High Solvent Concentrations Research Articles

Recent developments in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives.

Although electrochromatography in packed beds or monolithic columns has gained enormous interest, techniques based on charged pseudostationary phases like micelles are of high practical importance in electrically driven separation science. However, nonmicellar alternatives, e.g., using charged soluble polymers or smaller additives are still attractive, as they allow high concentrations of organic solvents, and their application is not limited by the critical micellar concentration. This review discusses the developments in the field of electrokinetic chromatography with these additives in the last three years, covering ionic polymeric pseudostationary phases, dendrimers and so-called micelle polymers, but also small molecules which implement separation selectivity due to their specific interaction with the analytes.

Procerain, a Stable Cysteine Protease from the Latex of Calotropis procera.

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.

Procerain, a stable cysteine protease from the latex of Calotropis procera

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0–9.0 and temperature 55–60 °C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala- p-nitroanilide but not l-Ala-Ala- p-nitroanilide, l-Ala p-nitroanilide and N-d-Benzoyl- dl-Arg- p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (ε 1% 280nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0–12.0 and temperatures up to 70 °C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.

Lyotropic Behavior of Diruthenium(II,III) Alkoxybenzoates in Dodecane

The lyotropic behavior of three mixed valence diruthenium(II,III) alkoxybenzoates in dodecane mixtures has been analyzed. The studied compounds, namely, Ru2(3,4,5-B3OC14)4Cl [1] (3,4,5-B3OC14 = 3,4,5-tri(n-tetradecyloxy)benzoate), Ru2(3,4-B2OC14)4Cl [2] (3,4-B2OC14 = 3,4-di(n-tetradecyloxy)benzoate), and Ru2(3,5-B2OC14)4Cl [3] (3,5-B2OC14 = 3,5-di(n-tetradecyloxy)benzoate), were investigated at three different temperatures (40, 70, and 100 °C) for variable solvent concentrations (between 0 and 70 wt %). Mixing the complexes with up to 40−60 wt % of dodecane did not alter the nature of the low-temperature mesophase exhibited by the pure compound still showing a columnar hexagonal phase. However, it resulted in a substantial decrease of the viscosity for all of them and a depression of the melting point for [2]. For solvent concentrations greater than 40−60 wt % and high temperatures, the mixtures exhibited also a nematic columnar mesophase prior to the transition to the isotropic solution. At a structural level, the incorporation of solvent resulted in the swelling of the columns, leading to an increase of the intercolumnar distance up to a point where the columns lost their hexagonal order to give the columnar nematic phase. At higher concentrations of solvent, the mesophase dissolved into the solvent.

Non‐Fickian nonisothermal model for drying of polymer coatings

AbstractAs a sequel to our study on experimental measurements of trapping skinning in a poly(methyl methacrylate)/acetone coating, a mathematical model of non‐Fickian diffusion that predicts trapping skinning is reported here including solvent transport due to a stress gradient that develops because of coating shrinkage. The predicted solvent concentration profile shows a sigmoidal shape and a front that moves into the coating. In the nearly constant rate period, the stress relaxes near the coating surface due to short relaxation times at high coating temperature. The stress relaxation causes negative non‐Fickian flux. At higher gas flow rates, the negative non‐Fickian flux causes the development of a thinner layer of low solvent concentration near the coating surface and a thicker layer of high solvent concentration inside the coating. Thus, more solvent remains in the coating dried at higher flows and trapping skinning occurs.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 μg l −1.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction.

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 micro g l(-1).

Arylsulfotransferase from Clostridium innocuum—A new enzyme catalyst for sulfation of phenol‐containing compounds

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.

