Title: Lab 2: Thin Layer Chromatography

Source: A. Franz and V. Samoshin, et al, Chem 121 Handout Part 1, University of the Pacific:
Stockton, CA, 2020, pp.17-26

Purpose: The first part of this lab is to determine the composition of an unknown mixture by
running chromatograms of the standards and the unknowns. The second part consists of setting
up your own extraction of Capsaicin from dried peppers; and determining which pepper contains
the most Capsaicin.

Chemical Properties and Safety:

Name of the Formula Structure Physical Properties Safety Information
Compound

Ibuprofen C13H18O2 ● Molecular weight: ● Eye irritant

206.29 g/mol ● May cause
● Boiling Point: 157 respiratory
°C irritation
● Melting Point: 75 ● Reproductive
°C toxicity
● Density: 1.03 g/cm3

Caffeine C8H10N4O2 ● Molecular weight: ● Harmful if

194.19 g/mol swallowed
● Boiling Point: 178
°C
● Melting Point: 234
to 235 °C
● Density: 1.23 g/cm3

Aspirin C₉H₈O₄ ● Molecular weight: ● Harmful if

180.158 g/mol swallowed
● Boiling Point: 140 ● Causes skin
°C irritation
● Melting Point: 136 ● Causes
°C serious eye
● Density: 1.40 g/cm3 irritation
● May cause
respiratory
irritation

Acetaminophen C8H9NO2 ● Molecular weight: ● May cause an

151.16 g/mol allergic
● Boiling Point: 420 reaction
 °C ● Acute toxicity
● Melting Point: 170.0 ● Skin irritant
°C
● Density: 1.26 g/cm³

Capsaicin C18H27NO3 ● Molecular weight: ● Toxic if

305.41 g/mol swallowed
● Boiling Point: ● Harmful if
210°C swallowed
● Melting Point: 62°C ● Causes skin
● Density: 1.02 g/cm³ irritation
● Serious eye
damage

EtOAc (Ethyl C4H8O2 ● Molecular weight: ● Highly

Acetate) 88.11 g/mol flammable
● Boiling Point: ● Toxic when
77.1°C ingested or
● Melting Point: inhaled
-83.6°C ● Irritant to
● Density: 0.90 g/cm³ eyes and skin

AcOH (Acetic CH₃COOH ● Molecular weight: ● Highly

Acid) 60.052 g/mol corrosive to
● Boiling Point: eyes and skin
117.9°C ● Do not inhale
● Melting Point:
16.6°C
● Density: 1.05 g/cm³

Methanol CH3OH ● Molecular weight: ● Highly

32.04 g/mol flammable
● Boiling Point: and toxic
64.7°C ● Do not inhale
● Melting Point:
-97.6°C
● Density: 0.792g/cm³

Hexane C6H14 ● Molecular Weight: ● Flammable

86.18 g/mol ● Irritant
● Melting Point: ● Health
-95.3°C Hazard
● Boiling Point:
 68.7°C
● Density: 0.672g/cm³

Procedure and Observations:

Experiment 1 Overview: A mixture of several analgesics (which are solutions of pure
individual analgesics) will be provided. By running the chromatograms of the standards and the
unknowns on the same plate, the composition of the unknown mixture will be determined. If the
unknown has one or more spots that correspond to spots with the same Rf values as the
standards, then those substances are more likely to be present.
Experiment 1 Procedure: Observations:

1. Using two chromatographic plates, On each chromatographic plate, 3 X’s were

draw pencil lines about 1 cm from the drawn 1 cm away from the bottom.
bottom of the plates.

2. Using a separate capillary for each
unknown and standard, spot aspirin,
acetaminophen, and the unknown on
this line on the first plate. On the
second plate, spot ibuprofen, caffeine,
and the unknown.
The first chromatographic plate consisted of
aspirin, acetaminophen, and unknown #1 (on
each x, a small droplet of each substance was
placed). The second chromatographic plate
was aspirin, acetaminophen, and unknown #2.
The third chromatographic plate consisted of
ibuprofen, caffeine, and unknown #1. The
fourth chromatographic plate consisted of
ibuprofen, caffeine, and unknown #2.

3. To make sure enough of each
compound has been applied, examine
the plates under UV light.
Under UV light (which gave out a neon green
color), we checked that each compound was
applied enough and efficiently. Each droplet
that was applied to an X portrayed a lighted
circle under the light.

