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PubMed2001

Ab initio phasing of high-symmetry macromolecular complexes: successful phasing of authentic poliovirus data to 3.0 A resolution.

A genetic algorithm-based computational method for the ab initio phasing of diffraction data from crystals of symmetric macromolecular structures, such as icosahedral viruses, has been implemented and applied to authentic data from the P1/Mahoney strain of poliovirus. Using only single-wavelength native diffraction data, the method is shown to be able to generate correct phases, and thus electron density, to 3.0 A resolution. Beginning with no advance knowledge of the shape of the virus and only approximate knowledge of its size, the method uses a genetic algorithm to determine coarse, low-resolution (here, 20.5 A) models of the virus that obey the known non-crystallographic symmetry (NCS) constraints. The best scoring of these models are subjected to refinement and NCS-averaging, with subsequent phase extension to high resolution (3.0 A). Initial difficulties in phase extension were overcome by measuring and including all low-resolution terms in the transform. With the low-resolution data included, the method was successful in generating essentially correct phases and electron density to 6.0 A in every one of ten trials from different models identified by the genetic algorithm. Retrospective analysis revealed that these correct high-resolution solutions converged from a range of significantly different low-resolution phase sets (average differences of 59.7 degrees below 24 A). This method represents an efficient way to determine phases for icosahedral viruses, and has the advantage of producing phases free from model bias. It is expected that the method can be extended to other protein systems with high NCS.

Algorithms↗
PubMed1989

Ketamine enhances phase I and phase II neuromuscular block of succinylcholine.

The effect of intravenous injection of ketamine 2, 5 and 10 mg.kg-1 on the neuromuscular blocking action of succinylcholine was studied on the indirectly stimulated adductor pollicis muscle twitch of monkeys anaesthetized with 0.5-1.0 per cent halothane in oxygen. Neuromuscular block was quantified by single twitches evoked at 0.1 Hz. The changing nature of neuromuscular block from Phase I to Phase II was monitored periodically by train-of-four fade. In the absence of succinylcholine, ketamine had no consistent neuromuscular effect of its own. In the presence of succinylcholine, ketamine in a dose-dependent manner potentiated both the Phase I and the Phase II neuromuscular blocking effect of succinylcholine. In Phase I, 2 mg.kg-1 of ketamine reduced the ED50 of succinylcholine from 0.46 +/- 0.07 mg.kg-1 to 0.33 +/- 0.06 mg.kg-1 (P less than 0.01), and increased its 25-75 per cent recovery index from 4.0 +/- 0.4 min to 5.3 +/- 0.1 min (P less than 0.01). In Phase II, ketamine in the same dose deepened a steady neuromuscular block maintained by succinylcholine infusion from 48 +/- 3 per cent block to 71 +/- 2 per cent block (P less than 0.01). We concluded that ketamine potentiates the Phase I and the Phase II neuromuscular blocks of succinylcholine.

Anesthesia, Inhalation↗
PubMed2004

Comparison of breath-hold 2D phase-contrast with non breath-hold cine phase-contrast MRA in the assessment of azygos venous blood flow in portal hypertension.

