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Eluent Conditions for IEC-series and AXpak

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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.

pHIEC QA-825, DEAE-825, AXpak WA-624IEC SP-825, CM-825
6 20 mM Piperazine HCl 20 mM Sodium malonate
7 20 mM Bis-Tris propane HCl 20 mM Sodium phosphate
7.5 20 mM Tris HCl 20 mM Sodium phosphate
8 20 mM Tris HCl 20 mM HEPES
9 20 mM Ethanolamine HCl
10 20 mM 1,3-Diaminopropane HCl


HEPES : 4-(2-Hydroxymethyl)-1-piperazine ethanesulfonic acid

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.

CAUTION
1) Each column must be used in a pH range of 2 to 12.
2) Total concentration of the salts in the eluent is usually in a range of 20 mM to 1 M.
3) Water-soluble organic solvents1 such as ethylene glycol and isopropyl alcohol, must not be added in the quantity of more than 20 %.
4) 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.

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