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Articles Analysis of the Phase Diagram and Microstructural Transitions in Phospholipid Microemulsion Systems Using High-Resolution Ultrasonic Spectroscopy Sinead Hickey,† M. Jayne Lawrence,‡ Sue A. Hagan,§ and Vitaly Buckin*,† Department of Chemistry, UCD, Belfield, Dublin 4, Ireland, Molecular Biophysics, Pharmaceutical Science Research DiVision, King’s College London, U.K., and Aphton Biopharma, Loughborough, U.K. ReceiVed October 10, 2005. In Final Form: March 31, 2006 In the present work, high-resolution ultrasonic spectroscopy was applied to analyze a pseudoternary phase diagram for mixtures consisting of water/isopropyl myristate/Epikuron 200 and a cosurfactant (n-propanol). Changes in the ultrasonic velocity and attenuation in the megahertz frequency range were measured in the course of titration of the oil/surfactant/cosurfactant mixture with water at 25 °C. The ultrasonic titration profiles showed several phase transitions in the samples, which allowed the construction of an “ultrasonic” phase diagram. Quantitative analysis of the ultrasonic parameters enabled the characterization of various phases (swollen micelles, microemulsion, coarse emulsion, and pseudo-bicontinuous) as well as the evaluation of the state of the water and the particle size. The particle size obtained for the microemulsion region ranged from 5 to 14 nm over the measured concentrations of water/isopropyl myristate/ Epikuron 200 and n-propanol, which agreed well with the previous literature data.
Introduction Microemulsions, originally described by Hoar and Schulman in 1943,1 are homogeneous, transparent, thermodynamically stable dispersions of water and oil. They are stabilized by a relatively large amount of surfactant, frequently in combination with a cosurfactant (typically a short-chain alcohol).2 The cosurfactant allows additional steric flexibility for surfactant rearrangements, enabling the formation of droplets and other microemulsion structures rather than liquid crystals. The particles in these systems are much smaller than the wavelength of light (below 200 nm) and therefore appear transparent. Microemulsions, unlike emulsions, are thermodynamically stable. A microemulsion system can exist as either oil-in-water, water-in-oil, or bicontinuous. These systems can be either single-phase systems or part of a two- (or three-) phase system in which the microemulsion phase coexists with an excess of predominately dispersed (and continuous) phase.3 Microemulsions are considered to be ideal candidates for pharmaceutical drug delivery because of their ease of preparation and thermodynamic stability.4-7 However, to be used as drug delivery systems, extensive characterization of their physicochemical properties is required. This includes analysis of microemulsion phase diagrams (composition of the mixture at * Corresponding author. Address: Department of Chemistry, UCD, Belfield, Dublin 4, Ireland. E-mail:
[email protected]. Tel: + 353 1 716 2371. Fax: + 353 1 716 1178. † UCD, Belfield. ‡ King’s College London. § Aphton Biopharma. (1) Hoar, T. P.; Schulman, J. H. Nature 1943, 152, 102-103. (2) Aboofazeli, R.; Lawrence, M. J. Int. J. Pharm. 1993, 93, 161-175. (3) Winsor, P. A. J. Chem. Soc., Faraday Trans. 1948, 44 (1), 376-398. (4) Lawrence, M. J.; Rees, G. D. AdV. Drug DeliVery ReV. 2000, 45, 89-121. (5) Malcolmson, C.; Satra, C.; Kantaria, S.; Sidhu, A.; Lawrence, M. J. J. Pharm. Sci. 1998, 87 (1), 109-116. (6) Gao, Z.; Choi, H.; Shin, H.; Park, K.; Lim, S.; Hwang, K.; Kim, C. Int. J. Pharm. 1998, 161, 75-86. (7) Aboofazeli, R.; Lawrence, M. J. Int. J. Pharm. 1994, 106 (1), 51-61.
which the microemulsion is formed), size of the microemulsion droplet, and hydration (solvation effects). Various spectroscopic techniques have been employed for this characterization, including light scattering (dynamic and static), neutron scattering, pulsedfield gradient NMR spectroscopy, and others.2,8-11 In addition, microemulsions have been characterized by measuring their electrical properties using conductivity or electroacoustic techniques;12,13 however, these last techniques are often limited to charged particles in a conducting medium. To obtain a reliable estimate of particle sizes using light-scattering techniques, significant dilution of the samples or complicated calculations are required. The dilution of a microemulsion containing a cosurfactant often leads to a change in either the microstructure of the system or, in extreme cases, the disappearance of the microemulsion droplets.14,15 Traditional spectroscopy (light-based techniques, NMR, etc.) employs the electromagnetic wave. A second type of wave that propagates through materials is the ultrasonic wave (high frequency (above 100 kHz) acoustical waves). This wave probes the elastic rather than the electric and magnetic properties of materials. As it transverses a sample, compressions and decompressions in the ultrasonic wave change the distance between molecules within the sample, which, in turn, respond by intermolecular repulsions and attractions. This ability of ultrasonic waves to probe intermolecular forces allows access to molecular (8) Reynolds, P. A.; Gilbert, E. P.; White, J. W. J. Phys. Chem. B 2001, 105, 6925-6932. (9) Benjamins, J.; Thuresson, K.; Nylander, T. Langmuir 2005, 21, 28042810. (10) Soderman, O.; Nyden, M. Colloids Surf., A 1999, 158 (1-2), 273-280. (11) Giustini, M.; Palazzo, G.; Giomini, M.; Ceglie, A. J. Phys. Chem. 1996, 100, 3190-3198. (12) Jian, X.; Ganzuo, L.; Zhiqiang, Z.; Guowei, Z.; Kejian, J. Colloids Surf., A 2001, 191 (3), 269-278. (13) Dukhin, A. S.; Goetz, P. J.; Wines, T. H.; Somasundaran, P. Colloids. Surf., A. 2000, 173 (1-3), 127-158. (14) Hou, M. J.; Kim, M.; Shah, D. O. J. Colloid Interface Sci. 1988, 123 (2), 398-412. (15) Coupland, J. N.; McClements, J. D. J. Food Eng. 2001, 50 (2), 117-120.
