Formation, Characterization and Stability of ... - wseas.us
Mathematical Methods and Techniques in Engineering and Environmental Science
Formation, Characterization and Stability of Nanoemulsions Prepared by Phase Inversion P. SEVCIKOVA a), P. VLTAVSKA a), V. KASPARKOVA a,b), J. KREJCI a) a) Department of Fat, Surfactant and Cosmetics Technology b) Centre of Polymer Systems Tomas Bata University in Zlin nam. T. G. Masaryka 275, 76272 Zlin CZECH REPUBLIC [email protected] http://www.ft.utb.cz Abstract: Formation and stability of n-undecane in water nanoemulsions prepared by EIP method (Emulsion Inversion Point) in the presence of two nonionic surfactants were studied. In the study, the influences of surfactant concentration, HLB value and oil–to–water ratio on the particle size and distribution were investigated. The stability of emulsions was evaluated from changes in particle size distribution determined by photon correlation spectroscopy, and by visual and microscopy description of their appearance. The results obtained indicate that all the above mentioned variables significantly influence particle size and distribution. It is obvious that for given oil phase, the optimum HLB value of 10.5 exists, at which the emulsion particle size is the smallest at all studied oil-to-water ratios (5/95, 10/90, 15/85, 20/80, 25/75, 30/70). The HLB values above and below optimum then induce the increase of particle size of the studied emulsions. The best emulsion stability was observed for samples stored at the temperature of 4 °C. Key-Words: Emulsion, Hydrophile-lipophile balance, Nanoemulsion, Photon correlation spectroscopy, Stability
method. In this technique, the dispersed phase is simply added to the continuous phase in the presence of suitable surfactant under intensive agitation. [9,10,11,12] The nature of emulsion, i.e. oil in water (O/W) or water in oil (W/O), is mainly governed by the affinity of the surfactant towards the water or oil phase. In the case of nonionic surfactants, this affinity was first described by Griffin. [13] He suggested a semi-empirical hydrophile-lipophile balance (HLB) scale as a way of predicting emulsion type from surfactant molecular composition. Surfactants with a high HLB value usually form O/W emulsions, while surfactants with a low HLB value usually form W/O emulsions. [14] In the phase inversion method, the continuous phase is added to the disperse phase until phase inversion occurs and the required emulsion type is formed. [11,12] Phase inversion is the process whereby an O/W systems inverts into a W/O system, in the absence or presence of a surfactant, or vice versa. [14,15] There are two types of inversions: catastrophic phase inversion (CPI) and transitional phase inversion (TPI). [11] Catastrophic inversion is induced by increasing the fraction of the dispersed phase. [16] Transitional inversion occurs when the affinity of the surfactant
1 Introduction The increasing interest in the area of nanoemulsions is caused by growing number of their promising applications in material science, medicine, pharmacology or agriculture. In order to understand behavior of nanoemulsions and thus extend their application potential, studies on model systems are beneficial. By definition, nanoemulsions are transparent or bluish, kinetically stable, two-phase systems with a typical particle size range of 50-200 nm, as given in publications [1,2] or 50-500 nm, as given by authors. [3,4,5,6] They are non-equilibrium systems with a spontaneous tendency to phase separation [7], nevertheless, they may have a long kinetic stability and are reasonably resistant to creaming or sedimentation as well as to flocculation. [5,8] Due to their small particle size they are reported to break mainly by the Ostwald ripening mechanism in time. [7] All these properties have led to an increased use of nanoemulsions in many practical applications. [5,9] However, the main limitation for their application is their nonstraightforward preparation procedure and limited long-term stability. [7] Nanoemulsions can be prepared in different ways, one possibility is a direct emulsification
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for the water phase equilibrates its affinity for the oil phase. The variation in the affinity or hydrophile-lipophile balance (HLB) of the surfactant can be conducted by alteration in temperature [17-19] or by addition of a surfactant with a different HLB. [20-22] The best-known transitional phase inversion process is called the Phase inversion temperature (PIT) method and was first introduced and named by Shinoda and coworkers. [23,24] The method works with a surfactant system which is temperature sensitive, generally a non-ionic surfactant of the ethoxylate type. These surfactants become less hydrophilic on heating due to the dehydration of the polyethyleneoxide chain. [1,25,26] When an O/W emulsion prepared using a non-ionic surfactants is heated, then at a critical temperature (PIT) the emulsion inverts to a W/O emulsion. At this temperature (PIT) both particle size and interfacial tension are minimal. However, the small particles are unstable and coalesce rapidly, but by rapid cooling of the emulsion, which is produced at a temperature near PIT, very small and stable emulsion particles can be prepared. [11] The second type of transitional phase inversion can be induced by changing the HLB value of the surfactant at constant temperature using surfactant mixtures [1] and is known as Emulsion Inversion Point (EIP) method. [2] Change in HLB is performed by mixing „low“ and „high“ HLB surfactants. [12,15] In the current study, influence of emulsion composition and preparation conditions on the particle size of (nano)emulsion was investigated. Effects of different oil (O) to water (W) ratio, surfactant concentration and HLB value were elucidated. For this purpose, photon correlation spectroscopy was employed. In addition to the determination of particle size immediately after emulsion preparation, the stability of prepared emulsions stored at temperatures 4 °C, 25 °C and 35 °C was followed.
