Name: methane hydrate crystallization on sessile water droplets
Uploaded: 2021-05-26T15:00:00
Description: Watch this Scientific Journal Video about Methane Hydrate Crystallization on Sessile Water Droplets at JoVE.com

NASA Exobiology grant 80NSSC19K0477 funded this research. We thank William Waite and Nicolas Espinoza for valuable discussions.

1. Design, validate, and machine the pressure cell.
<ol>
	<li>Design the cell to allow direct visualization of hydrate formation from a water droplet. Ensure that the cell has a main chamber with a see-through sapphire window and four ports for fluid/gas inlet, outlet, light, and wires (Figure 1). Create the final design in engineering design software (Supplemental Figure S1).</li>
	<li>To check that the pressure cell is safe under working high pressure, conduct a finite e...

<ol>
	<li>Bohrmann, G., Torres, M. E., Schulz, H. D., Zabel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+hydrates+in+marine+sediments.">Gas hydrates in marine sediments.</a> Marine Geochemistry. , 481-512 (2006).</li><li>Ruppel, C. D., Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+climate+change+and+methane+hydrates.">The interaction of climate change and methane hydrates.</a> Reviews of Geophysics. 55 (1), 126-168 (2017).</li><li>Hammerschmidt, E. G. <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 and Engineering Chemistry. 26, 851-855 (1934).</li><li>Ke, W., Kelland, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+hydrate+inhibitor+studies+for+gas+hydrate+systems:+a+review+of+experimental+equipment+and+test+methods.">Kinetic hydrate inhibitor studies for gas hydrate systems: a review of experimental equipment and test methods.</a> Energy &amp; Fuels. 30 (12), 10015-10028 (2016).</li><li>Kelland, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+kinetic+hydrate+inhibitors+from+an+environmental+perspective.">A review of kinetic hydrate inhibitors from an environmental perspective.</a> Energy &amp; Fuels. 32 (12), 12001-12012 (2018).</li><li>Walker, V. K., 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=Antifreeze+proteins+as+gas+hydrate+inhibitors.">Antifreeze proteins as gas hydrate inhibitors.</a> Canadian Journal of Chemistry. 93 (8), 839-849 (2015).</li><li>Bruusgaard, H., Lessard, L. D., Servio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+study+of+structure+I+methane+hydrate+formation+and+decomposition+of+water+droplets+in+the+presence+of+biological+and+polymeric+kinetic+inhibitors.">Morphology study of structure I methane hydrate formation and decomposition of water droplets in the presence of biological and polymeric kinetic inhibitors.</a> Crystal Growth &amp; Design. 9 (7), 3014-3023 (2009).</li><li>Jung, J. W., Espinoza, D. N., Santamarina, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+phenomena+relevant+to+CH4-CO2+replacement+in+hydrate-bearing+sediments.">Properties and phenomena relevant to CH4-CO2 replacement in hydrate-bearing sediments.</a> Journal of Geophysical Research. 115 (10102), 1-16 (2010).</li><li>Chen, X., Espinoza, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostwald+ripening+changes+the+pore+habit+and+spatial+variability+of+clathrate+hydrate.">Ostwald ripening changes the pore habit and spatial variability of clathrate hydrate.</a> Fuel. 214, 614-622 (2018).</li><li>DuQuesnay, J. R., Diaz Posada, M. C., Beltran, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+gas+hydrate+reactor+design:+3-in-1+assessment+of+phase+equilibria,+morphology+and+kinetics.">Novel gas hydrate reactor design: 3-in-1 assessment of phase equilibria, morphology and kinetics.</a> Fluid Phase Equilibria. 413, 148-157 (2016).</li><li>Udegbunam, L. U., DuQuesnay, J. R., Osorio, L., Walker, V. K., Beltran, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+equilibria,+kinetics+and+morphology+of+methane+hydrate+inhibited+by+antifreeze+proteins:+application+of+a+novel+3-in-1+method.">Phase equilibria, kinetics and morphology of methane hydrate inhibited by antifreeze proteins: application of a novel 3-in-1 method.