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