August 29, 2020

Biochemistry Laboratory
Experiment 1: Qualitative Analysis of Amino Acids

Asynchronous Class: Submit this in the form of Google Document and submit it through Google Classroom.
Deadline: September 4, 2020 at 12 midnight.

Time Mode of Topic Platform url link Remarks

instructio
n
1:00- Lecture Qualitativ video https://www.youtube.com/watch?v=wmhmAESv72E&list=PLzju0_wImbUdrzsu0zd7pIZT7fQlucGtt&index=2 Observation
5:00 e Analysis and intensive
p.m. of Amino report writing
Acids

Laboratory Report Format:

Qualitative Analysis of Amino Acids

Name:_________Angela Nicole O. Mamawag__________________ Date:______September 4, 2020_______________

Course & Section:________Nursing 1A__________
Objectives:__This experiment aims to identify amino acids with a serious of 10 chemical tests, namely; Ninhydrin test, Xanthoproteic acid test, Pauly’s diazo test, Millon’s test,
Histidine test, Hopkin’s cole test, Sackaguchi test, Lead sulphide test, Folins McCarthy sullivan’s test and Isatin test. This solution can be identified through color, precipitation
reaction or reactions with terminal groups and their side chain radicals groups.

Materials:
Ninhydrin reagent, 40% sodium hydroxide, concentrated nitric acid, 1N hydrochloric acid, sulphanilic acid, Sodium nitrite, Sodium carbonate, Million’s reagent, Sodium nitrate
solution, Bromine, Ammonium carbonate, Acetic acid-glyoxilic acid reagent, Concentrated sulphuric acid, Alpha naphthol reagent, Hybromite solution, Urea solution, Lead
acetate solution, Glycine solution, 10% sodium nitropusside solution, Isatin reagent, Test tube, Test tube holder, Water bath, Spatula,
Droppers, Marble chips, Bunsen burner, Filter paper strip and Icebox.

Procedure:
1. To carry out Ninhydrin test, first transfer a few drops of unknown solution to a test tube using a dropper. Now add a few drops of ninhydrin reagent to the same test
tube. Keep the test tube in a boiling water bath for five minutes. After five minutes, take the test tube from the water bath. Now observe the color of the solution. If the
color of the solution is blue, it indicates the presence of alpha amino acids and if the color of the solution is yellow, then it indicates the presence of imino acids.
2. To perform Xanthoproteic acid test. Transfer 1 ml of unknown solution to a test tube using a dropper. Add a few drops of concentrated nitric acid into this uknown
solution in a fume hood. Put some marble chips into this solution in the test tube to avoid bumping of the solution while boiling. Then heat the solution containing test
tube under the bunsen burner and cool it by placing under the running tap water. Now add a few drops of 40% NaOH Solution into this test tube containing solution. On
adding 40% NaOH, the color of the solution changes to orange which indicates the presence of aromatic amino acids.
3. To do Pauly’s Daizo test. First transfer a few drops of unknown sample to a test tube and place it in a small ice bucket. Now take prechilled sulphanilic acid from the ice
bucket and add a few drops of sulphanilic acid to the same test tube. similarly add a few drops of prechilled sodium nitrate to this test tube. This is followed by dropwise
addition of sodium carbonate solution. The appearance of red color during the addition of sodium carbonate solution indicates the presence of histidine and tyrosine.
4. To perform Hopkin’s cole test. Add 1 ML of unknown solution in a test tube using a dropper. Then add 1ml of acetic acid- glyoxilic acid-reagent to the same test tube
containing solution. Mix the solution by shaking it. Now, carefully add a few drops of concentrated sulphuric acid down the sides of the test tube containing solution. We
can see that purple-violet ring appears in the test tube indicating the presence of tryptophan.
5. To carry out Lead sulphide test. Add 1ml of unknown amino acid solution to a test tube using a dropper. Now add a few drops of NaOH solution to the same test tube
containing solution. Then put some marble chips into this solution in order to avoid the bumping of the solution while heating. Now heat the solution under the bunsen
burner for 5-10 minutes using a test tube holder. After heating, keep the test tube under running tap water to cool the contents. Now, add a few drops of 10% lead
acetate solution into the test tube containing solution. On observation, a black precipitate appears in the test tube indicating the presence of cysteine.
 6. To do Isatin test. Apply a drop of unknown amino acid solution ona filter paper strip using a dropper and dry the spot using a hair dryer. Apply a drop of isatin reagent
again unto the dried spot. Then repeat the drying procedure for a few minutes. We can see a blue colored spot on the filter paper which indicates the presence of imino
acid.
7. To perform Folin’s McCarthy Sullivan’s test. Take 1ml of the unknown amino acid solution in a test tube and add a few drops of 40% NaOH followed by a few drops of
glycine and sodium nitroprusside solution. Now place the test tube in a hot water bath, maintained at 40% C for 15 minutes. Add 0.5 ml of 6N HCI into this test tube
taken from water bath. On adding HCI, the content of the test tube changes to red color indicating the presence of Methionine.
8. To carry out Sakaguchi test. Transfer a few drops of prechilled unknown amino acid to a test tube using a dropper. Add a few drops of 40% NaOH, alpha-naphthol
solution, 5% urea, hyprobomite solution respectively to the same test tube containing solution. The color of the solution in the test tube changes to red indicating the
presence of arginine.
9. To perform Histidine test. Transfer 2ml of unknown amino acid solution to a test tube using a dropper. Add a few drops of 5% bromine in 33% acetic acid solution into
this test tube containing solution. Keep the test tube at room temperature for 10 minutes. Then add 2ml of Ammonium carbonate solution to this test tube and keep the
test tube in boiling water bath for five minutes. After five minutes, we can see that the color of the solution in the test tube changes to blue. This indicates the presence
of Histidine.
10. To do Millon’s test. Transfer 1ml of amino acid solution to a test tube using a dropper. Add a few drops of millon's reagent and mix the solution by shaking it. Now heat
the test tube under the bunsen burner for 3-5 minutes and cool it under tap water. Carefully add a few drops of concentrated nitric acid solution into this test tube. We
can see that the color of the solution in the test tube changes to red which indicates the presence of tyrosine.

