The authors thank American Chemical Society - Petroleum Research Fund (ACS - PFR), grant number: PRF # 57216-UNI9, for financial support.

1. Hydrate formation on water droplet in cyclopentane
NOTE: The experimental procedure described below is for the study of hydrate formation on a water droplet in cyclopentane using the IMPd and hydrate visualization cell described in the introduction.
<ol>
	<li>Attach a 19 G needle to the 1 mL glass syringe (Figure 2b, C).</li>
	<li>Rinse the 1 mL glass syringe and 19 G needle 3x with DI water.</li>
	<li>Fill the syringe with DI water.</li>
	<li>Fill the hydrate visualization cell (Figure 2b, E) with 25 mL of cyclopentane.</li>
	<li>Using the syringe, insert a droplet of DI water (i.e., 50&#8722;100 &#181;L) at the bottom of the hydrate visualization cell. This water droplet is the seed hydrate. 
	NOTE: The drop should be placed at the bottom of the hydrate visualization cell. The purpose of the seed hydrate is to initiate the formation of the hydrate and to form consistent nucleation and tracking of the growth rate.</li>
	<li>Place the temperature sensor inside the hydrate visualization cell, close to the bottom of the cell.</li>
	<li>Put the acrylic cover on the hydrate visualization cell to prevent evaporation of the cyclopentane. Use screws to keep the cover in place.</li>
	<li>Adjust the lights and camera to focus. Adjust the focus on the seed hydrate.</li>
	<li>Set the temperature of the Peltier plate to -5 &#176;C in the temperature control device.</li>
	<li>Check the temperature values read by the temperature sensor.</li>
	<li>Once the temperature reaches -5 &#176;C, make sure the droplet at the bottom (seed hydrate) turns to ice.</li>
	<li>Set the temperature of the Peltier plate to 2 &#176;C in 0.5 &#176;C increments.</li>
	<li>When the temperature reaches 2 &#176;C, fill the plumbing with water using the syringe, and lower the brass hook into the cyclopentane to equilibrate for 5 min. 
	NOTE: This temperature ensures the solid ice is converted to hydrate, because the system is above the melting point of ice, yet below that of cyclopentane hydrates11.</li>
	<li>Start recording with the camera.</li>
	<li>Press the Start Measurement on the pressure transducer software to start the digital transducer recordings.</li>
	<li>Connect the syringe to the syringe pump.</li>
	<li>Set the syringe pump to inject a volume of 2 &#181;L and activate. The syringe will plunge the water into the cyclopentane bath to form the submerged droplet.</li>
	<li>Use a needle tip to remove a small piece of the seed hydrate.</li>
	<li>Bring the needle tip with the piece of seed hydrate (Figure 3a) into brief contact with the water droplet (Figure 3b) to initiate the formation of the hydrate on the water droplet.</li>
	<li>Press Record on the camera capture software. Record images of the crystallization process of the droplet hemisphere from the camera at 1 Hz.</li>
</ol>
2. Hydrate formation on water surfactant droplet in cyclopentane
NOTE: Hydrate crystallization experiments with surfactant solutions are performed in the same way as pure water. However, when using a surfactant solution to study the surfactant effect on hydrate crystallization there is a need to find the critical micelle concentration (CMC) of each surfactant. The CMC can either be found in the literature9 or using the method described below.
<ol>
	<li>Prepare 50 mL of standard solutions of sorbitane monolaurate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate by dissolving a measured mass of each surfactant into deionized water to prepare a series of 12 solutions of each surfactant, each representing a different concentration ranging from 10-4 g/100 mL&#8211;1 g/100 mL.</li>
	<li>Prepare solutions of sorbitane monooleate in cyclopentane at different concentrations. 
	NOTE: Cyclopentane is used due to the high level of hydrophobicity and low solubility of sorbitane monooleate in water. The same concentrations are used for sorbitane monooleate as well.</li>
	<li>Measure the surface tension of each surfactant solution using the stalagmometry method.
	<ol>
		<li>Place the syringe pump and syringe vertically as shown in Figure 4 in order to count falling drops.</li>
		<li>Program the pump to expel 1 mL of solution at a rate of 0.5 mL/min and release the drops into the air.</li>
		<li>Obtain the drop volume (V) as an average by dividing 1 mL by the number of observed drops.</li>
		<li>Test each solution at least 3x.</li>
		<li>Calculate interfacial tension using 
		<img alt="Equation 1" src="/files/ftp_upload/60391/60391eq1.jpg" /> 
		where g is the acceleration due to gravity, &#916;p is the density change at the interface (i.e., the density difference between the surfactant solution and air), V is the droplet volume, F is an empirical correction given by12 
		<img alt="Equation 2" src="/files/ftp_upload/60391/60391eq2a.jpg" /> 
		NOTE: Alternatively, surface tension of some surfactant solutions can be found in the literature9.</li>
		<li>Plot the surface tension as a function of concentration. The surface tension will decrease with increasing surfactant concentration until it flattens and becomes constant.</li>
		<li>Find the CMC for each surfactant (i.e., the concentration where the surface tension flattens) and use it in the experiments. 
		NOTE: Increasing the surfactant concentration will not change the surface tension.</li>
	</ol>
	</li>
	<li>Repeat the experimental procedure in section 1, but instead of water use surfactant solution at various concentrations compared to the CMC (i.e., 0.1x CMC, 1x CMC, and 10x CMC).</li>
</ol>
3. Image processing and interfacial stress measurements
