Taylor Dispersion Analysis Research Articles

Fast Characterization of Polyelectrolyte Complexes by Inline Coupling of Capillary Electrophoresis to Taylor Dispersion Analysis

The inline coupling of capillary electrophoresis (CE) to Taylor dispersion analysis was used for the characterization of polyelectrolyte complexes. The charge stoichiometry and the hydrodynamic radii of the two polyelectrolyte constituents were determined in a fully automated single run using standard commercial CE apparatus with a single detection point. The proposed methodology utilizes unusual high ionic strength (1.3 M) background electrolyte to obtain the dissociation and electrophoretic separation of polyelectrolyte constituents. Such highly saline conditions in combination with neutrally coated capillary were found to avoid any polyelectrolyte interactions onto the capillary surface. This innovative methodology should greatly contribute to simplify and accelerate the characterization of polyelectrolyte complexes or polyplexes.

Measurement of drug diffusivities in pharmaceutical solvents using Taylor dispersion analysis

Knowledge of drug diffusivity is of key importance in the understanding of a number of pharmaceutical and biological processes. However, experimentally determined diffusion coefficients and hydrodynamic radii are only reported for a limited number of drug substances. In this work, Taylor dispersion analysis conducted using capillary electrophoresis instrumentation coupled with a UV imaging detector, with two detection windows along the capillary, is introduced as a powerful method for the determination of drug diffusivities in nanoliter samples. Several potential advantages associated with applying two detection windows instead of one window as done in most previous studies were identified. Overall diffusion coefficient measurements performed using two detection windows are more robust and correction for changes in flow rate and sample volume is not required. The experimental conditions applied were suboptimal for performing single detection window measurements due to the relatively large sample volumes and may be optimized to alleviate the need for tedious correction procedures for this setup. The diffusivities of eleven aromatic compounds in water at 25 °C were determined, and showed a good agreement with the literature values. Furthermore, the diffusivities and hydrodynamic radii of four selected drug substances were determined in acetonitrile, methanol, isopropyl myristate, medium chain triglyceride, and propylene glycol in addition to water. The solvent viscosity was determined simultaneously along with the measurement of analyte diffusivity. Drug diffusivities decreased with increasing solvent viscosity. Taylor dispersion analysis is a robust, simple and automated method of quantification of diffusion coefficients even in media with a relatively higher viscosity than water.

A Taylor dispersion analysis method for the sizing of therapeutic proteins and their aggregates using nanolitre sample quantities

The growing number of Biosimilars now being approved for development lends the need to develop new analytical techniques for rapid, cost effective analysis of these high value biotherapeutics. The presence of aggregates in biopharmaceutical products is undesirable for many reasons. A major concern is the potential immunogeneic response that aggregates can induce on administration. The detection of low levels of aggregated proteins in solution may only be determined by a limited number of techniques, many of which require in-depth method development, multi-stage sample preparation and lengthy time of analysis. We explore the use of a novel analytical instrument using UV area imaging and Taylor dispersion analysis (TDA) to determine the hydrodynamic radius of BSA in an aggregated state and monitor it with time. Protein aggregation and its reversibility over time has been measured for a number of BSA samples (stressed and unstressed) by TDA with the results obtained being compared to those obtained from dynamic light scattering (DLS) and microcalorimetry. Correlations between the techniques for investigating protein aggregation behaviour were explored. The reproducibility of TDA measurements enabled the stability and reversibility of BSA aggregates to be more readily monitored than by using the other techniques.

Taylor Dispersion Analysis Compared to Dynamic Light Scattering for the Size Analysis of Therapeutic Peptides and Proteins and Their Aggregates

PurposeTo evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS).MethodsThe hydrodynamic radius of oxytocin, bovine serum albumin, various monoclonal antibodies (type IgG) and etanercept at concentrations between 0.05 and 50 mg/ml was determined by TDA and DLS. IgGs and etanercept were stressed (elevated temperatures) and analyzed by TDA, DLS and HP-SEC.ResultsTDA and DLS were comparable in sizing non-stressed peptides and proteins in a concentration range of about 0.5 to 50 mg/ml. TDA performed well even at lower concentrations, where DLS tends to provide theoretically high values of the Z-average radius. However, because of differences in the detection physics, DLS was more weighted towards the detection of aggregates in stressed formulations than TDA. Advantageously, TDA was also able to size the small peptide oxytocin, which was not feasible by DLS.ConclusionTDA allows the accurate determination of the hydrodynamic radius of peptides and proteins over a wide concentration range, with little interference from excipients present in the sample. It is marginally less sensitive than DLS in detecting size increase for stressed protein samples.

