Expt 3 intro similar to like dissolves like; a polar component is

attracted to a polar phase, while a non-polar

Chromatography is one of the classical
component is attracted to a non-polar phase
methods used widely for the separation,
(Williamson and Masters, 2011). Thus,
identification, and determination of chemical
components, which have different polarities, will
constituents in complex mixtures. Generally,
have different migration rates. Migration rate is
chromatographic processes involve unequal
measure in terms of retention factor, which is equal
distribution of components in two immiscible
to the quotient of the distance travelled by the
phasesmobile phase and stationary phase
component and solvent. (see equation 1).
(Gilbert and Martin, 2011). Through the flow of the
mobile phase, constituents of a mixture are carried distance travelled by the substance
Rf =
through the stationary phase. Here, the basis of distance travelled by the solvent
separation is the migration rates among the mobile- (1)
phase components (Skoog, et al., 2014).
A polar adsorbent, usually in the form of
Based on the type of equilibration involved, silica or alumina gel coated on the TLC plate, is
which is dependent on the nature of the mobile and used as a stationary phase in thin-layer
stationary phases, chromatographic processes are chromatography. Silica and alumina particles are
classified into adsorption, partition, ion exchange, very polar due to their extended network of
and pore penetration (Christian, 2004). covalent bonds and the difference in

This experiment used Thin-layer electronegativity of the Si-O or Al-O bonds

chromatography (TLC), a normal phase solid-liquid (Williamson and Master, 2011). Thus, a polar

adsorption type of chromatography, which is based component will not migrate far because it is

on different affinities of components to the strongly adsorbed on the plate, and will obtain a

stationary phase and mobile phase (Williamson and lower Rf value compared to the less polar

Masters, 2011). It is an analytical technique component.

commonly used in determining identity and number

of components, progress of a reaction, and
effectiveness of a purification, due to its practicality.
Small amounts of the sample, as little as 10 -9g, can
rapidly be analyzed through this method. However,
volatile substances cannot be analyzed through this
technique (Gilbert and Martin, 2011).

In TLC, partitioning happens through

migration of the components with the mobile phase
and past the stationary phase. Migration of the Figure 1. Silica and alumina structure

component is dependent on its attraction to the (Williamson and Masters, 2011)

mobile and stationary phase. It follows a principle

 On the other hand, the mobile phase is a
solvent which carries components that are attracted
to it. The type of solvent to be used is a crucial
choice in the success of chromatography. Since the
stationary phase is a very polar adsorbent, using a
non-polar solvent will not yield good results,
because polar components will not migrate and
non-polar components will move along with the
solvent. With that, it will be difficult to differentiate
the components. A better choice of solvent is a
mixture of non-polar and polar solutions. However,
the difference in polarities must be significant to
obtain comparable Rf values.

As mentioned before, TLC is tool to identify

an unknown and determine the effectiveness of a
Figure 2. Structure of components of
purification. This tool can be used to analyze
analgesic drugs (Williamson and Masters, 2011)
different organic substances like lycopene and
analgesics. Moreover, as seen in Figure 2, components
of analgesic drugs contains different functional
Analgesics are substances found in drugs
groups that contribute to their polarity. The
that can relieve pain. Common pain relievers
difference in polarities is taken advantage in this
contain different analgesics like aspirin,
experiment to analyze the different components of
acetaminophen, and Ibuprofen. Analgesics often
analgesic drugs. The components of the unknown
have sedative properties, so caffeine was added to
will be identified through comparing the Rf values to
counter its effects. These components are bounded
standard Rf values of analgesics.
through a binder usually made of starch or silica gel
(Fieser and Williamson, 1992). In addition, natural organic products like
plant pigments can also be analyzed through TLC.
This experiment will analyze lycopene, a C 40-
carotenoid found in tomatoes, through extraction
from tomato paste and subjecting it to TLC
(Williamson and Masters, 2011). The components
and effectiveness of extraction can be determined
through measuring its Rf value.

