INTRODUCTION Extraction and purification of naturally occurring molecules is of the most common methods of obtaining organic molecules. Locating and identifying molecules found in flora and fauna can provide a number of different molecules. These molecules are frequently used as feedstock for the chemical industry or as components of medicines and foods. Obtaining pure samples of these molecules is essential to our modern chemical and pharmaceutical industry. In this experiment you will use a common method of extraction to obtain a mixture of organic compounds. Extraction involves separation of selected components of a mixture through contact with a second insoluble phase. The ability to separate the desired components from the rest of the mixture will depend on the affinity of the desired components for one phase versus the other. Plants are known to produce many interesting and useful organic compounds; today we will be extracting Chlorophyll from spinach. A significant portion of any cellular material is water soluble; however the most of the useful organic components are more soluble in non-polar solvents than in water. We will take advantage of this trait by breaking down the cellular structure of a sample in the presence of an organic solvent. As a result, the components of interest will preferentially be dissolved in the organic solvent, and subsequent washing of the organic phase with water will remove any remaining water soluble components. We are then left with a mixture of organic compounds dissolved in our solvent, which will require further purification to isolate the desired compound(s). To further purify our Chlorophyll we will employ chromatography, another common laboratory technique. Chromatography involves use of a mobile phase, called the eluent, and a stationary phase. The mobile phase, typically a gas or liquid, is moved through the stationary phase, most often a solid, to separate the mixture. The more attracted a compound(s) is to the stationary
phase the slower it will move, while those more attracted to the mobile phase will move through the stationary phase much faster. Resulting in a separation of our mixture based on the characteristics of the compound(s) and that of the stationary and mobile phases. Most often compounds are separated based on their polarity, but other characteristics such as chirality can also be used. The measure of any particular compound s mobility in a chromatograph is Rf, Retention factor. The Rf of any particular compound is a ratio of how far/fast the compound has traveled over how far/fast the mobile phase has traveled. Because Rf is a ratio that is dependent on the nature of both the mobile and stationary phases, it can be used for identification purposes, provided the same mobile and stationary phases are used. In the case of spinach, our remaining organic solution will contain a mixture of carotenes, Chlorophyll s A and B, and Xanthophyll s. We will begin by performing Thin-Layer Chromatograph to determine the best mobile phase to use; in TLC the stationary phase is a thin-layer of silica(or alumina) powder adhered to a glass or plastic plate. Our mobile phase will travel up the plate (due to capillary action) through the silica separating our compounds. Once an optimum mobile phase is found we will perform larger scale Column chromatography by filling a burette tube with silica (or alumina) powder and flushing the column with our mobile phase to separate our compounds into fractions. A fraction is a set amount of mobile phase that has been put through our column. Ideally our purified compounds should now be collected in a few of our fractions, separated by other fractions which contain only solvent. To determine what fractions our desired compounds are in, we will then perform a TLC on each fraction.
PROCEDURE Extraction 1) Place 5g of raw spinach, torn up into small pieces, into a mortar and pestle. 2) Add 25mL of Petroleum Ether and 10mL of Methanol, and grind the leaves well. 3) Using a Buchner funnel, vacuum filter the resulting pulp making sure no solid particulate gets through the filter, rinse the solid with 10mL of Petroleum Ether and transfer the liquid to a separatory funnel. Dispose of the remaining solid in the trash can after observing the color. 4) Add 25mL of a saturated NaCl solution to the separatory funnel and stopper the funnel, then invert and vent the funnel by opening the stopcock. Carefully shake the funnel venting often. 5) Place the funnel in a ring-stand and drain the aqueous layer into a 100mL beaker and repeat step four again draining the aqueous layer into same beaker. 6) Collect the organic layer into a 50mL beaker, taking care to avoid collecting any water, add a small amount of anhydrous Na2SO4 (approx. 2g). If you still see two layers you will need to add more Na2SO4. After 5 minutes, transfer your dried extract to a properly labeled sample vial. Your instructor will direct you on where to store your sample. PROCEDURE Chromatography 1) Obtain your dried sample of Spinach Extract, Label and prepare 3 TLC chambers by filling them with about 5mL of the following solvents: a. Hexane/Acetone 90:10 (least polar mixture) b. Hexane/Acetone 70:30 c. Hexane/Acetone 50:50 (most polar mixture) 2) Obtain 3 TLC plates (avoid touching the silica [dull side] with your fingertips by holding the plates by the side). Draw, in PENCIL, an origin line at the base of the plates about 1-2 cm from the bottom, your origin must be ABOVE the level of your eluent (solvent) when the TLC is placed in the chamber. Make two crosshatch marks on each plate equidistant on the origin line. Each crosshatch represents the starting point of your samples.
