Visualizing Chemical Phenomena in Microdroplets - American

In the Classroom

Visualizing Chemical Phenomena in Microdroplets Sunghee Lee* and Joseph Wiener Department of Chemistry, Iona College, New Rochelle, New York 10801, United States *[email protected]

We introduce the intriguing world of chemical phenomena that occur in an aqueous microdroplet, which are droplets having a diameter in the micrometer range. Chemical phenomena can sometimes be dramatically influenced by the scale at which they take place. When chemical reactions take place on a microdroplet scale, numerous enhancements can occur, such as more efficient mass and heat transfer between droplets and their environment due to a greater interfacial availability and less total mass or energy required for transfers to reach completion. These features are a consequence of the extremely high surface area-to-volume ratio for the typical microdroplet. For example, a 25 μm diameter aqueous droplet has a ratio of external surface area to volume of about 2 105 m-1; in contrast, a 25 mL aqueous sample in a beaker has a ratio of about 20 m-1. Reactions that occur in a microdroplet have taken on an added significance in recent years, because of the increasing importance of microfluidic systems. Advances toward miniaturization in the fields of biology and chemistry have focused on the development of microdroplet microfluidic technology, which uses discrete volumes of reagent species carried within immiscible phases (1-3). This technique involves generation and manipulation of distinct microdroplets within devices, generally flowing within channels having about 1-100 μm length dimensions. These droplets are usually in the micrometer diameter range (e.g., nanoliter volume), often are monodisperse, and can be made to flow at a rate of many hundreds of droplets per second. The use of microdroplets offers the advantage of being able to compartmentalize reagents within isolated microdroplets, which can later be merged or divided as need be. Many types of reactions have been conducted within microdroplets in microfluidic systems (4). With the use of microfluidic systems, chemists have found it possible to achieve miniaturization of chemical reactions, as well as attaining high-throughput chemistry. The field of droplet microfluidics holds great promise, as one can perform typical laboratory operations using a fraction of the volume of reagent in significantly less time. This exciting and burgeoning field will undoubtedly be of increasing importance for chemistry and biochemistry. Consequently, there will be an increasing need to expose students to developments in this field. At least one article focusing on the application of microfluidics in chemical synthesis and analysis has appeared in this Journal (5). However, it is not likely that microdroplet microfluidics will soon become standard fare in the undergraduate curriculum. Furthermore, undergraduate students who may become familiar with microfluidics at an applications (or “black-box”) level may lose sight of some of the factors that make chemistry at the microdroplet level so uniquely advantageous. The purpose of this article is to provide visual evidence of the unique aspects of the chemical phenomena that take place in microdroplets. By means of video still images (and video clips in the supporting information), we hope to enhance student

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understanding of systems where high ratios of surface area to volume are important, one such example being the field of microdroplet fluidics. Although our system is not an example of a microfluidic device because we manipulate individual droplets that do not flow, we nevertheless believe it is an ideal model to showcase microfluidics. It has the added advantage of providing “stop-action” snapshots of the evolution of the system. We use the established technique of micropipet manipulation, combined with digital video microscopy, to show individual aqueous droplets containing different reagents, surrounded by immiscible oil. Real-time visualizations of the unfolding chemical phenomena are provided at the single, isolated droplet level. For institutions that are suitably equipped to reproduce some of our demonstrations, we provide experimental detail to permit this; however, doing so is not necessary for these educational purposes, because our still microscopic images can satisfactorily achieve this. Chemistry in a Microdroplet In our research into the chemistry of the oil-aqueous microdroplet interface (6, 7), we have encountered and recorded numerous examples. Many of these examples illustrate chemical and physical transformations that are made more notable owing to the small size of the droplets and especially their large surfaceto-volume ratio. Other examples are simply more striking by virtue of being viewed under real-time microscopic observation. These examples are presented as microscopic still images taken from recorded videos. The examples dramatize various phenomena that are prominent at the microdroplet level and are chosen to highlight some of the salient features in this size range. Each video image can be used in conjunction with a corresponding benchtop scale demonstration, if desired, to compare notable differences that arise as a consequence of the large surface-to-volume ratio. We encourage readers to adapt and use the images as appropriate for instructional purposes. In these video images, chemical reactions and physical phenomena are shown to occur in one or more individual water droplets usually having a diameter in the range of tens to hundreds of micrometers, surrounded by an immiscible liquid such as decane (Figure 1). These nanoliter volume droplets are delivered into the immiscible phase from micropipets employing digital video microscopic monitoring. Because microdroplets are created and manipulated one at a time, under continuous video monitoring, the precise outcomes of a chemical reaction or other physical phenomenon within or between microdroplets can be visualized, from start to finish. Generally, our images show a single droplet or, alternatively, a pair of droplets that are manipulated simultaneously and brought into contact. Upon the merger of a pair of droplets, different chemical reagents encapsulated in the respective droplets mix and subsequent chemical reactions occur (schematically depicted in Figure 2).

