FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

Experiment #: 2
Experiment Title: TLC and Column Chromatography

Group #: 1

Denilyn Madrid1, Renyrick Manaloto1, Carla Bianca Salutin1

1
FSTEM, CTD, Philippine Normal University, Taft Avenue, Malate, Manila

Date Performed: 05/18/2021

Date Submitted: 05/28/2021

A. Thin Layer Chromatography

I. Read: Williamson, K. L., & Masters, K. M. (2011). Macroscale and microscale

Organic experiments. In Macroscale and microscale organic experiments (6th ed.,
pp. 164-183). Boston, MA: Cengage Learning.
II. Watch the Pre-Lab Video Series
III. Explore and Perform the Simulation experiment for Thin Layer Chromatography.
IV. Answer the Guide questions.

a. Thin Layer Chromatography Simulator Procedure

1. Click “Start Experiment” to run the simulator with:

a. a 50:50 mixture of Hexane and Ethyl Acetate on Chamber 1.
b. A 75% Ethyl Acetate: 25% Hexane on Chamber 2.

Sketch the plate or paste screenshot in the data sheet (label the spots
red and blue).

2. Click on “reset experiment” and change the solvent system on one of the
plates to be:
a. a 50:50 mixture of Hexane and Ethyl Acetate on Chamber 1.
b. 25% Ethyl Acetate : 75% Hexane on Chamber 2.

Sketch the plate or paste screenshot in the data sheet (label spots).

3. Click on “reset experiment” and change the solvent systems to be:

a. 100% Ethyl Acetate on Chamber 1
b. 100% Hexane on Chamber 2.

Sketch the plates or paste screenshot in the data sheet (label spots).

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

b. Data Sheet
1. Draw a schematic diagram of the experiment.

2. Note any precautionary measures that need to be observed in doing

this experiment. Record your observation on the table provided below.
3. Briefly describe the polarity of the solvent system in each chamber and
its effect in the movement of the spots. Lastly, describe the Rf values of
the red and blue spots.
The development solvent is less polar compared to the stationary
phase which causes the solvent to travel up to the plate by capillary
action. As the solvent passes to the solute spots it carries some solute
with it, this is called mobile phase since there is an observed movement.
The movement of the mobile phase and the solute is based on its polarity,
likewise, more polar solutes are more attracted to the stationary phase
since silica gel is polar hence, the movement is slow. On the other hand,
the less polar solute sticks less often, hence, the movement is fast.

The Rf values depend on the polarity of the solvent used, likewise, if non
polar solvent is used, there would be no forces available to pull polar
compounds, hence Rf=0. Conversely, if the solvent is polar enough the
polar compounds will not be able to stick and all the spots will move as
fast, hence Rf=1. In the experiment, the blue spots pertain to less polar
molecules while the red spots pertain to more polar molecules. More
polar compounds (red) travel more slowly due to the strong affinity with
the silica gel. This explains why the more polar compounds will have
lower Rf values compared to less polar compounds.

OrgBioLabOnline
 FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

Steps Plate (draw or Screenshot) Observation Rf Value of

spots
1 The components in The Rf
the chamber 2 value of
move faster than spots in
those in chamber 1 chamber 1
probably because have higher
the polar solvent is values
greater in there. compared
to those in
Always put the chamber 2.
right amount of
solvent in each
chamber, and
close the lid when
experimenting.
2 Reducing the Reducing
amount of polar the amount
solvent in chamber of polar
2 made the solvent in
components in chamber 2
there move slowly made the
than those in components
chamber 1 which in there
has more polar move slowly
solvent. than those
in chamber
1 which has
more polar
solvent.
3 The spots in The spots in
chamber 1 have chamber 1
higher Rf values have higher
than those in Rf values
chamber 2. than those
in chamber
2.

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

c. Guide Questions:
1. Which of the 2 spots (red and blue) is more polar? How can you
say so?
The red spot is more polar as it represents ethyl acetate
that travels slowly than the blue spot which corresponds to the
hexane that moves faster and is less polar.
.
2. Draw the structure of ethyl acetate and hexane. Which is more
polar?

