TITLE : THIN LAYER CHROMATOGRAPHY

OBJECTIVES

1. Determine the Rf value.

2. To identify the components in the analgesic tablet by a TLC comparison 1 with standard
compounds.

INTRODUCTION

Chromatographic are used extensively in organic chemistry laboratories for routines analysis. Thin
layer chromotography (TLC) can be used to determine the purity of compound , to analyze the
composition of a mixture are differentiated by exposing to two competing phases , the stationary
phase is a polar adsorbent such as silica gel or alumina , which has been coated on plastic plate. The
mobile phase is an organic solvent .The solvent moves up the plate capillary action.

As the solvent moves past the spot that was applied an equilibrium was achieved for each
component of the mixture between the molecules of that component which are adsorbed on the
solid and the molecules which is are in solution . In principle , the components will differ in solubility
and in the strength of their adsorption to the adsorbent and some components will be carried
further up the plate than the others. When the solvent has reached on the top of the plate, the plate
will be removed from the solvent , it will dried and the developing components will be visualised .

UV lamp and iodine are used to visualized the components .In this experiment the TLC was used to
examine the composition of known analgesic which is the pain relieving drug such as paracetamol.
Several common analgesics are aspirin and acetomeniphen . Caffein is sometimes added to these
formulations to overcome drowsiness.

The distance travelled by each component is measured and this value is called the retardation factor
which is designated as Rf value.

Rf value for a component is calculated using the following expression :

Rf = Distance traveled by the component

Distance traveled by the solvent

PROCEDURE

Initial preparation was started with drew the line on the plate about 1 cm from the bottom and top of
the plate. Next, the scale was spotted every 1 cm interval. Thus, solution was spotted on reference
plate from left to right with a small spot each of them. The capillary differently used for every spot.
Then, the chamber was filled with 0.5% glacial acetic acid about 0.5-0.7 cm only below the spot.

For the development of the reference TLC plate was run with placing the spotted plate in chamber for
a few minutes. The plate removed when the solvent rise to a level 0.5 cm form top of the plate. Then,
the plate was observed under the ultraviolet for UV visualization. The outline observed spot was drew
on the plate. Thus, the distance from each spot travelled was measured.

Half of the analgesic tablet was crushed and transferred into a clean test tube. Besides, 15mL of
methlyne chloride and 15mL of the solvent was placed into the hot water bath. Next, the solution was
spotted on the sample plate and placed in the chamber in 0.5% glacial acetic acid. Lastly, from iodine
analysis, the plate was placed in the jar that containing iodine crystal. The cap was placed and it was
warmed on steam bath until the spot appeared.

RESULT

DEV.MIX (M) : ACETOMINOPHEN,ANALGESIC,CAFFEINE,ASPIRIN

DISTANCE SOLVENT(0.5% glacial acetate) TRAVELED : 4.3 CM

COMPOUND DISTANCE TRAVELED (CM) Rf value

MIX (M) 4.0 0.93
2.2 0.51
3.0 0.70
ACETOMINOPHEN (A) 4.2 0.97
ANALGESIC (B) 3.0 0.70
CAFFEINE (C) 2.2 0.51
ASPIRIN (D) 4.1 0.93

Calculation

Rf of M (MIXED) = 4.0 / 4.3 = 0.93 , 2.2 / 4.3 = 0.51 , 3.0 / 4.3 = 0.70

Rf of A (ACETOMINOPHEN) = 4.2 / 4.3 = 0.97

Rf of B (ANALGESIC) = 3.0 / 4.3 = 0.70

Rf of C (caffeine) = 2.2 / 4.3 = 0.51

Rf of D (ASPIRIN) = 4.1 / 0.43 = 0.93

The TLC reports that the drug containing is ANALGESIC,CAFFEINE and ASPIRIN
DISCUSSION

The unknown M compound was a mixture of ACETOMINOPHEN, ANALGESIC, CAFFEINE, ASPIRIN..

For this experiment, ACETOMINOPHEN, ANALGESIC, CAFFEINE, ASPIRIN which have different
polarity was mixtured. In the column chromatography, the separated mixture was collected. The
column chromatography allow the mixture to separate and being collected individually. Silica is used
as stationary phase in column chromatography. The least polar compound will elute from the
column first, then followed by each compound in order of increasing polarity. The yellow compound
is obviously than clear compound. This means that it must absorb more strongly to the silica gel than
the clear solution. The less polar spent most of its time in the solvent thus, washes through the
column much faster. Once the TLC plate finished developing, it was clear, visually the Rf values were
calculated for each compound using the equation

Rf =distance from baseline traveled by solute

distance from baseline traveled by solvent.

As shown in the above tables, the Rf values of the unknown M (0.93),(0.51),(0.70) came perfectly
with the Rf values of the ANALGESIC (A) (0.70) , ASPIRIN (D) (0.93) , CAFFEINE (C) (0.51).

The more polar the compound is, the slower it moves across the plate and the lower the Rf value will
be.

QUESTIONS

1. What happens if the spots are made too large when preparing a TLC plate for
development?

If the spot too large it may cause the shape of the final spot become skewed and elongates.

2. What happens if the spots are made too small when preparing a TLC plate for
development?

If the spots are too small the Rf value are hard to determined.

3. Why must the spots be above the level of the developing solvent in the development
chamber ?
Because the spot will move upward from the solvent level and carry the components of the spot
at constant rate to the top but if the spot is below the solvent level , then the solvent will wash
away the spot into the solvent and there will no development.

4. What would happen if the spotting line and positions marked on the plate with a ball-
point pen?
This will make the ink of the ball pen will contaminated the result , the ink will move with the
spot and we cannot get the correct result.

5. Is it possible to distinguish two spots that have the same Rf value but represent different
compounds? Give two different methods.
it is always possible if you can run another experiment: you may be able to resolve them using
another solvent or type of TLC plate often, "mixed spot" technique is useful or you can use
different method entirely and use infrared or MS.
 6. Name some advantages of using a acetaminophen (Tylenol) instead of aspirin as an
analgesic.
One of the advantages is that acetaminophen is less of an irritant to the stomach than aspirin. If
you want more detail then the main undesirable side effects of aspirin are gastrointestinal
ulcers, stomach bleeding, and tinnitus, especially in higher doses. Acetaminophen also has an
advantage in that it can be given at the same time as a number of other medications.

CONCLUSION
The Rf value was determined which is Rf value for aspirin is 0.93, analgesic is 0.70 , caffein is 0.51 and
acetaminophen is 0.97. From the calculation the compound in the TLC tablet trial is ANALGESIC,CAFFEINE
and ASPIRIN.

REFERENCES

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http://www.hbcpnetbase.com.proxyau.wrlc.org/

2. Williamson, K; Milnard, D; Masters, K.Column Chromatography. Macroscale and Microscale

Organic Experiments. Houghton Mifflin Company: Boston, NY, 2007; 184-200

3. Xiong Cariao; Zhou Xiayo, et al. Characteristics of column packing materials in high performance
liquid chromatography by charge detection. Analytical Chemistry 2011 [online]. 83, 13, 5400-5406.
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