DOCSLIB.ORG

Analysis of Whole Bacterial Cells by Flow Field-Flow Fractionation and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Analysis of Whole Bacterial Cells by Flow Field-Flow Fractionation and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry Anal. Chem. 2003, 75, 2746-2752 Analysis of Whole Bacterial Cells by Flow Field-Flow Fractionation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Hookeun Lee² and S. Kim Ratanathanawongs Williams* Chemistry and Geochemistry Department, Colorado School of Mines, Golden, Colorado 80401 Karen L. Wahl and Nancy B. Valentine Pacific Northwest National Laboratory, Richland, Washington 99352 The purpose of this study is to develop a novel bacterial be a more universal approach for microorganism analysis since analysis method by coupling the flow field-flow fraction- molecular weight information can be obtained regardless of the ation (flow FFF) separation technique with detection by type of bacterium present. matrix-assisted laser desorption/ionization time-of-flight Matrix-assisted laser desorption/ionization time-of-flight (MALDI- (MALDI-TOF) mass spectrometry. The composition of TOF) mass spectrometry has been demonstrated as a potentially carrier liquid used for flow FFF was selected based on rapid and accurate identification method for bacteria.5-15 The retention of bacterial cells and compatibility with the majority of bacterial analyses using MALDI-TOF are based on MALDI process. The coupling of flow FFF and MALDI- mass spectral patterns derived from cellular proteins. The mass TOF MS was demonstrated for P. putida and E. coli. spectral patterns of different bacteria are unique, which provides Fractions of the whole cells were collected after separation a basis for bacterial identification.16 Successful identification of by FFF and further analyzed by MALDI-MS. Each fraction, bacteria in a mixture of up to three components was demon- collected over different time intervals, corresponded to strated.16 However, this identification approach was challenged different sizes and possibly different growth stages of when one component was present in lower concentrations. Since bacteria. The bacterial analysis by flow FFF/MALDI-TOF MALDI is a competitive ionization process, it will be difficult to MS was completed within 1 h with only preliminary detect the signature peaks from complex sample matrixes unless optimization of the process. the mass spectrometric analysis is preceded by a separation or isolation method to simplify the sample. One approach we are Rapid and accurate identification of pathogenic bacteria is investigating is the preseparation of the intact bacterial cells prior extremely important in flagging food and beverage contamination, diagnosing disease, and signaling environmental biohazards. The (5) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; analysis of bacteria present in complex sample matrixes usually Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10, consists of a separation or isolation step followed by enumeration 1227-1232. and identification. However, conventional culturing techniques for (6) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996, 10, 883-888. enumeration take anywhere from hours to days. Thus, new (7) Welham, K. J.; Domin, M. A.; Scannell, D. E.; Cohen, E.; Ashton, D. S. Rapid methods for rapid detection and identification are being pursued Commun. Mass Spectrom. 1998, 12, 176-180. including nucleic acid sequence-based techniques such as poly- (8) Krishnamurthy, T.; Ross, P. Rapid Commun. Mass Spectrom. 1996, 10, 1992-1996. 1,2 merase chain reaction and antibody-based methods such as (9) Holland, R. D.; Burns, G.; Persons, C. C.; Rafii, F.; Sutherland, J. B.; Lay, J. enzyme-linked immunosorbent assays3,4 for specific detection of O., Jr. Proc. 45th ASMS Conf. Mass Spectrom. Allied Top., Palm Springs, known organisms. Many of the current approaches, e.g., antibody- CA, 1997; p 1353. (10) Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass based methods, have limited usefulness in a broad spectrum Spectrom. 1998, 12, 456-464. approach to microorganism identification since a priori knowledge (11) Holland, R. D.; Duffy, C. R.; Rafii, F.; Sutherland, J. B.; Heinze, T. M.; Holder, of the organisms of concern is required. Mass spectrometry may C. L.; Voorhees, K. J.; Lay, J. O., Jr. Anal. Chem. 1999, 71, 3226-3230. (12) Demirev, P. A.; Ho, Y.-P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, * Corresponding author. E-mail: [email protected]. Fax: (303) 273-3629. 