</a> The Journal of Chemical Thermodynamics. 117, 155-163 (2018).</li><li>Espinoza, D. N., Santamarina, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-CO2-mineral+systems:+Interfacial+tension,+contact+angle,+and+diffusion+-+Implications+to+CO2+geological+storage.">Water-CO2-mineral systems: Interfacial tension, contact angle, and diffusion - Implications to CO2 geological storage.</a> Water Resources Research. 46 (7537), 1-10 (2010).</li><li>Sloan, E. D., Koh, C. A. <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. 3rd edn. , (2007).</li><li>Makogon, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydrates of natural gas. , 125 (1981).</li><li>Johnson, A. M., 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=Mainly+on+the+plane:+deep+subsurface+bacterial+proteins+bind+and+alter+clathrate+structure.">Mainly on the plane: deep subsurface bacterial proteins bind and alter clathrate structure.</a> Crystal Growth &amp; Design. 20 (10), 6290-6295 (2020).</li></ol>

We have developed a method to form methane hydrate shells on sessile water droplets safely and share this method to machine and assemble a pressure cell rated to 10 MPa working pressure, as well as the pressurizing and cooling systems. The pressure cell is outfitted with a stage for the droplet containing embedded thermocouples, a sapphire window for visualizing the droplet, and a pressure transducer fixed to the top of the cell. The cooling system includes chilled ethylene glycol circulating through copper coils in a ta...

Gas hydrates are cages of hydrogen-bonded water molecules that trap guest gas molecules via van der Waals interactions. Methane hydrates form under high-pressure and low-temperature conditions, which occur in nature in the subsurface sediment along continental&#160;margins, under Arctic permafrost, and on other planetary bodies in the solar system1. Gas hydrates store several thousand gigatons of carbon, with important implications for climate and energy2. Gas hydrates can also be hazardous in the natural gas industry because conditions favorable for hydrates occur in gas pipelines, which can clog the pipes leading to fatal explosions and oil spills3.
Due to the difficulty of studying gas hydrates in situ, laboratory experiments are often employed to characterize hydrate properties and the influence of inhibitors and substrates4. These laboratory experiments are performed by growing gas hydrate at elevated pressure in cells of various shapes and sizes. Efforts to prevent gas hydrate formation in gas pipelines have led to the discovery of several chemical and biological gas hydrate inhibitors, including antifreeze proteins (AFPs), surfactants, amino acids, and polyvinylpyrrolidone (PVP)5,6. To determine the effects of these compounds on gas hydrate properties, these experiments have employed diverse vessel designs, including autoclaves, crystallizers, stirred reactors, and rocking cells, which support volumes from 0.2 to 106 cubic centimeters4.
The sessile droplet method used here and in previous studies7,8,9,10,11,12 involves forming a gas hydrate film on a sessile droplet of water inside a pressure cell. These vessels are made of stainless steel and sapphire to accommodate pressures up to 10-20 MPa. The cell is connected to a methane gas cylinder. Two of these studies used the droplet method to test AFPs as gas hydrate inhibitors compared to commercial kinetic hydrate inhibitors (KHIs), such as PVP7,11. Bruusgard et al.7 focused on the morphologic influence of inhibitors and found that droplets containing Type I AFPs have a smoother, glassy surface than the dendritic droplet surface without inhibitors at high driving forces.
