Column Chromatography ________________________________________________ You have already performed two chromatography experiments: gas chromatography and thin layer chromatography. All chromatography experiments involve passing a mixture of analytes through a system that includes a mobile phase and a stationary phase. The partitioning of the analytes between these two phases determines the rate at which they pass through the system, and (in theory) allows them to be separated from one another. Column chromatography is a technique routinely used by organic chemists to separate sometimes complicated mixtures of compounds. For example, a chemical reaction may produce more than one compound and the products must be separated. In other instances, if a reaction does not go to completion, i.e. the starting materials do not react completely, then leftover reactants must be separated from the products of interest. Column chromatography is also used to separate mixtures of naturally occurring compounds isolated from plants and other living organisms. Performing column chromatography involves packing a column, which is a glass cylinder, with the stationary phase. The stationary phase is typically silica “gel” or powdered alumina, depending on the types of compounds you wish to separate. The mixture is placed on top of the packing material and a steady, continuous flow of eluting solvent is passed through the column. Although in the TLC experiment only one developing solvent is used to develop a plate, this is not the case for column chromatography: we can switch solvents in the middle of the process. A typical column chromatography experiment would involve starting with a less polar solvent; then, as the non-polar components of the mixture are eluted from the column, one can change to a more polar solvent to move the more polar compounds off the column as well. The polarity of a solvent mixture can be easily adjusted simply by changing the proportions of each of the components. In today’s experiment, you will be separating the components of a spinach extract. Before you arrive at lab, the mixture of plant pigments will have already been extracted from spinach leaves for you and will be ready to separate. These brightly colored plant pigments include two major groups of compounds: the chlorophylls, which are green, and the carotenoids, which are yellow-orange. Both groups are involved in photosynthesis, the process by which plants absorb certain wavelengths of light and convert this energy to chemical energy.
CH3 CH3 CH3
H3C H3C CH3 CH3
Figure 1. β-Carotene.
Figure 1 shows the structure of one of the carotenoids, β-carotene. Notice the pattern of alternating single and double bonds in the molecule. This pattern is called conjugation; molecules with extended conjugated systems such as this show strong absorption of light in the visible region of the electromagnetic spectrum. β-Carotene absorbs light at wavelengths of 454 nm; since this wavelength of light appears blue, the molecule itself appears orange. You will likely see several different compounds today, particularly in your TLC analysis. In order of elution from the column (first to last), the carotenoids (α-carotene and βcarotene) will appear yellow-orange. You may also see the pheophytins (a and b); these gray compounds are degradation products of the chlorophylls. The next compound to elute, chlorophyll a, is a blue-green compound that will have a more intense appearance on the TLC plate than chlorophyll b, which is green. Finally, you may see three yellowish spots for the xanthophylls, oxygen-containing carotenoid derivatives.
Experimental Procedure Today you will use a 10 mL disposable pipet as a column. These will be available in the lab. You will use this pipet as a gravity column: no external pressure is used to force the solvent through the column. The mobile phase in column chromatography is the eluting solvent. There are two eluting...
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