Biochemistry lab manual II semester

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YASH INSTITUTE OF PHARMACY AURANGABAD
LABORATORY MANUAL
Subject Code: BP209P
Subject Name: Biochemistry
Class and Semester: I year II semester
Laboratory: Pharmaceutical Chemistry II
Name Role Signature Date
Prepared by: Mrs. D. M. Kulkarni Subject
Incharge
Checked by: Mr.A.S.Narute Head of
Department
Authorized
by:
Dr. S. S. Angadi Principal
Issued by: Dr. G. A. Vaishnav ISO
Coordinator
Document version history:
Version Issued on (Date) Reason for issue
00 01-01-2020 First issue

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INDEX
Expt. No. Aim Page No.
1 To introduce different tests used in qualitative analysis of
carbohydrates
2 To perform qualitative analysis of given unknown sample of
carbohydrates [Glucose/dextrose ]
carbohydrates [Starch ]
carbohydrates [Fructose ]
carbohydrates [Lactose ]
carbohydrates [Maltose ]
carbohydrates [Sucrose ]
8 To introduce different test used in qualitative analysis of proteins
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To perform Qualitative analysis of given unknown sample of protein
(albumin)
10
To perform Qualitative analysis of given unknown sample of protein
(CASEIN)
11 To perform Quantitative analysis of given unknown sample of protein
(Albumin)
12 To demonstrate estimation of quantity of creatinine in given
sample of urine by Alkaline picrate method (Jaffe's reaction)
13 To demonstrate estimation of quantity of glucose in given sample of
serum. (GOD/POD method ).
14 To detect abnormal constituents in given sample of urine by qualitative
tests.
15 To demonstrate Estimation of Cholesterol in serum by enzymatic
method

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EXPERIMENT NO. 1
AIM – To introduce different tests used in qualitative analysis of carbohydrates
REFERENCE: -
1. Kale S.R.,Kale R.R., “Practical Biochemistry And Clinical Biochemistry”, Nirali Prakashan,
24 th edition, 2015 page no 4 to 6
2. David T. Plummer “Introduction of Practical Biochemistry” 3rd Edition
3. Rajagopal and Ramakrishna “Practical Biochemistry for Medical students” 5 th edition .
THEORY: - A carbohydrate is an organic compound with the general formula Cn(H2O)n, that
is, consists only of carbon, hydrogen and oxygen, with the last two in the 2:1 atom ratio.
Carbohydrates make up the bulk of organic substances on earth and perform numerous roles in
living things.
The carbohydrates (saccharides) are divided into four chemical groups: monosaccharides,
disaccharides, oligosaccharides and polysaccharides. Polysaccharides serve for the storage of
energy (e.g., starch in plants and glycogen in animals) and as structural components (e.g.,
cellulose in plants and chitin in arthropods). Structural polysaccharides are frequently found in
combination with proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides). The
5-carbon monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD and
NAD) and the backbone of the genetic molecule known as RNA. The related deoxyribose is a
component of DNA. Saccharides and their derivatives include many other important
biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis,
blood clotting and development [1].
This experiment aims to introduce you with the identification of unknown carbohydrates. To
gain maximum benefit, observations should be related, as far as possible, to the structure of
the substances examined.
Some important points:
1. Most of the tests and reactions described are not quantitative and volumes are approximate,
despite these facts some tests do not work if quantities greatly in excess of those stated are used.
2. DO NOT place your pipettes in reagent bottles as this leads to contamination.
3. In most tests, it is important to apply a control test using water instead of the solution under
examination. If you are in doubt about the result of a test, perform the reaction with a suitable
known compound.
4. In this experiment, sugar samples are given in their solid state. To perform each procedure,
you should prepare your own sugar solution by taking very small amounts of solid sugars.

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5. When you need to boil your sample in a test tube, prepare a hot water in a large beaker and put
your test tube inside the beaker. DO NOT forget to put boiling chips in the beaker.