4. In a 150 mL beaker, put 4 mL of a Ethyl-acetate (a yellow-brown liquid), along

mobile phase (a mixture of 95% with filter paper and the first chromatographic
EtOAc and 5% AcOH) and a piece of plate was placed in a beaker; and covered with
filter paper. Cover it with a watch a watch glass.
glass.

5. Develop 2 spotted TLC plates.
After ethyl-acetate had reached the X’s drawn
1 cm from the bottom, we removed the first
chromatographic plate. This was repeated for
the other three plates as well. We made sure to
label each one to avoid any confusion.

6. When the solvent has risen to the top Each plate was marked with a pencil at the
of the plates, mark the solvent front point where the solvent had risen to.
 using a pencil. Then, remove the plates
from the developing beaker.
7. Let the plate dry.
8. Under UV light, check the plates.
Make sure to outline any spots.
9. Calculate Rf values for all spots and
identify the components in the
unknown.
Experiment 2 : Setting up your own extraction, but running the TLC with a lab partner.
Experiment 2 Procedure:
1. Put 0.5g of habanero pepper or
crushed pepper into a 25 mL round
bottom flask (wear gloves at all times
when dealing with capsaicin).
2. Add 10 mL of methanol and attach an
air cooled reflux condenser to the
flask.
3. Using the fume hood, heat the
suspension to reflux for 15 minutes
(on a hot plate).
4. After the suspension has cooled, use
the liquid extract for TLC analysis.
5. In three different spots on two
The plates were dried.
Under UV light, each spot was circled with a
pencil. This was done for all four plates. For
all four plates, there was only 1 spot for lane 1
and 2; however 2 spots for lane 3.
All the Rf values for each spot on each plate
are listed in the data section. Unknown #1 is
composed of aspirin and caffeine. Unknown
#2 is composed of acetaminophen and
ibuprofen.
Observations:
We weighed out 0.5g of habanero pepper (a
strong red powder) and chilli powder
(burgundy red powder) on a scale using
weighing paper (we neglected the weight of
the paper). Each substance was placed in a 25
mL round bottom flask.
10 mL of methanol (pure transparent liquid)
was measured in 2 graduated cylinders; one
for the habanero and one for the chilli powder.
When methanol was added to the chilli
powder, the burgundy color became a little
more vibrant and brighter. When it was added
to the habanero, the mixture became an
orange red. The air cooled reflux was first
attached to the habanero powder flask.
In the fume hood, the habanero powder flask
was heated. As the heat increased and 15
minutes had passed, the mixture became fuzzy
and bubly, it was boiling and had a dark red
color. This was the case with the chilli powder
too.
An hour later, the suspension for both
powders was cool.
On two different chromatographic plates, 3
 different TLC plates, spot the
capsaicin reference solution, the
extract from your pepper sample, and
that of your lab partner. Touch a spot
3-5 times.
6. Develop the plates using different
mobile phases: a.) 9:1 hexane-ethyl
acetate mixture; b.) 1:1 hexane-ethyl
acetate mixture.
7. Under UV light, mark the spots with a
pencil. Also, visualize the TLC plates
in an iodine chamber for 5 minutes.
8. If the spots of the compounds aren’t
clearly visible, perform the TLC
analysis on another TLC plate.
9. Record observations. Remember to
take a picture of the TLC plate, as the
iodine stain will fade in minutes.
Calculate the Rf values for all spots.
Based on observations, which type of
pepper contains more capsaicin.
Data:
Part 1:
X’s were drawn 1 cm from the bottom of the
plate. On the first X of the first plate, a small
drop of capsaicin acid was placed. On the
second X, a small drop of habanero extraction
was placed. On the third X, a small drop of
chilli powder extract was placed. The same
was done on the second chromatographic
plate.
The two mobile phases were developed in two
different beakers. In the first beaker, 9 mL of
hexane and 1 mL of ethyl acetate was mixed.
A filter paper was placed inside. In the second
beaker, 5 mL of both hexane and ethyl acetate
was mixed. After filter paper was placed in
the two beakers, one chromatographic plate
was dropped into the beaker with a 9:1
hexane-ethyl acetate mixture, and the other
plate was placed in the 1:1 hexane-ethyl
acetate mixture.
After taking out the plates from the beakers,
we analyzed them under UV light. With a
pencil, the spots were marked. In addition, the
TLC plates were placed in the iodine chamber
for 5 minutes (a yellowish powder). This
made it easier to see the spots. There was only
1 spot for the reference solution on both
plates. There were 2 spots of habanero extract
on both plates; and for the chilli extract there
were 2 spots on 1 plate (9:1), and 3 spots on
the second (1:1).
The spots were visible enough.
The Rf values for all spots are listed in the data
section below. Chilli powder contains more
capsaicin.
Chromatographic Chromatographic Chromatographic Chromatographic
Plate #1 Plate #1 Rf Values Plate #2 Plate #2 Rf Values

