NAME: GROUP NO.

: 6
BOLIVAR, Leonilyn SECTION: BSN1-A
LEGASPI, Ma. Lara Micaela INSTRUCTOR: SAMPOLLO, Leslie
SCORE:
OSUMO, Betiara Jazel
PELINGON, Robert Kris
PIO, Krea Joy
YSULAN, Cyky Mae DATE: December 4, 2020

Module 4: Qualitative Test for Proteins

I. MATERIALS
PART A. Different tests for proteins
 NINHYDRIN TEST

Test tube Unknown solution

Test tube holder Water bath

Droppers Ninhydrin reagent

 XANTHOPROTEIC ACID TEST

Test tube Unknown solution

Test tube holder Concentrated nitric acid solution

Droppers 40% sodium hydroxide

Marble chips Tap water

Bunsen burner
  PAULY’S- DIAZO TEST

Test tube Unknown sample

Small ice box or ice bucket Sulphanilic acid

Droppers Sodium nitrate

Sodium carbonate

 HOPKINS COLE TEST

Test tube Unknown solution

Dropper 1 ml of acetic acid- glyoxylic acid reagent

Concentrated sulfuric acid

 LEAD SULPHIDE TEST

Test tube 1 ml of unknown amino acid

Dropper NaOH solution

Marble chips Running tap water

Bunsen Burner 10% lead acetate solution

Test tube holder

 ISATIN TEST

Dropper Unknown amino acid solution

Filter paper strip Isatin reagent

Hair blower
  FOLINS MCCARTHY SULLIVAN’S TEST

Test tube Unknown amino acid solution

Dropper 40% NaOH solution

Test tube holder Glycine

Sodium nitroprusside solution

Water bath

6N HCI

 SACKAGUCHI TEST

Test tube Prechilled unknown amino acid

Dropper 40% NaOH solution

Alpha-naphthol solution

5% urea solution

Hypobromite solution

 HISTIDINE TEST

Test tube Unknown amino acid

Dropper Bromine

Ammonium carbonate solution

Water bath

 MILLON’S TEST
Test tube Amino acid solution

Dropper Millon’s reagent

Test tube holder

Bunsen Burner

Tap water

PART B. Qualitative test for proteins

 BIURET TEST

Dropper NaOH solution

Test Tubes 1% copper sulphate solution

Egg Albumin
Bunsen Burner
Gelatin Dispersion

 XANTHOPROTEIC TEST

Dropper Nitric acid

Egg Albumin
Test Tubes
Gelatin Dispersion
Bunsen Burner

 NINHYDRIN TEST

Dropper Ninhydrin solution

Egg Albumin
Test Tubes
Gelatin Dispersion
Bunsen Burner
  MILLON'S TEST

Dropper Millon’s reagent

Egg Albumin
Test Tubes
Gelatin Dispersion
Bunsen Burner

PART C. Determination of the unknown

 NINHYDRIN TEST

Dropper Ninhydrin solution

Unknown solution (Amino acid solution)
Test Tubes
Water bath
Test Tube Rack

 XANTHOPROTEIC ACID TEST

Dropper Unknown solution

Concentrated HNO3
Test Tubes
Water bath
Bunsen Burner
NaOH
Test Tube Rack

 PAULY’S DIAZO TEST

Dropper Sulphanilic acid

NaNO2
Test Tubes
Amino acid
Test Tube Rack
Na2Co3
  LEAD SULPHIDE TEST

Dropper Unknown solution (Amino acid solution)

NaOH
Test Tubes
Lead Acetate
Bunsen Burner

Test Tube Rack

II. PROCEDURES
PART A. Different tests for proteins
NINHYDRIN TEST
 First, transfer a few drops of unknown solution to a test tube using a dropper.
 Add a few drops of ninhydrin reagent to the same test tube.
 Next, keep the test tube in a boiling water bath for 5 minutes
 Then, after 5 minutes take the test tube from the water bath.
 Lastly, observe the color of the solution.

