Applications of Paper

Chromatography
Paper chromatography has evolved over the years and has found widespread
applications in separation of molecules of different polarities. Innumerable
applications have been reported in analysis of different classes of compounds such
as:

Amino acids and organic acids.

Alkaloids.
Polysaccharides.
Proteins and peptides.
Natural and artificial pigments.
Plant extracts.
Typical applications in key areas are briefly outlined here

THE APPLICATION OF PAPER

CHROMATOGRAPHY TO FORENSIC CHEMISTRY:-
Unknown substances left at a crime scene can be identified by separating the
molecules that make them up. Matching this unknown chromatogram to
chromatograms of known substances can help identify the unknown
substance providing a clue to the crime.
The ink used in a note left at a crime scene can be identified through paper
chromatography. The chromatogram showing the components of the ink
used in the note can be compared to chromatograms of known inks
hopefully identifying the type and brand of ink used providing a clue to the
crime.

This report summarizes many of paper chromatography uses in the field of

Forensic Chemistry. Paper chromatography since its rediscovery barely ten years
ago has been applied in every branch of chemistry for the separation, purification,
and identification of a great range of compounds. The number of subjects and
materials that fall within its sphere of influence increases daily. While there is no
general theory that covers all aspects of the separation of compounds on paper it is
useful to think of this separation as being due to differences in the partition
coefficients of the compounds between the eluent phase and the stationary phase
held in the paper fibers. The method offers a rapid means of comparison, and in
many cases an identification, on a few micrograms of material and is obviously a
very powerful tool in the hands of the forensic chemist. It is not possible here to
review all the literature that is of interest in this field; indeed, it is the intention
only to give a few examples in which from personal experience paper
chromatography has proved to be an invaluable aid, forced to resort in many cases
to such tests as empirical color reactions and physical comparisons in an effort to
compare or contrast his sample with supplied control substances or a sample of
known origin. It is in this field that paper chromatography provides a valuable
addition. Its advantages may be summarized as follows.

1. The method offers a ready separation and comparison of similar compounds on

a microgram scale. Consequently in the case of toxicological specimens, for
example, traces of drugs and their metabolites are easily separated from one
another and from such impurities as fats, and normal body breakdown products.

2. The Rf values, i.e. distance of travel of spot of the sample distance of travel of
solvent, pounds under investigation in several solvent systems provide a set of
numerical values that taken into account with other considerations are very often
specific.

3. Only micrograms of material are required and, while the method as usually
performed is only roughly quantitative, it is very easily made highly quantitative.

4. Where compounds having unknown physical properties are involved the active
fraction can easily be traced on the micro scale prior to a macro work up.

5. Because such a small quantity of material is used up the bulk of the sample can
often be retained-always a matter of great importance in forensic work. The
mechanics whereby the solvent is caused to flow over the paper are largely
governed by personal choice coupled with a desire to obtain maximum resolution
of the spots. The solvent can flow vertically ascending or descending-or
horizontally. Sufficient is it to say that no more than the paper, the solvent, and
some type of container are required. The Rf values and techniques for a vast
number of different classes of compounds have appeared in the literature, it is
essential to use published Rf values only as a guide and to run one's own control
specimens side by side with the unknown. This will have particular appeal to the
forensic chemist. The paper itself usually requires little pre-treatment although the
author invariably washes before use with acetic acid followed by distilled water, as
advocated by Hanes and Isherwood. Care must be taken in the interpretation of
chromatograms as it is not unknown for a pure compound to give more than one
spot or to change in Rf with the pH of the solution in which it is applied to the
paper. This emphasizes the importance of running exact controls. While the
solvents and types of paper used for the actual development are largely governed
by experience, it is possible here to give some general headings for the techniques
of identification. These are:

Natural color of substances under investigation.

Behaviour in ultraviolet light.
Use of chemical reagents designed to produce color or changes in behavior
under UV.
Biological tests on the paper or on eluted spots.
Radioactivity detected by counts or autoradiography.