Aspirin 1.3 Aspirin 1.2

=.76 =.75
1.7 1.6

Acetaminophen 1.0 Acetaminophen 0.8

=.59 =0.5
1.7 1.6

Unknown #1 0.4 Unknown #2 0.9

Spot #1: =.24 Spot #1: =.56
1.7 1.6
1.3 1.3
Spot #2: =.76 Spot #2: =.81
1.7 1.6

Chromatographic Chromatographic Chromatographic Chromatographic

Plate #3 Plate #3 Rf Values Plate #4 Plate #4 Rf Values

Ibuprofen 1.4 Ibuprofen 1.3

=.93 =.93
1.5 1.4

Caffeine 0.4 Caffeine 0.4

=.27 =.29
1.5 1.4

Unknown #1 0.4 Unknown #2 0.9

Spot #1: =.27 Spot #1: =.64
1.5 1.4
1.2 1.3
Spot #2: =.80 Spot #2: =.93
1.5 1.4
Identity for unknown #1: Aspirin and Caffeine
Identity for unknown #2: Acetaminophen and Ibuprofen
Part 2:
Compound 9:1 EtOAc and AcOH 1:1 EtOAc and AcOH
Rf Values Rf Values

Capsaicin Reference Solution 0 0.7

=0 =.47
1.5 1.5

Habanero Extract Sample 0.5 1.1

Spot #1: =.33 Spot #1: =.73
1.5 1.5
0.7 1.3
Spot #2: =.47 Spot #2: =.87
1.5 1.5

Chilli Powder Extract Sample 0.5 0.7

Spot #1: =.33 Spot #1: =.47
1.5 1.5
 0.7 1.1
Spot #2: =.47 Spot #2: =.73
1.5 1.5
1.3
Spot #3: =.87
1.5

Discussion:
The purpose of this experiment was to determine the composition of two unknowns by
the usage of TLC. The unknowns were a mixture of the standards that were given to us. The
second part of this experiment was to set up an extraction of capsaicin. Through this extraction
and the application of TLC, we were to determine which pepper (of the given to us) contained
more capsaicin. TLC, thin-layer chromatography is a sensitive, fast and inexpensive analytical
technique that separates two or more compounds or ions caused by their molecular interactions
with two phases; one moving and one stationary. With its numerous uses, the purpose of TLC in
this lab was to determine the identity of an unknown compound. TLC plates are outlined with a
thin layer of polar absorbent, which absorbs the solvent. A small line is made 1 cm from the
bottom of the plate, where the plate comes in contact with the solvent. This is the most polar area
of the plate. When the solvent travels to the top of the plate by absorption (this is also marked by
a line), it becomes less polar (or nonpolar). Due to this, compounds that are nonpolar leave spots
closer to the top; whereas, polar compounds usually remain closer to the bottom. In the first part
of our experiment, we used a 95%:5% ratio of acetic acid and ethyl acetate as our solvent. The
second part contained different ratios of hexane and ethyl acetate. If ethyl acetate was used
alone, it would be too polar and the compounds would shoot to the top. If hexane or acetic acid
was used alone (nonpolar or less polar solvents), the compound would remain to the bottom.
Therefore, a mixture of both was used as the solvent because it produced a nice medium to
provide us with more accurate results.

Experiment 1:

Experiment 1 consisted of determining the compositions of two unknowns by using TLC.

