Chemical Engineering Laboratory-1 

 
(CHE F312) 
 
Lab Report 
 
Engineering Chemistry Lab 
 
E-1 
 
Thin Layer Chromatography

Group 14
Vedant Desai
Pratyush Srivastava
Hritik Vinayak Lal
Apoorv Kaushik
Index Page

Exp.No Name of the Date Date of Instructor’s Sign Marks

Experiment of Submiss
Condu ion
ction

E2 Surface 15/09/ 20/09/20

Tension 20

E3 Colligative 17/09/ 22/09/20

Properties 20

E4 Titrations 22/09/ 24/09/20

20

E5 Organic 24/09/ 29/09/20

Synthesis 20

E6 Saponification 29/09/ 1/10/20

20

E1 Thin 1/10/2 6/10/20

Layer 0
Chromatograp
hy
OBJECTIVE:
To determine the composition of given over-the-counter analgesics (pain
medications) and to select the solvent best suitable for given components using thin
layer chromatography method.

THEORY:

TLC is one of the fastest and least expensive out of several chromatographic
techniques used. It is a separation process which is based on the principle of
differential adsorption, i.e. the difference in interaction of molecules i.e Basically the
relative polarities to an inert solid dipped in an organic solvent or solvent mixture.
The stationary phase used in this case is silica gel or alumina coated on a glass
plate.

The components are distributed among the stationary phase and the solvent
depending upon the relative polarities of the compound and the solvent. TLC takes
advantage of the fact that different compounds will have different solubility, different
extent of adsorption and different polarities as compared to each other. Hence, these
compounds travel at different speeds and hence cover different distances on the
plate.

Polar compounds are strongly attracted to the polar adsorbent while nonpolar
compounds are held weakly as compared to polar. When a nonpolar solvent is
passed through the adsorbent, nonpolar compounds are released easily whereas
because polar adsorbents have high affinity for polar compounds, they are retained.
Whereas when a moderately polar solvent is passed through the adsorbent, both
polar and nonpolar compounds are discharged but since there is still an attraction
between the polar compounds and polar adsorbent,the non polar counterpart moves
faster on the plate.

The distance travelled by each component is expressed in the terms of retardation

factor Rf or rate factor and it is calculated by the following formula :

In this experiment we use TLC to identify the components of an over the counter
analgesic.
APPARATUS REQUIRED: ​TLC
chamber ,TLC plates ,Micro
Capillaries,Ruler ,Test Tubes ,Spatula, Small Beaker ,Pencil

CHEMICALS REQUIRED :​Acetaminophen,Aspirin,Caffeine ,Ibuprofen,Standard

reference mixture ,Analgesics Tablet,99/1 mixture of ethyl acetate/glacial acetic acid
,50/50 ethanol/ethyl acetate solution

OBSERVATION:

S.No Distance Distance travelled by X​avg ​(cm) R​f

Travelled solute
by Solvent

(cm) X​1​(cm) X​2​(cm)

1. 3.5 2.2 2.3 2.25 0.642

2. 3.6 2.1 2.3 2.2 0.611

3. 3.5 2.1 2.2 2.15 0.614

Avg - - - 0.622

R​f, Avg​ =
​ (0.642+ 0.611 + 0.611)/3 = 0.62233
DISCUSSION:

PRATYUSH :

1.​Chromatography is an analytical technique commonly used for ​separating a

mixture of chemical substances into its individual components, so that the individual
components can be thoroughly analyzed.

2.There are four main types of chromatography:

●Liquid Chromatography
●Gas Chromatography
● Thin-Layer Chromatography
● Paper Chromatography

3.Thin Layer Chromatography is a useful method of quickly and efficiently analyzing

the main active ingredients of certain commercial analgesics which contain aspirin,
caffeine, acetaminophen, and ibuprofen etc. when they don’t have many other
organic compounds in significant proportions.

4.TLC is basically a chromatography technique used to separate ​non-volatile or

moderately volatile mixtures.It can be used to analyze the reaction, identify the
compounds present in a given mixture, and determine its purity.