2732-2738. ² Institute for Systems Biology, 1441N 34th St., Seattle, WA 98103. (13) Winkler, M. A.; Uher, J.; Cepa, S. Anal. Chem. 1999, 71, 3416-3419. (1) Chen, J.; Johnson, R.; Griffiths, M. Appl. Environ. Microbiol. 1998, 64, 147- (14) Rose, N. L.; Sporns, P.; McMullen, L. Y. Appl. Environ. Microbiol. 1999, 152. 65, 2238-2242. (2) Waller, D. F.; Ogata, S. A. Appl. Environ. Microbiol. 2000, 66, 4115-4118. (15) Hathout, Y.; Demirev, P. A.; Ho, Y.-P.; Bundy, J. L.; Ryzhov, V.; Sapp, L.; (3) Johnson, R. P.; Durham, R. J.; Johnson, S. T.; MacDonald, L. A.; Jeffrey, S. Stutler, J.; Jackman, J.; Fenselau, C. Appl. Environ. Microbiol. 1999, 65, R.; Butman, B. T. EHEC-Tek. Appl. Environ. Microbiol. 1995, 61, 386- 4313-4319. 388. (16) Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; (4) Milley, D. G.; Sekla, L. H. Appl. Environ. Microbiol. 1993, 59, 4223-4229. Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223. 2746 Analytical Chemistry, Vol. 75, No. 11, June 1, 2003 10.1021/ac020698u CCC: $25.00 © 2003 American Chemical Society Published on Web 05/30/2003 to MALDI-MS. Conventional separation techniques such as high- combinations of field and separation mode give rise to different performance liquid chromatography and gel electrophoresis have FFF techniques. been used for the analysis of bacterial cell extracts prior to MALDI- In this study, flow (or cross-flow) FFF was used to fractionate TOF MS.17,18 However, these techniques are not successful for bacterial cells prior to MALDI-TOF analyses. The cross-flow ªfieldº dealing with intact bacterial particles. In addition, size exclusion interacts with sample components through positive displacement. chromatography is one of the most widely used techniques for This leads to two features that make flow FFF unique among the macromolecular fractionation but is not a suitable technique for FFF family. First, flow FFF is universally applicable since the biological particles that would plug the column. Capillary elec- cross-flow sweeps everything to the accumulation wall, and thus, trophoresis, which separates bacteria on the basis of differences there is no requirement on sample properties; e.g., the sample in electrophoretic mobilities, has been shown to yield rapid (<10 species must be charged in order to use electrical FFF. Second, min) and high-efficiency separations (>106 plates/m).19-24 The in the absence of differentiating interactions between the cross- electrophoretic mobilities are dependent upon surface properties flow ªfieldº and the sample components, the flow FFF separation of bacteria and the solution in which the bacteria are sus- process occurs as a result of differences in diffusion coefficients. pended.20,22 Hence, care must be taken in the preparation of The smaller faster diffusing components occupy equilibrium microbes and microbial solutions.20,22 This can be a difficult positions further from the channel accumulation wall and are endeavor because of the complexity of microbes and the variety eluted prior to larger size components. Hence, flow FFF separa- of environments in which they are found. tions are based solely on the size of the sample species, making In the present study, we demonstrate the feasibility of a novel this technique particularly suited for complex samples that also approach of separating intact bacterial cells using flow field-flow have distributions in density, charge, etc. This simplifies the fractionation (flow FFF) as a precursor to MALDI-MS analysis. interpretation of flow FFF results. FFF is a family of elution techniques capable of performing high- Previous FFF studies for bacteria were performed using resolution separation and characterization of particles and mac- sedimentation FFF (centrifugal force is the external field).32-35 romolecules ranging in size from 1 nm to 100 µm.25,26 Retention These works involved submicrometer-sized bacteria and the in most types of FFF is based on differences in the physicochem- normal mode separation mechanism. High-resolution separations ical properties such as size, density, charge, and composition. were achieved, but each run required 50-100 min. Typically, these Field-flow fractionation uses a thin, ribbonlike, open channel times can be reduced by increasing the channel flow rate or through which sample components are transported by a carrier decreasing the field strength. Alternately, the hyperlayer mech- liquid. The separation occurs by differential migration in the anism can be invoked to yield high-speed separations.36 In the stream of the carrier flowing through the channel. The flow profile hyperlayer mode, high channel flow rates generate hydrodynamic