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Bioseparations Publications

Two-Dimensional Particle Focusing: Sheath Flow on Two Sides

2010

Authors: Shin, J. and M. Ladisch
Journal:
Book Chapter: From "The Microflow Cytometer", Frances S. Ligler and Jason S. Kim, Pan Stanford Publishing Pte. Ltd., Singapore (2010)

Abstract:

Research Area: Bioseparations    

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Targeted Capture of Pathogenic Bacteria Using a Mammalian Cell Receptor Coupled with Dielectrophoresis on a Biochip

2009

Authors: O. K. Koo, Y.S. Liu, S. Shuaib, S. Bhattacharyall, M. R. Ladisch, R. Bashir and A. K. Bhunia
Journal: Journal of Analytical Chemistry, 81(8), 3094-3101 (2009)
Book Chapter:

Abstract: Efficient capture of target analyte on biosensor platforms is a prerequisite for reliable and specific detection of pathogenic microorganisms in a microfluidic chip. Antibodies have been widely used as ligands, however, because of their occasional unsatisfactory performance, a search for alternative receptors is underway. Heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein is a receptor for Listeria adhesion protein (LAP) during Listeria monocytogenes infection. This paper reports application of biotinylated Hsp60 as a capture molecule for living (viable) L. monocytogenes in a microfluidic environment. Hsp60, immobilized on the surface of streptavidin-coated silicon dioxide exhibited specific capture of pathogenic Listeria against a background of other Listeria species, Salmonella, Eschericia, Bacillus, Psuedomonas, Serratia, Hafnia, Enterobacter, Citrobacter, and Lactobacillus. The capture efficiency of L. monocytogenes was 83 times greater than another Listeria receptor, the monoclonal antibody, mAb-C11E9. Additionally, the capture rate was further increased on a Hsp60-coated biochip by 60% when a dielectrophoresis force was applied for 5 min at the beginning of the final 1 h incubation step. Our data show that Hsp60 could be used for specific detection of L. monocytogenes on a biochip sensor platform.

Research Area: Bioseparations    

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A Multifunctional Micro-Fluidic System for Dielectrophoretic Concentration Coupled with Immuno-Capture of Low Numbers of Listeria monocytogenes

2006

Authors: Yang, L., P. P. Banada, M. R. Chatni, K. S. Lim, A. K. Bhunia, M. Ladisch, and R. Bashir
Journal: Lab on a Chip , 6, 896-905 (2006)
Book Chapter:

Abstract: In this study, we demonstrated a micro-fluidic system with multiple functions, including concentration of bacteria using dielectrophoresis (DEP) and selective capture using antibody recognition, resulting in a high capture efficiency of bacterial cells. The device consisted of an array of oxide covered interdigitated electrodes on a flat silicon substrate and a ~ 16 µm high and ~ 260 µm wide micro-channel within a PDMS cover. For selective capture of Listeria monocytogenes from the samples, the channel surface was functionalized with a biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Positive DEP (at 20 Vpp and 1 MHz) was used to concentrate bacterial cells from the fluid flow. DEP could collect ~90% of the cells in a continuous flow at a flow rate of 0.2µl min-1 into the micro-channel with concentration factors between 102 - 103 , in sample volumes of 5-20µl. A high flow rate of 0.6 µl min-1 reduced the DEP capture efficiency to ~65%. Positive DEP attracts cells to the edges of the electrodes where the field gradient is the highest. Cells concentrated by DEP were captured by the antibodies immobilized on the channel surface with efficiencies of 18 to 27% with bacterial cell numbers ranging from 101 to 103 cells. It was found that DEP operation in our experiments did not cause any irreversible damage to bacterial cells in terms of cell viability. In addition, increased antigen expression (antigens to C11E9 monoclonal antibody) on cell membranes was observed following the exposure to DEP.

Research Area: Bioseparations    

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Anomalous Resonance in a Nanomechanical Biosensor

2006

Authors: Gupta, A. K., P. R. Nair, D. Akin, M. R. Ladisch, S. Broyles, M. A. Alam, and R. Bashir
Journal: Proceedings of the National Academy of Sciences, 103, 13362-13367 (2006)
Book Chapter:

Abstract: The decrease in resonant frequency of a classical cantilever provides a sensitive measure of the mass of entities attached on its surface. This elementary phenomenon has been the basis of a new class of bio-nanomechanical devices as sensing components of integrated microsystems that can perform rapid, sensitive, and selective detection of biological and biochemical entities. Based on classical analysis, there is a widespread perception that smaller sensors are more sensitive, and this notion has motivated scaling of biosensors to nanoscale dimensions. In this work, we show that the response of a nanomechanical biosensor is far more complex than previously anticipated. Indeed, in contrast to classical microscale sensors, the resonant frequencies of the nanosensor may actually decrease or increase after attachment of protein molecules. We demonstrate theoretically and experimentally that the direction of the frequency change arises from a size-specific modification of diffusion and attachment kinetics of biomolecules on the cantilevers. This work may have broad impact on microscale and nanoscale biosensor design, especially when predicting the characteristics of bio-nanoelectromechanical sensors functionalized with biological capture molecules.

Research Area: Bioseparations    

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Surface Engineering of Microchannel Walls for Protein Separation and Directed Microfluidic Flow

2006

Authors: T. T. Huang, N. S. Mosier, and M. R. Ladisch
Journal: Journal of Separation Science, 29(12), 1733-1742 (2006)
Book Chapter:

Abstract: The preparation of surfaces in microfluidic devices that selectively retain proteins may be difficult to implement due to the incompatibility of derivatization methods with microdevice fabrication techniques. This review describes recently reported developments in simple and rapid methods for engineering the surface chemistries of microchannels bassed on construction of press-fit microdevices. These devices are fabricated by placing a glass fiber on a PDMS film and pressing the film on a silicon wafer or a microscope slide that has been derivatized with octadecyltrichlorosilane (ODS). The film adheres to the slide and forms an elliptically shaped channel around the fiber. The combination of surface wettability of a hydrophilic glass microfiber and the surrounding hydrophobic microchannel surfaces directs a narrow boundary layer of liquid next to the fiber in order to bring the sample in contact with the separation media and results in selective retention of proteins. This phenomenon may be exploited to enable microscale separation applications since there are a wide variety of fibers available with different chemistries. These may be used to rapidly fabricate microchannels that serve as stationary phases for separation at a microscale. The fundamental properties of such devices are discussed.

Research Area: Bioseparations    

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Surface-Directed Boundary Flow in Microfluidic Channels

2006

Authors: Huang, T. T., D. G. Taylor, K. S. Lim, M. Sedlak, R. Bashir, N. S. Mosier, and M. R. Ladisch
Journal: Langmuir, 22, 6429-6437 (2006)
Book Chapter:

Abstract: Channel geometry combined with surface chemistry enables a stable liquid boundary flow to be attained along the surfaces of a 12 µm diameter hydrophilic glass fiber in a closed semi-elliptical channel. Surface free energies and triangular corners formed by PDMS/glass fiber or OTS/glass fiber surfaces are shown to be responsible for the experimentally observed wetting phenomena and formation of liquid boundary layers that are 20-50 µm wide and 12 µm high. Viewing this stream through a 20 µm slit results in a virtual optical window with a 5 pL liquid volume suitable for cell counting and pathogen detection. The geometry that leads to the boundary layer is a closed channel that forms triangular corners where glass fiber and the OTS coated glass slide or PDMS touch. The contact angles and surfaces direct positioning of the fluid next to the fiber. Preferential wetting of corner regions initiates the boundary flow, while the elliptical cross-section of the channel stabilizes the microfluidic flow. The Young-Laplace equation, solved using fluid dynamic simulation software, shows contact angles that exceed 105° will direct the aqueous fluid to a boundary layer next to a hydrophilic fiber with a contact angle of 5°. We believe this is the first time that an explanation has been offered for the case of a boundary layer formation in a closed channel directed by a triangular geometry with two hydrophobic wetting edges adjacent to a hydrophilic surface.

Research Area: Bioseparations    

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Dielectrophoresis and Antibody Mediated Selective Capture of Microorganisms in Micro-Fluidic Biochips

2005

Authors: H. Li, L. Yang, D. Akin, T. Geng, A. Bhunia, T. T. Huang, M. Ladisch, R. Bashir
Journal: The 13th International Conference on Solid-State Sensors, Actuators and Microsystems, Seoul, Korea, June 5-9, 2005, Vol. 2, Papers 3A1.1-4D3.4, pp. 1103-2162.
Book Chapter:

Abstract: A dielectrophoretic (DEP) filter device was fabricated for antibody mediated specific capture of microorganisms. The device consists of a network of fluidic inlet/outlet ports and chamber etched into silicon substrate with bonded glass on the top and interdigitated electrodes at the bottom of the chamber. The whole electrode array was covered with a thin silicon oxide layer for preventing electroosmotic currents at the electrodes. For selective capture of Listeria monocytogenes from the mixture of L. monocytogenes and Escherichia coli, the channel surface of the DEP filter device was functionalized with biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Postive DEP (at 20Vpp and 1 MHz) was used to attract, capture and concentrate all bacteria in the sample from the fluid flow. About 8% Listeria bacteria were captured while no E. coli was captured with 40 min DEP. We demonstrated that this novel method combining dielectrophoresis, micro-fluidics, and antibody-antigen recognition can be used to selectively capture L. monocytogenes or other target bacteria in the microfluidic device with good efficiency and selectivity.

Research Area: Bioseparations    

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Mechanistic Study of Membrane Concentration and Recovery of Listeria monocytogenes

2005

Authors: Wan-Tzu Chen, Richard L. Hendrickson, Chia-Ping Huang, Deb Sherman, Tao Geng, Arun K. Bhunia, Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 89, 3, 263-273 (2005)
Book Chapter:

Abstract: Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) innoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95 x with up to 95% recovery of living microorganisms by concentrating 50 mL of food samplejinto a volume of 500 uL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 um pores showed the 0.2 um polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 um. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems.

Research Area: Bioseparations    

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Microfiber-Directed Boundary Flow in Press-Fit Microdevices Fabricated from Self-Adhesive Hydrophobic Surfaces

2005

Authors: T. T. Huang, D. G. Taylor, M. Sedlak, N. S. Mosier, and M. R. Ladisch
Journal: Analytical Chemistry, 77, 3671-3675 (2005).
Book Chapter:

Abstract: We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (poly(dimethylsiloxane) (PDMS)), and microfibers to form patterns of hydrophobic channels, wells, elbows, or orifices that direct fluid flow into controlled boundary layers. Tweezers are used to place glass microfibers in a defined pattern onto an elastomeric (PDMS) hydrophobic film. The film is then manually pressed onto a hydrophobic silicon wafer, causing it to adhere to the silicon wafer and form a liquid-tight seal around the fibers. Completed in 15 min, the technique results in an operable microdevice with micrometer-scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by use of the surface wetting properties of the hydrophilic glass fiber and the hydrophobicity of surrounding surfaces. The simplicity of this technique allows quick prototyping of microfluidic components, as well as complete biosensor systems, such as we describe for the detection of pathogenic bacteria.

Research Area: Bioseparations    

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Separation of Denatured Proteins in Free Solution on a Microchip Based on Differential Binding of Alkyl Sulfates with Different Carbon Chain Lengths

2005

Authors: Chang Lu, Aaron E. Smith and Harold G. Craighead
Journal: Chemical Communication, 183-185 (2005).
Book Chapter:

Abstract: Electrophoresis of polyelectrolytes such as DNA and denatured proteins is usually performed in chemical or physical gels instead of in free solution, except in some special cases. It has been generally accepted that proteins with molecular weights more than 10 kDa have a constant free solution mobility that is independent of their molecular weights, after they are fully denatured by sodium dodecyl sulfate (SDS) and a reducing agent. This phenomenon is generally attributed to the constant charge density along the polypeptide chain. The coating of the negatively charged surfactant makes the intrinsic charge of the proteins insignificant. For similar reasons, DNA fragments longer than 10–20 bp have the same free solution mobilities regardless of fragment size or base composition. In this report, we demonstrate that denatured proteins have different electrophoretic mobilities in free solution after the denaturation is carried out using a mixture of alkyl sulfates with different carbon chain lengths. Furthermore, the free solution mobilities are not correlated with the molecular weights of the proteins. In this work, the free solution electrophoresis was carried out on a glass microchip.

