Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures

The overall goal of this experiment is to determine the glass or vitreous state densities of aqueous solutions at cryogenic temperatures. This method can help answer key questions in the fields of Cryobiology and Cryocrystallography, such as, how much do aqueous cryo protected mixtures contract or expand when cooled into a vitreous state at cryogenic temperatures. The main advantage of this technique is that both cryogenic temperature sample density and phase, can be determined using small samples that can be cooled rapidly.

Though this method was developed for cryoprotecting solutions, it can also be used to determine cryogenic temperature densities of tissues, cell aggregates and other biological samples. I first had the idea for this method when I learned from Thomas Lutin, how to make a cryogenic liquid with a tunable density and the range between 0.9 and 1.3 grams per milliliter. To begin this procedure, place a disk of neoprene rubber on the bottom of a 4.5 liter glass dewar flask, to protect the dewar flask from damage.

Carefully insert a high thermal conductivity copper chamber into the flask, until it rests on the rubber disk. Adjust struts projecting outward from the chamber to the dewar flask walls, so that the chamber centered and has no tendency to rock. Insert the outlet of a gas tube with dry nitrogen gas flowing at approximately two liters per minute, down to the bottom of the copper chamber and purge the chamber of moist air.

Now, slowly pour liquid nitrogen into the dewar flask, outside the copper chamber, allowing time for nitrogen boil off. Remove the dry nitrogen gas tube from the copper chamber and insert it into a matching opening in a foam insulating lid. Then cover the outer portion of the dewar flask with the lid.

Secure the lid in place with elastic straps. Slowly pour liquid nitrogen into the copper chamber, so that the final fill level after boiling has ceased, is within approximately five centimeters of the top of the copper chamber. Following this, place a thin optically transparent plastic sheet over the central opening in the lid.

Reduce the nitrogen gas flow rate to approximately 0.2 liters per minute, leaving a slight overpressure of nitrogen gas within the gas spaces above the cryogenic liquids. Determine the apparent mass of a one gram 0.4 milliliter PTFE test mass, in the air, at 298 Kelvin, by placing it on the pan of a calibrated analytical micro balance. Next, suspend the test mass using a 50 micron monofilament line strung from the hook on the underside of the micro balance and through a hole in the test mass.

Then, determine the apparent mass in air and compare to the mass measurement, correcting as needed for the mass of the line. To determine the volume of the test mass at 77 Kelvin, lower the test mass into the copper chamber containing liquid nitrogen until it is fully submerged. When boiling has ceased, measure the apparent mass.

Flow Argon gas at a flow rate of approximately two liters per minute through a coiled tube to its outlet. Place the coiled tube on top of the upper struts that stabilize the position of the copper chamber, just above the liquid nitrogen level and below top surface in the dewar flask. After allowing the coiled tube to cool for five minutes, place the outlet and the tube in the copper chamber at least 10 centimeters below the surface and the liquid nitrogen.

Then, cover the dewar flask with the annular lid and transparent sheet. Following this, adjust the Argon flow rate until bubbles rise from the tube outlet to the top surface of the liquid nitrogen. Then, reduce the flow rate until the bubbles form at the outlet but dissolve or liquefy just before breaking the liquid nitrogen surface.

Periodically, mix the liquid by inserting a thin circular sheet of copper foil attached to a thin insulating rod into the copper chamber and slowly moving it up and down like a piston. After measuring the apparent mass of the test mass in air, remove the insulating dewar cover and cool the mass to 77 Kelvin by lowering it into the liquid nitrogen outside the copper chamber. Raise the cool test mass into the cold layer of nitrogen gas above the liquid nitrogen and wait for residual liquid nitrogen to evaporate off the test mass.

Then, lower the cool, dried test mass into the liquid nitrogen Argon mixture until it is fully submerged and within two centimeters of the liquid surface. After any boiling and surface waves have disappeared, measure the apparent mass of the test mass. Immediately prior to drop dispensing in cooling, mix the nitrogen argon cryogenic liquid, as previously described.

After removing the sample tubes cap, extract up to one milliliter solution using a clean one milliliter syringe. Attach a 27 to 33 gauge needle onto the syringe and then push a small amount of sample through the needle to expel air and any residues from previous dispensing. For samples that require faster cooling for vitrification, place the outlet of a gas tube connected to a vacuum generator, in the gas space above the liquid nitrogen argon mixture and gently suction away the cold gas layer that forms.

For samples with large non-aqueous component concentrations that can be vitrified with modest cooling rates, lightly press the syringe to displace a small diameter, 10 nanoliter to one microliter drop that hangs from the needle tip by surface tension. Gently tap the needle to detach and project the drop towards the liquid nitrogen argon mixture. Using a long working distance binocular microscope, and bright, cool illumination from an LED illuminator, carefully examine the drop while keeping it immersed in the liquid nitrogen argon mixture.

Vitrified drops should appear clear. Reject drops are cloudy or show an optical imperfections including cracks. The drop floats, decrease the density of the liquid nitrogen argon mixture by adding liquid nitrogen using a 1.8 milliliter cryo vial.

Gently mix the liquid nitrogen argon mixture, using a thin perforated copper foil sheet and moving it up and down in the copper chamber. After each liquid nitrogen addition, move a small prequel rod beneath the floating drop to gently displace it downward into the liquid and observe its speed as it returns to the surface. Density measurements at 77 Kelvin for vitrified drops of aqueous glycerol and ethylene glycol, versus cryo protecting concentration, are shown here.

The corresponding change in specific body in between 298 and 77 Kelvin, calculated using previously determined 298 Kelvin densities is displayed here. Near 20%to 25%solutions of both cryo protectants are predicted to show no net expansion or contraction. The slope of the volume change versus concentration has the largest magnitude below 40%where the effects of additional cryo protectant on waters, tetrahedral low temperature structure are most pronounced.

Once mastered, the initial high density liquid nitrogen argon cryogenic solution can be prepared in two to two and a half hours and the densities of individual drops can be determined in 10 to 15 minutes. While attempting this procedure, it's important to remember to watch for icing and remove as much of the cold gas layer that forms on the liquid nitrogen argon mixture surface as possible, using a vacuum generator. Following this procedure, drops can be collected, stored in liquid nitrogen and later measured using cryocrystallography to verify the phase of a drop.

Don't forget, that working with liquid cryogens can be extremely hazardous and precautions such as, wearing appropriate gloves, face shields, clothing and footwear, should always be taken while performing this procedure.