Adsorption HPLC Research Articles

HPLC Characterization of cis and trans Mixtures of Double-Decker Shaped Silsesquioxanes

HPLC was used as the quantitative analysis technique in determining the molar ratio of cis and trans isomers in the double-decker shaped silsesquioxanes (DDSQ). Different experiments were performed to analyze the effects in the retention times of the polarity of moieties bonded to the DDSQ, as well as other possible adsorption driving forces. As expected, the use of adsorption HPLC was successful in separating cis and trans DDSQ isomers with the resolution of elution better than 1.5. Interestingly, the molecular size of moiety attached to the DDSQ resulted in significant reduction of the retention time suggesting the sterics constraint plays a critical role in the separation of these cage-like structures along with the strength of hydrogen bonding. In partition HPLC using Si bonded with CN groups as a normal phase resulted in a partial separation for one of the selected systems, which indicates the extent of polarity plays a secondary role in the separation mechanism.

Entropy characteristics of adsorption of benzene derivatives from aqueous-methanol solutions on the graphite-like adsorbent

Thermodynamics of adsorption of benzene and its various functional derivatives from aqueous-methanol solutions of different compositions was investigated by equilibrium adsorption HPLC on columns with porous graphitized carbon Hypercarb. A method was proposed for the determination of the molar standard entropy of the adsorptive in the state of a infinitely dilute solution in the eluent. The method makes use of the data of molecular statistical calculations of the equilibrium parameters of adsorption on the basal plane of graphite. Good agreement between values of entropy of the adsorptive in the eluent and entropy of pure liquid compounds is shown. It is concluded that solvation effects appear upon the interaction of molecules of the adsorptives and components of a binary solution.

Growth inhibition of Microcystis aeruginosa by a Glycolipid-type compound from Bacillus subtilis C1

We attempted to identify the compound responsible for the growth inhibition of Microcystis aeruginosa occurring when a culture broth of Bacillus subtilis C1 was added to the medium. The active compound was purified from B. subtilis C1 culture broth by adsorption chromatography and HPLC, and was identified as a type of glycolipid based on 1H NMR and MS analyses. The purified active compound completely inhibited the growth of M. aeruginosa at a concentration of 10 microgram/ml. This is the first report of a glycolipid produced by a Bacillus strain that has potential as an agent for the selective control of bloom-forming M. aeruginosa.

Isolation of adenosine, iso-sinensetin and dimethylguanosine with antioxidant and HIV-1 protease inhibiting activities from fruiting bodies of Cordyceps militaris

According to previous studies, a close relationship between oxidative stress and AIDS suggests that antioxidants might play an important role in the treatment of AIDS. Cordyceps militaris was selected from nine edible mushrooms by assay of inhibition of erythrocyte hemolysis. Macroporous adsorption resin and HPLC were used to purify three micromolecular compounds named L3a, L3b and L3c. L3a was identified to be adenosine with the molecular formula C(10)H(13)N(5)O(4); L3b was 6,7,2',4',5'-pentamethoxyflavone with the molecular formula C(20)H(20)O(7), and L3c was dimethylguanosine with the molecular formula C(12)H(17)N(5)O(5). The compound 6,7,2',4',5'-pentamethoxyflavone was first isolated from C. militaris. The assay of inhibition of HIV-1 protease (HIV-1 PR) was based on the fact that the expression of this enzyme can inhibit the growth of E. coli. This is a new screening system for HIV-1 PR inhibitors. Both L3a and L3b showed high inhibition to HIV-1 PR. These compounds could be new anti-HIV-1 PR drugs.