Udegbunam et al.11 used a method developed to assess KHIs in a previous study10, which allows for the analysis of morphology/growth mechanisms, the hydrate-liquid-vapor equilibrium temperature/pressure, and kinetics as a function of temperature. Jung et al. studied CH4-CO2 replacement by flooding the cell with CO2 after forming a CH4 hydrate shell8. Chen et al. observed Ostwald ripening as the hydrate shell forms9. Espinoza et al. studied CO2 hydrate shells on various mineral substrates12. The droplet method is a relatively simple and cheap method to determine the morphologic effect of various compounds and substrates on gas hydrates and requires small amounts of additives due to the small volume. This paper describes a method for forming such hydrate shells on a droplet of water using a stainless-steel cell with a sapphire window for visualization, rated up to 10 MPa working pressure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CAMERA AND LAPTOP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera Body</td>
			<td>Nikon</td>
			<td>D7200</td>
			<td>Name in Protocol: camera</td>
		</tr>
		<tr>
			<td>Camera Control Pro 2 Software</td>
			<td>Nikon</td>
			<td></td>
			<td>Name in Protocol: camera software</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>HP Pavilion</td>
			<td>hp-pavilion-laptop-14-ce0068st</td>
			<td>Needs to be PC with plenty of storage (~ 1 Tb) 
			Name in Protocol: laptop</td>
		</tr>
		<tr>
			<td>Macrophotography Lens</td>
			<td>Nikon</td>
			<td>AF-S MICRO 105mm f/2.8G IF-ED Lens</td>
			<td>Name in Protocol: lens</td>
		</tr>
		<tr>
			<td>CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: DI water</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>VWR or grocery store</td>
			<td></td>
			<td>Buy just before nucleation 
			Name in Protocol: dry ice</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: ethanol</td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: ethylene glycol</td>
		</tr>
		<tr>
			<td>COOLING SYSTEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 in. O.D. x 3/8 in. I.D. x 25 ft. Polyethylene Tubing</td>
			<td>Everbilt</td>
			<td>Model # 301844</td>
			<td>For circulating coolant from chiller to copper coils in aquarium 
			Name in Protocol: 3/8&#8221; (inner diameter) plastic tubing</td>
		</tr>
		<tr>
			<td>Circulating chiller</td>
			<td>Polyscience</td>
			<td></td>
			<td>Name in Protocol: chiller</td>
		</tr>
		<tr>
			<td>Economical Flexible Polyethylene Foam Pipe Insulation</td>
			<td>McMaster-Carr</td>
			<td>4530K162</td>
			<td>3/4&#34; thick wall; 1/2&#34; inner diameter; R Value 3; 6&#39; long 
			Name in Protocol: foam pipe insulation</td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td>use any tubing that fits the airline connection in the lab and long enough to travel from the airline connection to the front of the aquarium</td>
		</tr>
		<tr>
			<td>DATALOGGER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Armature Multiplexer Module for 34970A/ 
			34972A, 20-Channel</td>
			<td>Keysight Technologies</td>
			<td>34901A</td>
			<td>Name in Protocol: datalogger multichannel</td>
		</tr>
		<tr>
			<td>Benchvue or Benchlink software</td>
			<td>Benchvue or Benchlink</td>
			<td></td>
			<td>Name in Protocol: temperature transducer software</td>
		</tr>
		<tr>
			<td>Data Acquisition/Switch Unit. GPIB, RS232</td>
			<td>Keysight Technologies</td>
			<td>34970A</td>
			<td>Name in Protocol: datalogger</td>
		</tr>
		<tr>
			<td>USB/GPIB interface</td>
			<td>Keysight Technologies</td>
			<td>82357B</td>
			<td>Name in Protocol: datalogger USB</td>
		</tr>
		<tr>
			<td>datalogger multichannel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schott Fostec -Llc 20510 Ace Fiber Optic Light Source</td>
			<td>Schott Fostec</td>
			<td>A20500</td>
			<td>3115PS-12W-B20 115 V ~AC 50/60Hz 5/4.5 W 
			Name in Protocol: light source unit</td>
		</tr>
		<tr>
			<td>Schott Fostec light source guide - single bundle</td>
			<td>Schott Fostec</td>
			<td>A08031.40</td>
			<td>Name in Protocol: fiber optic light source cable</td>
		</tr>
		<tr>
			<td>METHANE GAS AND REGULATOR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 OD in. x 20 ft. Copper Soft Refrigeration Coil</td>
			<td>Everbilt</td>
			<td>Model # D 04020PS</td>
			<td>For pressurizing ISCO pressure pump. An additional pack is needed for coolant circulation, as listed below. 
