Angela Leong Feng Ping

M13608
Date of Experiment: 18 July 2013
Partner: Yang Qian Asarelah

Experiment #2
Thin Layer Chromatography
Results & Calculations
Choosing a Solvent
Solvent +
Visualisation
method
TLC plate
[A: aspirin; P: paracetamol; I: ibuprofen; C:
caffeine]
Rf
100% methanol
+ UV
Solvent front = 3.20 cm
Rf of spot A =


= 0.688
Rf of spot P =


= 0.719
Rf of spot I =


= 0.656
Rf of spot C =


= 0.531
35%
hexane/65%
methanol + UV
Solvent front = 3.10 cm
Rf of spot A =


= 0.581
Rf of spot P =


= 0.516
Rf of spot I =


= 0.500
Rf of spot C =


= 0.468
35% ethyl
acetate/65%
methanol + UV
Solvent front = 3.00 cm
Rf of spot A =


= 0.733
Rf of spot P =


= 0.733
Rf of spot I =


= 0.667
Rf of spot C =


= 0.633
65% ethyl
acetate/35%
methanol + UV
Solvent front = 3.10 cm
Rf of spot A =


= 0.484
Rf of spot P =


= 0.613
Rf of spot I =


= 0.581
Rf of spot C =


= 0.419
90% ethyl
acetate/10%
methanol + UV
Solvent front = 3.30 cm
Rf of spot A =


= 0.803
Rf of spot P =


= 0.697
Rf of spot I =


= 0.940
Rf of spot C =


= 0.485
50% ethyl
acetate/30%
hexane/20%
methanol + UV
Solvent front = 3.40 cm
Rf of spot A =


= 0.735
Rf of spot P =


= 0.691
Rf of spot I =


= 0.706
Rf of spot C =


= 0.706
The first TLC plate was developed in 100% methanol, the most polar solvent system tested. The Rf
values of all four spots were between 0.2 0.8, showing that all four compounds are polar (albeit to
varying degrees) and can be moved up the TLC plate by a polar solvent in mobile phase. However, the Rf
values are too close for reliable distinction. Making the solvent system less polar, by increasing the
composition of hexane and/or ethyl acetate, increases the disparity in Rf values of the four compounds,
making it easier to tell that the four spots have moved different distances from the baseline. As the
composition of ethyl acetate (against methanol) increases from 35% to 65% to 90%, making the solvent
system less polar, the less polar substances move further from the baseline while the more polar
substances move a shorter distance. Some TLC plates exhibit a horizontal smear below the spots, in the
shape of an upward crescent. This is common in TLC of compounds with strongly acidic or basic groups.
The TLC plate developed in 50% ethyl acetate/30% hexane/20% methanol solvent system showed long
spots for all four lanes, with nearly identical Rf values. Because the solvent system has comparable
amounts of each solvent, the more polar compounds (caffeine and paracetamol) would be primarily
soluble in and carried up by methanol, the most polar solvent; while the least polar compounds (aspirin
and ibuprofen) would be carried up by hexane, the least polar solvent. All four compounds thus travel
roughly the same distance up the plate, carried by the solvent that was most alike to them in terms of
polarity, but some molecules would be left behind due to poorer solubility in the other solvents of less
complementary polarity, leading to the long, smeared spots.
The final solvent system chosen was 80% ethyl acetate/20% methanol. The TLC plate developed in 90%
ethyl acetate/10% methanol solvent system showed the best results, with the four spots at distinctly
different distances. We can deduce that caffeine is the most polar substance, having migrated the least,
followed by paracetamol, then aspirin; ibuprofen is the least polar. However, the ibuprofen spot appears
too close to the solvent front (Rf > 0.8), which may lead to errors. This indicates that such a solvent
system is too non-polar, therefore we selected a solvent system that was slightly more polar, by
reducing the composition of ethyl acetate from 90% to 80%.
Analysis of Unknown Powder
- Take one-quarter to one-third spatula, each, of Powders A, B and C, and add into three labeled test
tubes
- To each test tube, add sufficient 50% ethanol to dissolve the powder completely to form a colourless
solution. If you are unable to dissolve all the powder, allow the mixture to rest so the powder sinks
to the bottom, and draw using a capillary tube from the solution above.
- Prepare a TLC plate: draw a baseline about 1 cm from one end and mark seven crosses on the
baseline lightly with a pencil. Spot, in order, the solutions of aspirin, paracetamol, ibuprofen, caffeine
and Powders A, B, and C.
- Place a small amount of solvent 80% ethyl acetate/20% methanol in a TLC jar, as well as a
semicircle of filter paper soaked in solvent. Develop the TLC plate, ensuring that the solvent is below
the baseline and the TLC jar remains undisturbed while developing.
- When the solvent front is about 1 cm from the top of the plate, take it out and mark the solvent front
using pencil. Allow the solvent to evaporate and view the plate under UV light. Use pencil to circle
the spots.
Solvent +
Visualisation
method
TLC plate
[A: aspirin; P: paracetamol; I:
ibuprofen; C: caffeine; PA: Powder A;
PB: Powder B; PC: Powder C]
Rf
80% ethyl
acetate/ 20%
methanol + UV
Solvent front = 3.50 cm
Rf of spot A =


