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PubMed1984

Preparative and analytical separation of amygdalin and related compounds in injectables and tablets by reversed-phase HPLC and the effect of temperature on the separation.

Previous HPLC procedures for amygdalin and related compounds in injectables and tablets were either time consuming or produced inadequate separations of D-amygdalin and its epimer. A study of the effects of temperature on the separation resulted in development of an HPLC method for amygdalin and some related compounds, using water as the mobile phase at 15 degrees C. Multimilligram quantities of amygdalin and related compounds were separated by this preparative procedure. The aqueous mobile phase allows the compounds to be recovered by simple lyophilization of the sample after elution. This permitted the carbon-13 NMR spectrum of the isolated aglyconic epimer of amygdalin to be reported for the first time. D-amygdalin, its L-mandelonitrile epimer (D-epiamygdalin), their hydrolysis products (the epimeric amides and epimeric acids), and the sugar gentiobiose were separated by the method.

Amygdalin↗

PubMed1990

Liquid chromatographic retention behavior and separation of chlorophenols on a beta-cyclodextrin bonded-phase column, Part II. Monoaromatic chlorophenols: separation.

The liquid chromatographic separation of 19 monoaromatic chlorophenols on a beta-cyclodextrin bonded-phase column is investigated in both an isocratic and a gradient elution mode. Even though the isocratic mode can be employed for the separation of the member components of each category of chlorophenols (e.g., mono-, di-, etc.), significant overlaps between the retention times of the various categories prevent the separation of all chlorophenols on a single chromatogram. Gradient elution can be used, however, for the separation of 15 of the 19 chlorophenol isomers. The unique features of gradient elution as applied to beta-cyclodextrin bonded-phase columns are discussed. The detection of chlorophenols with UV and electrochemical methods is also discussed and the two are compared.

Chlorophenols↗

PubMed1992

Separation of glucuronides from urine by coupled-column separation using underivatized silica as precolumn.

Glucuronides are separated from urine by coupled-column separations (CCSs). The fraction containing the glucuronide(s) is transferred on-line from a silica precolumn to the analytical column (octadecyl derivatized silica), enriched, and separated by ion-pair chromatography. The retention and selectivity on the precolumn are controlled by pH, buffer components, organic modifier, and ion-pair agent. After the injection of filtered urine samples, glucuronides with different chemical properties can be separated. The total analysis of morphine-3-glucuronide and morphine-6-glucuronide is accomplished in less than six minutes, with UV detection at 210 nm.

Buffers↗

PubMed1976

Separation of tumor cells by density gradient centrifugation: recent work with human tumors and a discussion of the kind of quantitation needed in cell separation experiments.

The application of centrifugation to the purification of individual kinds of cells from tumors has been reviewed. Recent applications of a previously described isokinetic gradient to the purification of subpopulations of cells from human tumors are presented. Individual kinds of cells have been separated from Hodgkin's disease by a method described previously, and the cells from Hodgkin's disease which contain the tumor associated antigen described by Order and his colleagues have been identified for the first time. The importance of the thorough documentation of cell separation procedures is discussed, and we have specifically listed several kinds of data which we believe are important in the documentation of cell separation techniques.

Adenocarcinoma↗

PubMed1992

Effective concentration of bone marrow mononuclear cells using density gradient separation within an automated cell separator.

An effective method for concentrating bone marrow is described. Concentration was achieved using an intermittent flow cell separator. Elimination of mature haemopoietic cells was enhanced by the addition of a density separation medium (Ficoll-metrizoate) which was then removed by washing. All procedures were undertaken using the cell separator, this allowed for standardization of procedure and less manipulation which is associated with enhanced mononuclear cell recovery and less risk of microbial contamination. Mature granulocytes were depleted by 86.8% and red cells by 97.7% whilst mononuclear cells showed a 49% recovery. Marrows processed in this way and subsequently purged and/or cyropreserved were shown to successfully engraft when reinfused.

Bone Marrow Transplantation↗

PubMed2006

Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells.

Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm Ø) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm Ø) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl alpha-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.

Blood Proteins↗

PubMed2004

ACSD labelling and magnetic cell separation: a rapid method of separating antibody secreting cells from non-secreting cells.

Several new ways of selecting cells have recently been developed. These include magnetic separation of cells by labelling with magnetic beads against the recombinant product, gel microdrop technology which encapsulates the cells in gelatine beads and matrix-based secretion assays. Affinity capture surface display (ACSD) is a matrix-based assay for the enrichment of high producing cells and relies on the strong affinity between biotin and avidin derivatives. Matrix-based assays have previously only been used for the enrichment of recombinant cells. Here, we have optimised this assay and developed a method of separating antibody producing cells from non-producing cells in a recombinant myeloma cell line using ACSD combined with MACS magnetic separation. The method is rapid, simple enough to become routine and adaptable to many different secreted products from recombinant mammalian cells.