A nanolitre method to determine the hydrodynamic radius of proteins and small molecules by Taylor dispersion analysis

The escalating number of new therapeutic biopharmaceuticals being developed and their high value increases the need for the development of novel analytical technologies. Faster analysis time, high accuracy, low sample consumption and the ability to monitor process flow are all essential prerequisites. We evaluate a novel analytical instrument that combines UV area imaging and Taylor dispersion analysis (TDA) to determine the hydrodynamic radius of proteins and small molecules in solution. Benchmarking the results against dynamic light scattering, we report the influence of injection system, injection volume, flow rates, analyte concentration and highlight the importance of washing procedures. Issues arising from the manual injection valve in the alpha laboratory system that led to high standard deviations were eliminated by incorporating an automated injector in a beta system. The hydrodynamic radii obtained show good correlation with literature values and in most cases a relative standard deviation of less than 5%. The system is fully automated after coupling to the CE which allows for multiple injections and sample/buffer changes without operator intervention. The small sample size (approx. 60 nL), the lack of sample preparation required, and the speed of analysis (approx. 2–3 mins) makes this instrument highly applicable to the real-time analysis of inherently unstable, high cost biopharmaceutical materials where understanding their aggregation state and size is important.

Physicochemical characterization of a PEGylated liposomal drug formulation using capillary electrophoresis

In this work, the applicability of using CE to perform a physicochemical characterization of a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was examined. Characterization of the liposomal drug formulation using CE instrumentation encompassed: determination of the electrophoretic mobilities, size determination by Taylor dispersion analysis and interaction studies. Electrophoretic mobilities determined by CE were compared with the results obtained by laser Doppler electrophoresis, which were found to be subject to larger variation. Average hydrodynamic diameters of the liposome preparations, as determined by Taylor dispersion analysis, were in the range of 61-84 nm and were compared with the results obtained by dynamic light scattering. Interactions between oxaliplatin (and paracetamol) and the PEGylated liposome were non-detectable by CE frontal analysis as well as by liposome electrokinetic chromatography. In contrast, for the more lipophilic compound propranolol, apparent liposome-aqueous phase distribution coefficients (D(lip) ) were successfully determined by both electrokinetic chromatography (log D(lip) =2.10) and by CE frontal analysis (log D(lip) =2.14). It is envisioned that CE and capillary-based techniques, including Taylor dispersion analysis, will be useful tools for the characterization of nanoparticulate (e.g. liposomal) drug formulations.

Characterization of Asphaltenes by Nonaqueous Capillary Electrophoresis

Nonaqueous capillary electrophoresis was used for the separation and characterization of asphaltene samples from different sources. For the separation medium (background electrolyte), mixtures of tetrahydrofuran and a high-permittivity organic solvent could be used. The best results were obtained with an 80:20 mixture of tetrahydrofuran and acetonitrile, containing 1−10 mM of lithium perchlorate. In this separation medium, asphaltene samples were found to be composed of two fractions that could be clearly separated: one fraction of neutral species and a fraction that carries a positive charge in the solvent mixture employed. Between samples of different origin, differences were found in the relative amounts of the neutral and the charged fractions and in the average electrophoretic mobility of the charged components. Taylor dispersion analysis was applied to estimate the average diffusion coefficient of the asphaltene species in the solvent mixture used. From the results, it is concluded that the asphaltenes are present as nanoaggregate clusters of 3000−4000 Da and that the charged aggregates carry a net charge of approximately +1. The possible correlation between the electrophoretic properties of asphaltenes in crudes of different origin and their field behavior is discussed.