= 0.743
Rf of spot P =


= 0.829
Rf of spot I =


= 0.943
Rf of spot C =


= 0.629
Rf of spot A =


= 0.743
Spot PA: Rf of upper spot =


= 0.857
Rf of lower spot =


= 0.571
Rf of spot PB =


= 0.857
Rf of spot PC =


= 0.943
The compound with the greatest Rf value (travelled the longest distance from baseline) is ibuprofen,
followed by paracetamol, aspirin, and lastly caffeine this is the order of solubility of the four compounds
in this solvent system of 80% ethyl acetate/ 20% methanol.
Lane PA has two spots; the upper spot is roughly the same distance from the baseline and with an Rf value
approximately equal to spot P, while the lower spot has an Rf value close to spot C. Thus, Powder A is a
mixture of paracetamol and caffeine. Spot PB has travelled about the same distance and has an Rf value
equal to spot P, so Powder B is paracetamol. Spot PC is equal distance from the baseline and has the same
Rf value as spot I, hence Powder C is ibuprofen.
Melting point determination is another method. Draw a small amount of each unknown powder into a
capillary tube used for melting point analysis and place into a melting point apparatus, set to a
temperature range of 70C to 200C. As aspirin, paracetamol, ibuprofen and caffeine have different
melting points (136C, 170C, 75C and 238C respectively), determining the temperature at which each
powder melts will point to the identity of each powder, and whether the powder is a mixture of more
than one compound (mixtures will start melting at the melting point of the compound with the lower
melting point, and melt over a large temperature range, and become fully liquid after reaching the
melting point of the compound with the higher melting point).
Post-Lab Questions
1.



2. Chromophores are chemical groups in a molecule that gives it its colour. The energy difference between
two molecular orbitals in a chromophore corresponds to the energy of electromagnetic (EM) waves of a
certain wavelength usually visible light or UV light, in this experiment. When light with a spectrum of
wavelengths falls on the molecule, certain wavelengths excite electrons in the chromophore from
ground state to excited state, and are thus absorbed, while other wavelengths are transmitted or
reflected. This absorption of certain wavelengths and reflection of others gives the molecule its colour.
The silica on the TLC plates contains a fluorophore that fluoresces when irradiated with UV. The
compounds in this experiment contain aromatic groups, which are UV active (other UV active functional
groups include , -unsaturated carbonyls, and any other compounds containing extensively -
conjugated systems). The spots on the TLC plate absorb UV light, masking the fluorescence of the silica,
thus appearing as dark spots.

3. This is to prevent overloading the TLC plate; when too much or too concentrated solution is spotted on
the plate, the amount of solvent in the mobile phase is insufficient to pull the compound up, creating a
streak that makes measurements to measure and inaccurate. Also, if the diameter of the initial spot is
too large, the spot may merge with adjacent spots as it runs up the plate, creating a smear. Lastly, we
want the resultant spot to be as small as possible, so that we can accurately measure the distance the
compound has travelled from the baseline. It is troublesome and difficult to accurately mark the centre
of the spot as the diameter increases.

4. The student might have overloaded the TLC plate, spotting too much solution initially at the baseline, or
the concentration of the solution spotted may be too high. The capillary force of the solvent in the
mobile phase as it moves up the TLC plate is not strong enough to overcome the force of gravity and
intermolecular forces of attraction between the large number of compound molecules in the spot and
the silica substrate. Thus the solvent is unable to pull all of the molecules of the compound upward,
and some molecules are left behind while others move ahead with the mobile solvent through the silica
substrate, creating the streak.

5. Ibuprofen has the highest Rf value, followed by aspirin, and paracetamol has the lowest. The solvent
acetone is a slightly polar molecule, with only one polar C=O bond. Aspirin, paracetemol and ibuprofen
each contain a non-polar benzene ring, but with different numbers of polar functional groups and bonds
that can form hydrogen bonds, dipole-dipole forces and London dispersion forces (LDF) with acetone
molecules. Ibuprofen is the least polar molecule, with only one polar carboxylic acid group (containing
two polar bonds: O-H and C=O). The strength of the intermolecular forces (IMF) between ibuprofen and
acetone is similar to that in ibuprofen and in acetone, thus ibuprofen dissolves the best in acetone.
Aspirin contains more polar groups and bonds (a carboxylic acid group and an ester group to give 2
polar C=O bonds, 1 O-H bond and polar C-O bond) than ibuprofen, so is more polar. The IMF between
aspirin and the less polar acetone is weaker than that between aspirin molecules, thus aspirin dissolves
more poorly in acetone. Paracetamol, with a polar hydroxyl group and a polar amide group, containing 1
O-H bond, 1 C=O bond, and polar N-H and C-N bonds (nitrogen is a highly electronegative), has the most
number of polar bonds and is the most polar. The IMF between acetone and the much more polar
paracetamol is far weaker than that between paracetamol molecules, thus paracetamol thus dissolves
most poorly in acetone. On a TLC plate developed using acetone, the most soluble ibuprofen would
travel the greatest distance, and the least soluble paracetamol the shortest distance.

6. The Rf values would increase for paracetamol, and decrease for ibuprofen, such that the order of Rf
values is likely reversed in comparison to Question 5: paracetamol > aspirin > ibuprofen. This is because
ethanol is a more polar molecule, with a polar O-H bond that can form strong hydrogen bonds, thus the
more polar compounds would dissolve better in ethanol. Paracetamol, being the most polar, is likely the
most soluble in ethanol and travel the greatest distance on the TLC plate. However, the results for the
TLC plate developed in 100% methanol show that the spots are rather close together. It is also possible
that aspirin may have a greater Rf value than paracetamol when ethanol is used, as ethanol is a less polar
solvent than methanol (as ethanol has a longer non-polar hydrocarbon chain).