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PubMed1998

Capillary zone electrophoresis at subzero temperatures. III. Operating conditions and separation efficiency.

The advantages of carrying out capillary zone electrophoresis at subambient rather than ambient temperatures are examined. They include the possibility of using higher electric field strength and/or buffer concentration as well as wider capillary lumen without untoward effects of Joule heating on the separation. Furthermore, the separation efficiency is enhanced as molecular diffusivity decreases with temperature. A Beckman P/ACE unit with an auxiliary cooling system was used at temperatures down to -20 degrees C and, based on the dependence of axial temperature gradient on the capillary length and the temperature of the coolant, an average capillary temperature was defined. The day-to-day migration times of proteins were reproducible with an R.S.D. better than 2.3%. The improvements in the separation efficiency upon lowering the temperature from 40 to 1 degree C are illustrated by the electropherograms of four closely related peptides. Generally, the plate efficiency is enhanced at low temperatures and the analysis time is prolonged due to the increased viscosity. However, the current at a fixed electric field also decreases with temperature and the capillary can be shortened to speed up the analysis. With benzenesulfonic acids, the rate of the generation of theoretical plates was two- to three-times higher using a 27 cm long capillary at -15 degrees C than a 47 cm long capillary at 40 degrees C. By using a 180 microns instead of a 50 microns I.D. capillary for the separation of the four peptides, the sample loading could be increased about 15 times without loss of resolution. In the capillary zone electrophoresis of proteins, it was found that protein interactions with the capillary wall can be significantly reduced by increasing buffer concentration at low temperatures where salt mediated hydrophobic interactions are attenuated. This is illustrated by the separation of beta-lactoglobulins A and B in a raw fused-silica capillary using 400 mM sodium borate buffer, pH 8.4. The results suggest that at subambient or even subzero temperatures, the scope of CZE can be extended by several ways so that the benefits are great enough to justify the use of a cooling system and temperature control appropriate for psychroelectrophoresis with fused-silica capillaries.

Buffers↗

PubMed1999

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments.

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2 X or 4 X 48 base pair repeats) of the dopamine D4 receptor gene (D4DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.

DNA↗

PubMed1999

Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment.

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis-capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of alpha-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(-)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at approximately 10 nmol/l (approximately 1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 microliters sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3-6 microliters volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis-capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte (L(-)-tryptophan).

Anions↗

PubMed1999

Improvement of EPA method 8330: complete separation using a two-phase approach.

Complete separation of the 14 nitroaromatic and nitramine analytes targeted in EPA Method 8330 was achieved using a 30 x 4.6 mm Bondesil CN guard column in series with a 250 x 4.6 mm Bondesil C18 column (5 microns particles). Consistent with Method 8330 specifications, the mobile phase in the separation was 1:1 methanol:water and the flow-rate was 1.5 ml min-1. The success of this two-phase system proved to be largely a consequence of the unique resolution afforded by the Bondesil C18 column: complete separation of the 14 explosives could not be achieved using other CN/C18 column combinations. Additionally, while Method 8330 calls for a 100-microliter injection loop, separation on the Bondesil CN/C18 system was possible only using a 20-microliter injection loop. The loss of resolution with larger injection volume appears to be a result of the injection solvent, methanol, modifying the composition of the mobile phase both in the CN guard column and in the initial portion of the C18 column. The current method nevertheless represents an improvement over Method 8330, which calls for both a screening and confirming analysis. By separating the 14 Method 8330 analytes in a single run, the two-phase approach can increase sample throughput by decreasing analysis times.

Aniline Compounds↗

PubMed1999

Separation and characterization of octylphenol ethoxylate surfactants used by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns.

The separation and characterization of octylphenol ethoxylate surfactants were carried out by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns. For Triton X-100, simultaneous separation of octylphenol ethoxylate oligomers, positional isomers of octylphenyl group and butylphenol ethoxylate oligomers was achieved. These oligomers were completely separated and identified by means of MS spectra. Ethoxylated oligomers are eluted in the sequence from small to large oligomers. Fifty-five oligomers of Triton X-405 could be separated by using gradient elution. To separate octylphenol ethoxylate surfactant, non-end-capped branched fluorinated silica gel columns were superior to end-capped columns. The relationship between ln k' and methanol concentration was linear, indicating that branched fluorinated silica gel columns were operating in the reversed-phase mode. As Van 't Hoff plots of capacity factor for all oligomers gave straight lines, the equilibrium of conformation for the ethylene oxide chain might lay to one side of either zigzag or meander conformers.

