Paper Chromatography in Chlorophyll Analysis
Translated by
ScribdTranslationsPaper Chromatography in Chlorophyll Analysis
Translated by
ScribdTranslationsNARIÑO UNIVERSITY
DEPARTMENT OF CHEMISTRY
GENERAL CHEMISTRY
PRACTICE 8. PAPER CHROMATOGRAPHY
1. OBJECTIVES
Acquire experience in the use of paper chromatography as a method of separation and identification of
the components of a mixture.
Separate the main components of chlorophyll using paper chromatography.
2. THEORETICAL FOUNDATION
2.1. PAPER CHROMATOGRAPHY
Chromatography is a chemical analysis technique used to separate pure substances from complex mixtures.
Paper chromatography is a technique used for the separation and identification of the components of a mixture,
by progressive adsorption of each of the components onto a special paper (stationary phase) as they are
transported by a solvent (mobile phase).
This is one of the simplest chromatographic techniques and requires simple equipment. It is used as a phase
stationary a sheet of pure cellulose paper and as the mobile phase pure solvents or mixtures of solvents, contained in
a closed camera.
The mixture to be separated must be in solution and is applied to one end of the strip of paper. It is then inserted into the...
camera and comes into contact with the solvent, which will ascend by capillarity through the paper, carrying with it the
components of the mixture at different speeds, in a multiple distribution process, thus causing separation.
The appropriate solvent must be used for separation, for which several tests must be conducted selecting the
solvent or mixture of solvents that achieves the best separation of the components of the mixture.
When the solvent has completed its path, the paper is removed from the chromatographic chamber and the front is marked.
solvent. The identification of the components of the mixture is based on the Rf, which express the relative distance traveled
the components of the mixture regarding the solvent's path, this value is constant and characteristic of each substance in
similar separation conditions.
If the components of the sample are colorless, it is necessary to proceed to the development, which consists of applying a reagent.
color developer with which a colored complex will be formed.
3. MATERIALS
2 beakers of 50 mL Glass capillaries
Graduated pipette 10 mL Mortar and pestle
Balance Absorbent paper
Chromatography paper or filter paper (Whatman Scissors
No.1 or Whatman 3 MM Scalpel
Chromatography chamber (100 mL graduated cylinder with aluminum foil (students)
cork
4. REAGENTS
Fresh spinach or Swiss chard leaves (students) Acetone
Magnesium sulfate Hexane:acetone mixture (8.5:1.5 v/v)
Washed Arena
5. PROCEDURE:
Wash and dry the spinach or Swiss chard leaves with paper towels. Remove the ribs and cut into small pieces.
grams of spinach leaves and deposit in a mortar. Macerate for a few minutes with 2 grams of MgSO.4and 4 grams
washed sand.
Transfer the obtained paste to a 50 mL beaker, add 15 mL of acetone, completely wrap with aluminum foil and
Shake intermittently for 10 minutes. Decant the extract into a 50 mL beaker, cover with aluminum foil.
leaving a small opening at the top, and evaporate at room temperature until half the volume is reached
initial of the extract.
¿Qué es la clorofila? ¿Para qué se utiliza MgSO4And the washed sand? What function does acetone serve and why is it left?
evaporate? What kind of pigments would be present in this extract?
Preparation of chromatographic chamber
Secure the chromatographic paper at one end to the groove of the cork. At the other end, at a distance of 2 cm, trace
with a pencil, a solid line and a second dotted line 1 cm from the edge. In the center of the solid line, the
sample (start of the process) and up to the dotted line, the paper will be immersed in the mobile phase. Insert the paper
chromatographic in the vial and determine the volume of mobile phase required to reach the level of the line
dotted.
What characteristics does the paper used in chromatography have?
Immerse a fine glass capillary in the extract and carefully apply the extract to the center of the sample point;
let it dry and apply more sample again, until a clear and well-defined stain of approximately 0.5 cm is formed.
diameter.
Why does this procedure have to be carried out several times?
Using a 10 mL graduated pipette, fill the graduated cylinder with the volume of mobile phase (hexane:acetone mixture)
Required, be careful not to spill the mobile phase onto the internal walls of the test tube. Carefully introduce the paper.
in the mobile phase up to a height of 1 cm from the bottom edge (see graph), fit the cork and seal it hermetically to
to prevent the evaporation of the mobile phase. Previously, make an aluminum foil cover and carefully cover the chamber.
chromatographic.
D fm
Distance traveled
through the mobile phase d3
d2
Sample d1
Mobile phase
d1, d2y d3they are the distances traveled
for each of the substances
Wait the necessary time for the mobile phase to rise up to 1 cm below the base of the cork, taking with it the
pigments and separate them. When it has reached that height, remove the paper from the test tube and mark the point with a pencil.
up to which the mobile phase arrived.
Follow the paper with pendulum movements, define the contour of the obtained spots, measure the distance traveled by
each substance from the center of the stain to the point of application of the mixture. Also determine the distance
traveled through the mobile phase. Determine the Rf of each of the separated substances.
Distance traveled by the substance
Rf =
Distance traveled by the moving phase
Table 1. Rf values for identification of separated pigments
Compounds Rf
Chlorophyll a 0.40
Chlorophyll b 0.46
Violaxanthin 0.56
Lutenina 0.76
beta-carotene and alpha-carotene 0.99
What role do the mobile phase and the stationary phase play in separation? Why is it possible to separate the components of
What is the utility of Rf in paper chromatography?
6. COMPLEMENTARY WORK
A. What do thin layer chromatography and column chromatography consist of?
B. What are the main solvents and solvent mixtures used in chromatography?
REFERENCES
VILLA, M. Manual of General Chemistry Practices, University of Medellín, Medellín 2005.
TORRISO, F., D. Separation of photosynthetic pigments using paper chromatography. Annals of Chemistry, 2007, 104(4)
45-51.