Research Area: Bioseparations    

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Bioseparation Techniques in Microfluidic Devices Using Micro-Bead

2004

Authors: W.-J. Chang, K. W. Ro, T. T. Huang, Y.-M. Koo, J. H. Hahn, M. R. Ladisch, D. Akin, R. Bashir
Journal: Theories and Applications of Chemical Engineering, 10, 1, 203-206, (2004)
Book Chapter:

Abstract: There has been a significant increase of interest on microfluidic device as miniaturized analytical system, recently. Micron-size fluidic paths and other components are integrated in microfluidic device, performing essential procedures for the analysis of chemical and/or biological materials (Harrison et al., 1993, Jacobson et al., 1994). The device that has dimensions of a few centimeters is capable of providing rapid identification of molecules and enhanced sensitivity with reduced consumption of reagents and samples (Stone and Kim, 2001). Various separation processes have been applied to microfluidic device such as zone electrophoresis, gel electrophoresis, isoelectric focusing, micellar electrokinetic chromatography (MEKC) and electrochromatography, resulted in the increase of its applications. The applications using polymer-based microfluidic devices are increasing due to their ease of fabrication, inexpensive fabrication costs and increasing versatility (Becker and Locascio, 2002). These devices have been fabricated from various polymers including poly(methyl- methacrylate), polycarbonate, polystyrene, and poly(dimethylsiloxane) (PDMS). The usages of PDMS are increasing among these polymers because of relatively lower expense and simpler procedures for fabrication than others. The control of minute volume of liquid in PDMS microfluidic device using electroosmotic flow (EOF) enhanced the increase of its application. In this work, capillary electrochromatography (CEC) and preconcentration of neutral compounds have been realized on poly(dimethylsiloxane) (PDMS) microfluidic devices. The micro-structures of PDMS were fabricated in micro-channel for the packing of micro-beads. In addition, rapid prototyping technique for the fabrication of micro-channels using micro-fiber and PDMS slab was developed. The flexible characteristic of PDMS makes simple fabrication of micro-channel possible, by directed placement of glass micro-fibers on solid substrate. The applications of developed microfluidic device are tested.

Research Area: Bioseparations    

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Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

2004

Authors: Geng, T., Morgan, M. T., and Bhunia, A. K.
Journal: Applied and Environmental Microbiology, 70, 10, 6138-6146 (2004)
Book Chapter:

Abstract: Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 x 104 or 2.8 x 107 CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.

Research Area: Bioseparations    

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Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells

2004

Authors: J. L. Wampler, K.-P. Kim, Z. Jaradat, and A. K. Bhunia
Journal: Infection and Immunity, 72, 2, 931-936 (2004)
Book Chapter:

Abstract: The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Research Area: Bioseparations    

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Impedance Microbiology-on-a-Chip: Microfluidic Bioprocessor for Rapid Detection of Bacterial Metabolism

2004

Authors: R. Gomez-Sjoberg, D. T. Morisette, and R. Bashir
Journal: Journal of Microelectromechanical Systems, 1-8, (2004)
Book Chapter:

Abstract: Detection of a few live bacterial cells in many industrial or clinical samples is a very important technological problem. We have developed a microscale technique for concentrating bacterial cells from a dilute sample, by factors on the order of 10 to the 4th power to 10 to the 5th power, and detecting their metabolic activity by purely electrical means. The technique was implemented on a silicon-based microfluidic chip where the cells are concentrated and incubated in a chamber with a volume of 400 pl. Concentration and capture are obtained by the use of dielectrophoresis on the bacterial cells, and metabolism detection is achieved by means of impedance measurements of the medium in which the bacteria are incubated. Performing impedance-based detection at the microscale results in drastically reduced detection times for dilute bacterial samples, thanks to the ability to efficiently concentrate and capture the cells in an extremely small volume. Such concentration eliminates the need to amplify the bacterial population by long culture steps. This detection technique can be used for a wide variety of applications.

Research Area: Bioseparations    

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Investigating Pathogen-Specific TLR Signaling of Innate Immune Cells for Biosensor Applications

2004

Authors: A. Lottes, H. Oh, H. HogenEsch, M. Ladisch, J. Hutchcroft, A. Rundell
Journal: 30th NE Bioengineering Conference, April 17-18, 2004, Western New England College
Book Chapter:

Abstract: The goal of this project is to develop a real-time cell-based biosensor using Toll-like receptors (TLRs) for pathogen detection. Existing biosensors rely on technologies that recognize only specific target analytes, requiring prior knowledge of the possible contaminating agents. Innate immune cells express TLRs that recognize conserved pathogen-associated molecular patterns on bacteria, viruses, parasites and fungi. Using TLRs as the receptor element in this biosensor will eliminate the need for a priori knowledge of the threat. At least 10 different members of the TLR family are expressed on cells of the innatejimmune system, each responding to different attributes of pathogenic organisms. Through flow cytometry, TLRs 2, 4 and 9 have been identified on THP-1 cells, and TLRs 2, 3, 4, 5 and 9 have been detected on J774 cells. Western blotting has identified Erk activation upon lipopolysacharide (LPS), E. coli and Poly(I):(C) exposure in J774 cells, and upon LPS and E. coli exposure in THP-1 cells. Cellular model systems are being developed to distinguish between bacteria and virus by selective stimulation of TLR3 and TLR5 (TLR3 specifically recognizes double-stranded viral RNA and TLR5 detects bacterial flagellin). A target application of this technology is point--of-care diagnostics. Real-time detection of viruses in nasal or throat swabs could help decrease the inappropriate use of antibiotics.

Research Area: Bioseparations    

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Optimization of a Rapid Dot-Blot Immunoassay for Detection of Salmonella entrica serovar Enteritidis in Poultry Products and Environmental Samples

2004

Authors: Z. W. Jaradat, J. H. Bzikot, J. Zawistowski, A. K. Bhunia
Journal: Food Microbiology, 21, 761-769, (2004)
Book Chapter:

Abstract: An immunoassay was developed for the detection of Salmonella serovar enteritidis in poultry and environmental samples. This assay consisted of a two-step procedure that involved an enrichment step using whole egg homogenate (EH) as the enrichment medium and detection by a monoclonal antibody (MAb)-based dot-blot assay. Egg homogenate enriched Salmonella enteritidis was heated to 100 C for 10 min in the presence of cholic acid, a detergent, to liberate the lipopolysaccharide (LPS) antigen in gelled egg matrix. This was subsequently transferred onto a nitrocellulose membrane for detection with MAb 2F11. Several commercially available media were compared with egg homogenate for their relative ability to resuscitate and propagate Salmonella enterititis to detectable levels. Incubation in EH, trypticase soy broth (TSB), and lactose broth (LB) resulted in comparable levels of Salmonella Enteritidis as demonstrated by viable plate counts. Salmonella enteritidis grown in TSB exhibited the greatest visual intensity showing a positive test when tested by the dot-blot assay. Incubation time necessary to detect one cfu of Salmonella enteritidis was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of ferrous sulphate or ferrioxamine E or cholic acid in the enrichment broth had negligible negative effects on the growth of Salmonella. Salmonella enteritidis when incubated with a mixture of naturally contaminated or artificially innoculated competitive micro-organisms in environmental samples at a ratio of 1:10 to the 2nd power, was able to reproduce to detectable numbers for the immunoassay. This method was able to detect all phage types (PT 1, 6, 7, 8, 13, 13a, 14b, 21 and 28) with unique ribopatterns. The results demonstrated that Salmonella enteritidis, when pre-enriched in a medium containing ferrous sulphate or cholic acid, could be readily detected in the presence of 100-fold higher competition of other microorganisms.

Research Area: Bioseparations    

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Biotextiles - Monoliths with Rolled Geometrics

2003

Authors: Jeremiah Bwatwa, Yiqi Yang, Chenghong Li, Craig Keim, Christine Ladisch, and Michael Ladisch
Journal: Journal of Chromatography, (2003)
Book Chapter:

Abstract: Stationary phases that are formed from textiles are a continuous, interconnected fibrous matrix in the form of yarns and fabric. The fibers are assembled into yarns and the yarns are woven into fabric. Since individual fibers have exhibited poor flow properties when used as stationary phases, rolled fabric stationary phases have been developed. Rolled stationary phases enable a long bed length to be attained while retaining food flow properties [1,2]. This kind of stationary phase orients the fabric into a three-dimensional structure through contact between adjacent layers of fabric where the fabric [1,3,4] supports the fibers (Fig. 11.1(a)) [5] assembled into the yarns (Fig. 11.1(b)) [6], and the woven fabric (Fig. 11.1(c)) [6]. This is a type of monolithic material since there are no distinct or individual particles packed into the column. Further, since the material is a textile, and it is used to fractionate biomolecules, we have called this material a biotextile.

Research Area: Bioseparations    

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Characterization and Application of a Listeria monocytogenes Reactive Monoclonal Antibody C11E9 in a Resonant Mirror Biosensor

2003

Authors: Amanda A. Lathrop, Ziad W. Jaradat, Tim Haley, Arun K. Bhunia
Journal: Journal of Immunological Methods, 281, 119-128, (2003)
Book Chapter:

Abstract: Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44–3.58 and for L. innocua 0.22–1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6–0.99) and low (0.18–0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 108 cells/ml; however, it showed binding (85–150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 Ag/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly ( p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10–20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite crossreaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Research Area: Bioseparations    

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Composite Surface for Blocking Bacterial Adsorption on Protein Biochips

2003

Authors: Huang, T. T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, M. R. Ladisch
Journal: Biotechnology and Bioengineering, 81(5), 618-624, (2003)
Book Chapter:

Abstract: The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO2 surfaces of a biochip that had been modified with a C18 coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C18-derivatized SiO2 surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.

Research Area: Bioseparations    

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Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

2003

Authors: T. Geng, K. P. Kim, R. Gomez, D. M. Sherman, R. Bashir, M. R. Ladisch, A. K. Bhunia
Journal: Journal of Applied Microbiology, 95, 762-777 (2003)
Book Chapter:

Abstract: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments.

Research Area: Bioseparations    

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Lysozyme for Capture of Microorganisms on Protein Biochips

2003

Authors: Tom Huang, Tao Geng, Jennifer Sturgis, Haibo Li, Rafael Gomez, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, Michael R. Ladisch
Journal: Enzyme and Microbial Technology, 33, 958-966 (2003)
Book Chapter:

Abstract: Lysozyme placed on the SiO2 surfaces that have previously been derivatized with C18 coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C18 coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO2 surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO2 surfaces coated with C18 to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.

Research Area: Bioseparations    

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Micro-assembly of Functionalized Particulate Monolayer on C18-Derivatized SiO2 Surfaces

2003

Authors: Tom T. Huang, Tao Geng, Demir Akin, Woo-Jin Chang, Jennifer Sturgis, Rashid Bashir, Arun K. Bhunia, J. Paul Robinson, and Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 83, 4, 416-427, 2003.
Book Chapter:

Abstract: This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C18-coated SiO2 substrate via specific or non-specific biomediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C18 surface was used to mediate the surface assembly of streptavidin-coated microbeds (2.8 um), while a bare C18 surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 um) through hydrophobic interactions. For a C18 surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) and positively charged dimethylamino microbeads (0.8 um) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads preadsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.

