Bulk Aqueous Phase Research Articles

Novel interface-binding chloroperoxidase for interfacial epoxidation of styrene

A unique interface-binding chloroperoxidase (CPO) was developed and examined for interfacial biocatalysis. Native CPO was conjugated with polystyrene (PS) to form a surfactant-like structure that self assembled at oil–water interfaces. While enantioselectivity of the enzyme was maintained, the interfacial assembly of the enzyme improved its overall catalytic efficiency as compared to that observed with the enzyme contained in the bulk aqueous phase. The PS conjugated CPO (PS-CPO) showed a 2.5-fold enhancement of enzyme productivity versus native CPO under batch reaction conditions for the epoxidation of styrene, whereas a 25-fold improvement was realized in a continuous feeding reaction to reach a productivity of 10 μmol h −1 mg protein −1. The interface-binding enzyme also demonstrated several other advantages such as suppressing unwanted side reactions including the hydrolysis of styrene epoxide products, stabilizing the enzyme by limiting its exposure to both the oxidant H 2O 2 and epoxide products, and alleviating the deactivating effect of interfacial stress on enzymes by functioning as a surfactant.

Concentration of polyaromatic hydrocarbons in water to sodium dodecyl sulfate-γ-alumina admicelle

Polyaromatic hydrocarbons (PAHs) in water were concentrated into sodium dodecyl sulfate (SDS)-γ-alumina and di-2-ethylhexyl sodium sulfosuccinate (Aerosol-OT, AOT)-γ-alumina admicelles. The comparison of the binding constants ( K ad[={adsorbed concentration of the solute (mol/g surfactant)}/{the concentration in the bulk aqueous phase (mol/ml)}] indicated almost the same extraction abilities of the both admicelles. However, better and more reproducible recovery was obtained in the concentration of PAHs into the SDS-γ-alumina admicelle. PAHs in tobacco smoke that were trapped in water were successfully concentrated into SDS-γ-alumina admicelle for the HPLC analysis.

Photophysics in Motionally constrained Bioenvironment: Interactions of Norharmane with Bovine Serum Albumin¶

ABSTRACTSteady‐state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (ΔG) for the probe‐protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein‐bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water‐dioxane mixture with varying composition.

New magnetic nano-absorbent for the determination of n-octanol/water partition coefficients

A novel and generic miniaturization methodology for the determination of partition coefficient values of organic compounds in n-octanol/water by using magnetic nanoparticles is, for the first time, described. We have successfully designed, synthesised and characterised new colloidal stable porous silica-encapsulated magnetic nanoparticles of controlled dimensions. These nanoparticles absorbing a tiny amount of n-octanol in their porous silica over-layer are homogeneously dispersed into a bulk aqueous phase (pH 7.40) containing an organic compound prior to magnetic separation. The small size of the particles and the efficient mixing allow a rapid establishment of the partition equilibrium of the organic compound between the solid supported n-octanol nano-droplets and the bulk aqueous phase. UV–vis spectrophotometry is then applied as a quantitative method to determine the concentration of the organic compound in the aqueous phase both before and after partitioning (after magnetic separation). log D values of organic compounds of pharmaceutical interest (0.65–3.50), determined by this novel methodology, were found to be in excellent agreement with the values measured by the shake-flask method in two independent laboratories, which are also consistent with the literature data. It was also found that this new technique gives a number of advantages such as providing an accurate measurement of log D value, a much shorter experimental time and a smaller sample size required. With this approach, the formation of a problematic emulsion, commonly encountered in shake-flask experiments, is eliminated. It is envisaged that this method could be applicable to the high throughput log D screening of drug candidates.

