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Dye aggregates as new stabilizers for (mini)emulsions Susanne Sihler, and Ulrich Ziener Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04160 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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Dye aggregates as new stabilizers for (mini)emulsions Susanne Sihler and Ulrich Ziener* Institute of Organic Chemistry III-Macromolecular Chemistry and Organic Materials, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany ABSTRACT: Water-soluble organic dyes like fluorescein are widely used, mainly for coloration of e.g. biological samples, groundwater tracing etc., and they are not obviously amphiphilic by molecular structure like surfactants. Here, we show that the dyes alone stabilize oil-in-water emulsions. Exemplarily, fluorescein is compared with the classical surfactant sodium dodecyl sulfate (SDS) by means of surface/interfacial tension, concentration of stabilizer and electrolyte as well as pH. The principle can be extended to further classes of water-soluble dyes, which keep up with or exceed SDS by efficiency. Various organic liquids of different polarities can be employed and be polymerized in the case of styrene as disperse phase. Thus, surfactant free latex solely stabilized by water-soluble dyes is accessible. The emulsions can be destabilized by absorption of the dyes to hydrogels and their complete removal is easily followed visually. The stabilization mechanisms are different for SDS and the dyes: The latter stabilize droplets not as monomers but by their aggregates as molecular scale Pickering stabilizers, which is a new concept of stabilization.

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INTRODUCTION Water-soluble organic dyes are well-known for hundreds of years. Azo dyes such as congo red, xanthene dyes (e.g. fluorescein) or phenothiazine dyes (e.g. methylene blue) and many others can be easily synthesized and are widely used for various coloration applications. Many of these dyes are used to stain biological samples for microscopy1,2 or employed as colorants in heterophase systems like emulsions to selectively stain the polar aqueous phase3–8. For most applications it is commonly wanted that the dyes do only dye but not further interact with other components or with interfaces. Otherwise, other functions of the heterophase systems could deteriorate which is undesired. The chemical structures of some water-soluble dyes are shown in Figure 1; on close inspection, it is noticeable that none of these dyes displays classical amphiphilic topology. SDS (sodium dodecyl sulfate) as a classical surfactant exhibits a flexible aliphatic tail in combination with a charged head group but there is no hydrophilic part and separate hydrophobic part in the dyes. Therefore one would not assume that these molecules are able to adsorb to an oil-water interface as classical surfactants do; instead, they should stay in the water phase as they are hydrophilic. Thus, the aforementioned request not to interact significantly with oil-water interfaces should be easily fulfilled for the compounds in Figure 1, especially as the working concentrations of the dyes are very low. Hence the dyes are not expected to stabilize emulsions. In contrast, it was reported9 that fluorescent dyes such as rhodamine B can stabilize emulsions temporarily. The authors attribute this property to the decrease in the interfacial tension between water and oil and claimed that rhodamine B acts as a classical surfactant. Additionally, the authors state that many other fluorescent dyes act similarly, but fluorescein does not.


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Here, we demonstrate that fluorescein and many other (also non-fluorescing) water-soluble dyes (see Figure 1) can act as stabilizers of emulsions. For this purpose we have chosen miniemulsions as basic systems because they use surfactants in a more efficient way than macroemulsions10–13. All of the dyes are able to stabilize o/w miniemulsions, but we focus on fluorescein and discuss the influence of dye and electrolyte concentration and pH value on the emulsion stability. The behavior of the sodium salt of fluorescein (in the following called fluorescein) is compared to that of SDS with respect to the influence of these parameters on the emulsion stability. A new aggregation based stabilization mechanism is proposed; in dependence on the stabilization mechanism reported for metallacarboranes14 we call it “molecular scale Pickering stabilization” because the stabilizing species offer a similar size.


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Figure 1. Chemical structures of water-soluble organic dyes, which are used to stabilize o/w miniemulsions. Dyes from five different classes were employed with up to three members of each class.

EXPERIMENTAL SECTION


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Materials The dyes alizarin yellow GG (AY, Merck), eosin Y disodium salt (E, Sigma Aldrich), fluorescein sodium salt (Fl, Aldrich), congo red (CR, Merck), nile blue A (NB, Sigma), malachite green-oxalate (MG, Merck), methyl orange (MO, Merck), methylene blue (MB, Merck), rhodamine B (RhB, Fluka), crystal violet (CV, Merck), the solvents IsoparM (CALDIC Deutschland), n-hexadecane (HD, >98 %, TCI), isooctane (>99.5 %, Merck), cyclohexane (VWR), diethyl adipate (99 %, Sigma Aldrich), toluene (VWR), chloroform (99 %, VWR), perfluorodecalin (cis+trans, 98 %, ABCR), silicon oil (Baysilone M3, Bayer), the surfactants sodium dodecyl sulfate (SDS, AppliChem) and Lutensol AT 50 (BASF), the initiator 2,2’azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70, 98 %, Wako pure chemical industries Ltd.), hydrochloric acid (37 %, VWR), sodium hydroxide solution (1 M, Merck), sodium chloride (99.5 %, Merck) and D-(+)-glucono-1,5-lactone (99 %, Alfa Aesar) were used as received. Styrene (99 %, Merck) was passed through an Al2O3-filled column before use. FmocLG was prepared as described in literature15. Demineralized water with Milli-Q grade (resistivity: 18 MΩ) was used for all experiments.