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TESTS ON CARBOHYDRATES:
1) Molisch’s Test:
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid
to produce an aldehyde (either furfural or a derivative), which then condenses with the phenolic
structure resulting in a red or purple-colored compound.
PROCEDURE:
- Apply this test two different carbohydrate solutions of your own choice, preferably to one
monosaccharide and one polysaccharide.
- Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s reagent
(10% α-naphthol in ethanol).
- Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a layer at the
bottom of the tube.
- Observe the color at the interface between two layers and compare your result with a control
test.
A brown color due to charring must be ignored and the test should be repeated with a
more dilute sugar solution.
2) Carbohydrates as Reducing Sugars:
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The
enolization of sugars under alkaline conditions is an important consideration in reduction tests.
The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde
or ketone group for reduction reactions. A number of sugars especially disaccharides or
polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar)
molecule to another one, and hence there is no reducing group on the sugar; like in the case of
sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence of alkali
causes extensive enolization especially at high pH and temperature. This leads to a higher
susceptibility to oxidation reactions than at neutral or acidic pH. These sugars, therefore, become
potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort. Most commonly used
tests for detection of reducing sugars are Fehling’s Test, Benedict’s Test and Barfoed’s Test.
a) Fehling’s Test:
Fehling’s Solution (deep blue colored) is used to determine the presence of reducing sugars and
aldehydes. Perform this test with fructose, glucose, maltose and sucrose.
Procedure:
- To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling solution B
(solution of potassium tartrate).

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- Add 2 mL of the sugar solution, mix well and boil.
Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.
b) Barfoed’s Test:
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that is can
only be reduced by monosaccharides but not less powerful reducing sugars. Disaccharides may
also react with this reagent, but the reaction is much slower when compared to monosaccharides.
Perform this test with glucose, maltose and sucrose.
Procedure:
- To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar solution.
- Boil for 5 min. in a water bath and allow to stand.
You will observe a brick-red cuprous oxide precipitate if reduction has taken place.
c) Seliwanoff’s Test:
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are distinguished
from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a
ketose and if it contains an aldehyde group, it is an aldose. This test is based on the fact that,
when heated, ketoses are more rapidly dehydrated than aldoses. Perform this test with glucose,
fructose, maltose and sucrose.
Procedure:
- Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10%
HCl) in boiling water.
In less than 30 seconds, a red color must appear for ketoses.
Upon prolonged heating, glucose will also give an appreciable color.
.
d) Bial’s Test:
Bial’s Test is to determine the presence of pentoses (5C sugars). The components of this reagent
are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to form furfural
and the solution turns bluish and a precipitate may form. Perform this test with ribose and
glucose.
Procedure:
- To 5 mL of Bial’s reagent, add 2-3 drops of sugar solution and boil.
Upon boiling, note the green-blue color formed.
3) Action of Alkali on Sugars:
Procedure:

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- Heat 1 mL glucose solution with 1 mL 40% NaOH for 1 min.
- Cool and apply test for reducing sugars (e.g.; Fehling’s Test).
- Apply a control test with glucose solution to observe the difference.
4) The Inversion of Sucrose:
Sucrose is a disaccharide, which means that it is a molecule that is derived from two simple
sugars (monosaccharides). In the case of sucrose, these simple sugars are glucose and fructose.
Inverted sugar is a mixture of glucose and fructose. It is obtained by splitting sucrose into these
two components. The splitting of sucrose is a hydrolysis reaction which can be induced simply
by heating an aqueous solution of sucrose. Acid also accelerates the conversion of sucrose to
invert.
Procedure:
- Add 5 mL of sucrose solution to two test tubes.
- Add 5 drops of conc. HCl to one test tube.
- Heat both tubes in boiling water bath for 10 min.
- Cool and neutralize with diluted NaOH (use litmus paper).
- Test both solutions for the presence of reducing sugar with Fehling’s Test.
5) Iodine Test:
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an
aqueous solution of potassium iodide) reacts with starch producing a blue-black color. Apply
this test to all the polysaccharides provided.
Procedure:
- To 2-3 mL of polysaccharide solution, add 1-2 drops of iodine solution.
- Observe the different colors obtained for each of the polysaccharide solutions.
6) Unknown Part:
- Take an unknown solid from your assistants and please DO NOT forget to write your
unknown number in your lab reports.
- Carry out the carbohydrate tests in a reasonable sequence to determine your

