Photosynthetic Pigments

Jessica Tietgens

Botany Lab

2 May 2008

Photosynthetic Pigments

Introduction: In this lab we were to investigate photosynthetic pigments using paper chromatography and then using the spectrophotometer determine the absorption for each pigment. Since the lab before had a difficult time getting all 5 pigments to show, it was expected we might, as well, not get all 5 pigments using the same procedure. As for the absorption, I figured that brighter, lighter; colors would have a higher absorption of light.

Method: In this lab, first we had to extract pigment from 1 gram of fresh spinach leaves. Once the spinach was macerated we added 10ml of 100% methanol in the fume hood. The pigment extract had a green, slimy appearance. We then pipetted a small about of the liquid into 2 centrifuge tubes, and weighed them out, we had to add or take out until their weights were the same. We centrifuged at 10,000g for 1 minute. We took our supernatant back to our table and placed a small dot onto a piece of chromatography paper using a capillary tube. We marked the spot with a pencil so we could measure later on. We then placed a small amount of the 100% methanol in a tube (not high enough to touch our dot) and placed the chromatography paper in the tube and caped it. We observed as the solvent traveled up the paper, before it reached the top we had to take it out of the tube and place it in the fume hood to dry. We then measured out the distant the pigments traveled and with these numbers we figured out our Rf values. After this we scrapped our pigments and put them in the appropriate centrifuge tube with the rest of the class’s pigment as well. Once all pigment was gathered, we added methanol and placed in centrifuge at 10,000g for 1 minute. We then blanked the spectrophotometer, then placed the supernatant in the spectrophotometer cuvette, we placed it in the spectrophotometer and read the pigment and methanol absorption. With this data graphs were printed and we followed this procedure for each pigment.

Results: After the chromatography bands had finished drying each group observed their bands. Some groups saw as many as 5 colors while others saw only 2. My group only saw 2 colors, they were very light almost hard to make out. At this point each group measured each components distant traveled on the band from the origin. With this data we could now calculate each components retention factor. The retention factor is defined by the ratio traveled from the original spot by the solute component to that of the distance traveled from the origin by the solvent. The equation is:

Rf=distance traveled by sample

distance traveled by solvent

The PM lab calculated the following Rf values:

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

.41 - - .125 .11 .146

.47 .692 - .2875 .457 .213

.65 .754 - - - -

.73 .785 .81 - - -

.86 .846 .86 - - -

With this information we did our best to identify the pigments using the following scale:

Pigments Rf Values

Carotente 0.98

Chl a 0.60

Chl b 0.42

Xanthophyll 1 0.28

Xanthophyll 2 0.15

Once we figured out which pigment our chromatography strip contained we scraped, and gathered all the same pigments together. Using the spectrophotometer we gathered graphs for each pigment. For the green pigment 3 peaks were observed from a 390.0 to a 740.0 nm wavelength. All 3 of these peaks were decent in size, 2 were close together at a lower wavelength and the other was at a higher wavelength. All 3 nearly reaching the 0.1 absorption level. For the yellow pigment 2 peaks were observed from a 390.0 to a 740.0 nm wavelength. Both peaks were very small; the one at the lower wavelength was carried out longer than that at the higher wavelength. They both were stayed around the 0.01 absorption level. In the third tube there were no peaks observed, the absorption just stayed at about 0.08 through a 390.0 to a 740.0 nm wavelength. So I guess it could be considered one consistant peak. The forth tube was the more prominent, where 5 large peaks were observed from a 390.0 to a 740.0 nm wavelength. The first reached 0.58 absorption, the second reached 0.70 absorption, third reached 0.45 absorption, the forth reached about 0.1 absorption, and the fifth reached 0.41 absorption.

Discussion: Visible light and other forms of electromagnetic energy emit through space as waves of various lengths. We distinguish different wavelengths of visible light, which range from about 380 to 750 nm, as different colors. White light is a mixture of all wavelengths of visible light. A prism can sort white light into its component colors by bending light of different wavelengths at different angles. Visible light is what drives photosynthesis. A spectrophotometer measures the relative amounts of light of different wavelengths absorbed and transmitted by a pigment solution; it plots this data onto graphs. The first graph (attached) shows the results of the scrapings of the green pigment. The absorption remains low, with 3 small peaks. The absorption peaks arose at wavelengths; 400 nm, 430 nm, and 670 nm. This proves that the pigment was green and also indicates that this pigment is chlorophyll b. Chlorophyll b is known for having its highest peak between the 400 nm and 500 nm wavelengths and then a smaller peak between the 600 nm and 700 nm wavelengths, just as is shown in graph 1. The second graph (attached) shows the results of the scrapings of the yellow pigment. The absorption remains very low, with one small peak. The absorption