NOTE: Tracking the conical and planar hydrate growth is performed with visual analysis methods. The software programs used are described in the Table of Materials. An example of the contour detection and coloring can be found in Figure 5. Because the camera only captures 2D projection of the spherical droplet, a 3D reconstruction needs to be created.
<ol>
	<li>Tracking the hydrate growth
	<ol>
		<li>Open the first image of the image sequence using image processing software.</li>
		<li>Use the Length tool in the software to measure the length of the brass tube in the image.</li>
		<li>Set the scale of the brass tube in the image based on the known diameter of 1/16 in. (1.588 mm).</li>
		<li>Select 10 equally spaced snapshots from each sequence. The snapshots should capture the full process, from the point of nucleation to full droplet conversion.</li>
		<li>Repeat the scale setting (steps 3.1.1&#8722;3.1.3) for the 10 chosen snapshots.</li>
		<li>Use the software to manually detect the contour of the drop in every frame. Mark the contour in red (Figure 5b).</li>
		<li>Use the software to manually detect the contour of the hydrate in every frame. Color the entire area of the entire area of the hydrate in black (Figure 5b).</li>
		<li>Use mathematical modelling software to form a 3D reconstruction of the drop as a correction to the surface area. 
		NOTE: Full details on the construction of the 3D surface area is described in Dann et al.13.</li>
	</ol>
	</li>
	<li>Apparent average interfacial stress measurements 
	NOTE: Apparent average interfacial stress is calculated using the internal pressure data collected from the pressure transducer.
	<ol>
		<li>Use the recorded data from the pressure transducer (&#916;P).</li>
		<li>For every data point, use the Young-Laplace relation14 to determine the apparent average interfacial stress (y), 
		<img alt="Equation 3" src="/files/ftp_upload/60391/60391eq3.jpg" /> 
		where R1 and R2 are the droplet radii of curvature and &#916;P is the change in pressure within the droplet relative to t = 0. 
		NOTE: In the initial period following droplet formation, the two radii are approximately equal, hence R1 and R2 in the Young-Laplace equation can be replaced with the radius of the predetermined 2 &#181;L drop equal to R = 782 &#181;m.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Graham, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Deep water: The Gulf Oil disaster and the future of offshore drilling. Report to the President. , (2011).</li><li>Hammerschmidt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+gas+hydrates+in+natural+gas+transmission+lines.">Formation of gas hydrates in natural gas transmission lines.</a> Industrial &amp; Engineering Chemistry. 26, 851-855 (1934).</li><li>Sloan, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+changing+hydrate+paradigm-from+apprehension+to+avoidance+to+risk+management.">A changing hydrate paradigm-from apprehension to avoidance to risk management.</a> Fluid Phase Equilibria. 228-229, 67-74 (2005).</li><li>Xiaokai, L., Latifa, N., Abbas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-agglomeration+in+cyclopentane+hydrates+from+bio-+and+co-surfactants.">Anti-agglomeration in cyclopentane hydrates from bio- and co-surfactants.</a> Energy &amp; Fuels. 24, 4937-4943 (2010).</li><li>Sloan, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+principles+and+applications+of+natural+gas+hydrates.">Fundamental principles and applications of natural gas hydrates.</a> Nature. 426, 353-363 (2003).</li><li>Sloan, E. D., Koh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clathrate Hydrates of Natural Gases. , (2007).</li><li>Lee, J. D., Englezos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+kinetic+inhibitor+effects+on+gas+hydrate+formation.">Unusual kinetic inhibitor effects on gas hydrate formation.</a> Chemical Engineering Science. 61, 1368-1376 (2006).</li><li>Daimaru, T., Yamasaki, A., Yanagisawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surfactant+carbon+chain+length+on+hydrate+formation+kinetics.">Effect of surfactant carbon chain length on hydrate formation kinetics.</a> Journal of Petroleum Science and Engineering. 56, 89-96 (2007).</li><li>Karanjkar, P. U., Lee, J. W., Morris, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+effects+on+hydrate+crystallization+at+the+water-oil+interface:+hollow-conical+crystals.">Surfactant effects on hydrate crystallization at the water-oil interface: hollow-conical crystals.</a> Crystal Growth &amp; Design. 12, 3817-3824 (2012).</li><li>Leopercio, B. C., de Souza Mendes, P. R., Fuller, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+kinetics+and+mechanics+of+hydrate+films+by+interfacial+rheology.">Growth kinetics and mechanics of hydrate films by interfacial rheology.</a> Langmuir. 32, 4203-4209 (2016).</li><li>Karanjkar, P. U., Lee, J. W., Morris, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetric+investigation+of+cyclopentane+hydrate+formation+in+an+emulsion.">Calorimetric investigation of cyclopentane hydrate formation in an emulsion.</a> Chemical Engineering Science. 68, 481-491 (2012).</li><li>Mori, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harkins-brown+correction+factor+for+drop+formation.">Harkins-brown correction factor for drop formation.</a> AIChE Journal. 36, 1272-1274 (1990).</li><li>Dann, K., Rosenfeld, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+effect+on+hydrate+crystallization+at+oil-water+interface.">Surfactant effect on hydrate crystallization at oil-water interface.</a> Langmuir. 34 (21), 6085-6094 (2018).</li><li>Ibach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physics of Surfaces and Interfaces. , (2006).</li></ol>