2.2 Preparation of nanoemulsions Preparation of oil in water nanoemulsions was performed using Emulsion Inversion Point technique (EIP). Emulsifications were carried out using a simple laboratory equipment consisting of a RZR 2020 stirrer (Heidolph), a glass vessel and a burette. The pairs of nonionic surfactants were used to prepare mixtures with a required range of HLB values. Depending on the initial HLB to be used, Igepal 210 (Igepal 520) and Igepal 720 were predissolved in the appropriate ratios in oil phase and water phase, respectively. The water phase containing surfactant was added drop wise at a rate of 1ml/min to oil phase. The constant stirring rate of 1050 rpm was used during emulsifications. All the experiments were performed at the room temperature (25 °C). Oil to water ratios of 5/95, 10/90, 15/85, 20/80, 25/75 and 30/70 were used. Two different concentrations of surfactants of 3 and 5 wt. % and HLB values of 9.5, 10, 10.5, 11, 11.5 were applied. Selection of HLB values for surfactant mixtures was based on the assumption that for a preparation of stable emulsion, a surfactant with HLB recommended for given oil phase is the most suitable. [27] To produce a stable emulsion with the undecane as an oil phase, HLB value between 10 and 11 is recommended. [28] For comparison, emulsions with an O/W ratio 5/95, 15/85, 30/70 containing only one surfactant, Igepal 520 were prepared.
2.3 Measurement distribution
particle
size
Particle size and particle size distribution of nanoemulsions were determined by photon correlation spectroscopy (PCS) in terms of zaverage diameter using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at 25 °C.
2.4 Emulsion stability Stability of emulsions was evaluated by following a phase separation both visually and by microscopy as well as by following the particle size and distribution by PCS. For stability evaluation, the emulsions were stored at three different temperatures of 4 °C, 25 °C and 35 °C and observed at regular time intervals. Microscopic observation was performed using a microscope OLYMPUS CX 41. Photographic records were made by a computer program Quick PHOTO PRO 2.0.
2 EXPERIMENTAL 2.1 Materials n-Undecane (Sigma Aldrich) and purified water were used as an oil and water phase, respectively. Three nonionic surfactants, supplied by Aldrich, Igepal 210 (HLB 4), Igepal 520 (HLB 10) and Igepal 720 (HLB 14.2) with nonyl phenol ethoxylate chain length of 2 (NPE2), 5 (NPE5) and 12 (NPE12) were used throughout the study.
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optimal value. Particle sizes lower than 200 nm were observed for HLB 10, 5 % content of surfactants and the O/W ratios 15/85, 20/80, 25/75. Though the HLB of 9.5 is generally considered to be low for formation of O/W nanoemulsions, they can be successfully prepared as documented for example for O/W 25/75, which provides emulsions with the particle size of 116 nm, but these nanoemulsions are unstable and brake soon into two phases. Nanoemulsions with good stability were also obtained at HLB 11 (64 nm) and HLB 11.5 (43 nm) at the concentration of surfactant of 5 % and O/W ratio 5/95 as well as at HLB 11.5, 5 % surfactant concentration and the O/W ratio 30/70. Particle size of emulsions were 140 nm, they were stable and showed a negligible phase separation for a period of 12 days.
3 Results and discussion 3.1 Particle size The results of influence of HLB and O/W ratio on emulsion particle size are summarized in Fig. 1 and 2. From the results it is clear that the particle size in emulsions is influenced by the O/W ratio, HLB value of surfactant and its concentration. The figure 1 shows that the particle size of emulsions is growing with increasing amount of oil phase. This is particularly visible on emulsions containing more than 15 % oil phase, that implies emulsions with O/W 15/85, 20/80, 25/75, and 30/70. Differences in particle sizes were also observed when using different amount of surfactants, 3 % and 5 %. According a theoretical assumption, particle size should decrease with increasing amount of surfactant. [29] However this trend is not clearly demonstrated by the present data. For example, for emulsions prepared at HLB 10.5 (data not presented), there was a slight decrease in particle size due to the increase in the amount of surfactant from 3 % to 5 %, as well as in case of emulsions with an O/W ratio 15/85, 20/80, 25/75, and 30/70 prepared at HLB 11.5. The results also illustrate influence of HLB value on particle size in studied emulsions. Figure 2 shows the relationship between the HLB and particle size for the observed O/W ratios and 5 % surfactant concentration. This figure proves that for the given system, an optimal HLB exists, in which the particle size is smallest in all ratios of O/W (5/95, 10/90, 15/85, 20/80, 25/75, 30/70). In this case it occurs at HLB of 10.5 and particle sizes in emulsions then increase with HLB higher and lower than the optimal one. Moreover, with decreasing HLB, emulsions become unstable and a low content of oil phase (O/W 5/95, 10/90) at HLB 9.5 even lead to the formation of inverted water in oil (W/O) emulsions. Similar results were observed for emulsions prepared with surfactant concentration of 3 %. Also in this case, the increase in particle size of emulsions was observed on either side of the optimum HLB. Correspondingly to surfactant content of 5 %, optimum with the smallest emulsion particles was showed at the HLB of 10.5. In both figures it can be seen that true nanoemulsions with a particle size smaller than 200 nm, prepared by the EIP method have been formed for all studied O/W ratios in the case of HLB value of 10.5 irrespective of surfactant content. However, nanoemulsions can be also prepared with HLB values lower and higher than the
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Fig. 1 Particle size of emulsions determined immediately after preparation. Influence of O/W ratio.
Fig. 2 Particle size of emulsions determined immediately after preparation. Influence of HLB value (5 % surfactant).