Research Area: Bioseparations    

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Microfiber Assisted Fabrication of Microfluidic Channels Using Poly(dimethylsiloxane)

2003

Authors: Tom T. Huang, Woo-Jin Chang, Demir Akin, Rafael Gomez, Rashid Bashir, Nathan Mosier, Michael R. Ladisch
Journal: AIChE Journal, 49, 11, 2984-2987 (2003)
Book Chapter:

Abstract: A microfluidic device is typically formed using bulk silicon etching techniques on a silicon substrate (Kovacs et al., 1998). A photolithography step defines the desired pattern on the silicon substrate with photoresist. Etching using acids or gases, followed by a solvent and acid cleaning process to remove residual photoresist, leaves micron-scale features. Such devices are capable of providing rapid identification of nucleic acids, proteins, drugs, or other important biological compounds with enhanced sensitivity and time-to-result, while reducing consumption of expensive reagents compared to microtiter plates or test tube scale analyses (Stone and Kim, 2001; Khandurina and Guttman, 2002; Meldrum and Holl, 2002). Polydimethylsiloxane (PDMS), created by mixing a silicone elastomer base and a curing agent in a 10:1 ratio, gives an alternate material for fabricating microfluidic devices (McDonald and Whitesides, 2002). The liquid pre-polymer is poured over a master generated either from photolithography using a high resolution transparency as a photomask, or by laser ablation or Solid-Object Printing to form the device (Grzybowski et al., 1998; McDonald et al., 2002; McDonald and Whitesides, 2002). We report formation of a master by directed placement of glass microfibers on silicon or glass substrates, followed by pressing a preformed PDMS sheet onto the substrate to form microfluidic channels. Wells are formed by criss-cross fiber patterns, while functionalized microbeads coated onto fibers result in microscale channels that separate proteins. We believe this approach is an attractive research tool, because it places rapid prototyping capability within the reach of laboratories that have access to glass slides, an optical microscope, digital camera, tweezers, and PDMS.

Research Area: Bioseparations    

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Model for Temperature Profiles in Large Diameter Electrochromatography Columns

2003

Authors: Craig Keim and Michael Ladisch
Journal: AIChE Journal, 49, 2, 402-410 (2003)
Book Chapter:

Abstract: Scale-up of electrochromatographic separations has been problematic due to electrically induced heating. A two-dimensional transient temperature model for electrochromatography was developed, which accounts for physical properties of the stationary and mobile phase, and the column wall. The model also accounts for both the temperature effect on the electrical conductivity and a nonuniform, radially variant current density. This model was compared to experimental data from two electrochromatography systems with different cylindrical-column dimensions, packing materials, and operating conditions. In all cases, the model predicts the temperature to within 3 C of the actual temperature, both for column heatup and cooldown. Separation of a mixture of model proteins on the 3.81-cm-ID scale was used as the basis for scale-up calculations. The model identifies equipment parameters that control heating characteristics and can be scaled up to process 75 mL of sample per run.

Research Area: Bioseparations    

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Pathogen Detection, Food-Borne

2003

Authors: R. Bashir
Journal: McGraw-Hill Yearbook of Science and Technology, 1-3 (2003)
Book Chapter:

Abstract: The presence of microorganisms in food is a natural and unavoidable occurrence. Cooking generally destroys most harmful bacteria, but undercooked foods, processed ready-to-eat foods, and minimally processed foods can contain harmful bacteria that are serious health threats. Meat, dairy, and poultry products are important reservoirs for many of the food-borne pathogens, including Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7. Animal by-products, such as feed supplements, may also transmit pathogens to food animals (for example, Salmonella and bovine spongiform encephalopathy). Seafood is another potential source of food-borne pathogens, such as Vibrio, Listeria, and Hepatitis A. Infectious doses of many of these pathogens are very low (~10 bacterial cells), increasing the vulnerability of the elderly, infants, and people with immunological deficiencies or organ transplants. Researchers are continuously searching for sensitive tools that are fast, accurate, and ultrasensitive. In recent years, there has been much research activity in the area of sensor development for detecting pathogenic microorganisms.

Research Area: Bioseparations    

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Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture

2003

Authors: Woo-Jin Chang, Demir Akin, Miroslav Sedlak, Michael R. Ladisch and Rashid Bashir
Journal: Biomedical Microdevices, 5, 4, 281-290 (2003)
Book Chapter:

Abstract: In this study, a novel PDMS/silicon hybrid microfluidic biochip was fabricated and tested for the long-term batch culture of bacterial cells. The PDMS (poly(dimethylsiloxane)) cover with 3-dimensional micro-channels for flow was fabricated using Teflon tubing and hole-punch techniques, without photolithographic methods. The PDMS/silicon hybrid biochip was prepared by bonding of PDMS cover and a silicon chip that had electrodes and micro-fluidic channels defined. The absorption of liquid into PDMS cover was characterized and conditions to prevent drying of nutrient media within the micro-chamber were shown. The absorption of liquid from micro-chambers into the PDMS cover was reduced up to 2.5 times by changing the mixing ratio of PDMS and curing agent from 10:1 to 2.5:1. In addition, pre-saturation of the PDMS cover with media prior to the incubation resulted in the preservation of liquid in the micro-chambers for up to 22 hours. Optimization of the mixing ratio and pre-saturation of the PDMS cover reduced the drying time 10 times when compared to the unsaturated PDMS cover composed of 10:1 ratio of PDMS and curing agent. Listeria innocua and a strain of Escherichia coli, expressing green fluorescent protein (GFP), were successfully cultured in batch mode within the PDMS/silicon hybrid biochip.

Research Area: Bioseparations    

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Biotechnology for Future Army Applications

2002

Authors: Ladisch, M. R., J. J. Valdes, and LTC R. J. Love
Journal: Army AL&T, July-August, 36-37, (2002)
Book Chapter:

Abstract:

Research Area: Bioseparations    

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Genetic Homogeneity Among Listeria monocytogenes Strains from Infected Patients and Meat Products from Two Geographic Locations Determined by Phenotyping, Ribotyping and PCR Analysis of Virulence Genes

2002

Authors: Jaradat, Z. W., G. E. Schutze, and A. K. Bhunia
Journal: International Journal of Food Microbiology, 76, 1-10, (2002)
Book Chapter:

Abstract: Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinterR database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92–99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.

Research Area: Bioseparations    

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Microfabricated Device for Impedance-Based Detection of Bacterial Metabolism

2002

Authors: Gomez, R., M. R. Ladisch, A. K. Bhunia, and R. Bashir
Journal: Materials Research Society Symposium Proceedings, 729 (2002)
Book Chapter:

Abstract: We present the use of a microfabricated device for impedance-based detection of a few live bacterial cells. Impedance-based detection relies on measuring changes in the AC impedance of two electrodes immersed in a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing bacterial cells. Rapid detection of a few cells (1 to 10) is possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to test this miniaturized assay. The conductance of the bacterial suspensions is extracted from measuring their complex impedance in a 5.27 nl chamber in the biochip, at several frequencies between 100 Hz and 1 MHz. Measurements on suspensions of the bacteria Listeria innocua, Listeria monocytogenes, and Escherichia coli in a low conductivity buffer demonstrate that, under the current experimental conditions, the minimum detection level is between 50 and 200 live cells, after two hours of off-chip incubation. Work is in progress to develop techniques for selective capture of bacteria inside the chip, and to minimize background changes in impedance during on-chip incubation.

Research Area: Bioseparations    

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Microscale Electronic Detection of Bacterial Metabolism

2002

Authors: Rafael Gomez, Rashid Bashir, and Arun K. Bhunia
Journal: Sensors and Actuators B, 86, 198-208 (2002)
Book Chapter:

Abstract: In this paper, we present a microscale impedance-based technique for detecting the metabolic activity of a few live bacterial cells. Impedance-based detection relies on measuring changes in the ac impedance of two electrodes in contact with a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing cells. Rapid detection of a few live cells (1-10) is, in theory, possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to explore this technique, consisting of a network of channels and chambers etched in a crystalline silicon substrate. The complex impedance of bacterial suspensions is measured with interdigitated platinum electrodes in a 5.27 nl chamber in the biochip at frequencies between 100 Hz and 1 MHz. After 2 h of off-chip incubation, the minimum number of live cells suspended in a low conductivity buffer that could be easily distinguished from the same number of heat-killed cells was on the order of 100 Listeria innocua, 200 L. monocytogenes, and 40 Escherichia coli cells, confined into the 5.27 nl chamber. A number on the order of 100 live L. innocua cells suspended in Luria-Bertani (LB) broth produced a significantly higher signal than the same number of heat-killed cells, and a difference is evident even down to ~5-20 cells. To the best of our knowledge, this is the first demonstration of microscale impedance-based detection of bacterial metabolism.

Research Area: Bioseparations    

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Modeling Pore Size Distribution in Cellulose Rolled Stationary Phases

2002

Authors: Keim, C., C. Li, C. M. Ladisch, and M. R. Ladisch
Journal: Biotechnology Progress, 18, 317-321, (2002)
Book Chapter:

Abstract: Rolled stationary phases are fabrics (i.e., nonparticulate phases) that rapidly separate proteins from salts on the basis of size exclusion. Pore size and pore size distributions in the stationary phase determine how different size molecules distribute between the stationary and mobile phases in liquid chromatography columns. The potential for size exclusion chromatography by fabrics is not initially obvious because their interlaced structures are atypical for size exclusion supports. A simple logistic model fits the pore size distribution of a rolled stationary phase when pore sizes were measured using PEG, Dextran, D2O, glucose, and NaCl probes. When the fabric is treated with cellulase enzymes, the water-accessible pores uniformly decrease and peak retention is lower. The logistic function model captures this result and enables comparison of pore size distribution curves between enzyme-treated and untreated fabrics in rolled stationary phase columns.

Research Area: Bioseparations    

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Optimal Packing Characteristics of Rolled, Continous Stationary-Phase Columns

2002

Authors: Li, C., C. M. Ladisch, Y. Yang, R. Hendrickson, C. Keim, N. Mosier, M. R. Ladisch
Journal: Biotechnology Progress, 18, 309-316, (2002)
Book Chapter:

Abstract: Rolled, continuous stationary phases were constructed by tightly rolling and inserting a whole textile fabric into a chromatography column. This work reports the column performance, in terms of plate height, void fraction, and resolution, of 10 cellulose based fabrics. The relation between fabric structural properties of yarn diameter, fabric count, fabric compressibility, and column performance are quantitated. General requirements, including reproducibility of packing, for choosing fabrics to make a good SEC column are identified. This research showed that the packed columns have an optimal mass of fabric that minimizes plate height and maximizes resolution, in a manner that is consistent with chromatography theory. Mass of material packed is then an important column parameter to consider when optimizing columns for the rapid desalting of proteins. Proteins were completely separated from salt and glucose in less than 8 min at a pressure drop less than 500 psi on the rolled, continuous stationary-phase columns. These results, together with stability and reproducibility, suggest potential industrial applications for cellulose-based rolled, continuous stationary-phase columns where speed is a key parameter in the production process.

Research Area: Bioseparations    

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Adsorption of Avidin on Microfabricated Surfaces for Protein Biochip Applications

2001

Authors: Bashir, R., R. Gomez, A. Sarikaya, M. R. Ladisch, J. Sturgis, and J. P. Robinson
Journal: Biotechnology and Bioengineering, 73(4), 325-328 (2001)
Book Chapter:

Abstract: The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radio frequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.

Research Area: Bioseparations    

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Adsorption of Water from Liquid-Phase Ethanol-Water Mixtures at Room Temperature Using Starch-Based Adsorbents

2001

Authors: Beery, K. E., M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 40, 2112-2115, (2001)
Book Chapter:

Abstract: The kinetically controlled, selective removal of water from ethanol vapors by desiccants is well documented. However, studies on the removal of water by liquid-phase contacting of water- ethanol mixtures with starch-based material are limited. This research presents a screening study that shows that starch-based adsorbents remove liquid-phase water between 1 and 20 wt% from ethanol without the adsorbent being dissolved. The mass of water adsorbed per gram of dry adsorbent increases with increasing water content. Side by side comparisons of these starch-based adsorbents to silica gel and molecular sieves show that, in a kinetically controlled adsorption scheme below 10 wt % water, the inorganic desiccants have a greater operational (nonequilibrium) adsorption capacity per gram. At water concentrations at or above 10% water, however, the operational adsorptive capacity per gram of the starch-based adsorbents is roughly equivalent to the inorganic adsorbents, when using the same regeneration and adsorption conditions. The starch-based adsorbents adsorb water by forming hydrogen bonds between the hydroxyl groups on the surface of the adsorbent and the water molecules.

