Development of Aqueous Two-Phase Separations by Combining High-Throughput Screening and Process Modelling
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Protocols and Tips in Protein Purification
Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11. -
Ultracentrifugation Techniques for the Ordering of Nanoparticles
nanomaterials Review Ultracentrifugation Techniques for the Ordering of Nanoparticles Xufeng Xu 1,† and Helmut Cölfen 2,* 1 Laboratory of Physical Chemistry, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5612AE Eindhoven, The Netherlands; [email protected] 2 Physical Chemistry, University of Konstanz, Universitätsstraße 10, Box 714, 78457 Konstanz, Germany * Correspondence: [email protected] † Present address: Institute of Materials, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. Abstract: A centrifugal field can provide an external force for the ordering of nanoparticles. Especially with the knowledge from in-situ characterization by analytical (ultra)centrifugation, nanoparticle ordering can be rationally realized in preparative (ultra)centrifugation. This review summarizes the work back to the 1990s, where intuitive use of centrifugation was achieved for the fabrication of colloidal crystals to the very recent work where analytical (ultra)centrifugation is employed to tailor-make concentration gradients for advanced materials. This review is divided into three main parts. In the introduction part, the history of ordering microbeads in gravity is discussed and with the size of particles reduced to nanometers, a centrifugal field is necessary. In the next part, the research on the ordering of nanoparticles in analytical and preparative centrifugation in recent decades is described. In the last part, the applications of the functional materials, fabricated from centrifugation-induced nanoparticle superstructures are briefly discussed. Keywords: centrifugation; sedimentation; nanoparticle; concentration gradient; non-equilibrium process; superstructure; functional material Citation: Xu, X.; Cölfen, H. Ultracentrifugation Techniques for 1. Introduction the Ordering of Nanoparticles. 1.1. The Era of Microbeads in Gravity Nanomaterials 2021, 11, 333. -
Continuous-Flow Centrifugation to Collect Suspended Sediment for Chemical Analysis
1 Table 6. Compounds detected in both equipment blank samples and not in corresponding source blank samples or at concentrations greater than two times the corresponding source blank sample concentration. Prepared in cooperation with the National Water Quality Monitoring Council and [Source data: Appendix A, table A1; Conn and Black (2014, table A4); and Conn and others (2015, table A11). CAS Registry Number: Chemical Abstracts Service Washington State Department of(CAS) Ecology Registry Number® (RN) is a registered trademark of the American Chemical Society. CAS recommends the verifi cation of CASRNs through CAS Client ServicesSM. Method: EPA, U.S. Environmental Protection Agency’s SW 846; SIM, select ion monitoring. Unit: µg/kg, microgram per kilogram; ng/kg, nanogram per kilogram. Sample type: River samples were from the Puyallup River, Washington. Q, qualifi er (blank cells indicate an unqualifi ed detection). J, estimated, result between the Continuous-Flow Centrifugationdetection level and reporting level; toNJ, result Collect did not meet all quantitation Suspended criteria (an estimated maxiumum possible concentration is reported in Result column) U, not detected above the reporting level (reported in the Result column); UJ, not detected above the detection level (reported in the Result column). Abbreviations: na, not Sediment for Chemicalapplicable; Analysis PCBs, polychlorinated biphenyls] Sample type Chapter 6 of CAS River River Commercial Commercial Section D, Water Quality Parameter name Registry Method Unit source equipment -
Nanosep® Centrifugal Devices - Protocols for Use Nanosep® Centrifugal Devices - Protocols for Use
Contact Us: www.pall.com/contact Nanosep® Centrifugal Devices - Protocols for Use Nanosep® Centrifugal Devices - Protocols for Use Ultrafiltration Fundamentals Purification and Handling of DNA Fragments PCR: Before and After Protein Purification and Handling Miscellaneous Protocols Appendices References Ordering Information Ultrafiltration Fundamentals Background Ultrafiltration (UF) is a membrane separation technique used to separate extremely small particles and dissolved molecules in fluids. The primary basis for separation is molecular size, although other factors such as molecule shape and charge can also play a role. Molecules larger than the membrane pores will be retained at the surface of the membrane (not in the polymer matrix as they are retained in microporous membranes) and concentrated during the ultrafiltration process. Compared to non-membrane processes (chromatography, dialysis, solvent extraction, or centrifugation), ultrafiltration: Is far gentler to the molecules being processed. Does not