DOCSLIB.ORG

Reversed-Phase and Hydrophilic Interaction Chromatography Columns for Size

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Reversed-Phase and Hydrophilic Interaction Chromatography Columns for Size Reversed-Phase and Hydrophilic Interaction Chromatography Columns for Size- Exclusion Chromatography A Thesis Submitted to the Faculty of Drexel University by Anna Maria Caltabiano in partial fulfillment of the requirements for the degree of Doctor of Philosophy March 2017 © Copyright 2017 Anna M. Caltabiano. All Rights Reserved ii Dedications This thesis is dedicated to seven wonderful people who mean the world to me: To my son Greg Matthews and my future daughter-in-law Paulina Iniewicz, who have always been my sources of encouragement and my greatest inspirations! To my loving husband Joe Miscavage, who has always been supporting, helping and standing by me. To my parents, Nadiya and Gregory Matevosyan, without whose unconditional and endless support this work would not have been possible. To my sister Marianna Zimmerli and to my nephew Andrei Polyschuk, who encouraged me to pursue my dreams and finish my journey towards PhD. iii Acknowledgments First and foremost, I am grateful to God, whose many blessings have formed me into the person I am today and allowed me to see this long journey through against all odds. I am very thankful to Father Aleksandr and Father Mark from Saint Andrew’s Cathedral in Philadelphia, and Father Aleksiy from Holy Trinity Church in Odessa, Ukraine, for their constant prayers and blessings. There have been so many people who have contributed to my success. I would like to acknowledge and thank each of them for their kindness, guidance and support. First of all, I would like to express my sincere gratitude and appreciation to my research advisor, Professor Joe. P. Foley for his fundamental role in my PhD studies and research. My success would not have been possible without his expertise, guidance, support, and encouragement. I greatly value the close professional rapport that Prof. Foley and I have acquired over the years and feel privileged to have been his student. I would like to express my special appreciation and thanks to my committee chair, Prof. Kevin Owens, for taking the time and effort to chair both my Oral Candidacy and PhD defense committees. I gratefully acknowledge the members of my PhD committee, Prof. Anthony Addison, Prof. Peter Wade, Prof. Karl Sohlberg, Prof. Daniel King, and Prof. Alexander Tsygankov (Temple University), for taking time out to participate and for providing their great scientific insight to my research. They all have contributed to my success as brilliant teachers that went above and beyond their instructor duties. iv My gratitude also goes to Prof. Alan Bandy and Prof. Jean Claude Bradley who left a mark on my research and who served on my Oral Candidacy Exam committee, but regretfully were not able to see the completion of this thesis. They have finished their earth journeys and went on to join our Father in heaven. Scientific research was everything to Prof. Bandy, it was his life, and it was the learning part of it that fascinated him the most. He taught his students that it is not just theories and laws from books, but hands on experiences are the key to success in the professional career. In sync, an innovative researcher Prof. Bradley has always emphasized the importance of publically sharing the research results, even if unsuccessful. I am honored to have known these outstanding teachers and true research scientists. Eternal Memory. I would like to acknowledge contributions of Dr. Howard Barth from Analytical Chemistry Consultants Ltd. and Dr. André Striegel from NIST to my research work. I’m very thankful to Dr. Barth for sharing his extensive experience in polymer analysis and co-authoring my first publication in size-exclusion chromatography research. During that period, Dr. Barth was facing very challenging time with his health, where he was in and out of hospitals, receiving extensive pain therapy, but still eager to see research results and to engage with novel experiments. I cannot thank Dr. Striegel enough for providing his invaluable guidance on the manual calculations of polymer molar masses, review of accuracy results and comments on solvent, temperature screens. Without his prospects and scrupulous analysis of the data, it would be very difficult to arrive to the right v conclusions. I feel privileged to have my work reviewed by such an advanced and outstanding researcher of our time. I cannot express enough gratitude to my employer GlaxoSmithKline for the financial support, use of laboratory, equipment, and reagents. I would like to sincerely thank my current and past leadership team and managers at GlaxoSmithKline: Dr. Al Kearney, Mr. Paul Cummings, Dr. Alireza Kord, Dr. Suwimon Asawasiripong, Dr. Gerald Terfloth, Dr. Fran Muller, Dr. Philip Floyd, Dr. Leon Zhou and Dr. Anne Payne, who have supported me throughout this quest. I’m especially thankful to Mr. Paul Cummings and Dr. Suwimon Asawasiripong for sending their recommendation letters on my behalf to Drexel University to support my application and then acceptance into the PhD program. I’m deeply grateful to Dr. Gerald Terfloth who set the direction for my PhD research and provided an outstanding coaching for my Oral Candidacy Exam. I feel honored to work/ have worked with these true scientists who make significant contributions to science on the daily basis and fulfill GSK mission to help people do more, feel better, live longer. Last, but not least, I would like to thank all my family for their love, commitment and sacrifices. There are no words to express the feelings I have for them, who are my source of strength and inspiration, for their constant unconditional love and support. I would not embark on this endeavor if it was not for them. vi TABLE OF CONTENTS List of Tables ...................................................................................................................... x List of Figures .................................................................................................................. xiv Abstract .......................................................................................................................... xxiii CHAPTER 1: INTRODUCTION ....................................................................................... 