Research Area: Bioseparations    

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Chemistry and Properties of Starch Based Dessicants

2001

Authors: Kyle E. Beery, Michael R. Ladisch
Journal: Enzyme and Microbial Technology, 28, 573-581 (2001)
Book Chapter:

Abstract: Desiccants currently used in industry include molecular sieves, lithium chloride, silica gel, and corn grits. Of these, only corn grits (a form of ground corn) are biodegradable and derived from a renewable resource. A major component of the corn grits, starch, is the primary adsorptive material in the corn grits. Other polysaccharides, including cellulose and hemicellulose also have adsorptive properties. The use of alpha-amylase (EC 3.2.1.1) to modify porosity and surface properties of starch resulted in materials with enhanced water sorption properties compared to the native material. This paper reviews the chemical and structural properties of starch, corn grits, and cellulose-based scaffolds on which starch can be affixed, in order to attain structures that might someday find uses in a range of desiccant applications for industrial, commercial, and residential processes.

Research Area: Bioseparations    

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Impedance Spectroscopy and Biochip Sensor for Detection of Listeria monocytogenes

2001

Authors: Bhunia, A. K., Z. W. Jaradat, K. Naschansky, M. Shroyer, M. Morgan, R. Gomez, R. Bashir, and M. Ladisch
Journal: Proceedings of SPIE, (4206) 32-39 (2001)
Book Chapter:

Abstract: Listeria monocytogenes is a deadly foodborne human pathogen. Its ubiquitous nature and its ability to grow at refrigeration temperatures makes this organism a difficult one to control. High-volume processed, ready-to-eat (RTE) foods. Improved processing along with real-time detection could reduce the incidence of this pathogen. Conventional methods can detect this pathogen accurately, but take several days (2-7d) to complete, which is not practical considering the short shelf-life and cost fiber optic and microelectrical-mechanical system (MEMS) biochips were designed and examined for direct detection of L. monocytogenes from liquid samples. Also, interdigitated microsensor electrode (IME) chip and spectrofluorometer were used to measure L. monocytogenes interaction with mammalian cells (cytopathogenic activities) for indirect detection. Preliminary data generated using laboratory cultures of Listeria species indicated that L. monocytogenes could be detected in 30 min to 1 h 30 min depending on the techniques used.

Research Area: Bioseparations    

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Microfluidic Biochip for Impedance Spectroscopy of Biological Species

2001

Authors: Gomez, R., R. Bashir, A. Sarikaya, M. Ladisch, J. Sturgis, J. P. Robinson, T. Geng, A. Buhnia, H. Apple, S. Wereley
Journal: Biomedical Microdevices, 3(3), 201-209, (2001).
Book Chapter:

Abstract: This paper describes the fabrication and characterization of a microelectronic device for the electrical interrogation and impedance spectroscopy of biological species. Key feature of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the nonplanar topography of the chip using spin-on-glass as an intermediate bonding layer. The total fluidic path volume in the device is on the order of 30 nl. Flow fields in the closed chip were mapped by particle image velocimetry. Electrical impedance measurements of suspensions of the live microorganism Listeria innocua injected into the chip demonstrate an easy method for detecting the viability of a few bacterial cells. By-products of the bacterial metabolism modify the ionic strength of a low conductivity suspension medium, significantly altering its electrical characteristics.

Research Area: Bioseparations    

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New System for Preparative Electrochromatography of Proteins

2000

Authors: Keim, C., and M. R. Ladisch
Journal: Biotechnology and Bioengineering, 70, 72-81, (2000)
Book Chapter:

Abstract: Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 °C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 °C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.

Research Area: Bioseparations    

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Rolled Stationary Phases: Dimensionally Structured Textile Adsorbents for Rapid Liquid Chromatography of Proteins

1999

Authors: Hamaker, K., S-L. Rau, R. Hendrickson, J. Liu, C. M. Ladisch, and M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 38, 865-872 (1999)
Book Chapter:

Abstract: A woven textile fabric, consisting of 60% cotton/40% polyester, tightly rolled in a cylindrical configuration, has a three-dimensional structure with sufficient hydrodynamic stability to withstand interstitial eluent velocities of up to 300 cm/min when packed into standard liquid chromatography column assemblies. Demonstration of the pressure stability of the cotton/polyester fabric was followed up with experiments in which the cotton (cellulose) portion was derivatized and the fabric evaluated for chromatography of proteins. When derivatized to give a (diethylamino) ethyl (DEAE) anion exchanger, a velocity independent plate height of 2 mm, a static capacity of 115 mg of bovine serum albumin/g of stationary phase, and a dynamic protein loading capacity which decreases only 25% over an 800% increase in mobile-phase velocity from 6.7 to 54 cm/min was achieved. The fibers that make up the stationary phase have a relatively nonporous structure which minimizes pore diffusional effects. A protein separation of Cytochrome C from B-lactoglobulin a is shown to be completed by ion-exchange chromatography in less than 10 min using an NaCl step gradient. Gradient chromatography of a hen egg white shows resolution of the proteins into two major components (lysozyme and ovalbumin) as well as two minor ones. A size exclusion separation of PEG 20000 from glucose requires only 90 s. These characteristics, together with the ability of the cellulose-based stationary phase to withstand rapid flow rates, indicate that this type of stationary phase has potential for applications where chromatography using DEAE-cellulose particles has proven successful.

Research Area: Bioseparations    

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Bioseparations

1998

Authors: M. Ladisch
Journal: Encyclopedia of Chemical Technology, John Wiley & Sons, p89-122 (1998)
Book Chapter:

Abstract: The large-scale purification of proteins and other bioproducts is the final production step, prior to product packaging, in the manufacture of therapeutic proteins, specialty enzymes, diagnostic products, and value-added products from agriculture. These separation steps, taken to purify biological molecules or compounds obtained from biological sources, are referred to as bioseparations. Large-scale bioseparations combine art and science. bioseparations often evolve from laboratory-scale techniques, adapted and scaled up to satisfy the need for larger amounts of extremely pure test quantities of the product. Uncompromising standards for product quality, driven by commercial competition, applications, and regulatory oversight, provide many challenges to the scale-up of protein purification. The rigorous quality control embodied in current good manufacturing practices, and the complexity and lability of the macromolecules being processed provide other practical issues to address.

Research Area: Bioseparations    

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Effect of Enzyme Modification of Corn Grits on Their Properties As An Adsorbent in Skarstrom Pressure Swing Cycle Drier

1998

Authors: Beery, K., M. Gulati, E. P. Kvam, and M. R. Ladisch
Journal: Adsorption, 4, 321-335 (1998)
Book Chapter:

Abstract: Corn grits have been tested as a desiccant in a pressure swing adsorption (PSA) system to produce dry air. Two sizes of unmodified corn grits were tested in the PSA system: 2.16 and 0.978 mm in diameter, which dried corn grits that gives an increase in the operational adsorptive capacity in a pressure swing, adsorption system, so that grits dry moist air to a – 56 °C dew point and the 0.978 mm corn grits dry air to a – 80 °C dew point. The modification process creates surface modifications on the corn grits apparently making more adsorption sites easily available. The modification procedure increases the specific surface area of the grits and possibly decreases the crystallinity, which would make more hydroxyl groups available for adsorption of water. Possible applications of using corn grits in the pressure swing adsorption system include industrial gas dryers, sorptive cooling air conditioners, and recycling equipment for industrial solvents.

Research Area: Bioseparations    

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Transport Properties of Rolled, Continuous Stationary Phase Columns

1998

Authors: Hamaker, K., J. Liu, C. Ladisch, and M. R. Ladisch
Journal: Biotechnol. Progr., 14(1), 21-30 (1998)
Book Chapter:

Abstract: Continuous stationary phase columns consist of woven textile matrixes of fibers rolled into a cylindrical configuration and inserted into a liquid chromatography column. This configuration allows separations to be carried out at interstitial mobile phase velocities in excess of 100 cm/min and pressures of up to 700 psig for stationary phases based on cellulose. Ordinarily, these conditions would cause compaction of a Cellulosic stationary phase to the point where flow is no longer possible. The packing of the column with cellulose as a continuous stationary phase enables these linear velocities to be achieved. Most importantly, this type of column allows the study of momentum transport and mass transfer in a media in which the mobile phase explores almost all of the void volumes in the column. The analysis of flow patterns in these columns has been modeled using elution patterns of both retained and unretained components, and plate height has been correlated as a function of velocities in the range of 1-100 cm/min. Engineering analysis of this type of chromatography column based on visual representation of the packed fibers by scanning electron microscopy, analysis of porosities using unretained (nonadsorbing) molecular probes, and application of momentum and mass transport equations is discussed.

Research Area: Bioseparations    

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Biobased Adsorbents for Drying of Gases

1997

Authors: Ladisch, M. R.
Journal: Enzyme Microb. Tech., 20, 162-164 (1997)
Book Chapter:

Abstract: Fundamental, structural and compositional studies on the properties of corn grits and their ability to selectively adsorb water from organic vapors have resulted in new bio-based adsorbents. Structure/function relationships of these bio-based adsorbents are reviewed.

Research Area: Bioseparations    

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Chromatography for Rapid Buffer Exchange and Refolding of Secretory Leukocyte Protease Inhibitor

1996

Authors: Hamaker, K. H., J. Liu, R. J. Seely, C. M. Ladisch, and M. R. Ladisch
Journal: Biotechnol. Progr., 12, 184-189 (1996)
Book Chapter:

Abstract: A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20oC, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

Research Area: Bioseparations    

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Intraparticle Flow and Plate Height Effects in Liquid Chromatography Stationary Phases

1996

Authors: Hamaker, K. H. and M. R. Ladisch
Journal: Separation and Purification Methods, 25(1), 47-83 (1996)
Book Chapter:

Abstract: Velocity independent plate heights were apparently first recognized for hydrodynamic chromatography columns, packed with nonporous, 115 micron glass beads which were run at reduced mobile phase velocities of 10 to 10,000. Hydrodynamic chromatography separates based on the tendency of small molecules (or particles) to associate with slower moving fluid streamlines near the surfaces of particles, compared to larger molecules which seek faster streamlines. Consequently, the larger molecules elute first. Velocity independent plate heights in liquid chromatography have also been observed for nonadsorbed solutes in particulate and fibrous stationary phases. These stationary phases have pores which exceed 10-4 to 10-5 cm in dimension. The flat plate height is attributed to flow in the channels formed by these large intraparticle spaces. The development of plate height expressions which represent dispersion at interstitial velocities above 10 cm/min are discussed. Explanations of the uncoupling of dispersion from eluent flow rate in continuous stationary phases, membranes, and gigaporous particles is shown to have their origins in the studies of distribution of particles and molecules in hydrodynamic chromatography columns, and to be adequately described by modifications of the van Deemter equation.

Research Area: Bioseparations    

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Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

1996

Authors: N. Cao, J. Du, C. S. Gong, and G. T. Tsao
Journal: Applied and Environmental Microbiology, p. 2926-2931 (Aug. 1996)
Book Chapter:

Abstract: An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The production of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.

Research Area: Bioseparations    

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Synthesis and Optimization of a New Starch Based Adsorbent for Dehumidification of Air in a Pressure Swing Drier

1996

Authors: Anderson, L., M. Gulati, P. Westgate, E. Kvam, K. Bowman, and M. R. Ladisch
Journal: Ind. & Eng. Chem. Res., 35, 1180-1187 (1996)
Book Chapter:

Abstract: Corn grits selectively adsorb water from many types of organic vapors and are used commercially to dry 2.8 billion L of fuel-grade fermentation ethanol annually. Evaluation of grits in a pressure-swing dryer at 308 kPa, combined with analyses of their physical properties, showed that the specific surface of the grits (0.5 m2/g) limited steady-state drying of air to a dewpoint of -20 °C. By selectively taking advantage of the best features of the natural material, a new class of natural adsorbents with a higher affinity for water was then synthesized using material derived from corn: starch and cob flour. The chemical composition of the synthesized adsorbent was determined, as well as specific physical properties. Scanning electron microscopy showed the synthesized adsorbent surface had a large number of macropores (10-25 um in diameter) unlike corn grits which have limited porosity. This material gave reasonable and reproducible results, similar to those obtained with molecular sieves using a commercially available pressure-swing air dryer. After 70 h of operation at 30 psi, the new adsorbent provided air at a dewpoint of -63 °C. The methods for preparing this material and an explanation of its performance in terms of macroscopic and microscopic structural characteristics are described.