Mechanism of herbicidal action of the component I from Pythium aphanidermatum

The herbicidal mechanism of the components extracted from Pythium aphanidermatum was examined in this study. Component I was isolated using the HPD500 macroporous adsorption resin and HPLC. Its impact on seed germination and plant growth of weeds was determined and the contents of MDA, superoxide anion radical, and the activities of hills and roots were examined. The root length of weed plants was inhibited under illumination while the stem height was inhibited evidently under darkness. The relative electric conductivity of Digitaria sanguinalis and Amaranthus retroflexus under illumination was 94.55 μS·cm−1 and 58.75 μS·cm−1, respectively, whereas that under darkness was 85.25 μS·cm−1 and 36.25 μS·cm−1, respectively. The MDA contents of Digitaria sanguinalis and Chlorella pyrenoidosa were 0.08385 μmol·L−1 and 0.1742 μmol·L−1 under illumination, respectively, while those were 0.0129 μmol·L−1 and 0.01935 μmol·L−1 under darkness, respectively. Simultaneously, superoxide anion radical content was higher under illumination than under darkness. These results showed the photosynthesis was affected by component I extracted from Pythium aphanidermatum.

I. Adsorption mechanism of chlorophenols on iron oxides, titanium oxide and aluminum oxide as detected by infrared spectroscopy

The adsorption of 2-chlorophenol, 2,3- and 2,4-dichlorophenols and 2,4,6-trichlorophenol in liquid and gas phase on iron, titanium and aluminum oxides seem to proceed in a similar way. Higher adsorption of chlorophenols either from gas phase or from aqueous solution was observed on α-Fe2O3 than on α-FeOOH. The low adsorption of chlorophenols from aqueous solution on oxide surfaces suggests that hydrophobic chlorophenols cannot effectively compete with water for the absorption on hydrophilic oxide surface sites. The adsorption of chlorophenols on iron, titanium and aluminum oxides was followed by the adsorption isotherm, HPLC and diffuse reflectance FT-IR (DRIFT) spectroscopy. The adsorption of the chlorophenols on the oxides under study is related to the amount of interfacial water content on the iron oxide. The alumina–chlorophenolate surface complex was found to be weak when compared with either the iron or titanium analogs as seen by the CO stretching vibrations, leading to a lower adsorption on alumina than on iron and titanium oxides.

Study of the behaviour of azinphos-methyl in a clay mineral by batch and column leaching

Research efforts dealing with the processes affecting the transport of pesticides in soils are needed in order to prevent further damage of surface and groundwater reserves. Although organic matter has been recognised as the most important contributor to the adsorption of non-ionic organic pesticides in soils, in some cases clay minerals may have an important role in the retention of these compounds. The present study was designed to improve the knowledge of the behaviour of azinphos-methyl in soils. Coefficients from adsorption isotherms and HPLC analysis of soil column leachates were used in this work for predicting pesticide mobility in soils. The studied clay mineral was a Spanish bentonite with a predominant montmorillonite fraction. The results showed that azinphos-methyl was adsorbed on the clay mineral and demonstrated the catalytic effect of bentonite on the hydrolysis of the pesticide.

Purification and characterisation of haemolymph 3-dehydroecdysone 3 beta-reductase in relation to ecdysteroid biosynthesis in the cotton leafworm Spodoptera littoralis.

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (approximately 82%), with lesser amounts of ecdysone (approximately 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable. 3-Dehydroecdysone 3 beta-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and Km values for NADPH and NADH were determined to be 0.94 microM, and 22.8 microM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 microM). The results suggest that the 3-dehydroecdysone 3 beta-reductase has a high-affinity (low Km) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site. The 3 beta-reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.

High-performance liquid chromatographic analysis of polydisperse ethoxylated non-ionic surfactants in aqueous samples

An adsorption HPLC method using traditionally reversed-phase solvents and a hybrid column/precolumn has been developed for the quantitative separation and analysis of ethoxylated non-ionic surfactants on the basis of the number of ethoxylate (EO) groups per molecule. This method is demonstrated to separate ethoxylated homologues of broadly distributed linear alcohol ethoxylates and alkylphenol ethoxylates, with numbers of EO groups ranging from 3 to 50 or greater. This method presents a significant advance in non-ionic surfactant analysis because it permits direct injection of aqueous samples, eliminating the need for extensive sample preparation. The method discussed here is optimized for use with an evaporative light scattering detector (ELSD), but works equally well with UV absorbance or fluorescence detectors for the analysis of surfactants with chromophores. ELSD and UV detector operating conditions and calibration methods are discussed.