Preparation of emulsions. The aqueous phase (continuous phase, CP) and oil phase (disperse phase, DP) were prepared separately. If not stated otherwise, the CP was prepared by dissolving a certain amount of dye in water at pH 6 to reach the desired concentration of dye (normally between 0.5 and 1 mg/mL). The oil phase consists of 0.5 mL of a liquid, which is immiscible with water and 20 µL of HD as osmotic agent if required. 9.5 mL of the aqueous phase were mixed with the oil phase in a 30 mL screw cap jar and the mixture was ultrasonicated with a Branson W450 digital sonifier for 3 minutes under ice cooling (70 % amplitude, 1/4" tip).


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Polymerization and washing of the polystyrene particles. 5 mL of the emulsion containing a mixture of styrene, HD and V-70 as the disperse phase (P-1 and P-2) were transferred to a screw cap jar. The closed bottle was placed in a pre-heated oil bath. The polymerizations were continued for 20 h at 40 °C under magnetic stirring. Thereafter, 250 µL of the dispersion was mixed with 500 µL of ethanol in an Eppendorf tube and centrifuged at 5 000 rpm for 2 min. The supernatant was removed and the particles were washed 5 times with the following procedure: the particles were dispersed in 500 µL of 0.1 M sodium hydroxide solution and the dispersion was placed in an ultrasonic bath for 30 minutes. Afterwards, 500 µL of ethanol was added and the dispersion was centrifuged at 3500 rpm for 5 minutes. In a final step, the particles were washed with a mixture of 500 µL of Milli-Q water and 500 µL of ethanol to get rid of sodium hydroxide. The particles were then centrifuged at 13000 rpm for 2 minutes and dried at room temperature. Breaking an emulsion stabilized with nile blue with an FmocLG hydrogel. The FmocLG hydrogel was prepared as described in literature15. 5.8 mg of FmocLG was dissolved in 146 µL of 0.1 M sodium hydroxide solution and 883 µL of water in an ultrasonic bath. Afterwards, 5.2 mg of D-(+)-glucono-1,5-lactone was added to induce gelation. Some pieces of the hydrogel were added to 1 mL of NB-1 and the mixture was allowed to stand for 5 hours to complete destabilization of the emulsion. To show the phase separation more clearly, the hydrogel was prepared in an NMR tube and some NB-1 emulsion was added.

Characterization Dynamic light scattering (DLS). Both the size and the size distribution (polydispersity index (PDI)) of the emulsion droplets were measured by DLS on a Nano-Zetasizer (Malvern


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Instruments) at 20 ºС with a scattering angle of 173º and a wavelength of λ = 633 nm. Droplet sizes and PDIs are given as the average of five measurements. 10 µL of the emulsion were diluted with 1.5 mL of water in a polystyrene cuvette before the measurement. Zeta potential measurements. The zeta potential of the samples was measured on a NanoZetasizer (Malvern Instruments) at 20 °C under the mode of zeta potential. A total of 20 µL of the emulsion was diluted with 980 µL of water. For the polystyrene particles, a total of 0.3 mg of the dried particles was dispersed in a Lutensol solution in diluted PBS. The concentration of Lutensol was 1 mg/mL and the PBS was diluted 1:10 with MilliQ water. Zeta potentials are given as the average of three measurements. Conversion measurements. The styrene conversion after the polymerization was calculated from the solid content of the dispersion. Surface tension measurements. All surface tension measurements were performed on a tensiometer (DCAT21, DataPhysics) using the Du Noüy ring method. The Pt-Ir-ring (wire radius: 0.185 mm, ring radius: 9.4425 mm, height: 25 mm) was annealed prior to every measurement. The measurement itself was performed in the push–pull mode with each 10 push and pull operations per measurement. Surface tensions are given as the average of at least four measurements. Interfacial tension measurements. The measurement of the interfacial tension between an aqueous dye solution and IsoparM was performed on the same instrument with the same ring as the surface tension measurements. The measurement itself was performed in the pull mode with each 10 push and pull operations per measurement. Interfacial tensions are given as the average of at least three measurements.


