CHE-029L: Analytical Chemistry Laboratory

Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

ACTIVITY NO.10
THIN LAYER CHROMATOGRAPHY

Learning Objectives
At the end of the activity, you will be able to:
• Demonstrate Thin Layer Chromatography (TLC) technique.
• Explain the principles of TLC.
• Compute for the Rf values of the samples.

Materials
• ball pen, pencil, ruler, scientific calculator, pre-coated TLC plate, spot applicator or toothpick, 250-mL
beaker, 10-mL graduated cylinder, filter paper, watch glass, tweezer, methylene blue solution, methyl red
solution, methyl orange solution, dye mixture (methylene blue + methyl red + methyl orange)

Introduction

Chromatography is a technique used for the analysis and separation of chemical mixtures. The
technique is based on a polarity interplay between the sample and two other substances called the solid or
stationary phase and the mobile phase or eluent, which can be a liquid or a gas. As their names indicate, the
stationary phase does not move, whereas the mobile phase flows across, or through the solid phase.

In this “love triangle,” the sample to be separated first becomes adsorbed onto the surface of the solid
phase through polar interactions. More polar samples adhere (or bind) more strongly than less polar ones. The
mobile phase then comes in and sweeps across the stationary phase, competing for the sample. If the mobile
phase is a solvent, it is called the eluting solvent. The more polar the solvent, the greater its capacity to carry
the components of the mixture with it, “yanking” them away from the solid phase as it moves. Different
components with different polarities will travel at different rates as the solvent moves, causing their separation.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

The most used solid adsorbents as stationary phases in laboratory are silica gel and alumina due to
their versatile polarity range. In Thin Layer Chromatography, the solid phase (silica gel or alumina) is applied
as a thin coating on a plastic sheet or glass slide, called a TLC plate. Using a capillary tube, spot applicator or
toothpick, a solution of the sample is applied on the solid support as a spot, a technique known as spotting.
When the sample dries it becomes adsorbed onto the solid support. This process can be repeated several
times to produce a more concentrated spot.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

Procedure:
1. Using a pencil, draw a horizontal line about 1 cm from the bottom of the TLC plate to mark the
starting point of the sample for elution. Precaution: Do not apply too much pressure, or you will
break the coating. The sample spots are to be applied on this horizontal line later.

2. Add 4 hash marks (reference lanes) perpendicular to line. Make sure each hash mark or reference
lane must be equidistant to one another. Name the reference lanes as to Reference Lane A, B, C,
and D.

3. Carefully spot small amounts of analytes (sample solutions) on the reference lanes.
a. Ask for four (4) sample solutions from your instructor. Each reference lane will be spotted by
each solution (Reference Lane A = methylene blue solution, Reference Lane B = methyl red
solution, Reference Lane C = methyl orange solution, Reference Lane D = dye mixture).
b. Dip the tip of a spot applicator or toothpick in the first sample solution to be analyzed. Capillary
action will cause the liquid to rise to the spot applicator or toothpick.
c. Lightly touch the solid support with the tip of a spot applicator or toothpick as if using a fountain
pen. Transfer a small amount of first solution to the plate forming a spot to its designed
reference lane. Precaution: Avoid making big or large spots, therefore just “peck” the coating
and withdraw the spot applicator or toothpick.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

d. Wait until the spot dries before applying the next one, which should be done on the same place
or reference lane. Three (3) or four (4) applications of the same solution should suffice to
produce a good spot. Precaution: A spot that is too dense or too big can cause problems during
separation.
e. Repeat the same procedures from b to d for the second, third, and fourth solution to their
designated reference lane.
f. Let all spots dry completely.

