Taylor Dispersion Analysis Research Articles

Rapid determination of the critical micelle concentration by Taylor dispersion analysis in capillaries using both direct and indirect detection.

The critical micelle concentration is a basic characteristic of surfactants. In this article, the process of formation of micelles is studied by Taylor dispersion analysis using capillary electrophoresis instrumentation as a substituent of capillary liquid chromatography. New methods for determination of critical micelle concentration are presented. Sodium octylbenzene sulfonate and sodium dodecylsulfate are used as model compounds with and without chromophore. Two novel approaches based on indirect ultraviolet detection and indirect Taylor dispersion analysis with direct ultraviolet detection are introduced for the determination of critical micelle concentration value of surfactant without any chromophore. The determined critical micelle concentration values are in correspondence with the tabulated values at 95% confidence level.

Characterisation of aggregates of cyclodextrin-drug complexes using Taylor Dispersion Analysis

There is a need to understand the nature of aggregation of cyclodextrins (CDs) with guest molecules in increasingly complex formulation systems. To this end an innovative application of Taylor dispersion analysis (TDA) and comparison with dynamic light scattering (DLS) have been carried out to probe the nature of ICT01-2588 (ICT-2588), a novel tumor-targeted vascular disrupting agent, in solvents including a potential buffered formulation containing 10% hydroxypropyl-β-cyclodextrin. The two hydrodynamic sizing techniques give measurement responses are that fundamentally different for aggregated solutions containing the target molecule, and the benefits of using TDA in conjunction with DLS are that systems are characterised through measurement of both mass- and z-average hydrodynamic radii. Whereas DLS measurements primarily resolve the large aggregates of ICT01-2588 in its formulation medium, methodology for TDA is described to determine the size and notably to quantify the proportion of monomers in the presence of large aggregates, and at the same time measure the formulation viscosity. Interestingly TDA and DLS have also distinguished between aggregate profiles formed using HP-β-CD samples from different suppliers. The approach is expected to be widely applicable to this important class of drug formulations where drug solubility is enhanced by cyclodextrin and other excipients.

Taylor dispersion analysis, resonant mass measurement and bioactivity of pepsin-coated gold nanoparticles

Immobilized enzyme reactors based on nanoparticulate carriers are becoming increasingly popular. A toolbox of methods is usually utilized for their characterization in order to be capable of assessing their suitability for the intended purpose. In this work, as a model system pepsin was conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process. The success of the immobilization procedure was monitored by Vis spectroscopy via shifts of the localized surface plasmon resonance (LSPR) band, size characterization by dynamic light scattering (DLS), and ζ–potential determinations by electrophoretic light scattering. DLS revealed a significantly different hydrodynamic diameter for unmodified GNPs and protein-coated GNPs. However, the hydrodynamic diameters of pepsin-coated GNPs obtained with various concentrations of pepsin in the coating solution were not significantly different. In contrast, Taylor dispersion analysis allowed measuring the slight differences in the hydrodynamic radius. It provided also information on the viscosity of GNP suspensions and diffusion coefficients for the various pepsin@GNP preparations. For the determination of the pepsin surface coverage on the GNPs results from indirect protein quantitation of non-immobilized pepsin by Lowry assay were compared to direct measurement of immobilized pepsin by resonant mass measurements. Reasonable agreement was found. Accurate information on enzyme coverage is of utmost importance for a representative comparison of the turnover numbers and catalytic efficiencies of nanoparticulate immobilized enzyme reactors, which is shown by the adsorptively immobilized pepsin@GNP as model system.

Particle sizing methods for the detection of protein aggregates in biopharmaceuticals.

Protein aggregation is a common biological phenomenon which is responsible for degenerative diseases and is problematic in the pharmaceutical industry. According to the rules provided by regulatory agencies, industry is supposed to assess the product quality regarding the presence of subvisible particles. Also, they should evaluate the technologies that are used to measure these particles. Therefore, US FDA and industry have been looking for methods capable of accurately characterizing the protein products. Four sizing techniques reviewed here are good candidates to be used for characterization of protein and their aggregates: dynamic light scattering, size-exclusion chromatography, electron microscopy and Taylor dispersion analysis. The first three are more established techniques while the last one is a more recent and growing technique.

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

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 (μep ) 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 (Rh ), 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 (μep ; S and Rh ) 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.