5.In TLC, we use a ​stationary phase (in this case silica gel) which is deposited over
a glass or aluminium support.Here, 99/1 mixture of ethyl acetate/glacial acetic acid
was used as the ​mobile phase​.The solution used to dissolve the analgesic here is
50/50 mixture of Ethanol/Ethyl acetate

6.​When we dip the TLC plate in an organic solvent, different compounds will move
upwards at different rates due to capillary action ,according to their relative polarities
and concentrations, allowing the separation of different components.

7.TLC works on the differential adsorption of compounds on the absorbent and the
polarity of the solvent used. The relative polarity of the compounds plays an
essential role in the mobility of compounds in the solvent (especially if it is nonpolar).
The compounds travel different distances on the plate; these distances are marked
and the Rf values are calculated and compared with the standard literature values to
identify the compounds.Due to different polarities, the binding forces result in
accumulation of non polar materials upwards from the origin point on the sheet.

8. ​Rf ​= distance travelled by the substance / distance travelled by the solvent

From literature we found the various Rf values

Aspirin = 0.45
Caffeine = 0.08
​Acetaminophen = 0.24
Ibuprofen = 0.60

9.​Applications​ of TLC:

1.TLC method is used for separation and identification of colors,

preservatives, sweetening agents, and various other cosmetic products.

2.TLC is used in separation of multicomponent pharmaceutical formulations.

3.It is being used in chemistry for the separation and identification of compounds that
resemble each other to a large extent.

4.It is also used for identification of cations and anions in inorganic chemistry.

5.TLC is also useful in separation of biochemical constituents from its body fluids,
blood plasma,urine etc

10.​Limitations​ of TLC:

1.It fails to operate as efficiently as some other chromatographic techniques like

paper chromatography or liquid chromatography to detect impurities in lake water
etc.

2.One of its limitations is that it cannot tell the difference between enantiomers and
isomers of some compounds.

3.Another disadvantage of TLC is that to identify the compounds, the Rf values for
them must be known beforehand.

4.This technique of chromatography is only applicable for non-volatile or moderately

volatile substances and also its results are very difficult to reproduce and reanalyze
VEDANT:

1. Chromatography is a seperation technique based on adsorption, this method

enables the separation, identification, and purification of the components of a
mixture, which helps in qualitative analysis of a component/mixture.
2. The 3 main branches of chromatography based on the phase of mobile fluid
are:
a. Gas Chromatography
b. Liquid Chromatography
c. Supercritical Fluid Chromatography
3. These branches can be further divided into many types of chromatography.
Liquid chromatography can be divided into 4 main types:
a. Ion Exchange - Aqueous medium( Water Based)
b. Exclusion - Aqueous medium (Water Based)
c. Partition- Include Paper Chromatography. ( Organic Solvent)
d. Liquid solid Absorption - Include Thin Layer Chromatography (Organic
Solvent)
4. TLC is one of the easiest and fastest of several chromatographic techniques.
The mobile phase is an organic solvent and the stationary phase is an
alumina plate coated with an even & uniform layer of silica. The separation is
based on differential adsorption. It is used to test purity of a compound among
mixture. In general non-polar molecules move higher than the polar molecules
due to strong bonding of molecules with stationary phase.
5. The retardation factor is calculated which is ratio of:
Distance travelled by compound/ The Distance Travelled by Solvent.

The unknown compound here is identified as ​ibuprofen whose experimental

value of Retardation factor is ​0.622 which is closest to its literature value of
0.6.
R​f​ values for other compounds from referring to literature are:
Compound R​f