10.1021/la052735t CCC: $33.50 © 2006 American Chemical Society Published on Web 05/23/2006
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levels of organization. Measurements of the scattering effects of ultrasonic waves allow particle size analysis. Traditionally, the application of ultrasonic techniques has been problematic because of the necessity of having large sample volumes and limited resolution. This was overcome recently with the introduction of high-resolution ultrasonic spectrometers (HR-US).16-18 This technique allows fast, nondestructive, and highly sensitive measurements to be performed on small volumes for real-time material analysis, and eliminates the need for dilution and complicated calculations. HR-US has successfully been used for the characterization of a number of pharmaceutical and biochemical systems. These include the monitoring of immunochemical reactions,19 gelatinization of starch,20 crystallization of proteins,21 the characterization and determination of particle size of emulsions,22,23 and other micelle systems.24-26 This technique enables the application of a titration regime, which involves stirring the sample within the ultrasound cell while adding aliquots of the dispersed phase, thereby saving time and reducing errors in sample preparation. Here, we report the application of the HR-US technique for analysis of a pseudoternary phase diagram for a system consisting of an oil (isopropyl myristate (IPM)), a surfactant (Epikuron 200), a cosurfactant (n-propanol), and water. Changes in the ultrasonic velocity and in the ultrasonic attenuation were measured when the oil/surfactant/cosurfactant mixture was titrated with water. These changes were then related to changes in the supramolecular architecture. An “ultrasonic” phase diagram was constructed from the ultrasonic titration profiles. The particle sizes of the swollen micelles and the microemulsion region in the system were calculated using the thermophysical properties of the dispersed water phase and continuous oil and surfactants phase. Experimental Section Materials. Epikuron 200 (E200; soy lecithin, minimum 95 wt % phosphatidylcholine; fatty acid content: palmitic and stearic, 1620%; oleic acid, 8-12%; linoleic acid, 62-66%; linolenic acid, 6-8%)2 was supplied by Lucas Meyer (Germany) (King’s College London). IPM (cat. no. 110-27-0) and n-propanol (cat. no. 71-23-8) were obtained from the Sigma-Aldrich Chemical Co. Ltd. All reagents were of the highest purity available and were used as received. Water was obtained from a Millipore Super-Q System. All samples were prepared by weight. Ultrasonic Measurements. Ultrasonic velocity (u) and attenuation (R) measurements were performed over a frequency range between 2 and 12 MHz using a HR-US 102 instrument (Ultrasonic Scientific Ltd.). This device is equipped with two cells enabling measurements in single-cell or differential regimes. The differential regime allows a resolution of 0.2 mm/s for the ultrasonic velocity and 0.2% for ultrasonic attenuation. Single-cell measurements were performed in this work since the peak resolution was not required. This gave a (16) Buckin, V.; O’Driscoll, B.; Smyth, C. Spectrosc. Eur. 2003, 15 (1), 2025. (17) Kudryashov, E.; Smyth, C.; Duffy, G.; Buckin, V. Prog. Colloid Polym. Sci. 2000, 115, 287-294. (18) Buckin, V.; O’Driscoll, B. Lab Plus Int. 2002, 16, 17-21. (19) Buckin, V.; Kudryashov, E. Biochemist 2002, 24 (4), 25-27. (20) Lehmann, L.; Kudryashov, E.; Buckin, V. Prog. Colloid Polym. Sci. 2004, 123, 136-140. (21) Smyth, C.; O’Driscoll, B.; Lawrence, M. J.; Hickey, S.; O’Regan, T.; Buckin, V. Pharm. Technol. Eur. 2004, 16 (6), 31-34. (22) Buckin, V.; O’Driscoll, B. Labmate UK & Ireland 2003, 8, 7-8. (23) Kudryashov, E.; Smyth, C.; O’Driscoll, B.; Buckin, V. Pharm. Technol. Eur. 2005, 1, 40-45. (24) Buckin, V.; Kudryashov, E.; O’Driscoll, B. Pharm. Technol. Eur. 2002, 14 (12), 33-37. (25) Kudryashov, E.; Kapustina, T.; Morrissey, S.; Buckin, V.; Dawson, K. J. Colloid Interface Sci. 1998, 203, 59-68. (26) Smyth, C.; Dawson, K.; Buckin, V. Prog. Colloid Polym. Sci. 1999, 112, 221-226.
Hickey et al. reproducibility of 1 cm/s for ultrasonic velocity and 0.4% for ultrasonic attenuation. All measurements were performed at a constant temperature of 25 °C controlled by a Haake F8 waterbath, which provided a temperature stability of ( 0.01 °C. For ultrasonic velocity and attenuation measurements, the appropriate amounts of E200, n-propanol (1:1 ratio) and IPM were weighed into screw-capped vials. The samples were stirred until a clear solution was obtained. The samples were then placed into a syringe along with a magnetic stirrer and degassed under vacuum over a stirring plate. A 1.3 mL portion of each sample was placed in the HR-US 102 cell using a calibrated Hamilton syringe. An automated Hamilton dispenser series 500 and a 100 µL Hamilton syringe were used for titrations. Measurements of the ultrasonic velocity and the attenuation were performed while the sample was titrated with aliquots of degassed water through a septum in the lid of the cell. The samples were stirred at the bottom of the cell using the built-in HR-US digital stirring system, and at the top of the cell using a mechanical micromotor-based stirrer. This provided fast, effective, and homogeneous stirring. The samples were stirred until no further change in ultrasonic velocity and attenuation occurred, indicating equilibrium had been reached. The titration regime was used for water concentrations up to 50 wt % in the phase diagram. Above this concentration, a detailed titration profile was not required, and therefore measurements in individually prepared samples were used. In addition, the titration measurements were replicated in the whole concentration range using samples that were individually prepared and equilibrated before being placed in the HR-US cell. The results showed very good agreement between the two methods, and revealed that the titration method gave a more refined picture and was less time-consuming. Density. The density of the various IPM-E200/n-propanol solutions were measured using a vibrating tube densimeter (DMA602, Anton Paar, Austria) with a resolution of (1.5 × 10-6 g cm-3. A 1 mL portion of each solution was degassed and placed in the densimeter. The temperature was controlled using a Haake F8 waterbath with a stability of (0.01 °C. Measurements were made at 15, 25, and 35 °C, and the density and thermal expansion coefficient were calculated from these data. Differential Scanning Calorimetry. The specific heat capacity of the various IPM-E200/n-propanol solutions were measured using a DSC 2010 differential scanning calorimeter (TA Instruments). A 10.5 mg portion of the degassed sample was weighed into an aluminum crucible and placed in the sample holder. Measurements of specific heat capacity were carried out for each ratio of IPME200/n-propanol using water as a reference from 0 to 40 °C at a ramp rate of 1 °C per minute. Thermal Conductivity. Thermal conductivity measurements were carried out using a thermal conductivity probe constructed by Dr. James Lyng of the Department of Food Science, UCD, Dublin 4.27 The probe was placed into a plastic cup containing approximately 50 mL of the sample. The ratio of heat flux density to temperature gradient (over a 10 °C temperature range) was measured. Viscosity. The viscosity of the various IPM-E200/n-propanol solutions at 25 °C were measured using a Rheometric Scientific SR2000 rheometer equipped with an HAAKE F8 (Karlsrue, Germany) programmable water circulator. Cuette geometry was used with a gap size of 5 mm and at a frequency of 1 rad/s. Construction of Pseudoternary Phase Diagrams. A phase diagram was constructed (Figure 4) indicating phase transition points (abrupt changes in ultrasonic profile) and a microemulsion region for various water/oil/surfactant concentrations. To show variation in IPM/E200/n-propanol/water on the phase diagram, the top apex of the triangle represents the water, with the other apexes representing oil and the lecithin/alcohol concentrations. Particle Size. In homogeneous materials, velocity and attenuation are determined by intrinsic properties of the medium. Heterogeneous dispersions produce an additional “scattering” contribution to ultrasonic velocity and attenuation, which is a function of particle (27) Lyng, J. G.; Scully, M.; McKenna, B. M. J. Muscle Foods 2002, 13 (2), 123-142.
