The use of poly(2-acrylamido-2-methyl-1-propanesulfonic acid) polymers as spacers for isotachophoresis in sieving gel matrices

The electric field strength gradients generated in isotachophoresis (ITP) may be used for the separation of biomolecules. Poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS) polymers of a uniform distribution of molecular mass were synthesized and used as novel spacers in ITP. Since these polymeric spacers are strongly acidic species, their ionic charges remain constant over a wide pH range, so that their ionic mobilities are governed solely by their molecular masses and not by the pH of the milieu. A modification of ITP known as telescope electrophoresis was used to separate a number of acidic dyes of varying ionic mobility, using polyAMPS polymers as spacers. The resolution obtained was superior to that obtained by polyacrylamide gel electrophoresis (PAGE), due to the focusing effect of the electric field strength gradient. Since these novel polymeric spacers are designed to operate within sieving medium, it was decided to test their suitability for the separation of DNA molecules. DNA molecules up to 1000 bp long were successfully resolved, with a similar resolution to that obtained with conventional PAGE.     

High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve the detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, the authors review recent developments in LC-FAIMS-MS and its application to MS-based proteomics.     

Study of charged particle motion in fields of different configurations for developing the concept of plasma separation of spent nuclear fuel

The concept of plasma separation of spent nuclear fuel in a plane perpendicular to the magnetic field in an electric potential of special configuration is developed. A specific feature of the proposed approach consists in using an accelerating potential for reducing energy and angular spread of plasma ions at the entrance to the separator chamber and a potential well for the spatial separation of ions with different masses. The trajectories of ions of the substance imitating spent nuclear fuel are calculated. The calculations are performed for azimuthal and axial magnetic fields and model electric field configurations corresponding to different geometries of the separator chamber. It is shown that, using magnetic fields with a characteristic strength of 1 kG and electric potentials of up to 1 kV inside a region with a linear size less than 100 cm, it is possible to separate ions of spent nuclear fuel with energies from 0.2 to 3 eV. The calculations were performed for a collisionless mode in the single-particle approximation. Possible variants of the experimental facility for plasma separation of spent nuclear fuel are proposed.     

Oscillation spectrum of an electron gas with a small density fraction of ions

The problem is solved of the stability of a nonneutral plasma that completely fills a waveguide and consists of magnetized cold electrons and a small density fraction of ions produced by ionization of the atoms of the background gas. The ions are described by an anisotropic distribution function that takes into account the characteristic features of their production in crossed electric and magnetic fields. By solving a set of Vlasov-Poisson equations analytically, a dispersion equation is obtained that is valid over the entire range of allowable electric and magnetic field strengths. The solutions to the dispersion equation for the m = +1 main azimuthal mode are found numerically. The plasma oscillation spectrum consists of the families of Trivelpiece-Gould modes at frequencies equal to the frequencies of oblique Langmuir oscillations Doppler shifted by the electron rotation and also of the families of “modified” ion cyclotron (MIC) modes at frequencies close to the harmonics of the MIC frequency (the frequencies of radial ion oscillations in crossed fields). It is shown that, over a wide range of electric and magnetic field strengths, Trivelpiece-Gould modes have low frequencies and interact with MIC modes. Trivelpiece-Gould modes at frequencies close to the harmonics of the MIC frequency with nonnegative numbers are unstable. The lowest radial Trivelpiece-Gould mode at a frequency close to the zeroth harmonic of the MIC frequency has the fastest growth rate. MIC modes are unstable over a wide range of electric and magnetic field strengths and grow at far slower rates. For a low ion density, a simplified dispersion equation is derived perturbatively that accounts for the nonlocal ion contribution, but, at the same time, has the form of a local dispersion equation for a plasma with a transverse current and anisotropic ions. The solutions to the simplified dispersion equation are obtained analytically. The growth rates of the Trivelpiece-Gould modes and the behavior of the MIC modes agree with those obtained by numerical simulation.     

Liquid chromatography/mass spectrometry of fatty acids as their anthrylmethyl esters

The mass spectra of a series of saturated and unsaturated fatty acids have been recorded as their anthrylmethyl esters using a liquid chromatographic mass spectrometric interface. The spectra show an intense peak for the aromatic nucleus, and a molecular ion. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of acetone + acetonitrile. While a complete separation of the fatty acids known to occur in man was not achieved, the recognition of all of these acids is possible using a scanning mode or by ion monitoring.     

