Analytical Lab Manual Edited Final 2.0-5

CERTIFICATE
This is to Certify that _____________________________ of Pharm-D 4th

Professional (7th semester) has carried out the necessary Practical work of
__________________________ lab as prescribed by Faculty of Pharmacy
Federal Urdu University of Arts Sciences and Technology, Karachi.
________________ ________________
Signature of Remarks
Course In-charge
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FEDERAL URDU UNIVERSITY OF ARTS SCIENCE AND TECHNOLOGY
FACULTY OF PHARMACY
4th Professional (7th Semester)
CONTENT LIST
Object Page no. Remarks Sign
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PAPER CHROMATOGRAPHY
WHAT IS PAPER CHROMATOGRAPHY?

Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary phase through
which a solution is made to pass is called paper chromatography. It is an inexpensive method of separating
dissolved chemical substances by their different migration rates across the sheets of paper. It is a powerful
analytical tool that uses very small quantities of material. Paper chromatography was discovered by Synge
and Martin in the year 1943.
PRINCIPLE OF PAPER CHROMATOGRAPHY:

The principle involved can be partition chromatography or adsorption chromatography. Partition
chromatography because the substances are partitioned or distributed between liquid phases. The two phases
are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper.
When the mobile phase moves, the separation of the mixture takes place. The compounds in the mixture
separate themselves based on the differences in their affinity towards stationary and mobile phase solvents
under the capillary action of pores in the paper. Adsorption chromatography between solid and liquid phases,
wherein the solid surface of the paper is the stationary phase and the liquid phase is the mobile phase.
PAPER CHROMATOGRAPHY DIAGRAM:
APPLICATIONS OF PAPER CHROMATOGRAPHY:

There are various applications of paper chromatography. Some of the uses of Paper Chromatography in
different fields are discussed below:
➢ To study the process of fermentation and ripening.
➢ To check the purity of pharmaceuticals.
➢ To inspect cosmetics.
➢ To detect the adulterants.
➢ To detect the contaminants in drinks and foods.
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➢ To examine the reaction mixtures in biochemical laboratories.
➢ To determine dopes and drugs in humans and animals.
TYPES OF PAPER CHROMATOGRAPHY:

1. Ascending Paper Chromatography – The techniques goes with its name as the solvent moves in an
upward direction.
2. Descending Paper Chromatography – The movement of the flow of solvent due to gravitational pull
and capillary action is downwards, hence the name descending paper chromatography.
3. Ascending – Descending Paper Chromatography – In this version of paper chromatography,
movement of solvent occurs in two directions after a particular point. Initially, the solvent travels
upwards on the paper which is folded over a rod and after crossing the rod it continues with its travel
in the downward direction.
4. Radial or Circular Paper Chromatography – The sample is deposited at the centre of the circular
filter paper. Once the spot is dried, the filter paper is tied horizontally on a Petri dish which contains
the solvent.
5. Two Dimensional Paper Chromatography – Substances which have the same rf values can be
resolved with the help of two-dimensional paper chromatography.
IMPORTANCE OF PAPER CHROMATOGRAPHY:

Paper chromatography has traditionally been used to analyze food colors in ice creams, sweets, drinks and
beverages, jams and jellies. Only edible colors are permitted for use to ensure that no non-permitted coloring
agents are added to the foods. This is where quantification and identification come into play.
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EXPERIMENT # 01
OBJECT
Separation of through paper chromatography using & separately
as solvent systems.
INTRODUCTION:
The purpose of this experiment is to observe how chromatography can be used to separate mixtures of
chemical substances. Chromatography serves mainly as a tool for the examination and separation of mixtures
of chemical substances. Chromatography is using a flow of solvent or gas to cause the components of a mixture
to migrate differently from a narrow starting point in a specific medium, in the case of this experiment, filter
paper. It is used to the purification and isolation of various substances. A chromatographically pure substance
is the result of the separation. Because purification of substances is required to determine their properties,
chromatography is an indispensable tool in the sciences concerned with chemical substances and their
reactions. Chromatography is also used to compare and describe chemical substances. The chromatographic
sequence of sorbet substances is related to their atomic and molecular structures. A change in a chemical
substance produced by a chemical or biological reaction often alters the solubility and migration rate. With
this knowledge, alterations or changes can be detected in the substance.
In all chromatographic separations, there is an important relationship between the solvent, the chromatography
paper, and the mixture. For a particular mixture, the solvent and the paper must be chosen so the solubility is
reversible and be selective for the components of the mixture. The main requirement, though, of the solvent
is to dissolve the mixture needing to be separated. The porous paper used must also absorb the components of
the mixtures selectively and reversibly. For the separation of a mixture, the substances making up the mixture
must be evenly dispersed in a solution, a vapor, or a gas. Once all of the above criteria have been met,
chromatography can be a simple tool for separating and comparing chemical mixtures.
HYPOTHESIS:
Paper can be used to separate mixed chemicals.
MATERIALS:
The materials used for this lab are paper, pencil, eraser, filter paper, beaker/measuring cylinder/test tube, petri
dish to cover or aluminum foil, paper clip, metric ruler, black ink, ethanol.
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PROCEDURE:
PART A: PREPARATION OF CHROMATOGRAPHY PAPER:
1. Wash your hands thoroughly to remove excess oils from your skin. Obtain a ruler and a piece of
chromatography paper from your instructor. Handle the paper only on the edges to avoid leaving
fingerprints, as these may hinder the elution process.
2. Place the chromatography paper on a sheet of clean notebook paper or paper towel to avoid picking
up dirt or contaminants from the bench top. Orient the paper into a "landscape" position and write your
name on the top edge of the paper in one comer. Using a pencil and ruler to measure accurately, draw
a straight line across the paper, about 1.5 cm above the bottom edge. This is the starting line. Draw
another line about 10 cm above the bottom edge. This is the finish line.
3. On the starting line, measure in from one side about 2.5 cm and lightly draw a small "X" centered on
the starting line. Draw seven more, 1.5 cm apart.
4. In the center of each X, make a small spot of ink color.
5. Go back over each ink spot a second time to ensure there is enough in the spot.
PART B: ACQUISITION OF CHROMATOGRAM:

1. Take two mL beakers and pour about mL of each and into
the separate beakers. Obtain a piece of plastic wrap to cover the top.
2. Gently place the paper cylinder into the beaker and cover the top with the plastic wrap. Remember that
the spots must be above the liquid level for the experiment to work. Watch the eluent creep up the
paper until it begins to move some of the inks. It will take about 45-90 minutes for the solvent front to
reach the finish line.
3. When the solvent front reaches the finish line, remove the paper from the beaker, being careful to touch
only the top. Let excess eluent drip into the beaker. Gently remove the tape and lay the chromatogram
on a piece of paper towel in the hood. Leave the paper in the fume hood, where it will dry completely.
If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp. allow
5-10 minutes to dry.
PART C: INTERPRETATION OF CHROMATOGRAM:

1. Write the names of the original ink colors bath each "X" mark (from the order you added the colors).
Each ink sample should no longer be on the "X" mark, and have travelled up the paper, becoming one
or more separate color spots between the starting and finish lines. Circle around each color spot.
2. Use u ruler and draw a plus sign in the center of each spot. Measure the distance from the starting line
to each plus sign. Record this distance for each spot on your lab report. These are DD values, in cm.
3. Measure the distance between the starting line and the finish line or, the farthest up the solvent front
reached. Record this distance. This is the FF value, in cm.
4. Calculate the retention factor (Rf) for each spot and record the values in your lab report.
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5. You and your lab part will hand in your lab reports at the same time, with the paper chromatogram
stapled to the lab reports
RESULT:
The results of the experiment are shown in a graph and a chart.
OBSERVATION:
COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf

(LISTED IN COMPONENTS Value)= Distance travelled by
ORDER) solute(color)/ Distance travelled by
solvent front
DATA ANALYSIS:
1. How many colors are separated from the ink?
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2. What served as the solvent for the ink?
3. List the color in order, from top to bottom, which separated from the blue ink
4. In millimeters, how far did the solvent travel?
5. From your result, what can you conclude is true about blue ink?
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6. Why did the ink separated?
7. Why did some colors move greater distance?
ERROR ANALYSIS:
Possible errors could include inaccurate measurements of the distances travelled by the inks and
miscalculation of the ratio travelled by solvent and colors. If a longer chamber was used, a longer strip of filter
paper could have been used. This may have changed the ratios. Another color may have been present, but not
detected because small length of filter paper.
CONCLUSION:
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EXPERIMENT # 02
OBJECT
Separation of through paper chromatography using mixture as
a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:

1. Take an mL beaker and pour about mL of mixture of and into
the beaker. Obtain a piece of plastic wrap to cover the top.
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If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp. allow

