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NASA NTRS1996

Binary Oscillatory Crossflow Electrophoresis

We present preliminary results of our implementation of a novel electrophoresis separation technique: Binary Oscillatory Cross flow Electrophoresis (BOCE). The technique utilizes the interaction of two driving forces, an oscillatory electric field and an oscillatory shear flow, to create an active binary filter for the separation of charged species. Analytical and numerical studies have indicated that this technique is capable of separating proteins with electrophoretic mobilities differing by less than 10%. With an experimental device containing a separation chamber 20 cm long, 5 cm wide, and 1 mm thick, an order of magnitude increase in throughput over commercially available electrophoresis devices is theoretically possible.

Molloy, Richard F.↗

NASA NTRS1997

Binary Oscillatory Crossflow Electrophoresis

Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that solute is drawn into the cell from reservoirs at both ends of the cell leading to a large mass build up. As a consequence, any initially induced mass flux will vanish after short times. This effect was not captured by the infinite channel model and hence numerical and experimental results deviated significantly. The revised model including finite cell lengths and reservoir volumes allowed quantitative predictions of the time history of the concentration profile throughout the system. This latter model accurately describes the fluxes observed for both oscillatory flow modes in experiments using single protein species. Based on the results obtained from research funded under NASA grant NAG-8-1080.S, we conclude that binary separations are not possible using purely oscillatory flow modes because of end effects associated with the cos((omega)t) mode. Our research shows, however, that a combination of cos(2(omega)t) and steady flow should lead to efficient separation free of end effects. This possibility is currently under investigation.

Molloy, Richard F.↗

NASA NTRS2012

Automated Lab-on-a-Chip Electrophoresis System

Capillary electrophoresis is an analytical technique that can be used to detect and quantify extremely small amounts of various biological molecules. In the search for biochemical traces of life on other planets, part of this search involves an examination of amino acids, which are the building blocks of life on Earth. The most sensitive method for detecting amino acids is the use of laser induced fluorescence. However, since amino acids do not, in general, fluoresce, they first must be reacted with a fluorescent dye label prior to analysis. After this process is completed, the liquid sample then must be transported into the electrophoresis system. If the system is to be reused multiple times, samples must be added and removed each time. In typical laboratories, this process is performed manually by skilled human operators using standard laboratory equipment. This level of human intervention is not possible if this technology is to be implemented on extraterrestrial targets. Microchip capillary electrophoresis (CE) combined with laser induced fluorescence detection (LIF) was selected as an extremely sensitive method to detect amino acids and other compounds that can be tagged with a fluorescent dye. It is highly desirable to package this technology into an integrated, autonomous, in situ instrument capable of performing CE-LIF on the surface of an extraterrestrial body. However, to be fully autonomous, the CE device must be able to perform a large number of sample preparation and analysis operations without the direct intervention of a human.

Willis, Peter A.↗

NASA NTRS2019

The Non-Destructive Separation of Diverse Astrobiologically Relevant Organic Molecules By Customizable Capillary Zone Electrophoresis and Monolithic Capillary Electrochromatography

The in situ detection of organic molecules in space is key to understanding the variety and the distribution of the building blocks of life, and possibly the detection of extraterrestrial life itself. Gas chromatography mass spectrometry (GC-MS) has been the most sensitive analytical instrument for organic analyses in flight, and was used on missions from NASA’s Viking, Phoenix, Curiosity missions to ESA’s Rosetta space probe. While pyrolysis GC-MS revealed the first organics on Mars, this step alters or degrades certain fragile molecules that are excellent biosignatures including polypeptides, oligonucleotides and polysaccharides, rendering the intact precursors undetectable. We have identified a solution tailored to the detection of biopolymers and other biomarkers by the use of liquid-based capillary electrophoresis and electrochromatography. In this study, we show that a capillary electrochromatography (CEC) approach using monolithic stationary phases with tailor-made surface chemistry can separate and identify various polycyclic aromatic hydrocarbons (PAHs), nucleobases and aromatic acids that could be formed under astrophysically relevant conditions. In order to simulate flyby organic sample capture, we conducted hypervelocity impact experiments which consisted of accelerating peptide-soaked montmorillonite particles to a speed of 5.6 km/sec, and capturing them in an amorphous silica aerogel of 10 and 30 mg/cc bulk density. Bulk peptide extraction from aerogel followed by capillary zone electrophoresis led to the detection of only two stereoisomeric peptide peaks. The recovery rates of each step of the extraction procedure after the hypervelocity impact suggest that major peptide loss occurred during the impact. Our study provides initial exploration of feasibility of this approach for capturing intact peptides, and subsequently detecting candidate biomolecules during flight missions that would be missed by GC-MS alone. As the monolith-based electrochromatography technology could be customized to detect specific classes of compounds as well as miniaturized, these results demonstrate the potential of the instrumentation for future astrobiology-related spaceflight missions.

