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PubMed2000

Cross-linked glucose isomerase crystals as a liquid chromatographic separation material.

Cross-linked crystals of glucose isomerase (CLGI) were characterized as a liquid chromatographic separation material. The experiments were done with crystals having an average diameter of 83 µm. Porosity (epsilon(p)) and pore size distribution of the CLGI crystals were measured with size exclusion chromatography using D(2)O and polyethylene glycols as probes. CLGI material was capable of separating <1000 g/mol polyethylene glycols. Fifty two percent (epsilon(p) = 0.47) of the total crystal volume was in pores. Pore size measurement showed that CLGI crystals were microporous material, having an average apparent pore diameter of 29 +/- 0.08 Å. CLGI material separated n-alcohols C(1) to C(8) based on the hydrophobic interaction between the protein material and the carbon chain of the alcohols. Height equivalent to a theoretical plate (HETP, in millimeters) ranged from 1.6 to 0.89 for the C(1) to C(7) n-alcohol series. Despite the large crystal size, CLGI as a chirally active phase effectively separated D- and L-arabitol (R(s) = 0.58) and showed potential for chiral separation of amino acids.

Journal Article↗
PubMedDate not supplied

The long-term psychobiological consequences of intermittent postnatal separation in the squirrel monkey.

Long-term effects of early experiences have been well documented in rodents, non-human primates and humans. The influence of early experiences on the regulation of the hypothalamic-pituitary-adrenal has demonstrated that this biological system is highly plastic and is permanently modified by early experiences. The effects of brief periods of maternal separation in rodents tend to result in a reduction of the stress response, whereas longer periods of maternal separation produce an animal that is hyperresponsive. The effect of disruptions of mother-infant interactions on the HPA axis in non-human primates has not been clearly established. However, the primate research has primarily focused on more permanent separation paradigms and thus it is difficult to compare the rodent studies with those conducted in primates. The present study attempted to use a separation procedure in monkeys that more closely resembled the rodent model. Intermittent separation during infancy followed by reunion with the mother were shown to lead to a significant reduction in both the cortisol response and the response of the noradrenergic system following social isolation in juvenile squirrel monkeys at 2 and 3 years of age. The behavioral response (vocalizations) was also reduced.

Animals↗
PubMed1986

Maternal separation alters later consumption of novel liquids in the squirrel monkey.

The consumption of novel, fruit-flavored drinks was measured in juvenile squirrel monkeys that had either had their mother removed from the home cage for 2 h on 80 occasions prior to weaning or had not undergone this separation procedure. Among previously separated animals, males drank more than did females during the first 30 min following presentation, whereas there was no difference in the drinking of nonseparated males and females at this time. Further, the amount of liquid consumed during the 20 h following presentation increased across days of exposure in the nonseparated, but not in the previously separated monkeys. These results show that brief maternal separation can affect the later behavior of squirrel monkeys, and are consistent with findings in rhesus macaques that maternal separation alters later exploratory behavior.

Animals↗
PubMed2001

Chiral separations of 1,3,4-thia- and 1,3,4-selenadiazine derivatives by use of non-aqueous capillary electrophoresis.

Our aim was to establish suitable conditions for the chiral separation of 12 1,3,4-thia- and 1,3,4-selenadiazine derivatives; some of them were identified in screening tests as potential antituberculotics. To overcome possible problems with the water insolubility of most analytes, we profited by the advantages of non-aqueous capillary electrophoresis. Methanol, formamide, and a mixture of formamide with acetonitrile (1:2, v/v) were used as separation media. Hydroxyethyl-, hydroxypropyl-, and methyl-beta-cyclodextrin were applied as chiral selectors in concentrations of 200 mM. Besides the effect of these different electrophoretic media and selectors, we also investigated the consequences of using different electrolytes (25 mM ammonium acetate/1 M acetic acid and 25 mM citric acid/12.5 mM TRIS). Distinct differences of the separation factors in the different separation media were observed. Depending on structure characteristics of the analytes, we established clear classifications to these cyclodextrins (CD), which were most appropriate for the separation of the enantiomers of the particular analytes.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1999

Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis.

