Use of ion exchange chromatography |
High resolution mode
(gradient elution) | Group separation mode
(step elution) |
| Separates proteins, peptides, and oligonucleotides according to net charge. | Concentrates dilute samples of proteins, peptides and oligonucleotides. |
| Suitable for intermediate and polishing steps in multi step purification protocols. | Suitable for capture steps in multistep purification protocols. |
Experimental |
 | High resolution mode
(gradient elution) | Group separation mode
(step elution) |
IEX medium | Use strong IEX media only
Use media with 10 - 30 mm beads for purification work.
Use media with 3 - 5 mm beads for analytical work. | Use strong IEX media only.
Use media with 30 mm beads or larger to allow higher flow rates. |
Column | Typical column lenghts are 1 - 10 cm.
Short columns may prevent the use of very shallow gradients (large gradient volumes). | Column length is less important. Short and "fat" columns, however, will allow higher flow rates. |
Eluents | Buffering ion should be of the same change sign as the ion exchanger.
Buffer pKa should not deviate more than 0.5 pH units from the running pH.
A buffer concentration of 20 - 50 mM is usually sufficient. | Buffering ion should be of the same sign as the ion exchanger.
Buffer pKa should not deviate more than 0.5 pH units from the running pH.
A buffer concentration of 20 - 50 mM is usually sufficient. |
Sample volume | With a properly designed gradient the target molecule will bind and concentrate at the top of the column.
The sample volume is thus not important. | The sample volume is not important since the sample will bind to the column. |
Sample amount | 5 - 10 % of the total loading capacity of the column used can be applied without loss of resolution. | Around 40 % of the total loading capacity of the column used can be applied. |
