Chromatography Lab
AP Biology 2011
Radha Dave, Chelsea Mbakwe, and Navya Kondeti

Abstract:

Chromatography is a technique for separating and identifying substances in a mixture based upon their solubility in a solvent. It can be used in the evaluation of photosynthesis, separating colors in chloroplast. Green plant pigments, that contain both chlorophyll a and b, can be separated through this process by testing the solubility in a solvent. In addition to chlorophyll, the leaves of many green plants also contain one or more other pigments including carotene (orange) and xanthophylls (yellow). Through this process we can test and see which color travels fastest.

Introduction:

The purpose of this lab is to separate and identify pigments using paper chromatography. Chromatography is a technique for separating and identifying substances in a mixture. When you put the paper chromatography into a certain solvent, the solvent travels up the paper by capillary action. Capillary action occurs when cohesion and surface tension bind a liquid together and these forces that attracted the liquid together bond it onto another surface (adhesion), in this case the paper. As the solvent moves up the paper it takes the various pigments along with it. You start to see different colors such as orange, green, ect. at different points in the paper because they are attracted to different points on the paper through the formation of intermolecular bonds, such as hydrogen bonds. To measure the rate of the pigments migration on a chromatography is the Rf value. You can figure out the Rf value by dividing the distance solute travels(D unknown) by distance solvent traveled (Dsolvent).

Methods:

When you receive your strip of chromatography paper you will need to cut a pointed end at the bottom end of the paper. After doing so you will draw a faint line with your pencil right where you started to cut the paper. Once you have completed that you will need to obtain a test tube and pour out 2 mL of “chromatography solvent”. You place this test tube on a test tube rack and then with a quarter you will roll a portion of the spinach leave onto the pencil line that you have made on the paper strip. After that you will take a paper clip, unfold it, and the pierce it into a cork. After doing so you will hook the other end of the paper clip onto the chromatography paper and carefully place it into the test tube so that the pointed end of the paper just touches the bottom. You will need to adjust the height of your piece of paper if needed because you want to make sure that the solvent doesn’t touch the sample line (spinach line). When solvent has reached about 1 cm from the top of the vial you will carefully take out the chromatograph paper and lay it down on the table to air dry for a bit. You will then need to record the various plant pigment colors you see. By using a pencil you will need to make the location of each pigment color and the solvent. You many need to hold the paper up to the light to get a better look at the colors because some may be faint. After doing so measure the total distance the solvent moved up the paper(Dsolvent). This variable is measured from where the spinach pigment is to where the it ended and you can no longer see the pigment. You will also need to measure the distance each pigment traveled(Dunknown). This variable is measured from the pigments starting point to the end point for each individual color you identify. Record your measurements and the Rf value for each pigment identified. Once you have done so ask you teacher what to do with chromatography paper strip.

Results:


Solute Color
Dsolvent (cm)
Dunknown (cm)
Rf=Duknnown/Dsolvent
Orange
(carotene)
7.6 cm
7.3 cm
1
Yellow
(xanthophylls)
7.6 cm
3.2 cm
0.42
Light Green
(Chlorophyll a)
7.6 cm
1.7 cm
0.22
Green
(chlorophyll b)
7.6 cm
0.7 cm
0.09



Discussion:

1. How many pigments were you able to identify on your spinach chromatogram?
The four pigments we were able to identify on on our spinach chromatogram were orange, yellow, light green, and green.

2. what purpose does the chromatography paper have in this experiment?
The chromatography paper served as a visual aid to the reaction taking place with the solvent (acetone) and the pigment. Through the duration of the experiment we observed the green pigment break off into a variety of colors showing us that a reaction was taking place and we were viewing it with our own eyes with help from the chromatography paper.

3. Do some extra investigating to answer the following question: Green pigments are essential for photosynthesis to take place. How do you think that a Japanese maple tree, which only has red leaves, can carry on photosynthesis?
When the experiment was conducted we observed that, although, green was the most dominant color, there was still traces of other color pigments found in the green. Using this information to answer this question,there would still be traces of green pigments so that photosynthesis can still take place, it might be as fast as it would have been in a green plant but photosynthesis is still happening nonetheless.

Conclusion:

Although we were not testing for anything, the results were essential for confirmation of a known technique. By using chromatography for our green plant pigments we were able toidentify four pigments, green, light green, yellow, orange other than that of JUST green. These colors displayed on the chromatography paper showed the pigments and solubility of each. Through this view we were able to depict which pigment color was lighter and which was heavier by the distance travelled up the chromatography paper of each individual color. We now understand the use of chromatography in identifying different compounds and the pigments of green leaves.

Literature Cited:

"LabBench." Prentice Hall Bridge Page. Web. 16 Nov. 2011. <http://www.phschool.com/science/biology_place/labbench/lab4/intro.html>.