Column In Conjunction Research Articles

Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis

Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes. Strains were isolated from arthropods collected in South Moravia, Czech Republic. Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one “ Spironema culicis” strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies. Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA). Results obtained on the basis of cluster analysis of FAME profiles showed, that the B. burgdorferi sensu lato isolates could be well separated from other spirochetal isolates. We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans.

Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria.

Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis/transformation, membrane transport, translation, protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.

Computer visualisation of the moving human lumbar spine

Disorders of the spine which lead to back pain are often mechanical in origin and, despite extensive research, diagnosis of the underlying cause remains problematical, yet back pain is one of the most common rheumatological symptoms presented to the general practitioner. Diagnosis must frequently be based upon evidence gathered at the segmental level which invariably means that imaging is used in the process. In addition, surgical fixation is increasingly used when the spinal column is considered to exhibit instability. A solid model of the spine creates the possibility of visualising spine motion, of assessing the effects of loading of the spinal column in conjunction with finite element analysis to investigate the consequences of vertebral fusion, and of planning surgical intervention. Such a model could also be valuable in medical education and for demonstrating spine motion to a patient to highlight abnormalities or the effects of treatment. This paper describes a three-dimensional visualisation of the human lumbar spine which runs on a personal computer operating under the Windows environment. The user interface enables the clinician to select the viewpoint for the spine model to allow the motion to be studied from different angles. Motion data are currently acquired from fluoroscopic image sequences but the model could be used to display data from different imaging modalities when they are developed sufficiently for spine motion studies.

Hydrologic analysis of the Fort Collins, Colorado, flash flood of 1997

On 28 July 1997, an unusually moist air mass, driven westward towards the foothills of the Rocky Mountains near Fort Collins, Colorado, produced torrential rainfall. The nearly saturated atmospheric column in conjunction with light upper-level winds resulted in a “warm rain” process convective storm with little net motion. On the evening of 28 July over 200 mm of rain fell in western Fort Collins. This extreme rainstorm was observed by three S-band weather radars, including two National Weather Service WSR-88D radars and the dual-polarization CSU-CHILL radar. Fourteen recording rain gages in and near the affected area recorded the event. The US Geological Survey, Colorado District, performed indirect peak discharge measurements. In our analysis, the two-dimensional, physically-based hydrologic model CASC2D is applied to examine the influence of rainfall and land surface data uncertainty on runoff predictions in the 25 km 2 Spring Creek watershed. Soil saturated hydraulic conductivity values are calibrated in simulations of the rise in nearby Horsetooth Reservoir. Results of simulations driven by polarimetric and single-polarization radar-rainfall estimates and recording rain gage data show that for this extreme event in an urbanized watershed, rainfall estimation errors give rise to the most significant errors in runoff predictions. Hydrologic simulations with various levels of land-surface detail reveal that uncertainty in watershed characteristics has a considerably smaller effect on runoff predictions than uncertainty in the space/time distribution of rainfall. The soil saturated hydraulic conductivity, fraction of impervious area, and the retention depth are the most sensitive land-surface parameters.

Fluid Dynamics of Radial-Flow Ion Exchange in Partially Filled Columns

An experimental and numerical study demonstrated that completely filling the resin bed is essential if a radial-flow ion-exchange column is to work properly. The first phase of the study developed and verified a finite-element model of both an infinitely long column and finite-height column that was completely filled with resin. The porosity and tortuosity of the support columns of the ion-exchange column were calculated using a least-squares analysis of experimental pressure drop versus flow rate data in an empty column in conjunction with a theoretical analysis based on a layered-cylinder model. Numerical analysis of a partially filled column (i.e., one in which the top 2% of the resin bed was empty headspace) demonstrated that approximately 9% of the fluid would flow through the headspace. This fluid would never come in contact with the ion-exchange resin and would thus leave the column untreated. Modifying the column so that the top 10% of the support membranes was impermeable changed the flow lines such that the headspace was filled with a stagnant fluid and that, after this space was filled, all subsequent fluid entering the column flowed through the ion-exchange bed. This provides a method in which one could ensure that all the fluid would flow through the ion-exchange resin and be treated even if the column could not be or was not completely filled.

