Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures

Summary

A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

Abstract

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid cooling, to prepare the desired cryogenic temperature phase. Microliter to picoliter size drops are cooled by projection into a liquid nitrogen-argon (N₂-Ar) mixture. The cryogenic temperature phase of the drop is evaluated using a visual assay that correlates with X-ray diffraction measurements. The density of the liquid N₂-Ar mixture is adjusted by adding N₂ or Ar until the drop becomes neutrally buoyant. The density of this mixture and thus of the drop is determined using a test mass and Archimedes principle. With appropriate care in drop preparation, management of gas above the liquid cryogen mixture to minimize icing, and regular mixing of the cryogenic mixture to prevent density stratification and phase separation, densities accurate to <0.5% of drops as small as 50 pL can readily be determined. Measurements on aqueous cryoprotectant mixtures provide insight into cryoprotectant action, and provide quantitative data to facilitate thermal contraction matching in biological cryopreservation.

Introduction

The physical properties of water and aqueous mixtures in their various phases are of fundamental interest, and are important to in vivo and in vitro understanding of biological systems. In contemporary cryobiology and biological cryopreservation, the vitreous or amorphous phases of aqueous cryoprotectant mixtures are of particular interest^1,2. Nucleation and growth of ice crystals can disrupt cells and tissues, and promote protein denaturation and aggregation, so cryopreservation protocols that vitrify the solvent have become increasingly popular. In biomolecular crystallography, the crystall....

Protocol

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Please use all appropriate safety practices when using compressed gases, including appropriate calibrated gas handling regulators and valves, and approved gas tubing. Contact with liquid cryogens can cause  severe frostbite and necrosis. Use appropriate personal protective equipment (face shield, gloves, lab coat, full length pants, closed-toe shoes), all of which must be impermeable to liquid nitrogen. Remain standing and ensure an unobstructed exit path from the apparatus when using liquid cryogens. Be aware of asphyxiation hazards when using compressed gases and liquid cryogens, and....

Results

Density measurements at T = 77 K for vitrified drops of aqueous glycerol and ethylene glycol versus cryoprotectant concentration are shown in Figure 1A and Figure 1B respectively, and the corresponding change in specific volume between T = 298 K and 77 K, calculated using previously determined T = 298 K densities, is shown in Figure 2. At h.......

Discussion

The present apparatus and methods, developed primarily by undergraduates with limited access to instrument-building tools and machinery, nevertheless delivers highly accurate density measurements for individual liquid drops as small as 50 pL. In the concentration range near and above 50% w/w, where small cooling rates are sufficient to obtain vitrified samples, the densities agree with those obtained in previous measurements on bulk samples. Extrapolations of the present densities to 0% concentration – pure water – also .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
centrifuge tube	Falcon	6029236	15 mL conical centrifuge tube
glycerol, >99.5%	Sigma	G9012-100 mL
ethylene glycol, >99.8%	Sigma	324558-100 mL
analytical microbalance	Mettler	AE240	Analytical balance, 0.01 mg resolution, has hook on bottom for weighing below the balance
vortexer	Scientific Industries	SI-0236	Vortex-Genie 2
Apparatus enclosure framing	Unistrut	1-5/8" metal framing	48" wide x 24" deep x 40" tall
Apparatus enclosure air barrier			any clear plastic sheeting
neoprene rubber disk			4" diameter, 1/8" thick
dewar flask	Scilogix Dilvac	SS333	4.5 liter dewar flask with steel case and clamp lid
copper chamber			This fabricated part is comprised of a 1.43" diameter, 0.017" wall thickness copper tube with a solid cylindrical copper base soldered to seal one end.  The copper base is 0.87" tall and the overall chamber height is 7".
nitrogen gas	Airgas	NI HP300	99.998% pure N2 gas
argon gas	Airgas	AR HP300	99.998% pure Ar gas
rotameter	Omega	FL3692ST	2.52 L/min max flow rate
foam insulating lid			This part is fabricated from 4 lb/ft3 crosslinked polyethylene foam (supplied by Technifab, 1355 Chester Industrial Parkway, Avon, OH), and has an OD of 2.42", and ID of 1.52", and a thickness of 0.79".
PTFE test mass			This fabricated part is a 0.246" diameter, 0.580" tall cylinder with a 0.060" diameter hole running perpendicular to and intersecting the cylinder axis ~0.10" from one end.
microbalance platform	Unistrut	1-5/8" metal framing	11" wide x 24" long x 24" high rectangular frame with an top aluminum sheet containing a hole for the monofilament and hanging test mass
2 mil (50 um) monofilament line	Berkley	NF1502-CM	Nanofil fishing line
Argon precooling coil tubing	VWR	60985-512	1/8" ID x 1/4" OD PVC tubing
perforated copper foil mixer			1.4" diameter,  35 micron thick copper disk, cut from 1 ounce/ft2 copper sheet and perforated with holes using an awl or other sharp pointed tool.  Insert 1-2 mm diameter rigid thermally insulating (plastic or wood) rod into the center and fix using epoxy as needed.
syringe	BD	309628	1 mL Luer-Lok tip syringe
vacuum generator	Gast	VG-015-00-00	compressed air venturi single stage vacuum generator
hydrophobic coating spray	RainX	620036	plastic water repellent
long focal length stereo microscope	Bausch and Lomb Stereozoom 6		0.67-4 x zoom pod with 20X eyepieces, 10 cm working distance
LED ring illuminator	Amscope	LED144S
LED spot illuminator	Newhouse Lighting	NHCLP-LED	3W LED gooseneck clamp lamp
1.8 ml cryo vial	Nunc	V7634-500EA	Any 1.8 or 2 mL cryovial is adequate