The authors have nothing to disclose.

In this article we describe an experimental technique to study hydrate crystallization at the oil-water interface in the presence of nonionic surfactants. The apparatus is comprised of a temperature control system and a visualization cell that includes a brass chamber with windows, CMOS camera, and pressure transducer. The temperature control system is comprised of a microcontroller, powerful Peltier plate, 120 mm CPU cooler as the heatsink, and a waterproof digital temperature sensor. A hydrate visualization brass cell ...

The incentive to understand the mechanism of hydrate crystallization and inhibition comes from the fact that hydrates occur naturally in oil pipelines and can result in difficulties in flow assurance. For example, the 2010 Gulf of Mexico oil spill1 was a result of hydrate accumulation in an underwater oil piping system, causing contamination to the environment. Hence, understanding hydrate formation and inhibition is crucial in order to prevent future environmental disasters. Much of the driving force for the study of hydrate crystallization in the past years is the oil industry&#39;s effort to prevent hydrate plug agglomeration and the subsequent blockage of flow. The first study to determine that hydrates were responsible for plugged flowlines was done by Hammerschmidt in 19342. To this day, oil producers find it highly important to understand and inhibit hydrate formation for flow assurance3.
One way to prevent hydrate formation is to insulate deep water pipelines so that ice does not form. However, it is expensive to adequately insulate the pipelines, and the additional costs can be in the order of $1 million/km3. Thermodynamic inhibitors, such as methanol, can be injected into wellheads to prevent the formation of hydrates. However, large volumetric ratios of water to alcohol, as great as 1:1, are needed in order to adequately prevent the formation of hydrates4. Recently, the global cost to use methanol for hydrate prevention has been reported as $220 million/year. This is not a sustainable amount of alcohol usage5. In addition, the use of methanol is problematic because it is environmentally hazardous, and cannot be used for large-scale transport5. Alternatively, kinetic inhibitors, such as surfactants, can suppress hydrate growth at small quantities and temperatures of up to 20 &#176;C6. Hence, surfactant presence can reduce the large amount of alcohols needed for hydrate prevention.
Surfactants are considered good inhibitors for hydrate crystallization due to two main reasons:
1) They can inhibit hydrate formation through surface property changes; and 2) They initially help the formation of hydrate cells but prevent further growth and agglomeration of the crystal down the pipeline7. Although surfactants have proved to be efficient inhibitors, there is still a large amount of information missing regarding the crystallization process in the presence of surfactants. While some studies have shown that the use of surfactants can extend the initial hydrate crystallization time at certain subcoolings, other studies have found exceptions at low surfactant concentrations. At low surfactant concentrations, the water droplets tend to coalesce and accelerate the process of hydrate formation8. The inhibition process has been explained by surfactant molecules interrupting planar hydrate growth, forcing the hydrate into hollow-conical crystal formation. The conical crystals form a mechanical barrier for crystal growth9, and thus inhibit the growth.
In this study we designed and implemented a low-cost, integrated modular Peltier device (IMPd) along with a hydrate visualization cell and used them to study cyclopentane hydrate formation in the presence of nonionic surfactants. The reason for using cyclopentane instead of low molecular weight gases (e.g., CH4 and CO2) that usually form hydrates in deep sea reservoirs, is that these gases require higher pressures and lower temperatures to form stable hydrates. Because cyclopentane forms hydrates at ambient pressure and temperatures up to ~7.5 &#176;C, it is often used as a model material for hydrate formation10.
