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Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 1 of 16
Methane Hydrate Crystallization on Sessile Water
Droplets
Abigail M. Johnson1, Yumeng Zhao2, Jongchan Kim2, Sheng Dai2, Jennifer B. Glass1
1Earth and Atmospheric Sciences, Georgia Institute of Technology 2Civil and Environmental Engineering, Georgia Institute of Technology
Corresponding Authors
Sheng Dai
Sheng.Dai@ce.gatech.edu
Jennifer B. Glass
Jennifer.Glass@eas.gatech.edu
Citation
Johnson, A.M., Zhao, Y., Kim, J.,
Dai, S., Glass, J.B. Methane Hydrate
Crystallization on Sessile Water
Droplets. J. Vis. Exp. (), e62686,
doi:10.3791/62686 (2021).
Date Published
May 21, 2021
DOI
10.3791/62686
URL
jove.com/t/62686
Abstract
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 °C to -9 °C. During depressurization, a 0.2 °C to 0.5 °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.
Introduction
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 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
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 2 of 16
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.
Protocol
1. Design, validate, and machine the pressure cell.
1. 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).
2. To check that the pressure cell is safe under working
high pressure, conduct a finite element analysis using
simulation software.
1. Input the full-size pressure cell model from the
engineering design software into the simulation
software.
2. Assign a Young's modulus of 400 GPa and a
Poisson's ratio of 0.29 to the sapphire window.
3. For all stainless-steel parts, assign stainless steel
316 with a Young's modulus of 190 GPa and
Poisson's ratio of 0.27.
4. In a step-by-step manner, apply 0 to 1, 2, 3, 4 5, 6,
7, 8, 9, and 10 MPa air pressure to the inside of the
cell (Supplemental Video S1 and Supplemental
Video S2). Treat each loading step as a static
problem by ignoring the time-dependent terms in
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 3 of 16
the governing equations and consider only elastic
deformation during pressurization.
5. Use the direct linear equation solver in simulation
software to calculate the stress distribution
and the deformation of the cell under various
pressure conditions (Supplemental Table S1 and
Supplemental Table S2).
3. Once the pressure cell design is verified to be safe,
have all parts machined based on the engineering design
software blueprint.
2. Assemble the pressure cell (Figure 1).
1. Screw the four National Pipe Tapered (NPT) threads into
the respective ports on the pressure cell with plumber's
tape.
2. Assemble the illumination port using the blueprint design
(Supplemental Figure S1, parts C, D, and E) and
connect to the top left NPT screw.
3. Connect the pressure transducer to the top port NPT
using the branch tee fitting and port connector fitting.
4. Connect the inlet needle valve in the left side NPT screw
using a port connector fitting.
5. Install a pressure seal connector into the right-side port
of the pressure cell. Insert three K-type thermocouple
wires through the pressure seal connector with 3" of slack
inside the cell and 3' slack outside the cell.
6. Polish the stage surface with sandpaper (Supplemental
Figure S1, Part F).
7. Insert the thermocouples into the respective holes in
the stage so that the tips are flush with the top of the
stage. Use a small drop of glue in each hole to fix the
thermocouples in place and allow them to dry.
8. Fit the acrylic disc on the back wall of the pressure cell to
enhance light reflection. Fit the stage in the pressure cell.
9. Install the sapphire window.
1. Apply vacuum grease to two static sealing O-rings
(one 1" and one 1-1/5"). Fit the O-rings into the
grooves around the window hole on the pressure
cell.
2. Insert the sapphire window. Cover the sapphire
window with a 2-1/4" rubber washer and screw on
the stainless-steel washer (Supplemental Figure
S1, Part B) using eight M8 stainless steel screws
(Figure 2C).
3. Assemble the equipment in a large fume hood
( Figure 2).
NOTE: As methane is a flammable gas under pressure,
keep all methane-related tubing and vessels away from heat,
sparks, open flame, and hot surfaces. Set all equipment up
inside a well-ventilated area (e.g., a fume hood). Don safety
glasses and lab coat before working with methane gas.
1. Carefully lift the pressure pump into a fume hood large
enough for all the equipment to fit (Figure 2A). Place the
pump controller on top of the pump base. Connect the
pump controller to the pump and plug it into a power strip.
