Chromatography of Photosynthetic Pigments
In this experiment a process of chromatography was used to separate chlorophyll a, chlorophyll b, xanthophyll, and beta carotene - Chromatography of Photosynthetic Pigments introduction. When these pigments were obtained they were used to measure the wavelengths by way of spectrophotometer of each and the total of all the pigments wavelength. Introduction
Photosynthesis is a process by which plants use the sunlight to convert it from light energy into chemical energy. This equation shows us how it is done, 6CO2 + 6H2O + light energy –> C6H12O6 + 6O2. The pigments called chlorophylls in the plants are what trap the light energy. Carotenoids also help aid the chlorophylls by also absorbing light. The two chlorophylls found in green plants are chlorophyll a and chlorophyll b. The purpose of this lab is to extract the pigments from spinach leaves and separate them by using chromatography to determine each of the pigments absorption spectra. Methods and Materials
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For this experiment I used a 10mL pipette, a 100mL graduated cylinder, a rubber stopper, chromatography paper, forceps, cuvettes, spinach leaves, hexane-acetone, acetone, and the spectrophotometer to record the wavelength.
To start the experiment I first pipette 10mL of hexane-acetone into a 100mL graduated cylinder and put the rubber stopper on to prevent any of the solution from evaporating. While letting the hexane-acetone set I then prepared my chromatography strip but first cutting it to 20 centimeters and then cutting one end to a point. Then I used 2-3 spinach leaves and a quarter to leave a green residue line containing the pigments of the spinach leaves. I then inserted the strip with the spinach pigments into the cylinder I prepared earlier with the hexane-acetone. When the chromatography strip was properly into the cylinder I then put the rubber stopper onto the cylinder checking the progress of how far the solvent moved every 5 minutes. After 20 minutes the solvent reached to about 1 cm from the top of the paper I unstopped the cylinder and used the forceps to pull out the chromatography strip and quickly marked the solvent front. After I traced the chromatography strip for each shade I then marked them each as chlorophyll a, chlorophyll b, xanthophylls, and beta-carotene accordingly. To determine the absorption spectrum for each pigment I cut the chromatography strip into pieces according to the color and what it is. When the 5 groups of people had also cut up the strips we put each into their separate beakers with 8mL of pure acetone for 3 minutes. Then each group assigned with the pigment of chlorophyll a, chlorophyll b, xanthophylls, beta-carotene, and the total of all of the pigments. Then using each of the solutions each group pipetted each pigment into a curvette and measuring each in the spectrophotometer to get the lambda max. These procedures where preformed as described by ‘Wilson and Mayer (2012)’.
When the absorbance values were achieved the results were that chlorophyll b had a larger absorbance value of 0.121189 at a wavelength of 420 than chlorophyll a, which had an absorbance value of 0.074254 at a wavelength of 430. The absorbance value for xanthophylls was 0.117486 at a wavelength of 700, and Beta-carotene had an absorbance value of 0.018409 at a wavelength of 440. Finally for the total of all of them the absorbance value was 0.696251 at a wavelength of 430. The Rf value for chlorophyll a was 0.618, chlorophyll b was 0.448, xanthophylls were 0.679, and beta-carotene was 0.709. These results are shown in the graph below.
Chlorophyll a and chlorophyll b mostly absorb in the red and blue in the absorption spectrum. They are very similar in molecular structure and chlorophyll b helps chlorophyll a by absorbing slightly different wavelengths broadening the work that can be done. Therefore chlorophyll a in a blue-green color and chlorophyll b is an olive green color. Carotenoids or accessory pigments such as Xanthophylls and Beta-carotene help prevent damage to the chlorophylls by absorbing the damaging light. These accessory pigments absorb violet and blue-green light and are various shades of yellows and oranges. Xanthophylls and beta-carotene also help to absorb the light energy and transfer it to chlorophyll a so it can be converted from light energy into chemical energy. The effect of these accessory pigments is to broaden the range of wavelengths that can be used in photosynthesis and to protect the chlorophylls from photo oxidation.
Paper chromatography proved to be an accurate method of separating and observing the various colors of plant pigments. The pigments dissolved in the solvent and migrated upward. The colors were observed and their migration distances measured & recorded. The Rf value of each pigment was determined by dividing its migration by the migration of the solvent. It was determined that 4 pigments were present in the original spot beta-carotene, xanthophylls, chlorophyll a, and chlorophyll b. Beta-Carotene was the most soluble, while chlorophyll b was the least soluble.
* W.R.Wilson, PhD, G. Mayer. PhD. (2012). Gorton cell biology. (pp. 33-43). United States of America: Lansing Community College. * Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson. (2012).Campbell biology. United States of America: Pearson learning company.