Influence of Molecule Size on Its Transport Properties through a Porous Medium

The mass transfer kinetics of toluene and polystyrenes (of which the M(w) varies from 162 to 1.85 x 10(6) g mol(-1)) through columns filled with silica porous spheres were studied by inverse size exclusion chromatography. The mass transfer parameters were measured by modeling the band broadening of the chromatograms. The experimental height equivalent to a theoretical plate (HETP) data were analyzed using the general rate model in order to determine the effective diffusion coefficient in porous particles as a function of molecular size. The bulk molecular diffusion coefficients were experimentally determined by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA). The topological tortuosity of the porous particles was determined by electrical measurements. The effective molecular diffusion coefficient through porous particles was modeled taking into account exclusion, friction, and at last tortuosity effects. A phenomenological law is proposed to model the evolution of the tortuosity experienced by a molecule in a porous particle as a function of its size. It gives a good prediction of the evolution of effective diffusion coefficient with the molecule/pore size ratio.

Determination of Individual Diffusion Coefficients in Evolving Binary Mixtures by Taylor Dispersion Analysis: Application to the Monitoring of Polymer Reaction

This study demonstrates that it is possible to get valuable information on the individual populations of a binary mixture from the signal obtained by Taylor dispersion analysis (TDA). In the case of mixtures composed of two populations of different sizes (such as a monomer/polymer mixture), the information available from TDA is not restricted to an average diffusion coefficient or an average hydrodynamic radius calculated on the entire binary mixture. In this work, TDA was used to monitor a polymerization reaction. In this scope, it has been possible to determine the degree of conversion and the weight average hydrodynamic radius of the polymer at different reaction times. Three different methods are proposed for the data processing of taylorgrams derived from polymerization mixtures or, more generally, for taylorgrams of binary mixtures. These three methods, either based on deconvolution or on integration of the signal, were found to give similar results. TDA results obtained for a model binary mixture of acrylamide and standard polyacrylamide were consistent with DLS experiments provided that the differences in the type of average hydrodynamic radius values between the two methods are taken into account. An example of application to the monitoring of acrylamide radical polymerization is shown.

Simultaneous Evaluation of Ligand Binding Properties and Protein Size by Electrophoresis and Taylor Dispersion in Capillaries

The interplay between biophysical characteristics such as protein size and shape and protein function is difficult to ascertain using simple methods. Here, we present an approach for characterizing both protein-ligand binding as well as protein hydrodynamic radius in one operation combining electrophoresis and size measurement by dispersion using capillaries. The methodology is based on the integration of Taylor dispersion analysis and capillary electrophoresis and is here demonstrated using commercially available capillary electrophoresis instrumentation modified with a pixel sensor UV area imager, allowing two detection points along the capillary. Analytes are the human serum proteins alpha(1)-acid glycoprotein and albumin interacting with the drug propranolol in a frontal analysis mode. Upon introduction of the propranolol-protein sample, voltage is initially applied to facilitate electrophoretically mediated separation of ligand and protein and frontal analysis. Then a pressure mobilization step is used whereby Taylor dispersion can be characterized online based on the signal from the UV area imager. Estimates of ligand binding and values for hydrodynamic radii agree with values obtained by independent methods.

Size-Based Characterization of an Ionic Polydiacetylene by Taylor Dispersion Analysis and Capillary Electrophoresis

This work focuses on the size-based characterization of a water-soluble ionic polydiacetylene with a polycationic structure, poly-[(1,6-bis(N-methylimidazolium)hexa-2,4-diyne)dibromide]. This polymer could not be characterized using classical analytical techniques such as size-exclusion chromatography and MALDI−TOF mass spectrometry due to problems of purification, low quantities available, and difficult laser desorption. The work presented here demonstrates the interest and the complementarity of two independent analytical methods, Taylor dispersion analysis (TDA) and capillary electrophoresis (CE), that require only very small amounts of sample (only a few nanoliters are injected) and that can be easily implemented on commercially available capillary electrophoresis apparatus. TDA is a nonseparative method that allows the absolute determination of the average hydrodynamic radius of the polymer. This method does not require the determination of the polymer concentration in the sample and is not perturbed by the presence of residual monomer. Since the average hydrodynamic radius determined by this method is a weight average value, it also gives information complementary to the average value derived from dynamic light scattering measurements. Simple hydrodynamic modeling allows estimation of a minimal value for the average degree of polymerization. Free solution CE can be used for monitoring the polymerization process and quantifying the degree of conversion. Furthermore, entangled polymer solution CE was used as a size-based separation technique for the characterization of the molar mass distribution using calibration with polyvinylpyridine standards. Number and weight molar mass distributions of the sample were obtained relative to this calibration.