Chromatography, High Pressure Liquid↗

PubMed1999

Isocratic separations of closely-related mono- and disaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection using dilute alkaline spiked with barium acetate.

Mixtures of closely related mono- and disaccharides may be efficiently separated by high-performance anion-exchange chromatography (HPAEC) only when relatively dilute alkaline eluents are employed (i.e., < 20 mM NaOH). The main drawbacks of these eluent solutions are (i) column regeneration between runs, (ii) poor reproducibility of the retention times, and (iii) the need for post-column base addition for enhancing sensitivity. Here, we describe some examples of isocratic separations of carbohydrates by HPAEC coupled with pulsed amperometric detection (PAD) accomplished by carbonate-free alkaline eluents (i.e., 5-20 mM NaOH) obtained upon addition of Ba(OAc)2 (1-2 mM). These separations include aldohexoses (i.e., galactose, glucose, and mannose), aminohexoses (i.e., glucosamine and galactosamine) and their N-acylated derivatives (i.e., N-acetylglucosamine and N-acetylgalactosamine) along with some isomeric disaccharides (i.e., lactose, lactulose and epilactose). The separation of closely related isomers of trehalose, alpha,alpha, alpha,beta, and beta,beta, is also presented. It is recommended to add Ba(OAc)2 to NaOH solutions several hours before using the alkaline eluent (i.e., 12-24 h) to ensure complete barium carbonate precipitation in the eluent reservoir. Adopting such a simple strategy can be especially useful for performing carbohydrate separations under isocratic conditions in which no regeneration and or re-equilibration of column between runs is required. Excellent repeatability of retention data throughout a three-day working session was observed, with relative standard deviations ranging from 2.0 to 3.7%, and from 0.5 to 2.0%, as day-to-day and within-day values, respectively. In addition, there was no need for postcolumn addition of strong bases to the eluent, and successful applications of the present approach confirmed its validity and practicability with detection limits of simple carbohydrates in the picomole range.

Acetates↗

PubMed1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time II. Two-run assay procedures.

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15-20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Chromatography, Liquid↗

PubMed2000

Separation of polyesters by gradient reversed-phase high-performance liquid chromatography on a 1.5 microm non-porous column.

Efficient separation of polyesters composed of a large number of individual oligomers was achieved on a 1.5 microm "non-porous" octadecylsilyl (ODS) silica support by gradient high-performance reversed-phase liquid chromatography (gRP-HPLC) with a mobile phase of acetonitrile, aqueous trifluoroacetic acid (0.2%) and tetrahydrofuran at ambient temperature and signal monitoring by UV absorption at 280 nm. Substantial signal splitting of oligomers in the low molecular weight (Mr) region is indicative that separation not only occurs with respect to molecular weight distribution (MWD) but also to chemical composition distribution (CCD) and functionality type distribution (FTD). Although separation according to CCD and FTD decreases with increasing number of oligomers, co-elution of species with identical number of repeat units but differing in either structure of repeat units or end-groups can be assumed from the relatively broad signals succeeding the aforementioned peaks showing at least partial resolution. Despite the observation that high Mr oligomers elute as sharp signals, the preceding observations suggest that each of these peaks presumably composes of more than one individual component. The polyester oligomers are eluted in the range of increasing Mr and therefore, either separation according to MWD or CCD/FTD was at least achieved for the low Mr sample constituents. Some principal mechanistic aspects of separation are discussed and adsorption seems to play the dominant role. The detection limit, defined as that sample amount yielding an unequivocal recognition on the base of its characteristic chromatographic fingerprint pattern was about 5,000 ppm for the pair Alftalat 3258 - Alftalat 3352 and 10,000 ppm for the pair Crylcoat 430 - Crylcoat 801.

Chromatography, High Pressure Liquid↗

PubMed2000

Separation of some platinum group metal chelates with 8-hydroxyquinoline by various high-performance liquid chromatographic methods.

