EXPERIMENT 2 – THIN LAYER
CHROMATOGRAPHY AND FUNCTIONAL
GROUPS
Question 1: Draw the structure for each of the substances in the label by hand.
Benzoic acid:
Phenyl benzoate:
Acetyl salicylic acid
Question 2: Why
never touch the coated surface of a TLC plate with your fingers?
should you
Touching the coated surface can transfer oils, dirt, fingerprints, and other
contaminants onto the silica plate, which creates uneven spots and damages the
coating. This leads to the integrity of the TLC plate being compromised and causes
inaccurate results, as it stops the samples from separating in the right way.
Furthermore, some TLC coatings can be hazardous, and so touching the surface
could lead to skin irritation or absorption of harmful substances (Unknown, 2026).
Question 3: Why must you use a pencil and not a pen to mark your TLC plate?
A pencil is made out of graphite, which does not dissolve in the mobile phases and
stays fixed at the origin point. Pen ink, however, contains organic dyes and pigments
that could dissolve in the solvent. Dissolved ink travels up the silica gel, mixing with
the sample components and obscuring the actual results. (Sandtorv, n.d.)
Additionally, a sharp pen tip can scrape off the layer of silica gel, impacting how the
liquid moves up the plate.
Question 4: What is silica gel?
Silica gel is an amorphous, porous form of silicon dioxide. In TLC, it serves as a
polar stationary phase (Rabel & Sherma, 2005).
Polar compounds stick tightly to the silica gel and move slowly up the plate, while
less polar compounds travel faster with the liquid solvent.
Question 5: Why do you use the narrow end of the capillary tube to spot your
sample on the TLC plate?
The narrow end of the capillary tube is used to ensure a small, concentrated sample
spot, which stops the different sample components from bleeding together and
cross-contaminating each other. (Miller, 2012)
It also allows for more accurate placement of the sample onto the origin line without
overloading the silica layer (Sandtorv, n.d.).
Question 6: Why should the beaker be covered during the development of the
chromatogram?
The beaker should be covered during chromatography development to prevent
evaporation, control the atmosphere, and to prevent contamination.
Covering the beaker prevents the solvent from evaporating, which could lead to an
uneven development of the chromatogram. It also aids in controlling the atmosphere
inside the beaker by keeping the solvent vapour consistent throughout the
development. Additionally, covering the beaker prevents any particles that could
interfere with the chromatogram from entering. (Moore, 2016).
Question 7: Explain what forces the mobile phase to move up the TLC plate?
Capillary action forces the mobile phase to move up the plate. Adhesion between the
solvent molecules and the functional group creates a pressure difference that draws
the liquid upwards. Cohesive forces, such as hydrogen bonding, pull the solvent
molecules up the plate. (Gierak, Skorupa, & Lazaska, 2015)
Question 8: It is important that the developing solvent in the beaker is below the line
on which the samples were spotted. Why is this the case?
The solvent level must stay below the origin line so that samples move up through
the medium via capillary action, rather than dissolving straight into the solvent. If the
developing mixture was above the line, the mixture would wash off in the bottom of
the beaker, and no meaningful chromatogram can form on the plate. (Organic
Chemistry at CU Boulder)
Question 9: What is effervescence and what is causing it here?
Effervescence is the rapid escape of gas from an aqueous solution, seen as fizzing,
bubbling, or foaming. (Helmenstine, 2019).
During the test for a carboxylic acid, the reaction between the carboxylic acid and a
base – in this case, sodium hydrogen carbonate – forms a carboxylate salt, water,
and carbon dioxide gas. The sudden production of this carbon dioxide gas creates
the effervescence. ((NCERT))
Table 1: Table showing the distance of solvent advance and sample advance, as well as the retention factor for
each sample
Sample
A
B
C
AS (authentic
aspirin)
Distance of solvent Distance of
advance (mm)
sample advance
(mm)
64.9
48.2
64.9
60.4
64.9
44.9
64.9
44.8
Retention factor
(Rf)
0.743
0.931
0.692
0.690
Where 𝑅𝑓 =
distance travelled from spotting line by substance (mm)
distance travelled from spotting line by solvent front (mm)
The distance travelled from the spotting line by the substance was measured with a
ruler from the origin line to the centre of each sample’s marking spot. The distance
travelled from the spotting line by the solvent front was measured with a ruler from
one marked edge of the plate to the other (from bottom origin point to top marked
point).
