Practical 2 Extraction of Glycogen From Liver Cells

Titles:
1. Solubility test for amino acid
2. Ninhydrin test
3. Xanthoproteic test
4. Millon’s test
5. Nitroprusside test
6. Biuret test

Objectives:
1. To investigate the solubility of selected amino acid in various solutions
2. To detect the presence of aromatic acids in the given samples by Ninhydrin Test.
3. To detect the presence of aromatic amino acids in the given samples by Xanthoproteic Test.
4. To detect the presence of amino acids containing hydroxybenzene radical in the given samples
by Millon’s Test.
5. To detect the presence of amino acids containing sulfhydryl group (-SH) in the given samples
by Nitropruside Test.
6. To detect the presence of peptide bonds in the given samples by Biuret Test.

Result:
Solubility test for amino acid
Amino acid Glycine tyrosine Glutamic acid Cysteine
Solvent
Water Soluble Insoluble Soluble Soluble
Absolute alcohol Insoluble Insoluble Insoluble Insoluble
Dilute HCI Soluble Soluble Soluble Soluble
Dilute NaOH Soluble Soluble Soluble Soluble

Amino acids Observation

Glycine No changes
Tyrosine No changes
Glutamic acid Blue litmus paper changed to Red
Cysteine No changes
Ninhydrin Test
Amino acids Observation
Tyrosine Violet-blue color is formed
Glycine Violet-blue color is formed
Phenylalanine Violet-blue color is formed
BSA Violet-blue color is formed

Xanthoproteic Test
Amino acids Observation
Tyrosine Yellow precipitate is formed
Glycine Yellow precipitate is formed
Phenylalanine Remain unchanged (colorless)
Lysine Remain unchanged (colorless)

Millon’s Test
Amino acids Observation
Tyrosine Brick-red precipitate is formed
Glycine Remain unchanged (colorless)
Casein Remain unchanged (colorless)
BSA Brick-red precipitate is formed

Nitropruside Test
Amino acids Observation
Cysteine Changed from yellow to red
Methionine Remain unchanged (yellow)

Biuret Test
Amino acids Observation
Gelatin Violet-blue color is formed
Casein Remain unchanged (colorless)
BSA in NaOH Remain unchanged (colorless)
Discussion
1. Solubility test for amino acid
The result showed that Glycine, Glutamic acid and Cysteine are soluble in water. Tyrosine
is the only amino acid in this test to show insolubility in distilled water. The solubility of amino
acid can be affected due to the number of the carbon presence in different amino acid. In this case,
Tyrosine has a higher carbon ratio compare to other amino acids in this test. Glycine is the most
soluble compound among all of the amino acid because of its absence of carbon atom in the side
chain. Next, Glutamic acid is able to dissolve in polar solvent (distilled water) due to its polar side
chain properties. Cysteine also have a side polar chain therefore it is able to dissolve in polar
solvent. All the amino acids in the test are not able to dissolve in absolute alcohol. The reason
behind this is because amino acids have dipolar side. Amino acids are polar compound that exhibit
low solubility towards non-polar solvent (Absolute alcohol). Next, the result showed that all amino
acids dissolve in dilute acidic (HCI) and alkaline (NaOH) solution in room temperature. The
expected result should show that all amino acids are able to dissolve in both acidic (HCI) and
alkaline (NaOH) solution. Amino acid is an amphoteric compound that are able to react with acid
and base. At certain pH, zwitterion is formed in amino acid which consists of deprotonated
carboxylic acid group and protonated amine group. Isoelectric point is the point where amino acids
show no net charge in certain pH value. At low pH value, both groups are protonated to form
cationic amino acid, which is positive charge. The deprotonation of zwitterion as the pH of the
solution increases causing the formation of anionic amino acid, which is negatively charged. The
result showed that glutamic acid is acidic in aqueous medium when tested with litmus paper.
Glutamic acid has an acidic side chain containing carboxyl acid group in which the pKa value is
low and one proton is lost. Therefore, the total net charge for acidic amino acids are negative. In
the other hand, glycine and tyrosine have a neutral side chain which cannot donate proton hence it
is neutral (Litmus paper does not have any changes). Thus, cysteine shows slight changes in litmus
paper, turning blue litmus paper to slightly red. It is not stated in the result because cysteine has a
sulfhydryl group as its side chain which will likely to donate proton in aqueous medium. Therefore,
it can be seen that a low concentration of cysteine is deprotonated.

