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Investigations into the preparation of silica hydride intermediate in supercritical carbon dioxide (sc-CO(2)) that avoids the use of organic solvents such as toluene or dioxane are described. The effects of reaction temperature, pressure and time on the surface coverage of the supercritical fluid generated silica hydride intermediate were studied. Under optimised supercritical conditions of 120°C, 483 bar and 3 h reaction time, silica hydride (Si-H) conversion efficiencies of ca. 40% were achieved for the hydride intermediate prepared from a monofunctional silane reagent (dimethylmethoxysilane). Si-H conversion efficiencies (as determined from (29)Si CP-MAS NMR spectral analysis) for the hydride intermediate prepared from triethoxysilane (TES) in sc-CO(2) were found to be comparable to those obtained using a TES silanisation approach in an organic solvent. (13)C and (29)Si CP-MAS-NMR spectroscopy was employed to provide a complete structural assignment of the silica hydride intermediates. Furthermore, supercritical CO(2) was subsequently employed as a reaction medium for the heterogenous hydrosilation of silica hydride with octadecene and with styrene, in the presence of a free radical initiator. These supercritical fluid generated reversed-phase materials were prepared in a substantially reduced reaction time (3 h) compared to organic solvent based methods (100 h reaction time). Silica functionalisation in sc-CO(2) presents an efficient and clean alternative to organic solvent based methods for the preparation of important silica hydride intermediate and silica bonded stationary phases via a hydrosilation approach., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A very simple and fast method for the fabrication of poly(dimethylsiloxane) (PDMS) microfluidic devices is introduced. By using a photocopying machine to make a master on transparency instead of using lithographic equipment and photoresist, the fabrication process is greatly simplified and speeded up, requiring less than 1.5 h from design to device. Through SEM characterization, any micro-channel network with a width greater than 50 microm and a depth in the range of 8-14 microm can be made by this method. After sealing to a Pyrex glass plate with micromachined platinum electrodes, a microfluidic device was made and the device was tested in FIA mode with on-chip conductometric detection without using either high voltage or other pumping methods.

On-chip separation of inorganic anions by ion-exchange chromatography was realized. Micro separation channels were fabricated on a silicon wafer and sealed with a Pyrex cover plate using standard photolithography, wet and dry chemical etching, and anodic bonding techniques. Quaternary ammonium latex particles were employed for the first time to coat the separation channels on-chip. Owing to the narrow depths of the channels on the chip, 0.5-10 microm, there were more interactions of the analytes with the stationary phase on the chip than in a 50-microm I.D. capillary. With off-chip injection (20 nl) and UV detection, NO2-, NO3-, I-, and thiourea were separated using 1 mM KCl as the eluent. The linear ranges for NO2- and NO3- are from 5 to 1000 microM with the detection limits of 0.5 microM.

The fabrication of components for a miniaturised liquid chromatography system on silicon has recently been reported by our research group [J. Cap. Electrophoresis Microchip Technol. 6 (1999) 33; Analyst 125 (2000) 25]. To date, inlet and outlet connection ports, separation micro-channels (20-200 microm in width, 0.5-10 microm in depth, 15-60 cm in length), and an intersection for picolitre injection have been etched on a silicon wafer and then sealed with a Pyrex cover plate on which platinum electrodes for on-chip detection have been patterned. The platinum electrodes have been used for the amperometric detection of phenol, using 20 nl off-chip injection. In this work we present our latest results obtained with on-chip pressure driven picolitre injection, designed to realize the full capabilities of this micro-LC system. The injection volume is dependent on the micro-channel depth, width, and also on the intersection length, allowing injection in the low nanolitre to picolitre range.

A robust procedure has been developed to overcome the instability problems experienced with the fluorescent derivative of eprinomectin. The procedure involves addition of acetic acid, together with the typical reagents methylimidazole and trifluoroacetic anhydride, to produce a fluorescent molecule that can be determined by high performance liquid chromatography (HPLC) with fluorescence detection. Derivatisation is completed in 30 min at 65 degrees C. This derivatisation procedure was shown to be suitable, also, for the related compounds, moxidectin, abamectin, doramectin and ivermectin. A multi-residue method for these compounds in bovine liver has been developed using the derivatisation procedure. Samples are extracted with acetonitrile; followed by clean-up on deactivated alumina and C18 solid phase extraction (SPE) cartridges. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery ranged between 73 and 97%. The intra- and inter-assay variations showed relative standard deviations typically of < 6% and < 14%, respectively. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).

