Capillary electrophoresis sequencing of small ssDNA molecules versus the Ogston regime: fitting data and interpreting parameters

We study the mobility of short ssDNA fragments (approximately 30-500 bases) separated by capillary electrophoresis in entangled polymer solutions. Although this corresponds to what is commonly called the Ogston regime, the corresponding sieving concept has never been defined properly nor tested quantitatively. We consider three formulas that have been suggested to fit data in this range of ssDNA sizes, and we discuss how their free parameters are related to actual physical parameters. We test these formulas with new data obtained in our laboratory using a commercial poly-N,N-dimethylacrylamide sieving matrix. Our results show that all three formulas provide decent fits. However, the traditional Ogston equation produces fitting parameters that appear to lack physical meaning. Surprisingly, all three approaches predict that the effective pore size and fiber radius are almost equal. This is the first step towards the development of a systematic approach to optimizing sequencing systems for this size range.     

Dynamic light‐scattering measurement of sieving polymer solutions for protein separation on SDS CE

We evaluated the mesh size and homogeneity of polymer network by dynamic light scattering and discussed the relationship between the physical properties of polymer network and the protein separation behavior by capillary polymer electrophoresis. We compared three kinds of sieving polymers in solutions with a wide range of molecular weights and concentrations: polyacrylamide and polyethylene oxide as flexible polymers, and hydroxyethyl cellulose as a semiflexible polymer. We found that the mobility of protein was dominated primarily by the mesh size ξ, irrespective of the type of sieving polymers, and the peak spacing between protein peaks increased drastically in the range of ξ<10 nm, where the mobility also decreased. And the peak widths were dependent on the molecular species of sieving polymers and their homogeneity of polymer network. We proposed that a polymer network with a homogenous mesh size of less than 10 nm is the best sieving medium for separation of the proteins in the molecular weight range 14 300–97 200 Da from the view point of the resolution in protein separation.     

Separation performance of single-stranded DNA electrophoresis in photopolymerized cross-linked polyacrylamide gels

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models.     

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.     

Theoretical and experimental study of the achievable separation power in resistive‐glass atmospheric pressure ion mobility spectrometry

We present a detailed investigation of the performance of our previously reported nanoelectrospray high‐resolution resistive‐glass atmospheric pressure drift tube ion mobility spectrometer constructed with monolithic resistive‐glass desolvation and drift regions. Using experimental spectral data and theoretical pulse width and diffusion variables, we compare theoretical and experimental resolving powers achievable under a variety of field strengths and ion gate pulse widths. The effects of instrumental and operational parameters on the resolution achievable in chromatographic terms are also discussed. Following characterization of the separation power of the instrument, experimental spectral peak width data is fitted by a least‐squares procedure to a pre‐existing semi‐empirical model developed to study contributions to peak width other than initial pulse width and diffusional broadening. The model suggests possible contributions to the final measured peak width from electric field inhomogeneity and minor contributions from instrumental parameters such as anode size, anode‐to‐anode grid distance and drift gas flow rate. The model also reveals an unexpected ion gate width dependence on the final measured peak width that we attribute to non‐ideal performance of the Bradbury‐Nielsen ion gate and limitations in the design of our pulsing‐electronics. Copyright © 2010 John Wiley & Sons, Ltd.     

DNA electrophoresis in agarose gels: effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.     

Identification of peaks in capillary zone electrophoresis based on actual mobilities.

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.     

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.     

Modeling the electrophoretic mobility and diffusion of weakly charged peptides

A bead model to determine the electrophoretic mobilities and translational diffusion constants of weakly charged peptides is developed that is based on a approximate structural model of peptides and is also grounded in electrohydrodynamic theory. A peptide made up of X amino acids is modeled as N=2X beads with 2 beads representing each amino acid in the chain. For the two beads representing a particular amino acid in a peptide, the radius of one bead is set to one-half the nearest neighbor Calpha-Calpha distance, and the radius of the other bead is chosen on the basis of the diffusion constant of the free amino acid. Peptide conformations, which are defined by a set of psi-phi dihedral angles, are randomly generated by using the transformation matrix approach of Flory (Flory, P. Statistical Mechanics of Chain Molecules; John Wiley: New York, 1969) and rejecting conformations which result in bead overlap. The mobility and diffusion constants are computed for each conformation and at least 100 independent conformations are examined for each peptide. In general, the mobility is found to depend only weakly on peptide conformation. Model and experimental mobilities are compared by examining the data of Janini and co-workers (Janini, G.; et al. J. Chromatogr. 1999, 848, 417-433). A total of 58 peptides consisting of from 2 to 39 amino acids are considered. The average relative error between experimental and model mobilities is found to be 1.0% and the rms relative error 7.7%. In specific cases, the discrepancy can be substantial and possible reasons for this are discussed. It should be emphasized that the input parameters of the peptide model are totally independent of experimental mobilities. It is hoped that the peptide model developed here will be useful in the prediction of peptide mobility as well as in using peptide mobilities to extract information about peptide structure, conformation, and charge. Finally, we show how simultaneous measurements of translational diffusion and mobility can be used to estimate peptide charge.     

Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes. The model assumes that the electrophoretic mobility can be represented by a product of three functions that return the contributions of peptide charge, length and width, respectively to the mobility. The model relies on accurate experimental determination of the electrophoretic mobilities of a diverse set of peptides, by capillary zone electrophoresis (CZE), at 22 degrees C, with a 50 mM phosphate buffer, at pH 2.5. The electrophoretic mobilities of a basis set of 102 peptides that varied in charge from 0.65 to 16 and in size from two to 42 amino acid residues were accurately measured at these fixed experimental conditions using a stable 10% linear polyacrylamide-coated column. Data from this basis set was used to derive the peptide charge, length, and width functions respectively. The main purpose of this endeavor is to use the model for the prediction of peptide mobilities at pH 2.5, and for simulation of CZE peptide maps of protein digests. Excellent agreement was obtained between predicted and experimental electrophoretic mobilities for all categories of peptides, including the highly charged and the hydrophobic. To illustrate the utility of this model in protein studies it was used to simulate theoretical peptide maps of the digests of glucagon and horse cytochrome c. The resulting maps were compared and contrasted with their experimental counterparts. The potential of this approach and its limitations are discussed.     

Temperature dependence of charge mobility in model discotic liquid crystals

We address the calculation of charge carrier mobility of liquid-crystalline columnar semiconductors, a very promising class of materials in the field of organic electronics. We employ a simple coarse-grained theoretical approach and study in particular the temperature dependence of the mobility of the well-known triphenylene family of compounds, combining a molecular-level simulation for reproducing the structural changes and the Miller-Abrahams model for the evaluation of the transfer rates within the hopping regime. The effects of electric field, positional and energetic disorder are also considered. Simulations predict a low energetic disorder (~0.05 eV), slightly decreasing with temperature within the crystal, columnar and isotropic phases, and fluctuations of the square transfer integral of the order of 0.003 eV(2). The shape of the temperature-dependent mobility curve is however dominated by the variation of the transfer integral and barely affected by the disorder. Overall, this model reproduces semi-quantitatively all the features of experimentally measured mobilities, on one hand reinforcing the correctness of the hopping transport picture and of its interplay with system morphology, and on the other suggesting future applications for off-lattice modeling of organic electronics devices.     

Capillary electrophoresis of RNA in dilute and semidilute polymer solutions

We report separations of RNA molecules (281-6583 nucleotides) by capillary electrophoresis in dilute and semidilute solutions of aqueous hydroxyethylcellulose (HEC) ether in varying buffers. RNA mobility and peak band widths are examined under both nondenaturing and also denaturing conditions. From studies of sieving polymer concentration and chain length, it is found that good separations can be obtained in semidilute solutions as well as in dilute solutions. The dependence of RNA mobility on its chain length is consistent with separation by a similar to transient entanglement mechanism in dilute solutions. In semidilute entangled solutions the separation proceeds by segmental motion.     

Low-conductivity background electrolytes in capillary zone electrophoresis--myth or reality?

The asymmetric triangle (fronting or tailing) concentration profiles and their broadening are the typical results of the electromigrational zone dispersion characterizing a system of the analyte in the background electrolyte (BGE). The present contribution suggests the parameter named the relative velocity slope, SBGE,X, which was introduced here as a quantity characterizing the peak broadening and the asymmetry. SBGE,X VS. analyte ionic mobility diagrams are suitable for the comparison of BGEs of given pH and the conductivity composed of electrolytes of different pKaS and ionic mobilities. The concept of SBGE,X diagrams is verified by capillary zone electrophoresis of the model analytes, which involve (i) the series of sulfobenzoylated poly(ethylene glycols) as examples of the strong electrolytes with different ionic mobilities and (ii) the series of monobasic phenols as weak electrolytes with different pKaS and similar ionic mobilities. It follows from both theoretical predictions of peak symmetry and their experimental verification that the optimum composition of BGEs is determined mostly by the suitable ionic mobility of the coion in dependence on the ionic mobility of the analyte. The low-conductivity BGEs based on low-molecular carrier ampholytes are at best only comparable with the properly chosen monobasic electrolytes.     

Calculation of electrophoretic mobility in mixed solvent buffers in capillary zone electrophoresis using a mixture response surface method

The electrophoretic mobilities of three beta-blocker drugs, practolol, timolol and propranolol, have been measured in electrolyte systems with mixed binary and ternary water-methanol-ethanol solvents with acetic acid/sodium acetate as buffer using capillary electrophoresis. The highest mobilities for the analytes studied have been observed in pure aqueous, the lowest values in ethanolic buffers. The measured electrophoretic mobilities have been used to evaluate the accuracy of a mathematical model based on a mixture response surface method that expresses the mobility as a function of the solvent composition. Mean percentage error (MPE) has been computed considering experimental and calculated mobilities as an accuracy criterion. The obtained MPE for practolol, timolol and propranolol in the binary mixtures are between 0.9 and 2.6%, in the ternary water-methanol-ethanol solvent system the MPE was about 2.7%. The MPE values resulting from the proposed equation lie within the experimental relative standard deviation values and can be considered as an acceptable error.     