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UV-vis measurements. For the recording of the UV-vis spectra, 100 µL of the aqueous sample were put in one well of a Greiner 96 Flat Bottom Transparent Polystyrene plate and the measurement was performed on a Tecan infinite M1000. A step size of the wavelength of 2 nm as well as 1 flash was used. To quantify the absorption at one specific wavelength, the multiple reads per well mode was used with a boarder of 1000 µm, 5 reads per well and 30 flashes. In order to determine the fluorescein content in the polystyrene particles after washing them, 5 mg of the dried particles was dissolved in 800 µL of chloroform and 200 µL of ethanol was added. The solution was transferred to a quartz glass cuvette and analyzed with a SPECORD 50 (Analytic Jena AG). For the calibration curve, polystyrene was produced in bulk by heating a mixture of 2.2 mL of styrene, 100 µL of HD and 70 mg of V-70 to 40 °C for 20 h. 5 mg of this polymer was dissolved in 800 µL of chloroform and 200 µL of a fluorescein solution in ethanol was added. The final concentration of fluorescein in the mixture was between 0.01 and 0.0625 mg/mL and the samples were analyzed as described above for the polymer particles. Subsequently, the bands at 457 and 487 nm were taken to calculate the content of fluorescein in the polymer particles with help of the calibration curve and application of the Lambert-Beer law. NMR spectroscopy. NMR spectra were recorded on a Bruker AMX 400. Transmission electron microscopy (TEM). TEM measurements were performed on a Zeiss EM10 microscope with an acceleration voltage of 120 kV. The samples were prepared by putting 5 µL of the dispersion on a copper grid, subsequently, they were allowed to dry over night at room temperature. Optical microscopy. Optical microscopy images were recorded with a Zeiss Axio Vert.A1. One drop of the emulsion was placed on an object plate and covered with a cover slip prior to analyzing the sample with the microscope.


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RESULTS AND DISCUSSION Preparation of miniemulsions stabilized by various amounts of fluorescein and SDS. In a first series of experiments, the influence of the concentration of fluorescein on the miniemulsion stability is investigated. The amount of aqueous dye solution was kept constant at 9.5 mL and 0.5 mL of IsoparM (a mixture of linear and branched hydrocarbons, mainly C12 to C15) was used as the disperse phase. Because of the high hydrophobicity of the oil no additional osmotic agent is required. Experimental details as well as the characterization of the emulsions by DLS can be found in the supporting information (see Table S 1). With fluorescein concentrations below 0.05 mg/mL, no long-term stable emulsions can be obtained indicating that the concentration of the stabilizing species is too low. The optical microscope image of an emulsion prepared with 0.025 mg/mL fluorescein shows large droplets in the µm-range with a broad distribution (see Figure S 1 B). Already a few minutes after preparation, some oil is released from this emulsion, which shows that it is not long-term stable. Above 0.05 mg/mL (Fl1), a pale yellow emulsion with an average droplet size of about 9 µm and a zeta potential of -70 mV is obtained. This behavior is surprising as the molecular structure of fluorescein gives no indication for the dye as classical amphiphile to stabilize emulsions. Several days after preparation, the emulsion shows creaming by separating into a turbid pale yellow orange layer on the top (“cream”) and a transparent bright yellow layer below (“solution”) caused by density differences of the disperse and the continuous phases. Nevertheless, this emulsion is stable because creaming is reversible and redispersion can be simply induced by shaking. Even ten months after preparation, no oil has been released from the emulsion, which confirms that Fl-1 is


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long-term stable. It should be noted that the concentration of fluorescein in Fl-1 is in the range or even below the concentrations employed for water-soluble dyes to color emulsions and double emulsions5–8. An increase in the amount of dye from 0.075 mg/mL up to 15 mg/mL (Fl-2 – 9) results in long-term stable emulsions (see photograph in Figure S 1 A and optical microscope images in Figure S 1 B and C in SI) with decreasing droplet sizes down below 200 nm (see Figure 2 (blue circles) and Table S 1 in SI) as known from classical surfactants like SDS (see Figure 2 (red squares) and Table S 4 in SI (SDS-2 – 9)). For SDS the droplet diameter does not decrease further but stays fairly constant at about 180 nm for concentrations above 10 mg/mL. In contrast, when the concentration of fluorescein is increased to above 20 mg/mL, the droplet diameter is increasing. However, those emulsions (Fl-10 – 13) are long-term stable and no oil phase separates from the emulsions even 15 months after preparation (see Table S 1 and Figure S 1 in SI). This different behavior of fluorescein and SDS raises the question on the mechanism of stabilization in both cases and will be discussed in more detail later.

Figure 2. Correlation between the amount of stabilizer and average droplet size of the emulsion. The emulsions stabilized by SDS (red squares) display the expected decline with increasing


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concentration of SDS while in the case of fluorescein (blue circles) the sizes go through a minimum. The lines are only a guide to the eye.

Interfacial tension measurements between aqueous solution of stabilizer and IsoparM. In order to clarify why fluorescein is able to stabilize o/w miniemulsions, both surface tension and interfacial tension (versus IsoparM) of aqueous solutions of fluorescein were measured. It turns out that the surface tension of a fluorescein solution with a concentration of 1 mg/mL (0.0027 mol/L) is still above 70 mN/m and the lowest value that could be obtained is 54 mN/m for a concentration of 60 mg/mL (see Figure S 2 in the supporting information). If these results are compared with the classical surfactant SDS (about 35 mN/m at a concentration of 3 mg/mL (0.01 mol/L) which is above the cmc), it can be concluded that fluorescein is only weakly surface active. The interfacial tensions of aqueous fluorescein solutions versus IsoparM behave comparably. Again, very high interfacial tensions can be observed for low concentrations of fluorescein (above 30 mN/m for 1 mg/mL and even 36 mN/m for concentrations below 0.1 mg/mL). The lowest value reached with fluorescein is still much higher than the one obtained with SDS at its cmc (above 18 mN/m for a 60 mg/mL fluorescein solution against 5.7 mN/m for a 3 mg/mL SDS solution). The results of the interfacial tension measurements are summarized in Figure 3 A.