Azygos venous blood flow as an index of blood flow through the gastroesophageal collaterals and varices is of value in the prediction of gastrointestinal bleeding. Measurement of azygos venous blood flow has been achieved by non breath-hold (NBH) cine phase-contrast magnetic resonance imaging. The objective of this study was to compare the faster breath-hold (BH) phase-contrast technique with the standard (NBH) cine phase-contrast technique in the measurement of azygos blood flow. Thirty-two cirrhotic patients with esophageal varices were examined by magnetic resonance imaging using a BH technique and a NBH cine phase-contrast technique to measure the flow velocity, flow volume and calibre of the azygos vein at the mid-right atrial level. The flow values were obtained on the velocity image of the phase-contrast study. Values obtained from the two methods were evaluated statistically for the strength and significance of correlation by the Pearson test. Measurement by the BH method performed at full-inspiration as well as end-expiration was also obtained in 15 healthy volunteers. The breath-hold phase-contrast method has significant but weak correlation with non BH cine phase-contrast method in the measurement of azygos venous blood flow volume (r = 0.55, p < 0.01) and flow velocity (r = 0.43, p = 0.01). However, the calibre of the azygos vein gave a strong correlation in these two methods (0.82). In the subgroup of patients whose azygos blood flow velocity was greater than 7.4 cm/s, the correlation of azygos blood flow volume is strong (r = 0.80, p < 0.01). The azygos vein calibre remains highly correlated between the BH and NBH method, in both high flow velocity (r = 0.73) and low flow velocity (r = 0.83) groups. Breath-hold sequence leads to higher values for flow velocity and flow volume in the cirrhotic patients and also the control group. In patients with portal hypertension, BH 2D phase-contrast (PC) magnetic resonance angiography (MRA) could give a comparable estimation of the calibre of the azygos vein as the NBH 2D cine PC MRA but not for azygos flow volume. In patients with high azygos flow velocity, the strong correlation in flow volume between the BH and NBH method suggests that the BH method may be a time-saving alternative to the NBH method.

Azygos Vein↗
PubMed1990

Phase transition from a gel to a fluid phase of cubic symmetry in dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) bilayers.

Aqueous dispersions (pH 4.0) of a 2:1 (mol/mol) mixture of myristic acid with dimyristoylphosphatidylcholine undergo a sharp transition at 45-47 degrees C from a lamellar gel phase to a fluid phase which is optically isotropic. This fluid phase gives rise to 31P-NMR spectra, and 2H-NMR spectra of the chain-deuterated components, which are also isotropic. X-ray diffraction studies of the fluid phase at 49 degrees C, reveal reflections with spacings in the ratio square root of 2: (square root of 3): square root of 4: square root of 6: square root of 8, accompanied by a strong diffuse scatter. These reflections index on a cubic lattice of primitive space group Pn3 or Pn3m, or possibly the body-centered group Im3m, with a lattice constant of 21.2 nm. The dimensions of the phase are consistent with a structure composed of two systems of tetrahedrally (octahedrally) oriented inverted lipid cylinders, found for other cubic lipid phases with Pn3m (Im3m) symmetry. At higher temperatures the cubic phase gradually converts, with increasing temperature, to a coexisting inverted hexagonal phase.

Dimyristoylphosphatidylcholine↗
PubMed1992

Retention of C60 and C70 fullerenes on reversed-phase high-performance liquid chromatographic stationary phases.

The separation of C60 and C70 fullerenes on four different polysiloxane stationary phases was examined. It was determined that polar solvents can be used as mobile phases effectively for the separation of fullerene molecules. Unlike previously published work, a polymeric octadecyl siloxane (ODS) stationary phase provided higher separation factors for C70/C60 than did monomeric ODS stationary phases or phenyl substituted stationary phases. For example, for a methanol-diethyl ether (50:50, v/v) mobile phase and C60, k' approximately 5.0 separation factors, alpha = 3.3, were achieved with polymeric ODS compared to alpha = 2.2, with a monomeric ODS stationary phase. A linear solvation energy relationship (LSER) was used to model the importance of solvent interactions and stationary phase interaction to solute retention.

Carbon↗
PubMed1993

Effect of stationary phase solvation on shape selectivity in reversed-phase high-performance liquid chromatography.

The effect of stationary phase solvation on reversed-phase chromatographic shape selectivity has been investigated using n-hexanol as an additive to methanol-water mobile phases. A wide range of mobile phase compositions was evaluated to normalize for solvent strength selectivity differences. Monomeric C18 stationary phases of both high and low bonding density were synthesized and used to correlate selectivity changes caused by stationary phase ordering with those seen by the addition of n-hexanol. The temperature dependence of retention and selectivity was also investigated using Van 't Hoff plots, which provided insight into the nature of selectivity behavior for estrogens and polyaromatic hydrocarbons. The results showed that using n-hexanol as a mobile phase additive did not provide higher shape selectivity, suggesting that changes in the solvation of the stationary phase did not impart a significant change in the level of surface ordering or morphology. However, n-hexanol did impart solvent selectivity changes in the separation of estrogen diastereomers that could prove useful in future methods development schemes.

