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PubMed1997

Isolation of ceramide fractions from human stratum corneum lipid extracts by high-performance liquid chromatography.

This report describes a procedure to isolate ceramides from human stratum corneum lipid extracts using preparative liquid chromatography (LC) and semi-preparative high-performance liquid chromatography (HPLC) methods, which is economical in construction and operation. The composition of isolated individual 'peaks' of separated ceramide fractions is reported. These peaks were quantified by scanning of the chromatographic plates stained after high performance thin-layer chromatography (HPTLC). The HPTLC was done by automated multiple development (AMD). The enrichment of the ceramides by liquid chromatography turned out to be necessary for their separation from more polar lipids of the human stratum corneum lipid extract. Only the evaporative light scattering detector gave a stable baseline, reproducible results and eliminated the 'solvent front' in which peaks of interest could coelute. Fluorescence measurements of extracted human stratum lipids indicated lipofuscin-like substances.

Ceramides↗
PubMed2003

A review of environmental toxicant analysis by using multidimensional gas chromatography and comprehensive GC.

The purpose of this review is to highlight the developments in coupled-column gas chromatography methods for qualitative analysis of selected environmental toxicants such as dioxin, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), etc. In particular, the new technique of GC x GC will be introduced, and its role, and the promise it offers to this field is discussed. The benefits of enhanced separation to quantitative analysis will be considered. In order to perform an accurate risk assessment, both the dose and biological effects of environmental toxicants have to be determined with a high degree of certainty. This is most often achieved by using chromatographic methods. Given the complexity of most environmental sample extracts, single-column gas chromatography is unable to fully resolve all the components of interest frequently leading to a positive bias in the reported concentrations. Advanced separation tools, such as multidimensional gas chromatography (MDGC), were investigated quite early and demonstrated improvements in separation. However, limitations in the number of target analytes that could be analyzed in a single run as well as insufficient robustness lead to a continued interest in alternative solutions. The parallel development of mass spectrometric identification and quantification strategies proved useful in many cases, although it frequently failed to provide positive identification of chromatographically unresolved isomeric compounds. More recently, comprehensive two-dimensional gas chromatography (GC x GC) may offer a solution to that problem, especially because it offers enhanced resolution for complex mixtures containing trace level environmental toxicants.

Chromatography, Gas↗
PubMed1996

Validation of candidate reference methods based on ion chromatography for determination of total sodium, potassium, calcium and magnesium in serum through comparison with flame atomic emission and absorption spectrometry.

OBJECTIVES: We report on the validation of candidate reference methods based on ion chromatography for the determination of total sodium, potassium, calcium, and magnesium in serum. The current study is a continuation of previous investigations in which we were able to show the potential of ion chromatography to serve as a reference method principle. The validation consisted of comparison of the ion chromatographic methods with generally recognized reference methods based on flame atomic emission (for sodium/ potassium) and absorption (for calcium/magnesium) spectrometry. METHODS: Sample preparation consisted of acidic dilution and pre-purification with reversed phase cartridges. Ion chromatography with suppressed conductivity was performed with polymeric stationary phases chemically modified with carboxylate and sulfonate functional groups for sodium/potassium and calcium/magnesium, respectively. Single-point calibration was used in combination with individual adaptation of serum sampling and diluting to give similar responses in samples and standards. In the study, more than 25 control sera were analyzed in parallel with the ion chromatographic methods and the traditional reference methods. The measurement protocol consisted of quadruplicate analysis of each sample per run, in 3 different runs. In each measurement series, internal quality control with certified reference materials from the National Institute of Standards and Technology and the Bureau Communautaire de Référence was applied. The performance criteria to fulfill were pre-defined (i.e., the maximum within-day and overall coefficient of variation, the confidence interval, the daily and overall deviation from the certified value and the maximum total analytical error). RESULTS: The comparison is presented as difference plot (i.e., % deviation of the results obtained with ion chromatography from those obtained with the respective traditional reference methods). The mean deviations were +0.9% for sodium, +1.0% for potassium, +/- 0.0% for calcium, and +0.1% for magnesium. With the exception of 2 values each for sodium and potassium, all deviations were within the limit of 2 times the allowed total analytical error. From the data obtained for internal quality control during the different measurement campaigns, we further estimated the long-term precision and accuracy of our methods. The overall coefficient of variation (respectively the bias) amounted to 0.7% (+0.1%) for sodium, 0.9% (-0.3%) for potassium, 1.3% (-0.1%) for calcium, and 1.0% (+/- 0.0%) for magnesium. CONCLUSION: From the precision and accuracy reached by our ion chromatographic methods, we conclude that they can be proposed as candidate reference methods for the determination of total sodium, potassium, calcium, and magnesium in serum.

