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Chromatography - Extrac'on Purifica'on and Characteriza'on...

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Unformatted text preview: 9/11/10 Extrac'on, Purifica'on and Characteriza'on Felix Hoffmann 1 9/11/10 How do we get at these compounds?? How do we isolate them? How do we purify them? How do we know we have them? Extrac'on: "pulling out" a compound from one phase to another "cup of tea" In the case of Natural Product isola'on, for example, begin by grinding up "source" then allow to stand in a solvent. Solvents "like dissolves like" Polar compounds dissolve in polar solvents; non-polar compounds dissolve in non-polar solvents benzene nonpolar dichloromethane Ethyl acetate polar Non-polar fats in hexanes; Salts in the water phase 2 9/11/10 Let's look at extrac'ng compound X from water into dichloromethane. In prac'ce, substances generally par$$on themselves between solvents, although one is oLen favoured. You can extract the compound into the favoured solvent (coffee into water). Use a separatory funnel: We define a par''on coefficient K for X. K = c oncentra'on in solvent A concentra'on in solvent B Extrac'on of X from water into CH2Cl2: Suppose we have 1 gram of a compound X that has K = 9 between 100 mL each of dichloromethane and water: 0.9 g /100 ml (in CH2Cl ) K = 9 = 0.1 g /100ml (in w2ater) Upper layer has less dense solvent one extrac'on (par''oning) results in 90% of X moving to the organic phase, dichloromethane. two extrac'ons of the aqueous phase remove 99% of X 3 9/11/10 Simple Dis'lla'on Dis'lla'on is the main technique for purifying vola$le liquids. Involves vaporizing a material by hea'ng it and subsequently condensing the vapour back to a liquid. Dis'lla'on Adaptor Thermometer Rubber tubing Liebig Condensor Receiving Adaptor Dis'lla'on Flask cold water Open to atmosphere! Collec'on Flask Frac'onal Dis'lla'on Crystalliza'on Simplest technique for purifying a solid Dissolve in suitable solvent Filter, if required Crystallize: e.g. by cooling or slow evapora'on Collect: e.g. on Buchner funnel Dry to remove residual solvent Columns should be packed with stainless steel sponge typically measure mel'ng point (mp) to test purity 4 9/11/10 Chromatography: Separa'on of the components of a mixture based on the differences in the rates at which they are carried through a sta'onary phase by a mobile phase. There are many different types of chromatography but all are based on the same principles. Separa'on of ink on paper Why and how does it separate? The sta'onary phase is fixed in place either in a column or on a planar surface (e.g. silica, alumina, etc.) The mobile phase moves over or through the sta'onary phase carrying with it the analyte mixture. Can be a gas or a liquid. The solute is distributed between the mobile and sta'onary phases We define a distribu$on constant K = cs / cm Where cs and cm are the molar concentra'ons in the sta'onary and mobile phases, respec'vely. 5 9/11/10 Ideally, every compound will have a unique K (or affinity for sta'onary versus mobile phase). The net result is that different compounds move at different rates through the chromatograhic medium and hence can be separated! Column Chromatography: Sta'onary phase is generally a porous solid like silica (SiO2) or alumina (Al2O3). Mobile phase is a solvent of par'cular polarity that will dissolve the compounds of interest and carry them through the column. Sample A + B B A Packed Column Add Mobile Phase Notes: Reten'on 'me (tr) is a measure of the 'me the compound takes to pass through the column. B A B A B Detector t0 t1 t2 t3 t4 Detector signal A B Solvent and sta'onary phase must be carefully chosen and matched with the polarity of the compounds to be separated. t0 t1 t2 t3 t4 Elu'on Time 6 9/11/10 If affinity for mobile phase too great, compound passes through the column with solvent front (i.e. is not retained) If affinity for sta'onary phase too great, compound retained on top of column and does not move. Intermediate situa'on desired. Solvents benzene nonpolar dichloromethane Ethyl acetate polar For Reference only, no need to remember... Solvents arranged in order of increasing polarity index, a rela've measure of the degree of interac'on of the solvent with various polar test solutes. Polarity Index Pentane 1,1,2-Trichlorotrifluoroethane Cyclopentane Heptane Hexane Iso-Octane Petroleum Ether Cyclohexane n-Butyl Chloride Toluene Methyl t-Butyl Ether o-Xylene Chlorobenzene o-Dichlorobenzene Ethyl Ether Dichloromethane Ethylene Dichloride n-Butyl Alcohol Isopropyl Alcohol 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.2 1.0 2.4 2.5 2.5 2.7 2.7 2.8 3.1 3.5 3.9 3.9 n-Butyl Acetate Isobutyl Alcohol Methyl Isoamyl Ketone n-Propyl Alcohol Tetrahydrofuran Chloroform Methyl Isobutyl Ketone Ethyl Acetate Methyl n-Propyl Ketone Methyl Ethyl Ketone 1,4-Dioxane Acetone Methanol Pyridine 2-Methoxyethanol Acetonitrile Propylene Carbonate N,N-Dimethylformamide Dimethyl Acetamide N-Methylpyrrolidone Dimethyl Sulfoxide Water Polarity Index 4.0 4.0 4.0 4.0 4.0 4.1 4.2 4.4 4.5 4.7 4.8 5.1 5.1 5.3 5.5 5.8 6.1 6.4 6.5 6.7 7.2 10.2 For chromatography on silica gel (BOTH column and TLC), increasing solvent polarity: makes ALL compounds travel faster elutes increasingly polar molecules e.g. an alkane: elutes in any solvent An ether: does NOT elute in hexane An alcohol: requires ether to elute it A diol (two OH groups): might require methanol/ CHCl3 mixture to elute Solvent Choice hjp://macro.lsu.edu/howto/solvents/Polarity %20index.htm 7 9/11/10 Thin Layer Chromatography. Excellent for analysis of mixtures. Adsorbent is supported as a thin coa'ng on a flat surface (glass, aluminum, or plas'c). Compound is spojed on to the plate Solvent travels up the plate via capillary ac'on. Ideally, each component has a different Rf Other types of chromatography include Prepara've Layer Chromatography (PLC), High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). 8 ...
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