Miscible Organic Solvents Research Articles

Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-Proteobacterium isolated from the marine environment of the Sundarbans

In this communication we report purification and characterization of a single salt, solvent, detergent and bleach tolerant alkaline serine protease produced by a truly marine bacterium. Based on the 16S rRNA gene sequence, absence of polyhydroxybutyrate accumulation, growth with very high NaCl levels as well as with hydrocarbons as sole carbon source, the isolate was classified as a new bacterium belonging to the gamma-Proteobacteria family. A 69-fold purification (specific activity 791.7 U/mg protein, unit expressed as μmole of tyrosine liberated per minute) was achieved by a three-step purification procedure. The enzyme is active over a broad range of pH (6.0–11.0), the optimum being at 9.0. This protease enzyme shows activity from 30 to 70 °C and Ca 2+ increase the thermostability. This enzyme exhibits appreciable activity in presence of up to 30% NaCl and is highly stable even after 18 h pre-incubation with 35% (w/v) NaCl. While Ba 2+ and Ca 2+ enhance the enzyme activity, heavy metals like Co 2+, Zn 2+, Hg 2+ inactivate the enzyme. The enzyme is completely stable in presence of laboratory detergents (Tween 80 and Triton X-100), oxidizing agents, reducing agents, commercial detergents and bleaches (hydrogen peroxide and sodium perborate) after 1 h of pre-incubation. Water miscible and immiscible organic solvents like ethylene glycol, ethanol, butanol, acetone, DMSO, xylene and perchloroethylene enhance as well as stabilize the enzyme activity. Reflectance measurements during wash performance studies show that our enzyme is capable of almost complete removal of recalcitrant blood and egg stains in both wet and dry wash operations. Enzymatic activity against a wide variety of substrates indicates that our enzyme can be investigated for a range of commercial applications especially for soy protein and gelatin hydrolysis in the food processing industry as well as for the dehairing process in the leather industry in addition to catalyzing hydrolysis reactions at high salt concentrations.

Complete degradation of anthracene by Manganese Peroxidase in organic solvent mixtures

The goal of this study is the development of a system based on the use of the ligninolytic enzyme Manganese Peroxidase (MnP) for the degradation of polycyclic aromatic hydrocarbons (PAHs), of which anthracene was selected as an example. A main problem of these compounds is their poor solubility in aqueous media. Therefore, the addition of different water miscible organic solvents (acetone, methyl-ethyl-ketone, methanol and ethanol) was considered as a previous step to increase the bioavailability of anthracene. Due to the maximal solubilisation of anthracene and the minimum loss of MnP activity, acetone was selected as the optimal cosolvent, allowing to enhance 140-fold the anthracene solubility for an acetone concentration of 36% (v/v). The in vitro degradation of anthracene by MnP was investigated for different concentrations of the main cofactors and substrates that affect the catalytic cycle of MnP (Mn 2+, H 2O 2 and organic acids) as well as for other environmental parameters (temperature, air/oxygen atmosphere and light source). The system attained a nearly complete degradation of anthracene, around 100%, after 6 h of operation under optimal conditions.

Gels of zirconium phosphate in organic solvents and their use for the preparation of polymeric nanocomposites

Gels of α-zirconium phosphate (α-ZrP) in water have been prepared by exfoliation of crystalline α-ZrP induced by intercalation–deintercalation of propylamine. Although these gels are X-ray amorphous, their Raman spectra indicate that the α-type layers of the starting material remain essentially unaltered. Replacement of gel water with water miscible organic solvents has allowed the formation of α-ZrP gels in a great variety of solvents, such as alkanols, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), acetone, tetrahydrofuran (THF) and even chloroform. Particular attention has been devoted to α-ZrP gels in DMF. On the basis of the surface area of the dry gels, the average thickness of the particles of exfoliated α-ZrP has been estimated between 25 and 80 nm for α-ZrP percentages in the starting gels between 2.5 and 10 wt%, respectively, in agreement with SEM images. α-ZrP gels in THF have been used for the preparation of polystyrene/α-ZrP nanocomposites with filler loadings up to 8 wt%. The presence of exfoliated α-ZrP delays the beginning of thermal decomposition of the polymer by a maximum of 45 °C for 4 wt% filler loading. The state of filler exfoliation has been confirmed by transmission electron microscopy.

