Date:

Experiment No. 1
Name of The Experiment : TLC plate preparation and adjustment of solvent system.
Basic Principle :
Chromatography: Chromatography is a separation method that exploits the
differences in partitioning behavior between mobile phase and a stationary phase to
separate the components in a mixture. Components of a mixture may be interacting
with the stationary phase based on charge, relative solubility or adsorption.
Classification of Chromatography:
Techniques by chromatographic bed shape
1.

Column chromatography.

2.

Paper chromatography.

3.

Thin Layer chromatography.

Techniques by physical state of mobile phase

1.

Gas chromatography.

2.

Liquid chromatography.

3.

Affinity chromatography.

Techniques by separation mechanism

1.

Ion exchange chromatography.

2.

Size exclusion chromatography.

Thin Layer chromatography :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, or cellulose. This
layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as
the mobile phase) is drawn up the plate via capillary action. Because different
analytes ascend the TLC plate at different rates, separation is achieved.

Purpose :
1. To check purity of given samples.
2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines,
antibiotics etc.
3. To evaluate reaction process by assessment of intermediates, reaction course etc.
4. To purify samples i.e for purification process.
5. To keep a check on the performance of other separation processes.

Aparatus :

Capillary/ Spotter/ Pastor pipette.

Aluminium plate / TLC plate.

A jar with a lid.

Pencil.

Scale.

UV lamp.

Chemical Reagents :

Ethyl acetate.

Toluene.

Vanillin spray (containing vanillin in concentrated sulfuric acid)

Material &Methods :
For Preparing Solvents :
%

Methanol(ml)

Chloroform(ml)

19

10

18

15

17

For Preparing TLC Plate :

A.
At first we prepare TLC plates measuring 20cm x5cm sheets. Each large sheet
is cut horizontally into plates which are 5 cm tall by various widths. Handle the plates
carefully so that do not disturb the coating of adsorbent or get them dirty.
B.
Using a pencil, draw a line across the plate at the 0.5 cm mark.This is the
origin: the line on which we will spot the plate. We will take care not to press so hard
with the pencil that doesnt disturb the adsorbent. Under the line, mark lightly the
name of the samples we will spot on the plate, or mark numbers for time points.
Leave enough space between the samples so that they do not run together.
Procedure :
1)
At first desired solvent (15%MC) system was prepared by combining two
solvents at correct proportion.
2)
Then TLC plate was taken and spot the samples (which samples to be
analyzed)into the dot line with the help of spotter.( The sample was picked up just by
touching sample solution by the spotter).
3)
Don't allow the spot to become too large - if necessary, we can touch it to the
plate, lift it off and blow on the spot. If we repeat these steps, the wet area on the plate
will stay small.
4)
The plate was then placed in the jar and the liquid mobile phase was allowed
to phase through the plate which carried the compounds of the sample along with.
5)
Let the mobile phase run until it reached to dot line of the upper part of the
plate.
6)
Once the running completed the plate was removed from the jar and it was
allowed to air dried.
7)
When the plate had air dried there are any colored spots, circle them lightly
with a pencil.
8)
Most samples are not colored and need to be visualized with a UV lamp. Hold
a UV lamp over the plate and circle any spots we see.

9)
If samples color are not visualized by UV lamp or we also want to show the
clear spots then we also sprayed by vanillin spray and burnt at oven.
10)
Finally, the spot of the plate was observed for the determination of purity of
compound.

Result:
As there were more spots appeared in a vertical series on the plate so the compound
present in the sample solution was impure .

Precaution :

TLC plate should be handled carefully.

The jar should be cleaned and preferably with alcohol.

The solvent system in the jar must remain below the spot placed.

Solvents should be handled and measured carefully.

Vanillin spray must be used carefull.

