Experiment 4: Separation of Spinach Pigments by Thin Layer
Chromatography
Theoretical Background
Thin layer chromatography, known as TLC, is another chromatographic technique that is used
to separate mixtures of compounds and to identify any particular chemical compound. It is often
used to monitor the progress of organic reactions and to check the purity of products. It also
permits the optimization of the solvent system for a given separation problem. In comparison
with column chromatography, it only requires small quantities of the compound (~ ng) and is
much faster as well [1,2].
The stationary phase is special finely ground matrix (such as silica gel) coated on a glass plate
or a metal plate as a thin layer. In many cases, a fluorescent powder is mixed with the ground
matrix to simplify the visualization of the samples under UV light.
Thin layer chromatography separates molecules on the basis how well the molecules absorb to
silica. The reason for coating silica on the plate is its large surface area, therefore the binding
capacity of the molecule. The surface consists of Si-OH groups that are very closely spaced,
these groups contain the active sites. Silica is polar and the oxygens can hydrogen bond to the
molecule and the water. The activity of the silica depends on the number of these sites.
Figure 1: The bonding on the surface of silica [1,3]
In this technique the mobile phase is the solvent that carries the sample. The solvent has two
functions, to carry the component through the chromatographic bed, and to compete with the
sample for space in the absorbed layer of molecules. The choice of solvent depends on working
these two solvent functions against one another. If the solvent is very polar, it will prevent the
absorption of sample to the silica. In this case the sample will move on the chromatographic bed
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rapidly. With a less polar solvent, the sample spends more time to be absorbed to the silica,
therefore moves slower.
In thin layer chromatography, the sample is dissolved in the mobile phase solvent. The resulting
solution is then spotted near to the bottom of the rectangular sheet of glass or plastic coated
with silica. The sheet is then immersed in a chamber containing a small amount of solvent,
keeping the spots above the surface of the solvent. Once the sheet is put in the chamber, the
solvent begins to increase on the sheet through capillary action. The coating on the sheet is the
stationary phase and the solvent is the mobile phase, since it moves up the sheet.
The distance travelled by each compound from the origin, or the base line, relative to the
solvent front itself is called as retention factor, R f.
Rf =
distance compound traveled from base line
distance solvent traveled from base line
The retention factor of a substance is constant under the same experimental conditions [1,4].
Apparatus
Equipment
- TLC chamber
- TLC silica gel plate
- Separatory funnel
- Mortar and pestle
- Graduated cylinder, 100 mL
- Graduated cylinder, 25 mL x 5
- Erlenmeyer flask, 50 mL x 2
- Funnel
- Beaker, 100 mL
- Pipettes, 10 mL x 2
- Stand
- Clamps
- Ring
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Chemicals
- Petroleum ether
- Acetone
- Calcium carbonate, CaCO3
- Sodium chloride, NaCl
- Sodium sulfate, Na2SO4
Procedure
1. Weigh about 0.5 – 1.0 g spinach leaves and cut the leaves into small pieces.
2. Put the spinach into a mortar; add 22 mL of acetone, 3 mL of petroleum ether and spatula
tip of calcium carbonate (CaCO3).
3. Grind the leaves until the pieces are too small to be seen and then filter the pigment extract
if needed.
4. Put the filtrate into a separatory funnel and add 20 mL of petroleum ether and 20 mL of 10%
NaCl solution.
5. Shake the separatory funnel and drain the lower layer into a beaker.
6. Wash the upper layer twice with 5 mL distilled water.
7. Then put the extract into a 50 mL Erlenmeyer flask and add a spatula tip of sodium sulfate,
Na2SO4, then decant the solution in another 50 mL Erlenmeyer flask.
8. Concentrate the leaf extract by evaporating the solvent under the hood or placing the flask
on a water bath which was set to 60 0C.
9. Put small amount of petroleum ether in the TLC chamber and cover the chamber for 15
minutes to equilibrate the conditions inside the chamber.
10. On the 4 cm x 8 cm TLC plate, draw a line with a pencil approximately 1 cm from the bottom
of the plate.
11. Using a capillary, apply the leaf extract on the plate as a line.
12. Wait until the line dries, then again draw the line with the leaf extract using capillary, repeat
this until a dark green line is obtained.
13. Check the depth of the solvent in the chamber, the surface of the solvent should below the
extract line.
14. Immerse the TLC plate into the solvent, seal the chamber and allow the chamber
undisturbed.
15. When the solvent surface reaches the 3 / 4th of the plate, remove the plate from the
chamber and mark the position of the solvent and the developed lines immediately.
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References
1. Yeditepe University, Biyoorganics Laboratory Experiments, 2006, Istanbul.
2. Bacher, A., Chem30BL Lecture Notes, Thin Layer Chromatography, 2003
http://www.chem.ucla.edu/bacher/General/30BL/tips/TLC1.html
3. Chemistry Department, CH 223 Organic Chemistry, John Carroll University, 2002
http://chemistry.jcu.edu/nicholsweb/ch223f01/spinachextraction.pdf
4. Chemistry Department, Chem 1020 Resources, Anoka-Ramsey Community College, 2005
http://www.ar.cc.mn.us/chemistry/Chem1020/
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