Aubrie Russell
CHM2210L-026
TA: Nathaniel Johnson
9/24/20
Post Lab Report Experiment #3: Chromatography – Analyzing Analgesics by TLC and
Isolation of β-Carotene by Column Chromatography
Introduction
The purpose of the first preformed experiment was to review 4 different analgesics
through thin layer chromatography (TLC) and to identify an unknown chemical through the
same process. The second experiment was done with the intent to apply chromatography to
separate β-carotene from spinach. Chromatography is the separation of two or more compounds
or ions caused by their molecular interactions with two phases – one moving and one
stationary1.TLC is used by placing different chemicals along a plate coated with silica gel and
then placed into a solvent. Through capillary action the solvent will ride to the top of the plate
and will carry the different chemicals different distances based upon their differences in
solubility. The main purpose of this technique is to identify unknown substances. The second
experiment was conducted through column chromatography, which can isolate compounds, such
as β-carotene. Major function of β-carotene in human nutrition is that of a provitamin A2. This
molecule can be found in various vegetables, but spinach was used for the experiment due to the
molecule being the least polar in spinach, making it the easiest to isolate.
Experimental Procedures
Part 1
1. Obtain the silica plates with fluorescent indicator from the stockroom. Draw a light pencil
line about 1 cm from the end of each chromatographic plate.
2. Using a micropipette, place 4 spots of known standards. Spot the ibuprofen 10-12 times
and 2-3 times for other compounds.
3. Examine the plate under UV light to see if the compound is applied.
4. Add approximately 10 mL of 95% ethyl acetate and 5% acetic acid in a chromatography
chamber and place the TLC inside the chamber vertically using tweezers.
5. Close the chamber. Once the solvent front has moved to about 1/2 cm from the top edge,
mark it by drawing a line.
6. After the plate has dried, examine the plate under UV light and outline the spots.
7. Record the color of the spots and Rf. If necessary, place the plate in an iodine chamber
and let it remain for about 2 minutes.
8. After the two minutes is up, remove the plate from the chamber and record the necessary
information.
9. Obtain another TLC. Apply the 4 standards as before on the plate.
10. Dissolve an unknown tablet in a test tube using ethanol as a solvent and apply the
unknown sample on the TLC plate on which the four known tablets were applied.
11. Place the TLC into the TLC chamber. Record the color of the spots under UV light and
iodine chamber and calculate the Rf value of the unknown.
12. Finally, identify the unknown.
Part 2
1.
2.
3.
4.
To prepare the micro-column, fill the column first with a little bit of cotton.
Then, fill the column half-way with silica gel.
Add about 1/2 cm of sand on top of the silica gel
To extract the pigment mixture from the spinach leaves, weigh out about 2 g of spinach
leaves and transfer it to a small Erlenmeyer flask.
5. Add 15 mL of ethyl acetate and using a spatula, mash the spinach for 10-15 minutes.
6. Decant the liquid and add anhydrous sodium sulfate to dry it.
7. Remove the solvent by using an aspirator over the steam bath.
8. Use an aspirator trap.
9. Dissolve the solid in 5-6 drops of 1:1 ethyl acetate-petroleum ether solution. Save some
liquid for TLC in a capped vial.
10. Label 6 test tubes and place them in a test tube rack. Wet the column with petroleum
ether and allow it to drip through.
11. Use a gentle stream of air to push the liquid through quicker.
12. Add spinach residue to the column and add more drops of petroleum ether.
13. Repeat to keep the column wet. Continue to push air through the column and collect the
fractions in the test tubes.
14. Record the color of the fractions and its volume.
15. Transfer the same colored fractions to a 50 mL Erlenmeyer flask and evaporate the
solvent using an aspirator.
16. Use heat from the steam bath if necessary.
17. Dissolve the residue (saved from spinach extraction) in 3 drops of hexane and TLC the
crude and purified mixture against standard β-carotene.
18. Use hexane for the chamber solvent.
19. Spot the pure compound after evaporation repeatedly.
20. Determine whether the β-carotene was successfully isolated and purified by visualizing
both by UV light and I2.
Chemical Table of Contents
Chemical
Formula
Molar
mass
Density
Acetaminophen
C8H9NO2
151.16
g/mol
1.26
g/mL
Aspirin
C9H8O4
180.16
g/mol
1.40
g/mL
Structure
Caffeine
C8H10N4O2
194.19
g/mol
1.23
g/mL
Ibuprofen
C13H18O2
206.28
g/mol
1.03
g/mL
β-Carotene
C40H56
536.87
g/mol
940.0
kg/m3
Results
Compound
Distance traveled
Rf
Acetaminophen
3.35 cm
0.644
Caffeine
1.7 cm
0.327
Aspirin
4.00 cm
0.769
Ibuprofen
4.80 cm
0.923
Unknown
4.10 cm
0.788
Rf =
Acetaminophen:
3.35 cm
R f = 5.20 cm = 0.644
Caffeine:
1.7 cm
R f = 5.20 cm = 0.327
Aspirin:
4.00 cm
R f = 5.20 cm = 0.769
Ibuprofen:
distance traveled by compound
distance traveled by solvent
4.8 cm
R f = 5.20 cm = 0.923
Unknown:
4.10 cm
R f = 5.20 cm = 0.788
Compound
Distance traveled
Rf
Isolated β carotene
1.3cm
0.289
Standard β carotene
1.3cm
0.289
1.30 cm
R f = 4.50 cm =0.289 & final mass was 0.2g
Discussion
Conclusion
References
1. Weldegirma, Solomon. “Experimental Organic Chemistry: Laboratory manual for
CHM 2210L and CHM 2211L.” Lab handbook. University of South Florida.
2. Grune, T., Lietz, G., Palou, A., Ross, A. C., Stahl, W., Tang, G., Thurnham, D., Yin, S.
A., & Biesalski, H. K. (2010). Beta-carotene is an important vitamin A source for
humans. The Journal of nutrition, 140(12), 2268S–2285S.
https://doi.org/10.3945/jn.109.119024