In chromatography, a high retention factor means that a substance travels a long
distance with a weak stationary phase interaction, usually representing non-polar
substances. The opposite is true for substances with a low retention factor.
With this being said, and from the above table, the following assumptions can be
made:
1. Sample C is acetyl salicylic acid (authentic aspirin) due to the low difference in
retention factors (ΔRf = 0.002).
2. Sample B has the highest retention factor and is thus the most non-polar of all
of the substances. Out of the three samples, phenyl benzoate is the least
polar, as it only contains an ester with no free acidic groups.
3. Sample A has an intermediate retention factor, meaning it is more polar than
sample B, but less than sample C. This indicates that sample A is benzoic
acid, as it contains a single carboxylic acid group.
However, retention factor is not a definitive test for identifying samples. Thus, two
additional tests were run in conjunction with chromatography to further justify the
aforementioned assumptions.
Table 2: Table showing the observational results from the test for carboxylic acid
Sample
A
B
C
Observation
Active bubbling and fizzing
No effervescence/clear solution
Rapid bubbling and effervescence
The presence of effervescence indicated the presence of an acidic functional group.
Since both samples A and C presented bubbles when interacting with sodium
hydrogen carbonate, it indicated that they are acidic. Thus, by deduction, sample B
is phenyl benzoate.
Table 3: Table showing the observational results from the test for a carboxylic ester
Sample
Observation
A
B
C
Light yellow solution
Dark orange/reddish precipitate
Deep red solution
From the test for a carboxylic ester, it can be seen that a dark red colour indicates
that the sample contains an ester group. This furthers the deduction that sample B is
phenyl benzoate, as the dark orange colour shows that sample B contains an ester
group, which phenyl benzoate does.
It can be seen that, due to its light colouring, sample A does not contain an ester
group. On the contrary, sample C displayed an extremely dark red colour, meaning
that it does contain an ester group. That being the case, it can be assumed that
sample A is benzoic acid, while sample C is acetyl salicylic acid. This mimics the
assumptions made previously.
Table 4: Table summarizing the findings of the experiment
Sample
A
B
C
Final Bottle Assignment
Benzoic Acid
Phenyl Benzoate
Acetyl Salicylic Acid
Works Cited
(NCERT), N. C. (n.d.). Test for Carboxyl Group. Laboratory Manual Chemistry for
Class XII.
Gierak, A., Skorupa, A., & Lazaska, I. (2015, August). ResearchGate. Retrieved from
Capillary Action Liquid Chromatography: New Chromatographic Technique for
the Separation and Determination of Colour Substances:
https://www.researchgate.net/publication/282899691_Capillary_Action_Liquid
_Chromatography_New_Chromatographic_Technique_for_the_Separation_a
nd_Determination_of_Colour_Substances
Helmenstine, A. M. (2019, February 4). ThoughtCo. Retrieved from Effervescence
Definition in Chemistry: https://www.thoughtco.com/definition-ofeffervescence-604435
Miller, S. (2012, July 3). Tips and Tricks for the Lab: How to Make a Capillary TLC
Spotter. Retrieved from
https://www.chemistryviews.org/details/education/2102991/Tips_and_Tricks_f
or_the_Lab_How_to_Make_a_Capillary_TLC_Spotter/
Moore, M. (2016). Quoara. Retrieved from Why must you cover a beaker using a lid
during paper chromatography?: https://www.quora.com/Why-must-you-covera-beaker-using-a-lid-during-paper-chromatography
Organic Chemistry at CU Boulder. (n.d.). Retrieved from Thin Layer Chromatography
(TLC):
https://www.orgchemboulder.com/Technique/Procedures/TLC/TLC.shtml
Rabel, F., & Sherma, J. (2005, May 28). Thin-Layer Chromatography | Plate
Technology. Retrieved from ScienceDirect:
https://www.sciencedirect.com/topics/medicine-and-dentistry/silica-gel
Sandtorv, A. (n.d.). LibreTexts. Retrieved from TLC - Analysis:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book%3A_How_t
o_be_a_Successful_Organic_Chemist_(Sandtorv)/02%3A_COMMON_ORGA
NIC_CHEMISTRY_LABORATORY_TECHNIQUES/2.04%3A_TLC_ANALYSIS
Unknown. (2026). Manasa Life Sciences. Retrieved from Thin Layer
Chromatography: https://manasalifesciences.com/blog/thin-layerchromatography