Size-Based Characterization by the Coupling of Capillary Electrophoresis to Taylor Dispersion Analysis

In this work, a new methodology is presented for performing capillary electrophoresis (CE) coupled to Taylor dispersion analysis (TDA). The CE step allows the separation of the different compounds of the injected mixture, while the diffusion coefficient related to each sample zone can be derived from the subsequent TDA step. TDA is an absolute and straightforward nonseparative method allowing the determination of the diffusion coefficient (or hydrodynamic radius) from the peak dispersion obtained in an open tube under Poiseuille laminar flow conditions. With a mass concentration sensitive detector, the hydrodynamic radius derived from TDA is a weight average value calculated upon all the molecules present in the sample zone. Since CE can be hardly coupled to light scattering detection for technical reasons (low volumes, short detection path length), TDA represents an interesting alternative for the size characterization, without calibration, of sample mixtures using CE-based separation techniques. The coupling of CE to TDA can be implemented on a commercial CE apparatus.

Ballistic dispersion in temperature gradient focusing

Molecular dispersion is caused by both molecular diffusion and non-uniform bulk fluid motion. While the Taylor–Aris dispersion regime is the most familiar regime in microfluidic systems, an oft-overlooked regime is that of purely kinematic (or ballistic) dispersion. In most microfluidic systems, this dispersion regime is transient and quickly gives way to Taylor–Aris dispersion. In electrophoretic focusing methods such as temperature gradient focusing (TGF), however, the characteristic time scales for dispersion are fixed, and focused peaks may never reach the Taylor limit. In this situation, generalized Taylor dispersion analysis is not applicable. A heuristic model is developed here which accounts for both molecular diffusion and advective dispersion across all dispersion regimes, from pure diffusion to Taylor dispersion to pure advection. This model is compared to results from TGF experiments and accurately captures both the initial decrease and subsequent increase in peak widths as electric field strength increases. The results of this combined analytical and experimental study provide a useful tool for estimation of dispersion and optimization of TGF systems.

Taylor Dispersion Analysis of Mixtures

Taylor dispersion analysis (TDA) is a fast and simple method for determining hydrodynamic radii. In the case of sample mixtures, TDA, as the other nonseparative methods, leads to an average diffusion coefficient on the different molecules constituting the mixture. We set in this work the equations giving, on a consistent basis, the average values obtained by TDA with detectors with linear response functions. These equations confronted TDA experiments of sample mixtures containing different proportions of a small molecule and a polymer standard. Very good agreement between theory and experiment was obtained. In a second part of this work, on the basis of monomodal or bimodal molar mass distributions of polymers, the different average diffusion coefficients corresponding to TDA were compared to the z-average diffusion coefficient (D(z)) obtained from dynamic light scattering (DLS) experiments and to the weight average diffusion coefficient (D(w)). This latter value is sometimes considered as the most representative of the sample mixture. From these results, it appears that, for monomodal distribution and relatively low polydispersity (I = 1.15), the average diffusion coefficient generally derived from TDA is very close to Dw. However, for highly polydisperse samples (e.g., bimodal polydisperse distributions), important differences could be obtained (up to 35% between TDA and D(w)). In all the cases, the average diffusion coefficient obtained by TDA for a mass concentration detector was closer to the Dw value than the z-average obtained by DLS.

