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PubMed1998

Effects of chronic social separation on cardiovascular disease risk factors in female cynomolgus monkeys.

A lack of social support is associated with increased risk of coronary heart disease morbidity and mortality in human beings. Similarly, chronic social separation (single cage housing) potentiates atherosclerosis in female monkeys. Under the hypothesis that autonomic arousal and/or ovarian impairment may mediate this effect (as both are associated with increased atherosclerosis), heart rate and luteal phase plasma progesterone concentrations were measured in 12 female cynomolgus monkeys that were first socially housed, then individually housed, and finally returned to their original social groups. Afternoon heart rates increased during social separation compared to the social groupings (P < 0.001). Increased heart rates could not be explained by activity levels, which were lower during social separation than in social groupings (P < 0.001). Ovarian function (i.e. luteal-phase progesterone concentrations) was not influenced by housing condition. Single caging reduced the extent of social signaling, even though animals were in visual and auditory contact. Rates of affiliative behaviors increased and time spent alone decreased in post-reunion social groups compared to pre-separation social groups (P's < 0.01). The results indicate that chronic social separation in this group-living species may exacerbate atherosclerosis via altered autonomic activity, as evidenced by higher heart rates during social separation.

Animals↗

PubMed2000

Separation and direct UV detection of lanthanides complexed with cupferron by capillary electrophoresis.

Separation and detection of lanthanides by capillary zone electrophoresis in the presence of cupferron (N-nitroso-N-phenylhydroxylamine) as UV absorbing complexing agent were investigated. The resolution of partially complexed positively charged cupferron complexes is improved by using a buffer ligand competing with cupferron for metal ions. When hydroxyisobutyric acid (HIBA) is used as buffer and competing ligand, it provides complete separation of all 14 lanthanides with good peak shapes. An on-column separation of 14 lanthanides was achieved in only 7 min using 0.1 mmol/l cupferron, 15 mmol/1 HIBA at pH 4.9. The separation efficiencies for the optimum separation condition are between 77,000 and 208,000 theoretical plates. Determination of lanthanide complexes was performed by direct UV detection at 210 nm. Detection limits (signal-to-noise ratio=3) are ca. 0.24-0.47 microg/ml for lanthanides. Under optimum conditions, the complete separation of thorium and uranium from mixed lanthanides was achieved.

Metals, Rare Earth↗

PubMed2000

Separation of bisphenol A and three alkylphenols by micellar electrokinetic chromatography.

Analytical conditions of pH, surfactants, and additives were investigated for the simultaneous separation of bisphenol A and alkylphenols by micellar electrokinetic chromatography. Reproducibility of migration time was improved at higher pH (pH 8.0). When five surfactants having linear alkyl chains or four bile salts were used, the separation of hydrophobic phenols and 4-nonylphenol isomers was not achieved. In order to improve the separation, the use of additives with sodium dodecyl sulfate solution was investigated. The separation of hydrophobic phenols was improved by the addition of organic solvents, however, isomers were not separated. Their separation was achieved by the addition of beta- or gamma-cyclodextrin.

Benzhydryl Compounds↗

PubMed2001

Mixed triblock copolymers used as DNA separation medium in capillary electrophoresis.

A polymer solution, formed by mixing two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B10E270B10 and B6E46B6), was tested as a new separation medium for double-stranded DNA separation in capillary electrophoresis. The mixture of B10E270B10 and B6E46B6 has a viscosity-adjustable property and a dynamic coating ability, which makes the medium very easy to handle. The performance of the mixture on the DNA separation is greatly affected by the mass ratio of the two constituents. There is a minimum amount of concentration for B10E270B10, below which the medium will lose its performance. The addition of B6E46B6 increases both the selectivity and the separation efficiency. The optimal concentration, with 3% (w/v) B10E270B10 and 5% (w/v) B6E46B6, is determined with the consideration of both speed and resolution. A resolution of 1.3 was achieved on the separation of 123/124 base pairs in the pBR322/HaeIII digest within 20 min by using a 10 cm column of 75 microm I.D., demonstrating the potential use of mixtures of amphiphilic block copolymers as an effective DNA separation medium.

DNA↗

PubMed2001

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column.

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.

Buffers↗

PubMed2001

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives.

