Column Chromatography of a Spinach Extract
Objectives Experimental Procedure
Introduction Write-up Prelab Preparation References
i. To learn the technique of column chromatography. ii. To learn how molecular polarity determines both the solubility and the relative column migration rates (Rf) of different compounds. iii. To determinethe relative polarity and colours of chlorophylls versus carotenoids.
Organic chemists use a variety of techniques to prepare, isolate and analyze organic compounds. Synthetic chemists make new molecular compounds by performing reactions, for example inter-converting functional groups and making new arrangements of carbon chains and rings.
Unlike the many elegant enzyme-catalyzed biological reactions that produce a pure product, synthetic reactions in the laboratory usually produce a mixture of product, additives, side products and/or unreacted reactants. It is therefore necessary to perform a “work-up” of the product mixture in which one or more of a variety of standard lab techniques is used to separate and purify the desired products. These techniques include: selective solvent washes, recrystallization, distillation, liquid-liquid extraction and column chromatography to obtain the pure product. These techniques were introduced in CHEM 2020, with the exception of column chromatography which will be learned in this lab.
Chromatography is a very common technique used often in synthetic chemistry labs and is also used for instrumental analyses of organic compounds to establish purity. In this lab, students will learn how to separate an organic mixture using the technique of column chromatography which is essentially a large scale version of TLC and allows the separation of large multi-gram amounts of compound mixtures. The amount of purified compound obtained is then enough to perform detailed chemical analyses and to be used for further synthetic reactions. A detailed explanation of these techniques is presented in the “Background theory - chromatography and TLC” file posted to the Expt2 Blackboard folder.
Analogous to a synthetic reaction mixture, a biological extract usually contains a mixture of organic compounds that must be separated and purified in order to identify and characterize them. In this experiment you will isolate a set of compounds from spinach leaves, and then purify them using column chromatography with silica gel. There are two classes of compounds that will be isolated from the spinach leaves: chlorophylls and carotenoids. Chlorophylls are central to photosynthesis in plants, while the carotenoid class of compounds includes many nutrients such as -carotene. The structures of the compounds you will isolate and purify in this experiment are depicted in Figure 1 below.
There are two chlorophylls that you will isolate and purify in this experiment: chlorophyll-a and chlorophyll-b. The structure of each chlorophyll is depicted in Figure 1, and the difference between the two structures is indicated; chlorophyll-a has a methyl group (-CH3) at the same position where chlorophyll-b has an aldehyde group (-CHO). This small structural difference has a significant effect on the separation of these two chlorophylls. First, the presence of the aldehyde group in chlorophyll-b extends the number of “conjugated” double bonds.1 This difference in the degree of conjugation
between chlorophyll-a and chlorophyll-b causes the two compounds to be slightly different colours; one is green, while the other is blue-green. Secondly, the presence of either a methyl group or an aldehyde group affects the polarity of the chlorophylls. An aldehyde group is more polar than a methyl group, and this makes chlorophyll-b slightly more polar than chlorophyll-a. This difference in polarity is what permits the separation of these two chlorophylls by column chromatography over silica gel. Besides the presence of either a