Cyclosporin Nanoparticulate Lipospheres for Oral Administration

Cyclosporin is a first line immunosuppressive drug used to prevent transplant rejection and to treat autoimmune diseases. It is a hydrophobic cyclic peptide built from nonmammalian amino acids with low oral bioavailability. The aim of this study was to develop an oral delivery system for cyclosporin A (CyA) and investigate the effect of composition and particle size of the CyA lipid nanoparticles (lipospheres) on the oral bioavailability of this drug. Dispersible concentrated oil formulations that upon mixing in water spontaneously form a nanodispersion were developed. The concentrated oil formulations were clear solutions composed of the drug, a solid triglyceride, a water miscible organic solvent, and a mixture of surfactants and emulsifiers. The activity of the formulated cyclosporin was determined in vitro following the effect on the proliferation of T cells. The oral bioavailability was determined on humans following the cyclosporin blood levels after oral intake of formulated cyclosporin. Cyclosporin dispersion systems resulting in particle size of 25 to 400nm were prepared from acceptable pharmceutical components. The composition of the surfactants and emulsifiers, the lipid core component, and the amount and type of the water miscible organic solvent N-methylpyrrolidone (NMP) and alcohols had a strong effect on the particle size of the dispersions. All formulations were reproducible and stable at room temperature for at least 6 months, with full activity of cyclosporin retained. Human oral bioavaiability study indicated a correlation between the AUC and Cmax and the particle size of the dispersion. A Cmax of ∼1300 ng/mL was found after 2h of oral intake of four capsules, each loaded with 50mg cyclosporin. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1264–1270, 2004

Core−Shell Structure of PLA−PEG Nanoparticles Used for Drug Delivery

Small-angle neutron scattering (SANS) has been used to study the internal structure of poly(lactic acid)−poly(ethylene glycol) (PLA(d)−PEG) block copolymer assemblies, which are being investigated as particulate drug carriers. Three PLA(d)−PEG copolymers with a fixed PEG of 5 kDa and a fully deuterated PLA(d) block of either 3, 15, or 45 kDa were synthesized by the ring opening polymerization of d8-d,l-lactide, using stannous octoate as a catalyst. These copolymers assembled to form nanoparticles in aqueous media, following precipitation from a water miscible organic solvent. The hydrodynamic radius of the PLA(d)−PEG nanoparticles increased with the molecular weight of the PLA(d) block. SANS data obtained at three different solvent contrasts were analyzed simultaneously using core−shell models, which assumed a homogeneous core of uniform scattering length density and a simple functional form for the scattering length density profile of the shell. The thickness and structure of the stabilizing PEG layer were found to depend on the molecular weight of the PLA(d) block. The splayed PEG chains of the PLA(d)−PEG 3:5 assemblies were characteristic of those found in polymeric micelles. However, as the molecular weight of the PLA(d) block was increased, the PEG brush became more radially homogeneous, in accord with recent Scheutjens−Fleer mean-field theory predictions.

Transient pseudo-isotachophoresis for sample concentration in capillary electrophoresis.

We show that many water miscible organic solvents such as acetonitrile, acetone and small alcohols can function as a terminating ion in transient isotachophoresis, which leads to sample concentration on the capillary. It is suggested that this method could be termed transient "pseudo-isotachophoresis" (pseudo-ITP). Because of their low conductivity, these water miscible organic solvents provide the high field strength necessary for band sharpening similar to that provided by the terminating ion. Salts, when present in such samples act briefly as leading ions, migrating rapidly in the organic solvent until they are slowed at the interface of the separation buffer. When the organic solvents are added to the sample, both the migrations as well as the stacking of the analytes are affected by the concentration of salts (leading ions) in the sample, similar to that observed in isotachophoresis. Our results show that this type of stacking offers good reproducibility and reliability for practical analysis. In practice, pseudo-ITP stacking is much easier to perform compared to that of true ITP with several added practical advantages as discussed.

Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents

A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91×10 −8 M paraoxon and 1.24×10 −9 M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile.

