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Contribution of Eigenmobility Shifts to the Separation of Peptides in Capillary Electrophoresis with Aqueous–Acetonitrile Background Electrolytes

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journal contribution
posted on 05.12.2016, 20:07 by Chadin Kulsing, Reinhard I. Boysen, Milton T. W. Hearn
In this investigation, the mobility of system eigenpeaks in capillary electrophoresis (CE) was experimentally found to decrease when the background electrolyte (BGE) contained higher percentages of acetonitrile. In order to explain this observation, the effects of changes in the pH and ionic strength of the BGE on the pKa and actual mobility of each constituent in the system were determined, and the results evaluated in terms of their theoretical basis. Utilizing the derived values of each of these parameters, the software Peakmaster was then applied to simulate the eigenpeak mobility. Although general trends for BGEs with different acetonitrile contents could be simulated, these simulations did not exactly match the experimental results. To account for this divergence between theory and experimental practice, the consequences of tube radial distribution of the organic solvent in an aqueous–organic system within the capillary and the effects of radial ion distribution leading to the electro-osmotic flow mobility (EOF) are proposed to be the cause of this deviation. Consequently, the Debye–Hückel approximation and Boltzmann distribution function were employed to calculate the amount of each constituent across the radius of the capillary. The inhomogeneous radial distributions of the constituents in the BGE and the organic solvent were simplified to a 1-dimensional problem based on a 4-constituent BGE approximation. A high level of correlation was then achieved between the experimental results and the corresponding CE separations simulated using Peakmaster. In addition, cancellation or suppression of the peak broadening was experimentally and theoretically demonstrated by taking advantage of the influence of a second independent system eigenpeak. The outcome from these studies was a new way to achieve sharpening of specific peaks in the CE separations of peptides.