THIN-LAYER CHROMATOGRAPHY OF

ACETAMINOPHEN, ASPIRIN AND CAFFEINE

IP6 – GROUPS 4, 5 and 6
I. INTRODUCTION
Chromatography

• From Greek “chroma” (color) and “graphein” (to

write)

• A group of methods used for the separation,

identification, and determination of chemical
components in a complex mixture

• Can be defined as a procedure by which solutes are

separated by a differential migration process in a
system consisting two or more phases, one of which
moves continuously in a given direction and in which
the individual substances exhibit different motilities
by reason of differences in adsorption, partition,
solubility, vapor pressure, molecular size, or ionic
charge density
Chromatography

• Stationary Phase
 one of the phases; a fixed bed of large surface area
 it must be small and homogenous as possible to allow frequent and
efficient sorption and desorption

• Mobile Phase
 a fluid that moves through or over the surface of the stationary phase

• Involves an equilibrium between a vapor enriched in the more-

volatile component and a liquid condensate of the same
composition

• Mikhail Tswett – a Russian-Italian botanist who pioneered

adsorption chromatography, used it for separation of plant pigments
 Classification of Chromatographic Techniques

A. Adsorption Chromatography C. Ion-Exchange Chromatography

Examples: Examples:

a. Gas-Solid Chromatography a. Ion-Exchange Chromatography

b. Liquid Column Chromatography b. High-Performance Liquid Chromatography
c. High-Performance Liquid Chromatography D. Permeation Chromatography
d. Thin-Layer Chromatography Example:

a. Size Exclusion Chromatography

B. Partition Chromatography
Examples: E. Affinity Chromatography
a. Gas-Liquid Chromatography Example:

b. Supercritical Fluid Chromatography a. DNA Affinity Chromatography

c. Liquid-Liquid Chromatography F. Electrophoresis

Example:
d. Paper Chromatography
a. Capillary Electro-Chromatography
e. High-Performance Liquid Chromatography
 Theory of Chromatography
• Plate theory
• By Martin and Singe
• Considers the chromatographic system as a
series of discrete layers of theoretical plates.
At each of these, equilibration of the solute
between the mobile and stationary phases
occurs. The movement of the solute is
considered as a series of stepwise transfers
from plate to plate.
• Rate theory
• By Giddings
• Considers the dynamics of the solute
particle as it passes through the void spaces
between the stationary phase particles in the
system as well as its kinetics as it is
transferred to and from the stationary phase
 Retardation Factor (Rf)

• Chromatographic systems achieve their

ability to separate mixtures of chemicals by
selectively retarding the passage of
compounds to through the stationary phase
while permitting others to move more freely

• Used for qualitative evaluation

• Different chromatographic techniques have

different ways of calculating Rf

• The ratio of the distance from the origin

traveled by the solute band to the distance
traveled by the mobile phase in a particular
time (for TLC)
Thin-Layer Chromatography

• Differs from other techniques because

separation occurs on a planar surface, not in
an enclosed column and the mobile face is
drawn by capillary action

• Not as effective in separation but has

advantages of speed, versatility, and simplicity

• Normal Phase – relatively polar stationary

phase and relatively non-polar mobile phase

• Reverse Phase – relatively non-polar

stationary phase and relatively polar mobile
phase
 Stationary Phase

• Finely divided solid spread as a thin layer • Alumina (aluminum oxide) – preferred for
on a rigid supporting plate (plastic, steel, separation of basic and weakly polar
or aluminum) compounds

• Silica gel – most frequently used • Polyamide (nylon) – strong H-bonding

stationary phase
• Surface is acidic due to the presence of
many silanol hydroxyl groups therefore
best suited for polar and acidic compound
analysis
abilities
• Other less frequently used sorbents:
calcium phosphate, calcium carbonate,
diatomaceous earth (kieselguhr)
cellulose.