Calcium↗
PubMed2000

Effects of DNA topology, temperature and solvent viscosity on DNA retardation in slalom chromatography.

Slalom chromatography is a unique size-fractionation method applicable to large DNA molecules [>5 kilobase pairs (kbp)]. The method was first developed by using columns packed with microbeads (diameter, <20 microm) used for high-performance liquid chromatography and by applying a relatively fast flow-rate (>0.3 ml/min). Previous studies suggested that the separation is attributed to a hydrodynamic rather than to an equilibrium phenomenon (J. Hirabayashi and K. Kasai, Anal. Biochem. 178 (1989) 336; J. Hirabayashi, N. Itoh, K. Noguchi and K. Kasai, Biochemistry, 29 (1990) 9515). In the present report, the results of a systematic study on the effects of DNA topology, temperature, and solvent viscosity on DNA retardation are described. Firstly, the behaviour of circular (super-coiled) and linearized forms of charomid DNAs (20-42 kbp) was studied. Circular-form DNA molecules were found to be fractionated size-dependently similarly to linear forms in a flow-rate dependent manner. However, the extent of retardation of the circular form DNA was apparently less than that of the corresponding linear forms. Circular DNAs showed almost the same retardation (e.g., 42 kbp) as DNA fragments (e.g., 20 kbp) having approximately half of the size of the former. This observation indicates that DNA retardation is basically related to physical length, not to mass. Secondly, to study the effect of temperature with special reference to solvent viscosity, we carried out chromatographic analysis at various temperatures ranging from 6 to 65 degrees C in both the absence and presence of sucrose (10 or 20%, w/v). The results showed that it is the solvent viscosity that determines the extent of retardation. Taken together, all of physicochemical parameters that define hydrodynamic properties, i.e., particle size, flow-rate and solvent viscosity, proved to be critical in slalom chromatography as well as the potential physical length of the DNA, thus supporting the concept that slalom chromatography is based on a hydrodynamic principle.

Chromatography, High Pressure Liquid↗
PubMed1977

Dry column chromatography of phospholipids.

Separation of common phospholipids can be effected by dry column chromatography on silica gel. The method involves packing the column with dry gel and developing it in solvent mixtures used for thin-layer chromatography of the same lipids. Solvent is allowed to migrate only to the end of the column; access to the bands of separated material is obtained by using columns with a removable glass front. RF values of lipids on development columns and those on thin-layer plates are nearly identical when the column is packed with thin-layer chromatography gel. Such columns, however, develop very slowly. Columns packed with fine silica gel designed for elution column chromatography develop very rapidly and yield separations that are still quite comparable to those obtainable from thin-layer plates. Such columns are convenient for the purification of phospholipids in amounts of 10 mg to about 10 g. Column design and construction are described in detail.

Chromatography, Gel↗
PubMed1977

Determination of the rodenticide difenacoum in biological materials by high-pressure liquid chromatography with confirmation of identity by mass spectrometry.

A method for determining difenacoum in liver, plasma, urine and feedingstuffs by high-pressure liquid chromatography is described. Samples are cleaned up by molecular exclusion chromatography on porous glass. In some cases this also serves for determination; if not, the separated difenacoum is determined on an adsorption column. Identity is confirmed by chemical ionisation mass spectrometry. Recoveries at levels of 0.025-5 ppm from plasma were 101-113% by exclusion chromatography alone and 93-101% after adsorption chromatography. Recoveries from liver after both chromatographic steps were 62-86%. Reasons for the lower recoveries from liver are suggested.

4-Hydroxycoumarins↗
PubMed1988

Isolation of biologically active plant constituents by liquid chromatography.

Strategies are outlined for the separation of biologically active products of plant origin. The techniques involved include low-pressure liquid chromatography, semi-preparative high-performance liquid chromatography, flash chromatography and droplet counter-current chromatography. Their application to the isolation of compounds from Sesamum angolense (Pedaliaceae), Psorospermum febrifugum (Guttiferae) and Cordia goetzei (Boraginaceae) is described.

