Advanced Analytical Intro & What Is Chromatography? (PDF)

28/09/2023 Advanced Analytical Intro & What is Chromatography? Introduction Nikola P. Chmel L1 CH3F2 1 Overview of CH3F2 24 Lectures 6 workshops Chromatography Mass Spectrometry NMR Data analysis lab (33%) Exam (67%) Module leader: Mark Barrow [email protected] https://moodle.warwick.ac.uk/co...

28/09/2023 Advanced Analytical Intro & What is Chromatography? Introduction Nikola P. Chmel L1 CH3F2 1 Overview of CH3F2 24 Lectures 6 workshops Chromatography Mass Spectrometry NMR Data analysis lab (33%) Exam (67%) Module leader: Mark Barrow [email protected] https://moodle.warwick.ac.uk/course/view.php?id=57391 2 1 28/09/2023 Preparing for exam/revision Lecture slides will be colour coded: Green = Must Know – likely to be on the exam (basic material) Yellow/Amber = Should know – might be on the exam (moderate material) Red = Useful, but will not be on the exam (Advanced material, necessary for those who end up doing an MChem project using those techniques or MSc/PhD) 3 Overview of CH3F2 Chromatography 4 Lectures 2/10 2pm; 5/10 1pm; 9/10 2pm;12/10 1pm Calculations workshop (group work) 6/10 1pm Working with Drylab software (computer lab) (HPLC and GC simulation) 9/10 3pm & 4pm, 10/10 10am Nikola P. Chmel Office C517 (5th floor) Ext. 23234 [email protected] 4 2 28/09/2023 Vevox polling Chromatography lectures use Vevox polling via a web browser or the Vevox app (Android, iPhone). In your browser go to: vevox.app Meeting number: 126-545-180 6 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Lecture 2 Lecture 3 Normal phase chromatography (column, TLC) Reverse phase chromatography (HPLC) Workshop 1 Group calculation workshop Lecture 4 Gas chromatography Other chromatographic techniques Workshop 2 Simulation of HPLC/GC (DryLab) 8 3 28/09/2023 Why is Analytical Science important? Analytical Science develops and uses instruments and methods used to separate, identify, and quantify matter. In 2012 (salary survey data), analytical chemistry was the most popular field of work for American Chemical Society chemists. The furthest man-made object in space, Voyager 1, contains, amongst others, IR and UV-Vis spectrometers on board. Estimated 600 million people fall ill due to food contamination, resulting in over 400,000 deaths annually. Estimated 2 billion people drink contaminated water, resulting in nearly 0.5 million deaths annually. An estimated 1 in 10 medical products in low- and middle-income countries is substandard or falsified, leading to up to 5% of total malaria deaths in the world. 9 Why are units important? When you measure anything, your result is almost completely meaningless unless you provide a unit! http://squirrelcomics.com/units-of-measurement/ 12 4 28/09/2023 Why are significant figures and errors important? Significant figures (s.f.) and decimal places (d.p.) give you an idea of measurement certainty where it is not explicitly stated. e.g. 132.1 (assumed error ±0.05) 5.562×10-7 (assumed error ±5×10-11)* Where stated, errors give you an idea of how significant (trustworthy) the result is! e.g. 100 ±0.05** versus 100 ±25 * (0.0005×10-7 = 5×10-11) ** 100 ±0.05 can be also expressed as 100.0 14 Why are significant figures and errors important? Similarly to units, measurement result without stating the error can be almost completely meaningless. 15 5 28/09/2023 Precision and accuracy Precision gives you an idea of how reproducible a measurement is. Precision is limited by the random error. Precision is often describe using the standard deviation. Accuracy gives you an idea of how close to the true value a measurement is. Accuracy is limited by the systematic error. Accuracy is often describe using the absolute or the relative error. 16 18 6 28/09/2023 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History 19 What is Chromatography? Solvent extraction: Extraction is the transfer of a solute from one phase to another Phase 2 Phase 1 S(in phase 1) S(in phase 2) Extraction of Uranyl Nitrate from water into ether Partition coefficient: K [S]2 [S]1 Chromatography: Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it. 20 7 28/09/2023 What is Chromatography? Chromatography: Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it. Air flow 21 What is Chromatography? Fresh solvent (eluent) Initial band A and B solutes Column packing (stationary phase) Suspended in solvent (mobile phase) Solvent flowing out (eluate) A B B emerges A emerges 22 8 28/09/2023 What is Chromatography? It is the collective term for a family of laboratory techniques for the separation of mixtures by distribution of its components between a mobile and stationary phase over time Mobile phase: solvent moving through the plate or column (liquid or gas) Fresh solvent (eluent) Initial band A and B solutes Column packing (stationary phase) Suspended in solvent (mobile phase) A B Solvent flowing out (eluate) B emerges A emerges Stationary phase: substance which is fixed in place for the chromatographic procedure The partitioning of solutes between the mobile and stationary phases gives rise to separation All chromatographic separations depend on the reversible sorption and desortpion of the components of the mixture in the stationary and mobile phase 23 Chromatographic principle Mobile phase Sample mixture Equilibrium established at each point (ideally) Stationary phase The molecules of the mixture interact with