Lecture 32. Chromatography Intro

Lecture 32. Chromatography Intro - Chapter 21:...

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Unformatted text preview: Chapter 21: Chromatography Chapter Daniel C. Harris, Exploring Chemical Analysis, 2nd ed., W.H. Freeman and Company, New York, 2001. Freeman Analytical Chemisty Analytical • What is it? How much is there? What • Two approaches: (1) Measure with high selectivity. (2) Separate and measure with low selectivity. (2) • Different approaches to separation: Different (1) Membrane separation (1) (2) Separation based on different mobilities (electrophoresis) (2) (3) Separation based on distribution between a stationary and a mobile phase (chromatography) http://www.studyhplc.com/chromatographyanimation.php Animation of a separation Excel Demo Gaussian Peak Profile, Separation, Tailing, Leading Leading • Fill in 1024 molecules into E40; this will be injjected into the column at ected the first evaluation. the • Inject the 1024 molecules into the first mobile phase element, move the content of all mobile phase elements to the next element (shown before equilibration in column B), and equilibrate the stationary with the mobile phase with “cmd =“. phase • Start feeding the mobile phase with eluent by entering 0 into E40. Start • Move the content of all mobile phase elements to the next element (shown before equilibration in column B), and equilibrate the stationary with the mobile phase with “cmd =“; repeat this process until all sample has left the “c repeat column. column. Low sample concentration Low High sample concentration => Why the strange peak shape? Paper Chromatography Paper QuickTimeª and a decompressor are needed to see this picture. QuickTimeª and a decompressor are needed to see this picture. QuickTimeª and a decompressor are needed to see this picture. Thin Layer Chromatography: Dependence of Rf Value on Sample Concentration Dependence Stationary phase: Silica gel; Mobile phase: Benzene BG: butter yellow, SR: Sudan Red, IP: Indophenol, SS: Sudan Black Peak Shape Peak Ideal: • Sample distribution between stationary and mobile phase does not depend on concentration. depend • Symmetrical peak (Gaussian shape). Symmetrical • Retention time does not depend on sample concentration. Retention Normal: • Sample concentration high; stationary phase saturated. Sample • What is the expected peak shape? What • How does the retention time depend on the sample concentration? How Poor Chromatogram: • Sample concentration high; sample condenses in stationary phase. Sample • What is the expected peak shape? What • How does the retention time depend on the sample concentration? How HPLC separation of benzodiazepines. Conditions: sample: 40 ng each; column: 3cm× 4.6mm i.d.; stationary 3cm 4.6mm phase: ChromSphere UPP C18, 1.5 µ m (non-porous); (non-porous); mobile phase: 3.5 ml·min-1 mobile water-acetonitrile (85:15); temperature: 35 °C; UV C; detector 254 nm. Typical applications of ion chromatography. (a) Separation of anions on an anion-exchange column. Eluent: 0.0028 M NaHCO3/0.0023 M Na2CO3. NaHCO Sample size: 50 µ L. (b) L. Separation of alkaline earth ions on a cation-exchange column. Eluent: 0.025 M phenylelendiamine dihydrochloride/0.0025 M HCl. Sample size: 100 µ L Gas Chromatography of Amino Acids Gas Liquid Chromatography (1) (2) (3) (4) (5) p-Xylene Methoxybenzene Nitrobenzene Acetophenone Dipropyl Phthalate Diode Array Detector Diode ...
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