Determination of Dendrigraft Poly-l-Lysine Diffusion Coefficients by Taylor Dispersion Analysis

This work focuses on the physicochemical characterization of dendrigraft poly-L-lysines (DGLs) obtained by polymerization of N-carboxyanhydride in buffered water (pH 6.5). Diffusion coefficients (D) and hydrodynamic radii (Rh) of five successive DGL generations were determined by Taylor dispersion analysis (TDA). To our knowledge, this is the first experimental work using TDA for the characterization of dendrimer-like structures. Experimental Rh values obtained by TDA were compared to those derived from dynamic light scattering and size exclusion chromatography coupled to a triple detection (refractive index, viscosimetry, and static light scattering). Significant differences were obtained, especially for the highest generations, as a result of the inherent contribution of aggregates to the light scattering intensity. For that reason, TDA was found to be the most appropriate technique for determining the D values of these hyperbranched macromolecules. Regarding their physicochemical behavior, the experimental results confirm that DGLs are very similar to trifunctional dendrimers (exponential growth of the molar mass, almost linear variation of the hydrodynamic radius, high branching density, and maximum of the intrinsic viscosity or of the free volume fraction for generation 4).

Dynamics of field-amplified sample stacking

Field-amplified sample stacking (FASS) uses conductivity gradients and resulting non-uniform electromigration fluxes to effect concentration increases of analyte ions. For cases where the initial sample concentration is much smaller than the background electrolyte (BGE) concentration, the ideal maximum concentration enhancement is equal to , the ratio of conductivity of the sample solution to that of the BGE. However, in practice both molecular diffusion and convective dispersion limit concentration enhancement. We present a theoretical and experimental study of concentration enhancement using FASS. We model the FASS process as electromigration, diffusion, and advection of two background electrolyte ions and multiple sample species across a known initial concentration gradient. Regular perturbation methods and a generalized Taylor dispersion analysis are used to derive area-averaged species conservation and electric field equations. The model predicts the spatial and temporal development of background electrolyte concentration field, electric field, and sample-ion distribution of the FASS process. We have validated this model using on-chip FASS experiments. We use an acidified poly(ethylene oxide) (PEO) coating to minimize dispersion due to electro-osmotic flow (EOF), and thereby evaluate the low- (but finite) dispersion regime of most interest. We have used CCD-based quantitative epifliuorescence imaging to quantify unsteady concentration fields and validate the model. This experimentally validated model is useful in developing optimal designs of sample stacking assay devices.

Comparison of methods for the determination of diffusion coefficients of polymers in dilute solutions: The influence of polydispersity

A comparison between various methods to determine diffusion coefficients of polymers in dilute solutions has been made. It is shown that Taylor dispersion analysis (TDA), dynamic light scattering (DLS), hydrodynamic chromatography (HDC), and size exclusion chromatography (SEC) can all be used to accurately determine diffusion coefficients when the polymer samples have low polydispersities. By the analysis of a series of practically representative styrene acrylonitrile copolymer (SAN) samples, it is shown that polydispersity of the samples and the presence of low-molecular-mass material cause considerable differences between the methods. It was found that TDA is mostly disturbed by the presence of low-molecular-mass material, whereas DLS is more sensitive to the polydispersity of the polymer. With broad samples, DLS gives the Z-average diffusion coefficient. SEC can be used to obtain a diffusion coefficient distribution as well as an average diffusion coefficient of a polydisperse sample. Although, the same was expected for HDC, it was found that this method could only be successfully used for polymer samples having low polydispersities. Deviations between SEC, HDC, and TDA found for narrow samples were not related to the chemical composition of the samples.