Structure—Function Relationship among Quillaja Saponins Serving as Excipients for Nasal and Ocular Delivery of Insulin

AbstractThe purpose of this investigation was to explore the structure–function relationship among naturally occurring Quillaja saponins and derivatives for their ability to stimulate insulin delivery from nosedrops and eyedrops and to test the hypothesis that stimulation of peptide drug delivery was correlated with surfactant strength. Native saponins, including QS-21, were purified from an aqueous extract of Quillaja saponaria bark by adsorption chromatography and HPLC. Native saponins were then deacylated by mild alkaline hydrolysis to form DS-1 and DS-2, derivatives that are smaller and more hydrophilic than their parent compounds. DS-1 was further treated either to reduce an aldehyde residue to form DS-1(R) or to remove the fucose-containing oligosaccharide to form QH-957. Rats receiving eyedrops or nosedrops formulated with insulin, but without any Quillaja saponins, showed no hypoglycemic response. Rats receiving eyedrops or nosedrops formulated with insulin plus saponins showed a dose-dependent hypoglycemic response, with the following rank order: QS-21>DS-1>DS-1(R)>DS-2>QH-957. Surfactant strength was determined by measurement of the critical micellar concentration (cmc) and hemolysis of sheep erythrocytes. The cmc was lowest for the parent saponins QS-21 and QS-18, and increased for the deacylated saponin derivatives DS-1, DS-2, and QH-957; hemolysis of sheep erythrocytes was observed at low concentrations (∼0.006mM) of the parent saponins, QS-21 and QS-18, at intermediate concentrations (0.06–0.08mM) of DS-1 and DS-2, and at higher concentrations of DS-1(R) (0.45mM) and QH-957 (1.5mM). Hence, efficacy as an absorption-enhancing agent was greatest in those saponins with the lowest hemolytic titers and cmc values. However, this relationship was not a strict one, because DS-1, which differs from DS-2 only in the absence of one glucose residue, was significantly more potent than DS-2 in stimulating the absorption of insulin. DS-1 and DS-2 share a similar cmc and hemolytic titer, so this difference in efficacy must be due to some specificity beyond simple surfactant strength. Furthermore, DS-1 does not trigger an immune response when administered to animals, whereas QS-21 is a strong immune system activator. Therefore, DS-1 has emerged as an interesting candidate for inclusion in an eyedrop or nosedrop formulation.

1206 The role of neurotrophin for the neurite extension and cell migration of cerebellar granule cells in vitro

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Microcalorimetric characterization of the anion-exchange adsorption of recombinant cytochrome b 5 and its surface-charge mutants

The adsorption of recombinant soluble tryptic fragment of rat cytochrome b 5 on the strong anion exchanger Mono Q was studied using isothermal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed protein show increasingly endothermic (unfavorable) enthalpies of binding with increasing surface coverage, confirming the heterogeneous nature of binding. The enthalpy of adsorption declines toward zero at higher loadings. At low surface coverage, enthalpies increase linearly with temperature, giving rise to a positive value of ΔC p. Enthalpies of adsorption depend strongly on the history of the adsorbent. DSC is used to show that cytochrome b 5 is stable in both free and adsorbed states at all temperatures used in the titration calorimetric experiments. Site-directed mutants of recombinant cytochrome b 5 carrying single charge-neutralaizing substitutions are used to test the contributions of particular residues to the thermodynamics of adsorption. Like those derived from van 't Hoff analysis of equilibrium adsorption isotherms and HPLC retention data, calorimetric enthalpies of adsorption are positive, confirming the dominant role of entropic effects in ion-exchange adsorption in this system.

Production and characterization of a new specific antiserum against the taurine putative biosynthetic enzyme cysteine sulfinate decarboxylase.

We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were immunostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.

Solubilization and characterization of dehydrodolichyl diphosphate synthase from the yeast Saccharomyces carlsbergensis.