1 1.1. The Birth of Chromatography .................................................................................. 1 1.2. The Evolution of Chromatography .......................................................................... 3 1.3. Development of Bonded Phases for LC ................................................................... 7 1.4. The LC Column ........................................................................................................ 9 1.4.1. Properties of LC Column Packings ................................................................ 10 1.4.1.1. Physical Properties of Column Packings .................................................... 10 1.4.1.2. Column Chemistry ...................................................................................... 13 1.4.1.2.1. Silica Supports......................................................................................... 13 1.4.1.2.1.1. Silica-Based Bonded Phases ................................................................... 14 1.4.1.2.2. Polymeric Packings ................................................................................. 17 1.5. Size-exclusion Chromatography ............................................................................ 18 1.6. Reversed-Phase and HILIC Columns for Size-exclusion Chromatography .......... 21 CHAPTER 2: NON-AQUEOUS SIZE-EXCLUSION CHROMATOGRAPHY OF SYNTHETIC POLYMERS ON COMMON REVERSED-PHASE AND HILIC COLUMNS ....................................................................................................................... 25 2.1. Introduction ............................................................................................................ 25 2.2. Theory .................................................................................................................... 29 2.3. Experimental .......................................................................................................... 34 vii 2.3.1. Materials ......................................................................................................... 34 2.3.2. HPLC .............................................................................................................. 35 2.3.3. Sample Preparation ......................................................................................... 37 2.4. Results and Discussion ........................................................................................... 37 2.4.1. Non-aqueous SEC on RP Columns ................................................................ 37 2.4.2. Effect of Column Surface Chemistry on Elution Behavior ............................ 41 2.4.3. Effect of Solute, Eluent and Stationary Phase Polarities on Elution Behavior .. ........................................................................................................................ 43 2.4.4. Effect of Pore Size on Elution Behavior ........................................................ 56 2.5. Conclusions ............................................................................................................ 60 CHAPTER 3: SOLVENT AND TEMPERATURE EFFECTS IN NON-AQUEOUS SIZE-EXCLUSION CHROMATOGRAHPY ON REVERSED-PHASE COLUMNS ... 64 3.1. Introduction ...........................................................................................................
Load more

Recommended publications
  • I. Chromatography
    Chromatography & Electrophoresis I. Chromatography • Chroma: “color” + graph: “measure” Separation Techniques – Developed in 19th century to separate pigments from plants and dyes Purifying & analyzing macromolecules – Refined technique now used to separate, (e.g., proteins & DNA) from a mixture analyze, & purify wide range of compounds I. Chromatography • Still called “chromatography” even though no longer limited to separating colored molecules II. Electrophoresis Mixture of macromolecule solutes from Chromatography sample dissolved in mobile phase Stationary phase: gel, beads, or other solid medium with specific absorptive characteristics Mobile phase: fluid solvent that flows over/through the stationary phase Separation of macromolecule solutes Solutes interact with stationary phase from mixture in original sample — ↑ interaction Æ Ø migration rate based upon relative rates of migration through the stationary phase Ø interaction Æ ↑ migration rate •Solutes that interact strongly with the stationary phase do not migrate very far Types of interactions: Interactions may be modified by manipulating •Solutes that interact with the stationary phase • Size migrate slower than the mobile phase • Charge the mobile phase: ∆ pH • Polarity/hydrophobicity ∆ Ionic strength • Non-specific binding ∆ Polarity •Solutes that do not interact with the stationary phase • Specific binding migrate at the same rate as the mobile phase ∆ Competitive binding Bruce Heyer 1 Chromatography & Electrophoresis Column chromatography Column chromatography • Solid phase as beads or particles packed in a cylinder • Solid phase as beads or particles packed in a cylinder Load sample containing mixture of macromolecules onto top of solid phase bed Pattern of bands reveals which • Elution macromolecules were in the sample mixture – Allows collecting separated fractions containing purified macromolecules Elute mobile phase fluid from bottom of column while adding fresh mobile phase to top Other types of chromatography II.