Phase Transitions for Phospholipid Microemulsions
Figure 1. Ultrasonic titration profile for 75% IPM and 25% E200/ n-propanol (1:1) mixture at 25 °C. (A,B) Ultrasonic velocity profile at 5.2 MHz. The filled symbols represent direct titration of the oil and surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell. (C,D) Ultrasonic attenuation profile at four different frequencies: (9,0) 2.7 MHz, (b,O) 5.2 MHz, ([,]) 8.5 MHz, and (f,g) 11.7 MHz. The filled symbols represent direct titration of the oil and surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell.
Figure 2. Ultrasonic titration profile for 50% IPM and 50% E200/ n-propanol (1:1) mixture at 25 °C. (A,B) Ultrasonic velocity profile at 5.2 MHz. The filled symbols represent direct titration of the oil and surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell. (C,D) Ultrasonic attenuation profile at four different frequencies: (9,0) 2.7 MHz, (b,O) 5.2 MHz, ([,]) 8.5 MHz, and (f,g) 11.7 MHz. The filled symbols represent direct titration of the oil & surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell. size. In the long wavelength limit, that is, when the wavelength of ultrasound (λ) is much greater than the particle radius (r), explicit expressions for the ultrasonic scattering in dispersions have been derived.28-31 The basic mechanism of interaction of the ultrasonic wave with particles in dispersions in this regime contains two major contributors, thermoelastic and viscoinertial scattering, which are associated with the heat wave and shear wave generated in the ultrasonic field. The Psize 2.27 software module provided with the HR-US 102 spectrometer (Ultrasonic Scientific Ltd.) is based on the most widely used multiple-scattering theoretical approaches.28-31 This software utilizes the theoretical relationship between the complex wave vector and the scattering coefficients, which are determined by the physical properties of the spherical particles and the continuous medium.30,31 (28) Epstein, P. S.; Carhart, R. R. J. Acoust. Soc. Am. 1953, 25 (3), 553-562. (29) Waterman, P. C.; Truell, R. J. Math. Phys. 1961, 2 (4), 512-537. (30) Allegra, J. R.; Hawley, S. A. J. Acoust. Soc. Am. 1972, 51 (5 (2)), 15451564. (31) Povey, M. J. Ultrasonic Techniques for Fluid Characterisation; Academic Press: London, 1997.
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Figure 3. Ultrasonic titration profile for 25% IPM and 75% E200/ n-propanol (1:1) at 25 °C. (A,B) Ultrasonic velocity profile at 5.2 MHz. The filled symbols represent direct titration of the oil and surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell. (C,D) Ultrasonic attenuation profile at four different frequencies: (9,0) 2.7 MHz, (b,O) 5.2 MHz, ([,]) 8.5 MHz, and (f,g) 11.7 MHz. The filled symbols represent direct titration of the oil and surfactants with water in the ultrasonic cell, and the clear symbols represent preparation of individual samples before being placed into the ultrasonic cell.
Figure 4. Pseudoternary phase diagram of water/IPM/E200 and n-propanol (1:1) at 25 °C. The dotted line represents results obtained by Aboofazeli et al.2 for the transition from microemulsion to the pseudo-bicontinuous region. The points connected by the full line represent phase changes observed in the ultrasonic titration profiles. It allows calculation of particle sizes in emulsions, suspensions, and other colloidal systems based on the measured attenuation values and the thermophysical parameters of the continuous medium and the particles. In addition, it allows calculations of the changes in ultrasonic velocity and attenuation caused by the scattering effects for particles of a given size. The required thermal parameters for the dispersed and continuous phases were measured at 25 °C (see Table 1). Using the physical parameters for the continuous medium and water phase, the particle sizes (diameter) of the microemulsion droplets were determined (Figure 5 A-C), and the predicted attenuation and velocity profiles were generated (Figure 6). The two highest frequencies employed in our measurements, 8.5 and 11.7 MHz, which provide the highest attenuation response and the shortest length of the penetration depth for the heat wave (see discussion below) were used in our calculations of particle sizes.
