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At least 991 records · Page 55Linked to original sources
PubMed1992

Separation of closely related large peptides by micellar electrokinetic chromatography with organic modifiers.

Large peptides with similar electrophoretic mobilities were separated by micellar electrokinetic chromatography (MEKC) with organic modifiers. [Leu13]motilin and [Met13]motilin differ by only one neutral amino acid residue. Because the electrophoretic mobilities of these peptides are almost identical, these peptides were not separated by capillary zone electrophoresis (CZE). Such large peptides have not been separated by conventional MEKC either, because they interacted strongly with the micelle. However, they were completely separated by MEKC when an organic solvent was added to the micellar solution. Some insulins, larger peptides than motilin, from different origins, which have very similar electrophoretic mobilities, were also successfully separated by the same technique. The size of peptides which were separated without organic modifiers was examined.

Amino Acid Sequence↗
PubMed2000

Microchip devices for high-efficiency separations.

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.

Journal Article↗
PubMed1995

Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.

Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupled-column systems in particular offer the advantages of on-line detection and automation. With the development of rapid two-dimensional (2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column 3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only after the final CZE separation, by laser-induced fluorescence detection. Analysis data from this system consist of a series of 2D "slices" of the SEC effluent, which when stacked together give the 3D separation "volume".

Chromatography, Gel↗
PubMed2001

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2003

A 15-s protein separation employing hydrodynamic force on a microchip.

We report here a novel pressurization technique for microchip electrophoresis that enables 15-s separation of protein mixtures extracted from biological samples. Although pressure-driven flow is usually parabolic flow, pressurization prior to electrophoresis separation produced a plug flow and achieved a dramatic migration time reduction without compromising resolution. Sample plugs were pushed forward by pressurization after loading the sample but before electrophoresis separation, in the absence of an electric potential. Higher pressures enabled higher speed separation; furthermore, the resolution could be easily controlled using an optimal pressure. In addition, the slow medium-pressurization technique enabled 2-D separation in only a single channel on a microchip. Utilizing this technique, 12 samples of complex protein mixture extracted from a human T lymphoblastic cell line, Jurkat cells, were separated within 15 s in a single run using a 12-microchannel array. In addition, target proteins from Jurkat cells were detected within this time. This novel pressurization technique on a microchip will offer enormous advantages for proteome analysis over commonly used 2-D electrophoresis.

Computer Simulation↗
PubMed2003

Separation of plutonium and neptunium species by capillary electrophoresis-inductively coupled plasma-mass spectrometry and application to natural groundwater samples.

Capillary electrophoresis (CE) was coupled to ICPMS in order to combine the good performance of this separation technique with the high sensitivity of the ICPMS for the analysis of plutonium and neptunium oxidation states. The combination of a fused-silica capillary with a MicroMist AR 30-I-FM02 nebulizer and a Cinnabar small-volume cyclonic spray chamber yielded the best separation results. With this setup, it was possible to separate a model element mixture containing neptunium (NpO2(+)), uranium (UO2(2+)), lanthanum (La3+), and thorium (Th4+) in 1 M acetic acid. The same conditions were also suitable for the separation of various oxidation states of plutonium and neptunium in different aqueous samples. All separations were obtained within less than 15 min. A detection limit of 50 ppb identical with 2 x 10(-7) M (3-fold standard deviation of a blank) was achieved. To prove the negligible disturbance of the plutonium and neptunium redox equilibria during the CE separations, plutonium and neptunium speciation by CE-ICPMS in acidic solutions was compared with the results of UV/visible absorption spectroscopy and was found to be in good agreement. The CE-ICPMS system was also applied to study the reduction of Pu(VI) in a humic acid-containing groundwater at different pH values.

Journal Article↗
PubMed2004

Dual microcoil NMR probe coupled to cyclic ce for continuous separation and analyte isolation.

Capillary electrophoresis (CE)-nuclear magnetic resonance (NMR) spectroscopy combines the separation efficiency of CE and the information-rich detection capabilities of NMR. However, the temporally narrow CE peaks reduce NMR sensitivity and prevent on-line multidimensional NMR acquisitions. In this work, cyclic CE with multicoil NMR instrumentation is developed to perform CE in multiple closed loops. As a proof of concept, a two-loop five-junction capillary configuration creates two connected yet independently operable fluidic loops. With appropriate voltage switching, analytes can be directed as desired around or between the loops, and a particular analyte band can be parked in one NMR detector coil while CE continues in the second loop and monitored with a second NMR detector coil. The separation of a mixture of amino acids (Ala, Val, Thr) is achieved in two cycles. After one CE cycle, Ala is separated and COSY data are recorded in one loop while Val and Thr are separated in the second loop. At the end of the second cycle, both Val and Thr are separated and multidimensional NMR spectra acquired. With this instrumentation and appropriate protocols, two-dimensional NMR data acquisition and CE separation are achieved simultaneously.