3. How does the polarity of a compound relate to its Rf value?

The polarity and Rf value of a compound have an indirect
relationship to each other. The more polar the compound is the
lower its Rf value.

4. How does the polarity of solvent affects the Rf value?

The polarity and Rf value of a solvent have an indirect
relationship to each other; just like with the compounds. The more
polar the compound is the lower its Rf value.

5. What will be the appearance of a TLC plate if a solvent of too low

polarity is used for the development? A solvent of too high
polarity?
If the polarity of solvent used were too low, there would be
no forces available to pull any polar compounds away from the silica
gel, hence, there would be no movement observed. Conversely, if
the solvent of too high polarity, the polar compounds would not be
able to stick to silica gel and all the spots will move as fast as
possible.

6. What will happen if spots are very near the edge of the plate?
If the spots are very near to the edge this will lead to the
evaporation of solvent from sides and this will give you an
inconsistent results.

7. What problem will ensue if the level of the developing liquid is

higher than the applied spot in a TLC analysis?
It will end up with the sample or the applied spot being
dissolved in the developing solvent.

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

8. What will be the result of applying a very concentrated compound

to a TLC plate?
If the samples are too concentrated, the spots will run
together or will be streaked because the solvent cannot affect the
concentrated sample that might affect the analysis.

9. Why is it necessary to run TLC in a closed container and to have

the interior vapor saturated with the solvent?
To ensure maximum resolution between components and
to prevent solvent evaporating off.

10. In carrying out an analysis of a mixture, what do you expect to see

when the TLC plate has been allowed to remain in the developing
chamber too long, so that the solvent front has reached the top of
the plate?
If the TLC plate has been allowed to remain in the
developing chamber too long it will end up with large diffused spots.

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

B. Column Chromatography

I. Read: Williamson, K. L., & Masters, K. M. (2011). Macroscale and microscale

Organic experiments. In Macroscale and microscale organic experiments (6th ed.,
pp. 185-194). Boston, MA: Cengage Learning.
II. Watch the Pre-Lab Video Series
III. Answer the Guide questions.

a. Data Sheet

1. Draw a column Chromatography set up and label the parts.

2. Based from the TLC simulation, design a column chromatography

experiment. Use the red and blue compounds in the simulation.
Choose the best solvent system from what you observed in the
TLC experiment.

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

a. Draw a schematic diagram of the designed experiment.

b. Note any precautionary measures that need to be

observed in doing this experiment.

 Wearing of protective gears to protect yourself (e.g

gloves, laboratory coat, goggles...etc.)
 Silica and alumina are highly toxic when inhaled
 Handle absorbents in the hood

c. Write a short narrative of your expected results.

Column chromatography is a technique that uses a pack

column to separate compounds dissolved in the mixture by
using the interaction of solvent to the stationary phase.
Opening the column allows the solvent to flow through the
stationary phase. When the mixture is applied on the top of
the pack column followed by the solvent, the mixture will

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

move to the mobile phase and flow through the stationary

phase. Each component of the mixture will interact with the
stationary phase in a different way, some will have less
interaction with the stationary phase, thus it will move
quickly through the column. While others will have strong
interactions with the stationary phase, thus it will move
slowly through the column. The compounds will be
separated into bands and will be collected into small
fractions.
b. Guide Questions:
1. How does intermolecular forces present in compounds in a
mixture and in the stationary phase (silica gel) drive its separation
in a column chromatography?
It is said that the higher the intermolecular forces are
present in a mixture and in the stationary phase, the longer
the compound or mixture retained in the column.

2. Column length tells us the difficulty of separation of compounds in

the stationary phase. What kind of compounds can the following
column description separate better?
a. Long column: silica gel or natural compounds
b. Shorter column: alumina
3. What kind of compound are the following solvent system used to
purify?
a. Diethyl ether/pentane: nonpolar compounds

b. Methanol/dichloromethane: polar compounds

4. With the ideal Rf value of approx. 0.3, what does the following Rf
value imply?
a. Rf is too low: the substance is very polar.

b. Rf is too high: the substance is less polar.

