Cassandra Shank Lab station A1

CHEM 2125-003 2/10/2020

Experiment 2: Thin Layer Chromatography

Abstract

Thin Layer Chromatography (TLC) is a technique that can be used for checking the purity of a

sample, determining the components in a mixture, and monitoring the progress of a reaction. The

main goal of the experiment was to learn about testing polarity with chromatography techniques

while calculating the R​f values.

​ To conduct this experiment, three TLC chambers were set up in

order to run each step of this experimental set. TLC plates were also set up in order to test

different samples to an unknown sample. The TLC plates were placed in the TLC chambers in

order to let the developing solvent absorb onto the plates which demonstrated the polarity of

each sample. Unknown analgesic #2 and unknown dye #2 were found to be caffeine and a

combination of red 3 and yellow 5. This was found because of the calculated R​f values
​ which can

be found using the distance from the baseline to the spot of the sample divided by the distance

the developing solvent traveled on the TLC plate as well as the visual appearance of the samples.

This experiment demonstrated how polarity can be used in order to identify an unknown

substance.

Theory

Chromatography is a technique that separates compounds based on their polarity. There are four

main types of chromatography: paper, high-performance liquid, gas, and thin-layer

chromatography. The different uses of Thin Layer Chromatography (TLC) include checking the
purity of a sample, determining the components in a mixture, and monitoring the progress of a

reaction. When using thin-layer chromatography, the samples are dotted on the same line along a

thin-layer chromatography plate. This plate will eventually be placed into a thin-layer

chromatography chamber which has a specific solution at the bottom. The solution will absorb

into the plate and cause the samples to travel up the plate based on its polarity. The distance of

the solvent absorption and how far it traveled will be used in the R​f calculations.
​ The formula to

calculate for R​f is

​ the distance it traveled over the total distance that the solvent in the chamber

absorbed into the TLC plate. Ultraviolet light is a tool that can be utilized in order to enhance the

detection of samples after the TLC plate is removed from the chamber. This technique can be

useful when examining colorless samples. (Stockton University Chemistry Faculty, 2019)

Objective

The main goal of this experiment was to learn about testing polarity with chromatography

techniques while calculating the R​f values.

​ (Stockton University Chemistry Faculty, 2019)

Procedure

In order to perform this experiment, a thin layer chromatography (TLC) plate was collected and

set up. In order to set the TLC plate up, a one-centimeter line was drawn in pencil from the top

and bottom of the powdered side of the plate. After this, segments were drawn on one of the two

lines that were drawn half of a centimeter apart. These were then labeled in the following order
(from left to right) for the first experiment: Red 3, Red 40, Yellow 5, unknown, Yellow 6, and

Blue 1. Using a clean scintillation vial, a drop of each of these dyes was dropped onto their

corresponding segmented line. After this was completed, the Thin Layer Chromatography

chamber was assembled. In order to prepare the chamber, a 150-milliliter beaker was required as

well as five milliliters of the developing solvent consisting of a 25:25:25:10:1 ratio of 1-butanol,

ethyl acetate, ethanol, water, and glacial acetic acid, a clean watch glass, and one piece of

circular filter paper that was folded in half. The original procedure called for two pieces of filter

paper, but the one piece that was utilized was folded in half to emulate the function of the

original requirement for the filter pieces. Once the solvent and filter paper were placed into the

beaker, the watch glass was placed on top acting as a lid for two to three minutes. Once the time

passed, the TLC plate, with the dye samples toward the bottom, was placed inside of the beaker

against the filter paper and was leaning against the side of the beaker with the powdered side

facing the ceiling. The solvent at the bottom of the beaker will have absorbed into the TLC plate.

After the solvent was absorbed to the top of the plate, it was removed from the chamber and

placed on the watch glass until it dried. While waiting for this to dry, the second experiment for

the lab was prepared. The chamber was prepared in the same way but the developing solvent was

composed of 95% ethyl-acetate with 5% acetic acid. The TLC plate was set up the same way but

different samples were being tested so the lined segments were labeled as follows: aspirin,

acetaminophen, unknown, ibuprofen, and caffeine. All of these samples were colorless. Then,

scintillation vials were used to place a drop of each sample onto their corresponding sections.

This TLC plate was placed into a separate beaker with the five milliliters of the developing

solvent after the chamber equilibrated for two to three minutes. This TLC plate sat in the
chamber until the solvent absorbed onto the top line like the previous experiment. Once this

happened, the plate was removed and sat on the watch glass to dry. The third part of this lab

consisted of testing the same colorless samples that were tested using a different developing

solvent. This solvent was made using one milliliter of hexane to four milliliters ethyl acetate.