Direct high-performance liquid chromatographic determination of the enantiomeric purity of levodopa and methyldopa: comparison with pharmacopoeial polarimetric methods

Chiral high-performance liquid chromatography was employed for determination of the enantiomeric purity of levodopa and methyldopa. The determination of d-DOPA in levodopa was accomplished using a chiral ligand-exchange chromatograpy with an ordinary C 18 column and a chiral mobile phase containing N, N-dimethyl- l-phenylalanine and Cu(II) acetate or by means of LC on a teicoplanin column in conjunction with ethanol–water (65:35, v/v). Both methods gave good performance, however, the latter was faster and more convenient and suitable for routine analyses. For the determination of d-methyldopa a LC method based on the use of a teicoplanin column in polar organic mode with methanol-acetic acid-triethylamine (1000:0.05:0.05, v/v/v) mobile phase was developed. The precision, accuracy, linearity and selectivity were satisfactory. In comparison with pharmacopoeial polarimetric methods (according to the European Pharmacopoeia and the Pharmacopoea Bohemoslovaca), the LC methods proved to be much more sensitive giving detection limits 0.04% of d-DOPA and 0.3% of d-methyldopa.

A Single-Step Purification of Assembled Insulin on a Resource RPC Column

ABSTRACT We have designed an efficient single step purification process using Resource RPC column in conjunction with triethyl amine/acetonitrile solvent system at similar pH at which the in vitro assembly of insulin is carried out from its two chains. The conditions have been optimized to separate native insulin from its single chain precursors as well as from other by-products. This method obviates the need of acidification, centrifugation, gel filtration, and concentration steps before HPLC purification of insulin from assembly mixture. The system offers wide pH stability, rapid regenerability, excellent pressure/flow characteristics, and high loading capacity. The authenticity and purity of human insulin purified by this procedure was evaluated by analytical reverse phase HPLC, peptide mapping, and isoelectric focusing.

A QUANTITATIVE HPLC DETECTION METHOD FOR WHI-P154 [4-(3′-BROMO-4′-HYDROXYLPHENYL)-AMINO-6,7-DIMETHOXYQUINAZOLINE

WHI-P154 [4-(3′-bromo-4′hydroxylphenyl)-amino-6,7 di-methoxyquinazoline] is a novel anti-tumor agent with unique cytotoxic activity against human glioblastoma cells (Clin. Cancer. Res. 4:1405-1414, 1998). Further development of WHI-P154 will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method for WHI-P154. This method allows the measurement of WHI-P154 levels in plasma, as well as in target human glioblastoma cells. Plasma and cell lysates were extracted with chloroform, dried with nitrogen gas and reconstituted in methanol : water (9:1, v/v). An aliquot was injected into a Hewlett Packard HPLC system employing a 250 × 4mm Lichrospher 100, RP-18 (5 μm) analytical column in conjunction with a 4 × 4 mm Lichrospher 100, RP-18 (5 μm) guard column. The eluted compounds were detected by a diode array detector set at a wavelength of 335 nm. Acetonitrile/water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (28:72, v/v) was used as a mobile phase. The average extraction recovery of WHI-P154 was 78.3% for plasma and 96.0% for U373 human glioblastoma cells. The assay was linear (r>0.999) within the concentration range of 0.1 – 20 μM in 100 μL plasma and within the quantity range of 0.025 – 5 nmol per 2.5 million U373 glioblastoma cells. The intra- and inter-assay variabilities were less than 6% and the lowest detection limit of WHI-P154 was 0.05 μM in plasma and 0.01 nmol in U373 cells, respectively. The practical utility of this new HPLC method was confirmed in pilot pharmacokinetic studies using rats as well as cellular uptake studies using U373 human glioblastoma cells.

