CHEM 2010: Chemical Analysis A Chromatography Lecture (PDF)

CHEM 2010: Chemical Analysis A Chromatography Lecture 1 of 8 Dr. Joyann Marks [email protected] First Half of the Semester Date (Monday) 2-Sep 9-Sep 16-Sep 23-Sep 30-Sep 07-Oct Week # 1 2 3...

CHEM 2010: Chemical Analysis A Chromatography Lecture 1 of 8 Dr. Joyann Marks [email protected] First Half of the Semester Date (Monday) 2-Sep 9-Sep 16-Sep 23-Sep 30-Sep 07-Oct Week # 1 2 3 4 5 6 Course Tues 2pm Introduction Chromatography JM (8) VRR Thur 10am TEST 1 EChem Fri 1pm EChem PN (7) /Chrom Problem Solving Sessions Tues 4-6pm C7 Wed 10am-12pm C7 EChem2 Chrom1 EChem1 Chrom2 Thur 6-8pm (C7/ONLINE)  Reference Texts: Fundamentals of Analytical Chemistry Skoog, West, Holler and Crouch 10th Ed Chapters 29 - 32 Quantitative Chemical Analysis 7th Ed Daniel C. Harris Chapters 22 - 26 VLE: for lecture outlines, tutorial sheets and notifications, and additional information sources… but don’t forget the texts! Content/ Structure of Course Section: 8 Lectures Lecture 1: Introduction and history of chromatography Lecture 2: Chromatographic principles Lecture 3: Chromatographic principles II Lecture 4: Chromatographic Principles III Lectures 5 and 6: Liquid Chromatography (LC) Lecture 7: Gas Chromatography (GC) Lecture 8: GC detectors and Qualitative and Quantitative analyses in Chromatography Chemical Analysis Chromatography practicals will be in weeks 3 and 5 Course test ~week 6 Office Hours Mondays 2-3 PM Wednesdays 2-3 PM Thursdays 1-2 PM Please make an appointment to meet with me virtually or otherwise: https://calendly.com/joyannmarks/uwichem Email me to discuss appointments outside of these times [in the case of scheduling conflicts]. Email: [email protected] Chromatography Chroma -"colour" and graphein - "to write”. Colour bands - separation of individual compounds Chromatography is a technique used to separate and identify the components of a mixture based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Detection of the separated analytes may be achieved though electro-, spectro- or mass methods History of Chromatography 1903 - Mikhail Tswett 1930’s - Edgar Lederer Austrian Chemist PhD 1930, moved to Germany Read translation of Tswetts work α and β carotenes Russian Botanist Warsaw University Plant pigments (chlorophyll) Adsorption Column Chromatography Died 1919 age 47 Exponential growth in chromatography publications in 1930s  History 1940s - Archer Martin & Richard Synge Chromatography 1903 Tswett - plant pigments separated on chalk columns 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 Reverse phase LC 1952 Martin & Synge (Nobel Prize) 1959 Gel permeation British Chemists 1965 instrumental LC (Waters) Partition Chromatography (proteins) Nobel Prize in Chemistry 1952 Purpose of Chromatography ❑Analytical - Determine Chemical composition of a sample ❑Preparative - Used to purify sufficient quantities of a substance The separated analytes can be identified and their concentrations determined by comparison with standards QUALITATIVE QUANTITATIVE ANALYSIS ANALYSIS Chromatography terms ❑Chromatograph - equipment that enables a sophisticated separation eg. Gas chromatograph or Liquid chromatograph GC Chromatograph HPLC Chromatograph https://www.hitachihightech.com/global/product_detai https://www.kutztown.edu/Departments- l/?pn=ana-chromasterultra Offices/MR/PhysicalSciences/Chemistry%20Images/GC/GC_ 6890_Labeled.jpg Chromatography terms ❑Elution- Process by which analytes are washed from a chromatograph during chromatography ❑Eluent- Fluid entering column/ solvent that carries the analyte. ❑Eluate - Mobile phase leaving the column. Chromatography terms ❑Stationary phase - Immobilized phase Immobilized on the support particles or on the inner wall of the column tubing. Examples : Silica layer - Thin Layer Chromatography ❑Mobile phase - Moves in a definite direction. Liquid (LC), Gas (GC). The mobile phase moves through the chromatography column (the stationary phase) where the analyte(s) interact(s) with the stationary phase and is separated. ❑Analyte-Substance analyzed in chromatography. ❑Solvent -Any substance capable of solubilizing another substance. Chromatography terms ❑Retention: The process of retardation (delay) of the elution of a chemical species. Direction of mobile phase flow The red analyte molecules are more attracted to the blue stationary phase than the green molecules and are therefore retained on the column for a longer time. Chromatography terms ❑Retention time (tr): Time taken after sample injection for an analyte peak to elute and be detected at the peak maximum. Because of varying affinities of different analytes for the stationary phase, retention times of different analytes will vary. ❑Retention volume (Vr): Volume of mobile phase required to carry a band of a species through the system to the detector. Chromatogram ❑Visual output of the chromatograph. ❑Separation - Different peaks or patterns on the chromatogram correspond to different components of the separated mixture. Chromatography terms ❑Dead time (tM) - Time for an unretained species to reach the detector. It is also the mobile phase hold up time Injection time ❑ Adjusted retention time (tS or tR’)- Difference between tR and tM Classification of Chromatography There are two main classification schemes: ❑Mobile phase ❑Attractive forces Mobile Phase: Attractive forces: Gas (Gas Chromatography) Adsorption Aqueous solvent (Liquid Chromatography) Ion Exchange Organic solvent (LC) Partition Size