From these few examples it will be seen that while the separation of compounds on
paper in several solvent systems, in one and two dimensions, provides highly
characteristic Rf values, there can be coupled with these values an ever increasing
number of spot tests so that the degree of final identification is very high indeed.
To turn from the general field to the subjects of interests in forensic chemistry is
not a big jump as the following examples will prove. The original experiments in
paper chromatography took place nearly one hundred years ago and were used to
separate dyestuffs. This provides a link with present day practice where
compounds that have no color are converted into colored derivatives either in
solution before development or actually on the paper itself. The separation of the
individual cresols and phenols in that favourite suicide draught-disinfectant may be
accomplished by diazotisation followed by a separation of the coloured diazo
derivatives

It is, however, to the toxicologist that the applications of paper chromatography

provide a very material aid. The advent of new drugs having a very high specific
activity and consequently a relatively low lethal dose has presented the toxicologist
with the need for the classification of poisonous material not on a milligram but on
a microgram scale. The use of paper chromatography provides the answer. The
identity of the poison can be found with a higher degree of certainty and using at
the very most only one tenth the quantity by paper chromatography than by the
older means of arbitrary color tests. This is because one has not only a set of color
tests on the pure material on the paper but also a set of very highly characteristic
numerical Rf values. The importance of a paper chromatographic separation of the
wanted material from all impurities, before the color tests are applied, cannot be
overestimated. Some measure of the resolving power of paper chromatography is
revealed by a study of the amino-acids and peptides separated on a single two
dimensional chromatogram by Dent

6. where no less than 61 very similar compounds are resolvable using this
technique. Another example is the separation of the 2' and 3' phosphates of various
nucleosides on paper

Soon no doubt examples will appear of this type of work being used by the
forensic chemist. In the inorganic field there have been many papers on the
separation of both anions and cations, and there are many applications of this type
of separation that will interest the forensic chemist and the toxicologist. It is hoped
that these few examples and suggestions will draw attention to the potentialities of
the technique and the horizon of the forensic chemist will by its use be even further
extended.

Reaction monitoring
In a chemical reaction over a period of time the concentration of reactants
decreases whereas the concentration of products increases. Developing the
chromatogram over different time intervals by spotting the reactants can give a fair
idea on the progress of reaction. Traditionally the technique was used for
qualitative monitoring but availability of densitometers made quantitative
estimations possible. However, rapid methods using spectroscopic techniques are
limiting the application of paper chromatography as a reaction monitoring option.

Isolation & Purification

Paper chromatography has been put to use as a purification and isolation technique
for components of mixtures. The separated components on the paper are cut,
dissolved in suitable solvents and their absorption characterized at specific
wavelengths using spectrophotometric methods. This approach is being superseded
by techniques such as Preparative HPLC.

Foods

Foods & Beverages

Paper chromatography has been primarily used for analysis of food colors in
synthetic drinks and beverages, ice creams, jams &jellies, sweets etc. Colors both
natural and synthetic are added to foods to improve their acceptability and to make
them more popular. Only edible colors are permitted for use so identification and
quantification becomes all the more important to ensure that no non- permitted
coloring agents are added to the foods.

Forensics

Forensic
A forensic scientist is often faced with a severe limitation on the quantity of
sample available for analysis. Paper chromatography offers a viable analysis option
for samples available in milligrams or microlitre quantities.

The major applications are for identification and comparison against reference
standards for drugs and their metabolites in viscera, explosive residues from blast
sites, inks used in forgery of documents and paint pigments investigations in hit
and run road accident cases.

Pharmaceuticals

Pharmaceutical Products

Paper chromatography provides useful information on development of new drug

molecules, reaction completion and progress of manufacturing processes. It also
offers a cost-effective alternative to monitoring the active ingredients in drug forms
administered in bio analytical studies. Its main contribution is when the quantity of
sample available is in minute amounts. In a subsequent article some reasons will be
presented for justification of continued popularity of paper chromatography in the
face of tough competition from more advanced chromatographic techniques.