A supercritical fluid extraction procedure has been developed for the extraction of beta-agonists in bovine liver samples. The method is suitable for compounds of different beta-agonist classes: the substituted aniline-type compounds (e.g. clenbuterol) and the phenolic-type compounds (e.g. salbutamol), including conjugated forms of the latter. The developed procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol and salbutamol residues at the low ppb level. Addition of methanol modifier and removal of sample moisture are necessary for the extraction of more polar analytes such as salbutamol. Method validation incorporating intra- and inter-assays was carried out on fortified liver tissue and showed good recovery and low variation (RSD < 15%). An enzyme hydrolysis procedure was incorporated into the method for the deconjugation of conjugated residues. The developed procedure was shown to be successful for the determination of both clenbuterol and salbutamol in incurred liver tissue.

The development of a supercritical fluid extraction procedure for the extraction of clenbuterol from bovine liver tissue is described. The procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol residues at the low ppb level. Method validation incorporating inter- and intra-assays was carried out on fortified liver tissue and showed good recoveries and low variations (RSD < 15%) for levels of clenbuterol of 0.5, 2 and 5 ppb. The developed procedure was shown to be successful for the determination of clenbuterol in incurred liver tissue. The effects of organic modifiers and of inherent sample moisture on analyte extractability are also presented.

Calixarenes are synthetic macrocyclic compounds, described as "molecular baskets" as they possess high ionophoric selectivity and form inclusion complexes with many important ionic guests. In our initial work, hexameric and tetrameric tert-butylcalixarenes, unfunctionalized at the lower rim, are shown to be separable on a diol column using supercritical fluid chromatography with methanol/chloroform-modified CO(2) as mobile phase. The variation in capacity factors for these calixarenes was studied as a function of modifier composition. However, the solubility of these molecular baskets in unmodified supercritical CO(2) is enhanced by fluorination at the upper rim. For example, when p-allylcalix[4]arene is derivatized by a thiol-ene addition reaction with heptadecafluorodecanethiol, CF(3)(CF(2))(7)(CH(2))(2)SH, a solubility of >0.12 mol L(-)(1) in supercritical CO(2) is measured for the p-heptadecafluorodecylthio-n-propylcalix[4]arene at 60 °C and 200 atm. However, subsequent lower rim functionalization to form the tetrahydroxamate derivative, while reducing the solubility, allows supercritical fluid extraction of Fe(III) by the fluorinated calix[4]arene ligands to be studied as a function of temperature and pressure and monitored using UV/visible and atomic absorption spectrometry. In particular, the visible absorption spectra obtained for the extracted Fe(III)-calix[4]arene tetrahydroxamate complex, collected in dimethyl sulfoxide, are indicative of octahedral Fe(III) complexation in a manner similar to that displayed by water-soluble siderophores. Studies on the efficiency and selectivity of Fe(III) extraction are also reported.

A new (-)-menthyl bonded silica phase has been prepared by hydrosilation of a hydride silica intermediate. The hydride silica intermediate was synthesized by the reaction of a monoalkoxysilane (CH(3))(2)SiH(OEt) with silica gel, yielding a relatively high surface coverage (4.4 μmol/m(2)) of SiH groups. This intermediate was then used successfully in the preparation of a monomeric (-)-menthyl bonded silica phase. The bonded phase produced has been used for the chromatographic separation of enantiomers in a reversed phase mode (chiral separations). Solid state (13)C and (29)Si CP-MAS NMR spectroscopy and DRIFT spectroscopy provides valuable information on the structure of the different surface species formed on silica after modification. The surface coverage of the hydride silica intermediate and of the final bonded silica phase produced are also determined. It is found that this modification procedure can exclusively produce a monomeric coverage of SiH groups on the silica surface and can further produce a final monomeric bonded organic silica phase for the separation of enantiomers.

The functionalisation of macrocyclic p-tertbutylcalix[4]arenes at the lower rim with hydroxamic acid and proline hydroxamic acid groups gives the calixarenes chelating properties similar to siderophores. The (1)H NMR spectrum of p-tertbutylcalix[4]arene tetrahydroxamic acid shows a broad band at 10.8 ppm in DMSO-d(6) attributed to NHOH protons. Diffuse reflectance spectral analysis of PVC membranes containing the calixarene hydroxamic acids show absorption bands at 485 nm and 504 nm following contact with aqueous solutions of Fe(III) and V(V) respectively. The pH dependency of this extractive selectivity is examined for Fe(III), Co(II), Pb(II), Mn(II), Cu(II) and Ni(II) using a solid phase extraction approach. Spectrophotometric evidence for the complexation of Cu(II) in methanol by p-tertbutylcalix[4]arene tetraproline acid is also provided.

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12 degrees C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.

