The eluent buffer must be high in buffering
capabilty for a given eluent pH and the buffering
ions must have the same electric charge as
the ion-exchanger. The following table gives
some examples of such buffers.
|pH||IEC QA-825, DEAE-825, AXpak WA-624||IEC SP-825, CM-825|
|20mM Piperazine HCl||20mM Sodium malonate|
|20mM Bis-Tris propane HCl||20mM Sodium phosphate|
|20mM Tris HCl||20mM Sodium phosphate|
|20mM Tris HCl||20mM HEPES*|
|20mM Ethanolamine HCl|
Generally, the eluent pH is higher than the
isoelectric point of the sample in the case
of the QA-824 and DEAE-825 column and lower
the case of SP-825 and CM-825.
- Each column must be used in a pH range
of 2 to 12.
- Total concentration of the salts in the
eluent is usually in a range of 20 mM to
- Water-soluble organic solvents1 such as
ethylene glycol and isopropyl alcohol, must
not be added in the quantity of more than
- Proteins are generally separated by gradient
elution in which the ionic strength or pH
of the eluent is changed for their separation.
Normally, however, only the ionic strength
is changed or increased for the separation
while the pH is kept at a given level because
it is difficult to change the pH. Almost
all proteins are eluted from the column by
increasing the salt concentration in a 0.02
to 0.05 M buffer from 0 to 0.5 M.