Sephacryl Gels: Physical Properties and Evaluation of Performance in Gel Filtration

Abstract

Sephacryl is a gel filtration medium composed of allyl dextran and N,N'-methylene bisacrylamide. Sephacryl S-200 SF has a fractionation range similar to that of Sephadex G-150. Sephacryl S-300 SF has a fractionation range more similar to Sephadex G-200 or Sepharose 6B, with an exclusion limit for globular proteins of about 10⁶ daltons. In typical applications, excellent resolution of protein mixtures was obtained at flow rates up to 30 cm/hr. Good correlation was established between elution volumes of proteins and their molecular weights. The gels contain few ionic groups, as determined by titration. Studies using Sephacryl S-200 at different pH values indicate that, above pH 3, protein does not interact with Sephacryl S-200 at moderate ionic strengths. However, below pH 3, protein binds strongly to the gel through hydrophobic interactions and/or hydrogen bonding. Most separations using Sephadex G-150 or Sephadex G-200 could be duplicated using Sephacryl S-200 or S-300 at much higher flow rates.     

Separation of xanthones and a biphenyl fromKielmeyera coriacea by centrifugal partition chromatography

Summary Eight xanthones and a biphenyl have been isolated from the dichloromethane extract ofKielmeyera coriacea (Guttiferae) stem bark by a combination of centrifugal partition chromatography and gel filtration on Sephadex LH-20, with HPLC-UV monitoring of fractions. The structures of the isolated compounds (Figure 1) were established by spectroscopic methods (UV, EI-MS, D/CI-MS,¹H NMR,¹³C NMR).     

New insights into the transitional behaviour of methyl-6-O-(n-dodecanoyl)-α-D-glucopyranoside using variable temperature FTIR spectroscopy and X-ray diffraction

The liquid crystalline behaviour of methyl-6-O-(n-dodecanoyl)-α-D-glucopyranoside, 1, has been characterised using X-ray diffraction and variable temperature Fourier transform infrared (FTIR spectroscopy). 1 exhibits a monotropic interdigitated smectic A phase consisting of bilayers in which the alkyl chains are overlapped. The crystal–isotropic transition is accompanied by a pronounced decrease in the strength of the hydrogen bonding network involving the sugar groups resulting in a marked change in the environment of the alkyl chains. The isotropic phase consists of disordered smectic-like domains stabilised via hydrogen bonding between the sugar groups. At the transition to the smectic A phase, a subtle change in hydrogen bonding is observed which is manifested by a change in the temperature dependence of the OH stretching peak position in the FTIR spectrum. On crystallisation, the strong hydrogen bonding network is re-established accompanied by a change in the conformational distribution of the alkyl chains. A model is proposed in which a combination of hydrogen bonding (enthalpic effects) and conformational arrangements (entropic effects) promotes initially the formation of smectic-like domains in the isotropic phase and subsequently stabilises the smectic A phase by inhibiting the microphase separation leading to the crystal phase.     

Improved High-Pressure Liquid Chromatographic Assay of Serum 25-Hydroxycholecalciferol and 25-Hydroxyergocalciferol After Reverse-Phase Sep-Pak C18 Cartridge Preparation of Sample

Abstract

A simplified method is described for extracting and purifying 25-hydroxycholecalciferol and 25-hydroxyergocalciferol from serum for quantitation by high-pressure liquid chromatography. The method involves extracting and purifying these metabolites from serum (1–10 ml) with a reverse-phase octadecylsilane bonded silica cartridge (Sep-Pak C₁₈). This method is faster than a previously described method involving extraction with dichloromethane and purification by Sephadex LH-20 chromatography. The correlation between the two methods was excellent (r² = 0.96, p≤0.0001). The coefficient of variation for the new method is 4.3%. The new method allows measurement of 25-hydroxyergocalciferol from human serum since both 25-hydroxycholecalciferol and 25-hydroxyergocalciferol are extracted equally. This allows the use of [³H] 25-hydroxycholecalciferol to monitor the recovery of both the D₂ and the D₃ forms of the metabolite.     

