Millon's Test

Indian Journal of Drugs, 2019, 7(1), 30-33 ISSN: 2348-1684
QUALITATVE ANALYSIS OF PROTEIN: EGG ALBUMIN AND MILK

Somendra Kumar Maurya1 Amrita Asthana2, Satya Prakash Maurya3, Pooja Maurya4, Anita Maurya5,
1,2,4,5 Department of Pharmacy, Prasad Institute of Technology, Jaunpur-222001 (U.P.)
3 R.D.S. college of pharmacy, Kusanw , Jaunpur U.P.222136.
*
For Correspondence: ABSTRACT
Department of Pharmacy, Prasad As the name suggests amino acids are organic compounds that contain
Institute of Technology, Jaunpur amino and carboxyl groups. The above formula stands for different
U.P 222001 chemical groups and this determines the characteristics of the amino
Received: 29.09.2018 acids. The color tests have frequently been used for qualitative
Accepted: 22.03.2019 detection of amino acids. Not all amino acids contain the same reactive
Access this article online groups. For this reason, the various color tests yield reactions varying in
intensity and type of color according to the nature of groups contained
Website: in the particular amino acid under examination. Ninhydrin test, Biuret
www.drugresearch.in test, Xanthoproteic acid test and Millon’s test were present in egg
Quick Response Code: albumin and milk. This is essential substances in human body for the
maintenances of protein.
KEY WORDS: Egg Albumin and Milk, Ninhydrin test, Biuret test,
Xanthoproteic acid test and Millon’s test.
P
INTRODUCTION
ROTEIN: The word “protein” is derived from the Greek word “proteios”, which means “of
primary importance”. In fact, proteins play an important role in all biochemical and
physiological body processes; they act as enzymes, hormones, receptors, antibodies and are
required for the structural integrity of cells. Proteins are the most essential for the maintenance of
human life. Tissue, muscles, organs, enzymes and hormones are protein in nature. The body can’t
store excess protein. Protein requirement is expressed in terms of grams/ kg body weight. The daily
body requirement of protein for an adult is about 1g/kg body weight. It is desirable that one fifth of it
should be animal protein. Protein deficiency amounts to amino acids deficient and is accompanied by
deficiency of mineral and vitamins. Protein calorie malnutrition (PCM) means deficiency of calories
due to deficiency of protein, it is also known as protein Energy Malnutrition (PEM). [1] A protein is said
to be all the essential amino acids are present in the required proportion by the human body that is
egg albumin.
Essential and non-essential amino acids: There are 20 standard amino acids which differ in their side
chain. Some of them are considered “essential” since they cannot be synthesized in our body and
must be therefore provided in the diet (e.g. tryptophan & phenylalanine), while others are “non-
essential” and can be synthesized in the body (e.g. alanine & cysteine). A good source of protein is
often a combination of various foods, because different foods are rich in different amino acids. A
IJOD, 2019, 7(1), 30-33 www.drugresearch.in
good source of dietary protein meets two requirements: [2] The requirement for the nutritionally
indispensable amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valine) under all conditions and for conditionally indispensable amino
acids (cystine, tyrosine, taurine, glycine, arginine, glutamine, proline) under specific physiological and
pathological conditions
 The requirement for nonspecific nitrogen for the synthesis of the nutritionally dispensable amino
acids (aspartic acid, asparagine, glutamic acid, alanine, serine) and other physiologically important
nitrogen-containing compounds such as nucleic acids, creatine, and porphyrins.
Protein deficiency and malnutrition (PEM) can lead to variety of ailments including mental
retardation and kwashiorkor. Symptoms of kwashiorkor include apathy, diarrhea, inactivity, failure to
grow, flaky skin, fatty liver and edema of the belly and legs. This edema is explained by the action of
lipoxygenase on arachidonic acid to form leukotrienes and the normal functioning of proteins in fluid
balance and lipoprotein transport. [3,4] PEM is fairly common worldwide in both children and adults
and accounts for 6 million deaths annually. In the industrialized world, PEM is predominantly seen in
hospitals, is associated with disease, or is often found in the elderly. [5] Albumin and globulin are two
types of protein in your body. The total protein test measures the total amount albumin and globulin
in your body. It’s used as part of your routine health checkup. It may also be used if you have
unexpected weight loss, fatigue or the symptoms of a kidney or liver disease.
Purpose of the total protein test
A total protein test is completed as part of your routine health checkup. It’s one of the tests that
make up your comprehensive medical panel (CMP). It may be ordered if you have:
 unexplained weight loss
 fatigue
 edema, which is swelling caused by extra fluid in your tissues
 symptoms of kidney or liver disease
The total protein test measures the total amount of protein in your blood and specifically looks for
the amount of albumin and globulin. This test will also look at the ratio of albumin to globulin in your
blood. This is known as the “A/G ratio.”
MATHODS
Using the provided solutions of albumin (egg white), casein (milk protein) and gelatin (animal
collagenous material), perform the following [6,7,8].
A. General tests
B. Color reactions
C. Precipitation reactions
A. GENERAL TESTS FOR PROTEINS
1. Ninhydrin test: Ninhydrin is most commonly used as a forensic chemical to detect “fingerprints”, as
amines left over from proteins sloughed off in fingerprints react with ninhydrin giving a characteristic
purple color.
 Procedure & observation:
To 1 ml amino acid solution in a test tube, add 1 drop of ninhydrin. Put in a boiling water bath and
observe the formation of a purple color.
2. Biuret test: The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins
to form a blue to violet complex known as the “biuret complex”.

