Thin
Layer Chromatography: An Analysis of Analgesics
Student’s
Name
Institutional
Affiliation
Date
Table of Contents
Analysis
of Analgesics in a Compound Using TLC
Purpose
The main objective of
this laboratory report is to identify analgesics in a compound containing a
mixture of known analgesics by separating the mixtures using Thin Layer
Chromatography (TLC) and determining the individual Rf values of the
separated mixtures. The individual Rf values of the mixtures are
then compared with the Rf values of known analgesics. Besides, the industrial
purpose of the laboratory report is to understand the application of the
procedure in the different contemporary fields of professions such as colour
separation in textile industries and forensic criminal investigations.
Introduction
Chromatography is
routinely used in organic chemistry laboratories as a technique of separation
of molecules and subsequent analysis. For instance, TLC is usually used to find
the degree of purity of molecules or compounds and to analyze the components of
a compound or mixture through exposing them to both stationary (Mohrig et al.
2014). In principle, the identity of molecules is made possible due to the different
solubility capacity of molecules and their strength of attraction to the
stationary phase.
In this laboratory experiment, TLC was applied
in the determination of unknown components in a mixture by comparing their Rf
values with known analgesic compounds. The analgesics used in this
experiment were; Acetaminophen, Asprin, Caffeine, Phenacetin, and salicylamide
which are compounds used for pain-relieving processes. This TLC technique is
applied in several professional fields of study. For instance, forensic
detectives use TLC in matching known illegal drugs with blood samples of
criminal suspects.
Procedure
Preparation of TLC Plastic Plate
Using a clean pair of
forceps, two separate TLC plates (Silica gel) were placed on a clean surface
and marked using a pencil as TLC plate 1 and TLC plate 2. Using a clear pencil
marker, baseline and finishing line were drawn 1cm from each edge of both
bottom and top of the TLC plates (Silica gel) respectively. On the first plate,
spots were drawn and labeled as Ace, Asp, Caf, and Unkn for the first plate
representing Acetaminophen, Asprin, Caffeine and Unknown mixture. On the second
plate, spots were drawn and labeled as Phen, Sal and Unkn to represent
Phenacetin, Salycylamide and unknown mixture. Simultaneously, with intermittent
cleaning of TLC capillary tube, 0.25ml of each molecule was measured and transferred
to its respective spots and the resulting TLC plates transferred to the developing
chamber using clean forceps.
Developing Chamber
Before the transfer of
the TLC Silica gel plates using clean forceps into the developing chambers, the
ethyl acetate solvent was poured into the chamber at the level of 0.8cm just
below the 1cm mark measured using a ruler. The reason for this was to reduce
the chances of diffusion of the molecule with solvent, creating an unmeasurable
movement due to scattering of diffused molecules. The developing chamber was enclosed
with a clear watch glass to allow the environment within the chamber to be
saturated. The experiment was then left to run and after 25 minutes, the plates
were removed using forceps for visualization. This time was used to ensure that
the solvent had traveled the furthest distance to TLC plates
Visualization
The plates could be seen
immediately using UV light and each spot from both plates circled using a
pencil to avoid fading of the colours due to UV light fluorescence of the
spots.
Analysis
The distance in
millimeters (mm) from the baseline to the center of each circle for each
molecule movement was measured using a ruler and recorded. Additionally, the
distance of the movement of the solvent was measured known as the solvent front
as shown in table 1.
|
Molecule
|
Distance
Travelled (mm)
|
|
Acetaminophen
|
30
|
|
Asprin
|
45
|
|
Caffeine
|
17
|
|
Phenacetin
|
40
|
|
Salicylamide
|
46
|
|
Unknown spot 1(Both
plates)
|
17
|
|
Unknown spot 2 (Both
plates)
|
30
|
|
Unknown spot 3 (Both
plates)
|
45
|
|
Solvent
|
65
|
Table
1.
Distance covered by each molecule on the First and Second TLC plates from the
baseline to the middle of the spots circled.
Results and Discussion
The results
recorded were used to calculate individual Rf values. In TLC plate
analysis, Rf value could be
determined by identifying the ratio spot to solvent travel (Balshan, 2017;
Singh et al. 2016). The formula is as represented below.
|
Molecule/Spot
|
Rf
Values
|
|
Acetaminophen
|
0.46154
|
|
Asprin
|
0.69230
|
|
Caffeine
|
0.26154
|
|
Phenacetin
|
0.61538
|
|
Salicylamide
|
0.70769
|
|
Unknown spot 1(Both
plates)
|
0.26154
|
|
Unknown spot 2 (Both
plates)
|
0.46154
|
|
Unknown spot 3 (Both
plates)
|
0.69230
|
|
Solvent
|
N/A
|
Table 2:
Rf values of different
molecules/spots as recorded from TLC plates in the experiment.
