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At least 199 records · Page 11Linked to original sources
PubMed1999

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography.

Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.

Amino Acid Sequence↗
PubMed1999

In-house packing and testing of capillaries for capillary electrochromatography using simple equipment.

The feasibility of producing packed capillaries for capillary electrochromatography (CEC) is evaluated. Emphasis is put on the fact that only material was used that is already available in any LC/CE orientated laboratory. An experimental set-up is developed for filling the capillaries by use of an ordinary LC pump, and frits are sintered with a glowing resistance wire, fed by a d.c. power supply. Electrochromatography was carried out in an in-house built capillary electrophoresis apparatus, without pressurizing the vials. Under these conditions, the capillaries performed well, producing up to 190,000 plates per meter. Bubble formation did not appear, on condition that the mobile phase was thoroughly helium degassed. Even at ambient temperature, electrophoresis obeyed Ohm's law up to a voltage of 30 kV, proving that Joule heating was not a major concern.

Anti-Inflammatory Agents↗
PubMed2004

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis.

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.

DNA↗
PubMed2006

Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass spectrometry.

In this study, we report a novel procedure for fabricating internally tapered capillary columns suitable for the coupling of capillary electrochromatography (CEC) to electrospray mass spectrometry (ESI-MS). The internal tapers were prepared by slowly heating the capillary end in a methane/O2 flame. Due to continuous self-shrinking of the inner channel of the capillary, the inside diameter of the opening was reduced to 7-10 microm. The procedure is easy to handle, with no requirement for expensive equipment as well as elimination of problematic grinding of the tip. Several advantages of these new internal tapers, as compared to using externally tapered columns, are described. First, the problems of poor durability and tip breakage associated with external tapering were successfully overcome with the internal taper. A comparison of the online CEC/ESI-MS between external versus internal tapers showed that the latter provides enhanced electrospray stability, resulting in significantly lower short-term noise and very short-term noise values. In turn, the more rugged design of internal tapers allows performing CEC/MS utilizing a harsh polar organic mobile phase, which was not previously successful using an external taper due to higher operating current and electrospray arcing. Next, data on the reproducibility of the internally tapered CEC/MS column using warfarin and beta-blockers as model analytes are presented. For example, when comparing the reproducibility for separation of warfarin under reversed-phase conditions, the internal taper demonstrated superior intraday % RSD (1.6-3.4) as compared to the external taper intraday % RSD (5-6). Last, the applicability of performing quantitative CEC/MS with internally tapered capillaries is demonstrated for simultaneous enantioseparation of beta-blockers. Impressive quantitative results include good linearity of calibration curves (e.g., R2 = 0.9940-0.9988) and limit of detection as low as 30 nM. The sensitive detection of a minor impurity of one enantiomer at the 0.1% level in a major chiral entity buttresses the suitability of compliance with FDA guidelines.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2000

Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions.

The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.

Dihydroxyacetone Phosphate↗
PubMed2000

KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation.

Hyperkeratotic capillary-venous malformations (HCCVMs) are rare cutaneous lesions that occur in a small subgroup of patients with cerebral capillary malformation (CCM). CCMs cause neurological problems that range from headaches to life-threatening intracranial bleeding. CCMs and HCCVMs have a similar histopathological appearance of dilated capillary-venous channels. Genetic linkage of inherited CCMs has been established to three chromosomal loci, 3q25. 2-27, 7p13-15 and 7q21-22. The first mutations were identified in the CCM1 gene (located on 7q21-22), which encodes KRIT1 protein (KREV1 interaction trapped 1), presumably a membrane-bound protein with signalling activity. Although KRIT1 is known to interact with KREV1/RAP1A, a Ras-family GTPase, the exact function of KRIT1 in the formation of cerebral capillaries and veins is poorly understood. In this study, we screened five families with CCM for mutations in the KRIT1 gene. In one of the families, CCMs co-segregated with HCCVMs. We identified a KRIT1Delta(G103)mutation in this family, suggesting that this rare form of the condition is also caused by mutations in the CCM1 gene and that KRIT1 is probably important for cutaneous vasculature. Interestingly, this deletion introduces the earliest stop codon among identified mutations, suggesting a possible correlation between the molecular alteration and the cutaneous phenotype. Another novel mutation, KRIT1(IVS2+2(T-->C)), was found in a family with only cerebral capillary-venous malformations.

Brain↗
PubMedDate not supplied

Capillary ultrastructure and functional capillary density.