in such a channel is parabolic with the fastest flow velocity at the lift forces that propel sample away from the accumulation wall.37 center of the channel. Different transverse distributions of sample The distance between the particle and the wall is determined by components in the parabolic flow profile are induced by counter- the balance between lift forces and the cross-flow-induced force action between diffusion of components and an external field as shown in Figure 1. Larger particles experience greater lift forces applied perpendicular to the separation axis. The external field and elute earlier than the smaller particle. Compared to the normal drives the sample to the accumulation wall of the channel while mode mechanism that was used in the previous FFF studies, this the diffusion causes migration of the sample away from the wall. hyperlayer mode is capable of high-resolution separations in very Each component will be transported at a different velocity short times. Hence, this is the separation mechanism of choice depending on its equilibrium position in the channel.
Load more

Recommended publications
  • Gas Chromatography-Mass Spectroscopy
    Gas Chromatography-Mass Spectroscopy Introduction Gas chromatography-mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical techniques. As the name implies, it is actually two techniques that are combined to form a single method of analyzing mixtures of chemicals. Gas chromatography separates the components of a mixture and mass spectroscopy characterizes each of the components individually. By combining the two techniques, an analytical chemist can both qualitatively and quantitatively evaluate a solution containing a number of chemicals. Gas Chromatography In general, chromatography is used to separate mixtures of chemicals into individual components. Once isolated, the components can be evaluated individually. In all chromatography, separation occurs when the sample mixture is introduced (injected) into a mobile phase. In liquid chromatography (LC), the mobile phase is a solvent. In gas chromatography (GC), the mobile phase is an inert gas such as helium. The mobile phase carries the sample mixture through what is referred to as a stationary phase. The stationary phase is usually a chemical that can selectively attract components in a sample mixture. The stationary phase is usually contained in a tube of some sort called a column. Columns can be glass or stainless steel of various dimensions. The mixture of compounds in the mobile phase interacts with the stationary phase. Each compound in the mixture interacts at a different rate. Those that interact the fastest will exit (elute from) the column first. Those that interact slowest will exit the column last. By changing characteristics of the mobile phase and the stationary phase, different mixtures of chemicals can be separated.
    [Show full text]
  • Qualitative and Quantitative Analysis
    qualitative and quantitative analysis Russian scientist Tswett in 1906 used a glass columns packed with divided CaCO3(calcium carbonate) to separate plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one. Tswett was the first to use the term "chromatography" derived from two Greek words "Chroma" meaning color and "graphein" meaning to write. Invention of Chromatography by M. Tswett Ether Chromatography Colors Chlorophyll CaCO3 5 *Definition of chromatography *Tswett (1906) stated that „chromatography is a method in which the components of a mixture are separated on adsorbent column in a flowing system”. *IUPAC definition (International Union of pure and applied Chemistry) (1993): Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction. *Principles of Chromatography * Any chromatography system is composed of three components : * Stationary phase * Mobile phase * Mixture to be separated The separation process occurs because the components of mixture have different affinities for the two phases and thus move through the system at different rates. A component with a high affinity for the mobile phase moves quickly through the chromatographic system, whereas one with high affinity for the solid phase moves more slowly. *Forces Responsible for Separation * The affinity differences of the components for the stationary or the mobile phases can be due to several different chemical or physical properties including: * Ionization state * Polarity and polarizability * Hydrogen bonding / van der Waals’ forces * Hydrophobicity * Hydrophilicity * The rate at which a sample moves is determined by how much time it spends in the mobile phase.