Research Area: Bioseparations    

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Correlation of Electrophoretic Mobilities of Proteins and Peptides with Their Physicochemical Properties

1995

Authors: Basak, S. and M. R. Ladisch
Journal: Anal. Biochem., 226, 51-58 (1995)
Book Chapter:

Abstract: Electrophoretic mobilites, u, of nine proteins (Mr 14,200 to 70,000) in 28 mM Tris/47 mM glycine buffer at pH 8.77 and 5 mM ionic strength were measured by laser Doppler velocimetry and correlated to ratios of charge (q) to molecular weight (Mr) and shape factor (f/f0) by the equation u(f/f0) = (Aq/Mr –B). This correlation was previously reported for peptides and proteins for u measured at 100 mM ionic strength. When A = 6.048 x 10-3, B = 1.13 x 10-5, and p = 2/3, the correlation fitted 51 measured and literature values over the molecular weight range of 178 to 140,000 for components whose electrophoretic Mobilities ranged from + 13.35 x 10-5 to -19.7 x 10-5 cm2/ (V.s). The experimental measurements confirm the general suitability of p=2/3 and show that the familiar charge/mass relation for electrophoresis is applicable to proteins in low-ionic-strength buffers which are typical of electrochromatography systems. Extrapolation of the correlation to different ionic strengths indicates that a low-ionic-strength buffer amplifies differences of electrophoretic mobility as a function of charge/mass, while high ionic strength diminishes such differences.

Research Area: Bioseparations    

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Effect of Modulator Sorption on Gradient Shape in Ion Exchange Chromatography

1995

Authors: Velayudhan, A. and M. R. Ladisch
Journal: Ind. Eng. Chem. Res., 34(8), 2805-2810 (1995)
Book Chapter:

Abstract: Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Research Area: Bioseparations    

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Electrochromatographic Separations of Proteins

1995

Authors: Basak, S., A. Velayudhan, K. Kohlmann and M. R. Ladisch
Journal: Journal of Chromatography A., 707, 69-76 (1995)
Book Chapter:

Abstract: We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

Research Area: Bioseparations    

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Mechanistic Description and Experimental Studies of Electrochromatography of Proteins

1995

Authors: Basak, S. and M. R. Ladisch
Journal: AIChE J., 41(11), 2499-2507, (1995)
Book Chapter:

Abstract: Electrochromatography is a form of gradient liquid chromatography in which an axial electric potential is applied to columns packed with gel-filtration media. Experimental methodology and a mechanistic model are further developed for a system that minimizes Joule heating at electric field strengths of 100 V/cm by dissipating heat through a cooling jacket and use of a cooled, low ionic strength eluting buffer. Focusing of proteins can be achieved in a 15-mm-dia. Column by the interplay of eluent velocity, electrophoretic migration rate, and electrically induced concentration polarization when the stationary phase is more conductive than the mobile phase. Voltage gradients of up to 125 V/cm for eluent velocities at 18-25 cm/h separate binary protein mixtures of Bhb-a–lactalbumin, BSA-myoglobin, and a-lactalbumin-myoglobin over Sephadex G-100 and G-50. Retention times are consistent with values obtained from a mechanistic nonlinear model.

Research Area: Bioseparations    

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Scale-up Techniques in Bioseparation Processes

1995

Authors: Ladisch, M. R., and A. Velayudhan
Journal: Bioseparation Processes in Foods, Dekker, (1995)
Book Chapter:

Abstract: Chromatography plays a major role in the downstream processing of biological materials encountered in the manufacture of food, pharmaceutical, and biotechnology products. Here, we offer approaches to scaling up linear chromatography in chromatographic separations based on isocratic elution, as well as illustrate a nonideal, gradient-induced peak deformation effect, which may occur upon scale-up of gradient chromatography.

Research Area: Bioseparations    

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Simultaneous Concentration and Purification Through Gradient Deformation in Gradient Elution Chromatography

1995

Authors: Velayudhan, A., R. L. Hendrickson, and M. R. Ladisch
Journal: AIChE J., 41(5), 1184-1193 (1995)
Book Chapter:

Abstract: Mobile-phase additives, commonly used to modulate absorbate retention in gradient elution chromatography, are usually assumed to be either linearly retained or unretained. Previous theoretical work from our laboratory has shown that these modulators, such as salts in ion-exchange and hydrophobic interaction chromatography and organic modifiers in reversed-phase chromatography, can absorb nonlinearly, giving rise to gradient deformation. Consequently, adsorbate peaks that elute in the vicinity of the head of the deformed gradient may exhibit unusual shapes, form shoulders, and/or be concentrated. These effects for a reversed-phase sorbent with aqueous acetonitrile (CAN) as the modulator are verified experimentally. Gradient deformation is demonstrated experimentally and agrees with simulations based on CAN isotherm parameters that are independently determined from batch equilibrium studies using the layer model. Unusual adsorbate peak shapes were found experimentally for single-component injections of phenylalanine, similar to those calculated by the simulations. A binary mixture of tryptophan and phenylalanine is used to demonstrate simultaneous concentration and separation, again in agreement with simulations. The possibility of gradient deformation in ion-exchange and hydrophobic interaction chromatography is discussed.

Research Area: Bioseparations    

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Characterization of Buffers for Electrokinetic Separations

1994

Authors: Basak, S. K., A. Velayudhan, and M. R. Ladisch
Journal: Appl. Biochemistry Biotechnol., 44, 243-261 (1994)
Book Chapter:

Abstract: Buffers used in electrophoresis and electrochromatography must have a relatively low ionic strength in order to minimize ohmic heating in the presence of an applied potential. Calculation of pH, ionic strength, and the van Slyke buffer capacity, B, is therefore important. This paper describes the a priori calculation of these parameters for tris buffer made up with either glycine (a zwitterions) or HCl. A quadratic expression for pH, valid over wide ranges, is obtained for both buffer systems. The calculated values of pH, ionic strength, and buffer capacity are shown to agree with experimental results as a function of tris, HCl, and glycine concentrations ranging from 1 to 50 mM. A new parameter, the electrokinetic buffer effectiveness factor, is introduced to characterize buffers being considered for use in electrokinetic systems such as electrochromatography, and is used to determine the appropriate composition ranges for the buffer components.

Research Area: Bioseparations    

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Air Drying Using Corn Grits as the Sorbent in a Pressure Swing Adsorber

1993

Authors: Westgate, P. J., and M. R. Ladisch
Journal: AIChE J., 39(4), 720-723 (1993)
Book Chapter:

Abstract: Vapor streams containing organics and water can be dried in an energy-efficient manner by being passed over a cellulose or starch adsorbent such as corn grits. The success of this dehydration method appears to be related to differences in the rates of adsorption, as well as differences in the strength of interaction between each species and the adsorbent. Hence, compounds which exhibit either weak interactions or slow rates of adsorption are expected to be readily separated from those that exhibit strong, relatively fast interactions with the adsorbent.

Research Area: Bioseparations    

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Liquid Chromatography Using Cellulosic Continuous Stationary Phases

1993

Authors: Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
Journal: Advances in Biochemical Engineering and Biotechnology, 49, 147-160, (1993)
Book Chapter:

Abstract: A novel type of continuous stationary phase based on fabric materials is described. This column packing utilizes the continuous character of a cellulose (cotton) stationary phase, and the chemistry of the derivatized forms of the adsorbent, to obtain separations of proteins and small molecules based on cation and anion exchange, hydrophobic interactions, and size. The mechanical stability of the stationary phase facilitates chromatographic velocities in excess of 70 cm min-1. The influence of eluent properties on the adsorption of sample proteins is discussed in this chapter. Sequential stepwise desorption is used to separate 100 ul mixtures of BSA, IgG, B-galac10sidease, and insulin in 10 minutes or less, using 10 mm i.d. x 500 mm length columns.

Research Area: Bioseparations    

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Modulator Sorption in Gradient Elution Chromatography

1993

Authors: Velayudhan, A., and M. R. Ladisch
Journal: Bioproducts and Bioprocesses, 2, 217-232, (1993)
Book Chapter:

Abstract: Introduction Theoretical Development-Literature Survey Unretained modulator Linearly Sorbed Modulator Finite Sample Concentration Modulator Sorption Motivation Modulator Sorption Isotherm Gradient Deformation and Shock Formation Implications for Separations Conclusions and Outlook Nomenclature References Gradient elution chromatography is widely used to separate both small molecules and macromolecules. The mobile phase additive (modulator) used to modify adsorbate retention is usually considered to be either unretained or linearly retained. In both cases, the shape of the gradient does not change as it moves down the column. However, the high mobile phase concentrations at which such a modulator is commonly used makes it likely to adsorb according to its nonlinear sorption isotherm. Here the quantitative consequences of such nonlinear modulator sorption are reviewed. Nonlinear sorption deforms the shape of the gradient during its passage through the column; ultimately a shock (or shock layer) could be formed. The condition for shock formation is discussed, and numerical simulations using representative parameter illustrate the magnitude of gradient deformation and the consequences for separation.

Research Area: Bioseparations    

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Plates Models in Chromatography: Analysis and Implications for Scale up

1993

Authors: Velayudhan, A., and M. R. Ladisch
Journal: Advances in Biochem. Engr. Biotech., 49, 123-145, (1993)
Book Chapter:

Abstract: Detailed chromatography rate theories from the literature can be used to determine the appropriate plate count for a plate model of linear chromatography so that the bandspreading generated by the detailed rate model is reproduced by the plate model. This process provides a link between the plate count and the physical parameters that cause bandspreading. Each sample component can be assigned an appropriate plate count, thus allowing the accurate simulation of multicomponent separations even for widely differing adsorbates. Analytical solutions are presented for the Craig distribution and the continuous plate model for both finit-pulse elution and frontal chromatography. The Craig model is widely considered unsuitable because it assumes discontinuous flow; it is shown that, for a suitably corrected plate count, the Craig model is as accurate as the continuous-flow plate theory (expect for the case of an unretained solute). Direct calculation of effluent histories from these plate models show excellent agreement between themselves and with results from complex rate models available in the literature. Reasonable agreement is also found when the plate models are used a priori to predict experimental scale-up results.

Research Area: Bioseparations    

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Solute Retention in Electrochromatography by Electrically Induced Sorption

1993

Authors: Rudge, S. R., S. Basak and M. R. Ladisch
Journal: AIChE J., 39(5), 797-808 (1993)
Book Chapter:

Abstract: Column chromatography and electrophoresis are combined in electrochromatography, where an electric potential is applied to a chromatography column in the axial direction. These studies utilized a dextran gel stationary phase and an eluent of low ionic strength, which were chosen to minimize electric current and therefore column heating and undesirable dispersion effects. The gel, with a small ion exchange capacity of several microequivalents per mL, turned out to be more conductive than the eluent and was able to concentrate macromolecules in the presence of combined electric and flow fields. The model presented describes solute retention due to electrically induced concentration polarization of solute on the resin surfaces, as well as electrophoresis in the mobile and stationary phases. The polarization effect explains differences between retention of high-molecular-weight solutes with exclusion coefficients of less than 1 and that of a charged low-molecular-weight solute, which is hypothesized to pass through the gel matrix in the presence of an electric field and does not experience concentration polarization. It also shows the application of this effect for protein separation in a liquid chromatography system with a superimposed electric potential.

Research Area: Bioseparations    

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Sorption of Organics and Water on Starch

1993

Authors: Westgate P., and M. R. Ladisch
Journal: Ind. Eng. Chem. Res , 32(8), 1676-1680 (1993)
Book Chapter:

Abstract: Starch is a well-established adsorption agent for drying ethanol. This work examines its potential for other gas-phase drying applications. Results from gas chromatography studies confirm that starch separates water from organic acids, alcohols, ketones, ethers, and aromatics, many of which from azeotropes with water. Trends in organics with respect to size and functional group show that the efficiency of this separation is related to both transport properties and strength of interaction between the organic components and starch. Small, polar molecules such as methanol and formic acid that have rapid mass-transfer characteristics and relatively strong interactions with starch are retained to a greater degree and are more difficult to separate from water than either compounds of higher molecular weight or decreased polarity. The large number of possible separations indicates that starch is a versatile material for use in sorbents for vapor-phase separations.

