The effect of operating conditions on separation of soybean proteins in a home-made free-flow electrophoresis apparatus was investigated. Measurement of the pH, conductivity, and UV-absorbance (280 nm) were carried out at each run and the purity of the sample was tested with SDS-PAGE analysis.
The soybean extract pretreated with Tris and boric acid was mixed with the amino acids composed of glutamic acid, histidine, arginine, glycine(1 mM each) with glycyl-glycine(2mM) and KCl(1mM).
When the cellulose acetate was used as a compartment between the electrode and the buffer solution in the cell, pH distribution in the separation cell varied from 3.0 at the anodic side to 8.0 at the cathodic side and had two inflection point. The applied voltage was from 300V to 1000V and the separation was better at a higher voltage but the voltage was limited by the capability of the cooling system due to Joule heat. The proteins focused near the middle of the channel. From the change of pH and conductivity it was found that the ions in the channel moved out to the electrodes through the membrane. In the case when the concentration of the buffer solution was increased 5 times, proteins were focused at 300V. We could not increase up to the ten times of the concentration since the temperature difference between inlet and outlet was more than 25°C and denaturation of proteins was expected. When ion-exchange membranes were used U-type pH distribution was set up due to the ionic polarization near the membrane. The commercial ampholytes, instead of the mixed amino acids showed not much improvements in purity of the separated sample.
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