The integrated modular Peltier device (IMPd) consists of an open-source microcontroller, Peltier plate, CPU cooler (heat sink), and waterproof digital temperature sensor. The device can deliver a maximum temperature differential of 68 &#176;C. The minimum temperature resolution is 1/16 &#176;C. The entire system, including the electrical circuitry and hardware, can be constructed for less than $200. The temperature sensor reports to the microcontroller, which sends output signals to the transistor. The transistor then passes current from the DC power source through the Peltier element. The heat sink helps cool the Peltier element by convecting the heat coming from the hot side of the Peltier to the ambient air. The assembled hardware components of the IMPd system are shown in Figure 1a,b. Figure 1c shows the wiring schematic with all the components of the control loop (proportional-integral-derivative [PID] controller) and the pin-outs. The output current of the microcontroller was limited with the gate resistor R1 to a maximum current of 23 mA (I = 5 V/220 W). The pull-down resistor R2 in Figure 1c allows the gate charge to dissipate and to turn the system off. To tune the PID controller, Ziegler-Nichols based methods combined with an iterative process are used11. Microcontroller integrated development environment (IDE) software is used to monitor and send commands to the microcontroller for temperature regulation.
Along with the IMPd, we applied a novel approach using visualization techniques and internal pressure measurements. The hydrate visualization cell, which is placed on top of the IMPd, is comprised of a brass cell equipped with two double-paned observation windows. The windows allow video recording of the hydrate formation process on the water droplet in cyclopentane. The complementary metal-oxide semiconductor (CMOS) camera is placed outside the window and the pressure transducer is connected to the water injection line in order to get the internal pressure measurements of the drop. A digital transducer application is used to get the readings from the pressure transducer. A camera viewer is used to capture the videos and images from the CMOS camera. The software controls the exposure and snapshot frequency. Image processing software programs are used to track the growth of the hydrate. Figure 2a shows a schematic description of the hydrate visualization cell and Figure 2b shows an overview of the entire experimental system. The seed hydrate (Figure 2a) is required for consistent nucleation and tracking of the hydrate growth rate. The seed hydrate is a small volume (e.g., 50&#8211;100 &#181;L) of pure water deposited on the floor of the hydrate cell. As the temperature decreases, the drop forms ice, which then turns to hydrate as the temperature increases. The small piece of the seed hydrate then contacts the water droplet. This process controls the initiation of the hydrate in the submerged water droplet. Silica desiccant is inserted into the gap between the two glass slides (Figure 2c), which serve as viewing windows. The silica desiccant helps reduce the amount of frosting and fogging on the windows. Anti-fog is also applied to the outer window to reduce fogging. Images are captured with a CMOS camera and a 28&#8211;90 mm lens. A 150 W fiber optic goose-neck lamp is used for illumination. An acrylic cover is placed on top of the brass cell in order to limit evaporation of cyclopentane. Plumbing consists of a combination of flexible polytetrafluoroethylene (PTFE) tubing and rigid brass tubing. A syringe pump with a 1 mL glass syringe and a 19 G needle control the flow of water and surfactant solution. A pressure transducer monitors the pressure changes inside the water surfactant solution droplet. 19 G PTFE tubing connects the syringe to the T-fitting and 1/16 in. (1.588 mm) brass tubing connects the transducer and brass hook to the T-fitting (Figure 2d). A brass hook, approximately 5 cm in length with a 180&#176; bend, generates the water/surfactant solution droplet. The bend ensures that the droplet generated by the syringe sits on top of the tube throughout the experiment. A 1/16 in. stainless steel T-fitting in conjunction with PTFE crush ferrules and PTFE thread tape seal the fittings.
Using this apparatus, we examined four different nonionic surfactants with different hydrophilic-lipophilic balances (HLB) that are commonly used in the oil industry: sorbitane monolaurate, sorbitane monooleate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate.