Precipitation of Sulfur Dioxide from Gaseous Feed by Oxidative Chemisorption

Several sorption processes based on precipitation of sulfur dioxide by absorption with chemical reaction have been investigated. The target of this study was the evaluation of process boundaries and boundaries of application for the following processes: • Absorption of sulfur dioxide with caustic soda. • Catalytically supported oxidation of sulfur dioxide in the liquid phase. • Absorption of sulfur dioxide with dichromate. • Absorption of sulfur dioxide with sulfate acidic hydrogen peroxide. The processes were investigated in a lab‐scale falling‐film absorption column. Mass transfer area of the film was 0.22 m2. In absorption of sulfur dioxide with aqueous solution of dichromate below pH values of 2.5, the rate of reaction of dichromate with sulfur dioxide is very fast. As a consequence, absorption of sulfur dioxide is mainly controlled by mass transfer in the gaseous phase. Absorption of sulfur dioxide with caustic additives from an oxygen‐containing gaseous phase is followed by oxidation in the liquid phase. Oxidation is accelerated by heavy metal catalysis. The role of iron(II) and manganese(II) in oxidation of sulfur dioxide has been investigated. Parallel to the oxidation of sulfur dioxide, the catalyst iron(II) is subject of irreversible deactivation by oxidation. Manganese(II) accelerates oxidation. Hydrogen peroxide, sometimes suggested for application in technical scale absorption of sulfur dioxide, causes instantaneous formation of sulfuric acid in the aqueous bulk phase. As long as the concentration of sulfuric acid can be kept below 20% by weight, absorption of sulfur dioxide is again controlled by the gas phase mass transfer resistance.

Micellar Counterion Binding Probed by Fluorescence Quenching of 6-methoxy-N-(3-sulfopropyl)quinolinium

The halide sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), provides a direct approach for measurement of halide concentrations dissociated from the surfactant monomer and micelle. The gel filtration method confirms that the water-soluble SPQ probe will tend to partition in aqueous bulk phase without trapping in cationic micelles. The fluorescence of SPQ is quenched by free halide ions with a linear Stern-Volmer relation below the CMC of cationic surfactants. The Stern-Volmer plot gave a distinct break at CMC owing to the counterion binding to micelles. The CMC and degree of micellar counterion binding (β) are generally in agreement with those obtained by conductivity method. The SPQ fluorescence quenching had high sensitivities with Stern-Volmer constants of 125 M-1 (chloride) and 223 M-1 (bromide), while it was insensitive to nitrate ion. The selective quenching behavior shows that the nitrate ions bind to micelles displacing bromide for tetradecyltrimethylammonium micelles. The increase in counterion binding to micellar surface lower the CMC of surfactants, TTAOAc (tetradecyltrimethylammonium acetate) > TTAC (tetradecyltrimethylammonium chloride) > TTAB (tetradecyltrimethylammonium bromide) > TTAN (tetradecyltrimethylammonium nitrate).

Formation of Molecular Bromine from the Reaction of Ozone with Deliquesced NaBr Aerosol: Evidence for Interface Chemistry

The reaction of ozone with aqueous sodium bromide particles is investigated with a combination of aerosol chamber experiments, kinetics modeling, and molecular dynamics simulations. The molecular bromine production in the chamber experiments is approximately an order of magnitude greater than that predicted by known chemistry in the gas and bulk aqueous phases with use of a comprehensive computer kinetics model. Molecular dynamics simulations indicate that ozone has significant residence time at the air−solution interface, while making frequent contacts with bromide ions for as long as 50 ps in the surface layer of a 6.1 M NaBr solution. The formation of a complex between ozone and bromide ion, [O3···Br-], which can lead to production of Br2 by reaction at the air−water interface, is therefore feasible. Experimentally observed Br2 is well predicted by including an interface process with a reaction probability of [1.9 ± 0.8] × 10-6 (1 s) as the first step in a surface mechanism to produce additional gas-ph...

Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as Δ A/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 μT, respectively), and at different temperatures (16, 18 and 24 °C) was studied. Only the exposure of proteoliposomes to 18-Hz ( B acpeak and B dc=70 μT) magnetic field for 60 min at 16±0.4 °C resulted in a significant enhancement of the hemi channel permeability from Δ A/min=0.0007±0.0002 to Δ A/min=0.0010±0.0001 ( P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.

Interfacial properties of acidified skim milk

The purpose of this study is to investigate the tension properties and dilatational viscoelastic modulus of various skim milk proteins (whole milk, EDTA-treated milk, β-casein, and β-lactoglobulin) at an oil/water interface at 20 °C. Measurements are performed using a dynamic drop tensiometer for 15,000 s. The aqueous bulk phase is a skim milk simulated ultrafiltrate containing 11 × 10 −3 g L −1 milk protein. At pH 6.7, β-casein appears as the best to decrease the interfacial tension, whereas β-lactoglobulin leads to the highest interfacial viscoelastic modulus value. Whole milk was almost as surface-active as individual β-casein in terms of the final (steady-state) lowering of the interfacial tension, but the rate of tension lowering was smaller. EDTA treatment improved the rate of tension lowering of whole milk. The acidification of milk, from previous measurements, would lead to the enhancement of surface activity. At t = 15 , 000 s , the order of effectiveness is pH 4.3 > pH 5.3 = pH 5.6 > pH 6.7 whole milk, suggesting that pH 4.3 whole milk is the best surface active. As compared to pH 6.7 whole milk, the use of pH 5.3 and pH 5.6 milk as surface active would result in the use of milk containing more free β-casein born of pH-dissociated casein micelles.