Results The change in the ultrasonic velocity and attenuation due to the addition of water to various ratios of IPM-E200/n-propanol (1:1) at 25 °C is shown in Figures 1-3. The ultrasonic velocity titration profiles (Figures 1-3, panels A and B) did not show a significant dependence on frequency within the resolution of measurement, and therefore, for clarity, data for one frequency, 5.2 MHz, are shown. The ultrasonic attenuation shows significant
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Table 1. Thermophysical Properties of the Dispersed Phase (Water) and the Continuous Phase (IPM-E200/n-Propanol (1:1)) at 25 °C Used in the Particle Size Calculations and Concentration Dependence of Ultrasonic Velocity in Ideal Mixtures physical property
watera
75:25 IPM-E200/ n-propanol
50:50 IPM-E200/ n-propanol
25:75 IPM-E200/ n-propanol
density (kg m-3) (specific volume (m3 kg-1)) ultrasonic velocity (m s-1) ultrasonic attenuation coefficient (R/f2) (m-1s2) viscosity (Pa s) thermal conductivity, J (m s K)-1 thermal expansion coefficient (104 K-1) (specific thermal expansion (106 m3 K-1)) specific heat capacity (kJ kg-1K-1)
997 (1.003 × 10-3) 1497 2.3 × 10-14
861.3 ( 0.1 (1.161 × 10-3) 1321.1 ( 0.5 2.2 × 10-13 ( 0.1 × 10-13
872.9 ( 0.1 (1.161 × 10-3) 1323.8 ( 0.5 3.3 × 10-13 ( 0.10 × 10-13
888.4( 0.1 (1.161 × 10-3) 1328.2 ( 0.5 5.5 × 10-13 ( 0.2 × 10-13
0.000895 0.60
0.0063 ( 0.0002 0.19 ( 0.01
0.0099 ( 0.0002 0.19 ( 0.01
0.0130 ( 0.0002 0.19 ( 0.01
2.57
8.77 ( 0.07
8.42 ( 0.07
8.38 ( 0.07
(0.258)
(1.02)
(0.965)
(0.943)
4180
1.25 ( 0.04
1.54 ( 0.04
1.99 ( 0.04
a
Reference 43.
Figure 6. (A) The predicted change in ultrasonic parameters with increasing size of water droplets for the mixture 75:25 IPM-E200/ n-propanol (1:1) and water (4 wt %) at 5.2 MHz and 25 °C, calculated with the HR-US Psize 2.27 particle size module using the physical properties for the continuous and dispersed phases given in Table 1. (B) Zoom to the initial part of the attenuation curve.
Figure 5. Evolution of particle size with the addition of water at 25 °C, calculated from ultrasonic attenuation measured at various frequencies: ([) 8.5 MHz; (9) 11.7 MHz. (A) Particle size of 75% IPM and 25% E200/n-propanol (1:1). (B) Particle size of 50% IPM and 50% E200/n-propanol (1:1). (C) Particle size of 25% IPM and 75% E200/n-propanol (1:1). Solid line marked by symbols (0) represents the size obtained previously using PCS, corrected for the length of the surfactant chain on the surface of the particle.35
frequency dependence, and a number of different frequencies between 2 and 12 MHz are plotted (Figures 1-3, panels C and D). Figures 1 and 2 show a comparison of the ultrasonic concentration profiles for two types of sample preparation. One of the curves in each profile represents the titration technique, and the second curve is obtained from the measurement of individual samples that have been prepared and equilibrated before being placed in the HR-US 102 cell. Good correlation is observed between the two preparation techniques. A number of stages of microstructural rearrangement can be resolved in the ultrasonic velocity and attenuation profiles in Figures 1-3. The ultrasonic velocity profiles exhibit a number of abrupt changes in slope. At low levels of added water, the
ultrasonic attenuation remains constant. Upon further addition of water, it begins to increase at all frequencies, with higher frequencies exhibiting a higher magnitude of increase. Following this stage, the ultrasonic attenuation plateaus briefly and then scatters at higher concentrations of water. Similar profiles are observed for each ratio of IPM-E200/n-propanol studied; however, the water concentration at which these transitions occur increases with an increasing amount of surfactant present in the system.
Concentration Dependence of Ultrasonic Velocity. Basic Relationships. In liquids with low ultrasonic attenuation (attenuation, R, per wavelength, λ, is significantly smaller than 1, Rλ , 1), the relationship between ultrasonic velocity and the physical characteristics of a liquid is described by a well-known equation:32
u)
1
xβSF
(1)
where F is the density of the liquid, and βS is the coefficient of adiabatic compressibility, which represents a relative change of volume, V, per unit of pressure applied, P, at constant entropy, S:
βS ) -
1 ∂V V ∂P S
( )
(2)
When analyzing the concentration dependence of ultrasonic (32) Urick, R. J. J. Appl. Phys. 1947, 18, 983-987.
Phase Transitions for Phospholipid Microemulsions
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velocity, it useful to express the values of βS and F in eq 1 through the parameters (which have an additive thermodynamic sense) specific volume, V (≡ 1/F, volume of unit of mass), and specific adiabatic compressibility, kS (≡ VβS ) -(∂V/∂P)S, compressibility of a unit of mass). This provides the following relationship for ultrasonic velocity:
u)
V
xkS
(3)
For a liquid mixture consisting of a solute (or particle) and a solvent (continuous medium), the specific volume and compressibility can be expressed in terms of the apparent specific volume of the solute, φV, and the apparent specific adiabatic compressibility of the solute, φKS, defined as
φV )
V - V0 m
(4a)
φKS )
K - K0 m
(4b)
where V is the volume of the mixture, V0 is the volume of the pure solvent before mixing, KS (≡ -(∂V/∂P)S) is the adiabatic compressibility of the mixture, KSo (≡ -(∂Vo/∂P)S) is the adiabatic compressibility of a pure solvent, and m is the mass of the solute in the mixture. Following the above definitions of φV and φKS, the specific volume and adiabatic compressibility of a mixture can be expressed as a sum:
solvent will affect the measured adiabatic compressibility of the mixture and therefore ultrasonic velocity. The thermal low-frequency limit suggests that the oscillations of pressure (and temperature) are so slow that complete heat equilibration between the solute and the solvent occurs, and therefore the temperature in the solute is always the same as that in the solvent. At this limit, all parts of the compressed volume are effectively at thermal equilibrium. Therefore, the measured adiabatic compressibility of the mixture at this limit is not additive, and the specific apparent compressibility of the solute in this limit, φK 0S, depends on the concentration of the solute. Analysis of the concentration dependence of φK 0S requires its relation to thermodynamic parameters, which are additive at these conditions of thermal equilibrium. A well-known thermodynamic relationship can be used for this purpose:
kS ) kT - T
e2 cP
(8)
where T is the temperature (K), kT (≡-(∂V/∂P)T) is the specific isothermal compressibility, e (≡(∂V/∂T)P) is the specific heat expansion, and cP is the specific heat capacity at constant pressure. The values of kT, e, and cP of the mixtures can be represented as the sum of the contributions of the pure solvent, kT0, e0, and cP0, and the solute, φKT, φE, and φCP, defined as represented by eqs 4:
kT ) kT0(1 - w) + wφKT
(9a)
V ) Vo(1 - w) + wφV
(5a)
e ) e0(1 - w) + wφE
(9b)
kS ) kSo(1 - w) + wφKS
(5b)
cP ) kP0(1 - w) + wφCP
(9c)
where w is the mass fraction of the solute in the mixture. The combination of eqs 5 and 3 provides the following relationship for the analysis of the concentration (w) dependence of ultrasonic velocity in a mixture:
u)
Vo + w(φV - Vo)
xkSo + w(φKS - kSo)
(6)
φK 0S )
(7)
Thermal Low- and Thermal High-Frequency Limits for Ultrasonic Velocity. A. Thermodynamic Relationships. The apparent specific adiabatic compressibility, in general, is not additive and exhibits more complex behavior, which depends on the frequency at which compressibility is measured. The basics of this behavior could be discussed in terms of thermal highfrequency and thermal low-frequency limits. Adiabatic compressions are accompanied by a heating of the compressed volume (cooling at decompression), which results in a change in the temperature. The amplitude of the temperature change is determined by the thermophysical properties of the material. A difference in the thermophysical properties of the solute and the solvent will result in a difference in their temperatures during a course of compression and decompression in ultrasonic wave and therefore the heat flow between them. The extent of the heat exchange between the solute and the
(
(
2
)
( ))
φCP φE φCP φE φE +w -2 + e0 T e 0 cP0 cP0 e0 e0 φKT φCP cP0 1-w+w cP0 2
Because volume as a thermodynamic characteristic is additive, that is, the volume of a system is the sum of the volumes of subsystems, the specific apparent volume of the solute in ideal mixtures does not depend on concentration, w, and is equal to the specific volume of the solute:
φV ) Vsolute
A combination of eqs 8 and 9 provides the following relationship for the concentration dependence of the apparent specific adiabatic compressibility at a thermal low-frequency limit, φK 0S (superscript 0 stands for zero frequency):
2
(10)
Because of the additivity of the isothermal compressibility, heat expansion, and heat capacity, the specific apparent characteristics φKT, φE, and φCP in ideal mixtures do not depend on concentration, and are equal to the corresponding specific characteristics of solute:
φKT ) kT solute; φE ) esolute; φCP ) cP solute
(11)
Equations 6, 7, 10, and 11 allow calculation of the concentration dependence of ultrasonic velocity in ideal mixtures for the thermal low-frequency limit. The thermal high-frequency limit implies that oscillations of pressure (and therefore of temperature) are too fast for a heat flow between the subsystems, the solute, and solvent. In this case, the subsystems are effectively thermally insulated, and the adiabatic compressibility is an additive sum of adiabatic compressibilities of subsystems. Therefore, in ideal mixtures, the specific apparent compressibility of the solute at the thermal high-frequency limit, φK ∞S (superscript ∞ stands for infinite frequency), does not depend on concentration, w, and is equal
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to the specific adiabatic compressibility of the solute:
φK ∞S ) kS solute
(12)
A combination of this equation and eq 5 is equivalent to a wellcited31 expression for the compressibility of a two-component mixture:
β ) β0x0 + βsolutexsolute
(13)
where x0 and xsolute are the volume fractions of the solvent and the solute, respectively, and β0 and βsolute are the coefficients of compressibility of the solvent and the solute, respectively. Equations 6, 7, and 12 allow calculation of the concentration dependence of ultrasonic velocity in ideal mixtures for the thermal high-frequency limit. The calculated dependence of ultrasonic velocity on the concentration of water in our mixtures (thermophysical parameters of Table 1) at the thermal low-frequency and high-frequency limits at 25 °C and ideal conditions is presented in Figure 7. As can be seen from the figure, the ultrasonic velocity curves for the thermal high-frequency limit for all three systems are nearly the same and show a monotonic (nearly linear) increase from the value corresponding to the pure solvent to the value of pure solute (water). In contrast to this, the thermal low-frequency curves are significantly different from each other and exhibit nonmonotonic behavior. At the thermal low-frequency limit in our systems, the addition of water, at first, leads to a decrease in velocity in the mixture, although the velocity in the water is higher than that in the solvent. This effect as well as the large difference in the ideal behavior of three systems could be explained by the abnormally high specific heat capacity of water, which results in a large ratio of φCP/cP0 in eq 10. At low concentrations (w) this leads to a large value of apparent adiabatic compressibility, φKS, and therefore low ultrasonic velocity. The large heat capacity of the solute in our systems provides a significant heat exchange between the solvent and the solute in adiabatic compressions and decompressions, thus decreasing the amplitude of temperature changes. This affects the elastic response of the solvent to changes in pressure, making it effectively more compressible. B. Structural Aspects. Realization of the thermal low- and high-frequency limits of ultrasonic velocity in our systems can be understood from considerations on the length scale of the heat exchange, which allow linking the thermodynamic definitions with the structural ones. In the case of an oscillating temperature gradient between two contacting media, the effective heat exchange between them occurs within the scale of the penetration depth of the heat wave, δT (distance over which the amplitude of the heat wave decays by e times). The value of δT is a function of the thermal properties of the medium and the frequency, f:
δT )
x
γ πfFcP
(14)
where γ is the thermal conductivity of the medium, and f is the frequency at which the measurements are performed.44 For our systems, at a frequency of 5.2 MHz, the value of δT is between (33) Madigosky, W. M.; Warfield, R. W. J. Chem. Phys. 1987, 86 (3), 14911497. (34) Aboofazeli, R.; Barlow, D. J.; Lawrence, M. J. AAPS PharmSci 2000, 2 (13), article 1. (35) Aboofazeli, R.; Barlow, D. J.; Lawrence, M. J. AAPS PharmSci 2000, 2 (3), article 19. (36) Savazyan, A. P. Annu. ReV. Biophys. Chem. 1991, 20, 321-342. (37) Buckin, V. Biophys. Chem. 1988, 29, 283-292. (38) Buckin, V.; Kankiya, B. I.; Kazaryan, R. L. Biophys. Chem. 1989, 34, 211-223.
Figure 7. Predicted change in ultrasonic velocity with the concentration of water for the high-frequency (H) and the lowfrequency (L) regimes in our mixtures at 25 °C and a frequency of 5.2 MHz. 75% IPM and 25% E200/n-propanol (1:1): H1 and L1; 50% IPM and 50% E200/n-propanol (1:1): H2 and L2; 25% IPM and 75% E200/n-propanol (1:1): H3 and L3. Equations 6-8, 9, and 10 for ideal mixtures were used as described in the text.
