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PubMedDate not supplied

Evaluation of eluents from separations of CD34+ cells from human cord blood using a commercial, immunomagnetic cell separation system.

Human CD34+ cells from cord blood were separated in a two-step process using a commercial, immunomagnetic cell retention system. The performance of the system was evaluated by analyzing a number of eluents from the separations with a number of analytical techniques. In addition to cell counts and flow cytometry analysis, a new experimental technique that is undergoing development, cell tracking velocimetry (CTV), was used. CTV measures the degree to which a cell is immunomagnetically labeled, known as the magnetophoretic mobility, of a population of cells on a cell-by-cell basis and presents the results in the form of a histogram similar to flow cytometry data. The average recovery and purity of CD34+ cells from 10 separations was 52% and 60%, respectively. CTV analysis indicated that the mean magnetophoretic mobility of the positively enriched CD34 cells was 9.64 x 10(-5) mm3/T-A-s, while the mean mobility from negative eluents was -2.02 x 10(-6) mm3/T-A-s, very similar to the mobility of unlabeled cells. Within the positive eluents, the range of magnetophoretic mobility was approximately 50-fold, representing a plausible 50-fold range in surface CD34 antigen expression. CTV analysis also indicated that in some separations, positive cells were not retained by the immunomagnetic cell retention system. Finally, preliminary studies indicate that monocytes might be a primary cause in the lower purities and recoveries seen in this study. It is suggested that the monocytes phagocytose the magnetic nanobeads and become sufficiently magnetized to be retained within the Miltenyi column, reducing the purity of the positive eluent.

Antigens, CD34↗

PubMed1981

Separation of haemopoietic cells for biochemical investigation. Preparation of erythroid and myeloid cells from human and laboratory-animal bone marrow and the separation of erythroblasts according to their state of maturation.

The separation of haemopoietic bone-marrow cells by centrifugation through discontinuous density gradients of Percoll is described. This method was used to prepare fractions enriched in erythroblasts, myeloid blast cells or reticulocytes from bone marrow of anaemic and non-anaemic rabbits, from the marrow of other anaemic laboratory animals and from human samples. It is a simple, rapid, reproducible and inexpensive technique that can be readily adapted to suit individual requirements. Secondly, a convenient method is presented for the separation of large quantities of bone-marrow cells into fractions enriched in erythroblasts at different stages of maturation, by velocity sedimentation through a linear gradient of 1-2% sucrose at unit gravity. In vitro, erythroblasts adhere together strongly via a mechanism almost certainly involving a beta-galactoside-specific surface lectin termed erythroid developmental agglutinin. Since the efficiency of cell-separation techniques depends heavily on the maintenance of a single cell suspension in which each unit can move independently, the presence of an adhesive molecule at the cell surface is of considerable significance. The effect of washing the marrow with a lactose-containing medium, which has been shown to remove the agglutinin, was therefore investigated in relation to both methods. The separation on Percoll gradients is considerably enhanced by this treatment. In addition, the unit-gravity sedimentation gradient can be loaded with 5-10 times more cells after lactose extraction in comparison with intact marrow. Although enrichment is less, a useful fractionation according to maturation is still obtained.

Anemia↗

PubMed1999

Separation of double-stranded and single-stranded DNA in polymer solutions: I. Mobility and separation mechanism.

We have studied the separation of single-stranded and double-stranded DNA in a matrix of entangled, linear poly-N,N-dimethylacrylamide. Our results give better insight into the mechanisms involved during separations in polymer solutions. The dependence of different parameters on DNA size, electric field, pore size and the polymer chain length are evaluated and compared to theoretical predictions. Striking differences between experimental data and predicted scaling laws are found. Our data should help to optimize DNA separation in capillary electrophoresis and to improve existing models for DNA separation in porous matrices.

Acrylamides↗

PubMed2001

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.

Chromatography, Gas↗

PubMed2003

Tailoring the separation selectivity of metal complexes and organometallic compounds resolved by capillary electrophoresis using auxiliary separation processes.

The use of auxiliary separation mechanisms to manipulate the separation selectivity of metal complexes and organometallic species is reviewed. Auxiliary separation mechanisms included in the review are micellar electrokinetic capillary chromatography, ion-pairing and ion-exchange electrokinetic chromatography. This paper discusses how these secondary mechanisms can be effectively employed to tailor separation selectivity.

Chromatography, Ion Exchange↗

PubMed2005

Separation and on-column preconcentration of some nonsteroidal anti-inflammatory drugs by microemulsion electrokinetic capillary chromatography using high-speed separations.

