RESUMEN
Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of â¼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).
Asunto(s)
Electroforesis Capilar , Polielectrolitos/química , Electroforesis Capilar/métodos , Proteínas/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
Aggregation mechanisms of amyloid ß peptides depend on multiple intrinsic and extrinsic physicochemical factors (e.g., peptide chain length, truncation, peptide concentration, pH, ionic strength, temperature, metal concentration, etc.). Due to this high number of parameters, the formation of oligomers and their propensity to aggregate make the elucidation of this physiopathological mechanism a challenging task. From the analytical point of view, up to our knowledge, few techniques are able to quantify, in real time, the proportion and the size of the different soluble species during the aggregation process. This work aims at demonstrating the efficacy of the modern Taylor dispersion analysis (TDA) performed in capillaries (50 µm i.d.) to unravel the speciation of ß-amyloid peptides in low-volume peptide samples (â¼100 µL) with an analysis time of â¼3 min per run. TDA was applied to study the aggregation process of Aß(1-40) and Aß(1-42) peptides at physiological pH and temperature, where more than 140 data points were generated with a total volume of â¼1 µL over the whole aggregation study (about 0.5 µg of peptides). TDA was able to give a complete and quantitative picture of the Aß speciation during the aggregation process, including the sizing of the oligomers and protofibrils, the consumption of the monomer, and the quantification of different early- and late-formed aggregated species.
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Péptidos beta-Amiloides , Fragmentos de Péptidos , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/química , MetalesRESUMEN
Taylor dispersion analysis (TDA) allows the determination of the molecular diffusion coefficient (D) or the hydrodynamic radius (Rh) of a solute from the peak broadening of a plug of solute in a laminar Poiseuille flow. The main limitation plaguing the broader applicability of TDA is the lack of a sensitive detection modality. UV absorption is typically used with TDA but is only suitable for UV-absorbing or derivatized compounds. In this work, we present a development of the TDA method for non-UV absorbing compounds by using a universal detector based on refractive index (RI) sensing with backscattering interferometry (BSI). BSI was interfaced to a capillary electrophoresis-UV instrument using a polyimide coated fused silica capillary and an in-house designed flow-cell assembly. Polysaccharides were selected to demonstrate the application of TDA-BSI for size characterization. Under the conditions of validity of TDA, D and Rh average values and the entire Rh distributions were obtained from the (poly)saccharide taylorgrams, including non-UV absorbing polymers.
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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.
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Polilisina/química , Reología/métodos , Difusión , Hidrólisis , Cinética , Reología/instrumentaciónRESUMEN
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.
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Difusión , Nanoestructuras/química , Polímeros/química , Algoritmos , Cromatografía en Gel , Hidrodinámica , Modelos TeóricosRESUMEN
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.
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In this work, we investigate the possibility of optimizing the operating conditions, namely mobilizing pressure, capillary length and capillary radius, for performing Taylor dispersion analysis on solutes having hydrodynamic diameter, 2Rh, between 1 and 100 nm. Optimizing Taylor dispersion analysis means finding the set of operating conditions that verify the conditions of validity of this method, and finding the most appropriate conditions that may enhance or maximize the separation performances. Our conclusion is that the performances of Taylor dispersion analysis are independent of the operating conditions, as far as the conditions of validity of the method are verified. The inequalities defining the set of acceptable operating conditions are given in this work as a function of the maximal relative error on the diffusion coefficient, D, fixed by the user. These inequalities define operating zones that were represented for three typical capillary diameters (25, 50 and 100 µm). Within these zones, all experiments should lead to similar results on D (or Rh) and similar separation performances. It was concluded that assuming a 3% relative error on the determination of D, a 60 cm × 50 µm i.d. capillary can be used by default for performing TDA of analytes in the 1-100 nm diameter range with mobilizing pressure in the 50-100 mbar range.
Asunto(s)
Electroforesis Capilar/métodos , Algoritmos , Difusión , Electroforesis Capilar/instrumentación , PresiónRESUMEN
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.
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Polímeros/química , DifusiónRESUMEN
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.
RESUMEN
The hydrolysis of valine N-carboxyanhydride (NCA) in aqueous phosphate buffers was shown to proceed through nucleophilic catalysis via an aminoacyl phosphate intermediate that displays phosphorylating capabilities through a potentially prebiotic process that simulates modern biochemical metabolic pathways.
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Aminoácidos/química , Anhídridos/química , Organofosfatos/química , Hidrólisis , Cinética , Soluciones , Agua/químicaRESUMEN
We propose a scenario for the dynamic co-evolution of peptides and energy on the primitive Earth. From a multi component system consisting of hydrogen cyanide, several carbonyl compounds, ammonia, alkyl amine, carbonic anhydride, borate and isocyanic acid, we show that the reversibility of this system leads to several intermediate nitriles, that irreversibly evolve to alpha-amino acids and N-carbamoyl amino acids via selective catalytic processes. On the primitive Earth these N-carbamoyl amino acids combined with energetic molecules (NOx) may have been the core of a molecular engine producing peptides permanently and assuring their recycling and evolution. We present this molecular engine, a production example, and its various selectivities. The perspectives for such a dynamic approach to the emergence of peptides are evoked in the conclusion.