Moving and stationary boundaries in immobilized pH gradients.
The relations describing the concentration changes at moving boundaries in a medium containing bound, buffering group are derived for a system which, except for hydrogen and hydroxyl ions, contains one anionic and one cationic mobile constituent. The relations found have been used to calculate concentrations and conductivities in zones developing in immobilized pH gradients. Assumptions used in the calculations as well as conductivity ratios between zones have been experimentally controlled and were found to reasonably agree with expectations. It is also shown how difference in transference numbers between sample droplet and gel will cause concentration and pH changes at the gel-sample droplet interfaces and it is explained how these changes are related to ionic concentrations in the gel. The high concentration zone generated at one of the interfaces will be transported into the gel. This transport has been numerically simulated and experimentally verified. The low concentration generated at the opposite interface will cause titration impeding sample entrance in the gel through this interface even when the gel contains ions other than H+ or OH- transported towards the interface. The described phenomena explain the dependence of lateral spreading, precipitation at the application site as well as streaking and smearing along sample lanes, on the type and concentration of low molecular weight ions originally present in the gel.
Bjellqvist
B
B
Pharmacia-LKB Biotechnology AB, Bromma, Sweden.
Linderholm
M
M
Ostergren
K
K
Strahler
J
J
D002021:Buffers; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing / Q000379:methods*; D008433:Mathematics; D011506:Proteins / Q000302:isolation & purification*
D002021:Buffers; D007202:Indicators and Reagents; D011506:Proteins