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PubMed2007

Separation of C2-naphthalenes by gas chromatography x fourier transform infrared spectroscopy (GC x FT-IR): two-dimensional separation approach.

A two-dimensional separation approach involving gas chromatography (GC) and Fourier transform infrared spectroscopy (FT-IR) is used to separate C2-naphthalene isomers at or near baseline resolution. In addition to GC separation, the FT-IR also plays an important role in the separation, as well as its traditional role of detection and identification. This two-dimensional separation approach for the analysis of a C2-naphthalene isomeric mixture is a good example of separation design based on molecular difference and the characteristics of an analytical instrument. The details of this two-dimensional separation are discussed, along with the advantages and limitations of this approach. While two-(or multiple-)dimensional separations have demonstrated superior capabilities in the characterization of complex, largely unknown mixtures, the development of GCxFT-IR illustrates the applicability of this analytical approach in the separation of simpler but still challenging, mixtures. The GCxFT-IR results have extended this approach toward its application to the analysis of samples of more complicated composition.

Journal Article↗
PubMed2001

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Antigen-Antibody Reactions↗
PubMed1979

Separation of functional subpopulations of murine and human lymphoid cells on colloidal silica density gradients. I. Preparation of the Ludox AM gradient material and characterization of separation capacities.

This report describes a unique modification of an isopycnic density gradient system utilizing as a separating menstrum colloidal silica (Ludox AM). The primary advantages of this preparation are: (1) It is chemically defined, allowing extremely reproducible cell separation employing different lots of material; (2) the physical parameters (pH, density, salt concentration) of the final gradient suspension can be manipulated over a wide range of values, allowing for the separation of many different biological materials; (3) it allows separation of very large numbers of lymphoid cells with greater than 95% recovery of applied cells; (4) separated cellular subpopulations can be easily washed free of silica and cellular function is retained. This paper is a report of the preparation and functional characteristics of the gradient material as it relates to the separation of very large numbers of lymphoid cell subpopulations in both mouse and man. Subpopulations of murine and human lymphocytes separated by this gradient material were assayed for IgM synthesis, T-cell mediated cytotoxicity, and lymphokine production.

Animals↗
PubMed1980

Separation of blood cells from normal blood and blood of patients with various malignant blood diseases using separation media.

Differential counts of normal and leukemic cells separated from human peripheral blood were compared using three different separation media a) Verografin, b) Verografin-Ficoll, c) Isopaque-Ficoll (Lymphoprep), density 1.077 g/ml. In the separation the solution of Verografin (SPOFA) or Verografin-Ficoll yields analogous cell counts to Isopaque-Ficoll solution. The separation of leukemic cells is similar on all three separation media with considerable packing of some cells of the myeloid line. The blood cells separated from peripheral blood of normal donors on three various separation media correlate also in T and B lymphocyte counts, as demonstrated by the results of E and EAC rosette tests.

Cell Separation↗
PubMed2001

Separation selectivity patterns in the capillary electrophoretic separation of anionic enantiomers by octakis-6-sulfato-gamma-cyclodextrin.

The capillary electrophoretic separation of anionic enantiomers with multiply-charged, single-isomer, 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 (micro0RCD/micro0SCD), and parameter a, the complexation-induced alteration of the analyte's mobility (micro0SCD/micro0). Function analysis of the model indicates that there are six unique separation selectivity vs. resolving agent concentration patterns: in two of the patterns, separation selectivity asymptotically increases to the limiting value set by parameter b; in two other patterns, separation selectivity passes a local maximum and asymptotically decreases to the limiting value set by parameter b; and in the last two patterns, separation selectivity passes a local maximum, decreases to unity, then, after reversal of the intrinsic migration order, asymptotically increases to the limiting value set by parameter b. Though the patterns with asymptotically increasing selectivities were observed in earlier work, this paper reports the first experimental verification of the existence of the local selectivity maximum during the capillary electrophoretic separation of the enantiomers of several weak acids in high pH background electrolytes with octakis-6-sulfato-gamma-cyclodextrin as the resolving agent.

Cyclodextrins↗
PubMed2001

Isotachophoresis separations of enantiomers on a planar chip with coupled separation channels.

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line coupled separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. A 94-mm separation path in ITP with the tandem-coupled separation channels made possible a complete resolution of a 1.5 nmol amount of the racemic mixture of the enantiomers. However, this led only to a moderate extension of the concentration range within which the enantiomers could be simultaneously quantified. The best results in this respect were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels. Here, a high production rate, favored in the first separation channel, was followed by the ITP migration of the enantiomers in the second channel under the electrolyte conditions enhancing their detectabilities. In dependence on the migration configuration of the enantiomers, this technique made possible their simultaneous determinations when their ratios in the loaded sample were 35:1 or less (D-tryptophan a major constituent) and 70:1 or less (L-tryptophan a major constituent).

