Reverse Micellar System Research Articles

Do ionic and hydrophobic probes sense similar microenvironment in Triton X-100 nonionic reverse micelles?

Rotational diffusion of two structurally similar ionic probes, rhodamine 110 and fluorescein, has been examined in nonionic reverse micellar system of Triton X-100/benzene-n-hexane/water as a function of mole ratio of the water to surfactant, W. This study has been undertaken to find out whether ionic and hydrophobic probes experience similar microenvironment in these reverse micelles. Experimental results indicate that, from W=0 to 3, the average reorientation time, which is a measure of the microviscosity experienced by the probe molecule, increases by 90% and 40% for rhodamine 110 and fluorescein, respectively, and from W=3 to 8, it decreases by 20% for both the probes. The increase in the average reorientation time with W has been rationalized on the basis of the flexible oxyethylene chains of the TX-100 surfactant being hydrogen bonded by the water molecules, which makes the core region less fluid. However, once the hydration of the oxyethylene chains is complete, further addition of water results in formation of water droplet; which renders the micelle-water interface in the core region less compact leading to a marginal decrease in the average reorientation time of the probe molecules. These explanations are consistent with the location of the probes and the structure of the Triton X-100/benzene-hexane/water reverse micelles. To compare how the microenvironment experienced by these ionic probes is different from the hydrophobic ones, results from our earlier work [J. Phys. Chem. B 108, 7944 (2004)] have been considered. Such a comparison revealed that both ionic and hydrophobic probes experience similar microenvironment in these reverse micelles until the hydration of the oxyethylene chains is complete. In case of hydrophobic probes, however, the onset of water droplet formation does not alter their microenvironment, which is due to their location in the reverse micellar cores.

Microemulsion approach to neodymium, europium, and ytterbium oxide/hydroxide colloids—Effects of precursors and preparation parameters on particle size and crystallinity

Colloids based on lanthanides or their oxides have a great potential in the areas of optical and magnetic materials. In this study the confined space of reverse micellar systems formed by water in cyclohexane was used to precipitate particles based on neodymium, europium, and ytterbium. The morphology and structural properties of the prepared colloids were determined by transmission electron microscopy, IR spectroscopy, and X-ray diffraction and absorption measurements. The size of the obtained systems as determined by dynamic light scattering ranged from a few nanometers to several hundreds of nanometers in diameter, depending on the reaction conditions. The employed surfactant was found to have a major influence on the particle size and morphology. In contrast to the ionic surfactants sodium dodecyl sulfate and cetyltrimethylammonium bromide, the nonionic surfactant Triton X-100 generally delivered very small and unagglomerated particles. The precursor counterion had a similar effect, depending on its ability to coordinate to the particle surface, and prevented particle growth and agglomeration. The influence of further parameters such as the pH of the aqueous starting solutions, the mixing methodology, and the preparation temperature was also investigated. Applying increased temperatures the particles exhibited a higher crystallinity, and at the same time the particle size was drastically increased.

Photophysics of the phenoxazine dyes resazurin and resorufin in direct and reverse micelles

The photophysics of the phenoxazin-3-one dyes resazurin and resorufin was studied in a micellar solution of cetyltrimethylammonium chloride and in reverse micelles of 1,4-bis(2-ethylhexyl)sulfosuccinate and benzylhexadecyldimethylammonium chloride. Absorption and fluorescence emission spectra, as well as fluorescence lifetimes and T–T transient absorption spectra were determined as a function of surfactant concentration. In the presence of direct micelles of cetyltrimethylammonium chloride, both dyes displayed red shifts in the absorption and fluorescence spectra together with a simultaneous fluorescence lifetime increase. The electrostatic attraction between the anionic dyes and the positive micellar interface favors the location of the dyes closer to the head groups of the surfactant molecules. In reverse micellar systems the spectral properties depended upon the charge of the surfactant and water content. In the case of 1,4-bis(2-ethylhexyl)sulfosuccinate, at low water content both dyes were incorporated into the interface; as the water content increased their spectral properties tended to those in pure water. In contrast, in the case of cationic surfactant, the dyes were located in the interfacial pseudophase as a result of electrostatic interactions.

