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. Thin-

Layer 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 TraveledAcetaminophen 3.5 cm 5.3 cmAspirin 4.2 cm 5.0 cmCaffeine 2.0 cm 4.4 cmCo-Spot 2.0 cm 4.0 cm 5.0 cmSolvent Front 5.8 cmTable1: 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 cmAspirin 1.3 cm 3.9 cmCaffeine 4.0 cm 1.3 cmCo-Spot 0.9 cm 4.5 cmSolvent 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 cmAspirin 3.8 cmCaffeine 0.4 cm 1.5 cmUnknown 1.9 cm 4.4 cmCo-Spot 1.5 cm 4.0 cmSolvent 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 1Substance Rf Value

Acetaminophen 0.603 0.914Aspirin 0.724 0.862Caffeine 0.345 0.756Co-Spot 0.345 0.690 0.862

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

Table 5: Rf Values of Trial 2Substance Rf Value

Acetaminophen 0.181Aspirin 0.236 0.709Caffeine 0.073 0.236Co-Spot 0.164 0.818

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

Table 6: Rf Values of Trial 3Substance Rf Value

Acetaminophen 0.2

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Aspirin 0.76Caffeine 0.08 0.3Unknown 0.38 0.88Co-Spot 0.3 0.8

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

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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. Thin-

Layer 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 aren’t heavily affected.

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Post-Lab Q’s:

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:

BIBLIOGRAPHY 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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