Anions↗
PubMed2003

Electrophoretic separations on chips with hydrodynamically closed separation systems.

This review focuses on capillary electrophoretic separations performed on capillary electrophoresis chips (CE chips) with hydrodynamically closed separation systems in a context with transport processes (electroosmotic flow (EOF)) and hydrodynamic flow (HDF)) that may accompany the separations in these devices. It also reflects some relevant works dealing with conventional CE operating under such hydrodynamic conditions. The use of zone electrophoresis (ZE), isotachophoresis (ITP) and their on-line combination (ITP-ZE) on the single-column and column-coupling CE chips with the closed separation systems and related problems are key topics of the review. Some attention is paid to sample pretreatment in the separations performed on the CE chips. Here, mainly potentialities of the ITP-ZE combination in trace analysis applications of the miniaturized systems are discussed in a broader extent. Links between the ZE separation and detection provide a frame for the discussion of current status of the detection on the CE chips. Analytical applications illustrate potentialities of the CE chips operating with the closed separation systems (suppressed HDF and EOF) to the determination of small ions present in various matrices by ZE, ITP and ITP-ZE.

Electrophoresis, Capillary↗
PubMed2006

A comparative investigation of methods for logistic regression with separated or nearly separated data.

In logistic regression analysis of small or sparse data sets, results obtained by classical maximum likelihood methods cannot be generally trusted. In such analyses it may even happen that the likelihood meets the convergence criteria while at least one parameter estimate diverges to +/-infinity. This situation has been termed 'separation', and it typically occurs whenever no events are observed in one of the two groups defined by a dichotomous covariate. More generally, separation is caused by a linear combination of continuous or dichotomous covariates that perfectly separates events from non-events. Separation implies infinite or zero maximum likelihood estimates of odds ratios, which are usually considered unrealistic. I provide some examples of separation and near-separation in clinical data sets and discuss some options to analyse such data, including exact logistic regression analysis and a penalized likelihood approach. Both methods supply finite point estimates in case of separation. Profile penalized likelihood confidence intervals for parameters show excellent behaviour in terms of coverage probability and provide higher power than exact confidence intervals. General advantages of the penalized likelihood approach are discussed.

Amniotic Fluid↗
PubMed2005

Application of a gas-solid fluidized bed separator for shredded municipal bulky solid waste separation.

A laboratory-scale gas-solid fluidized bed separator able to separate fractions of 5.6-50mm was used for separation of shredded municipal bulky waste (SBW) into combustibles and incombustibles. In batch-scale tests, it was found that accumulation of SBW in the bottom of the bed significantly reduced the separation efficiency. In this study, stirring was shown to be effective in preventing this accumulation. Flexible sheet materials such as paper and film plastics also significantly decreased the separation efficiency. In batch-scale tests, an overall efficiency of 90% was obtained when flexible materials such as film plastics and paper were excluded from the feed SBW. In continuous feeding tests, purities of the float and sink fractions attained 95% and 86% efficiencies, respectively, with an overall efficiency of 79%. The effect of feedstock shape on separation efficiency was also investigated. This study revealed that large particles can be properly separated on the basis of density, while the shape of the material significantly influenced behavior in the fluidizing bed.

Incineration↗
PubMed2003

Free-flow electrophoresis in a microfabricated chamber with a micromodule fraction separator. Continuous separation of proteins.

Continuous free flow electrophoresis of proteins was carried out in a microfabricated free flow electrophoresis (mFFE) module with the 30-microm thick slit of the separation. The newly developed micromodule fraction separator (MFS) was attached to the down-stream end site of the separation chamber of mFFE. By using the MFS, electrolyte solution from the separation chamber was introduced to the peristaltic pump without disturbing the electrolyte solution flow at the bottom side of the chamber. The separation of protein mixture samples was achieved by a hydroxypropylmethylcellulose pretreatment coating of the separation chamber. The pretreatment of the sample chamber effectively suppressed electroosmotic flow. All fractionated samples were collected using the MFS after continuous elecrophoresis and analyzed by reversed-phase HPLC. From the results of HPLC analyses none of the cytochrome c fractions at the other ports revealed cross talk phenomena at adjacent ports. A similar result occurred for the myoglobin. This means that these proteins were completely separated from each other by continuous mFFE, and the MFS functioned efficiently during continuous electrophoresis.

