Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: theoretical approach and in situ measurement.
Electrode reactions during the electrophoretic process may change the pH of the buffer and subsequently the migration behavior of solutes with resultant loss of reproducibility. A theoretical treatment of pH variations due to electrolytic processes is presented. The choice of buffer appears to have a dramatic influence on the pH variations observed, even if substantial buffer action is expected at the pH chosen. The experimental evaluation of the separation of 4-hydroxy-3-methoxycinnamic acid and 3-hydroxybenzoic acid reveals that the quality of the separation decreases continuously from a baseline separation observed in the first experiment to a comigration of the two solutes (resolution = 0) in the ninth experiment. A pH decrease of about 0.05 pH units accounts for the observed changes in mobility. A novel in situ pH measurement approach is presented, in which the mobility, peak area, and peak height of an indicator dye are related to the pH in the capillary. This enables the identification and quantitation of pH variations during electrophoretic runs: the pH decreases at the anodic side already after the first experiment and pH variations as small as 0.02 pH units can be measured. The variations in peak height appear to be less suited. The calculated pH variations are in close agreement with the ones obtained experimentally.
Department of Biochemical Engineering, Delft University of Technology, The Netherlands.
H
H
Corstjens
H A
HA
Billiet
J
J
Frank
K C
KC
Luyben
D002021:Buffers; D004566:Electrodes; D004573:Electrolytes; D019075:Electrophoresis, Capillary; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D015203:Reproducibility of Results
D002021:Buffers; D004573:Electrolytes