Different HPLC methodologies are employed to evaluate the separation and determination of some platinum metals (Pt, Pd, Ir and Rh) after the formation of 8-hydroxyquinolate chelates. With the aim of reducing the number of steps in treating the samples, the method developed did not include the elimination of excess chelating reagent before the analysis of metal chelates. Reversed-phase (RP), non-aqueous reversed-phase (NARP) and normal-phase (NP) HPLC are compared. The RP-HPLC method only permits the quantitative separation of Rh and Pd from the excess reagent. A silica column can be used to separate Ir and Rh by NP-HPLC. The NARP-HPLC method allows for the effective separation of the four elements tested, but the high detection limit (90 ng) for platinum and the peak width do not favour its application for quantitative measurement. Platinum group metals can be quantitatively separated and determined by NP-HPLC using a cyano column in less than 15 min. The broad linear range of all the elements (between 1 and 500 ng) is superior to that which has been previously reported and the detection limits (1.0 ng for Pt, 0.3 ng for Pd, 1.0 ng for Ir and 0.3 ng for Rh) are slightly lower.

Chelating Agents↗

PubMed2000

Separation of peptides by strong cation-exchange capillary electrochromatography.

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.

Buffers↗

PubMed2000

Chiral separation of amino acids and peptides by capillary electrophoresis.

Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.

Amino Acids↗

PubMed2000

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds.

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

Growth Hormone↗

PubMed1988

Staging the separation of ischiopagus twins.

A pair of ischiopagus tetrapus twins were successfully separated in Calcutta in 1986. The separation was staged. The gastrointestinal tracts were separated at a preliminary operation, and definitive separation carried out 3 months later. Staging permitted us to perform the final separation on healthier babies with the exclusion of the intestinal contents from the wounds and with reduced operating time.

Digestive System Surgical Procedures↗

PubMed1998

Preliminary evidence that cell death may contribute to separation of the trachea from the primitive foregut in the rat embryo.

BACKGROUND/PURPOSE: The embryogenesis of many congenital tracheoesophageal abnormalities has been elucidated poorly, mainly because of an incomplete understanding of the normal mechanism of separation of the primitive foregut into the trachea and esophagus. There has been controversy about the existence and significance of the so-called tracheoesophageal septum. This study investigates the normal mechanism of tracheoesophageal separation in the rat. METHODS: Timed-pregnant Sprague-Dawley rats were killed on gestational days 11 to 16, respectively (the day on which the vaginal smear showed sperm was considered day 0 of gestation). Thirty-six embryos (six embryos from each age group) were fixed in 10% formalin. Fixed embryos from gestational day 13, 12, and 11 were sandwiched in melted agar to facilitate proper orientation before being processed. Half of the fetuses from each age group were sectioned serially in a transverse plane and the other half in a sagittal plane. The histological sections were stained with H&E. RESULTS: On gestational day 11, the foregut appears as a ventrodorsal slit in transverse section. Below the primitive pharynx, the epithelial cells on the ventral part of the foregut are actively proliferating, producing two lateral epithelial bulges (primitive bronchial buds). The epithelial cells of the dorsal aspect of the foregut show no sign of active growth. On day 12, the proximal trachea still shares a common lumen with the foregut. The distal trachea, the tracheal bifurcation, and both primary bronchi are recognized easily. At the point of tracheoesophageal separation, there is obvious debris of dead epithelial cells and condensed nuclei. These appearances were specifically located around the so-called tracheoesophageal septum and nearby grooves, resulting in enfolding cristae. There was no cell proliferation or inflammatory response in this region. On gestational day 13, the separation of the trachea and esophagus was almost complete. CONCLUSION: The present study demonstrates that a specific and consistent pattern of cell death in the region of the "tracheoesophageal septum" produced enfolding cristae that may be part of the mechanism of separation of the trachea and esophagus.

Animals↗

PubMed1998

In vivo accuracy of two radiographic systems in the detection of Björk-Shiley convexo-concave heart valve outlet strut single leg separations.