XANTHOPROTEIC ACID TEST

 First, transfer the 1 ml of unknown solution to a test tube using a dropper.
 Add a few drops of concentrated nitric acid solution into the unknown solution in
a fume hood.
 Next, add some marble chips into the solution in the test tube to avoid bumping
of solution while boiling.
 Then, heat the solution containing the test tube under the Bunsen burner and
cool it by placing under the running tap water.
 Lastly, add a few drops of 40% of NaOH solution into the test tube containing
solution.
LEAD SULPHIDE TEST
 Add 1 ml of unknown amino acid solution to a test tube using a dropper.
 Add also a few drops of NaOH solution to the same tube containing solution.
 Next, put some marble chips into the solution in order to avoid the bumping of the
solution while heating.
 Then, Heat the solution under the Bunsen Burner for 5-10 minutes using a test
tube holder.
  After heating, keep the test tube under a running tap water to cool down the
contents.
 Now, add few drops of 10% lead acetate solution into the test tube containing
solution and observe.
ISATIN TEST
 First, apply a drop of unknown amino acid solution on a filter paper strip using a
dropper and dry the spot using a hair dryer.
 Then, Apply a drop of Isatin reagent again on the dried spot. Repeat the drying
procedure for a few minutes.
 Lastly, observe the solution.
FOLINS MCCARTHY SULLIVAN’S TEST
 First, Add 1 ml of the unknown amino acid solution in a test tube
 Next, add a few drops of 40% of NaOH solution and a few drops of glycine and
sodium nitroprusside solution.
 Then, place the test tube into a hot water bath at 40’C for 15 minutes.
 Lastly, add 0.5 ml of 6N HCI into the test tube taken from water bath then
observe.
SACKAGUCHI TEST
 First, 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, hypobromite
solution to the same test tube containing solution and observe.
HISTIDINE TEST
 First, transfer a 2 ml of unknown amino acid solution into a test tube using a
dropper.
 Next, add a few drops of 5% bromine in 33% acetic acid solution into this test
tube.
 Then, keep the test tube at a room temperature for 10 minutes.
 Lastly, add a 2 ml of ammonium carbonate solution to the test tube and keep the
test tube in a boiling water bath for 5 minutes and observe.
MILLON’S TEST
 First, transfer 1 ml of amino acid solution into a test tube using a dropper.
 Next, add a few drops of Millon’s reagent and mmx the solution by shaking it.
 Then, heat the test tube under the Bunsen burner for 3-5 minutes and cool it
under a tap water.
 Lastly, carefully add a few drops of concentrated nitric acid solution into the test
tube and observe.
PART B. Qualitative test for proteins
BIURET TEST
 Drag the dropper to the test tubes to drop NaOH solution into tubes A and B
respectively.
 Drag the dropper to the test tubes to drop 1% copper sulphate solution into tubes
A and B respectively.
 Click on the knob of the burner to turn it on.
 Drag test tube A to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
 Drag test tube B to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
XANTHOPROTEIC TEST
 Drag the dropper to the test tubes to drop Conc. Nitric acid into tubes A and B
respectively.
 Click on the knob of the burner to turn it on.
 Drag test tube A to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
 Drag test tube B to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
NINHYDRIN TEST
 Drag the dropper to the test tubes to drop Ninhydrin solution into tubes A and B
respectively.
 Click on the knob of the burner to turn it on.
 Drag test tube A to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
 Drag the test tube B to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
MILLON'S TEST
 Drag the dropper to the test tube A to drop Millon’s reagent into it.
 Click on the inference icon to see the inference.
 Drag the dropper to the test tube B to drop Millon’s reagent into it.
 Click on the knob of the burner to turn it on.
 Drag the test tube A to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
 Drag the test tube B to the burner to heat the contents of the test tube.
 Click on the inference icon to see the inference.
PART C. Determination of the unknown
NINHYDRIN TEST
 Drag and drop down unknown solution (amino acid solution) in a test tube, and
add few drops of ninhydrin reagent.
 After adding these solutions, place the test tube in a boiling water bath for 5
minutes and cool to room temperature.
XANTHOPROTEIC ACID TEST
 Add unknown solution (amino acid solution) taken in a test tube and add few
drops of HNO₃.
 Boil the contents over a Bunsen flame, using a test tube holder, for few minutes.
 After that, cool the test tube under running tap water and add few drops of NaOH
to the solution.
PAULY’S DIAZO TEST
 Drag and drop down sulphanilic acid reagent in a test tube and add few drops of
of prechilled NaNO2 solution.
 Add immediately few drops of pre chilled amino acid solution.
 This is followed by dropwise addition of Na2CO3 solution until the color appears.
LEAD SULPHIDE TEST
 Drag and drop down unknown solution (amino acid solution) taken in a test tube
and add few drops of NaOH.
 Boil the contents for 5-10 mins. over a bunsen burner.
 After that, add few drops of Lead Acetate to this solution.
 Black precipitate will appear in the solution.