This
technique that uses the concepts of stationary and mobile phase’s components
based on polarity differences to separate and identify mixture in compounds is
known as chromatography. A good example used in this experiment is TLC. It is
essential in the separation and identification of unknown molecules in a
mixture. More polar compounds have strong intermolecular forces resulting in
strong affinity to the polar phase of TLC thus will move a shorter distance up
the plate.
From the above
tabulations of the Rf calculations, the unknown mixture spots had Rf values of
0.26154, 0.46154, and 0.69230 correspondings to the Rf values of
Caffeine molecule (0.26154), Acetaminophen molecule (0.46154), and Asprin
molecule (0.69230). This indicates that the constituents of the mixture were Caffeine,
Asprin, and Acetaminophen since they moved an equal distance up the TLC Silica
gel plate under the influence of the mobile solvent phase. Besides, they had
same Rf values after corresponding calculations. The unknown mixture was also
able to separate into 3 spots due to different levels of intermolecular forces
of attraction holding the molecules together and the different level of
solubility in the mobile phase (DCM). The stationary phase containing silica
gel has a hydroxyl group in its structure making a stronger polar compound
(Mohrig et al, 2014). Conversely, ethyl acetate is the mobile phase since it is
a less polar molecule compared to polar silica gel resulting in movement up the
TLC plate noted by the higher solvent front. In this case, its affinity for
polar compound (Silica gel) is lower. However, using a more non-polar solvent
such as 100% hexane reducing the affinity of other less polar compounds in the
mixture to it and hence spots/molecules staying closer to the origin/baseline.
This separation based on the affinity or polarity can be understood by the
structure of the molecules shown below.
List of Figures
Figure 1: Acetaminophen Figure 2: Asprin
Figure 3: Caffeine Figure 4: Phenacetin
Figure
5:
Salicylamide
The structure of the TLC plate is a
polar SiO2 making any molecule that is more polar to move a less
distance up the plate due to attraction/affinity due to the same polarity
strengths (Filipic et al, 2016). From our experiment, salicylamide moved the
furthest up the TLC plate because of less affinity to the stationary phase.
Salicylamide structure, Figure 5, indicates that the molecule is made up of a
carboxyl functional group apart from the hydroxyl group creating weak
intermolecular forces. As a result, it becomes less polar and traveling further
the TLC plate hence a larger Rf value of 0.70769.
Figure 2 indicates the structure of
Asprin. The structure has both a carboxyl group and ester functional groups
which are similarly attractive to the stationary phase making it travel up the
TLC plate further. However, esters are relatively polar making it travel
slightly a lesser distance than Salicylamide, The result is an Rf
value of 0.69230. Figures 1 and 4 are structures of Acetaminophen and
phenacetin. Looking closely, the hydroxyl group in Acetaminophen is replaced by
an ethoxide. Therefore, since Acetaminophen structure has a strong hydrogen
bond with SiO2 than phenacetin, creating a high affinity to the TLC plate than
phenacetin. This makes it to move a lesser distance than phenacetin and Asprin
on the TLC Silica gel plate and hence an Rf value of 0.46154 compared to Rf
value of 0.61538. However, Caffeine traveled the list distance of the molecules
under study. Based on Figure 3, the structure of caffeine is made up of several
amine functional groups with numerous oxygen and Nitrogen lone pairs reactively
strongly to the SiO2 of the stationary phase (Maia, Batista, &
Fernandes, 2017). This makes it travel the list and hence an Rf value of
0.26154.
Conclusion
In summary, the
main objective of this laboratory experiment was to identify the analgesics
present in the unknown mixture using TLC. Acetaminophen, Asprin, Caffeine and
the unknown mixture were placed using the capillarity tube on the spots drawn
using pencil on the first TLC plate and the same was done on the second plate
with Phenacetin, Salycylamide and two unknown mixtures. The resulting
preparations were then developed and visualized under Uv light. The resulting
spotted were used to find the Rf value. The results indicated that the unknown
mixture contained Caffeine, Acetaminophen, and Asprin with Rf values of
0.26154, 0.46154, and 0.696230 correspondingly. Besides, the Rf values obtained
for each molecule were theoretically close to the standard values, signifying
that the experiment was conducted successfully with minimal experimental
errors. This technique is an important procedure as it is applied in the field
of forensic criminal investigation and in separation of colours in textile industries.
References
Balshan, Y., 2017. Performing 1D Thin Layer
Chromatography | Protocol. Retrived from
https://www.jove.com/science-education/5499
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Maia, J. C., Batista, F. S., &
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