Lab 2 - Gaurav Singh Brooke Leslie/Dr Chad Landrie Lab Two...

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Gaurav Singh Brooke Leslie/Dr. Chad Landrie February 11, 2009 Lab Two: Chromatographic Methods, Separation of Dyes, and Spinach Pigments by Column and TLC Introduction There are two parts to this experiment. The purpose of the first is to set up an efficient column chromatography that allows for 100% separation of methyl orange and methylene blue in a reasonable amount of time and with a reasonable solvent. The second part serves to develop an effective set of conditions (more specifically, mobile phase composition) that will allow complete separation and visualization of chlorophyll A/B, pheopytin, carotenes, and xanthophylls. Column chromatography is used to separate a mixture of two or more compounds, and makes use of the differences in polarity (which is essentially a partial charge or separation of charge) between substances. More polar compounds will become more attracted to polar stationary phases (like silica gel). When a mixture is added, both compounds in it (after it has been eluted with methanol) spend a portion of their time absorbing into the silica, and a portion in the mobile phase (methanol). This is called dynamic equilibrium. Less time in the stationary phase means more time in the mobile phase, and therefore less retention time. If a compound spends more time to travel through, it must be more polar. Methyl orange and methylene blue, shown in figure one, are used because they are not colorless and the separation can be easily determined.
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Part two of the experiment involves TLC, or thin-layer chromatography. It is essentially similar to column chromatography, except the stationary phase is connected as a thin layer to a solid structure. In addition, less compound can be separated because the amount of adsorbent is less. Beyond that, the mobile phase is at the bottom of the TLC plate and travels up the plate via capillary action. These key differences make this method better for comparing a spot from a reactant to a spot from the reaction mixture. But since sometimes TLC produces poor results that are difficult to interpret because two spots are too close, a co-spot is placed between the other two which contains overlapping components of both. If the co-spot separates, we can assume that the spots are different; the converse applies if it does not. The pigments we are trying to separate are chlorophylls a and b, carotenoids, and pheophytins. They have varying degrees of polarity, as is later discussed, which make the TLC very effective. The basic idea, however, is that the pigments are different in composition and can be determined from one another by color and Rf value. Pigments containing polar functional groups are absorbed more strongly and therefore travel slower. The compounds used in this experiment are displayed in figure two.
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