Various strategies have been investigated for separating a group of nonsteroidal anti-inflammatory drugs (NSAIDs) by microemulsion electrokinetic capillary chromatography (MEEKC) using high-speed separations. The parameters that of affect the separation, such as the nature of the oil droplet and the buffer, and the surfactant concentration have been studied. In addition, several organic solvents were used to decrease the retention of the analytes in the oil droplet phase and to improve the resolution of the NSAIDs. The optimum microemulsion background electrolyte (BGE) solution made of 0.8% w/w ethyl acetate, 6.6% w/w butan-1-ol, 6.0% w/w acetonitrile, 1.0% w/w sodium dodecyl sulfate (SDS), and 85.6% w/w of 10 mM sodium tetraborate at pH 9.2 resolved the drugs within 8 min. The short-end injection procedure is an alternative for reducing the analysis time. When this procedure was used, the microemulsion BGE solution consisted of 0.8% w/w ethyl acetate, 6.6% w/w butan-1-ol, 17.0% w/w methanol, 1.0% w/w SDS, and 74.6% w/w of 10 mM sodium tetraborate, pH 9.2, and the NSAIDs were separated within 3 min. The reversed electrode polarity stacking mode (REPSM) technique was applied to the on-line concentration of the NSAIDs. In this technique, the sample matrix was pumped out of the capillary using a polarity-switching step. When this technique was applied, the sensitivity was enhanced up to 40-fold and the limits of detection (LODs) were in the low microg.L(-1) levels.

2-Propanol↗

PubMed2005

Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions.

Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.

Chromatography, High Pressure Liquid↗

PubMed2006

A 150 micron id packed column for the separation of soybean proteins by elution gradient mu-HPLC: simultaneous separation of soybean proteins from cereal and milk proteins.

Mu-HPLC has previously been used to increase the resolution and sensitivity of protein separations but never for the analysis of soybean proteins. In this work, soybean proteins were, for the first time, separated using a capillary column with an internal diameter of 150 microm packed with a Genesis C18 stationary phase (4 microm, 300 angstroms) and UV detection. TFA and acetic acid were investigated as ion-pairing reagents in order to optimise water-ACN gradients to achieve this separation. The column showed good selectivity enabling the separation of soybean proteins from other vegetable proteins such as cereal (wheat, rice and corn) and also from milk proteins. The developed method was applied to the detection of soybean proteins in commercial products elaborated with mixtures of vegetable proteins.

Chromatography, High Pressure Liquid↗

PubMed2000

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations.

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.

Animals↗

PubMed1989

Sequence of centromere separation: separation in a quasi-stable mouse-human somatic cell hybrid.

A quasi-stable mouse-human hybrid cell line, HR61, containing between one and ten human chromosomes was analyzed for the sequence of centromere separation. The purpose was to determine which genome of the two initiates centromere separation first. The data clearly indicate that the separation of centromeres of the human genome is not only initiated but is completed before any centromeres from the mouse chromosomes start splitting into daughter units. The information on whether uniparental chromosome loss results from a lack of deposition of kinetochore proteins was equivocal. The human genome also completes its DNA replication before the mouse genome does. Our studies, therefore, show that the timing of centromere separation is tightly linked to the completion of replication of DNA. At least in this cell line the segregant genome is not the one which exhibits delayed DNA replication.

Animals↗

PubMed1995

Sb(III) and Sb(V) separation and analytical speciation by a continuous tandem on-line separation device in connection with inductively coupled plasma atomic emission spectrometry.

A sensitive, precise and automated non-chromatographic method for Sb(III) and Sb(V) analytical speciation based on a continuous tandem on-line separation device in connection with inductively coupled plasma-atomic emission (ICP-AES) detection is proposed. Two on-line successive separation steps are included into this method: a continuous liquid-liquid extraction of Sb(III) with ammonium pyrrolidine dithiocarbamate (APDC) into methylisobuthylketone (MIBK), followed by direct stibine generation from the organic phase. Both separation steps are carried out in a continuous mode and on-line with the ICP-AES detector. Optimization of experimental conditions for the tandem separation and ICP-AES detection are investigated in detail. Detection limits for Sb(III) were 3 ng.mL(-1) and for Sb(V) 8 ng.mL(-1). Precisions observed are in the range +/- 5%. The proposed methodology has been applied to Sb(III) and Sb(V) speciation in sea-water samples.

Journal Article↗

PubMed1986

Sequence of centromere separation: a mechanism for orderly separation of dicentrics.

Stable dicentric chromosomes from three mouse cell lines (viz., SEWA Rec4, brain tumor, and L-cells), as well as a human t(9;11) line were analyzed for the sequence in which the two centromeres separate. At prometaphase, as well as in many cells at midmetaphase, the dicentrics express the two centromeres in the form of two primary constrictions. As the cell advances to late metaphase, one of the constrictions loosens the two chromatids so that eventually there is no connection between them. The other centromere stays intact during this period and separates into two units at the metaanaphase junction along with the rest of the genome. The centromere that separates prematurely (out-of-phase) usually is the same in a given dicentric. It is proposed that such a prematurely separating centromere does not function as active element during chromatid migration. Apparently, in dicentrics some sort of control is exerted to eliminate the functioning of one centromere. The nature of such control is not understood at this time. The mouse dicentrics "synthesize" only one kinetochore as definable by antikinetochore antibody studies.

Animals↗

PubMed2004

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids.

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.

Amino Acids↗

PubMed2004

Liquid chromatographic separation of the enantiomers of trans-chlordane, cis-chlordane, heptachlor, heptachlor epoxide and alpha-hexachlorocyclohexane with application to small-scale preparative separation.

