EXPERIMENT 2: THIN-LAYER CHROMATOGRAPHY: ANALYSIS

OF ANALGESIC DRUGS
Philip Gabriel Gimotea, Eden Cabana, Nia Dominguez
Locker No. 21
Chemistry Department, Xavier University Ateneo de Cagayan, Cagayan de Oro
City
Date Performed: November 20, 2014
Date Submitted: November 24. 2014

ABSTRACT
Chemical Analysis is a body of procedures and techniques used to identify
and quantify the chemical composition of a sample of a substance. A chemist
executing a qualitative analysis seeks to identify the substances in the sample. A
quantitative analysis is an attempt to determine the quantity or concentration of a
specific substance in the sample. The determination of the identity or quantity of a
constituent of such materials is preceded by a sampling step and by the separation
from the sample of either the desired constituent or the undesired, interfering
constituents. Most common technique in chemical analysis is thin-layer
chromatography. Thin Layer Chromatography or TLC is a technique used as a
separation and identification technique. There are many forms of chromatography,
but one thing that remains constant throughout all of the types of chromatography
is that there is a stationary phase, silica gel, and a mobile phase, ethyl acetate in
hexane. The mobile phase is the mixture that is being separated. It is a useful
application upon finding an unknown sample of a drug and of which drug its
components may be more likely close to. In this case, the sample to be proven is
Ponstan. After the development of the TLC plate, comparing of R f values of the
sample analgesic to those of the standard drug was done. From this information, the
determination and verification sample analgesics identity is possible but it is
generally an unreliable procedure for it shows unclear results and human error may
cause a huge difference.

INTRODUCTION
Chromatography is analytical technique used for the chemical separation of
mixtures and substances. The technique depends on the principle of selective
adsorption a type of adhesion. Chromatography is essentially a physical method of
separation in which components of a material are distributed between two phases.

One phase is stationary (stationary phase) while the other (the mobile phase)
percolates through it in a definite direction. As the mobile phase moves, the
components are displaced from the origin and separate from each other. A
distinction between chromatographic techniques can be made based on the nature
of the mobile and stationary phases.
Chromatography can be carried out in several ways. Thin layer
chromatography (TLC) is a chromatographic technique used to separate the
components of a mixture using a thin stationary phase supported by an inert
backing. It may be performed on the analytical scale as a means of monitoring the
progress of a reaction, or on the preparative scale to purify small amounts of a
compound. TLC is an analytical tool widely used because of its simplicity, relative
low cost, high sensitivity, and speed of separation. TLC functions on the same
principle as all chromatography: a compound will have different affinities for the
mobile and stationary phases, and this affects the speed at which it migrates. The
goal of TLC is to obtain well defined, well separated spots.
Chromatography is essential to the separation of pure substances from
complex mixtures and is widely used in the analysis of foods, drugs, blood,
petroleum products, and radioactive-fission products.
MATERIALS AND METHODS
The apparatuses used were capillary micropipette, TLC plate, beaker,
aluminium foil, watch glass, and iodine-vapor chamber while the chemicals used
were ethyl acetate (C4H8O2), hexane (C6H14), ibuprofen (C13H8O2), mefenamic acid
(C15H15NO2), acetaminophen (C8H9NO2), aspirin (C9H8O4) and a sample Ponstan.
There were two parts of the experiment the preparation of the sample and
the development of the chromatogram.
In preparing the sample, fill a capillary micropipette with the clear solution of
the analgesic drug sample. Apply the sample to the adsorbent side of the TLC plate
at about 1cm from the bottom edge by gently touching the tip of the capillary
micropipette to the plate. Remove the tip from the plate before the dot solute grows
more than 3 mm in diameter. Let the solute evaporate and reapply more sample to
exactly the same spot. Repeat the process to apply other analgesic drugs.
During the development of the chromatogram, place the thin-layer in a
beaker covered with aluminium foil containing a small amount of solvent ethyl
acetate in hexane. Place watch glass over the beaker. The TLC plate must be
positioned so that the spot of your sample is above the solvent. The elution solvent
climbs the plate by capillary action, eluting the sample up the plate. Stop by
removing the plate from the jar or beaker when the solvent front nears the top of
the TLC plate. Quickly mark the position of the solvent front on the plate. Place the

TLC plate in an iodine-vapor chamber for a minute or two. When dark spots appear,
mark them with a pencil, after removing the TLC plate from the iodine chamber.
Identify the spots in the chromatogram by calculating their R f (retention factor).

RESULTS AND DISCUSSION

Results:

Distance Travelled by the Compund (mm)

60
50
40
30
20
10
0

Calculations:

Drug

Mefenamic acid

Distance
Travelled by
Compound
40 mm

TLC Plate A
Distance
Travelled by
Solvent
Front
60 mm

Rf

Rf

Calculation

Rf =

40 mm
60 mm

0.67

Ponstan

45 mm

60 mm

Ibuprofen

50 mm

60 mm

Drug

Distance
Travelled by
Compound

Acetaminophen

32 mm

TLC Plate B
Distance
Travelled by
Solvent
Front
70 mm

Ponstan

50 mm

70 mm

Aspirin

52 mm

70 mm

Rf =

45 mm
60 mm

0.75

Rf =

50 mm
60 mm

0.83

Rf

Rf

Calculation

Rf =

32mm
70mm

0.46

Rf =

50mm
70mm

0.71

Rf =

52mm
70mm

0.74

Discussions:
Chromatography is the most generally applicable of the separation methods
and has many variants according to the nature of the column packing and the
sample-constituent interaction. One of the many variants of chromatography is thinlayer chromatography. TLC is carried out on glass plate coated on one side with a
thin layer of adsorbent. The adsorbent contains an amount of silica gel which acts
as a binder to give an adherent coating and as the stationary phase in the TLC.
To load the plate, very small samples of the analgesic drugs in some volatile
solvent, in the identity of a non-polar hexane and a polar ethyl acetate, are applied
as spots near one end and the solvent is allowed to evaporate. The plate is then
placed, sample end down, in a closed vessel containing a shallow pool of the
developing solvent. The solvent rises on the plate by capillary action, passing over
the sample and causing the compounds to move at varying rates depending on
their relative affinities for the adsorbent and solvent. As the solution percolated
through the column the individual components of the mixture migrated downward
at different rates of speed, so that the column became marked with horizontal
bands of colors, called a chromatogram. Each band corresponded to a different
pigment. When the solvent has risen to the top of the plate, the plate is removed
and the solvent is allowed to evaporate. The zones or spots containing then
detected at various points along the plate by treating the plate with a reagent,
iodine vapour, that causes the color to develop.

Upon the visualization of colors is obtained, measure the distance travelled

by the compound from the starting point of application. With the data obtained in
the experiment, it is possible to solve for the retention factor. Since some factors
like solvent system, adsorbent, and temperature are difficult to keep constant from
experiment to experiment, relative Rf values are generally considered. Relative R f
means that the values are reported to a standard, or it means that you compare the
Rf values of compounds run on the same plate at the same time. The larger an R f of
a compound, the larger the distance it travel on the TLC plate. The R f can provide
corroborative evidence a to the identity of a compound. If the identity of a
compound is suspected but not yet proven, an authentic sample of the compound,
or standard, is spotted and run on a TLC plate side by side with the compound on
question, like the Ponstan. If the two substances have the same R f, they are likely
but not necessarily the same compound. As for the sample, Ponstan, which is a
mefenamic acid, there is a great difference between R f values of the two. This might
be because of the TLCs limitations and disadvantages like TLC plates do not have
long stationary phases. Therefore, the length of separation is limited compared to
other chromatographic techniques. Also, the detection limit is a lot higher. If you
would need a lower detection limit, one would have to use other chromatographic
techniques. TLC operates as an open system, so factors such as humidity and
temperature can be consequences to the results of your chromatogram. There are
also problems like over-large spot which could cause overlapping of other
component spots with similar Rf values on your TLC plate and uneven advance
which could cause inaccurate Rf values due to the uneven advance of sample origin
spots.

CONCLUSION
Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two
phases are a solid, stationary phase and a liquid, mobile phase. The solid phase that
will be used in the lab is a glass plate covered with an adsorbent, in this case, silica
gel. Because silica is a polar molecule, the components of the solution use in the lab
will be separated based on their relative polarities. The more polar a molecule is,
the higher affinity it will have for the more polar silica plate and will therefore spend
less time in the mobile phase. As a result, it will move up the plate more slowly.
Conversely, a less polar molecule will spend more time in the mobile phase and will
therefore move up the plate more quickly. The speed at which the molecules will
move up the plate thus depends on the relative difference in polarity between the
stationary and mobile phases, and will vary depending on the nature of the
stationary and mobile phases used for separation. On the other hand, using TLC for
analysing and comparing analgesics is possible with the use of calculating the
retention factor. The sample, Ponstan, is near to the possible value of mefenamic

acid which is the generic name of the sample but its value is still different from each
other. Thus, it is not an ideal way for verifying and comparing the analgesics for a
more thorough study for it is subject to different errors and limitations.

REFERENCES
1.) Thin Layer Chromatography. (n.d.). Retrieved November 22, 2014, from
http://chemwiki.ucdavis.edu/Reference/Lab_Techniques/Thin_Layer_Chromato
graphy#Disadvantages_of_TLC
2.) Thin Layer Chromatography. (n.d.). Retrieved November 22, 2014, from
http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html
3.) Thin Layer Chromatography. (n.d.). Retrieved November 23, 2014, from
http://static.sewanee.edu/chem/Chem
%26Art/Detail_Pages/Projects_2005/Holladay/index.htm
4.) Microsoft Encarta 2008. 1993-2007 Microsoft Corporation. All rights
reserved.
Conclusion:
The major similarity between an aldehyde and a ketone is the carbonyl group.
A carbonyl group is a carbon atom doubly bonded to an oxygen atom. Both
molecules have a carbonyl group, the aldehyde and ketone differ in what
atom is bonded to the carbonyl carbon. The carbonyl carbon of an aldehyde
is bonded to two hydrogens or one hydrogen and one carbon. The carbonyl
carbon of a ketone is bonded to two carbons. Three test reagents will be used
in this experiment. Dinitrophenylhydrazine and Tollens reagent.
Dinitrophenylhydrazine, DNP for short, reacts with aldehydes and ketones to
form a light orange precipitate. Both chromic acid and Tollens reagent are
used to differentiate between aldehydes and ketones. Aldehydes may be
oxidized relatively easily, while ketones are not easily oxidized. The chromic
acid will change in color from orange to green when it is reduced by, in this
case by an aldehyde. The Tollens reagent, composed of a silver-ammonium
ion, will have its silver reduced by an aldehyde. This silver will plate a glass
surface creating a mirror.