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