    [Show full text]
  • Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry of Biomolecules Yu-Chen Chang Iowa State University
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1996 Laser desorption/ionization time-of-flight mass spectrometry of biomolecules Yu-chen Chang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Analytical Chemistry Commons Recommended Citation Chang, Yu-chen, "Laser desorption/ionization time-of-flight mass spectrometry of biomolecules " (1996). Retrospective Theses and Dissertations. 11366. https://lib.dr.iastate.edu/rtd/11366 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been rqjroduced fix>m the microfihn master. UMI fihns the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, w^e others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afifect reproduction. In the unlikely event that the author did not send IMl a complete manuscript and there are misang pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps.
    [Show full text]
  • Separations Development and Application (WBS 2.4.1.101) U.S
    Separations Development and Application (WBS 2.4.1.101) U.S. Department of Energy (DOE) Bioenergy Technologies Office (BETO) 2017 Project Peer Review James D. (“Jim”) McMillan NREL March 7, 2017 Biochemical Conversion Session This presentation does not contain any proprietary, confidential, or otherwise restricted information. Project Goal Overall goal: Define, develop and apply separation processes to enable cost- effective hydrocarbon fuel / precursor production; focus on sugars and fuel precursor streams; lipids pathway shown. Outcome: Down selected proven, viable methods for clarifying and concentrating the sugar intermediates stream and for recovering lipids from oleaginous yeast that pass “go” criteria (i.e., high yield, scalable, and cost effective). Relevance: Separations are key to overall process integration and economics; often represent ≥ 50% of total process costs; performance/efficiency can make or break process viability. Separations this project investigates – sugar stream clarification and concentration, and recovery of intracellular lipids from yeast – account for 17-26% of projected Minimum Fuel Selling Price (MFSP) for the integrated process. 2 Project Overview • Cost driven R&D to assess/develop/improve key process separations - Sugar stream separations: S/L, concentrative and polishing - Fuel precursor recovery separations: oleaginous yeast cell lysis and LLE lipid recovery • Identify and characterize effective methods - Show capability to pass relevant go/no-go criteria (e.g., high yield, low cost, scalable) • Exploit in situ separation for process intensification - Enable Continuous Enzymatic Hydrolysis (CEH) • Generate performance data to develop / refine process TEAs and LCAs 3 Separations Technoeconomic Impact $1.78 $2.18 $1.43 $0.79 4 Quad Chart Overview Timeline Barriers Primary focus on addressing upstream and Start: FY 15 (Oct., ‘14) downstream separations-related barriers: End: FY 17 (Sept., ‘17; projected) – Ct-G.
    [Show full text]
  • Rubber-Modified Epoxies: in Situ Detection of the Phase Separation by Differential Scanning Calorimetry
    Rubber-modified epoxies: in situ detection of the phase separation by differential scanning calorimetry Dong Pu Fang*, C. C. Riccardi and R. J. J. Williamst Institute of Materials Science and Technology (INTEMA), University of Mar de/Plata and National Research Council (CONICET), J. B. Justo 4302, 7600 Mar de/Plata, Argentina (Received 18 May 1993) The phase separation of a rubber in the course of a thermosetting polymerization is associated with heat release. Mixing is endothermic as revealed by the positive value of the interaction parameter while demixing is exothermic. Differential scanning calorimetry may be used to detect heat effects related to phase separation. Experimental results are reported for an unreactive system (castor oil/epoxy resin) and reactive systems (modifier/epoxy resin/diamine) using either castor oil or epoxy-terminated copolymers of acrylonitrile and butadiene as modifiers. (Keywords: rubber-modified epoxies; phase separation; d.s.c.) Introduction represents the residual free energy of mixing (part or all A common method to toughen epoxy resins is to of it may be considered as an enthalpic contribution). dissolve rubbers or other modifiers that become phase- The interaction parameter A, expressed in units of energy separated in the course of polymerization. The resulting per unit volume, is usually a positive value. This means two-phase structure exhibits an increase in the fracture that mixing occurs due to the prevailing influence of the resistance at the expense of lower values of the elastic combinatorial part of the equation over the interaction modulus and yield stress. term. Dissimilar molecules (positive A) are thus obliged Monitoring the phase separation process is important to mix due to an entropic driving force; their intrinsic in two respects: (i) for unreactive epoxy-rubber repulsion is manifested by an endothermic effect in the formulations, i.e.
