Biuret Test

• The biuret test is a chemical test that can be used to check for the presence
of pep琀椀de bonds in a given analyte.
• Posi琀椀ve result: a deep blue/purple color due
to the copper ion complex with the amide
group of the protein.
• Nega琀椀ve result: if the sample solu琀椀on
remains a light blue color, the protein
concentra琀椀on is low and considered a
nega琀椀ve result.
Reagents: hydrated copper sulfate, sodium
hydroxide, and Rochelle salt (sodium-potassium
tartrate)
Biuret Test Manual/ Materials
• 1% alanine and 5% albumin or egg white (as positive control)
• Biuret reagents.
• De-ionized water (as negative control)
• Dry test tubes.
• Water bath.
• Pipettes.
Biuret Reagent Preparation
It is formed by mixing NaOH in a solution of CuSO4, turning it alkaline. Following are the steps
to yield 1000mL of Biuret reagent.

• Take distilled water (500 ml) and dissolve pentavalent copper sulphate (1.5gm) and
sodium-potassium tartrate (6gm).

• Sodium potassium tartrate, a chelating substance that stabilizes the copper ions.

• Now take 2 molar hydroxide (375 ml)

• Now take a volumetric flask and mix two solutions.

• Finally, make it to 1000 ml by pouring distilled water.

Procedure
Using the following steps, you can easily conduct this test.

• First, take 3 dry and clean test tubes.

• Now add 1 or 2 mL of the test solution, albumin and de-ionized water in the test tubes.

• Add Biuret reagent (1-2 mL) in each test tube.

• Now shake the solution well and let it stand for 5 minutes.

• Finally, observe how the color changes.

Xanthoprotein Test
• Xanthoproteic test is used to detect amino acids containing an aroma琀椀c nucleus
(tyrosine, tryptophan and phenylalanine) in a protein solu琀椀on which gives yellow
color nitro deriva琀椀ves on hea琀椀ng with conc. HNO3. The aroma琀椀c benzene ring
undergoes nitra琀椀on to give yellow colored product.
• Posi琀椀ve result: The appearance of a dark yellow or orange-colored
solu琀椀on represents a posi琀椀ve test. This indicates the presence of aroma琀椀c groups in
the proteins and amino acids
• Nega琀椀ve result: The absence of a dark yellow or orange-colored solu琀椀on represents
a nega琀椀ve test.

Reagents: 1 % tyrosine, 1 % tryptophan, 1 % phenylalanine, 5 % egg white (albumin), Nitric

acid and 40 % NaOH
Material Required
• Test tubes
• Test tube stand
• Pipettes
Procedures
• Take 1ml test solution in dry test tube.
• Similarly, take 1ml distilled water in another test tube as control.
• Add 1ml of conc. HNO3 in all test tubes and mix well.
• Cool the solution under tap water.
• Now add 2ml of 40 % NaOH to all test tubes.
• Look for the color development.

Ninhydrin Test
• The ninhydrin test is a chemical test useful to iden琀椀fy
ammonia, primary/secondary amines, or amino acids.
• Posi琀椀ve result: If the solu琀椀on develops a deep blue or
purplish color, we have a posi琀椀ve ninhydrin test. If our
test sample contains ammonia, a primary or
secondary amine or any amino acid heteroatom, then
the ninhydrin test reac琀椀on will yield a Ruhemann's
purple colora琀椀on.
 • Nega琀椀ve result: The absence of the complex in the tube represents a nega琀椀ve result
and indicates the lack of amino acids in the sample.
Reagents: In 100 mL of ethanol, dissolve 0.35g of ninhydrin. Solvent for dilu琀椀on (for
quan琀椀ta琀椀ve tes琀椀ng): Combine equal parts of water and n-propanol, Standard solu琀椀on (1 per
cent protein solu琀椀on), and Sample solu琀椀on.
Materials required
• Test tubes
• Test tube stand
• Pipette
Equipment
• Water bath
• Spectrophotometer

The procedure of the Ninhydrin Test

For qualita琀椀ve analysis

• Combine 1 ml of the test sample and 1 ml of the standard protein solution in a

single dry test tube.

• Add a few drops of the ninhydrin reagent to each test tube.

• After five minutes in the water bath, let the test tubes cool to room temperature.

• Pay attention to how colours are created and how they turn out.
For quan琀椀ta琀椀ve analysis

• Pipette increasing volumes of the protein solution (10 l, 20 l, etc.) from the given
stock solution into a series of test tubes, then distil the liquid to 1 mL.

• Take a blank tube, 1ml of distilled water, and the remaining tubes labelled 2 to 9
for the construction of a standard curve. Unidentified samples are stored in tubes
10-15.

• Add 1 ml of the ninhydrin reagent and 5 mL of the diluent solvent to each tube
and fully mix by vertexing.

• Allow time for the tubes to cool.

• Cover the tubes and incubate at 90°C for 17 minutes or 20 minutes in a boiling
water bath.

• Cool the tubes to room temperature before measuring the optical density of the
solutions against a blank at 570 nm (440 nm for proline and hydroxyproline).

