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PubMed1998

A physical method for separating spermatozoa from epithelial cells in sexual assault evidence.

The analysis of genetic markers for the purpose of individualization of semen specimens is extremely important in cases of sexual abuse and assault. The serological analysis of sexual assault evidence can sometimes be complicated because stains are often composed of a mixture of spermatozoa, vaginal epithelial cells and white and red blood cells. A filtration method has been developed to cleanly separate spermatozoa from epithelial cells based upon differences in size and shape. Nylon mesh filters of the appropriate pore size can be used to separate the smaller oval shaped spermatozoal cells from the larger and flatter epithelial cells. The former pass freely through the membrane while the latter are retained on the filter. In this study, cell separation was demonstrated by (a) microscopic observation of stained cells, (b) amplified fragment length polymorphism analysis of DNA obtained from separated cells. The results of these analyses indicate that: (1) Approximately 70% of spermatozoa in the mixed cell sample will penetrate the 10 microns pore size filter, (2) Only about 1-2% of intact epithelial cells will do so, and (3) A small number of nuclei from spontaneously lysed epithelial cells will cross the filter. Experimental results using mixtures of spermatozoa and vaginal epithelial cells prepared in different ratios support the conclusion that the filtration process is an efficient and reliable method to separate spermatozoa from epithelial cells in casework specimens for subsequent DNA analysis.

Autoradiography↗
PubMed2001

Optic disc, foveal, and extrafoveal damage due to surgical separation of the vitreous.

OBJECTIVE: To evaluate the morphologic outcomes resulting from surgical vitreoretinal separation in young adult primates. MATERIALS AND METHODS: Vitrectomy and mechanical separation of the vitreous from the internal limiting lamina (ILL) of the posterior retina and surface of the optic disc were performed on 25 young adult cynomolgus monkey eyes in vivo. Lectin histochemical studies were used to evaluate the vitreoretinal interface. Morphologic outcomes were tabulated. RESULTS: In 11 of 25 eye regions, residual vitreous remained attached to the ILL in some of the regions. Localized ILL breaks or separation of the ILL from the neural retina was noted in 9 eyes. Retinal tissue loss, including avulsion of the ganglion cell, inner plexiform, or inner nuclear layers, was observed in 7 eyes. Avulsion of axon bundles in the optic disc was noted in 9 eyes. Significantly, partial- or full-thickness foveal tears were noted in 11 eyes. Based on the surgeons' intraoperative observations, small superficial optic disc or retinal hemorrhages were observed in 3 of 25 eyes. None of the eyes on which a vitrectomy alone was performed showed ILL damage, or retinal or optic disc tissue loss. CONCLUSION: Damage may occur to the optic disc, fovea, and extrafoveal retina as a result of surgical separation of the vitreous from the retina in young adult primates. CLINICAL RELEVANCE: These data support the contention that surgically induced damage at the level of the vitreoretinal interface may help explain the visual field defects noted after surgery to close full-thickness macular holes. These data also support the need for developing additional modalities to assist in vitreous separation, thereby reducing the risk of traumatic complications associated with purely mechanical procedures.

Animals↗
PubMed1998

Rapid identification and screening of proteins from whole cell lysates of human erythroleukemia cells in the liquid phase, using non-porous reversed phase high-performance liquid chromatography separations of proteins followed by matrix-assisted [correction of multi-assisted] laser desorption/ionization mass spectrometry analysis and sequence database searching.

Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed extraction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.

Calmodulin↗
PubMed1998

Selective separation of proteins from mixtures using polysaccharides.

The phase behavior of ternary (protein + dextran + solvent) and quaternary (protein 1 + protein 2 + dextran + solvent) aqueous solutions have been monitored at various ionic strengths and pHs by turbidity measurements. For the ternary systems, phase separation was favored when the solvent conditions promoted protein self-association. Bovine serum albumin (BSA) and gamma-globulin were believed to self-associate in the presence of dextran at their respective isoelectric points through the electrostatic interactions between positive and negative charged patches at their surface. The fact that phase separation for BSA became less evident on increasing the ionic strength supported this concept. In the case of lysozyme alone, self-association was promoted by the addition of electrolyte as was phase separation in the lysozyme + dextran ternary system. For this biopolymer self-association is presumably as a consequence of van der Waals and/or hydrophobic forces. The influence of dextran has been discussed in terms of polymer depletion theory where an additional attractive force is created due to the exclusion of the dextran molecules from the immediate vicinity of the protein surface. For the quaternary systems, by subtle choice of solvent conditions, BSA and globulin could be exclusively partitioned into separate liquid layers through a segregative mechanism, while BSA and lysozyme could be separated into a single highly concentrated layer through an associative mechanism.

