Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.     

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. I. Theoretical studies

Distortion of the starting zone upon its electrophoretic migration toward the detection window gives rise to both symmetrical zones caused by diffusion, sedimentation in the horizontal section of the capillary and the curvature of the capillary, and asymmetrical zones having their origin in Joule heating, sedimentation in the vertical section of the capillary, pH and conductivity differences between the sample zone and the surrounding buffer, solute adsorption onto the capillary wall, and association-dissociation of complexes between the analyte and a buffer constituent or between analytes. Interestingly and importantly a theoretical study shows that moderate pH and conductivity differences as well as adsorption and all of the above interactions when they are characterized by a fast on/off kinetics do not increase the zone broadening (or only slightly), because the sharpening of one boundary of the zone is about the same as the broadening of the other boundary. In addition the peak symmetry caused by a conductivity difference is in most experiments counteracted by a pH difference. The experimentally determined plate numbers in the absence of electroosmosis exceeded one million per meter in some experiments (Part II). These plate numbers are among the highest reported [Z. Zhao, A. Malik, M.L. Lee, Anal. Chem. 65 (1993) 2747; M. Gilges, K. Kleemiss, G. Schomburg, Anal. Chem. 66 (1994) 2038; H. Wan, M. Ohman, L.G. Blomberg, J. Chromatogr. A 924 (2001) 591 (plate numbers determined in the presence of electroosmosis may be higher, although the width of the zone in the capillary may be larger) [p. 680 in S. Hjertén, Electrophoresis 11 (1990) 665]). Capillary free zone electrophoresis is perhaps the only separation method, which, under optimum conditions, gives a plate number not far from the theoretical limit. A prerequisite for this high performance is that the polyacrylamide-coated capillary is washed with 2 M HCl between the runs and stored in water over night (Part II). The difference between the experimentally determined total variance and the sum of the calculated variances originating from the width of the starting zone, longitudinal diffusion, Joule heating, sedimentation in the vertical section of the capillary, curvature of the capillary (i.e., the sum of all other variances) was in our most successful experiments about 28% of the variance of diffusion. The zone broadening, 2sigma, caused by diffusion was estimated at 0.77 mm. The total zone width (2sigma) calculated from the experimentally determined plate number was as small as 1 mm when the migration distance was 40 cm. Accordingly, the only efficient way to reduce drastically the total zone width is to decrease the analysis time and, thereby, the diffusional broadening. An important finding was that the variance originating from the loops of the capillary is not always negligible in high-performance runs. Therefore, one should employ straight capillaries and avoid CE apparatus with cartridges that require a strong curvature of the capillary, common in most commercial instruments. Mathematical formulas have been derived for the sedimentation of the solute zone, the enrichment factor, and the migration time in experiments where the solute is dissolved in a dilute running buffer. This zone sharpening method gave very narrow starting zones (0.04-0.4 mm). However, upon high dilution of the buffer the enrichment becomes so strong that part of the sample zone probably sediments out of the capillary; the almost inevitable change in pH may decrease the mobility of the proteins and, thus, cause the enrichment factor to become still lower than expected. Diffusion of the protein in the very narrow starting zone (located close to the tip of the capillary) and sometimes the thermal expansion of the buffer in the capillary contributes to additional loss of protein in the enrichment step. In some buffers, the interaction between the protein and the buffer constituents is so slow that the peaks become broad. Therefore, different types of buffers should be tested when high resolution is required. The relation sigma2 (the variance of the interaction between a protein and the buffer constituents) = constant x u (the mobility) seems to be valid for all proteins in the applied sample, at least when they have similar molecular masses. To facilitate the understanding of the progress of a free zone electrophoresis experiment, we have discussed in simple terms how the concentrations of the background electrolytes become rearranged during a run and why the difference between the mobilities of the proteins and the mobilities of the background electrolyte determines whether a peak exhibits fronting or tailing. A theoretical analysis of zone broadening in capillary zone electrophoresis, chromatography, and electrochromatography indicates that electrochromatography in homogeneous gels might be the only chromatographic technique which can compete in performance with free electrophoresis. Using an equation, valid not only for electrophoresis, but also for chromatography and centrifugation, the mobility of a concentration boundary has been calculated for the first time and was, as expected, low. Equations based on the Kohlrausch regulating function do not permit such calculations. Another regulating function (the H function) and some of its characteristics are briefly discussed. The theoretical discussions in this paper and the experimental studies in Part II show that high-performance electrophoresis deserves its prefix when the runs are designed to give minimum zone broadening. Some guidelines are given to facilitate this optimization. The plate numbers are so high that the resolution cannot be increased by more than 30% even if they approach the theoretically maximum values.     

Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii–vii) and how to overcome them, which also may help to overcome the difficulty (ii), which in turn minimizes difficulty (i).     

Agarose gels in HPLC separation of biopolymers

Agarose gels of high concentration (above 6–8%) are both highly permeable to macromolecules and very rigid, particularly after cross-linking. Gel spheres of agarose can therefore be used with advantage as matrices for HPLC. Owing to the well-known good chromatographic properties of agarose gels they have been used for 20 years as packing materials in various conventional low-pressure chromatographic techniques. The way is now open to use these methods in the HPLC mode with attendant improvements in run times and resolution. Agarose based HPLC matrices will be commercially available.     