80 and 100 nm for the three continuous media. Therefore, our microemulsions with a droplet size less than 10 nm are described by the thermal low-frequency limit of ultrasonic velocity and emulsion, whereas those with droplet sizes of 1 µm and higher are described by the thermal high-frequency limit. The evolution of ultrasonic velocity from the thermal low- to high-frequency limits at a growing particle size can be described by general ultrasonic scattering theories. Figure 6 represents an example of calculated dependence of ultrasonic velocity on the size of water droplets in our mixtures (thermophysical parameters of Table 1) at a constant volume fraction at 4% of water, at 5.2 MHz and 25 °C, using the described above Psize 2.27 software module provided with the HR-US 102 spectrometer. The total difference in ultrasonic velocity between the low- and highfrequency limits, which corresponds to zero and an infinite size of water droplets (Figure 6), agrees well with the values calculated using eqs 6, 7, and 10 for the low and eqs 6, 7, and 12 for the high-frequency limits for concentrations below 5%. We found that, at concentrations higher than 10%, the scattering theory progressively overestimates this difference, compared with our thermodynamic calculations. This overestimation should be expected since the utilized theory30 does not account for the heat exchange between the particles.31,45 The contribution of the interparticle heat exchange increases with concentration as the portion of volume within the distance δT around the particle, occupied by neighboring particles, rises. This is of special importance for dispersions of small, nanometer-range particles characterized by a short average interparticle distance (8 nm of surface-to-surface distance for 10% w/w of a 10 nm diameter dispersion), compared with large, micrometer-range particles of the same concentration (800 nm of surface-to-surface distance for 10% w/w of a 1 µm diameter dispersion). The concentration profile for ultrasonic velocity at the thermal high-frequency limit predicted by the scattering theory agrees well with our thermodynamic calculations in the whole concentration range. As (39) Buckin, V.; Kankia, B. I.; Bulichov, N. V.; Lebedev, A. V.; Gukovsky, I. Ya.; Chupnna, V. P.; Sarvazyan, A. P.; Williams, A. R. Nature 1989, 340, 321-322. (40) Stuehr, J.; Yeager, E. In Physical Acoustics; Mason, W. P., Ed.; Academic Press: San Diego, CA, 1965; Vol. 2A, pp 351-358. (41) Nemethy, G.; Scheraga, H. A. J. Chem. Phys. 1962, 36 (12), 3382-3400. (42) Binks, B. P.; Meunier, J.; Langevin, D. Prog. Colloid Polym. Sci. 1989, 79, 208-213. (43) Herrmann, N.; McClements, D. J. Langmuir 1999, 15, 7937-7939. (44) Thurston, R. N. In Physical Acoustics; Mason, W. P., Ed.; Academic Press: New York, 1964; Vol. 1A, pp 2-110. (45) McClements, J.; Hemar, Y.; Herrmann, N. J. Acoust. Soc. Am. 1998, 105 (2), 915-918.
Phase Transitions for Phospholipid Microemulsions
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discussed in the previous sections, the scattering theory utilized includes several “scattering” effects (viscous-inertial, etc.), in addition to the thermal one, which affect the ultrasonic parameters.31 It is expected that the contribution of other than thermal effects to ultrasonic parameters in emulsions is small,46 which is especially true for our systems because of the low viscosity of the continuous medium and the small difference in the density of the continuous medium and that of the particles.30,31,46 This is well supported by the agreement between our thermodynamic calculations and the prediction of the scattering theory (Figures 6 and 7). The predicted concentration dependence of ultrasonic velocity in ideal mixtures with thermophysical parameters of our systems (Figure 7) can be used for assessment of the state of water in our mixtures. For this purpose, the partial concentration increment of the ultrasonic velocity, a, was used:
a)
δu uδw
(15)
where δu is the change in ultrasonic velocity caused by a change in concentration (addition of water in our systems), δw. The value of a represents the slope of the concentration dependence of ultrasonic velocity. Comparison of experimental and calculated values in different concentration ranges allows one to assess a difference between the ideal (bulk) state of added water and the state of water in the real system.
Particle Size The particle sizes (diameter) of the dispersed water droplets were determined at the various concentrations of IPM/E200/npropanol/water (Figure 5A-C). The particle size calculations were based on a two-phase model system composed of a dispersed water phase in a continuous medium of IPM, E200, and n-propanol, assuming that all the water added to the system is incorporated into the dispersed phase. These calculations provide an effective particle size, as some differences in the physical properties of the bulk and hydration water as well as the existence of an intermediate layer of surfactant tails between both phases are not included in the model. Therefore, the real particle size may deviate partially from the calculated one. This uncertainty can also be attributed to a complexity of the definition of the particle size in a system where the size of the intermediate layer is comparable with the whole particle. The effect of the alcohol cosurfactant present in the water dispersed phase on the values of ultrasonic attenuation in water droplets used in the particle size calculations was also considered. According to Madigosky,33 an increase in the concentration of n-propanol in water leads to some increase in attenuation, beginning from a mole fraction of 0.01 of propanol. This peaks at a propanol mole fraction of 0.14 and decreases steeply until a mole fraction of 0.5 where it plateaus to a low level and remains constant. The concentration range of n-propanol used in our systems corresponds to the concentration range above this peak, thus indicating that the effect of n-propanol in the water phase on the total ultrasonic attenuation could be neglected.
Discussion Ultrasonic Phase Diagram. The ultrasonic velocity and attenuation profiles clearly show the existence of several stages. Transitions between the stages are marked as an abrupt change in the slope of the ultrasonic velocity and attenuation curves (marked by gray columns in Figures 1, 2, and 3). (46) McClements, J. Langmuir 1996, 12, 3454-3461.
Figure 8. Illustration of various proposed phases present in the IPM-E200/n-propanol (1:1)-water mixture at 25 °C.