Research Area: Bioseparations    

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A New Approach to the Study of Textile Properties by Liquid Chromatography, Comparison of Void Volume and Surface area of Cotton and Ramie Using a Rolled Fabric Stationary Phase

1992

Authors: Ladisch, C. M., Y. Yang, A. Velayudhan, and M. R. Ladisch
Journal: Textile Res. J., 62(6), 361-369 (1992)
Book Chapter:

Abstract: A novel rolled stationary phase using whole fabric has been developed for liquid chromatography. This paper describes the column and its properties and use in characterizing pore volumes of the whole fabric. The logistic model gives a good fit of the measured void volume data obtained by size exclusion chromatography. With the help of this function, both pore size and surface area distribution of cotton and ramie fabrics can be obtained. The relationship between pore shape and surface area is discussed. Cotton has 100 to 200% more void volume and surface area than ramie (for all sizes of pores). Increasing the temperature from 30 to 60 °C does not significantly influence either the total void volume or surface area of cotton and ramie. The void volume and surface area of cotton as determined by this rolled fabric column method are comparable to previously reported data.

Research Area: Bioseparations    

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Effect of Modulator Sorption in Gradient Elution Chromatography: Gradient Deformation

1992

Authors: A. Velayudhan, and M. R. Ladisch
Journal: Chemical Eng. Sci. J., 47. No. 1, 233-239 (1992)
Book Chapter:

Abstract: In gradient elution chromatography, the mobile phase composition at the column inlet is a function of time. The consequences of accounting for the adsorption of the mobile phase components themselves are investigated for the experimentally common situation of a linear inlet gradient. Surface excess adsorption data from the literature for water-acetonitrile mobile phases in reversed-phase chromatography using different octadecyl stationary phases are shown to be in good internal agreement. The resulting individual isotherms as calculated by Tani and Suzuki (1989) are fitted to Langmuir and BET forms; it is shown that the Langmuirian form fits the data well except at extremely high acetonitrile concentrations. Using these isotherm parameter, it is found that a linear inlet gradient could suffer significant distortion while moving down the column. In the extreme case, a shock front of the mobile phase modulator could result. Analytical expressions are derived describing the conditions under which such a shock could occur within the column and its subsequent path, assuming Langmoirian sorption.

Research Area: Bioseparations    

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Modeling of Equilibrium Sorption of Water Vapor on Starch Materials

1992

Authors: Westgate, P. J., J. Y. Lee, and M. R. Ladisch
Journal: Transactions ASAE, 35(1), 213-219 (1992)
Book Chapter:

Abstract: The equilibrium behavior of corn grits and corn starch at temperatures above 70o C, a region for which limited data and theory are available, are compared and found to be similar. Sircar’s model and potential theory accurately represent isotherm data for both adsorption systems as well as data for desorption from corn. Values of the model parameters indicate that physical properties of these starch-based sorption materials exhibit a temperature dependence that is likely related to the breaking of hydrogen bonds as water interacts with the sorbent. Modification of the two models with an exponential temperature relation is proposed to account for the experimentally measured temperature dependence of model parameters. The resulting modified Sircar’s model and potential theory equations are shown to fit the data for both starch and corn grits in the high temperature range.

Research Area: Bioseparations    

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Modeling of Non-Linear Elution Chromatography for Preparative-Scale Separations

1992

Authors: Velayudhan, A., M. R. Ladisch, and J. Porter
Journal: AIChE Symp. Ser., 88, (1992)
Book Chapter:

Abstract: Many models are available for describing elution profiles in preparative-scale overloaded chromatography. These range from empirical to theoretical and describe experimental data with varying success. This chapter reviews models and approaches to analyzing isocratic and gradient elution. When the loading is low-to-moderate in nonlinear isocratic elution, analytical solutions are available from which productivity can be calculated as a function of operating parameters. For higher loadings, optimization studies have been carried out on binary mixtures obeying Langmuirian isotherms. Gradient elution separations have been successfully scaled-up using a simplified theory.

Research Area: Bioseparations    

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Protein Chromatography Using a Continuous Stationary Phase

1992

Authors: Yang, Y., A. Velayudhan, C. M. Ladisch, and M. R. Ladisch
Journal: J. Chromatogr., 598, 169-180 (1992)
Book Chapter:

Abstract: A continuous stationary phase consisting of yarns woven into a fabric is rolled and packed into mechanically stable liquid chromatography columns. This work utilized yarns have a characteristic width of 200 400-um, made from 10 20-um fibers consisting of 95% poly(m-phenylene isophthalamide) and 5% poly(p-phenylene terephthalamide). Although loadings on this stationary phase were low at 4 mg/g for bovine serum albumin and 6 mg/g for B-galactosidase, this material shows the interesting characteristic of a leveling off of plate height at mobile phase velocities of 30 80-cm/min. This phenomenon is explained on the basis of a coupling argument whereby a fraction of the mobile phase flows through the intramatrix pore space, and convective transport through the pore space dominates transport by diffusion. A modified Van Deemter expression is derived and shown to lit plate height data for polyethylene glycol standards having molecular weights of 200 and 20 000. The characteristic of this continuous stationary phase at high eluent velocities are discussed and conditions which give separation of immunoglobulin G, bovine serum albumia, insulin and B-galactosidase in 12 min are described.

Research Area: Bioseparations    

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Recombinant Human Insulin

1992

Authors: Ladisch, M. R., and K. Kohlmann
Journal: Biotechnol. Prog., 8(6), 469-478 (1992)
Book Chapter:

Abstract: Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.

Research Area: Bioseparations    

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Analytical and Preparative Scale Chromatography of Phenylalanine from Aspartame Using a New Polymeric Sorbent

1991

Authors: Ladisch, M. R., R. L. Hendrickson, and E. Firouztale
Journal: J. Chromatogr. (540), 85-101 (1991)
Book Chapter:

Abstract: A new, large-pore, cross-linked, polymethacrylate stationary phase separates Phe from Aspartame in 10% aqueous ethanol by reversed-phase chromatography. Batch equilibrium data at 30, 50, and 70 °C, obtained with 165-um particle size material showed linear sorption at loadings of up to 140 mg/g stationary phase, and corresponded to a mobile phase adsorbate concentration approaching the solubility limits. Column runs with 40-, 60-, 117-, and 165-um particle size materials at 30-70 °C showed retention behavior that was predictable from batch equilibrium data, and was independent of particle size at sample volumes as high as 80% of the column void volume and at outlet concentrations of 5-10 mg/ml. The relatively large pores (250 A) of the stationary phase allowed free access of small molecules, with the methacrylate structure promoting strong sorption of aromatic amino acids. These characteristics permitted the ready calculation of column retention times, and facilitated extrapolation of analytical-scale results obtained with small particle size material to reparative-scale separations carried out under volume overload conditions with a larger particle size stationary phase.

Research Area: Bioseparations    

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Ion-Exchange and Affinity Chromatography Costs in a-Galactosidase Purification

1991

Authors: Jill E. Porter and Michael R. Ladisch
Journal: Biotechnology and Bioengineering, 39, 717-724 (1991)
Book Chapter:

Abstract: The purification of a-galactosidase from soybean seeds is a five to six-step procedure consisting of cryoprecipitation, acid precipitation and ammonium sulfate fractionation followed by two or three chromatography steps. The procedures, while not optimized, were carried out in a manner that resulted in 414-515-fold purification, as reported previously. The costs of two purification sequences were compared. In the best case, the preparative-scale costs of stationary phase, reagents, and hardware were $790 per million enzyme units, excluding labor. Stationary phase costs predominated over extraction, chromatography reagent and eluent costs when the stationary phase is replaced after 10-40 cycles of use. However, if stationary phase life exceeds 50-200 cycles, stationary phase costs become similar in magnitude to eluent and reagent costs. Labor costs, which are process-specific and difficult to estimate, exceed all other costs by a factor of 10-50 at a small scale of operation and constitute a major cost, regardless of scale. This case study provides equations and a framework for carrying out a first comparison of costs for multistep purification sequences. Column life, throughput, and scale of operation were found to determine not only the magnitude, but also the relative contributions of the different components that make up purification costs. This analysis shows that there are major opportunities for reducing purification costs through the development of less expensive stationary phases and the implementation of intelligent process control and automation for process scale chromatography.

Research Area: Bioseparations    

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Purification and Characterization of an Extracellular Protease Produced by Pseudomonas fluorescens M3/6

1991

Authors: Kohlmann, K. L., S. S. Nielsen, and M. R. Ladisch
Journal: J. Dairy Sci., 74, 4125-4136 (1991)
Book Chapter:

Abstract: Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 °C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the culture’s life cycle. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The isolated protease had activity on azocasein, a-, B-, and k-caseins and a plasmin substrate but did not have plasminogen activator activity. The protease had a molecular weight of 45 kDa, an isoelectric point of pH 8.25, a broad temperature and pH range for activity, and was less heat stable in the isolated form than in the cellfree extract.

Research Area: Bioseparations    

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Role of Modulator in Gradient Elution Chromatography

1991

Authors: Velayudhan, A. and M. R. Ladisch
Journal: Anal. Chem., 63(18), 2028-2032 (1991)
Book Chapter:

Abstract: Mobile-phase additives are frequently used in gradient elution chromatography to modulate adsorbate retention. These additives are known to adsorb themselves onto the stationary phase, resulting in solvent dernixing. In this paper, the concentration of the mobile-phase additive in the mobile phase is assumed to be high enough for it to lie in the nonlinear region of the its own adsorption isotherm. Then the shape of the gradient would become deformed while passing down the column and could ultimately form a shock layer. A series of numerical simulations of a binary feed mixture is presented under conditions where a shock layer is formed, and the possible consequences are discussed. Depending on the location of the adsorbate peak with respect to the mobile-phase shock layer, leading or trailing shoulders can result, along with significant peak sharpening. The implications of these effects on separation are presented, and conditions under which the present analysis might be tested experimentally are indicated.

Research Area: Bioseparations    

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Water and Ethanol Sorption Phenomena on Starch

1991

Authors: Lee, J. Y., P. J. Westgate, and M. R. Ladisch
Journal: AIChE J., 8(37), 1187-1195 (1991)
Book Chapter:

Abstract: The sorption behavior of water and ethanol on starch material has been investigated in relation to the adsorptive separation of water from ethanol. The adsorption isotherms of water-starch, ethanol-starch and water-ethanol-starch were measured using a Cahn electrobalance. Careful examination of the many sorption isotherm models resulted in selection of Sircar’s model and the potential theory to best represent the isotherm data of water-starch and ethanol-starch adsorption. Experimental results showed that ethanol as well as water can adsorb on starch. The adsorption rate of ethanol, however, is much slower than that of water. This suggests that the selective removal of water from ethanol vapor in a packed-bed adsorber is likely a rate-dependent, not equilibrium-dependent, process.

Research Area: Bioseparations    

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Analysis of Sub-microgram Quantities of Cellodextrins by Aqueous Liquid Chromatography Using a Differential Refractometer

1990

Authors: A. N. Pereira, K. L. Kohlmann, and M. R. Ladisch
Journal: Biomass, 23, 307-317 (1990)
Book Chapter:

Abstract: The analysis of water-soluble cellodextrins using liquid chromatography is readily achieved with a variety of packings. Direct injection of enzyme incubation mixtures allows quantitation of 10 mM cellodextrins in hydrolysis mixtures, resulting in a method which is useful for kinetic studies. Reported here are operating procedures for a 4% cross-linked, styrene-divinyl benzene cation exchanger (Aminex 50W-X4 (Bio Rad Lab., Griffin, CA, USA), 20-30 um particle size) in the Ca++ form, packed in a column of dimensions 6 mm i.d. x 60 cm long. Using this column, resolution of the cellodextrins, celloheptaose through cellobiose and glucose was possible with 91 mM HsSO4 as the eluent. Requirements of the separation system included use of a pulsation free syringe pump to minimize baseline fluctuations, the use of Ca++ as the counterion to give a column operational life of 500-1000 injections, and injection of sample volumes of up to 25 uL. Cellodextrins were quantified at sub-microgram (nmole) levels using a differential refractometer as the detector. Examples of this technique for analysis of the acid hydrolysis of cellodextrins and enzymatic hydrolysis of cellodextrins and carboxymethylcellulose are described.

