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ADVANCED BIOCHEMICAL INSTRUMENTATION (BCH 582)

UNIVERSITY OF NIGERIA, NSUKKA


PAPER CHROMATOGRAPHY
BY
AYOLOTU MUYIWA AUGUSTINE

PAPER CHROMATOGRAPHY
Chromatography is a technique that is used to separate and to identify
components of a mixture. This analytical technique has a wide range of applications in
the real world since many substances are mixtures of chemical compounds. There are
different types of chromatography, but all of them are based on the same principle: the
different molecules or ions in the mixture will interact differently with the stationary
phase of the chromatograph, and get separated in the process. The different techniques
of chromatography use different substances as the stationary and mobile phases.
Chromatography in broad term can either be analytical or preparative. Analytical
chromatography is used for determining the relative proportions of the different
components in the mixture, while the separation of the different components is what
preparative chromatography is used for.
Chromatography techniques are generally classified on the basis of the
mechanism of separation. The types of chromatography, based on the mechanism of
separation, are adsorption chromatography, partition chromatography, ion exchange
chromatography, molecular exclusion chromatography, and affinity chromatography.
Paper chromatography is based on the principle of partition chromatography.
The discovery of paper chromatography in 1943 by Martin and Synge provided, for
the first time, the means of surveying constituents of plants and for their separation and
identification. There was an explosion of activity in this field after 1945. Paper
chromatography is an analytical method technique for separating and identifying
mixtures that are or can be coloured, especially pigments. This can also be used in
secondary or primary colours in ink experiments. This method has been largely replaced
by thin layer chromatography, but is still a powerful teaching tool. Double-way paper
chromatography, also called two-dimensional chromatography, involves using two
solvents and rotating the paper 90 in between. This is useful for separating complex
mixtures of compounds having similar polarity, for example, amino acids. If a filter paper
is used, it should be of a high quality paper. The mobile phase is developing solutions that
can travel up to the stationary phase carrying the sample alongside with it

In paper chromatography, the sample mixture is applied to a piece of filter paper,


the edge of the paper is immersed in a solvent, and the solvent moves up the paper by
capillary action. Components of the mixture are carried along with the solvent up the
paper to varying degrees, depending on the compound's preference to be adsorbed onto
the paper versus being carried along with the solvent. The paper is composed of cellulose
to which polar water molecules are adsorbed, while the solvent is less polar, usually
consisting of a mixture of water and an organic liquid. The paper is called the stationary
phase while the solvent is referred to as the mobile phase. Performing a
chromatographic experiment is basically a three-step process:
1) application of the sample,
2) "developing" the chromatogram by allowing the mobile phase to move up the paper,
and
3) calculating Retardation Factor (Rf) values and making conclusions.
In order to obtain a measure of the extent of movement of a component in a
paper chromatography experiment, we can calculate an "Rf value" for each separated
component in the developed chromatogram. An Rf value is a number that is defined as:
distance traveled by component from application point

Rf = ---------------------------------------------------distance traveled by solvent from application point

Several factors explain why the different parts of the mixture separate out as they do:
1. Solubility: If the components of the mixture are soluble in the solvent being used,
the mixture will be carried up the paper strip as the solvent travels. If the material
is soluble, the mixture will dissolve as the solvent front moves through it. If the
material is a mixture of substances, some of these substances will likely be more
or less soluble than others. The more soluble substances will move faster and to a
greater distance than those that are less soluble.
2. Molecular Weight: Those substances of lighter molecular weight will move higher
up the paper than those substances having a higher molecular weight.