Methodology

Thin-Layer chromatography involves 3

major steps, namely: sample preparation, TLC
plate spotting, solvent picking, and
developing.
To identify unknown components of an
analgesic drug, 1% 10mL solutions of standard
analgesics was prepared. 0.1 g of crushed Aspirin,
acetaminophen, ibuprofen, caffeine, and unknown
analgesic were dissolved separately in ethanol and
was placed in separate beakers. Ethanol was used
in order to dissolve the polar organic compounds,
and allow it to migrate with the solvent front. The
solutions were spotted 1cm from the bottom of a 6
cm x 10 cm TLC plate using a capillary tube (see
figure 3). The spotting of each analgesic was five
times with spots only having a diameter of 1-2 mm.
The solvent used for elution was made by
mixing 9.9 mL of ethyl acetate and 0.1 mL of
ethanol. This resulted to polar solution, but it is
significantly less polar than the stationary phase.
This solvent was used to allow the
components migrate. Enough solution
transferred to chamber allowing only less than 1 cm
of the TLC plate be submerged in the solution. The
chamber was covered, shaken, and allowed to
stand in order to saturate the atmosphere inside the
chamber. Saturation is important to prevent drying
of the solvent while rising through the TLC plate.
plate After preparation of the chamber, the TLC
plate was placed inside the chamber. The solvent
was allowed to rise in the TLC plate until it reached
1 cm from the top of the plate, and then was
removed and dried.
After drying, the plate was developed
through placing it in a beaker containing anhydrous
iodine pellets. The beaker was heated and the plate
was allowed to develop until all the spots were
visible. The distance travelled by the spots and
solvent were measured and the Rf values were
computed.
In the analysis of lycopene, the lycopene
was first extracted from the tomato paste. Five
grams of tomato paste placed in a 50-mL beaker
and was dissolved in 10 mL acetone and 10 mL
ethanol. The solution was heated for five minutes.
After heating, the sample was filtered, and it
polar
was ensured that all the filtrate is collected through
was
pressing the crude product. The filtrate was stored
in a 125-mL Erlenmeyer flask.
The crude product and 10 mL
dichloromethane were placed in a round-bottomed
flask and was then refluxed for 5 minutes. The
supernatant was collected. Reflux and collection of
supernatant was repeated for two more times.

All the filtrate was collected and placed in a

separatory funnel. The sample was extracted using
Unknown
Ibuprofen
Cafeine
Aspirin
Acetaminophen

sodium chloride solution. After shaking, the solution

was allowed to stand and separate into two layers.
The lower layer was collected.

One teaspoon of anhydrous Na2SO4 was

added and the solution was allowed to stand for
five minutes. After five minutes, the solution was

Figure 3. TLC setup

filtered. The filtrate was kept in a dark bottle to
preserve the color of the extracted lycopene.
Lycopene was isolated through TLC with the
same procedures used in TLC of analgesics;
however, the eluent used was a mixture of 8 mL
hexane and 2 mL acetone.
Learning.
Fieser, L. & Williamson, K. (1992). Organic
Experiments. (7th Ed., pp.117-131). MA,
Canada: D.C. Heath and Company.
Christian, G. D. (2004). Analytical chemistry (6th
ed.). United States of America: John Wiley &
Sons, Inc.

After developing the plate, the distance travelled by Williamson, K. L., & Masters, K. M. (2011).
the solvent and lycopene was measured and the Rf Macroscale and microscale organic
experiments (6th ed.). Belmont, CA:
value was computed. The computed Rf value was Brooks/Cole, Centrage Learning
compared to the theoretical Rf value of lycopene to Harris, D. C. (2007). Quantitative chemical analysis
(7th ed.). United States of America: W. H.
determine the effectiveness of extraction. Freeman and Company.

Gilbert, J. & Martin, S. (2011). Experimental Skoog, D. A., West, D. M., Holler, F. J., & Crouch,
Organic Chemistry: A Miniscale and S. R. (2014). Fundamentals of analytical
Microscale Experiment Approach chemistry (9th ed.). Belmont, CA: Brooks/Cole,
(pp.180-188). Boston: USA: Cengage Centrage Learning.