Isolation by Means of Preparative Reversed-Phase Liquid Chromatography of Epimeric Alcohols Formed Upon Reduction of Pregnenolone and Progesterone

Abstract

A method is described which permits complete separation on a preparatory scale of the 20R and 20S epimeric alcohols obtained from lithium aluminium hydride and sodium borohydride reduction of pregnenolone and progesterone, respectively. The retention behaviour and resolution obtained on chromatography of the epimers on C-18 bonded phase material and elution with different acetonitrile/water and methanol/water mobile phases were studied. The order of retention is in both cases in accordance with ¹H-NMR chemical shift data which indicate a stable conformation with a more exposed 20-OH group in the 20S (=20α) epimer. Deviations from the elution order expected for true reversed-phase retention mechanisms were found on elution with mobile phase systems of reduced water content.     

Simplified Method for the Isolation and Analysis of Ethynyl Steroids in Urine

Abstract

A simplified procedure for selective isolation of ethynyl steroids from urine is described. Urinary steroids are extracted with Sep-Pak^R C₁₈ and subjected to enzyme hydrolysis. The liberated steroids are extracted with Lipidex 1000 and the eluate from this column is passed through a bed of sulfohydroxypropyl Sephadex LH-20, partly converted into silver form by a wash with aqueous silver nitrate. Ethynyl steroids are eluted with a solution of acetylene in methanol.     

Adsorption Effects Observed in Gel Permeation Chromatography of Thiouracil

Abstract

In gel permeation chromatography (GPC), several compounds deviate from the molecular volume/elution count relationship which is prepared using satured hydrocarbons. In this paper, this problem is investigated in detail using thiouracil in aqueous solution as a model chromatographic adsorbate. The concentration dependences of elution counts and peak heights prove the adsorption of thiouracil on Sephadex G-25 when water is the solvent. Thus to investigate further the mechanisms of adsorption responsable for the chromatographic behaviour, thiouracil-Sephadex interac—tions were investigated by studying equilibrium adsorption. Isotherms of type IV of BDDT classification were found which are typically associated with a weak adsorption such as physisorption, on a porous solid. The effect of water structure perturbants, ionic strength and pH on this adsorption was consistent with the-hypothesis that with water as a solvent both aromatic adsorption and electrostatic interaction are the determinants of the affinity of this gel for a thiouracil compound. This may be particularly useful since results of equilibrium adsorption isotherms are frequently used to develop liquid chromatographic theories.     

Use of Sephadex LH-20 for solid-phase synthesis of oxytocin

The solid-phase synthesis of oxytocin on commercial Sephadex LH-20 is described. The biological activity of the product obtained was 132 AU/mg, and its oxytocin content, according to HPLC, was 28.2%. It was shown that the amount of oxytocin and the biological activity do not depend on the degree of addition of Gly⁹ to the polymer matrix within the range of 0.6–1.6 mmoles of Gly⁹/g of resin; after regeneration, the Sephadex LH-20 can be re-used for solid-phase synthesis of oxytocin.All-Union Scientific-Research Institute of the Technology of Blood Substitutes and Hormone Preparations, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 106–113, January–February, 1989.     

Hydrogen-Bonded Complexes of Fluorophenylacetylenes: To Fluoresce or Not?

The hydrogen-bonded complexes of fluorophenylacetylenesexhibit unusual and interesting fluorescence turn ON/OFF behaviour following excitation to ¹ππ* (S₁) state. The fluorescence switching behaviour can be realized by (i) “change in the intermolecular structure, (ii) change in the position of fluorine substitution and (iii) change in the hydrogen bonding partner or a combination thereof. Experiments indicate that the ≡C−H⋅⋅⋅X (X=O, N) hydrogen bonding with the acetylenic group plays a pivotal role in this switching behaviour. Intriguingly, weaker ≡C−H⋅⋅⋅X hydrogen bonding leads to fluorescence OFF state, which is turned ON by stronger hydrogen bonding. The observed fluorescence this switching behaviour is rationalized on the basis of a phenomenological model which suggests a coupling between the initially excited S₁ state and a dark S_n state in the Franck-Condon region with limited window controlled by the ≡C−H⋅⋅⋅X hydrogen bonding as a crucial parameter. Such fluorescence switching behaviour in hydrogen-bonded complexes is unprecedented and these intriguing results hopefully will stimulate theoreticians to test ′state of the art′ theories to explain these observations in a consistent manner.     

