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Lab 5 - Spinach Chromatography

Lab 5 - Spinach Chromatography - Column and Thin-Layer...

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Column and Thin-Layer Chromatography of Spinach Extracts Chemistry 0330 Hikmat Daghestani Lauren Allen 10/8/07
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Introduction The purpose of this experiment was to explore column chromatography using a solid adsorbent as the stationary phase and a liquid as the mobile phase, and to then use TLC to determine the composition of the extracts. Reactions/Mechanism/Theory Chromatography is the separation of a mixture based on the affinities the components have for mobile and stationary phases. Both column and thin layer chromatography (TLC) separate mixtures based on polarity. Column Chromatography and TLC used a powder (such as alumina) for the stationary phase and liquids of varying polarities for the mobile phase of the experiment. The stationary phase in both chromatography techniques is polar. A polar stationary phase is chosen because other polar molecules will bind to the stationary phase. The more polar the molecule, the more likely it is to bind to the stationary phase, or absorption. Desorption is the solvated mobile phase of the chromatography. As in gas chromatography, a partition coefficient (Kp) can be calculated. Kp is equal to the concentration of the component in the stationary phase divided by the concentration of the component in the mobile phase. If Kp increases, retention factor increases and if the Kp is large, then the component is a polar one. The larger and closer to one the retention factor is, the more nonpolar the component of the mixture is. Column chromatography purifies mixtures. It will separate mixtures and therefore the pure component will be “in hand” at the end. Column chromatography relies on the force of gravity to carry the eluent down. For the mobile phase, the eluent is
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a pure solution or a mixture of miscible solvents. Generally, the eluent is changed throughout the experiment, for example: from a nonpolar solvent to a polar solvent. This is because the mobile phase will carry along some of the components of the mixture based on the polarity of the molecules in the fraction. Each eluent is added in a stepwise gradient (from more nonpolar to more polar solvents.) This is because if the most polar eluent is added first, all the molecules (from most nonpolar to most polar) will come out with it leaving nothing for the least polar eluent to take with it - instead of just the least polar molecules with the least polar eluent and medium polarity molecules with the medium polar eluent and etc. After column chromatography is completed, several components of the original mixture have been separated with different eluent mixtures. Thin layer chromatography (TLC) is the comparison of the separated components to a standard to determine which of the components each separated solution actually has.
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