RESULT:
OBSERVATION:
COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

(LISTED IN ORDER) COMPONENTS Distance travelled by solute(color)/
Distance travelled by solvent front
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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 03
OBJECT
Separation of through paper chromatography using mixture of Acetone and Methanol
as a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:

1. Take an mL beaker and pour about mL of mixture of and into
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If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp,allow

stapled to the lab reports.
RESULT:
OBSERVATION:

(LISTED IN COMPONENTS Distance travelled by solute(color)/
ORDER) Distance travelled by solvent front
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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 04
OBJECT
Separation of and through paper chromatography using as
a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:

1. Take an mL beaker and pour about mL of into the beaker. Obtain a piece of
plastic wrap to cover the top.
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RESULT:
OBSERVATION:

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DATA ANALYSIS:
3. List the color in order, from top to bottom, which separated from the red and black ink
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5. From your result, what can you conclude is true about red and black ink?
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 05
OBJECT
Separation of and through paper chromatography using as
a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:

1. Take a mL beaker and pour about mL of into the beaker. Obtain a piece of
plastic wrap to cover the top.
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5. You and your lab part will hand in your lab reports at the same time, with the paper chromatogram.
RESULT:
OBSERVATION:

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DATA ANALYSIS:
3. List the color in order, from top to bottom, which separated from the black and red ink
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5. From your result, what can you conclude is true about black and red ink?
ERROR ANALYSIS:
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CONCLUSION:
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EXPERIMENT # 06
OBJECT
Separation of and through paper chromatography using a mixture of
Acetone & Methanol as a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:
2. Place the chromatography paper on a sheet of clean notebook paper or paper towel to avoid picking up
dirt or contaminants from the bench top. Orient the paper into a "landscape" position and write your

1. Take an mL beaker and pour about mL of mixture and into the
beaker. Obtain a piece of plastic wrap to cover the top.
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If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp, allow

RESULT:
OBSERVATION:

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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 07
OBJECT
Separation of and through paper chromatography by using different
percentage of mixture of & and setting up them as solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:
up dirt or contaminants from the bench top. Orient the paper into a “landscape” position and write your
3. On the starting line, measure in from one side about 2.5 cm and lightly draw a small “X” centered on

1. Take an mL beakers and pour about mL of and mL of into a
single beaker. Obtain a piece of plastic wrap to cover the top.
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If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp. Allow
4. Repeat the process with gradual increase in the ratio of solvent system and see which turned best.

1. Write the names of the original ink colors bath each “X” mark (from the order you added the colors).
Each ink sample should no longer be on the “X” mark, and have travelled up the paper, becoming one
RESULT:
OBSERVATION:

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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 08
OBJECT
Separation of and through paper chromatography by using different
percentage of mixture of & and setting up them as solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:
up dirt or contaminants from the bench top. Orient the paper into a “landscape” position and write your
3. On the starting line, measure in from one side about 2.5 cm and lightly draw a small “X” centered on

1. Take a mL beakers and pour about mL of and mL of into a
single beaker. Obtain a piece of plastic wrap to cover the top.
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If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if using the heat lamp. Allow
4. Repeat the process with gradual increase in the ratio of solvent system and see which turned best.

1. Write the names of the original ink colors bath each “X” mark (from the order you added the colors).
Each ink sample should no longer be on the “X” mark, and have travelled up the paper, becoming one
RESULT:
OBSERVATION:

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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
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EXPERIMENT # 09
OBJECT
Separation of and through paper chromatography using Water as a
solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:
7. Place the chromatography paper on a sheet of clean notebook paper or paper towel to avoid picking up
dirt or contaminants from the bench top. Orient the paper into a "landscape" position and write your

4. Take an mL beaker and pour about mL of and into the beaker. Obtain a
piece of plastic wrap to cover the top.
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RESULT:
OBSERVATION:

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DATA ANALYSIS:
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ERROR ANALYSIS:
CONCLUSION:
The paper strip and solvent molecules adsorbed by the cellulose form the stationary phase. The mobile phase
is a liquid solvent. There are several way to increase the separation of components. One way is to use a longer
paper strip.The minimum number of components is the maximum number of colors observed using any
solvent. It is possible that not all components are separated. Ink from the pen we tested
contains at least components. Results will vary. Color changes may be due to reactions of
some dyes with acid or base or to hydration states of organic dyes. Order of appearance of color is determined
by attraction of the dye to the solvent. In some cases, different molecules may have about the same attraction
and appear as a single color spot. Reaction with acid or base may change the polarity of molecules. The degree
of separation and the order in which colors appear can vary with the concentration of the solvent.
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EXPERIMENT # 10
OBJECT
Separation of different colored pointers/markers (dollar) through paper chromatography using a mixture
of Acetone ( ) and Methanol ( ) as a solvent system.
HYPOTHESIS:
MATERIALS:
PROCEDURE:
up dirt or contaminants from the bench top. Orient the paper into a "landscape" position and write
your name on the top edge of the paper in one comer. Using a pencil and ruler to measure accurately,
draw a straight line across the paper, about 1.5 cm above the bottom edge. This is the starting line.
Draw another line about 10 cm above the bottom edge. This is the finish line.
3. On the starting line, measure in from one side about 2.5 cm and lightly draw a small "X" centered
on the starting line. Draw five more, 1.5 cm apart.

1. Take an mL beaker and pour about mL of and ml of into
2. Gently place the paper cylinder into the beaker and cover the top with the plastic wrap. Remember
that the spots must be above the liquid level for the experiment to work. Watch the eluent creep up
the paper until it begins to move some of the inks. It will take about 45-90 minutes for the solvent
front to reach the finish line.
3. When the solvent front reaches the finish line, remove the paper from the beaker, being careful to
touch only the top. Let excess eluent drip into the beaker. Gently remove the tape and lay the
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chromatogram on a piece of paper towel in the hood. Leave the paper in the fume hood, where it
will dry completely. If needed, use a heat lamp (in the fume hood) to dry the chromatogram, if
using the heat lamp, allow 5-10 minutes to dry.

1. Write the names of the original ink colors bath each "X" mark (from the order you added the
colors). Each ink sample should no longer be on the "X" mark, and have travelled up the paper,
becoming one or more separate color spots between the starting and finish lines. Circle around
each color spot.
2. Use u ruler and draw a plus sign in the center of each spot. Measure the distance from the starting
line to each plus sign. Record this distance for each spot on your lab report. These are DD values,
in cm.
3. Measure the distance between the starting line and the finish line or, the farthest up the solvent
front reached. Record this distance. This is the FF value, in cm.
RESULT:
OBSERVATION:
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COLOR OF SEPARATION OF RETENTION FACTOR (Rf
MARKER COMPONENTS Value)= Distance travelled by
(LISTED IN solute(color)/ Distance
ORDER) travelled by solvent front
DATA ANALYSIS:
1. How many colors are separated from each marker?
2. What served as the solvent for the respective pointers/markers?
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3. List the color in order, from top to bottom, which separated from each pointer
ERROR ANALYSIS:
Possible errors could include inaccurate measurements of the distances travelled by the inks of the pointers
and miscalculation of the ratio travelled by solvent and colors. If a longer chamber was used, a longer strip of
filter paper could have been used. This may have changed the ratios. Another color may have been present,
but not detected because small length of filter paper.
CONCLUSION:
paper strip. The minimum number of components is the maximum number of colors observed using any
solvent. It is possible that not all components are separated. Ink from the different color pointers/markers i.e.
pink we tested, contains number of components
i.e. .Results will vary. Color changes
may be due to reactions of some dyes with acid or base or to hydration states of organic dyes. Order of
appearance of color is determined by attraction of the dye to the solvent. In some cases, different molecules
may have about the same attraction and appear as a single color spot. Reaction with acid or base may change
the polarity of molecules. The degree of separation and the order in which colors appear can vary with the
concentration of the solvent.
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EXPERIMENT # 11
OBJECT
Separation of different colored pointers/markers (dollar) through paper chromatography using 1%NaCl
Solution as a solvent system
HYPOTHESIS:
MATERIALS:
PROCEDURE:
on the starting line. Draw more, 1.5 cm apart.

1. Take mL beaker and prepare solution of NaCl to be used as solvent by taking about
0.1 g NaCl and 10 mL of water into the beaker. Obtain a piece of plastic wrap to cover the top.
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1. Write the names of the original ink colors bath each "X" mark (from the order you added the
colors). Each ink sample should no longer be on the "X" mark, and have travelled up the paper,
becoming one or more separate color spots between the starting and finish lines. Circle around
each color spot.
in cm.
RESULT:
OBSERVATION:
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COLOR OF SEPARATION OF RETENTION FACTOR (Rf
MARKER COMPONENTS Value)= Distance travelled by
(LISTED IN solute(color)/ Distance
ORDER) travelled by solvent front
DATA ANALYSIS:
1. How many colors are separated from each marker?
2. What served as the solvent for the respective markers/pointers?

