Dataset Information

Separation and Characterization of Highly Charged Polyelectrolytes Using Free-Solution Capillary Electrophoresis.

ABSTRACT: The characterization of statistical copolymers of various charge densities remains an important and challenging analytical issue. Indeed, the polyelectrolyte (PE) effective electrophoretic mobility tends to level off above a certain charge density, due to the occurrence of Manning counterion condensation. Surprisingly, we demonstrate in this work that it is possible to get highly resolutive separations of charged PE using free-solution capillary electrophoresis, even above the critical value predicted by the Manning counterion condensation theory. Full separation of nine statistical poly(acrylamide-co-2-acrylamido-2-methylpropanesulfonate) polymers of different charge densities varying between 3% and 100% was obtained by adjusting the ionic strength of the background electrolyte (BGE) in counter electroosmotic mode. Distributions of the chemical charge density could be obtained for the nine PE samples, showing a strong asymmetry of the distribution for the highest-charged PE. This asymmetry can be explained by the different reactivity ratios during the copolymerization. To shed more light on the separation mechanism, effective and apparent selectivities were determined by a systematic study and modeling of the electrophoretic mobility dependence according to the ionic strength. It is demonstrated that the increase in resolution with increasing BGE ionic strength is not only due to a closer matching of the electroosmotic flow magnitude with the PE electrophoretic effective mobility, but also to an increase of the dependence of the PE effective mobility according to the charge density.

SUBMITTER: Desvignes I

PROVIDER: S-EPMC6401714 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Separation and Characterization of Highly Charged Polyelectrolytes Using Free-Solution Capillary Electrophoresis.

Desvignes Isabelle I Chamieh Joseph J Cottet Hervé H

Polymers 20181202 12

The characterization of statistical copolymers of various charge densities remains an important and challenging analytical issue. Indeed, the polyelectrolyte (PE) effective electrophoretic mobility tends to level off above a certain charge density, due to the occurrence of Manning counterion condensation. Surprisingly, we demonstrate in this work that it is possible to get highly resolutive separations of charged PE using free-solution capillary electrophoresis, even above the critical value pre ...[more]

PMID: 30961256

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Project description:Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Dataset Information

Separation and Characterization of Highly Charged Polyelectrolytes Using Free-Solution Capillary Electrophoresis.

Publications

Separation and Characterization of Highly Charged Polyelectrolytes Using Free-Solution Capillary Electrophoresis.

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