Flyby sample return↗

NASA NTRS1973

Electrophoresis separator combining centrifugal separation

Centrifugal force causes buffer, chosen to be denser than particles, to move outward and particles to move inward. Electrophoresis force can be made to equal centrifugal force. System tends not to be affected by convection and other disturbances that are so troublesome in conventional electrophoresis systems.

Semon, H. W.↗

NASA NTRS1974

Development of coatings to control electroosmosis in zero gravity electrophoresis

A major problem confronting the operation of free fluid electrophoresis in zero gravity is the control of electrokinetic phenomena and, in particular, electroosmosis. Due to the severity of counter flow, as a result of electroosmosis, the electrical potential developed at the surface of shear must be maintained at near, or as close to, zero millivolts as possible. Based upon this investigation, it has been found that the amount of bound water or the degree of hydroxylation plays a major role in the control of this phenomena. Of necessity, factors, such as adhesion, biocompatibility, protein adsorption, and insolubility were considered in this investigation because of the long buffer-coating exposure times required by present space operations. Based upon tests employing microcapillary electrophoresis, it has been found that gamma amino propyl trihydroxysilane produced a coating which provides the lowest potential (minus 3.86 mv) at the surface of shear between the stationary and mobile layers. This coating has been soaked in both borate and saline buffers, up to three months, in a pH range of 6.5 to 10 without deleterious effects or a change in its ability to control electrokinetic effects.

Krupnick, A. C.↗

NASA NTRS1974

Role of gravity in preparative electrophoresis

Electrophoresis has contributed significantly to the methodology of biological sciences, and shows the potential for large scale fractionation of a wide range of medically important substances, including living cells. Gravity plays an important role in the electrophoretic process, and hence the importance of the NASA effort to develop a zero-gravity separation facility as part of its shuttle program. The current state of art in electrophoresis is reviewed with particular emphasis on the role of gravity and the possible use of istachophoresis. This technique utilizes a discontinuous buffer system, and appears to be the only high resolution electrophoretic technique currently available for separation of living cells.

Bier, M.↗

NASA NTRS1974

Development of coatings to control electroosmosis in zero gravity electrophoresis

Krupnick, A. C.↗

NASA NTRS1975

Electrophoresis of biological materials

The selection of biological products was studied for electrophoresis in space. Free flow electrophoresis, isoelectric focusing, and isotachophoresis are described. The candidates discussed include: immunoglobulins and gamma globulins; isolated islet of langerhans from pancreas; bone marrow; tumor cells; kidney cells, cryoprecipitate; and column separated cultures.

Source record↗

NASA NTRS1977

Automatic multiple applicator electrophoresis

Easy-to-use, economical device permits electrophoresis on all known supporting media. System includes automatic multiple-sample applicator, sample holder, and electrophoresis apparatus. System has potential applicability to fields of taxonomy, immunology, and genetics. Apparatus is also used for electrofocusing.

Grunbaum, B. W.↗

NASA NTRS1975

Fluid flow electrophoresis in space

Four areas relating to free-flow electrophoresis in space were investigated. The first was the degree of improvement over earthbound operations that might be expected. The second area of investigation covered the problems in developing a flowing buffer electrophoresis apparatus. The third area of investigation was the problem of testing on the ground equipment designed for use in space. The fourth area of investigation was the improvement to be expected in space for purification of biologicals. The results of some ground-based experiments are described. Other studies included cooling requirements in space, fluid sealing techniques, and measurement of voltage drop across membranes.