<table><tbody><tr><td>1/16 in. Swagelok 316 stainless steel T-fitting</td><td>Swagelok</td><td></td><td></td></tr><tr><td>19 gauge PTFE tubing</td><td>Scientific Commodities, Inc.</td><td></td><td></td></tr><tr><td>19-gauge needle (model: 1001 LTSN SYR)</td><td></td><td></td><td></td></tr><tr><td>1-Wire DS18B20 - waterproof digital temperature sensor</td><td></td><td></td><td></td></tr><tr><td>Anti fog</td><td>RainX</td><td></td><td></td></tr><tr><td>Arduino Leonardo open-source microcontroller</td><td></td><td></td><td></td></tr><tr><td>Brass tubing 1/16 in.</td><td>K&amp;amp;S Precision Metals</td><td></td><td></td></tr><tr><td>Chemyx Fusion 100 Infusion Pump</td><td>Chemyx</td><td></td><td></td></tr><tr><td>cMOS camera acA640-750um</td><td>Basler</td><td></td><td></td></tr><tr><td>Cyclopentane 98% extra pure</td><td>ACROS organics</td><td>AC111481000</td><td></td></tr><tr><td>Fiber optic goose-neck lamp 150W</td><td>AmScope</td><td></td><td></td></tr><tr><td>Fotodiox macro extension tubes, 35 mm</td><td></td><td></td><td></td></tr><tr><td>Hamilton glass syringe 1 mL</td><td>Hamilton</td><td></td><td></td></tr><tr><td>ImageJ software</td><td></td><td></td><td></td></tr><tr><td>Kipon EOS to C-mount adapter</td><td>Kipon</td><td></td><td></td></tr><tr><td>Lens 28-90 mm</td><td>Canon</td><td></td><td></td></tr><tr><td>Mathematica software</td><td>Mathematica</td><td></td><td></td></tr><tr><td>OMEGA PX409-10WGUSBH pressure transducer</td><td>OMEGA</td><td></td><td></td></tr><tr><td>Peltier plate TEC1-12715</td><td>Amazon</td><td></td><td></td></tr><tr><td>Pluronic L31 (PEG-PPG-PEG)</td><td>Sigma Aldrich</td><td>9003-11-6</td><td></td></tr><tr><td>Pylon Viewer v5.0.0.6150</td><td>Basler</td><td></td><td></td></tr><tr><td>Span 20 (Sorbitan laurate, Sorbitan monolaurate)</td><td>Sigma Aldrich</td><td>1338-39-2</td><td></td></tr><tr><td>Span 80 (Sorbitan Monooteate)</td><td>Sigma Aldrich</td><td>1338-43-8</td><td></td></tr><tr><td>Thermaltake NiC C4 120mm CPU cooler</td><td>Thermaltake</td><td></td><td></td></tr><tr><td>Tween 65 (Polyoxyethylenesorbitan Tristearate)</td><td>Sigma Aldrich</td><td>9005-71-4</td><td></td></tr><tr><td>variable Tooluxe DC power supply</td><td></td><td></td><td></td></tr></tbody></table>