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated gamma-cyclodextrin (CM-gamma-CD) as pseudostationary phase mixed with beta-cyclodextrin (beta-CD) or permethylated beta-cyclodextrin (PM-beta-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-gamma-CD, 10 mM beta-CD or 20 mM PM-beta-CD, and 2 M urea at a temperature of 45 degrees C and an applied voltage of 20 kV.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed1976

Separation of nucleic acid hydrolysis products, purines, pyrimidines, nucleosides, nucleotides, ribonucleic acid hydrolyzates, and mixtures from nucleotide syntheses by column chromatography on amberlite XAD-4.

Amberlite XAD-4 resin has been studied as a support for liquid-solid column chromatography. By coating the resin with triethylammonium bicarbonate, a new and unique separation of nucleic acid components has been achieved. Separations are accomplished with a linear gradient of this buffer from 0.1 to 0.4 M. Separation occurs in the following order: inorganic phosphate, purine or pyrimidine bases, 5'-monophosphates, nucleosides and 5'-diphosphates or 5'-triphosphates; the 2'(3')-monophosphates are eluted after either the 5'mono-, di-or triphosphates. The bases and nucleosides are separated in the order: cytosine, uracil, guanine and adenine. Inorganic phosphate and the nucleotides are eluted in the order: inorganic phosphate 5'-mono, di- and tri-phosphates. Excellent separation of the 5'-monophosphates and the 2'(3')-monophosphates is now possible. In each series of 5'-mono-, di- and tri-phosphates or 2'(3')-monophosphates, the elution order is generally cytidine, uridine, guanosine and adenosine. By use of water instead of coating the resin with triethylammonium bicarbonate, the nucleotides and inorganic phosphate are found in the void volume; adenine is eluted very slowly, whereas adenosine is not eluted. Adenosine is eluted only with ethanol-water (1:3). The method is advantageous in that the recovery is quantitative, the buffer is easily removed, the capacity of the column is large (35 mugmoles pergram of resin), flow-rates are high, the time required is short and separations of combinations of inorganic phosphate, bases, nucleosides and nucleotides are now possible that previously could not be accomplished.

Buffers↗

PubMed1981

Use of mixed-mode, high-performance liquid chromatography for the separation of peptide and protein mixtures.

The packing material recently introduced for use in a radial compression chamber, with a partially flexible cartridge, has a low C18-coating (5% w/s) combined with an absence of secondary capping. This report demonstrates that such a support, which contains significant concentrations of both free silanol and hydrocarbon groups, can allow a mixed-mode separation to occur via adsorption and reversed-phase separation mechanisms. In any given separation, the predominant mechanism depends both on the nature of the sample and the mobile phase. For efficient peptide and protein separations, it was necessary to suppress most silanol group interactions by the use of a mobile phase which contained a high concentration of an amine phosphate, e.g., 0.17 M triethylammonium phosphate, pH 3.2. In addition, it was necessary to deactivate further the silanol groups by an initial column wash of at least 20 column volumes of methanol. Samples which contained strongly basic groups, for example the guanidino group of arginine, can still exhibit poor separation efficiencies on such a support. These problems were largely overcome, however, with the use of isopropanol as an organic modifier. If these precautions were followed, the packing material gave excellent selectivities in the separation of closely related materials, as well as allowing increased sample capacities. These observations will be supported by an examination of the chromatographic properties of a range of small peptides, the C-apolipoprotein mixture present in human very-low-density lipoproteins and the purification of an 8-mg sample of a synthetic pentadecapeptide in a single chromatographic run.

Apolipoproteins↗

PubMed1988

Tandem separation schemes for preparative high-performance liquid chromatography of proteins.

Preparative chromatography of protein mixtures was carried out by tandem separation schemes involving frontal chromatography followed by stepwise desorption or displacement. In this way, with columns and instruments generally employed in analytical high-performance liquid chromatography, proteins could be purified in quantities similar to those typically separated by a preparative-scale system. A mixture of beta-lactoglobulin A and B was loaded onto an anion-exchange column, and, in the process, a large fraction of the less-retained beta-lactoglobulin B was recovered in pure form. The column was then flushed with the carrier, and subsequent desorption of the substances bound on the stationary phase was carried out by single-step desorption, two-step desorption, or displacement. With this mixture, the last two methods yielded approximately the same results in terms of the amount of product obtained per unit column volume. Whereas stepwise desorption is a simpler technique than displacement, the latter is required for the separation of components having similar adsorption behavior. In another set of experiments, a protein mixture obtained by heat treatment of human growth hormone was fractionated on a reversed-phase column. After loading the column by frontal chromatography, which separated a large fraction of the main product from the other components retained by the column, four desorption steps were applied to recover the individual components. These separation schemes offer an approach to preparative chromatography of proteins that is superior to conventional linear elution in terms of column load capacity, low mobile phase consumption, simultaneous separation and concentration, as well as enrichment of trace components.