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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 2 February 2011 10.1021/ed100518k Published on Web 11/03/2010

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Journal of Chemical Education

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In the Classroom

Figure 1. (I) Individual aqueous microdroplet held at tip of a micropipet in surrounding oil. (II) For size comparison, an image of a human hair is shown. Typical micropipet used here has a 20-30 μm diameter.

Figure 3. Held by a micropipet, a single water droplet (95 μm) in 1-octanol (I), 1-decanol (II), and 1-dodecanol (III) carrier fluid at varying times after droplet formation. Figure 2. Merger of two aqueous microdroplets containing dissolved reactants, A and B, induces the mixing of the reactants to produce the product.

Solubility of Liquids We begin our discussion of microdroplets with one of the more dramatic effects of a large surface area-to-volume ratio, namely, the visualization of solubility phenomena. Solubility phenomena are important in many applications including pharmaceutical, food industry, and chemical engineering processes, but they can be difficult to see with the naked eye. If solubility is extremely small, as in the case of water and a hydrocarbon or a long-chain alcohol, then visual observation of the dissolution process on a benchtop scale can take days or months to demonstrate. However, by using a microdroplet, a liquid-liquid dissolution process at an observable laboratory timescale (seconds to minutes) can be seen. Aqueous microdroplet shrinkage can be conceptualized as a mass transport process of water molecules from a microdroplet, across an interface into a medium having slight solubility for water. The relatively large interface can lead to droplet shrinkage on a more convenient timescale. The images in Figure 3 show the correlation between the relative solubility of an aqueous phase and the hydrocarbon chain length of the oil phase. A series of water droplets held in longchain alcohols of varying hydrocarbon chain lengths is shown in Figure 3. Sequence I shows a water droplet in 1-octanol, sequence II shows a water droplet 1-decanol, and sequence III shows a water droplet 1-dodecanol. The series shows that a water microdroplet dissolves faster in a shorter chain length alcohol than in a longer chain length alcohol, correlating with the expected chemical solubility of water in the various members of this class of solvents. The solubility of water in straight-chain alcohols of C8, C10, and C12 hydrocarbon chain lengths is 4.4%, 3.3%, and 2.9%, respectively. These results can be used in a classroom as confirmation of the expectations set by a lecture. These images allow students to 152


