PRELAB ASSIGNMENT 1 Purpose of experiment 2 Structures of Plant Pigments 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 Also include a diagram of your column set up 3 Procedure Outline Make sure you have an explanation for every step in your procedure outline 4 Chemical Data Table 5 Read Background Information a An article by P Keusch Organic Chemistry Demonstration Experiments on Video Chemistry Visualized www uniregensburg de Fakultaeten nat Fak IV Organische Chemie Dida ktik Keusch D TLC e htm b The Chemical Pigments of Plants J Alkema and S L Seager J Chem Ed 59 1982 pgs 183 186 6 PreLab Exercise a Give one advantage and one disadvantage of using 8g of alumina instead of 4 5 g as in Procedure 2 of the experiment b What is meant by the expression TLC is used to monitor the progress of the reaction c What is the purpose of tapping the column as you add the alumina If you didn t do this what might be the effect on your separation 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 dependent INTRODUCTION Thus far you have utilized two techniques to isolate pure organic compounds recrystallization and distillation Column chromatography is another valuable isolation technique that is often employed when very small quantities of sample are available 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 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 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 C C H3H 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 H C H3 H C H C C C H C H3 C H3 C H3 lycopene C4 0H56 m p 173 C all trans C H3 H C C H C C H3 H C H C C H C C H H C C H C C C H3 H H C 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 N H 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 carotene both of which are precursors to Vitamin A PROCEDURE ISOLATING PLANT PIGMENTS BY COLUMN CHROMATOGRAPHY 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 3 mL of petroleum ether and a spatula tip of calcium carbonate CaCO3 Grind the leave pieces with a pestle until the solvent in the mixture becomes highly colored Filter the pigment mixture into a separatory funnel Add 20 mL of petroleum ether and 20 mL of saturated aqueous NaCl solution into the separatory funnel 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 anhydrous sodium sulfate Na2SO4 Gravity filter into an Erlenmeyer flask and concentrate the organic layer to 3 mL by blowing a stream of nitrogen into the flask Packing the Column While concentrating the organic layer set up the microscale column located in your red kit Refer to Figure 8 1 on page 196 for the set up of the microscale column You will be using silica gel as your stationary phase and the slurry method to pack the column 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 Also review the polarity of silica gel as a stationary phase using Figure 7 5 on page 174 Before assembling your column check the small plug that fits into the bottom of the column to make sure that it has a small fritted disk inside Next make sure that the plug fits snugly into the glass column and is not easy to pull out If it is loose use a new bottom plug from your Organic Lab Equipment Kit Finish assembling the chromatography column as depicted in Figure 8 1 Be sure to clamp the column securely CH3 and vertically to a ring stand Then place an Erlenmeyer flask or small beaker under the stopcock of the column to catch any dripping solvent if you should spring a leak while packing the column It is important to get into the habit of always having a beaker or flask under the column to catch any solvent that may accidentally drip out of the tip of your column Weigh out approximately 3 g of silica in a 50 mL beaker and then add 10 mL of the 7 3 petroleum ether acetone solvent mixtures While stirring the mixture pour it onto column as carefully as possible using a spatula to scrape out the solid as you pour the liquid The most important thing to remember when you are packing a column is to make sure you are creating an evenly distributed …