require an organic extraction which may denature labile proteins. Maintains the ionic and pH milieu. Is fast and relatively inexpensive. Can be performed at low temperatures (for example, in the cold room). Is very efficient and can simultaneously concentrate and purify molecules. The retention properties of ultrafiltration membranes are expressed as Molecular Weight Cutoff (MWCO). This value refers to the approximate molecular weight (MW) of a dilute globular solute (i.e., a typical protein) which is 90% retained by the membrane. However, a molecule’s shape can have a direct effect on its retention by a membrane. For example, linear molecules like DNA may find their way through pores that will retain a globular species of the same molecular weight. There are three generic applications for ultrafiltration: 1. -
Centrifugal Hyperfiltration
TO: Technology Innovation Program Mail Stop 4750 National Institute of Standards and Technology 100 Bureau Drive Gaithersburg, MD 20899-4750 FM: Dirk Forman 125 S. St. Louis Lafayette Louisiana 70506 [email protected] Centrifugal Hyperfiltration A means to economically recover resources from saltwater and wastewater streams with lower the energy cost of producing freshwater and potable water from marginalized water resources. Introduction Reverse Osmosis (RO) is a filtration process for the removal of ionic and organic pollutants from wastewater. Today’s technology of utilization of this filtration process is by large array of high-pressure piping and pressure pumps. This process yields low volumes of filtrated output (permeate), utilizes large areas for pipe array and components and the concentration polarization and membrane fouling hinders the wide application of RO filtration process. Utilizing centrifugal forces and cross flow membranes has potential to change the standard (RO) process to one of portable and high volume water purification. AN AREA OF CRITICAL NATIONAL NEED Ensuring Future Water Supply: As the Nation’s population and economy grow, greater demands are being placed on freshwater resources. At the same time, temporary or permanent drought conditions and water access rights affect regional freshwater availability. Water needs threaten to outstrip available freshwater, now and in the future. Emerging contaminants that must either be removed from distributed water or converted to harmless forms of waste is also pressuring water quality, both in terms of decontamination and disinfection of water supplies. Food contaminations are often traced back to water contaminations, either in the field or in processing. Municipal waste streams and irrigation runoff waste resources that are not recovered.1 Over 97 percent of the Earth's water -- seawater and brackish groundwater -- is too salty to use for drinking water or agriculture. -
Coagulation-Flocculation As a Submerged Biological Filter Pre-Treatment with Landfill Leachate
Coagulation-flocculation as a submerged biological filter pre-treatment with landfill leachate A. Gálvez Perez1,2, A. Ramos1,2,3, B. Moreno1,2,3 & M. Zamorano Toro1,2 1Department of Civil Engineering, Granada University, Spain 2MITA Research Group 3Institute of Water Research Abstract Landfill leachate may cause environmental problems if it is not properly managed and treated. An appropriate treatment process of landfill leachate often involves a combination of physical, chemical and biological methods to obtain satisfactory results. In this study, coagulation-flocculation was proposed as a pre- treatment stage of partially stabilized landfill leachate prior to submerged biological filters. Several coagulants (ferric, aluminium or organic) and flocculants (cationic, anionic or non-ionic) were assayed in jar-test experiments in order to determine optimum conditions for the removal of COD and total solids. Among the cationic flocculants, that of highest molecular weight and cationicity (CV/850) showed highest removal efficiencies (15% COD and 8% TS). Organic and aluminium coagulants showed better results than ferric coagulants. Coagulant removal efficiencies were between 9% and 17% for COD and between 10% and 15% for TS. Doses of 1 ml/l of coagulant were preferred. Some combinations of coagulant and flocculant enhanced the process. The best combinations obtained were FeCl3+A30.L, Ferriclar+A20.L, SAL8.2+A30.L and PAX-18+A30.L, which presented COD removal efficiencies between 24% and 37% with doses between 10 and 18 ml/l. Keywords: landfill leachate, coagulation-flocculation, submerged biological filter pre-treatment. Waste Management and the Environment II, V. Popov, H. Itoh, C.A. Brebbia & S. -
404 Sedimentation Handout