Diffusion of H +, H 2O and D 2O in polymer/silicate gels

General features of H +, H 2O and D 2O diffusion in polymer/silicate gels applied in different industrial areas were analyzed. It was found that the cumulative mass transport curves consist of two sections: an unsteady-state and a steady-state one. The length of the transient period is strongly dependent on the silicate content of the gel and the concentration gradient of H + ions. Using the length and the intersection point of these periods the effective diffusion coefficient, break-through time and the ion retention capacity of gel could be calculated. The obtained effective diffusion coefficients were very close to the values characteristic in aqueous solutions. The diffusion mass transport could be described by the modified Fick's I law, however, it was stated that on account of the high ion retention in all gels the one-dimensional random-walk equation may not be used for prediction of the mean diffusion distance (break-through time). Using the formation factor of gel (effective “porosity” and tortuosity) the effective diffusion coefficient can be predicted with good accuracy and vice versa, the gel structure can be determined by the absolute and the effective diffusion coefficients characteristic in bulk aqueous phase and gel, respectively. The laboratory experiments provided valuable new information and data to design and formulation of the industrial “gel” technologies in both the enhanced oil recovery and the environmental protection.

Factors Controlling the Formation and Stability of Air Bubbles Stabilized by Partially Hydrophobic Silica Nanoparticles

Air bubbles have been formed using partially hydrophobic silica nanoparticles as the stabilizer. The particles were of primary particle size 20 nm, chemically treated to different degrees with dichlorodimethylsilane to render them partially hydrophobic. Above a certain bubble size range (typically 80-microm diameter), the bubbles seemed to be almost indefinitely stable, while for any size above 20 microm their stability against disproportionation is far better than bubbles stabilized by any protein film investigated in previous studies. A possible theoretical justification for this observation is presented. Bubbles could be formed by shaking water with the particles, but a much higher volume fraction of bubbles was obtained by pressurizing the aqueous phase to 5 atm overnight followed by suddenly releasing the pressure to nucleate bubbles within the silica dispersion. Sonicating the silica dispersion before nucleation also gave more bubbles, which were also found to be more stable. There appeared to be an optimum degree of surface hydrophobicity that gave maximum foamability and foam stability, where around 20-33% of the silanol groups on the silica surface had been converted to dimethylsilane groups. However, a sharp increase in stability occurred when between 1.8 and 2 mol dm(-3) NaCl was also included in the aqueous phase. The change in stability due to inclusion of salt can be rationalized in terms of changes occurring in the value of the particle contact angle. The effects of increasing sonication and an optimum surface chemical treatment can be explained by the need to make the particles sufficiently hydrophobic so that they adsorb strongly enough, while at the same time minimizing their tendency to aggregate in the bulk aqueous phase, which hinders their adsorption. Furthermore, confocal laser scanning microscopy of the bubble dispersions suggests that a large volume fraction of stable bubbles is only formed when the particles adsorbed to the bubbles are also part of a spanning silica particle network in the bulk aqueous solution, forming a weak gel with a finite yield stress.

Polymer-enzyme conjugates can self-assemble at oil/water interfaces and effect interfacial biotransformations.

Native water-soluble enzymes were transformed into interface-binding enzymes via conjugation with hydrophobic polymers, thus enabling interesting interfacial biocatalysis between immiscible chemicals at oil/water interfaces. Such interfacial biocatalysis demonstrated a significantly improved catalytic efficiency as compared to traditional biphasic reactions with enzymes contained in the bulk aqueous phase. Particularly, polystyrene-conjugated beta-galactosidase showed a catalytic efficiency that was more than 145 times higher than that of the native enzyme for a transgalactosylation reaction. It is believed that the improved accessibility of the biocatalysts to chemicals held in both phases across the interface is the key driver for the enhancement of enzyme activity.