Rapid determination of hydrodynamic radii beyond the limits of Taylor dispersion

Taylor dispersion analysis (TDA) is an absolute method for determining the diffusion coefficients, and hence the hydrodynamic radii, of particles by measuring the dispersion in a carrier medium flowing within a capillary. It is applicable under conditions which allow the particles to radially diffuse appreciably across the cross-section of the flow before the measurement and therefore implies long measurement times are required for large particles with small diffusion coefficients. In this paper, a method has been developed by which the diffusion coefficients of large particles can be rapidly estimated from the shapes of the concentration profiles obtained at much earlier measurement times. The method relies on the fact that the shapes of the early-time concentration profiles are dependent on the diffusion coefficient, flow rate and the capillary radius through the dimensionless residence time which, theoretically, is a measure of the amount of radial diffusion undergone by the particles. The amount of radial diffusion for nanospheres of varying sizes was estimated by quantifying the relative change in the shapes of concentration profiles obtained at two points in the flow and a correlation was obtained with the variation of the dimensionless residence time to confirm the theory. This correlation was then tested by applying it to another set of measurements of solutes and solute mixtures of different sizes including a protein. The estimated diffusion coefficients were found to be in good agreement with the expected values. This demonstrates the potential for the method to extend dispersion analysis to regimes well outside the TDA limits to enable the rapid characterization of large particles.

Hydrodynamic size characterization of a self-emulsifying lipid pharmaceutical excipient by Taylor dispersion analysis with fluorescent detection

In this work, the sizing of microemulsion droplets of a lipid-based pharmaceutical excipient (Labrasol® ALF) is performed by Taylor dispersion analysis (TDA) using fluorescent detection. An hydrophobic fluorescent marker is used to tag the microemulsion droplet and to increase the sensitivity of detection (compared to UV detection). Combined with the frontal TDA mode, fluorescent detection was mandatory for an accurate sizing of microemulsions containing large coacervates. Microemulsion sizing of Labrasol was performed at various concentrations from 1 to 70g.L−1 and at two different temperature (25°C and 37°C). Results obtained by TDA are compared to those derived from DLS measurements. The combination of both techniques allows estimating the size and proportion of coacervates in the microemulsion, as well as the polydispersity in size of the sample.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions

Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations.

Theoretical Comparisons of the Concentration-Dependent Diffusion Coefficients from Dynamic Light Scattering and Taylor Dispersion Analysis

Dynamic Light Scattering and Taylor Dispersion Analysis are two methods employed for the measurement of the diffusion coefficients and interaction parameters of solutes. For self-associating solutes, the interaction parameter provides a measure for the degree of association and hence the stability of the solutes. Due to the characteristics, peculiar to each method, the measured values are weighted averages and are therefore different from the intrinsic or unweighted values. In this paper, using a simple model for self-association, theoretical expressions for the DLS, TDA and intrinsic diffusion coefficients are derived for self-associating solutes. The corresponding interaction parameters are also derived and compared. As expected, at low concentrations, it was found that the DLS gives higher diffusion coefficients than TDA and vice versa at high concentrations. More interestingly, points of inflection were found in the DLS and TDA diffusion coefficient-concentration curves which imply the presence of minima in measured interaction parameters with the TDA minimum occurring at a lower concentration than for DLS. These are absent in the intrinsic curves. Furthermore, it was found that whilst the intrinsic interaction parameter tends to a non-zero value in the limit of low concentrations, the measured interaction parameters vanish. These trends are important for the interpretation of the results obtained from both measurement methods especially when compared to the intrinsic values. With the interaction parameter being increasingly used as a measure of stability, consideration of these expected trends could prove valuable for explaining experimental data measured at low concentrations. Furthermore, they could prove important when comparisons are made between results from the two measurements which are increasingly being used as orthogonal methods for protein analysis.

Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C12E8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

Methodologies for the rapid determination of the diffusion interaction parameter using Taylor dispersion analysis

Using Taylor dispersion analysis to determine diffusion coefficients as a function of concentration in a single measurement.

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.

Size-based characterization of nanoparticle mixtures by the inline coupling of capillary electrophoresis to Taylor dispersion analysis

Separation of closely related nanoparticles is still a challenging issue for the characterization of complex mixtures for industrial/research applications or regulatory purposes. In this work, the remarkable separating performances of CE were complemented with the absolute size-based determination provided by Taylor dispersion analysis (TDA) for the characterization of nanoparticle mixtures. The inline hyphenation of CE to TDA was successfully implemented for the baseline separation followed by a size-based characterization of a bimodal mixture containing two closely size-related nanolatexes (70nm and 56nm radii). A pixel sensor UV area imager providing three detection points along the capillary was used for a differential measurement of the peak broadening during the Taylor dispersion step. Comparison of this new technique with dynamic light scattering and hydrodynamic chromatography is also discussed.

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 z-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.

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.

Application of the Exact Dispersion Solution to the Analysis of Solutes beyond the Limits of Taylor Dispersion.