Effect of organic solvents on stability and activity of two related alcohol dehydrogenases: a comparative study

A comparative study was performed regarding the catalytic activity and stability of two related enzymes (thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii and its mesophilic counterpart from yeast) in the presence of a number of miscible and immiscible organic solvents. The study was performed in view of the practical usefulness of organic solvents for alcohol dehydrogenases which have been shown to catalyse a variety of industrially-important dehydrogenation reactions. A number of organic solvents of different physicochemical characteristics were used and substantial stabilization was achieved. The non-polar solvents utilized showed the ability to enhance thermal stability of both proteins. Protection against thermal denaturation was especially pronounced by n-dodecane, the solvent with the highest log P used in the present study. Dimethylformamide and dioxane, employed as two miscible organic solvents, showed the ability to cause substrate inhibition and changes in protein conformation as indicated by kinetic and fluorescence studies. A higher resistance of the thermophilic protein to the deleterious effect of pyridine and thermostabilization of the mesophilic enzyme by non-polar solvents are especially emphasized. Combined differences in protein structure and nature of organic solvents are suggested to explain the differences in stability and catalytic activity observed in the present investigation.

Microencapsulation of dissociable human growth hormone aggregates within poly( d, l-lactic-co-glycolic acid) microparticles for sustained release

For the sustained release formulation of recombinant human growth hormone (rhGH), dissociable rhGH aggregates were microencapsulated within poly( d, l-lactic-co-glycolic acid) (PLGA) microparticles. rhGH aggregates were first produced by adding a small volume of aqueous rhGH solution into a partially water miscible organic solvent phase (ethyl acetate, EtAc) containing PLGA. These rhGH aggregates were then microencapsulated within PLGA polymer phase by extracting EtAc into an aqueous phase pre-saturated with EtAc. Release profiles of rhGH from these microparticles were greatly affected by changing the volume of incubation medium. The released rhGH species consisted of mostly monomeric form having a correct conformation. This study reveals that sustained rhGH release could be achieved by microencapsulating reversibly dissociable protein aggregates within biodegradable polymers.

Disposable cholinesterase biosensor for the detection of pesticides in water-miscible organic solvents

A disposable cholinesterase biosensor based on screen-printed electrodes was assembled and used to assess the effect of miscible organic solvents on the acetylcholinesterase activity and on the inhibitory effect of organophosphorus pesticides on acetylcholinesterase activity. Acetonitrile, ethanol and DMSO were tested in a range 0–30% mixed with phosphate buffer (0.1 M, pH 7). With 5% acetonitrile and 10% ethanol, an increase of the recorded current was observed. The addition of 0.2% polyethylenimine to the enzyme preparation, before immobilisation, allowed the utilisation of 15% acetonitrile without negative effect on the enzyme activity. An inhibition calibration curve was obtained using chlorpyrifos-ethyl-oxon, a compound widely used for agricultural purposes. The lowest detectable amount was 1 ppb following an incubation time of 10 min. The use of 5% acetonitrile and 0.2% polyethylenimine did not interfere with the enzyme-inhibitor interactions.

Increased transglycosylation activity of Rhodotorula glutinis endo-beta-glucanase in media containing organic solvent.

The transglycosylation of p-nitrophenyl-beta-D-cellotrioside to cellotetraose catalyzed by endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50 degrees C in the presence of 40% acetone.

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.

Screen-printed electrodes, with genetically modified cholinesterase, for detection of pesticides in water-miscible organic solvent.

A disposable cholinesterase biosensor based on screen-printed electrodes (SPE) was assembled and used to assess the effect of miscible organic solvents on the acetylcholinesterase activity and on the inhibitory effect of organophosphorus pesticides on acetylcholinesterase activity. Acetonitrile, ethanol and DMSO were tested in a range of 0 to 30% mixed with phosphate buffer (0.1M, pH7). With 5% acetonitrile and 10% ethanol, an increase of the recorded current was observed. The addition of 0.2% polyethylenimine to the enzyme preparation, before immobilization, allowed the utilization of 15% acetonitrile without negative effect on the enzyme activity. An inhibition calibration curve was obtained using chlorpyrifosethyl-oxon, a compound widely used for agricultural purposes. The lowest detectable amount was 1ppb following an incubation time of 10min. The use of 5% acetonitrile and 0.2% polyethylenimine did not interfere with the enzyme-inhibitors interactions. The second part is focused on the evaluation of the activity of several genetically modified acetylcholinesterases obtained from Drosophila melanogaster and their inhibition constant face to the methamediphos. The selection of one of them and its immobilization on a SPE allowed a detection of 1.4ppb methamediphos.