Resonant TE Transmission Through a Continuous Metal Film: Perspectives for Low-Loss Plasmonic Elements

Whereas resonant transverse magnetic transmission across an undulated continuous metal film is achieved with the mediation of plasmon modes excited by the undulation, it is shown here that transverse electric (TE) resonant transmission through a continuous metal film can also be achieved with the mediation of the second-order TE₁ mode of a dielectric slab waveguide having the metal film sandwiched at its middle. The demonstration is made by using the materials currently used in the domain of optical security and counterfeit deterrence: ZnS is shown to possibly be a lossless interface/adhesion layer between a polymer and a noble metal for plasmonic resonant elements.     

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.     

Isolating Influenza RNA from Clinical Samples Using Microfluidic Oil-Water Interfaces

The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAX^TM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample.     

Migration and Stabilization of Multiple Heavy Metals in an Aged Contaminated Soil under a Constant Voltage Electric Field

With the aim of metal decontamination, migration and stabilization of multiply heavy metals in an aged contaminated soil under a constant 1 V cm^?1 parallel-plate electric field were investigated through monitoring the metal migration in the anolyte, as well as analyzing their species distribution residual in soil. Besides anionic Cr(VI), cationic metals were also found in the anolyte, primarily by the concentration-gradient-driven diffusion of free ions, especially when the produced H⁺ considerably increased their levels in the soil. After 295 h, parts of Cu, Cr, Ni, and Zn were found to electro-migrate into the intermediate area, but no obvious Pb migration was observed, likely ascribed to its own great inertia and precipitation with the present Cr(VI). However, in the whole, only 5.3% of Zn and 2.7% of Ni were separated, while the release of other heavy metals was almost ignorable. Although Pb mobility in the soil near the anode even increased three times, the overall metal mobility in all sample locations was found to significantly reduce under the electric field, indicating an effective stabilization approach. Moreover, compared with the bottom soil, the top soil near the anode was found to have a lower pH, higher moisture, lower heavy metal concentrations, and less soil oxidant demand; these phenomena may be due to a faster electro-migration of charged ions, especially H⁺, in the top soil. Therefore, such a divergence may considered to improve the current simulation approach for a realistic estimation of the actual metal and H⁺ electro-migration rate and the associated behavior under an electric field.     

Effect of elution modes on protein separation by cross-axis coil planet centrifuge with two different types of coiled columns

Effect of elution modes on protein separation was investigated using cross-axis coil planet centrifuge (cross-axis CPC) with two different types of coiled columns, i.e., eccentric coil and toroidal coil assemblies. Myoglobin and lysozyme were separated with an aqueous two-phase solvent system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. The substantial effect of elution modes was observed by the toroidal coil, while the negative result was given at the eccentric coil. Using the toroidal coil, higher peak resolution of proteins was attained at the tail to head elution mode. In the outward lower phase mobile elution mode, the satisfactory separation was obtained by both eccentric coil and toroidal coil assemblies. However, in the inward upper phase mobile elution mode, the toroidal coil produced a broad and asymmetric myoglobin peak. The analysis using polyacrylamide gel electrophoresis revealed that the toroidal coil partially separated the components originally present in the myoglobin sample. As the result, a modified equation was devised to express the peak resolution (Rs) using the lysozyme peak. The overall results indicated that the toroidal coil produced better partition efficiency than the eccentric coil under the optimized experimental condition including the direction of the Coriolis force acting on the mobile phase in the toroidal coil.     

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).     

Protein fractionation according to molecular size in constant pH media with immobilized charges in colinear porosity gradient

A new method of protein electrophoresis is described here. Electrophoretic separation is performed in gel media with uniform concentration of immobilized charges, combined with porosity gradient directed against protein movement. Successful separation becomes possible due to the effect of strong sample zone compression; the latter effect is connected with complex conductivity profile dynamics in a gel system containing immobilized charges. Immobilized buffers combined with porosity gradient provide an opportunity of protein discrimination based on molecular size, while in the case of uniform gel concentration the separation is based on mobility differences and strongly affected by non-uniform electric field strength profile. The proposed method does not require ionic detergent for protein separation according to their molecular weight.     

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.     

Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions

We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (B?ttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]⁺ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.     

