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At least 775 records · Page 43Linked to original sources
PubMed1981

Separation of 5-hydroxyindole-3-acetic acid and indole-3-acetic acid in urine by direct injection on a reversed-phase column containing a hydrogen-accepting stationary phase.

The separation of 5-hydroxyindole-3-acetic acid and indole-3-acetic acid from urine is demonstrated. The urine is injected directly on a liquid chromatographic column which is coupled to a flow-through fluorescence detector or a UV detector. A hydrogen-accepting stationary phase, tributyl phosphate (TBP), which retains carboxylic acid selectivity, was more selective than more common reversed-phase systems with alkyl-bonded stationary phases. The mobile phases were buffers, sometimes buffers containing methanol or acetonitrile. A simple method of removing late peaks in the chromatogram, in order to reduce analysis time, is also presented.

Chromatography, Liquid↗
PubMed1980

Reversed-phase ion-pair partion chromatography of biogenic catecholamines and their alpha-methyl homologues with tributylphosphate as stationary phase.

Ion-pair partition chromatography is applied to the separation of the biogenic catecholamines and their alpha-methyl homologues. A useful selectivity has been obtained using an adduct-forming organic stationary phase (tributylphosphate). The retention of the compounds can be regulated easily by means of the concentration of the counter-ion (the perchlorate ion) in the mobile phase. The selectivity for separation of amines from amino acids can be influenced by changing the pH of the aqueous phase. The phase system shows a good long-term stability and reproducibility with respect to the capacity ratios and the efficiency.

5-Methoxytryptamine↗
PubMed1984

Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases. IV. Molecular interaction forces and retention behaviour in chromatography on bovine serum albumin as a stationary phase.

Chromatography with the use of immobilized bovine serum albumin as a stationary phase and aqueous buffer systems as eluents has proved to be a highly selective method, capable of separating structurally very closely related compounds. Retention can be effectively regulated by changes in at least three independent parameters of the mobile phase, which may be used for an optimization of separation factors. Particularly, the enantioselective properties of the chiral stationary phase have been demonstrated to be useful for the analytical resolution of a variety of racemates into enantiomers. From the variation of the retention behaviour with substituent effects, as well as the mobile phase composition, some indications regarding the molecular interaction forces regulating the substrate-protein equilibria have been obtained.

Buffers↗
PubMed2001

Comparison of chromatographic properties of cyanopropyl-, diol- and aminopropyl- polar-bonded stationary phases by the retention of model compounds in normal-phase liquid chromatography systems.

Polar-bonded stationary phases, such as CN-, diol- and NH2-silica, have been characterised by the retention of model solutes (phenols, aromatic amines and quinoline bases) in normal-phase systems using n-heptane--polar modifier (2-propanol, tetrahydrofuran or dioxane) mixtures as eluents. The selectivity of separation for the particular groups of substances has been analysed by the log kI versus log kII relationships for CN- and diol, CN- and NH2- and NH2- and diol phases in examined eluent systems by the plotting of correlation lines. The values of regression coefficient r indicate either the similarity of the retention mechanisms of model solutes in some examined systems where r>0.9, or differences among various systems where r<<0.9. The values of slopes of correlation lines show the selectivity of separation for particular group of compounds. The selectivity of separation has also been characterised by deltalog k values. The effect of modifier (2-propanol, tetrahydrofuran and dioxane) on selectivity of model solutes on these phases has also been discussed.

Chromatography, Gel↗
PubMed2001

Reversed-phase separation of basic tricyclic antidepressants using buffered and fluoroform-enhanced fluidity liquid mobile phases.

In an effort to expand the range of applications of enhanced-fluidity liquid chromatography (EFLC) to strongly polar and basic analytes, fluoroform (CHF3) was investigated as a fluidity-enhancing agent. Fluoroform was chosen due to its high polarity, low viscosity and chemical inertness toward water and basic analytes. A group of representative basic compounds, tricyclic antidepressants, covering a wide range of polarity was chosen as model compounds. Their retention behavior on a C18 stationary phase in methanol/phosphate buffer and methanol/phosphate buffer/CHF3 mobile phases was characterized. The chromatographic performance with mobile phase conditions of different pH, with and without CHF3 addition and with addition of triethylamine was studied. The advantages of using CHF3 enhanced and buffered mobile phases were shown in the much improved chromatographic performance, such as shortened analysis time, increased efficiency, lower pressure drop and improved selectivity. Furthermore, this study demonstrated for the first time, that a commercial instrument could be readily utilized for EFLC separations which greatly expands the application range of the EFLC technique and chromatographic instrumentation.

Antidepressive Agents, Tricyclic↗
PubMed2001

Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues.

