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ACTIVITY 2B

PRECIPITATION TEST FOR PROTEIN

GROUP 6
MARIMON, Dave Michael C.
MACION, Ayn Rand Louise V.
TANDUYAN, Maria Cristalia
RUIZO, Roichinne
DATUMANONG, Madatu Datudick Jr.
BAID, Ivy Zea
MALIRONG, Chiezyl

OBJECTIVES:

To observe the reactivity of reagent and solutions in each test.


To know the practical reactions in each test.
To identify and observe the precipitate in each test which yield a positive result.

BMLS 2H
April 21, 2014

RESULTS AND OBSERVATIONS:


NAME OF TEST
A. Precipitation reaction by
concentrated mineral and
organic acids

SUBSTANCE TO
BE TESTED
Proteins

B. Precipitation by metallic
salts

Proteins

C. Precipitation by alkaloidal
reagents

Proteins

D. Precipitation by alcohol

Proteins

E. Coagulation by heat

Proteins

F. Test for
denatured/Coagulated
Proteins

Proteins

REAGENTS USED
A. Concentrated Sulfuric Acid
B. Concentrated HCl
C. Concentrated HNO3
D. Glacial Acetic Acid
A. Dilute Mercuric Chloride
B. Dilute Lead Acetate
C. Dilute Copper Sulfate
D. Dilute Ferric Chloride
F. Dilute Barium Chloride
A. Picric Acid Solution
B. Trichloroacetic Acid
C. Tannic Acid
D. Phosphotungstic Acid
A. 95% alcohol + Dilute HCl
B. 95% alcohol + 10% NaOH
C. 95% alcohol
Acetic Acid
MILLONS TEST
Millons Reagent
XANTHOPROTEIC TEST
Concentrated Nitric Acid
Ammonium Hydroxide
BIURET TEST
10% Sodium Hydroxide
0.5% Copper Sulfate
HOPKINS COLE TEST
Hopkins Cole Reagent
Pure Concentrated Sulfuric Acid

VOLUME OF
REAGENTS USED
5 drops
5 drops
5 drops
5 drops
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
5 mL + 2 drops
5 mL + 2 drops
5 mL
2 drops

OBSERVATIONS/RESULTS
A.
B.
C.
D.
A.
B.
C.
D.
E.
A.
B.
C.
D.
A.
B.
C.

Whitish solution with precipitate at the bottom


Whitish solution with precipitate at the bottom
Clear solution ; NO PRECIPITATE
Little cloudy solution ; NO PRECIPITATE
(NOT PERFORMED)
Whitish solution ; NO PRECIPITATE
Light Bluish solution ; NO PRECIPITATE
Clear Orange solution ; NO PRECIPITATE
Clear solution ; NO PRECIPITATE
Yellowish solution with precipitate at the bottom
Whitish solution with precipitate at the top
Brownish solution with precipitate at the bottom
Whitish solution with precipitate at the top
Clear solution ; NO PRECIPITATE
Clear solution ; NO PRECIPITATE
Little cloudy solution ; NO PRECIPITATE
Coagulated whitish substance

Cloudy faded flesh solution ; NO PRECIPITATE


2 drops
Yellow precipitate at the top
5 drops
5 drops
Cloudy light bluish solution ; NO PRECIPITATE
1 mL
1 mL
Cloudy whitish solution ; NO PRECIPITATE
1 mL
10 drops

PROOF/DOCUMENTATION:

A. PRECIPITATION REACTION
BY CONCENTRATED MINERAL
AND ORGANIC ACIDS
FROM LEFT TO RIGHT:
A. CONCENTRATED SULFURIC ACID
B. CONCENTRATED HCl
C. CONCENTRATED HNO3
D. GLACIAL ACETIC ACID

B. PRECIPITATION BY
METALLIC SALTS
FROM LEFT TO RIGHT:
A. DILUTE LEAD ACETATE
B. DILUTE COPPER SULFATE
C. DILUTE FERRIC CHLORIDE
D. DILUTE BARRIUM CHLORIDE