Animals↗

PubMed2000

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns.

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.

Animals↗

PubMed1996

Crying in separated and non-separated newborns: sound spectrographic analysis.

During the first 1-2 h after birth crying occurs during separation from the mother and stops on reunion. In rats, such "separation distress calls" have distinct phonetic properties. We examined this early crying by sound spectrography in 29 healthy, full-term, vaginally delivered babies, randomly assigned either to be kept in a cot or to be placed in body contact with the mother during the 90 min following birth. The former babies cried almost 10 times more than the latter ones. The duration of the cry signal (the smallest element of a cry analysed by spectrography) in both groups was 0.8-09s; the melody contour was flat or slightly rising--falling with a fundamental frequency of around 500 hertz. The cry is provisionally characterized as a discomfort cry, elicited mainly by separation from the mother.

Animals↗

PubMed1976

Analysis of velocity sedimentation techniques in cell separation. Influence of apparative and sample properties on separative power, resolution and sensitivity.

In the present investigation the velocity sedimentation technique was analysed with respect to separation resolution, power and sensitivity. It was found that apparative modifications do not influence the resolution, which is a function of the contribution of apparative errors to the dispersion. A surprisingly small parameter of 0.15 was determined and it seems unlikely that this value can be improved. On the other hand an apparative modification is presented which improves the separation power and makes sample loading independent of the gradient filling. If cells (from rat bone marrow) were separated, a several times higher dispersion for a given cell volume was observed than was due to the apparative error. It was concluded that density variations were the major source of this dispersion. Since cell volume and density apparently show independent variations within a biological cell population the cell density cannot be disregarded if velocity sedimentation profiles are discussed in physical terms as is often done.

Cell Separation↗

PubMed1991

[Stem cell separation with different cell separators].

For some years, there has been an increasing success in transplanting peripheral blood stem cells (PBSC) instead of autologous bone marrow in patients suffering from different malignancies. While collecting PBSC for autologous transplantation, we compared four different separation techniques and three different cell separators (COBE Spectra, Fresenius AS 104, Haemonetics V50) routinely used for platelet production. Our results suggest that continuous flow separators seem to have some advantage over discontinuous flow machines in harvesting PBSC.

Blood Cell Count↗

PubMed1995

Preparation and storage in Plasma-Lyte A of platelets collected with the cell separator CS3000 Plus equipped with the PLT30-separation and TNX6 collection chambers.

The last five years have been characterized by the presentation of new cell separators the main task of which is the collection of high yield-high quality platelets. The CS3000 is an old apparatus which has undergone rejuvenation to sustain the assault of its new competitors. The CS3000 Plus Omnix system is the latest version to be offered along with a combination of TNX-6 separation/PLT30 collection chambers for optimal platelet collection. In this paper we present our results with this apparatus and configuration after its adaptation to the collection and storage of platelets in a non-plasma medium, the Baxter Plasma-Lyte A. After separation the platelet product (PC) was left in the collection chamber and resuspended with 200 ml of Plasma-Lyte A instead of being resuspended in autologous plasma as usual. Plasma was collected in a separate bag (400-450 ml) for transfusion or fractionation purposes. PC quality was assessed by evaluating the platelet yield (4.17 +/- 1.8 x 10(11), and the WBC contamination (4.8 +/- 2.6 x 10(5)). The presence of platelet aggregates (platelet count after aggregate fixation with formalin/platelet count after disaggregation in EDTA), the aggregation induced by ADP, collagen and ristocetin, the hypotonic shock response and the stability of membrane glycoproteins (CD 62 - 62 - 63 - 36- 42b - 51) were measured in the preapheresis samples and in the PC immediately after, 24 and 72 hours after collection. These results were totally satisfactory as was the post-transfusion survival measured as corrected count increment in 10 transfusions to non refractory patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1994

Responses of free-ranging rhesus monkeys to a natural form of social separation. I. Parallels with mother-infant separation in captivity.

Observations of 23 free-ranging rhesus monkey infants on Cayo Santiago, Puerto Rico, indicated that mothers' first postpartum estrous periods were marked by large increases in the amount of time infants were separated from their mothers, by disturbances in mother-infant relationships, and by increases in infant distress behavior. When their mothers resumed mating, most infants showed signs of agitation; a few briefly showed indications of depression. Male infants responded to their mothers' resumption of mating by playing more, whereas females engaged in less play and more allogrooming. The results suggest (a) that basic parallels exist between the behavioral responses of rhesus infants to their mothers' resumption of mating in the field and to forcible separation from their mothers in captivity and (b) that early separation experiences may play a role in the normal development or manifestation of sex differences in behavior.

