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A large-scale magnetic separator for selective cell separations with paramagnetic microbeads.

An improved magnetic separator has been developed for use in large-scale cell separations. This separation method uses paramagnetic microbeads coated with antibodies that selectively bind to target cells. The magnetic separator attracts the microbead-target cell aggregates and holds these aggregates at its surface while the suspending fluid and nontarget cells flow past. The optimum separator design was determined to be two magnetic assemblies in series along with a peristaltic pump. The assemblies consist of neodymium-iron-boron magnet bars sandwiched between steel bars (magnetic pole pieces). The size and pole spacing of the two magnetic assemblies are designed to be different, so that the first assembly, which captures greater than 99.99% of the microbeads, has good magnetic reach-out and a high magnetic holding force at its surface, while the second assembly has an even higher magnetic holding force at its surface. Studies show that the separator can remove 1 x 10(10) microbeads from a suspension of red blood cells processed at a flow rate of 9 ml/min, so that no microbeads are detected in the effluent.

Animals↗

Separation of X- and Y-chromosome-bearing murine sperm by free-flow electrophoresis: evaluation of separation using PCR.

The effectiveness of separation of murine X- and Y-bearing sperm by free-flow electrophoresis was evaluated by the polymerase chain reaction (PCR). The ratio of X- and Y-bearing sperm from cauda epididymis was analyzed before and after free-flow electrophoresis. A Y-chromosome-specific sequence (pY353/B) and an autosomal sequence (myogenin) were used to estimate the ratio between X- and Y-sperm in the separated fractions. Cauda epididymal mice sperm were separated into two peak fractions under the electric field. Each peak fraction contained sperm of normal shape, however, the motility of the sperm was extremely diminished after separation by electrophoresis. DNA was extracted from 10(4) sperm from each fraction and from the unseparated sperm, and Y-chromosome specific PCR was performed. The PCR experiment revealed that fraction No. 16 (the peak near the cathode) was a Y-sperm rich fraction, whereas fraction No. 22 (the peak near the anode) was a Y-sperm poor one. These results suggested that murine X- and Y-sperm could be successfully separated by free-flow electrophoresis. Analysis of the chromosome-specific sequence by PCR was demonstrated to be a direct and adequate method to evaluate the separation of X- and Y-sperm.

Animals↗

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.

Albuterol↗

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.

Cyclodextrins↗

Increased responsiveness of presumed 5-HT cells to citalopram in adult rats subjected to prolonged maternal separation relative to brief separation.

RATIONALE: Certain adverse events in childhood, such as loss of a parent or sexual abuse, are associated with an increased vulnerability to develop depression later in life. Prolonged, daily maternal separation of rat pups induces several behavioral, endocrine and neurochemical changes similar to those observed in human depression. OBJECTIVES: Because dysfunction of brain serotonergic systems has been implicated in the pathophysiology of depression, the effects of neonatal maternal separation on these systems was studied in adult rats. METHODS: Male rat pups were subjected to daily maternal separation for 180 min (HMS180) from postnatal day 2 to day 14. Neonatal handled rats, i.e., pups undergoing daily 15-min separations during the same time period (HMS15), were chosen as a control group, since the 180-min separations involved handling of the pups, i.e., the pups were removed from the home cage during the separations. As adults, the effect of citalopram (0.05-0.80 mg/kg, intravenous) on the firing rate of 5-HT neurons in the dorsal raphe nucleus (DRN) was studied. RESULTS: The inhibitory effect of citalopram on serotonergic cell firing was significantly enhanced at doses of 0.1 mg/kg and 0.4 mg/kg in the HMS180 compared with that in the HMS15 rats. However, the number of binding sites and mRNA expression of the 5-HT transporter and 5-HT(1A) receptors in the DRN did not differ between the two rearing groups. CONCLUSION: These findings suggest that early life stress gives rise to persistent changes in the function, but not the density or mRNA expression of central 5-HT(1A) receptors and/or 5-HT transporters.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

Magnetic aqueous two-phase separation: a new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles.

A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-micron iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.

Cell Fractionation↗

Molded continuous poly(styrene-co-divinylbenzene) rod as a separation medium for the very fast separation of polymers. Comparison of the chromatographic properties of the monolithic rod with columns packed with porous and non-porous beads in high-performance liquid chromatography of polystyrenes.

Gradient elution separations of polystyrene standards in a monolithic molded 50 x 8 mm I.D. poly(styrene-co-divinylbenzene) rod column and in 50 x 8 mm I.D. and 30 x 4.1 mm I.D. columns packed with porous and non-porous poly(styrene-co-divinylbenzene) beads has been carried out. All of these separation media differ in shape and porosity. Excellent separations of eight polystyrene standards were achieved with both the molded monolithic rod and porous beads at moderate flow-rates. However, the monolithic medium proved to be superior for high-speed separations using very steep gradients at a flow-rate of 20 ml/min. Three polystyrene standards were separated in the rod column within 4 s. The separation in the column packed with non-porous beads was poor at flow-rates of 2-8 ml/min, while higher flow-rates led to an unacceptably high back pressure.