OBJECTIVE: Modified cineradiographic systems have been used clinically to detect partially broken outlet struts in normally functioning Björk-Shiley convexo-concave heart valves. Almost all such valves were explanted, presuming that full failure would likely follow. Inasmuch as the clinical setting only rarely permits examination of normally rated valves, the accuracy of radiographic detection cannot be clinically defined. This study uses the clinical radiographic technique in sheep implanted with known-status convexo-concave valves, comparing its accuracy and that of a newly developed, geometric image magnification radiography system. METHODS: Twenty-one sheep with mitral convexo-concave valves were studied on both systems. Five were used for extensive training. When operators were expert with both systems, images of four intact valves and 12 valves with outlet strut single leg separations, along with a seventeenth single leg separation valve used for calibration, were integrated into 112 image sets organized into a balanced incomplete block design for evaluation by eight trained, blinded reviewers. RESULTS: Cineradiography sensitivity was 24% versus 31% for direct image magnification. The odds ratio for detection of single leg separation by direct image magnification versus cineradiography was 2.0 (95% confidence interval, 0.76 to 5.9; p = 0.13). Cineradiography specificity was 93% versus 90% for direct image magnification. Sensitivity and specificity varied markedly by reviewer, with sensitivity ranging from 8% to 55% and specificity from 51% to 100% for the combined technologies. CONCLUSIONS: The data support the need for more intensive training for convexo-concave valve imaging and further investigation of unconventional radiographic technologies. Clinical cineradiography of convexo-concave valves may detect as little as 25% of valves having a single leg separation, underestimating the prevalence of single leg separations and thereby implying more rapid progression to full fracture than is actually the case.

Animals↗

PubMed2000

Application of high-power ultrasound to enhance fluid/solid particle separation processes

The separation of fine particles from gases or liquids is a topic of permanent industrial attention. The use of ultrasonic energy to assist conventional separation techniques seems to be very promising. The adequate applications of high-intensity ultrasonic fields may contribute to improve the efficiency and capacity of the separation methods presently used. The specific mechanisms to ultrasonically enhance separation processes basically depend on the medium to be treated. In gas suspensions, where very fine particles have to be removed, ultrasonic action involves agglomeration of particles in order to increase their size and, consequently, to improve collection efficiency of conventional filters. In liquid suspensions, agglomeration is, in general, less efficient than in gases. Nevertheless, the ultrasonic energy is useful to dewater fine-particle high-concentration suspensions such as slurries and sludges. This paper deals with the application of acoustic energy to assist fluid/solid separation processes in gas and liquid suspensions and presents some theoretical and experimental results in specific applications.

Journal Article↗

PubMed2000

Can cytoplasm exist without undergoing phase separation?

Studies on such systems as the lens of the eye and theoretical considerations suggest that phase separation may well occur in cytoplasm. In this chapter, several issues relevant to this question are raised. It is suggested that while the interaction between water and the macromolecules in a mixture is proving crucial to their phase separation behavior, the abnormal water that is widely observed in cytoplasm and concentrated protein solutions is unlikely to constitute a thermodynamic phase in the sense of phase separation studies. The role of fixed structures in the cytoplasm, the likelihood that the volume of separated phases would be small and subject to spreading over the fixed structures and the expectation that much of the phase volume could be occupied and dominated by properties of the interface are also discussed. Finally, some experimental approaches to studying the existence of liquid-liquid phases in cytoplasm are proposed. While there is no proof that phase separation exists in cytoplasm, application of some of the techniques outlined might well provide more positive evidence for its presence.

Animals↗

PubMed1998

Early maternal separation increases NGF expression in the developing rat hippocampus.

Nerve Growth Factor (NGF) is a neurotrophin involved in growth and differentiation of central cholinergic neurons. In this study a maternal separation paradigm was used to test whether levels of NGF might be affected by brief manipulations of rat pups early during ontogeny. The expression of NGF mRNA was examined in 3-day-old rat pups following 45 min maternal separation using in situ hybridization. Early maternal separation in neonatal rats resulted in increased expression of NGF mRNA in the dentate gyrus and the hilus of the hippocampus. NGF protein levels measured (by means of a sensitive ELISA assay) in the whole hippocampus the day following the separation procedure did not differ in separated vs. nonseparated pups. These data indicate that brief manipulations performed early during development can affect hippocampal NGF expression.

Animals↗