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Simultaneously with the visual description, a microscopic observation of all emulsions was also carried out. Using microscopy, different ways of emulsion destabilization were observed such as changes in particle sizes, flocculation, etc. Similarly to visual and microscopic observations, particle size of emulsions measured by PCS was also significantly influenced by the storage temperature and the most prominent variations were observed in samples stored at 35 °C. For example, particle size of emulsions with O/W ratio of 25/75 (HLB=11.5, 3 % of surfactant) gradually increased during storage and after the phase of growth, the complete disintegration of emulsions into the oil and aqueous phase occurred. An increase in particle size and shift of particle size distribution is evident from the Figure 4. This figure compares the particle size distributions of the above mentioned emulsion during the storage at 35 °C. Immediately after preparation, a monomodal particle size distribution with the z-average diameter of 550 nm (red curve) was determined. After three-day storage, the particle size increased to 620 nm (green curve) and elevendays treatment at 35 °C increased the emulsion zaverage diameter to 1300 nm (blue curve).
3.2 Emulsion stability Emulsion stability was evaluated by visual and microscopic observation as well as by following the changes in their particle size. All these methods showed that emulsion stability is influenced by the composition and primarily by storage temperature. Visual assessment of samples stored at 4 °C, 25 °C and 35 °C was performed daily and the height of separated phase in cm was observed and measured as a function of time. Results from visual observation are shown in Figure 3 for emulsion with O/W 5/95 stored at 35 °C. This figure describes the time-dependent progress of phase separation of emulsion into two emulsions, one of which was richer in the disperse phase (white layer) than the other (semitransparent layer). At this temperature, separation has already occurred during the first day of storage at all ratios O/W regardless of the HLB value. Temperature of 35 °C was hence the worst for preservation of emulsions. Storage temperature of 4 °C was evaluated as the best for the preservation of homogeneous, stable emulsions. Though phase separation also occurred at this temperature, it was far slower than at higher storage temperatures. The best stability, in terms of visual assessment, showed emulsion with oil-in-water ratio 5/95 prepared with HLB of 10.5 and surfactant concentration of 3 %. This emulsion was homogenous or showed a negligible phase separation for a period of 43 days. Phase separation at room temperature (25 °C) occurred faster than at temperature of 4 °C. However samples with oil-in-water ratio of 5/95 prepared with HLB value of 10.5 (3 %), showed also sufficiently good stability with homogeneous emulsions and a minimal phase separation for 36 days. Emulsions with the same O/W ratio, HLB value of 10.5 and increased surfactant concentration of 5 % were stable for 20 days.
Fig. 4 Changes in particle size distribution of an emulsion with 3 % of surfactant, HLB value 11.5 and the O/W ratio (25/75) stored at 35 °C. Red curve: distribution obtained immediately after preparation, green curve: distribution obtained after 3 days of storage, blue curve: distribution obtained after 11 days of storage. On the contrary, changes in particle size during the storage at 4 °C were minimal. The best stability showed the emulsions with the O/W ratio of 5/95 (HLB values 10.5 and 11, surfactant concentration 3 % and 5 %), in which only negligible changes in the particle size during storage were detected. According to expectations, these emulsions were also smallest in particle size.
Fig. 3 Separation process of emulsion stored at 35 °C (5/95, HLB 11.5, 5 % of surfactant).
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inverted W/O emulsions even occurred. It was found that the stability of emulsions is significantly dependent on the ratio of oil and aqueous phase, temperature, the HLB value of surfactant and its concentration. The stability of emulsions was most affected by storage temperature. The best stability was observed in emulsions stored at the temperature of 4 °C. Some of these emulsions were stable for more than a month and only small changes in their particle size were observed.
Changes in particle size of emulsions stored at temperature of 25 °C were significantly influenced by their composition. For example, the particle size of emulsions with O/W 5/95 (HLB 11, 3 % of surfactant) increased during eleven days of storage from 59 nm to 91 nm, whilst for the same emulsion with 5 % of emulsifier remained the particle size during the storage unchanged. On the contrary, the particle size of emulsions with the O/W ratios 15/85 and 20/80 with 3 % and 5 % of surfactant gradually decreased in size from the first day of their preparation. Particle sizes of emulsions prepared with O/W ratios 5/95, 15/85, 30/70, HLB 11.5 and at both surfactant concentrations (3 %, 5 %) increased depending on composition. As example, emulsions with O/W ratio 15/85 (5 % of surfactant) and O/W ratio 5/95 (3 % of surfactant) which increased in size during eleven days of storage from 460 nm to 630 nm and from 180 nm to 390 nm, respectively can be given. For comparison, particle sizes of emulsions prepared by direct emulsification with single surfactant, Igepal CA 520 and O/W ratios of 5/95, 15/85 and 30/70 were studied. All these emulsions were highly heterogeneous and PCS measurements showed the presence of three fractions with distinctly different particle sizes. The particles of thus prepared emulsions were also larger than particles produced by EIP method using a mixture of two surfactants. This is especially evident at the emulsion with O/W ratio 5/95, whose particle sizes were of 618 nm and 112 nm in the case of direct emulsification and EIP method, respectively.
Acknowledgement: This article was created with support of Operational Program Research and Development for Innovations co-funded by the European Regional Development Fund (ERDF) and national budget of Czech Republic, within the framework of project Centre of Polymer Systems (reg. number: CZ.1.05/2.1.00/03.0111). The work was also supported by an internal grant of UTB in Zlín IGA/15/FT//D financed from funds of specific academic research.
