36B BioOrganic Modifications for Technique Experiments Technique of Column Chromatography Experiment Title Applying Column Chromatography As A Method to Isolate Plant Leaf Pigments Introduction Thus far you have utilized two techniques to isolate pure organic compounds recrystallization and distillation Another very valuable isolation technique that is often employed when very small quantities of sample are available is column chromatography This separation method is very similar in principle to thin layer chromatography and can be used for samples of many different sizes TLC is used to determine an effective solvent system for the separation and then column chromatography is used to separate and isolate the desired compound s This laboratory assignment demonstrates the utility of column chromatography to separate natural products such as leaf pigments You will need to bring leaf samples with you to complete this lab Background The orange and red pigments in fruits and vegetables such as tomatoes and carrots are hydrocarbons known as carotenoids The two most common carotenoids which incidentally are precursors to Vitamin A are lycopene and b carotene the structures of which are shown below C H3 H C H3 H C C C H3H C C H C C H3 C C H C H C H2OH C H3 Vitamin A C2 0H3 0O C H3 C H3 H C C C H3 H C H3 C H3 H C H3 H C C C H C C H C H C C H H C C H C C C H3 H H C C H C C H3 C C H3 H lycopene C4 0H56 m p 173 C all trans C H3 C H3 H C H3 H C C C H3H C H3 C C H C C H3 H C H C C H C H C C H C H C H C C C H3 H C H3 HC H 3 C C C C H3 H C H3 carotene C4 0H56 m p 183 C all trans The carotenoid pigments also occur in the leaves of plants but they are not obvious because of the presence of other pigments The green pigments in leaves are principally from chlorophyll a and chlorophyll b which have the structures shown below CH2 CH N H3C O CH3 N CH2 CH2CH3 H3C H N H N CH2CH3 Mg CH3 N C N H3C Mg CH H3C H H N CH3 N H CH2 CH2 O COOCH3 CH3 CH2COOCH2 CH3 chlorophyll a CH3 CH3 CH3 O COOCH3 CH3 CH2COOCH2 CH3 CH3 CH3 chlorophyll b Chlorophyll b differs from chlorophyll a in that it has a formyl CHO group in place of a methyl group CH3 at the position shown in the above structure The leaves of plants contain not only the chlorophylls a and b but also other pigments whose colors are masked by the chlorophylls in live healthy leaves The other pigments become visible in the fall when the leaf dies and the chlorophylls rapidly decompose Among the other pigments are the carotenoids the two commonest being lycopene and bcarotene both of which are precursors to Vitamin A Activities Read the Introduction section of the Column Chromatography Experiment found in Chapter 8 of the Chem 36 Lab Guide pgs 177 182 and also read an article by P Keusch Organic Chemistry Demonstration Experiments on Video Chemistry Visualized www uniregensburg de Fakultaeten nat Fak IV Organische Chemie Didaktik Keusch D TLC e htm Also read for background information on plant pigments The Chemical Pigments of Plants J Alkema and S L Seager J Chem Ed 59 1982 pgs 183 186 In the first lab period you will extract the plant pigments from fresh leaves In the second lab period you will separate the pigments using column chromatography and characterize the plant pigments by UV VIS CH3 PreLab Follow the prelab sheets when preparing your prelab and complete your Chemical Data Table with any reagents not found in the Common Shelf Data Table and include the structures of the plant pigments listed in Table 1 below Highlight with colored pens or pencils the portion of the molecule that determines the class of the pigment The classes are listed in Table 1 and the background article on plant pigments highlights the structures of these classes Hand in a copy of the completed Common Shelf Chemical Date Table and a copy of your Chemical Data Table with your Prelab When completing the procedure outline make sure you have an explanation for every step in your procedure outline You do not need to include a mortar and pestle in your diagrams but you do need to include a picture of your column set up Prelab Exercises Answer questions a b and c of the Prelab Exercises on page 194 of your Lab Guide Post Lab Questions Answer question Why is chlorophyll green Table 1 Plant Pigments Class Compound Colors Produced Carotenoid Carotenes Golden Porphyrin Pheophytin Olive Green Porphyrin Chlorophyll a Blue Green Porphyrin Chlorophyll b Yellow Green Carotenoid Lutein Yellow Carotenoid Xanthophylls Yellow Flavonoid Anthocyanin Red Blue or Purple pH dependent Column Chromatography Experiment Separation of Plant Pigments Taken from P Keusch University of Regensburg Separation of Plant Pigments by Column Chromatography CC Organic Chemistry Demonstration Experiments on Video Chemistry Visualized www uniregensburg de Fakultaeten nat Fak IV Organische Chemie Didaktik Keusch D TLC e htm Extraction of Plant Pigments Working in a hood rip two leaves into pieces and then add them to a mortar with 22 mL of acetone and 3 mL of petroleum ether and a spatula tip of CaCO3 Grind the leave pieces with a mortar until the solvent in the mixture becomes highly colored Filter the pigment mixture into a separatory funnel and add 20 mL of petroleum ether and 20 mL of 10 aqueous NaCl solution Remove the water layer and wash the organic layer 4 times with 5 mL of distilled water Dry the organic layer with 4 spatula tips of Na2SO4 Gravity filter and concentrate the organic layer to 3 mL Column Chromatography Packing the Column You will be carrying out a microscale column separation for the first step of this synthesis The column you will need is found in your microscale lab kit and it looks just like the one found in Figure 8 1 on page 196 of your Lab Guide When using this column you will be separating no more than 50 mg of material at a time Please keep this in mind because you do not want to overload your column You will be using silica gel as your stationary phase You will be using the slurry method to pack your column so make sure you review this procedure on page 197 of the your Lab Guide The solvent system you will use for the separation is a 7 3 mixture of petroleum ether and acetone Review the polarity of these solvents using Figure 7 8 on page 176 of your Lab Guide Also review the polarity of silica gel as a stationary phase using Figure 7 5 on page 174 of your Lab Guide Before assembling your column check the small plug that fits into the bottom of the column to …