    [Show full text]
  • F:\IJCAS\Sept 2010 Ijcas\3Aasth
    Aastha Ahuja et al. /International Journal of Chemical and Analytical Science 2010, 1(9),205-207 Review Article Available online through ISSN: 0976-1209 www.ijcas.info Advances In HPLC Column Packing *Aastha Ahuja, Rahul Chib, Mukul Sonker, Geet Sethi, Shweta Gupta , Anju Hooda Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), University of Delhi Pushp vihar, sector-3, New Delhi-110017 Received on: 20-05-2010; Revised on: 16-06-2010; Accepted on:15-07-2010 ABSTRACT High Performance Liquid Chromatography (HPLC) is a highly improved form of column chromatography in which instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres(500-5000 p.s.i). The sensitivity and range of the technique depends on the choice of column and on the efficiency of the overall system. Column technology has seen great developments over the years. The transition from large porous particles and pellicular materials to small porous particles occurred in the early 1970s, when micro particulate silica gel (10 -mm dp) came into light and appropriate packing methods were developed. Monolithic columns are a promising alternative to packed columns in the future but much of the research is yet to be done. The use of CAPILLARY COLUMNS (4 mm) has increased in recent years, in part because small-diameter columns use much less solvent and provide higher sensitivity. While speaking of the future of packaging development, silica gel with chemically bonded phases will be around for a long time. The field is a promising one with great scope for research and development.
    [Show full text]
  • Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), Ion Exchange Chromatography (IC), Super Critical Fluid Chromatography (SFC)
    Chromatography For S. Y. B. Sc. Sem-IV By: Dr Vipul B. Kataria The technique was invented by Russian botanist Mikhail tswett in 1906. The word Chromatography is derived from latin language and in this word chroma means “colour” and graphein means “to write”. It can be defined as a method of separating a mixture of components into individual component through equilibrium distribution between two phases. Essentially, it is based upon the differences in rate at which the components of mixture move through a porous medium (stationary phase) under the influence of gas or liquid (mobile phase). Some basic terminology Analyte: substance that to be analysed. Chromatogram: Visual output of chromatography. Stationary phase: Non-moving phase that provides support for mixture to move. Mobile phase: Moving phase to specific direction. Eluent: Solvent that carries analyte. Retention time: it is specific time required for a particular analyte to pass through the system. Classification of chromatography Chromatography can be classified into different way based upon different aspects. 1 | Page 1. Based upon geometry On the basis of geometry, chromatography can be classified into following. a. Planar: Paper chromatography and thin layer chromatography (TLC) b. Column: Gas chromatography (GC), High performance liquid chromatography (HPLC), Ion exchange chromatography (IC), Super critical fluid chromatography (SFC). 2. Based upon mobile phase On the basis of mobile phase, chromatography can be classified into following. a. Gas chromatography: Mobile phase is gas. b. Liquid chromatography: Mobile phase is liquid. c. SFC: Supercritical fluid chromatography. Mobile phase is CO2 in liquid form. 3. Based upon stationary phase On the basis of stationary phase, Chromatography can be classified into following.