2. Ninhydrin test
Ninhydrin test is used to detect the presence of amino acid by reacting with the α.-amine
group of amino acid. This amino acid is a very powerful oxidizing agent that oxidizes amino acid
and liberate ammonia, CO2, corresponding aldehyde and a reduced form of ninhydrin in the
process. The ammonia reacts with ninhydrin and the reduced product form a purple color dye
called Ruhemann’s purple. The result showed that all amino acids which includes tyrosine,
glycine, phenylalanine and BSA react positively with the ninhydrin by producing a changes from
colorless to violet-blue color.
3. Xanthoproteic test
Xanthoproteic test is used to detect the presence of aromatic amino acids. It helps in
detecting the presence of phenyl group by reacting with concentrated nitric acid. The nitrated
aromatic amino acids show a positive result when the phenolic group is neutralized by alkali,
forming a yellow colored nitro-derivatives. The result showed that tyrosine and glycine gave a
positive result, yellow in color; while phenylalanine and lysine showed negative result, colorless.
Among all the amino acid used in this test only tyrosine and phenylalanine contain aromatic ring
in the side chain. In theory glycine should give a negative result due to absence of the aromatic
ring in its side chain. This is possibly due to the alkaline react with the nitric acid in the solution.
The reason Phenylalanine gives negative or weakly positive reaction though this amino acid
contains aromatic nucleus because it is difficult to nitrate under normal condition1. It’s benzene
ring is not activated and remain highly stable. In contrast, the phenolic group in the side chain of
tyrosine is activated due to presence of hydroxyl group on the benzene ring. The one pair of proton
present in hydroxyl group delocalizing the pI system of benzene ring, enhancing the reactivity
toward electrophilic substitution.

4. Millon’s test
Millon’s test is used to detect presence of amino acids containing hydroxybenzene radical,
In the sample solution, the phenol group of tyrosine is first nitrated with nitric acid2. Then the
nitrated tyrosine complexes ions of mercury (I) and mercury (II) to form a pink to brick red
precipitate or a solution2. Initially, some tyrosine-containing proteins form a white precipitate that
turns red when heated while others immediately form a red solution2. Both findings were
considered to be positive2. Tyrosine is the only amino acid that contain hydroxybenzene ring in its
side chain. The result showed that tyrosine forms a yellow precipitate of mercury amino acid
complex when reacted with 15% mercuric sulphate. Upon heating and addition of sodium nitrate
solution, the yellow complex converts to mercury phenolate which is a positive result, (brick-red
color). BSA also showed positive result which conclude that tyrosine-containing protein is present.
Glycine and casein showed a negative result, (colorless) in the test which prove there is no presence
of hydroxybenzene side chain in the amino acids.

5. Nitroprusside test
Nitropruside test is used to detect the presence of amino acids containing sulfhydryl group
(-SH). The test is involving reaction between sodium nitroprusside and the sulfhydryl group in
alkaline medium upon heating. This test is specific for cysteine, the only amino acid that contain
sulfhydryl group(-SH). The result showed positive result, changed from yellow to red for cysteine
and negative result, remained yellow in color for methionine.
6. Biuret test
The purpose for biuret test is to detect the presence of peptide bonds or amino acid residues
in peptides. The result showed positive result for gelatin, which a violet-blue is formed; casein and
BSA showed negative result, which the color remains unchanged. The expected result should be
all the amino acid listed above showed a positive result. The factor for casein and BSA to show
negative result could be inadequate amount of 1% CuSO4.5H2O, causing the amino acid residues
cannot form a colored complex with CU2+ion.

Reference
1. Xanthoproteic test: Objective, Principle, Reagents, Procedure and Result - Online Biology
Notes [Internet]. Online Biology Notes. 2019 [cited 8 November 2019]. Available from:
https://www.onlinebiologynotes.com/xanthoproteic-test-objective-principle-reagents-procedure-
and-result/
2. [Internet]. Himedialabs.com. 2019 [cited 8 November 2019]. Available from:
http://himedialabs.com/TD/R097.pdf