2. Run a high-pressure-rated 1/4" copper pipe from the
regulator on the methane gas cylinder to the fume hood
next to the inlet of the pressure pump (Figure 2A,B).
3. Place the data logger next to the pressure pump and set
the laptop on the data logger (Figure 2A). Plug both into
a power strip. Connect the data logger to the laptop via
the data logger USB.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 4 of 16
4. On the laptop, install the proper software to control the
data logger, camera, and pressure transducer on the
pressure cell.
5. Set the aquarium beside the data logger and place non-
leaching padding in the bottom of the aquarium to limit
vibrations to the pressure cell (Figure 2C).
6. Using a new 1/4" copper pipe, coil the copper pipe twice
into an oval to fit in the aquarium, leaving room for the
pressure cell to sit inside (Figure 2D). Ensure that the
coil does not block the sapphire window in the pressure
cell. Elevate the pressure cell in the aquarium to view the
sapphire window.
7. Place the circulating chiller on the floor near the fume
hood (Figure 2A). Fill the chiller with 50/50 v/v ethylene
glycol/water.
NOTE: As ethylene glycol is hazardous, use appropriate
safety attire, including gloves, lab coat, and goggles
when pouring.
8. Cut two lengths of a 3/8" (inner diameter) plastic tubing
to connect the chiller inlet and outlet to the copper pipe
ends in the aquarium. Ensure there will be enough slack
for the foam pipe insulation to fit before cutting.
9. Slide the plastic tubing through the foam pipe insulation.
10. Connect the insulated plastic tubing from the inlet and
outlet on the circulating chiller to the ends of the copper
coil inside the aquarium. Secure the seals by wrapping
plumber's tape around the metal parts and tightening
the connections with worm drive hose clamps. Turn the
chiller on and set it to circulate at high speed. Ensure
there are no leaks.
11. Apply underwater sealant around the copper coil/plastic
tubing connections inside the aquarium. Allow the
sealant to cure. Wrap the sealant with duct tape.
12. Install pressure pump tubing (Figure 2E).
NOTE: Always hand-tighten connections before using
tools and never detach the NPT connections with
plumber's tape because they will not re-seal well.
1. Install a 1/8" stainless steel pipe on either side of the
pressure pump with the company fittings that came
with the pump using plumber's tape (Figure 2F).
2. With a tube bender, bend the 1/8" pipe forward at a
90° angle, approximately 2" away from the pump, to
avoid bending at the connection.
3. With a tube bender, bend the 1/8" pipe downward
at a 90° angle, approximately 2" away from the first
bend.
4. Attach 1/8" to 1/4" adapter fitting to the 1/8" pipe on
both sides (Figure 2G).
5. Attach 1/4" pipe to adapter fitting on both sides.
NOTE: To affix the valve to the side of the pump, trim
the 1/4" tubing so that the attached valve will sit next
to the two screw holes.
6. Install the 1/4" needle valves (Figure 2H). If affixing
valves to the pressure pump, machine a steel or
plastic plate with two 1/16" holes for screws and
one 1/2" hole to secure between needle valve
connections. Insert the plate between the valve
connections and screw the plate to the side of the
pump.
NOTE: Ensure that arrows on the needle valves
point from high pressure (inside the pressure pump)
to low pressure (outside the pressure pump).
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 5 of 16
7. Connect one end of the 1/4" braided stainless steel
flexible pressure-rated hose to the outlet valve on the
pressure pump and the other end to the side valve
of the pressure cell.
8. Connect thermocouples from the pressure cell
to data logger channels using the data logger
multichannel. Connect an additional thermocouple
wire to measure the temperature of the tank solution
and put the other end in the tank.
9. Connect the pressure transducer on the pressure
cell to the laptop.
10. Set the pressure cell inside the aquarium, close to
the front, for clearer imaging.
13. To insulate the aquarium, wrap the outside of the
aquarium with foil-lined fiberglass, with a hole/slit for the
camera to view the sapphire window of the pressure cell.
Cover the top of the aquarium with insulating material to
prevent evaporation during experiments.
NOTE: Avoid tightly sealing the aquarium top to avoid the
buildup of heat from the light source.
14. To prevent the condensation of moist air on the front
of the aquarium, run plastic tubing from the closest air
valve to the front of the aquarium where the camera will
be pointing so that the tubing will not be visible in the
photographs.