Phase behavior of poly( N-isopropylacrylamide) in binary aqueous solutions

In this work, the phase behavior of linear poly( N-isopropylacrylamide) (PNIPA) in water–solvent mixtures was investigated. Several solvents, including low molecular weight alcohols, were selected and phase separation temperatures were determined through cloud point measurements. All the studied systems exhibited the cononsolvency effect, i.e. lower PNIPA compatibility within definite ranges of composition in water-rich mixtures. However, it was first detected that the coexistence of phase separation temperatures—a lower critical solution temperature (LCST) with an upper critical solution temperature (UCST)—at higher solvent concentrations in most systems, depend on the hydrophobic nature of the solvent. The change from a LCST to a UCST was correlated with the competition between polymer–water and polymer–solvent interactions mediated by compositional factors. The effects produced by the different solvents tested were qualitatively compared, considering aspects related to their particular molecular structures, such as the potential to form hydrogen bonds and the implications of the size and shape of non-polar groups for hydrophobic hydration.

Modified enzymes for reactions in organic solvents.

Recent studies on biocatalysis in water-organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a(w)) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene.

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production.

Rapid evolution of novel traits in microorganisms.

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

Improved Network Development in Bidisperse AB/ABA Block Copolymer Gels

Blending a B-selective solvent and an ordered ABA triblock copolymer can, at sufficiently high solvent concentrations, produce a physical gel composed of a network of B midblocks stabilized by A-rich micelles. We demonstrate here that addition of short AB diblock copolymer molecules to such a gel at constant copolymer composition can enhance the solid-like character of the gel, as indicated by an increase in the magnitude of the dynamic elastic modulus. Similarly, addition of AB molecules to a nongelled ABA/solvent system can induce gelation. In light of recent theoretical predictions and related experimental evidence, these results are consistent with a scenario in which tail-induced volume exclusion (due to the presence of AB molecules) within the micellar coronas improves network development through an increase in the population of bridged B midblocks.

Structural change and catalytic activity of horseradish peroxidase in oxidative polymerization of phenol.

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.

Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32.

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanase activity during the late trophophase. The low molecular mass (35 kDa) endoglucanase purified from cultured broths works optimally at 37 degrees C and pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures of 50 degrees C or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergents, by EDTA, and by the divalent cations Hg(2+) and Ag(2+). It is also inhibited to some extent by 10 mM Zn(2+), Fe(2+), and Mg(2+), but it is stimulated by Mn(2+). Enzyme activity is not affected by reducing agents. In the presence of low concentrations of water miscible organic solvents (20%) endoglucanase activity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activity is not affected by the water immiscible solvent ethyl acetate. Carboxymethylcellulose is the preferred substrate (K(m(app)) = 8.3 g/L; V(max(app)) = 1.1 microM/min). Hydrolysis of crystalline cellulosic substrates is very limited, but it is greatly enhanced by phosphoric acid swelling. The purified enzyme shows no activity toward disaccharides or aryl-glucosides. Its activity is inhibited by cellobiose.

Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus.

An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8. 5 and 60 degrees C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA approximately 3'AMP >> RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5'dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5'dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5'dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3' or the 5' end, indicates that C-linkages are resistant to cleavage by nuclease Bh1.

Determination of Carbaryl in Vegetables Using an Immunosensor Working in Organic Media

Sensitive and rapid analysis of carbaryl using a flow immunosensor is described. An azlactone polymeric gel with covalently bound Protein A/G was used as immunofiltration support. The immunosensor was able to work at high concentrations of organic solvents (e.g. up to 50% MeOH) using a competitive enzyme-immunoassay protocol, with an analysis rate of a complete assay cycle in 22 min. Good sensitivity (I 50 = 9.05 μgl−1) was achieved in organic media and the reusability of the sensor was at least 300 cycles without loss of performance. The sensor was applied to the analysis of carbaryl extracted by both multiresidue method and methanol in fresh and processed vegetable samples. The analytical results were compared with those obtained by ELISA and high-performance liquid chromatography (HPLC) methods. A good agreement was achieved by both immunological methods (r = 0.98) and those obtained by HPLC technique. The results indicated the suitability of the immunosensor for carbaryl analysis in vegetable samples due to its reproducibility, rapidity and compatibility with organic solvents.