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EXPERIMENT 2
AIM – To perform qualitative analysis of given unknown sample of carbohydrates
[Glucose/dextrose ]
REFERENCE: -
Chemicals Requirement
Sr. Chemical Grade Qty/ group Qty/Batch Qty/ 3 batches
1 Molish reagent LR 2ml 40 ml 120ml
2 felhing A reagent LR 2ml 40 ml 120ml
3 felhing B reagent LR 2ml 40 ml 120ml
4 Barford reagent LR 2ml 40 ml 120ml
5 Tommers Reagent LR 2ml 40 ml 120ml
6 selwinof’s reagent LR 2ml 40 ml 120ml
8 sodium hydroxide LR 2ml 40 ml 120ml
9 potassium ferricyanide LR 2ml 40 ml 120ml
10 Dextrose LR 1gm 20 gm 60gm
Glassware Requirement
Sr. Glassware Qty/ group Qty/Batch
1 Test tubes 5 20
2 Test tubes holder 4 20
3 Test tubes stand 1 5
4 Beaker 2 10
5 Glass rod 5 20

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1 Microscope 1 4
Observation table:
Sr.No. Test Observation Inference
1 Molish test
Place 2 mL of a carbohydrate solution in a test
tube, add 1 drop of Molisch’s reagent. Add 1-2 mL
of conc. H2SO4 down the side of the test tube, so
that it forms a layer at the bottom of the tube.
Violet color ring is
formed at the
interface between two
layers.
Carbohydrates
may be present.
2. Solubility Test
Compound + 2 ml water
Soluble Monosaccharide
/disaccharide may
be present
3 Fehling test
To 1 mL of Fehling’s solution A add 1 mL of
Fehling solution B Add 2 mL of the sugar solution,
mix well and boil.
Red, orange, violet
precipitate of cuprous
oxide
Reducing sugar
may be present
4 Benedicts test
To 1-2 ml of benedicts reagent, add an equal
volume of sugar solution.
brick-red precipitate Reducing sugar
may be present
5 Tommers test
To 1-2 ml of Tommers reagent, add an equal
volume of sugar solution. Boil for 5 min. in a water
bath and allow to stand.
Orange /yellow
precipitate
Reducing sugar
may be present
6 Barfoed’s test
To 1-2 mL of Barfoed’s reagent, add an equal
brick-red cuprous
oxide precipitate at
the bottom of test
tube
Monosaccharide
may be present
7 Selwinof’s test
To 1-2 mL of Selwinof’s reagent, add an equal
bath and allow to stand
No red precipitate Aldoses may be
present

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8 Rapid ferfural test
To 1-2 mL of sugar solution add 1 ml of 1% alpha
naphthol solution +5 ml conc. HCl
No deep purple
colour or precipitate
Aldoses may be
present
C.T. of Glucose /Dextrose
1 Test solution +NaOH solution Brown resinous
formed
Glucose present
2 3 ml of water + drop of methylene blue + NaOH
solution+ sugar solution
Blue colour
decolorized
Glucose present
3 3 ml of potassium ferricyanide + 1 ml NaOH
solution+ sugar solution drop by drop keep
solution boiling
Yellow colour of
ferricynide is
discharged
Glucose present
4 3 ml of sugar solution + 1 ml of NaOH solution +
heat
Red colour developed Glucose present
5 Osazone test
0.2 g of sugar sample + 0.4 g of phenyl hydrazine
hydrochloride + 0.6 g of sodium acetate + 4 ml
water+ heat on boiling water bath for 30 min ,
allow to cool and crystals developed observe them
under microscope
Greenish, brownish
fan shaped crystals
were observer
Glucose confirmed
Structure:

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Conclusion: All the naturally occurring carbohydrates are dextrorotatory in nature, hence glucose
and dextrose are used simultaneously.
Result : Qualitative tests of given unknown sample of carbohydrates was performed and it was
found to be glucose / dextrose

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EXPERIMENT 3
AIM – To perform qualitative analysis of given unknown carbohydrate sample [Starch ]
REFERENCE: -
REQUIREMENT:
Chemicals required :
2 Sstarch solution LR 2ml 40 ml 120ml
3 Iodine Solution LR 2ml 40 ml 120ml
Glassware required :
Sr.no. Glassware Qty/ group Qty/Batch
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20
Observation table:
Sr.No. Test Observation Inference
1 Molish test
formed at the
layers.
Carbohydrates
may be present.
2. Iodine test
Carbohydrate sample + 2 ml water + drop of
iodine solution
Blue colour obtained starch may be
present

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3 C.T. for starch
Carbohydrate sample + 2 ml water + drop of
iodine solution+ heat
Blue colour
disappear
And reappears on
cooling
Starch confirmed
Structure:
Resul : Qualitative tests of given unknown sample of carbohydrates was performed and it was
found to be Starch

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EXPERIMENT 4
AIM – To perform qualitative analysis of given unknown sample of carbohydrates [fructose]
REFERENCE: - Kale S.R.,Kale R.R., “practical biochemistry and clinical biochemistry”,
Nirali prakashan 24 th edition 2015 page no 4 to 6
REQUIREMENT:
Chemicals:
10 Fructose LR 1gm 20 gm 60gm
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20

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Observation table:
1 Molish test
Place 2 mL of a carbohydrate solution in a test tube,
add 1 drop of Molisch’s reagent. Add 1-2 mL of
conc. H2SO4 down the side of the test tube, so that it
forms a layer at the bottom of the tube.
formed at the
layers.
Carbohydrates
may be present.
2. Solubility
soluble Monosaccharide
/disaccharide may
be present
3 Fehling test
mix well and boil.
Red, orange, violet
oxide
Reducing sugar
may be present
4 Benedicts test
To 1-2 ml of benedicts reagent, add an equalvolume
of sugar solution.
may be present
5 Tommers test
To 1-2 ml of Tommers reagent, add an equal volume
of sugar solution. Boil for 5 min. in a waterbath and
allow to stand.
Orange /yellow
precipitate
Reducing sugar
may be present
6 Barfoed’s test
brick-red cuprous
oxide precipitate at
the bottom of test
tube
Monosaccharide
may be present
7 Selwinof’s test
red precipitate Ketoses may be
present
8 Rapid ferfural test
To 1-2 mL of sugar solution add 1 ml of 1% alpha
naphthol solution +5 ml conc. HCl
deep purple colour or
precipitate
Ketoses may be
present

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under microscope
crystals were
observer
Structure:
Result : Qualitative tests of given unknown sample of carbohydrates was performed and it was
found to be fructose
9 Osazone test
Greenish, yellowish
needle shaped
fructose confirmed

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EXPERIMENT 5
AIM – To perform qualitative analysis of given unknown sample of carbohydrates [Maltose ]
REFERENCE: - Kale S.R.,Kale R.R., “practical biochemistry and clinical biochemistry”, nirali
prakashan 24 th edition 2015 page no 4 to 6
REQUIREMENT:
10 Lactose LR 1gm 20 gm 60gm
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20

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Observation Table:
Sr.N
o.
Test Observation Inference
1 Molish test
formed at the
layers.
Carbohydrates
may be present.
2. Solubility
/disaccharide may
be present
3 Fehling test
mix well and boil.
Red, orange, violet
oxide
Reducing sugar
may be present
4 Benedicts test
may be present
5 Tommers test
Orange /yellow
precipitate
Reducing sugar
may be present
6 Barfoed’s test
greenish precipitate Disaccharide may
be present
7 Osazone test
under microscope
Greenish, yellowish
sunflower shaped
crystals were
observer
Maltose confirmed