Comparison of methods for extraction of flavanones and xanthones from the root bark of the osage orange tree using liquid chromatography

This study compares conventional solid–liquid extraction, supercritical fluid extraction (SFE), and pressurized fluid extraction (PFE) for their efficiency in extracting xanthones and flavanones from the root bark of the osage orange tree ( Maclura pomifera). Seven compounds were extracted from the plant material by solvent extraction at room temperature for 48 h. The same compounds were removed from the root bark by 45- and 35-min extractions using SFE and PFE, respectively, and under optimized conditions, in same or higher yields than those obtained by the conventional 48-h solvent extraction. Although all seven compounds were present in the SFE extracts when only CO 2 was used as the fluid, the addition of 20 vol.% methanol (MeOH) to the CO 2 proved essential for achieving high yields. Use of SFE with CO 2–MeOH also led to the recovery of an additional flavanone from a wet sample of root bark. This flavanone is absent from the conventional solvent extracts and appears in small amounts in the PFE extracts. An optimized LC separation for the analysis of the different extracts is presented, and it is demonstrated that the separation of xanthones and flavanones is considerably improved by the use of deactivated C 18 columns in conjunction with a mobile phase containing acetonitrile and a weak organic acid.

New branched chain tricycloquinazoline derivatives: a room temperature electron deficient discotic system

Tricycloquinazoline (TCQ) based discotic liquid crystals have recently received increasing attention due to their interesting physical and electronic properties. Three new branched chain TCQ derivatives 6a–c were prepared and characterized. One of them, 6c, was found to be mesogenic at room temperature, having a very broad mesophase range. The small angle X-ray scattering of this compound shows a centre to centre distance of 3.29 Å, one of the lowest core–core separations known in discotic liquid crystal systems. This very close packing of the molecules in the column in conjunction with the stability of the mesophase well below and above the ambient temperature makes it very suitable for carrying out physical studies.

Three-fluid retention in porous media involving water, PCE and air

A classical way to obtain three-fluid retention curves in porous media from measured two-fluid retention curves is based on the Leverett concept, which states that the total volumetric liquid content in a water-wet porous medium, containing water, a nonaqueous-phase liquid (NAPL) and air, is a function of the capillary pressure across the interface between the continuous NAPL and air. This functional relationship results from the assumed condition that in a three-fluid porous medium, the intermediate wetting fluid spreads over the water-air interface. Application of Leverett's concept may not be valid, however, for nonspreading NAPLs like perchloroethylene (PCE). This paper discusses measurements of both PCE-air and water-PCE-air retention curves using a long vertical column in conjunction with a dual-energy gamma radiation system. The data indicate that the Leverett concept was applicable only until a critical PCE saturation had been reached.

Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.

Methodology for Bioassay-Directed Fractionation Studies of Air Particulate Material and Other Complex Environmental Matrices

A normal phase HPLC methodology using a semi-preparative polyaminocyano column in conjunction with a selection of short-term genotoxicity assays has been developed for bioassay-directed fractionation studies of complex environmental mixtures. To illustrate the effectiveness of this methodology, an organic extract prepared from respirable air particulate samples collected in Hamilton, Canada was separated into a non-polar aromatic fraction and a polar aromatic fraction using a combination of alumina and Sephadex LH20 chromatography. These fractions were evaluated for their genotoxic potential using the Salmonella/microsome (Ames) assay with six different strains of Salmonella. The non-polar aromatic fraction was analyzed by normal phase HPLC and the eluent was collected in one-minute subfractions ; these subfractions were bioassayed in three different Salmonella strains (YG1021 -S9. YG1024 -S9 and YG1029 +S9) to afford three different mutation profiles of this sample. Some subfractions which exhibited high mutagenic responses were subjected to further chemical analyses using GC/MS in order to identify those compounds responsible for the genotoxic responses. The nitroarene compounds 2-nitrofluoranthene, 1-nitropyrene and 2-nitropyrene and higher molecular weight polycyclic aromatic hydrocarbons such as benzo[a]pyrene and indeno[1,2,3-cd]pyrene were identified and quantified in some of the biologically active subfractions. The normal phase gradient conditions afforded very reproducible retention times for a series of polycyclic aromatic standards with a broad range of compound polarities. In addition, polycyclic aromatic hydrocarbons (PAH) were observed to elute from the normal phase HPLC column in a series of peaks ; successive peaks contained PAH of increasing molecular weight while any individual peak was shown to contain PAH of the same molecular weight.