III. RESULTS AND DISCUSSION

PART A. Different tests for proteins

TEST EXPECTED RESULTS INTERPRETATION

Ninhydrin test  The color of the  If the color of the solution is

solution will turn BLUE, it has the presence of
BLUE. alpha amino acids.
 The color of the  If the color of the solution is
solution will turn YELLOW, it has the presence
YELLOW. of imino acids.
Xanthoproteic acid  The color of the  It indicates the presence of
test solution changes to aromatic amino acids.
 ORANGE
Pauly’s- diazo test  The color of the  It indicates the presence of
solution turn RED. histidine and tyrosine.
Hopkins cole test  Purple-Violet ring  It indicates the presence of
appears in the test tryptophan.
tube
Lead sulphide test  Black precipitate  It indicates the presence of
appears in the test cysteine.
tube.
Isatin test  Blue coloured spot  It indicates the presence of
on the filter paper imino acid.
Folins mccarthy  The content of the  It indicates the presence of
sullivan’s test test tube turns from Methionine
yellow color into red
color
Sackaguchi test  The color turns from  It indicates the presence of
clear to dark red arginine
color
Histidine test  The color of the  It indicates the presence of
solution changes Histidine
into blue
Millon’s test  The color of the  It indicates the presence of
solution changes tyrosine.
from clear to red
 

PART B. Qualitative test for proteins

Test Sample Observation Conclusion

Biuret test Egg Albumin  Bluish violet

colouration
Bluish violet colour is
indicates the
formed.
presence of
protein.
  In an alkaline
solution, the
copper (II) ion
forms a violet
colored
coordination
complex with
the peptide
bond present in
the protein.
This complex is
called biuret.

 Bluish violet
colouration
indicates the
presence of
protein.
 In an alkaline
solution, the
copper (II) ion
Bluish violet colour is
Gelatin forms a violet
formed.
colored
coordination
complex with
the peptide
bond present in
the protein.
This complex is
called biuret.

Xanthoproteic Test Egg Albumin A yellow precipitate  Yellow

is formed. precipitate
indicates the
presence of
protein.
 The yellow
precipitate is
due to
xanthoproteic
acid. It is formed
by the nitration
 of certain amino
acids, such as
tyrosine and
tryptophan,
present in the
protein.

 Yellow
precipitate
indicates the
presence of
protein.
 The yellow
precipitate is
due to
A yellow precipitate
Gelatin xanthoproteic
is formed.
acid. It is formed
by the nitration
of certain amino
acids, such as
tyrosine and
tryptophan,
present in the
protein.

Ninhydrin Test Egg Albumin Intense blue colour is  Intense blue

formed. colouration
confirms the
presence of
protein.
 This test is
mainly used for
the detection of
amino acids and
proteins with
free
‾NH2 group.
When such an
‾NH2 group
reacts with
ninhydrin, an
intense blue
 colored imino
derivative is
formed.

 Intense blue
colouration
confirms the
presence of
protein.
 This test is
mainly used for
the detection of
amino acids and
Intense blue colour is proteins with
Gelatin
formed. free
‾NH2 group.
When such an
‾NH2 group
reacts with
ninhydrin, an
intense blue
coloured imino
derivative is
formed.

Millon's Test Egg Albumin White precipitate  White

which changes to precipitate
brick red on boiling. which changes
to brick red on
boiling
indicates the
presence of
protein.
 In Millon's test,
the phenol
group of
tyrosine (amino
acid) is first
nitrated
by nitric acid in
the test solution.
The nitrated
 tyrosine then
complexes
mercury(I) and
mercury(II) ions
in the solution to
form a red
precipitate.

No characteristic
changes.  The test is not
Gelatin
applicable.

PART C. Determination of the unknown

Number of
Amino Acid Test Conducted Structure
attempts

Tyrosine or Pauly’s Diazo Test

Histidine
1

Alpha Amino
Ninhydrin test 2
Acid

Cysteine Lead sulphide test 1

Aromatic
Xanthoproteic acid test 1
Amino Acid