BIOLOGY INVESTIGATORY PROJECT

PROJECT: – To Seprate amino acid from

mixture using paper chromatography

Name – Shruti. M. Garje

Roll No – 28
Std – 12th Science (Biology)
Subject Head – Miss Radhika Korane (PGT
Biology)
 ACKNOWLEDGEMENT
The success and final outcome of this project
required a lot of guidance and assistance from
many people and I am extremely privileged to
have got this all along the completion of my
project. All that I have done is only due to such
supervision and assistance and I would not forget
to thank them. I respect and thank our Chemist
I respect and thank our biology teacher, Miss
Radhika Korane for providing me an opportunity
to do the project work in VSG International
School and giving us all support and guidance,
which made me complete the project duly. I am
extremely thankful to her for providing such a nice
support and guidance, although she had a busy
schedule. I provide a special mention to my
parents for their constant support. Also, I am
greatly thankful to my team members for their
humble co-operation
 Introduction

Chromatography is used to separate mixtures of

substances into their components. All forms of
chromatography work on the same principle. They
all have a stationary phase (a solid, or a liquid
supported on a solid) and a mobile phase (a liquid
or a gas). The mobile phase flows through the
stationary phase and carries the components of the
mixture with it. Different components travel at
different rates. We'll look at the reasons for this
further down the page. In paper chromatography,
the stationary phase is a very uniform absorbent
paper. The mobile phase is a suitable liquid solvent
or mixture of solvents.
Producing a paper chromatogram: -
You probably used paper chromatography as one of
the first things you ever did in chemistry to separate
out mixtures of coloured dyes - for example, the dyes
which make up a particular ink. That's an easy
example to take, so let's start from there.
Suppose you have three blue pens and you want to find
out which one was used to write a message. Samples of
each ink are spotted on to a pencil line drawn on a
sheet of chromatography paper. Some of the ink from
the message is dissolved in the minimum possible
amount of a suitable solvent, and that is also spotted
onto the same line. In the diagram, the pens are
labelled 1, 2 and 3, and the message ink as M.

The paper is suspended in a container with a shallow

layer of a suitable solvent or mixture of solvents in it. It
is important that the solvent level is below the line with
the spots on it. The next diagram doesn't show details
of how the paper is suspended because there are too
many possible ways of doing it and it clutters the
diagram. Sometimes the paper is just coiled into a loose
cylinder and fastened with paper clips top and bottom.
The cylinder then just stands in the bottom of the
container.
The reason for covering the container is to make sure
that the atmosphere in the beaker is saturated with
solvent vapour. Saturating the atmosphere in the
beaker with vapour stops the solvent from evaporating
as it rises up the paper.
As the solvent slowly travels up the paper, the different
components of the ink mixtures travel at different rates
and the mixtures are separated into different coloured
spots.
The diagram shows what the plate might look like after
the solvent has moved almost to the top.

It is fairly easy to see from the final chromatogram that

the pen that wrote the message contained the same dyes
as pen 2. You can also see that pen 1 contains a mixture
of two different blue dyes - one of which might be the
same as the single dye in pen 3.
Rf values
Some compounds in a mixture travel almost as far as
the solvent does; some stay much closer to the base line.
The distance travelled relative to the solvent is a
constant for a particular compound as long as you keep
everything else constant - the type of paper and the
exact composition of the solvent, for example.
The distance travelled relative to the solvent is called
the Rf value. For each compound it can be worked out
using the formula:

For example, if one component of a mixture travelled

9.6 cm from the base line while the solvent had
travelled 12.0 cm, then the Rf value for that component
is:

In the example we looked at with the various pens, it

wasn't necessary to measure Rf values because you are
making a direct comparison just by looking at the
chromatogram.
You are making the assumption that if you have two
spots in the final chromatogram which are the same
colour and have travelled the same distance up the
paper, they are most likely the same compound. It isn't
necessarily true of course - you could have two
similarly colored compounds with very similar
Rf values. We'll look at how you can get around that
problem further down the page.

