Characterization of Photopigments by TLC Dan Wolfgram, BSCI105, Section 5271, Spring 2013 Thin layer chromatography is used to separate

and purify extracts of photopigments. In this lab, we separated photopigments extracted from spinach. The primary task was to identify the photopigments in different types of plants, especially chlorophyll-B. We used different wavelengths of light and the corresponding absorptions to identify the pigments. In the scenario given, identifying the different pigments assists in determining how the herbicide will effect different plants. We used a porous matrix that carries pigments as a solvent is run through it. (Keller 2013). The solubility determines how far the pigments will travel down the matrix; the higher the solubility the farther the pigment moves. This process resulted in separation of seven pigments and calculation of absorption of dark green, light green, and orange pigments. These results will help determine which photopigments were extracted and thus which plants the herbicide will effect. Introduction Chlorophyll-B is a vital pigment used in plants to absorb light that is used for photosynthesis. If the herbicide is interfering with this pigment, it explains why all the plants have died. A way to tell if it is indeed chlorophyll-B that is being effected, plants that lack the pigment need to be tested with the herbicide to see if they still die or not. Thin layer chromatography is a method that can be used to separate pigments so they can later be identified. TLC is accomplished by running a solvent through a matrix that carries the pigments. Polarity plays a large role in TLC because of its solubility properties. If a pigment is soluble in the solvent it will be carried up the plate as the solvent moves up (Keller 2013). If a pigment is not soluble then it will not move at all because it will not be carried with the solvent. Polar solvents dissolve polar pigments and non-polar solvents dissolve non-polar pigments. In our experiment we separated pigments extracted from a spinach plant. We used a solution of acetone and hexane to separate the non-polar pigments from other polar molecules. Next, we created a concentrated line of pigment on the TLC plate. Then put the plate in a solution that was mostly nonpolar but also had some polar parts. This means the more non-polar pigments moved farther on the plate because they were most soluble in the solution. The more polar pigments did not move as far because they were not as soluble in the solvent solution. Thin layer chromatography is used in this scenario to discover what photopigments are in spinach plants that may or may not contain chlorophyll-B. The professor's hypothesis is that his herbicide interferes with chlorophyll-B function but he cannot be sure because most plants use chlorophyll-B for photosynthesis and thus to live. Thin layer chromatography is useful in this scenario because using the technique in the previous paragraph, it is possible to find which plants do not have chlorophyll-B so the herbicide can be tested on them. After TLC is complete, samples of each separated pigment is made into a solution, whose light absorption is tested at different wavelengths. These absorption results can be compared to known data about chlorophyll-Bs absorption levels to see if it is present in the plant.

Materials and Methods Preparation of Pigment and Solvent solution Following the procedure of (Keller 2013), non-polar pigment molecules and other more polar molecules were separated using a mixture of hexane and acetone in a separatory funnel. Using the separatory funnel helps avoid any contamination by water. (Qauch, Steeper, Griffin 2004). Next, using the separatory funnel separate the acetone and hexane solutions. Create a concentrated line on a TLC plate with the hexane solution. Next create a 120mL hexane/60mL diethyl ether/40mL acetone solvent solution. This is where the TLC plate will be placed so the solvent can run up the plate. Calculating Absorption Levels of Pigments Scrape off the separated pigments and add 100% acetone to create a solution that can be added to the spectrophotometer. Check absorption of each pigment at wavelengths of 430nm, 440nm, 460nm, 640nm, and 660nm. Results There were 7 pigments separated using TLC. They were in order of increasing Rf values: light yellow, darker yellow, darkest yellow, dark green, darker green, gray, yellow. The calculated Rf values are determined by how far the pigment traveled on the plate (Figure 1). The absorption levels were taken of the dark green, light green, and orange. The light green had the highest absorbtion, second was orange, and then dark green. None of these pigments exactly matched the absorbtion graphs provided (Keller 2013), but the light green pigment was the closest. Discussion The main purpose of this lab was to determine whether the spinach plant has the pigment chlorophyll-B. The professor needs to find a plant without chlorophyll-B so he can test his herbicide on it and see if it is indeed inhibiting the pigment from performing its functions in photosynthesis. We used TLC to separate the pigments of spinach and then took absorption levels to see if the spinach did contain chlorophyll-B. In order to determine if the chlorophyll-B pigment was present, the absorbance levels determined from the pigments separated in TLC were compared to the absorbance levels known of chlorophyll-B. The absorbance levels of light green, orange, and dark green pigments were taken and recorded. While none of the levels matched exactly the absorbance levels given of chlorophyll-B, the light green pigment (.696OD), peaked at 460nm, the same wavelength chlorophyll-B (.800OD) also has peak absorbance. The difference may be accounted to the fact that the chart provided is absorbance of photopigments in 80% acetone while we used 100% acetone. Based solely on the data received from our experiment, it cannot be stated conclusively that there is chlorophyll-B present. However, spinach is known to have chlorophyll-B (Kidmose 2005). The discrepancy may also be due to human error, or the fact that we only took absorption levels of three pigments. If the professor was to test his herbicide on this spinach plant because the results did not show conclusively the presence of chlorophyll-B, he would most likely be disappointed when the plant dies. What remains to be determined is to find a plant that does not have chlorophyll-B so the professor can test his herbicide. The TLC method, is very useful in separating pigments and would be appropriate to use on a new plant to test whether the pigment is present.

Literature Cited Quach, Hao T., Robert L. Steeper, and William G. Griffin. "An Improved Method for the Extraction and Thin-Layer W Chromatography of Chlorophyll a and B from Spinach."Journal of Chemical Education 18.3 (2004): 385-87. Print. Kidmose, U., Edelenbos, M., Christensen, L. P., & Hegelund, E. (January 01, 2005). Chromatographic determination of changes in pigments in spinach (Spinacia oleracea L.) during processing. Journal of Chromatographic Science, 43, 9, 466-72. Forgacs, E., & Cserhati, T. (January 01, 2002). THIN-LAYER CHROMATOGRAPHY OF NATURAL PIGMENTS: NEW ADVANCES. Journal of Liquid Chromatography & Related Technologies, 25, 10, 1521. Keller, M.J. 2013. Introduction to Experimental Biology. Hayden-McNeil Publishing: Plymouth, MI.

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Color/Rf Light Yellow/ .022 Darker Yellow/ .067 Darkest Yellow/ .098 Dark Green/ .125 Darker Green/ .150 Gray/ .188 Yellow/ 1.00

Figure 1. Image of TLC plate after separation of pigments using hexane/diethyl ether/acetone. The table shows the color and calculated Rf values of the corresponding numbers. Rf values indicate how far the pigment travel due to it level of solubility in the solvent.