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Structure:
Result: Qualitative analysis of given unknown sample of carbohydrates was performed
and it was found to be Maltose

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EXPERIMENT 6
AIM – To perform qualitative analysis of given unknown sample of carbohydrates [Lactose ]
REFERENCE: - Kale S.R.,Kale R.R., “practical biochemistry and clinical biochemistry”, Nirali
REQUIREMENT:
6
selwinof’s reagent LR 2ml 40 ml 120ml
10 Maltose LR 1gm 20 gm 60gm
Glassware:
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20

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Observation Table:
Sr.N
o.
1 Molish test
formed at the
layers.
Carbohydrates
may be present.
2. Solubility
/disaccharide may
be present
3 Fehling test
mix well and boil.
Red, orange, violet
oxide
Reducing sugar
may be present
4 Benedicts test
may be present
5 Tommers test
Orange /yellow
precipitate
Reducing sugar
may be present
6 Barfoed’s test
Greenish cuprous
oxide precipitate
disaccharide may
be present
7 Osazone test
under microscope
Thin needle shaped
crystals were
observed all are in
prickles
lactose confirmed

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Structure:
Result: Qualitative analysis of given unknown sample of carbohydrates was performed and
it was found to be maltose

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EXPERIMENT 7
AIM – To perform qualitative analysis of given unknown sample of carbohydrates [Sucrose]
REFERENCE: - Kale S.R.,Kale R.R., “practical biochemistry and clinical biochemistry”, nirali
REQUIREMENT:
Chemicals:
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20
10 Sucrose LR 1gm 20 gm 60gm

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Observation table:
Sr.N
o.
1 Molish test
formed at the
layers.
Carbohydrates
may be present.
2. Solubility
/disaccharide may
be present
3 Fehling test
No Red, orange,
violet precipitate of
Nonreducing sugar
may be present

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mix well and boil.
cuprous oxide
4 Benedicts test
No brick-red
precipitate
Nonreducing sugar
may be present
5 Tommers test
No orange /yellow
precipitate
Nonreducing sugar
may be present
6 Acid Hydrolysis Test/Inversion Test:
To 5 mL of sugar solution to two test tubes. Add 5
drops of conc. HCl to one test tube. - Heat both
tubes in boiling water bath for 10 min.
- Cool and neutralize
with diluted NaOH
(use litmus paper).
Test both solutions
for the presence of
reducing sugar
with Fehling’s
Test
7 Fehling test
Fehling solution B Add 2 mL of solution,from
above test. mix well and boil.
Red, orange, violet
oxide
Sucrose confirmed
8 Selwinof’s test
Red precipitate Ketoses may be
present
(fructose)
Sucrose confirmed
9 Osazone test
under microscope
Greenish, yellowish
needle shaped
crystals were
observer
Sucrose confirmed
Structure:
Result: Qualitative analysis of given unknown sample of carbohydrates was performed and
it was found to be sucrose

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Experiment no 8
Aim: To introduce different tests used in Qualitative analysis of proteins
Theory:
Amino acids are molecules containing an amine group, a carboxylic acid group and a
side chain that varies between different amino acids. Amino acids of the general formula R-
CH(NH2)COOH are amphoteric, behaving as amines in some reactions and as carboxylic acids
in others.
At a certain pH known as the isoelectric point an amino acid has no overall charge, since
the number of protonated ammonium groups (positive charges) and deprotonated carboxylate
groups (negative charges) are equal. Since the amino acids at their isoelectric points have both
negative and positive charges, they are known as zwitterions.
Amino acids are critical to life. They have particularly important functions like being the
building blocks of proteins and being the intermediates in metabolism.
Amino acids are generally classified by the properties of their side chain into four groups.
The side chain can make an amino acid a weak acid or a weak base, and a hydrophile if the side
chain is polar or a hydrophobe if it is nonpolar.