Electrophoresis, Capillary↗
PubMed2006

Surfactant gradient methods using mixed systems of cethyltrimethylammonium chloride and nonionic surfactants possessing polyoxyethylene chains for electrokinetic separation of benzoate anions as model analytes.

Surfactant gradient methods for electrokinetic separation of 10 benzoates as model organic anions were investigated using mixed micellar solutions of cetyltrimethylammonium chloride (CTAC) and nonionic surfactants possessing polyoxyethylene chains, polyoxyethylene sorbitan monolaurate (Tween 20) or polyoxyethylene lauryl ether (Brij 35). Electroosmotic flow (EOF) was eliminated virtually by a coating of the inner wall of the capillaries, and then the benzoates were detected fundamentally in the order of their hydrophobicity. In a pure CTAC system, the synergistic influences of attractive electrostatic and hydrophobic interactions gave rise to quite large retention factors of many of the benzoate anions, resulting in their coelution. Addition of an adequate amount of Tween 20 to the pure CTAC system decreased the electrostatic interaction significantly to give remarkably improved separation of the analytes, but long analysis time was required. A surfactant gradient method would be useful to decrease analysis time and to improve separation simultaneously. Under slight EOF, the micelles in the inlet reservoir can pass through and, thus, interact with all of the analytes before they were detected. In the present system, surfactant gradient separations could be performed simply by changing compositions of the surfactant solutions in the inlet reservoir during a single run. Additionally, we carried out continuous gradient separation using a simple device. Brij 35 gave an effect parallel to that by Tween 20 in migration behavior of the analytes. A practically negligible change in the level of the baseline was observed in a stepwise gradient elution with the CTAC/Brij 35 system because of the small absorbance at the detection wavelength, while that with the CTAC/Tween 20 was considerable. All the benzoates were separated completely within reasonable analysis times using both stepwise and continuous gradient programs for the concentrations of Tween 20 or Brij 35 in the presence of 100 mM CTAC.

Benzoates↗
PubMed1996

Micellar electrokinetic chromatography separations and analyses of biological samples on a cyclic planar microstructure.

Micellar electrokinetic capillary chromatography (MECC) separations and analyses of biological samples on a planar glass microchip capillary electrophoresis device with laser-induced fluorescence solute detection are discussed. A cyclic channel system which permits dead volume free repeated column switching and thus the use of various channel lengths together with a relatively low applied separation voltage is described. It features an unbiased, dead volume free electrokinetic sample inlet system of approximately 12 pL. Because of the small cross section and favorable heat dissipation in glass microstructures, MECC separations with an electric field strength of up to 2000 V/cm achieving efficiencies of submicrometer plate heights can be performed. After a separation length of 2 cm, six fluorescein isothiocyanate labeled amino acids are shown to be separable within a few seconds and with an imprecision for peak areas (or heights) and detection times of < 2% and < 0.5%, respectively. Without application of electrokinetic solute stacking, the detection limit of fluorescein isothiocyanate labeled arginine is 3.3 nM, corresponding to approximately 40 zmol injected. Furthermore, the feasibility of directly applying human urine and serum samples onto the uncoated channel system is demonstrated and first data of the successful performance of a chip-based MECC immunoassay for serum theophylline are presented. Compared to MECC in conventional fused-silica capillaries, MECC analyses on microchips can be performed 1-2 orders of magnitude faster, with higher efficiency and at no expense of accuracy and precision. Furthermore, versatility is shown to be much increased with the use of a cyclic rather than a single-path channel system. The MECC separation efficiency of fluorescein isothiocyanate labeled amino acids is shown to be comparable to that obtained by gel electrophoresis performed in the same chip layout.

Amino Acids↗
PubMed1996

Two-dimensional separations: capillary electrophoresis coupled to channel gel electrophoresis.

Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, high-concentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 microns thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20,000-50,000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over one-dimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and < 0.05% of an individual B2 neuron from the marine mollusk Aplysia californica are presented.

Animals↗
PubMed1997

Peptide separation in normal phase liquid chromatography.

