Faculty of Science

Bachelor of Science (Hons) Chemistry Year

1 Trimester 3

UDEC 1224
Chemistry Laboratory II

Name: Melanie Ho Yenn Ting

Student ID: 21ADB06296
Lab Partner : Phylicia Liu
Practical Group: 1
Experiment No.: 9
Experiment Title: Thin-Layer Chromatography
Experiment Date: 10/08/2022
Submission Date: 31/08/2022
Lecturer’s Name: Dr. Ooi Mei Lee
Title : Thin-Layer Chromatography

Objective :
1. To learn the technique of TLC and the visualization of colourless components.
2. To identify an unknown drug by a TLC comparison with Standard compounds.

Introduction :

I. General Concepts
Chromatography is a common and powerful method used to separate and analyze complex
mixtures. Using this technique, the course of a reaction can be followed, and the products can be
separated and isolated. In this method, the components within the mixture are distributed between
two phases: a stationary phase and a mobile phase which moves through the stationary.
Chromatography works on the principle that different compounds will have different solubilities
and adsorption to the two phases between which they are to be portioned. The material to be
separated is place onto the stationary phase and is then carried along by the mobile phase. The
components of the mixture are absorbed by the stationary phase to different degrees, and it is thus
the various rates of migration for each component on the adsorption the slower the material passes
through the system. In this experiment you will learn the techniques of two types of
chromatography: column chromatography and thin layer chromatography

II. Principles of Chromatography

The separation of the components of a mixture depends on the phase each component
remains in and the rate at which each travel. The stationary phase does not move, while the other
mobile phase travels past the fixed phase. Due to various functional groups, each compound travel
past the stationary phase at a different speed. The strength of the following types of interactions:
ion-dipole and van der Waals forces.

Different types of chromatography use various types of stationary and mobile phases. In
this experiment, the solid phase is silica gel while the mobile phase is an organic solvent (may be
a single type or a mixture of solvents). This means that the compounds to be separated must choose
between being absorbed to the solid silica gel or moving along in the organic solvent. The silica
gel is either pack into a column or adhered to a sheet of glass, plastic, or aluminium, depending on
the type of chromatography.

When a column is used, the compound mixture is placed on top, and the solvents are run
down the column separating the mixture along the way. With the silica gel on a plate, the
compounds are placed close to the bottom and the mixture is separated as the solvent travels up
by means of capillary action. Since silica gel is a porous form of SiO2 , the surface of gel contains
Si-OH and Si-O-Si functional groups. With silica gel, the dominant interactive force between the
adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules
interact strongly with the polar Si-O bonds of these adsorbent and will tend to stick or adsorb onto
the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar
molecules thus generally tend to move through the adsorbent more rapidly than the polar species.
Figure 1 illustrates the general column chromatography setup and Figure 2 illustrates the
movement of a compound mixture in a column and their separation and isolation.

Another factor that establishes the rate at which a compound travels past silica gel is the
polarity of the solvent. A polar solvent will compete for silica absorption sites, disallowing the
compounds to do so. This promotes a faster rate at which all compounds travel. The order in which
the compounds to do so. This promotes a faster rate at which all compounds travel. The order in
which the compounds move remains the same, while moving faster as the polarity of the eluent
(solvent system) increases.

III. Thin Layer Chromatography (TLC)

TLC involves the same principles of separation as column chromatography but the apparatus and
technique for development is different. Instead of a column, the silica (or alumina) is adhered to a
plate of glass, plastic, or aluminium. A capillary spotter I used to apply the dissolved sample onto
the plate about 1cm from the bottom (a line with pencil is drawn). Once the solvent has evaporated,
only the sample remains. The plate is carefully placed into a closed developing chamber, which as
a shallow layer of solvent that does not submerse the spot. The chamber is lined with a folded
piece of filter paper to ensure a uniform and saturated atmosphere of solvent vapour.