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Figure 3. Interfacial tension between oil (IsoparM) and water for SDS and fluorescein and representative emulsions. A: Logarithmic plot of the water-IsoparM interfacial tension as a function of fluorescein (blue circles) and SDS concentration (red squares). B: Photograph of o/w emulsions prepared under the same conditions from aqueous solutions of SDS (1, left) and fluorescein (2, right) with the fluorescein solution having an even higher interfacial tension versus IsoparM than the SDS solution (see 1 and 2 in A). Note that the yellow emulsion on the right is not transparent and no oil droplets can be seen on top while emulsion number 1 phase separates after a few minutes as shown in the magnification in the black box.

It has been reported earlier that rhodamine B can stabilize emulsions temporarily. This behavior has been attributed to the lowering of the interfacial tension9. Nevertheless, stable emulsions can be obtained even at fluorescein concentrations where the interfacial tension between the dye solution and IsoparM is clearly above 30 mN/m (see Figure 3 B emulsion 2 and Fl-1 in Table S 1 in SI). In contrast, an emulsion prepared under the same conditions with an SDS solution exhibiting an interfacial tension of about 34 mN/m is not stable (see Figure 3 B emulsion 1 and SDS-1 in Table S 4 in SI). While Fl-1 is stable for several months SDS-1 shows phase separation a few minutes after preparation and turns transparent (see pencil in the background of Figure 3


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B). In addition, an emulsion prepared from an aqueous solution of fluorescein with the same interfacial tension as the one for Fl-1 but a lower concentration of fluorescein is not stable (see blue dotted line in Figure 3 A). Thus, the (slightly) lowered interfacial tension cannot be the only reason why fluorescein is able to stabilize (mini)emulsions. The logarithmic plot of the interfacial tension versus the stabilizer concentration (Figure 3 A) shows another difference between SDS and fluorescein. For low concentrations, a plateau is obtained for both stabilizers. After the following decay in the range of intermediate concentrations, the graph of SDS shows a sharp bend which is attributed to the total coverage of the interface with surfactant molecules and the formation of micelles while the graph of fluorescein shows only a very slight bend and the interfacial tension continues decreasing. This behavior indicates that the interface is not fully covered by fluorescein molecules even at very high concentrations of up to 60 mg/mL and that there is no formation of classical micelles. Effect of electrolyte. In the next series of experiments, the effect of electrolyte on droplet size and emulsion stability is investigated by adding sodium chloride to the aqueous phase of both fluorescein (0.5 mg/mL) and SDS (0.1 mg/mL) stabilized emulsions before preparing the emulsions. For the charged surfactant SDS, which stabilizes emulsion droplets in its monomeric form, low concentrations of sodium chloride (below 0.025 mol/L) do not affect the emulsion stability (see Figure 4 and emulsions SDS-2 and SDS-10 - 13 in Table S 5 in the supporting information). At higher electrolyte concentrations, the average droplet size increases with an increase in the amount of added sodium chloride (see Figure 4 and emulsions SDS-14 - 19 in Table S 5 in the supporting information); at the same time, the emulsion stability decreases as expected from DLVO theory16,17. The emulsions prepared with fluorescein as the stabilizer behave completely


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different. In the range of low sodium chloride concentrations, the average diameter of the emulsion droplets decreases with an increase in the electrolyte concentration, which indicates that the amount of the stabilizing species is increasing in this range (see Figure 4 and emulsions Fl-4 and Fl-14 - 17 in Table S 2 in the supporting information). The surface tension of 0.5 mg/mL fluorescein solutions, which contain up to 0.1 mol/L sodium chloride, do not differ from the one of a fluorescein solution without the electrolyte within the error of measurement (data not shown). This observation is another indication of the difference in the stabilization mechanism between SDS and fluorescein and gives rise to the assumption that fluorescein aggregates are the stabilizing species in the sense of molecular scale Pickering stabilizers. It is known that the dimerization constant of xanthene dyes increases with an increase of electrolyte concentration18,19. Thus, it is assumed that small fluorescein aggregates (see next subsection) are formed as stabilizing species and its amount increases with an increase of sodium chloride concentrations up to 0.05 mol/L. For a salt concentration of 0.1 mol/L, the stability reaches its maximum and at higher electrolyte concentrations the emulsions are no longer long-term stable by showing phase separation after one month (see Figure S 3 in SI). This behavior can be attributed to the formation of bigger fluorescein aggregates, which cannot stabilize the emulsion any longer presumably because of too high charge density and repulsion between the aggregates. The assumption that at high sodium chloride concentrations not only more small aggregates but also bigger aggregates are formed than at low electrolyte concentrations is affirmed by DLS measurements of aqueous fluorescein solutions with a dye concentration of 0.5 mg/mL and different sodium chloride concentrations (see Figure S 4 in SI). For sodium chloride concentrations below 0.1 mol/L, objects with a size of about 1 nm can be detected. Presumably, this signal belongs to fluorescein dimers or small fluorescein aggregates, which are able to


