Perales, Kent Benedict B.

– 3CHEM1

DATE: September 10, 2020

Experiment no. 2

Examining intact protein and acid and base hydrolyzate samples for their color reactions.

OBJECTIVE(S)

 To assess what would be the composition of the intact protein, and acid and base

hydrolyzate samples in the provided color reactions

 To observe the different effects that the five different color reactions would produce on

intact protein, acid, and base hydrolyzate samples

MATERIALS AND INSTRUMENTS

A. Materials

 Acid hydrolysis solution  Concentrated NaOH

 Base hydrolysis solution  Hopkins-Cole reagent

 Intact protein solution  Concentrated H2S

 10% (2.5M) NaOH  Large test tubes

 0.01M CuSO4  Test tube rack

 0.02 % α-naphthol solution  Masking tape

 2% NaOBr  Serological pipette

 0.1% Ninhydrin solutionN  Pasteur pipette

 Concentrated HNO3  Styrofoam

PROCEDURE

For the Biuret test, a 3-mL of intact protein or hydrolyzate filtrates, both acid and

base, were used to mix the reagents of a 1-mL of 10% (2.5M) NaOH and 1-mL of 0.01

CuSO4 as shown in Figure 1.

Figure 1. The reagents that were used for Biuret test were 1-mL of 10%
(2.5M) NaOH and 1-mL of 0.01 CuSO4

For the Sakaguchi test, a 5-mL of the protein intact suspension or hydrolyzate

samples, both acid and base, were used to mix the reagents of a 1-mL of 10% (2.5M)

NaOH, 1-mL of 0.02% α-naphthol solution, and 1-mL of 2% NaOBr as shown in Figure

2.

Figure 2. The reagents that were used for Sakaguchi test were 1-mL of 10% (2.5M)
NaOH, 1-mL of 0.02% α-naphthol solution, and 1-mL of 2% NaOBr
 For the Ninhydrin test, a 1-mL of the protein intact suspension or hydrolyzate

samples, both acid and base, were used to mix the reagent of a 0.5mL of 0.1% Ninhydrin

solution. Afterward, the test was heated for 2-3 minutes as shown in Figure 3.

Figure 3. The reagents that was used for Ninhydrin test was
0.5mL of 0.1% Ninhydrin solution

For the Xanthoproteic test, a 1-mL of the protein intact suspension or hydrolyzate

samples, both acid and base, were used to mix the reagent of a concentrated HNO 3 where

three (3) drops of the said sample were added. It was heated for 1 minute. Next, the test

was subjected to the sink to be cooled down by running water. Afterward, another

reagent, a 0.5mL of concentrated NaOH, was added in the intact protein or hydrolyzate

sample as shown in Figure 4.

 Figure 4. The reagents that were used of Xanthoproteic test were 3 drops of HNO3
and 0.5mL of concentrated NaOH
For the Hopkins-Cole test, two (2) drops of protein intact suspension or

hydrolyzate samples, both acid and base, were used to mix to a 1-mL of Hopkins-Cole

reagent. The said reagent was composed of oxalic acid and acetic acid. Afterward,

another reagent, 1-mL of concentrated H2SO4, was slowly added using the inclined tube

in the protein intact suspension or hydrolyzate sample as shown in Figure 5.

Figure 5. The reagents that were used for Hopkins-Cole test were 1-mL of Hopkins-Cole
reagent and 1-mL of concentrated H2SO4

RESULTS

For the Beirut test, color observations were noted before and after intact protein

was subjected to a 1-mL of 10% (2.5 M) NaOH solution and 1-mL of 0.01M CuSO 4 as
shown in Figure 6. The color turned from a white or clear solution to a light purple or

lavender solution. Same with hydrolyzate samples, their color changed right after the said

reagents were mixed. For acid hydrolyzate, it went from a light-yellow solution to a

yellow solution. While for base hydrolyzate, it went from a yellow solution to an almost

similar color of intact protein's solution which has a shade of violet.

Figure 6. Three samples were observed from Biuret test

For the Sakaguchi test, color observations were noted before and after intact

protein suspension was subjected to a 1-mL of 10% (2.5M) NaOH solution, 1-mL of

0.02% α-naphthol solution, and 1-mL of 2% NaOBr as shown in Figure 7. The color

turned from a white or clear solution to a light-red solution. Same with hydrolyzate

samples, their color changed right after the said reagents were mixed. For acid

hydrolyzate, it went from a clear yellow solution to a pale-yellow solution. While for

base hydrolyzate, it went from a dark-yellow solution to a gold color solution.