On four different TLC plates, we drew three X’s (one to represent each substance) 1 cm from the
bottom. The first plate had aspirin, acetaminophen, and unknown #1. Using hexane ethyl acetate
as the solvent, we dipped the plate into the solvent with filter paper. The absorbed solvent
allowed for the three substances to travel and leave spots. To examine these spots, the plates
were examined under UV light. UV light at 254 nm wavelength produces a dark bluish light
which makes it easier to see the spots. With a pencil (pen will leave stains and provide inaccurate
results), we marked the spots. Knowing that the unknown was a mixture of the compounds given
us to, the Rf values helped us classify what the composition of each unknown was. The Rf value
is the ratio of the distance the spot travels to the distance the solvent travels. We identified the
unknown by matching its Rf value with that of the standards. If two spots have the same Rf value,
it is concluded that they are the same molecule. For the first unknown, the Rf values were 0.24,
0.76, 0.27, and 0.80. The second unknown had the Rf values of 0.56, 0.81, 0.64, and 0.93. The
standards had the following Rf values: aspirin was around 0.75, acetaminophen was around 0.5,
ibuprofen was around 0.93, and caffeine was around 0.30. Ibuprofen had the highest Rf value,
meaning it traveled the greatest distance, because it is the least polar of the four. Due to there
being only one functional group, carboxylic acid, ibuprofen was tied together through the least
amount of hydrogen bonding and dipole-dipole forces. In contrast, caffeine had a Rf value that
was the lowest among the four, 0.30. This was due to its strong polarity displayed by two amine
and two amide functional groups. It contained most of the hydrogen bonding and dipole-dipole
forces among the four compounds. Based on these Rf values, we know that unknown #1 is
composed of caffeine and aspirin because the values of the unknown are very similar to those of
the standards. On the other hand, we know that unknown #2 is composed of acetaminophen and
ibuprofen because the values are very closely tied.

Experiment 2:

The second part of the experiment included setting up an extraction. Among the many
uses of TLC, the main purpose of its usage in this part of the lab was to determine the number of
components (capsaicin) in a mixture. We were to determine which peper, habanero or chilli,
contained more capsaicin through TLC. The extraction consisted of two round bottom flasks
(each one had a type of pepper and 10 mL of methanol) attached to a reflux heating system. To
be cautious and safe, the heating was done in the fume hood. After the heated mixtures were
cooled, TLC was performed on each pepper. Similar to the first part of the experiment, three X’s
were drawn on TLC plates about 1 cm from the bottom. On both plates, a drop of capsaicin
reference solution, the heated chilli powder extract sample, and the heated habanero pepper
extract sample was placed. Another difference in this part of the experiment was the solvent. We
used two different ratios of hexane and ethyl acetate. The first beaker contained a 9:1 hexane-
ethyl acetate mixture and the second mixture contained a 1:1 hexane-ethyl acetate mixture. After
setting up the beakers with the TLC plates, the solvent, and the filter paper, we waited 5 minutes
for the spots to travel. Under UV light, using a pencil, we circled the spots. In addition, the TLC
plates were placed in an iodine chamber. Through this visualization technique, iodine vapors can
be absorbed and detected by alkanes, alcohols, and ethers. Usually, UV light resists catching
onto these functional groups. Due to our solvent being a mixture of an alkane (the hexane) and
an ester, the iodine chamber better helped visualize the spots. After being absorbed, the
compounds left brown spots, making it easier to calculate the Rf values. For the plate with the
solvent ratio 9:1, the capsaicin reference solution had a Rf value of 0. Both the habanero solution
and chilli powder solution left two spots with the Rf values of 0.33 and 0.47 (for the plate with
the solvent ratio 9:1). For the second plate with solvent ratio as 1:1, the capsaicin reference
solution had a Rf value of 0.47. The habanero extract sample left two spots with the Rf values of
0.73 and 0.87. Lastly, the chilli powder extract sample left three spots with the Rf values of 0.73,
0.87, and 0.47. Based on these values, we could identify which pepper had more capsaicin.
Knowing that equivalent Rf values for any two spots means they are the same substance, we
identified the chilli powder to contain more capsaicin. Shown on the second TLC plate with the
1:1 solvent ratio, the capsaicin reference solution had the same Rf value as one of the spots for
the chilli powder extract sample. Because the Rf values were the same (0.47), chilli powder
contained more capsaicin.

Conclusion:
Part 1 of the lab gave us the compositions of the unknown compounds. Running TLC and
examining the spots under UV light provided us with Rf values. Equal Rf values of the unknowns
and standards identified unknown #1 to be aspirin and caffeine. Unknown #2 was a composition
of acetaminophen and ibuprofen. With similar techniques and the addition of extraction, we
classified which pepper, chilli or habanero, contained more capsaicin in part 2. Because the Rf
value for the chilli powder matched with that of the capsaicin reference solution, we concluded
that chilli powder had more capsaicin.

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