3) Using a small capillary tube, draw up some of your Spinach Extract and carefully deposit your solution on one of your crosshatches (the spot should be as small as possible, with practice this can be around 1-2mm). Reapply your solution to your plate 1-2 more times in the same location allowing the spot to dry each time. 4) Repeat step 9 using the KNOWN CHLOROPHYLL sample on the second crosshatch. Be sure to gently label each crosshatch below your origin line. 5) Place one TLC in each of your chambers and allow your TLC s to develop undisturbed. Remove your plates when the solvent has traveled up the plate within about 1cm from the top, and immediately make a line where the solvent stopped. DO NOT allow the solvent to reach the top of the plate. 6) Closely outline each spot on the TLC plates and identify which spot is Chlorophyll. Determine which solvent (eluent) system displayed the best separation of your mixture AND calculate the Rf for all of the compounds in that solvent. (Our compounds are easily visible; however, organic compounds often are not visible on a TLC plate to the naked eye and additional equipment may be needed to make your compounds visible, such as UV light, I2(g), or other chemical indicator.) 7) Obtain a prepared Chromatography Column from your instructor. Your column should have a small glass wool plug at the bottom of a burette followed by a layer of sand, on top of the sand is a column of silica gel with a small amount of sand on top of that. There should be about 2-3 ml of solvent ABOVE the sand in your column. 8) Carefully place your column in your hood so that you can collect the eluent drained from it in small test tubes when placed in a test-tube rack. Drain the column into a beaker until the solvent just reaches the top of the FIRST sand layer. FROM THIS POINT ON DO NOT LET THE ELUENT (SOLVENT) GO BELOW THE TOP LAYER OF SAND.
9) With a pipet add 1mL of your Spinach Extract to top sand layer (try to make sure it doesn t run down the sides of the column), and again drain your column to the top of the FIRST layer of sand. Obtain 50mL of your selected eluent (solvent), add 1 ml of it to your column, and again drain to the top of the sand. 10) Fill a test-tube rack with small test tubes to collect your fractions (test-tubes). When collecting fractions you will want to collect approximately 2-3 ml of eluent per fraction. 11) Fill your column with as much of your eluent (solvent) as you can and begin collecting fractions by allowing the eluent to run into the test-tubes. When a test-tube reaches 2-3 ml, quickly move to the next fraction (test-tube). You will have to carefully add more eluent (solvent) to your column until you have used all 50mL of your chosen eluent (solvent). Again DO NOT LET THE ELUENT (SOLVENT) GO BELOW THE TOP LAYER OF SAND. (You should end with around 15-18 fractions.) 12) As your fractions are finishing, prepare a TLC plate with 3 crosshatches. On the first crosshatch place 1-2 spots of your Spinach Extract, on the others place 5-6 spots of your first two fractions, respectively. Develop the plate in a TLC Chamber using your chosen eluent (solvent). Prepare a similar plate for 3 rd and 4 th fractions and for any remaining fractions. Use your developed TLCs to determine which fraction(s) contain only purified Chlorophyll.