Differentiation of chemical components in a binary solvent vapor mixture using carbon/polymer composite-based chemiresistors

We demonstrate a "universal solvent sensor" constructed from a small array of carbon/polymer composite chemiresistors that respond to solvents spanning a wide range of Hildebrand solubility parameters. Conductive carbon particles provide electrical continuity in these composite films. When the polymer matrix absorbs solvent vapors, the composite film swells, the average separation between carbon particles increases, and an increase in film resistance results, as some of the conduction pathways are broken. The adverse effects of contact resistance at high solvent concentrations are reported. Solvent vapors including isooctane, ethanol, diisopropylmethylphosphonate (DIMP), and water are correctly identified ("classified") using three chemiresistors, their composite coatings chosen to span the full range of solubility parameters. With the same three sensors, binary mixtures of solvent vapor and water vapor are correctly classified; following classification, two sensors suffice to determine the concentrations of both vapor components. Poly(ethylenevinyl acetate) and poly(vinyl alcohol) (PVA) are two such polymers that are used to classify binary mixtures of DIMP with water vapor; the PVA/carbon particle composite films are sensitive to less than 0.25% relative humidity. The Sandia-developed visual-empirical region of influence (VERI) technique is used as a method of pattern recognition to classify the solvents and mixtures and to distinguish them from water vapor. In many cases, the response of a given composite sensing film to a binary mixture deviates significantly from the sum of the responses to the isolated vapor components at the same concentrations. While these nonlinearities pose significant difficulty for (primarily) linear methods such as principal component analysis, VERI handles both linear and nonlinear data with equal ease. In the present study, the maximum speciation accuracy is achieved by an array containing three or four sensor elements, with the addition of more sensors resulting in a measurable accuracy decrease.

Theoretical studies of the reduction of ethylene carbonate

A mechanism for the reduction of ethylene carbonate proposed by D. Aurbach, M.D. Levi, E. Levi and A. Schechter [J. Phys. Chem. B 101 (1997) 2195] is analyzed using quantum ab initio and classical transition state theory methods. The reduction reaction leads to open-chain anion products. The two-electron transfer reduction mechanism forming carbonate and ethylene di-carbonate radical anions is thermodynamically feasible. The first electron transfer is the rate-determining step. Further reaction of the carbonate ion with lithium ion or with another ethylene carbonate molecule yields Li 2CO 3 as the most probable product, with lithium ethylene di-carbonate most likely to be present at high solvent concentrations.

Proteases Entrapped in Hydrogels Based on Poly(N-Vinyl Caprolactam) as Promising Biocatalysts in Water/Organic Systems

Chemico-enzymatic methods are a promising approach to obtain various biologically active compounds, including enantiomerically pure substances. Entrapment in gels is one of the most convenient methods to stabilize enzymes for their application in water/organic media. In this study, proteases (trypsin and chymotrypsin) were entrapped into a composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogel, and properties of the biocatalysts obtained were studied. Both entrapped trypsin and α-chymotrypsin retained high activity (up to 85% of the initial value for the immobilized trypsin) and displayed high storage stability. The entrapped proteases were active over broad temperature ranges, and their heat tolerance was approximately 20-25°C higher than that of the soluble enzymes. The dependence of the activities of the soluble and entrapped enzymes on the organic solvent concentration was studied. It appeared that gel entrapment provided retention of enzyme activity in the media with high concentrations of organic solvents (up to 0.5% water content) and ensured high operational stability of α-chymotrypsin in a cyclic process (270 h in total). The entrapped α-chymotrypsin was used as a biocatalyst for obtaining optically pure L-phenylalanine (e.e. 87.8%) by enantioselective hydrolysis of Schiff's base of amino acid ethyl ester in an acetonitrile/water system.