Emulsion Templating of Poly(lactic acid) Particles: Droplet Formation Behavior

Monodisperse poly(dl-lactic acid) (PLA) particles of diameters between 11 and 121 μm were fabricated in flow focusing glass microcapillary devices by evaporation of dichloromethane (DCM) from emulsion droplets at room temperature. The dispersed phase was 5% (w/w) PLA in DCM containing 0.1-2 mM Nile Red and the continuous phase was 5% (w/w) poly(vinyl alcohol) in reverse osmosis water. Particle diameter was 2.7 times smaller than the diameter of the emulsion droplet template, indicating very low particle porosity. Monodisperse droplets have only been produced under dripping regime using a wide range of dispersed phase flow rates (0.002-7.2 cm(3)·h(-1)), continuous phase flow rates (0.3-30 cm(3)·h(-1)), and orifice diameters (50-237 μm). In the dripping regime, the ratio of droplet diameter to orifice diameter was inversely proportional to the 0.39 power of the ratio of the continuous phase flow rate to dispersed phase flow rate. Highly uniform droplets with a coefficient of variation (CV) below 2% and a ratio of the droplet diameter to orifice diameter of 0.5-1 were obtained at flow rate ratios of 4-25. Under jetting regime, polydisperse droplets (CV > 6%) were formed by detachment from relatively long jets (between 4 and 10 times longer than droplet diameter) and a ratio of the droplet size to orifice size of 2-5.     

Novel method for obtaining homogeneous giant vesicles from a monodisperse water-in-oil emulsion prepared with a microfluidic device

A novel technique called the "lipid-coated ice droplet hydration method" is presented for the preparation of giant vesicles with a controlled size between 4 and 20 microm and entrapment yields for water-soluble molecules of up to about 30%. The method consists of three main steps. In the first step, a monodisperse water-in-oil emulsion with a predetermined average droplet diameter between 4 and 20 microm is prepared by microchannel emulsification, using sorbitan monooleate (Span 80) and stearylamine as emulsifiers and hexane as oil. In the second step, the water droplets of the emulsion are frozen and separated from the supernatant hexane solution by precipitation, followed by a removal of the supernatant and followed by the replacement of Span 80 by using a hexane solution containing egg yolk phosphatidylcholine, cholesterol, and stearylamine (5:5:1, molar ratio). This procedure is performed at -10 degrees C to keep the water droplets of the emulsion in a frozen state and thereby to avoid extensive water droplet coalescence. In the third step, hexane is evaporated at -4 to -7 degrees C and an external water phase is added to the remaining mixture of lipids and water droplets to form giant vesicles that have an average size in the range of that of the initial emulsion droplets (4-20 microm). The entrapment yield and the lamellarity of the vesicles obtained depend on the lipid/water droplet ratio and on the composition of the external water phase. At high lipid/water droplet ratio, the giant vesicles have a thicker membrane (indicating multilamellarity) and a higher entrapment yield than in the case of a low lipid/water droplet ratio. The highest entrapment yield ( approximately 35%) is obtained if the added external water phase contains preformed unilamellar egg phosphatidylcholine vesicles with an average diameter of 50 nm. The addition of these small vesicles minimizes the water droplet coalescence during the third step of the vesicle preparation, thereby decreasing the extent of release of water-soluble molecules originally present in the water droplets. The GVs prepared can be extruded through polycarbonate membranes to yield large unilamellar vesicles with about 100 nm diameter. This size reduction, however, leads to a decrease in the entrapment yield to about 12% due to solute leakage from the vesicles during the extrusion process.     

Fast transfer process of pyrene between oil-in-water miniemulsion droplets

Recently phase formation mechanisms have been estimated by using various fluorescent probes. In this report, the mixing process between internal phases of oil-in-water miniemulsions is discussed for two-dimensional color graphics data (two-dimensional fluorescence images) based on the excimer formation of pyrene as a hydrophobic fluorescent probe. Just after miniemulsion solution B (water, oil, and nonionic surfactant) was gradually added to miniemulsion A (water, oil, surfactant, and trace amount of pyrene) with gentle and careful stirring, the fluorescence spectra and the two-dimensional image of pyrene were measured. The decreasing of the excimer peak of pyrene was observed as soon as miniemulsion solution B was added. The result showed that pyrene initially located in miniemulsion droplets was smoothly diluted by the addition of miniemulsion droplets which contain only oil in the internal phase. The internal phases of miniemulsion droplets are miscible without changing the droplet diameter, and it is declared that pyrene transfers smoothly to the interface between droplets stabilized by the nonionic surfactant because the droplet diameter showed no significant difference throughout this mixing process. Received: 7 December 1999 Accepted: 11 April 2000     