The plate is removed when the solvent front has reached about 0.5cm from the top and is quickly
marked with pencil. The capillary action of the solvent causes the initial spot to be separated into
individual components that may be visualized by colour identification or with the following
techniques for colourless compounds:

(a) Irradiation with ultraviolet light

(b) Reversible staining with iodine vapour (formation of brown spot which fade)
(c) Spraying with a reagent that irreversibly colours the spots, e.g.: H2SO4 , KMnO4

Figure 1 illustrates the general principles showing the separation of the same mixture of 3
components, A, B and C.

The rate at which a compound moves in respect to the solvent front, Rf , is characteristic of that
compound under standard conditions. The Rf value is calculated by dividing the distance each spot
has travelled (measures from the pencil line to the middle of the spot) by the distance the solvent
front travelled from the pencil line. This is illustrated in Figure 2. The advantages of TLC are the
very small quantities of sample required and the great ease and rapidly with which it is resolved.
For these reasons, it is often used to monitor the progress of a reaction by running the crude sample
beside the reaction sample on the same plate. The following are some common uses of TLC:

(a) To determine the number of components in a mixture.

(b) To determine the identity of two substances
(c) To monitor the progress of a reaction.
(d) To determine the effectiveness of a purification
(e) To determine the appropriate conditions for a column chromatographic separation.
(f) To monitor column chromatography
 Figure 2: Illustration of a developed TLC plate and Rf calculation

IV. Factors affecting resolution of separations

(a) Adsorbents: Weight ratio of adsorbent to sample is important to obtain accurate separation.
If too many samples is applied, the active adsorbing sites will be saturated and the column
will be flooded, resulting in poor separations. In most cases, a ratio of 20 to 1 is satisfactory
but sometimes up to 100 to 1 is necessary. Even the ratio of column height to diameter is
important; about 8 to 1 is considered optimal.
(b) Solvents: The solvent (or solvent mixture) is important to the compound separation, keeping
in mind that the more polar the solvents, the faster the compound move. In some cases, a
solvent system may increase in polarity by gradually changing the composition of the
solvent mixture.

Cyclohexane
n-hexane (non-polar)
Benzene
Toluene
Dichloromethane
Chloroform
Diethyl ether Increasing Increasing eluting
Ethyl acetate polarity power with polar
Acetone stationary phases
Ethanol
Methanol
Acetonitrile
Water (polar)
 (c) Functional group: Compounds with highly polar groups are strongly adsorbed and eluted
less readily than less polar (or polarizable) compounds. The strengths of adsorption for
compounds having the following types of polar functional groups decreases in the order
listed below. However, variations may occur depending on the overall structure of each
specific compound.

RH, R-X, alkenes, R-OCH3 , R-CO2R’, RCOR’, RNH2 , R-OH, R-CO2H

Increasing absorption on polar stationary phases

Part 1: TLC Analysis of Analgesic Drugs

In this experiment, TLC will be used to examine the composition of various analgesic (pain
relieving) drugs. Among these are the aspirin and acetaminophen. Caffeine is sometimes added to
these formulations to overcome drowsiness.

Aspirin Acetaminophen Caffeine

Apparatus and Materials :

UV Lamp, Capillary tube, 250mL beaker, Aspirin, Acetaminophen, Caffeine, Unknown A,

Unknown B, TLC plates, Ethyl acetate, Hexane, Acetic acid and Iodine.

Procedures :

A. Spotting of the TLC plates

1) A TLC plate was obtained from instructor.
2) The plate was set down on a clean, dry surface then a line was drawn lightly across the plate
about 1cm from the bottom of the plate with the aid of a 2B PENCIL.
3) Next five 2-3mm lines were made, spaced about 0.8cm apart and running perpendicularly
through the lines across the bottom of the TLC plate. The first and the last line were about
 0.8cm from the edge of the plate. These lines marked the points where the samples will be
spotted or applied to the TLC plate.
4) The plate was spotted with the 5 different analgesics by sharing with the people around. A
separate capillary tube was used for each sample. First the acetaminophen solution was
spotted, then the caffeine, unknown A, aspirin, and lastly the unknown B. The plate was
examined under the ultraviolet (UV) light to see that enough of each compound had been
applied; if not, more was added.

