Module 4

Qualitative Test for

Proteins
PART A
 Millon’s Test
Materials:
• 1 mL Amino acid solution
• Millon’s Reagent
• Concentrated Nitric Acid Solution

Procedure:
1. Transfer 1 mL of amino acid solution to a test tube using a
dropper.
2. Add few drops of Millon’s reagent and mix by shaking it.
3. Carefully add few drops of concentrated nitric acid solution
into the test tube.
Results and Discusions:
 Histidine Test
Materials:
• 2 mL unknown solution
• 5% Bromine in 33% acetic acid ssolution
• 2 mL ammonium carbonate solution
• Test tube
• Dropper
• Water Bath

Procedure:
1. Transfer 2 mL of unknown amino acid solution to a test tube using
a dropper.
2. Add few drops of 5% Bromine in 33% Acetic acid solution into the
test tube.
3. Keep the test tube at room temperature for 10 minutes.
4. Add 2 mL Ammonium Carbonate solution to this test tube and keep
it in a boiling water bath for 5 minutes.
Results and Discusions:
 Hopkin’s Cole Test
Materials:
• 1 mL unknown solution
• 1 mL Acetic acid – Glyoxilic acid reagent
• Concentrated Sulfuric acid
• Test tube
• Dropper

Procedure:
1.Add 1 mL of unknown solution in a test tube using a dropper.
2.Add 1 mL of Acetic acid – Glyoxilic acid reagent to the same test
tube containing solution.
 3.Mix the solution by shaking.
 4.Carefully add few drops of concentrated Sulfuric Acid down
the side of the test tube containing solution

Results and Discusions:

 Lead Sulfide Test
Materials:
 1 mL unknown amino acid solution
 NaOH solution
 Marble Chips
 10% Lead acetate
 Bunsen burner

Procedure:
1.Add 1 mL of unknown amino acid solution to a test tube using a
dropper.
2.Add few drops of NaOh solution to the same test tube-containing
solution.
3.Put some marble chips into the solution in order to avoid the
bumping of the solution while heating.
 4.Heat the solution on the Bunsen Burner for about 5-10 minutes.
 5.After heating, keep the test tube under running tap water to
cool the contents.
 6.Add few drops of 10% Lead Acetate solution into the test tube-
containing solution.

Results and Discusions:

 Isatin Test
Materials:
 Unknown amino acid solution
 Isatin reagent
 Filter paper strip
 Dropper

Procedure:
 Apply drop of unknown amino acid solution on the
filter paper strip using a dropper, and dry the spot
using hair dryer.
 Apply a drop of Isatin reagent again and repeat drying
procedure for s few minutes.
Results and Discusions:
 Folin’s McCarthy Sullivan’s Test
Materials:
 1 mL unknown amino acid solution
 40% NaOH
 Glycine solution
 Nitroprusside solution
 0.5 mL 6N HCl
 Test tube
 Water Bath

 Procedure:
 Take 1 mL of the unknown amino acid solution in a test tube.
 Add a few drops of 40% NaOH.
 Add few drops of glycine and sodium nitroprusside solution.
 Place the test tube in a hot water bath maintained at 40⁰C for 15 minutes.
 Add 0.5 mL of 6N HCl into the test tube
Results and Discusions:
 Sackaguchi Test
Materials:
 Pre-chilled Unknown amino acid solution
 40% NaOH
 Alpha-naphtol solution
 5% Urea
 Hypobromite solution

Procedure:
 Transfer few drops of pre-chilled unknown amino acid to a test
tube.
 Add few drops of 40% NaOH solution.
 Add few drops of Alpha-Naphtol Solution.
 Add few drops of 5% Urea.
 Add few drops of Hypobromite Solution
Results and Discusions:
PART B
 Biuret Test

Materials:
 Egg albumin
 Gel dispersion
 NaOH solution
 1% CuSO4 solution
 Bunsen Burner

Procedure:
 Place a small amount of egg albumin on a test tube labeled with “A”
while place a small amount of gel dispersion on a test tube labeled
with “B”.
 Use the dropper to drop NaOH solution into each of the test tubes
respectively.
 Use another dropper to drop 1% CuSO4 solution into each of the
test tubes respectively.
 Place a small amount of egg albumin on a test tube labeled with “A”
while place a small amount of gel dispersion on a test tube labeled
with “B”.
 Use the dropper to drop NaOH solution into each of the test
tubes respectively.
 Use another dropper to drop 1% CuSO4 solution into each of the
test tubes respectively.

