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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
(pH3.0) 50mM Acetic acid
(pH6.7) 50mM Ammonium acetare
(pH10.0) 50mM Ammonium acetare
Flow rate : 1.0mL/min
Detector : UV(220nm)
Column temp. : 30°C