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stabilize o/w emulsions. For electrolyte concentrations between 0.1 and 0.25 mol/L, the average diameter of particles increases to 5 – 10 nm. In this range, emulsions can be stabilized temporarily, but they exhibit no long-term stability. An increase of the sodium chloride concentration to around 0.4 mol/L causes an increase in the size of aggregates to about 20 nm; these aggregates are apparently inappropriate to stabilize the emulsion droplets, probably because the wetting behavior changes when the aggregates grow larger (see Fl-22 in Table S 2). When the behavior of fluorescein in a saturated sodium chloride solution is finally studied, the size of the aggregates that are detected by DLS is in the µm-range and the aggregates can be even seen with the naked eye.

Figure 4. Development of the average droplet diameter with an increase of the sodium chloride concentration. While for SDS (red squares, 0.1 mg/mL) a steady increase of droplet sizes with increasing salt concentration is found in accordance with the DLVO theory the emulsions with fluorescein (blue circles, 0.5 mg/mL) go through a minimum.


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Considering that presumably an aggregated species of fluorescein is stabilizing the emulsion droplets and not the monomeric form, the behavior shown in Figure 2 can be further discussed. In the first section, it was pointed out that for SDS, the droplet diameter decreases with an increase of the surfactant concentration until a minimum is reached. For fluorescein, however, an increase of the droplet diameter can be observed at high concentrations. This indicates that the concentration of the stabilizing species is increasing in the first range (for concentrations up to 15 mg/mL) followed by a decrease in the second range (for concentrations above 15 mg/mL). Supposing that small aggregates are the stabilizing species, this suggests that at concentrations above 15 mg/mL another species arises, which is less effective in stabilizing the emulsion droplets than the small aggregates. One possibility is that, comparable to the addition of salt, at higher concentrations bigger aggregates are formed which are not able to stabilize the emulsion as good as the small aggregates. Nevertheless, these emulsions are stable, which means that either the concentration of the small aggregates is still high enough or the bigger aggregates can also stabilize the oil droplets, but are not as effective as the di- and/or trimers. This might be also related to higher charge density and stronger repulsion between the aggregates (see above). UV-vis spectroscopy. A direct experimental proof of the presence of aggregates can be found in optical spectra. Therefore, we have performed UV-vis measurements of o/w miniemulsions with 0.05 mg/mL fluorescein as stabilizer (Fl-1, see Table S 1 in SI) because it can be clearly differentiated between the monomeric (λmax = 492 nm) and aggregated (λmax,1 < 492 nm and λmax,2 > 492 nm) form of the molecule20. Fl-1 is long-term stable although the oil droplets are in the micrometer range; moreover, the concentration of dye is so low that the emulsion does not have to be diluted to record the UV-vis spectra. The emulsion was allowed to cream for several weeks and UV-vis


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spectra of both the “solution” and the “cream” diluted 1:1 with the “solution” were recorded. The strong scattering of the emulsion droplets in the “cream” cause a baseline-shift but still it can be seen even in the raw spectra that there is a huge difference in the absorption behavior of the “cream” compared to the solution (the raw spectra are shown in Figure S 5 in the SI). To eliminate the influence of the scattering oil droplets on the spectrum, the UV-vis spectrum of an emulsion stabilized by SDS was recorded and subtracted from the spectrum of the cream of Fl-1. The scattering of the emulsion droplets is the same for emulsions stabilized with SDS and the cationic surfactant cetyltrimethylammonium bromide and seems to be independent from the droplet size; therefore, it can be assumed that this kind of correction excludes the influence of the oil droplets. After a baseline correction and the normalization of the spectra to their maximum it turns out that the spectrum of the “solution” shows the characteristic band at 492 nm which is attributed to the monomeric form of fluorescein. In contrast, the spectrum of the “cream” shows a broad band at about 460 nm as well as one shoulder at 506 nm and another weaker one at about 430 nm (see Figure 5). The spectrum of the dimeric form of fluorescein is known to be composed of 2 bands at smaller and larger wavelengths than the monomer maximum at 492 nm, respectively. Fluorescein trimers show even three bands in the UV-vis spectrum, two at higher energies and one at lower energy than the one of the monomeric form20. Thus, the spectrum of the cream can be regarded as the overlay of the monomer and dimer and potentially trimer spectrum of fluorescein. Therefore, an aggregate species of fluorescein is located at the oil-water interface while the monomeric form dominates in the surrounding solution.