Animals↗
PubMed2001

Growth substrates made from duck excreta enriched wood shavings and source-separated municipal solid waste compost and separates: physical and chemical characteristics.

Production and use of compost is an effective means to reduce wastes, and offers a large potential as growth substrates and source of nutrients. The objective of this study was to determine the physical and chemical characteristics of duck excreta enriched wood shavings (DMC) and source-separated municipal solid waste (MSW) composts and separates, and to assess the physical characteristics of growth substrates made from these two composts and selected substrates. MSW compost separates were the following sizes: F1 > 4 mm diameter, 2 mm < F2 < 4 mm, 1 mm < F3 < 2 mm and F4 < 1 mm. Growth substrates were prepared by mixing DMC and F2 and F3 MSW separates (M/M ratios). Growth substrates A-E consisted exclusively of 10-60% DMC and 20-60% of MSW separates F2 and F3. Growth substrates F-J, and K-O were the same as substrates A E, with 15% M/M brick fragments or shredded plastic added as porosity agents, respectively. Growth substrates (BE/S) made of black earth (BE) and sandy loam soil (Ls) in a 1:4 (M/M) ratio, commercially available peat substrate (Pr) and an in-house sphagnum peat-based substrate (Gs) were used for comparison. Principal component analysis (PCA) showed that DMC was a better material than MSW with respect to porosity and water field capacity. MSW compost and separates differed by their relatively high levels of water-soluble and HCl-hydrolyzable N and increased advantageous water retention capacity. PCA also showed that substrates A-E exhibited porosity and water field capacity similar to those of Pr. Substrates F-J had porosity and water field capacity similar to those of BE/S, whereas substrates K O were more similar to Pr and to substrates A and B. The presented data indicate that DMC and MSW separates were complementary in providing good physical and chemical characteristics to the growth substrates.

Agriculture↗
PubMed2001

Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?

BACKGROUND: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre-anaphase separation is continuous or only transient and whether the transient separation has any physiological significance. RESULTS: Time-lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty-three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time-lapse observation of the double-colour marked cells by GFP-CEN and CFP-SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established. CONCLUSIONS: In the budding yeast, once sister centromeres separate, they rarely reassociate in pre-anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.

Anaphase↗
PubMed2002

Effects of separation and separation with supplemental stroking in BALb/c infant mice.

The purpose of this pilot study was to investigate selected stress, immune, and growth consequences of maternal separation and separation with supplemental stroking in neonatal BALB/c infant mice and their dams. Three groups of 5 litters each (7 pups per litter) were studied. Control litters were undisturbed. Separated litters experienced 3 h of daily maternal deprivation on postnatal days 6 to 10. Separated/stroked litters were separated also, but for 2 h, which was then followed by 1 h of stroking with a wet paintbrush to simulate maternal tactile stimulation. After the experimental period, all animals were returned to the nest and left undisturbed for 5 additional days. One pup from each litter was sacrificed on postnatal days 6, 8, 10, and 15. Spleens and thymuses were removed, weighed, and homogenized for cell sorting, cytokine analysis, and proliferation studies. Blood was drawn for corticosterone levels and hematocrit. Hematocrits and thymus weights were lower in separated mice, suggesting decreased growth and protein synthesis. Separated/stroked pups had increased splenic proliferation responses to conconavalin A and phytohemagglutinin at day 15. Separated dams' proliferative response to ConA was lower than control dams at day 15. Day 15 decreases in thymic CD8 cells occurred in pups, with an increased thymic H:S ratio in separated pups. CD90 cells were higher at day 15 in separated/stroked pups as were CD25s at day 10 in spleen and thymus. However, gene expression of cytokines was not measurable in spleen and thymic cells, with the exception of gamma-IFN in separated/stroked animals. Pooled organ homogenates were used in this preliminary work, and further studies are needed to more precisely analyze the stress, immune, and growth effects of these interventions.

Animals↗
PubMed2006

Loss of Rec8 from chromosome arm and centromere region is required for homologous chromosome separation and sister chromatid separation, respectively, in mammalian meiosis.

