a magnet, it'll stick and stay there. To elute the bound proteins, the column is flushed with a salt, usually excess NaCl. In cation exchange chromatography the Na+ ion will compete with the bound protein for the negative functional group, and in anion exchange chromatography, the Cl- ion will compete to bind the columns. Another way to flush the system would be with a low pH buffer. The more acidic conditions will lower the net charge (or make it more positive) of the protein. Since the protein now bears a positive net charge, it no longer feels compelled to be around the like-charged resin (since like charges repel), and it'll come out of the column pure.
If there are impurities in the sample that have a similar charge of the protein being isolated, a pH gradient buffer solution is needed. Unless the proteins have exactly the same amino acids, it is unlikely that they will have exactly the same charge at the same exact pH. Raising (or lowering) the pH, which is in effect causing more molecules to be deprotonated (or protonated), will cause the molecule to have a slight change in charge negatively (or positively). This will affect the ionic interaction between the molecule and the resin, causing some of the molecules to elute from the column. By changing the pH, different molecules will have different charge densities (or degree of negative charge; -2,-1,-3, etc.). So at a certain pH, a protein might have a higher or lower charge density and will thus bind to the resin differently, and those with a lower charge density will elute first.
For another example, say we are analyzing an air sample that has been collected onto an air filter and put through filter extraction (adding water to the filter, purifying by putting through another filter, and extracting the water to be the sample). The samples are then further prepared to put into the IC (ion chromatograph) by adding a given amount of the sample and a given amount of a water. A series of standard solutions and water are first put through the IC in order to calibrate the instrument. The standard solutions consist of certain cation or anion, depending on which ion chromatography is being performed, that are to be detected in the samples. Once all the samples have been put through the IC an ion chromatrogram (see image)is created for each standard and sample solution. In the ion chromatogram the analyte separation can been seen. Each analyte travels through the column at a different rate due to the positively or negatively charged resin. In the ion chromatogram the time at which it takes each analyte to pass through as well as the amount present can be seen. Each analyte will travel through the column at a consistent time in each sample thus each peak can be determined to be certain analytes.