    [Show full text]
  • Advanced Separation Techniques Combined with Mass Spectrometry for Difficult Analytical Tasks - Isomer Separation and Oil Analysis
    FINNISH METEOROLOGICAL INSTITUTE Erik Palménin aukio 1 P.O. Box 503 FI-00101 HELSINKI 131 CONTRIBUTIONS tel. +358 29 539 1000 WWW.FMI.FI FINNISH METEOROLOGICAL INSTITUTE CONTRIBUTIONS No. 131 ISBN 978-952-336-016-7 (paperback) ISSN 0782-6117 Erweko Helsinki 2017 ISBN 978-952-336-017-4 (pdf) Helsinki 2017 ADVANCED SEPARATION TECHNIQUES COMBINED WITH MASS SPECTROMETRY FOR DIFFICULT ANALYTICAL TASKS - ISOMER SEPARATION AND OIL ANALYSIS JAAKKO LAAKIA Department of Chemistry University of Helsinki Finland Advanced separation techniques combined with mass spectrometry for difficult analytical tasks – isomer separation and oil analysis Jaakko Laakia Academic Dissertation To be presented, with the permission of the Faculty of Science of the University of Helsinki, for public criticism in Auditorium A129 of the Department of Chemistry (A.I. Virtasen aukio 1, Helsinki) on May 12th, 2017, at 12 o’clock noon. Helsinki 2017 1 TABLE OF CONTENTS ABSTRACT 4 Supervisors Professor Tapio Kotiaho TIIVISTELMÄ 5 Department of Chemistry LIST OF ORIGINAL PAPERS 6 Faculty of Science ACKNOWLEDGEMENTS 7 ABBREVIATIONS 8 and 1. INTRODUCTION 10 Division of Pharmaceutical Chemistry and Technology 1.1.1 Basic operation principles of ion mobility 12 spectrometry 11 Faculty of Pharmacy 1.1.2 Applications of ion mobility spectrometry 16 University of Helsinki 1.2 Basic operation principles and applications of two-dimensional gas 19 chromatography – time-of-flight mass spectrometry Docent Tiina Kauppila 1.3 Aims of study 23 Division of Pharmaceutical Chemistry and Technology
    [Show full text]
  • Electrification Ionization: Fundamentals and Applications
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 11-12-2019 Electrification Ionization: undamentalsF and Applications Bijay Kumar Banstola Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Analytical Chemistry Commons Recommended Citation Banstola, Bijay Kumar, "Electrification Ionization: undamentalsF and Applications" (2019). LSU Doctoral Dissertations. 5103. https://digitalcommons.lsu.edu/gradschool_dissertations/5103 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. ELECTRIFICATION IONIZATION: FUNDAMENTALS AND APPLICATIONS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by Bijay Kumar Banstola B. Sc., Northwestern State University of Louisiana, 2011 December 2019 This dissertation is dedicated to my parents: Tikaram and Shova Banstola my wife: Laxmi Kandel ii ACKNOWLEDGEMENTS I thank my advisor Professor Kermit K. Murray, for his continuous support and guidance throughout my Ph.D. program. Without his unwavering guidance and continuous help and encouragement, I could not have completed this program. I am also thankful to my committee members, Professor Isiah M. Warner, Professor Kenneth Lopata, and Professor Shengli Chen. I thank Dr. Fabrizio Donnarumma for his assistance and valuable insights to overcome the hurdles throughout my program. I appreciate Miss Connie David and Dr.