References: [1] TADROS T., IZQUIERDO P., ESQUENA J., SOLANS C., Formation and stability of nanoemulsions, Colloid Interface Sci., 108, 2004, pp. 303-318. [2] SADTLER V., RONDON-GONZALEZ M, ACREMENT A., CHOPLIN A., MARIE E., PEO-Covered Nanoparticles by Emulsion Inversion Point (EIP) Method, Macromol. Rapid Commun., 31, 2010, pp. 998-1002. [3] FERNANDEZ P., ANDRÉ V., RIEGER J., KÜHNLE A., Nano-emulsions formation by emulsion phase inversion, Colloids Surf., 251, 2004, pp. 53-58. [4] FORGIARINI A., ESQUENA J., GONZÁLES C., SOLANS C., Formation of Nano-emulsions by Low-Energy Emulsification Methods at Constant Temperature, Langmuir, 17, 2001, pp. 2076-2083. [5] SOLÉ I., MAESTRO A., GONZÁLES C., SOLANS C., GUTIÉRREZ J.M., Optimization of Nano-emulsion Preparation by Low-Energy Methods in an Ionic Surfactant System, Langmuir, 22, 2006, pp. 8326-8332. [6] EL-AASER M., LACK C., VANDERHOFF J., FOWKES F., The miniemulsification process different form of spontaneous emulsification, Colloids Surf. A, 29, 1988, pp. 103-118. [7] GOTIÉRREZ J.M., GONZÁLES C., MAESTRO A., SOLÉ I., PEY C.M., NOLLA
4 Conclusion In this work, the Emulsion Inversion Point technique was used to prepare the water/surfactants/n-undecane nanoemulsions and emulsions. The influence of oil-to-water ratio, surfactant concentration and HLB value on emulsion particle size and emulsion stability at three different temperatures was investigated. Generally, the smallest particle sizes were obtained for emulsions with 5/95 O/W ratio, HLB value of 10.5, 11, 11.5 and 5 % of surfactant. Their sizes were in the range of 40-120 nm. Emulsions of these sizes may be, due to their method of preparation, classified as true nanoemulsions. Most of the emulsions with remaining O/W ratios can be classified as classical macro-emulsions, whose sizes lay depending on the composition mainly between 300-700 nm. At HLB values 9.5, 10 and lower content of oil phase (5/95, 10/90) formation of
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J., Nano-emulsions: New applications and optimization of their preparation, Colloid Interface Sci, 13, 2008, pp. 245-251. [8] VILLIERS M.M., ARAMWIT P., KWON G.S., Nanotechnology in Drug Delivery, Published by Springer, New York, 2009, ISBN 978-0-387-77667-5. [9] SOLANS C., IZQUIERDO P., NOLLA J., AZEMAR N., GARCIA-CELMA M.J., Nanoemulsions, Colloid Interface Sci, 10, 2005, pp. 102-110. [10] AUBRUN O.S., SIMONNET J.T., ALLORET F.L., Nanoemulsions: a new vehicle for skincare products, Colloid Interface Sci, 108109, 2004, pp. 145-149. [11] FERNANDEZ P., ANDRÉ V., RIEGER J., KÜHNLE A., Nano-emulsion formation by phase inversion emulsification, Colloids Surf. A, 251, 2004, pp. 53-58. [12] SAJJADI S., Effect of mixing protocol on formation of fine emulsions, Chem. Eng. Sci, 61, 2006, pp. 3009-3017. [13] W. C. GRIFFIN, Classification of surface active agents by HLB, J. Soc. Cosmet. Chem., 5, 1954, pp. 249. [14] SAJJADI S., ZERFA M., BROOKS B.W., Phase inversion in p-xylene/water emulsions with the non-ionic surfactant pair sorbitan monolaurate/polyoxyethylene sorbitan monolaurate (Span 20/Tween 20), Colloids Surf. A, 218, 2003, pp. 241-254. [15] SAJJADI S., Formation of fine emulsions by emulsification at high viscosity or low interfacial tension; A comparative study, Colloids Surf. A, 299, 2007, pp. 73-78. [16] BINKS B.P., Modern aspects of emulsion science, Royal Society of Chemistry, 1998, ISBN 0854044396. [17] FRIBERG S., SOLANS C., Emulsification and the HLB-temperature, Colloid Interface Sci, 66, 1978, pp. 367. [18] SHINODA K., HANRIN M., KUNIEDA H., SAITO H., Principles of attaining ultra-low interfacial tension: The role of hydrophilelipophile balance of surfactant at oil/water, Interface Colloids Surf., 2, 1981, pp. 301. [19] SHINODA K., FRIEBERG S., Emulsion and Solubilization, John Wiley, New York, 1986 ISBN 0-471-03646-3. [20] SMITH D., LIM K.H., An experimental test of catastrophe and critical-scaling theories of emulsion inversion, Langmuir, 6, 1990, pp. 1071.
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[21] BROOKS B.W., RICHMOND H.N., Dynamics of liquid-liquid phase inversion using non-ionic surfactants, Colloids Surf., 58, 1991, pp. 131. [22] SAJJADI S., JAHANZAD F., YIANNESKIS M., Catastrophic phase inversion of abnormal emulsions in the vicinity of the locus of transitional inversion, Colloids Surf. A, 240, 2004, pp. 149. [23] SHINODA K., SAITO H., The effect of temperature on the phase equilibria and the types of dispersions of the ternary system composed of water, cyclohexane, and non-ionic surfactant, Colloid Interface Sci, 26, 1968, pp. 70. [24] SHINODA K., SAITO H., The Stability of O/W type of emulsions as functions of temperature and the HLB of emulsifiers: The emulsification by PIT-method, Colloid Interface Sci, 30, 1969, pp. 258. [25] SALAGER J-L., FORGIARINI A., MÁRQUEZ L., PEÑA A., PIZZINO A., RODRIGUEZ M.P., RONDÓN-GONZÁLEZ M., Using emulsion inversion in industrial processes, Colloid Interface Sci, 108-109, 2004, pp. 259-272. [26] MILLER D.J., HENNING T., GRÜNBEIN W., Phase inversion of W/O emulsions by adding hydrophilic surfactant - a technique for making cosmetics products, Colloids Surf. A, 183-185, 2001, pp. 681-688. [27] FLORENCE, A. T., ATTWOOD, D., Physicochemical Principles of Pharmacy. 4th Edition, Chapter 7. Emulsions, Suspensions and other disperse systems, Publisher by the Pharmaceutical Press, 2006, London, ISBN 085369608X. [28] YAMAGUCHI S., Correlation between the Mixing Ratio of Surfactants and the Water/Oil Ratio in the Middle Microemulsions in Water/Mixed Surfactant/Hydrocarbon Systems, Langmuir, 14, 1998, pp. 7183–7188. [29] CHANADA, G.D., SHETH, B.B., Particle size reduction of emulsions by formulation designII: effect of oil and surfactant concentration, J Pharm Sci Technol., 49, 1995, pp. 71-76, ISSN 1079-7440.
