p. 14
Elution with stepwise ionic strength gradients
Stepwise elution is th e sequential use of t he same buffer a t different ionic
strengths. It is tech nically simple and fa st, and is suitab le for syringe
operation. It is ofte n used for sample con centration and sa mple clean-up.
Stepwise elution give s small peak volume s and the resolut ion depends on
the difference in elution power between each step.
1. Choose starting conditions as outlined under Optimizing starting
conditions.
2. Equilibrate the column, see Purification.
3. Adjust t he sample to the choo sen starting p H and ionic streng th, see
Sample preparation.
4. Apply t he sample at 1 or 5 ml/mi n for the HiTrap 1 ml or 5 ml colum n
respectively. Collect eluate.
5. Wash wit h at least 5 column vol umes of start bu ffer or until no mat erial
appears in effluent. Collect eluate.
6. Elute wit h the first ste p ionic strengt h buffer. The volumes r equired for
stepwise elution de pend on the operat ing conditions . However, 3–5
column volumes is usual ly sufficient . Collect elua te.
7. Elute wi th next ionic str ength buffer. Coll ect eluate.
8. After completed elution, regenerate the column by washing with 5
column volumes of regene ration buffer ( start buf fer with 1 M NaCl)
followed by 5–10 column volumes of s tart buffer. The c olumn is now
ready for a new sample.
Elution with continuous ionic strength gradients
Continuous salt grad ient elution is the m ost frequently u sed type of
elution in ion exchang e chromatography . It is very reproduci ble and leads
to improved resolutio n, since zone sharp ening occurs duri ng elution.
Continuous gradient s can be prepared in di fferent ways, de pending on
available equipment.