Chromatography, High Pressure Liquid↗

The effects of parental separation, the timing of separation and gender on children's performance on cognitive tests.

The relationship between exposure to parental separation and measures of cognitive ability and school achievement was examined in a birth cohort of New Zealand children studied to the age of 13 years. Children who were exposed to parental separation following the point of school entry had mean test scores that were between .1 and .3 standard deviations lower than children who had not been exposed to separation even when due allowance was made for potentially confounding variables. Separation prior to school entry was found to be unrelated to later outcomes after adjustment for confounding factors. There was no evidence to suggest that the effects of separation varied with the child's gender. The association between parental separation after school entry and cognitive test scores was similar for children whose custodial parents remarried and those who did not remarry.

Adolescent↗

A separation chamber to sort cells and cell organelles by weak physical forces. V. A sector-shaped chamber and its application to the separation of peripheral blood cells.

A separation chamber of 90 ml effective volume is described, that incorporates a sector shape (to prevent wall sedimentation) and anti-vortex lamellae (to prevent swirling upon acceleration and deceleration) as well as a flow-diverter for undisturbed layering and fractionation. The chamber is run in an ordinary table centrifuge and possesses a resolving power for equilibrium density centrifugation that seems unsurpassed. Peripheral human blood cells were separated in a Percoll gradient yielding in 1 single run the concurrent isolation of 99% pure monocytes, 99% pure lymphocytes and 99% pure granulocytes. Recoveries varied from 94% to 102%. By modifying the shape of the density gradient, either 99% of all monocytes collected were more than 92.5% pure and at the same time separation of small versus large lymphocytes was obtained, or substantial purification of neutrophils and eosinophils was achieved. The potential for simultaneous separation of monocytes, B, T cells and granulocytes was established. Good separations were obtained with buffy coats from 1 U of blood. The chamber also functioned well in the standard Böyum method for mononuclear cell preparation. In addition, a method for the rapid removal of erythrocytes (within 9 min) at unit gravity is described with nearly quantitative yield of white cells.

Cell Separation↗

A monoamine oxidase inhibitor reverses the 'separation syndrome' in a new hamster separation model of depression.

A broad spectrum monoamine oxidase inhibitor, tranylcypromine sulfate (Parnate) was tested in a new hamster separation model of depression. In this paradigm, male dwarf hamsters show increases in body weight, decreases in exploratory behaviors, and decreases in social interactions, when separated from female mates. Tranylcypromine (10 mg/kg s.c. daily for 14 days) effectively reduced body weight, increased exploration, and increased social interaction, in the separated males. Subsequent treatment with saline restored the separation-induced changes in body weight, exploratory behaviors, and other social behaviors. The 'separation syndrome' in dwarf hamsters appears to be completely reversed by at least one antidepressant treatment.

Animals↗

Migration behaviour and separation of tramadol metabolites and diastereomeric separation of tramadol glucuronides by capillary electrophoresis.

Capillary electrophoresis with UV detection was used to separate tramadol (TR), a centrally acting analgesic, and its five phase I (M1, M2, M3, M4, M5) and three phase II metabolites (glucuronides of M1, M4 and M5). Several factors were evaluated in optimisation of the separation: pH and composition of the background electrolyte and the influence of a micellar modifier, sodium dodecyl sulfate. Baseline separation of TR and all the analytes was obtained with use of 65 mM tetraborate electrolyte solution at pH 10.65. The lowest concentrations of the analytes that could be detected were below 1 microM for the O-methylated, below 2 microM for the phenolic and ca. 7 microM for the glucuronide metabolites. The suitability of the method for screening of real samples was tested with an authentic urine sample collected after a single oral dose (50 mg) of TR. After purification and five-fold concentration of the sample (solid-phase extraction with Oasis MCX cartridges), the parent drug TR and its metabolites M1, M1G, M5 and M5G were easily detected, in comparison with standards, in an interference-free area of the electropherogram. Diastereomeric separation of TR glucuronides in in vitro samples was achieved with 10 mM ammonium acetate-100 mM formic acid electrolyte solution at pH 2.75 and with basic micellar 25 mM tetraborate-70 mM SDS electrolyte solution at pH 10.45. Both separations showed that glucuronidation in vitro produces glucuronide diastereomers in different amounts. The authentic TR urine sample was also analysed by micellar method, but unambiguous identification of the glucuronide diastereomers was not achieved owing to many interferences.

