Ch. Körber

Interaction of particles and a moving ice-liquid interface

Abstract A cryomicroscope was used to determine critical interface velocities marking the transition between repulsion and entrapment of spherical latex particles by an advancing ice-liquid interface. The employed freezing stage yields a planar ice front which propagates with increasing velocity into a region of decreasing thermal gradients. It was found that the critical velocity associated with the transition is inversely proportional to the particle radius as suggested theoretically under the assumption of the flat front (no cusping behind the particle). The critical velocity increases about linearly with the imposed thermal gradient which seems to stabilize the front against the perturbation induced by the particle. On the other hand, concentration gradients did not show a major effect: the addition of solute (0.56 mol% NaMnO4) did not result in a significant change of the transition points as compared to the results obtained in pure water.

Solute polarization during planar freezing of aqueous salt solutions

Abstract An analysis of solute redistribution in binary aqueous salt solutions during unidirectional freezing with a planar solid-liquid interface is presented. A cryomicroscope was equipped with a specially designed freezing stage yielding a well-defined temperature distribution and interface propagation kinetics similar to large systems of plate-like geometry. A microscope spectrophotometer was attached for concentration measurements by means of transmitted light. Aqueous solutions of NaMnO4, exhibiting a maximum absorption at 525 nm and having a phase diagram, as well as mass diffusion properties, very similar to NaCl in water, were used as sample solutions. A major advantage of the method is the combination of quantitative measurements and observation allowing a visual control of the shape of the ice front, and hence of the transition from planar freezing to higher structures. Photometricscans of concentration profiles were recorded duringone-dimensional freezing with interface propagation rates between 1 and 10 μm s−1. It was shown that the ‘steepness’ of the concentration distributions increased with both time and growth velocity. During the initial transient, the results agree well with amathematical model describing pure mass diffusion at constant ice front velocity. Instabilities of the planar interface resulting in dendritic breakdown always occurred before eutectic or steady-state conditions were reached.

Extended phase diagrams for the ternary solutions H2O-NaCl-glycerol and H2O-NaCl-hydroxyethylstarch (HES) determined by DSC

Abstract Phase transformations occurring in the solutions H 2 O-NaCl-glycerol and H 2 O-NaCl-HES under nonequilibrium cooling and heating conditions were examined using the DSC method. Mainly, glass transition, devitrification, eutectic melting, and primary ice melting were evaluated to yield data for the stability of the amorphous phases as well as for the composition inside the residual solution which remains after crystallization. It was found that for a lot of initial compositions and cooling/heating conditions used in cryobiology only primary ice crystallization occurs. Therefore the description of the ternary phase-change behavior may become even more simple for nonequilibrium conditions than in the case of assumed equilibrium. The results were used to modify and extend the equilibrium phase diagrams to make them applicable to problems with boundary conditions which exclude thermodynamic equilibrium and cause formation of metastable phases. The application of the phase diagrams to obtain information about ice fraction and concentration during the cooling/heating process is demonstrated.

The influence of the freezing process on vapour transport during sublimation in vacuum-freeze-drying

Abstract The influence of the freezing process on mass transfer during subsequent sublimation in an aqueous polymer solution is investigated. Different textures of the frozen sample depending on the solidification parameters and the texture dependent diffusion coefficient for vapour transport out of the frozen, drying sample are studied. Experiments are performed, using a controlled directional solidification method (Bridgman technique) and isothermal sublimation, analysed through a light microscope. The dependence of the ‘ice finger’ primary spacing λ1 with the solidification parameters v il −1 4 · G −1 2 (solidification interface velocity and temperature gradient, cf. Hunt, Solidification and Casting of Metals, Proc. Int. Conf. on Solidification, Sheffield, U.K., pp. 3–9. The Metals Society (1979); Kurz and Fisher, Acta Metall.29, 11–20 (1981)) is shown. A proportionality between this combination of freezing parameters and the diffusion coefficient for vapour transport during sublimation is confirmed by the experiments.

The influence of the freezing process on vapour transport during sublimation in vacuum-freeze-drying of macroscopic samples

Abstract The influence of freezing on mass transfer during subsequent sublimation was examined in macroscopic samples (cm scale). A matrix with regions of pure ice and regions of concentrated solution forms during freezing. In macroscopic samples, resulting from local differences in solidification conditions, this matrix is irregular, leading to local differences in mass transfer properties in the drying sample. A characteristic local distribution of diffusion coefficients in frozen, drying macroscopic samples could be analysed with differences amounting up to 425%. Sample segments at positions where solidification started yield small diffusion coefficients, the value increasing with the distance from this position, reaching a maximum in a layer right below the sample surface. A covering layer forms a limiting barrier against vapour transport. In macroscopic samples, a decrease of the applied cooling rate leads to a significant shift of the profile of space dependent diffusion coefficients to higher values and therefore to reduced drying times.