15. Set the light source unit beside the aquarium and plug it
into the power strip.
16. Set the camera in front of the aquarium, with the lens
pointing towards the sapphire window. Plug the camera
into the laptop and power strip.
17. Elevate all electronics from the hood surface to prevent
potential leak damage. Double-check that power is
distributed for the power capacity of the outlets.
4. Leak-test the pressure cell with water.
NOTE: To ensure all connections were sealed properly, leak-
test the pressure cell with water any time the cell has been
reassembled, especially after disconnecting the NPT screws.
This is not necessary after removing the sapphire window or
top valve. Water is safer under pressure than gas.
1. Open the pressure transducer software on the laptop and
start collecting data at a scanning interval of 1 s.
2. Turn on the pressure pump and controller. Press Pump A
on the pressure pump controller to monitor the pressure.
3. If there is pressure in the pump, decrease the pressure
by pressing Refill on the pressure pump controller while
both the pump inlet and outlet valves are still closed.
4. With both pressure cell valves open, open the pump
outlet valve slightly by ~1/16" to slowly release the
remaining pressure.
5. If connected, disconnect the 1/4" copper pipe from the
inlet valve on the pressure pump.
6. Attach 1/4" flexible tubing to the pump inlet valve using a
nut and ferrule set. Place the end of the tubing in a gallon
of water.
7. Close the pump's outlet valve and open the pump's inlet
valve.
8. Press Refill on the pressure pump controller to fill the
pump piston with water.
9. Set the pressure cell in a shallow empty container outside
of the aquarium.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 6 of 16
10. Purge the air out of the pressure cell until water comes
out of the top port and fills the pressure cell completely.
1. Close the pump's inlet valve and open the pump's
outlet valve.
2. Ensure the valves on the pressure cell are still open.
3. Set the maximum (max) flow to 100 mL/min: on
the pressure pump controller, press Limits; press 3
for max flow; press 1 to set max flow; punch in 100;
press Enter.
4. Press D to reach the previous page.
5. Set the constant flow rate to 100 mL/min: on the
pressure pump controller, press Const Flow; press
A for flowrate; punch in 100; press Enter. Press
Run.
6. If water does not come out or if the volume in the
piston is insufficient, refill the piston again by closing
the pump outlet valve, opening the pump inlet valve
with tubing in water, and press Refill. Then, purge
the air out by closing the pump inlet valve, opening
the pump outlet valve, setting the flow rate to 100,
and pressing Run.
7. Once water comes out of the top port of the
pressure cell, check for leaks and tighten any leaking
connections. Press Stop. Close the pressure cell
outlet (top) valve.
11. Pressurize the pressure cell.
NOTE: Don safety glasses before pressurizing the
pressure cell.
1. Set the max flow limit to 10 mL/min to prevent fast
pressurization of the cell: on the pressure pump
controller, press Limits; press 3 for max flow; press
1 to set max flow; punch in 10; press Enter.
2. Pressurize the cell to 100 kPa: on the pressure pump
controller, press Const Press; press A; punch in
100; press Enter. Press Run.
3. Check for leaks. If there is a leak, press Stop on
the pump controller, tighten the leaking components,
press Run, and repeat until there are no leaks at
100 kPa. Ensure there are no leaks by closing the
pump outlet valve and monitoring the pressure cell's
pressure in the pressure transducer software.
NOTE: If the pressure decreases consistently and
is not normal fluctuation due to room temperature
variation, there is a leak.
4. Increase the pressure in increments of 50 kPa from
100 kPa to 500 kPa, then in increments of 100 kPa
from 500 kPa to 1,000 kPa, and finally in increments
of ~1,000 kPa from 1,000 kPa to ~10,000 kPa. Do
this by changing the Const Press setting as before.
Between pressure settings, close the pump outlet
valve and monitor the cell's pressure like before to
ensure that the pressure is constant. If the pressure
drops, carefully tighten the leaking components.
12. Upon reaching 10,000 kPa, close the pump outlet valve
and observe how well the pressure cell holds pressure
according to the pressure transducer. As a consistent
drop in pressure indicates a leak, tighten connections at
a lower pressure, ~1,000 kPa.
