p. 13

Further optimization

The recommendation s given above will giv e a sound basis for dev eloping an
efficient purif ication step. De tails of how flow ra te, sample loading , particle
size and elution schem e may be optimized to m eet the special ne eds can
be found in the handbook , Ion Exchange Chr omatography & Chro mato-
foucsing, Principle s and Methods, Cod e No. 11-0004-21.
GE Healthcare supplie s a wide range of ion exch ange chromatogra phy
media for purificat ion of biomolecu les at all scales. Se e Ordering inform ation
and visit www.gehealthcare.com/hitrap.
5. Choice of gradient type
1. Stepwis e gradients are eas y to produce and requi re minimal equipme nt.
Eluted peaks are very sh arp and elution vo lumes minimal. Ho wever,
care must be exercised in t he design of the ste ps and the interpret ation
of results for substan ces eluted by a sharp c hange in pH or small
differences in ionic s trength. Peak s tend to have sharp fro nts and
pronounced tailing since they frequently contain more than one
component.
2. Continu ous salt gradient s are the most frequ ently used type of el ution.
Many types of gradient for ming systems are ava ilable. Two buffer s of
differing ionic st rength, the star t and elution bu ffer (star t buffer
+ 1 M NaCl or higher buffer s alt concentratio n), are mixed tog ether and if
the volume ratio is chan ged linearly, th e ionic strength c hanges linearl y.
Note: Another, but less c ommon, method to de sorb bound mate rial is to
increase (SP) or decrea se (Q) the pH of the e luent. Continu ous pH
gradients are diff icult to produce at co nstant ionic st rength, since
simultaneous changes in ionic strength, although small, also occur
(buffering capacities are pH dependent).