CE determination of the thermodynamic pKa values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation
Language English Country Germany Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
Research Gift No. 4135
Agilent Foundation - International
Grant No. 18-11776S
Grantová Agentura České Republiky - International
- Keywords
- Capillary isoelectric focusing, Limiting ionic mobility, Thermodynamic acid dissociation constant, pH Gradient linearity, pI Marker,
- MeSH
- Ampholyte Mixtures chemistry MeSH
- Electrophoresis, Capillary methods MeSH
- Isoelectric Focusing methods MeSH
- Hydrogen-Ion Concentration MeSH
- Osmolar Concentration MeSH
- Computer Simulation MeSH
- Buffers MeSH
- Thermodynamics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Ampholyte Mixtures MeSH
- Buffers MeSH
Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pKa values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pKa values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pKa and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download).
Agilent Technologies Deutschland GmbH Waldbronn Germany
Chemistry Department Texas A and M University College Station TX USA
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