studying surfactant effects on hydrate crystallization at oil-water interfaces using a low-cost integrated modular peltier device

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a temperature regulator, visualization techniques, and inner pressure measurements. The temperature control system contains a low-cost, programmable temperature regulator made with solid-state Peltier components. Along with the temperature control system, we incorporated visualization techniques and internal pressure measurements to study hydrate formation and inhibition in the presence of nonionic surfactants. We studied the hydrate-inhibiting ability of nonionic surfactants (sorbitane monolaurate, sorbitane monooleate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate) at low (i.e., 0.1 CMC), medium (i.e., CMC), and high (i.e., 10 CMC) concentrations. Two types of crystals were formed: planar and conical. Planar crystals were formed in plain water and low surfactant concentrations. Conical crystals were formed in high surfactant concentrations. The results of the study show that conical crystals are the most effective in terms of hydrate inhibition. Because conical crystals cannot grow past a certain size, the hydrate growth rate as a conical crystal is slower than the hydrate growth rate as planar crystal. Hence, surfactants that force hydrates to form conical crystals are the most efficient. The goal of the protocol is to provide a detailed description of an experimental system that is capable of investigating the cyclopentane hydrate crystallization process on the surface of a water droplet in the presence of surfactant molecules.

Using this experimental system one can examine the hydrate formation at the oil-water interface and measure the interfacial stress associated with the crystallization process. Figure 6 shows a representative set of results that include both crystal formation and interfacial stress. In the planar shell growth (Figure 6a), the crystal grew from the two poles towards the equator. For that reason, in the planar crystal, the hydrate shell grew consta...

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Studying Surfactant Effects on Hydrate Crystallization at Oil-Water Interfaces Using a Low-Cost Integrated Modular Peltier Device

We present a protocol to study the formation of hydrates in the presence of nonionic surfactants on the interface of a water droplet submerged in cyclopentane. The protocol consists of building a low-cost, programmable, temperature regulator. The temperature control system is combined with visualization techniques and internal pressure measurements.

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a ...

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6.3K Views.  San José State University. We present a protocol to study the formation of hydrates in the presence of nonionic surfactants on the interface of a water droplet submerged in cyclopentane. The protocol consists of building a low-cost, programmable, temperature regulator. The temperature control system is combined with visualization techniques and internal pressure measurements.

Video: Studying Surfactant Effects on Hydrate Crystallization at Oil-Water Interfaces Using a Low-Cost Integrated Modular Peltier Device

Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are resistant to low molecular-weight oil components, unlike traditional polydimethylsiloxane (PDMS) devices. Here, we demonstrate a facile method for making a device with this property, and we use the product of this protocol for examining the pore-scale mechanisms by which foam recovers crude oil. A pattern is first designed using computer-aided design (CAD) software and printed on a transparency with a high-resolution printer. This pattern is then transferred to a photoresist via a lithography procedure. PDMS is cast on the pattern, cured in an oven, and removed to obtain a mold. A thiol-ene crosslinking polymer, commonly used as an optical adhesive (OA), is then poured onto the mold and cured under UV light. The PDMS mold is peeled away from the optical adhesive cast. A glass substrate is then prepared, and the two halves of the device are bonded together. Optical adhesive-based devices are more robust than traditional PDMS microfluidic devices. The epoxy structure is resistant to swelling by many organic solvents, which opens new possibilities for experiments involving light organic liquids. Additionally, the surface wettability behavior of these devices is more stable than that of PDMS. The construction of optical adhesive microfluidic devices is simple, yet requires incrementally more effort than the making of PDMS-based devices. Also, though optical adhesive devices are stable in organic liquids, they may exhibit reduced bond-strength after a long time. Optical adhesive microfluidic devices can be made in geometries that act as 2-D micromodels for porous media. These devices are applied in the study of oil displacement to improve our understanding of the pore-scale mechanisms involved in enhanced oil recovery and aquifer remediation.