Chromosome separation in meiosis I is different from those in mitosis and meiosis II in that homologs separate from each other in the former while sisters do so in the latter. We show here that meiosis-specific cohesin subunit Rec8 in mouse oocytes shows essentially the same pattern of localization to those reported in yeasts and mammalian spermatocytes; Rec8 along chromosome arm (armRec8) is lost at the metaphase I-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until the onset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8 antibody into the oocytes inhibits homolog separation but not the first polar body emission (cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase II oocytes prevents sister separation in anaphase II after oocyte activation. These data demonstrate that the loss of armRec8 and cenRec8 is required for separation of homologs and sisters, respectively, but both are not required for other late mitotic events such as spindle elongation and cytokinesis in mouse oocytes. Further, by using some inhibitors for spindle assembly, proteasome and Topoisomerase II and overexpression of Securin, we propose that loss of armRec8 (homolog separation) and cytokinesis are suppressed until anaphase I by Securin whose destruction is regulated by spindle checkpoint-proteasome pathway, and that Topoisomerase II is required for homolog separation independently from such pathway.

Animals↗
PubMed2001

A new continuous-flow cell separation method based on cell density: principle, apparatus, and preliminary application to separation of human buffy coat.

With the recent progress in transfusion medicine, separation and isolation of cells in a large quantity is becoming increasingly important. At present, the continuous cell separation method in a preparative scale is limited to apheresis and elutriation: the former is mainly used for collection of platelet and buffy coat from the whole blood, while the latter separates cells virtually according to their size. Here we introduce a continuous flow method that separates cells entirely based on cell density. The method is gentle and capable of processing a large number of cells. The potential capability of the method was demonstrated on separation of lymphocytes and granulocytes from human buffy coat. Lymphocytes were enriched to 90% in the fraction at density = 1.065 and granulocytes are isolated in fractions at density = 1.075-1.080 while red cells were completely retained at the periphery of the channel. CD34 cells were distributed around 1.065 and coeluted with lymphocytes, suggesting that further enrichment requires focusing the density gradient around 1.070. The method could process 10(9) nucleated cells in 2 hours. Our preliminary results suggest that the present method is an effective and efficient means to separate blood cells.

Blood Cell Count↗
PubMed2006

The relationship of maternal separation anxiety and differentiation of self to children's separation anxiety and adjustment to kindergarten: a study in Druze families.

The study explores relationships between preschoolers' separation anxiety and adjustment to kindergarten and their mothers' separation anxiety and levels of differentiation. It also examines the universality of several theoretical and clinical notions in Bowen's [Bowen, M. (1978). Family therapy in clinical practice. Aronson: New York] Family Systems Theory, by testing them in the Eastern-collectivist Israeli-Druze society. The main findings were positive correlations between mothers' and children's separation anxiety, as well as negative correlations between children's separation anxiety and maternal differentiation (for total DSI score and for emotional cutoff), so that lower differentiation and higher cutoff were associated with higher levels of anxiety. Another interesting finding was that teachers perceived the children of Druze working mothers as having more problematic behaviors than non-working mothers, pointing to poorer adjustment to kindergarten. Results suggest that a crucial balance of separation and closeness provides an optimal context for meeting the needs and promoting the healthy development of both mother and child. The present study is the first to indicate relations between mothers' differentiation and preschoolers' separation anxiety among Druze participants, partially supporting the universality of Bowen's theory "in all families and in all cultures" [Kerr, M., & Bowen, M. (1988). Family evaluation. New York: Norton, p. 202].

Adult↗
PubMed1998

Separation of a process from the neuronal cell body using a thin aluminum foil separator.

A new technique using a simple-structured separator is described to separate the end of the process from the cell body (soma) of cultured cells. By this method, stimulation limited to neuronal processes is experimentally possible. The separator is of an oblong shape and consists of two parts: a square bracket-shaped frame made of Teflon and a thin aluminum foil which is stuck on both free ends of frame (bracket) coated with epoxy-resin. The separator is horizontally placed on the dish under microscope to divide the process on the way, so that the free end of the process is outside the oblong separator. This separator is simple and low cost, and does not need a long process, unlike other methods requiring a special culture dish with a micro-groove for growth of the neuronal process (neurite). The new technique could be useful in basic pain research and neurophysiology.

Aluminum↗
PubMed2003

Novel method for continuous cell separation by density gradient centrifugation: evaluation of a miniature separation column.

A compact bench-top model of the centrifuge enables continuous cell separation based on density differences. The apparatus holds a small separation disk equipped with a circular channel (8 mL capacity) separated by a septum. A set of isotonic Percoll media with different densities is continuously introduced at one terminal and collected from the other. Under a centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated with the separation of human buffy coat containing nucleated cells (>10(8)) among a large population (10(10)) of RBC. The results indicated that the method is capable of separating a large number of nucleated cells, with minimum damage, for a few hours of operation wherein neutrophils are well resolved from lymphocytes. The method may be applied to other types of samples including cord blood, blood from small animals, cultured cells, pancreatic beta cell islets, malaria parasites, sperm cells, etc.

Blood Cell Count↗