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14- lab 14- R-HPLC for detn of caffeine

14- lab 14- R-HPLC for detn of caffeine - C hem 223...

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Chem 223: Experiment 14 Determination of caffeine in soft drinks by High Performance Liquid Chromatography (HPLC) . References: 1. Harris, 7 th ed. Chap. 25 2. Harvey: 578-589 3. Kealey: pp. 155-164 4. Experimental procedure. The University of Adelaine. Australia. Department of Chemistry. 5. Experiment 5. University Kuala Lumpur. Malaysian Institute of Chemical And BioEngineering Technology 6. Experiment 5. Chem 426. Washington State University. 7. Expriment . Chem 427. Portland State University I. Purpose of the experiment The traditional method for the determination of caffeine is via extraction with spectrophotometric quantification. Use of the liquid chromatography permits a fast and easy separation of caffeine from other substances such as tannic acid, caffeine acid, and sucrose found in these beverages. The amount present in soft drinks is controlled by the manufacturer, and can be obtained upon request. The following experiment illustrates the utility of high performance liquid chromatography (HPLC) as an analytical tool for the determination the amounts of caffeine in various commercially available soft drinks. From the resulting chromatograms, measurements of retention time t R and peak areas are made. If the flow rate and pump pressure are held constant throughout the entire experiment, t R may be used as qualitative measure and the peak area as a quantitative measure. A calibration curve for peak area vs. concentration of the caffeine standards can then be employed to determine the concentration of caffeine in the beverages. II. Introduction Chromatographic methods are based on differential affinities of solutes for two phases, a stationary phase and a mobile phase. Molecular interactions leading to these affinities are polar forces (dipole-dipole and H-bonding) and dispersion forces (induced dipoles). Thus, in a chromatographic system, solute molecules will be attracted to the phase of similar polarity. Mobile phases in chromatography are either liquid or gaseous. Gas chromatography (GC) is the only technique that uses a gas as a mobile phase, while many techniques employ liquids (liquid chromatography, LC). High Performance Liquid Chromatography (HPLC) is a modern (late 60's) modification of the classical open column techniques that established chromatography as the ultimate separation technique. The name is derived from the fact that much higher column efficiencies are possible when the particle size of the stationary phase is small (3-10 mm in HPLC versus 40 mm conventional open column LC). As a result of these smaller
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particles, large back-pressures require the mobile phase to be pumped through the column under high pressures. HPLC has several advantages over GC: 1. HPLC is not limited to volatile compounds. 2. A greater control and wider selection of stationary and mobile phases.
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