Anthracenes↗
PubMed1980

Affinity chromatography of cells and cell membranes.

This article attempts to introduce the concept of cellular affinity chromatography as it departs from molecular affinity chromatography. Special emphasis is placed on the selectio of a solid support as well as on the role of lectins as affinity ligands. Our major goal was to bring to light the basic principles involved, multiple options of ligands and matrices, and different techniques, which may be applied to separate the complex cell population as well as cell membranes. It is hoped that further developments in the field, especially in the selection of proper experimental conditions, ligands and matrix material may provide better results. We have tried to identify some of the potential problems which should be considered before these approaches can be used on a routine basis. Although the review deals primarily with the affinity chromatography of cells and cell membranes, examples are presented for diverse systems such as cell organelles, viruses and phages. A table summarizing the use of cellular affinity chromatography is included. It lists more than 80 examples covering the literature up to December, 1979.

Animals↗
PubMed1989

Ion-pair, anion-exchange and ligand-exchange high-performance liquid chromatography of tenuazonic acid and 3-acetyl 5-substituted pyrrolidine-2,4-diones.

The ion-pair, ligand-exchange and anion-exchange chromatography of the fungal metabolic tenuazonic acid (TA) and its related 3-acetyl 5-substituted pyrrolidine-2,4-diones were studied. Ion-pair chromatography was performed on a C18 column with a mobile phase composed of cetrimide, phosphate buffer in water-methanol and a metal complexant (ethylenediamine) to improve the peak sharpness. Addition of the same metal complexant to the mobile phase of the anion-exchange chromatographic system also improved its efficiency. TA and its 5-substituted analogues derived from valine and leucine were separated with the ion-pair and anion-exchange chromatographic systems. With ligand-exchange chromatography, TA could only be separated from its valine analogue. These chromatographic systems were used for the detection of TA in the culture filtrates of the fungus Pyricularia oryzae and in infected rice leaves. Deproteinated culture filtrates could be rapidly analysed for their TA content by anion-exchange chromatography. However, this system was not suitable for the detection of TA in the infected rice leaf as interfering compounds were coeluted with TA. Ion-pair and ligand-exchange chromatographic systems allowed the efficient quantification of TA in infected leaves.

Antibiotics, Antineoplastic↗
PubMed1984

Analysis of steroids in bulk pharmaceuticals by liquid chromatography with light-scattering detection.

A detector for liquid chromatography based on light-scattering was evaluated to determine its application to pharmaceutical analysis. It performed adequately as a mass detector in liquid-solid chromatography of steroids, since response factors were equal to within 20%. The detection limit was approximately 0.5 micrograms. In reversed-phase chromatography response factors varied from 0.13 to 1.0 because of partial vaporization of some steroids. Stable baselines were obtained in gradient elution chromatography, but response varied with solvent composition. This detector was useful for determining impurities in bulk pharmaceuticals.

Chromatography, Liquid↗
PubMed1990

Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography.

The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3289-3293]. This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide: N-glycosidase F (PNGase F) or endo-beta-N-acetylglucosaminidase H digestion of glycoproteins. Separations were carried out at a constant base concentration (0.1 M NaOH) using linear gradients from 0 to 0.2 M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating "oligosaccharide maps" of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering salt-free oligosaccharides after this chromatography were also investigated. On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of 0.15-0.2 M at a flow-rate of 1 ml/min. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversion to permethyl derivatives or after reductive amination with rho-aminobenzoic acid ethyl ester.

Animals↗
PubMed1990

Micropellicular stationary phases for rapid protein analysis by high-performance liquid chromatography.

The high separating speed, efficiency and operational stability of various micropellicular stationary phases are demonstrated in the high-performance liquid chromatography (HPLC) of biopolymers. The micropellicular sorbents were prepared from 2-microns fluid-impervious silica microspheres as the support, with a thin layer of different retentive materials at the surface. These include a molecular fur of octyl or stearyl chains for reversed-phase chromatography as well as a hydrophilic layer with amino groups and polyethyleneglycol chains for anion-exchange and hydrophobic interaction chromatography, respectively. The use of appropriate micropellicular stationary phases for protein separation by metal-interaction and affinity chromatography is also illustrated. In most cases, operation at elevated column temperature was found to be preferable for rapid separations. Preliminary results show that the stability of micropellicular columns compares very favorably with that of columns conventionally used in HPLC and that they are easy to maintain.