			Name in Protocol: high pressure-rated 1/4&#8221; copper pipe</td>
		</tr>
		<tr>
			<td>Methane cylinder regulator</td>
			<td>Airgas</td>
			<td>Y11N114G350-AG</td>
			<td>Name in Protocol: methane cylinder regulator</td>
		</tr>
		<tr>
			<td>Methane gas cylinder</td>
			<td>Airgas</td>
			<td>ME UHP300</td>
			<td>Name in Protocol: methane gas cylinder</td>
		</tr>
		<tr>
			<td>PRESSURE PUMP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in.&#160; flexible tubing, ~ 3 ft.</td>
			<td></td>
			<td></td>
			<td>Connect to pump inlet for leak test 
			Name in Protocol: 1/4&#34;&#160; flexible tubing</td>
		</tr>
		<tr>
			<td>260D Syringe Pump W/Controller</td>
			<td>Teledyne Instruments Inc.</td>
			<td>67-1240-520</td>
			<td>Name in Protocol: pressure pump</td>
		</tr>
		<tr>
			<td>Controller &#8722; Ethernet/USB</td>
			<td>Teledyne Instruments Inc.</td>
			<td>62-1240-114</td>
			<td>Purchase if you would like to install Labview onto computer and control pressure pump remotely. We did not do this.</td>
		</tr>
		<tr>
			<td>Smooth-Bore Seamless 316 Stainless Steel Tubing, 1/4&#34; OD, 0.035&#34; Wall Thickness, 1 Foot Long (x5)</td>
			<td>McMaster-Carr</td>
			<td>89785K824</td>
			<td>Name in Protocol: 1/4&#34; pipe</td>
		</tr>
		<tr>
			<td>Smooth-Bore Seamless 316 Stainless Steel Tubing, 1/8&#34; OD, 0.02&#34; Wall Thickness, 1 Foot Long (x4)</td>
			<td>McMaster-Carr</td>
			<td>89785K811</td>
			<td>Name in Protocol: 1/8&#34; pipe</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Reducing Union, 1/4 in. x 1/8 in. Tube OD (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-6-2</td>
			<td>Name in Protocol: 1/8&#8221; to 1/4&#8221; adapter</td>
		</tr>
		<tr>
			<td>PRESSURE CELL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>316 Stainless Steel Nut and Ferrule Set (1 Nut/1 Front Ferrule/1 Back Ferrule) for 1/4 in. Tube Fitting (20)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-NFSET</td>
			<td>Used for fitting connections where necessary 
			Name in Protocol: ferrule set</td>
		</tr>
		<tr>
			<td>316L Stainless Steel Convoluted (FM) Hose, 1/4 in., 316L Stainless Steel Braid, 1/4 in. Tube Adapters, 60 in. (1.5 m) Length</td>
			<td>Swagelok</td>
			<td>SS-FM4TA4TA4-60</td>
			<td>Connects pressure pump to pressure cell 
			Name in Protocol: 1/4&#34; braided stainless steel flexible pressure-rated hose</td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>ABAQUS FEA</td>
			<td></td>
			<td>Name in Protocol: simulation software</td>
		</tr>
		<tr>
			<td>Abrasion-Resistant Cushioning Washer for 7/8&#34; Screw Size, 0.875&#34; ID, 2.25&#34; OD, packs of 10 (x1)</td>
			<td>McMaster-Carr</td>
			<td>90131A107</td>
			<td>Name in Protocol: 2.25&#34; rubber washer</td>
		</tr>
		<tr>
			<td>Abrasion-Resistant Sealing Washer, Aramid Fabric/Buna-N Rubber, 3/8&#34; Screw Size, 0.625&#34; OD, packs of 10 (x1)</td>
			<td>McMaster-Carr</td>
			<td>93303A105</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>Acrylic Sheet | White 2447 / WRT31 
			Extruded Paper-Masked (Translucent 55% (0.118 x 12 x 12)</td>
			<td>Interstate Plastics</td>
			<td>ACRW7EPSH</td>
			<td>Machine a circle of acrylic to fit in the inner chamber of the pressure cell to serve as the background for imaging 
			Name in Protocol: acrylic disc</td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>AutoCAD</td>
			<td></td>
			<td>Name in Protocol: engineering design software</td>
		</tr>
		<tr>
			<td>Conax fitting</td>
			<td>Conax Technologies</td>
			<td>311401-011</td>
			<td>TG(PTM2/)-24-A6-T, OPTIONAL 1/4&#34; NPT 
			Name in Protocol: pressure seal connector</td>
		</tr>
		<tr>
			<td>High Accuracy Oil Filled Pressure 
			Transducers/Transmitters for General 
			industrial applications (x2)</td>
			<td>Omega Engineering, Inc.</td>
			<td>PX409-3.5KGUSBH</td>
			<td>Buy two so there is a backup. 