4. While the spots are being applied and allowed to dry, prepare a developing chamber for the
separation.
a. Prepare an eluting solvent (eluent). Mix 3.6 mL of hexane and 3.6 mL of ethyl acetate in a 10-
mL graduated cylinder.
b. Transfer the eluting solvent into the 250-mL beaker carefully.
c. Take a piece of filter paper and place it around the inside walls of the beaker that it is soaked
with the solvent, or you may squirt some of the eluting solvent on the filter paper to make it wet
so that it adheres to the beaker walls better.
d. Cover the beaker with a watch glass while waiting for the TLC plate.
e. Once the TLC plate has been completely spotted and dried, carefully place it inside the beaker
using a tweezer. The TLC plate should be placed upright, slightly inclined against the wall of the
beaker. Precaution: the solvent should not touch the spots, that’s why its level should be below
the spots by at least 0.5 cm.
f. Cover the beaker with a watch glass and wait for the elution to proceed. As the solvent moves
up to the TLC plate, it will cause the spots to move as well.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

5. When the solvent front is about 0.5 to 1 cm from reaching the top of the TLC plate, it is time to stop
the elution. Do not allow the solvent to reach the top of the plate. Take the TLC plate out using a
tweezer and mark the solvent front line with a pencil as soon as possible. Precaution: If you wait too
long, it will fade, and you will miss a crucial part of information needed to calculate the Rf values.

Note: The elution of the spots are sample illustrations only. Results may vary.

6. Compute the retardation or retention factor values (Rf values) of the following spots. These can be
calculated using the formula below.

Some spots are large, while others “tail”. When this happens, the center of mass of the spot is used
to measure the distance travelled by the spot. Note: If there are multiple colors appear in the spot
(especially in the Reference Lane D since the sample is a dye mixture), compute the Rf values
separately depending on the distance traveled by each colored spot. For example, if there are 3
different colored spots appeared for that specific reference lane, 3 distance of the spots will be
measured and 3 Rf values will be computed for that specific reference lane.

This document is the property of PHINMA EDUCATION

 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

7. Write your measurements and calculations in the OBSERVATION SHEET.

8. Take a picture of your output (Picture A) and make sure to also take a picture of your group with the
output (Picture B). Place these pictures in the DOCUMENTATION SHEET.

This document is the property of PHINMA EDUCATION

 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

OBSERVATION SHEET

1. Illustrate the following:

a. Draw the set-up of the TLC.

b. Label the name of the dye/sample solution for every spot present in the TLC plate.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

2. Complete the data.

Distance traveled…
Reference Sample by spot (cm) by Rf
Lane solution 1 2 3 solvent 1 2 3
spotted (If applicable) (If applicable) front (If applicable) (If applicable)
(cm)
A Methylene
blue
B Methyl
red
C Methyl
orange
D Dye
mixture

Calculations of Rf values:

Reference Lane A:

Reference Lane B:

Reference Lane C:

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

Reference Lane D:

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

DOCUMENTATION SHEET

Place the pictures needed in their designated boxes.

PICTURE A PICTURE B

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

ACTIVITY QUESTIONS

Instruction: Answer the following questions.

1. What is chromatography?

2. Describe the following types of chromatography?

a. Liquid Chromatography

b. Gas Chromatography

c. Paper Chromatography

d. Thin-Layer Chromatography

3. Explain the principles of elution in TLC.

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 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

4. What are the visualization techniques that you can use if your sample solutions or analytes are
colorless or have “invisible spots” in the TLC plate?

5. How would a developed TLC plate appear if a sample was a mixture of 4 compounds? Illustrate
the appearance.

6. What is Rf and what does its value signify?

This document is the property of PHINMA EDUCATION

 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

7. What are the uses of Rf values in TLC?

8. What are the factors that can affect Rf values?

9. Were the Rf values of the spots from dye mixture (Reference Lane D) comparable to or are the
same with the Rf values of the spots from every single component of the dye (Reference Lane A,
B, and C)?

10. Consider a nonpolar compound, N, and a polar compound, P. In a typical TLC experiment using
alumina as the stationary phase and hexane as the mobile phase.
a. Which compound will display a higher Rf value? N or P and why?

b. Which compound will interact more with the alumina? N or P and why?

This document is the property of PHINMA EDUCATION

 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

11. Calculate the Rf value of the compound on the TLC plate shown below.

This document is the property of PHINMA EDUCATION

 CHE-029L: Analytical Chemistry Laboratory
Laboratory Activity Sheet #10

Name: _______________________________________________ Class number: ______

Section: ____________ Schedule: ________________________ Date: _____________

References:
• West, D. M., Skoog, D. A., Holler, J. F., & Crouch, S. R. (2014). Fundamentals of Analytical Chemistry.
Belmont: Cengage Learning.

This document is the property of PHINMA EDUCATION