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3. List the color in order, from top to bottom, which separated from each marker
ERROR ANALYSIS:
Possible errors could include inaccurate measurements of the distances travelled by the inks of different
pointers and miscalculation of the ratio travelled by solvent and colors. If a longer chamber was used, a longer
strip of filter paper could have been used. This may have changed the ratios. Another color may have been
present, but not detected because small length of filter paper.
CONCLUSION:
paper strip. The minimum number of components is the maximum number of colors observed using any
solvent. It is possible that not all components are separated. Ink from the different color pointers/markers i.e.
we tested, contains number of components i.e. Results
will vary. Color changes may be due to reactions of some dyes with acid or base or to hydration states of
organic dyes. Order of appearance of color is determined by attraction of the dye to the solvent. In some cases,
different molecules may have about the same attraction and appear as a single color spot. Reaction with acid
or base may change the polarity of molecules. The degree of separation and the order in which colors appear
can vary with the concentration of the solvent.
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EXPERIMENT NO # 12
OBJECT:
Separation of the inks (red and black) with paper chromatography by using different concentrations of
Ethanol Solution i.e. 5%, 10%, 20%, 50% as a solvent system.
REQUIREMENTS:
❖ Chromatographic chamber, Covers lids , Distilled H20 , Strips of filter paper ,Pencil , Ruler ,Scissor
Solvent ,Different pointers.
PROCEDURE:
1) Label the beakers with the following 5%,10%, 20%,and 50%

2) Using the 100% Ethanol and distilled H20, make the following ethanol solutions 5%,
10%, 20%, and 50% Ethanol Solutions for Paper Chromatography.
Ethanol Solutions for Paper Chromatography 5% 10% 20% 50%
5% 10% 20% 50%

Ethanol 7.5ml of 10% 2.5 ml 3 ml 7.5 ml
ethanol
Distilled 7.5 ml 22.5 ml 12 ml 7.5 ml
water
Total 15 ml 25 ml 15 ml 15 ml
Volume
3) Pour each of these solutions into their labeled beakers just enough to cover the bottom. Cover them
immediately because the Ethanol will evaporate.
4) Cut the filter paper into strips that are about 5 cm x 10 cm. If the beakers are tall enough you can make
the strips longer to allow for more separation. The longer the strips are the longer it will take to develop
them.
5) Draw a line lightly with the pencil across the strips 1 cm above the bottom edge
6) Label the strip with the corresponding solution that it will be placed in.
7) Use the pointers to spot the color onto the strips. Try to keep the spots small and spaced apart. Spot 2
or 3 times for each spot.
8) Suspend the filter paper vertically in the chromatographic chamber containing the Solvent, in such a
way that a pencil line remains about 1 cm above the Solvent.
9) Close the jar with its lid and keep it standing.
10) Notice the rising solvent, along with the ink component .After the solvent has risen up to the finish
line (Solvent front).
11) Take the filter paper out of the jar and use the pencil mark at the distance that the solvent has risen on
the filter paper, this is the solvent front.
12) Dry the filter paper and put the pencil mark at the center of the separated spots.
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13) Measure the distance of different spots from the original line, and the distance of solvent from the
original line.
14) Calculate the Rf value of different components by using formula:
Rf =Distance travelled by solute/ Distance travelled by solvent.
OBSERVATION & CALCULATIONS:
FOR 5% ETHANOL SOLUTION:
COLOR SEPARATION DISTANCE DISTANCE Rf =VALUE a/b

OF INK OF TRAVELLED TRAVELLED
(Listed In COMPONENTS BY BY
Order) SOLUTE(a) SOLVENT(b)
COLOR SEPARATION DISTANCE DISTANCE Rf VALUE a/b

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COLOR SEPARATION DISTANCE DISTANCE Rf =VALUE a/b

COLOR SEPARATION DISTANCE DISTANCE Rf=VALUE a/b

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RESULT:
We have successfully separated the components of Black and Red ink with paper chromatography using
different concentration of ethanol solution i.e; 5%, 10%, 20%, 50%
DATA ANALYSIS:
1. How many colors are separated from the inks?
2. What served as the solvent for the inks?
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3. List the color in order, from top to bottom, which separated from the black and red ink.
5. Which concentration of ethanol solution gives best separation of black and red ink?
ERROR ANALYSIS:
Possible errors could include inaccurate measurements of the distances travelled by the inks of different
pointers and miscalculation of the ratio travelled by solvent and colors. If a longer chamber was used, a longer
strip of filter paper could have been used. This may have changed the ratios. Another color may have been
present, but not detected because small length of filter paper.
CONCLUSION:
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EXPERIMENT # 13
OBJECT:
Separation of different food dyes colors through paper chromatography using 1% NaCl solution as a
solvent system.
HYPOTHESIS:
MATERIALS:
dish to cover or aluminum foil, paper clip, metric ruler, black and red ink,1% NaCl solution.
PROCEDURE:
on the starting line. Draw more, 1.5 cm apart.

1. Take mL beaker and prepare 1% solution of NaCl to be used as solvent by taking about
0.1g NaCl and 10 mL of water into the beaker. Obtain a piece of plastic wrap to cover the top
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1. Write the names of the original dye colors bath each "X" mark (from the order you added the
colors). Each dye color sample should no longer be on the "X" mark, and have travelled up the
paper, becoming one or more separate color spots between the starting and finish lines. Circle
around each color spot.
in cm.
RESULT:
OBSERVATION:
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COLOR OF FOOD SEPARATION OF RETENTION FACTOR (Rf Value)=
DYES(LISTED IN COMPONENTS Distance travelled by solute(color)/
DATA ANALYSIS:
1. How many colors are separated from the dyes?
2. What served as the solvent for the dyes?
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3. List the color in order, from top to bottom, which separated from the each dye.
ERROR ANALYSIS:
CONCLUSION:
solvent. It is possible that not all components are separated. Different food dyes i.e. we
tested contains number of components respectively. Results will vary. Color changes may
be due to reactions of some dyes with acid or base or to hydration states of organic dyes. Order of appearance
of color is determined by attraction of the dye to the solvent. In some cases, different molecules may have
about the same attraction and appear as a single color spot. Reaction with acid or base may change the polarity
of molecules. The degree of separation and the order in which colors appear can vary with the concentration
of the solvent.
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EXPERIMENT # 14
OBJECT:
Separation of different food dye colors/yums through paper chromatography using 1% NaCl/ 1%Sugar
solution as a solvent system.
HYPOTHESIS:
MATERIALS:
dish to cover or aluminum foil, paper clip, metric ruler, black and red ink,1% NaCl solution.
PROCEDURE:
the starting line. Draw more, 1.5 cm apart.

1. Take mL beaker and prepare 1% solution of NaCl/Sugar to be used as solvent by taking about
0.1g NaCl/Sugar and 10 mL of water into the beaker. Obtain a piece of plastic wrap to cover the top
69

1. Write the names of the original dye colors bath each "X" mark (from the order you added the colors).
Each dye color sample should no longer be on the "X" mark, and have travelled up the paper, becoming
one or more separate color spots between the starting and finish lines. Circle around each color spot.
RESULT:
OBSERVATION:
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COLOR OF FOOD SEPARATION OF RETENTION FACTOR (Rf Value)=
DYES(LISTED IN COMPONENTS Distance travelled by solute(color)/
DATA ANALYSIS:
5. How many colors are separated from the dyes/yums?
6. What served as the solvent for the dyes/yums?

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7. List the color in order, from top to bottom, which separated from the each dye.
ERROR ANALYSIS:
Possible errors could include inaccurate measurements of the distances travelled by the inks/food dyes/yums
and miscalculation of the ratio travelled by solvent and colors. If a longer chamber was used, a longer strip of
filter paper could have been used. This may have changed the ratios. Another color may have been present,
but not detected because small length of filter paper.
CONCLUSION:
solvent. It is possible that not all components are separated. Different food dyes/Yums i.e.
green, we tested contains number
of components respectively. Results will vary. Color changes may be due to reactions of some dyes with acid
or base or to hydration states of organic dyes. Order of appearance of color is determined by attraction of the
dye to the solvent. In some cases, different molecules may have about the same attraction and appear as a
single color spot. Reaction with acid or base may change the polarity of molecules. The degree of separation
and the order in which colors appear can vary with the concentration of the solvent.
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PAPER CHROMATOGRAPHY
SOME GENERAL QUESTIONS OF PAPER CHROMATOGRAPHY:
Which type of solvents are generally employed in chromatography?

Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate
of flow of a solvent varies inversely as its viscosity.
How does the liquid rise through the filter paper?
Ans. By means of capillary action.