3. The chromatography paper is made of cellulose, a polar substance, and the


compounds within the mixture travel farther if they are non-polar. More polar
substances bond with the cellulose paper more quickly, and therefore do not
travel as far.
Types of Paper Chromatography
1. Descending Paper Chromatography-In this type, development of the chromatogram is
done by allowing the solvent to travel down the paper is called Descending
Chromatography. Here the mobile phase is present in the upper portion.
2. Ascending Paper Chromatography-Here the solvent travel upward direction of the
Chromatographic paper. Both the Descending and Ascending Paper Chromatography are
used for separation of Organic and Inorganic substances.
3. Ascending-Descending Paper Chromatography-It is the hybrid of both the above
technique. The upper part of the Ascending chromatography can be folded over a rod
and allowing the paper to become descending after crossing the rod.
4. Horizontal Paper Chromatography-It is also called as Circular chromatography. Here a
circular filter paper is taken and the sample is given at the center of the paper. After
drying the spot the filter paper tied horizontally on a Petridish containing solvent. So that
Wick of the paper is dipped inside the solvent. The solvent rises through the wick and the
component get separated in form of concentrate circular zone.
5. Two-Dimensional Paper Chromatography-In this technique a square or rectangular
paper is used. Here the sample is applied to one of the corners and development is
performed at right angle to the direction of first run.
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
A non-polar solvent such as hexane could be used to develop a chromatogram.
Non-polar molecules in the mixture of separation 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.
Uses of Paper Chromatography
Given below are some important uses of paper chromatography.
Separating Colored Pigments
Paper chromatography is an effective technique for separating colored pigments from a
mixture. A few drops of the mixture of colored pigments are placed on the filter paper
(stationary phase) and it is then slowly submerged into a jar of solvent (mobile phase). As
the solvent rises up the paper, it dissolves the molecules present in the mixture, their
solubility depending on their polarity. Because of different polarity, molecules of each
pigment leaves the solution at different places, as the solvent continues to rise up the

stationary phase. Thus, each pigment rises up to a particular level on the


chromatography paper, and gets separated in the process.
Obtaining Pure Compounds
Paper chromatography is used to obtain pure compounds from a mixture. This is done by
cutting out and redissolving the patterns formed by each constituent. Also, this technique
can be effectively used to remove impurities from chemical compounds. Due to the
process of paper chromatography, the impurities get separated from the compound and
the pure compound can be obtained.
Qualitative Analysis
Paper chromatography is one of the methods of qualitative analysis, to analyze or
separate the different constituents of a mixture. It is a useful tool for separating polar as
well as nonpolar solutes. Pharmaceutical companies use this technique to analyze the
different compounds in drugs.
Pathology and Forensic Science
Paper chromatography is useful in the field of forensic science, for investigation of crime.
This is because this process can be successfully carried out even with very small
quantities of material. Samples from crime scenes are collected to be analyzed and
identified, using this technique.
Analyzing Complex Mixtures
Paper chromatography is used to detect the presence of, or identify certain organic
compounds such as carbohydrates and amino acids, from a complex mixture of organic
compounds.
How Does Paper Chromatography Work
To understand the principle of paper chromatography, we must learn what partition
chromatography is. As the name suggests, partition chromatography is a method of
separating the components of a mixture in which the constituents of the mixture are
partitioned or separated between two liquid phases, one of which is supported by a solid
and is termed as the stationary phase. The other liquid phase is the solvent in which a
small amount of the mixture (that is to be separated) is dissolved. In paper
chromatography, the solid in question is a filter paper, and the stationary phase is water
in the pores of the filter paper. The following are the steps to perform paper
chromatography.
Step 1: Take a long rectangular piece of filter paper and draw a straight line on it using a
pencil, a few centimeters above one of its shorter edges. This is your start line. Place a
drop of the mixture on the start line, using a capillary tube.

Step 2: Take a glass jar and pour a small amount of the solvent liquid into it. Now, place
the filter paper inside the glass jar such that the part of it below the start line, is
submerged in the solvent. Do not disturb the setup and you shall see that the solvent in
the jar slowly rises up due to the capillary action of the paper. Wait for around 15-20
minutes, till the solvent nearly reaches the top of the paper.
Step 3: Remove the filter paper from the jar and mark the highest point on the filter
paper till which the solvent has risen. You shall see that the different components of the
mixture have been carried to different levels, by the solvent. In the above diagram, you
can see two colored spots formed by two different solutes, A and B. This is due to the
difference in the affinity of the solutes present in the mixture, to the filter paper
(stationary phase). So, while one solute (solute B) is easily carried farther away by the
solvent, the other is not. This results in the solutes getting separated from the mixture.
Step 4: When the filter paper has dried, note the distance covered by each constituent
from the start line. Now, calculate the retardation factor (Rf value) by the following
formula. This value can never be more than 1, which implies that a solute can never
travel ahead of the solvent.
Retardation Factor (Rf) = Distance traveled by the solute from the start lineDistance
traveled by the solvent from the start line
Note: If the spot formed by a component is irregular, you need to measure the distance
from the middle of the spot to the start line.

REFERENCES
Haslam, Edwin (2007). "Vegetable tannins Lessons of a phytochemical lifetime".
Phytochemistry 68 (2224): 271321. doi:10.1016/j.phytochem.2007.09.009. PMID
18037145
http://4.bp.blogspot.com
http://www.buzzle.com/articles/paper-chromatography-uses.html
https://en.wikipedia.org/wiki/Paper_chromatography