Polymerisable Miniemulsions Using Rotor‐Stator Homogenisers

The use of a rotor‐stator mixer as a homogenisation device to make miniemulsion droplets with industrially pertinent solid contents was investigated. Methyl methacrylate/butyl acrylate (50:50 w/w ratio) miniemulsions with droplet diameters from 2 µm to 300 nm and polydispersity indices from 1.2 to 3.6 were used. Miniemulsions with three different mean droplet diameters (300, 400, 600 nm) were polymerised and the evolution of particle size was observed. When 300 nm droplets were polymerised they yielded particles of similar diameter to the original droplets, whereas particle coalescence of the growing particles with a loss of control over the particle size distribution was observed for the 400 and 600 nm droplets. The influence of costabiliser, agitation speed, solid content, colloidal protectors and surface coverage on the evolution of the droplet size and size distribution as well as on the evolution of the average particle size and its distribution were examined. It was observed that changing the above parameters had no impact on the evolution of the particle size, suggesting we have a very robust miniemulsion system.

     

Influence of oil polarity on droplet growth in oil-in-water emulsions stabilized by a weakly adsorbing biopolymer or a nonionic surfactant

The influence of oil type (n-hexadecane, 1-decanol, n-decane), droplet composition (hexadecane:decanol), and emulsifier type (Tween 20, gum arabic) on droplet growth in oil-in-water emulsions was studied. Droplet size distributions of emulsions were measured over time (0-120 h) by laser diffraction and ultrasonic spectroscopy. Emulsions containing oil molecules of low polarity and low water solubility (hexadecane) were stable to droplet growth, irrespective of the emulsifier used to stabilize the droplets. Emulsions containing oil molecules of low polarity and relatively high water solubility (decane) were stable to coalescence, but unstable to Ostwald ripening, irrespective of emulsifier. Droplet growth in emulsions containing oil molecules of relatively high polarity and high water solubility (decanol) depended on emulsifier type. Decanol droplets stabilized by Tween 20 were stable to droplet growth in concentrated emulsions but unstable when the emulsions were diluted. Decanol droplets stabilized by gum arabic exhibited rapid and extensive droplet growth, probably due to a combination of Ostwald ripening and coalescence. We proposed that coalescence was caused by the relatively low interfacial tension at the decanol-water boundary, which meant that the gum arabic did not absorb strongly to the droplet surfaces and therefore did not prevent the droplets from coming into close proximity.     

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

We investigated how chemical equilibria are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing number of publications in which electrospray ionization mass spectrometry is used for binding constant determination. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, we found an average initial droplet diameter of 10 microm in the electrospray. The PDA allowed us to follow the evolution of these droplets down to a size of 1 microm. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equilibrium of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equilibrium. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concentration increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the average pair distance.     

Hydrophobic and ionic interactions in nanosized water droplets

A number of situations such as protein folding in confined spaces, lubrication in tight spaces, and chemical reactions in confined spaces require an understanding of water-mediated interactions. As an illustration of the profound effects of confinement on hydrophobic and ionic interactions, we investigate the solvation of methane and methane decorated with charges in spherically confined water droplets. Free energy profiles for a single methane molecule in droplets, ranging in diameter (D) from 1 to 4 nm, show that the droplet surfaces are strongly favorable as compared to the interior. From the temperature dependence of the free energy in D = 3 nm, we show that this effect is entropically driven. The potentials of mean force (PMFs) between two methane molecules show that the solvent separated minimum in the bulk is completely absent in confined water, independent of the droplet size since the solute particles are primarily associated with the droplet surface. The tendency of methanes with charges (M(q+) and M(q-) with q(+) = |q(-)| = 0.4e, where e is the electronic charge) to be pinned at the surface depends dramatically on the size of the water droplet. When D = 4 nm, the ions prefer the interior whereas for D < 4 nm the ions are localized at the surface, but with much less tendency than for methanes. Increasing the ion charge to e makes the surface strongly unfavorable. Reflecting the charge asymmetry of the water molecule, negative ions have a stronger preference for the surface compared to positive ions of the same charge magnitude. With increasing droplet size, the PMFs between M(q+) and M(q-) show decreasing influence of the boundary owing to the reduced tendency for surface solvation. We also show that as the solute charge density decreases the surface becomes less unfavorable. The implications of our results for the folding of proteins in confined spaces are outlined.     