What if the substances you are interested in

are colourless?
In some cases, it may be possible to make the spots
visible by reacting them with something which
produces a coloured product. A good example of this is
in chromatograms produced from amino acid mixtures.
Suppose you had a mixture of amino acids and wanted
to find out which particular amino acids the mixture
contained. For simplicity we'll assume that you know
the mixture can only possibly contain five of the
common amino acids. A small drop of a solution of the
mixture is placed on the base line of the paper, and
similar small spots of the known amino acids are placed
alongside it. The paper is then stood in a suitable
solvent and left to develop as before. In the diagram,
the mixture is M, and the known amino acids are
labelled 1 to 5.
The position of the solvent front is marked in pencil
and the chromatogram is allowed to dry and is then
sprayed with a solution of ninhydrin. Ninhydrin reacts
with amino acids to give coloured compounds, mainly
brown or purple.
The left-hand diagram shows the paper after the
solvent front has almost reached the top. The spots are
still invisible. The second diagram shows what it might
look like after spraying with ninhydrin.
There is no need to measure the Rf values because you
can easily compare the spots in the mixture with those
of the known amino acids - both from their positions
and their colours. In this example, the mixture contains
the amino acids labelled as 1, 4 and 5. And what if the
mixture contained amino acids other than the ones we
have used for comparison? There would be spots in the
mixture which didn't match those from the known
amino acids. You would have to re-run the experiment
using other amino acids for comparison.

Two-way paper chromatography: -

Two-way paper chromatography gets around the
problem of separating out substances which have very
similar Rf values. I'm going to go back to talking about
coloured compounds because it is much easier to see
what is happening. You can perfectly well do this with
colourless compounds - but you have to use quite a lot
of imagination in the explanation of what is going on!
This time a chromatogram is made starting from a
single spot of mixture placed towards one end of the
base line. It is stood in a solvent as before and left until
the solvent front gets close to the top of the paper.
In the diagram, the position of the solvent front is
marked in pencil before the paper dries out. This is
labelled as SF1 - the solvent front for the first solvent.
We shall be using two different solvents.

If you look closely, you may be able to see that the large
central spot in the chromatogram is partly blue and
partly green. Two dyes in the mixture have almost the
same Rf values. They could equally well, of course, both
have been the same colour - in which case you couldn't
tell whether there was one or more dye present in that
spot.
What you do now is to wait for the paper to dry out
completely, and then rotate it through 90°, and develop
the chromatogram again in a different solvent.
It is very unlikely that the two confusing spots will have
the same Rf values in the second solvent as well as the
first, and so the spots will move by a different amount.
The next diagram shows what might happen to the
various spots on the original chromatogram. The
position of the second solvent front is also marked.
You wouldn't, of course, see these spots in both their
original and final positions - they have moved! The
final chromatogram would look like this:

Two-way chromatography has completely separated

out the mixture into four distinct spots. If you want to
identify the spots in the mixture, you obviously can't do
it with comparison substances on the same
chromatogram as we looked at earlier with the pens or
amino acids examples. You would end up with a
meaningless mess of spots. You can, though, work out
the Rf values for each of the spots in both solvents, and
then compare these with values that you have measured
for known compounds under exactly the same
conditions.
How does paper chromatography work?
Although paper chromatography is simple to do, it is
quite difficult to explain compared with thin layer
chromatography. The explanation depends to some
extent on what sort of solvent you are using, and many
sources gloss over the problem completely. If you
haven't already done so, it would be helpful if you
could read the explanation for how thin layer
chromatography works (link below). That will save me
a lot of repetition, and I can concentrate on the
problems.

The essential structure of paper: -

Paper is made of cellulose fibres, and cellulose is a
polymer of the simple sugar, glucose.

The key point about cellulose is that the polymer chains

have -OH groups sticking out all around them. To that
extent, it presents the same sort of surface as silica gel
or alumina in thin layer chromatography.
It would be tempting to try to explain paper
chromatography in terms of the way that different
compounds are adsorbed to different extents on to the
paper surface. In other words, it would be nice to be
able to use the same explanation for both thin layer and
paper chromatography. Unfortunately, it is more
complicated than that!
The complication arises because the cellulose fibres
attract water vapour from the atmosphere as well as
any water that was present when the paper was made.
You can therefore think of paper as being cellulose
fibres with a very thin layer of water molecules bound
to the surface.
It is the interaction with this water which is the most
important effect during paper chromatography.

Paper chromatography using a non-polar

solvent :-
Suppose you use a non-polar solvent such as hexane to
develop your chromatogram.
Non-polar molecules in the mixture that you are trying
to separate will have little attraction for the water
molecules attached to the cellulose, and so will spend
most of their time dissolved in the moving solvent.
Molecules like this will therefore travel a long way up
the paper carried by the solvent. They will have
relatively high Rf values.
On the other hand, polar molecules will have a high
attraction for the water molecules and much less for
the non-polar solvent. They will therefore tend to
dissolve in the thin layer of water around the cellulose
fibres much more than in the moving solvent.
Because they spend more time dissolved in the
stationary phase and less time in the mobile phase, they
aren't going to travel very fast up the paper.
The tendency for a compound to divide its time
between two immiscible solvents (solvents such as
hexane and water which won't mix) is known
as partition. Paper chromatography using a non-polar
solvent is therefore a type of partition chromatography.