75:25 IPM-E200/n-Propanol. At the first stage, there is a linear increase in the ultrasonic velocity up to 5 wt % of water added (Figure 1A). At 5 wt % water, there is a break in the linear dependence, and the slope decreases significantly. The ultrasonic attenuation (Figure 1C) shows no change at this stage. The beginning of stage II is marked by an increase in the ultrasonic attenuation at 5 wt % water. There is a linear increase in ultrasonic velocity upon the addition of water from 5 to 11 wt % in Figure 1A. At 11 wt % water, there is another break in the linear dependence of the ultrasonic velocity, marking a second transition to stage III. At this stage, the ultrasonic velocity increases linearly from 11 to 19 wt % water. At 19 wt % water, the linear dependence clearly breaks again, and there is an increase in the slope, marking another transition to stage IV (Figure 1B). The ultrasonic attenuation increases at this stage followed by a break and a plateau after the transition. At stage IV, the ultrasonic attenuation remains nearly constant from 20 wt % water until 33 wt % water (Figure 1D). At 33 wt % water, there is a change in the attenuation profile, marking another transition. There is no change in the ultrasonic velocity profile at this transition. The beginning of stage V is marked by the random scatter of the ultrasonic attenuation followed by a jump in ultrasonic velocity. The ultrasonic velocity increases in this stage until 85 wt % water where it begins to decrease, which could be considered another transition to stage VI. In the final stage, the ultrasonic velocity decreases (from 85 wt % water in Figure 1B). The transition at 85 wt % water is unclear in the attenuation profile (Figure 1D) because of the large amount of scattering of the acoustic wave at this concentration of water. 50:50 IPM-E200/n-Propanol and 25:75 IPM-E200/nPropanol. The same (as in 75:25 IPM-E200/n-propanol) transitions take place for 50:50 and 25:75 IPM-E200/n-propanol systems (Figures 2 and 3); however, they occur at different concentrations of water. The points representing the concentrations of the components corresponding to the center of the transition areas between the stages are plotted on the pseudoternary phase diagram in Figure 4. The dotted line represents data of Aboofazeli et al.2 on the transition between microemulsion and coarse emulsion states obtained by crossed-polarized light microscopy and visual inspection. This line coincides favorably with the transition between stages III and IV on the ultrasonic phase diagram (Figure 4). Detailed microscopic interpretations of the stages of the ultrasonic phase diagram are outlined in the following section and are illustrated in Figure 8. Microscopic Interpretation of Ultrasonic Profiles. Stage I. The addition of water at this stage does not cause any significant
5582 Langmuir, Vol. 22, No. 13, 2006
Hickey et al.
Table 2. The Partial Concentration Increment of the Ultrasonic Velocity, a, of the Various Ratios of IPM-E200/ n-Propanol-Water at 25 °C 75% oil 25% surf
50% oil 50% surf
measured
0.06 ( 0.03
.0.8 ( 0.04
predicteda measured predicteda measured predicteda measured predictedb
-0.3 0.03 ( 0.02 -0.20 0.05 ( 0.03 -0.09 0.13 ( 0.03 0.1
-0.1 0.13 ( 0.03 -0.03 0.09 ( 0.04 0.025 0.14 (0.03 0.1
stage I II III IV
25% oil 75% surf (0.28 ( 0.05)c 0.14 ( 0.03d -0.01 0.17 ( 0.03 0.03 0.11 ( 0.03 0.08
a Calculated from the ultrasonic velocity curve for ideal mixtures in the thermal low-frequency regime (Figure 7) at a concentration of water corresponding to the middle of the stages, as presented in Figures 1-3. b Calculated from the ultrasonic velocity curve for ideal mixtures in the thermal high-frequency regime (Figure 7) at a concentration of water corresponding to the middle of the stage. c The value at the beginning of the stage. d The value at the end of the stage.
increase in attenuation, which indicates an absence of droplets or aggregates scattering the ultrasonic waves. This is in good agreement with previous results (total intensity light scattering and photon correlation spectroscopy (PCS)), which showed an absence of aggregates at low concentrations of water.34,35 It can be concluded that water at this stage hydrates the hydrophilic atomic groups of the surfactant and cosurfactant. The partial concentration increment of ultrasonic velocity (the slope of concentration dependence), a, at this stage is significantly higher (0.1-0.3) than the increment expected for the ideal mixture at the thermal low-frequency limit, -0.3 to 0 (Table 2). This could be explained by hydration effects. Hydration typically leads to a significantly lower compressibility of hydration water compared with that of the bulk water.36-39 This results from the abnormally high compressibility of the bulk water caused by a large structural contribution (structural changes associated with a change in pressure).37,40 The structural contribution to compressibility is attributed to a “unique” structure of bulk water, where each molecule forms four hydrogen bonds with its neighbors.41 The hydration process partially “destroys” this structure, thus decreasing the structural contribution to compressibility and increasing the velocity.37 In addition, the same hydration effects should decrease the large heat capacity of water and therefore, according to eq 10, decrease the apparent adiabatic compressibility of water at the thermal low-frequency limit, thus increasing the ultrasonic velocity and its partial concentration increment, a. The partial concentration increment of the ultrasonic velocity of water increases with the concentration of surfactant and cosurfactant. This is expected since the hydration level should rise with the amount of hydrophilic atomic groups in the mixture. The increase of a is also predicted in the ideal mixtures for the thermal low-frequency limit, as seen in the Figure 7. Stage II. The attenuation profile begins to increase at this stage (Figure 1C). This could be explained by the formation of “microstructures” that scatter the ultrasonic wave. The particle size calculations show a growth in diameter up to 6 nm for 25 wt % surfactants, up to 9 nm for 50 wt % surfactants, and up to 12 nm for 75 wt % surfactants. We suggest that, at this stage, the headgroups of the surfactants are drawn together by hydrogen bonding, which involves water molecules bridging, thus aggregating them into structures similar to inverted swollen micelles (see Figure 8). The structure of these aggregates and their compressibility should highly depend on the amount of surfactant, cosurfactant, and water in the system. This could
explain the high dependence of the partial concentration increment of ultrasonic velocity for water, which ranges from 0.051 for 25 wt % surfactants to 0.26 for 75 wt % surfactants at stage II. The effective particle size calculated at this stage is based on a simple model of ideal mixtures of the continuous and dispersed particles of water. Since the water is not free but exists in a hydrating form at this stage, the exact physical properties of the dispersed phase are unclear. Hence, the calculated particle sizes for this phase (Figure 5A-C) may differ from the true values. However, the trend of increasing size of aggregates with an increase in the concentration of water is still observed. The partial concentration increment of ultrasonic velocity at this stage significantly exceeds the predicted values for the ideal mixtures at the thermal low-frequency limit. This shows that the state of added water at this stage is different from the state of pure (bulk) water. Stage III. The attenuation continues to increase during this stage. This demonstrates a growth of microaggregates that scatter the ultrasonic wave. Overall, the particle sizes calculated for this stage (Figure 5A) agree well with those obtained using light scattering measurements