Analytical high-performance liquid chromatographic separations of the individual enantiomers of five polychlorinated compounds were obtained on polysaccharide stereoselective HPLC columns. The enantiomers of the pesticides trans-chlordane, cis-chlordane and heptachlor were separated on CHIRALCEL OD using a hexane mobile phase. The enantiomers of the heptachlor metabolite, heptachlor epoxide, were separated on CHIRALPAK AD using a methanol mobile phase. The enantiomers of alpha-hexachlorocyclohexane (alpha-HCH), were separated on CHIRALCEL OJ using a hexane/2-propanol mobile phase. Similar chromatographic conditions using preparative columns were used to isolate approximately 250 mg of each of the individual enantiomers. The purified individual enantiomers have been submitted for testing of their endocrine disruptor (ED) activity.

Chlordan↗

PubMed2004

Liquid chromatographic-mass spectrometric separation of oligoalanine peptide stereoisomers: influence of absolute configuration on enantioselectivity and two-dimensional separation of diastereomers and enantiomers.

This contribution describes the chromatographic separation of peptide stereoisomers. Thereby, one focus is laid on the influence of the absolute configurations of peptide enantiomer pairs on their enantioselective separation. Three different N-terminal protecting groups and three different chiral stationary phases (CSPs) based on cinchona alkaloid derivatives were employed and oligoalanine di-, tri- and tetra-peptides were used as model set. The absolute configurations of the individual enantiomeric pairs were found to profoundly influence both the elution order and the enantioselectivity. The stereoselective molecular recognition mechanism was observed to be dependent on the combination of configuration and the chosen protecting group and CSP. As the CSPs on their own exhibited insufficient diastereoselectivity, a two-dimensional liquid chromatography-mass spectrometry (LC-MS) system was developed for the separation of both diastereomers and enantiomers of peptides in the second part of this study. Diastereomers were separated by reversed phase (RP) and the resulting enantiomeric pair fractions were transferred to a CSP for enantioseparation. All eight stereoisomers of a tripeptide (Ala-Ala-Ala) and 9 out of 10 stereoisomers of a tetrapeptide (Ala-Ala-Ala-Ala) could be successfully resolved.

Alanine↗

PubMed2006

Separation stress, litter size, and the rewarding effects of low-dose morphine in the dams of maternally separated rats.

Potential differences in sensitivity to the rewarding effects of morphine as a function of litter separation stress were assessed in post-weaning rat dams. During the first two weeks postnatal, Sprague-Dawley rat litters were subjected to daily 15- or 180-min sessions of dam-pup separation while control litters only experienced twice-weekly animal facility care. One week after weaning, the dams (n=7 per group) underwent a fully unbiased conditioned place preference (CPP) procedure to 1 mg/kg subcutaneous morphine. CPP responses after each conditioning cycle were recorded. Rates of acquisition and asymptotic levels of CPP were comparable in all groups; however, an inverse relationship between litter size and magnitude of morphine CPP was revealed. Although these initial data indicate no differential sensitivity to the rewarding effects of low-dose morphine produced by the stress of litter separation, this assessment of litter size and drug-induced place conditioning in post-weaning litter-separated dams is the first of its kind. Potential effects of other doses, drugs of abuse and post-partum manipulations remain to be evaluated within this emerging etiological model.

Analysis of Variance↗

PubMed2002

Two dimensional reversed-phase-reversed-phase separations isomeric separations incorporating C18 and carbon clad zirconia stationary phases.

Informational theory and a geometric approach to factor analysis were employed to evaluate the degree of orthogonality of a two-dimensional reversed-phase-reversed-phase chromatographic system. The system incorporated a C18 column as one dimension and a carbon clad zirconia column as the second dimension. In order to study the resolving power of this system, the separation of a sample matrix containing an artificial mix of 32 isomers (structural and diastereoisomers) was evaluated. Using this system, between 25 and 28 of the 32 isomers could be separated, depending on the mobile phase combinations--with resolution that could not possibly be achieved in a single one dimensional separation. The results from this study indicate that in order to fully evaluate the resolving power of a 2D system multiple methods of analysis are most appropriate. This becomes increasingly important when the sample contains components that are very closely related and the retention of solutes is clustered in one quadrant of the 2D space. Ultimately, the usefulness of the 2D separation is determined by the goals of analyst.

Carbon↗

PubMed2002

Novel surface modification of polymer-based separation media controlling separation selectivity, retentivity and generation of electroosmotic flow.

Uniformly sized packing materials based on synthetic polymer particles for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) have been prepared from polymerization mixtures containing methacrylic acid (MAA) as a functional monomer and by using a novel surface modification method. This "dispersion method" affords effectively modified separation media. Both the amount of MAA utilized in the preparation and reaction time affect the selectivity of chromatographic separation in both the HPLC and the CEC mode and electroosmotic flow. This detailed study revealed that the dispersion method effectively modified internal surface of macroporous separation media and, based on the amount of MAA introduced, exclusion mechanism for the separation of certain solutes could be observed.

Chromatography, High Pressure Liquid↗