The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported.

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are ...

Environment

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Engineering

Methane Hydrate Crystallization on Sessile Water Droplets

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa working pressure, containing a stage for sessile droplets, a sapphire window for visualization, and temperature and pressure transducers. A pressure pump connected to a methane gas cylinder is used to pressurize the cell to 5 MPa. The cooling system is a 10 gallon (37.85 L) tank containing a 50% ethanol solution cooled via ethylene glycol through copper coils. This setup enables the observation of the temperature change associated with hydrate formation and dissociation during cooling and depressurization, respectively, as well as visualization and photography of the morphologic changes of the droplet. With this method, rapid hydrate shell formation was observed at ~-6 &#176;C to -9 &#176;C. During depressurization, a 0.2 &#176;C to 0.5 &#176;C temperature drop was observed at the pressure/temperature (P/T) stability curve due to exothermic hydrate dissociation, confirmed by visual observation of melting at the start of the temperature drop. The "memory effect" was observed after repressurizing to 5 MPa from 2 MPa. This experimental design allows the monitoring of pressure, temperature, and morphology of the droplet over time, making this a suitable method for testing various additives and substrates on hydrate morphology.

We describe a method to form gas hydrate on sessile water droplets to study the effects of various inhibitors, promoters, and substrates on the hydrate crystal morphology.

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Bioengineering

Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO2/Si/SiO2 Wafers for Green Desalination

Desalination through direct contact membrane distillation (DCMD) exploits water-repellent membranes to robustly separate counterflowing streams of hot and salty seawater from cold and pure water, thus allowing only pure water vapor to pass through. To achieve this feat, commercial DCMD membranes are derived from or coated with water-repellent perfluorocarbons such as polytetrafluoroethylene (PTFE) and polyvinylidene difluoride (PVDF). However, the use of perfluorocarbons is limiting due to their high cost, non-biodegradability, and vulnerability to harsh operational conditions. Unveiled here is a new class of membranes referred to as gas-entrapping membranes (GEMs) that can robustly entrap air upon immersion in water. GEMs achieve this function by their microstructure rather than their chemical make-up. This work demonstrates a proof-of-concept for GEMs using intrinsically wetting SiO2/Si/SiO2 wafers as the model system; the contact angle of water on SiO2 is &#952;o &#8776; 40&#176;. Silica-GEMs had 300 &#956;m-long cylindrical pores whose diameters at the (2 &#956;m-long) inlet and outlet regions were significantly smaller; this geometrically discontinuous structure, with 90&#176; turns at the inlets and outlets, is known as the &#34;reentrant microtexture&#34;. The microfabrication protocol for silica-GEMs entails designing, photolithography, chrome sputtering, and isotropic and anisotropic etching. Despite the water loving nature of silica, water does not intrude silica-GEMs on submersion. In fact, they robustly entrap air underwater and keep it intact even after six weeks (&#62;106 seconds). On the other hand, silica membranes with simple cylindrical pores spontaneously imbibe water (&#60; 1 s). These findings highlight the potential of the GEMs architecture for separation processes. While the choice of SiO2/Si/SiO2 wafers for GEMs is limited to demonstrating the proof-of-concept, it is expected that the protocols and concepts presented here will advance the rational design of scalable GEMs using inexpensive common materials for desalination and beyond.

Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO2/Si/SiO2 Wafers for Green Desalination

Presented here is a stepwise protocol for realizing gas-entrapping membranes (GEMs) from SiO2/Si wafers using integrated circuit microfabrication technology. When silica-GEMs are immersed in water, the intrusion of water is prevented, despite the water-loving composition of silica.

Desalination through direct contact membrane distillation (DCMD) exploits water-repellent membranes to robustly separate counterflowing streams of hot and ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the ...