OKANYA, VIRGINUS .K.

GROUP .2. REPORT

BSPH – 2A

01/21/2017

EXPERIMENT NUMBER .2.

ANALYSIS OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

ABSRACT

Chromatography is a common technique for separating chemical substances. The prefix

“chroma,” which suggests “color,” comes from the fact that some of the earliest
applications of chromatography were to separate components of the green pigment,
chlorophyll. You may have already used this method to separate the colored components
in ink. In this experiment you will use chromatography to separate and identify amino
acids, the building blocks of proteins. The proteins of all living things are composed of
20 different amino acids.

The experiment aimed to separate amino acids using the technique of paper
chromatography and to identify the color reactions of different amino acids which helps
to identify the unknown Amino acids using detection reagent. In this experiment, we
investigate the different properties of Amines, Amides, Aromatics and Thiols of Amino acids.
We also investigate their solubilities in water and in aqueous acid; how are they prepared and the
different chemical reactions they undergo.

The mixture of the unknown is found to contain Leucine, Alanine, and Tryptophan. We
know that the unknown had leucine as there was a spot in the same line of the chromatogram as
leucine section. There was a similar stain(in shape and height) in the unknown to that of Alanine
therefore we know that Alanine was in unknown. There was also another stain in unknown of the
same height and shape as Tryptophan so obviously this amino acid was also present in the
unknown.

i. OBJECTIVES
1. To separate amino acids using the
technique of paper chromatography.
2. To identify the color reactions of different
amino acids with the detection reagent.
ii. INTRODUCTION
Chromatography is a common technique
for separating chemical substances. The
prefix “chroma,” which suggests “color,”
comes from the fact that some of the
earliest applications of chromatography
were to separate components of the green
pigment, chlorophyll. You may have
already used this method to separate the
colored components in ink. In this
experiment you will use chromatography
to separate and identify amino
acids, the building blocks of proteins.
The proteins of all living things are
composed of 20 different amino acids.
Chromatography is a technique for
analyzing or separating mixtures of
substances into their components. there are
various forms of chromatography
techniques. Most of these techniques involve
two distinct phases; the stationary phase and
the mobile phase. In paper chromatography,
the stationary phase is a liquid which is
adsorbed on separating the components in a
sample. The relative affinity of a substance
for each phase depends on properties such as
molecular weight, structure and shape of the
molecule, and the polarity of the molecule.
In this experiment, very small volumes of
solutions containing amino acids will be
applied using capillary tube. After the
solutions have been applied, the paper will
be placed inside the chromatographic
chamber for development. As the mobile
phase rises on the paper it will eventually
encounter the “spots” of amino acids. The
fate of each amino acid in the mixture now
depends on the affinity of each substance for
the mobile and stationary phases. It is these
differences in the amino acid affinities that
lead to the separation. Identification of the
amino acid is done by spraying with
Ninhydrin solution. The Ninhydrin forms a
blue violet complex with an amino acid
except for proline/ hydroxyl proline which
gives a yellow color complex
Amino acids play central roles both as
building blocks of proteins and as
intermediates in metabolism. The 20 amino
acids that are found within proteins convey a
vast array of chemical versatility.
In this experiments students will identify
amino acids by qualitative analysis and
paper chromatography.
iii. MATERIAL
A. Equipment
(1) 250-mL Beaker
(1) Capillary tube
Aluminum foil
Whatman # 1 paper
Gloves
(5) 10-mL Test tubes
B. Reagents
(1mg/mL) Alanine
Ninhydrin solution (4mg/mL)
(1mg/mL) Leucine
Unknown amino acid mixture
(1mg/mL) Lysine
Solvent system (1-butanol, acetic acid, water
(12:3:5)
(1mg/mL) Cystein
(1mg/mL) Arginine
(1mg/mL) Tryptophan
(1mg/mL) Glycine
(1mg/mL) Tyrosine
iv. PROCEDURE
A. Preparation of Detection Reagent
100 mL Ninhydrin solution was prepared
with a concentration of 2 mg/mL.
B. Preparation of Amino Acid solution.
20 mL (1mg/mL) solution each of the amino
acid was prepared
C. Quantitative analysis of amino acids
1. Test for Amines
The following test tubes were prepared
Test tube 1: 10 drops of Ninhydrin solution
and 5 drops of Alanine were mixed.
Test tube 2: 10 drops of Ninhydrin solution
and 5 drops of Lysine were mixed.
Test tube 3: 10 drops of Ninhydrin solution
and 5 drops of Tyrosine were mixed.
The three test tubes were heated in a water
bath and the color change was observed.
2. Test for Amides
The following test tubes were prepared.
a. Test tube 1: 10 drops of 20% NaOH and 5
drops of Glutamine were mixed.
b. Test tube 2: 10 drops of 20% NaOH and 5
drops of Alanine were mixed.
The test tubes were heated in a water bath .
While heating, a strip of red litmus paper
was placed at the mouth of the tube. Record
your observations.
3. Test for substituted benzene rings.
The following test tubes were prepared.
Test tube 1: 5 drops of Tyrosine and 5 drops
of concentrated nitric acid were mixed.
Test tube 2: 5 drops of Tryptophan, and 5
drops of concentrated nitric acid were
mixed.
Test tube 3: 5 drops of Alanine, and 5 drops
of concentrated nitric acid were mixed.
The test tubes were heated in a water bath
for 3 minutes. 10 drops of 20% NaOH was
added and the observations were recorded.
4. Test for Thiol groups
The following test tubes were prepared.
Test tube 1: 5 drops of Cystein and 10 drops
of DTNB(5,5’-dithiobis (2- nitrobenzoic
acid) were mixed.
Test tube 2: 5 drops of Leucine and 10 drops
of DTNB (1mg/mL) were mixed.
It were allowed to stand for 5 minutes and
the observations were recorded.
5. Paper Chromatography.
a. A 8 x 6 cm paper chromatogram using Test Tube 2: Yellow color observed
whatman # 1 paper was prepared. Lysine
Test Tube 3: Pink color observed
b. A pencil line 1.5cm was drawn from the Tyrosine
edge of the paper.