Data Analysis (present it in the tabular form):

Chemical Test Composition Reagent Color

Ninhydrin test Unknown solution, ninhydrin reagent Blue(alpha amino

acids)/yellow (imino
acids)

Xanthoproteic acid test Unknown solution, concentrated nitric acid Orange(aromatic

amino acids)
Pauly’s Daizo test Unknown solution, sulphanilic acid Red( histidine and
tyrosine)
 Hopkin’s cole test Unknown solution, acetic acid- glyoxilic acid-reagent Purple( tryptophan)

Lead sulphide test Unknown amino acid solution, NaOH solution Black (cysteine)

Isatin test Unknown amino acid solution, isatin reagent Blue(imino acid)

Folin’s McCarthy Sullivan’s test Unknown amino acid solution, NaOH, glycine and sodium nitroprusside solution Red(methionine)

Sakaguchi test prechilled unknown amino acid, 40% NaOH, alpha-naphthol solution, 5% urea, hyprobomite solution Red(arginine)

Histidine test unknown amino acid solution, 5% bromine in 33% acetic acid solution, 2ml of Ammonium carbonate solution
(Blue)(histidine)

Millon’s test amino acid solution, millon's reagent

(Red ) (tyrosine)

Observation:
In this observation, the Ninhydrin test states that if the color of the solution is blue, it indicates the presence of alpha amino acids and if the color of the solution is yellow, then it
indicates the presence of imino acids. The xanthoproteic acid test states that if the color of the solution changes to orange which indicates the presence of aromatic amino acids.
The Pauly’s Diazo test states that if the appearance of red color during the addition of sodium carbonate solution indicates the presence of histidine and tyrosine. The millon’s
test is that the color of the solution in the test tube changes to red which indicates the presence of tyrosine. Histidine test states that if that the color of the solution in the test
tube changes to blue. This indicates the presence of Histidine. The Hopkin’s cole test can see that purple-violet ring appears in the test tube indicating the presence of
tryptophan.
The Sackaguchi test states that if the color of the solution in the test tube changes to red indicating the presence of arginine. The Lead Sulphide test states that on observation, a
black precipitate appears in the test tube indicating the presence of cysteine. The Folins McCarthy Sullivan’s test states that on adding HCI, the content of the test tube changes
to red color indicating the presence of Methionine and lastly in the Isatin test states that we can see a blue colored spot on the filter paper which indicates the presence of imino
acid.
Conclusion:

Therefore in conclusion in each solution containing amino acids with a serious of 10 chemical tests, there are different types of amino acids in appearance of different colors
mixed from different solutions.