Exclusion Supercritical fluid (SCFC) Affinity Classification based on Mobile Phase Gas Chromatography- mobile phase is a GAS Name of GC Method Type of Stationary Phase Gas-solid chromatography (GSC) solid, underivatized support Gas-liquid chromatography (GLC) liquid-coated support Bonded-phase gas chromatography chemically-derivatized support  Classification based on Mobile Phase Liquid Chromatography - mobile phase is a LIQUID Name of LC Method Type of Stationary Phase Adsorption chromatography (LSC) solid, underivatized support Partition chromatography (LLC) liquid-coated or derivatized support Ion-exchange chromatography support containing fixed charges Size exclusion chromatography porous support Affinity chromatography support with immobilized ligand HPLC: High Performance Liquid Chromatography ❑Uses columns packed with smaller particle size materials for better performance (but requiring high pressure for elution) Supercritical Fluid Chromatography (SFC) ❑Uses a supercritical fluid as the mobile phase, i.e. a substance above its critical pressure and temperature. Under these conditions the physical properties of the mobile phase are intermediate between those of a gas and liquid. ❑Supercritical fluid viscosities are relatively low compared with those of liquids and diffusion rates are much higher than in liquids. ❑This allows for fast, efficient separation to be done (as in GC) but using a mobile phase that has some solvating ability (as in HPLC). ❑Carbon dioxide is the most common mobile phase used in SFC. Classification based on Attractive Forces Adsorption Chromatography ❑The mobile phase may be a liquid or a gas (LSC Adsorption or GSC). Accumulation of the ❑The stationary phase is a molecular species at the solid and sample surface rather than in the components are bulk of the solid or liquid adsorbed on its surface. Mobile Phase Stationary Phase Adsorption The more strongly a solute is adsorbed, the slower it travels through the column. Liquid Solid Chromatography Thin Layer Chromatography (LSC - Adsorption Chromatography) ❑The adsorbents (e.g. silica gel, alumina) are packed in a column or coated on a planar support and the analytes transported by mobile phase flow. ❑Thin Layer Chromatography (TLC) is a type of An example of Planar Liquid-Solid (Adsorption) Chromatography planar liquid-solid chromatography (LSC). The column may be thought of as being cut open and spread out flat. Partition (Absorption) Chromatography ❑The mobile phase may be a liquid or a gas (LLC or GLC). Absorption ❑The stationary phase is a liquid Assimilation of supported on an inert solid. molecular species throughout the bulk of the liquid Mobile Phase Stationary Phase Liquid-Liquid Chromatography (LLC -Partition Chromatography) ❑ The analytes are retained by partitioning (distributing) between the liquid mobile phase and the liquid stationary phase. ❑ One important requirement is that the liquid mobile phase must not be a solvent for the liquid stationary phase. ❑ Paper chromatography is a type of partition chromatography in which the stationary phase is a layer of water adsorbed on a sheet of paper. https://en.wikipedia.org/wiki/Paper_chromatography#/media/File:Chro matography_tank.png Ion Exchange Chromatography An ion exchange resin is the stationary phase. The mechanism of separation is based on ion exchange equilibria. The resin contains anions or cations that attract oppositely charged solute ions. The mobile phase is a liquid Ion Exchange Chromatography ❑ Uses zeolites and synthetic organic and inorganic resins (including polymer matrix with permanently attached charges) to perform chromatographic separations by an exchange of ions between the sample and the resins. https://www.linkedin.com/pulse/ion-exchange-resin-characteristics-magesh-john/ ❑ Compounds which form ions that have different affinities for the resins can be separated. ❑ This is commonly used for separation of amino acids and inorganic ions. Ion Exchange Chromatography Separation based on attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. http://upendrats.blogspot.com/2013/05/ion-exchange-chromatography.html Size Exclusion Chromatography ❑Solvated molecules are separated according to their size, by their ability to penetrate a sieve-like structure ❑Also called Gel permeation/Gel filtration Chromatography ❑Large molecules elute first Size Exclusion Chromatography ❑ A uniform, highly porous, non-ionic gel is used to separate materials according to their molecular size. ❑ Gel filtration chromatography is generally applied to the use of Sephadex (a polysucrose or dextrin polymer which swells in water) as a sieving agent. ❑ Gel permeation chromatography describes separations performed with polymers that swell in organic solvents.  Affinity Chromatography Basis: Highly specific interactions between a solute molecule and a second molecule covalently attached (immobilised) to the stationary phase (eg enzyme/substrate) This is the most selective type of chromatography. Chromatographic techniques may also be classified based on the type of support material ❑Column Chromatography- Stationary phase is held in a cylindrical tube and the mobile phase is forced through it by gravity or pressure. ❑Planar Chromatography- Stationary phase is supported on a flat plate or in the pores of paper and the mobile phase moves through the stationary phase by capillary action or gravity.  End of Lecture # 1 Family of Chromatographic Techniques Next time: Introduction to Basic Chromatographic Theory