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Figure 5. Baseline corrected and normalized UV-vis spectra of both the “cream” and the “solution” of Fl-1 emulsion. The spectra look different and show that the “cream” contains clearly a higher concentration of the aggregated dye molecules (λmax = 460 nm and a shoulder at 506 nm) than the “solution” which shows only the band of the monomeric form of fluorescein at 492 nm.

Determination of contact angle, aggregation number, area per molecule and surface coverage of fluorescein on oil droplets. Apart from surfactants, solid particles are able to stabilize emulsions. For the stability of these so-called Pickering emulsions, one of the most essential parameters is the particle wettability expressed by the three-phase contact angle θ between water, oil and particle21–24. It should be off from 0° (totally hydrophilic) and 180° (totally hydrophobic) and should take values slightly smaller (for o/w emulsions) and higher (for w/o emulsions) than 90°25–28. When these particles adsorb to the oil-water interface, the interfacial tension is not necessarily lowered significantly, but stable emulsions can be obtained by steric stabilization and also electrostatic stabilization if the particles are charged. We suggest that the fluorescein aggregates act as kind of Pickering stabilizers. To roughly estimate whether these aggregates are able to adsorb to the oil-water


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interface based on their “wetting”, the macroscopic wetting behavior of bulk fluorescein relative to water and IsoparM was investigated by measuring the three-phase contact angle θ with the compressed disk method described by Yan and Masliyah29. The photograph of a water drop on a fluorescein pellet under IsoparM is shown in Figure S 6. A three-phase contact angle of (58±1)° was determined by analyzing three water droplets on fluorescein pellets. This value might not be easily transferred to the “wetting” behavior of fluorescein aggregates but it shows that fluorescein in its bulk form is neither totally hydrophilic nor totally hydrophobic and, thus, may be able to stabilize direct emulsions as the contact angle is smaller than 90°. Theoretical calculations on the wetting behavior and the affinity of different fluorescein species to the oilwater interface are running currently; presumably, the wetting behavior of the aggregated form differs from the one of the monomers and makes aggregates more appropriate to stabilize o/w emulsions. To further verify the assumption that fluorescein aggregates rather than single fluorescein molecules are the stabilizing species, the area per molecule was calculated from the linear part of both the surface and the interfacial tension isotherm shown in Figure S 2 and Figure 3. The maximum excess solute per unit area at the surface or interface, Γmax, was

calculated using the Gibbs equation Γ = −

,

with temperature T, universal gas

constant R, surface or interfacial tension γ and bulk concentration of fluorescein c. The area per molecule Ai is then given by A = (Γ N )−1 with Avogadro’s constant NA and was calculated to be 0.21 nm2 (by surface tension) and 0.22 nm2 (by interfacial tension). In literature, there are detailed studies on the space requirement of different perylene derivatives30–32, which exhibit a size that is fairly comparable to that of fluorescein. By means of Langmuir and LangmuirBlodgett films, areas per perylene molecule in the range of 0.4 – 0.6 nm² were obtained, which indicates that the molecules are arranged in the edge-on or the head-on configuration at the


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interface and not in the flat-on configuration. As the space requirement of fluorescein at the IsoparM-water interface is in the same order of magnitude, it can be assumed that the fluorescein molecules are not arranging flatly as monomers on the interface either. On the other hand, in contrast to Langmuir-Blodgett films, there is no force applied to the molecules at the interface when the area per molecule is calculated from interfacial tension measurements and presumably, the interface is not fully covered by fluorescein. To get an idea of which percentage of the interface is really covered by fluorescein in emulsion, Fl-4 and Fl-5 (0.5 and 1 mg/mL fluorescein, respectively) were allowed to cream for several weeks and the “solutions” were diluted until their absorption at a wavelength of 492 nm was in the Lambert-Beer range. Subsequently, the concentration of fluorescein in the “solution” was determined from the data obtained by UV-vis and the amount of fluorescein at the interface was calculated (details see Calculation S 1 in the supporting information). The data obtained with UV-vis were reproduced with NMR spectroscopy. Therefore, another emulsion with the composition of Fl-5 but with deuterated water as the basis for the continuous phase was prepared and allowed to cream. Afterwards, 1 µL of THF as a standard was added to 700 µL of the “solution” and the mixture was analyzed by NMR spectroscopy. The percentage of fluorescein adsorbed to the interface obtained by this method (16 % of the applied amount) is in good agreement with the result of the UV-vis measurements (18 %). Together with the area per molecule and the average droplet diameter, the percentage of the droplets’ surface covered by fluorescein was estimated to be 5 % for Fl-4 and 7 % for Fl-5. These values confirm the assumption that the interface is not fully covered with fluorescein molecules. Together with the space requirement per molecule of about 0.2 nm² without the application of any force in comparison with the values from literature, where a surface coverage of nearly 100 % is assumed, this leads to the presumption that the fluorescein


