About ion exchange chromatography - (May/08/2007 )

Since there are two different methods to elute product we wanted, one is step gradient elution and other is linear gradient elution (Both are in changing of salt concentration), i want to know,

1. When will we choose the step gradient or linear gradient for elution?

2. What is the purpose for each one?

Thanks

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step gradient is often a surrogate for linear gradient, f.i. if you are not eqipped with gradient mixing and pumping HPLC, or if you use LPLC w/o using pumps

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With a linear gradien you will elute proteins from the ion exchange in broader peaks. The proteins start to elute with the salt concentration where the exchange on the resins begins, but not all the protein will elute at the same time, there is rebinding, mixing of the eluent. There is a possibility to coelute proteins just before or after your protein and the peaks could intermix.

With a step gradient you change the concentration rapidly and when you chose the right concentration above the recomendet salt-concentration, all your protein should elute in one sharp peak. But with this method you could also elute contaminations with an equal affinity to the resin.

And there is no rule, which method you should use for a special problem, try and fault is the answer!!!

ms-olli

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As a rule, when you start to work with your protein mixture and don't know conditions of elution you should make non-sharp gradient from 0 to 100% NaCl to see elution profile of your mixture , then you should choose salt concentration when interest protein start elute and choose salt conc below this ( for ex your protein elute at 20% so you choose 15%) and elute your protein. it is so called development of purification method. Also it is important, before IEC It will be better to know pI of your protein and bind your protein on resin in pH with 0.5 pH shifting of your protein pI. This procedure helps you to elute impurities during binding.

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