What is Paper Chromatography?

Principle and Procedure

Paper chromatography has proved to be very successful in the analysis of

chemical compound and lipid sample in particular. 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.
What is Amino acid and its Structural Chemistry?
Color reactions of Amino acids
Components of the mixture are carried along with the solvent up the paper
to varying degrees, depending on the compounds 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.
Protein Fundamental molecules are Amino acids
Why Proteins are Very Important? How to Explain?
The paper is called the stationary phase while the solvent is referred to as
the mobile phase. 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.

NATURE OF THE PAPER:

1
2
3
o
o
o

4
o
5

Table of Contents [hide]

Nature of the paper:
Apparatus:
Paper development:
3.1 1) Ascending techniques:
3.2 2) Descending techniques:
3.3 3) Two dimensional chromatography (3D):
3.3.1 Locating the compounds:
3.3.2 Identifying the compounds:
Detection:
4.1 Ninhydrin test:
Applications of Paper Chromatography:

The paper commonly used consists of highly purified cellulose. Cellulose, a

homopolysaccharide of glucose. Contains several thousand anhydro-glucose
units-linked through oxygen atoms. The paper exhibits weak ion exchange
and adsorptive properties. Modified forms of paper have been produced
in which the paper has been impregnated with alumina, silica gel, and ionexchange resin etc.
Estimation of Blood Glucose level by Folin-Wu method
Estimation of Tyrosine by Folin-Ciocalteau Method
The chemical composition of whatmann filter paper no: 1 is: a-cellulose (98
to 99%), b-cellulose (0.3 to 1%), Pentosans (0.4 to 0.8%), Ash (0.07 to 0.1%)
& ether soluble matter (0.015 to 0.1%).

APPARATUS:
The apparatus required for paper
chromatography are
1.
2.
3.
4.
5.
6.
7.
8.

Support for paper

Solvent trough
Airtight chamber
Whattmann filter paper number 1
Capillary tubes
Samples Amino acids (or) Pigments
Solvents
Platinum loop

PAPER DEVELOPMENT:
There are two main techniques, which may be
employed for the development of paper Chromatograms.
1.
2.
3.

Ascending techniques
Descending techniques
Radial development

4.

Two-dimensional chromatography

1) ASCENDING TECHNIQUES:
The filter paper is then dried and equilibrated by putting it into on airtight
cylindrical jar, which contains an aqueous solution of a solvent. The most
widely applicable solvent mixture is n-Butanol: Acetic acid: Water (4:1:5),
which is abbreviated
as BAW.
The sheet of paper is
supported on a frame with
the button edge in contact
with a trough with solvent.
The arrangement is
contained in an airtight
tank lined with paper
saturated with the solvent
to prove a constant
atmosphere and
separations are carried out
in a constant temperature
room. Thus, the solvent will
ascend into the paper this process is, therefore, termed Ascending
Chromatography

2) DESCENDING TECHNIQUES:

The end of the filter paper may be put into the solvent mixture contained in
a narrow trough mounted near the top of the container. In this
chromatography, the solvent will descend into the paper and this process is
then termed Descending Chromatography.
This method is convenient for compounds, which have similar Rf values since
the solvent drips off the
bottom of the paper, thus
giving a wider separation.

3) TWO
DIMENSIONAL

CHROMATOGRAPHY (3D):

The mixture is separated then the first solvent, which should be volatile: then
after drying, the paper is turned through 900 and separation is carried out in
the second solvent.
After locating the migrated unknown sample along with standard known
sample, a map is obtained and comparing their position with a map of known
compounds can identify compounds.

LOCATING THE COMPOUNDS:

Strip is removed when the solvent has migrated over most of the available
space. The distance to which the solvent has run is marked. In most cases,
the completed Chromatogram is colorless with no indication of the presence
of any compounds. Such a chromatogram is said as Undeveloped for
locating the various compounds.
The filter paper strip is first dried, then sprayed with 0.5% Ninhydrin in
acetone and at least heated for a few minutes at 80 to 1000 C. the reaction
occurs and the colored spots appear at the sites of the amino acids, such as
Chromatogram is now called Developed.
In paper chromatography, the stationary cellulose phase is more polar than
the mobile organic phase.

IDENTIF
YING THE COMPOUNDS:

The ratio of the distance travelled by a component (i.e. amino acid) to that
travelled by the solvent front, both measured from the marked point of the
application of the mixture, is called the Resolution front (Rf) value for that
component.
Distance from origin run by the compound

Rf =
Distance from origin run by the solvent

DETECTION:
The filter paper strip may be sprayed with ninhydrin and heated so that the
colored spots indicating the location of amino acids may develop. The color
densities of these spots may be measured with a recording transmittance
(or) reflectance photometer device.

Ninhydrin test or Ninhydrin reagent

NINHYDRIN
TEST:

Amines (including -amino acids) react with ninhydrin to give a

coloured product.
It can be used qualitatively (e.g. for chromatographic visualization) or
quantitatively (e.g. for peptide sequencing).
The -amino acids typically give a bluepurple product.
Proline, a secondary amine, gives
a yellow-orangeproduct.
The test is sensitive enough that
ninhydrin can be used for the visualization of
fingerprints.

APPLICATIONS OF PAPER
CHROMATOGRAPHY:
By using this technique

1.
2.
3.
4.
5.
6.
7.

To
To
To
To
To
To
To

check the control of purity of pharmaceuticals,

the detection of adulterants,
detect the contaminants in foods and drinks,
the study of ripening and fermentation,
the detection of drugs and dopes in animals & humans
the analysis of cosmetics
the analysis of the reaction mixtures in biochemical labs.

2 Comments
1.

Lidya

very helpful information

Reply

2.

Rachana

For circular chromatography from where do we calculate the solvent

front.from the wick or from the line where the samples are added? plz
answer me
Reply

Leave a Reply

Sign me up for the newsletter!

Post Comment

Notify me of follow-up comments by email.

Notify me of new posts by email.

Search

SUBSCRIBE AND GET FREE STUDY MATERIAL

Name:

Email address:
Sign up

MOST POPULAR POSTS

Plasma Proteins: Types and Functions (Basic Notes)

What is Paper Chromatography? Principle and Procedure
Hexose MonoPhosphate Shunt and its Significance (HMP Shunt)
Determination of Carbohydrate by Anthrone Method
Ebooks: Biochemistry By U.Satyanarayana & U. Chakrapani

CATEGORIES
Categories

LAST UPDATED

Ebooks: Biochemistry By U.Satyanarayana & U. Chakrapani

Titration Curve of Glycine : The zwitter ionic changes
What are the Basics of Biochemical Techniques?
Carbohydrates MCQ Online Examination- Part 2
Multiple Choice Question - MCQ on Carbohydrates

BiochemDen.com Copyright 2016.

Shares

44

44