Analysis of wheat and kidney samples for ochratoxin a using immunoaffinity columns in conjunction with HPLC

Ochratoxin A is a highly nephrotoxic mycotoxin commonly associated with cereals and cereal products. Residues of the toxin are also frequently found in animal tissues, e.g. pig kidney, when animals are fed with contaminated feed. The analysis of foodstuffs for ochratoxin A has been simplified by the introduction of immunoaffinity methods such as the Biocode ochratoxin EASI EXTRACT column. These columns contain monoclonal antibodies raised against ochratoxin A and allow purification and concentration of the toxin from a foodstuff. Analysis involves a solvent extraction stage prior to application to the ochratoxin EASI EXTRACT column, with detection of the toxin by high‐pressure liquid chromatogra‐phy. Several solvent extraction systems have been evaluated for both wheat and kidney. Percentage recoveries from spiked material ranged between approximately 35 and 80% for kidney samples and between approximately 65 and 80% for wheat samples depending on the extraction solvent used. Immunoaffinity analysis for o...

The Use of High-Performance Electrophoresis Chromatography for the Micropurification of Cerebrospinal Fluid Proteins in the Rat

Using reversed-phase high-performance liquid chromatography (HPLC) on a Vydac C8 column in conjunction with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining, we have identified more than 30 proteins in cerebrospinal fluid collected from adult rats by cannulation of cisterna magna. When these partially purified cerebrospinal fluid proteins were further fractionated by high-performance electrophoresis chromatography (HPEC) on an Applied Biosystems 230A HPEC system using a 10% T SDS-polyacrylamide gel with a phosphate base running buffer system under nonreducing conditions, we have purified more than 10 proteins to apparent homogeneity from a pool of 10 ml of rat cerebrospinal fluid as verified by silver staining and direct N-terminal amino acid sequencing. Two additional series of experiments using rat cerebrospinal fluid over a 12-month period yielded virtually identical results. A major advantage of HPEC over conventional HPLC is that the recovery of protein is almost quantitative and is in the range of 90-95% using as little as 1 μg of protein. The purified proteins from HPEC are ready for direct protein sequencing following a buffer exchange to remove residual Tris and phosphate without additional manipulation. The potential use of HPEC for micropurification of proteins was discussed.

Role of a microbore capillary column in the development of a portable gas chromatograph‐mass spectrometer

AbstractFor the development of a portable gas chromatograph‐mass spectrometer (GC‐MS) system, drastic reduction in the carrier gas load is necessary in order to minimize the pumping requirements of the mass spectrometer. Two approaches were investigated for achieving this. The first involved the use of a palladium‐hydrogen interface and was found not to be viable due to its chemical reactivity. The second utilized a short microbore capillary column (3−m × 50−μm i.d.). The small cross‐sectional area of the column reduced the gas load into the MS and yielded sharp chromatographic peaks possessing full‐widths at half‐height in the range of 200–600 ms from a mixture of priority pollutants. Good column efficiency (> 2.3 × 104 theoretical plates/meter for k of 10) and resolution were observed, and analyses were completed within a few minutes. Mass spectral measurements were performed with a miniaturized focal plane mass spectrograph having an integrating array detector. Because this system detects (integrates) ions of all masses simultaneously and continuously, short data acquisition times can be used to measure several spectra per gas chromatographic peak without loss in sensitivity. Such time‐resolved mass spectral measurements were applied to the separation and identification of benzene and carbon tetrachloride which coeluted from the column. Our results show that a short microbore capillary column in conjunction with a miniaturized mass spectrograph and an array detector is eminently suited for the development of a field portable GC‐MS system of high performance.

