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QuynhTrang Dao
Hanh Nguyen
February 6, 2015

Thin Layer Chromatography and Its Uses

Introduction
Thin Layer Chromatography or TLC is a simple, quick and common analytical
technique that researchers use to identify the components present in a mixture by
comparing its Rf values (retention factor), which is the proportion of distance spot
traveled and distance of solvent traveled.1,2 Therefore, this technique is also used to
determine a compounds purity.1 Moreover, it helps monitor the progress of a reaction.3
Thin Layer Chromatography technique
Running a TLC plate involves stationary phase the absorbent Silica (SiO 2)
coating on TLC plate which separates spots on the same plate and mobile phase the
polar solvent inside the developing chamber.4 A TLC plate works base on the concept of
polar and nonpolar molecules.3 When a mixture of compounds is spotted on the plate
which is placed in a polar solvent chamber, this is when Like Dissolves Like concept is
applied. The polarity of the solvent will quickly push the non-polar compounds far away
from the solvent to the solvent front. In contrast, polar compounds will stick to the
bottom. Therefore, the selection of polar solvent is very important. A too polar solvent
will push the spots out of the plate and a too non-polar solvent will not affect the sample.

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A typical solvent should be the mixture of two far away solvents like Hexanes and Ethyl
Acetate so that the spots will stop in the middle of the plate.3
Identifying compounds by comparison
According to the FDA, there are two categories of color additives for foods:
Certifiable and Exempt from certification.3 Certified food coloring or dyes are polar and
soluble in water.3 Various combinations of 4 certified dyes Yellow #5, Red #40, Blue #1,
and Red #3 are often used in Store Food coloring. In this experiment, student will run a
TLC procedure to find what dyes are in Store Food coloring sample by comparing R f
values.
Determining Purity of a compound.
Base on the Like dissolve like concept, a pure compound always leaves only
one spot after running TLC while an impure compound will leave several spots on the
plate.3 However, choosing which solvent is suitable to use in the experiment so that it will
give out the number components is also very important. In this experiment, student will
try different proportion of Ethyl Acetate : Hexanes solvents and decide which solvent is
best fit. Moreover, calculating and comparing the R f values of those spots, student will be
able to identify the compounds in the sample. The more polar the compound, the smaller
the Rf value and vice versa.2
Purposes
After the experiment, students are able to use Thin Layer Chromatography
technique to calculate the Rf value and comparing the Rf value to: 1) Identify the dye or
compound present in a sample; and 2) Determine the purity of a compound.
Other techniques used in this lab

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In order to determine which compound is which spot, Rf value of each spot need
to be calculated and compare with each other.

Rf =

Distance spot traveled

Distance solvent traveled

(1)

Both distances have to be in the same unit to get an accurate value.

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Experimental Procedures and Observations

Part 1: Identifying compounds by comparison
First, poured a 0.5cm tall of 1%NaCl into a developing chamber. Then, cut a 2x5cm TLC
plate and drew a line about 1cm from the bottom. Used pencil to mark 4 even spots on
the line and named them as SD1, Red, Blue and Yellow. Used a capillary to spot a 1-2mm
of the sample into the mark places in the order of unknown #3, Red, Blue, and Yellow
color. Put the bottom of the plate into the chamber so that the solvent did not touch the
base line. Closed top of the chamber with a lid so that the solvent would not evaporate.
Waited until the solvent front raise to about 1cm from the top; pulled the TLC plate out
and immediately drew a front line. Used a ruler to measure the distance the spots traveled
(from base line to the center of the spot) and the distance the solvent traveled (from the
base line to the solvent front). Used equation (1) to calculate R f for each spot and
recorded the values. The Store Dye #3 was found to be Yellow #5 (Rf = 0.56)
Part 2: Determining Purity of a compound
Repeated the same TLC procedure. Marked 3 spots for the mixture of three
compounds: Benzoic Acid, Benzophenone, and 3-nitrobenzoic acid. Since normal eyes
could not see this mixture, we used UV light to locate the spots after running the trials
and circled the spots for calculation. Ran 8 trials with different proportion of Ethyl
Acetate : Hexanes solvents: 50:50, 40:60, 60:40, 30:70, 20:80, 80:20, 0:100, and 100:0.
Calculateed Rf for each spot and recorded the data. The 20E:80H was found to be the
only solvent with 3 distinctive spots.
Cleaning up

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Used capillaries were disposed in the broken glass box, and papers were disposed
in the wastebasket. The equipment were washed with water, rinsed with acetone and
placed back to the equipment kit.