    [Show full text]
  • LC Columns and Accessories
    Thermo Scientific Chromatography Columns and Consumables Thermo Scientific Chromatography Columns and Consumables 2014-2015 2014-2015 Your world of chromatography predictability LC Columns and Accessories As a leader in LC column technology including silica, polymer and porous graphitic carbon manufacturing, bonded phase production and column packing for over 35 years, you can rely on the quality of Thermo Scientific HPLC products. Here we 2014-2015 showcase our latest and most comprehensive range of innovative columns, accessories and equipment for fast LC, analytical HPLC and biomolecule separations. Chromatography Columns and Consumables Thermo Scienti c Thermo Scienti 4 LC Columns and Accessories Section Contents Introduction . 4-001 LC Accessories. 4-158 Column Selection. 4-002 LC Equipment . 4-186 Column Protection . 4-016 LC Reagents . 4-193 Columns for Fast and Analytical LC . 4-022 LC and LC-MS Instrument Parts . 4-209 Columns for Biomolecules . 4-110 Technical Information. 4-231 Featured Products Accucore Columns Syncronis Columns Hypersil GOLD Columns Ultimate core performance - Consistent, reproducible separations, Outstanding peak shape for speed and selectivity combined column after column, time after time your separations PAGE 4-042 PAGE 4-074 PAGE 4-024 MAbPac Columns Acclaim PepMap Columns Viper Connectors Unrivalled resolution and ef ciency The standard for peptide Simple, dead volume free plumbing in the analysis of protein variants separations in proteomics of HPLC and UHPLC systems PAGE 4-126 PAGE 4-149 PAGE 4-158 www.thermoscienti c.com/chromatography 4-001 LC Column Selection Information in the following section will help you make an informed decision on the appropriate HPLC column for your application, based on stationary phase use, analyte properties, LC-MS requirements or USP specifications.
    [Show full text]
  • Basic Principles of Chromatography
    27 chapter Basic Principles of Chromatography Baraem Ismail ∗ Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108-6099, USA [email protected] and S. Suzanne Nielsen Department of Food Science, Purdue University, West Lafayette, IN 47907-2009, USA [email protected] 27.1 Introduction 475 27.2.3 Countercurrent Extraction 475 27.2 Extraction 475 27.3 Chromatography 475 27.2.1 Batch Extraction 475 27.3.1 Historical Perspective 475 27.2.2 Continuous Extraction 475 27.3.2 General Terminology 476 S.S. Nielsen, Food Analysis, Food Science Texts Series, DOI 10.1007/978-1-4419-1478-1_27, 473 °c Springer Science+Business Media, LLC 2010 474 Part V • Chromatography 27.3.3 Gas Chromatography 476 27.4.3 Ion-Exchange Chromatography 483 27.3.4 Liquid Chromatography 477 27.4.4 Size-Exclusion Chromatography 485 27.3.4.1 Paper Chromatography 477 27.4.5 Affinity Chromatography 488 27.3.4.2 Thin-Layer 27.5 Analysis of Chromatographic Peaks 489 Chromatography 478 27.5.1 Separation and Resolution 490 27.3.4.2.1 General 27.5.1.1 Developing a Separation 490 Procedures 478 27.5.1.2 Chromatographic 27.3.4.2.2 Factors Affecting Resolution 491 Thin-Layer 27.5.1.2.1 Introduction 491 Separations 478 27.5.1.2.2 Column 27.3.4.3 Column Liquid Efficiency 492 Chromatography 479 27.5.1.2.3 Column 27.3.5 Supercritical Fluid Chromatography 480 Selectivity 494 27.4 Physicochemical Principles of Chromatographic 27.5.1.2.4 Column Capacity Separation 481 Factor 494 27.4.1 Adsorption (Liquid–Solid) 27.5.2 Qualitative Analysis 495 Chromatography 481
    [Show full text]
  • Mechanism of Paper Chromatography of Inorganic Ions on Impregnated Papers: Separation of Ag(I), Cu(II), Ni(II), Co(II), Hg(II), Pbcii) & Feciii)
    indian Journal or Chemistry Vol. 15A, December 1977, pp. 1089-109l Mechanism of Paper Chromatography of Inorganic Ions on Impregnated Papers: Separation of Ag(I), Cu(II), Ni(II), Co(II), Hg(II), PbCII) & FeCIII) R. P. BHATNAGAR & N. P. BHATNAGAR Department of Chemistry. Jiwaji University. Gwalior 2 Received 24 September 1976; revised 28 March 1977; accepted 16 July 1977 Migration of ions on filter papers impregnated with aqueous glycine (2%, w/v) and ammonium thiocyanate (4%, w{v) solutions has been studied. Organic solvents like alcohols, ketones and chloroform have been used as mobile phases to get the RJ data. The migration tendencies of different ions have been explained on the basis of a new mechanism of paper chromatography with impregnated papers using polar and non-polar solvents. The migration of inorganic ions in polar solvents (with donor properties) on untreated papers has also been studied. ANY attempts have been made to explain the tapering free end (width 2 mm; length 2-3 em) were mechanism of paper chromatography+P, Re- prepared as described earlier13,14 from untreate d Mcently janardhan and Paul-l have tried to as well as treated (impregnated) papers. Spotting explain the migration of inorganic ions by proposing of the metal ions was done by micropipette (",,0·02 a stepwise mechanism involving (i) adsorption of the ml or 30-50 fLgper drop). After the spots had been metal ion on the cellulose through ion association dried, development was carried out by irrigating leading to ion-associated metal-cellulose complex, with the required solvent system (--2 hr) in airtight (ii) desorption of the ion due to its displacement chambers previously saturated with the vapours by the proton of the acid present in the solvent of the solvent concerned and water.