13. To depressurize, open the pump outlet valve and set the
pressure to 100 kPa. Once the pressure plateaus, slightly
open the pressure cell outlet valve.
14. To remove water from the pressure pump, close the
pump inlet valve, change the max flow and Const Flow
settings to 100 mL/min, and press Run until the pump
is empty.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 7 of 16
15. Disconnect the 1/4" flexible tubing from the pump inlet.
Disconnect the braided stainless-steel hosing from the
pressure cell. Open both valves and drain the water.
Remove the sapphire window to allow the cell to
completely dry.
5. Form a methane hydrate shell on the droplet
surface.
1. Prepare the equipment.
1. Connect the methane cylinder regulator to the pump
with the 1/4" copper pipe using a new nut and ferrule
set. Ensure that the gas cylinder is closed.
2. Practice droplet insertion technique.
1. Glue a flexible tip, such as IV tubing, cut at an
angle to the end of the cannula to help direct
the droplet toward the sapphire window. Attach
a 1 mL syringe to the cannula and pull in the
desired volume of deionized water (~50-300
µL). Without the needle valve or sapphire
window attached, insert the end of the cannula
into the top port and practice expelling the
droplet onto the center stage. After practicing
droplet insertion, remove the droplet and dry the
stage.
NOTE: In this protocol, 250 µL of deionized
water was taken into the syringe.
3. Reattach the sapphire window and washers with M8
screws. 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 (side valve), and set the
pressure cell in the aquarium. Insert a fiber optic light
source cable into the pressure cell illumination port.
4. Add 50/50 ethanol/water (v/v) to the aquarium until
it is level with the top of the pressure cell, just
below the light source connection. Ensure that the
hood flow is turned on. When the solution level falls
before future trials in the following weeks, add more
ethanol. Replace the solution monthly.
5. Set the chiller to the temperature that will achieve ~0
°C to 3 °C inside the cell (~-4 °C) and start circulating
through coils. Turn on the airflow to the front of the
aquarium to prevent condensation on the aquarium
surface.
6. Start a temperature log in the data logger software.
Set the scanning interval to 30 s. Wait until the
temperature inside the pressure cell is stable at 2 °C
(~6-24 h).
2. Add a water droplet into the pressure cell using the
camera view on the laptop.
1. Turn on the light source to ~80%. Open the camera
software. In live view, focus the camera lens at the
cell's inner chamber. Adjust the light source for best
imaging.
2. Start a new temperature log with a 1 s scanning
interval.
3. If attached, detach the outlet needle valve in the top
port of the pressure cell. Attach a 1 mL syringe to the
cannula and pull in the desired volume of deionized
water (~50-300 µL).
NOTE: In this protocol, 250 µL of deionized water
was pulled into the syringe.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 8 of 16
4. Insert the cannula through the top port until the tip
is visible in the camera software in live view mode.
Expel the fluid droplet from the syringe over the
central thermocouple. Reattach the needle valve.
3. Focus the camera on the droplet in the pressure cell.
Begin time-lapse imaging every ~60 s.
4. Open the pressure transducer software on the laptop
and start collecting data on the chart and the data log
at a scanning interval of 1 s (same as the temperature
scanning interval). Wait until the droplet temperature is
stable between 0-3 °C.
5. Pressurize the pressure cell to the desired pressure.
NOTE: Don safety glasses before pressurizing the cell.
1. Turn on the pump and the controller. Close the
pressure pump's inlet valve.
2. Open the pump's outlet valve and the pressure cell's
valves.
NOTE: The pressure cell inlet valve should always
be open.
3. Tare the pump pressure by pressing Zero on the
pressure pump controller. Select Pump A on the
pressure pump controller to monitor the pressure.
4. 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 Const
Flow to 100 mL/min and pressing Run. Leave it
running until the pump is empty. Close the pump
outlet valve and open the pump inlet valve.
5. Open the gas cylinder and set the gas cylinder
regulator to 1,000 kPa.
6. Press Refill on the pressure pump controller. When
the pump is full and near 1,000 kPa, close the pump
inlet valve and the gas cylinder.
7. Slightly open (~1/16" turn) the pump outlet valve to
the cell. Monitor the pressure cell pressure in the
pressure transducer software as the pressure may
decrease due to the relatively lower temperature in
the pressure cell.
