On-chip coupling of isoelectric focusing and free solution electrophoresis for multidimensional separations

We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73x) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 microm/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was 'parked' during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was approximately 1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.     

Determination of fexofenadine in tablets by capillary electrophoresis in free solution and in solution with cyclodextrins as analyte carriers

Capillary electrophoresis (CE) methods for the determination of fexofenadine (FEX) in commercial pharmaceuticals were developed. It was demonstrated that FEX could be effectively analyzed in free solution cationic CE at low pH. Another analytical approach studied was based on cyclodextrin (CD) modified CE where highly charged CD derivatives served as analyte carriers. In this way, the separation range was spread to physiological pH region and a CE analysis of FEX, present actually in its zwitterionic form, could be accomplished. Several parameters affecting the separations were studied, including the type and concentration of carrier ion, counterion, analyte carrier, and pH of the buffer. The methods based on the free solution CE and CD-modified CE were compared each other, validated, and applied for the determination of FEX in tablets.     

High-throughput single-molecule spectroscopy in free solution

A high-speed high-throughput single-molecule imaging technique for identifying molecules in free solution based on differences in their fluorescence emission spectra is presented. Unlike previous reports, the entire spectrum, rather than selected wavelengths through optical filters, is recorded. Furthermore, the millisecond data acquisition time means that the molecules do not need to be immobilized or spatially confined. In one example, individual lambdaDNA molecules labeled with YOYO-I, POPO-III, or a combination of the two dyes can be distinguished from one another. In another example, biotinylated 2.1-kb DNA labeled with YOYO-I was reacted with avidin-conjugated R-phycoerythrin. The two different reactant molecules and the product molecule can be simultaneously imaged and identified by their spectroscopic characteristics. This technique can therefore be used for screening single molecules for disease markers and for monitoring individual molecular interactions at a rate of thousands of molecules per second.     

On completeness of RFM solution structures

 The R-Function Method (RFM) solution structure is a functional expression that satisfies all given boundary conditions exactly and contains some undetermined functional components. It is complete if there exists a choice of undetermined component that transform the solution structure into an exact solution. Such a structure was used by Kantorovich (Kantorovich and Krylov, 1958) and his students to solve boundary value problems with homogeneous boundary conditions on geometrically simple domains. RFM is based on the theory of R-functions (Rvachev, 1982) that allows construction of a set of functions vanishing on the boundary and can be applied to problems with arbitrarily complex domains and boundary conditions. The resulting solution method is essentially meshfree, in the sense that the spatial discretization no longer needs to conform to the geometry of the domain, and can be completely automated. This paper summarizes the main principles of RFM, proves its completeness, and presents numerical results for several simple test problems.     

Enhanced separation of membranes during free flow zonal electrophoresis in plants

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.     

Exponential Basis Functions in solution of laminated structures

In this paper a new method for solution of static two-dimensional laminates has been proposed. A series of Exponential Basis Functions (EBFs) have been used to satisfy the governing equations within each layer. Compatibility and equilibrium conditions at the interfaces of the layers, with isotropic or orthotropic properties, are rigorously satisfied through defining a characteristic problem. The solution of such a characteristic problem yields to a set of spectral basis functions through which one may obtain a clear insight to the modes governing the displacement field. For further use, we present simplified relations for single-layer and two-layer laminates. The boundary conditions at the ends of the laminate are satisfied by a suitable discrete transformation in a collocation style. With such an approach one can solve problems with variety of end conditions. Excellent agreement is observed between the results of this work and some benchmark problems. Some more problems are solved as new benchmarks for further studies.     

Characterizing the structures and folding of free proteins using 2-D gas-phase separations: observation of multiple unfolded conformers

Understanding the 3-D structure and dynamics of proteins and other biological macromolecules in various environments is among the central challenges of chemistry. Electrospray ionization can often transfer ions from solution to gas phase with only limited structural distortion, allowing their profiling using mass spectrometry and other gas-phase approaches. Ion mobility spectrometry (IMS) can separate and characterize macroion conformations with high sensitivity and speed. However, IMS separation power is generally insufficient for full resolution of major structural variants of protein ions and elucidation of their interconversion dynamics. Here we report characterization of macromolecular conformations using field asymmetric waveform IMS (FAIMS) coupled to conventional IMS in conjunction with mass spectrometry. The collisional heating of ions in the electrodynamic funnel trap between FAIMS and IMS stages enables investigating the structural evolution of particular isomeric precursors as a function of the intensity and duration of activation that can be varied over large ranges. These new capabilities are demonstrated for ubiquitin and cytochrome c, two common model proteins for structure and folding studies. For nearly all charge states, two-dimensional FAIMS/IMS separations distinguish many more conformations than either FAIMS or IMS alone, including some with very low abundance. For cytochrome c in high charge states, we find several abundant "unfolded" isomer series not distinguishable by IMS, possibly corresponding to different "string of beads" geometries. The unfolding of specific ubiquitin conformers selected by FAIMS has been studied by employing their heating in the FAIMS/IMS interface.     

Accurate solution describing axisymmetric gas-dynamic structures

An accurate solution describing the dynamics of complex gas-dynamic structures arising in the adiabatic compression of material in conditions with aggravation is constructed.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 51, No. 6, pp. 952–955, December, 1986.     