Survival of directionally solidified B-lymphoblasts under various crystal growth conditions

Reduction of temperature during freezing brings about two complex and interrelated phenomena: (1) crystal nucleation and subsequent growth processes and (2) change in biophysical properties of a biological system. The purpose of this investigation is to relate the morphology of the solid phase with the survival of a cell. To this end, B-lymphoblasts were exposed to directional solidification in phosphate-buffered saline + 0.05 M dimethyl sulfoxide. Directional solidification is a freezing technique which allows the morphology of the interface to be varied without varying the chemical history that a cell would experience during a constant cooling rate protocol. Results indicated that, for the range of experimental conditions tested, a maximum survival of approximately 78% could be achieved using a temperature gradient of 25(10)3 K/m and an interface velocity of 23(10)-6 m/s (cooling rate: 35 K/min). Survival dropped off sharply for freezing at faster cooling rates with little or no variation in survival for different crystal growth conditions. Survival at slower cooling rates decreased with decreasing cooling rate. It was observed, however, that the presence of secondary branches in the ice phase correlated with lower survival for a given cooling rate. These results indicated that not only is the redistribution of solute during freezing a potential source of damage during freezing but ice/cell interactions are also. Thus, the cooling rate alone may not be adequate to describe the freezing process.

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ϑ = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ϑ determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures Td which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.

Redefining cooling rate in terms of ice front velocity and thermal gradient: First evidence of relevance to freezing injury of lymphocytes

A freezing process and the resulting injury or survival of biological cells is commonly characterized in terms of the cooling rate, B. Under certain circumstances, the cooling rate can be expressed as B = G.v, where G denotes the thermal gradient at the ice-liquid interface and v its velocity, respectively. To determine the influence of G and v on the morphology of the ice-liquid interface and on cell survival, a gradient freezing stage was designed. Flat capillaries could be pushed with constant velocity from a warm to a cold heat reservoir. With this setup both parameters, G and v, are independently adjustable and the resulting process of directional solidification can be observed dynamically in a light microscope. Human lymphocytes in phosphate-buffered saline with 10 vol% of dimethyl sulfoxide were used as biological test material. Viability was assessed by a membrane integrity test with fluorescein diacetate and ethidium bromide. All cells were cooled down to a final temperature of -196 degrees C and then rapidly thawed. The results obtained with this technique show that the viability determined after freezing and thawing with a certain cooling rate, B = G.v, may vary considerably depending on the imposed values of the thermal gradient, G, and the ice front velocity, v. In addition, the data seem to suggest that, first, the maximum viability which can be reached is governed by the cooling rate, and, second, this maximum for a given cooling rate could be achieved by establishing small temperature gradients and high interface velocities (about 30 degrees K/cm and 500 microns/sec, respectively, for the range of values of G and v tested).

Observations of the non-planar freezing of aqueous salt solutions

Abstract A light microscope equipped with a special freezing stage and a spectrophotometer was employed to study the non-planar solidification of the binary model system H2O-NaMnO4. Instabilities of the initially planar solid-liquid interface were detected visually and could be related to the constitutional supercooling criterion. The redistribution of solute in the interstices between the dendrite arms was measured densitometrically. The concentration profiles scanned along the interdendritic midlines were generally about linear. This result is in agreement with Flemings dendritic solidification model which assumes local equilibrium according to phase diagram relations in small volume elements.

Basic investigations on the freezing of human lymphocytes

Human lymphocytes were frozen at constant cooling rates in the range 2.4 to 1000 degrees K/min without cryoadditive on the cold stage of a thermally defined cryomicroscope. The volume loss due to water efflux was quantified optically for the cooling rates 2.4, 12, 48, and 120 degrees K/min. The likelihood of the formation of intracellular ice was determined as function of the cooling rate. Intracellular crystallization temperatures were obtained for ice formation during both cooling and rewarming. A theoretical analysis of the cell volume loss during freezing was compared to the experimental data and used for an indirect determination of the water permeability of the cells. A relative optimum of the cooling rate is predicted theoretically under the assumption of a critical level of intracellular salt concentration near the eutectic temperature. The dependence of survival and cooling rate was determined cryomicroscopically by simultaneously applying the FDA/EB fluorescence viability test. The optimal cooling rate of about 35 degrees K/min was also found for 2-ml samples frozen within the range of cooling rates of interest. The results show that for freezing in physiological saline solution (1) the optimum of the cooling rate is theoretically predictable, (2) cryomicroscopical data are significant for freezing of samples of larger volume, and (3) the lethal type of intracellular crystallization is cooling rate dependent and distinguishable from innocuous types.