Analgesics, Opioid↗

Silica monolithic membrane as separation medium. Summable property of different membranes for high-performance liquid chromatographic separation.

We studied an applicability of a silica monolithic membrane as separation medium for high-performance liquid chromatography (HPLC). We prepared porous monolithic silica membranes having a three-dimensional network structure to cut and shape into a membrane separation medium. We evaluated chromatographic properties of a variety of solutes using a column containing the membranes with HPLC to elucidate summable property of the membrane separation media. In addition, we made brief study on separation of HbA1c in whole blood with the stacked" membranes having different surface characteristics in one column, which is a membrane column. We confirmed that the membrane column was able to separate HbA1c from other matrix in whole blood to some extent, and it also had an excellent ability for hydrophobic and ion exchange adsorption.

Chromatography, High Pressure Liquid↗

Optimization of ion-exchange displacement separations. II. Comparison of displacement separations on various ion-exchange resins.

A variety of stationary-phase materials are currently available for the chromatographic purification of biomolecules. However, the effect of various resin characteristics on the performance of displacement chromatography has not been studied in depth. In Part I, a novel iterative scheme was presented for the rapid optimization of displacement separations in ion-exchange systems. In this article, the optimization scheme is employed to identify the optimum operating conditions for displacement separations on various ion-exchange resin materials. In addition, the effect of different classes of separation problems (e.g., diverging, converging or parallel affinity lines) on the performance of displacement separations is also presented. The solid film linear driving force model is employed in concert with the Steric Mass Action isotherm to describe the chromatographic behavior in these systems. The results presented in this article provide insight into the effects of resin capacity and efficiency as well as the type of separation problem on the performance of various ion-exchange displacement systems.

Algorithms↗

Harnessing electrical forces for separation. Capillary zone electrophoresis, isoelectric focusing, field-flow fractionation, split-flow thin-cell continuous-separation and other techniques.

A simple analysis, first presented twenty years ago, showed that the effectiveness of a field-driven separation like electrophoresis, as expressed by the maximum number of theoretical plates (N), is given by the dimensionless ratio of two energies N = -delta mu ext/2RT in which -delta mu ext is the electrical potential energy drop of a charged species and RT is the thermal energy (R is the gas constant and T is the absolute temperature). Quantity -delta mu ext is the product of the force F acting on the species and the path length X of separation. The exceptional power of electrophoresis, for which often N approximately 10(6), can be traced directly to the enormous magnitude of the electrical force F. This paper explores the fundamentals underlying several different means for utilizing these powerful electrical forces for separation, including capillary zone electrophoresis, gel electrophoresis, isoelectric focusing, electrical field-flow fractionation and split-flow thin continuous separation cells. Remarkably, the above equation and its relatives are found to describe the approximate performance of all these diverse electrically driven systems. Factors affecting both the resolving power and separation speed of the systems are addressed; from these considerations some broad optimization criteria emerge. The capabilities of the different methods are compared using numerical examples.

Chemical Fractionation↗

Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms.

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS-PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.

Cations, Divalent↗

Assessing adult adjustment to relationship separation: the Psychological Adjustment to Separation Test (PAST).

Relationship separation is associated with substantial adult adjustment problems. The Psychological Adjustment to Separation Test (PAST) was developed as a self-report measure of 3 key dimensions of separation adjustment problems: lonely negativity, ex-partner attachment and coparenting conflict. Two independent samples (n = 219 and n = 169, respectively) of recently separated adults, 60% of whom had children, completed the PAST and other measures of general adjustment. Exploratory and confirmatory factor analyses demonstrated a replicable 3-factor structure, with each factor showing satisfactory test-retest and internal reliability and good convergent and discriminant validity. The PAST meets initial criteria for a potentially useful new measure of adult separation adjustment.

Adaptation, Psychological↗

A new concept for separability problems in blind source separation.

The goal of blind source separation (BSS) lies in recovering the original independent sources of a mixed random vector without knowing the mixing structure. A key ingredient for performing BSS successfully is to know the indeterminacies of the problem-that is, to know how the separating model relates to the original mixing model (separability). For linear BSS, Comon (1994) showed using the Darmois-Skitovitch theorem that the linear mixing matrix can be found except for permutation and scaling. In this work, a much simpler, direct proof for linear separability is given. The idea is based on the fact that a random vector is independent if and only if the Hessian of its logarithmic density (resp. characteristic function) is diagonal everywhere. This property is then exploited to propose a new algorithm for performing BSS. Furthermore, first ideas of how to generalize separability results based on Hessian diagonalization to more complicated nonlinear models are studied in the setting of postnonlinear BSS.

Algorithms↗