Alcohols↗
PubMed2004

Monolithic columns with mixed modes of reversed-phase and anion-exchange stationary phase for capillary electrochromatography.

A capillary electrochromatography (CEC) monolithic column with mixed modes of reversed-phase and anion-exchange stationary phases was prepared by in situ polymerization of 2-(methacryloxy)ethyltrimethylammonium methyl sulfate (MEAMS) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting 1-propanol and 1,4-butanediol. The ammonium groups on the surface of the stationary phase generate an electroosmotic flow (EOF) from cathode to anode, and serve as a strong anion-exchange stationary phase at the same time. The EOF of the stationary phase can be determined by the amount of MEAMS monomer in reaction mixtures during the polymerization. The monolithic stationary phases exhibited reversed-phase chromatographic behavior toward neutral solutes. For charged solutes, hydrophobic as well as electrostatic interaction/repulsion with the monoliths was observed. Separations of aromatic compounds and basic compounds on the prepared column were performed under the mode of CEC. Peak tailing of basic compounds was avoided and the efficient separation of aromatic acids was achieved using neutral mobile phase due to the same direction of EOF and electrophorestic mobility of negatively charged solutes.

Anion Exchange Resins↗
PubMed2005

Synthesis and characterization of a novel resorcinarene-based stationary phase bearing polar headgroups for use in reversed-phase high-performance liquid chromatography.

A novel silica-bonded stationary phase containing a functionalized resorcinarene selector was prepared by a straightforward synthesis. The complete modification of all resorcinic hydroxyl groups was achieved by reaction with isopropyl isocyanate. The derivatized resorcinarene selector was subsequently immobilized via the four alkenyl chains containing a terminal double bond by a free radical-induced reaction on mercaptopropyl-functionalized silica. A comprehensive characterization of the resulting bonded stationary phase was carried out by solid state NMR, IR and elemental analysis. The resulting selector is defined as a "polar headed" reversed phase since the highly ordered polar carbamate groups of the new stationary phase are located, compared to conventional polar embedded stationary phases, at a greater distance from the silica surface. Thus a new concept is introduced in the field of polar modified reversed-phase HPLC. The properties of the novel stationary phase are demonstrated by comparison with commercially available reversed phases.

Anthracenes↗
PubMed2005

Microfabricated refractive index gradient based detector for reversed-phase liquid chromatography with mobile phase gradient elution.

Typical refractive index (RI) detectors for liquid chromatography (LC) are not well suited to application with mobile phase gradient elution, due to the difficulty in correcting for the detected baseline shift during the gradient. We report a sensitive, highly reproducible, microfabricated refractive index gradient (micro-RIG) detector that performs well with mobile phase gradient elution LC. Since the micro-RIG signal remains on-scale throughout the mobile phase gradient, one can apply a baseline correction procedure. We demonstrate that by collecting two mobile phase gradient blanks and subtracting one of them from the other, a reproducible, flat baseline is achieved. Therefore, subtracting a blank from a separation provides a baseline corrected chromatogram with reasonably high signal-to-noise ratio for eluting analytes. The micro-RIG detector uses a collimated diode laser beam to optically probe a RIG formed perpendicular to the laminar flow direction within a microfabricated borosilicate glass chip. The chip-based design of the detector is suitable for either traditional bench-top or LC-on-a-chip technologies. We report reversed phase high performance liquid chromatography (RP-HPLC) separations of proteins and polymers, over mobile phase gradient conditions of 67% A:33% B to 3% A:97% B by volume, where A is 96% methanol:3.9% water:0.1% trifluoroacetic acid (TFA), and B is 3.9% methanol:96% water:0.1% TFA. The separations were performed on a Jupiter 5 mu C4 300 A 150 mm x 1.0 mm Phenomenex column at a flow rate of 20 microl/min. Viscosity changes during the mobile phase gradient separation are found to shift the on-chip merge position of the detected concentration gradient (i.e., RIG), in a reproducible fashion. However, this viscosity effect makes detection sensitivity vary throughout the mobile phase gradient, due to moving the optimized position of the probe beam in relation to the analyte concentration gradient being probed. None-the-less, consistent limits of detection (LODs) were achieved. The 3-sigma deflection angle LOD was 16 microrad for micro-RIG detection, corresponding to an injected concentration LOD of 7 ppm (mass/mass) for cytochrome c.

