Thin-Layer Chromatography

Joseph Personelli
Lab Partners: Dominic Gonzlez & Benjamin Lowry
Chem. 2400
Dr. Joseph Kappel
Lab Assistant: Stacey Mendigutia
January 27, 2015

Abstract: The purpose of this experiment was to use a thin-layer chromatographic

technique to calculate retention factors in order to determine the identity of two
unknowns. Thin-Layer chromatography is a technique used to separate a sample so that
the sample can be analyzed and the purity determined. It was hypothesized that thin-layer
chromatography could be used to calculate Rf values that can be compared to Rf constants
to determine the identity of unknown substances. The hypothesis was accepted. The
unknowns of trial three were determined to be caffeine and aspirin based on the TLC
technique used.

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The purpose of this experiment was to use a thin-layer chromatographic technique

to calculate retention factors in order to determine the identity of two unknowns. ThinLayer chromatography is a technique used to separate a sample so that the sample can
analyzed and the purity determined.
Chromatographic techniques utilize absorptive and distributive properties of
different compounds and solvents. There are two phases, a stationary solid phase and a
mobile liquid phase. The stationary phase is the surface that is stained with a compound
you want to determine, in this case, the polarity of. The mobile phase is the solvent that
the stationary phase is placed into and is absorbed. As this solvent rises up the stationary
phase, it moves with it the blotted compounds on the stationary phase. The distance from
the solvent front is then used to calculate the Rf value of the compound.
Retention factors (Rf) are the distance traveled by the substance divided by the
distance traveled by the solvent front1. The retention factors of substances are a constant
and can be compared to help determine the substance and the purity of that substance.

(acetaminophen)

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(caffeine)

(Aspirin)
Materials and Methods:
Please refer to Chapter 6, Experiment 1 on pages 97-99 of Microscale Organic
Laboratory with Multistep and Multiscale Syntheses by Mayo, Pike, and Forbes for
information regarding the method and utilization of Thin Layer Chromatography (TLC).
There were no adjustments made to the experimental procedure.
Results:
Table 1: Trial 1 Thin-Layer Chromatography (Ethyl Acetate)
Substance
Distance Traveled
Acetaminophen
3.5 cm
5.3 cm
Aspirin
4.2 cm
5.0 cm
Caffeine
2.0 cm
4.4 cm
Co-Spot
2.0 cm
4.0 cm
5.0 cm
Solvent Front
5.8 cm
Table1: This table shows the distance that each substance traveled due to the solvent
ethyl acetate carrying it up the stationary phase plate. Each substance had two different
spots. The co-spot distance represent acetaminophen, aspirin, and caffeine respectively.

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Table 2: Trial 2 Thin-Layer Chromatography (Hexane)
Substance
Distance Traveled
Acetaminophen
1.0 cm
Aspirin
1.3 cm
3.9 cm
Caffeine
4.0 cm
1.3 cm
Co-Spot
0.9 cm
4.5 cm
Solvent Front
5.5 cm
Table 2: This table shows the distance that each substance traveled due to the solvent
hexane carrying it up the stationary phase plate.
Table 3: Trial 3 Thin-Layer Chromatography (Hexane)
Substance
Distance Traveled
Acetaminophen
1.0 cm
Aspirin
3.8 cm
Caffeine
0.4 cm
1.5 cm
Unknown
1.9 cm
4.4 cm
Co-Spot
1.5 cm
4.0 cm
Solvent Front
5.0 cm
Table 3: This table shows the distance that each substance traveled due to the solvent
hexane carrying it up the stationary phase plate. This trial had the addition of two
unknown added.
Table 4: Rf Values of Trial 1
Substance
Acetaminophen
Aspirin
Caffeine
Co-Spot

Rf Value
0.603
0.724
0.345
0.345

0.690

0.914
0.862
0.756
0.862

Table 4: This table shows the calculated Rf values for trial 1.

Table 5: Rf Values of Trial 2
Substance
Acetaminophen
Aspirin
Caffeine
Co-Spot

Rf Value
0.181
0.236
0.073
0.164

0.709
0.236
0.818

Table 5: This table shows the calculated Rf values for trial 2.

Table 6: Rf Values of Trial 3
Substance
Acetaminophen

Rf Value
0.2

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Aspirin
Caffeine
Unknown
Co-Spot

0.76
0.08
0.38
0.3

Table 6: This table shows the calculated Rf values for trial 3.