Reverse Micellar Extraction of β-Galactosidase from Barley (Hordeum vulgare)

The reverse micellar system of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane was used for the extraction and primary purification of beta-galactosidase (EC 3.2.1.23) from the aqueous extract of barley (Hordeum vulgare) for the first time. The process parameters such as the concentration of the surfactant, the volume of the sample injected, and its protein concentration, pH, and ionic strength of the initial aqueous phase for forward extraction, buffer pH, and salt concentration for back extraction are varied to optimize the extraction efficiency. Studies carried out with both phase transfer and injection mode of reverse micellar extraction confirmed the injection mode to be more suitable for beta-galactosidase extraction. The extent of reverse micellar solubilization of proteins increased with an increase in protein concentration of the feed sample. However, back extraction efficiency remained almost constant (13-14.4%), which indicates the selectivity of AOT reverse micelles for a particular protein under given experimental conditions. beta-Galactosidase was extracted with an activity recovery of 98.74% and a degree of purification of 7.2-fold.

Kinetic Study of the Reaction of Leuco Methylene Blue with 2,6-Dimethyl-p-benzoquinone in a Reverse Micellar System

The kinetics of the reaction of leuco methylene blue (MBH) with 2,6-dimethyl-p-benzoquinone (DMBQ) were studied in a heptane/bis(2-ethylhexyl)-sulfosuccinate (AOT)/water reverse micellar system. The pseudo-first-order rate constant (k (obsd)) obtained in the presence of excess of DMBQ was found to be proportional to the initial concentration of DMBQ for W (0)=3, 5, 10, 15 and 20 (W (0)=[H2O]/[AOT]). The second-order rate constant (k (2)=k (obsd)/[DMBQ](0)) increased with an increase in the W (0) value, but was almost independent of the concentration of the water pool. A mechanism involving the distribution of DMBQ between the reverse micellar interface and bulk organic solvent was proposed to explain these findings.

Monomer Formation and Function of p‐Hydroxybenzoate Hydroxylase in Reverse Micelles and in Dimethylsulfoxide/Water Mixtures

It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the K(M) value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.

Reverse micellar extraction of bromelain from Ananas comosus L. Merryl

AbstractBACKGROUND: A reverse micellar system (RMS) of ionic surfactants is used for the first time for the extraction and primary purification of fruit bromelain (EC 3.4.22.33) from the aqueous extract of pineapple (Ananas comosus L. Merryl). The effect of various process parameters on both forward and back extraction of bromelain is studied to improve the extraction efficiency of RMS. Most of the reverse micellar extraction (RME) studies reported so far are on model systems and its application to enzyme extraction from a natural source is rarely reported.RESULTS: Studies carried out with ionic surfactants sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) confirmed that electrostatic interaction was the main driving force for the extraction of fruit bromelain. Among the two surfactants studied, CTAB was found to be the most suitable for the extraction of fruit bromelain with respect to activity recovery (97.56%) and degree of purification (4.54 fold) when employed as a 150 mmol L−1 CTAB/iso‐octane/5% (v/v) hexanol/15% (v/v) butanol system. Activity recovery with a counterionic system is higher (94.30%) in comparison with isopropyl alcohol added system (85.35%).CONCLUSION: RME could be used as an efficient primary purification step for the recovery of bromelain from pineapple juice. Reverse micellar phase components can easily be recovered and efficiently reused for fresh or subsequent extraction, which contributes favorably to the process economics and environmental issues. Copyright © 2007 Society of Chemical Industry

Detection of bicontinuous phase in some reverse micellar systems by positron lifetime spectroscopy

