Quantitative Analysis in Capillary Electrophoresis: Transformation of Raw Electropherograms into Continuous Distributions

Quantitative analysis in capillary electrophoresis based on time-scale electropherograms generally uses time-corrected peak areas to account for the differences in apparent velocities between solutes. However, it could be convenient and much more relevant to change the time-scale electropherograms into mass relative distribution of the effective mobility or any other characteristic parameter (molar mass, chemical composition, charge density, ...). In this study, the theoretical background required to perform the variable change on the electropherogram was developed with an emphasis on the fact that both <i>x</i> and <i>y</i> axes should be changed when the time scale electropherograms are modified to get the distributions. Applications to the characterization of polymers and copolymers by different modes of capillary electrophoresis (CE) are presented, including the molar mass distribution of poly-l-lysine oligomers by capillary gel electrophoresis (CGE), molar mass distribution of end-charged poly-l-alanine by free solution CE, molar mass distribution of evenly charged polyelectrolytes by CGE, and charge density distribution of variously charged polyelectrolytes by free solution CE

Investigating the Influence of Phosphate Ions on Poly(l‑lysine) Conformations by Taylor Dispersion Analysis

In this work, the influence of the ionic strength and phosphate ions on poly­(l-lysine) hydrodynamic radius, conformation and persistence lengths has been studied for molar masses comprised between 3000 and 70 000 g/mol. Mark–Houwink coefficients have been obtained via the determination of poly­(l-lysine) hydrodynamic radius using Taylor dispersion analysis. The influence of phosphate ions and ionic strength on the solvent quality (poor, Θ, or good solvent) for poly­(l-lysine) have been studied in details. Quantitative data on hydrodynamic radius, persistence length, Mark–Houwink coefficients are provided at pH 7.4, in the range of 10 mM to 1 M ionic strength, and for different phosphate ion concentrations from 0.1 mM to 50 mM under physiological conditions (154 mM ionic strength, pH 7.4). The strong influence of phosphate ions on poly­(l-lysine) properties was finally illustrated by studying the interactions (stoichiometry, binding constant, and cooperativity) between poly­(l-lysine) of DP 50 and human serum albumin, in the absence and in the presence of phosphate ions at pH 7.4

Extracting Information from the Ionic Strength Dependence of Electrophoretic Mobility by Use of the Slope Plot

The effective mobility (μ_ep) is the main parameter characterizing the electrophoretic behavior of a given solute. It is well-known that μ_ep is a decreasing function of the ionic strength for all solutes. Nevertheless, the decrease depends strongly on the nature of the solute (small ions, polyelectrolyte, nanoparticles). Different electrophoretic models from the literature can describe this ionic strength dependence. However, the complexity of the ionic strength dependence with the solute characteristics and the variety of analytical expressions of the different existing models make the phenomenological ionic strength dependence difficult to comprehend. In this work, the ionic strength dependence of the effective mobility was systematically investigated on a set of different solutes [small mono- and multicharged ions, polyelectrolytes, and organic/inorganic (nano)­particles]. The phenomenological decrease of electrophoretic mobility with ionic strength was experimentally described by calculating the relative electrophoretic mobility decrease per ionic strength decade (<i>S</i>) in the range of 0.005–0.1 M ionic strength. Interestingly, the “slope plot” displaying <i>S</i> as a function of the solute electrophoretic mobility at 5 mM ionic strength allows for defining different zones that are characteristic of the solute nature. This new representative approach should greatly help experimentalists to better understand the ionic strength dependence of analyte and may contribute to the characterization of unknown analytes via their ionic strength dependence of electrophoretic mobility

Monitoring Biopolymer Degradation by Taylor Dispersion Analysis

This work aims at demonstrating the interest of modern Taylor dispersion analysis (TDA), performed in narrow internal diameter capillary, for monitoring biopolymer degradations. Hydrolytic and enzymatic degradations of dendrigraft poly-l-lysine taken as model compounds have been performed and monitored by TDA at different degradation times. Different approaches for the data processing of the taylorgrams are compared, including simple integration of the taylorgram, curve fitting with a finite number of Gaussian peaks, cumulant-like method and Constrained Regularized Linear Inversion approach. Valuable information on the kinetics of the enzymatic/hydrolytic degradation reactions and on the degradation process can be obtained by TDA

Measuring Arbitrary Diffusion Coefficient Distributions of Nano-Objects by Taylor Dispersion Analysis

Taylor dispersion analysis is an absolute and straightforward characterization method that allows determining the diffusion coefficient, or equivalently the hydrodynamic radius, from angstroms to submicron size range. In this work, we investigated the use of the Constrained Regularized Linear Inversion approach as a new data processing method to extract the probability density functions of the diffusion coefficient (or hydrodynamic radius) from experimental taylorgrams. This new approach can be applied to arbitrary polydisperse samples and gives access to the whole diffusion coefficient distributions, thereby significantly enhancing the potentiality of Taylor dispersion analysis. The method was successfully applied to both simulated and real experimental data for solutions of moderately polydisperse polymers and their binary and ternary mixtures. Distributions of diffusion coefficients obtained by this method were favorably compared with those derived from size exclusion chromatography. The influence of the noise of the simulated taylorgrams on the data processing is discussed. Finally, we discuss the ability of the method to correctly resolve bimodal distributions as a function of the relative separation between the two constituent species