DNA and RNA

It is used in the sequencing of DNA and RNA.
 To separate the plant leaves pigments
In our lab, the paper was placed in a solution of plant pigment and acetone and
methanol, the solvent. As the acetone solution traveled up the paper, it took the
plant pigment with it which dispersed and climbed at different rates. When the
solvent finished moving, blatant separations in the colors of the pigment were seen
and then could be calculated. By dividing the distance one component traveled by
the distance the solvent traveled, we could obtain the Rf value for the pigment.
These values are constant for any and every solution so this type of
chromatography is useful in concluding the composition of an unidentified
substance. The Rf value for olive green was 0.391, green was 0.641, yellow was
0.953.
ABSTRACT:-
Chromatography is used widely throughout the world of science from the field
of forensics to molecular biology as well as chemistry.

Paper chromatography, as the term implies, is carried out on paper; for example,
an ordinary filter paper can be used for this purpose. The main characteristics of
paper chromatography are the flat-bed arrangement and the choice of
heterogeneous phases. This is achieved in paper chromatography by immobilizing
a liquid (referred to as the stationary phase) on paper. It is important to recognize
at this point that paper contains a significant amount of water.

As mentioned above, paper chromatography is a flat-bed or an open-bed technique

that is similar to thin-layer chromatography, where a thin layer of sorbent is
coated onto a glass plate or foil. Both of these techniques are related in terms of
sample application, development, detection, and quantification.
The main advantages that paper chromatography offers are simplicity, low cost,
and unattended, hassle-free operation. It can be run in various modes, and
quantification may be achieved without the use of expensive instrumentation.
The purpose of this lab report is to explain what exactly happened to the labs, the
time that the experiment was executed and apply the results and conclusions that
were deduced. Also, specify the errors that might have happened during the
execution of the experiment.

Paper chromatography is gaining increasing importance in synthetic organic

chemistry.

Simplicity and availability of materials and equipment

High efficiency of separation
The possibility of working with microgram quantities
The characterization of substances in mixtures without prior separation of
individual components good separation of homologues
Study of reaction Kinetics
Paper chromatography is supplemented by thin-layer chromatography
which has an even higher resolving power but often uses more complicated
equipment.

Several technical points of general interest have arisen in the course of the work.
 ACKNOWLEDGMENT
Assalam u Alaikum!

For this report, I would like to acknowledge my lab partner Sara because working
with others in a lab allows you to compare data and possibly avoid any errors. I
would also like to acknowledge the professor, Dr. Noshab and Miss. Aasma for
doing such a great job of instructing all of us on what to do, and how to properly
work in a lab. Having a good professor that knows the material and is willing to
help you always makes things go by quicker and easier, and this lab was no
exception.

And also I would like to thank specially to my mother who put forward to me in
the way to research and doing practical work. Without her corporation I would not
be able to do research work and make this report.

Thank you
.INTRODUCTION

Paper chromatography has the widest field of application of all the

chromatographic laboratory methods. It has gained its significance and popularity
as an analytical method through the revolution achieved by its aid in the fields of
biochemistry and the organic chemistry of natural substances.

Although paper chromatography is not alone in its ability to separate homologues,

since it shares this property with other forms of partition chromatography such as
column chromatography and gas-liquid chromatography, the simplicity and
rapidity of the technique, together with its wide applicability, commend the use of
paper chromatography for this purpose.

HISTORY
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.

PREPARATIVE PAPER CHROMATOGRAPHY

The advantage of paper chromatography is that it gives good separation of
microgram quantities of material. Its separative ability led to the use of the method
for preparative purposes, i.e. for the chromatography of milligram and gram
quantities of mixtures of substances. Various modifications of technique were
made and the field of preparative paper chromatography was created. The method
is tedious, however, and can often lead to the concentration of impurities which are
eluted from the paper along with the main component. Sometimes losses of
material occur (especially of peptides and amino-acids) and then it is better to use
other methods, especially separation on ion-exchange columns.
 EQUIPMENTS OF PAPER CHROMATOGRAPHY
The equipment for paper chromatography consists basically of five items:

i. Pure filter paper

ii. Solvents
iii. A container
iv. A capillary pipette, and an atomizer for spraying the chromatogram with the
detection reagent. The atomizer is not essential, as the chromatogram can be
dipped into the reagent.
v. Chromatographic papers, of which there are many types, are best kept in
hermetically closed cabinets with removable shelves; one for each type of
paper. The papers should be excluded. Excess of humidity should be
avoided.
SPOTTING DEVICES FOR PAPER CHROMATOGRAPHY
Preparation of the chromatograms and application of the samples may be carried
out on a chromatographic table. Its surface is covered with the sheet of transparent
plastic with a longitudinal slit over the starting line. Below this slit there is a
corresponding slit in the table top. A heating device is placed under the slits and
hot air streams upwards and accelerates drying of the solutions applied. There are
several designs for spotting devices, and some of them can be built in to the
laboratory bench and drawn out when they are to be used. The solutions are usually
applied by means of a micro-pipette..

PAPER CROMATOGRAPHY VESSELS

It is possible to use glass cylinders or aquarium tanks for paper chromatography, or
large desiccators for circular chromatography. Tanks specially designed for
chromatography are commercially available and will usually hold several
chromatograms. Chromatography cabinets for the simultaneous development of a
large number of chromatograms have also been designed. They often have a steel
or wood frame, and glass or metal shelves. As the solvents may corrode the metal
parts of the cabinets, the glass plates forming the walls are placed close together so
that their edges touch. The glass is sometimes sealed to the metal with a cement
made of water-glass and talc. When correctly made this cement consists of a paste
which quickly solidifies into a very hard mass resistant to most solvents. Stainless
steel is recommended for the metal parts that come into contact with the vapors of
the solvents.
 MATERIAL FOR PAPER CHROMATOGRAPHY
The paper used for chromatography is chosen according to the problem to be
investigated. Factors to be taken into account are whether the paper is to be used
for qualitative or quantitative analysis, for analytical or preparative,
chromatography and whether the substances are hydrophilic or lipophilic neutral or
charged. For many purposes almost any kind of paper will do. Sometimes,
however the problem will require a careful selection of the paper to be used.
Chromatographic papers are commercially available and are pure, homogeneous in
structure, and have suitable capillarity.

The best type of chromatography papers are made from linters and consist of
practically pure cellulose. The papers are very often washed with various solvents
(including dilute acids) to remove inorganic and other impurities. The surface of
papers satisfactory for chromatography should not be too rough and should be
uniformly opaque. The paper should not contain oxy cellulose. The purest type of
paper is required for analytical paper chromatography.
 SOLVENTS FOR PAPER CHROMATOGRAPHY
The solvents were formerly chosen on the basis of tradition but since paper
chromatography is so widely used for almost all groups of organic and inorganic
substances, suitable solvents can generally be chosen from the data given in the
literature.

For example; Methanol, HCl, formalin, ethanol, water, methanol+ Acetic

acid(1:1), acetic acid+ ethyl acetate, acetone, methanol, hexane etc.
 PAPER CHROMATOGRAPHY
Paper chromatography (PC) is a form of plane chromatography in which a piece
of filter paper or cellulose is used as the stationary phase and a liquid solvent is
used as the mobile phase. Of the several forms of paper chromatography,
ascending PC in figure is the most popular. In ascending PC, a rectangular strip of
paper is used as the stationary phase and the mobile phase flows upward through
the paper. Generally, the strip is between 3 and 5 cm wide and about 20 to 30 cm
long. A notch is often cut in one end of the strip, as shown in figure, and an eye
dropper, syringe, pipette, or glass tube is used to place a spot of sample solution at
the top of the V in the notched end of the paper.

