Separation of Photosynthetic Pigments by Chromatograph
Introduction:
The distance traveled by a particular compound can be used to identify the
compound. The ratio of the distance traveled by a compound to that of the
solvent front is known as the Rf value; unknown compounds may be identified by
comparing their Rf value to the Rf value of known standards.
Rf equation:
Rf = distance traveled by compound
distance traveled by solvent
Purpose:
A. To identify plant pigments by separation and isolation of the pigments
using thin layer paper chromatography
B. To observe the difference between the pigments found in a green leaf
(spinach) and one that has lost its chlorophyll (sugar maple)
Materials:
2 test tubes and rack
filter paper strips
15ml - 95% ethanol
sand glass rod
fresh green spinach leaves
safety goggles
4 toothpicks
2 - 50 ml beakers
mortar and pestle
10ml Graduated cylinder
plastic wrap
scissors
pencil
orange-red sugar maple leaves
Perform Procedures A and B Simultaneously
Procedure A:
1. Cut the spinach leaves into small pieces with the scissors and place
them into the mortar and grind them with the pestle for 1 minute.
2. Put on your safety goggles. Add a pinch of sand (as an abrasive) and 10
ml of 95% ethanol to the mixture and continue to grind for 2 minutes.
Note: ethanol is highly flammable.
3. Pour the resulting solution into a 50ml breaker, cover with plastic wrap
and leave for 10 minutes.
4. Wrap a filter paper strip around a toothpick and suspend the paper in a
test tube so that the end of the paper is about 1 cm above the bottom.
5. Remove the paper, place it flat on the counter and draw a pencil line 2
cm above the end.
6. Measure 5ml of 95% ethanol and pour it into a test tube.
7. Use a glass rod to place a small drop of the green liquid onto the pencil
line. Suspend the paper in the test tube. Be sure that the bottom of the
paper is touching the alcohol, but the line is well above the alcohol
level.
8. Cover the test-tube with plastic wrap and leave it for 20 minutes or until
the alcohol gets within 1 cm of the top of the paper.
9. Remove the paper and place it flat on the table.
10. Identify the different coloured pigments. Measure the distance that the
alcohol travelled and the distance of each identified pigment (IF
POSSIBLE).
11. Calculate the Rf value for the pigments that you identify (IF POSSIBLE).
Procedure B:
Repeat the above procedure for the sugar maple leaves and compare the
results.
What do you submit?
A. Raw data with a picture or drawing of your filter paper strips after the
chromatography is complete. Include qualitative observations.
Often the results are not clear enough to calculate an RF Value, but if
they are do parts b & c.
B. Calculation of Rf values for all pigments identified in each of the
samples.
C. Discussion of the identity of the pigments in each sample based on a
comparison of your calculated Rf values with published values.
You may use the following document as a resource. It contains Rf values of
a number of pigment.
http://soyroothair.org/images/education/pigmentchromatographytanaka-2014.pdf