Lab 2: Extraction of Spinach Pigments and Thin Layer Chromatography (TLC)
-To learn the highly useful thin layer chromatography technique.
-To study the chemical composition of plant leaves.
One of the most modern methods of separating mixtures in chemistry is chromatography. Chromatography utilizes a mobile phase and a stationary phases. Much as the name implies, the mobile phase is a solvent that flows sample and the stationary phase (also known as the adsorbent). The stationary phase is generally a solid powdery material with a polar surface. Silica (SiO2) and alumina (Al2O3) are the two most common stationary phase materials.
In thin layer chromatography (TLC) the stationary phase is the adsorbent silica, which is bound to a plastic-backed plate called a TLC plate. Silica is considered a polar substance since the surface of the crystals consists of polar hydroxyl (OH) groups. The mobile phase is an organic solvent mixture that, by capillary action, will move up the stationary silica coated plate. The solvent is relatively non-polar compared to the silica adsorbent.
The sample mixture is usually applied as a small spot near the base of the TLC plate (this process is called spotting). The plate is then put into a solvent reservoir where, by capillary action, the solvent will rise up the plate. As the solvent ascends the plate, the compounds in sample will dissolve into the mobile phase. This process is called developing the TLC plate.
When developing a TLC plate, the various components in the mixture are separated based upon polarity. Polar molecules will tend to spend a greater amount of time clinging the polar stationary phase than nonpolar molecules. The more strongly molecules cling the stationary phase, the slower the mobile phase will transport them across the place. Thus polar molecules tend to move across the plate more slowly than nonpolar molecules.
Compound Distribution Equilibrium
In this experiment you will separate a mixture of colored molecules extracted from spinach leaves and then separate this mixture into its individual components using TLC.
Pre-Lab: (Answers submitted at the beginning of lab)
- Read the short article “The Chemistry of Autumn Colors” and write a 1-2 paragraph summary of this article. Article can also be found at this web address:
1. On a balance weigh 1.0 grams of fresh spinach and combine with 1.0 gram of anhydrous magnesium sulfate and 2.0 grams of sand. Transfer the materials to a mortar and using a pestle grind the mixture until a fine powder is obtained (if the leaves are wet to begin with, you may not get a dry powder).
2. Transfer the powder to a large test tube and combine with 2.0 ml of acetone. Stopper the test tube and shake vigorously for approximately one minute. You need to make sure that the solvent and solid are well mixed. Allow the mixture to stand for 10 minutes.
3. Use a pipette to carefully transfer the solvent above the solid (should be green) into a small test tube. Add 1 or 2 mL of acetone if necessary. Do not suck the solid into the pipette. Cover the tube to avoid evaporation. Do not discard this solution because it will be needed again in step 10.
4. Obtain a TLC chamber (a glass jar with a cover) and add developing solvent (a mixture of pet ether, acetone, cyclohexane, ethyl acetate and methanol). The solvent should completely cover the bottom of the chamber to a depth of approximately 0.5 cm or 1/8”.
5. Obtain a TLC plate (a silica gel coated plastic sheet) which has been precut and make a dot with a pencil on the matte powder-coated side approximately 1.0 cm from the bottom of the strip. Do not use pen because the ink will run when exposed to the solvent.
6. Fill a capillary tube (the very small glass tubes) by placing it in the leaf extract. Apply the extract to the center of the dot on the matte powder-coated side of the TLC plate by quickly touching the end of the TLC applicator to the plate. You want the spot to be approximately 0.5 cm or 1/8” across. Allow to dry. Repeat several times to make a concentrated dot of extract (your instructor will demonstrate this process). Be sure to let the spot dry between applications and before you place it in the chamber.
7. Carefully place the TLC plate in the TLC chamber. The TLC plate should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract dot). Screw the lid on the TLC chamber.
8. Allow the TLC plate to develop (separation of pigments) until the solvent is close to the top of the plate (roughly 1 cm away). As the solvent moves up the TLC plate you should see the different colored pigments separating.
TLC Development Chamber
9. Remove the TLC plate from the chamber when the solvent is approximately 1.0 cm from the top of the TLC plate. With a pencil, mark the level of the solvent front (highest level the solvent moves up the TLC plate) as soon as you remove the strip from the chamber. Gently mark the location of all visible spots on the TLC plate with a pencil because they will fade over time.
10. Use the solution to prepare a TLC plate for each member of the group.
10. Calculate and record the Rf values (see below).
Edited by Brent Nannenga4/30/09
Rf Values: Rf= distance traveled by substance/distance traveled by solvent front. Calculate Rf values for each observed spot. Note that this example has two spots but the spinach should show 3 to 6 spots.
Put all extra organic solvent into the organic waste bottle in the hood. Aqueous solutions can go down the drain. Spinach residue can go into the trash.
Lab Report Guide:
-1. Results (5 pts)
- Create a table containing following information about each spot
- Distance traveled
- Rf value
- Sketch your TLC plate
- Attempt to identify each of the spots using the below information:
The colored pigments from a plant fall into 2 categories, Chlorophylls and Carotenoids. Caroteniods are yellow pigments that are involved in the photosynthesis process. Xanthophylls are oxygen-containing carotenes (OH and C=O) The carotenes are shown below. The Green pigments are the Chlorophylls that act as the principal photoreceptor molecules of plants. There are two different forms, chlorophyll a and chlorophyll b. The two forms are identical except that the methyl group that is shaded in the structural formula of Chlorophyll a is replaced by an aldehyde (C=O) group in chlorophyll b. Pheophytin a and pheophytin b are identical to chlorophyll a and b except that in each case the magnesium ion (Mg2+) has been replaced by two hydrogen ions.
On your TLC place you will see in the following in order of decreasing Rf value (top of plate to bottom); Carotenes (yellow), Pheophytin a (grayish), Pheophytine b (grey, may not be visible), Chlorophyll a (blue-green), Chlorophyll b (green), Xanthophylls (up to 3 spots, yellow)
-2. Post Lab Questions (5 pts)
- Typed answers to the Post Lab questions. Note that single sentence answers will not suffice. State the answer to the question followed by a brief description of the evidence supporting that answer.
- Why are the chlorophylls less mobile than the carotenes on the TLC plate?
- Is it possible to have an Rf value greater than 1? Explain.
- There are a number of differently colored molecules present in the spinach. Explain what role these molecules might serve in photosynthesis.
- You may have noticed that many of the spots on the TLC faded upon exposure to air. Does the speed with which the spots faded suggest anything about their strength as antioxidants? You may wish to research antioxidants before answering this question.
- Very polar compounds are sometime purified by reverse phase chromatography where the stationary phase is very nonpolar while the solvent is very polar. Why might one want to use this technique with extremely polar compounds?
Edited by Brent Nannenga4/30/09