    [Show full text]
  • PAPER and THIN LAYER CHROMATOGRAPHY (TLC) Objectives
    PAPER AND THIN LAYER CHROMATOGRAPHY (TLC) Objectives • Understand the principle of Paper and Thin Layer Chromatography (TLC). • Diagnose two samples of urine for Phenylketonuria and Cystinuria, using paper chromatography and TLC. Chromatography • Is the collective term for a set of laboratory techniques for the separation of mixtures. • Chromatography is a method by which a mixture is separated by distributing its components between two phases. The stationary phase remains fixed in place while the mobile phase carries the components of the mixture through the medium being used • -The separation of materials is based on differential partitioning [retardation]between the mobile and stationary phases. Chromatography • The mobile phase (also known as solvent) may be either liquid or gas. • The stationary phase (also known as sorbent) can be either a solid or liquid, a liquid stationary phase is held stationary by a solid (support or matrix). • The molecules in the mixture to be separated are the solutes Uses of Chromatography Uses of chromatography in general Identifying To know the Can be used as a Establishing purity unknown property of a purification of a sample compounds. specific compound process Types of Chromatography will be studied in this course • Thin layer chromatography • Paper chromatography • Gel filtration • Ion exchange chromatography Paper and Thin layer chromatography • Method for testing the purity of compounds and identifying substances. • Useful technique because it is relatively quick and requires small quantities of material. 1-Paper chromatography - is one of the types of chromatography procedures which runs on a piece of specialized paper. Phases in which substance will be separated The stationary phase Mobile phase is a water molecules is the solvent (in our bound to an (inert) lab butanol with cellulose matrix acetic acid) • [solutes are separated according to their solubility in mobile phase, or their attraction to the stationary phase] -(Depend in the polarity).
    [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]
  • Laboratory Manual
    Biology Laboratory Manual Twelfth Edition Darrell S. Vodopich Baylor University Randy Moore University of Minnesota vod00720_fm_i-xii.indd 1 10/16/18 1:57 PM BIOLOGY LABORATORY MANUAL, TWELFTH EDITION Published by McGraw-Hill Education, 2 Penn Plaza, New York, NY 10121. Copyright © 2020 by McGraw-Hill Education. All rights reserved. Printed in the United States of America. Previous editions © 2017, 2014, and 2011. No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of McGraw-Hill Education, including, but not limited to, in any network or other electronic storage or transmission, or broadcast for distance learning. Some ancillaries, including electronic and print components, may not be available to customers outside the United States. This book is printed on acid-free paper. 1 2 3 4 5 6 7 8 9 LMN 21 20 19 ISBN 978-1-260-20072-0 (bound edition) MHID 1-260-20072-8 (bound edition) ISBN 978-1-260-41330-4 (loose-leaf edition) MHID 1-260-41330-6 (loose-leaf edition) Portfolio Manager: Andrew Urban Product Developer: Donna Nemmers Marketing Manager: Kelly Brown Content Project Managers: Jessica Portz & Sandra Schnee Buyer: Laura Fuller Design: David W. Hash Content Licensing Specialist: Lorraine Buczek Cover Image: ©Darrell S. Vodopich Compositor: MPS Limited All credits appearing on page are considered to be an extension of the copyright page. Some of the laboratory experiments included in this text may be hazardous if materials are handled improperly or if procedures are conducted incorrectly.