C. PRECIPITATION BY ALKALOIDAL
REAGENTS
FROM LEFT TO RIGHT:
A. PICRIC ACID SOLUTION
B. TRICHLOROACETIC ACID
C. TANNIC ACID
D. PHOSPHOTUNGSTIC ACID

D. PRECIPITATION BY
ALCOHOL

E. COAGULATION BY HEAT

F. TEST FOR DENATURED/


COAGULATED PROTEINS

FROM LEFT TO RIGHT:


A. 95% ALCOHOL + DILUTE HCl
B. 95% ALCOHOL + 10% NaOH
C. 95% ALCOHOL

ANALYSIS AND CONCLUSION: GUIDE QUESTIONS

FROM LEFT TO RIGHT:


A.
B.
C.
D.

MILLONS TEST
XANTHOPROTEIC TEST
BIURET TEST
HOPKINS COLE TEST

1. GIVE THE PRINCIPLES INVOLVED IN EACH OF THE TEST.

Precipitation reaction by concentrated mineral and organic acids.


Salt bridges results from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium
group and the negative acid group. Any combination of various acidic or amine amino acid side chains will have this effect. As might be expected,
acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative
ions in the salts change partners with the positive and negative ions in the new acid or base added.
Precipitation by metallic salts.
Proteins are precipitated by salts of heavy metals, such as mercuric chloride, zinc sulfate, etc. in weak alkaline solution, protein molecules
carry negative charges and combine with positively charged metal ions to form insoluble salts which precipitate from the solution. The precipitated
proteins are denatured and this process is irreversible.
Precipitation by alkaloidal reagents.
Alkaloidal reagents (e.g. tannic acid and trichloroacetic acid) are high molecular weight anions. The negative charge of these anions
counteracts the positive charge of the amino group in proteins in giving a precipitate.
Precipitation by Alcohol.
Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bond is formed instead between the
new alcohol molecule and the protein side chains.
Where does the maximum precipitation occur? 3 rd tube.
Coagulation by Heat.
The result was coagulation by albumin solution. Egg- white is faintly alkaline. Complete precipitation takes place only in faintly acid solution.
The temperature in which coagulation takes place depends on a large extent on the amount of acid and of salts present.
Test for denatured/Coagulated Proteins.
The term denaturation is used more frequently than coagulation by scientific investigators at the present time to denote certain changes in
proteins. Definite characteristics of the proteins are changed when they are coagulated, among which is loss of solubility in water and dilute salt
solutions. In some instances and under certain conditions the coagulation process may be reversible. Manner in which denaturation may be bought
about. Coagulation of proteins may be brought about by a variety of processes. But in addition to heat the action of acids, akalies, salts, alcohol,
mechanical agitation, radiation, and ultra-sonic vibrations may denature the protein and convert it from soluble into insoluble form weight anions. The
negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate. Therefore, even a protein is
denatured it will still give a positive result from the Qualitative of proteins.

2. USING MILK AS TEST SOLUTION, HOW WILL YOU PROVE THAT MILK IS A PROTEIN.

Biuret solution is used to identify the presence of proteins. Biuret reagent is a blue that, when it reacts to protein, will change color to pink- purple.

Procedure:

To a test tube, add 40 drops milk solution


Add 3 drops of Biuret reagent
Shake gently to mix
Note color change
Proteins will turn the solution into pink- purple

3. SHOW A SCHEMATIC DIAGRAM TO ARRIVE AT YOUR ANSWER.


TO A TEST TUBE, ADD 40 DROPS OF MILK SOLUTION

ADD 3 DROPS OF BIURET REAGENT


(SODIUM HYDROXIDE 10% AND COPPER SULFATE 0.5%

SHAKE GENTLY TO MIX

CHANGE IN COLOR OF SOLUTION TO


PINK-PURPLE CONFIRMS PRESENCE OF PROTEIN