			Name in Protocol: pressure transducer</td>
		</tr>
		<tr>
			<td>HIGH PRESSURE CHAMBER&#160; PARTS</td>
			<td>Wither Tool, Die and Manufacturing Company</td>
			<td></td>
			<td>Machining for pressure cell parts as listed in CAD drawings (Figure S1) 
			Name in Protocol: Part B = stainless steel washer</td>
		</tr>
		<tr>
			<td>High-Strength 316 Stainless Steel Socket Head Screw, M5 x 0.80 mm Thread, 14 mm Long (x20)</td>
			<td>McMaster-Carr</td>
			<td>90037A119</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>High-Strength 316 Stainless Steel Socket Head Screw, M8 x 1.25 mm Thread, 25 mm Long (x20)</td>
			<td>McMaster-Carr</td>
			<td>90037A133</td>
			<td>Name in Protocol: M8 stainless steel screws</td>
		</tr>
		<tr>
			<td>Oil-Resistant Hard Buna-N O-Ring, 3/32 Fractional Width, Dash Number 120, packs of 50 (x1)</td>
			<td>McMaster-Carr</td>
			<td>5308T178</td>
			<td>Name in Protocol: 1&#34; o-ring</td>
		</tr>
		<tr>
			<td>Oil-Resistant Hard Buna-N O-Ring, 3/32 Fractional Width, Dash Number 128, packs of 50 (x1)</td>
			<td>McMaster-Carr</td>
			<td>5308T186</td>
			<td>Name in Protocol: 1.5&#34; o-ring</td>
		</tr>
		<tr>
			<td>Omega Inc. pressure transducer software</td>
			<td>Omega Engineering, Inc.</td>
			<td></td>
			<td>Name in Protocol: pressure transducer software</td>
		</tr>
		<tr>
			<td>Polycarbonate Disc</td>
			<td>McMaster-Carr</td>
			<td>8571K31</td>
			<td>Listed in CAD drawings for illumination port, Fig. S1 Part E</td>
		</tr>
		<tr>
			<td>Sapphire windows (x3)</td>
			<td>Guild Optical Associates, Inc.</td>
			<td>Optical Grade Sapphire Window, C-Plane 
			Diameter: 1.811&#8221; &#177;.005&#8221; 
			Thickness: .590&#8221; &#177;.005&#8221; 
			Surface Quality: 60/40 
			Edges ground and safety chamfered</td>
			<td>Buy three so there are two backups. 
			Name in Protocol: sapphire window</td>
		</tr>
		<tr>
			<td>Solid Thermocouple Wire FEP Insulation and Jacket, Type K, 24 Gauge, 50 ft. Length (x1)</td>
			<td>McMaster-Carr</td>
			<td>3870K32</td>
			<td>Name in Protocol: thermocouples</td>
		</tr>
		<tr>
			<td>Stainless Steel Integral Bonnet Needle Valve, 0.37 Cv, 1/4 in. Swagelok Tube Fitting, Regulating Stem (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-1RS4</td>
			<td>Two will be used for the pressure pump as well. 