8. Set the max flow to 10 mL/min: on the pressure
pump controller, press Limits; press 3 for max flow;
press 1 to set max flow; punch in 10; press Enter.
9. Set the max pressure to 5,000 kPa: on the pressure
pump controller, press Limits; press 1; punch in
5000; press Enter.
10. Set the constant pressure to 1,000 kPa: on the
pressure pump controller, press Const Press; press
A; punch in 1000; press Enter. Press Run.
11. When 1,000 kPa 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.
12. If the cell is stable, open the pump outlet and set the
Const Press to 2,000 kPa. Press Stop and monitor.
If stable at 2,000 kPa, set Const Press to 3,000 kPa.
Press Stop and monitor. If stable at 3,000 kPa, set
Const Press to 4,000 kPa. Press Stop and monitor.
If stable at 4,000 kPa, set Const Press to 5,000 kPa.
Press Stop and monitor.
13. If the pressure is stable, close the pump outlet.
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NOTE: If the pump volume runs out, close the pump
outlet and slightly open the pump inlet. Slowly open
the gas cylinder and set the gas regulator to 1,000
kPa. Press Refill on the pump controller. When the
pump is refilled, close the gas cylinder and the pump
inlet. Pressurize the pump to match the pressure cell
pressure.
14. Wait for ~12-24 h for the gas to permeate the droplet.
6. Nucleate the hydrate shell using dry ice.
1. Switch the time-lapse to take images every 2-5 s.
2. 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.
7. Observe the progress of the methane hydrate formation
through time-lapse photos for ~2-6 h.
8. Depressurize the cell to 2,000 kPa by opening the pump
outlet and setting the Const Press to 2,000 kPa. Note
when melting occurs.
NOTE: Bubbling in the sessile droplet may occur due to
the escape of the dissolved gas.
9. After ~30 min, repressurize the pressure cell to 5,000 kPa
to observe the memory effect. Note when a hydrate shell
begins to reform. Allow the shell to form for ~30 min to
2 h.
10. Depressurize the cell by opening the pump outlet and
setting the Const Press to 0 kPa. If there is residual
pressure in the pressure cell, slightly open the pressure
cell top valve by ~1/16".
11. Save the pressure and temperature data as .csv files.
12. Remove the droplet by removing the top pressure
cell valve as before and extracting the droplet with
the syringe/cannula/IV tube. If there is a concern
for contamination between trials, remove the sapphire
window and sanitize the stage and replace the vacuum
grease. Use a suction cup to remove the sapphire
window once the pressure cell has warmed to room
temperature.
6. Analyze the data.
1. Open the temperature and pressure .csv files.
2. Make a new spreadsheet. Copy the time and pressure
columns from the pressure .csv and the time and
temperature from the temperature .csv file into the new
spreadsheet.
3. Make a scatter plot with time on the x-axis and two y-axes
with temperature and pressure (Supplemental Figure
S2).
4. Make two more columns for the hydrate stability curve.
In the first column, input the temperatures from 273.15
K to ~279.15 K at 0.1 K intervals. In the second column,
calculate the pressure by using formula (1) from Sloan
& Koh13 .
P [kPa] = exp(a+b/T [K]) where a = 38.98 and b =
-8533.80 (1)
5. Make a scatter plot of the hydrate stability boundary,
with temperature (K) on the x-axis and pressure (kPa) on
the y-axis. Add a second series on the scatter plot with
experimental temperature and pressure on the x and y
axes, respectively (Figure 4).
6. Note on the graphs where a hydrate shell became visible,
according to the time-lapse imaging.
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7. Maintain the equipment.
1. Top off the tank solution with ethanol before every trial to
replace evaporated ethanol. Completely replace the tank
solution monthly.
2. Change the o-rings and rubber washer every 2 months
of regular use.
3. Replace port connections if persistent leaking occurs that
is not fixed by tightening.
Representative Results
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 (Figure 3A) to an opaque hydrate shell with
a slightly dendritic surface (Figure 3B). The addition of 100 µg
mL-1 Type I AFP altered the hydrate morphology by inducing
ridged edges along the droplet and protrusions from the top
of the droplet (Figure 3C,D).