Analysis of free intracellular nucleotides using high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) with UV absorbance detection (254 nm) has been applied for analyzing intracellular free ribonucleotides. The nucleotide profiles obtained from peripheral blood lymphocytes differ from those obtained from Molt4 human leukemic cells. With a 140 mM borate buffer, pH 9.4, a nearly complete profile can be obtained in 25 min. HPCE has comparable resolution to that of high-performance liquid chromatography (HPLC) but is faster in terms of time per sample run (25 min vs 45 min) and requires much less sample (nanoliter range for HPCE vs microliter range for HPLC).     

Tailoring of free standing microchannels structures via microtemplating

The preparation of ceramic microchannel structures from powder based material is a time consuming, expensive or inflexible process. This work demonstrates microtemplating - a new reliable and economical fabrication method for preparing ceramic alumina self-supporting microchannels with diameters from 10 to 1000 μm. The basis for this method is the controlled dip-coating of a sacrificial polymeric fiber template with suspensions containing sinterable particles. Subsequent sintering results in self-supporting ceramic microchannels. Thermal behavior of a microtemplate is critical for achieving self-supporting microchannels. We used polyamide, keratin, para-aramide, polypropylene, and polyimide fibers to set models of microtemplate thermal behavior. We found that the negative influence of thermal expansion and gas production could be eliminated by microtemplates with hollow and porous internal structure. Furthermore we show flexibility of the green stage procedure on an alumina microfluidic loop and a multichannel system with gradient porosity. The microtemplating is demonstrated to be an inexpensive and flexible process.     

Perturbed solution of free non-linear vibrations of composite beams

The aim of this study is to characterize the free non-linear vibration behavior of composite beams by using polynomial finite element method with shape functions based on Legendre polynomials or sinusoidal functions. The beams are subjected to different boundary conditions. Non-linear frequencies and modes shapes are obtained using asymptotic linearization method applied on the fundamental vibrating mode. The different composite beams are subjected to different layer dispositions. The nature of the interface between layers is of no object in this study. Navier–Bernoulli hypothesis on the transverse sections is adopted. The study concerns the influence of geometrical non-linearities of the free dynamical behavior. The analysis is performed thus by considering the influence of the vibration amplitude on the modal frequencies of the structure. The solutions are obtained by numerical simulation and by semi-analytical method using asymptotic linearization applied in the real temporal modal space. The numerical values are compared to the different ones obtained in literature.     

圆形薄膜自由振动的理论解

本文研究圆形薄膜的自由振动。首先根据哈密顿原理建立薄膜横向振动的动力学方程,然后采用分离变量法,导出时间t\、径向坐标r和环向坐标 变量分离的2个二阶常微分方程和1个贝塞尔方程并分别求解,求得周边固定圆形薄膜、扇形薄膜自由振动的理论解,从而得到固有频率及其振型的解析表达式。最后,应用ANSYS有限元计算软件计算上述几种类型自由振动的频率及其模态并与理论解比较。ANSYS有限元数值解与理论解二者十分接近,理论解是有限元数值解的下限。     

Role of charge suppression and ionic strength in free zone electrophoresis of proteins.

The free zone electrophoretic mobility of proteins can be predicted from the protein's amino acid content by applying a model based on the Debye-Hückle-Henry theory and Henderson-Hasselbalch equation. Calculated mobilities are always greater than actual mobility but a pH-independent proportionality (described by the constant FZ) is found between the two. Thus, determination of a protein's mobility at one pH allows, with the use of the model and FZ, calculation of its mobility at other pH conditions. This leads directly to optimum conditions for the electrophoretic resolution of proteins in capillary zone electrophoresis. The fundamental nature of FZ is examined and found to be a function of a proteins molecular weight, charge, and solution ionic strength. This work aids in explaining the form of previously proposed empirically based equations for peptide and protein mobility.     

A solution method for problems of free convective motion in liquids

The solution of the problem of free convective motion of liquids with high Prandtl and Schmidt numbers is obtained in the form of inner and outer asymptotic solutions which are joined together. Boundary conditions of the first or second kind are considered.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 29, No. 5, pp. 857–863, November, 1975.     

Geometric solution strategy of Laplace problems with free boundary

This paper introduces a geometric solution strategy for Laplace problems. Our main interest and emphasis is on efficient solution of the inverse problem with a boundary with Cauchy condition and with a free boundary. This type of problem is known to be sensitive to small errors. We start from the standard Laplace problem and establish the geometric solution strategy on the idea of deforming equipotential layers continuously along the field lines from one layer to another. This results in exploiting ordinary differential equations to solve any boundary value problem that belongs to the class of Laplace's problem. Interpretation in terms of a geometric flow will provide us with stability considerations. The approach is demonstrated with several examples. Copyright © 2015 John Wiley & Sons, Ltd.     

A solution to the multiple set-up problem

This note finds the optimal order quantity for the multiple set-up problem. The solution is obtained by a direct method.     

General boundary elements solution for ageing viscoelastic structures

A general boundary element formulation for the stress analysis of bodies with ageing viscoelastic constitutive relations such as concrete or biological materials is presented. It is shown that in the case of a constant Poisson modulus, the domain integral vanishes. Otherwise, the domain integrals are taken to the boundary by means of the dual reciprocity method. Computational details and examples are given. Copyright © 2001 John Wiley & Sons, Ltd.     

NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.