Creaming Stability of Flocculated Monodisperse Oil-in-Water Emulsions

The influence of droplet flocculation on the creaming stability of monodisperse n-hexadecane oil-in-water emulsions was studied. The creaming velocity of emulsions with different droplet radii (0.43 and 0.86 μm), droplet concentrations (1-67 vol%), and sodium dodecyl sulfate (SDS) concentrations (7-80 mM) were measured. Depletion flocculation was observed in the emulsions when the aqueous phase SDS concentration exceeded a particular level ( approximately 40 mM for 0.43-μm droplets and approximately 15 mM for 0.86-μm droplets). Creaming was monitored by measuring the back-scattered light from an emulsion as a function of its height. The creaming velocity increased with increasing flocculation and decreased with increasing droplet concentration. These results have important implications for the formulation of emulsion-based materials. Copyright 2000 Academic Press.     

Microfluidic separation of satellite droplets as the basis of a monodispersed micron and submicron emulsification system

Emulsions are widely used to produce sol-gel, drugs, synthetic materials, and food products. Recent advancements in microfluidic droplet emulsion technology has enabled the precise sampling and processing of small volumes of fluids (picoliter to femtoliter) by the controlled viscous shearing in microchannels. However the generation of monodispersed droplets smaller than 1 microm without surfactants has been difficult to achieve. Normally, the generation of satellite droplets along with parent droplets is undesirable and makes it difficult to control volume and purity of samples in droplets. In this paper, however, several methods are presented to passively filter out satellite droplets from the generation of parent droplets and use these satellite droplets as the source for monodispersed production of submicron emulsions. A passive satellite droplet filtration system and a dynamic satellite droplet separation system are demonstrated. Satellite droplets are filtered from parent droplets with a two-layer channel geometry. This design allows the creation and collection of droplets that are less than 100 nm in diameter. In the dynamic separation system, satellite droplets of defined sizes can be selectively separated into different collecting zones. The separation of the satellite droplets into different collecting zones correlates with the cross channel position of the satellite droplets during the breakup of the liquid thread. The delay time for droplets to switch between the different alternating collecting zones is nominally 1 min and is proportional to the ratio of the oil shear flows. With our droplet generation system, monodispersed satellite droplets with an average radius of 2.23 +/- 0.11 microm, and bidispersed secondary and tertiary satellite droplets with radii of 1.55 +/- 0.07 microm and 372 +/- 46 nm respectively, have been dynamically separated and collected.     

Interfacial oil droplets

Very small, discrete oil droplets can form at the solid-liquid interface. We demonstrate this effect through formation of decane droplets at the interface between an aqueous ethanol solution and silicon wafers that have been made hydrophobic through self-assembly of octadecyltrichlorosilane (OTS). The droplets have a lens-like shape; the shape is approximately a spherical cap with a contact angle < 25 degrees. The heights of the droplets are about 2-50 nm, and diameters at the three-phase boundary are about 100-600 nm in 25% ethanol solution. The size and contact angle can be varied by changing the ethanol concentration. The contact angle of the very small droplets (height < 20 nm) is similar to the contact angle of macroscopic droplets (height approximately equal to 1 mm), so the line tension is very small. The droplets are only stable for a few hours: they gradually lose mass, presumably through Ostwald ripening. The drop perimeter is not pinned during ripening but retreats across the solid. We form the droplets by direct adsorption from an emulsion; evidence for adsorption is obtained by comparing the drop volumes in bulk to the volumes at the interface. The droplet sizes are obtained by dynamic light scattering and atomic force microscopy.     