After the hydrate shell developed for ~1 h, the cell was
depressurized to 2 MPa (Supplemental Video S3). During
depressurization, there was a 0.2 °C to 0.5 °C drop in
temperature near the P/T stability curve13 (Figure 4) due
to exothermic hydrate dissociation. Hydrate dissociation was
confirmed by visual melting through time-lapse imaging at
the beginning of the decrease in temperature, noted by
stars in Figure 4. After complete hydrate dissociation, we
repressurized the cell to observe the morphology and melting
temperature with the "memory effect"14 , the phenomenon
in which hydrate forms faster after hydrate has already
formed in the system (Supplemental Video S4). Upon re-
pressurization, a hydrate shell reformed within a couple
of minutes after reaching 5 MPa, and we observed the
same temperature decrease at the stability curve during
dissociation.
Negative controls with no droplet and with a droplet that did
not form a hydrate shell (Figure 4, Trials 4 and 5) showed
no decrease in temperature during depressurization. Upon
depressurization below 2 MPa, we observed gas bubbling
within the droplet from rapid degassing. Because the apex
of each temperature decrease was above the previously
established P/T stability curve13 (hydrate stability curve #1 in
Figure 4), a regression curve was calculated based on the
apex P/T of these trials (P [kPa] = EXP(38.98+-8533.8/T [K]),
hydrate stability curve #2 in Figure 4).
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 11 of 16
Figure 1: Pressure cell. The stage on which the droplet sits and the embedded thermocouples are revealed by removing
the sapphire window and overlying rubber and steel washers. All parts and connections are labeled. Top left inset: stage
shown from above with central and side stage embedded thermocouples. Please click here to view a larger version of this
figure.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 12 of 16
Figure 2: Methane hydrate experimental setup. (A) The fume hood in which the experimental setup is located. (B) The
gas cylinder is connected via a copper coil to the pressure pump. Highlighted from panel (A) are (C) the assembled pressure
cell, (D) the 10-gallon (37.85 L) tank without the insulation or solution, (E) the pressure pump, and (F, G, H) zoomed-in
images ofpressure pump connections. Please click here to view a larger version of this figure.
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 13 of 16
Figure 3: Methane hydrate shells. Representative images of the droplet before (A) and after (B) a methane hydrate shell
formed on a deionized water droplet and before (C) and after (D) a hydrate shell formed on a droplet containing 100 µg
mL-1 Type I antifreeze protein. Scale bars = 5 mm. Please click here to view a larger version of this figure.
Figure 4: Pressure-temperature stability diagram. Pressure and temperature data during depressurization are shown with
P/T stability curves of methane hydrate (#1 from Sloan and Koh 200713 and #2 calculated from taking a regression curve
from hydrate melting peaks from this study). Trials with successfully formed hydrate shells on DI water droplets are Trials
1, 2, and 3. Trial 4 was a negative control with no droplet on the stage. The droplet in trial 5 was another negative control in
which no hydrate shell was formed. Stars indicate when visual hydrate melting began during depressurization. Trial 1 has
a resolution of 30 s (a data point every 30 s); other trials have a resolution of 1 s. Abbreviations: T = trial; M.E. = memory
effect; P/T = pressure-temperature; DI = deionized; res = resolution. Please click here to view a larger version of this figure.
Supplemental Figure S1: CAD images for machining the
pressure cell. Parts A-F of the pressure cell are labeled
with their part letter and dimensions. Abbreviation: CAD =
Copyright © 2021 JoVE Journal of Visualized Experiments jove.com May 2021 • • e62686 • Page 14 of 16
computer-aided design. Please click here to download this
File.
Supplemental Figure S2: Pressure and temperature data
over time for Trials 2-4. Trials 2 and 3 were regular deionized
water droplets that formed hydrate shells. Trial 4 was a
negative control in which no droplet was present. The trials
are lined up at the first depressurization, which occurs at time
zero. A small drop in temperature occurs at the beginning
of depressurization due to the gas mixing with the pressure
pump. A larger temperature drop occurs due to the hydrate
melting after the initial pressure drop, as shown in trials 2 and
3. The temperature fluctuation at the end of trial 4 is due to
the opening of the valve leading to complete depressurization,
which also occurs at the end of trials 2 and 3. Please click
here to download this File.
Supplemental Table S1: Allowable stress (MPa) of the
machined pressure cell. Abbreviation: FS = factor of safety.