Two high-performance liquid chromatography (HPLC) procedures, a rapid normal-phase isocratic method for the analysis primarily of retinol and retinoic acid on a 3 mu silica column, and a reversed-phase gradient method for the simultaneous analysis of retinoids and very polar to nonpolar carotenoids on a 3 mu C18 column, are described. The normal-phase isocratic HPLC procedure is rapid (12 min), requires a sample size of 100 microl or less of serum, and is suitable for routine analysis of retinol in any serum, and of retinol and retinoic acid in serum after administration of retinoic acid. The reversed-phase gradient method is suitable for the simultaneous analysis of very polar to nonpolar carotenoids such as epoxy-xanthophylls and xanthophyll esters, along with other carotenoids and retinoids that occur normally in human serum and other plant and animal tissues. A run time of 30-70 min is necessary, depending on the presence or absence of xanthophyll esters in the sample.

Animals↗
PubMed2002

Retention of ionizable compounds in high-performance liquid chromatography. IX. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition with acetonitrile-water mobile phases.

The influence of pH and solvent composition of acetonitrile-water mobile phases on the retention of acids and bases on a polymeric stationary phase is studied. Very good relationships between retention and mobile phase pH are obtained if the pH is measured in the proper pH scale. The fit of retention to pH for a particular solvent composition provides the pKa values of the equilibria between the different acid-base species and the retention parameters of these species at this solvent composition. Several models are tested that relate these parameters to solvent composition and properties in order to propose a general model to predict retention for any mobile phase pH and composition.

Acetonitriles↗
PubMed1988

Retention in reversed-phase liquid chromatography: solvatochromic investigation of homologous alcohol-water binary mobile phases.

The mechanism of retention in reversed-phase liquid chromatography (RPLC) has been further investigated using the ET(30) solvatochromic solvent polarity scale. The retention behavior of a variety of solutes was measured using a homologous series of normal alcohols as the organic modifiers in hydroorganic mobile phases. The results imply that a systematic change in the extent of solvation of the stationary phase occurs with respect to the size of the organic modifier. It was also found that a linear extrapolation of the log k' versus ET(30) plots for different mobile phases using methanol, ethanol and acetonitrile (but not n-propanol) as modifiers, intersected at approximately the ET(30) value of pure water. This intersection is further evidence that the ET(30) model of solute retention is a useful tool with which to study the mechanism of retention in RPLC. The extrapolated retention value in water, log k'w, from the ET(30) plots should then prove to be a more reliable means of estimating solute lipophilicity using RPLC than the percent organic modifier model.

Alcohols↗
PubMed1984

The separation of collagen alpha-chains by reversed-phase high-performance liquid chromatography. Comparison of column alkyl stationary phases and temperature effects.

Procedures for the separation of alpha 1(I), alpha 2(I), alpha 1(II) and alpha 1 (III) chains of human collagen by reversed-phase high-performance liquid chromatography are described. The influence of several different chromatographic parameters (stationary phase, mobile phase and temperature) has been examined and procedures to optimise resolution presented. These reversed-phase high-performance liquid chromatographic conditions also permit the separation of collagen alpha 1(I), alpha 2(I), alpha 1(II) and alpha 1(III) monomers from their corresponding dimeric beta- and gamma-components.

Amino Acids↗
PubMed1991

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase.

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Acetates↗
PubMed1991

Non-ideal behaviour of silica-based stationary phases in trifluoroacetic acid-acetonitrile-based reversed-phase high-performance liquid chromatographic separations of insulins and proinsulins.

Several C18 stationary phases were found to behave non-ideally when insulins and proinsulins were eluted with shallow acetonitrile gradients in 0.1% trifluoroacetic acid, resulting in poor peak shapes or no elution at all. With triethylammonium phosphate or ammonium sulphate as buffer components, the insulins and proinsulins were eluted with excellent peak shapes, presumably owing to better masking of residual silanol groups on the stationary phases. Similar use of trifluoroacetic acid-acetonitrile gradients on the less hydrophobic C4 or C3 stationary phases resulted in excellent peak shapes. The difficult separation of rat proinsulin I and II, which are important for the study of rat insulin biosynthesis, was only achieved with two different stationary-mobile phase combinations.

Acetonitriles↗
PubMed1989

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry.

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.

Adenine↗
PubMed1991

Chemically bonded phases for the reversed-phase high-performance liquid chromatographic separation of basic substances.