Chromatography, High Pressure Liquid↗

PubMed2001

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode.

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.

Chromatography, Gel↗

PubMed2002

Effects of substituted cyclodextrins on the separation of aromatic sulphonic acids by capillary zone electrophoresis.

Effects of the addition of various additives in the working electrolyte on the selectivity of capillary electrophoretic separation of naphthalenesulphonic acids used as intermediates in the production of synthetic dyes were investigated. Cyclodextrins form inclusion complexes with various compounds and are not only excellent chiral selectors, but have been also successfully applied for separations of positional isomers. In this work, methyl-beta-cyclodextrin, heptakis(2,6-di-O-methyl)-beta-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin and (2-hydroxypropyl)-gamma-cyclodextrin were studied as isomeric selector additives and compared with unsubstituted beta-cyclodextrin. In addition to the size of the cyclodextrin cavity, the number and type of the substituents in the cyclodextrin molecules strongly affect the separation of isomeric naphthalenesulphonic acids, but the effect of the substituted cyclodextrins on the separation selectivity is different for various types of sulphonic acids. Best separations of non-substituted naphthalenesulphonic acids were achieved in a borate buffer with methyl-beta-cyclodextrin, whereas the running buffer with non-substituted beta-cyclodextrin provides superior separation of amino and hydroxynaphthalenesulphonic acids.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1989

High-performance liquid chromatography and capillary supercritical-fluid chromatography separation of vegetable carotenoids and carotenoid isomers.

Carotenoids from carrots and tomatoes were separated with high-performance liquid chromatography (HPLC) and capillary supercritical fluid chromatography (SFC). All trans alpha- and beta-carotene were separated from their respective cis-isomers with capillary SFC. Carotenoids extracted from tomatoes included xanthophyll, lycopene and beta-carotene, while alpha- and beta-carotene were extracted from carrots. The HPLC separations were accomplished isocratically with a 25-cm column containing 5-microns ODS and methanol-acetonitrile-chloroform (47:47:6) or acetonitrile-dichloromethane (80:20). beta-Carotene cis-isomers were separated with SFC with a SB-cyanopropyl-25-polymethylsiloxane column, while alpha-carotene isomers were separated with two SB-cyanopropyl-50-polymethylsiloxane columns. Carotenoids from carrots and tomatoes were separated with a SB-phenyl-50-polymethylsiloxane column. Carbon dioxide with 1% ethanol was the SFC mobile phase. The eluent was monitored at 461 nm for HPLC and either 453 or 461 nm for SFC.

Carotenoids↗

PubMed1985

Separation of flavivirus membrane and capsid proteins by multistep high-performance liquid chromatography optimized by immunological monitoring.

Complete separation of the three structural proteins, E, C and M, of an enveloped virus (tick-borne encephalitis virus) was achieved by means of a two-step high-performance liquid chromatography (HPLC) technique in less than 1 h. The hydrophobically associated membrane proteins E and M were successfully separated by high-performance gel permeation chromatography (TSK-3000 SW column) in the presence of sodium dodecyl sulphate (SDS), whereas the separation of M and C as well as desalting and removal of SDS was achieved by subsequent reversed-phase chromatography on a C3 column. With regard to further characterization by peptide mapping, analysis of the amino acid composition and aminoterminal sequencing, the second step was performed with volatile buffer systems. Quality control of the separation was achieved by a combination of HPLC with a highly sensitive dot immunoassay by the use of polyclonal as well as monoclonal antibodies. This method proved extremely sensitive and revealed strong tailing effects and cross-contaminations of peaks well-separated in reversed-phase chromatography, which were neither apparent in the absorbance curve at 214 nm nor in the analysis by SDS-polyacrylamide gel electrophoresis. By visualization of the peak-tailing effect, the chromatographic conditions could be modified in order to achieve an optimum separation of proteins.