and PCS (the method used by Aboofazeli et al).35 This technique provides information on the diffusion of particles, which is related to the hydrodynamic diameter of the particles consisting of the water core and the volume occupied by the hydrocarbon chains of the surfactant adsorbed at the wateroil interface. It is believed that the hydrocarbon chain of the surfactant is approximately 80% of its extended chain length. Opposite to this, ultrasonic scattering is determined by the change of thermophysical properties on the border between the continuous medium and the particle, and therefore the ultrasonic size shall correspond to the size of the particle core. The size of the droplets measured by PCS for the 75:25 system is 15 nm at 13 wt % water, while the ultrasonic particle size is 7 nm at the same concentration of water. The difference corresponds approximately to the length of the surfactant chains, which is the difference between the hydrodynamic and core sizes (see Figure 5). The partial concentration increment of ultrasonic velocity at this stage is 0.05, 0.09, and 0.11 for 75:25, 50:50, and 25:75 systems, respectively. The increase in the partial concentration increment of ultrasonic velocity with concentration of surfactant is well predicted by the behavior of ideal mixtures (Figure 7). The absolute value of the partial concentration increment of ultrasonic velocity at this stage is much closer than that for the previous stages, to the increment expected for the ideal mixtures at the thermal low-frequency limit, -0.09, 0.025, and 0.08 for 75:25, 50:50, and 25:75 systems, respectively. This suggests that the state of the water added at this stage is approaching the state of pure (bulk) water. One of the possible reasons for the higher values of the measured partial concentration increment of ultrasonic velocity compared with the predicted values for the ideal systems could be the effect of ‘rigidity’ of the surfactant interface between water in the droplets and continuous medium. It was discussed earlier that the lecithin forms rigid films at water-oil interfaces.42 We could expect that this effect will make the microdroplets of water slightly less compressible compared with water droplets in the ideal mixture, which would increase the partial concentration increment of ultrasonic velocity. Aboofazeli et al. previously calculated that, at 9 wt % water, all the surfactant molecules present in the 75:25 IPM-E200/ n-propanol system were in the hydration state.35 The phosphatidylcholine headgroup is hydrated by 12 water molecules with an additional 2 water molecules hydrating each propanol molecule. Therefore, since the surfactants are fully hydrated, it is believed that a tight, closely packed “microaggregate” is formed at this
Phase Transitions for Phospholipid Microemulsions
concentration of water for 75:25 IPM-E200/n-propanol, and further addition of water leads to a free water core in the micelle. This agrees well with the transition from microaggregates to microemulsion (stage II to III at ultrasonic titration curves). Stage IV. The ultrasonic attenuation nearly plateaus at this stage. This suggests that the microemulsion droplets are no longer increasing in size or number with the addition of water. Therefore, the water added is being incorporated elsewhere. It was proposed that, at this stage, there is an insufficient amount of surfactant to stabilize the microemulsion droplets; therefore, other structures exist. The formation of a coarse emulsion with a droplet size in the micron range can explain the attenuation profile. Droplets with this size will lie far to the right of the maximum on the predicted attenuation (versus particle size) curve given in Figure 6. Therefore the contribution of these droplets to the measured ultrasonic attenuation should be low. If we suggest that all water in the sample at this stage exists in the form of a coarse emulsion, the attenuation level would correspond to a particle size in the range of 10 µm, which is not an unreasonable size given the conditions of agitation present during the measurement. This large particle size corresponds to the thermal high-frequency limit behavior for ultrasonic velocity. The concentration curves for velocity in ideal mixtures (Figure 7) predict the partial concentration increment of ultrasonic velocity for this stage of 0.10 for all three systems. This is very close to the measured values in 75:25 and 50:50 systems at this stage (0.13 and 0.14, respectively) if experimental error (0.03) is taken into consideration. This indicates that, as expected, the state of added water at this stage is nearly the same as that of pure water. In the 25:75 system, stage IV occupies only a narrow concentration range, and therefore the partial concentration increment of ultrasonic velocity is difficult to determine. Stage V. At this stage, similar amounts of oil and water are present in the system. It is believed a cloudy pseudo-bicontinuous structure exists, containing flexible and highly disorganized internal interfaces with no separation into continuous or dispersed phases. This could explain the observed random scattering of the ultrasonic attenuation. We should expect that the structure of this phase is very sensitive to stirring, and the scattering of attenuation at this stage can be explained by the shear effects which change the position of the random interfaces causing the scattering of the ultrasonic waves (Figure 1D). We can also expect that the sizes of the structures in the system at this stage are
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comparable to the wavelength (520 µm at 2 MHz to 116 µm at 12 MHz), which also should have an additional effect on ultrasonic attenuation and its sensitivity to shearing. The structures present in the system at this stage are too complicated to enable the prediction of the partial increment of ultrasonic velocity. Stage VI. Phase separation is visually observed at this stage. This limited concentration range occurs in the water-rich part of the phase diagram. The separation could explain the reversed concentration dependence of ultrasonic velocity. It was previously suggested that the cause for the phase separation is the limited solubility of IPM in water,2 and hence the formation of a waterin-oil system is not favorable at this ratio of oil/water/surfactant/ cosurfactant.
Conclusions The application of HR-US has enabled the construction of a pseudoternary phase diagram of an oil/water/phospholipid/alcohol system, indicating phase transitions from surfactant hydration to swollen micelles, microemulsions, and other structures (a coarse emulsion and pseudo-bicontinuous phase). The previously available literature data for the end of the microemulsion region agree well with our results. Changes in the ultrasonic velocity and attenuation measurements along with calculation of the partial concentration increment of ultrasonic velocity allowed characterization of various structures present within the system. This included analysis of the state of water and particle size. The particle size obtained for microemulsion region agrees well with the previous literature data. Overall, HR-US has proven to be a powerful technique for the characterization of microemulsion systems. The ability to analyze these systems in titration mode is another advantage of this technique for microemulsion characterization. Acknowledgment. We thank Ultrasonic Scientific Ltd. for providing instrumentation for this work and consultation on the optimal measuring regimes, Jim Lyng and Earl Waghorne for assistance in the measurements of the rheological and thermophysical properties in our continuous media, and E. Kudryashov for assistance with data handling. This work was supported by a combined grant from the Irish-American Partnership, Ultrasonic Scientific Ltd., and Aphton Biopharma. LA052735T
