Ion exchange chromatography
Ion exchange chromatography is a technique used to separate molecules according to their charge, for example, it can be used to purify charged molecules such as proteins, amino acids and nucleotides.
Ion exchange chromatography is based on the attraction that positively or negatively charged ions and molecules have for anything with an opposite charge.
Preparing the sample
Proteins, amino acids, and nucleotides each carry a charge that is dependent on the pH of the surrounding solution. Therefore, the sample must first be put into a solution that buffers the mixture at a particular pH.
Ion exchange chromatography apparatus
The gel matrix is positively or negatively charged, attracting the ions of interest in the solution that is being separated.
Preparing the ion exchange column
An ion exchange column is composed of a gel matrix made from beads with charged functional groups. The functional group that is used will depend on the molecule being targeted for separation. For example, if the molecules to be extracted from the sample have a positive charge, the functional groups in the column will have a negative charge.
Separating the sample
The sample is poured slowly into the ion exchange column. As the fluid drains through the gel matrix, the ions bind to the functional groups in the column while the rest of the solution passes through and can be collected as it exits the bottom of the column.
Collecting the sample of interest
In order to collect the molecules that are bound to the column, they must first be released from the functional groups in the gel matrix. This is called elution. It can be done in one of two ways:
Changing the charge of the molecules in the sample – this is done by running a buffer of a different pH through the column. This affects the shape of the bound molecule and, therefore, which parts of the molecule are exposed, changing the overall charge.
Displacing the bound molecules with something else. If ions that are more attracted to the functional groups in the gel matrix are run through the column, they will displace the molecules already bound.
The purified molecule from the sample can then be collected as it comes off the matrix in the column.
Uses for ion exchange chromatography
Ion exchange chromatography has many uses including:
separation of proteins from foods, for example, to investigate the effects of individual food components on health – this type of analysis is used in nutrition research
separation of high value proteins from substances
drinking water analysis for pollution and other constituents
determination of water chemistries in aquatic ecosystems
determination of sugar and salt content in foods.
Find out more about Mining milk for lactoferrin.
Written by Sara Loughnane – a 2006 New Zealand Science, Mathematics, and Technology Teacher Fellow.