 Figure 7. Three samples were observed from Sakaguchi test

For the Ninhydrin test, color observations were noted before and after intact

protein suspension was subjected to a 0.5 mL of 0.1% Ninhydrin solution as shown

in Figure 8. The color stayed the same which is a clear and transparent solution. Same

with hydrolyzate samples, their color changed right after the said reagents were mixed.

Both the acid and base hydrolyzates had a color of the dark-brown solution that went

from a yellow solution and dark yellow, respectively.

Figure 8. Three samples were observed from Ninhydrin test

For the Xanthoproteic test, color observations were noted before and after intact

protein suspension was subjected to three (3) drops of concentrated HNO3 and 0.5-mL of
 concentrated sodium NaOH as shown in Figure 9. The color turned from a clear or

transparent solution to a bright-yellow solution. Same with hydrolyzate samples, their

color changed right after the said reagents were mixed. For acid hydrolyzate, it went from

a yellow solution to an orange solution. While for base hydrolyzate, it went from a dark-

yellow solution to an almost similar color of acid hydrolyzate which has a shade of

orange.

Figure 9. Three samples were observed from Xanthoproteic test

For the Hopkins-Cole test, color observations were noted before and after intact protein

suspension was subjected to a 1-mL Hopkin’s Cole reagent and 1-mL concentrated H2SO4 as

shown in Figure 10. The color turned from a clear and transparent solution to a light purple or

lavender solution. Same with hydrolyzate samples, their color changed right after the said

reagents were mixed. For acid hydrolyzate, it went from a yellow solution to a bright yellow

solution with a clear and transparent on the bottom of the test tube. While for base hydrolyzate, it

went from a dark-yellow solution to a bright orange solution with a clear and transparent on the

bottom of the test tube.

 Figure 10. Three samples were observed from Hopkins-Cole test

POSTLAB ANSWERS

1. Give the principle behind each chemical test done.

A. Biuret Test

o For the Biuret test, NaOH is used to deprotonate the nitrogen in the amide

group. This negatively charged nitrogen can now form a complex with the

Cu2+ ions in the solution from the CuSO 4. This copper complex is

lavender, confirming the presence of the peptide bonds and therefore

confirming the presence of proteins.

B. Sakaguchi Test

o For the Sakaguchi test, it is mainly used for amino acids specifically,

Arginine that produces a red color in the solution. This implies that there

is the presence of an oxidizing agent like NaOBr.

C. Ninhydrin Test

o For the Ninhydrin test, it is useful because it can react with unhindered

amines to yield a colored product. It is reactive toward almost any

 sterically unhindered amine. It won't distinguish an unhindered amine and

lysine, where the amino acid that usually reacts with other groups.

D. Xanthoproteic Test

o For the Hopkins-Cole test, this gives a yellow color of the solutions.

Aromatic amino acids, specifically phenylalanine, tyrosine, and

tryptophan, will fall under this test. Since there is a presence of

concentrated HNO3, the aromatic phenyl ring is nitrated to give yellow-

colored nitro-derivatives.

E. Hopkins-Cole Test

o For the Hopkins-Cole test, this gives positive results with proteins

containing the essential amino acid specifically Tryptophan. The indole

group of Tryptophan reacts with glyoxylic acid in the presence of

concentrated H2SO4 to give a light-purple or lavender colored product.

2. Compare the results with the acid and base hydrolyzates. Explain the differences.

A. Biuret Test

o For acid hydrolyzate, it went from a light-yellow solution to a yellow

solution. While for base hydrolyzate, it went from a yellow solution to an

almost similar color of intact protein's solution which has a shade of

violet.

B. Sakaguchi Test
 o For acid hydrolyzate, it went from a clear yellow solution to a pale-yellow

solution. While for base hydrolyzate, it went from a dark-yellow solution

to a gold color solution.

C. Ninhydrin Test

o Both the acid and base hydrolyzates had a color of the dark-brown

solution that went from a yellow solution and dark yellow, respectively.

D. Xanthoproteic Test

o For acid hydrolyzate, it went from a yellow solution to an orange solution.

While for base hydrolyzate, it went from a dark-yellow solution to an

almost similar color of acid hydrolyzate which has a shade of orange.

E. Hopkins-Cole Test

o For acid hydrolyzate, it went from a yellow solution to a bright yellow

solution with a clear and transparent on the bottom of the test tube. While

for base hydrolyzate, it went from a dark-yellow solution to a bright

orange solution with a clear and transparent on the bottom of the test tube.