			Name in Protocol: 1/4&#34; needle valves</td>
		</tr>
		<tr>
			<td>Stainless Steel Pipe Fitting, Hex Nipple, 1/4 in. Male NPT (x2)</td>
			<td>Swagelok</td>
			<td>&#160;SS-4-HN</td>
			<td>Used for illumination port</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Female Branch Tee, 1/4 in. Tube OD x 1/4 in. Tube OD x 1/4 in. Female NPT (x2)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-3-4TTF</td>
			<td>Used with pressure transducer 
			Name in Protocol: branch tee fitting</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Male Connector, 1/4 in. Tube OD x 1/4 in. Male NPT (x4)</td>
			<td>Swagelok</td>
			<td>&#160;SS-400-1-4</td>
			<td>Used on top port and side port leading to needle valves 
			Name in Protocol: NPT screws</td>
		</tr>
		<tr>
			<td>Stainless Steel Swagelok Tube Fitting, Port Connector, 1/4 in. Tube OD (x8)</td>
			<td>Swagelok</td>
			<td>&#160;SS-401-PC</td>
			<td>Use as tube connections between NTP and valve connections 
			Name in Protocol: port connector fitting</td>
		</tr>
		<tr>
			<td>TANK</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 OD in. x 20 ft. Copper Soft Refrigeration Coil</td>
			<td>Everbilt</td>
			<td>Model # D 04020PS</td>
			<td>For circulating coolant 
			Name in Protocol: 1/4&#34; copper pipe</td>
		</tr>
		<tr>
			<td>10 gallon aquarium</td>
			<td>Tetra</td>
			<td></td>
			<td>Name in Protocol: 10 gallon tank</td>
		</tr>
		<tr>
			<td>2 oz. Waterweld</td>
			<td>J-B Weld</td>
			<td>Model # 8277</td>
			<td>Name in Protocol: underwater sealant</td>
		</tr>
		<tr>
			<td>3 in. x 25 ft. Foil Backed Fiberglass Pipe Wrap Insulation</td>
			<td>Frost King</td>
			<td>Model # SP42X/16</td>
			<td>For wrapping around aquarium 
			Name in Protocol: foil-lined fiberglass</td>
		</tr>
		<tr>
			<td>3/8 7/8 in. Stainless Steel Hose Clamp (10 pack)</td>
			<td>Everbilt</td>
			<td>Model # 670655E</td>
			<td>Name in Protocol: worm drive hose clamps</td>
		</tr>
		<tr>
			<td>Styrofoam</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: insulating material</td>
		</tr>
		<tr>
			<td>TOOLS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-1/8 in. Ratcheting Tube Cutter</td>
			<td>Husky</td>
			<td>Model # 86-036-0111</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 in. to 1 in. Pipe Cutter</td>
			<td>Apollo</td>
			<td>Model # 69PTKC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable wrench (x2)</td>
			<td>Steel Core</td>
			<td>Model # 31899</td>
			<td>Need two wrenches with jaw at least 1&#34;</td>
		</tr>
		<tr>
			<td>Allen wrench set</td>
			<td>Home Depot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duct tape</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: duct tape</td>
		</tr>
		<tr>
			<td>Flexible tubing, like an IV line, to fit on the end of grainger probe (canula)</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: IV tube</td>
		</tr>
		<tr>
			<td>Grainger 18 gauge probe</td>
			<td>Grainger</td>
			<td></td>
			<td>For inserting droplet 
			Name in Protocol: cannula</td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow corning</td>
			<td></td>
			<td>Apply to o-rings before inserting sapphire window 
			Name in Protocol: vacuum grease</td>
		</tr>
		<tr>
			<td>Klein Tools Professional 90 Degree 4-in-1 Tube Bender</td>
			<td>Klein Tools</td>
			<td>Model # 89030</td>
			<td>Name in Protocol: tube bender</td>
		</tr>
		<tr>
			<td>Snoop liquid leak detector</td>
			<td>Swagelok</td>
			<td>MS-SNOOP-8OZ</td>
			<td>To detect leaks when pressurized when methane 
			Name in Protocol: liquid leak detector</td>
		</tr>
		<tr>
			<td>Suction cup</td>
			<td>Home Depot</td>
			<td></td>
			<td>For removing tight fitting sapphire window 
			Name in Protocol: suction cup</td>
		</tr>
		<tr>
			<td>Teflon Tape</td>
			<td></td>
			<td></td>
			<td>Name in Protocol: plumber&#39;s tape</td>
		</tr>
		<tr>
			<td>Temflex 3/4 in. x 60 ft. 1700 Electrical Tape Black</td>
			<td>3M</td>
			<td>Model # 1700-1PK-BB40</td>
			<td>Name in Protocol: electrical tape</td>
		</tr>
	</tbody>
</table>

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.