Paper chromatography is an analytical method used to separate colored

chemicals or substances. It is primarily used as a teaching tool, having been
replaced by other chromatography methods, such as thin-layer chromatography. A
paper chromatography variant, 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.
The setup has three components. The mobile phase is a solution that travels up the
stationary phase, due to capillary action. The mobile phase is generally an alcohol
solvent mixture, while the stationary phase is a strip of chromatography paper, also
called a chromatogram. A chromatographic method is called adsorption
chromatography if the stationary phase is solid.
Types of paper chromatography
Descending paper chromatography - Development of the chromatogram is
done by allowing the solvent to travel down the paper. Here, mobile phase is
placed in solvent holder at the top. The spot is kept at the top and solvent flows
down the paper from above.
Ascending paper chromatography - Here the solvent travels up the
chromatographic paper. Both descending and ascending paper chromatography
are used for the separation of organic and inorganic substances.
Ascending-descending paper chromatography - This is the hybrid of both of
the above techniques. The upper part of ascending chromatography can be
folded over a rod in order to allow the paper to become descending after
crossing the rod.
Radial paper chromatography - This is also called circular chromatography.
A circular filter paper is taken and the sample is deposited at the center of the
paper. After drying the spot, the filter paper is tied horizontally on a petri dish
containing solvent, so that the wick of the paper is dipped in the solvent. The
solvent rises through the wick and the components are separated into concentric
circles.
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 a right angle to the direction of the first run.
 PHENOMENON OF PAPER CHROMATOGRAPHY
In order to minimize the spot size after development, the sample is placed on the
paper in a spot of as small an area as possible, as the chromatogram develops, the
sample components diffuse to the regions of lower concentration bordering the
spot, and there is a resulting increase in the size of the developed spots. After the
sample solution has been spotted on the paper and the sample solvent has been
removed by evaporation, the pointed tip of the paper strip is dipped into a vessel
containing the mobile phase. The paper strip and mobile phase reservoir are
usually enclosed in an airtight glass vessel. The air within the glass vessel is kept
saturated with vapor from the mobile phase in order to prevent evaporation of the
mobile phase from the paper strip. The saturation is sometimes accomplished by
hanging pieces of filter paper which are soaked with the mobile phase inside the
glass vessel.

The mobile phase is pulled upward through the paper strip by capillary action. The
V in the end of the paper strip directs the mobile phase over the sample spot. As
the mobile phase passes over the sample, the sample components are partitioned
between the mobile phase solvent and the stationary liquid phase which is attached
to the paper.
With other types of paper chromatography, a notch is normally not cut into the end
of the paper stationary phase which dips into the mobile phase reservoir. In
descending PC shown in figure, the mobile phase is added to the top of the strip
(rather than the bottom) and descends through the paper. The major advantage of
descending Pc chromatography as compared to ascending PC is the greater
distance the mobile phase can move during the development and hence the better
the separation between the sample components. The upward movement of solvent
during ascending PC is limited by the downward pull of gravity on the solvent.

PC can also be done by using radial development. The sample is spotted in the
center of a circular piece of the stationary phase, and the mobile phase solvent is
slowly added with a pipet, syringe, or eye dropper to the dried spot. The outward
motion of the mobile phase from the sample spot results in rings of separated
sample components. A variation of the radial development method is a ring oven-
technique, in which the circular paper is supported at its outer edge on an oven in
the shape of a ring. The oven is electrically heated to a temperature high enough to
evaporate the mobile phase as it reaches the edge of the filter paper. The
evaporation can also be used to concentrate the sample components on the edge of
the paper.
 R value, solutes, and solvents
The retardation factor (R) may be defined as the ratio of the distance traveled by
the solute to the distance traveled by the solvent. R values are usually expressed as
a fraction of two decimal places.
Rf Value = Distance from Baseline travelled by Solute

Distance from Baseline travelled by Solvent (Solvent Front)

Calculation
If R value of a solution is zero, the solute remains in the stationary phase and
thus it is immobile. If R value = 1 then the solute has no affinity for the stationary
phase and travels with the solvent front. To calculate the R value, take the
distance traveled by the substance divided by the distance traveled by the solvent
(as mentioned earlier in terms of ratios).
For example, if a compound travels 9.9 cm and the solvent front travels 12.7 cm,
(9.9/12.7) the R value = 0.779 or 0.78. R value depends on temperature and the
solvent used in experiment, so several solvents offer several R values for the
same mixture of compound. A solvent in chromatography is the liquid the paper is
placed in, and the solute is the ink which is being separated.
A substance may be characterized by determination of a physical constant, called
the Rf value, which is a function of the partition coefficient and is constant for a
given compound in a given chromatogram system.