c. 6 marks were made (0.5 cm apart) b. Test for Amines

d. A capillary tube were used to draw the Sample Observations

sample from the 5 samples of amino acids ( Test Tube 1: Changes red litmus
Ala, Leu, Cys, Trp) and the unknown Arginine paper to blue litmus
paper
mixture of amino acid.
Test Tube 2: No change observed
e. It were applied to the marks made. Alanine
Samples applied were small drops, which
was a wet spot of 1/4 to 3/8 inch in c. Test for Aromatic Amino Acids
diameter. The sample was applied 3x and
the spot was dried between applications. Sample Observations
Test Tube 1: Orange
f. The ends of the paper were stapled and the Tyrosine
main area of the chromatogram was not Test Tube 2: Yellow
touched (Gloves were wore). The paper was Tryptophane
transferred into a wide-mouth quart jar Test Tube 3: Alanine Negative
containing 15mL of solvent mixture (1-
butanol, acetic acid, and water in 12:3:5 d. Test for Thiols
ratio). The chromatogram was developed for
1 hour. Sample Observations
Test Tube 1: Yellow
g. After 1 hour, the chromatogram was Cysteine
removed from the chamber and was left to Test Tube 2: Negative
dry inside the hood. Leucine

h. The chromatogram was sprayed with

Ninhydrin solution (2mg/mLin ethanol) and e. Paper Chromatogram
it was placed in the hotplate for 30 minutes
to develop.

v. RESULTS

a. Test for Amines

Samples Observations
Test Tube 1: Purple color observed
Alanine
 A. Test For Amine

In the pH range of 4-8, all α- amino acids

The unknown has three spot and they are
calculated below:
Distance from Baseline travelled
Rf Value =
by Solute
Distance from Baseline travelled
by Solvent (Solvent Front)
react with ninhydrin (triketohydrindene
hydrate), a powerful oxidizing agent to give
a purple colored product (diketohydrin)
termed Rhuemann’s purple. All primary
amines and ammonia react similarly but
without the liberation of carbon dioxide. The
imino acids proline and hydroxyproline also
react with ninhydrin, but they give a yellow
colored complex instead of a purple one.
Besides amino acids, other complex
structures such as peptides, peptones and
proteins also react positively when subjected
to the ninhydrin reaction. Here, Test tubes
1,2, and 3 were with 10 drops of Ninhydrin
solution and 5 drops of alanine, lycine, and
tyrosine in each Test tube. A purple color
was observed for Alanine, yellow color for
Lysine, and pink color for Tyrosine which
are all positive test for Amines in the various
Amino acid samples.