Animals↗
PubMed1996

Polylactide macroporous biodegradable implants for cell transplantation. II. Preparation of polylactide foams by liquid-liquid phase separation.

Potential of thermally induced phase separation as a porogen technique has been studied in an effort to produce a surgical implant suitable for cell transplantation. Emphasis has been placed on the liquid-liquid phase separation of solutions of amorphous poly DL-lactide and semicrystalline poly L-lactide in an 87/13 dioxane/water mixture. The related temperature/composition phase diagrams have been set up by turbidimetry, and the possible occurrence of a gel has been discussed. Freeze-drying of some phase-separated polylactide solutions can produce flexible and tough foams with an isotropic morphology. Interconnected pores of 1-10 microns in diameter are expected to result from the spinodal decomposition of the polylactide solutions with formation of co-continuous phases. Thermodynamics of the polymer/solvent pair has a decisive effect on the final macroporous foams, as shown by the dependence of their porosity, density, porous morphology, and mechanical behavior on molecular weight and crystallinity of polylactide and concentration of the original solutions. On the basis of the foam characteristics, potential of the liquid-liquid phase separation (spinodal decomposition) has been compared with the solid/liquid phase separation (solvent crystallization) as a porogen technique.

Cell Transplantation↗
PubMed1998

Ontogeny and stability of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life.

Twenty-six rhesus monkeys were tested repeatedly at 4, 8, and 12 months of age to characterize the expression and development of their defensive responses induced by separation from their mothers and exposure to a potential threat. Results demonstrated that by 4 months of age infant monkeys engaged in adult-like context-dependent responses and adaptively regulated these responses in relation to the changing context. When separated from their mothers and alone, infants at 4 months of age were active and emitted frequent coo vocalizations. However, when exposed to the profile of a human face, infants responded by becoming behaviorally inhibited and freezing. At 8 months of age, a dramatic reduction in infants' separation-induced coos was observed, whereas their duration of threat-induced freezing remained unchanged. At 12 months of age, a further decrease in cooing occurred, while freezing duration was maintained. No sex differences were found in the expression of these behaviors or their developmental patterns. Individual differences in separation-induced cooing and threat-induced freezing were apparent and remained stable over the three ages studied. However, within animals no relation was found between individual differences in cooing and freezing. These data demonstrate important differences in the developmental patterns for the expression of cooing and freezing over the first year of life. Marked individual differences in separation-induced cooing and threat-induced freezing were apparent and remained stable from 4-12 months of age. The data support the hypothesis that these different defensive responses reflect different adaptive responses that likely have different underlying mechanisms. The similarities between these defensive responses in rhesus monkeys and humans suggests that understanding the factors that promote the development of individual differences in monkeys will illuminate important factors that promote individual differences in humans.

Age Factors↗
PubMedDate not supplied

Separation of porphyrins by capillary electrophoresis in fused-silica and ethylene vinyl acetate copolymer capillaries with visible absorbance detection.

A mixture of 5 porphyrins were separated by capillary electrophoresis. A UV-VIS detector was used to detect the separated components. Separation was performed in 2 types of capillaries, i.e. fused-silica and ethylene vinyl acetate copolymer (EVA) plastic capillaries. The concentration limit of detection (CLOD), mass limit of detection (MLOD), and relative standard deviations (RSD) of migration time and area were investigated. LOD was comparable to that of epi-fluorescence detection. LOD was lower when separation was performed in EVA capillary compared to fused-silica capillary. RSD of migration times of the porphyrins when separated in fused-silica capillary ranges between 0.5 to 1.6% and in EVA capillary ranges between 0.3 to 1.2%. Area RSD in fused-silica capillary ranges between 7 to 20% and in EVA capillary ranges between 4.7 to 12.3%. Urine spiked with porphyrins was also analysed by CE using fused-silica and EVA capillaries. Analysis of urine sample spiked with porphyrins showed that stacking effect of porphyrins was observable only in fused-silica and not in EVA capillary. This led to similar LOD in fused-silica capillary to those for EVA capillary.

Electrophoresis, Capillary↗
PubMed1999

Enantioseparation of amfepramone (rac-diethylpropion): preparative separation of the enantiomers and enantioselective analysis.

The enantiomers of the anorectic drug amfepramone [rac-diethylpropion, rac-2-(diethylamino)-1-phenyl-1-propanone; rac-DEP] were separated in the preparative scale by crystallization. With enantiopure di-O-benzoyltartaric acid as salt-forming chiral selector, diastereoisomeric salts of DEP enantiomers with a final purity of more than 97.5% were obtained. Analytical liquid chromatographic and electrophoretic methods for the control of the enantiomeric purity and the stoichiometry of the salts were developed. The enantioseparation of rac-DEP by capillary electrophoresis (CE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral discriminator and phosphate buffer (pH 3.3) as run buffer led to good separations. HPLC methods were developed using polysaccharide chiral stationary phases (CSP). The separation of the two enantiomers and the two main degradation products (1-phenyl-1,2-propanedione and propiophenone), known from solid and liquid pharmaceutical preparations, was attained in one run on the silica-based CSP cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). The conditions which might affect the enantioselectivity and the quality of the enantiomeric separation were investigated for Chiralcel OD and the related CSP amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). Both CSPs showed very similar chromatographic properties. The separation factors could be influenced significantly by varying the polar organic modifier added to the mobile phase.