Antibodies, Monoclonal↗

PubMed1990

Separation and partial characterization of Maillard reaction products by capillary zone electrophoresis.

Capillary zone electrophoresis proved useful for separating small amounts of both charged and uncharged solutes that are otherwise difficult to analyse. A typical complex mixture that had previously resisted all analytical approaches, including reversed-phase separations, is the products arising from the reaction of free amino acids with aldehydic sugars (Maillard reaction products). By using capillary zone electrophoresis [untreated capillary 50 cm x 75 microns I.D., 18 kV, 0.02 mol/l phosphate buffer (pH 7.5)], a number of products resulting from the reaction of glucose or ribose with glycine, alanine and isoleucine were separated and partially characterized. They were separated (1) without derivatization (and profiles of compounds absorbing at 220 nm were obtained), (2) as phenylthiocarbamyl derivatives in a search for reactive amino groups and (3) after derivatzation with 2,4-dinitrophenylhydrazine in a search for a method for compounds with a free aldehydic group. Phenylthiocarbamyl derivatives were separated in 0.005 mol/l borate buffer (pH 9.6) at 20 kV and 25 microA. Separation of 2,4-dinitrophenylhydrazones was effected by electrokinetic micellar chromatography in the same apparatus using a 50 cm x 75 microns I.D. capillary at 10 kV in 0.01 mol/l Na2HPO4-0.006 mol/l tetraborate, 0.050 mol/l with respect to sodium dodecyl sulphate. The results are compared with those given by high-performance liquid and thin-layer chromatography.

Amino Acids↗

PubMed1977

High-performance liquid chromatographic separation and photometric detection of phospholipids.

A rapid and efficient method for the separation of (phospho)lipids by high-performance liquid chromatography using n-hexane-2-propanol-water mixtures as the solvent system is described. The lipid separation occurs on silica gel columns and the individual components are monitored directly by UV absorption at 206 nm. Of a total lipid extract from erythrocytes as well as suboesophageal ganglia of the snail Helix pomatia, a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are partly separated under these circumstances. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some instances, as is shown for phosphatidylcholines and sphingomyelins.

Animals↗

PubMed1979

Separation of small DNA and RNA oligonucleotides by high-performance anion-exchange liquid chromatography.

Small oligonucleotides from DNA and RNA have been separated according to their base composition by high-performance anion-exchange liquid chromatography on Partisil-10 SAX using triethylammonium acetate buffer as the eluent. Fifteen of the 16 possible deoxydinucleoside monophosphates and all 16 dinucleoside monophosphates have been separated. All pairs of sequence isomers were all resolved. The 15 commercially available deoxydinucleotides were resolved into 13 fractions. A good resolution of deoxytrinucleoside diphosphates isolated from an alkaline phosphatase-Mg2+-activated DNase I digest of calf thymus DNA was achieved by this technique. A large number of sequence isomers could be fully separated. The base sequence of the eluted individual constituents has been determined by their hydrolysis with snake venom and spleen phosphodiesterase followed by high-performance liquid chromatographic analysis of the nucleotides released. The eight trinucleoside diphosphates isolated from an alkaline phosphatase-pancreatic RNase digest of yeast RNA have also been separated according to base composition. Their sequence was determined as above. The described technique is fast and gave very good separation. Most of the sequence isomers could be separated. Moreover, the eluent triethylammonium acetate can easily be removed from column effluents by freeze-drying in order to facilitate subsequent sequence analysis of the eluted compounds. The observed elution orders of the sequence isomers obey certain rules which are discussed in detail.

Animals↗

PubMed1985

Separation of gangliosides by anion-exchange chromatography on Mono Q.

A new type of strong anion-exchange resin, Mono Q, has been used in the separation of brain gangliosides. The resin consists of monodisperse particles (9.8 micron) and was used in prepacked columns with a bed volume of 1 ml. The gangliosides were separated into mono-, di-, tri- and tetrasialoganglioside fractions by a discontinuous gradient of potassium acetate in methanol. The separation was complete in a volume of 50 ml. The major advantages of the new procedure compared to conventional methods are the shorter separation time, higher loading capacity and recovery of separated ganglioside fractions in small solvent volumes. The procedure was applied to the separation of gangliosides from normal human and GM2-gangliosidosis brain.

Aged↗

PubMed2002

Super/subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases.

The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31 degrees C and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.

Chromatography, Liquid↗