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Formation, Characterization and Stability of Nanoemulsions Prepared by Phase Inversion P. SEVCIKOVA a), P. VLTAVSKA a), V. KASPARKOVA a,b), J. KREJCI a) a) Department of Fat, Surfactant and Cosmetics Technology b) Centre of Polymer Systems Tomas Bata University in Zlin nam. T. G. Masaryka 275, 76272 Zlin CZECH REPUBLIC [email protected] http://www.ft.utb.cz Abstract: Formation and stability of n-undecane in water nanoemulsions prepared by EIP method (Emulsion Inversion Point) in the presence of two nonionic surfactants were studied. In the study, the influences of surfactant concentration, HLB value and oil–to–water ratio on the particle size and distribution were investigated. The stability of emulsions was evaluated from changes in particle size distribution determined by photon correlation spectroscopy, and by visual and microscopy description of their appearance. The results obtained indicate that all the above mentioned variables significantly influence particle size and distribution. It is obvious that for given oil phase, the optimum HLB value of 10.5 exists, at which the emulsion particle size is the smallest at all studied oil-to-water ratios (5/95, 10/90, 15/85, 20/80, 25/75, 30/70). The HLB values above and below optimum then induce the increase of particle size of the studied emulsions. The best emulsion stability was observed for samples stored at the temperature of 4 °C. Key-Words: Emulsion, Hydrophile-lipophile balance, Nanoemulsion, Photon correlation spectroscopy, Stability
method. In this technique, the dispersed phase is simply added to the continuous phase in the presence of suitable surfactant under intensive agitation. [9,10,11,12] The nature of emulsion, i.e. oil in water (O/W) or water in oil (W/O), is mainly governed by the affinity of the surfactant towards the water or oil phase. In the case of nonionic surfactants, this affinity was first described by Griffin. [13] He suggested a semi-empirical hydrophile-lipophile balance (HLB) scale as a way of predicting emulsion type from surfactant molecular composition. Surfactants with a high HLB value usually form O/W emulsions, while surfactants with a low HLB value usually form W/O emulsions. [14] In the phase inversion method, the continuous phase is added to the disperse phase until phase inversion occurs and the required emulsion type is formed. [11,12] Phase inversion is the process whereby an O/W systems inverts into a W/O system, in the absence or presence of a surfactant, or vice versa. [14,15] There are two types of inversions: catastrophic phase inversion (CPI) and transitional phase inversion (TPI). [11] Catastrophic inversion is induced by increasing the fraction of the dispersed phase. [16] Transitional inversion occurs when the affinity of the surfactant
1 Introduction The increasing interest in the area of nanoemulsions is caused by growing number of their promising applications in material science, medicine, pharmacology or agriculture. In order to understand behavior of nanoemulsions and thus extend their application potential, studies on model systems are beneficial. By definition, nanoemulsions are transparent or bluish, kinetically stable, two-phase systems with a typical particle size range of 50-200 nm, as given in publications [1,2] or 50-500 nm, as given by authors. [3,4,5,6] They are non-equilibrium systems with a spontaneous tendency to phase separation [7], nevertheless, they may have a long kinetic stability and are reasonably resistant to creaming or sedimentation as well as to flocculation. [5,8] Due to their small particle size they are reported to break mainly by the Ostwald ripening mechanism in time. [7] All these properties have led to an increased use of nanoemulsions in many practical applications. [5,9] However, the main limitation for their application is their nonstraightforward preparation procedure and limited long-term stability. [7] Nanoemulsions can be prepared in different ways, one possibility is a direct emulsification
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Mathematical Methods and Techniques in Engineering and Environmental Science
for the water phase equilibrates its affinity for the oil phase. The variation in the affinity or hydrophile-lipophile balance (HLB) of the surfactant can be conducted by alteration in temperature [17-19] or by addition of a surfactant with a different HLB. [20-22] The best-known transitional phase inversion process is called the Phase inversion temperature (PIT) method and was first introduced and named by Shinoda and coworkers. [23,24] The method works with a surfactant system which is temperature sensitive, generally a non-ionic surfactant of the ethoxylate type. These surfactants become less hydrophilic on heating due to the dehydration of the polyethyleneoxide chain. [1,25,26] When an O/W emulsion prepared using a non-ionic surfactants is heated, then at a critical temperature (PIT) the emulsion inverts to a W/O emulsion. At this temperature (PIT) both particle size and interfacial tension are minimal. However, the small particles are unstable and coalesce rapidly, but by rapid cooling of the emulsion, which is produced at a temperature near PIT, very small and stable emulsion particles can be prepared. [11] The second type of transitional phase inversion can be induced by changing the HLB value of the surfactant at constant temperature using surfactant mixtures [1] and is known as Emulsion Inversion Point (EIP) method. [2] Change in HLB is performed by mixing „low“ and „high“ HLB surfactants. [12,15] In the current study, influence of emulsion composition and preparation conditions on the particle size of (nano)emulsion was investigated. Effects of different oil (O) to water (W) ratio, surfactant concentration and HLB value were elucidated. For this purpose, photon correlation spectroscopy was employed. In addition to the determination of particle size immediately after emulsion preparation, the stability of prepared emulsions stored at temperatures 4 °C, 25 °C and 35 °C was followed.