Date :
Experiment No. 02
Name of the experiment : Identification of existing drugs by TLC (Caffeine, Aspirin,
Ciprofloxacin ).
Basic Principle :
Thin layer chromatography (TLC) is a method for identification substances and
testing the purity of compounds. TLC is a useful technique because it is relatively
quick and requires small quantities of materials.
Separations in TLC involve distribution a mixture of two or more substances between
a stationary phase and a mobile phase. The stationary phase is a thin layer of
adsorbent coated on a plate. The mobile phase is a developing liquid which travels up
the stationary phase ,carrying the samples with it.
Thin layer chromatography is an extremely useful technique for monitoring reactions.
It is also used to determined the proper solvent system for preforming separation
using column chromatography . TLC use a stationary phase, usually alumina or silica,
that is highly polar or non-polar and a mobile phase, some solvent ehose polarity we
will close. When we need to determine the best solvent or mixture of solvents to
develop a TLC plate or chromatography column loaded with an unknown mixture,
vary the polarity of the solvent in several trial runs: a process of trial and error. Ehen
we will apply our reactions mixture in solution to the plat then run the place by
allowing a solvent to move up the plate by capillary action. More polar compounds
will stick to the polar silica gel and travel short distance on the plate. The measure
of the distance a compounds travels in called Rf. Thus number, between zero and one,
is determined by measuring the distance the compound moved from baseline divided
by the distance the solvent moved form the baseline.
The Rf Value
The retention factory, or Rf, is define as the distance traveled by the compound
divided by the distance traveled by the solvent.
Rf= distance traveled by the compound/ distance traveled by the solvent front.

Rf value is a characteristics of a compounds in a specific solvent system. It helps in

the identification of the component.
The Rf for a compound is a constant from one experiment to the next only if the
chromatography conditions below are also constant:
.- Solvent system
- Absorbent
- Thickness of the adsorbent
. Amount of the material spotted
. temperature
Purpose :
The purpose of the experiment is to identify an unknown proprietary drug using thin
layer chromatography. The unknowns behavior in thin layer chromatography will be
comparer with that of its possible component analgesics. The possible unknown and
their analgesic ingredients will be Ciprofloxacin, Caffeine, Aspirin.
Apparatus :
. Capillary/spotter/pastor plate
. Aluminium plate/TLC plate
. A jar with a lid
. Pencil
. Scale
. UV lamp
Chemical Reagents :
. Methanol
. Chloroform
. Ciprofloxacin
. Caffeine
. Aspirin
. Vanillin spray
Material & Methods :

15% MC system : Take 3ml of menthol into the jar and make up the volume by 17ml
chloroform.
For preparing TLC plate :
A )At first we prepare TLC plates measuring 20cm5cm sheets. Each large sheet is
cut horizontally into plates which are 5cm tall by various widths. Handle the plates
carefully so that do not disturb the coating of adsorbent or get them dirty.
B) Using a pencil , draw a line across the plate at the0.5cm mark. We will take
care not to press so hard with the pencil that doesnt disturb the adsorbent.
Under the line, make lightly the name of the samples we will spot on the plate,
or make number for time points. Leave enough space between the samples so
that they do not run together.
Procedure :
1-At first desired solvent (10%) system was prepared by combining two
solvent at correct proportion.
2-Then TLC plate was taken and spot the samples into the dot line with the
help of spotter.
3-Dont allow the spot to become too large- if necessary, we can touch it to
the plate, lift it off and blow on the spot. If we repeat these steps, the wet area on the
plate will stay small.
4-The plate was then placed in the jar and the liquid mobile phase was allow
to phase through the plate which carried the compounds of the sample along with.
5-Let the mobile phase run until it reached to dot line of the upper part of the
plate.
6-Once the running completed the plate was removed from the jar and it was
allowed to air dried.
7-When the plate had air dried there are any colored spots, circle them lightly
with a pencil.
8-Most samples are not colored and need to be visualized with a UV lamp.
Hold a UV lamp over the plate and spots we see.
9-If sample are not visualized by UV lamp or we also want to show the clear
spots then we also sprayed by vanilline spray and burnt at over.
10-Finally, the spot of the plate was observed for the determination of compound.
Results :

As there were more spots appeared in a vertical series on the plate so the compound
present in the sample solution was impure.
We know,
Rf= D travel for compound/Distance travel for solvent.
1-Aspirin in

2-Caffein in
3-Ciprofloxacin in

Precaution :
1-TLC plate should be handled carefully.
2-The jar should be cleaned and preferable with alcohol.

Date: 7-6-2015
Experiment no: 03
Name of the experiment:. Drawing of different chemical structure using chemdraw.