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.

2-Propanol↗
PubMed1999

Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography.

In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl-3H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some 'tailing' by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.

Choline↗
PubMed1999

Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods.

Cost-efficient umbilical cord blood (UCB) banking requires well-standardized methods of volume reduction and storage. To compare UCB fractionation using a technique of hydroxyethyl starch (HES) sedimentation with the Ficoll (double) and Percoll methods, 50 whole units was allocated randomly to each procedure. HES resulted in a significantly better recovery of mononuclear cells (87.5%), granulocyte/macrophage colony-forming units (CFU-GM) (88.4%), and CD34- cells (87.4%) and lesser volume reduction (85.5%). HES was the least laborious, time consuming, and expensive of the three procedures, costing 3.4- and 4.4-fold less than the Ficoll and Percoll methods, respectively. Five units processed by each method was frozen in 4.5-mL cryotubes under optimal conditions. After thawing, the greatest degree of recovery of viable nucleated cells and number of CFU-GM per unit were obtained using the HES procedure. Using 4.5-mL cryotubes, the calculated number of units that could be stored in 600-L containers was 3.8- and 2.2-fold higher for Ficoll- and Percoll-separated than for HES-separated units, respectively. Nevertheless, the higher direct costs of the density gradient separation procedures outweighed their lower storage cost. For long-term cryopreservation, we assessed the freezing of HES-processed units in 50-mL cryobags and their specifically designed canisters. We found cell recoveries similar to those obtained with cryotubes, but storage capacity was decreased. Special racks designed for these canisters resulted in a 5-fold increase over the number of units stored in standard cryobags. This system also is feasible for Percoll- and Ficoll-separated units, resulting in comparable storage costs for the three separation methods. We conclude that this HES procedure and the 50-mL cryobags constitute a cost-efficient system for large-scale UCB banking.

Blood Preservation↗
PubMed1998

Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization.

Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (Tg). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the Tg. Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage.

Chemical Phenomena↗
PubMed2000

Capillary electrophoretic separation of proteins and peptides using Pluronic liquid crystals and surface-modified capillaries.

Separation of model mixtures of peptides/proteins carried out in a hydrophilically coated capillary in 10 mmol/l Tris and 75 mmol/l phosphate buffer containing 7.5% (w/w) Pluronic F127 copolymer (apparent pH 2.9) revealed that the separation is predominantly driven by the charge/mass ratio with little or no sieving effect. Using a coated capillary helped to remove current fluctuations that are observed in the fused-silica capillaries in the presence of the Pluronic copolymer. With peptides bearing distinct positive charge (polylysine of Mr around 3300) molecular sieving helps more detailed separation of individual species. Polyamino acids carrying negative charge can be brought to the detector window in the reversed polarity mode, however, no detailed separation of the individual species involved was observed under the conditions used. With a naturally occurring mixture of collagen fragments released by CNBr treatment of the protein the sequence of emerging peptides (positive polarity mode) with no relation to the rel. mol. mass could be revealed. It is concluded that separation of proteins/peptides in the presence of Pluronic in the background electrolyte occur on the charge/mass ratio basis with molecular sieving effects acting as a secondary partition mechanism.

Electrophoresis, Capillary↗
PubMed2000

External electric field control of electroosmotic flow in non-coated and coated fused-silica capillaries and its application for capillary electrophoretic separations of peptides.

The influence of an external electric field on the electroosmotic flow in the noncoated (bare) fused-silica capillaries and in the fused-silica capillaries with covalent coating of the inner surface by the polymer of a new acrylamido derivative, N-(acryloylaminoethoxy)ethyl-beta-D-glucopyranose, has been tested in the capillary electrophoretic separations of peptide analytes. The effect of magnitude and polarity of the external electric field on the flow-rate of the electroosmotic flow, the migration times of charged analytes and the separation efficiency and resolution of separations of synthetic oligopeptides, diglycine, triglycine, glycyl-proline and prolyl-glycine, by capillary zone electrophoresis has been evaluated. The effect of the external electric field on the velocity of the electroosmotic flow was much higher in the bare fused-silica capillaries than in the coated capillaries. Better separation of the analyzed peptides was achieved in the coated fused-silica capillaries. An external electric field proved to be an effective tool for control of the electroosmotic flow and for optimization of the speed and resolution of capillary electrophoretic separations of synthetic peptides.

