In capillary electrophoresis (CE), artifactual peak splitting causes a single analyte peak to be split into multiple peaks which can lead to misidentification of analytes and errors in quantification. Additional peaks created by such a phenomenon can be wrongly attributed to impurities or interactions with the background electrolyte. Here we demonstrate how the overlap of the sample plug with the analyte peak causes peak splitting and how certain experimental parameters such as pH, separation voltage and sample plug length can be altered to overcome this problem. To study this effect, a FITC tagged monoclonal antibody is detected using fluorescence.  The same laser beam used to excite fluorescence also measures refractive index changes in the detection volume using backscatter interferometry (BSI).  This universal detector senses the sample plug which is invisible to the fluorescence detector.  Since detection is performed in the same detection volume, both the analyte position and sample plug are precisely correlated in the two electropherograms.  The degree of overlap between the analyte and sample plug was evaluated and the resulting peak splitting was studied.  Comparing experimental data with COMSOL simulations helps understand the influence of separation voltage, pH of the background electrolyte (BGE), and the sample plug length in the peak splitting process.  For the antibody used here, the analyte peak begins to split in the pH range from 7 to 8, which is closer to the isoelectric point of the protein. Peak splitting was also observed at higher pH levels with larger sample plug lengths and lower separation voltages suggesting the importance of keeping a sufficient resolution between slow moving analytes and the sample plug during CE experiments.

Study of sample plug induced peak splitting in capillary electrophoresis using backscatter-interferometry coupled with fluorescence detection

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