Appetite Depressants↗
PubMed1999

Simultaneous separation and enantioresolution of racemic local anesthetic drugs by capillary zone electrophoresis with Tween 20 and methyl-beta-cyclodextrin as selectors, employing a double plug technique.

A new approach for simultaneous chiral and achiral separations by capillary zone electrophoresis is described. Two adjacent selector plugs, consisting of Tween 20 as an achiral and methyl-beta-cyclodextrin (CD) as a chiral selector, are employed and four related local anesthetics are used as model compounds. The principles of the partial filling technique, whereby the capillary is filled with the chiral selector solution followed by the micellar solution at different plug lengths and concentrations, prior to application of the solutes, was employed. During the run both capillary ends were dipped in a simple buffer, i.e., one without additives. The two separation media worked independently without any interaction. Separation of the solutes and their enantiomers was regulated by adjusting both the concentration and plug length (PL) of the micellar solution in the capillary, employing methyl beta-CD as chiral selector either at 38 or 76 mM. The solutes were separated on the basis of their affinity towards the micellar phase before they reached the methyl-beta-CD plug for enantioseparation. In the absence of the micellar plug, the enantiomers of prilocaine overlapped those of bupivacaine. The solutes and their enantiomers were completely separated by employing two adjacent plugs consisting of 100 mM Tween 20 solution (PL approximately 10 cm) and methyl-beta-CD solution at either 38 or 76 mM (PL approximately 30 cm).

Anesthetics↗
PubMed1999

Nonaqueous media for separation of nonionic organic compounds by capillary electrophoresis.

For the separation of neutral compounds by micellar electrokinetic chromatography, separations are usually carried out in predominantly aqueous solution in order to preserve the charged micelle necessary for the separation. We now show that polycyclic aromatic hydrocarbon (PAH) compounds can be separated efficiently by capillary electrophoresis in pure methanol or in aqueous-organic mixtures containing a high percentage of methanol. Sodium tetradecyl sulfate was the preferred surfactant. The effects of pH, solvent composition, surfactant structure, and surfactant concentration on the separations were studied. Reproducible migration times and linear calibration plots were obtained.

Electrophoresis, Capillary↗
PubMed2000

Separation of D-lysergic acid diethylamide derivatives using micellar electrokinetic capillary chromatography.

By adjusting column temperature and applied electric field, a fast separation in micellar electrokinetic capillary chromatography was developed for the separation of D-lysergic acid diethylamide derivatives. A baseline separation of nine derivatives was accomplished with a run time of less than 12 min by utilizing elevated column temperature (60 degrees C) and an applied electric field of 387 V/cm. The number of plates generated per unit time for the separations completed at elevated temperatures was significantly higher when compared to separations at the same applied electric field but at lower temperatures (20 degrees C).

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2000

Separation of related opiate compounds using capillary electrochromatography.

Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m.

Analgesics, Opioid↗
PubMed2000

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection.

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.

Anti-Bacterial Agents↗
PubMed2000

Subatmospheric electrospray interface for coupling of microcolumn separations with mass spectrometry.

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.

Animals↗
PubMed2000

Enantiomeric separation of a group of chiral dihydropyridines by electrokinetic chromatography.

Electrokinetic chromatography (EKC) was employed to achieve the enantiomeric separation of a group of chiral 1,4-dihydropyridines (DHPs) with pharmacological activity. Micelles of bile salts alone or mixed with neutral cyclodextrins, micelles of sodium dodecyl sulfate (SDS) mixed with neutral cyclodextrins, and anionic cyclodextrin derivatives, i.e., carboxymethyl-gamma-cyclodextrin (CM-gamma-CD), carboxymethyl-beta-cyclodextrin (CM-beta-CD), and succinylated beta-cyclodextrin (Succ-beta-CD), were employed as pseudostationary phases. The enantiomeric separation ability of these chiral selectors with respect to DHPs was studied in different experimental conditions. CM-beta-CD was shown to be the best chiral selector to perform the enantiomeric separation of DHPs by EKC. Next, the influence of the CM-beta-CD concentration, the pH and nature of the buffer, the temperature, and the applied voltage on the enantiomeric resolution of DHPs was studied. The use of a 50 mM ammonium acetate buffer, pH 6.7, 25 mM in CM-beta-CD together with an applied voltage of 15 or 20 kV, and a temperature of 15 degrees C enabled the individual enantiomeric separation of twelve DHPs, each one into its two enantiomers, and their separation in multicomponent mixtures of up to six DHPs into all their enantiomers.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed1993

Separation-individuation difficulties and the distinction between bulimia nervosa and anorexia nervosa in college women.