Research Area: Bioseparations    

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Characterization of the Swelling of a Size Exclusion Gel

1990

Authors: Monke, K., A. Velayudhan, and M. R. Ladisch
Journal: Biotechnol. Progress, 6(5), 376-382 (1990)
Book Chapter:

Abstract: The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated at 6.3x10-7cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 um, while the hydrated particles had diameters of 40-350 um. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.

Research Area: Bioseparations    

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Displacement Effect in Multicomponent Chromatography

1990

Authors: Gu, T., G. T. Tsao, G-J. Tsai, and M. R. Ladisch
Journal: AIChE J. 36(8), 1156-1162 (1990)
Book Chapter:

Abstract: The study of interference effects is of fundamental importance in understanding multicomponent chromatography. In this work, a displacement effect is examined and shown to be able to explain the dominating interference effects in three major modes of chromatography-frontal, elution, and displacement-involving competitive isotherms. It is concluded that the concentration profile of a component usually becomes sharper due to the displacement effect from another component, while the concentration from of the displacer is usually diffused. Five factors that escalate the displacement effect in multicomponent elution were investigated. A binary elution with a competing modifier in the mobile phase was also discussed. This study was carried out using computer simulations based on a general nonlinear multicomponent rate equation model that considers axial dispersion, external mass transfer intraparticle diffusion, and Langmuir isotherms. The use of the general model helps the visualization of the multicomponent interactions in chromatography under mass transfer conditions.

Research Area: Bioseparations    

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Freeze Concentration of Dyes

1990

Authors: Yang, Y., C. M. Ladisch, and M. R. Ladisch
Journal: Textile Res. J., 12(60) 744-753 (1990)
Book Chapter:

Abstract: Concentration of water soluble direct, acid, basic, and reactive dyes occurs when dilute solutions are frozen at a temperature below the melting point and above the eutectic point of the solution. When freezing is done in still solution, a concentration of up to 500% is achieved in one step, with the dye solution collecting in an oblately spheroidal liquid pocket surrounded by clear ice containing voids, formed from air pockets, radiating outward. Three repetitive freezing cycles concentrate the dye during freezing. The freezing rate of four acid and direct dyes had no close relationship with the size of the dyes studied. Over a larger molecular weight range, an effect was observed for other kinds of molecules. Freeze concentration of salt solution (MW = 58.5) gives almost a 700% concentration, detergent solution (sodium dodecyl sulfate, MW = 288.4) gives 400%, and bovine serum albumin, a large water soluble macro-molecule (MW = 66,200), gives 160%. A theory is presented suggesting that the concentration effect and the formation of the central sphere are consistent with minimizing of the free energy of the overall system. This simple technique may find application in the concentration of heat sensitive, labile dyes for analytical purposes, as well as in the recovery of dyes and other chemicals on a bench scale.

Research Area: Bioseparations    

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Ion Exchange and Affinity Chromatography in the Scaleup of the Purification of a-Galactosidase from Soybean Seeds

1990

Authors: Jill E. Porter, Michael R. Ladisch, and Klaus M. Herrmann
Journal: Biotechnology and Bioengineering, 37, 356-363 (1990)
Book Chapter:

Abstract: Soybeans (Glycine max) contain an a-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed a-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl a-D mannoside gradient gave a-galactosidase with an average specific activyt of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column in the laboratory procedure) thereby making scaleup practical.

Research Area: Bioseparations    

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Bioseparations of Milk Proteins

1989

Authors: Ladisch, M. R., S. R. Rudge, K. W. Ruettimann, and J. K. Lin
Journal: Bioproducts and Processes (1989)
Book Chapter:

Abstract: Milk is a complex biological fluid consisting of lipids, phospholipids, carbohydrates, proteins, sugars, salts and vitamins. Casein proteins exist in the milk serum (non-fat milk) as micellar structures stabilized by colloidal calcium phosphate and protein surfactant. Raw milk also contains microorganisms and somatic cells, which contribute small amounts of protcolytic enzymes and nucleic acids to milk in the course of processing. Since milk is a suitable carrier for a diverse range of biological molecules, its fractionation is considerable interest. Knowledge of these fractionation methods may further facilitate recovery of proteins of therapeutic value or of enhanced nutritional qualities from milk. The purpose of this chapter is to show separation strategies aimed at recovering selected molecules found in milk serum, and to indicate possible approaches for larger scale fractionations.

Research Area: Bioseparations    

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Cellulosic Adsorbents for Treating Textile Mill Effluents

1989

Authors: Yang, Y., C. M. Ladisch, and M. R. Ladisch
Journal: Enzyme Microb. Technol., 10(10), 632-636 (1988)
Book Chapter:

Abstract: The textile industry is one of the top 10 water consuming industries with much being used in chemical treatment of textiles. Effluent treatment for pollution control is important due to the color and chemistry of the dyes and additives used during dyeing. Textile effluents vary in composition, and, thus, numerous approaches have been developed for treatment of dyeing waste water with several steps being combined to obtain effluent suitable for discharge. For example, treatment of typical dyes used on synthetic fibers entails four stages: calcium chloride reaction, electrolysis, activated carbon decolorization, and filtration.

Research Area: Bioseparations    

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Hydrophobic Interaction Chromatography

1989

Authors: Roettger, B. F., and M. R. Ladisch
Journal: Biotech. Adv., Vol. 7, 15-29 (1989)
Book Chapter:

Abstract: Hydrophobic interaction chromatography (HIC) is emerging as a useful technique for the separation of biological compounds. Advances in the past two years in HIC applications, stationary phase, eluents, and theory are reviewed. Recent applications of HIC processes include analytical and semi-preparative separations of a variety of proteins, such as isolectins, hemoglobins, calmodulin, and cardiotoxins. Additionally, HIC is being employed as a tool to investigate protein properties and mechanisms. Advances in HIC stationary phases include development of non-porous, microparticulate supports as well as supports with pore sizes up to 1000 Angstroms. Studies of HIC eluents have further shown the effects of mobile phase pH, water-structuring characterization, and surface tension increments on retention. Various retention mechanisms which have been presented are reviewed; and a correlation relating resolution to column and solute parameters is presented. Protein conformational effects at specific sites have been shown to have a significant impact on retention and specific examples illustrating such effects are discussed.

Research Area: Bioseparations    

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Mechanisms of Protein Retention in Hydrophobic Interaction Chromatography

1989

Authors: Roettger, B., J. Myers, M. R. Ladisch, and F. Regnier
Journal: American Chemical Society, 80-92, (1989)
Book Chapter:

Abstract: Protein retention in hydrophobic interaction chromatography (HIC) depends on surface hydrophobicity of the support and solute and the kosmotropic nature and concentration of the salt used in the mobile phase. Wyman’s linkage theory, extended to provide a unifying model of HIC retention, relates protein retention to the preferential interactions of the mobile phase salt with the support and protein. Preferential interactions of ammonium salts with HIC supports were determined by extremely sensitive densimetric measurements. Chromatographic retention of lysozyme was also determined on a column packed with hydrophilic polymoric supports and retention of myoglobin was determined on butyl-derivatized polymeric sobenis. Mobile phases containing ammonium salts of SO4, C2H3O2, Cl-, and I- at several concentrations were used to probe retention behavior of lysozyme and myoglobin with respect to these supports. Preferential interactions of the salts with the supports and proteins were found to explain adsorption behavior in hydrophobic interaction chromatography, and results in an equation which predicts capacity factor as a function of lyotropic number and salt molality.

Research Area: Bioseparations    

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Electrochromatography

1988

Authors: Rudge, S. R., and M. R. Ladisch
Journal: Biotechnol. Prog., 4(3), 123-133 (1988)
Book Chapter:

Abstract: Chromatography is a separation process that exploits different chemical and/or physical affinities between the different components of a mixture and a fixed solid sorbent or gel matrix. The solid or gel may be chemically modified to enhance its affinity for a particular component. A source of energy, in the form of a temperature or chemical gradient, is often used to drive or improve the separation. Chemical gradients are usually used to specifically desorb a component, or to focus that component in a small volume of eluent. The scale-up of chromatography of protein mixtures has progressed along the lines of other fixed-bed operations, with special attention being paid to sorbent chemistry, sorbent particle size, and bed dimensions.

Research Area: Bioseparations    

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Liquid Chromatography of Carbohydrate Monomers and Oligomers

1988

Authors: Lin, J. K., B. J. Jacobson, A. N. Pereira, and M. R. Ladisch
Journal: Biomass Handbook: Academic Press, Inc., Methods in Enzymology 160, (1988)
Book Chapter:

Abstract: Cellodextrins, water-soluble B-1,4 oligomers of glucose with degree of polymerization (DP) between 2 and 7 (Table 1), are important substrates for the characterization of cellulases. There contribution to understanding the kinetics of cellulose hydrolysis has been reviewed. Cellodextrins can be used to infer features of the active site of carbohydrates and to study induction and repression of enzyme synthesis during fermentation.

Research Area: Bioseparations    

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Cause and Correction of Baseline Interruptions Observed for Small-Bore Liquid Chromatography Columns Using Cation Exchange Resin in the H+ Form

1987

Authors: Lin, J. K., S. J. Karn, and M. R. Ladisch
Journal: Biotechnol. Bioeng, 30, 331-333 (1987)
Book Chapter:

Abstract: Liquid chromatography of oligo- and monosaccharides using cation exchange resin in the H+ form with water or aqueous buffer as the eluent has proven utility in the analysis of hydrolysates obtained from biomass. As smaller diameter (2 mm i.d.) micro-columns come into use, greater sensitivities of sugar analysis and faster analysis times will result. Experience with such columns in our laboratory has suggested several procedures which minimize operating instabilities unique to 2 mm i.d. columns and not observed when larger bore (3.2 to 8 mm i.d.) columns are used. While the causes and solutions for these problems seem almost trivial, in retrospect, the details are briefly mentioned here to aid other biomass researchers who may have observed similar phenomena.

Research Area: Bioseparations    

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Polysaccharides as Adsorbents- An Update on Fundamental Properties and Commercial Prospects

1987

Authors: Lee, J. Y., and M. R. Ladisch
Journal: Annals of the New York Academy of Sciences, 506, 491-498, (1987)
Book Chapter:

Abstract: Ethanol is produced by fermentation of starch or sugars using yeast. The concentration of ethanol produced by the fermentation process is usually low (less than 10%). Thus, economically feasible separation processes to remove water from ethanol are important. Distillation has been widely used to separate ethanol from water. Because water forms an azeotropic mixture with ethanol, an additional step to break the azeotrope is required. This step needs a third solvent such as benzene. The energy consumption in this conventional process is about 22,000 Btu/gallon ethanol, in which 10,000 Btu/gallon ethanol is used to break the azeotrope and to recover the benzene. An alternative, energy-efficient way of breaking the azeotrope is with an adsorption process. Previous studies have indicated that a polysaccharide material such as corn is an excellent absorbent for this purpose. Selective adsorption of water is carried out by feeding water-ethanol vapor to a fixed bed column packed with corn grits. After the adsorption, regeneration is carried out by allowing hot inert gas to pass through the column. A schematic representation of this process is shown in Figure 2. Advantages of this process are: (1) corn is less expensive (10 cents/lb) than other commercial adsorbents; (2) regeneration is possible at a relatively low temperature (100 °C); and (3) the heat of adsorption is retained inside the column during the adsorption so that the heat can be utilized for the regeneration.