Taylor dispersion analysis (TDA) is a fast and simple method for determining hydrodynamic radii. The method is applicable under conditions that allow the solute molecules to diffuse appreciably across the cross section of the flow before its measurement. This mitigates the effects of early stage convection on the dispersion and thus imposes a lower bound on the value of the diffusion coefficient measurable at a given flow speed. In this paper, we use the exact solution to the dispersion problem to analyze solutes outside the limits of TDA. Furthermore, by modeling the early stage convection, we analyze a mixture of two solutes with significantly different sizes that mimics heavily aggregated samples. The results obtained from the fits in both cases were in good agreement to the expected values. This demonstrates the potential of the model to extend dispersion analysis to regimes well outside the TDA limits such as the analysis of large molecules and the use of high flow-rates.

Truncation effect on Taylor–Aris dispersion in lattice Boltzmann schemes: Accuracy towards stability

The Taylor dispersion in parabolic velocity field provides a well-known benchmark for advection–diffusion (ADE) schemes and serves as a first step towards accurate modeling of the high-order non-Gaussian effects in heterogeneous flow. While applying the Lattice Boltzmann ADE two-relaxation-times (TRT) scheme for a transport with given Péclet number (Pe) one should select six free-tunable parameters, namely, (i) molecular-diffusion-scale, equilibrium parameter; (ii) three families of equilibrium weights, assigned to the terms of mass, velocity and numerical-diffusion-correction, and (iii) two relaxation rates. We analytically and numerically investigate the respective roles of all these degrees of freedom in the accuracy and stability in the evolution of a Gaussian plume. For this purpose, the third- and fourth-order transient multi-dimensional analysis of the recurrence equations of the TRT ADE scheme is extended for a spatially-variable velocity field. The key point is in the coupling of the truncation and Taylor dispersion analysis which allows us to identify the second-order numerical correction δkT to Taylor dispersivity coefficient kT. The procedure is exemplified for a straight Poiseuille flow where δkT is given in a closed analytical form in equilibrium and relaxation parameter spaces. The predicted longitudinal dispersivity is in excellent agreement with the numerical experiments over a wide parameter range. In relatively small Pe-range, the relative dispersion error increases with Péclet number. This deficiency reduces in the intermediate and high Pe-range where it becomes Pe-independent and velocity-amplitude independent. Eliminating δkT by a proper parameter choice and employing specular reflection for zero flux condition on solid boundaries, the d2Q9 TRT ADE scheme may reproduce the Taylor–Aris result quasi-exactly, from very coarse to fine grids, and from very small to arbitrarily high Péclet numbers. Since free-tunable product of two eigenfunctions also controls stability of the model, the validity of the analytically established von Neumann stability diagram is examined in Poiseuille profile. The simplest coordinate-stencil subclass, which is the d2Q5 TRT bounce-back scheme, demonstrates the best performance and achieves the maximum accuracy for most stable relaxation parameters.

Analytical mitigation of solute–capillary interactions in double detection Taylor Dispersion Analysis

Taylor Dispersion Analysis (TDA) in the presence of interactions between solutes and capillary walls yields inaccurate results for the diffusion coefficients of the solutes because the resulting concentration profiles are broadened and asymmetric. Whilst there are practical ways of mitigating these interactions, it is not always possible to eradicate them completely. In this paper, an analytical method of mitigating the effects of the adsorptions is presented. By observing the dispersion of the solute molecules at two detection points and using the expected relations between measured parameters, such as the standard deviations and peak amplitudes, the dispersive components of the profiles were isolated with a constrained fitting algorithm. The method was successfully applied to lysozyme and cytochrome C which adsorb onto fused silica capillary walls. Furthermore, this illustrates an advantage of using the fitting method for Taylor Dispersion Analysis.

Size characterization of commercial micelles and microemulsions by Taylor dispersion analysis

In this work, Taylor dispersion analysis was applied to the measurement of micelles (or microdroplets) molecular diffusion coefficient in micellar (or microemulsion) systems based on neutral/anionic/cationic or zwitterionic surfactants. The choice of the micellar marker and the influence the surfactant/marker concentrations on this determination are studied. Experimental results are compared to those derived from the literature using other experimental techniques. Taylor dispersion analysis, experienced in narrow capillaries, was found to be an efficient and suitable method for micelle (or microdroplet) size measurement due to: the low sample consumption, the absence of filtration requirement of the sample, the broad range of size determination (with no lower limit down to angstroms), the simplicity of the protocol, the possibility to measure the viscosity of surfactant solutions in given conditions and the determination of the weight-average micelle hydrodynamic radius. Application to the size-characterization of commercial microemulsions (Gelucire® 44/14), used as an excipient in the pharmaceutical formulation, is provided with a comparison to DLS measurements. It was found that the polydispersity in size of the micelle did not influence the Gaussian peak shape of the taylorgram due to rapid surfactant exchange compared to the time-scale of the experiments (a few minutes).