2.2 Preparation of nanoemulsions Preparation of oil in water nanoemulsions was performed using Emulsion Inversion Point technique (EIP). Emulsifications were carried out using a simple laboratory equipment consisting of a RZR 2020 stirrer (Heidolph), a glass vessel and a burette. The pairs of nonionic surfactants were used to prepare mixtures with a required range of HLB values. Depending on the initial HLB to be used, Igepal 210 (Igepal 520) and Igepal 720 were predissolved in the appropriate ratios in oil phase and water phase, respectively. The water phase containing surfactant was added drop wise at a rate of 1ml/min to oil phase. The constant stirring rate of 1050 rpm was used during emulsifications. All the experiments were performed at the room temperature (25 °C). Oil to water ratios of 5/95, 10/90, 15/85, 20/80, 25/75 and 30/70 were used. Two different concentrations of surfactants of 3 and 5 wt. % and HLB values of 9.5, 10, 10.5, 11, 11.5 were applied. Selection of HLB values for surfactant mixtures was based on the assumption that for a preparation of stable emulsion, a surfactant with HLB recommended for given oil phase is the most suitable. [27] To produce a stable emulsion with the undecane as an oil phase, HLB value between 10 and 11 is recommended. [28] For comparison, emulsions with an O/W ratio 5/95, 15/85, 30/70 containing only one surfactant, Igepal 520 were prepared.
2.3 Measurement distribution
particle
size
Particle size and particle size distribution of nanoemulsions were determined by photon correlation spectroscopy (PCS) in terms of zaverage diameter using a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at 25 °C.
2.4 Emulsion stability Stability of emulsions was evaluated by following a phase separation both visually and by microscopy as well as by following the particle size and distribution by PCS. For stability evaluation, the emulsions were stored at three different temperatures of 4 °C, 25 °C and 35 °C and observed at regular time intervals. Microscopic observation was performed using a microscope OLYMPUS CX 41. Photographic records were made by a computer program Quick PHOTO PRO 2.0.
2 EXPERIMENTAL 2.1 Materials n-Undecane (Sigma Aldrich) and purified water were used as an oil and water phase, respectively. Three nonionic surfactants, supplied by Aldrich, Igepal 210 (HLB 4), Igepal 520 (HLB 10) and Igepal 720 (HLB 14.2) with nonyl phenol ethoxylate chain length of 2 (NPE2), 5 (NPE5) and 12 (NPE12) were used throughout the study.
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optimal value. Particle sizes lower than 200 nm were observed for HLB 10, 5 % content of surfactants and the O/W ratios 15/85, 20/80, 25/75. Though the HLB of 9.5 is generally considered to be low for formation of O/W nanoemulsions, they can be successfully prepared as documented for example for O/W 25/75, which provides emulsions with the particle size of 116 nm, but these nanoemulsions are unstable and brake soon into two phases. Nanoemulsions with good stability were also obtained at HLB 11 (64 nm) and HLB 11.5 (43 nm) at the concentration of surfactant of 5 % and O/W ratio 5/95 as well as at HLB 11.5, 5 % surfactant concentration and the O/W ratio 30/70. Particle size of emulsions were 140 nm, they were stable and showed a negligible phase separation for a period of 12 days.
3 Results and discussion 3.1 Particle size The results of influence of HLB and O/W ratio on emulsion particle size are summarized in Fig. 1 and 2. From the results it is clear that the particle size in emulsions is influenced by the O/W ratio, HLB value of surfactant and its concentration. The figure 1 shows that the particle size of emulsions is growing with increasing amount of oil phase. This is particularly visible on emulsions containing more than 15 % oil phase, that implies emulsions with O/W 15/85, 20/80, 25/75, and 30/70. Differences in particle sizes were also observed when using different amount of surfactants, 3 % and 5 %. According a theoretical assumption, particle size should decrease with increasing amount of surfactant. [29] However this trend is not clearly demonstrated by the present data. For example, for emulsions prepared at HLB 10.5 (data not presented), there was a slight decrease in particle size due to the increase in the amount of surfactant from 3 % to 5 %, as well as in case of emulsions with an O/W ratio 15/85, 20/80, 25/75, and 30/70 prepared at HLB 11.5. The results also illustrate influence of HLB value on particle size in studied emulsions. Figure 2 shows the relationship between the HLB and particle size for the observed O/W ratios and 5 % surfactant concentration. This figure proves that for the given system, an optimal HLB exists, in which the particle size is smallest in all ratios of O/W (5/95, 10/90, 15/85, 20/80, 25/75, 30/70). In this case it occurs at HLB of 10.5 and particle sizes in emulsions then increase with HLB higher and lower than the optimal one. Moreover, with decreasing HLB, emulsions become unstable and a low content of oil phase (O/W 5/95, 10/90) at HLB 9.5 even lead to the formation of inverted water in oil (W/O) emulsions. Similar results were observed for emulsions prepared with surfactant concentration of 3 %. Also in this case, the increase in particle size of emulsions was observed on either side of the optimum HLB. Correspondingly to surfactant content of 5 %, optimum with the smallest emulsion particles was showed at the HLB of 10.5. In both figures it can be seen that true nanoemulsions with a particle size smaller than 200 nm, prepared by the EIP method have been formed for all studied O/W ratios in the case of HLB value of 10.5 irrespective of surfactant content. However, nanoemulsions can be also prepared with HLB values lower and higher than the
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Fig. 1 Particle size of emulsions determined immediately after preparation. Influence of O/W ratio.