Aspirin 0.45

Caffeine 0.08

Acetaminophen 0.24
6. There is possibility of ​many errors and problems ​as well during TLC such
as:
a. Non uniform thickness of silica layer on plate can cause significant
amount of errors. Ideal thickness should be around 0.2mm.
b. Streaking: If the sample spot is too concentrated, the substance will
travel up the stationary phase as a streak rather than a single
separated spot.
c. Spotting: the sample spot will be washed off into the solvent before it
travels up the TLC plate if the sample is not above the solvent level.
7. Applications Of TLC :
● Purity of sample can be carried out with TLC. Direct comparison is
done between the sample and the standard or authentic sample; if any
impurity is detected, then it shows extra spots and this can be detected
easily.
● Thin layer chromatography can be employed in purification, isolation
and identification of natural products like volatile oil or essential oil,
fixed oil, waxes, alkaloids, glycosides, steroids etc.
● In food and cosmetic industry: TLC method is used for separation and
identification of colors, preservatives, sweetening agents, and various
cosmetic products.
● TLC is used in separation of multicomponent pharmaceutical drugs and
detection of its impurities.
● Analyzing the dye composition of fibers in forensics.
8. Disadvantages
a. Only qualitative analysis is possible, not quantitative.
b. Stationary phase layer thickness cannot always be maintained
uniformly all over the plate while preparing it in lab
c. It cannot tell the difference between enantiomers and some isomers.
d. Since TLC operates as an open system, factors such as humidity and
temperature can affect results.
e. It is difficult to replicate the results precisely in the experiment as we
get slightly different values every time.
HRITIK:

1. Chromatography is a laboratory technique for the separation of a mixture. The

mixture is dissolved in a fluid (gas, solvent, water etc.) called the mobile
phase, which carries it through a system (a column, a capillary tube, a plate,
or a sheet) on which is fixed a material called the stationary phase.
2. There are four main types of chromatography:
●Liquid Chromatography
●Gas Chromatography
● Thin-Layer Chromatography
● Paper Chromatography
3. Thin-layer chromatography (TLC) is a ​chromatography technique used to
separate non-volatile mixtures.Thin-layer chromatography is performed on a
sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of
adsorbent material, usually ​silica gel​, ​aluminium oxide (alumina), or ​cellulose​.
This layer of adsorbent is known as the ​stationary phase​.
4. These branches can be further subdivided into various types of
chromatography.
a. Ion Exchange
b. Exclusion
c. Partition
d. Liquid solid Absorption
5. Different ​compounds in the sample mixture travel at different rates due to the
differences in their attraction to the stationary phase and because of
differences in solubility in the solvent. By changing the solvent, or perhaps
using a mixture, the separation of components (measured by the ​Rf​ value)
can be adjusted. Also, the separation achieved with a TLC plate can be used
to estimate the separation of a ​flash chromatography​ column.
6. Separation of compounds is based on the competition of the solute and the
mobile phase for binding sites on the stationary phase.For instance, if
normal-phase silica gel is used as the stationary phase, it can be considered
polar. Given two compounds that differ in polarity, the more polar compound
has a stronger interaction with the silica and is, therefore, better able to
displace the mobile phase from the available binding sites.As a consequence,
the less polar compound moves higher up the plate (resulting in a higher ​R​f
value)
7. Distance travelled by the component (ibuprofen) was calculated by analysing
the chromatogram under UV light and value of retardation factor was
calculated.
8. R values of various given compounds: Aspirin - 0.45, Caffeine - 0.08,
Acetaminophen - 0.24, Ibuprofen - 0.60

9. Applications Of TLC :

Purity of any sample:​ Purity of sample can be carried out with TLC. Direct
comparison is done between the sample and the standard or authentic
sample; if any impurity is detected, then it shows extra spots and this can be
detected easily.

Identification of compounds​:​ Thin layer chromatography can be employed

in purification, isolation and identification of natural products like volatile oil or
essential oil, fixed oil, waxes, alkaloids, glycosides, steroids etc.

Examination of reactions​:​ Reaction mixture can be examined by Thin layer

chromatography to access whether the reaction is complete or not. This
method is also used in checking other separation processes and purification
processes like distillation, molecular distillation etc.

Biochemical analysis:​ ​ TLC is extremely useful in isolation or separation of

biochemical metabolites or constituents from its body fluids, blood plasma,
serum, urine etc.