A new method is established for separating peptides in normal phase liquid chromatography using TSK gel Amide-80, carbamoyl groups bonded to a silica gel matrix, and an acetonitrile-water solution containing 0.1% trifluoroacetic acid. Peptide retention time increased with acetonitrile concentration in the initial eluent. Hydrophilic peptides with no retention in a reversed phase column were retained and separated in the present method. Separation selectivities in the present and reversed phase methods differed significantly. Two-dimensional separation of protein digest using reversed and normal phases was conducted, taking advantage of the differences in selectivities. All peptides obtained from the digest could be separated completely. The present method is useful for separating peptide mixtures in conjunction with reversed phase liquid chromatography. Peptide recovery from the Amide-80 column exceeded 80%, as with the reversed phase column, and repeatability and reproducibility were satisfactory.

Amino Acid Sequence↗
PubMed1999

Independent optimization of capillary electrophoresis separation and native fluorescence detection conditions for indolamine and catecholamine measurements.

Separation conditions in capillary electrophoresis with native fluorescence detection often represent a compromise in terms of the separation and detection figures of merit. As both the separation and fluorescence properties greatly depend on pH, the ability to independently optimize pH in the separation capillary and the detection region can improve many complex separations. When using a sheath flow cell, the pH at the detection zone can be adjusted independently of the electrophoresis buffer pH. Using capillary electrophoresis with 257-nm excitation and native fluorescence detection, more than an order of magnitude improvement in the limits of detection for dopamine (from 1400 to 120 nM) and epinephrine (from 850 to 60 nM) is achieved by maintaining the basic separation conditions and an acidified sheath buffer. The detection of dopamine in an individual Aplysia californica cerebral ganglion neuron is demonstrated.

Animals↗
PubMed1999

Separating microbes in the manner of molecules. 1. Capillary electrokinetic approaches.

Selective, high-efficiency separations of intact bacteria may, in some cases, allow them to be identified and quantified in much the same way that molecules are done today. Two different capillary electrokinetic approaches were utilized. The first approach used a dissolved polymer-based CE separation that may be affected by size and shape considerations. Another approach uses capillary isoelectric focusing to separate bacteria by their surface charge or isoelectric point. Good peak shapes and extremely high efficiencies are observed (up to approximately 1,600,000 theoretical plates/m). Careful sample preparation and separation runs are essential in order to obtain reproducible separations. Expansion of these types of rapid, efficient microbial separations could have profound effects on many branches of science and technology.

Electrophoresis, Capillary↗
PubMed2000

Capillary electrophoresis of supercoiled DNA molecules: parameters governing the resolution of topoisomers and their separation from open forms.

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis. This technique is expected to be applied in the research of novel anticancer molecules targeting the activity of topoisomerase I. The separation of a plasmid mixture containing fully supercoiled molecules, single topoisomers, and their relaxed and open circular forms was tested in an electric field of 200 V/cm using Tris/borate buffer with the addition of magnesium ions at low concentrations and various sieving polymers. The resulting separation is quite simple to achieve and is clearly comparable to that obtained in agarose gels run at low voltage, but with an improved resolution, a higher quantitativity, and a higher speed of analysis. We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a good resolution of topoisomers. (II) Cellulose derivatives can be used as sieving polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.

Base Sequence↗
PubMed2000

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

Chromatography, High Pressure Liquid↗
PubMed1985

Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid containing bilayers.

The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations.

Calcium↗
PubMed1988

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy.

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.

1,2-Dipalmitoylphosphatidylcholine↗
PubMedDate not supplied

Development of a segmented model for a continuous electrophoretic moving bed enantiomer separation.

With the recent demonstration of a continuous electrophoretic "moving bed" enantiomer separation at mg/h throughputs, interest has now turned to scaling up the process for use as a benchtop pharmaceutical production tool. To scale the method, a steady-state mathematical model was developed that predicts the process response to changes in input feed rate and counterflow or "moving bed" velocities. The vortex-stabilized apparatus used for the separation was modeled using four regions based on the different hydrodynamic flows in each section. Concentration profiles were then derived on the basis of the properties of the Piperoxan-sulfated beta-cyclodextrin system being studied. The effects of different regional flow rates on the concentration profiles were evaluated and used to predict the maximum processing rate and the hydrodynamic profiles required for a separation. Although the model was able to qualitatively predict the shapes of the concentration profiles and show where the theoretical limits of operation existed, it was not able to quantitatively match the data from actual enantiomer separations to better than 50% accuracy. This is believed to be due to the simplifying assumptions involved, namely, the neglect of electric field variations and the lack of a competitive binding isotherm in the analysis. Although the model cannot accurately predict concentrations from a separation, it provides a good theoretical framework for analyzing how the process responds to changes in counterflow rate, feed rate, and the properties of the molecules being separated.

Computer Simulation↗