Sedimentation for Analysis and Separation of Macromolecules A ‘centrifugal force’ as experienced by particles in a centrifuge actually reflects the natural tendency of the particle to move in a straight line in the absence of external forces. If we view the experiment in the (fixed) reference frame of the laboratory, we observe that the particle is deflected from a linear trajectory by a centripetal force (directed toward the central axis of the rotor). However, when we consider the motion of the particle with respect to the (rotating) reference frame of the rotor, it appears that at any instant the particle experiences an outward or centrifugal force: Instantaneous Velocity (vtang) Centrifugal Acceleration Centripetal Acceleration As seen in laboratory As seen in (rotating) reference frame reference frame of rotor Ignoring buoyancy effects for the moment, the magnitude of the centrifugal force (which, in the rotating reference frame of the rotor, is as real as any other force) is m ⋅ v2 F = objecttan g =⋅mrω 2 centrif r object [1] where r is the distance from the axis of rotation, vtang is the linear velocity at any instant (in the direction perpendicular to the radial axis) and ω is the angular velocity (in radians sec-1 – note that 1 rpm = 2π///60 rad sec-1). The magnitude of this force can be compared to that of the gravitational force at the earth’s surface: Gm⋅()⋅() m = earth object Fgrav 2 re [2] -8 -1 3 -2 where G is the universal gravitational constant (= 6.67 x 10 g cm sec ) and re and me are the earth’s radius (= 6.37 x 108 cm) and mass (5.976 x 1027 g), respectively. -
PROTEIN a CHROMATOGRAPHY – the PROCESS ECONOMICS DRIVER in Mab MANUFACTURING
PROCESS Application Note PROTEIN A CHROMATOGRAPHY – THE PROCESS ECONOMICS DRIVER IN mAb MANUFACTURING THE OPTIMIZATION OF THE PROTEIN A CAPTURE STEP IN DOWNSTREAM PROCESSING PLATFORMS CAN CONSIDER- ABLY IMPROVE PROCESS EFFICIENCY AND ECONOMICS OF INDUSTRIAL ANTIBODY MANUFACTURING. PARAMETERS LIKE RESIN REUSE AND ITS CAPACITY CONTRIBUTE CONSIDERABLY TO THE PRODUCTION COSTS. THE USE OF A HIGH CAPACITY PROTEIN A RESIN CAN IMPROVE THE PROCESS EFFICIENCY AND ECONOMICS. THIS PAPER PRESENTS THE KEY FEATURES OF A NEW CAUSTIC STABLE PROTEIN A RESIN PROVIDING EXTREMELY HIGH IgG BINDING CAPACITIES. Biopharmaceuticals represent an ever growing important Protein A affinity resins are dominating the Cost of Goods part of the pharmaceutical industry. The market for recom- (COGs) of mAb manufacturing. Bioreactors at the 10.000 L binant proteins exceeded $ 100 billion in 2011 with a scale operating at a titer of about 1g/L typically generate contribution of 45% sales by monoclonal antibodies (mAbs) costs of $ 4-5 million (2). Therefore the Protein A capturing (1). The introduction of the first mAb biosimilars in Europe step is the key driver to improve process economics. Besides raised the competitive pressure in an increasingly crowded the capacity of the resin, life time and cycle numbers market place. The industry faces challenges, such as patent significantly contribute to the production costs in mAb expirations accompanied by approvals of corresponding manufacturing. biosimilars, failures in clinical trials/rejections or the refusal of health insurers to pay for new drugs. Today, the IgG binding capacities of most Protein A resins are in the range of 30-50 g/L, offering significant advantages These challenges force the industry to minimize risk and time- for the processing of high-titer feedstreams when compared to-market and to proceed more cautiously. -
Integrated Leaching and Separation of Metals Using Mixtures of Organic Acids and Ionic Liquids
molecules Article Integrated Leaching and Separation of Metals Using Mixtures of Organic Acids and Ionic Liquids Silvia J. R. Vargas, Helena Passos , Nicolas Schaeffer * and João A. P. Coutinho CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] (S.J.R.V.); [email protected] (H.P.); [email protected] (J.A.P.C.) * Correspondence: nicolas.schaeff[email protected] Academic Editors: Magdalena Regel-Rosocka and Isabelle Billard Received: 4 November 2020; Accepted: 25 November 2020; Published: 27 November 2020 Abstract: In this work, the aqueous phase diagram for the mixture of the hydrophilic tributyltetradecyl phosphonium ([P44414]Cl) ionic liquid with acetic acid (CH3COOH) is determined, and the temperature dependency of the biphasic region established. Molecular dynamic simulations of the [P44414]Cl + CH3COOH + H2O system indicate that the occurrence of a closed “type 0” biphasic regime is due to a “washing-out” phenomenon upon addition of water, resulting in solvophobic segregation of the [P44414]Cl. The solubility of various metal oxides in the anhydrous [P44414]Cl + CH3COOH system was determined, with the system presenting a good selectivity for CoO. Integration of the separation step was demonstrated through the addition of water, yielding a biphasic regime. Finally, the [P44414]Cl + CH3COOH system was applied to the treatment of real waste, NiMH battery black mass, being shown that it allows an efficient separation of Co(II) from Ni(II), Fe(III) and the lanthanides in a single leaching and separation step. Keywords: critical metals; process intensification; metal leaching; solvent extraction; solvometallurgy 1. Introduction Solvometallurgy, the extraction of metals from primary and secondary sources using solutions containing less than 50 vol. -
A Basic Chloride Method for Extracting Aluminum from Clay
(k Bureau of Mines Report of investigations/l984 A Basic Chloride Method for Extracting Aluminum From Clay By P. R. Bremner, L. J. Nicks, and D. J, Bauer UNITED STATES DEPARTMENT OF THE INTERIOR Report of Investigations 8866 A Basic Chloride Method for Extracting Aluminum From Clay By P. R. Bremner, L. J. Nicks, and D. J. Bauer UNITED STATES DEPARTMERIT OF THE INTERIOR William P. Clark, Secretary BUREAU OF MINES Robert C. Worton, Director Library of Congress Cataloging in Publication Data: Bremner, P, R. (Paul R,) A basic chloride method for extracting aluminum from clay. (Bureau of Mines report of investigations ; 8866) Bibliography: p. 8. Supt. of Docs. no.: I 28.23:8866. 1. Alumit~urn-Metallurgy. 2. Leaching. 3. Chlorides. 4, Kao- linite, I. Nicks, 1;. J. (Larry J.), 11. Bauer, D. J. (Donald J,). 111. Title. IV. Series: Report of investigations (United States. Bureau of TN23.U43 [TN776] 622s [669'.722] 84-600004 CONTENTS .Page Abstract ....................................................................... 1 Introduction ................................................................... 2 Materials. equipment. and procedures .......O.O...........,............... 3 Results and discussion........................................................b 3 Effects of calcination time and temperature .................................. 3 Single-stage leaching and crystallization... ................................. 4 Countercurrent leaching and crystallization.................................. 5 Purification and solubility studies ........................*................ -
Lecture 08: Leaching and Extraction
Mass transfer Lecture 08: Leaching and extraction Jamin Koo 2019. 10. 10 1 Learning objectives • Understand when leaching would be a more preferred, effective separation method. • Analyze cascades leaching using the material balance equation and associated graphs. • Determine when liquid extraction will be preferred versus distillation. 2 Today’s outline • Leaching Introduction Leaching equipment, dispersed solid leaching Cascades leaching, equilibrium analysis Variable underflow Ex. 23.1 • Liquid extraction Introduction Terminology and types 3 23.1 Introduction to Leaching • Definition: method of removing one constituent from a solid or liquid by means of a liquid solvent • Examples In chemical process In daily life 4 23.1 Mass transport • Generally there are five rate steps in the leaching process. (1) The solvent is transferred from the bulk solution to the surface of the solid. (fast) (2) The solvent penetrates or diffuses into the solid. (slow) (3) The solute dissolves from the solid into the solvent. (slow) (4) The solute diffuses through the mixture to the surface of the solid. (5) The solute is transferred to the bulk solution. 5 23.1 Leaching equipment • Generally, two types are used with respect to whether or not the mixture (often solid) is permeable. When working with permeable mass, percolation through stationary solid beds may be used. What will impact percolation? What will impact leaching? 6 23.1 Leaching equipment • Generally, two types are used with respect to whether or not the mixture (often solid) is permeable. When working with impermeable mass, solids are dispersed into the solvent and are later separated from it. e.g., moving-bed leaching 7 23.1 Dispersed-solid leaching • Solid is dispersed in the solvent by mechanical agitation in a tank or flow mixer. -
Manufacturing of Agarose-Based Chromatographic Media with Controlled Pore and Particle Size
MANUFACTURING OF AGAROSE-BASED CHROMATOGRAPHIC MEDIA WITH CONTROLLED PORE AND PARTICLE SIZE by Nicolas Ioannidis A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Chemical Engineering College of Engineering and Physical Sciences The University of Birmingham 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Chromatography remains the most commonly employed method for achieving high resolution separation of large-sized biomolecules, such as plasmid DNA, typically around 150-250 nm in diameter. Currently, fractionation of such entities is performed using stationary phases designed for protein purification, typically employing pore sizes of about 40 nm. This results into a severe underexploitation of the porous structure of the adsorbent as adsorption of plasmid DNA occurs almost exclusively on the outer surface of the adsorbent. In this study, the effect of two processing parameters, the ionic strength of agarose solution and quenching temperature, on the structure of the resulting particles was investigated. Three characterization methods, Atomic Force and cryo-Scanning Electron microscopy, as well as mechanical testing of single particles where used to quantify the effect of these parameters on the pore size/size distribution and mechanical properties of the adsorbent.