Select the optimal pH for your separation.
The basis for understanding the electrophoresis of your samples can be used to determine pKa and pI of Samples.
Since mobility of ions are a function of pH, ionic strength and the sample matrix, using multiple buffers can be a source of errors. The use of borate-phosphate buffers reduces some of these variables yet produces good buffering capacity.
- Prepare a buffer solution that is 25mM Sodium Tetraborate and 25mM Phosphoric Acid.
- Adjust the pH with either 1M NaOH or 1M Phosphoric Acid to yeild nine different pH buffers ranging from pH 2.5 to 11.0.
- Fill and equilibrate a capillary with the lowest pH buffer.
- Perform a run with your samples and include a neutral marker in your injection sample.
- Determine EOF with the use of the neutral marker.
- Perform another run with a different concentration of your solutes. This will aid in “peak tracking” by observing differing peak heights for respective changes in concentrations.
- If no peaks appear at a particular pH for any solute, try injecting with reversed polarity: the solute charge could reverse.
- Repeat the above for each of the nine different pH buffer solutions you prepared in step one.
- Measure the solute’s migration velocity (cm/s) and add or subtract the EOF.
- Divide the migration velocity by the field strength and plot this calculated Mobility (cm2/Vs) against pH. A narrow pH range using a titrated single buffer to various pH can be used to fine tune the mobility of your solutes. This is usually more accurate than a wide range because one buffer is used throughout the mobility test range.
The following Mobility Plot shows that performing a mobility plot makes is obvious to select a pH between 7 and 8. Any experimental changes will have only a slight effect on selectivity in the separation of Glutamate and Acetate.