Electroosmotic flow variations caused by the volatility of buffer components: Diagnosis and therapy

In order to separate a polar amine pharmaceutical and its potential impurities, a micellar electrokinetic chromatography method was developed. The main compound and 11 other substances were completely separated using a 20 mM Tris buffer, pH 8.0, containing 50 mM sodium dodecylsulfate (SDS) and 24% (v/v) acetonitrile. However, a strong, continuous reduction in the EOF occurred and quantification was not possible. The EOF reproducibility could not be improved by suitable rinsing procedures. Surface effects or interactions did not cause the EOF changes, but the evaporation of acetonitrile was identified as the major source for EOF instability. However, a high concentration of acetonitrile was decisive for selectivity. Thus a reliable protection against the evaporation of this buffer constituent had to be found. Paraffin and various silicon oils were tested as covering film. In order to quickly test buffer systems if the evaporation of electrophoresis solutions is acceptable, an alternative experimental design without doing CE experiments had to be found. Electrical conductivity was chosen as parameter, because it can be determined simply and fast. The buffers under investigation were placed in a 50-ml beaker with a magnetic stirring rod, placed on a magnetic stirrer. The buffer solution was kept in motion at 120 rev./min. The beaker was covered around the measuring head with laboratory film and in addition to this with paraffin or silicon oil. An acetonitrile content up to 10% (v/v) was acceptable if a coverage was used. The various cover liquids had a similar effect. A content of 15% (v/v) already increased the evaporation effect significantly. Higher acetonitrile contents are not acceptable. A buffer similar to the originally transferred method, 20 mM Tris (pH 8.0), 50 mM SDS containing 10% (v/v) acetonitrile as well as 10% (v/v) isopropanol showed an acceptably low evaporation in the conductivity experiments. With this buffer, a stable EOF was also obtained. Conductivity measurements are generally applicable to quickly test buffers that contain organic solvents.