Animals↗
PubMed2001

Analysis of non-saponifiable lipids by super-/subcritical-fluid chromatography.

Because of the particular properties of carbon dioxide or carbon dioxide/modifier mobile phases, super- or subcritical-fluid chromatography (SFC) can be an alternative to more classical chromatographic methods such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) for the separation of unsaponifiable lipids. These fluids can also be helpful in the extraction and/or the concentration steps of sterols, tocopherols or carotenoids from complex samples. Supercritical extraction, off-line prefractionation or semi-preparative supercritical fluid chromatography, carried out before the analysis are described. The effects on separation of analytical parameters such as pressure, nature of and modifier percentage or stationary phase nature are also reported. The performance of capillary, packed or capillary packed columns is discussed, as well as the consequences of their use (choice of stationary phases, type of coupled detector). Numerous examples of fine separations are reported.

Chromatography, Supercritical Fluid↗
PubMed2002

Preparative liquid chromatography.

The status of the theory and the main methods of implementation of preparative liquid chromatography are reviewed. On the theory front, the focus has recently shifted. The theory of non-linear, non-ideal chromatography has given rise to numerous models whose advantages, disadvantages and ranges of application are now well understood. Interest now resides in investigating the equilibrium thermodynamics of complex new systems, in the study of the kinetics of mass transfers in conventional chromatographic systems, and in the application of the various models of chromatography to optimize the experimental conditions. Progress in computer technology allows the use of sophisticated models, provided their parameters can be measured. This allows the detailed investigation of separations for which the mass transfer kinetics is slow such as chiral separations, the purification of basic compounds, and the extraction of recombinant proteins. On the applied front, in addition to numerous incremental improvements in reliability and economic performance, a few essential new features should be noted, i.e. the availability of instruments for simulated moving bed separations at the scale needed for preparative chiral separations, the use of expanded beds for the extraction of recombinant proteins from fermentation broths, and the attention given to improvements in the performance of packed beds. A survey of the literature dealing with practical applications and recent meetings shows that preparative chromatography is becoming a well established separation and purification method in the pharmaceutical industry.

Chromatography, Liquid↗
PubMed1985

Quantitative affinity high-performance liquid chromatography of neuroendocrine polypeptides using porous and non-porous glass derivatives.

Analytical affinity HPLC was developed to isolate and characterize neuroendocrine peptide/protein components. Bovine neurophysin II (NP-II) was covalently immobilized on succinamidopropyl derivatives of both controlled-pore glass (CPG) and non-porous glass (NPG). These derivatives were packed into 25 X 0.46 cm I.D. stainless-steel columns and incorporated into a high-performance liquid chromatograph. Interaction of [3H]Arg8-vasopressin ([3H]AVP) with NP-II was examined by chromatography of AVP on both CPG and NPG affinity matrices. Zonal elution profiles of [3H]AVP on NPG matrix showed, as predicted theoretically, a linear dependence of retardation on the concentration of hormone injected. The data permit calculation of the equilibrium dissociation constant for the NP-II/AVP interaction. Elution characteristics also were measured by frontal analysis of large-zone chromatography experiments, the results of which were in good agreement with the zonal elution analysis. Affinity resulting from dimerization also was studied by chromatography of [125I]NP-II on the NPG matrix. In this case, concentration dependence of retardation was non-linear, again as predicted theoretically. Off-rate kinetic constants for dissociation of the mobile interactant from the stationary phase also were obtained. The studies illustrate the utility of analytical affinity HPLC on non-porous beads for measuring relative affinities for various soluble ligands with small amounts of material. Chromatography on the CPG column proved useful for purification of microscale amounts of [3H]AVP.

Animals↗
PubMed1985

Investigation of operating parameters in high-performance displacement chromatography.