• Silica gel + silicone oil or octadecylsilyl • Binders to the backing plate: calcium
sulfate (gypsum), starch
(ODS) – used for reverse phase carboxymethylcellulose, or polyvinyl
alcohol
 Mobile Phase

• A liquid allowed to migrate across the • Can be altered (largely empirical)

surface of the plate
• Chosen using eluotropic series and
depends largely on the nature of the
solutes and stationary phase
• Preferable to use a single solvent
rather than a multicomponent mixture
because as it moves up the plate the
concentration changes since it is
adsorbed preferentially
• Should be volatile so the set-up can
be equilibrated and it can be
evaporated after plate development
• Substances with functional groups
similar to those of the solutes may be
added to increase the Rf value by
promoting the solubility in the mobile
phase.
• Acids or bases may be added to affect
the charges on the solutes to prevent
tailing (the substance trails a line
behind it like a tail)
 Preparation of Plates

• Larger dimensions = more effective separation

• Scrupulously cleaned

• Fluorescent indicators such as zinc silicate is added to

aid in detection

• Two different sorbents may be applied to form a

gradient of both to yield separations that otherwise
would be impossible (ex. silica gel and alumina)

• Thicker layer = quantitative (greater volume but may

cause band-broadening)

• Thinner layer = qualitative (more effective separation)

Sample Application and Development

• Plates should be dried

• Spots should be evaporated after every application

and should be as small as possible

• Saturate the atmosphere of the jar with mobile phase

• Mobile phase should be evaporated after

development

• Multiple development – to increase resolution, the

plate is redeveloped in the same direction after it is
dried

• Two-dimensional development – complex

development used for complex mixtures
Detection Methods

• Obvious highly colored samples

• Fluorescence Quenching – zinc silicate

II. METHODOLOGY
Procedure

A. Preparation of TLC Plates

Procedure

B. Preparation of Samples and Standards

• Prepare 1% (w/v) ethanolic solution for each
sample and standard

C. Preparation of the Mobile Phase

• 200 mL of 95% ethyl acetate and 5% acetic
acid

D. Spotting the Plate

RFIS

• Preparing the sample

Small amount of the material was used (1% solution)

• Spotting the TLC plate

 TLC plate heated before spotting (Activation) to evaporate
 So that any water molecule adsorbed by the Silica is removed. When water is absorbed, the site
of attachment for the sample is blocked.
 Pencil is used
 Since graphite is inert.
 TLC plate handled gently and by the edge
 Since the 100-mm-thick coating of silica gel can be easily scratched off or contamination of the
surface can occur with the oil, dead skin cells, etc. that our hands contain.
 Spotted a couple of times
 So as to ensure that sufficient amount of material is present.
 Spot made as small as possible
 So as to avoid streaking, spotting and other common errors which may affect the result.
RFIS

• Picking a solvent
Ethyl acetate has a polarity of 4.4 while acetic acid
has a polarity of 6.2 in accordance with the polarity index.
(A higher value refers to a more polar substance). Thus,
the mobile solvent used was relatively non-polar.
Stays at the bottom = Add more polar solvent
Runs with the mobile phase = Add more non polar solvent
Procedure

E. Developing the TLC Plate

F. Visualization of the Spots

RFIS

• Developing the plate

 Covered with aluminum foil
 So as to prevent evaporation of the solvent
 Saturate the atmosphere within the container
 So as to ensure better separation
 Spots should be above the developing liquid and not submerged
 So as to avoid washing off the sample
 Solvent shouldn’t reach the top end of the plate
 So that uncertainty in measurements for Rf will be avoided
 Immediately draw a line across where the solvent can be seen
For a more accurate Rf value
 RFIS

Visualizing the results

 Use of UV light
o Short wave UV: 280 – 100 nm
o Long wave UV: 400 – 315 nm
o TLC plates normally contain a fluorescent
indicator that makes them glow green under
UV light of wavelength 254 nm
o Quench the green fluorescence
o Yield dark purple or blue spots

Use of Iodine Chamber

o Adsorb iodine vapors
o Yield brown spots
Process of Thin-Layer Chromatography

1. Preparing the Sample

2. Spotting the TLC Plate

3. Picking a Solvent

4. Developing the TLC Plate

5. Visualizing the TLC Plate

6. Calculating the Rf Values

III. RESULTS AND
DISCUSSIONS
 Retardation Factor (Rf)

• A more polar substance will have a lower Rf

value while a less polar substance will have
a higher Rf value. This can be inferred from
the fact that the mobile phase used was
relatively non-polar.