To 2 ml of protein solution in a test tube, add 3 drops of 10% sodium hydroxide solution and 3-6
drops of 0.5% copper sulfate solution. Mix well; a blue to violet color is obtained with albumin, casein
& gelatin.
B. COLOR REACTIONS OF PROTEINS
1. Xanthoproteic acid test: Nitric acid gives a color when heated with proteins containing tyrosine
(yellow color) or tryptophan (orange color); the color is due to nitration.
To 2 ml of protein solution in a test tube, add 2 drops of concentrated nitric acid. A white precipitate
is formed and upon heating in a boiling water bath, it turns yellow with “tyrosine” and orange with
the essential amino acid “tryptophan” indicating a high nutritive value.
2. Millon’s test: Millon's reagent (Hg/HNO3) gives positive results with proteins containing the
phenolic amino acid “tyrosine”.
Took 2 ml of protein solution in a test tube, add 3 drops of Millon’s reagent. Mix well and heat
directly on a small flame. A white ppt is formed with albumin and casein (but not gelatin); the ppt
gradually turns into brick red.
RESULTS
S.No. Tests Egg Albumin Milk
1. Ninhydrin test: Test solution and added Present Present
2drops Ninhydrin reagent and heat on
water bath- Purple colour
2. Biuret test: Test sample and added Present Present

Biuret Reagent-Purple or pink colour
3. Xanthoproteic acid test: Test solution Present Present

and added 2 drops of nitric acid then
heat on water bath- Yellow colour
4. Millon’s test: test sample +2ml millon’s Present Present

reagent and boil for 10min on water
bath + 5drops NaNO2- Brick red colour
Ninhydrin test, Biuret test, Xanthoproteic acid test and Millon’s test were present in egg albumin and
milk. This is essential substances in human body for the maintenances of protein.
CONCLUSION
The normal range for total protein is between 6 and 8.3 grams per deciliter (g/dL). This range may
vary slightly among laboratories. Low albumin is considered albumin below 3.4 g/dL. It’s associated
with decreased effectiveness of medications used for ulcerative colitis. Low albumin levels may result
in complications during or after surgery.

REFERENCES
1. Jain.N.K; “ health education and community pharmacy” 1st edition 1996; reprint 2010; CBS
publisher and distributers, page no.15.
2. Young VR, Pellett PL (May 1994). "Plant proteins in relation to human protein and amino acid
nutrition" (PDF). The American Journal of Clinical Nutrition. 59 (5 Suppl).
3. Young VR (August 1994). "Adult amino acid requirements: the case for a major revision in
current recommendations" (PDF). The Journal of Nutrition. 124 (8 Suppl).
4. Young VR, Pellett PL (May 1994). "Plant proteins in relation to human protein and amino acid
nutrition" (PDF). The American Journal of Clinical Nutrition. 59 (5 Suppl)
5. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol,
Protein and Amino Acids, Institute of Medicine. National Academy Press, 2005.
6. satyanaryana U; chakarpani U;” Biochemestry, 3rd edition published by arunbha sen book and
allied and page no.765.
7. Sindhe Rana chattergea M.N,” Atext book of medical biochemistry”,8th edition published by
Jaypee brothers’ medical publishers (P) Ltd. Page no.156.
8. Murray K. Robert, Granner K.; Dryl Rod well W., v ictor, horpers Illustrated “Biochemistry “2nd
edition, published by the MC Graw, Hill companies page no.146.

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Indian Journal of Drugs, 2019, 7(1), 30-33 ISSN: 2348-1684

QUALITATVE ANALYSIS OF PROTEIN: EGG ALBUMIN AND MILK

IJOD, 2019, 7(1), 30-33 www.drugresearch.in

2. Biuret test: Test sample and added Present Present

3. Xanthoproteic acid test: Test solution Present Present

4. Millon’s test: test sample +2ml millon’s Present Present

IJOD, 2019, 7(1), 30-33 www.drugresearch.in

IJOD, 2019, 7(1), 30-33 www.drugresearch.in

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