This study investigated whether patterns of late adolescent separation-individuation from parents, as measured by the Psychological Separation Inventory (PSI), are different for anorexic-like and bulimic-like young women in college. It was hypothesized that anorexic-like women would display a pattern of underseparation from both mother and father. Bulimic-like women were expected to show a pattern of underseparation on the conflictual dimension of the PSI but overseparation on the other dimensions. A total of 198 college women completed the PSI, the Eating Disorder Inventory, and a DSM-III-R symptom checklist. Using that checklist, 19 women were classified as bulimic-like (BUN), 8 as like restricting anorexics (RAN), 11 as showing a pattern resembling both bulimia and restricting anorexia (BULAN), 109 as showing limited bulimic or anorexic behaviors (SOME), and 25 as having no eating disorder symptoms (NED). Results indicated some support for the hypotheses. BUN women were overseparated in terms of attitudinal independence from both parents and underseparated in that they experienced more guilt and conflict concerning separation than did the SOME or NED women. The anorexic-like women reported considerable guilt and conflict about separation and tended to be underseparated in general. The results point to the potential value of separation-individuation issues in the distinction between restricting anorexia nervosa and bulimia nervosa.

Adult↗
PubMed2002

Polymeric membranes for aromatic/aliphatic separation processes.

For the membrane-based separation of benzene/cyclohexane mixtures, the pervaporation properties of different 6FDA (4,4'-hexafluoroisopropylidene diphthalic anhydride) based copolyimide membranes have been investigated. In order to obtain high permeability as well as high selectivity copolyimides were synthesised using a combination of 4MPD (2,3,5,6-tetramethyl-1,4-phenylene diamine) and 6FpDA (4,4'-hexafluoro-isopropylidene dianiline) as well as DABA (3,5-diaminobenzoic acid) as monomers. Cross-linking is possible with this type of copolyimides and necessary to reduce swelling effects, which often occur with polymeric membranes and lead to a deterioration of the separation characteristics in aromatic/aliphatic separation processes. In order to find the most suitable membrane material, the polymer structure, the crosslinking agents, as well as the crosslinking methods have been varied. The separation characteristics have been determined by sorption and pervaporation experiments. Sorption experiments have been carried out at 60 degrees C using benzene, toluene and ethylbenzene as aromatic components and cyclohexane, cyclohexene, hexane and heptane as aliphatic solvents. Pervaporation experiments have been performed at 60 degrees C using benzene/cyclohexane mixtures with benzene concentrations covering the whole concentration range. It has been found that crosslinked copolyimide membranes show excellent chemical resistance, strongly reduced swelling behaviour and higher selectivity in aromatic/aliphatic separation compared to conventional membrane materials.

Chemistry, Physical↗
PubMed2000

Dual-opposite injection electrokinetic chromatography for the unbiased, simultaneous separation of cationic and anionic compounds.

The concept of dual opposite injection in capillary electrophoresis (DOI-CE) for the simultaneous separation, under conditions of suppressed electroosmotic flow, of anionic and cationic compounds with no bias in resolution and analysis time, is extended to a higher pH range in a zone electrophoresis mode (DOI-CZE). A new DOI-CE separation mode based on electrokinetic chromatography is also introduced (DOI-EKC). Whereas conventional CZE and DOI-CZE are limited to the separation of charged compounds with different electrophoretic mobilities, DOI-EKC is shown to be capable of separating compounds with the same or similar electrophoretic mobilities. In contrast to conventional EKC with charged pseudostationary phases that often interact too strongly with analytes of opposite charge, the neutral pseudostationary phases appropriate for DOI-EKC are simultaneously compatible with anionic and cationic compounds. This work describes two buffer additives that dynamically suppress electroosmotic flow (EOF) at a higher pH (6.5) than in a previous study (4.4), thus allowing DOI-CZE of several pharmaceutical bases and weakly acidic positional isomers. Several DOI-EKC systems based on nonionic (10 lauryl ether, Brij 35) or zwitterionic (SB-12, CAS U) micelles, or nonionic vesicles (Brij 30) are examined using a six-component test mixture that is difficult to separate by CZE or DOI-CZE. The effect of electromigration dispersion on peak shape and efficiency, and the effect of surfactant concentration on retention, selectivity, and efficiency are described.

Anions↗