    [Show full text]
  • Electrochemical Separation Is an Attractive Strategy for Development of Radionuclide Generators for Medical Applications
    Current Radiopharmaceuticals, 2012, 5, 271-287 271 Electrochemical Separation is an Attractive Strategy for Development of Radionuclide Generators for Medical Applications Rubel Chakravarty, Ashutosh Dash and M.R.A. Pillai* Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai – 400 085, India Abstract: Electrochemical separation techniques are not widely used in radionuclide generator technology and only a few studies have been reported [1-4]. Nevertheless, this strategy is useful when other parent-daughter separation techniques are not effective or not possible. Such situations are frequent when low specific activity (LSA) parent radionuclides are used for instance with adsorption chromatographic separations, which can result in lower concentration of the daughter radionuclide in the eluent. In addition, radiation instability of the column matrix in many cases can affect the performance of the generator when long lived parent radionuclides are used. Intricate knowledge of the chemistry involved in the elec- trochemical separation is crucial to develop a reproducible technology that ensures that the pure daughter radionuclide can be obtained in a reasonable time of operation. Crucial parameters to be critically optimized include the applied potential, choice of electrolyte, selection of electrodes, temperature of electrolyte bath and the time of electrolysis in order to ensure that the daughter radionuclide can be reproducibly recovered in high yields and high purity. The successful electrochemi- cal generator technologies which have been developed and are discussed in this paper include the 90Sr/90Y, 188W/188Re and 99Mo/99mTc generators. Electrochemical separation not only acts as a separation technique but also is an effective concen- tration methodology which yields high radioactive concentrations of the daughter products.
    [Show full text]
  • Separation Science - Chromatography Unit Thomas Wenzel Department of Chemistry Bates College, Lewiston ME 04240 [email protected]
    Separation Science - Chromatography Unit Thomas Wenzel Department of Chemistry Bates College, Lewiston ME 04240 [email protected] LIQUID-LIQUID EXTRACTION Before examining chromatographic separations, it is useful to consider the separation process in a liquid-liquid extraction. Certain features of this process closely parallel aspects of chromatographic separations. The basic procedure for performing a liquid-liquid extraction is to take two immiscible phases, one of which is usually water and the other of which is usually an organic solvent. The two phases are put into a device called a separatory funnel, and compounds in the system will distribute between the two phases. There are two terms used for describing this distribution, one of which is called the distribution coefficient (DC), the other of which is called the partition coefficient (DM). The distribution coefficient is the ratio of the concentration of solute in the organic phase over the concentration of solute in the aqueous phase (the V-terms are the volume of the phases). This is essentially an equilibration process whereby we start with the solute in the aqueous phase and allow it to distribute into the organic phase. soluteaq = soluteorg [solute]org molorg/Vorg molorg x Vaq DC = --------------- = ------------------ = ----------------- [solute]aq molaq/Vaq molaq x Vorg The distribution coefficient represents the equilibrium constant for this process. If our goal is to extract a solute from the aqueous phase into the organic phase, there is one potential problem with using the distribution coefficient as a measure of how well you have accomplished this goal. The problem relates to the relative volumes of the phases.