PEC films prepared from Chitosan-Alginate coacervates.

Chitosan-alginate polyelectrolyte complex (PEC) have been prepared in situ in beads and microspheres. This study examines the preparation of suitable chitosan-alginate coacervates for casting into homogeneous PEC films for potential applications in packaging, controlled release systems and wound dressings. Coacervation between chitosan and alginate was rapid, but the rate may be controlled with the addition of water miscible organic solvents. Compared with ethanol and PEG200, acetone was the more promising solvent moderator. Suspensions of fine, uniformly dispersed coacervates were produced by a dropwise addition of 0.25% w/v chitosan solution (solvent: 1: 1 v/v of 2% acetic acid and acetone) into 0.25% w/v sodium alginate solution in water under rapid agitation. The PEC films were transparent and flexible. They exhibited high permeability to water vapor, but resisted complete dissolution in 0.1 M HCI, distilled water and pH 7.4 phosphate buffer solution. Microscopic heterogeneity in the films could be reduced by immersion in aqueous media, but this was accompanied by modifications in the thickness, permeability and mechanical property of the films.

Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering of reaction conditions.

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1, 2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.

Irreversible thermoinactivation of α-chymotrypsin in buffer and water miscible organic solvent. Comparison with a reverse micellar system

The mechanism of irreversible thermoinactivation of α-chymotrypsin in buffer medium, dimethylformamide, dimethylsulfoxide/buffer and reverse micelles was studied. Experiments for the enzyme thermoinactivation in the different systems were accompanied by studies on protein structure alterations. The formation of free SH groups was followed during the stability tests. This formation is due to the cleavage of the enzyme primary structure which leads to small peptides. These were identified by SDS-PAGE, in the case of the buffer medium, and also by HPLC in the three other systems. An increase of SH groups with the residence time of the enzyme in the buffer and organic solvent/buffer system was observed. This was corroborated by the disappearance of the electrophoresis bands of the protein and also by HPLC analysis. In the latter technique, the peaks corresponding to α-chymotrypsin disappeared with the concomitant appearance of small peaks in the chromatogram. For the enzyme encapsulated in reverse micelles, the formation of free SH groups was not detected and the HPLC analysis revealed that the protein peak stayed intact during the residence time in this system. The thermoinactivation of chemically modified α-chymotrypsin by the introduction of dianhydride pyromellitic on the Lys residues of the protein was also studied in some of the systems. The results showed that the chemical modification stabilized the enzyme when the system used was buffer or organic solvent mixed with buffer being this stabilization partially due to a slower process of cysteine bond cleavage.

Effect of Water Miscible Organic Solvents on Kinetics of a Thermostable β-Glycosidase

A thermostable β-D-glycosidase from Sulfolobus solfataricus was used to determine the effect of water-miscible solvents on enzyme activity and kinetics. Various o- and p-nitrophenyl glycosides were used as representative substrates for hydrolysis. Initial enzyme activity was enhanced in the presence of methyl acetate, butan-2-one and acetonitrile. The activation by solvent was found to be transitory. 1,4-Dioxane, formamide and tetrahydrofuran were inhibitory at all concentrations with all substrates used. Kinetic studies in the presence of solvents suggested that KM values for all substrates were unaffected by methyl acetate, but increased or decreased in the presence of 1,4-dioxane or tetrahydrofuran, in a substrate dependent manner. Studies using an competitive inhibitor supported these results. Methyl acetate did not change competitive-type inhibition by D-gluconic acid lactone, while in the presence of 1,4-dioxane altered the inhibition kinetics to mixed-type. Tetrahydrofuran affected the mode of inhibition but not in a identifiable manner. These results suggest that both 1,4-dioxane and tetrahydrofuran may have a direct influence on the active site of the catalyst.