Effect of Dendrimer Generation on the Interactions between Human Serum Albumin and Dendrigraft Polylysines

This work aims at studying the interaction between human serum albumin and different generations of dendrigraft poly-l-lysine (DGL) in physiological conditions. The binding constants and stoichiometry of the interaction were successfully determined using frontal analysis continuous capillary electrophoresis. The effect of generation on the interaction was evaluated for the five first generations of DGL. An increase of the binding constant accompanied with a decrease of the HSA:DGL (1:<i>n</i>) stoichiometry and a decrease of the cooperativity with dendrimer generation was observed. These findings were in good agreement with the increase of ligand (DGL) size, the increase of electrostatic ligand–ligand repulsion, and the localization of two negatively charged interaction sites on the HSA. The effect of the ligand topology (linear vs dendrigraft) on the HSA interaction revealed that linear poly­(l-lysine) leads to much lower stoichiometry compared to DGL of similar molar mass due to much higher flexibility and contour length

Polydispersity Analysis of Taylor Dispersion Data: The Cumulant Method

Taylor dispersion analysis is an increasingly popular characterization method that measures the diffusion coefficient, and hence the hydrodynamic radius, of (bio)­polymers, nanoparticles, or even small molecules. In this work, we describe an extension to current data analysis schemes that allows size polydispersity to be quantified for an arbitrary sample, thereby significantly enhancing the potentiality of Taylor dispersion analysis. The method is based on a cumulant development similar to that used for the analysis of dynamic light scattering data. Specific challenges posed by the cumulant analysis of Taylor dispersion data are discussed, and practical ways to address them are proposed. We successfully test this new method by analyzing both simulated and experimental data for solutions of moderately polydisperse polymers and polymer mixtures

Prediction of Polyelectrolyte Complex Stoichiometry for Highly Hydrophilic Polyelectrolytes

The interaction between two hydrophilic polyelectrolytes of opposite charges was investigated using poly­(l-lysine) (PLL) as the polycation and a library of copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (P­(AM-<i>co</i>-AMPS)) with various chemical charge densities as polyanions. The formation of polyelectrolyte complexes (PECs) was comparatively studied by varying different parameters, such as the mixing order, the P­(AM-<i>co</i>-AMPS) chemical charge density and the initial polycation to polyanion molar ratio. PECs were then characterized in terms of charge stoichiometry and of stability toward ionic strength. The results showed a strong dependency of precipitated PEC stoichiometry on the P­(AM-<i>co</i>-AMPS) chemical charge density and the initial polycation to polyanion molar ratio. In contrast, PEC stoichiometry was not affected by the mixing order of the two polyelectrolyte partners. A general rule capable of predicting the PEC stoichiometry is proposed

Effective Charge Determination of Dendrigraft Poly‑l‑lysine by Capillary Isotachophoresis

In this work, capillary isotachophoresis (ITP) was used to determine the effective charge of the first five generations of dendrigraft poly-l-lysines. This approach, which is based on the linear dependence of ITP zone length of the solute on its concentration and effective charge, offers a simple and straightforward method for effective charge determination. The cationic ITP system employed in this work yields good linearity, repeatability and sharp zones. The value of effective charge number per one lysine residue obtained for long linear poly-l-lysine is in a good agreement with the Manning theoretical value (0.5). Results obtained for dendrigraft poly-l-lysines show a dramatic decrease in the effective charge number per lysine residue with increasing generation number, from 0.84 for short oligolysines (generation 1) down to 0.08 for the fifth generation. This decrease in effective charge is due to the proximity of charged groups in the dendrigraft structure of higher generation number

Fast Characterization of Polyplexes by Taylor Dispersion Analysis

In a single procedure, Taylor dispersion analysis (TDA) was used for the size characterization of polyplexes and the quantification of free polycation contained in excess within the polyplex sample. TDA analysis was carried out in frontal mode for a better sensitivity of detection. The proof of concept was established using a model polyplex generated from the mixture of linear polylysine (DP 20) and DNA from salmon testes at nitrogen to phosphate (N/P) ratio of 12. Polyplex hydrodynamic radius was compared to the values obtained by dynamic light scattering measurements. TDA was found to give access to the weight-average hydrodynamic radius, while DLS basically gives an intensity-average (harmonic <i>z</i>-average) value. The method was next applied to the study of various polyplexes issued from polylysines of various DP (50, 100) and different topologies (dendrigraft polylysines of generation 2 and 3). This new methodology should greatly contribute to the physicochemical characterization of polyplexes used for gene transfection