Development of field modulation in a split-field drift tube for high-throughput multidimensional separations

A field modulation approach for high-throughput ion mobility/time-of-flight analyses of complex mixtures has been developed using a split-field drift tube. In this approach, complex mixtures of peptides, such as those that arise from tryptic digestion of protein mixtures, are separated by nanocolumn liquid chromatography, ionized by electrospray ionization, and analyzed by ion mobility/time-of-flight techniques. The split-field drift tube allows parent ions to be separated based on differences in their low-field mobilities through the first-field region before entering the second region. For increased throughput, the magnitude of the field in the second region can be modulated throughout an LC separation in order to favor transmission of different types of ions: parent ions at low fields; fragments from primarily [M+3H]3+ peptides at moderate fields; or, fragmentation of [M+3H]3+ and [M+2H]2+ species at higher fields. We demonstrate the approach with two examples: a mixture of tryptic peptides from digestion of hemoglobin; and a complex mixture of tryptic peptides from digestion of human plasma.     

New food sources of essential trace elements produced by biotechnology facilities

Population satiety with trace elements (TE) is a problem that is widely discussed in nutrition science. For optimal nutrition, the form of TE eaten in food is very important. Organic forms of TE in nutrition are appropriate as human metabolism has adapted to these kinds of nutrients during species evolution. This is now considered a reason for the beneficial use of biotechnologically produced TE sources in human food. Advanced matrixes for TE incorporation are unicellular organisms such as yeast, lactobacilli and Spirulina. Addition of inorganic salts at certain concentrations into cultivation media enables the mineral ions to incorporate into the microbial biomass. As a consequence, the biomass becomes enriched with organic forms of incorporated TE, which are presented by their complexes with amino acids, proteins and probably lipids and polysaccharides. In addition, a new direction of research has made good advances, in which technology has been developed for production of organic forms of TE through complex formation between transition metals (zinc, copper, manganese, chromium, iron) with amino acids and peptides formed during enzymatic hydrolysis of food protein. This brief review discusses the results demonstrating the advances in the biotechnological production of new TE sources, to obtain food components destined for wide prophylaxis of TE deficiency or for dietary treatment of the adverse consequences of these deficiencies.     

A CPG component LE generates depolarization of buccal neurons by producing constant plateau potentials during feeding responses of Aplysia kurodai

In the buccal ganglia of Aplysia kurodai we have identified neurons (here termed LE neurons, or LE) producing plateau potentials lasting several seconds by application of short depolarizing currents. Results obtained from experiments using various bath solutions suggest that generation of these plateau potentials may be an endogenous property of LE. Application of various intensities or lengths of depolarizing currents induced in LE almost constant plateau potentials with fixed duration and depolarizing size. LE spikes produced monosynaptic EPSPs in the ipsilateral multi-action neuron (MA) and the jaw-closing motor neuron (JC) in the buccal ganglia. Conversely, MA spikes produced monosynaptic IPSPs in LE. There was electrical coupling between LE and both MA and JC. During the feeding-like response elicited by electrical stimulation of the nerve, LE showed rhythmic depolarization almost simultaneously with MA and JC, and firing on the plateau potentials occurred during the period of JC firing, the later phase of radula retraction. Hyperpolarization of LE during the feeding-like response suppressed generation of plateau potentials, though rhythmic small depolarization was still induced. During LE hyperpolarization, the duration of the depolarization of MA and JC was shortened. These results suggest that LE may be an element of the feeding CPG circuit and may contribute to part of the depolarization of MA and JC by generating constant plateau potentials during the feeding response, though LE may not have rhythm-generating ability.     

G2 restriction point within populations of hepatocytes and tongue keratinocytes in young, growing mice

The authors studied the effect of either extracts from liver (LE) or the malignant tumour ES2 (TE) or plasma from intact mice (PI) or tumour-bearing animals (PT) on the mitotic activity of the hepatocytes and tongue keratinocytes in young, growing C3H/s male mice (28+/-1 days old). Animals standardized for periodicity analysis were injected intraperitoneally with either TE, LE, PI, PT, or saline (S) at 16:00 h with 0.01 ml of sample/g of body weight and were then killed at (time of day/h post-injection) 20:00/04, 00:00/08, and 04:00/12. Colchicine (2 microg/g) was injected 4 h before death. Samples of the liver and tongue from each animal were processed for histology and assessment of mitotic index. The results were expressed as colchicine-arrested metaphases/1000 nuclei. The TE and LE stimulated the mitotic activity of hepatocytes and tongue keratinocytes. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.