In chemistry:​ TLC methodology is increasingly used in chemistry for the

separation and identification of compounds which are closely related to each
other. It is also used for identification of cations and anions in inorganic
chemistry.

10. Limitations Of TLC

● ​One of its limitations is that TLC cannot tell the difference between
isomeric and enantiomeric forms of a compound​. This is because the
chiral pairs of a molecule​ exhibit the same physical properties.

●Stationary phase layer thickness cannot be maintained uniformly all over the
plate.

●Since TLC operates as an open system, factors such as humidity and

temperature can affect results.

●Only qualitative analysis is possible, not quantitative analysis.

11. Disadvantages of Thin Layer Chromatography:

● Its separation length is generally insufficient in comparison to other
chromatographic techniques.
 ● Results obtained from TLC are difficult to reproduce.
● Only soluble components of the mixtures are possible.
12. Wavelength of UV rays used in this experiment was 254 nm.

APOORV:

Thin layer Chromatography is a lab technique used to identify different compounds

that are present in a mixture. It is useful for solid-liquid separation.There are 4 main
types of chromatography, 1) liquid chromatography, 2) gas chromatography, 3) thin
layer chromatography, 4) paper chromatography. The stationary phase is Polar and
the mobile phase is non polar. The TLC is a filter paper coated with a solvent. A
small amount of solid is placed near the bottom of the plate and the plate is placed in
the solvent developing chamber. As the solvent passes to the spots, equilibrium is
obtained for each component. Some components are carried further than others.
The separation components are viewed under UV light where we can see how far
each component has moved. Weak polar molecules and nonpolar molecules move
more quickly through the plate than the polar molecules. In the industry it can be
used in production for Quality Assurance. It is popular because it is inexpensive and
can be completed within a short period of time. It can also be used to monitor the
progress of the reaction by monitoring the disappearance and appearance of certain
compounds. It's done in 3 steps spotting, development and visualisation. Finally, Rf
Is calculated to quantify movement of materials. In the food and cosmetic industry
thin layer chromatography is used for separation and identification of colours,
preservatives, sweetening agents and various other cosmetic products.
TLC has various applications like determining purity of a sample, identifying
composition of compounds, in the pharmaceutical industry, etc.
The ​advantages​ of TLC are-

1. rapid analysis time because many samples can be analyzed simultaneously.

2. low solvent usage on a per-sample basis.
3. A high degree of accuracy and precision for instrumental TLC.
4. sensitivity in the nanogram or picogram range.
Precautions​-
1. Sample should be dissolved in a nonpolar solvent as polar solvent has a
tendency to spread out the starting spot.
2. Solvent used for dissolving sample should be volatile.
3. While applying sample, the surface of the adsorbent should not be disturbed
as this distorts the shapes of the spots on subsequent developed
chromatogram, hindering the accuracy of quantitative measurements.
4. The sample spot should be within 2-5 mm in diameter.
5. Use pencil to make marks and not pen
CONCLUSION:

The average ​Rf​ ​ v​ alue of the compound on which the TLC experiment was conducted
was found to be 0.622. When we compare this to the standard literature values
of the four substances present which are:
1.Aspirin - 0.45
2​.Caffeine - 0.08
3.Acetominphen - 0.24
4.Ibuprofen - 0.60

We can conclude that the substance on which the experiment was conducted
was ​Ibuprofen​,because the obtained ​Rf​ v​ alue i.e 0.622 is closest to the ​R​f
value of Ibuprofen i.e 0.60.

REFERENCES :

● https://chrominfo.blogspot.com/2018/10/advantages-and-disadvantages-
of-thin.html#:~:text=1%20There%20is%20a%20no%20longer%20stationa
ry%20are,Usually%2C%20it%20is%20not%20automatic.%20More%20ite
ms...%20
● https://en.wikipedia.org/wiki/Thin-layer_chromatography#:~:text=Thin-la
yer%20chromatography%20%28TLC%29%20is%20a%20chromatograph
y%20technique%20used,usually%20silica%20gel%2C%20aluminium%20
oxide%20%28alumina%29%2C%20or%20cellulose.