Background
As described in paper chromatography there is what is known as
the stationary phase which is the absorbent Chromatography paper and
the mobile phase which is a liquid solvent (or mixture of solvents) used
to carry the sample solutes under analysis along the paper. Usually, one
uses chromatography to find out the components of a sample which are
seperated depending how much soluble these are in particular solvents
and hence how far they travel along the chromatography paper. Samples
are usually of organic matter (not ionic salts) which dissolve in certain
polar solvents (namely water) or non-polar (organic) solvents.

Principle
In order to make the technique more scientific rather than a mere interpretation by
sight, what is called the Retention Value (Rf value for short) was applied in
chromatography. A particular compound will travel the same distance along the
 stationary phase by a specific solvent (or solvent mixture) given that other
experimental conditions are kept constant. In other words, every compound (dye,
pigment, organic substance etc) have a specific Rf value for every specific solvent
and solvent concentration. Rf values come very handy for identification because
one can compare Rf values of the unknown sample with Rf Values of known
compounds.

Rf Values for Identification

Note that different compounds can have the SAME Rf value for a particular
solvent, but unlikely to have similar Rf for a number (2-4) of different
solvents. Therefore the more different solvents (or mixtures) are used, the
more RF values are obtained, and so the more concise the identification is.
Identification relies on comparing a number of Rf values of the unknown
sample with known Rf values of a number of known dyes.

Environment Conditions
As mentioned before, the Rf value of a particular pure dye or analyte in a
particular solvent (or mixture) is constant if the following experimental
conditions are kept unaltered:
Temperature
Chromatography medium, i.e. same type and grade of
Chromatography Paper
Solvent concentration and purity
Amount of sample spotted on Chromatography medium

Pigments and polarity

Paper chromatography is one method for testing the purity of compounds and
identifying substances. Paper chromatography is a useful technique because it is
relatively quick and requires only small quantities of material. Separations in paper
chromatography involve the same principles as those in thin layer chromatography,
as it is a type of thin layer chromatography. In paper chromatography, substances
are distributed between a stationary phase and a mobile phase. The stationary
phase is the water trapped between the cellulose fibers of the paper. The mobile
phase is a developing solution that travels up the stationary phase, carrying the
samples with it. Components of the sample will separate readily according to how
strongly they adsorb onto the stationary phase versus how readily they dissolve in
the mobile phase.
When a colored chemical sample is placed on a filter paper, the colors separate
from the sample by placing one end of the paper in a solvent. The
solvent diffuses up the paper, dissolving the various molecules in the sample
according to the polarities of the molecules and the solvent. If the sample contains
more than one color, that means it must have more than one kind of molecule.
Because of the different chemical structures of each kind of molecule, the chances
are very high that each molecule will have at least a slightly different polarity,
giving each molecule a different solubility in the solvent. The unequal solubility
causes the various color molecules to leave solution at different places as the
solvent continues to move up the paper. The more soluble a molecule is, the higher
it will migrate up the paper. If a chemical is very non-polar it will not dissolve at
all in a very polar solvent. This is the same for a very polar chemical and a very
non-polar solvent.
It is very important to note that when using water (a very polar substance) as a
solvent, the more polar the color, the higher it will rise on the paper.
.
References
1. Haslam, Edwin (2007). "Vegetable tannins Lessons of a phytochemical
lifetime". Phytochemistry. Ettre,512.
2. Pakhomov, V. P. (2003). "Chromatography in Pharmaceutical Chemistry
(100 Years of the Discovery of Chromatography by M. S. Tswett)".
3. Chromatography: a century of discovery 1900-2000: the bridge to the
sciences/technology. Journal of Chromatography Library. Charles
4. Chromatographic theory and liquid chromatography by Franklin pirk
5. Partition chromatography
6. The essence of chromatography by Colin F.Poole
7. A manual of paper chromatography by Richard J.Block
8. Paper chromatography by Joseph Sherma
9. Paper chromatography by Fredrich Cramer