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Mobility Plot to Optimize your Separation
- 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.
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