Birefringence of high-pressure injection-molded high-density polyethylene at 70.5 μm wavelength

A newly constructed far-infrared laser interferometer was used to measure the birefringence of 1-mm-thick injection-molded high-density polyethylene test bars, manufactured at unusually high molding pressures. The long wavelength used, 70.5 μm, allowed nondestructive tests to be made, demonstrating the usefulness of far-infrared techniques for probing crystalline polymers. The birefringence was shown to increase with increasing molding pressure, supporting the belief that molecular orientation increases with increasing molding pressure. The tensile modulus was also measured and was found to increase linearly with molding pressure.     

Three registers in an untrained female singer analyzed by videokymography, strobolaryngoscopy and sound spectrography

There has been a lack of objective data on the singing voice registers, particularly on the so called "whistle" register, occurring in the top part of the female pitch range, which is accessible only to some singers. This study offers unique strobolaryngoscopic and high-speed (7812.5 imagess) videokymographic data on the vocal fold behavior of an untrained female singer capable of producing three distinct voice qualities, i.e., the chest, head and whistle registers. The sound was documented spectrographically. The transition from chest to head register, accompanied by pitch jumps, occurred around tones B4-C#5 (500-550 Hz) and was found to be associated with a slight decrease in arytenoids adduction, resulting in decrease of the closed quotient. The register shifts from head to whistle, also accompanied by pitch jumps, occurred around tones E5-B5 (670-1000 Hz) without any noticeable changes in arytenoids adduction. Some evidence was found for the vocal tract influence on this transition. The mechanism of the vocal fold vibration in whistle register was found principally similar to that at lower registers: vibrations along the whole glottal length and vertical phase differences (indicated by sharp lateral peaks in videokymography) were seen on the vocal folds up to the highest tone G6 (1590 Hz).     

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins)

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.     

Clustering by mixing flows

We calculate the Lyapunov exponents for particles suspended in a random three-dimensional flow, concentrating on the limit where the viscous damping rate is small compared to the inverse correlation time. In this limit Lyapunov exponents are obtained as a power series in epsilon, a dimensionless measure of the particle inertia. Although the perturbation generates an asymptotic series, we obtain accurate results from a Padé-Borel summation. Our results prove that particles suspended in an incompressible random mixing flow can show pronounced clustering when the Stokes number is large and we characterize two distinct clustering effects which occur in that limit.     

Global SO(3) × SO(3) × U(1) symmetry of the Hubbard model on bipartite lattices

In this paper the global symmetry of the Hubbard model on a bipartite lattice is found to be larger than SO(4). The model is one of the most studied many-particle quantum problems, yet except in one dimension it has no exact solution, so that there remain many open questions about its properties. Symmetry plays an important role in physics and often can be used to extract useful information on unsolved non-perturbative quantum problems. Specifically, here it is found that for on-site interaction U ≠ 0 the local SU(2) × SU(2) × U(1) gauge symmetry of the Hubbard model on a bipartite lattice with N_a^D sites and vanishing transfer integral t = 0 can be lifted to a global [SU(2) × SU(2) × U(1)]/Z₂² = SO(3) × SO(3) × U(1) symmetry in the presence of the kinetic-energy hopping term of the Hamiltonian with t > 0. (Examples of a bipartite lattice are the D-dimensional cubic lattices of lattice constant a and edge length L = N_aa for which D = 1, 2, 3,... in the number N_a^D of sites.) The generator of the new found hidden independent charge global U(1) symmetry, which is not related to the ordinary U(1) gauge subgroup of electromagnetism, is one half the rotated-electron number of singly occupied sites operator. Although addition of chemical-potential and magnetic-field operator terms to the model Hamiltonian lowers its symmetry, such terms commute with it. Therefore, its 4^N_aD energy eigenstates refer to representations of the new found global [SU(2) × SU(2) × U(1)]/Z₂² = SO(3) × SO(3) × U(1) symmetry. Consistently, we find that for the Hubbard model on a bipartite lattice the number of independent representations of the group SO(3) × SO(3) × U(1) equals the Hilbert-space dimension 4^N_aD. It is confirmed elsewhere that the new found symmetry has important physical consequences.     

Open and covered singing as studied by means of fiberoptics, inverse filtering, and spectral analysis

Covering the voice near the passaggio is a technique mainly used in male classic Western singing. The purpose is said to be to smooth the register transition and avoid register breaks. The physiological and acoustical differences between open and covered singing near the so-called passaggio were investigated by means of fiberoptics, inverse filtering, and spectral analysis. The study corroborated previous findings that covering physiologically means a widening and lengthening of the pharynx. Acoustically, covering implies a change of formant frequencies, and an elevated sound pressure level of the lowest source spectrum partial, i.e., the fundamental, resulting from an increased transglottal air flow. The findings indicate that covered singing also is associated with larynx lowering. Covered singing near the passaggio shows similarities to so-called phonation, and is probably preferable from the point of view of vocal hygiene.