On which factors, does the Rf value of a compound depend?
Ans.
• Nature of the compound.

• Nature of the solvent.
• Temperature.
What- is loading (or spotting) ?
Ans. The application of the mixture as a spot on the original line on the filter paper strip or addition of mixture
to the column, is called loading (or spotting).
What are the essential characteristics of the substance used as a developer ?
Ans.
• It should be volatile.
• It should impart colour to the different spots.
• It should not react with various compounds which are being separate
Why water is not used in paper chromatography?
Ans. In ambient conditions water is too polar to be efficient eluent in chromatographic analysis.
Is filter paper polar or non-polar?

Ans. Paper is comprised of cellulose, which is a polymer of the simple sugar glucose, and as such is very polar
due to the –OH groups present in glucose.
Why Rf value is always less than 1 in paper chromatography?
Ans. Rf values are always less than one because they are the ratios of migration distances of solutes (analytes)
and solvent fronts. As a general rule, the solvent front always travels more than that of solute, because the
solutes have to have some attractive properties with stationary phases.
Why spots are concentrated & small in chromatography?
Ans. When spots develop on the paper, they start to spread vertically, and to a little extent, horizontally. If the
spots are larger in size, there is a risk of spots merging into one another and hence comparison becomes
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difficult. Moreover, applied sample spot must be concentrated in order to make sure that loaded sample
contains all of it's possible components and ultimately a better separation is achieved. Otherwise nothing
would be visible on chromatogram in case applied sample spot is not concentrated enough to contain all the
possible components of sample.
Is ink Hydrophobic or Hydrophilic?

Ans. Ink sticks to paper because it has a high affinity for the medium it’s being applied to. To group things
into simple categories we could say, there are hydrophobic inks (non-polar inks that don’t like water) and
hydrophilic inks (polar inks that do like water).
Can Rf value be zero?

Ans. Rf values vary from 0 to 1, with 0 indicating extremely low solvent polarity and 1 indicating very high
solvent polarity.
How does polarity affect paper chromatography?
Ans. Polarity of the solvent affects the speed of the chromatography process. So, we can say that, if we increase
the polarity of the solvent all the other components present in the mixture move faster during the
chromatography experiment.
Which is more polar in paper chromatography?
Ans. The key to separation lies in the difference in polarity of the mobile and stationary phases. Usually, it is
the stationary phase that is more polar and the mobile is less or nonpolar.
Does increasing polarity will increase the Rf value?

Ans. The more polar the compound, the more it will adhere to the adsorbent and the smaller the distance it
will travel from the baseline, and the lower its Rf value.
What happens if solvent is too polar in chromatography?

Ans. If a development solvent of too high a polarity is used, all components in the mixture will move along
with the solvent and no separation will be observed (Rf's will be too large). If the solvent is of too low a
polarity the components will not move enough, and again separation will not occur (Rf's will be too small).
Which solvent appears to give a better separation?

Ans. The best separation is often achieved by using a mixture of a non-polar solvent with a polar solvent.
Which mobile phase is used in paper chromatography?
Ans. Generally polar compound is used as a mobile phase in paper chromatography.
Why are two solvents used in the process?

Ans. Different pigments will be soluble in one solvent but not another. Better separation of pigment bands
will result if a combination of solvents is used.
Can Rf value be same of the different components? If yes then what does it indicate?
Ans. Yes, Rf value of the different components can be same. Similar Rf value indicates that components are
identical.
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This lab employs chromatography to separate the components in ink. What other applications can we
use chromatography for?
Ans. Chromatography can be used to separate ink, dye, pigments in plants, in determination of shelf life of
food substance, presence of chemical additives in food, testing of water samples for purity, presence of toxic
contaminants in oil and pesticides. It can be used in pharmaceutical industry for identification of dye etc.
Which solvent gives best separation of following inks?
• Red ink = Water + Methanol

• Black ink = Acetone + Methanol
• Blue ink= Acetone + Methanol
Diverse solvents can be used in ink chromatography. For inks that are water soluble, water is the solvent of
choice. For inks that are not soluble in water, methanol, ammonium hydroxide, ethanol, acetone, or
hydrochloric acid can be used as solvents.
75
THIN-LAYER CHROMATOGRAPHY
INTRODUCTION:
TLC is one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in
qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds.
TLC is a form of liquid chromatography consisting of:
➢ A mobile phase (developing solvent) and

➢ A stationary phase (a plate or strip coated with a form of silica gel)
➢ Analysis is performed on a flat surface under atmospheric pressure and room temperature
DEFINITION:
Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of

components into individual components by using finely divided adsorbent solid/ (liquid) spread over a glass
plate and liquid as a mobile phase.
PRINCIPLE OF TLC:
“It is based on chromatography or partition chromatography or the principle of adsorption combination of

both, depending on adsorbent, its treatment and nature of solvents employed”.
• The components with more affinity towards stationary phase travel slower.
• Components with less affinity towards stationary phase travel faster
In TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of glass, plastic, and aluminum)
as a thin layer (about 0.25 mm thick). In many cases, a small amount of a binder such as plaster of Paris is
mixed with the absorbent to facilitate the coating.
Separation of mixtures in microgram quantities by movement of a solvent across a flat surface; components
migrate at different rates due to differences in solubility, adsorption, size or charge; elution is halted when or
before the solvent front reaches the opposite side of the surface and the components examined in situ or
removed for further analysis.
Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and
a mobile phase
The stationary phase:
Is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.
The mobile phase:
Is a developing liquid which travels up the stationary phase, carrying the samples with it.
SELECTION OF STATIONARY PHASE:
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The choice of the stationary phase for a given separation problem is the most difficult decision in TLC. The
choice of stationary phase in following characters considered.
➢ The chemical composition of the stationary phase and in particular that of its surface, must be
suitable for the task.
➢ To obtain satisfactory separation efficiency, the mean particle size, the particle size distribution and
the morphology of the particle must be considered.
STATIONARY PHASE FOR TLC:
Silica gel, modified silica gels , alumina ,cellulose powder , kieselguhr, modified celluloses e.g. DEAE and
CM, sephadex gel.
SELECTION OF MOBILE PHASE:

1. The choice of the mobile phase depends upon the following factors:-
➢ Nature of the substance to be separated
➢ Nature of the stationary phase used
➢ Mode of chromatography (normal phase or reverse phase)
➢ Separation to be achieved (analytical or preparative)
2. The organic solvent mixture of low polarity is used highly polar solvents are avoided to minimize
adsorption of any components of the solvent mixture use of water as a solvent is avoided as it may loosen
the adhesion of a layer on a glass plate.
3. Solvents with an increasing degree of polarity are used in liquid-solid or adsorption chromatography. The
solvents listed in elutropic series are selected
MOBILE PHASE FOR TLC:
n-Hexane , cyclohexene , toluene , benzene , diethyl ether , chloroform , dichloromethane ,1,2
dichloroethane , acetone, ethyl acetate , acetonitrile , propanol , methanol , acetic acid , water.
SELECTION OF ADSORBENTS:
• Solubility of compounds e.g. hydrophilic or lipophilic.
• Nature of substance to be separated i.e. whether it is acidic, basic or amphoteric
• Adsorbent particle size
• Adsorbent should not adhere to glass plate
• Reactivity of compound with the solvent or adsorbent
• Chemical reactivity of compounds with binders.
METHODS FOR APPLICATION OF ADSORBENT:

➢ Pouring
➢ Dipping
➢ Spraying
➢ Spreading.
1) POURING: The adsorbent of finely divided and homogeneous particle size is made into slurry and
is poured on a plate and allowed to flow over it so that it is evenly covered.
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2) DIPPING: This technique is used for small plates by dipping the two plates at a time, back to back
in a slurry of adsorbent in chloroform or other volatile solvents. Exact thickness of layer is not
known and evenness of layer may not be good.
3) SPRAYING: Slurry is diluted further for the operation of sprayer. But this technique is not used
now a days as it is difficult to get uniform layer.
4) SPREADING: All the above methods fail to give thin and uniform layers. Modern methods utilize
the spreading devices for preparation of uniform thin layers on glass plates. Commercial spreaders
are of two types (a) Moving spreader, (b) Moving plate type. It gives layer thickness from 0.2 to 2.0
mm.
APPLICATION OF SAMPLE:
• Sample solution in a non-polar solvent is applied.
• The concentration of a sample or standard solution has to be minimum of a 1% solution of either
standard or test sample is spotted using a capillary tube or micropipette.
• The area of application should be kept as small as possible for sharper and greater resolution.
DEVELOPMENT TECHNIQUES:
Different development techniques are:
1. ONE DIMENSIONAL DEVELOPMENT: In this the plate is placed in suitable TLC Development
.The Solvent rises up the layer transporting the sample mixture .Once solvent front reached remove
plate from the chamber and marked it with pencil .
2. HORIZONTAL DEVELOPMENT: In this the plate is positioned horizontally inside chamber