Amino Solvent Spot Rf

Acids Distance Distance value
Unknown 6.90 2.40, 0.34,
1.32, 2.20 0.19,
0.31

vi. DISCUSSION

In this experiment, we investigate the

different properties of Amines, Amides,
Aromatics and Thiols of Amino acids. We
also investigate their solubilities in water
and in aqueous acid; how are they prepared
and the different chemical reactions they
undergo
B. Test for Amides
For amides, 0.5 mL of 20% NaOH is mixed
with 5 drops of each amino acid solution and
a strip of wet red litmus paper is draped over
the mouth of the tube, held in place by a
rubber band. After heating in a boiling water
bath for 5 minutes (and being careful to keep
the litmus paper wet), evolution of ammonia
from side-chain amides turns the litmus D. Test for Thiol
blue. (It's obviously important not to allow
Ellman's reagent (5,5'-dithiobis-(2-
any of the NaOH solution to wet the top of
nitrobenzoic acid) or DTNB) is a chemical
the tube.) In this part of the experiment, 2ml
used to quantify the number or concentration
of aqueous samples were placed in a test
of thiol groups in a sample which was
tube. 2ml of 10% NaOH together with 1
developed by George L. Ellman. Thermo
drop of 2% CuSO4 and then the mixture was
Scientific Pierce Ellman's Reagent (DTNB)
examined. Here, properly labeled Test tubes
reacts with sulfhydryl groups to yield a
1 and 2 with 10 drops of 20% NaOH in each
colored product, providing a reliable method
Test tube and 5 drops of Glutamine and
to measure reduced cysteines and other free
Alanine which the Glutamine display its
sulfhydryls in solution. Ellman's Reagent
alkalinity properties by changing Red litmus
(5,5'-dithio-bis-[2-nitrobenzoic acid]) is
paper blue while Alanine had no reaction.
used to estimate sulfhydryl groups in a
sample by comparing to a standard curve of
a sulfhydryl-containing compound such as
C. Test For Aromatic Amino Acids. cysteine.
Aromatic amino acids, such as Phenyl alanine, For thiol groups, 5 drops of Cysteine
tyrosine and tryptophan, respond to this test. In and Leucine solution were mixed with 0.5
the presence of concentrated nitric acid, the mL of 1 mg/mL DTNB (2,2'-dithiobis-5,5'-
aromatic phenyl ring is nitrated to give yellow nitrobenzoic acid). A positive test is a
colored nitro-derivatives. At alkaline pH, the pronounced deepening of the reagent's pale
color changes to orange due to the ionization of yellow color for Cysteine and Negative test
the phenolic group. Here, Test tubes 1,2, and 3
for Leucine.
with 5 drops of nitric acid in each test tube and
5 drops of Tyrosine, Tryptophan, and Alanine E. Paper chromatogram.
respectively. An addition of 10 drops of 20%
NaOH which an orange color was observed for Results are pretty much what you would
Tyrosine, Yellow color for Tryptophan and expect with a couple of notes. I found that
Alanine showed negative reaction. This results cysteine and lysine didn't give a strong
implies that Tyrosine and Tryptophan are positive reaction with ninhydrin in the test
aromatic Amino acids while Alanine is not an tubes; the result for proline is yellow as
Aromatic amino acid. expected. If you use tyrosine for a known,
it's necessary to make it alkaline to stay in
solution; the alkaline solution gives a false Amino acid samples Color
positive in the DTNB test. Alanine, lysine, Tryptophan Yellow
and leucine don't give any identifying results Arginine Blue
in the test tube reactions, but are well Tyrosine Pink
separated by the chromatography. The Alanine Purple
Cysteine Yellow
unknown Amino acid has three spots which
the Rf value is calculated to be 0.34, 0.19
and 0.31 respectively. 2. Calculate the Rf values of the known and
unknown Amino acids.
CONCLUSION
Distance from Baseline travelled
Anomalies are however quite obvious here Rf Value =
by Solute
due to experimental error. The largest error
probably came from the measurement of the Distance from Baseline travelled
stains as they were not spots but elongated by Solvent (Solvent Front)
stains. The amino acids were also dabbed on
the origins with a large drop at times which
probably added to the inaccuracy of the Amino Solvent Spot Rf
acids Distance Distance value
corresponding stains. Some stains were
Alanine 6.90 1.32 0.19
longer than usual, probably because the Leucine 6.90 2.40 0.34
amino acids mixed. Other sources of error Lysine 6.90 0.90 0.13
include the fact that the amino acid solutions Cysteine 6.90 0.80 0.11
may have been impure. Blow drying the Tryptophan 6.90 2.20 0.31
amino acids may have interfered with the Unknown 6.90 2.40, 0.34,
amino acids. The chromatography paper 1.32, 0.19,
may have been little. The mixture of the 2.20 0.31
unknown is found to contain Leucine,
Alanine, and Tryptophan. We know that the The solvent in the unknown is Leucine,
unknown had leucine as there was a spot in Alanine and Tryptophan because the Rf-
the same line of the chromatogram as value of the three spots, 0.34, 0.19, 0.31 is
leucine section. There was a similar stain(in the same as that of the leucine Alanine and
shape and height) in the unknown to that of Tryptophan.
Alanine therefore we know that Alanine was
in unknown. There was also another stain in 3. Explain why there are two spots for
unknown of the same height and shape as cysteine.
Tryptophan so obviously this amino acid
Cysteine gives two spots on the
was also present in the unknown.
chromatogram, because of the presence of
ANSWER TO QUESTIONS its oxidized form, cysteine; I'm not sure
whether the cysteine I used was impure
1. Give the color reaction of the following (probably was,) or whether cysteine oxidizes
amino acids:
so readily in water which is difficult to avoid
unless made very fresh.

4. Identify the composition of your unknown

Amino acids mixture

The composition of the unknown Amino

acids are:

i. Leucine,

ii. Alanine

iii. Tryptophan.

5. Give the structure of Ninhydrin.