HPLC Products

Affinity Chromatography is essentially a sample purification technique, used primarily for biological molecules such as proteins.

Unlike other forms of HPLC, where most if not all components of the sample have an attraction to the column, in Affinity Chromatography the column contains a ligand which is specific to a sample component (or group of components) of interest. This component (or group of components) alone is bound to the column and everything else elutes unretained. Then by changing the eluent composition using a gradient, the adsorbed species (or group of species) is eluted.

Column Specificity. Since columns have quite highly specific active sites, a wide variety of columns is required, even to separate groups of compounds. It is not normally commercially viable to produce columns for individual compounds. However there are many groups of compounds which can be isolated using affinity chromatography, and details of these is given on the table below. It should be noted that if a column is used to isolate a group of compounds from a mixture, they will usually be all retained and then eluted together.

Process of Affinity Chromatography. First a column must be found containing a ligand which will specifically bind the sample component of interest. Then two sets of eluent conditions must be developed. The first is used to introduce the sample, allow it to bind to the ligand, and wash out all other components from the column. The second eluent is used to elute the sample from the column without denaturing it. It is normal to use a gradient, although a stepwise process can also be used.

Group-Specific Columns.  These are the most widely available, as per the list below. Each contains a rigid gel, onto which is bonded a spacer group to minimise steric hindrance when a larger molecule approaches, and onto that is bonded the ligand. Because the columns have an "on-off" retention sequence, the columns need only be short. 8mm diameter is often used to give a good loading capacity, although smaller bore columns are also available.