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species, which adsorbs to the IsoparM-water interface is not the monomeric form of the dye, but (small) aggregates. In addition, both the aggregation constant and aggregation number of fluorescein under the given conditions were determined by NMR spectroscopy as described in literature33 (see Figure S7 in SI). It turns out that the aggregation number is between 2 and 3, that means di- and trimers of fluorescein are formed. This value fits quite well to the space requirement of about 0.2 nm² per molecule and the UV-vis spectrum of the “cream” in Figure 5. The dimerization constant was calculated to be 5 L/mol, which is in very good accordance with literature34. This finding together with the UV-vis spectrum of the “cream” in Figure 5 gives rise to the assumption that the interface between the aqueous solution and oil has a huge impact on the aggregation of fluorescein. At a fluorescein concentration of 0.05 mg/mL as it is used for Fl1 only 0.03 % of the molecules is in the dimeric form and the rest of the fluorescein molecules are monomers (see Calculation S 2 in the SI). In the UV-vis spectrum, however, the aggregated form is the dominant species in the “cream” although it has been diluted 1:1 with the “solution” prior to measuring. This means that the apparent aggregation constant is much higher in the presence of the interface than it is in the pure aqueous solution. Effect of pH. As pH has a strong effect on the charge state of the dye molecules (see Figure 6 A)35 it is expected to have a significant influence on aggregation and emulsion stability, too. Indeed, below pH 4, no stable emulsions can be obtained, which is assigned to the low water solubility of the neutral species (see Fl-23 and 24 in Table S 3 in SI). In the range between pH 4 and 7, emulsions with droplet sizes between 200 and 500 nm can be stabilized (Fl-25 – 29 in Table S 3 and Figure 6 B) and pass a maximum stability (minimum droplet size) between pH 5 and 6 as expected from the pKa value. In the latter range the monoanionic form dominates. Although it is


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reported that the interfaces in microemulsions have a significant effect on the protonation equilibria of fluorescein molecules we assume that the absence of charged surfactant and cosurfactant in our system justifies staying with the solution pKa values36. Interestingly, the droplet diameters depending on the concentration of the monoanion show a highly linear correlation (see Figure 6 C) further suggesting that the monocharged ions are mainly responsible for the stabilization of the oil droplets in water (see below).

Figure 6. A: Fluorescein protonation equilibria between the cationic (1), neutral (2), monoanionic (3) and dianionic (4) form. B: Influence of the pH value on both the percentage of the neutral, monoanionic and dianionic species and the droplet diameter (Fl-25 – 29, see Error! Reference source not found.). The cationic form of fluorescein has been neglected for the calculation because it plays a minor role in the given pH range. C: Correlation between the droplet diameter and the concentration of the monoanionic form of fluorescein.


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Miniemulsions stabilized by other water-soluble dyes. The stabilizing ability of dyes is not limited to fluorescein. Rhodamine B and eosin Y feature a very similar chemical structure as fluorescein (see Figure 1). Long-term stable o/w miniemulsions can be produced with rhodamine B and eosin Y as stabilizer (see RhB-1, RhB-2, E-1 and E-2 in Table S 10 in SI) and smaller droplets at even lower concentrations in comparison with fluorescein (see Fl-3 and Fl-5 in Table S 1 in SI) are obtained with eosin Y. On the basis of the findings above we attribute this to the about 20 times higher dimerization constant of eosin Y in comparison to fluorescein34,37 and thus higher concentration of aggregates. Besides xanthene dyes also azo dyes like congo red and alizarin yellow GG are excellent stabilizers and show the same correlation between the average droplet diameter and the dye concentration (see Table S 6, Table S 8 and Figure S 8). Especially congo red is a very effective stabilizer, as concentrations of only 1 mg/mL (1.4 mmol/L) lead to emulsion droplets of an average diameter of 174 nm. Remarkably, under the employed conditions congo red is more effective than SDS. Crystal violet, malachite green, methylene blue, methyl orange and nile blue are capable of stabilizing direct miniemulsions, too (see Table S 10 and Figure S 9 in SI). Variation of the oil and polymerization in dye-stabilized emulsion. With regard to any application of miniemulsions stabilized by dyes, it is desirable that not only IsoparM but other organic substances can be used as the disperse phase. Droplets of several different nonpolar (cyclohexane, isooctane, hexadecane), aromatic (toluene, styrene) and polar (chloroform, diethyl adipate) oils as well as perfluorodecalin and silicone oil can be stabilized in water by both congo red and alizarin yellow GG (see CR-11 – 19 in Table S 7 and AY-8 – 16 in Table S 9). The perfluorinated compounds find e.g. applications in the medical sector, which makes them also very attractive candidates for being stabilized in water by dye aggregates.