We briefly summarize our findings on alterations in capillary structure in skeletal muscle and heart in response to up to 30 min of ischemia. In frog sartorius muscle, reactive hyperemia was absent in atrophy. Increased spatial heterogeneity of red cell velocity in individual capillaries was observed, as were increases in the percentage of capillaries with damaged endothelium and white cell volume density in capillaries. Examination of the effect of aging on the response of the vascular bed to 30 min ischemia in extensor digitorum longus muscle of Fisher 344 rats suggested that the lack of postischemic hyperemia and structural alterations in frog muscle were related to disuse rather than aging per se. However, the specific study of disuse in rat extensor digitorum longus muscle after chronic application of tetrodotoxin revealed both capillary damage and a postischemic hyperemic response. It suggested an effect of the degree of tissue deterioration on the hyperemic response after short-term disuse in rat muscle, compared to longer-term atrophy in frog. Morphometric data in isolated rabbit heart suggested a link between microvascular compression as a result of tissue edema and decreased perfusion after 30 min total ischemia.

Aging↗
PubMed2004

Capillary coating for protein separation based on Si-O and Si-C covalent bond formation for capillary electrophoresis with laser-induced fluorescence detection.

Protein adsorption to capillary walls is one of the major complications in protein analyses with capillary electrophoresis (CE). Coating the capillary with different materials is used to reduce the adsorption. This chapter overviews different approaches used for capillary coating and concentrates on those utilizing Si-O and Si-C covalent bonds. The apparatus and methods are presented for capillary coating using Si-O and Si-C chemistry. Furthermore, procedures are described for monitoring the quality of coating.

Acrylic Resins↗
PubMed2002

Capillary electrophoretic separation by double-strand polyaniline-coate capillaries of the advanced glycation endproducts formed from N-alpha-acetyl-L-lysine with reducing sugars.

Capillary electrophoresis using a capillary coated with a double-strand coating of polyaniline:poly(methyacrylate-co-acrylic acid) (PAN:P[MA-AAI) was used to separate advanced glycation endproducts (AGEs) formed at 37 degrees C from model systems containing either glucose (Glc), fructose (Fru), or glyceraldehyde (GA) and N-alpha-acetyl-L-lysine (NALys). The presence of the P(MA-AA) as a second strand in the polymer allows the maintenance of the conductive state of the PAN at a wide pH range. Effects of buffer pH and coating concentration on the electroosmotic flow (EOF) were investigated. More AGE species can be detected for the GA/NALys mixtures using this coated capillary than upon an uncoated capillary. The coating procedure is simple and the stability of the coated capillary is good.

Carbohydrates↗
PubMedDate not supplied

[Serum protein electrophoresis: comparison of capillary zone electrophoresis Capillarys (Sebia) and agarose gel electrophoresis Hydrasys (Sebia)].

Since several years, serum proteins electrophoresis became a routine analysis, mainly performed by agarose gel electrophoresis, frequently semi-automated. We compared the new fully automated capillary electrophoresis system from Sebia, Capillarys, with our reference method, agarose gel electrophoresis Hydrasys (Sebia). This study focused on the evaluation of both the analytical performances and some practical aspects such as ease of use, rapidity, costs. It appears clearly from that study that both methods give similar results for the detection of monoclonal proteins. We notice that the capillary electrophoresis (Capillarys) displays higher sensitivity (97.2%) than the agarose gel electrophoresis Hydrasys (93.5%), however with a lower specificity (93.7 versus 98.9%). On the other hand, the Capillarys method displays obvious practical advantages such as full automation, ease of use and rapidity.

Blood Protein Electrophoresis↗
PubMed1979

Capillary diffusion capacity for I-131 and capillary filtration rate in female patients with idiopathic oedema.

In female patients with idiopathic oedema the capillary filtration rate and capillary diffusion capacity for I-131 were studied in lower-limb muscles and subcutaneous tissue. The capillary filtration rate averaged 0.276 ml/100 ml per minute, and the capillary diffusion capacity for I-131 averaged 9.8 mol/(mol/ml) per 100 g per minute in the gastroonemius muscle and 8.7 mol/(mol/ml) per 100 g per minute in the lower-limb subcutaneous tissue. All of these figures are statistically significantly higher than the corresponding control findings. The results are compatible with the concept of disturbed capillary permeability, and etiopathogenetic factor of idiopathic oedema.

Adolescent↗
PubMed2002

Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation.

On-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed using a microinjector as the interface for performing two-dimensional (2-D) protein/peptide separations of complex protein mixtures. The focusing effect of CIEF not only contributes to a high-resolution protein/peptide separation, but also may permit the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. The preparative capabilities of CIEF are much larger than most of capillary-based electrokinetic separation techniques since the entire capillary is initially filled with a solution containing proteins/peptides and carrier ampholytes for the creation of a pH gradient inside the capillary. The focused peptides which have a similar pI are coinjected into the second separation dimension and further resolved by their differences in hydrophobicity. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from a period beginning before steroid-triggered programmed cell death and extending to its completion. The separation mechanisms of CIEF and CRPLC are completely orthogonal and the overall peak capacity is estimated to be around approximately 1800 over a run time of less than 8 h. Significant enhancement in the separation peak capacity can be realized by further increasing the number of CIEF fractions and/or slowing the solvent gradient in CRPLC, however, at the expense of overall analysis time. The results of our preliminary studies display significant differences in the separation profiles of peptide samples obtained from salivary glands of animals staged at the 6 and 12 h following puparium formation.