Fig. 2 Particle size of emulsions determined immediately after preparation. Influence of HLB value (5 % surfactant).
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Simultaneously with the visual description, a microscopic observation of all emulsions was also carried out. Using microscopy, different ways of emulsion destabilization were observed such as changes in particle sizes, flocculation, etc. Similarly to visual and microscopic observations, particle size of emulsions measured by PCS was also significantly influenced by the storage temperature and the most prominent variations were observed in samples stored at 35 °C. For example, particle size of emulsions with O/W ratio of 25/75 (HLB=11.5, 3 % of surfactant) gradually increased during storage and after the phase of growth, the complete disintegration of emulsions into the oil and aqueous phase occurred. An increase in particle size and shift of particle size distribution is evident from the Figure 4. This figure compares the particle size distributions of the above mentioned emulsion during the storage at 35 °C. Immediately after preparation, a monomodal particle size distribution with the z-average diameter of 550 nm (red curve) was determined. After three-day storage, the particle size increased to 620 nm (green curve) and elevendays treatment at 35 °C increased the emulsion zaverage diameter to 1300 nm (blue curve).
3.2 Emulsion stability Emulsion stability was evaluated by visual and microscopic observation as well as by following the changes in their particle size. All these methods showed that emulsion stability is influenced by the composition and primarily by storage temperature. Visual assessment of samples stored at 4 °C, 25 °C and 35 °C was performed daily and the height of separated phase in cm was observed and measured as a function of time. Results from visual observation are shown in Figure 3 for emulsion with O/W 5/95 stored at 35 °C. This figure describes the time-dependent progress of phase separation of emulsion into two emulsions, one of which was richer in the disperse phase (white layer) than the other (semitransparent layer). At this temperature, separation has already occurred during the first day of storage at all ratios O/W regardless of the HLB value. Temperature of 35 °C was hence the worst for preservation of emulsions. Storage temperature of 4 °C was evaluated as the best for the preservation of homogeneous, stable emulsions. Though phase separation also occurred at this temperature, it was far slower than at higher storage temperatures. The best stability, in terms of visual assessment, showed emulsion with oil-in-water ratio 5/95 prepared with HLB of 10.5 and surfactant concentration of 3 %. This emulsion was homogenous or showed a negligible phase separation for a period of 43 days. Phase separation at room temperature (25 °C) occurred faster than at temperature of 4 °C. However samples with oil-in-water ratio of 5/95 prepared with HLB value of 10.5 (3 %), showed also sufficiently good stability with homogeneous emulsions and a minimal phase separation for 36 days. Emulsions with the same O/W ratio, HLB value of 10.5 and increased surfactant concentration of 5 % were stable for 20 days.
Fig. 4 Changes in particle size distribution of an emulsion with 3 % of surfactant, HLB value 11.5 and the O/W ratio (25/75) stored at 35 °C. Red curve: distribution obtained immediately after preparation, green curve: distribution obtained after 3 days of storage, blue curve: distribution obtained after 11 days of storage. On the contrary, changes in particle size during the storage at 4 °C were minimal. The best stability showed the emulsions with the O/W ratio of 5/95 (HLB values 10.5 and 11, surfactant concentration 3 % and 5 %), in which only negligible changes in the particle size during storage were detected. According to expectations, these emulsions were also smallest in particle size.
Fig. 3 Separation process of emulsion stored at 35 °C (5/95, HLB 11.5, 5 % of surfactant).
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inverted W/O emulsions even occurred. It was found that the stability of emulsions is significantly dependent on the ratio of oil and aqueous phase, temperature, the HLB value of surfactant and its concentration. The stability of emulsions was most affected by storage temperature. The best stability was observed in emulsions stored at the temperature of 4 °C. Some of these emulsions were stable for more than a month and only small changes in their particle size were observed.
Changes in particle size of emulsions stored at temperature of 25 °C were significantly influenced by their composition. For example, the particle size of emulsions with O/W 5/95 (HLB 11, 3 % of surfactant) increased during eleven days of storage from 59 nm to 91 nm, whilst for the same emulsion with 5 % of emulsifier remained the particle size during the storage unchanged. On the contrary, the particle size of emulsions with the O/W ratios 15/85 and 20/80 with 3 % and 5 % of surfactant gradually decreased in size from the first day of their preparation. Particle sizes of emulsions prepared with O/W ratios 5/95, 15/85, 30/70, HLB 11.5 and at both surfactant concentrations (3 %, 5 %) increased depending on composition. As example, emulsions with O/W ratio 15/85 (5 % of surfactant) and O/W ratio 5/95 (3 % of surfactant) which increased in size during eleven days of storage from 460 nm to 630 nm and from 180 nm to 390 nm, respectively can be given. For comparison, particle sizes of emulsions prepared by direct emulsification with single surfactant, Igepal CA 520 and O/W ratios of 5/95, 15/85 and 30/70 were studied. All these emulsions were highly heterogeneous and PCS measurements showed the presence of three fractions with distinctly different particle sizes. The particles of thus prepared emulsions were also larger than particles produced by EIP method using a mixture of two surfactants. This is especially evident at the emulsion with O/W ratio 5/95, whose particle sizes were of 618 nm and 112 nm in the case of direct emulsification and EIP method, respectively.
Acknowledgement: This article was created with support of Operational Program Research and Development for Innovations co-funded by the European Regional Development Fund (ERDF) and national budget of Czech Republic, within the framework of project Centre of Polymer Systems (reg. number: CZ.1.05/2.1.00/03.0111). The work was also supported by an internal grant of UTB in Zlín IGA/15/FT//D financed from funds of specific academic research.