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Styrene as the disperse phase is very intriguing, too, because it offers the possibility of polymerization in dye stabilized miniemulsions. Polymerization was performed with the oil soluble azo initiator V-70 and fluorescein and SDS as stabilizer, respectively (see P-1 and P-2 in Table S 11 in SI). For both polymerizations, a styrene conversion of more than 90 % and particles with sizes in the range of 300 nm were achieved (see TEM of P-1 in Figure S 10 in SI). The particles were washed with sodium hydroxide solution and ethanol. The removal of the dye down to minimum traces could be easily followed by the fading color of the washing solutions while in the case of SDS other techniques are required (e. g. conductometry). Nevertheless, a remaining slightly yellow appearance of the particles from P-1 after washing suggests that there is some dye bound in or to the polymer particles. UV-vis spectroscopy of the dissolved particles revealed that about 6 wt% of the employed amount of dye is bound to the particles. Such simple analysis is impossible with colorless surfactants like SDS. Thus, troublesome rests of surfactants in polymer particles from (mini)emulsion polymerization can be easily detected for dyes as stabilizers while it is much more costly for colorless surfactants like SDS. Controlled destabilization. Another important aspect relevant for potential applications is the ability to destabilize the emulsion into water and oil phase. Similar to SDS-stabilized emulsions, phase separation of the dye-stabilized emulsions can be induced by the addition of an adequate amount of salt (e.g. NaCl, CaCl2 etc.). Furthermore, they can be destabilized by changing the pH value. Emulsions stabilized with cationic dyes are destabilized in the acidic and anionic dye systems in the basic range. Temperature plays an important role for the stability of dye-stabilized emulsions too. Some emulsions with e.g. congo red, eosin Y, fluorescein, methyl orange or alizarin yellow GG, are stable up to at least 90 °C, while emulsions with e.g. nile blue, methylene blue, crystal violet,


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malachite green or rhodamine B phase separate quite quickly when they are heated. Thus, the broad variety of stability ranges of dye-stabilized emulsions offers the possibility to choose the “right” dye for the respective application. Nevertheless, in none of the cases mentioned above the stabilizing species (dye or SDS) is separated from the aqueous and the oil phase. Nile blue offers a very interesting way to destabilize the emulsion and get rid of the stabilizer in one simple step by the application of a hydrogel from the protected dipeptide FmocLG15. As the affinity of nile blue to this gel is much higher than for the interface, an emulsion stabilized by nile blue can be easily broken into two colorless phases by the addition of FmocLG gel (see Figure 7 and Figure S 11 in the SI). This process is fast and quantitative, the dye cannot be detected in the aqueous phase afterwards by UV-vis any more. In our opinion, this way of destabilization displays the greatest potential for any application.

Figure 7. Photographs of NB-1 with FmocLG-gel in an NMR tube. A: Shortly after addition of the emulsion to the hydrogel. On close inspection, first oil droplets migrating from the gel to the top can be observed. B-D: Development within 3 weeks. At the beginning, the gel is colorless


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and the emulsion is blue while within 3 weeks the emulsion is slowly broken (limitation by diffusion; the process can be easily accelerated if some pieces of the hydrogel are added to the emulsion (see Figure S 10 in the SI)) into a decolorizing aqueous and a colorless oil-phase (upper phase) and the hydrogel has turned blue.

CONCLUSION A new concept of emulsion stabilization by small dye aggregates as molecular Pickering stabilizers is presented. The concept is applicable to many classes of water-soluble dyes. Furthermore, oil phases with highly variable polarity can be employed and polymerizations performed within the (mini)emulsions. Despite the high stability of the emulsions and dispersions they can be easily broken by temperature, salt or addition of a (specific) dye scavenger (e.g. hydrogel) depending on the employed dye. In contrast to common surfactants like SDS even traces of remaining dye, which might be harmful for applications of the dispersed phase are easily detectable. The high variability of the dyes allows a fine-tuning and adjustment of the stability of the emulsions to the desired application. Thus, surfactant free stable miniemulsions and lattices are accessible which offer a great potential as new materials for various fields.

ASSOCIATED CONTENT Supporting Information. Tables with recipes and characteristics of all emulsions; Photographs of emulsions prepared with different concentrations of fluorescein; Optical microscope images of selected emulsions; Plot of the surface tension of aqueous fluorescein solutions as a function of


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the concentration of fluorescein; Photographs of emulsions stabilized with fluorescein in sodium chloride solution; Development of the diameter of fluorescein aggregates in aqueous solution with different sodium chloride concentrations; Raw UV-vis spectra of both the “cream” and the “solution” of Fl-1; Photograph of a water droplet on a fluorescein pellet under IsoparM to determine the three-phase contact angle; NMR-study of fluorescein to determine the aggregation number; Photographs of emulsions prepared with different concentrations of congo red; Photographs of emulsions stabilized by different dyes; TEM image of the particles from P-1; Photographs of NB-1 with (right) and without (left) FmocLG-gel; Calculation on the percentage of fluorescein adsorbed at the oil water interface in emulsion; Calculation on the percentage of fluorescein dimers at a given concentration of fluorescein. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions

S.S. and U.Z. designed the experiments and wrote the manuscript. U.Z. designed and supervised the study.

ACKNOWLEDGMENT Financial support from Fonds der Chemischen Industrie (FCI) for S. S. and technical support by Raphael Koch and Payam Ajami are gratefully acknowledged. The authors also thank Stefanie Sieste for the preparation of FmocLG.


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