Electrophoresis, Capillary↗
PubMed2000

Optimization of the high-performance liquid chromatographic separation of a complex mixture containing urinary steroids, boldenone and bolasterone: application to urine samples.

An HPLC separation of a complex mixture containing 13 urinary anabolics and corticoids, and boldenone and bolasterone (synthetic anabolics) has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation was studied. The optimum separation was achieved by using a water-acetonitrile (60:40, v/v) mobile phase in reversed-phase HPLC at 30 degrees C, allowing the separation of all the analytes in about 24 min. Calibration graphs were obtained using bolasterone or methyltestosterone as internal standards. Detection limits were in the range 0.012-0.107 microg ml(-1). The optimized separation was applied to the analysis, after liquid-liquid extraction, of human urine samples spiked with steroids.

Adrenal Cortex Hormones↗
PubMed1986

Separation of phospholipids by high-performance liquid chromatography: all major classes, including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine.

High-performance liquid chromatographic methods for the separation and quantitation of phospholipids were developed and shown to give sensitive, reliable measurements of tissue phospholipids, including difficult-to-resolve pairs such as choline plasmalogen (plasmenylcholine) and phosphatidylcholine, choline glycerophospholipids and sphingomyelin, phosphatidylinositol and phosphatidylserine, and phosphatidylserine and lysophosphatidylethanolamine. Separations of most phospholipids including those mentioned above are more complete than in existing procedures, and require only 40 min per injection. Utilization of the hexane-2-propanol-water system has an advantage over separation techniques that employ acidic solvents in that the plasmalogens are not hydrolyzed and a less degradative environment for labile lipids is provided. Further, a rapid high-performance liquid chromatographic procedure for the separation of intact ethanolamine plasmalogen (plasmenylethanolamine) from phosphatidylethanolamine was developed. Previous procedures have required derivatized samples or acid hydrolysis of the plasmalogen vinyl ether linkage. A slight modification of the primary method (method I) increases the resolution of lysophosphatidylethanolamine from other classes (method II). A third modification (method III) can replace the standard silicic acid column separation of lipids into neutral, glycolipid, and phospholipid fractions.

Animals↗
PubMed2001

Separation methods for alkylating antineoplastic compounds.

The separating method for alkylating neoplastic compounds were reviewed based on the classification of the Merck Index (12th Edition). Each section, whenever available or relevant, was subdivided according to the following approach: stability studies, extraction methods, gas chromatography, high-performance liquid chromatography and capillary electrophoresis. At the end of each chapter a separate table summarizing the main characteristics of the separating method were established. In particular LODs and/or LOQs were expressed as quantity to facilitate comparison between methods. This review highlights the problems to measure trace levels of these compounds into biological fluids with respect to their instability, adsorption to glass and plastic or derivatization requirements. Over the last decades, HPLC seems to be more popular than GC for separating the alkylating agents. The development of narrow- or microbore LC coupled to MS is certainly the way to further improve both separation and sensitivity obtained in the different papers surveyed for this review.

Antineoplastic Agents, Alkylating↗
PubMed1997

Separation of the stereoisomers of the main metabolite of a non-steroidal anti-inflammatory drug, flobufen, by chiral high-performance liquid chromatography.