This solvent was placed in another beaker with another piece of folded filter paper. The watch

glass was placed on top of the beaker and this chamber sat for two to three minutes. After this

time, the TLC plate was placed in the container akin to the plates in the other two chambers.

When the solvent was absorbed at the top pencil line, it was removed from the chamber and

placed on the watch glass. While this dried, the first TLC plate with the colored dyes was

analyzed and the dye marks were circled and the corresponding dots were placed in the middle of

the circle. Then, the R​f ​values were calculated by taking the total length that the developing

solvent traveled as the denominator and the distance of the traced circles from the baseline, the

bottom pencil mark. The unknown dye was then to be compared to the other samples in terms of

circle location(s) as well as the R​f values.

​ This same method was used on the other two

experiments with the additional use of ultraviolet light in order to locate the circles. Finally, a

picture was taken of all three of the TLC plates in order to record the information. (Stockton

University Chemistry Faculty, 2019)

Research Data

Sample name Molar Chemical Structure

Mass Formula
(g/mol)
Red 3 / 879.86 C​20​H​6​I​4​Na​2​O​5
Erythrosine

Red 40 / Allura 496.42 C​18​H​14​N​2​Na​2​O​8​S​2

Red AC

Yellow 5 / 534.3 C​16​H​9​N​4​Na​3​O​9​S​2

Tartrazine

Yellow 6 / 452.4 C​16​H​10​N​2​Na​2​O​7​S​2

Sunset Yellow
FCF
Blue 1 / 792.85 C​37​H​34​N​2​Na​2​O​9​S​3
Brilliant Blue
FCF

Ethyl Acetate 88.11 C​4​H​8​O​2

Acetic Acid 60.052 CH​3​COOH

Hexane 86.18 C​6​H​14

 Aspirin 180.158 C​9​H​8​O​4

Acetaminophen 151.163 C​8​H​9​NO​2

Ibuprofen 206.29 C​13​H​18​O​2

Caffeine 194.19 C​8​H​10​N​4​O​2

Table 1: This table includes information on the different samples used in this set of lab

experiments. Information received from the online database PubChem.

Results

R​f​ formula: R​f​ = distance of dot from baseline / distance from baseline to solvent top line

Unknown for experiment 1: dye sample #2

Unknown for experiment 2 and 3: analgesic sample #2

Experiment 1 Experiment 2 Experiment 3

Calculations Red 3: Aspirin: Aspirin:

4.0 cm / 4.6 cm 3.6 cm / 4.8 cm 3.9 cm / 5 cm
= 0.913 = 0.75 = 0.35

Red 40: Acetaminophen: Acetaminophen:

2.25 cm / 4.6 cm 2.5 cm / 4.7 cm 1.5 cm / 4.9 cm
= 0.489 = 0.532 = 0.306

Yellow 5: Unknown: Unknown:

2.4 cm / 4.6 cm 0.5 cm / 4.75 cm 0.4 cm / 4.9 cm
= 0.521 = 0.105 = 0.082

1.2 cm / 4.6 cm
= 0.261

Unknown: Ibuprofen: Ibuprofen:

2.4 cm / 4.6 cm 2.5 cm / 4.75 cm 4.1 cm / 4.9 cm
= 0.522 = 0.526 = 0.837

1 cm / 4.6 cm
= 0.217
 Yellow 6: Caffeine: Caffeine:
2.4 cm / 4.5 cm 1.7 cm / 4.8 cm 1.5 cm / 4.9 cm
= 0.533 = 0.458 = 0.837

Blue 1:
1.75 cm / 4.5 cm
= 0.389
Table 2: R​f​ calculations for the three experiments conducted in lab two.

Conclusion

Based on the above experimental results as well as the visual appearance of the samples on the

TLC plates, the first experiment’s unknown was a mixture of red 3 and yellow 5. The unknown

from experiment two and three based on the above information and figures was caffeine. For the

first experiment, the unknown formed two dots and a streak of ink similar to the color of red 3.

The position of the spots were in close proximity to the location of the spots from yellow 5

which also means that they had similar R​f values.

​ For the second and third experiment, the

unknown had the closest similarity of its R​f​ value to the caffeine sample which means that the

spots positions were similar on the TLC plate.

Works Cited (5 points)

PubChem. (n.d.). Retrieved from ​https://pubchem.ncbi.nlm.nih.gov/

Stockton University Chemistry Faculty. (2019). Laboratory 2 Thin Layer Chromatography.

Retrieved from: ​https://blogs.stockton.edu/chem2/files/2019/01/TLClab_F19.pdf

General Requirements (14 points)

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