With this method, a gas hydrate shell on a droplet can be monitored visually through a sapphire window of the pressure cell and via temperature and pressure transducers. To nucleate the hydrate shell after pressurizing to 5 MPa, dry ice can be added to the top of the pressure cell to induce a thermal shock to trigger rapid hydrate crystallization. There is a clear morphologic difference upon dry ice-forced hydrate shell formation. The water droplet transitioned from a smooth, reflective surface (Figu...

Watch this Scientific Journal Video about Methane Hydrate Crystallization on Sessile Water Droplets at JoVE.com

Methane Hydrate Crystallization on Sessile Water Droplets

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 ...

The effect of various additives on gas hydrate morphology and on pressure temperature stability can be tested. But the main interest lies in finding out how biomolecules may interact and affect gas hydrates in situ. The main advantage of this protocol is that one can reproducibly form a hydrate shell on a sessile droplet safely.Leveling the droplet stage is challenging and needs practice as the droplet slides on an uneven stage. Also, familiarize with Swagelok connections and safety regarding high pressure flammable gases. Begin by connecting the methane cylinder regulator to the pump with a one-by-four inch copper pipe using a new nut and ferrule set.Glue a flexible tip of IV tubing, cut an angle, to the end of the cannula to help direct the droplet toward the sapphire window. Attach a one milliliter syringe to the cannula and pull in the desired volume of deionized water. Without the needle valve or sapphire window attached, insert the end of the cannula into the top board and practice expelling the droplet onto the center stage.Reattach the sapphire window and washers with M8 screws and attach top pressure cell valve. Connect the braided stainless steel hose from the pressure pump to the pressure cell. And double-check that all connections from the gas cylinder to the pressure cell are tight.Open the pressure cell inlet valve, and set the pressure cell in the aquarium. Then insert a fiber optic light source cable into the pressure cell illumination port. In an aquarium filled with a 50/50 ratio of ethanol and water, add more ethanol as the solution level falls in the following weeks, until it is level with the top of the pressure cell, just below the light source connection.Set the chiller to the temperature that will achieve approximately zero to three degrees Celsius inside the cell and start circulating through coils. Turn on the air flow to the front of the aquarium to prevent condensation on the aquarium surface. Start a temperature log in the data logger software.Set the scanning interval to 30 seconds and wait until the temperature inside the pressure cell is stable at two degrees Celsius. After the camera is placed, turn on the light source to approximately 80%and open the camera software. In Live View, focus the camera lens at the cell's inner chamber and adjust the light source for best imaging.Start a new temperature log with a one-second scanning interval. If attached, detach the outlet needle valve in the top port of the pressure cell. Attach a one milliliter syringe to the cannula and pull in the desired volume of deionized water.Insert the cannula through the top board until the tip is visible in the camera software in Live View mode. Expel the fluid droplet from the syringe over the central thermocouple. Then reattach the needle valve.Focus the camera on the droplet in the pressure cell. Begin time-lapse imaging every 60 seconds. Open the pressure transducer software on the laptop.Start collecting data on the chart and the data log at the scanning interval of one second, and wait until the droplet temperature is stable between zero to three degrees Celsius. Turn on the pump and the controller. Close the pressure pump's inlet valve.Open the pump's outlet valve and the pressure cell's valves. Tare the pump pressure by pressing zero on the pressure pump controller. Select Pump A on the pressure pump controller to monitor the pressure.Ensure that the pressure pump is empty if a