The effect of operational parameters of displacement chromatography was examined in the separation of various mixtures such as that of the main hydrolysis products of methylfurylbutyrolactone, a potential anticancer drug, the diastereoisomers benzoyl-D- and benzoyl-L-phenylalanyl-L-alanyl-L-proline, as well as polyethylene glycol homologues containing 1-10 ethylene oxide units. The chromatograph was assembled from modules generally used in analytical high-performance liquid chromatography (HPLC) and the column effluent was analyzed by an on-line HPLC unit at 30-sec intervals. Octadecyl-silica was used throughout as the stationary phase. Derivatives of ethylene glycol and propylene glycol as well as tetrabutylammonium bromide and n-butanol were used as displacers. The throughput was used as the measure of efficiency. In the absence of axial dispersion, for a given separation various displacers are expected to yield the same efficiency if the slope of the operating line is kept the same by appropriate adjustment of displacer concentrations. In practice, however, the optimum slope of the operating line has to be determined experimentally as most available chromatographic systems depart from ideal behavior. The dependence of the throughput on the flow-rate and feed load also indicated the presence of non-equilibrium phenomena and the optimum value of these parameters was established experimentally. In most cases water was used as the carrier solvent but the separation of poorly soluble peptides required the use of hydro-organic carriers. Results obtained with octadecyl-silicas of different origin and a given displacer were found to vary significantly suggesting that even for stationary phases of the same type the selection of displacer requires special consideration. Most experiments were carried out with columns having dimensions customary in analytical HPLC. Increasing the inner diameter of the column did not result in the expected increase in throughout probably due to poor distribution of the sample at the column entrance. Therefore scaling-up the process requires careful engineering of inlet conditions. Throughput can be increased by connecting a small inner diameter column to the outlet of a large diameter preparative column. As theoretical predictions for ideal displacement chromatography do not hold in practice when axial dispersion is significant, optimization of the process requires experimental support. The results obtained in the separation of a variety of mixtures shed light on the most important operational aspects of displacement chromatography and suggest approaches to find optimum conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Ascorbic Acid↗
PubMed1988

Separations of flavonoids and alkaloids in medicinal herbs by high-speed counter-current chromatography.

Counter-current chromatography is a new liquid-liquid partition chromatography without using solid support. Recently, the technique has been remarkably improved in both partition efficiency and separation time. In this paper the capability of this high-speed counter-current chromatography was demonstrated on separation of two sets of samples obtained from medicinal herbs: a synthetic mixture of 3'-hydroxygenkwanin, luteolin and apigenin was separated on a two-phase solvent system composed of chloroform-methanol-water (4:3:2, v/v/v) and a crude ethanol extract from Anisodus tangulicus (Maxin) Pasch on chloroform-0.07 M sodium phosphate (pH 6.4) (1:1, v/v). In the light of chromatograms obtained from these samples, advantages of high-speed counter-current chromatography over other chromatographic methods were discussed in terms of partition efficiency, peak resolution, separation time, sample loading capacity, etc.

Alkaloids↗
PubMed1990

Combination of positron emission tomography with liquid chromatography in neuropharmacologic research.

Positron emission tomography (PET) is an in vivo autoradiographic technique that determines the radioactive distribution and kinetics of a radiolabelled tracer in a tissue. By choice of tracer, it is possible to study physiological processes in living animals and man non-invasively. PET has certain disadvantages such as limited spatial resolution and simultaneous measurement of radiolabelled tracer with the metabolites formed. For an adequate interpretation of the data obtained, complementary techniques such as column liquid chromatography of radioactive composition in blood, plasma, urine and tissue samples have to be used. The prerequisites for any chromatographic technique used for the radioanalysis of substances are speed, high selectivity and high separation efficiency. Examples from PET studies in combination with chromatographic analysis will be given. The utilization of L-dopa in the brain constitutes several steps. Analysis by column liquid chromatography of metabolites in plasma and in monkey brain tissue will make it possible to elucidate different utilization processes of the tracer. Kinetic studies of 11C-labelled neuropeptides such as methionine-enkephalin and substance P revealed high radioactivities in the brain of monkeys. However, simultaneous determination plasma and urine radioactivities using liquid chromatography with radiochemical and photometric detection both indicated that the brain radioactivities emanated to a large extent from 11C-labelled metabolites formed in vivo. Studies with PET using radiotracers having a rapid and extensive metabolism require complementary techniques in the evaluation. High detection selectivity, by combination of photometric and radiochemical detection and rapid and efficient separation, will make liquid chromatography a most important complement in the analysis.

Chromatography, Liquid↗