Detailed studies are reported on the Ni(II)-binding site of human serum albumin (HSA) and the results are compared with those obtained from the N-terminal native-sequence peptide, l-aspartyl-l-alanyl-l-histidine N-methylamide (Asp-Ala-His-NHMe). Equilibrium dialysis of HSA and Ni(II) in 0.1m-N-ethylmorpholine/HCl buffer, pH 7.53, demonstrates a specific Ni(II)-binding site on the protein. l-Histidine, the low-molecular-weight Ni(II)-binding constituent of human serum, is shown to have a greater affinity for Ni(II) than does HSA. A small but significant amount of ternary complex HSA-Ni(II)-l-histidine is also present in the equilibrium mixture containing the three components. The log (association constant) values for the binary and ternary Ni(II) complexes are 9.57 and 16.23 respectively. The complex equilibria between Asp-Ala-His-NHMe and Ni(II) have been investigated by analytical potentiometry in aqueous solution (0.15m-NaCl, 25 degrees C). Several species, including MA, MA(2), MH(-2)A, and MH(-1)A(2) [where M and A represent Ni(II) ion and anionic peptide respectively], were detected in the system, MH(-2)A being the major complex species. Equilibrium studies involving Asp-Ala-His-NHMe, Ni(II) and l-histidine reveal the presence of a ternary complex MH(-1)AB (where B represents anionic l-histidine) at physiological pH. Detailed studies of visible-absorption spectra of HSA in the presence of Cu(II) and Ni(II) reveal that the two metal ions bind HSA at the same site. The visible-absorption spectrum of Ni(II)-HSA complex shows a highly absorbing peak at 420nm (epsilon(max.) = 137; with shoulder at 450-480nm) characteristic of a square planar or square pyramidal co-ordination arrangement about the metal ion. Similar visible-absorption characteristics were observed for the major species MH(-2)A in the Asp-Ala-His-NHMe-Ni(II) system (lambda(max.) = 420nm; epsilon(max.) = 135; with shoulder at 450-480nm). The combination of experimental results from the protein studies and the peptide analyses provides strong evidence for the structure of the Ni(II)-binding site of HSA as one that involves the alpha-amino nitrogen atom, two deprotonated peptide nitrogen atoms, the imidazole nitrogen atom and the side-chain carboxy group of the aspartic acid residue. On the basis of the results obtained from the individual ternary systems involving protein and peptide, a mechanism for the transportation of Ni(II) in the serum is proposed.

Aqueous solutions of the industrially important chelating agent N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine exhibit basic properties. The proton dissociation constants were determined to be 8.99 +/- 0.04 (pK(1)) and 4.30 +/- 0.04 (pK(2)) by potentiometric titration at 25 degrees in 0.15M sodium chloride.

The nonspecificity of dog serum albumin (DSA) for Ni(II) is mimicked by the simplest tripeptide, glycylglycyl-L-tyrosine-N-methyl amide, which forms a planar complex at high pH. In this study, the 1H and 13C nuclear magnetic resonance (nmr) spectra of the free and complexed peptide are reported. As the pH is increased for the free peptide, the deprotonation of the terminal amino group (pKa = 7.94) is reflected most strongly by the chemical shift changes of the NH2-terminal -CH2CO- unit. Large upfield and downfield shifts for the tyrosine C xi, C epsilon and C gamma carbon resonances occur on the ionization of the phenolic hydroxyl group. The planar Ni(II) complex is in slow exchange on the nmr time scale and is of 1:1 stoichiometry. The greater chemical shift changes on Ni(II) coordination are observed from the protons nearest the peptide and amino nitrogens:amide CH3 (-0.704), Tyr(3) alpha-CH (-0.667), Gly(1) alpha-CH2 (-0.382), and Gly(2) alpha-CH2 (-0.519, -0.487). In the 13C spectrum, the Gly(1) C alpha (+7.58) is most affected. The Ni(II) ion is therefore at the center of four coordinating nitrogens. Changes in the coupling constants for the Tyr(3) -CH-CH2- moiety suggests a mainly gauche conformation with the tyrosyl ring positioned above the plane of coordination and a weak bonding interaction with the Ni(II) ion is indicated. These results provide structural information regarding the reduced affinity of DSA for Ni(II).

New high-performance liquid chromatographic (HPLC) and gas-liquid chromatographic (GLC) methods for the measurement of the antidepressant cianopramine in human plasma are compared for precision, accuracy, selectivity, sensitivity and convenience. Important differences were found with regard to precision, selectivity and sensitivity when ultraviolet, fluorescence and electron-capture detection were used. The mean coefficients of variation for intra-assay variability of cianopramine were 1.0% (HPLC-UV), 1.5% (HPLC-fluorescence) and 5.3% (GLC) over the concentration range studied. The results obtained support the selection of HPLC as the method of choice for the analysis of cianopramine in plasma, based mainly on its merits of sensitivity and convenience, despite the enhanced selectivity of the GLC method.