Chromatography, High Pressure Liquid↗
PubMed2006

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography.

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.

Benzene Derivatives↗
PubMed2000

Low concentration of DMSO stabilizes the bilayer gel phase rather than the interdigitated gel phase in dihexadecylphosphatidylcholine membrane.

We have investigated effects of dimethylsulfoxide (DMSO) on the phase stability of multilamellar vesicles of the ether-linked 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC-MLV), which is known to be in the interdigitated gel (LbetaI) phase in excess water at 20 degrees C. The results of X-ray diffraction experiments indicate that the DHPC membrane was in the Lbeta, phase at X> or =0.12 (X=mole fraction of DMSO in DMSO/water mixture). The result of differential scanning calorimetry indicate that the gel to liquid-crystalline phase transition temperature increased, but the LbetaI to Pbeta, phase transition temperature decreased with an increase in DMSO concentration. These results show that DMSO stabilizes the bilayer gel phase rather than the LbetaI phase at its low concentration. The solubility of phosphorylcholine, which is the same structure as the headgroup of DHPC, decreased with an increase in DMSO concentration, indicating that the interaction free energy of the hydrophilic segments of the membrane with solvents increases with an increase in DMSO concentration. On the basis of the thermodynamic analysis, the mechanism of the stabilization of the bilayer gel phase of DHPC-MLV by DMSO is discussed. The decrease in the repulsive interaction between the headgroups of the phospholipid induced by the low concentrations of DMSO in water plays an important role in this stabilization.

Calorimetry, Differential Scanning↗
PubMed1989

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.

Animals↗
PubMed1998

Behavioral inhibition of light-induced circadian phase resetting is phase and serotonin dependent.

Circadian rhythms in Syrian hamsters can be phase shifted by light exposure during the subjective night and by a bout of wheel running induced during the subjective day. Interactions between photic and behavioral stimuli were examined by comparing phase shifts to 15 min, 50 lux light pulses with and without a bout of running induced by confinement to a novel wheel 30 min prior to and extending through light exposure. Light pulses 6 h after dark onset on the first night of constant dark induced phase advance shifts averaging 80 min. Wheel running attenuated these shifts by 45% on average (p<0.01). Light pulses 1 h or 2.25 h after dark onset induced phase delay shifts averaging 50 min and 20 min, respectively, that were not affected by stimulated running. A significant running response to the novel wheel was evident at all 3 time points, but was greater to wheel confinement at both times early in the night. Stimulated running alone early or late in the night did not produce significant phase shifts. Behavioral attenuation of phase advances to light late in the night was prevented by pretreatment with the general 5HT1 antagonist metergoline (2 mg/kg i.p.). Metergoline did not significantly attenuate running in novel wheels. These results indicate that modulation of light-induced phase shifts by behavior is phase dependent and may involve direct or indirect actions of serotonin within the circadian system.

Animals↗
PubMed2000

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of mobile phase composition.

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.

Chloroform↗
PubMed2003

Effects of alcohol mobile-phase modifiers on the structure and chiral selectivity of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

Following a previous publication, the present paper reports additional results on the effects of alcohol mobile-phase modifiers on the structure and chiral selectivity of amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) chiral stationary phase (CSP). Solid-state NMR (1H/13C CPMAS) was utilized to identify and compare structural differences in Chiralpak AD caused by the various alcohol mobile-phase modifiers, many of which were not studied in the previous publication. The influences of the various alcohol modifiers (in hexane-based mobile phase) on the structure and chiral selectivity of the CSP were studied and compared. CPMAS spectra of Chiralpak AD flushed with the mobile phases displayed clear evidence of solvent incorporation into the CSP. When alcohol modifiers with varying size and bulkiness were used in the mobile phase, differences in structure and chiral selectivity were observed on Chiralpak AD based on solid-state NMR and chromatographic data. The change of t-butanol concentration in the t-butanol/hexane mobile phase caused changes of structure and chiral selectivity of the Chiralpak AD. These data further support our belief that the different chiral selectivities of the CSP associated with the use of different alcohol modifiers are due to different alterations of the steric environment of the chiral cavities in the CSP by the different mobile-phase modifiers.