Animals↗

PubMed2001

Protein from pea mutants as a co-product in starch separation--isolates from wet and dry separation: yield, composition and solubility.

The dry separation behaviour of cotyledon material of certain pea genotypes (wild type and 5 r mutants) with different metabolic background in starch biosynthesis allowed satisfying protein recovery only with smooth seeded genotypes. Structural effects and protein distribution (starch granule size distribution, protein in non-starch part) play an important role. According to expectations protein composition remained unaltered during dry separation procedures. Independent of genotypes used the applied conditions in wet separation allowed to recover just 50% of seed proteins while approximately 10% got lost with fibres and 40% with unspecified process water streams. Globular vicilins remained dissolved during iso-electric precipitation and were discarded with waste water streams. The solubility of isolates was rather low under alkaline conditions, even for the round seeded cv. Odalett (0.4 to 0.7% dry substance).

Genotype↗

PubMed2002

Size-based separation of synthetic polyelectrolytes in entangled polymer solution capillary electrophoresis: the effect of binary mixtures of separating polymers differing in molecular mass.

The influence on the electrophoretic behavior of polystyrenesulfonates of the percentage of high-molecular-mass chains in an entangled poly(ethylene oxide) solution having a bimodal molecular mass distribution has been investigated and compared with the results obtained for similar solutions of unimodal molecular mass distribution. The comparisons between the different separating polymer solutions were made at a constant total mass concentration, so as to keep constant the mesh size and to highlight the sole effect of the network dynamics. The use of binary polymer mixtures of two different molecular masses but of same nature can be a convenient alternative to modulate the dynamics of the network and the viscosity of the separating medium. A 20-30% content of high-molecular-mass chains in an entangled poly(ethylene oxide) solution having a binary molecular mass distribution appears to be a good compromise for a moderate viscosity and a good separation selectivity in comparison with a solution containing only chains of high molecular mass at the same concentration.

Electrophoresis, Capillary↗

PubMed2004

Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK.

We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high-efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab-gel separation and electrospray ionization-mass spectrometry (ESI-MS) of LPS-derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O-deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse-stained LPS bands using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The quantities (30-40 microg) obtained from the two gel-resolved LPS bands were sufficient to allow MALDI-TOF-MS detection of O-deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high-molecular-weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low-molecular-weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI-TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal-to-noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI-TOF-MS led to a higher sensitivity of detection of less abundant low-molecular-weight LPS glycoforms. Taken together, this and our previous study on gel-micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel-separated LPS band, their relative abundance and oligosaccharide sequences.

Aliivibrio fischeri↗

PubMed2006

The promise of nanotechnology for separation devices--from a top-down approach to nature-inspired separation devices.

An overview is given of the possible applications of nanotechnology to optimise existing separation methods and to enable new methods. Attention is paid to nanotechnological contributions in the fields of HPLC, CEC, sieves, Brownian ratchets and preconcentration units. A brief description is also given of some selection/separation mechanisms that occur in biological (cell) structures and possible future applications of these mechanisms in separation devices are investigated. Especially the active transport in discrete events occurring in cells is mentioned as a potentially powerful separating mechanism.

Chromatography, High Pressure Liquid↗

PubMed1993

Novel affinity separations based on perfluorocarbon emulsions. Development of a perfluorocarbon emulsion reactor for continuous affinity separations and its application in the purification of human serum albumin from blood plasma.

Perfluorocarbon affinity emulsions are generated by the homogenisation of a perfluorocarbon oil with a polymeric fluorosurfactant previously derivatised with an affinity ligand and subsequently cross-linked in situ. This procedure gives rise to a novel liquid affinity adsorbent that can be used for continuous protein purification. Discrete emulsion droplets were found to be unstable when pumped for prolonged periods; however, when flocculated, the emulsion floccules with diameters of around 125 microns, were very stable and sedimented faster. A four-stage reactor unit (perfluorocarbon emulsion reactor for continuous affinity separations, PERCAS) was designed and constructed to carry out continuous separations, and exploited the unusual properties of the absorbent, i.e. liquid nature and high density. Each of the four stages of PERCAS consisted of a mixing tank, for contacting between emulsion phase and aqueous phase, adjacent to a settling tank for the subsequent separation of emulsion from the aqueous phase. Using PERCAS adsorption, washing, elution and re-equilibration of the emulsion could be carried out continuously with emulsion recycle. Using single-component adsorption of human serum albumin to a perfluorocarbon affinity emulsion derivatised with the triazine dye C.I. Reactive Blue 2, PERCAS was optimised with respect to flow-rates and input concentrations. The work was then extended to the continuous purification of essentially homogeneous human serum albumin from blood plasma.

Adsorption↗