AbstractPositron lifetime measurements have been made in some reverse micellar systems of cetyl tri methyl ammonium bromide (CTAB)/hexanol and tetra‐decyl‐tri‐methyl‐ ammonium‐bromide (TTAB)/pentanol by adding increasing amount of water in each system. For both the systems, the dependence of positron lifetime parameters on water concentration indicates the existence of bicontinuous phase in so called single reverse micellar phase. The results of this investigation have been presented in this paper. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effect of microemulsion variables on copper oxide nanoparticle uptake by AOT microemulsions

Ultradispersed metal oxide nanoparticles have applications as heterogeneous catalysts for organic reactions. Their catalytic activity depends primarily on their surface area, which in turn, is dictated by their size, colloidal concentration and stability. This work presents a microemulsion approach for in situ preparation of ultradispersed copper oxide nanoparticles and discusses the effect of different microemulsion variables on their stability and highest possible time-invariant colloidal concentration (nanoparticle uptake). In addition, a model which describes the effect of the relevant variables on the nanoparticle uptake is evaluated. The preparation technique involved solubilizing CuCl 2 in single microemulsions followed by direct addition of NaOH. Upon addition of NaOH, copper hydroxide nanoparticles stabilized in the water pools formed in addition to a bulk copper hydroxide precipitate at the bottom. The copper hydroxide nanoparticles transformed with time into copper oxide. After reaching a time-independent concentration, mixing had limited effect on the nanoparticle uptake and particle size. Particle size increased with increasing the surfactant concentration, concentration of the precursor salt, and water to surfactant mol ratio; while the nanoparticle uptake increased linearly with the surfactant concentration, displayed an optimum with R and a power function with the concentration of the precursor salt. Surface areas per gram of nanoparticles were much higher than literature values. Even though lower area per gram of nanoparticles was obtained at higher uptake, higher surface area per unit volume of the reverse micellar system was attained. A model based on water uptake by Wisor type II microemulsions, and previously used to describe iron oxide nanoparticle uptake by the same microemulsions, agreed well with the experimental results.

Synthesis, spectroscopic properties and photodynamic activity of a novel Zn(II) phthalocyanine substituted by fluconazole groups

A novel Zn(II) phthalocyanine derivative (ZnPcF) bearing four antifungal structures of fluconazole was synthesized by a two-step procedure starting from 4-nitrophthalonitrile. First, phthalonitrile-azole derivative was prepared by a nucleophilic ipso-nitro substitution reaction between 4-nitrophthalonitrile and fluconazole. The cyclotetramerization of phthalonitrile-azole with Zn(II) acetate in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) results in the formation of the ZnPcF as a mixtures of constitutional isomers with 27% yield. Absorption and fluorescence spectroscopic studies were analyzed in different media. The results show that ZnPcF is lowly soluble in polar solvents or in reverse micellar systems. However, addition of HCl produce an increase in the monomerization of ZnPcF in N, N-dimethylformamide (DMF)/water (10%, v/v) and in benzene/benzyl- n-hexadecyldimethyl ammonium chloride (BHDC, 0.1 M)/water ( W 0 = 10). A value of 0.19 was calculated for the fluorescence quantum yield ( ϕ F) of this photosensitizer in DMF/water (10%)/HCl 1.2 mM. The photodynamic activity of ZnPcF was evaluated using 9,10-dimethylanthracene (DMA). An enhancement in the singlet molecular oxygen, O 2( 1Δ g), production was obtained in acidified DMF/water or BHDC micellar system, which represent an appropriate system to induce monomerization of ZnPcF. Preliminary studies to evaluate the photodynamic activity of ZnPcF were tested against a typical yeast Candida albicans. These results indicated that ZnPcF is an interesting antifungal agent because it has antimycotic activity in dark and its efficiency increases in the presence of light due to the photodynamic inactivation caused by the photosensitizer moiety.

