Structural Biochemistry/Proteins/Purification/Ion-Exchange chromatography
Purpose: To separate a specific protein from its mixture by using the property of ion-charges.
General information[edit | edit source]
Ion Exchange Chromatography (IEC) is a purification method aimed at separating proteins based on charge, which is dependent on the composition of the mobile phase (a separation of mixtures that is dissolved). Adjusting the pH, or the ionic concentration, of the "mobile phase" allows for separation. For example, if a protein has a net positive charge of pH 7, it will bind to a column of negative charge beads. On the other hand a negatively charged protein would not. For example, if a proton has a net positive charge at pH of 7 then it will bind to a column of beads that contain the carboxyl groups, where as a negatively charged proteins will not. Once bound, the protein is eluted by increasing the ion concentration. The movement of a protein depends on the density of the net charge; the proteins that have a low density of net positive charge will emerge first. Proteins bind to ion exchangers due to the electrostatic forces between the surface of the protein charges and cluster of the charged group on the exchangers. A column is packed with a resin (usually cellulose or agarose) with a charged group bonded to it. This allows positively charged proteins, for example, to bind to the negatively charged beads on the column and the negatively charged proteins to flow through the column. Therefore ion exchange chromatography consists of cation exchange chromatography and anion exchange chromatography. In addition, a protein must displace the counterions and become attached; in other words, the net charge on the protein will be the same sign as that of the counterions displaced-therefore "ion exchange. The protein molecules in solution are neutralized by counterions also; the overall reaction must be electrically neutral. 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.
Anion-Exchange Chromatography[edit | edit source]
Anion-Exchange chromatography involves the use of positively charged beads. In the purification of acids, which often has the negative charge on its carboxyl group, anion-exchange chromatography is utilized. Anion-exchange chromatography mainly recollects biomolecules by the interaction of amine groups on the ion-exchange resin with aspartic or glutamic acid sidechains, which have pK of ~ 4.4. The mobile phase is buffered at pH > 4.4, below which acid sidechains start to protonate and retention declines.
Above pH 4.4, retention is fundamentally reliant on on the number of anionic sidechains existing in the protein. Proteins including the same number of anionic sidechains can often be separated by modification of the mobile phase pH between 7 and 10 where histidine is not protonated and lysine starts to deprotonate.
Delicate changes occur to proteins in this pH region which affect the interaction of the protein with the resin and which allow fine-tuning of the anion-exchange separation. A mobile phase, pH > 10, is not usually suggested because of possible protein deprivation, such as deamination, at higher pH's.
Techniques[edit | edit source]
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. Recall that a net 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 thus will come out of the column pure. Knowing the isoelectric point (pI) of the protein sample can be helpful in ion-exchange chromatography. Recall that pI is the pH at which a compound's net charge is zero. So if we have a compound with a high pI, for example 10, then to get the pH gown to 7 would cause the compound to become positive. Conversely, if the pH of the solution is higher than the pI, the protein becomes negative overall, thus more anion formation. Thus, depending on the pI of the protein, different solvents at specific pH's can be targeted to purify protein. This also implies that proteins with two significantly different pI's are the most successful in ion-exchange.
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.