Submicron polyethylene particles from catalytic emulsion polymerization

Particles of linear polyethylene (M(n) = (2-3) x 10(3) g x mol(-)(1); M(w)/M(n) = 2-4) obtained by catalytic emulsion polymerization of ethylene possess a nonspherical, lentil-like shape with an average aspect ratio of ca. 10 and diameters from 30 to >300 nm, as determined by TEM and AFM. The particle structure results from a stacking of the lamellae along the one shorter axis of the lentils (i.e., their height, by contrast to the diameter). In addition to these multilamellae particles, remarkably, a considerable number of the particles consist of only a single lamella. The thickness of the lamellae (spacing) as determined by TEM is only 9-11 nm, depending on the polymerization temperature during the synthesis of the dispersions. Crystallization in the dispersions during emulsion polymerization differs dramatically from the usual heterogeneous nucleation of bulk polyethylene samples. Each submicron droplet crystallizes individually, independently from the other droplets, resulting in large supercoolings of around 55 degrees C.     

Evolution of the solvent polarity in an electrospray plume

Solvent polarity plays an important role in electrospray ionization-mass spectrometry (ESI-MS), one of the most widely used analytical methods for biochemistry. To have a comprehensive understanding of how solvent polarity affects ESI-MS measurements, we systematically investigated the polarity change in the ESI plume formed from an ethanol solution using laser-induced fluorescence (LIF) spectroscopy. Two solvatochromic dyes (i.e., dyes whose fluorescence emission is sensitive to solvent polarity), Nile red and DCM (4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran), were used as probes. The peak emission wavelengths of these two dyes exhibited significant red shifts (8–12 nm) when the measuring spot was moved away from the spray tip and in radial direction in the plume, indicating a dramatic polarity change during shrinking of the droplets. The emission intensities were also measured with a polarity-insensitive dye as a reference. The results are consistent with the peak wavelength measurements. Two key mechanisms responsible for the change of solvent polarity in the plume were considered, water entrainment from the surrounding air and solvent evaporation. Furthermore, quantitative analysis of the solvent polarity change was performed by using the Lippert-Mataga polarity parameter Δf. The value of Δf reached 0.305–0.307 at the periphery of the ESI plume, which means that the solvent polarity in the smaller droplet is close to that of a mixture of 30% water and 70% ethanol (Δf = 0.307), even though the bulk solvent was ethanol containing less than 1% water as an impurity.     

Small-angle-neutron-scattering from giant water-in-oil microemulsion droplets. II. Polymer-decorated droplets in a quaternary system

Amphiphilic block copolymers of the type poly(ethylenepropylene)-co-poly(ethyleneoxide) dramatically enhance the solubilisation efficiency of non-ionic surfactants in microemulsions that contain equal volumes of water in oil. Consequently, the length scale of the microstructure of such bicontinuous microemulsions is dramatically increased up to the order of a few 100 nm. In this paper, we show that this so-called efficiency boosting effect can also be applied to water-in-oil microemulsions with droplet microstructure. Such giant water-in-oil microemulsions would provide confined compartments in which chemical reactions of biological macromolecules can be performed on a single molecule level. With this motivation we investigated the phase behavior and the microstructure of oil-rich microemulsions containing D(2)O, n-decane(d22), C(10)E(4) and the amphiphilic block copolymer PEP5-PEO5 [poly(ethylenepropylene)-co-poly(ethyleneoxide), weight per block of 5000 g/ mol]. We found that 15 wt % of water can be solubilised by 5 wt % of surfactant and block copolymer when about 6 wt % of surfactant is replaced by the block copolymer. Small-angle-neutron-scattering experiments were performed to determine the length scales and microstructure topologies of the oil-rich microemulsions. To analyze the scattering data, we derived a novel form factor that also takes into account the scattering contribution of the hydrophobic part of the block copolymer molecules that reside in the surfactant shell. The quantitative analysis of the scattering data with this form factor shows that the radius of the largest droplets amounts up to 30 nm. The novel form factor also yielded qualitative information on the stretching of the polymer chains in dependence on the polymer surface density and the droplet radius.     