5. With an ideal 6-7 inches tall of a column, what are the

consequence of using the following column?
a. Column which is too tall:
The different bands of compounds will disperse and
overlap resulting in poor separation.

b. Column which is too short:

The surface is not enough to have a good separation

6. How can you avoid or remove bubbles in the silica slurry in your
column?

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

After the slurry has been poured into the column, rap the
column with a rubber stopper for the silica gel to settle and remove
bubbles.

7. When is it advisable to use sample dry-loading in column

chromatography?
If the compound has poor solubility.

8. Why is it necessary to have a thin layer of sand on top of the

sample in the column?
To prevent disturbing the adsorbent bed during the
application of the sample. It will filter out insoluble material and
helps to distribute the samples evenly across the top of the
column.

9. Generally, 1-3 mL fractions should be collected, what will be the

consequence if the collected fractions are too large?
Overlapping fractions containing more than one compound
is the sign of failed separation.

10. What do the following result imply?

a. A maximum of 1 compound per fraction with a few clean
fractions between different compounds:
Good separation.
b. All material elutes in just a few fraction:
It is either the column was too small or the developing
solvent was too polar.

c. The compounds are spread out over many fractions:

The column may have been too tall or too much solvent
was used.

OrgBioLabOnline
 FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

TLC and Column Chromatography Simulator Worksheet

I.TLC Simulator: Follow instructions to explore the TLC simulator and answer the
questions below.

1. Click “Start Experiment” to run the simulator with a 50:50 mixture of Hexane and
Ethyl Acetate. Sketch the plate below (label the spots red and blue)

Is silica gel polar?

It is extremely polar
Which compound is more polar, red or blue? Explain briefly.
Red is more polar compared to the blue one. Since more polar compounds
travel more slowly, it has stronger affinity for the polar stationary phase
(silica gel).

Draw the structures for ethyl acetate (EtOAc): and hexane:

2. Click on “reset experiment” and change the solvent system on one of the plates to be
75% Ethyl Acetate. Sketch the plate below (label spots) and answer the given

questions. Is the new solvent (compared to 50:50) more or less polar? Explain

briefly.

The new solvent is more polar, because the amount of polar solvent is more.

What happened to the R of the spots in the new solvent? Explain briefly.
f

The Rf values of the spots increased because there’s also an increase in the solvent polarity as
substance which make it more attracted to solvent.

OrgBioLabOnline
 FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

3. Click on “reset experiment” and change the solvent system on one of the plates to be
25% Ethyl Acetate. Sketch the plate below (label spots) and answer the given

questions. Is the new solvent (compared to 50:50) more or less polar? Explain briefly.

The new solvent is less polar, because the amount of non-polar solvent is more.

What happened to the R of the spots in the new solvent? Explain briefly.
f

The Rf values of the spots decreased because there’s also a decrease in the solvent
polarity as substance which make the non-polar solvent move slower.

4. Click on “reset experiment” and change the

solvent systems to be 100% Ethyl Acetate on one
plate and 100% Hexane on the other. Sketch the
plates below and describe the R of the spots.
f

100% EtOAc 100% hexane

R =1
f R =0
f

II. Column Chromatography Simulator.

1. Click the ⍰ button in the set up to understand each part.

2. There are three samples given: red, blue and their mixture: purple.
3. Using the default value of Kred=10 and Kblue = 0, run each sample and observe their
separation.
4. Vary the values of K for red and blue and observe what will happen to the
separation of red and blue on the mixture.

Which K values give a better separation of red and blue from the purple sample?
The k values that give a better separation of red and blue from the purple sample
are 1 and 4.

OrgBioLabOnline
FACULTY OF SCIENCE, TECHNOLOGY AND MATHEMATICS OrganicxBiochemistry

How can you explain this?

As the K value increases the speed or time of the substance to move from
stationary phase to mobile phase decreases or it became faster.

OrgBioLabOnline