Separation of Chicken Egg‐White Lysozyme by High‐Speed Countercurrent Chromatography using a Reverse Micellar System

High‐speed countercurrent chromatography (HSCCC) was employed for the first time to separate lysozyme from chicken egg‐white using a reverse micellar system with 50 mM bis‐(2‐ethylhexyl) sulfosuccinate sodium (AOT) dissolved in isooctane and aqueous buffer. Lysozyme is separated from other main proteins through one step separation, and relatively high activity recovery (over 80%) can be achieved when applied to the purification of a preseparated sample, while a low recovery problem exists when applied to separate lysozyme from crude chicken egg‐white samples due to the interference of a large amount of other proteins. However, this method has advantages over traditional reverse micellar extraction (RME) for its simple continuous elution mode and high separation efficiency, and it is promising for scale‐up.

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis.

Kinetics of p-nitrophenyl Acetate Hydrolysis Catalyzed by Mucor javanicus Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W=([water]/[AOT])=6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

Thermostability of Cromobacterium viscosum lipase in AOT/isooctane reverse micelle

The thermostability of Cromobacterium viscosum lipase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was increased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60 degrees C was 28 h, ninefold higher than that in reverse micelles without PEG 400. The lipase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60 degrees C in PEG containing reverse micelles was 0.055 h!1, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lipase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.

The key role of support surface tuning during the preparation of catalysts from reverse micellar-synthesized metal nanoparticles

Abstract Silica-supported cobalt catalysts have been prepared by depositing cobalt nanoparticles synthesized in reverse micellar systems. Two different reverse microemulsions comprising both a neutral (Triton X114) or an ionic (AOT) surfactant were used for the synthesis of nanoparticles. The materials have been characterized by 29Si MAS NMR, XRD, FTIR, H2-TPR, and TEM. Protection of the hydroxyl groups on the silica surface by silylation prior to loading of the cobalt nanoparticles has been found crucial for attaining the desired metal dispersions in the final catalyst owing to a higher chemical compatibility between the hydrophobic support surface and the microemulsion during the deposition step.

Avoiding Inactivation of Proteins in the Process of AOT/Isooctane Reverse Micellar Extraction

Reverse micellar extraction has a potential application in proteins purification. In the process of AOT/isooctane reverse micellar extraction of proteins, a big problem is inactivation of interesting proteins when ionic surfactant AOT was used to form reverse micelles. In this study, it had been found that adding ethanol could stabilize the activity of protein, and it greatly improved extraction recovery of trypsin. When trypsin was extracted from crude material of pig pancreas by using AOT/isooctane reverse micellar system almost 90% and 100% of activity recovery of trypsin could be obtained in forward and backward extraction, respectively. The effects of ethanol concentration, organic salt types, AOT concentration, pH and temperature on the recovery of trypsin were investigated and the extraction process had been optimized. Finally, about 88% of the total yield was obtained, and the specific activity of trypsin purified was increased from 300 U/mg to over 1800 U/mg proteins with purification factor of 5 times more.

The influence of the nature of surfactants and dispersion medium on the catalytic effect of reverse micellar systems

The catalytic effect of reverse micellar systems in alkaline hydrolysis of phosphonic acid esters is to a considerable extent determined by the structure of the substrate and surfactant and the nature of the oil phase but weakly depends on the surface potential of the aggregates. AOT-butanol-decane-water, sodium dodecyl sulfate-butanol-decane-water, and cetyltrimethylammonium bromide-butanol-decane-water microemulsions exhibit specific catalytic properties rather than catalytic properties intermediate between those of the surfactant-alkane-water and surfactant-alkanol-water three-component systems.

Hydrolysis of phosphorus acid esters in the sodium bis(2-ethylhexyl)sulfosuccinate—n-nonane—polyethylene glycol—water supramolecular system before and after the percolation threshold

The kinetics of alkaline hydrolysis of O-ethyl O-p-nitrophenyl chloromethylphosphonate in a sodium bis(2-ethylhexyl)sulfosuccinate—n-nonane—polyethylene glycol—water reverse micellar system was studied in a wide range of concentrations of the surfactant and water. The sign of the catalytic effect of micelles is inverted in the presence of the polymer. A sharp change in the apparent rate constant of hydrolysis of the phosphonate was found in a region of the temperature percolation threshold.