• The non-polar substance experiences

stronger attraction to the non-polar solvent
than the polar stationary phase. Thus, they
spend most of their time in the mobile phase
and travel a larger distance.

distance travelled = Rf value = polarity

Retardation Factor (Rf) Values

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

Caffeine Excedrin 0.35 0.44 0.46 0.35 0.42 0.43
Caffeine Standard 0.37 0.43 0.38 0.35 0.42 0.43
Caffeine Sample 0.39 0.40 0.43 0.39 0.42 0.43
Aspirin Excedrin 0.84 0.86 0.91 0.80 0.82
Aspirin Standard 0.88 0.91 0.90 0.82 0.86
0.77 0.79
Aspirin Sample 0.84 0.90 0.87 0.81 0.82
0.76
Acetaminophen 0.70 0.75 0.83 0.66 0.73 0.68
Excedrin
Acetaminophen 0.70 0.77 0.78 0.68 0.75 0.68
Standard
Acetaminophen 0.70 0.77 0.78 0.68 0.75 0.68
Sample
 Retardation Factor (Rf)

• Experimental data show that aspirin is the least polar among the
three, followed by acetaminophen, then caffeine, the most polar. That
being said, aspirin travelled the greatest distance and has the highest
Rf value.
• Similar Rf values will not necessarily mean that the two components
are the same molecule. For it to be a valid comparison, the TLC
plates must be run under the exact same conditions for temperature,
stationary phase, and mobile phase.
It can be seen from the results that
the samples contain other
components (impurities) which may
mean that the purification process
done during the previous experiment
wasn’t efficient.

Aspirin

Acetaminophen

caffeine
IV. CONCLUSION AND
RECOMMENDATIONS
 Rf values vary with the structure and polarity of a
substance. The most polar yield the smallest value while
the least polar yield the greatest value. Experimental data
showed that caffeine is the most polar among the three,
followed by acetaminophen. Caffeine travelled the least
distance due to its affinity to the very polar stationary phase
and therefore interacted with it more. Aspirin is said to be
the least polar, having travelled the greatest distance due
to its high affinity to the slightly polar solvent.
 Through Thin-layer Chromatography with the use of
Silica as the stationary phase and a solvent system
composed of 95% ethyl acetate and 5% acetic acid as the
mobile phase, we were also able to determine the purity of
the extracts we yielded from the previous experiment. The
Rf values of the standard were used to identify the
components of the extracts. Through the collected data, it
was seen that most of the extracts weren’t exactly pure.
Aside from not having the exact Rf values that their
standards did, some spots were also present, marking
impurities.
 In the next future experiments, we recommend the use
of other solvent systems as mobile phases, other
adsorbents as stationary phases and chromatographic
techniques other than TLC.
 V. REFERENCES

Larcia, L. (ed.) (2014). Laboratory Manual in

Pharmaceutical Chemistry 125.1. Manila: UPMCP

Philadelphia College of Pharmacy (2013). Remington: The

science and practice of pharmacy (22nd ed.). Michigan:
Pharmaceutical Press.
Questions asked during the report:
1. What will happen if the TLC plates are left too long in the chamber?

-If the TLC plates are left too long in the chamber, all the spots will move up the top edge of the
plate, therefore making the Rf values inaccurate.

2. Where were the contaminants found in?

-Most of the contaminants were found in the extracts from the previous experiment.

3. What is the effect of contaminants on the Rf value?

- Contaminants do not affect the Rf value. They do however create additional spots and imply
impurity of the sample.

4. Why is the plate heated before spotting?

-So that any water molecule adsorbed by the Silica is removed. When water is absorbed, the
site of attachment for the sample is blocked.