.Development can be performed from one or both side of plate.
3. TWO DIMENSIONAL DEVELOPMENT: In this plate the normal chamber once from the bottom
to top after drying the plate is turned 90 degree and placed another chamber with different solvent and
developed again.
4. MULTIPLE DEVELOPMENT: In this TLC plate undergoes multiple development with drying
between each cycle .Solvent travel repeatedly through the layer re-concentrating and deforming spot
forming elliptical shape /narrow bands.
APPPLICATIONS OF TLC:
• It is used for separation of all classes of natural products and is established as an analytical tool in
modern pharmacopoeias.
-E.g. Acids, alcohols, glycols, alkaloids, amines, macromolecules like amino acids, proteins and
peptides, and Antibiotics
- For checking the purity of samples
- As a purification process
- -examination of reaction
- For identifying organic compounds
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• Extensively used as an identification test and test for purity.
• As a Check on process-checking of distillation fractions and for checking the progress of molecular
distillation.
• Applications of TLC for separation of Inorganic lons – Used for separating cationic, anionic, purely
covalent species and also some organic derivatives of the metals.
• Separation of Amino Acids- two dimensional thin-layer chromatography.
• Separation of vitamins-vitamin E, Vitamin D3, vitamin A.
• Application of TLC in quantitative analysis.
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EXPERIMENT #15
OBJECT:
To prepare your own TLC plate.
REQUIREMENTS:
Silica gel, Glass Slider, Solvent, Binder (Plaster of Paris or Carboxy Methyl Cellulose), Beaker, Water,
Oven.
PROCEDURE:
1. Grind the silica gel.
2. Weigh out and mix 4 grams of silica and 1 gram of plaster of paris.
3. Transfer these two in mortar and pestle and grind them again.
4. The mixture must be homogenous, fines the particles size, the better will be the result.
5. Now suspend particles in water and cover the bottle and shake it for 10 minutes.
6. Now fill uр the syringe with uniform suspension and place the thin layer of suspension across the
plate.
7. The thickness of layer should be less than 3 mm.
8. Now air dry the slide.
9. Activation is done by heating the plate in hot air oven at 120 C for 30-45 minutes.
10. Remove the plate from the oven. Now plate is ready for use.
RESULT:
We have successfully prepared our own TLC plate.
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EXPERIMENT #16
OBJECT:
Separation of Black ink / pointer (Dollar) through TLC using 1% NaCl solution as a solvent system.
REQUIRMENTS:
TLC plate, Pencil , NaCl ,Water, Scale, Beaker ,Capillary tube, Measuring cylinder, Ink/ pointer.
PROCEDURE:
SPOTTING:
• Take a TLC plate, using scale draw a line 1 cm from the bottom and top of the plate through pencil.
• By using capillary tube spot the ink on plate with 1 cm gap between each spot or by putting spot on
plate by pointer with 1 cm gap between each spot.
• Air dry the spots. Spot black ink on the same TLC plate.
DEVELOPMENT:
• Take a beaker and prepare 1% NaCl solution to be used as a solvent system by adding 1gm NaCl and
10 ml water in it.
• Place the TLC plate in a beaker in straight position.
• Cover the beaker with a glass plate immediately in order to avoid Loss of solvent.
• Allow the setup to develop.
• When the solvent reaches the solvent front, remove the TLC plate from the beaker and let it dry.
VISUALIZATION:
• Mark each spot on the plate after placing the plate on a flat surface by the help of a pencil.
• Calculate Rf value of each of the colors separated from each of the spot.
OBSERVATION:
The ink separated into following components:
• Black pointer:
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CALCULATION :
POINTER/INK COMPONENTS RETENTION FACTOR (Rf

SEPARATED Value)= Distance travelled by
solute(color)/ Distance
travelled by solvent front
RESULTS:
For Black ink/pointer:
CONCLUSION:
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EXPERIMRNT#17
OBJECT:
Separation of Black and Green ink / pointer (Dollar) through TLC using 50% Ethanol Solution/Mixture
of Chloroform and Methanol as a solvent system.
REQUIRMENTS:
TLC plate, Pencil , Chloroform, Methanol, Scale, Beaker ,Capillary tube, Pipette, Ink/ pointer.
PROCEDURE:
SPOTTING:
• Take a TLC plate, using scale draw a line 1 cm from the bottom and top of the plate through pencil.
• By using capillary tube spot the ink on plate with 1 cm gap between each spot or by putting spot on
plate by pointer with 1 cm gap between each spot.
• Air dry the spots. Spot green and black ink on the same TLC plate.
DEVELOPMENT:
• Take a beaker and add ml in it.
• Place the TLC plate in a beaker in straight position.
• Cover the beaker with a glass plate immediately in order to avoid Loss of solvent.
• Allow the setup to develop.
• When the solvent reaches the solvent front, remove the TLC plate from the beaker and let it dry.
VISUALIZATION:
• Mark each spot on the plate after placing the plate on a flat surface by the help of a pencil.
• Calculate Rf value of each of the colors separated from each of the spot.
• Compare Rf values obtained in separation of inks when using 50% ethanol solution to the Rf values
obtained by the separation of components of ink / pointer using mixture of chloroform and methanol
through thin layer chromatographic technique.
OBSERVATIONS:
The ink separated into following components:
• Black pointer:
• Green pointer:
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CALCULATION AND COMPARISON:
Solvent System Solvent System

(50% Ethanol solution) (Mixture of Chloroform + Methanol)
Pointer / Components Rf Value Pointer / Components Rf Value

Ink Separated Ink Separated
RESULTS:
For Green ink/pointer:
For Black ink/pointer:
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CONCLUSION:
85
COLUMN CHROMATOGRAPHY
INTRODUCTION:
In chemistry, Column chromatography is a technique which is used to separate a single chemical compound
from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to
the adsorbent as the compounds move through the column at different rates which allow them to get separated
in fractions. This technique can be used on small scale as well as large scale to purify materials that can be
used in future experiments. This method is a type of adsorption chromatography technique
PRINCIPLE:
When the mobile phase along with the mixture that needs to be separated is introduced from the top of the
column, the movement of the individual components of the mixture is at different rates. The components with
lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and
affinity with the stationary phase. The components that move fast are removed first whereas the components
that move slow are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of
the components is expressed as:
R(f) = the distance travelled by solute/ the distance travelled by solvent
R(f) is the retardation factor.
CHOOSING A STATIONARY PHASE:

As with TLC, alumina and silica are the two most popular stationary phases in column. Chromatography. For
these common phases, the partitioning works in an analogous manner. The more polar sample will be retained
on the stationary phase longer. Thus the least polar compound will elute from the column first, followed by
each compound in order of increasing polarity. Stationary phases for CC can come in a variety of sizes,
activities, acidic and basic variations for both alumina and silica. The type of adsorbent, the size of the column,
the polarity of the mobile phase as well as the rate of elution all affect the separation. These conditions can be
manipulated to get the best separation for your mixture.
CHOOSING SOLVENT SYSTEM:

Systems for use as mobile phases in CC can be determined from previous TLC experiments, the literature, or
experimentally. Normally, a separation will begin by using nonpolar or low polarity solvent, allowing the
compounds to adsorb to the stationary phase, then slowly switching the polarity of the solvent to desorb the
compounds and allow them to travel with the mobile phase. The polarity of the solvents should be changed
gradually. On a macroscale, the mixing of two solvents can create heat and crack the column leading to a poor
separation. Some typical solvent combinations are ligroin-dichloromethane, hexane-ethyl acetate and hexane-
toluene. Often an experimentally determined ratio of these solvents can sufficiently separate most compounds.
86
Solvents such as methanol and water are normally not used because they can destroy the integrity of the
stationary phase by dissolving some of the silica gel.
TYPE OF COLUMN CHROMATOGRAPHY:
There are two forms of column chromatography.
• Liquid chromatography (LC)

• Gas chromatography (GC)
The most widely used forms of column chromatography are:
1. Adsorption chromatography
2. Partition chromatography
3. Ion exchange chromatography
4. Gel chromatography
❖ Adsorption Column Chromatography:

Adsorption chromatography is a technique of separation, in which the components of the mixture are
adsorbed on the surface of the adsorbent.
❖ Partition column chromatography:

The stationary phase, as well as mobile phase, are liquid in partition chromatography.
❖ Gel column chromatography:

In this method of chromatography, the separation takes place through a column packed with gel. The
stationary Phase is a solvent held in the gap of a solvent.
❖ Ion exchange column chromatography:

A chromatography technique in which the stationary phase is always ion exchange resin.
PREPARATION OF THE COLUMN:

• The column mostly consists of a glass tube packed with a suitable stationary phase.
• A glass wool/cotton wool or an asbestos pad is placed at the bottom. Of the column before packing the
stationary phase.
• After packing, a paper disc kept on the top, so that the stationary. Layer is not disturbed during the
introduction of sample or mobile phase.
TYPES OF PREPARING THE COLUMN:

There are two types of preparing the column, they are:
1. Dry packing / dry filling:
In this the required quantity of adsorbent is poured as fine dry powder in .The column and the solvent
is allowed to flow through the column till Equilibrium is reached.
2. Wet packing / wet filling:
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In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It is
considered as the ideal technique for packing
B. INTRODUCTION OF THE SAMPLE:
• The sample which is usually a mixture of components is dissolved in minimum Quantity of the mobile
phase.
• The entire sample is introduced into the column at once and get adsorbed on the top portion of the
column.
• From this zone, individual sample can be separated by a process of elution.
C. ELUTION:
By elution technique, the individual components are separated out from the Column. It can be achieved by
two techniques:
• Isocratic elution technique:

Same solvent composition or solvent of same polarity is used throughout the process of separation.
E.g. Use of chloroform alone.
• Gradient elution technique:
Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of separation. E.g.
initially benzene, then chloroform, then ethyl acetate then chloroform
D. DETECTION OF COMPONENTS:
• If the compounds separated in a column chromatography procedure are colored, the progress of the
separation can simply be monitored visually.
• If the compounds to be isolated from column chromatography are colorless.
• In this case, small fractions of the eluent are collected sequentially in labelled tubes and the
composition of each fraction is analyzed by TLC.
APPLICATIONS:
Column chromatography is one of the most useful methods for the separation and purification of both solids
and liquids. Its major application includes:
• Column Chromatography is used to isolate active ingredients.

• It is very helpful in separating compound mixtures.
• It is used to determine drug estimation from drug formulations.
• It is used to remove impurities.
• Used to isolate metabolites from biological fluids.
ADVANTAGES:
• Any type of mixture can be separated by column chromatography.
• Any quantity of the mixture can also be separated.
• Wider choice of mobile phase.
• In preparative type, the sample can be separated and reused.
• Automation is possible.
88
EXPERIMENT#18
OBJECT:
Preparation of column for column chromatography.
Column chromatography is a commonly used purification technique in labs across the world. Done right it
can simply and quickly isolate desired compounds from a mixture. But like many aspects of practical
chemistry, the quick and efficient setting up and running of a column is something that can take years to
master. Here we present some of the tips and tricks of the trade to help you set up the perfect column.
In a typical column (Fig. 1), the stationary phase, a solid adsorbent normally silica gel (SiO 2) or alumina
(Al2O3), is placed in a vertical glass column. The mobile phase, a liquid, is added to the top of the column and
flows down through the column by either gravity or external pressure (flash chromatography). Separation of
compounds is achieved through the varying absorption on and interaction between the stationary
and mobile phases.
Figure 1. General column setup

The quality of the separation depends on a variety of factors not least of which is the absence of air bubbles
in the stationary phase. To prevent bubbles, the correct packing of a column is important.
CHOICE OF SILICA OR ALUMINA FOR THE STATIONARY PHASE:

Silica and alumina are both polar adsorbents so the more polar components in the mixture to be separated are
retained more strongly on the stationary phase and are therefore eluted from the column last. Silica is
recommended for most compounds, but as it is slightly acidic, it preferentially retains basic compounds
Alumina is slightly basic, so will retain acidic compounds more strongly. It is good for separation of
components that are weakly or moderately polar and the purification of amines.
89
Adsorbent particle size affects how solvent flows through the column. Silica or alumina are both available in
a variety of sizes.
The size is given by the mesh value which refers to the number of holes in the mesh that is used to sieve the
adsorbent. Thus higher mesh values such as “silica gel 230-400” have more holes per unit area and
correspondingly smaller particles than "silica gel 60”. Typically, 70-230 silica gel is used for gravity columns
and 230-400 mesh for flash columns.
Alumina is available in types I II & III. This refers to the water content of the alumina, with (I) having the
least water and III the most. A lower water content means there are more polar sites in the alumina free to
bind organic compounds, and polar compounds will remain on the column longer. Alumina of activity II or
III, 150 mesh, is most commonly employed.
The techniques for packing a column described below use silica as the stationary phase, but are equally suitable
for use with alumina.
PREPARING THE COLUMN:

Some colors have glass frits to prevent loss of the stationary phase out the bottom others do not and will need
to be plugged with either glass wool or cotton wool. Which you use is personal preference. Positioning the
cotton or glass wool can be awkward at first, but glass frits are harder to clean and may be a source of
impurities, such as silica leaking through the frit into the collected fractions. This can be prevented by adding
a layer of sand between the frit and the silica. The porosity of frits can also vary. This means that rate of
solvent flow can be different for different column ls. Very porous frits will leak more silica, but less porous
frits have slower flow rates, sometimes too slow and can lead to pressure build up in flash chromatography.
Figure 2. Fritted (left) and non-Fritted (right)

FRITTED COLUMN:
1. Find the clean, empty column of suitable size.

2. Clamp the column securely and close the tap or stopcock.
3. Add a layer of sand (approx. 0.5 cm, optional).
NON FRITTED COLUMN:

The ball of cotton or glass wool should be large enough to plug the bottom of the column, but not so large and
densely packed that it restrict solvent flow. A piece the size of the tip of your little finger should be suitable
for most columns.
90
1. Position the cotton or glass wool ball securely in the narrowest part of the column using a long glass
rod or other suitable device.
2. Clamp the column securely and close the tap or stopcock.
3. Add a layer of sand until it reaches the main body of the column (approx. 2cm). This will give the
stationary phase an even base and prevent concentration and streaking of the bands as they come off
the column and are collected.
Figure 3. Guidelines for the correct size of cotton or glass wool and sand for non-fritted columns.
FILLING THE COLUMN:

There are several method for filling columns. You may find one method easier or quicker than the others and
always fill a column that way, or you may find that different size columns require different methods. All
methods have their pros and cons and you may need to try all three to find the one that you prefer.
OPTION 1:
Dry-Pack Method 1:
You will need:
• Column prepared as in section 2 above
• Funnel suitable for dry solids
• Something to tap the column with (see box below)
• Solvent
• Silica or alumina
METHOD:
1. Fill the column with solvent, allowing some to run through the sand and cotton wool to remove air
bubbles (Fig. 4, step B).
2. Place a dry funnel in the top and gently pour the silica or alumina (stationary phase) into the solvent.
Allow the solvent to drain to prevent overflowing (Fig. 4, step C).
3. Let the stationary phase settle and gently tap the column (see box below) so that the silica or alumina
will pack tightly into the column (Fig. 4, step D).
4. Drain the solvent until the solvent level is just even with the surface of the phase (Fig. 4, step E).
91
Figure 4. Dry-pack method 1.
OPTION 2:
Dry Pack Method 2:
You will need:
• Column prepared as in section 2 above.
• Funnel for solvent.
• Vacuum line
• Solvent
METHOD:
1. Add dry silica gel to the column and apply house vacuum by attaching the vacuum tubing to the bottom
of the column (Fig. 5, step B). This will compress the silica gel and keep it compressed for the next
steps. Packing can be improved by tapping the column
2. With the vacuum still applied, pour in the solvent (Fig. 5, step D).
3. Allow the solvent to flow though the column until it is almost at the bottom. At this point, close the
stopcock and remove the vacuum line (Fig 5 step E).
4. Allow 5-6 columns worth of solvent to flow through the column to ensure complete packing Drain the
solvent until the solvent level is just even with the surface of the stationary phase (Fig 5, step F)
Figure 5. Dry-pack method 2.
OPTION 3:
92
Slurry method:
You will need
• Column prepared as in Section 2 above

• 2X beakers or conical flasks
• Glass rod or pasteur pipette
• Funnel suitable for wet solids
• Solvent
• Pasteur pipette
METHOD:
1. Fill the column about 1/3 with solvent

2. In the beaker, measure out the required amount of silica or alumina
3. In a separate flask or beaker measure solvent approximately one and a half times the volume of silica
4. Add the silica to the solvent, a little at a time, while swirling use the pasteur pipette or glass rod to mix
the slurry.
5. Pour or pipette some of the slurry into the column. Allow the solvent to drain to prevent overflowing
6. Tap the column gently to encourage bubbles to rise and silica to settle
7. Continue to transfer the slurry to the column until all the silica or alumina is added
8. Rinse the inside of the column by pipetting solvent down the inside edge
9. Drain the solvent until the solvent level is just even with the surface of the stationary phase.
Figure 6. The slurry method.