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see the “invisible” phenomena that generally take too long to demonstrate on a macroscale. In a classroom, the images can be used to emphasize the importance of surface-to-volume ratio in a small droplet. Thus far, our exposure of students in general chemistry to these images has led to fruitful classroom discussion of concepts including miscibility. The shrinkage of the water droplet was explained in lecture and discussion on the topic of solubility of molecules, in general. The relative rate of solubilization was explained in terms of the “like-dissolves-like” concept, in which the more nonpolar dodecanol was explained as having the least affinity for polar water molecules. The sequences of images also illustrate a feature of some microfluidic systems as well. In microdroplet-based microfluidic systems, microdroplets flow in a nominally “immiscible” carrier phase, but this may not always be strictly true. It has been found that droplets may sometimes suffer from rapid dissolution into their surrounding carrier phase, a phenomenon attributable to the surface-to-volume ratio. This has been reproduced at the single droplet level, which increases student awareness of some of the current problems in microfluidic technology. Crystallization Crystallization represents a delicate balance between thermodynamics, kinetics, electrostatic forces, geometric packing efficiencies, and chemical bonding environments. However, crystal chemistry is often underrepresented in the undergraduate laboratory because of the long time that is generally needed for the crystallization process. It has been found that if one of the aqueous droplets shown in the solubility experiments contains a crystallizable solute of interest, the solute becomes concentrated. Eventually, it can become concentrated enough to reach the critical saturation level for crystallization. The overall process takes only several

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r 2010 American Chemical Society and Division of Chemical Education, Inc.

In the Classroom

Figure 4. Video micrographs of in situ crystallization process in an aqueous microdroplet containing K2CrO4 (I) and K4Fe(CN)6 3 3H2O (II), both surrounded by 1-decanol. Typically crystallization is observed within 5 min.

minutes, which is another manifestation of the extremely high ratio of surface area to volume for microdroplets. As shown in Figure 4 (sequences I and II), video monitoring has been used to follow the crystallization and growth process for crystals of potassium chromate and of potassium hexacyanoferrate(II) trihydrate, respectively. In a period of usually 5 min or less, an aqueous droplet containing an undersaturated metal solution surrounded by 1-decanol shrinks in size to the point where a crystal dramatically appears. Simply “seeing” an individual single crystal as it appears and monitoring its shape can provide an invaluable means of understanding crystallization processes. Furthermore, images of the crystallization events shown in our general chemistry classes have prompted productive discussion of concepts including undersaturation, saturation, and supersaturation. On the basis of the initial concentration of the droplet and its initial and final size, students have been asked to calculate the final solute concentration at the point that the crystal appears, which is invariably higher than the equilibrium solubility value. This has encouraged students to think about crystallization phenomena in a more rewarding way. Cross-Droplet Reactions Microdroplets containing volatile reagents can sometimes undergo rapid reactions without even coming into contact. The rate at which a volatile solute diffuses out of a microdroplet can become magnified owing to the very large ratio of surface area to volume of the droplet. A series of video images shows a visualization of gaseous ammonia being transferred across the oilwater interface from one microdroplet to another, to form colored, aqueous coordination complexes. Although ammonia complexation reactions are often demonstrated on the benchtop scale with solutions, it is not always safe or convenient for students to handle gaseous ammonia. We simply bring into proximity (about 1020 μm apart) a droplet of aqueous ammonia (15%) and a droplet of a metal salt, both held in decane as the immiscible continuous phase (Figures 5-7). Gaseous ammonia diffuses from its droplet through the oil phase and into the droplet of metal salt. The resulting ammonia complexation reactions produce products of different colors and solubilities, depending upon the stage of reaction. Figure 5 depicts the interaction of CoCl2(aq) (A) with ammonia (B). At an initial stage of reaction, a green-blue precipitate is formed (frame II), which soon transforms into a soluble orange complex, presumably [Co(NH3)6]Cl2(aq), as more ammonia transfers into the metal-containing droplet (frames IV and V). In Figure 6, a droplet of CuSO4(aq) (A) is held within several tens of micrometers of a 15% aqueous ammonia droplet (B).


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Figure 5. Complexation reaction of ammonia (15% aq) (droplet B) with a droplet containing 0.4 M CoCl2 (droplet A), both in decane. The entire process takes place within 2-3 min.

Figure 6. Complexation reaction of ammonia (15% aq) (droplet B) with a droplet containing 0.2 M CuSO4 (droplet A), both in decane. An arrow in frame III indicates a point where turbidity in the droplet is clarified by reaction with further gaseous ammonia transferred from droplet B. Entire process from frame I to V takes place within 1 min.