Spectral study on the interaction of cryptophane-A and neutral molecules CHnCl4-n (n=0, 1, 2)

Cryptophane-A was synthesized from vanillin by a three-step method and its spectroscopic properties in different organic solvents were determined. Two absorption bands at about 240-250 and 280-290 nm were observed for cryptophane-A. A fluorescence emission peak was obtained at 320-330 nm using a solution of approximately 10(-5)M cryptophane-A. The interaction of cryptophane-A with chlorinated compounds CH(n)Cl(4 - n) (n=0, 1, 2) in dioxane and ethyl acetate solvents were studied in detail by fluorescence spectroscopy, respectively. The results show that cryptophane-A is well suited for inclusion of CH(2)Cl(2) to form a stable 1:1 complex and the binding constant was estimated to be 19+/-2M(-1). These results were also confirmed by (1)H NMR and CPK models. Larger similar molecules such as CHCl(3) and CCl(4) are unable to enter the cavity of cryptophane-A because of their bigger sizes. However, the fluorescence emission of cryptophane-A can be efficiently quenched by CHCl(3) and CCl(4), following the Stern-Volmer relationship.     

High-speed, clinical-scale microfluidic generation of stable phase-change droplets for gas embolotherapy

In this study we report on a microfluidic device and droplet formation regime capable of generating clinical-scale quantities of droplet emulsions suitable in size and functionality for in vivo therapeutics. By increasing the capillary number-based on the flow rate of the continuous outer phase-in our flow-focusing device, we examine three modes of droplet breakup: geometry-controlled, dripping, and jetting. Operation of our device in the dripping regime results in the generation of highly monodisperse liquid perfluoropentane droplets in the appropriate 3-6 μm range at rates exceeding 10(5) droplets per second. Based on experimental results relating droplet diameter and the ratio of the continuous and dispersed phase flow rates, we derive a power series equation, valid in the dripping regime, to predict droplet size, D(d) approximately equal 27(Q(C)/Q(D))(-5/12). The volatile droplets in this study are stable for weeks at room temperature yet undergo rapid liquid-to-gas phase transition, and volume expansion, above a uniform thermal activation threshold. The opportunity exists to potentiate locoregional cancer therapies such as thermal ablation and percutaneous ethanol injection using thermal or acoustic vaporization of these monodisperse phase-change droplets to intentionally occlude the vessels of a cancer.     

Influence of iota-carrageenan on droplet flocculation of beta-lactoglobulin-stabilized oil-in-water emulsions during thermal processing

The influence of thermal processing on droplet flocculation in oil-in-water emulsions stabilized by either beta-lactoglobulin (primary emulsions) or beta-lactoglobulin-iota-carrageenan (secondary emulsions) at pH 6 has been investigated. In the absence of salt, the zeta-potential of the primary emulsion was less negative (-40 mV) than that of the secondary emulsion (-55 mV) due to adsorption of anionic iota-carrageenan to the anionic beta-Lg-coated droplet surfaces. The zeta-potential and mean diameter (d(43) approximately 0.3 microm) of droplets in primary and secondary emulsions did not change after storage at temperatures ranging from 30 to 90 degrees C. In the presence of 150 mM NaCl, the zeta-potential of the primary emulsion was much less negative (-27 mV) than that of the secondary emulsion (-50 mV), suggesting that the latter was less influenced by electrostatic screening effects. The zeta-potential of the primary emulsions did not change after storage at elevated temperatures (30-90 degrees C). The zeta-potential of the secondary emulsions became less negative, and the aqueous phase iota-carrageenan concentration increased at storage temperatures exceeding 50 degrees C, indicating iota-carrageenan desorbed from the beta-Lg-coated droplets. In the primary emulsions, appreciable droplet flocculation (d(43) approximately 8 microm) occurred at temperatures below the thermal denaturation temperature (T(m)) of the adsorbed proteins due to surface denaturation, while more extensive flocculation (d(43) > 20 microm) occurred above T(m) due to thermal denaturation. In the secondary emulsions, the extent of droplet flocculation below T(m) was reduced substantially (d(43) approximately 0.8 microm), which was attributed to the ability of adsorbed carrageenan to increase droplet-droplet repulsion. However, extensive droplet flocculation was observed above T(m) because carrageenan desorbed from the droplet surfaces. Differential scanning calorimetry showed that iota-carrageenan and beta-Lg interacted strongly in aqueous solutions containing 0 mM NaCl, but not in those containing 150 mM NaCl, presumably because salt weakened the electrostatic attraction between the molecules.     