You are now ready to load your column and isolate the desired compound.
EMPTYING THE COLUMN:

Once you have your product isolated all that remains is to empty and clean the column ready for next time.To
speed up the process, elute all of the solvent using compressed air and allow air to flow through the column
for approximately 2 h. This will give dry, free flowing silica that is easily to pour into the silica waste container.
Alternatively, elute all the solvent and secure the column upside down over a large beaker and allow to dry
overnight into a fume hood.
Clean the column by rinsing it with water and acetone is usually sufficient
93
EXPERIMENT # 19
OBJECT:
Separation of ink / food coloring through column chromatography.
MATERIALS:
Chromatography column (burette), ink sample/ food colour, Silica Gel, beakers, Vials or small beakers cotton
plug.
PROCEDURE:
COLUMN PACKING AND SAMPLE APPLICATION:
• A small piece of cotton was placed at the bottom of burette.

• Followed by silica gel which must be compacted (dry with small blows on the burette or wet with
ethanol) to reach 2-4 cm height. Later, 2 drop of the sample previously diluted were gently placed on
the top of the column.
• Alternately, a clean toothpick can be dipped directly into a food coloring bottle and then submerged in
a small amount of mobile phase left in the packed column.
• The inked cotton can also be introduced directly to the column.
• The sample was pushed inside the Silica Gel by using an air device or the syringe plunger (smooth
rotations are needed to take out the plunger).
• Sample Elution and fraction collection. Mobile phase i.e water was slowly poured into the top of the
column and allowed to run forced by the air flow (using an air device or the plunger).
• The mobile phase is collected in fractions into small containers.
• The addition of mobile phase is continued until the separation of the colours is observed
(2 ml of mobile phase per 1 ml of column volume on average is needed).
• Green and yellow samples results in the best color separations, add a small amount of water to the
mobile phase in order to elute the more polar fractions.
• After collection of fractions, each were tested using paper/TLC for purity.
RESULT:
We have successfully separate sample of ink using column chromatography.
94
SOLVENT EXTRACTION
RECRYSTALLIZATION:
Recrystallization is a physical process. It is a laboratory technique used to purify solids based on their different
solubilities.The main reason behind recrystallization is the fact that substances will usually become more
soluble when it is cold. Therefore, a compound may dissolve in warm liquid but be insoluble at room
temperature. The difference in solubility at varying temperature allows an impure substance dissolved at a
higher temperature and then crystallize slowly at a lower temperature without re-trapping impurities
TYPES OF RECRYSTALLIZATION:
Following are the types of Recrystallization
• Single solvent recrystallization

• Double solvent recrystallization
• Hot-filtration Recrystallization
• Speeding
SINGLE SOLVENT CRYSTALLIZATION:
In single solvent method, the chosen solvent for crystallization will dissolve the compound at all when hot but
not at room temperature.
DOUBLE SOLVENT CRYSTALLIZATION:

Double solvent recrystallization is an alternative and very useful recrystallization method to single solvent
recrystallization. The first solvent is that in which desired compound is soluble at all temperatures. The second
solvent should not dissolve the desired compound at any Temperature or in hot solvent
The second solvent should induce crystallization when added to a saturated solution of Compound in the
primary solvent. The second solvent is added slowly until one of the compounds begin to crystallize from
solution and then the solution is cooled, Heating is not required for this technique but can be used. Desired
compound “A” or impurity” “B” will be insoluble in second solvent and precipitate. One solvent is good
solvent and other solvent is bad solvent. Good solvent is to dissolve aromatic compounds while bad solvent
is for crystal Formation.
Double solvent recrystallization is used when suitable solvent is not available.
95
SALICYLIC ACID:
CHEMISTRY:
• The chemical formula of salicylic acid is C7H6O3
• Melting point 158.6°C
• Boiling point 200°C
PHYSICAL PROPERTIES:
• Salicylic acid is lipophilic, monohydroxy-benzoic acid, a type of phenolic acid and a B- hydroxy acid.
• Colorless organic acid
• Greater solubility with organic solvent
• Insoluble in water
PHARMACEUTICAL USES:
• Salicylic acid is used as food preservative.
• Used as bacteriocidal, antiseptic.
• Used in production of different pharmaceutical products like aminos alicyclic acid.
• Used as anti-inflammatory.
• Used as keratolytic agent.
96
EXPERIMENT NO# 20
OBJECT:
Purification of salicylic acid by double solvent recrystallization.
REQUIREMENTS:
❖ Salicylic Acid
❖ Good solvent (ethanol)
❖ Bad solvent (water)
❖ Electrical balance
❖ Desiccator
❖ Filter Paper
❖ Spatula and Stirrer
PROCEDURE:
● Select the good and bad solvent.
● Dissolve small quantity of sample in ethanol first and then dissolve more until the sample is
completely dissolved.
● Be careful about the quantity of ethanol used.
● The quantity of ethanol should be as small as possible.
● After dissolution, check if solution requires filtration otherwise skip this step.
● Now add water drop by drop in solution.
● During addition of water, cloud like precipitates will occur. Continue adding water until this
formation become permanent.
● Now add 2 drops of ethanol to stabilize the cloud formation, if they don't disappear, don't
add more ethanol.
● Now filter the solution using simple filtration.
● During filtration, add water to that solution or crystal or precipitate to wash it.
● After filtration, let the residue dry at room temperature.
● Wrap filter paper around it and place it in a desiccator over weight.
● Next day, weigh the crystals and find the percentage recovery.
CALCULATION:
Percentage recovery = Weight of Salicylic Acid before Crystallization x 100
Weight of Salicylic Acid after Crystallization
Percentage recovery =
RESULT:
The percentage recovery of salicylic acid is found to be by double solvent recrystallization.
97
EXPERIMENT NO# 21
OBJECT:
Purification of Benzoic acid by Double Solvent Crystallization
MATERIALS:
• Benzoic acid
• Water
• Ethanol
• Electrical balance
• Filter paper
• Funnel
• Stirrer
• Desiccator
PROCEDURE:
• Select good and bad solvent
• Good solvent collected in ethanol, while bad Solvent collected in water.
• Weight about 1g of benzoic acid.
• Dissolve small quantity of sample in ethanol first and then dissolve more until the sample is completely
dissolved.
• Be careful about the quantity of ethanol used.
• The quantity of ethanol should be as small as possible.
• After dissolution, check if solution requires filtration, otherwise skip this step.
• Now add water drop by drop in solution.
• During addition of water, cloud like precipitates will occur. Continuous addition of water until this
formation become permanent.
• Now add 2 drops of ethanol to stabilise the cloud formation. If they don’t disappear don’t add more
ethanol.
• Now filter the solution, using simple filtration.
• During filtration add water to that solution or crystal or precipitate to wash it.
• After filtration let the residue dry it at room temperature.
• Wrap filter paper around it and place it in a desiccator overnight.
• Next day, weigh the crystals and find the percentage recovery.
OBSERVATION:
Weight of Benzoic acid before Crystallization =
Weight of Benzoic acid after Crystallization =
98
CALCULATION:
Percentage recovery = Weight of Benzoic Acid before Crystallization x 100
Weight of Benzoic Acid after Crystallization
RESULT:
The percentage recovery of benzoic acid (C7H6O2) is found to be by double solvent.
99
EXPERIMENT NO# 22
OBJECT:
Purification of Benzoic Acid by Single solvent method.
MATERIALS:
• Benzoic Acid 1 gm
• Analytical balance
• Filter paper
• Hot plate stirrer
• Thermostatic drying oven
• Beaker
• Hot water
• Glass rod
• Desiccator
• Tap water
THEORY:
RECRYSTALLIZATION
Recrystallization is a purification technique by dissolving both impurities and a compound in an appropriate

solvent, either the desired compound or impurities can be removed from the solution, leaving the other behind.
It is named for the crystals often formed when the compound precipitates out. Alternatively, recrystallization
can refer to the natural growth of larger ice crystals at the expense of smaller ones.
It works because:
• Different substances have different solubilities in the same solvent.

• Only molecules of the same compound will fit easily into the crystal lattice of that compound.
Impurities remain in solution or stick on the outside of the crystal lattice crystallization.
SINGLE SOLVENT RECRYSTALLIZATION:
The chosen recrystallization solvent will dissolve the compound when hot, but not at room temperature. It is
the most basic and commonly used but heating is also used. It involves choosing the solvent, dissolving the
sample, hot filtration, cooling, washing the crystals and drying the crystals.
BENZOIC ACID
Benzoic acid is a crystalline colorless solid and a simple aromatic carboxylic acid. The name is desired from
“Gum Benzoin” which was for a long time its only known source. Benzoic acid occurs naturally in many
plants and source as an intermediate in the biosynthesis of many secondary metabolities. Salt of benzoic acid
is used as food preservative and benzoic acid is an important precursor for the industrial synthesis of many
other organic substances. The salts and esters of benzoic acid are known as “Benzoates”.
100
PROPERTIES:
• Chemical formula:C7H6O2
• Molecular weight:132.12g/mol
• Appearance: Colorless crystalline solid
• Odor: faint, pleasant order
• Density: 1. 2659g/cm3
• Melting point: 122.410C
• Boiling point: 299.20C
• Solubility: insoluble in cold water but freely soluble in hot water.
CHARACTERISTICS:
• It should be faintly volatile.