Initially, a pale blue-green turbid precipitate is seen due to insoluble copper-ammine complex (frames II and III), which then dramatically is transformed into a soluble deep blue-purple solution, understood to be [Cu(NH3)4]SO4(aq), as the reaction


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Figure 7. Reaction of ammonia (15% aq) (droplet B) with a droplet containing 0.2 M Pb(NO3)2 (droplet A), both in decane. Entire process takes place within 1-2 min. Scale bar represents 200 μm (for frames I-IV) and 50 μm for frame V.

progresses further (frames IV and V). In particular, in frame III, the leading edge of the copper-containing droplet (shown by the arrow) begins to shed its turbidity and transforms into the clear blue color characteristic of the tetra-ammine complex. In Figure 7, Pb(NO3)2(aq) (A) is brought into proximity with 15% aqueous ammonia (B). Ammonia diffuses to droplet A and causes a reaction to form an insoluble white precipitate, understood to be Pb(OH)2. Upon close observation of the final product, it is clear that the white precipitate is composed of regularly shaped crystals (frame V). Images of these ammonia-complexation reactions shown in inorganic chemistry lectures have illustrated numerous chemical concepts. First, the videos images illustrate the color changes and precipitation reactions often associated with changes in coordination sphere of certain inorganic ions. Second, the detection of intermediate products (e.g., cloudiness in the Co2þ and Cu2þ reactions) sparks classroom discussion relating to stoichiometry and limiting reagent. Finally, more generally, the manner in which ammonia is spread from its droplet of origin into its neighboring droplet has been discussed in terms of vapor pressure, diffusion, and solubility, and ratio of surface area to volume of the droplet. Interfacial Reactions: Metal Extraction from Aqueous Phase Another example of the relatively more “invisible” chemical phenomena is the transfer of molecules across the boundary between phases such as hydrocarbon and water. Traditionally, organic chemistry students utilize extractions in shaken separatory funnels to transport a desired product from, for example, an aqueous phase to an organic phase. However, this type of process can suffer from the perception of being a black-box phenomenon. We demonstrate a droplet experiment that allows students to view the transport as it occurs. For example, a colorless microdroplet containing an organic chelating compound, dimethylglyoxime (dmg), dissolved in chloroform is held in a surrounding aqueous environment of 0.2 M NiCl2. A rapid appearance of acicular reddish-purplish 154


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Figure 8. Progression of chelation of Ni2þ by dimethylglyoxime (dmg): a droplet containing dmg dissolved in CHCl3, surrounded by NiCl2 aqueous solution. Metal ions (Ni2þ) in a continuous aqueous phase are transported into ligand-bearing oil microdroplet, forming needle-shaped crystal of the coordination complex, Ni(dmgH)2. The entire process takes place within 2-3 min.

crystals is seen. The nickel ions have been transported from the pale green surroundings into the colorless droplet with concomitant formation of the chelate complex Ni(dmgH)2 (frame II in Figure 8). Frames III and IV show the process of crystal growth. The sequence in Figure 8 demonstrates the molecular transport between two different phases, that is, metal extraction in the microdroplet. Furthermore, because no shaking or vibration of the droplet occurred, the relatively rapid rate at which the crystals are seen to form can be explained as a reflection of the large surface-to-volume ratio of the droplet. Aside from the unique features attendant to their size, we found that the reaction shown in this section can also have pedagogic value for illustrating other content as well. Particularly, this section has proven useful for enlivening lectures in inorganic chemistry classes pertaining to chelation and metal-complex formation. Two-Droplet Reaction with Color Change: Redox Reaction An exemplary type of microdroplet reaction, which reveals interesting effects when observed under continuous video microscopy, is the two-droplet redox reaction. Specifically, we show the merger of a droplet of 0.2 M aqueous K2CrO4 with a droplet of 0.2 M aqueous ascorbic acid (vitamin C). Each droplet is initially formed in a surrounding decane medium and held by its respective micropipet (frame I of Figure 9). Translation of the micropipets merges the droplet of potassium chromate into that of the ascorbic acid (frame II), causing a color change from yellow to the greenish color of Cr(III) within tens of seconds. There are two notable features visible at intermediate times. First, in the first seconds after droplet fusion, the outlines of the K2CrO4 droplet within the merged droplet can still be seen (frame III), presumably due to density effects. Second, the outermost areas of the outlines of the K2CrO4 droplet (shown by the arrow in frame IV) are seen to be turning green, a manifestation of kinetics at this position in the merged droplet. At a later time (frame V), more extensive interdiffusion occurs and this rapid reaction appears complete.