Animals↗
PubMed1991

Analysis of peptides and proteins by capillary electrophoresis/mass spectrometry using acidic buffers and coated capillaries.

The direct coupling of capillary electrophoresis (CE) and mass spectrometry, combined with ionspray ionization using a coaxial capillary arrangement, is described. The CE/mass spectrometer interface is shown to be effective for the analysis of native and tryptic peptides and of proteins of high molecular weight such as bovine serum apotransferrin (approximately 78 kDa). Adsorption of cationic analytes under acidic buffer conditions is minimized through the use of a non-covalent coated capillary possessing an overall positive charge. Since the direction of the electroosmotic flow is thus reversed, compared to that in conventional CE separation on uncoated capillaries, migration of cations is achieved by applying a negative voltage (typically -30 kV) at the injector end of the capillary. In addition to the inherent advantage of providing pre-formed cationic species for mass spectral detection, this arrangement permits analysis of proteins of high isoelectric points even at low pH. The ability to conduct electrophoresis of globular proteins under acidic conditions also provides a means of monitoring their conformational changes, as reflected both by the variation of migration times and by concurrent changes in the multiply charged ion envelopes.

Amino Acid Sequence↗
PubMed2006

Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis.

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.

Journal Article↗
PubMed1994

Optimization of the loading limit for capillary zone electrophoresis of synthetic opioid and tachykinin peptides: a study of the interactions among the amount of peptide, resolution, saturation, injection volume and capillary diameter.

The upper loading limit in the capillary zone electrophoresis (CZE) of a mixture of fourteen synthetic opioid peptides and one tachykinin peptide was determined for capillaries of 50-, 75-, 100-, 130-, 150-, and 200-microns internal diameter (I.D.) to optimize the separation of these neuropeptides. The loading limit is an important parameter for preparative post-CZE studies. Electrophoretic resolution is illustrated for each capillary I.D. as the injection volume and amount of injected peptide increase. Loading limit is evaluated, based on the considerations of resolution, saturation, and current-stability. Among the capillaries studied, the capillary with 100 microns I.D. gives the optimal loading limit (39-78 pmol of each peptide).

Electrophoresis↗
PubMed1998

New set-up for capillary isoelectric focusing in uncoated capillaries.

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.

Diabetes Mellitus↗
PubMed1991

Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity.

DNA↗
PubMed2000

Capillary electrochromatography with fused-silica capillaries packed with copolymeric reversed-phase adsorbent and ion exchangers.

Macroporous poly(styrene-divinylbenzene) (PSDVB), PRP-1, a reversed-phase adsorbent, and PSDVB-based strong acid cation exchangers and strong base and weak base anion exchangers were evaluated as stationary phases for capillary electrochromatography (CEC). Electroosmotic flow (EOF) for adsorbent and exchanger packed fused-silica capillaries for acetone as the marker increases with increasing ion exchange capacity, buffer organic solvent concentration, and applied voltage, is nearly independent of pH, and decreases with increased buffer ionic strength. For anion exchangers, EOF is reversed. Thiourea, acetone, acrylamide, nitromethane, propanal, and acetic acid were evaluated as EOF markers and undergo weak interaction with the PSDVB-based stationary phases. EOF in a basic buffer is greater than or equal to silica-based C-18 and cation exchanger packed capillaries. For an acidic buffer, EOF for a PRP-1 capillary is almost twice the C-18 packed capillary. As analyte hydrophobicity increases, retention and migration time increases for the PSDVB-based stationary phases. As exchange capacity increases, availability of the polymeric matrix for analyte partitioning decreases, causing analyte migration time to decrease. Increasing buffer organic solvent concentration decreases analyte retention. The PSDVB-based stationary phases provide good resolving power and reproducibility and are applicable to the CEC separation of neutral, weakly acidic, and basic analytes. Efficiency, however, is less than obtained with silica-based stationary phases. Because of stability in a strong acid buffer, the CEC separation of weak acids, where dissociation is suppressed, and weak bases as cations is possible. Separations of short-chain alkyl aldehydes, methyl ketones, aromatic hydrocarbons, substituted benzene derivatives, and short-chain carboxylic acids are described.

Journal Article↗