• To quantify the amount of amino acid in the unknown substance, draw a

standard curve of A570 on the Y-axis and the concentration of amino acid on the
X-axis.
Millon’s Test
• Millon's test is given by proteins containing phenolic amino acids. Gela琀椀n does not
give this test. First, a white precipitate is formed when proteins are treated with
millions reagent and then turns to brick-red color on boiling, this con昀椀rms the
presence of proteins.
• Posi琀椀ve result: any protein containing tyrosine will give a posi琀椀ve test of a pink to
dark-red color.
• Nega琀椀ve result: A nega琀椀ve result in the Millon's test is demonstrated by the absence
of colored precipitate in the test tube. This indicates the absence of tyrosine or
tyrosine-containing protein.
Reagent
• Millon’s reagent: Millon’s reagent consists of mercuric nitrate and mercurous nitrate
dissolved in nitric acid and distilled water.
Materials Required
• Test tubes
• Test tube stand
• Pipettes
• Water bath
Procedure
1. About 2 ml of the sample solu琀椀on or the 1% tyrosine solu琀椀on is taken in a test
tube.
2. To this, about 2 ml of Millon’s reagent is added. The test tubes are then kept in the
water bath for about 2 minutes if red colored precipitate is not observed
immediately.
3. The tubes are then observed for the forma琀椀on of the colored precipitate.
Lead Acetate or Sulfur Test
• to iden琀椀fy materials that contain sulfur,
because these materials might emit
gases that will tarnish silver, copper and
alloys that contain these metals (such as
sterling silver, brass and bronze).
• Posi琀椀ve result: if the paper turns dark
brown or black a昀琀er exposure to the
fumes from the sample and then turns
white a昀琀er exposure to hydrogen
peroxide
• Nega琀椀ve result: No forma琀椀on of black precipitate indicate the absence of sulfur-
containing amino acid.
Reagents: 2% lead acetate solution in water, 40% NaOH and Sample

Material Required
• Test tubes
• Test tube stand
• Pipettes
Procedure
1. In a test tube, 2 ml of the amino acid solution is taken. To this, 2 ml of NaOH is
added, and the solution is boiled for a minute.
2. Once the test tube cools down, a few drops of lead acetate are added to the
solution.
3. The test tube is then observed for the formation of a precipitate.

Hopkin’s Cole Test

• Hopkins Cole Test is one of the qualita琀椀ve test methods to determine di昀昀erences in
types of proteins and amino acids. This test is used to iden琀椀fy the presence of the
amino acid tryptophan, which is the only amino acid that has an indole ring group.
Tryptophan belongs to the group of essen琀椀al amino acids.
• Posi琀椀ve result: is represented by the
forma琀椀on of a purple-colored ring at the
junc琀椀on of two layers. This indicates the
presence of tryptophan-containing
proteins.
• Nega琀椀ve result: represented by the
absence of a purple-colored ring in the
test tube. This indicates the absence of
tryptophan-containing proteins.
Reagent
• Hopkin’s Cole reagent: Glyoxylic acid- It can be prepared by exposing glacial acetic
acid to sunlight for a few days.
• Concentrated H2SO4
• Sample: 0.1% amino acid solution
Material Required
• Test tubes
• Test tube stand
• Pipettes
Procedure
1. In a test tube, 2 ml of light-exposed glacial acetic acid and 2 ml of the sample liquid
are taken.
2. To this, concentrated H2SO4 is added along the sides of the test tube held at a
slanting position. Two distinct layers of liquid are to be formed without mixing.
(Note: Mouth of the tube must point away from the face, as we should be careful
while adding the sulfuric acid.)
3. The test tube should be observed for the formation of a colored ring at the
interface of two layers.

Sakaguchi Test
• The Sakaguchi test is a chemical test used to detect presence of arginine in proteins.
• Posi琀椀ve result: Red color (Arginine)
• Nega琀椀ve result: no red color (glycine, albumin)
Reagents:
• test solu琀椀on: 1 % arginine, 1 % glycine,
5 % egg white (albumin)
• 1 % α naphthol in alcohol
• Sodium hypochlorite
• 1 % urea solu琀椀on
• Dry test tubes
• Pipe琀琀es
Material required
• Test tubes
• Test tube stand
• Pipettes
Procedure
1. Take 1ml test solu琀椀on in dry test tube.
2. Similarly, take 1ml dis琀椀lled water in another test tube as control.
3. Add 2 drops of α naphthol and mix well.
4. Now add 2ml sodium hypochlorite to all test tubes.
5. Immediately add 1ml of urea solu琀椀on to establish the red complex formed.
References:
• h琀琀ps://www.vedantu.com/chemistry/biuret-test
• h琀琀ps://www.onlinebiologynotes.com/xanthoproteic-test-objec琀椀ve-principle-
reagents-procedure-and-
result/#:~:text=test%20solu琀椀on%3A%201%20%25%20tyrosine%2C,40%20%25%20
NaOH
• h琀琀ps://www.toppr.com/guides/chemistry/ninhydrin-
test/#:~:text=a%20brown%20colour.-
,Reac琀椀on%20Requirements,solu琀椀on)%2C%20and%20Sample%20solu琀椀on.
• h琀琀ps://microbenotes.com/ninhydrin-test/
• h琀琀ps://microbenotes.com/millons-test/
• h琀琀ps://microbenotes.com/lead-sul昀椀de-test/
• h琀琀ps://microbenotes.com/hopkins-cole-test/
• h琀琀ps://www.onlinebiologynotes.com/sakaguchi-test-objec琀椀ve-principle-reagents-
procedure-and-result/
• h琀琀ps://microbenotes.com/sakaguchi-test/