Research Area: Bioseparations    

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Separation by Adsorption

1987

Authors: Ladisch, M. R.
Journal: Advanced Biochemical Engineering, H. R. Bungay and G. Belfort, eds., J. Wiley & Sons, NY (1987)
Book Chapter:

Abstract: Many processes for manufacturing biochemicals have costs that are dominated by the expense of purification. Fermentation products are diluted by water and contaminated by debris, salts, proteins, and a variety of compounds that may have properties quite similar to those of the desired material. Purification usually starts with some way to increase the concentration of the product so that large volumes of water need not be handled during the more selective steps. Processes such as solvent extraction and ion exchange can accomplish severalfold concentration and considerable purification. Separation of the product from molecules with similar properties can be very difficult. This chapter will cover two methods that are in large-scale use: column procedures for vapor-phase adsorption of water and liquid chromatography.

Research Area: Bioseparations    

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Sulfuric Acid-Sugar Separation by Ion Exclusion

1987

Authors: Neuman, R. P., S. R. Rudge, and M. R. Ladisch
Journal: Reactive Polymers J., 5, 55-61 (1987)
Book Chapter:

Abstract: Biomass conversion continues to have significant potential in the production of fuel ethanol by fermentation. A major cost in acid hydrolysis of biomass to fermentable sugars is the acid itself. Separation and recycle of the acid could reduce ethanol production costs by $0.10/gallon or more. In this context, a process that separates sulfuric acid from glucose using ion-exclusion technology is presented. A 61 cm long, fixed-bed of Rohm and Haas Amberlite IR-118, strong cation-exchange resin in the hydrogen form was used. Samples containing 7.7% H2SO4 and 1.0% glucose, at sample volumes of 10% to 50% of the column void volume gave separation of H2SO4 from glucose at column temperatures ranging from 27 to 81 °C with water as eluent at a superficial velocity of 0.6 cm/min. Skewing of the H2SO4 peak was observed and traced to a sharp density gradient between the acid and the water eluent while the glucose peak was sufficiently symmetric to be fitted by an axial dispersion model. This work shows that a chromatographic resin having a particle size range of 300 to 1200 micrometers can give complete recovery of sulfuric acid with 94% recovery of glucose. This case study has interesting implications for both the practice of process chromatography, using a resin with large particle size at gross loading conditions, and the prospects of further reducing fermentable sugar costs in biomass conversion.

Research Area: Bioseparations    

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Leaching: A Separation Process for the Production of Fuels and Chemicals from Biomass

1986

Authors: Ladisch, M. R., G. T. Tsao, and K. Lin
Journal: Biotechnol. Bioeng. Symp. Ser., No. 15, 723-736 (1986)
Book Chapter:

Abstract: The application of biotechnology to the production of fuels and chemicals from biomass must include consideration of separation processes. One objective is to recover products of biomass processing in a concentrated form, if possible. The ability of biomass to absorb water will result in low fermentable sugar concentrations. A low moisture hydrolysis, coupled with leaching, can give fermentable sugar concentrations above 15%, and hence, circumvents this problem. Calculations required to design a sugar recovery process in a systematic manner are outlined.

Research Area: Biofuels/Bioproducts Bioseparations   

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Leaching: A Separation Process for the Production of Fuels and Chemicals from Biomass

1986

Authors: Ladisch, M. R., G. T. Tsao, and K. Lin
Journal: Biotechnol. Bioeng. Symp. Ser., No. 15, 723-736 (1986)
Book Chapter:

Abstract: The application of biotechnology to the production of fuels and chemicals from biomass must include consideration of separation processes. One objective is to recover products of biomass processing in a concentrated form, if possible. The ability of biomass to absorb water will result in low fermentable sugar concentrations. A low moisture hydrolysis, coupled with leaching, can give fermentable sugar concentrations above 15%, and hence, circumvents this problem. Calculations required to design a sugar recovery process in a systematic manner are outlined.

Research Area: Bioseparations    

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Process Considerations for Scale-up of Liquid Chromatography and Electrophoresis

1986

Authors: Rudge, S., and M. R. Ladisch
Journal: American Chemical Society symp. 314, 122-152, (1986)
Book Chapter:

Abstract: Chromatography is an important preparative and industrial process. Scale-up of chromatographic processes requires computation of mass transfer characteristics as a function of column area, support particle size and feed volume. In this context, an analytical solution for longitudinal diffusion in packed beds developed by Lapidus and Amundson, is used to demonstrate the characteristics of a typical size exclusion separation of proteins, including estimation of maximum sample size as allowed by support properties. Electrophoresis is also a powerful fractionation technique for proteins, but is subject to many microscopic effects. These include electric double layers, hydrodynamic drag, and electrical relaxation. In addition, macroscopic effects, such as electroosmosis and thermal gradients, also impact separation efficiency. These effects are discussed in relation to elution processes using selected examples. The combination of an electric field with a chromatographic process has recently been proposed to extend the power of electrophoresis separations. Analysis of such a process, referred to as electrochromatography, is also present.

Research Area: Bioseparations    

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Water Sorption Properties of a Polysaccharide Adsorbent

1986

Authors: Neuman, R., M. Voloch, P. Bienkowski, and M. R. Ladisch
Journal: I&EC Fundamentals, 25, 422-425 (1986)
Book Chapter:

Abstract: A flow method was used to determine equilibrium isotherms of the water-corn grit system in the temperature range of 343-373 K. A modified Henderson’s equilibrium equation was used to fit the experimental data. Calculated values of the heat of adsorption (10.8-14.6 kcal/g-mol) and surface area (170 m2/g) are similar to those previously reported based on data taken at lower temperatures. These data extend knowledge of the equilibrium behavior of water with respect to corn grits to the temperature range used for the selective adsorption of water from alcohol vapor by corn.

Research Area: Bioseparations    

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Breakthrough Behavior of 17.5 mol % Water in Methanol, Ethanol, Isopropanol, and t-Butanol Vapors Passed over Corn Grits

1985

Authors: Bienkowski, P., A. Barthe', M. Voloch, R. N. Neuman, and M. R. Ladisch
Journal: Biotechnol. Bioeng., 28(7), 960-964 (1985)
Book Chapter:

Abstract:

Research Area: Bioenergy Bioseparations   

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Cornmeal Adsorber for Dehydrating Ethanol Vapors

1984

Authors: Ladisch, M. R., M. Voloch, J. Hong, P. Bienkowski and G. T. Tsao
Journal: Ind. Eng. Chem. Process Des. Dev., 23(3), 437-443 (1984)
Book Chapter:

Abstract:

Research Area: Bioseparations    

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Adsorption of Ethanol/Water Mixtures by Biomass Materials

1982

Authors: Hong, J., M. Voloch, M. R. Ladisch, and G. T. Tsao
Journal: Biotechnol. Bioeng. 24, 725-730 (1982)
Book Chapter:

Abstract: The commercial production of biomass-derived ethanol is dependent on the energy balance which is defined as the ratio of the combustible energy obtained from the product to the energy necessary to its production. The recovery of ethanol in an anhydrous form from an 8-12% solution (wt. %) can be energy intensive.

Research Area: Bioseparations    

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Calcium Sulfate as a Selective Adsorbent of Water

1982

Authors: Pocium, D., M. Ladisch, G. Tsao, and P. Wankat
Journal: AIChE Annual Meeting (1982)
Book Chapter:

Abstract: The removal of water from fermentation ethanol in an energy efficient manner is of practical interest for a biomass conversion/fermentation process. The use of CaSO4 to adsorb water has been suggested and investigated many years ago. Our work has focused on removing water from 90 to 95.6% ethanol to give an anhydrous product. It is in this range that the separation becomes challenging because of energy considerations and the ethanol/water azeotrope. We examined the properties of CaSO4 and found that while it is a selective adsorbent of water from ethanol vapor (at 80 to 90 °C) and can be regenerated at 140 to 150 °C, it can also rapidly lose its capacity. Careful fundamental research in our laboratory shows that this loss of capacity is probably related to changes in the crystalline structure of CaSO4 which occur at 150o C when the partial pressure of the water vapor is relatively high. We found that increasing the temperature form 80 to 140 °C gradually during regeneration (instead of a step change to 140 °C) minimizes changes in crystalline structure. Hence, CaSO4 is quite stable in repetitive use. Using these conditions, CaSO4 was examined in a bench-scale adsorber. Breakthrough and temperature profiles were measured and then applied to characterize adsorber performance.

Research Area: Bioseparations    

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Separation of Meso and Racemic 2,3-Butanediol by Aqueous Liquid Chromatography

1981

Authors: Voloch, M., M. R. Ladisch, V. W. Rodwell, and G. T. Tsao
Journal: Biotechnol. Bioeng., 23, 1289-1296 (1981)
Book Chapter:

Abstract:

Research Area: Bioseparations Biofuels/Bioproducts   

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Vapor-Liquid Equilibria of the Water-Ethanol System at Low Alcohol Concentration

1981

Authors: Hong, J., M. R. Ladisch, and G. T. Tsao
Journal: J. Chem. Eng. Data, 26, 305-307 (1981)
Book Chapter:

Abstract: Vapor-liquid equilibrium data for the ethanol-water system at 760 mmHg are collected by using a Gillespie-type still. Data in the range of ethanol concentration from 0.01 to 1.0 wt % are scarce, and yet they are much needed in the design of efficient ethanol recovery systems.

Research Area: Bioseparations    

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Separation of Methanol Derivatives of Imidazolidinines, Urea and Carbonates by Aqueous Liquid Chromatography

1980

Authors: Beck, K. R., B. J. Leibowitz, and M. R. Ladisch
Journal: J. Chromatogr. 190, 226-232 (1980)
Book Chapter:

Abstract: Agents used to crosslink cellulose and produce durable press cotton fabrics include bis(1,3-hydroxymethyl) 4,5-dihydroxyimidazolidinone (1), hydroxymethyl derivatives of urea (II), carbamates (III), and other nitrogenous compounds. Since 100 million pounds of these chemicals are used annually by the U.S.A. textile industry, the analysis for these water soluble compounds is of significant interest. Previously reported methods of analysis include gas chromatography (GC), thinlayer chromatography (TLC), gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR).

Research Area: Bioseparations    

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Dehydration of Ethanol: New Approach Gives Positive Energy Balance

1979

Authors: Ladisch, M. R. and K. Dyck
Journal: Science, 205(4409), 898-900 (1979)
Book Chapter:

Abstract: Water was removed from aqueous ethanol by using cellulosic materials, starch, corn, and other agents. The combustion energy of the ethanol product can exceed the energy needed to carry out the dehydration by a factor of 10.

Research Area: Bioseparations    

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High-Speed Liquid Chromatography of Cellodextrins and Other Saccharide Mixtures Using Water as the Eluent

1978

Authors: Michael R. Ladisch, Aronson L. Huebner, and George T. Tsao
Journal: Journal of Chromatography, 147, 185-193 (1978)
Book Chapter:

Abstract: Ion-exchange resin AG 50W-X4 (Ca2+) separates the oligomers celloheltaose through glucose within 30 min when water is used as the eluent. The fractionation capabilities of this resin are a function of the procedures used in its preparation, packing, and operation. These procedures are described in detail. In addition to chromatograms showing separations obtained for cellodextrins and other saccharide mixtures, quantitative data relating concentrations of individual cellodextrin components to their respective peak areas are also presented.

Research Area: Bioseparations    

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Theory and Practice of Rapid Liquid Chromatography at Moderate Pressures Using Water as Eluent

1978

Authors: Ladisch, M. R. and G. T. Tsao
Journal: J. Chromatogr. 166, 85-100 (1978)
Book Chapter:

Abstract: The rapid separation of oligo- and monosaccharides on long, narrow columns packed with easily compressed, 4% cross-linked, cation-exchange resin adds a new dimension to the chromatography of carbohydrates on gel type supports: speedy analysis at low pressure. The discovery of a pressure-critical diameter effect in the packing of Aminex 50W-X4 (Ca2+) makes it possible to pack 60-cm long columns capable of separating malto- and cellodextrins in 12-15 min using water as the sole eluent. The surprising phenomena of a 50-fold increase in pressure due to an increase in column diameter from 6 to 8 mm is reported and reasons for this effect are explained. Equally noteworthy is the stability of the 6-mm columns. One column described in this report has been in continuous operation for over 2600 h. The application of low-pressure liquid chromatography to enzyme kinetics as well as to separation of oligo- and monosaccharides is also discussed.

Research Area: Bioseparations    

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