Influence of the ionic strength of acidic background electrolytes on the separation of proteins by capillary electrophoresis

Bekri, Samya; Leclercq, Laurent; Cottet, Hervé

doi:10.1016/j.chroma.2015.12.060

Cited by 14 publications

(14 citation statements)

References 60 publications

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“…The decrease of the electrophoretic mobility (μ ep ) with the ionic strength ( I ) of the surrounding BGE is highly dependent on the nature and the charge of the solute (mono or multicharged small ions , polyelectrolyte , nanoparticles, or particles ). This dependence on ionic strength is often used to optimize the separations as exemplified in the literature for small ions (SI) , pharmaceuticals , oligomers, peptides , proteins , polyelectrolytes (PE) , and nanoparticles (NP) . It can also be used to characterize the nature of complex/unknown samples, as exemplified recently on polymeric drug delivery systems using a new graphical representation called the slope‐plot .…”

Section: Introductionmentioning

confidence: 99%

On the ionic strength dependence of the electrophoretic mobility: From 2D to 3D slope‐plots

Cottet

Allison

2016

Electrophoresis

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Determining the charge and the nature (small ion, nanoparticle, or polyelectrolyte) of an unknown solute from its electrophoretic characteristics remains a challenging issue. In this work, we demonstrate that, if the knowledge of the effective electrophoretic mobility (μ ) at a given ionic strength is not sufficient to characterize a given solute, the combination of this parameter with (i) the relative decrease of the electrophoretic mobility with the ionic strength (S), and (ii) the hydrodynamic radius (R ), is sufficient (in most cases) to deduce the nature of the solute and its charge. These three parameters are experimentally accessible by CZE and Taylor dispersion analysis performed on the same instrumentation. 3D representation of the three aforementioned parameters (μ ; S and R ) is proposed to visualize the differences in the electrophoretic behavior between solutes according to their charge and nature. Surprisingly, such 3D slope plot in the case of small ions and nanoparticles looks like a "whale-tail," while polyelectrolyte contour plot represents a rather simple and monotonous map that is independent of solute size. This work also sets how to estimate the effective charge of a solute from a given experimental (S,Rh,μ ep 5 mM ) triplet, which is not possible to obtain unambiguously with only (Rh,μ ep 5 mM ) or (S,μ ep 5 mM ) doublet, where μ ep 5 mM is the effective electrophoretic mobility at 5 mM ionic strength.

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Section: Introductionmentioning

confidence: 99%

On the ionic strength dependence of the electrophoretic mobility: From 2D to 3D slope‐plots

Cottet

Allison

2016

Electrophoresis

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“…These figures of merit are very useful for estimating the impact of the ionic strength on the effective mobility and for the modeling of the effective and apparent selectivities according to f and I, as discussed in the next section. The S values are also very informative about the characteristics of the solute (charge, nature, or size) [25,26,27]. As a matter of comparison, proteins have typical S values between 0.3 and 0.4 [27], which are very similar to the values obtained for moderately charged PAMAMPS with 30% ≥ f ≥ 10%.…”

Section: Resultsmentioning

confidence: 83%

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

2018

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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.

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“…For the two proteins HSA, and ovalbumin several isoforms are observed in the separation . In this separation, a lower ionic strength was employed to reduce joule heating and facilitate a better separation of protein isoforms . It cannot be excluded that more than one isoform contributes to the signals of cytochrome c and myoglobin, which makes a detailed analysis based on theoretical plates challenging.…”

Section: Resultsmentioning

confidence: 99%

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

Poulsen

Østergaard

Petersen

et al. 2016

J of Separation Science

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Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in-capillary surface-initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4-8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4-8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.

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Influence of the ionic strength of acidic background electrolytes on the separation of proteins by capillary electrophoresis

Cited by 14 publications

References 60 publications

On the ionic strength dependence of the electrophoretic mobility: From 2D to 3D slope‐plots

On the ionic strength dependence of the electrophoretic mobility: From 2D to 3D slope‐plots

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

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

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