Separation of Plant Pigments Using Chromatography
By: Kasey Brent
Statement of the Problem
From prior knowledge, one knows that chromatography paper is a useful technique for identifying unknown compounds. According to The Biology Corner, chromatography paper is often used in crime scene investigations to match ink, lipstick, or colored fibers. By determining and comparing Rf value of unknown compounds, crime scene investigators are able to identify the compound.
Materials
· Strips of Chromatography paper (the number of strips depend on the desired number of test subjects)
· Pencil
· Beaker or flask
· English Ivy leaf
· Purple Plum tree leaf
· Acetone (fingernail polish remover)
· Popsicle sticks ( the number of popsicle sticks depend on the desired number of test subjects)
· Penny
· Tape
· Scissors
Procedure
In this lab, plants are rubbed onto chromatography paper and placed in acetone to determine the plant pigments in the plants. When the chromatography paper comes into contact with the acetone, the acetone begins to climb up the chromatography paper spreading the plant pigments up the chromatography paper. The following are the steps taken during the procedure:
1. Use the scissors to cut a strip of the chromatography paper.
2. Draw a horizontal line of one end of the paper with a pencil approximately ½ inch from the bottom.
3. Place a leaf on the line drawn during step 1 and roll the penny over the plant to create a line of pigment on the filter.
4. Repeat step three until the line is fairly dark.
5. Pour approximately one inch of acetone (fingernail polish remover) in a beaker.
6. Tape the top of the chromatography strip to a popsicle stick across the top of the beaker.
7. The bottom of the chromatography paper strip needs to be in the acetone, but the green line does not need to be in the liquid (one may need to adjust the length of the chromatography paper accordingly).
8. As the pigments move up the paper, take pictures to document the experiment.
9. Set the beaker aside and observe as the acetone moves up the chromatography paper and deposits the pigment components along the way.
10. Leave the chromatography paper in the acetone for ten to fifteen minutes.
11. Once the chromatography paper is taken out of the acetone, draw a line where the pigment ends (solute) and where the acetone stopped being absorbed by the chromatography paper (solvent).
12. Find the Rf value of the plants.
13. Repeat all steps for each subject being tested
Results (Data)
Table 1
Results of Experiment
Materials Tested: / Band Color: / Pigment: / Distance (mm): / Rf Formula (mm):English Ivy / Yellow / Xanthophyll / Solute- 17
Solvent- 30 / 0.57
Yellow Green to Olive Green / Chlorophyll b
Purple Plum Tree / Yellow / Xanthophyll / Solute- 30
Solvent- 30 / 1
Bright Green to Blue Green / Chlorophyll a
Yellow Green to Olive Green / Chlorophyll b
Pink/Magenta / Anthocyanin
Figure 1 Jordan Greer checks to make sure she is making a dark line on the chromatography paper.
Figure 2 The Chromatography Paper is placed in the acetone.
Figure 3 As the acetone travels up the chromatography paper, so does the plant pigments.
Conclusions
After drawing lines where the solute and solvent ended on the chromatography paper, one measured the distance from the starting point to the solute and from the starting point to the solvent. By using the Rf formula distance traveled by the solute from the original linedistance traveled by the solvent from the original line, one found the Rf formula of the English Ivy and the Purple Plum tree. This resulted in the Purple Plum tree having a slightly large Rf then the English Ivy. One also found the Purple Plum tree to have more pigments then the English Ivy.
During the experiment one learned human error does occur when conducting science experiments. The experiment had to be conducted twice due during the first trial the group did not mark the distance traveled by both the solvent and solute, which is essential in order to figure the Rf value of the plants. A second trial was then conducted.
After studying the results of the second trial, the group noticed that although the majority of the pigments of the English Ivy ended at seventeen millimeters, there was still a little bit of pigment that had ended at the solvent line. This may have occurred due to an error unobserved by the group during the experiment.
Also during the duration of the experiment, one learned plants all vary one from another in pigment color. Some plants have pigment colors that others do not and sometimes they share similar colors with just a slight difference in shade. Furthermore, it was interesting to know that plants have many pigments combined to create their color besides the colors seen by the naked eye.
References
The Biology Corner. (2001). Separation of plant pigments using chromatography (demo lab). Retrieved from http://biologycorner.com/worksheets/plant_pigments.html on September 29, 2013