Fehlerm?glichkeiten bei der quantitativen Analyse polycyclischer aromatischer Kohlenwasserstoffe aus Luftstaubextrakten mit der Capillar-Gas-Chromatographie

Zusammenfassung Ein vielfach angewandtes capillargas-chromatographisches Verfahren zur Bestimmung von polycyclischen aromatischen Kohlenwasserstoffen (PAH) in der Atmosphäre wurde auf seine Zuverlässigkeit bei der Gewinnung von quantitativen Analysendaten hin geprüft. Eine PAH-Fraktion, die in unserem Labor routinemäßig durch Extraktion von atmosphärischem Schwebstaub mit Methanol, Flüssig-Flüssig-Verteilung (Cyclohexan/Methanol-Wasser) und zwei säulenchromatographischen Trennungen (SiO₂/Cyclohexan und Sephadex LH-20/Propanol-2) gewonnen wird, wurde bei dem letzten chromatographischen Schritt (Sephadex LH-20/Propanol-2) weiter fraktioniert. Durch Untersuchung der so erhaltenen Fraktionen mit Hilfe der Capillar-Gas-Chromatographie und der GC-MS-Kopplung wurden für 14 ausgewählte PAH Überlagerungen durch Störsubstanzen qualitativ und quantitativ ermittelt. Dabei zeigte sich, daß ein Teil der Fehler vermieden werden kann, wenn die PAH mit zwei und drei Ringen exakt von den interessierenden PAH mit vier und mehr Ringen abgetrennt werden. Für einige PAH sind die quantitativen Ergebnisse jedoch auch bei richtiger Schnittpunktwahl mit Vorsicht zu bewerten; zu diesen Verbindungen gehört auch das oftmals als Maß für die cancerogene Potenz einer PAH-Fraktion angesehene Benzo(a)pyren.

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Pitfalls of quantitative analysis of polycyclic aromatic hydrocarbons in airborne particulate matter by capillary gas chromatography

Summary Glass capillary gas chromatography was checked with regard to the authenticity of quantitative results for the analysis of polycyclic aromatic hydrocarbons (PAHs) in airborne particulate matter. A PAH fraction, which was routinely separated from particulate matter by solvent extraction (methanol), liquid-liquid partition (cyclohexane/methanol-water) and two steps of column chromatography (SiO₂/cyclohexane and Sephadex LH-20/propanol-2), was further fractionated with the above mentioned Chromatographic system Sephadex LH-20/propanol-2. By analyzing the fractions obtained so far with glass capillary chromatography and g.c.-m.s. coupling, the interfering substances were determined qualitatively and quantitatively concerning 14 selected PAHs. It was shown that some errors in determining PAH values may be avoided, if PAHs with less than four rings are exactly separated from those with four and more rings in the last Chromatographic step; but quantitative data for some PAHs may be biased even in case of a well separated PAH fraction. One of these PAHs is the well known benzo(a)pyrene, which has often been considered as an indicator for the carcinogenic potency of the PAHs in general.

     

Analysis and behaviour of organophosphorus pesticides in a rice crop field

Summary A comprehensive analytical protocol for the determination of organophosphorus pesticides in the biotic and abiotic compartments of a rice crop field (Ebro delta, Spain) is described. This include two alternative clean-up procedures (Florisil column chromatography and Bio Beads SX-3 gel permeation chromatography) and gas chromatographic analysis with nitrogen-phosphorus and mass spectrometric detection. Among the pesticides identified, Fenitrothion was the most abundant with residue levels in sediment samples varying from 1–3 g/g in the first day of application to 0.5 g/g two days after, whereas in the water canals the concentrations were of 3–10 g/l, after aircraft application, with a rapid decrease several hours later. Half lives of Fenitrothion in water and sediment samples under natural conditions have been estimated to be lower than 1 day and between 1–1.5 days, respectively, with volatilization and microbial degradation accounting for this decay. The bivalves analysed in the coastal bays (Tapes semidecussatus, Ostrea edulis and Mytilus galloprovincialis) exhibited residue levels between 20–90 ng/g of organophosphorus pesticides, Fenitrothion being the most abundant organophosphorus pesticide. These levels are likely in the safe range for bivalves but not for other organisms such as crustacea, which are sensitive to water concentrations of 0.1 g/l.     