A photoactive molecular triad as a nanoscale power supply for a supramolecular machine

A tetrathiafulvalene-porphyrin-fullerene (TTF-P-C(60)) molecular triad, which generates electrical current by harnessing light energy when self-assembled onto gold electrodes, has been developed. The triad, composed of three unique electroactive components, namely, 1) an electron-donating TTF unit, 2) a chromophoric porphyrin unit, and 3) an electron-accepting C(60) unit, has been synthesized in a modular fashion. A disulfide-based anchoring group was tagged to the TTF end of the molecule in order to allow its self-assembly on gold surfaces. The surface coverage by the triad in a self-assembled monolayer (SAM) was estimated to be 1.4 nm(2) per molecule, a density which is consistent with hexagonal close-packing of the spherical C(60) component (diameter approximately 1 nm). In a closed electronic circuit, a triad-SAM functionalized working-electrode generates a switchable photocurrent of approximately 1.5 microA cm(-2) when irradiated with a 413 nm Kr-ion laser, a wavelength which is close to the porphyrin chromophore's absorption maximum peak at 420 nm. The electrical energy generated by the triad at the expense of the light energy is ultimately exploited to drive a supramolecular machine in the form of a [2]pseudorotaxane comprised of a pi-electron-deficient tetracationic cyclobis(paraquat-p-phenylene) (CBPQT(4+)) cyclophane and a pi-electron-rich 1,5-bis[(2-hydroxyethoxy) ethoxy]naphthalene (BHEEN) thread. The redox-induced dethreading of the CBPQT(4+) cyclophane from the BHEEN thread can be monitored by measuring the increase in the fluorescence intensity of the BHEEN unit. A gradual increase in the fluorescence intensity of the BHEEN unit concomitant with the photocurrent generation, even at a potential (0 V) much lower than that required (-300 mV) for the direct reduction of the CBPQT(4+) unit, confirms that the dethreading process is driven by the photocurrent generated by the triad-SAM.     

罗丹明B-PdI2-4缔合纳米粒子体系的极谱猝灭效应

缔合纳米微粒;共振散射;罗丹明B-PdI2-4缔合纳米粒子体系的极谱猝灭效应     

Use of helium nanodroplets for assembly, transport, and surface deposition of large molecular and atomic clusters

The utility of continuous beam of helium droplets for assembly, transport, and surface deposition of metal and molecular clusters is studied. Clusters of propyne having from about 10 to 10(4) molecules were obtained via sequential pickup of molecules by He droplets with average sizes in the range of 10(4)-10(7) atoms. The maximum attainable flux of the propyne molecules carried by He droplets was found to be in the range of (5-15)x10(15) molecules sr(-1) s(-1), being larger in larger droplets. The size of the clusters and the flux of the transported species are ultimately limited by the evaporative extinction of the entire helium droplet upon capture of particles. It is shown that the attenuation of the He droplet beam in the process of the cluster growth can be used in order to obtain the average size and the binding energy of the clusters. Furthermore, we used He droplets for assembling and surface deposition of gold and silver clusters having about 500 atoms. Typical deposition rate of metal atoms of about 3 x 10(15) atoms sr(-1) s(-1) is comparable to or larger than obtained with other beam deposition techniques. We propose that doping of He droplets by Au and Ag atoms in two separate pickup chambers leads to formation of the bimetal clusters having core-shell structure.     

Protein fluorescence measurements within electrospray droplets

The conformation of cytochrome c molecules within electrospray droplets is investigated by monitoring the laser induced fluorescence of its single tryptophan residue (Trp-59). By increasing the alcohol concentration of the electrosprayed solutions, protein denaturation is induced, giving rise to significant changes in the intensity of the detected fluorescence. Comparison with analogous denaturation experiments in solution provides information about the relative protein conformations and differences between the bulk-solution and droplet environments. Both electrospray-plume and bulk-solution fluorescence measurements using low methanol concentration solutions indicate the presence of folded protein structures. At high methanol content, fluorescence measurements are consistent with the presence of partly denatured or unfolded conformations. At intermediate methanol content, differences are observed between the extent of denaturation in solution and that within the droplets, suggesting electrosprayed proteins have more compact structures than those detected in bulk measurements using solutions of similar composition. This infers that some fraction of the proteins within the droplets have refolded relative to their bulk-solution conformation. Protein denaturation experiments using the low vapor pressure solvent 1-propanol indicate that differences between the droplet and solution measurements are not due to solvent evaporation effects. It is suggested that different droplet conformations are more likely the result of protein diffusion to the droplet surface and effects of the droplet/air interface. To our knowledge, these are the first reported measurements of protein fluorescence within electrospray droplets.