Ion Exchange Chromatography is the separation by HPLC of molecules which are or can be ionised to form positive or negative ions. Positively charge ions are known as Cations, and negatively charged ions are known as Anions. Such molecules will usually contain an amino group (quarternary amino groups are fully ionised, tertiary amino groups are ionised at low pH and not ionised at higher pH), a carboxyllic acid group (ionised at higher pH's, unionised at low pH), a sulphonic acid group (fully ionised) or a phosphate group (fully ionised at most pH's). Hence Ion Exchange Chromatography is used mostly for the analysis of biological compounds such proteins, peptides, nucleic acids, amino acids and glycoproteins.

To achieve separation, ions of the opposite charge are bonded to the column packing material, and the various ions in the sample travel through the column at different speeds, causing a separation to occur. Hence it follows that separate columns are required for anions and for cations.

Strength of the Ion Exchanger. Unlike inorganic ions which are small, densely charged molecules, organic species are often much larger, with a much lower charge density. Hence it is normal to offer both weak and strong anion and cation exchange columns. These columns usually contain the following bonded phases:

• SAX - Strong Anion Exchanger. Quarternary ammonium groups, (QA.)
• WAX - Weak Anion Exchanger. Tertiary amino groups, usually diethylaminoethyl. (DEAE)
• SCX - Strong Cation Exchanger. Sulphonic Acid, usually on a propyl chain. (SP)
• WCX - Weak Cation Exchanger. Carboxyllic acid, usually carboxymethyl. (CM)

Capacity of the Ion Exchanger. This is a measure of the loading capacity of the column, and is measure in milliequivalents per gram. For most applications, a capacity of around 0.5meq/g is fine, but for some applications a higher capacity is required.

Detection. Unlike ion chromatography, where detection is almost exclusively with a conductivity detector, in ion-exchange chromatography, detection is usually but not exclusively by UV.

Eluents for Ion Exchange Chromatography. Because of the polar nature of the substances to be analysed it is normal for eluents to be primarily aqueous buffers. pH control is vital, because changing the pH affects the ionisation of the species being analysed. In the case of proteins, particular care has to be taken to note and avoid the isoelectric point, where the number of positive and negative charges on the molecule is equal, the molecule has a net neutral charge, and is at its least soluble. To increase the strength of the eluent (to reduce retention times) it is necessary to increase the concentration of the buffer. It is quite common to use gradient elution, because often different molecules have quite different degrees of ionisation at a given pH. In this case, although an organic solvent such as methanol may be added to the stronger of the eluents, it is normal to run an ionic strength gradient. Care must be taken to watch the solubility of the buffer in any organic solvent used, and to watch that the UV cut-off of the buffer does not interfere with detection by UV.

pH Buffers. A pH buffer works in two ways. Firstly, because it is present in excess with respect to the sample, it holds the pH simply because the effect of the sample is minimal compared to the buffer. But pricipally a buffer works by changing the extent of its ionisation to cancel out the effect of any attempt to change the pH. To do this, the buffer must be used within +/-1pH unit of its pKa. Hence buffer choice is very important, once the optimum pH for the separation has been established. A search of the internet will reveal a list of papers recommending buffers to use at different pH, and a list is available from the Shodex website.

Why use Polymer-based Ion Exchange Columns? Because it is normal to need to work above pH7! Hence a polymer-based column will last much longer, and is much easier to clean. It can also withstand higher buffer concentrations. Silica columns are usually cheaper, but they are more restrictive in their application and they don't last as long, so it is a short term saving!

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