Calculations:
Rf Values from Table 6 from Trial 3
Acetaminophen: (distance compound traveled / distance solvent traveled)
(1.0 / 5.0) = 0.2
Aspirin: (distance compound traveled / distance solvent traveled)
(3.8 / 5.0) = 0.76
Caffeine: (distance compound traveled / distance solvent traveled)
Spot 1: (0.4 / 5.0) = 0.08
Spot 2: (1.5 / 5.0) = 0.3
Unknown: (distance compound traveled / distance solvent traveled)
Spot1: (1.9 / 5.0) = 0.38
Spot2: (4.4 / 5.0) = 0.88
Co-Spot: (distance compound traveled / distance solvent traveled)
Spot 1: (1.5 / 5.0) = 0.3
Spot 2: (4.0 / 5.0) = 0.8

0.3
0.88
0.8

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Discussion:
The purpose of this experiment was to use a thin-layer chromatographic technique
to calculate retention factors in order to determine the identity of two unknowns. ThinLayer chromatography is a technique used to separate a sample so that the sample can be
analyzed and the purity determined. Thin-Layer chromatography can also show the
polarity of a substance. The further distance that a spot traveled the less polar that
substance is. In order to see the spot and the distance it traveled the stationary phase
was placed under a black light and illuminated. After determining the distances of the
spots, the Rf values were calculated. It was hypothesized that thin-layer chromatography
could be used to calculate Rf values that can be compared to Rf constants to determine the
identity of unknown substances.
Three trials were performed during this experiment. In trial one the solvent that
was used is ethyl acetate and in trial two the solvent was hexane. In trial three the solvent
hexane was used again because it showed the most favorable results. In each of the trials
three known samples were used: acetaminophen, caffeine, and aspirin. In trial three,
there was an additional unknown compound added. Each of the samples were placed onto
the stationary phase plate and the plate was then added to the beaker containing the
solvents. The solvents began to travel up the stationary phase plate and when it reached
near the top the sheet was removed and noted where the solvent front reached. This
distance that the solvent front traveled was used at a later time in the experiment to
calculate Rf values. After evaporation the plates were placed in the UV light and
illuminated. This caused the spots to glow so that the distance from the initial drop spot

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can be compared to the new spot for calculation of the Rf value. Tables two and three
show the distance each spot traveled when placed into hexane.
After determining the distance that the spots traveled the retention factor values
(Rf) could be calculated using the Rf equation: Rf = (distance compound traveled /
distance solvent traveled). Tables 4, 5, 6 shows the calculated Rf values for each trial.
One reason that there are many spots on the stationary phase plate could be due to the
solvent being heavily concentrated. This could be eradicated during future experiments
by adding drops of another substance to dilute it.
Table three and table six show the distances and Rf values of trial three. In trial
three, two different unknowns were placed on the starting line along with the other
aforementioned substances. After placing the trial three stationary phase plate into the
UV light it could be seen that the unknown spot was a combination of Caffeine and
Aspirin. This was verified by comparing the Rf values of trial three to the Rf values of the
previous trials.

Conclusion:
After determining the Rf values for trial three and comparing them to trials two
and one the unknowns were caffeine and aspirin. The identity of the unknown spots was
able to be determined by way of a thin-layer chromatographic technique. The hypothesis
was accepted. One way to improve the results of this experiment is to have a more diluted
solvent so that the spots arent heavily affected.

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Post-Lab Qs:

5-35: Discuss the similarities and dissimilarities of TLC, paper, and column
chromatography.
A: In column chromatography, just like the name suggests, a column has a solid
stationary phase packed into it. The mobile phase flows up the column and is separated
due to solubility, polarity, and adsorption forces. In paper chromatography, cellulose
paper is used as an adsorbent unlike the column chromatography. However in thin-lay
chromatography a thin-layer of alumina or silica is used. In thin-layer chromatography
the mobile phase moves up the stationary phase while in column chromatography the
mobile phase moves down the column of silica. Similarities of TLC and paper is that both
use strips of a filter paper as a stationary phase.

5-39: In figure 5.41 why is a septum, not just a plain cap, used on the top of the gas
collection tube?
A: A septum is used because it allows for easier removal of the collected gaseous
butenes using a gastight syringe.

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References:
Mayo, D. W., Pike, R. M., & Forbes, D. C. (2011). Microscale Organic Laboratory with
Multistep and Multiscale Synthesis. Hoboken: John Wiley & Sons, Inc.

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