    [Show full text]
  • Transport Processes: Momentum, Heat, and Mass 01-P2401 1/9/03 12:14 PM Page 3
    01-P2401 1/9/03 12:14 PM Page 1 PART 1 Transport Processes: Momentum, Heat, and Mass 01-P2401 1/9/03 12:14 PM Page 3 CHAPTER 1 Introduction to Engineering Principles and Units 1.1 CLASSIFICATION OF TRANSPORT PROCESSES AND SEPARATION PROCESSES (UNIT OPERATIONS) 1.1A Introduction In the chemical and other physical processing industries and the food and biological process- ing industries, many similarities exist in the manner in which the entering feed materials are modified or processed into final materials of chemical and biological products. We can take these seemingly different chemical, physical, or biological processes and break them down into a series of separate and distinct steps that were originally called unit operations. How- ever, the term “unit operations” has largely been superseded by the more modern and de- scriptive term “separation processes.” These separation processes are common to all types of diverse process industries. For example, the separation process distillation is used to purify or separate alcohol in the beverage industry and hydrocarbons in the petroleum industry. Drying of grain and other foods is similar to drying of lumber, filtered precipitates, and wool. The separation pro- cess absorption occurs in absorption of oxygen from air in a fermentation process or in a sewage treatment plant and in absorption of hydrogen gas in a process for liquid hydrogena- tion of oil. Evaporation of salt solutions in the chemical industry is similar to evaporation of sugar solutions in the food industry. Settling and sedimentation of suspended solids in the sewage industry and the mining industry are similar.
    [Show full text]
  • HPLC: High Pressure Liquid Chromatography
    HPLC: High Pressure Liquid Chromatography 2013 Chem 413 Introduction Chromatography can be described as a mass transfer process involving adsorption using a nonpolar stationary phase and a mobile polar phase titrating through the column. The active component of the column, the sorbent or the stationary phase, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2-50 µm in size. The components of the sample mixture are separated from each other by means of mobile phase and different degrees of interaction with the sorbent particles based on their relative polarity. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol). Its composition and temperature plays a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical in nature, such as hydrophobic, dipole-dipole or ionic. High performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture. Before the invention of HPLC, chemists had column chromatography at their disposal, and column chromatography was time consuming. To speed up a classic column chromatography, chemists would have to use a short column for separation, however this lead to poor separation of molecular components held within solution. The basic setup of a classic column chromatography would include the column that varied in I.D. from 10 to 50nm and column lengths of 50-500cm. The column was then packed with the stationary phase ranging in particle size from 150 to 200 µm thick.
    [Show full text]
  • An Introduction to Liquid Chromatography Mass Spectrometry
    An Introduction to Liquid Chromatography Mass Spectrometry Dr Kersti Karu email: [email protected] Office number: Room LG11 Recommended Textbook: “Analytical Chemistry”, G. D. Christian, P. K. Dasgupta, K.A. Schug, Wiley, 7th Edition “Trace Quantitative Analysis by Mass Spectrometry”, R.K. Boyd, C.Basic, R.A. Bethem, Wiley Applications Lecture Overview • Rate theory - van Deemter equation for HPLC • Principle of LC analysis – HPLC subclasses – Stationary phases in HPLC – LC columns • Liquid chromatography-mass spectrometry (LC-MS) – Separation process in reversed-phase liquid chromatography – Electrospray ionisation (ESI) source – Taylor cone formation in ESI process – Quadrupole ion trap (QIT) analyser – TOF analyser – TOF analysers with V and W geometries – Orbitrap analyser – Tandem mass spectrometry • Sterolomics • Brief introduction to LC-MS practical Principle of LC-MS analysis • HPLC: flow rate 1-2 mL/min, 4.6 mm ID column, 3-or 5-µm silica-based particles, 5-30 µL injection. Basic components of an HPLC system • Capillary LC: 200 µL/min, 1 mm ID column, 1.5- to 3-µm silica-based particles, 1-5 µL injection • Nano LC: 200 nL/min, 0.1 mm ID column, 3-µm silica-based particles, 0.1-2 µL injection • UHPLC systems (ultra-high-pressure liquid chromatography): 0.6 - 1 mL/min, 1.5- to 3- µm silica-based particles capable of pumping at 15,000 psi. Particles in LC columns Analytes are separated based on their differential affinity between a solid stationary phase and a liquid mobile phase. The kinetics of distribution of analytes between the stationary and the mobile phase is largely diffusion-controlled.
    [Show full text]