    [Show full text]
  • Experiment 4: Thin Layer Chromatography (TLC) in LTOC, Read the Introduction to Part 4, Chromatography, on Pp 253-254, Then Read Pp 255-269, Chapter 18
    1 Experiment 4: Thin Layer Chromatography (TLC) In LTOC, read the introduction to Part 4, Chromatography, on pp 253-254, then read pp 255-269, Chapter 18. View video 18.1 Thin-layer Chromatography at http://www.macmillanlearning.com/Catalog/studentresources/mohrig4e. Thin layer chromatography (TLC) is an easy, convenient and inexpensive way to determine how many components are in a mixture and, in many instances, can be used to identify the components as well. In today’s experiments, you will gain experience with both paper and thin layer chromatography, you will work with a variety of developing solvents, and you will use several different techniques to visualize the spots of a chromatogram. Paper chromatography, which will be used to separate amino acids, is a form of partition chromatography (see LTOC page 253). Water, a component of the developing solvent, forms hydrogen bonds with the fibers of the paper and serves as the stationary phase. The organic liquids that are also present in the developing solvent serve as the mobile phase. The components of the mixture are drawn up the paper to different heights, depending on their solubility in the mobile phase. The compounds that are more soluble in the organic liquid remain dissolved in the mobile phase longer than those that are less soluble and thus travel further up the paper. Proteins, large molecules found in all living organisms, serve a variety of functions in metabolism, such as catalysis, transport, storage, control of growth and immune protection. Amino acids are the building blocks of proteins. Every amino acid has an amino group, a carboxyl group and a distinctive side-chain.
    [Show full text]
  • Chromatography Chromatography Is Usually Introduced As a Technique for Separating And/Or Identifying the Components in a Mixture
    116 Modern Chemical Techniques THE ROYAL Unilever SOCIETY OF CHEMISTRY 5. Chromatography Chromatography is usually introduced as a technique for separating and/or identifying the components in a mixture. The basic principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent. It is a powerful method in industry, where it is used on a large scale to separate and purify the intermediates and products in various syntheses. The theory There are several different types of chromatography currently in use – ie paper chromatography; thin layer chromatography (TLC); gas chromatography (GC); liquid chromatography (LC); high performance liquid chromatography (HPLC); ion exchange chromatography; and gel permeation or gel filtration chromatography. Basic principles All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The techniques rely on one of the following phenomena: adsorption; partition; ion exchange; or molecular exclusion. Adsorption Adsorption chromatography was developed first. It has a solid stationary phase and a liquid or gaseous mobile phase. (Plant pigments were separated at the turn of the 20th century by using a calcium carbonate stationary phase and a liquid hydrocarbon mobile phase. The different solutes travelled different distances through the solid, carried along by the solvent.) Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. The result is a separation into bands containing different solutes. Liquid chromatography using a column containing silica gel or alumina is an example of adsorption chromatography (Fig.
    [Show full text]
  • Table of Contents
    Table of Contents List of Figures and Tables 3 List of Abbreviations 4 Abstract 5 Introduction 6 Background 6 Phytochemical categorization 6 Extraction 10 Separation and detection techniques 11 Experimental Methods 15 Chemicals and materials 15 Extraction 15 Absorbance 16 Paper chromatography 16 Total phenolic content 17 Capillary electrophoresis 17 Isoquercetin standard solution preparation 18 Data Analysis 18 Results and Discussion 19 Paper Chromatography 19 Extraction Method Optimization 20 Capillary Electrophoresis Optimization 26 Capillary Electrophoresis Analysis 30 Conclusion 35 Future Work 36 Acknowledgements 37 References 38 2 List of Figures and Tables Figure 1: Phytochemical categorization based on structure 8 Figure 2: Chemical structures of isoquercetin and quercetin 9 Figure 3: Bioactivity of previously identified phytochemicals by category in Moringa 10 oleifera Figure 4: Bioactivity of previously identified flavonoids by category in Moringa 10 oleifera Figure 5: Basic setup of capillary electrophoresis 13 Figure 6: Separation of compounds by size and charge in CZE 14 Figure 7: Extraction method for Moringa oleifera leaves 16 Figure 8: Paper chromatograph of Moringa oleifera leaves extract 20 Figure 9: Absorbance spectra of whole Moringa oleifera leaves extracted in 80% 22 ethanol, acidified 80% ethanol, and 100% ethanol by refluxing Figure 10: Absorbance spectra of whole Moringa oleifera leaves extracted in 80% 22 ethanol and 100% ethanol by shaking Figure 11: Proposed mechanism of quercetin antioxidant activity in an
    [Show full text]