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Experimental Data
Table 1: Identifying the compounds by comparision
Standards

Commercial

Name

Rf values

Name

Rf values

Red

0.5

Store Dye #3

0.56

Blue

0.47

Yellow

0.56

SD#3 : Yellow
Chart 1:
Color
Red
Dye Standards
Blue
DyeYellow
name
Color
RedGreen
#40
Red
Blue
#1
Blue
Black
Yellow
#5
Yellow

Store Food coloring

Dye name
Red #40, Red #3
Blue #1, Red #40
Yellow #5
Yellow #5, Blue #1
Red #40, Blue #1, Yellow #5

Chart 2:

Table 2: Determining Purity of a compound

Trial #
1
2
3
4
5
6
7
8

% Ethyl Acetate
50
30
20
80
40
60
0
100

% Hexanes
50
70
80
20
60
40
100
0

0.97

Rf Value
0.98
0.83
0.56
0.91
0.92
0.93
0.23
0.9

0.14

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OH

OH

NO2

Benzoic Acid

Trial #1

Trial #4

Benzophenone

Trial #2

Trial # 5

3-nitrobenzoic Acid

Trial #3

Trial # 6

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Trial #7

Trial #8

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Result and Discussion

In part one of the experiment, 4 Rf values was found. The Rf value of red dye is
0.5, of blue dye is 0.47 and of yellow dye is 0.56. The R f value of the unknown store dye
is 0.56. Since the Rf value of yellow dye and the unknown are the same, the unknown is
Yellow #5. By comparing the Rf value of the unknown with given Rf values of certain
compounds, one can determine how many and what are the compounds present in
sample.
In part two of the experiment, different R f values of different solvents (Ethyl
Acatate : Hexanes proportions) were recorded. In trial 1, 50% Ethyl Acatate : 50%
Hexanes solvent, the Rf value of every spot is 0.98. In trial 2, 30% Ethyl Acatate : 70%
Hexanes solvent, the Rf value of every spot is 0.83. In trial 3, 20% Ethyl Acatate : 80%
Hexanes solvent, the Rf value of the first spot is 0.97, of the second spot is 0.56, and of
the third spot is 0.14. In trial 4, 80% Ethyl Acatate : 20% Hexanes solvent, the R f value of
every spot is 0.91. In trial 5, 40% Ethyl Acatate : 60% Hexanes solvent, the R f value of
every spot is 0.92. In trial 6, 60% Ethyl Acatate : 40% Hexanes solvent, the R f value of
every spot is 0.93. In trial 7, 100% Hexanes solvent, the R f value of every spot is 0.23. In
trial 8, 100% Ethyl Acatate solvent, the R f value of every spot is 0.90. Out of 8 trials,
there is only the trial of 20% Ethyl Acatate : 80% Hexanes gives 3 spots on the TLC
plate. It is because other solvent is too polar or too non-polar that it cannot give accurate
results. Therefore, the best fit polar solvent for this experiment is 20% Ethyl Acatate :
80% Hexanes. The sample contains 3 compounds: Benzoic Acid, Benzophenone the
least polar compound, and 3-nitrobenzoic Acid the most polar compound. The more
polar a compound is, the more it sticks to the bottom, and the smaller its R f value is.

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Thus, the highest spot (Rf value = 0.97) is Benzophenone; the middle spot (R f value =
0.56) is Benzoic Acid; and the lowest spot (Rf value = 0.14) is 3-nitrobenzoic Acid.

Conclusion
Thin Layer Chromatography is a widely used technique in many research labs
because of its simplicity and quickness. TLC is often used to determine the present and
concentration of active ingredients in drugs, separate amino acid, proteins, and peptides,
and many other applications in food analysis, environmental analysis, clinical chemistry,
biochemistry and so on.5

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References
1. Orgchem.colorado.edu,. 'Thin Layer Chromatography (TLC)'. N.p., 2015. Web. 7 Feb.
2015. http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html
2. Mendelset.com,. 'Thin Layer Chromatography (TLC) | Mendelset'. N.p., 2015. Web. 7
Feb. 2015. http://www.mendelset.com/articles/683/thin_layer_chromatography_tlc
3. Thaxton, A.; Lindsay, M.; Hickey, S. Organic Chemistry Lab 1; 2nd ed.; The
University of New Orleans: New Orleans, 2015; p. 21, 22, 25.
4.

Umich.edu,.

'How

TLC

Works'.

N.p.,

2015.

Web.

Feb.

2015.

http://www.umich.edu/~orgolab/Chroma/TLChow.html
5. Pharmainfo.net,. 'Application And Importance Of Thin Layer Chromatography In
Analysis

And

Research

Work'.

N.p.,

2015.

Web.

Feb.

2015.

http://www.pharmainfo.net/pharma-student-magazine/application-and-importance-thinlayer-chromatography-analysis-and-research--0

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Post-lab Question
1. Rank the following compounds from most polar to most non-polar: amide, ether,
carboxylic acid, hydrocarbon, and ketone.
2. Which of these compounds is NOT UV active? Circle all that qualify.
3. How would you fix this TLC plate?

CH3
H3 C

CH2

H3 C

4. Match the spots to the compounds

5. How would you fix this TLC plate?

6. How would you fix this TLC plate?

CH3

CH2

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