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4 Conclusion In this work, the Emulsion Inversion Point technique was used to prepare the water/surfactants/n-undecane nanoemulsions and emulsions. The influence of oil-to-water ratio, surfactant concentration and HLB value on emulsion particle size and emulsion stability at three different temperatures was investigated. Generally, the smallest particle sizes were obtained for emulsions with 5/95 O/W ratio, HLB value of 10.5, 11, 11.5 and 5 % of surfactant. Their sizes were in the range of 40-120 nm. Emulsions of these sizes may be, due to their method of preparation, classified as true nanoemulsions. Most of the emulsions with remaining O/W ratios can be classified as classical macro-emulsions, whose sizes lay depending on the composition mainly between 300-700 nm. At HLB values 9.5, 10 and lower content of oil phase (5/95, 10/90) formation of
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J., Nano-emulsions: New applications and optimization of their preparation, Colloid Interface Sci, 13, 2008, pp. 245-251. [8] VILLIERS M.M., ARAMWIT P., KWON G.S., Nanotechnology in Drug Delivery, Published by Springer, New York, 2009, ISBN 978-0-387-77667-5. [9] SOLANS C., IZQUIERDO P., NOLLA J., AZEMAR N., GARCIA-CELMA M.J., Nanoemulsions, Colloid Interface Sci, 10, 2005, pp. 102-110. [10] AUBRUN O.S., SIMONNET J.T., ALLORET F.L., Nanoemulsions: a new vehicle for skincare products, Colloid Interface Sci, 108109, 2004, pp. 145-149. [11] FERNANDEZ P., ANDRÉ V., RIEGER J., KÜHNLE A., Nano-emulsion formation by phase inversion emulsification, Colloids Surf. A, 251, 2004, pp. 53-58. [12] SAJJADI S., Effect of mixing protocol on formation of fine emulsions, Chem. Eng. Sci, 61, 2006, pp. 3009-3017. [13] W. C. GRIFFIN, Classification of surface active agents by HLB, J. Soc. Cosmet. Chem., 5, 1954, pp. 249. [14] SAJJADI S., ZERFA M., BROOKS B.W., Phase inversion in p-xylene/water emulsions with the non-ionic surfactant pair sorbitan monolaurate/polyoxyethylene sorbitan monolaurate (Span 20/Tween 20), Colloids Surf. A, 218, 2003, pp. 241-254. [15] SAJJADI S., Formation of fine emulsions by emulsification at high viscosity or low interfacial tension; A comparative study, Colloids Surf. A, 299, 2007, pp. 73-78. [16] BINKS B.P., Modern aspects of emulsion science, Royal Society of Chemistry, 1998, ISBN 0854044396. [17] FRIBERG S., SOLANS C., Emulsification and the HLB-temperature, Colloid Interface Sci, 66, 1978, pp. 367. [18] SHINODA K., HANRIN M., KUNIEDA H., SAITO H., Principles of attaining ultra-low interfacial tension: The role of hydrophilelipophile balance of surfactant at oil/water, Interface Colloids Surf., 2, 1981, pp. 301. [19] SHINODA K., FRIEBERG S., Emulsion and Solubilization, John Wiley, New York, 1986 ISBN 0-471-03646-3. [20] SMITH D., LIM K.H., An experimental test of catastrophe and critical-scaling theories of emulsion inversion, Langmuir, 6, 1990, pp. 1071.
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[21] BROOKS B.W., RICHMOND H.N., Dynamics of liquid-liquid phase inversion using non-ionic surfactants, Colloids Surf., 58, 1991, pp. 131. [22] SAJJADI S., JAHANZAD F., YIANNESKIS M., Catastrophic phase inversion of abnormal emulsions in the vicinity of the locus of transitional inversion, Colloids Surf. A, 240, 2004, pp. 149. [23] SHINODA K., SAITO H., The effect of temperature on the phase equilibria and the types of dispersions of the ternary system composed of water, cyclohexane, and non-ionic surfactant, Colloid Interface Sci, 26, 1968, pp. 70. [24] SHINODA K., SAITO H., The Stability of O/W type of emulsions as functions of temperature and the HLB of emulsifiers: The emulsification by PIT-method, Colloid Interface Sci, 30, 1969, pp. 258. [25] SALAGER J-L., FORGIARINI A., MÁRQUEZ L., PEÑA A., PIZZINO A., RODRIGUEZ M.P., RONDÓN-GONZÁLEZ M., Using emulsion inversion in industrial processes, Colloid Interface Sci, 108-109, 2004, pp. 259-272. [26] MILLER D.J., HENNING T., GRÜNBEIN W., Phase inversion of W/O emulsions by adding hydrophilic surfactant - a technique for making cosmetics products, Colloids Surf. A, 183-185, 2001, pp. 681-688. [27] FLORENCE, A. T., ATTWOOD, D., Physicochemical Principles of Pharmacy. 4th Edition, Chapter 7. Emulsions, Suspensions and other disperse systems, Publisher by the Pharmaceutical Press, 2006, London, ISBN 085369608X. [28] YAMAGUCHI S., Correlation between the Mixing Ratio of Surfactants and the Water/Oil Ratio in the Middle Microemulsions in Water/Mixed Surfactant/Hydrocarbon Systems, Langmuir, 14, 1998, pp. 7183–7188. [29] CHANADA, G.D., SHETH, B.B., Particle size reduction of emulsions by formulation designII: effect of oil and surfactant concentration, J Pharm Sci Technol., 49, 1995, pp. 71-76, ISSN 1079-7440.
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