• It should be non-toxic and inflammable.
• Boiling point of the solvent should be lower than the melting point of the compound to be crystallize.
USES:
• Used for the treatment of fungal skin diseases i.e. ringworm, athletes foot and tinea.
• It is used as expectorant, analgesic and antiseptic.
• It is common standard for calibrating a bomb calorimeter.
PROCEDURE:
1. Take 1gm of Benzoic acid, this sample of benzoic acid contains small impurities (Water soluble)
2. Now heat about 50 ml of water in a beaker
3. Now slowly add water to the impure benzoic acid until the benzoic acid is complete dissolve.
4. Then add additional 1-2 ml of hot water to keep it dissolve.
5. Skip filtration if the sample have no color full impurities or insoluble impurities.
6. Now allow the mixture to cool at room temperature
7. Use ice bath if available.
8. Filter the crystals using hot opacity filtration.
9. Let the crystal dry in an oven & weight them to find out percentage recovery.
CALCULATION:
Percentage recovery = Weight of Benzoic Acid before Crystallization x 100

Weight of Benzoic Acid after Crystallization
RESULT:
The percentage of Benzoic acid is found to be by single solvent recrystallization.

101
EXPERIMENT NO# 23
OBJECT:
Isolation of casein from milk by using solvent extraction technique.
REQUIREMENTS:
• Beaker
• Conical flask
• Graduated cylinder
• Glass watch
• Filter paper
• Burner
• Reagents (ethanol ,acetic acid and milk sample)
THEORY:
MILK:
Milk is a white liquid produced from mammary glands of mammal. It is the primary source of nutrition for
infant mammals before they are able to digest other types of food. Early lactation with contain colostrums
which carries the mother’s antibodies to its young and can reduce the risk of many disease. it contains other
nutrients including protien and lactose. Milk provide high quallity protein,vitamins and minerals and is a
sourse of energy.
PHSIOCHEMICAL PROPERTIES:
The gross properties of milk include:
Milk is an emulsion of fat globlues .A suspension of casein micelles (casien,calcium, phosphorous) all which
are suspended in aqeous phase. it contain solubilize lactose protein and minerals . Leucocyte in milk are a part
of suspended phase. Milk is an emulsion or colliode of butter fat globules, with in a water base fluid that
contain dissolved carbohydrate and protiens aggregate with minerals. Because it is produce as a food source
for the young , all of it contents benefits for growth. The princpal requiremet are energy (lipids ,lactose and
proteins), biosynthesis of non essential fatty acid , vitamins and inorganic elemnts and water .
PH:
6.4 to 6.8(it changes over time ).
LIPIDS:
Initially milk fat is secreted in the form of a fat globule surrounded by a membrane.
PROTEINS:
Normal bovine milk contains 30-35g of proteins per liter of which about 80% is arranged in casein micelles.
Total proteins in milk represent 3.2% of its composition.
102
SALTS, MINERALS AND VITAMINS:
Calcium, Phosphate, Magnesium, sodium, potassium, citrate and Chlorine are included as minerals at
concentration of 5-40 mM. The milk salts strongly interact with casein, most notably calcium phosphate. In
addition to calcium, milk is a good source of many other vitamins, Vitamin A, B6, B12, C, D, K, thiamine,
Niacin, Biotin, Riboflavin, Folate and Pantothemic acid, etc all is present in milk.
CARBOHYDRATES AND MISCELLANEOUS CONTENTS:

Milk contains several different carbohydrates including Lactose, Galactose, Glucose and other
Oligosaccharides. Lactose gives milk its sweet taste and contributes 40% of whole concentration, milk’s
calories. Lactose is a disaccharide composite of glucose and galactose. Other components found in raw cow
milk are living WBCs, mammary gland cells, various bacteria and large number of active enzymes. Milk is a
dispersion of fat globules (fat particles) and casein micelles (protein particles) in a Continuous phase of water,
sugar (lactose), whey proteins and minerals.
• Milk plasma: milk minus fat (skimmed milk)

• Milk serum: plasma minus micelles/cheese whey
• Milk permeate: serum less whey protein
USES OF MILK:
• Keep bones healthy.
• Helps in weight loss
• Beats stress
• Builds muscles
• Healthy body
• Milk is great appetizer and healthy snack
COMPOSITIONS:
The composition of milk in the V.S is:
• Water 87.77
• Lactose 4.9%
• Fat 34%
• Protein 3.3%
• Minerals 0.7%
Milk composition varies depending on the species, Breed, the animals feed and the steps of Lactation.
CASEIN:
Casein is a protein, which is mostly found in milk. Casein is a family of related phosphor proteins. It
comprising 20-45% of human milk and 80% of cow’s milk. It has a wide variety of Used, from being a major
component of cheese to uses from use as food additives. The most Common form casein is Sodium caseniate.”
As a food source casein supplies amino acid,Carbohydrate, two essential elements calcium & phosphorus,
Casein is composed of several similar proteins, which form a multi molecular, granular structure called a
Casein micelle. In addition to casein molecule, the casein micelle-contains salt and water (mainly calcium
dephosphorus). Some enzymes are associated with casein micelle too.The micelle structure of casein in milk
103
is an important part of mode of digestion of milk in the stomach of intestine. Cassin is one of the most basic
abundant organic components of milk. Individual molecules of casein alone are not very soluble in the aqueous
environment of milk However, the casein micelle granules are maintained as a colloidal suspension in milk.
If micelle structure is disturbed, the micelle may come apart and the casein may come out of the solution.
Forming the gelatinous material of the curd. This is part of the basis for formation of all non-fluid milk
products like cheese.
Casein has an appropriate amino acid composition that is important for growth and developing of the musing
young.
Casein are highly digest able in the intestine and highly source of amino acid. Casein is a slow digesting
protein while whey is a fast digesting protein
1. Rennet Casein:
Obtained by enzymatic precipitation
2. Acid Casein:
Obtained by acidifying skim milk to the isolation point. (Ph = 4.6-4.7)
In addition to these two main types, there is 2 other commercially available casein product of importance.
i. Co-precipitate: Made by heating skim milk to a high temperature and then precipitate.It with CaCl.
ii. Cascinates: Commercially commonly sodium casein obtained from acid casein dissolved in sodium
hydrochloride.
USES OF CASEIN:
• In making cheese.
• In formulating glue.
• In preparing plastics and glue.
• As a thickener and emulsifier.
• As intermediate in pharmaceutical.
• As adhesive agent and colorant in lather and paper making industry
• For encapsulation of probiotic or antibiotic.
• Casein paint is a fast drying, water soluble medicine used by artist.
• Use as protein supplement.
PROCEDURE:
• Measure 50ml milk by graduated cylinder and pour it in a beaker.

• Heat the solution i.e. milk for homogenization and add few drops of acetic acid with constant stirring.
Don't add it rapidly.
• Keep the beaker on burner until the liquid becomes transparent and casein layer precipitate.
• Collect casein through filter paper (filtration), and keep it in adhere beaker.
• Add 2ml of ethanol solution into precipitates and stir for few minutes.
• Keep the precipitate for few days to calculate percentage performances.
CALCULATIONS:
104
% Recovery: weight of casein/weight of milk x 100
RESULT:
By using solvent extraction technique, casein has been successfully isolated from milk. The percentage of
casein recovered was found to be __________
105

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CERTIFICATE

This is to Certify that _____________________________ of Pharm-D 4th

Object Page no. Remarks Sign

WHAT IS PAPER CHROMATOGRAPHY?

PRINCIPLE OF PAPER CHROMATOGRAPHY:

PAPER CHROMATOGRAPHY DIAGRAM:

APPLICATIONS OF PAPER CHROMATOGRAPHY:

TYPES OF PAPER CHROMATOGRAPHY:

IMPORTANCE OF PAPER CHROMATOGRAPHY:

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf

4. In millimeters, how far did the solvent travel?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

4. In millimeters, how far did the solvent travel?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=

2. What served as the solvent for the ink?

6. Why did the ink separated?

7. Why did some colors move greater distance?

PART B: ACQUISITION OF CHROMATOGRAM:

PART C: INTERPRETATION OF CHROMATOGRAM:

COLOR OF INK SEPARATION OF RETENTION FACTOR (Rf Value)=