Effect of Eluent pH (ODP)
A sample of different proteins and peptides was separated with a various pH values of the eluent. Under acidic conditions (pH2.3, pH3.0), Leu-Enkephalin and Leu-Trp-Met have been strongly retained and the elution times for the substances are quite similar; therefore, the separation is not satisfactory. In contrast, when an alkaline according to the fact that, under alkaline condition (pH10) with Asahipak ODP column, a better separation with sharper peaks of the substances has been obtained in a shorter time. In addition, ODP columns have higher theoretical plate numbers under alkaline conditions.
Refer to Analysis of Oligopeptides with Acidic and Alkaline Eluent.
Sample :
1. Glu-Gly-Phe
2. Met-Enkephalin
3. Leu-Enkephalin
4. Leu-Trp-Met
Theoretical plate number
No.
|
pH2.3
|
pH3
|
pH6.7
|
pH10
|
1
|
6100 | 1100 | 3800 | – |
2
|
7400 | 1100 | 4700 | 5700 |
3
|
7600 | 600 | 3800 | 6700 |
4
|
6100 | 500 | 2400 | 8000 |
Column : Shodex Asahipak ODP-50 6D (6.0mmID*150mm) Eluent : buffer/CH3CN=85/15 (pH2.3) 0.05% TFA aq. (pH3.0) 50mM Acetic acid aq. (pH6.7) 50mM Ammonium acetare buffer (pH10.0) 50mM Ammonium acetare buffer Flow rate : 1.0mL/min Detector : UV(220nm) Column temp. : 30deg-C