Please click here to download this Table.
Supplemental Table S2: Factor of safety for the machined
pressure cell. Abbreviation: FS = factor of safety. Please
click here to download this Table.
Supplemental Video S1: Strain. Video of the strain
simulation on machined pressure cell. Please click here to
download this Video.
Supplemental Video S2: Stress. Video of the stress
simulation on machined pressure cell. Please click here to
download this Video.
Supplemental Video S3: Trial 3 of hydrate shell
dissociation. Time-lapse video of hydrate shell dissociation
at 25x speed. Please click here to download this Video.
Supplemental Video S4: Trial 3 of memory effect
nucleation. Time-lapse video of hydrate shell formation by
memory effect after repressurizing from 2 MPa to 5 MPa at
10x speed. Please click here to download this Video.
Discussion
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 tank with
50% ethanol solution, in which the pressure cell is placed. A
pressure pump pressurizes the gas from the cylinder to the
pressure cell. The hydrate shell forms upon rapid temperature
decrease with the addition of dry ice to the top of the pressure
cell. We allow the shell to form for 2 h, during which we believe
the gas permeates through stochastic cracking of the hydrate
shell, and Ostwald ripening over a longer period. Indeed, this
device could be used to study these phenomena.
The critical steps for this protocol include: 1) leak-test the
pressure cell with water before pressurizing it with gas, 2)
practice adding the water droplet onto the stage before
inserting the sapphire window, 3) cool the droplet to be stable
at ~2 °C before pressurizing, 4) pressurize with a max flow
rate of 10 mL min-1 to 5 MPa in 1 MPa increments, 5) close
the outlet valve on the pressure pump (to the cell) to limit gas
exchange with the pressure pump, 6) set the temperature,
pressure, and time-lapse software to log every 1 s, 1 s, and
5 s (or less), respectively, before adding dry ice, 7) apply dry
ice to the top of the cell continuously until a hydrate shell
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is observed in the time-lapse, 8) allow the hydrate shell to
form for at least 1 h, 9) depressurize at the same speed as
pressurizing.
During method development, we optimized variables and
techniques, including the timing of cooling, pressurizing,
depressurizing, droplet size, and the droplet insertion
technique. There are a few limitations in using this method.
One limitation is the resolution of droplet imaging due to
the camera resolution and materials between the camera
and droplet (tank, ethanol solution, thick sapphire window).
Additionally, while other studies observe the surface droplet
on a microscale7, 9 , 10 , this method only allows for macro-
scale observations. A microscope lens attachment could be
installed if there was interest in micro observations.
Another limitation to this method is not being able to measure
the hydrate shell thickness precisely. However, the hydrate
thickness can be estimated by subtracting the cross-sectional
area before and after hydrate formation and calculating the
gas consumption using the change in temperature during
depressurization to determine the volume of hydrate formed.
Another limitation is that this droplet cannot be viewed in 3D
because there is only one side of the pressure cell containing
a sapphire window. In contrast, other studies have used cells
made entirely of sapphire to observe the droplet from multiple
angles7. We also did not install a temperature-controlling
stage10 or spectroscopic techniques; however, these could
certainly be installed using this setup.
With this method, the morphology, dissociation pressure
and temperature, and the change in temperature during
hydrate dissociation can be observed with droplets containing
additives or alternative stage substrates. This method is
relatively cheap, and there are few thorough protocols for
forming gas hydrate shells. Because high-pressure systems
can be dangerous, we include safety tips for pressurizing
and leak testing. Additionally, many setups do not allow the
visualization of gas hydrate formation, or do so on a much
smaller or much larger scale. Laboratory experiments are a
major contributor to the understanding of naturally occurring
gas hydrates and natural gas hydrates that can cause
lethal gas pipeline explosions. This method can be used to
quickly assess the effects of additives on the dissociation
temperature and morphology and the ability of additives to
eliminate the memory effect. Effective additives could be used
as inhibitors in natural gas pipelines or to study the biological
activity of deep-sea bacterial proteins6 , 15 .
Disclosures
There are no competing financial interests.
Acknowledgments
NASA Exobiology grant 80NSSC19K0477 funded this
research. We thank William Waite and Nicolas Espinoza for
valuable discussions.
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