B. Developing the TLC plate

1) A developing chamber was prepared. A 250mL beaker was used as a chamber, a half-piece
of filter paper was placed inside, and aluminium foil was used to cover.
2) The eluent, 2:1 mixture of ethyl acetate: hexane was poured, into the beaker to a depth of
under 1 cm. About 5 drops of acetic acid were added into the beaker and mixed. The
prepared TLC plate was placed in the developing chamber and the solvent level was ensured
to below the pencil line.
3) After the solvent had risen to near the top of the plate, the plate was removed, and the
solvent front was marked with a pencil. The solvent was allowed to evaporate from the plate
in the fume hood.

C. Visualization
1) The colourless compounds were visualized by illumination of the plate with an ultraviolet
(UV) lamp.
2) The spots were outlined with a 2B pencil. The spots were also visualized by putting the
plate in an iodine chamber for a couple minutes.

D. Comparison of the unknown with reference standards

1) The plate was sketched in notebook and the Rf values for each spot was calculated.
2) The unknown drug was determined based on the Rf values.
Results and Calculations :
Distance travelled by solvent = 18.7 cm

2.8
Rf for A, acetaminophen =
18.7
= 0.15
2.6
Rf for B, caffeine =
18.7
= 0.14
2.75
Rf for C, unknown A =
18.7
= 0.15
4.0
Rf for D, aspirin =
18.7
= 0.21
4.2
Rf for E, unknown B =
18.7
= 0.22
∴ Unknown A = Acetaminophen
Unknown B = Aspirin

Discussions :
It is important to indicate the polarity of non-polarity of the structure as they contribute to
the relative positions on the TLC Plate. The reason for this is because a compound that with more
polar functional groups present will be less attracted to the less polar filtrate so more than likely it
will be in the stationary phase, spending less time in the mobile phase which would then have a
lower Rf vale. But this can also be related to the intermolecular forces, IMF, of the functional
groups of the compound. A compound with a stronger IMF will spend more time in the stationary
phase which would also equate to having a lower Rf value (Nichols, Lisa, 2019).

Thin Layer Chromatography, TLC, is a laboratory technique in which the mixture is

dissolved within a solvent and placed near the bottom of a TLC plate. The TLC plate can be
compromised of glass, metal or plates which is covered in a thin layer of adsorbent serving as the
stationary phase. TLC also includes a mobile phase which a pure solvent or a mixture of solvents.
The configuration of the mobile phase depends on the polarities of the compounds that are being
separated. The stationary phase used the experiment is silica gel. It acts as a polar adsorbent of
partition of chromatography of organic compounds. Polar molecules with dipole-dipole bonding
and hydrogen bonding can be adsorbed by the stationary phase, silica gel.

Note, the more polar the compound, the more strongly the compound will bind to the
stationary phase. The mobile phase, ethyl acetate, polar solvent. A polar solvent, such as ethyl
acetate, allows better separation between polar and non-polar compounds since IMF’s and polarity
between compounds differ from one another. Polarity plays an important role. The concept of “like
dissolves like” comes into play as well. If the polar attraction between the samples and the silica is
high/more, then the samples will dwell more in the silica gel than eluting in the ethyl acetate. If
the solvent has more polarity, then the better identifying the mixtures/compounds would be.

On the TLC plate, there was a movement of different spots. There is capillary action of the
mobile phase where it can elute up the TLC plate. Yet, it is the IMF of the both the mobile and
stationary phase that determines how tightly held the components are to the silica gel while others
not held as tightly move up the TLC plate by the ethyl acetate. The portion of the plate near at the
solvent plate are less polar components where the more polar components are near the line of origin.
Compounds held more tightly to the silica gel are highly polar, so it stays as the near the line of
origin. The components that are less polar and more attracted to the solvent will elute up the TLC
plate meaning that these were not as attracted to the silica gel. With the experiment, both aspirin
and acetaminophen are polar. Acetaminophen contains two hydrogen bonding sites compared to
aspirin even though aspirin has more oxygen, hence, acetaminophen is more polar than aspirin.