Separation and Determination of 2,5-Dimethoxy-4-Methylamphetamine Enantiomers in Plasma by High-Performance Liquid Chromatography

Abstract

A method for quantitation of 2,5-dimethoxy-4-methylamphetamine (DOM) enantiomers in plasma by high-performance liquid chromatography (HPLC) is described, d- and l-DOM were readily converted to the amides by condensation with a newly developed chiral reagent, succinimidyl ester of l-α-methoxy-α-methyl-1-naphthaleneacetic acid. The yielded diastereomers were separated on the μPorasil column with cyclohexane/ethyl acetate (3:1) exhibiting satisfactory k' and R values. The clean-up procedure by use of Sep-pak C₁₈ and carboxymethyl Sephadex LH-20 (CM-LH-20) proved to be effective for determination of the drug in biological fluids by HPLC. The plasma levels of d- and l-DOM after administration of the racemate to the rabbit were determined by the method thus established.     

Gel Permeation Chromatography of Insulin

Abstract

Aquas gel permeation chromatography of insulin under denaturating conditions has been successfully performed on three different chromatographic supports. The separation pattern was identical to that obtained on soft gels (Sephadex, BioGel). The elution time was 10-20 min, recovery 98-100%.     

High performance liquid chromatography of aromatic carboxylic acids on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase

A new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) was used for the separation of some aromatic carboxylic acids by HPLC. The chromatographic behaviour of the solutes on CABS was studied in comparison with conventional ODS. The results show that the calix[8]arene-bonded phase exhibits a stronger retention and better selectivity than ODS for the aromatic carboxylic acids. The different elution order of the analytes was also observed on both packings, which show the existence of distinct retention mechanisms. According to chromatographic data, it can be concluded that the high selectivity of CABS for aromatic carboxylic acids ascribes to various interactions between CABS and the analytes, such as hydrophobic interaction, hydrogen bonding interaction, π-π interaction and inclusion interaction.     

Liquid crystalline behaviour of benzoic acid derivatives containing alkoxyazobenzene

Liquid crystal trimers based on the hydrogen bonding dimerization of 4‐{n‐[4‐(4‐m‐alkoxy‐phenylazo)phenoxy]alkoxy}benzoic acid (BAm‐n) have been synthesized and characterized. Temperature‐dependent FTIR spectroscopic studies showed that the carboxylic acid groups in BAm‐n are associated to form H‐bonded cyclic dimers both in their crystalline and liquid crystalline phases. The trimers exhibited enantiotropic liquid crystalline behaviour except for BA1‐3 which showed monotropic behaviour, and the mesophases changed from nematic to smectic phase, with the increase of length of the spacer and the terminal substituents. Pronounced odd–even effects in the melting temperatures, clearing temperatures and nematic–isotropic enthalpy changes were observed.     

Alkyl Ammonium Ion Selectivity of Hexahomotrioxacalix[3]arene Triamide Derivative having the Intramolecular Hydrogen-Bonding Group

The lower rim functionalized hexahomotrioxacalix[3]arene triamide 4 with cone-conformation was synthesized from triol 1 by a stepwise reaction. The different extractability for alkali metal ions, transition metal ions, and alkyl ammonium ions from water into dichloromethane is discussed. Due to the strong intramolecular hydrogen bonding between the neighboring NH and CO groups in triamide 4, its affinity to metal cations was weakened. Triamide 4 shows a single selectivity to n-BuNH₃⁺. The anion complexation of triamide 4 was also studied by ¹H NMR titration experiments. Triamide 4 binds halides through the intermolecular hydrogen bonding among the NH hydrogens of amide in a 1:1 fashion in CDCl₃. The association constants calculated from these changes in chemical shifts of the amide protons are K_a = 223 M⁻¹ for Cl⁻ and K_a = 71.7 M⁻¹ for Br⁻. Triamide 4 shows a preference for Cl⁻ complexation than Br⁻ complexation.     