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In the Classroom

Figure 10. Demonstration of precipitation reaction between two droplets held in decane: (I) droplet containing 0.2 M CaCl2 (A) solution and 0.2 M Na2CO3 (B) solution; (II) fusion of two droplets; and (III) formation of CaCO3 as product.

Figure 9. Fusion of two microdroplets containing 0.2 M aqueous K2CrO4 (A) and 0.2 M aqueous ascorbic acid (B) in a surrounding decane medium.

One can envision droplets A and B as being microscopic “containers” of their respective reagents. It is seen in Figure 9 that by merely bringing the two containers into contact, one droplet is absorbed into the other and reaction quickly proceeds, another manifestation of an enhanced ratio of surface to volume. Furthermore, these images can serve as a convenient introduction of how certain microdroplet fluidic reactions would appear when under observation. Precipitation Reaction: Crystal Formation To dramatize apparent differences between nanoliter-size droplet reactions as compared to those at a benchtop scale, a precipitation reaction was carried out by bringing together an aqueous droplet of CaCl2 with one of Na2CO3. This common precipitation reaction often gives an indistinct white precipitate in a flask, but can be seen to develop into well-formed crystals of CaCO3 when viewed with real-time microscopic observation. Interestingly, the initially perceptible product in the first few seconds of reaction (frame II, Figure 10) was an opaque precipitate, which minutes later ripened into euhedral crystals (frame III). Classroom Use of Demonstration Images The microdroplet chemical phenomena described have been used to supplement classroom learning in a number of chemistry courses. Thus far, images of the solubility phenomena and crystallization have been shown to general chemistry students, and those of the interfacial reactions and ammonia complexation have been shown to inorganic chemistry students. The relevant concepts that may be discussed when presenting these video images to students are summarized in Table 1. Experimental Setup Our system is an adaptation of commercially available micromanipulation equipment combined with an inverted microscope. This equipment, with minimal modification, can be used to


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form, hold, and manipulate microdroplets such that chemical phenomena can be imaged with the microscopy and video-capture equipment. Although the micropipet manipulation technique has been widely used in cell biology and biophysics research for studying cells and biomembranes (8), its application to chemical education is novel. Our system (Figure 11) is composed of (1) an experimental station (an inverted microscope with a micromanipulator); (2) data acquisition and analysis system (a digital camera-PC interface); (3) distribution system (digital library, images, and videos); and (4) display (projection system). The general experimental approach for our micromanipulation technique has been well established and described previously (6, 8). Briefly, our system was built on an inverted microscope (Olympus IX51) with hydraulic micromanipulators (Narishige) mounted on the opposing side of the microscope stage, supported on a vibrationisolated workstation (Newport) to minimize positional oscillations generated by vibration from external sources. Microdroplets were viewed by bright field optics with magnification of 20 to 40 (in air). The micropipet pressure was controlled by a simple syringe. A high-resolution digital camera (Olympus DP70) directly attached to the microscope allowed recording of experiment. Microdroplets were delivered into the immiscible phase from micropipets controlled via micromanipulators under threeaxis joystick control, with digital video microscopic monitoring. Micropipets used for manipulation of aqueous microdroplet were prepared by a commercially available micropipet puller and subsequently hydrophobized using hexamethyldisilazane, (CH3)3SiNHSi(CH3)3, to inhibit wetting of the glass surface by the aqueous solution. The progress of the reactions was visualized on a monitor and recorded in real time. Using a computer interface, still images or video clips were recorded and edited to appropriate video media for classroom use. Connecting the computer to a projection system allowed an entire class to observe the dynamics of reactions from a single camera on a microscope. Hazards Among the advantages of microdroplet chemistry are minimal use of reagent and low generation of waste. The typical quantity prepared for any demonstration is less than 1 mL of reagents. None of the substances used for these activities at the quantities described pose an unacceptable health hazard for students working in an academic laboratory with proper safety equipment. All chemicals need to be handled with care, with proper disposal of wastes. Alcohols used in this study are irritants and flammable. Potassium chromate is toxic, a suspected carcinogen, and a strong oxidizer. Chloroform is toxic and a suspected carcinogen. Nickel, lead, and copper salts are hazardous to ingest and eye irritants. Concentrated aqueous ammonia is irritating to the skin,