Solvation characteristics of pressurized hot water and its use in chromatography

The influence of temperature in the range 75–180 °C on the solvation properties of pressurized water is studied by use of chromatography for a varied group of compounds on a porous polymer sorbent PLRP-S 100. The solvation parameter model is used as the basis of a quantitative comparison of the influence of temperature and solvent composition variation on the elution strength and selectivity of the mobile phase. The predominant influence of increasing temperature on the solvation properties of water is to reduce its cohesion and capacity for hydrogen-bond interactions. Even at 180 °C, however, hot water retains sufficient of its room temperature properties to remain a relatively weak eluting solvent in reversed-phase chromatography compared with aqueous mixtures of miscible organic solvents. Changes in selectivity accompanying composition variation for acetonitrile, methanol, and propan-2-ol are quantitatively different from those demonstrated for temperature variation of pressurized water, indicating that for method development the two processes are complementary rather than redundant.

Activity of Free and Immobilized ExtracellularCerrena unicolorLaccase in Water Miscible Organic Solvents

The extracellular laccase of white rot fungus Cerrena unicolor was purified from culture by ion-exchange chromatography on DEAE-Toyopearl column and immobilized on silanized porous glass heads. During bonding procedure 94.35% protein and 100 % laccase activity were coupled to the support. Optimum pH for immobilized laccase compared to free enzyme was shifted from 5.5 to 5.7. The immobilized laccase was more resistant for thermal denaturation: at 70°C, the activity of immobilized enzyme was around 20 % higher than that of the free enzyme. It was also more stable during storage at 4°C. After about seven months the immobilized laccase retained 94.81 % of its initial activity, whereas free enzyme only 39.59 %. Nine water-miscible organic solvents tested in an anhydrous form caused an inhibitory effect on both laccase forms, but, in the mixture with water, the enzyme in all cases shows activity. It decreased, however, when the concentration of solvents increased. Among all solvents, the best results were obtained with ethylene glycol and methoxyethanol. When the reaction mixture contained 50 % of ethylene glycol, free and immobilized laccase retained 24 and 31 % respectively of their activity in the buffer. In the case of 50 % methoxyethanol these data were 6 and 36 %. Organic solvents shifted pH optima of both laccase forms to pH 6.0. The calculation from Lineweaver-Burk plot oxidizing capacity of laccase towards syringaldazine was slightly higher for the free enzyme than for immobilized one, 18.5 and 20.0 M/l respectively. These results show that Cerrena unicolor laccase both in free and immobilized form is able to catalyze the oxidation of syringaldazine in organic solvents. It may be usable in transformation of substrates insoluble or sparingly soluble in water. The immobilized laccase, as more stable and temperature resistant than free enzyme, seems to be more useful.

アクリル酸系ＳＡＰの親水性混合溶媒中に於ける膨潤，凝集挙動に関する研究

The writers have found the phenomenon that the low concentration of Microfibrillated Cellulose (MFC) has a high viscosity when evenly dispersed in a mixed solvent of water and water miscible organic solvent. In case that SAP (Super Absorbent Polymer) in a state of particles is dispersed in that dispersion solvent, it is possible to disperse SAP particles more stably in a high concentration condition due to the effect of the viscosity.Furthermore, it is possible to prevent the coagulation behavior caused by the mutual contact among SAP particles in the solvent due to the effect of dispersed MFC. Also it is possible to keep dispersed slurry having a long pot life.Consequently, in case of forming and removing the solvent from the above-mentioned slurry, it is possible to obtain the composite of SAP and MFC, whose SAP particles are combined tightly, due to the effect of strong hydrogen bonding. As the first report on the research regarding the composite of SAP and MFC, we have studied on the swelling & coagulation behaviors of acrylic acid base SAP in water, which does not contain MFC, and water miscible organic solvent mixture as a model condition. As a result of the study, we have found the following : 1. It is necessary to select an appropriate organic solvent and the most suitable mixing ratio with water that SAP does not show any swelling and coagulation behaviors to get a stable dispersion state of SAP ;2. Regarding SAP, specially the dispersion state is affected by the particle size and whether its surface has been done a surface cross linkage treatment or not : and3. Regarding the organic solvent, the dispersion state is affected by its dielectric constant, its mixture ratio with water, and its temperature.As a result, we have set to use the dispersion solvent of ethanol 60% and water 40% at 20°C as a standard condition