Cell separation with countercurrent chromatography and thin-layer countercurrent distribution in aqueous two-phase system
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Van Alstine, J. M.↗
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Cell separation with counter-current chromatography and thin-layer countercurrent distribution in aqueous two-phase system
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Van Alstine, J. M.↗
Hardware for studying the demixing of aqueous polymer two-phase systems in low gravity
The gravity-free demixing behavior of aqueous polymer two-phase systems (APTPS) has been studied on Earth, and onboard KC-135 aircraft and the Space Shuttle. Results suggest a good correlation between the demixing behavior of isopycnic systems on Earth and the low g behavior of systems exhibiting phase density differences. The short term (20 s) behavior of APTPS onboard KC-135 aircraft has been shown to mimic their initial demixing behavior in space. It appears that the few minutes of low g provided by parabolic rocket flight will be sufficient to study all but the later stages of demixing of APTPS. We are therefore turning our attention toward the design and use of hardware for studying the demixing of APTPS onboard sounding rockets. This seminar will discuss the evolution of hardware and research from hand-held KC-135 and Shuttle hardware through to automated hardware designed for utilization of KC-135, sounding rocket, and Space Shuttle Mid-deck Locker flight opportunities.
Vanalstine, J. M.↗
Demixing of aqueous polymer two-phase systems in low gravity
When polymers such as dextran and poly(ethylene glycol) are mixed in aqueous solution biphasic systems often form. On Earth the emulsion formed by mixing the phases rapidly demixes because of phase density differences. Biological materials can be purified by selective partitioning between the phases. In the case of cells and other particulates the efficiency of these separations appears to be somewhat compromised by the demixing process. To modify this process and to evaluate the potential of two-phase partitioning in space, experiments on the effects of gravity on phase emulsion demixing were undertaken. The behavior of phase systems with essentially identical phase densities was studied at one-g and during low-g parabolic aircraft maneuvers. The results indicate the demixing can occur rather rapidly in space, although more slowly than on Earth. The demixing process was examined from a theoretical standpoint by applying the theory of Ostwald ripening. This theory predicts demizing rates many orders of magnitude lower than observed. Other possible demixing mechanisms are considered.
Bamberger, S.↗
A theory of electrophoresis of emulsion drops in aqueous two-phase polymer systems
An electrophoresis study has been carried out in an emulsion formed from an electrically neutral aqueous mixture of dextran and polyethylene glycol equilibrated at sufficient concentrations in the presence of electrolytes. Electrophoresis of a drop of one phase suspended in the other is observed, and the direction of the drop's motion is reversed when the disperse phase and the continuous phase are interchanged. In the presence of sulfate, phosphate, or citrate ions, an electrostatic potential difference of the order of a few mV exists between the two phases. The potential implied by the direction of the electrophoretic motion is opposite to the Donnan potential observed between the two phases. The mobility of an emulsion drop increases with the drop radius and depends on ion concentration. These results are explained in terms of a model postulating an electric dipole layer associated with a mixture of oriented polymer molecules at the surface of a drop, with a potential difference between the interiors of the two phases resulting from the unequal ion distribution.
Levine, S.↗
Separation of aqueous two-phase polymer systems in microgravity
Phase separation of polymer systems in microgravity is studied in aircraft flights to prepare shuttle experiments. Short duration (20 sec) experiments demonstrate that phase separation proceeds rapidly in low gravity despite appreciable phase viscosities and low liquid interfacial tensions (i.e., 50 cP, 10 micro N/m). Ostwald ripening does not appear to be a satisfactory model for the phase separation mechanism. Polymer coated surfaces are evaluated as a means to localize phases separated in low gravity. Contact angle measurements demonstrate that covalently coupling dextran or PEG to glass drastically alters the 1-g wall wetting behavior of the phases in dextran-PEG two phase systems.
Vanalstine, J. M.↗
Cell separation by immunoaffinity partitioning with polyethylene glycol-modified Protein A in aqueous polymer two-phase systems
Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work it is shown that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, the paper provides a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.
Karr, Laurel J.↗
Demixing of aqueous polymer two-phase systems in low gravity
Experiments on the effect of gravity on phase emulsion demixing were performed in order to study the potential of two-phase partitioning in space. The behavior of phase systems possessing phase density differences and systems with essentially identical phase densities was investigated during one-g and low-g parabolic aircraft maneuvers. It is found that demixing can occur rather rapidly in space, although more slowly than on earth. The Ostwald ripening theory predicts demixing rates many orders of magnitude lower than the observed values.
Bamberger, S.↗
Cell separation in immunoaffinity partition in aqueous polymer two-phase systems
Two methods for immunoaffinity partitioning are described. One technique involves the covalent coupling of poly (ethylene glycol) (PEG) to immunoglobulin G antibody preparations. In the second method PEG-modified Protein A is used to complex with cells and unmodified antibody. The effects of PEG molecular weight, the degree of modification, and varying phase system composition on antibody activity and its affinity for the upper phase are studied. It is observed that both methods resulted in effective cell separation.