Interactions of polysaccharides with β-lactoglobulin adsorbed films at the air–water interface

In the present work we have studied the adsorption (dynamic surface pressure) of β-lactoglobulin (βLG), polysaccharides and their mixtures at the air–water interface at 20 °C and at pH 7. The measurements were performed on a automatic drop tensiometer. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied—Kelcoloid O (KO), Kelcoloid LVF (KLVF) and Manucol ester (MAN). Xanthan gum (XG) and λ-carrageenan (λ-C) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface-active and non-surface-active polysaccharides on dynamic characteristics of β-lactoglobulin adsorbed films. To explain the observed effects on the rates of diffusion, penetration, and rearrangement of these biopolymers at the air–water interface, three phenomena were taken into account: (i) the competitive adsorption; (ii) the complexation, and (iii) the existence of a limited thermodynamic compatibility between protein and polysaccharide at the air–water interface and in the bulk aqueous phase. Surface-active polysaccharides (MAN, KO) are less effective than non-adsorbing polysaccharides (XG) for increasing the surface pressure of protein films, because a competitive behaviour with protein. Highly hydrophilic polysaccharides that do not adsorb by their own at the interface (XG, λ-C) or surface-active polysaccharides with low hydrophobicity (KLVF) show a cooperative behaviour with protein that promotes a significant increase of surface pressure of adsorbed films.

Appearance of pure fluorocarbon micelles surveyed by fluorescence quenching of amphiphilic quinoline derivatives in fluorocarbon and hydrocarbon surfactant mixtures.

A halide-sensitive fluorescence probe was utilized to evaluate the miscibility of fluorocarbon and hydrocarbon surfactants in aqueous micellar systems. The fluorescence of 6-methoxy-N-1,1,2,2-tetrahydroheptadecafluorodecylquinolinium chloride, FC10MQ, was quenched by halide ions dissociated from the surfactant. The fluorescence in micellar solutions showed an initially rapid decay. This suggests that halide ions effectively quench FC10MQ fluorescence at the micellar surface. The subsequent slow decay corresponds to the quenching of FC10MQ fluorescence in the aqueous bulk phase by the free counterions. The Stern-Volmer plots for fluorescence quenching gave a distinct break at the critical micelle concentration of the cationic surfactants. The abrupt increase in fluorescence quenching is attributed to the solubilization of the probe in the micelles. The fluorescence quenching behavior provides direct information about the immiscibility of fluorocarbon and hydrocarbon species in micelles, and the results indicate that almost pure fluorocarbon micelles appear in surfactants mixtures.

A single-reagent method for the speciation of chromium in natural waters by flame atomic absorption spectrometry based on vesicular liquid coacervate extraction

The concept of liquid coacervate extraction (LCE) is deployed to accomplish metal speciation after appropriate derivatization of the target metallic species. The method involves the partitioning of the hydrophobic metal chelates, produced from the reaction of Cr with APDC, in the vesicular aggregates formed from an anionic surfactant with alkaline earth metals and a cosurfactant, which are separated from the bulk aqueous phase. Under the established experimental conditions, the method offers high reproducibility, very low detection limits and recoveries in the range of 95–104%. Moreover, the method affords high tolerance for EDTA, which is used as a masking agent in order to avoid interferences from co-extracted metals. Validation of the outlined method was performed by analysing a certified reference material (BCR 544) yielding recoveries higher than 94% for both Cr oxidation states.

Uranium Complexes Formed at Hematite Surfaces Colonized by Sulfate-Reducing Bacteria