Paper chromatography using a water and

other polar solvents: -
A moment's thought will tell you that partition can't be
the explanation if you are using water as the solvent for
your mixture. If you have water as the mobile phase
and the water bound on to the cellulose as the
stationary phase, there can't be any meaningful
difference between the amount of time a substance
spends in solution in either of them. All substances
should be equally soluble (or equally insoluble) in both.
And yet the first chromatograms that you made were
probably of inks using water as your solvent.
If water works as the mobile phase as well being the
stationary phase, there has to be some quite different
mechanism at work - and that must be equally true for
other polar solvents like the alcohols, for example.
Partition only happens between solvents which don't
mix with each other. Polar solvents like the small
alcohols do mix with water.
In researching this topic, I haven't found any easy
explanation for what happens in these cases. Most
sources ignore the problem altogether and just quote
the partition explanation without making any
allowance for the type of solvent you are using. Other
sources quote mechanisms which have so many strands
to them that they are far too complicated for this
introductory level. I'm therefore not taking this any
further - you shouldn't need to worry about this at UK
A level, or its various equivalents.

Contributions and Attributions:-

This page is licensed under a license and was authored,
remixed, and/or curated by Jim Clark. Page content has
been edited and updated to conform to the style and
standards of the Libre Texts platform; a detailed
versioning history of the edits to source content is
available upon request.

SEPARATION OF AMINO ACIDS BY

PAPER CHROMATOGRAPHY
AIM-
The aim of the experiment is to perform Ascending
paper chromatography for the separation of amino
acids present in the given sample.

BACKGROUND: -
Chromatography is used to separate mixtures of
substances into their individual components. All forms
of chromatography work on the same principle. They
all have basic requirements of stationary phase (a solid
or a liquid supported on a solid) and a mobile phase (a
liquid or a gas). The mobile phase flows through the
stationary phase and carries the components of the
mixture with it. Different components travel at
different rates based on their affinities toward
stationary phase and mobile phase. In paper
chromatography, the stationary phase is a very
uniform adsorbent paper. The mobile phase is a
suitable liquid solvent or mixture of solvents. Retention
(or) retardation factor (Rf)- Retention factor is defined
the ratio of the distance travelled by the solute to the
distance travelled by solvent. The distance travelled
relative to the solvent is called the Rf value.

REQUIREMENTS
Apparatus:
Glass beakers Whatman filter paper Petri dishes
Measuring cylinder Developing chamber and capillary
tubes etc. Chemicals: n-butanol Glacial acetic acid
Distilled water (4:1:5) Amino acids (Tryptophan and
threonine) Ninhydrin reagents. Solvent’s system and its
preparation methods 1. n-butanol and water are taken
in 4:5 ratios in a conical flask and allow it to saturate
for 24 hours. By using separating funnel separate
butanol and water. The saturated n-butanol and
Glacial acetic acid are taken in the ratio of 4:1 which
can be used as a solvent system (or) mobile phase.
Ascending paper chromatography The procedure for
ascending paper chromatography method is quite
simple as compared to other methods of
chromatography. The chromatography paper is cut
into rectangular strips and marks a line on the paper
with pencil at about 2 cm from the bottom. With the
help of capillary tube, the samples are applied at
different points on the starting line Now, place the
chromatography paper in the developing chamber,
which contains the mobile phase. While placing the
paper, it is important that the solvent level should not
reach the starting line or the sample spots and paper
shouldn’t touch the walls of the developing chamber.
After sometime the solvent rises up the paper or the
stationary phase by capillary action and dissolves the
sample. The components of the sample move along with
the solvent in upward direction. Check if the solvent
has reached near the top level of chromatography
paper. Then the paper is removed when it reaches the
top and marked the level with pencil. This level (or)
height is called the “solvent front”.

CONCLUSION
For ascending paper chromatography: -
The distance moved by tryptophan and threonine is cm
and cm respectively, and the solvent is cm Rf value of
tryptophan is Rf value of threonine is Rf value of
unknown mixture is & by performing the Ascending
paper chromatography, the distance moved by the
sample and unknown mixture is noted and by
substituting these values in the given Rf formula, the Rf
values of tryptophan, threonine and unknown samples
are known. By performing the Ascending paper
chromatography Rf values of both tryptophan and
threonine are found to be & respectively, and the
unknown samples are found to be tryptophan and
threonine. By performing the radial paper
chromatography Rf values of both tryptophan and
threonine was found to be & respectively, and the
unknown samples were found to be tryptophan and
threonine.