The major metabolite of a novel non-steroidal anti-inflammatory drug, DL-4-(2',4'-difluorobiphenyl-4-yl)-2-oxo-2-methylbutanoic acid (flobufen, I), namely 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-gamma-butyrolactone (4-dihydroflobufen lactone, III), has four stereoisomers consisting of two racemic pairs of enantiomers. Of three chiral stationary phases tested, Cyclobond I beta-RSP (Astec) (beta-cylodextrin derivatized with R,S-hydroxypropyl) was best able to separate the (+2)(--) racemate, with a liquid phase containing acetonitrile as modifier and triethylamine acetate as buffer. Using the Box-Wilson Central Composite Design for three factors, an optimum combination of pH and concentrations of the modifier and buffer was eventually obtained. A chromatographic response function based on a combination of the Kaiser peak separation function, Pi, and retention time of the second eluting enantiomer, tRL, served as a response criterion for the process of optimization. The optimum conditions developed for the (+2)(--) racemate were also found to be suitable for separating the (+-)(-+) racemate, for which earlier studies had shown the separation to be more facile. Separation of the four stereoisomers of III, for which the chiral chromatographic system optimized in this study is proposed as the second stage, is targeted at a biochemical study of the stereoisomeric metabolism of I.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Separation and detection of closely related peptides by micellar electrokinetic chromatography coupled with electrospray ionization mass spectrometry using the partial filling technique.

Closely related peptides such as neurotensin and angiotensin analogues were separated by capillary zone electrophoresis using a nonionic surfactant, sucrose monododecanoate, as a micelle forming reagent. These peptides were detected by an on-line coupled mass spectrometer using an electrospray ionization interface. However, the presence of the micelles in the separation solution drastically reduced the sensitivity of the mass spectrometer. Therefore, a partial filling technique was employed to prevent the micelles from entering the mass spectrometric interface. A part of the capillary from the injection end was filled with the micellar solution in this technique. Analytes passed through the micellar zone during the electrophoresis and when the separated analytes reached the detection end of the capillary, the micellar zone was still behind the analyte zones, because the nonionic surfactant moved very slowly in acidic conditions. Thus the technique was very useful for mass spectrometric detection for CE when the micellar solution was employed for separation. The optimization of separation and detection conditions was investigated.

Amino Acid Sequence↗
PubMed1997

Separation of hexokinase activity using different hydrophobic interaction supports.

Hydrophobic interaction chromatography (HIC) has been used extensively for the separation of proteins and peptides by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this paper we compare different hydrophobic interaction chromatographic media for the separation of multiple forms of hexokinase from rabbit reticulocytes. Among the different hydrophobic chromatographic media tested (Toyopearl Phenyl 650S, Ether 650S and Butyl 650S) Toyopearl Phenyl 650S offered the best separation of multiple forms of hexokinase, probably due to its intermediate hydrophobicity. In order to establish the optimal experimental conditions, we evaluated the effects of different salts, and the results obtained demonstrated that among the antichaotropic salts, ammonium sulphate is the most suitable for the separation of hexokinase sub-types. The sample loading capacity of the three Toyopearl supports was investigated and the recovery of enzymatic activity obtained ranged from 60% to 90%, depending on the different salts and hydrophobic media used. The chromatographic profiles of hexokinase activity from various mammalian and fungal tissues also demonstrate that Toyopearl Phenyl 650S can be successfully employed for the separation of multiple forms of enzymes from different biological sources.

Ammonium Sulfate↗
PubMed1998

Separation procedures for phosphatidylserines.

This paper reviews working procedures for the separation of phosphatidylserines (PS) in complex sample matrices. It begins with an introductory overview of important aspects of PS involvement in cellular lipid biochemistry. The main body of the review describes various procedures for the extraction, isolation, purification, and separation of the PS class and its molecular species in tissue samples. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques. Factors influencing normal-phase and reversed-phase separations are delineated. The last section covers selected chemical derivatization procedures useful for enhancing the separation efficiency and detection sensitivity and specificity.

Animals↗
PubMed1998

Protein analysis by membrane preconcentration-capillary electrophoresis: systematic evaluation of parameters affecting preconcentration and separation.

Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration-CE (mPC-CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC-CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO3H) or hydrophobic (C2, C8, C18, SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC-CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC-CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC-CE analysis of proteins using a C2 impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 mM ammonium acetate, 60 nl of 80% acetonitrile in H2O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.

Aqueous Humor↗