Effective interactions in lysozyme aqueous solutions: a small-angle neutron scattering and computer simulation study

We report protein-protein structure factors of aqueous lysozyme solutions at different pH and ionic strengths, as determined by small-angle neutron scattering experiments. The observed upturn of the structure factor at small wavevectors, as the pH increases, marks a crossover between two different regimes, one dominated by repulsive forces, and another one where attractive interactions become prominent, with the ensuing development of enhanced density fluctuations. In order to rationalize such experimental outcome from a microscopic viewpoint, we have carried out extensive simulations of different coarse-grained models. We have first studied a model in which macromolecules are described as soft spheres interacting through an attractive r(-6) potential, plus embedded pH-dependent discrete charges; we show that the uprise undergone by the structure factor is qualitatively predicted. We have then studied a Derjaguin-Landau-Verwey-Overbeek (DLVO) model, in which only central interactions are advocated; we demonstrate that this model leads to a protein-rich/protein-poor coexistence curve that agrees quite well with the experimental counterpart; experimental correlations are instead reproduced only at low pH and ionic strengths. We have finally investigated a third, "mixed" model in which the central attractive term of the DLVO potential is imported within the distributed-charge approach; it turns out that the different balance of interactions, with a much shorter-range attractive contribution, leads in this latter case to an improved agreement with the experimental crossover. We discuss the relationship between experimental correlations, phase coexistence, and features of effective interactions, as well as possible paths toward a quantitative prediction of structural properties of real lysozyme solutions.     

Impact of Fluoroalkylation on the n-Type Charge Transport of Two Naphthodithiophene Diimide Derivatives

In this work, we investigate two recently synthesized naphthodithiophene diimide (NDTI) derivatives featuring promising n-type charge transport properties. We analyze the charge transport pathways and model charge mobility with the non-adiabatic hopping mechanism using the Marcus-Levich-Jortner rate constant formulation, highlighting the role of fluoroalkylated substitution in α (α-NDTI) and at the imide nitrogen (N-NDTI) position. In contrast with the experimental results, similar charge mobilities are computed for the two derivatives. However, while α-NDTI displays remarkably anisotropic mobilities with an almost one-dimensional directionality, N-NDTI sustains a more isotropic charge percolation pattern. We propose that the strong anisotropic charge transport character of α-NDTI is responsible for the modest measured charge mobility. In addition, when the role of thermally induced transfer integral fluctuations is investigated, the computed electron–phonon couplings for intermolecular sliding modes indicate that dynamic disorder effects are also more detrimental for the charge transport of α-NDTI than N-NDTI. The lower observed mobility of α-NDTI is therefore rationalized in terms of a prominent anisotropic character of the charge percolation pathways, with the additional contribution of dynamic disorder effects.     

Capillary electrophoresis in sieving matrices: selectivity per base, mobility slope, and inflection slope

We compare the migration behavior of DNA sequencing fragments in hydroxyethyl cellulose (HEC) to the theoretical model of migration in the reptation mode. Good agreement was found for the mobility curve. We derived empirical equations for the relationship between selectivity per base and sieving matrix concentration and between the mobility slope and matrix concentration. We propose the inflection slope, i.e., the slope of the log-log mobility curve at its inflection point, as the quantitative parameter of sieving performance.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation of single-stranded DNAs using DNA conjugates having different migration properties in capillary electrophoresis

A probe-regulated simultaneous separation (PRESS) using capillary electrophoresis (CE) was developed for separating single-stranded (ss) DNAs. We synthesized two DNA conjugate probes, -(5'-TGTGTGTGT-3')p-AAm(q)- and -(5'-GCCACCAGC-3')m-AAm(n)-, by copolymerizing 5'-methacryloyl-modified ssDNA with acrylamide (AAm), and characterized them in detail. The two probes showed lower electrophoretic mobilities than 5'-methacryloyl-modified ssDNAs. Furthermore, -(5'-TGTGTGTGT-3')p-AAm(q)- showed slightly faster electrophoretic mobility toward the anode than -(5'-GCCACCAGC-3')m-AAm(n)- due to its higher molar fraction of negatively-charged ssDNA. We successfully separated target ssDNAs having the same chain length by using two ssDNA conjugate probes that showed different electrophoretic mobilities, although the separation of these ssDNAs was difficult in conventional capillary electrophoresis systems.     

Hydration, charge, size, and shape characteristics of peptides from their CZE analyses

A CZE model is presented for peptide characterization on the basis of well-established physicochemical equations. The effective mobility is used as basic data in the model to estimate relevant peptide properties such as, for instance, hydration, net and total electrical charge numbers, hydrodynamic size and shape, particle average orientation, and pH-microenvironment from the charge regulation phenomenon. Therefore 102 experimental effective mobilities of different peptides are studied and discussed in relation to previous work. An equation for the estimation of peptide hydration as a function of ionizing, polar, and non-polar amino acid residues is included in the model. It is also shown that the shape-orientation factor of peptides may be either lower or higher than one, and its value depends on a complex interplay among total charge number, molar mass, hydration, and amino acid sequence.