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In the Classroom Table 1. Demonstration Images and Relevant Concepts Demonstration Image

Selected Relevant Concepts

Solubility of Liquids (Figure 3)

Solubility, diffusion

Crystallization (Figure 4)

Undersaturation, saturation, supersaturation

NH3 Complexation Reaction (Figures 5-7)

Diffusion, stoichiometry, limiting reagents

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Interfacial Reactions (Figure )

Extraction, metal complex formation

Oxidation-Reduction Reaction (Figure 9)

Redox reaction, kinetics, stoichiometry, color change, diffusion

Precipitation Reaction (Figure 10)

Solubility of solids, nature of solid matter,

Figure 11. Schematic for the experimental system.

eyes, and respiratory system. Hexamethyldisilazane is a flammable volatile liquid, which may cause severe irritation or burns to skin, eyes, or respiratory tract. Conclusion An introduction to some of the interesting differences in chemical phenomena that occur due to the size effects attendant to performing chemistry in a microdroplet are presented. Using micropipet manipulation to generate and utilize aqueous microdroplets, we have captured these effects by video microscopy and can see some of their manifestations. Additionally, there is an inherent charm factor in viewing reactions under microscopy. Chemistry in a microdroplet is also relevant to the exciting advances in chemistry involving high-throughput techniques such as 156


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microdroplet microfluidics. These techniques often involve fascinating chemistry and represent the cutting-edge of today's research. We have generated video images that simulate some aspects of microdroplet microfluidics to bring these exciting results to the greater chemical education community. Acknowledgment The authors would like to thank and acknowledge the financial support for this research from: the National Science Foundation (NSF-CHE-0909978); the Donors of the American Chemical Society Petroleum Research Fund (Grant PRF#45241GB9); Camille and Henry Dreyfus Special Grant Program in Chemical Science (SG-07-016); The Patrick J. Martin Foundation; Iona College.


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In the Classroom

Literature Cited 1. Mukhopadhyay, R. Anal. Chem. 2006, 78, 1401–1404. 2. Huebner, A.; Sharma, S.; Srisa-Art, M.; Hollfelder, F.; Edel, J. B.; deMello, A. J. Lab Chip 2008, 8, 1244–1254. 3. Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Lab Chip 2008, 8, 198–220. 4. Song, H.; Chen, D. L.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2006, 45, 7336–7356. 5. Legge, C. H. J. Chem. Educ. 2002, 79, 173–178.


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6. Allain, K.; Bebawee, R.; Lee, S. Cryst. Growth Des. 2009, 9, 3183–3190. 7. Lee, S.; Sanstead, P. J.; Wiener, J. M.; Bebawee, R.; Hilario, A. G. Langmuir 2010, 26 (12), 9556–9564. 8. Needham, D.; Zhelev, D. V. In Giant Vesicles; Walde, P., Luisi, L., Eds.; John Wiley & Sons, Ltd.: New York, 2000; Vol. 6; pp 103.

Supporting Information Available Nine video clips demonstrating chemical reactions in a microdroplet. This material is available via the Internet at http://pubs.acs.org.


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Visualizing Chemical Phenomena in Microdroplets - American

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