Modeling uranium (U) transport in subsurface environments requires a thorough knowledge of mechanisms likely to restrict its mobility, such as surface complexation, precipitation, and colloid formation. In closed systems, sulfate-reducing bacteria (SRB) such as Desulfovibrio spp. demonstrably affect U immobilization by enzymatic reduction of U(VI) species (primarily the uranyl ion, UO2(2+), and its complexes) to U(IV). However, our understanding of such interactions under chronic U(VI) exposure in dynamic systems is limited. As a first step to understanding such interactions, we performed bioreactor experiments under continuous flow to study the effect of a biofilm of the sulfate-reducing bacterium Desulfovibrio desulfuricans attached to specular hematite (alpha-Fe2O3) surfaces on surface-associated U(VI) complexation, transformation, and mobility. Employing real-time microscopic observation and X-ray photoelectron spectroscopy (XPS), we show that the characteristics of the U(VI) complex(es) formed at the hematite surface are influenced by the composition of the bulk aqueous phase flowing across the surface and bythe presence of surface-associated SRB. The XPS data further suggest higher levels of U associated with hematite surfaces colonized by SRB than with bacteria-free surfaces. Microscopic observations indicate that at least a portion of the U(VI) that accumulates in the presence of the SRB is exterior to the cells, possibly associated with the extracellular biofilm matrix. The U4f7/2 core-region spectrum and U5f2 valence-band spectrum provide preliminary evidence that the SRB-colonized hematite surface accumulates both U(VI) and U(IV) phases, whereas only the U(VI) phase(s) accumulates on uncolonized hematite surfaces. The results suggest that mineral surfaces exposed to a continuously replenished supply of U(VI)-containing aqueous phase will accumulate U phases that may be more representative of those that exist in U-contaminated aquifers than those which accumulate in closed experimental systems. These phases should be considered in models attempting to predict U transport through subsurface environments.

Fluorophore Appended Saccharide Cyclophane: Self-Association, Fluorescent Properties, Heterodimers with Cyclodextrins, and Cross-Linking Behavior with Peanut Agglutinin of Dansyl-Modified Saccharide Cyclophane

A saccharide cyclophane bearing an environment-sensitive fluorophore (1) was prepared by introducing not only three branches with a terminal galactose residue but also one with a dansyl moiety into a tetraaza[6.1.6.1]paracyclophane skeleton. Self-association behavior of the dansyl-appended saccharide cyclophane was characterized in aqueous media by fluorescence spectroscopy and dynamic light scattering measurements. At least in the concentrations below 1.0 x 10(-5) M, saccharide cyclophane 1 existed in a monomeric state, whereas it tended to form self-aggregated complexes in the higher concentration. Solvent polarity dependency on the emission spectra of 1 was examined by fluorescence spectroscopy. With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 1 increased along with a concomitant blue shift of the fluorescence maximum (lambda(em)). In the monomeric state of 1 in water, the dansyl moiety of 1 was not fully included into its cyclophane cavity but partially exposed to the bulk aqueous phase. In the higher concentration ranges in an aggregate state, however, the dansyl group of 1 was located in the apolar cyclophane cavity whose microenvironment was equivalent to the polarity of 1-butanol evaluated on the basis of a correlation between lambda(em) and solvent polarity. This indicates an intermolecular inclusion of the dansyl moiety within the cyclophane. When cyclodextrin (CD) was mixed with 1, the dansyl group of 1 was bound to an internal cavity of CD such as gamma-CD, beta-CD, 6-O-alpha-glucosyl-beta-CD, and 6-O-alpha-maltosyl-beta-CD with binding constants of 7.5 x 10(2), 7.8 x 10(2), 7.7 x 10(2), and 6.0 x 10(2) M(-1), respectively. Such a supramolecular assembling of dansyl-modified cyclophane 1 and CDs caused changes of the fluorescence spectra as well as appearance of induced CD bands in aqueous media. Furthermore, saccharide cyclophane 1 was selectively bound to peanut agglutinin (PNA), galactoside-binding lectin, which was readily monitored by a visible turbidity of the solution due to a cross-linking agglutination of these components, as well as by fluorescence spectroscopy.

Interaction of Antiinflammatory Drugs with EPC Liposomes: Calorimetric Study in a Broad Concentration Range

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient ( K p), and/or the equilibrium constant ( K b). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, Δ H, and indirectly, Δ G and Δ S. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction—possibly the consequence of a modification of the membrane’s physical properties as it becomes saturated with the drug.

Photophysics in motionally constrained bioenvironment: Interaction of norharmane with bovine serum albumin

Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (DeltaG) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition.

Liquid-phase microextraction as an on-line preconcentration method in capillary electrophoresis.

A simple and efficient sample preconcentration method for capillary electrophoresis has been developed using liquid-phase microextraction (LPME). A thin layer of an organic liquid was used to separate a drop of the aqueous acceptor phase hanging at the inlet of a capillary from the bulk aqueous donor phase. The donor-phase pH was 1.0, and the acceptor phase pH was 9.5. This pH difference caused the preconcentration of the acidic compounds, fluorescein and fluorescein isothiocyanate, into the acceptor-phase drop. Enrichment factors of 3 orders of magnitude were obtained with 30-min LPME at 35 degrees C.