When the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-beta-(D)glucopyranoside at the concentration of 0.25-0.5% (10-15 mM) was used to solubilize the prenyltransferase. Both the membrane-bound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.

Structure of the asn-linked oligosaccharides of apolipophorin III from the insect Locusta migratoria. Carbohydrate-linked 2-aminoethylphosphonate as a constituent of a glycoprotein.

The primary structures of the N-linked carbohydrate chains of apolipophorin III from the insect Locusta migratoria have been determined. The glycoprotein was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100. Purification of the carbohydrate chains was achieved by a combination of FPLC anion-exchange chromatography on Mono-Q and amine adsorption HPLC on Lichrosorb-NH2. The structures of the carbohydrate chains were deduced with a combination of fast atom bombardment mass spectrometry, 1H- and 31P-NMR spectroscopy, and methylation analysis. The majority of the carbohydrate chains contains 2-aminoethylphosphonate (AEP), which is linked to the 6-position of Man and/or GlcNAc. L. migratoria apolipophorin III is the first example of a glycoprotein containing carbohydrate-linked 2-aminoethylphosphonate. The structures of the major oligosaccharides were established to be the following: [formula: see text]

Influence of Temperature on Separation of Metal Chelates by Adsorption HPLC

Effet de la temperature de la colonne sur les principaux parametres chromatographiques (k', H, R s ) des diethyldithiocarbamates de Ni, Co et Cr, des oxinates de Mo, Co et Cr et des thionates de Ni, V et Cr

The Fate of [3H]Ecdysone in Three Species of Annelids

Summary The metabolism of [3H]ecdysone was examined in 3 species of annelids: the bloodworm, Tubifex vulgaris (a freshwater oligochaete), the earthworm, Lumbricus terrestris (a terrestrial oligochaete) and the ragworm, Nereis divtrsicolor (a marine polychaete). One of these species, N. diversicolor, metabolised injected [3H]ecdysone into compounds which co-chromatographed on both reversed-phase and adsorption HPLC with authentic 20-hydroxyecdysone, 26-hydroxyecdysone and 20,26-dihydroxyecdysone, thus demonstrating the occurrence of 20-hydroxylation and 26-hydroxylation capability in the Annelida. Furthermore, [3H]ecdysonoic acid was also formed and excreted by N. diversicolor, suggesting that 26-oic acid formation is involved in ecdysteroid inactivation in this species. Other, as yet unidentified, radioactive metabolites were also excreted by N. diversicolor. Several metabolites of [3H]ecdysone were also detected in the other 2 species examined, T. vulgaris and L. terrestris.

Lectin affinity high-performance liquid chromatography: Interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH 4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Analysis of mutagens from cooked foods by directly combined liquid chromatography-mass spectrometry.

Directly combined high performance liquid chromatography-mass spectrometry (LC/MS) has been studied as a method of analysis of heterocyclic aromatic mutagens in cooked foods, in the parts per billion concentration range. Identification and semiquantitative estimation of mutagens is based on accurate measurement of chromatographic retention (k') and molecular weight-selective detection of mutagens, which are protonated during passage of the chromatographic eluant into a thermospray interface of a quadrupole mass spectrometer. Standard chromatographic retention (k') values in two reversed-phase systems and data from thermospray mass spectra from nine mutagens are reported. An isolation scheme employing CH3OH extraction, acid-base partition, cellulose-trisulfo-Cu-phthalocyanine adsorption, and normal-phase HPLC was used prior to LC/MS analysis. Initial applications have been demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon flesh. Levels measured were estimated to be in the range 0.2 to 0.4 microgram/kg IQ and 0.4 to 0.9 microgram/kg MeIQ. The method is judged to be generally applicable with minimal sample prefractionation to detection of mutagens at the parts per billion level in cooked foods.

Adsorption HPLC Investigation of Nitrogen-Bridged Compounds

Abstract Twenty two of tricyclic nitrogen bridged derivatives were examined by HPLC method in Zorbax SIL/chloroform–methanol system. Relationship between chemical structure (ring size, ring sequence, position of methyl substitution) and chromatographic behaviour is discussed.