Results and Discusions:

 Xanthoproteic Test
Materials:
 egg albumin
 gel dispersion
 Conc. Nitric Acid
 Bunsen Burner

Procedure:
 Place a small amount of egg albumin on a test tube labeled with “A”
while place a small amount of gel dispersion on a test tube labeled
with “B”.
 Use the dropper to drop Conc. Nitric Acid into each of the test
tubes respectively.
 Turn the knob of the Bunsen burner to turn it on.
 Using a test tube holder, place test tube A to the burner and heat
it for 5 minutes.
 Using a test tube holder, place test tube B to the burner and heat
it for 5 minutes.
Results and Discusions:
 Ninhydrin Test
Materials:
 egg albumin
 gel dispersion
 Ninhydrin solution
 Bunsen burner

Procedure:
 Place a small amount of egg albumin on a test tube labeled with “A”
while place a small amount of gel dispersion on a test tube labeled with
“B”.
 Use the dropper to drop Ninhydrin solution into each of the test
tubes respectively.
 Turn the knob of the Bunsen burner to turn it on.
 Using a test tube holder, place test tube A to the burner and heat it
for 5 minutes.
 Using a test tube holder, place test tube B to the burner and heat it
for 5 minutes.
Results and Discusions:
 Millon’s Test
Materials:
 egg albumin
 gel dispersion
 Millon’s Reagent
 Bunsen burner

Procedure:
 Place a small amount of egg albumin on a test tube labeled with “A”
while place a small amount of gel dispersion on a test tube labeled
with “B”.
 Use the dropper to drop Millon’s reagent into each of the test
tubes respectively.
 Turn the knob of the Bunsen burner to turn it on.
 Using a test tube holder, place test tube A to the burner and heat
it for 5 minutes.
 Using a test tube holder, place test tube B to the burner and heat
it for 5 minutes.
Results and Discusions:
PART C
 Ninhydrin Test

Materials:
 Unknown solution
 Ninhydrin reagent
 Water Bath

Procedure:
1. Few drops of the unknown solution were taken in a test tube 2.
2. Added a few drops of ninhydrin reagent, and vortex the
contents.
3. Place the tube in a boiling water bath for 5 minutes and cool to
room temperature.
Results and Discusions:

Yellow Result Blue Result

 Xanthoproteic acid Test

Materials:
 Unknown solution
 HNO
 NaOH
 Bunsen Burner

Procedure:
1. Few drops of the unknown solution taken in a testtube, add a
few drops of HNO​and vortex the contents.
2. Boil the contents over a Bunsen flame, using a test tube holder,
for a few minutes.
3. Cool the test tube by placing it on a beaker with water and add a
few drops of NaOH.
Results and Discusions:

No Change Result Orange Result

 Pauly’s- Diazo Test

Materials:
 Sulfanilic Acid
 NaNO
 Amino Acid
 NaCO

Procedure:
1. Add a few drops of Sulfanilic Acid, NaNO, Amino Acid, NaCO in
a test tube and vortex until the color appears
Results and Discusions:

No Change Result Red Result

 Millon’s Test
Materials:
• Unknown solution
• Millon’s Reagent
• HNO3
• Bunsen Burner

Procedure:
1. Put unknown solution into the test tube
2. Add Millon’s reagent
3. Put on burner for 3 to 5 minutes
4. And then add HNO3
Results and Discusions:
 Hopkins Cole test
Materials:
• Unknown solution
• Acetic acid
• Glyoxylic acid
• H2SO4

Procedure:
 Put unknown solution into the test tube
 2. Add acetic acid, Glyoxylic acid and, H2SO4
Results and Discusions:
 Lead Sulphide Test
Materials:
• Unknown solution
• NaOH
• Lead Acetate
• Bunsen Burner

Procedure:
1. Put unknown solution into the test tube
2. Add NaOH
3. Put on burner for a few minutes
4. Add Lead Acetate to the solution
Results and Discusions: