Vitrification of aqueous cryoprotectant mixtures is essential in cryopreservation of proteins and other biological samples. We report systematic measurements of critical cryoprotective agent (CPA) concentrations required for vitrification during plunge cooling from T=295 K to T=77 K in liquid nitrogen. Measurements on fourteen common CPAs including alcohols (glycerol, methanol, isopropanol), sugars (sucrose, xylitol, dextrose, trehalose), PEGs (ethylene glycol, PEG 200, PEG 2 000, PEG 20 000), glycols (DMSO, MPD), and salt (NaCl) were performed for volumes ranging over four orders of magnitude from ∼ nL to 20 µL, and covering the range of interest in protein crystallography. X-ray diffraction measurements on aqueous glycerol mixtures confirm that the polycrystalline-to-vitreous transition occurs within a span of less than 2% w/v in CPA concentration, and that the form of polycrystalline ice (hexagonal or cubic) depends on CPA concentration and cooling rate. For most of the studied cryoprotectants, the critical concentration decreases strongly with volume in the range from 5 µL to 0.1 µL, typically by a factor of two. By combining measurements of the critical concentration versus volume with cooling time versus volume, we obtain the function of greatest intrinsic physical interest: the critical CPA concentration versus cooling rate during flash cooling. These results provide a basis for more rational design of cryoprotective protocols, and should yield insight into the physics of glass formation in aqueous mixtures.
We present a lab-on-chip approach to the study of multiphase transport in porous media. The applicability of microfluidics to biological and chemical analysis has motivated much development in lab-on-chip methodologies. Several of these methodologies are also well suited to the study of transport in porous media. We demonstrate the application of rapid prototyping of microfluidic networks with approximately 5000 channels, controllable wettability, and fluorescence-based analysis to the study of multiphase transport phenomena in porous media. The method is applied to measure the influence of wettability relative to network regularity, and to differentiate initial percolation patterns from active flow paths. Transport phenomena in porous media are of critical importance to many fields and particularly in many energy-related applications including liquid water transport in fuel cells, oil recovery, and CO(2) sequestration.
When samples having volumes characteristic of protein crystals are plunge cooled in liquid nitrogen or propane, most cooling occurs in the cold gas layer above the liquid. By removing this cold gas layer, cooling rates for small samples and modest plunge velocities are increased to 1.5 × 10(4) K s(-1), with increases of a factor of 100 over current best practice possible with 10 μm samples. Glycerol concentrations required to eliminate water crystallization in protein-free aqueous mixtures drop from ∼28% w/v to as low as 6% w/v. These results will allow many crystals to go from crystallization tray to liquid cryogen to X-ray beam without cryoprotectants. By reducing or eliminating the need for cryoprotectants in growth solutions, they may also simplify the search for crystallization conditions and for optimal screens. The results presented here resolve many puzzles, such as why plunge cooling in liquid nitrogen or propane has, until now, not yielded significantly better diffraction quality than gas-stream cooling.
A method is presented for determining second virial coefficients (B(2)) of protein solutions from retention time measurements in size exclusion chromatography. We determine B(2) by analyzing the concentration dependence of the chromatographic partition coefficient. We show the ability of this method to track the evolution of B(2) from positive to negative values in lysozyme and bovine serum albumin solutions. Our size exclusion chromatography results agree quantitatively with data obtained by light scattering.
We report on new instabilities of the quasi-static equilibrium of water drops pinned by a hydrophobic inclined substrate. The contact line of a statically pinned drop exhibits three transitions of partial depinning: depinning of the advancing and receding parts of the contact line and depinning of the entire contact line leading to the drop's translational motion. We find a region of parameters where the classical Macdougall-Ockrent-Frenkel approach fails to estimate the critical volume of the statically pinned inclined drop.
A method of imaging ionomer in the catalyst layer (CL) of the catalyst coated membrane (CCM) in the proton exchange membrane (PEM) fuel cell was developed using scanning transmission X-ray microscopy (STXM). Component maps for ionomer in CL relative to the catalyst/carbon-support were obtained from STXM images at a few specifically pre-defined energies in the C 1s (on/off a peak characteristic for carbon support in the catalyst layer) and F 1s edges (on/off a peak characteristic for the fluorine in the ionomer). Comparing the different approaches, an optimized strategy to study the spatial distributions of chemical components in the catalyst layer was determined. C 1s multi-energy imaging was most informative for mapping all components of the carbon catalyst support. The F 1s maps offer a rapid and powerful way for visualization of ionomer in the catalyst layer.
We study how the microscale topography of a solid surface affects the apparent advancing and receding angles at the contact line of a liquid drop pinned to this surface. Photolithographic methods are used to produce continuous circular polymer rings of varying cross-sectional size and shape on hydrophilic silicon wafer surfaces. Drops of water and glycerol are dispensed into the areas bounded by these rings, and critical apparent advancing and receding angles are measured and correlated with the parameters that characterize the ring cross-section. For much of the examined parameter space, the apparent critical angles are independent of ring height and width and are determined primarily by the slope of the ring's sidewalls, consistent with a model due to Gibbs. For ring heights below a few micrometers, the critical angles decrease below the values predicted by the sidewall slopes alone. These results provide data for calculation of hysteresis on naturally rough surfaces, and demonstrate a simple method for controlling and enhancing contact line pinning on solid surfaces.
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