Alcohols↗
PubMed2000

Studies of retention and stability of a horizontally polymerized bonded phase for reversed-phase liquid chromatography.

We have studied the novel horizontally polymerized mixed trichloropropyl-trichlorooctadecyl silane bonded phase described by Wirth. These materials can be reproducibly prepared and give very high bonded phase density (>7.5 micromol m(-2)). They show significantly improved alkaline stability and chromatographic selectivity towards PAHs similar to conventional monomeric phases. Study of retention by the Linear Solvation Energy Relationship approach as well as measurement of dead volume and retention of methanol indicate that less mobile phase is sorbed by a horizontally polymerized phase than by conventional phases. Silanophilic interaction of amines are decidedly weaker on a silica modified by horizontal polymerization compared to a conventionally modified phase. In addition, this work provides additional support for the "partition-like" retention mechanism of bonded phase RPLC.

Chromatography, Liquid↗
PubMed1998

Aqueous two-phase systems for protein separation. Studies on phase inversion.

Phase equilibrium studies were done with the PEG 400-phosphate system, obtaining equilibrium binodal lines, tie lines and phase inversion points. A method of calculation of the critical point on the binodal curve is described. The influence of the presence of NaCl in solution was studied, and the comparative results are presented. It was found that in some range of concentration the shift produced in the binodal line can be important. The rate of phase separation can be used as an indication of which of the phases is continuous. Using this method the phase inversion point can be determined in the system for each tie line. A range of ambiguity was found, where the continuity of the phases is affected not only by the composition of the mixture, but also by the fluid dynamics. Within this range, gentle agitation produces a bottom-continuous suspension. while strong agitation produces a top-continuous suspension. Two inversion points exist therefore on each tie line, delimiting on the phase equilibrium plane a region where the phase continuity depends on fluid dynamics. The convergence of this region towards the critical point can be used to control of the consistency of the experimental data.

Chemistry Techniques, Analytical↗
PubMed2006

Structure-function relationships in high-density docosylsilane bonded stationary phases by Raman spectroscopy and comparison to octadecylsilane bonded stationary phases.

Raman spectroscopy is used to investigate the effects of temperature, surface coverage, polymerization method (surface or solution polymerized), and nature of the alkylsilane precursor on alkyl chain conformational order in a series of high-density docosylsilane (C22) stationary phases at surface coverages ranging from 3.61 to 6.97 mumol/m(2). The results of this study contribute to an enhanced understanding of the shape-selective retention characteristics of these phases at a molecular level. Conformational order is evaluated using the intensity ratio of the antisymmetric and symmetric nu(CH2) modes as well as the frequency at which these modes are observed. Alkyl chain order is shown to be dependent on surface coverage, alkyl chain length, and polymerization method. In general, alkyl chain order increases with surface coverage. Temperature-induced changes are observed between 250 and 350 K for the three phases with surface coverages between 3.61 and 4.89 mumol/m(2). These changes occur over a broad range of temperatures characteristic of two-dimensional systems, but in general adhere to the behavior predicted for a simple first-order transition. This change is not believed to be an abrupt cooperative disassociation characteristic of a phase transition in a bulk phase, but instead is thought to involve significant changes in conformational order in segments of the surface-tethered molecules, mostly those segments at the outer edge of the alkylsilane. In contrast to the changes observed in coverages below 5 mumol/m(2), a first-order change is not observed for the stationary phase with coverage of 6.97 mumol/m(2). A molecular picture of the temperature-induced disorder is proposed with disorder originating at the distal carbon and propagating only a short distance toward the proximal carbon. A comparison is made between these C22 stationary phases and similar high-density octadecylsilane (C18) bonded phases.

Journal Article↗