A chemically bonded phase with a peptide group (PB) for reversed-phase high-performance liquid chromatography (HPLC) is described. This packing was prepared by a two-stage modification of the surface of silica gel with mono- and trifunctional 3-aminopropylsilane and then with an appropriate derivative of a fatty acid. Packings prepared in this way were compared with standard C18 materials used in HPLC. Surface characteristics of the packings before and after chemical modification were determined by different physico-chemical methods, e.g., porosimetry, elemental analysis, 13C and 29Si cross-polarization magic angle spinning NMR and HPLC. Chromatographic properties of these packings were evaluated by comparison between log k' of one phase and log k' of a second phase for substances with different chemical natures. The PB packing was found to be especially useful for the separation of basic substances.

Amino Acid Sequence↗
PubMed2003

Evaluation of the two-dimensional reversed-phase-reversed-phase separations of low-molecular mass polystyrenes.

The resolving power of four reversed-phase-reversed-phase two-dimensional (2D) chromatographic systems was evaluated using information theory (IT) and a geometric approach to factor analysis. The first separation dimension employed a C18 column, while the second separation dimension employed a carbon clad zirconia column. Mobile phases in each dimension were either methanol or acetonitrile. The sample matrix that was employed in this study contained a mixture of 58 components, comprised of stereoisomers and structural isomers of a systematic variation in molecular weight. Each of the components were oligostyrenes, with between two and five configurational repeating units having either n-butyl, sec-butyl or tert-butyl end groups. In the two-dimensional systems employed in this study, between 46 and 49 of the 58 components could be separated, depending on the mobile phase combinations--with apparent resolution that could not possibly be achieved in a single one-dimensional separation. The results from this study indicate that in order to fully evaluate the resolving power of a 2D system multiple methods of analysis that evaluate the separation potential are most appropriate. This becomes increasingly important when the sample contains components that are very closely related and the retention of solutes displays a high degree of solute crowding.

Molecular Weight↗
PubMed1997

Relationship between stationary and mobile phase composition and its influence on retention factors of aromatic hydrocarbons in reversed-phase high-performance liquid chromatography.

The effect of the composition of the mobile phase on the composition of the stationary phase and its influence on the retention factor, k', of polynuclear hydrocarbons has been determined. The decreasing relationship between log k' and the concentration of modifier (methanol) in mobile and stationary phases has been proved. It has been determined that the solubility of examined aromatic hydrocarbons in stationary phase is a reason for changes in the k'. This fact can be used in order to improve the effectiveness of separation of some mixtures.

Adsorption↗
PubMed1997

Reversed-phase separations of nitrogenous phospholipids on an octadecanoyl poly(vinyl alcohol) phase.

Molecular species of nitrogenous phospholipids (PLs) phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-derivatives and sphingomyelin (SP) were separated on an octadecanoyl poly(vinyl alcohol) (ODPVA) column by reversed-phase HPLC with UV and evaporative light scattering detection (ELSD). Mobile phases employed variable proportions of acetonitrile, methanol and water. HPLC-UV of the polar lipids yield components with peak intensities somewhat different from those obtained by HPLC-ELSD despite discernible similarity in the peak profiles observed in the two detection systems. Incorporation of ammonium hydroxide in mobile phases resulted in a decrease in analyte retention. The mobile phase basicity effect on capacity factors of PE species was significantly greater than that of PC counterparts. The new ODPVA-HPLC-ELSD technique was applied to the analysis of PC and PE molecular species in vegetable oils.

Ammonium Hydroxide↗
PubMed1999

Electrostatic ion chromatography of polarizable anions in saline waters with N-[2-[acetyl(3-sulfopropyl)amino]ethyl]-N,N-dimethyldodecanaminium hydroxide (ammonium sulfobetaine-1) as the stationary phase and a dilute electrolytic solution as the mobile phase.

A new type of zwitterionic surfactant, N-{2-[acetyl(3-sulfopropyl)amino]ethyl}-N,N-dimethyldodecanaminium hydroxide (ammonium sulfobetaine-1), with a greater distance between the two charged groups, was used as the stationary phase for electrostatic ion chromatography (EIC) of polarizable anions (e.g., thiocyanate, iodide and nitrate) in saline water samples. The targeted species (polarizable anions) were baseline separated using this type of zwitterionic surfactant as the stationary phase, but the highly polarizable species (iodide and thiocyanate) were eluted faster (compared with the results obtained using N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, C12N3S, with a shorter distance between the two charged groups, as the stationary phase). In other words, the extent of binding of the highly polarizable anion (iodide and thiocyanate) was found to be smaller when using ammonium sulfobetaine-1 as the stationary phase. This provides a rapid but effective method for the analysis of highly polarizable anions in saline water samples. The results for the successful detection of iodide in seawater demonstrates the usefulness of this new type of zwitterionic surfactants for EIC.

Betaine↗
PubMed2000

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase.

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.

Amines↗