(E)- 2- hexenol

2-Tridecanone

Germacrene A

Caryophyllene

Hexenol

(Z)-3-hexenol

Spilantho

Ocimene (Trans)

Ocimene (Cis)

Cadinen

Veratrole

Methyxanthoxylin

Thymol (36)

Guajaco

Mycaminose

1,2-Dimethoxy benzene

Orsellinic Beta-D-glucopyranoside

Caffeic acid

Oresellinic acid

Syzygiol

2,5,7-Trihydroxy-6,8-dimethylflavone

2,4-Dihydroxy-6-methaylbenzoic acid

3-Friedelanol-4alpha,3-Ketone

8-Glucopyranosyl-5,7-dihydroxy-2-methyl4-benzopyran-4-one

3-Glucosyl-2,46-trihydroxy acetophenone

1,6-bis-O-galloyol-Beta-D-glucose

1-O-galloyol-Beta-D-glucose

1-galloyol-Beta-D-glucose

Alpha-terpinene

Alpha-Terpineol

ASC-7(38)

5,8-Dihydrixycalamenene

O-Hydroxycalamenene

Methyle-Beta-orsellinate

Date: 7-6-2015
Experiment No. 04
Name of The Experiment : Synthesis of Acetanilide and calculate yield value and
absorbance(lamdamax)
Principle:
Acetanilide can be synthesized from aniline by acetylating in presence acetic
anhydride, acetic acid and zinc dust as a catalyst.

Reagents & Apparatus:

Aniline, acetic anhydride, ethanol, distilled water, zinc dust, glacial acetic
acid etc.
Round bottom flask, reflux condenser, water bath, beaker, pipette, conical
flask, glass rod, filter paper, suction pump etc.

Procedure:
(1) Place6 gm aniline to a 150-250 ml round bottom flask.
(2) Add7ml acetic anhydride gradually to the reaction mixture followed by glacial
acetic acid and3g zinc dust.
(3) Heat the reaction mixture at 50-60 C for an hour.
(4) Ccol the reaction mixture at room temperature and then add ice-water (10
times of acetic anhydride) to it.
(5) Then filter the mixture
(6) Collect the precipitate of crude acetanilide by suction.

Precaution
Aniline and acetanilide are toxic chemicals. So care should be taken to avoid contact
with the skin.

Result:

Date: 17-6-2015
Experiment No. 05
Name of The Experiment: Synthesis of Methyl salicylate and calculate yield value
and absorbance(lamdamax).
Principle
Salicylic acid (o-hydroxybenzoic acid) upon esterficaition yields methyl salicylate (oil
of wintergreen). Esterfication proceeds slowly with methanol and requires the
presence of concentrated sulphuric acid as catalyst.

Reagents & Apparatus:

Salicylic acid, methanol, conc, sulphuriccid, ether or Ch2Cl2, NaHCO3

Solution anhydrous Na2So4 water etc.

Round Bottom flask, reflux condenser, water bath, beaker, pipette, filter paper,
separating funnel, suction pump etc.
Procedure:
1.
Place 5 gm of dry salicylic acid and 50 ml of anhydrous methanol in a round
bottom flask.
2.
Add 1.5 ml of concentrated sulphuric acid cautiously to the reaction mixture
and mix thoroughly.
3.

Reflux the reaction mixture for 5 hours

4.

Then distil off the excess methanol by evaporation.

5.
Cool the mixture at room temperature and then add ice-water (100-150ml) it
and extract with ether or CH2Cl2
6.
Wash the ethereal or CH2Cl2 extract with saturated NaHCO3 Solution and
then with water preferably in separating funnel.
7.

Dry the organic portion with anhydrous Na2SO4 and filter.

8.

Evaporate the ethereal solution at low heat to obtain the desired product.

Precaution:
1-Usually there are approved safety goggles that are worn while working in the lab.
These safety glasses protect anything from entering your eyes.
2-Make sure you read the bottle labels carefully and only use what you are supposed
to use during an experiment.
3-You should not try out anything on your own unless you are very sure of what
reagents or chemicals you need to use and their results
4-Do not touch anything that you are not authorized to touch especially if it is a
chemical or an acid that could cause harm to you
Results:

Date: 18-6-2015
Experiment No. 07
Name of The Experiment: Separation of compound using 2D-TLC plate
Basic Principle:
Thin Layer Chromatography (TLC) is an extremely useful technique for monitoring
reactions. It is also used to determine the proper solvent system for performing
separations using column chromatography. TLC uses a stationary phase, usually
alumina or silica, that is highly polar (standard) or non-polar (reverse phase), and a
mobile phase, some solvent whose polarity we will choose. When we need to
determine the best solvent or mixture of solvents (a "solvent system") to develop a
TLC plate or chromatography column loaded with an unknown mixture, vary the
polarity of the solvent in several trial runs: a process of trial and error. When we will
apply our reaction mixture in solution to the plate then "run" the plate by allowing a
solvent (or combination of solvents) to move up the plate by capillary action.
Depending on the polarity of the components of the mixture, different compounds
will travel different distances up the plate. More polar compounds will "stick" to the
polar silica gel and travel short distances on the plate, while non-polar substances will
diffuse into the solvent and travel large distances on the plate. The measure of the
distance a compound travels is called Rf. This number, between zero and one, is
determined by measuring the distance the compound moved from the baseline (where
it was originally spotted) divided by the distance the solvent moved from the baseline.
There is a list of some standard solvents and their polarityVery polar additives:
Methanol > Ethanol > Isopropanol
Moderately polar additives:
Acetonitrile > Ethyl Acetate > Chloroform >
Dichloromethane > Diethyl Ether > Toluene
Non-polar additives:
Cyclohexane, Petroleum Ether, Hexane, Pentane
Common solvent combinations:
Ethyl Acetate:Hexane - 0-30% most popular combination, sometimes tough to
remove solvents completely on rotary evaporator
Ether:Pentane - 0-40% very popular, easy to remove on the rotary evaporator

Ethanol:Hexane/Pentane - 5-30% useful for very polar compounds

Dichloromethane:Hexane/Pentane - 5-30% sometimes useful

Purpose:
1. To check purity of given samples.
2. Identification of compounds like acids, alcohols, proteins, alkaloids,
antibiotics etc.

amines,

3. To evaluate reaction process by assessment of intermediates, reaction course etc.

4. To purify samples i.e for purification process.
5. To keep a check on the performance of other separation processes.

Aparatus :
Capillary/ Spotter/ Pastor pipette.
Aluminium plate / TLC plate.
A jar with a lid.
Pencil.
Scale.
UV lamp.
Chemical Reagents :
Methanol.
Chloroform.
Vanillin spray (containing vanillin in concentrated sulfuric acid)

Material &Methods :
For Preparing Solvents :

15% MC system : Take 3ml of methanol into the jar and make up the volume
by 17ml chloroform.

For Preparing TLC Plate :

A. At first we prepare TLC plates measuring 20cmx5cm sheets. Each large sheet
is cut horizontally into plates which are 5 cm tall by various widths. Handle
the plates carefully so that do not disturb the coating of adsorbent or get them
dirty.
B. Using a pencil, draw a line across the plate at the 0.5 cm mark.This is the
origin: the line on which we will spot the plate. We will take care not to press
so hard with the pencil that doesnt disturb the adsorbent. Under the line, mark
lightly the name of the samples we will spot on the plate, or mark numbers for
time points. Leave enough space between the samples so that they do not run
together.
Procedure :
1) At first desired solvent (15%MC) system was prepared by combining two
solvents at correct proportion.
2) Then TLC plate was taken and spot the samples (which samples to be
analyzed) into the dot line with the help of spotter.( The sample was picked up
just by touching sample solution by the spotter).
3) Don't allow the spot to become too large - if necessary, we can touch it to the
plate, lift it off and blow on the spot. If we repeat these steps, the wet area on
the plate will stay small.
4) The plate was then placed in the jar and the liquid mobile phase was allowed
to phase through the plate which carried the compounds of the sample along
with.
5) Let the mobile phase run until it reached to dot line of the upper part of the
plate.
6) Once the running completed the plate was removed from the jar and it was
allowed to air dried.
7) When the plate had air dried there are any colored spots, circle them lightly
with a pencil.
8) Most samples are not colored and need to be visualized with a UV lamp. Hold
a UV lamp over the plate and circle any spots we see.
9) If samples color are not visualized by UV lamp or we also want to show the
clear spots then we also sprayed by vanillin spray and burnt at oven.
10) Finally, the spot of the plate was observed for the determination of purity of
compound.

Results:
As there were more spots appeared in a vertical series on the plate so the compound
present

Precaution :

TLC plate should be handled carefully.

The jar should be cleaned and preferably with alcohol.
The solvent system in the jar must remain below the spot placed.
Solvents should be handled and measured carefully.
Vanillin spray must be used carefull.

spots appeared in a vertical series on the plate so the compound present