Karr, Laurel J.↗
Phase partitioning in space and on earth
The influence of gravity on the efficiency and quality of the impressive separations achievable by bioparticle partitioning is investigated by demixing polymer phase systems in microgravity. The study involves the neutral polymers dextran and polyethylene glycol, which form a two-phase system in aqueous solution at low concentrations. It is found that demixing in low-gravity occurs primarily by coalescence, whereas on earth the demixing occurs because of density differences between the phases.
Van Alstine, James M.↗
Consort 1 sounding rocket flight
This paper describes a payload of six experiments developed for a 7-min microgravity flight aboard a sounding rocket Consort 1, in order to investigate the effects of low gravity on certain material processes. The experiments in question were designed to test the effect of microgravity on the demixing of aqueous polymer two-phase systems, the electrodeposition process, the production of elastomer-modified epoxy resins, the foam formation process and the characteristics of foam, the material dispersion, and metal sintering. The apparatuses designed for these experiments are examined, and the rocket-payload integration and operations are discussed.
Wessling, Francis C.↗
Application of partition technology to particle electrophoresis
The effects of polymer-ligand concentration on particle electrophoretic mobility and partition in aqueous polymer two-phase systems are investigated. Polymer coating chemistry and affinity ligand synthesis, purification, and analysis are conducted. It is observed that poly (ethylene glycol)-ligands are effective for controlling particle electrophoretic mobility.
Van Alstine, James M.↗
Rationale for two phase polymer system microgravity separation experiments
The two-phase systems that result when aqueous solutions of dextran and poly(ethylene glycol) are mixed at concentrations above a few percent are discussed. They provide useful media for the partition and isolation of macromolecules and cell subpopulations. By manipulating their composition, separations based on a variety of molecular and surface properties are achieved, including membrane hydrophobic properties, cell surface charge, and membrane antigenicity. Work on the mechanism of cell partition shows there is a randomizing, nonthermal energy present which reduces separation resolution. This stochastic energy is probably associated with hydrodynamic interactions present during separation. Because such factors should be markedly reduced in microgravity, a series of shuttle experiments to indicate approaches to increasing the resolution of the procedure are planned.
Brooks, D. E.↗
Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes
The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.
Non-NASA Center↗
Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana
Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.
Non-NASA Center↗
Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings
Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.
NASA Discipline Number 61-10↗
Application of capillary fluid management techniques to the design of a phase separating microgravity bioreactor
Manned space missions require the development of compact, efficient, and reliable life support systems. A number of aqueous biological conversion processes are associated with bioregenerative life support systems. Vessels, or bioreactors, capable of supporting these processes in microgravity must be developed. An annular flow bioreactor has been conceived. It has the potential to incorporate containment, phase separation, gas exchange, and illumination into a single vessel. The bioreactor utilizes capillary fluid management techniques and is configured as a cylindrical tube in which a two-phase liquid-gas flow is maintained. Vanes placed around the inner perimeter enhance capillary forces and cause the liquid phase to attach and flow along the interior surface of the tube. No physical barrier is required to complete phase separation. It is shown analytically that liquid film thickness is limited only by vane geometry and that an annular flow bioreactor capable of managing 284 liters would occupy 0.7 cubic m, less than half the volume of a Spacelab experiment rack.
Finger, Barry W.↗
Development of the Monolith Froth Reactor for Catalytic Wet Oxidation of CELSS Model Wastes
The aqueous phase oxidation of acetic acid, used as a model compound for the treatment of CELSS (Controlled Ecological Life Support System) waste, was carried out in the monolith froth reactor which utilizes two-phase flow in the monolith channels. The catalytic oxidation of acetic acid was carried out over a Pt/Al2O3 catalyst at temperatures and pressures below the critical point of water. The effect of externally controllable parameters (temperature, liquid flow rate, distributor plate orifice size, pitch, and catalyst distance from the distributor plate) on the rate of acetic acid oxidation was investigated. Results indicate reaction rate increased with increasing temperature and exhibited a maximum with respect to liquid flow rate. The apparent activation energy calculated from reaction rate data was 99.7 kJ/mol. This value is similar to values reported for the oxidation of acetic acid in other systems and is comparable to intrinsic values calculated for oxidation reactions. The kinetic data were modeled using simple power law kinetics. The effect of "froth" feed system characteristics was also investigated. Results indicate that the reaction rate exhibits a maximum with respect to distributor plate orifice size, pitch, and catalyst distance from the distributor plate. Fundamental results obtained were used to extrapolate where the complete removal of acetic acid would be obtained and for the design and operation of a full scale CELSS treatment system.
Fisher, John W.↗