Aspirin and acetaminophen both contain hydrogen bonding and polar. By comparing both
molecules, there are two true hydrogen bonding on acetaminophen. Whereas aspirin contains only
one true hydrogen bonding site even though hydrogen sites can be induced with aspirin and its
oxygen. This concludes that the silica gel holding tightly onto acetaminophen relative to the aspirin.
Aspirin will then more likely move with the ethyl acetate and is less tightly bound to the silica gel.

In this experiment, aspirin travelled further away than acetaminophen and caffeine. This
can be clearly shown by the calculation of the Rf values of each compound whereby the higher Rf
value indicate that the compound travel further away and thus is less polar (Flashcard Machine,
2012). The Rf value for aspirin is 0.21, followed by acetaminophen which is 0.15 and finally the
caffeine which is 0.14. Therefore, the polarity of the solvent increases from aspirin to
acetaminophen and finally to caffeine. This is due to the presence of large number of
electronegativity atom such as nitrogen and oxygen atom in the compounds, thus the polarity of
the caffeine compound increases. We can identify the unknown compounds since the compounds
with similar polarities will travel with the same distance along the TLC plate by comparing the Rf
values of the unknowns with the standard compounds. Unknown A and unknown B have the Rf
values of 0.15 and 0.22, respectively. Since unknown A has the nearest Rf value to acetaminophen,
thus we can conclude that unknown A is acetaminophen whereas for unknown B, it has the same
Rf value as aspirin, so we can deduce that unknown B belongs to the aspirin.

There are some precaution steps that had been taken in this experiment. First, be careful not
to bend the TLC plate excessively since the silica adsorbent may flake off. In addition, a TLC plate
should only be handled by the edges since our fingerprint contain UV active substances. Besides,
it is important that the spots be made as small as possible to avoid “tailing” when the plate is
developed. When handling the UV light, do not look directly at the ultraviolet light as it can cause
eye injuries. Lastly, do not interrupt the beaker after TLC plate has been introduced into the
developing chamber as it can cause error to the measurement.

Conclusion :
In conclusion, thin layer chromatography is a lab technique that can identify components that are
in found in a mixture. From the result obtained, the Rf values of unknown A is the same as
acetaminophen, whereas for unknown B, the Rf values is the nearest to aspirin. Hence, we can
conclude that unknown A is acetaminophen and unknown B is aspirin.

References :

1. Chemical Forum, (2015). Explain the elution order of RP-HPLC for aspirin, acetaminophen,
and caffeine. [Online]. Available at: https://www.chemicalforums.com/index.php?
topic=81206.0 [Accessed 30th June 2022].

2. Flashcard Machine, (2012). O-Chem Lab Quiz #2, 3/4 Flashcards. [Online]. Available at:
https://www.flashcardmachine.com/o-chemlabquiz234.html [Accessed 30th June 2022].
3. Nichols, Lisa. “2.2D: Separation Theory.” Chemistry LibreTexts, Libretexts. [Online].
Available at:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Techni
ques_(Nichols)/02%3A_Chromatography/2.03%3A_Thin_Layer_Chromatography_(TLC)/2.
3D%3A_Separation_Theory [Accessed 1st July 2022].
4. Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles
and Practice (Illustrated edition ed.). pp. 159–173 [Accessed 1st July 2022].
5. Department of Chemistry. (2013), CHEM 334 Thin Layer Chromatography. [Online].
Available at :
https://www2.chem.wisc.edu/deptfiles/OrgLab/handouts/CHEM%20344%20TLC%20info.p
df [Accessed 1st July 2022].