coromatography
PRACTICAL -5.12
Aim:
To perform Separation and identification of given amino acids (alanine
and
proline) by paper
chromatography.
Requirements:
Developing chamber, jar and petridish, mobile phase (n-butanol : glacial acetic
acid water (40 10:50 vIviv)], visualizing agent (ninhydrin in acetone).
dard amino acid solution (alanine. proline). unkhown amino acid mixture,
MWhatman ilter paper.
Principle:
Amino acids in a given mixture or sample aliquot are separated On
the basis of differences in their solubilities and hence differential
oartitioning coefticients in a binary solvent system.
The amino acids with higher solubilities in stationary phase move sloWIy
as compared to those with higher solubilities in the mobile phase. The
relative mobility of compounds during chromatography a function of
the partition coefficients of the compounds in to the solvent phases.
Thus, similar compounds which have different partition coefficients will
move to different extent. Since paper is composed of cellulose, a polar
substance, polar substances will have a greater affinity for the paper.
The separated amino acids are detected by spraying the air dried
chromatogram with ninhydrin reagent. All amino acids give purple or
bluish purple color on reaction with ninhydrin except proline and
hydroxyproline which gives a yellow colored product. The reaction
leading to the formation of purple complexes is given below:
Reaction:
OH
OH H,N + RCHO
OH OH
NH,
Amino acid Hydrindantin
Co,
257
Laboratory Handbook in Instrumental
Analysis
OH
OH
+ NH, +
OH
OH
Hydrindantin
Ninhydrin
Purple colouredproduct
t H,0
distance moved by a compound to the distance me.
The ratio of the
R, (retention factor) value
by the solvent front is known as its
characteristics of the compOlin4
For a given solvent, the R, value is identify unknown substances
and from this observation it is possible
substance. Paper chromatogranb
by their positions in relation to known
minutes to several hours.
takes anywhere from several
amino acids will be separated and
In this experiment, a mixture of
identified by ascending chromatography
(in which the solvent moves
the spot from a reservoir capillary and gravity forces). Ascendina
down
commonly employed specialy
(by capillary force) chromatography is also
in thin layer chromatography.
Procedure:
solutions:
Preparation of Standard amino acid
such as alanine, proline
Prepare standard solutions of amino acid
(1 mg/ml) in 10 % propanol.
2 Preparation of unknown amino acid solution:
in volumetric flask.
Mix equal amount of specified amino acid
3 Preparation of mobile phase:
Prepare a mobile phase as specified and pour it in developing Chanu
4 Preparation of visualizing agent:
0.2 g ninhydrin in 100 ml acetone.
258
chromatography
Application and detection of
sample:
5.
Select the Whatman filter paper of suitable size and shape.
Draw a straignt ine with pencil about 1 cm above the bottom edge
Make 3 points and name them (as S.. S. and M,). Place a sia
drop the sample on the points with the help of a capillary tuu
(S, = proline, S, = alanine, M, = mixture of proline and alanine)
Set up the paper in the chromatographic chamber previously
saturated with mobile phase (n-butanol: glacial acetic acid: water,
40:10:50 vviviv). Close the chamber firmly so that it is air tignt.
Let the solvent runs about 3/4th height of the paper.
Remove the paper from the chamber and mark the solvent front.
Dry the paper and spray Ninhydrin solution. Again dry the
chromatographic paper. Blue or purple colored spots would appear
on the paper. Mark the boundary of each spot with lead pencil.
Calculate the R, values for amino acids in the given mixture and
identify them by comparing with the R, values of the standards.
Precaution:
Do not touch the paper with naked hands due to sweat on hands
contains significant amount of amino acids.
The spots of the applied sample should be as compact as possible.
Larger the spot, poorer will be the resolution.
In paper chromatography base line on which the sample has been
applied does not dip into the solvent otherwise the sample might
get washed away in the solvent.
Solvent front should be in straight line and should not be zig-zag
or slopping.
reagent.
Dry the paper thoroughly before spraying with the detection
solvent
It is advisable to perform chromatography in three different
mixture or sample
systems before the identify of amino acids in the
can be established with any degree of certainty.
Observation:
Olationary phase: Whatman filter paper
acid: water = 40: 10: 50, vlv/y
ie phase: n-butanol: acetic
vnamber saturation time: 30 minutes
Praying reagent: Ninhydrin solution 259
Laboratory
Handbook in Instrumental
Analysis
Amino acid Distance Distance R value
Sr.
travelled by travelled by
No.
amino acid solvent front
1. Alanine (S,)
2 Proline (S,)
3 Unknown (M,)
Calculations:
R, value = Distance travelled by the sample spot (unknown amino ai.
distance travelled by the solvent front
Compare the R, value of unknown amino acid with standard sample (s, ar
S,) and find out unknown amino acid given in the mixture.
Result:
1 R, value of alanine =
2 R, alue of proline =
3 The R, value of amino acid present in the
given mixture was found
to be and therefore the unknown aminO acid
present in the given mixture was found to be
Study questions:
1 What is the basic fundamental behind
2
paper chromatography?
How colored compound can be
3
separated using paper chromatography
Explain R, value, chromatography.
4
What is the principle involved in
5
paper chromatography.
What are the factors
6
affecting R, value.
What is the composition
paper used in
7 What percentage of water is chromatography?
associated with paper?
260