different fluid other than methane gas was present in the pump. Do this by setting the max flow and constant flow to 100 milliliters per minute and pressing Run. Leave it running until the pump is empty.Close the pump outlet valve and open the pump inlet valve. Open the gas cylinder and set the gas cylinder regulator to 1, 000 kilopascals. Press Refill on the pressure pump controller.When the pump is full and near 1, 000 kilopascals, close the pump inlet valve and the gas cylinder. Slightly open the pump outlet valve to the cell. Monitor the pressure cell pressure in the pressure transducer software, as it may decrease due to the relatively lower temperature in the pressure cell.Set the max flow to 10 milliliters per minute, max pressure to 5, 000 kilopascals, and constant pressure to 1, 000 kilopascals on the pressure pump controller as described in the text manuscript. Press Run. When 1, 000 kilopascals is reached, press Stop on the pump controller and close the pump's outlet valve.Monitor the pressure in the pressure cell to ensure there are no leaks. If the pressure drops, use the liquid leak detector to find the leak at the connections and carefully tighten the leaking components. If the cell is stable, open the pump outlet and set the constant pressure gradually to 2, 000, 3, 000, 4, 000, and 5, 000 kilopascals, monitoring the cell stability by pressing Stop after each setting.If the pressure is stable, close the pump outlet and wait for around 12 to 24 hours for the gas to permeate the droplet. Switch the time-lapse to take images every two to five seconds. Add dry ice to the top of the cell until the hydrate shell is seen in time-lapse.If the dry ice slides, affix tape around the top of the cell. Observe the progress of the methane hydrate formation through time-lapse photos for two to six hours. Depressurize the cell to 2, 000 kilopascals by opening the pump outlet and setting the constant pressure to 2, 000 kilopascals, noting when melting occurs.After 30 minutes, repressurize the pressure cell to 5, 000 kilopascals to observe the memory effect. Note when a hydrate shell begins to reform and allow the shell to form for 30 minutes to two hours. Depressurize the cell by opening the pump outlet and setting the constant pressure to 100 kilopascals.If there is residual pressure in the pressure cell, slightly open the pressure cell top valve by 1/16 inches. Save the pressure and temperature data as CSV files. Remove the droplet by removing the top pressure cell valve and extracting the droplet with the syringe, cannula, or IV tube.Follow the directions in the text manuscript if there is a concern for contamination between trials. There's a clear morphologic difference upon dry ice forced hydrate shell formation, where the water droplet transitioned from a smooth reflective surface to an opaque hydrate shell with a slight dendritic surface. The addition of 100 micrograms per milliliter at type one antifreeze proteins altered the hydrate morphology by inducing ridged edges along the droplet and protrusions from the top of the droplet.After the hydrate shell developed for one hour, the cell was depressurized to two megapascals, leading to a 2 to 5 degree Celsius drop in temperature near the P/T stability curve. Due to exothermic hydrate dissociation, hydrate dissociation was confirmed by visual melting through time-lapse imaging at the beginning of the decrease in temperature. Negative controls with no droplet and with a droplet that did not form a hydrate shell showed no decrease in temperature during depressurization.Because the apex of each temperature degrees was above the previously established P/T stability curve, a regression curve was calculated based on the apex P/T of these trials. Two things are important to keep in mind when performing this protocol. One, check that all connections are secure before pressurizing, and two, practice expelling the droplet before attaching the sapphire window.Following this protocol, one can calculate gas consumption as well as analyze the images to calculate hydrate shell thickness, to determine the volume of hydrate formed.

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