Sampling for Liriomyza huidobrensis (Diptera: Agromyzidae) Larvae and Damage in Celery

A sampling plan to estimate population levels of Liriomyza huidobrensis (Blanchard) larvae and total mining damage was developed for use in celery. Dispersion indices from Taylor's power law were calculated from 1992 data obtained from whole plant samples collected from commercial fields at two times during the cropping season and utilizing one of three different leafminer management strategies. Analysis of covariance indicated that variance–to–mean relationships were consistent among petiole position among plants, pest management strategies, and time of growing season for live L. huidobrensis larvae and total numbers of mines per petiole. In an attempt to develop a presence–absence sampling plan, all equation was derived to relate the proportion of petioles infested, P(l), to the mean number of live larvae or the mean number of mines per petiole. However, predicted frequencies of infested petioles delivered from this equation were significantly greater than the frequencies of petioles infested with live larvae or infested with mines observed in validation studies. This lack of fit between predicted and expected frequencies precludes use of a presence–absence sampling plan to predict mean densities from the proportion of leaves infested. The results from Taylor's dispersion analysis were also used to evaluate a constant precision–level sampling plan. Validation tests of this sampling plan indicated that the formula used to ca1culate critical stoplines accurately predicted the observed cumulative totals of live larvae or total numbers of mines. Implementation of a sequential sampling plan for L. huidobrensis larvae with a 0.25 level of precision and with a possible maximum sample size of 100 petioles (to compensate for the prohibitively large sample sizes necessary at low densities) should greatly reduce the amount of labor necessary to achieve accurate estimates of population densities and to facilitate the use of action thresholds based upon these estimates.

Sedimentation and dispersion of Brownian particles in spatially periodic potential fields

A generalized Taylor dispersion analysis is presented of the combined diffusion and sedimentation of a Brownian particle subjected to a spatially periodic potential plus a uniform force. At long times this transport process is macroscopically characterized by a uniform mean drift velocity vector of the Brownian particle, and by a dispersion dyadic that quantifies stochastic ‘‘spreading’’ about the (traveling) mean position. A general explicit integral formula is derived for the dispersion coefficient in the one-dimensional case, which supplements known results for the mean velocity. Illustrative calculations show that, for sufficiently large values of the drift force, the dispersivity exceeds the molecular diffusivity, which characterizes dispersion in the absence of any potential barriers. These calculations also lead to the surprising conclusion that an increase in the height of the potential barriers can sometimes lead to an increase in the dispersivity. A general asymptotic analysis is developed for the limiting case where the drift force tends to infinity. Although in one dimension the dominance of the drift force over the potential leads to a spatially uniform, steady-state, periodic distribution, the same behavior does not generally obtain in higher-dimensional cases. Asymptotic expressions are derived for the mean velocity and dispersivity characterizing one-dimensional systems and a class of two-dimensional systems. The analysis is subsequently applied to the related problem of sedimentation and dispersion of a particle within a medium for which its molecular diffusivity is a periodic function of position (in the absence of any potential). Although a periodic potential and a periodic diffusivity lead to equivalent mathematical problems in the absence of a sedimentation force, the presence of drift leads to important qualitative differences.

Convective and dispersion models for dispersed phase axial mixing—the significance of polydispersity effects in liquid-liquid contactors

A non-uniform dispersed phase drop size distribution results in non-uniform drop rise velocities, which make a significant contribution to the dispersed phase axial mixing. The level of axial mixing is usually quantified using one of two commonly employed global models—the convective model and the dispersion model. The phenomenological coefficients appearing in each model have been determined by matching the first few global moments to the corresponding local ones in a generalized Taylor dispersion analysis. The convective model and the dispersion model are shown to be essentially equivalent in the sense that the same moments may be matched in either case. Simple asymptotic results are presented for the dispersion coefficients in terms of the local transport rates, which include the external mass transfer from the drop to the continuous phase, the internal diffusional resistance, and the drop terminal velocity. The analysis reveals the importance of interphase mass transfer in the dispersion process. If mass transfer is not possible, the dispersed phase exhibits non-Gaussian behavior in the limit of no coalescence or breakage. If the solute is soluble in both phases and mass transfer occurs, tracer curves are approximately Gaussian, but only for times long compared to the mass transfer time. The initial transient period required to reach this asymptotic behavior is not negligible for many systems of practical interest, indicating that a constant coefficient axial dispersion model is often inapplicable even when the long-time dispersion coefficient is constant. In addition, the long-time dispersion coefficient is shown to be sensitive to the mass transfer rate. These results raise serious questions about the procedures recommended in the past for measuring dispersion coefficients.