Techniques for resveratrol silylation

Summary Resveratrol, a wine stilbene phytoalexin with some pharmacological properties, was extracted from red wines by MeOH elution of a Sephadex LH-20 column, used for its purification. The column extract was dried and silylated by different methods to optimize resveratrol derivatization. The resveratrol analysis was by gas chromatography and gas cromatography-mass spectrometry allowing determination of its two isomers.     

New insights into the role of hydrogen bonding on the liquid crystal behaviour of 4-alkoxybenzoic acids: a detailed IR spectroscopy study

ABSTRACT

Hydrogen bonding is an efficient alternative to covalent bonding as a way to stabilise liquid crystallinity, by yielding symmetric and non-symmetric complexes with increased molecular anisotropy. In designing new hydrogen-bonded liquid crystals, HBLCs, it is crucial to account for the competing hydrogen bonds that can lead to different supramolecular species coexisting in a temperature-dependent equilibrium. Thus, as part of a systematic development of this area, in the present work we study with detail the relationships between the phase behaviour and hydrogen bonding in a series of 4-n-alkoxybenzoic acids, nOBAs, which are widely used as components in HBLCs. Five acids with alkyl chain lengths of n = 1, 4, 5, 7 and 8 have been investigated using Fourier transform infrared spectroscopy, FTIR, in a broad range of temperatures under two different experimental configurations: sandwiched between potassium bromide, KBr, windows and dispersed in KBr discs. The nematic phase is correlated with the amounts of closed dimers between acid molecules, through the formation of strong hydrogen bonds. Moreover, high concentrations of open dimers are found in samples sandwiched between KBr slides, which are linked to the appearance of smectic-like aggregates that perturb the local order of the nematic phase. The results are interpreted in terms of the ability of the 4-alkoxybenzoic acids to align due to surface interactions, which are less acute in samples dispersed in the discs. These effects must be taken into account in order to correctly interpret the information about the supramolecular species present in the samples, and thus to better understand the relationships between hydrogen bond strength and mesomorphism in HBLCs.     

A new anthraquinone and eight constituents from Hedyotis caudatifolia Merr. et Metcalf: isolation,purification and structural identification

Hedyotis caudatifolia Merr. et Metcalf. (HC), a folk medicine in Yao nationalities areas in China, was used to investigate the chemical constituents. Through silica gel and Sephadex LH-20 column chromatography, nine compounds were isolated and purified. By physical and chemical properties, IR, MS (EI-MS, high resolution EI-MS), 1D NMR (¹H NMR, ¹³C NMR) and 2D NMR (HSQC, ¹H–¹H COSY, HMBC), their structures were identified as β-sitosterol (1), stigmasterol (2), scopolin (3), 2-hydroxy-1,7,8-trimethoxyanthracene-9,10-dione (4), oleanolic acid (5), ursolic acid (6), methyl barbinervate (7), β-daucosterol (8) and p-Hydroxybenzoic acid (9). These compounds were isolated from HC for the first time, and 4 a new anthraquinone whose biological activities are worth to be investigated in future. These compounds may contribute to the HC’s pharmacological effects on treating diseases, and may be used as candidates for control index in establishing the quality control standard of HC.     

Separation and Configuration of Syn and Anti Isomers of Testosterone Oxime

Abstract

The syn and anti isomers of testosterone oxime, prepared with hydroxylamine hydrochloride in pyridine were separated on sulfoethyl Sephadex LH-20 in methanol. The predominant isomer had the highest mobility on the cation exchanger and the longest retention time when analyzed as the trimethylsilyl ether derivative on a glass capillary column coated with SE-30. Proton NMR spectra showed that the major isomer had a 4-anti and the minor isomer a 4-syn configuration.