THE MECHANISM OF THE INDUCTION OF IMMUNOLOGIC UNRESPONSIVENESS TO RAT CARDIAC ALLOGRAFTS BY RECIPIENT PRETREATMENT WITH DONOR LYMPHOCYTE SUBSETS

In continuation of our studies using UV-B-irradiated DST and donor leukocyte (DL) recipient pretreatment to induce specific unresponsiveness to organ allografts, we have examined the relative contributions of splenic lymphocyte populations and T lymphocyte subsets in the induction of immunologic unresponsiveness. Our data show that enriched populations of MHC class II-positive B lymphocytes and the W3/25+ T cell subset obtained from splenic leukocytes using immunoadsorbent columns in conjunction with mAbs led to indefinite graft survival (greater than 100 days) in the Lewis-to-ACI rat cardiac allograft model. In contrast, pretreatment with T lymphocytes or the Ox8+ T subset was relatively ineffective in prolonging cardiac allograft survival. In addition, third-party (W/F) W3/25+ T cell recipient pretreatment did not influence the survival of Lewis cardiac allografts in ACI recipients, thus confirming the specificity of pretreatment with the T cell subset in graft prolongation. Furthermore, we have examined the underlying mechanisms of donor-specific unresponsiveness induced by donor spleen cells, B lymphocytes, and W3/25+ T cells using adoptive transfer assays. Serial adoptive transfer studies demonstrated the presence of 0x8+ suppressor T cells in the spleens of unresponsive recipients bearing well-functioning cardiac allografts and of serum "suppressor factors" that have the capacity for specifically prolonging donor-type test graft survival in naive syngeneic rats. Our findings suggest that the induction of specific unresponsiveness in this model is dependent on a sequential collaboration between the appearance of donor-specific serum factor(s) (humoral phase) and donor-specific suppressor T cells (cellular phase). These results may be potentially useful in planning future strategies for the induction of unresponsiveness to clinical organ allografts by immunologic manipulation of the host with MHC class II-positive B cell and CD4+ T cell clones.

Fluorescence enhancement of PLP for application to HPLC.

In an HPLC analysis of vitamin B6 derivatives, the use of a reversed-phase ODS column in conjunction with a fluorescence detector has given promising results. However, PLP was rather insensitive to the analysis, making it difficult to obtain accurate results, especially in biological materials. Subsequently, a pre-column treatment that involved extracting the sample solution with 1 M perchloric acid, heating the extract for 3 hr at 50°C in the presence of 5mM KCN at pH 7.5, and then allowing it to stand for 24hr at room temperature served to overcome difficulties in the analysis. The fluorescence enhancement of PLP was over 10-fold and produced a single peak that was applicable to the quantification of PLP in biological materials.

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem. J. 122, 727–740) composed of KNO 3, MgCl 2, creatine, and ADP. The enzyme is a dimer composed of 2 M r 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of ϵ 280 nm = 2.22 ± 0.10 ml·mg −1·cm −1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.

Effect of pretreating samples with boric acid before liquid-chromatographic determination of urinary catecholamines.

Isolation of catecholamines on prepacked gravity-fed ion-exchange columns in conjunction with liquid-chromatographic separation is occasionally subject to disturbances of unknown origin, especially after injection of H3BO3 eluates from the ion-exchange columns onto the chromatography column. Peak spreading, sometimes even peak splitting, has been observed in many laboratories--effects easily confused with those sometimes caused by voided columns or by impurities. To elucidate the origins of these interferences, we performed a combined voltammetric and chromatographic investigation. We show that complexation of the catechol group by borate ions and dissociation of those pH-labile complexes in the course of the chromatographic separation is the cause of those peak distortions. Addition of a small volume of diluted HCI to the borate eluates eliminates such interference and leads to reproducible, easily interpretable chromatographic separations.