Griffin, R. N.↗

NASA NTRS1976

Electrophoresis technology experiment MA-011

Experiment MA-011, electrophoresis technology, was designed to test electrophoresis hardware that would continue the development of technology for electrophoretic separation of materials in the near zero g environment of space. The experimental hardware generally functioned as planned. Frozen live cells were successfully transported into space, electrophoretic processing was performed, and viable cells were returned to earth. A separation of the three types of fixed red blood cells (rabbit, human, and horse) was demonstrated. The human lymphocytes, however, showed no apparent migration. The separation of human kidney cells produced the most exciting data. Analysis shows electrophoretic separation throughout the entire column with at least four bands of viable cells. The isotachophoresis experiment definitely demonstrated the isotachophoretic separation of biological cells in a near zero g environment.

Allen, R. E.↗

NASA NTRS1976

Development of polymeric coatings for control of electro-osmotic flow in ASTP MA-011 electrophoresis technology experiment

The development of a methyl cellulose based coating system for control of electro-osmotic flow at the walls of electrophoresis cells is described. Flight electrophoresis columns were coated with this system, resulting in a flight set of six columns. In flight photography of MA-011 electrophoretic separations verified control of electro-osmotic flow.

Patterson, W. J.↗

NASA NTRS1976

Definition of performance specifications for automated Analytical Electrophoresis Facility (AAEF)

In order to provide specifications for the automated Analytical Electrophoresis Facility (AAEF) that would satisfy the broadest variety of demands of a future user community, a survey was carried out of all those people who were identified as having published papers on cell electrophoresis in the past four years. A computer search was conducted of the relevant literature from which a list of 87 investigators was derived and defined as the user community for purposes of the mailing. A questionnaire was developed covering the areas of performance which required definition which was subsequently circulated to the user community. Based on the response to this survey performance specifications were assembled.

Brooks, D. E.↗

NASA NTRS1977

Electrophoresis for biological production

Preparative electrophoresis may provide a unique method for meeting ever more stringent purity requirements. Prolonged near zero gravity in space may permit the operation of preparative electrophoresis equipment with 100 times greater throughput than is currently available. Some experiments with influenza Virus Antigen, Erythropoietin and Antihemophaliac Factor, along with process and economic projections, are briefly reviewed.

Mccreight, L. R.↗

NASA NTRS1977

Static free-fluid electrophoresis in space

The weightless environment onboard spacecraft in drifting flight has provided a unique opportunity to do experiments that cannot be done on the ground. High resolution free-fluid electrophoresis of particles proposed in the late 1960s to take advantage of reduced gravity began with brief experiments done during two Apollo flights. The recent Apollo Soyuz Test Project mission had two major experiments that accomplished the separation of viable biological cells. Experiments now are being planned for the Space Shuttle which will attempt to achieve high resolution of the separated species by using zone electrophoresis. These experiments will return a quantity sufficient for laboratory testing and establish the potential of fractionation and purification of biological materials in space.

Snyder, R. S.↗

NASA NTRS1977

Design, development, test, and evaluation of an automated analytical electrophoresis apparatus

An Automated Analytical Electrophoresis Apparatus (AAEA) was designed, developed, assembled, and preliminarily tested. The AAEA was demonstrated to be a feasible apparatus for automatically acquiring, displaying, and storing (and eventually analyzing) electrophoresis mobility data from living blood cells. The apparatus and the operation of its major assemblies are described in detail.

Bartels, P. A.↗

NASA NTRS1977

Automatic multiple-sample applicator and electrophoresis apparatus

An apparatus for performing electrophoresis and a multiple-sample applicator is described. Electrophoresis is a physical process in which electrically charged molecules and colloidal particles, upon the application of a dc current, migrate along a gel or a membrane that is wetted with an electrolyte. A multiple-sample applicator is provided which coacts with a novel tank cover to permit an operator either to depress a single button, thus causing multiple samples to be deposited on the gel or on the membrane simultaneously, or to depress one or more sample applicators separately by means of a separate button for each applicator.

Grunbaum, B. W.↗