Experiment 6: Chromatography

Objective:

This lab is about understanding the basis of chromatographic techniques and their procedures
in separating compounds using a stationary and a mobile phase. Understanding how polarity,
molecular weight, and boiling points influence the interaction between compounds and the
phases, impacting their separation.

Introduction:

Chromatographic techniques are a diverse set of methods used to separate and analyze the
components of mixtures based on their differing interactions with a stationary phase and a
mobile phase. There are several types of chromatographic methods used in organic chemistry
such as thin layer chromatography, column chromatography, gas chromatography and radial
chromatography, in experiment 6 we will be using the thin layer chromatography. The thin
layer chromatography (TLC) is cost efficient and gives favorable yields for students in organic
chemistry.

Materials:

• Lab book
• Ruler
• Acetaminophen
• Aspirin
• Ibuprofen
• Naproxen
• Acetone
• Ethyl acetate
• CAM staining solution
• Gloves
• Silica gel TLC plate
• Capillary tubes for TLC
• UV light
 • 500-mL beaker
• Filtering paper
• Aluminum foil

Procedure:

A. After putting on goggles and gloves, get a silica gel plate roughly 6.6x10cm, draw one of
two 10mm line (x-axis use a pencil only) from the bottom and another line (x-axis use a
pencil only) from the top ( solvent front) of the silicone gel plate. (this procedure vary
due to the solvent system able to migrant to top of plate.
B. Add 5 nodes 10mm apart from the bottom of the paper side to side for the samples in
the experiment.
C. Pour 1 mL of acetone and add it to the solvent.
D. Gether several capillary tubes for each of the sample used in this experiment (each
capillary tube use exclusively for samples)
E. Using the capillary tubes, dip the tube into the compound to apply this compound to the
TLC plate.
F. Using a UV light located in fume hood check if the compound was applied to the plate
properly.
G. Get a 500 mL beaker (properly cleaned and dried) place ½ of filter paper inside beaker.
H. Add 10 mL of acetate to beaker, then cover with aluminum foil, for roughly 10 mins for
the solvent to wet filter paper.
I. Using twizzers to place in beaker facing upward.

Questions/Discussion :

1. The toxicity of the solvents used:

In a laboratory setting, understanding the toxicity of chemicals like

acetaminophen, aspirin, ibuprofen, naproxen, acetone, and ethyl acetate is
essential for ensuring safety. For example, acetone is flammable and can cause
irritation to the eyes, skin, and respiratory issues. This is why lab safety techniques
are essential for students in organic chemistry. In this lab students are required to
gloves, goggles, and twizzers to avoid contact with samples.

2. Discuss the separation process and why compounds move up the plate at different
rates.
 In this experiment, the stationary phase will be silica gel, the mobile phase will consist of
a solvent or solvent mixture, to optimize the separation based on the polarity and
solubility of the target compounds. As the mobile phase travels up the TLC plate due to
applying samples in the capillary tubes, the compounds within the mixture will interact
differently with both phases, leading to their separation into distinct spots. The relative
positions of these spots, characterized by their retardation factor (Rf) values, will enable
the identification and characterization of the individual samples/components.

Conclusion:

Using the TLC plate, distinct spots were observed for each component in the mixture, reflecting
the number of different compounds present. These spots were located at different heights on
the plate, indicating varying Rf values for each compound. This variation in Rf values is a result
of the different polarities of the compounds, with more polar compounds interacting more
strongly with the polar stationary phase and thus traveling shorter distances, while less polar
compounds move further along with the less polar mobile phase.