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Order and linking steps
Sometimes it doesn't matter too much where in a sequence a particular unit operation occurs, but this is unusual. Normally there is a clear logic which determines the best position for solving a specific purification challenge. In addition there is a logic to linking techniques.
Try to choose the order of techniques in such a way, that the eluate from the first step is suitable for the start conditions of the next step. For example, if the sample has a low ionic strength it can be applied to an IEX column.
After elution from IEX the sample will usually be in a high ionic strength buffer and can be applied to a HIC column (if necessary the pH can be adjusted and further salt can be added).
In contrast, if sample is eluted from a HIC column, it is likely to be in high salt and will require dilution or a buffer exchange step in order to further decrease the ionic strength to a level suitable for IEX. Thus it is more straightforward to go from IEX to HIC than vice-versa.
Gel filtration is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation.
Logical combinations of techniques are shown below. |
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