the molecules of the Mobile and Stationary Phase Each molecule interacts differently with MP and SP Retardation of rate of movement of molecules Different distribution coefficients and different net rates of migration 24 9 28/09/2023 Purpose of Chromatography Analytical Chromatography – separate and identify or measure the relative proportions of components in a mixture Preparative Chromatography - purify and collect 300 L one or more components of a sample for further use The two are not mutually exclusive Scaling Up: Large column radius Large mass Small mass = Small column radius Purifies 1 Kg of material 25 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase 26 10 28/09/2023 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Cross section of open tubular column Solute adsorbed on surface of stationary phase Adsorption Chromatography Solute dissolved in liquid phase bonded to the surface of the column Partition Chromatography 27 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase Large molecules are excluded Mobile ions held near cations that are covalently attached to stationary phase Anion-exchange resin; only anions can be attracted to it Ion-exchange Chromatography Small molecules penetrate pores of particles Size-exclusion Chromatography 28 11 28/09/2023 Types of Chromatography Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase One kind of molecule in a complex mixture becomes attached to molecule that is covalently bound to stationary phase All other molecules simply wash through Affinity Chromatography 29 Terminology Analyte: substance that is going to be separated Eluent Eluate Mobile phase Stationary phase Chromatograph: equipment that enables a sophisticated separation Chromatogram: detector signal vs. retention time or volume Analytical chromatography Preparative chromatography Partition coefficient Retention time 31 12 28/09/2023 Classification of Chromatography Based on Stationary Phase Support Planar: Stationary Phase supported on a flat plate or in pores of paper. Mobile Phase moves through Stationary Phase by capillary action, gravity or electrosatic forces Column: Stationary Phase held in a tube and Mobile Phase forced through 32 Classification based on the physical properties of the analyte Molecular characteristic Polarity Ionic Size (mass) Shape Physical property Separation technique volatility Gas chromatography solubility Partition chromatography adsorptivity Adsorption chromatography charge Ion Exchange Electrophoresis diffusion Size Exclusion Dialysis sedimentation Ultracentrifugation ligand binding Affinity chromatography 33 13 28/09/2023 Classification based on the chromatographic principle Stationary Phase Adsorption Chromatography Competition between a solid adsorbent and the mobile phase Partition Chromatography Competition between a liquid stationary phase and the mobile phase Ion Exchange Chromatography Competition between an ionic exchange resin stationary phase and liquid mobile phase Permeation Chromatography Competition between a polymer matrix stationary phase and liquid mobile phase Mobile Phase Gas Stationary Phase Support Type of Chromatography Acronym Column Gas-Solid Chromatography GC/GSC Column Liquid column Chromatography High Performance Liquid Chromatography LC HPLC Planar layer Thin Layer Chromatography TLC Column Gas-Liquid Chromatography Supercritical Fluid Chromatography GC/GLC SFC Column Liquid-Liquid Chromatography High Performance Liquid Chromatography LC/HPLC Planar layer Paper Chromatography PC Liquid Column Ion-Exchange Chromatography High Performance Ion Chromatography IEC IEX IC/HPIC Liquid Column Liquid Gas Liquid Size Exclusion Chromatography Molecular Exclusion Chromatography Gel Permeation Chromatography Gel Filtration Chromatography SEC GPC GFC 34 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History 36 14 28/09/2023 Origins of Chromatography Chromatography: from Greek χρώμα: chroma, colour and γραφειν: grafein, to write Mikhail Tsvet was trying to solve the separation problem of green leaf pigments 1872-1919 30th Method first described on December 1901 at the Naturalists and Doctors in St. Petersburg. 11th Congress of First printed description was in 21st March 1903, in the Proceedings of the Warsaw Society of Natural Scientists, section of biology. “New category of adsorption phenomena and their applications to biochemical analysis” He first used the term chromatography in print in 1906 in his two papers on chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society. 37 Origins of Chromatography Mikhail Semyonovich Tsvet Glass cylinder with chalk powder Alcohol leaf extract on the top Brownish-green colour Add pure alcohol to the top The alcohol dissolved the pigments and moved them down the cylinder. Uniformly coloured bands of pure substances formed The bright-green and the yellowish-green bands were the two kinds of chlorophyll, and the yellow-orange band was from carotenoids. 38 15 28/09/2023 Milestones in Chromatography ✓ 1903 Tswett - plant pigments separated on chalk columns ✓ 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1941 Gas Chromatography 1950 reverse phase LC 1954 Martin & Synge (Nobel Prize) 1959 Gel permeation 1965 instrumental LC (Waters) 39 Recap What is chromatography? / Chromatographic principle. Division of chromatography based on the interaction type and the physical properties of the analyte Basic terminology. 40 16