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Proteins (also known as polypeptides) are organic compounds made of amino acids
arranged in a linear chain. The amino acids in a polymer are joined together by the peptide
bonds between the carboxyl and the amino groups of adjacent amino acid residues.
If large number of amino acid molecules combine, the product formed is called
polypeptide. A polypeptide having molecular mass greater than 10000 is called a protein.
Proteins differ from one another primarily in their sequence of amino acid. There are about more
than 20 amino acids. Some amino acids are not made by the body and are supplied through diet.
They are called essential amino acids.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins
are essential parts of organisms and participate in virtually every process within cells. Proteins
are important in:
1. catalyzing biochemical reactions (enzymes)
2. structural and mechanical functions (actin and myosin)
3. cell signaling
4. immune responses
5. cell adhesion
6. cell cycle
Biuret Test
This test is given by all peptides having at least two peptide bonds. So, it is given by all proteins.
Reagents:
10% NaOH: Take 10gm NaOH pellets and make it up to 100ml with DI water.
1% CuSO4: 1 gm of CuSO4 in 100 ml DI water.
Principle:

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Cupric ions of copper sulphate solutions in alkaline medium form coordinate complex with at
least two nitrogens of the peptide bonds to form purple colored complex. Thus color intensity is
proportionate to the presence of number of peptide linkages.
Minimum of 2 peptide bonds (3 amino acids) are required for binding of Cu2 + with peptide.
single amino acids and dipeptides do not give positive test.
The name of reaction is derived from organic compound biuret which is formed by condensation
of 2 urea molecules at high temperature.
Figure of Biuret
Biurat is formed when solid urea powder is heated in a tube. The resultant Biurat is solid at room
temperature and soluble in water.
The test produces color proportionate to number of peptide bonds which can be correlated with
amount of protein. Similar reagent is used for estimation of serum proteins quantitatively.
Ninhydrin Test
This test is given by all compounds having free α-Amino groups. ex: peptides, proteins, free α-
Amino acid. Different Proline and hydroxyproline give yellow color in this test.
Prepare reagent:
1 % Ninhydrine solution : 1 gm of Ninhydrine powder disolved in 100 ml DI water.
Principle:
Ninhydrine +α- Amino acid hydrindantin + aldehyde + CO2 + NH3
Hydrindantin + NH3 + Ninhydrine blue colored complex
Ninhydrin oxidises an α-amino acid to an aldehyde liberating NH3 and CO2 and is itself
reduced to hydrindantin. Hydrindantin then react with NH3 and another molecule of ninhydrine
to form a purple colored complex.
All amino acids that have a free amino group will give positive result (purple color) .While not
free amino group-proline and hydroxy-proline (amino acids) will give a (yellow color).

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Note: Many substances other than amino acids, such as amines will yield a blue color with
ninhydrin, particularly if reaction is carried out on filter paper.
Xanthoproteic Test:
This test is answered by aromatic amino acids. ( Tyrosine, Tryptophane)
Reagent:
Concentrated HNO3
40 % NAOH : 40 gm NAOH in 100 ml DI water.
Principle
Concentrated nitric acid causes nitration of activated benzene ring of tyrosine and tryptophan.
(Benzene ring is considered activated when additonal groups are attached to it) The nitrated
activated benzene is yellow in color. It turns to orange in alkaline medium.. Phenylalanine also
contains benzene ring, but ring is not activated, so it does not undergo nitration. The reaction can
be hastened by heating. The heat may be produced by dilution of concentrated HNO3 with OS or
may require heating.
Aldehyde Test
Reagents
1:500 Formaldehyde Reagent:
Take 1 ml of Formaldehyde solution (37-41 % W/V) and make upto 500 ml with DI water.Use
only for 1 week. Old Formaldehyde may not give test.
1 % Sodium Nitrite solution :
Take 1 gm sodium nitrite powder and make upto 100 ml with DI water. Use only for 1 week. Old
Sodium nitrite may not give test.

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Sulphuric acid AR :
Use sulphuric acid Bottle directly for use as reagent. Use for 1 week.Old Sulphuric acid may not
give test
Principle
Indole ring is present in tryptophan. Formaldehyde react with indole ring to give
violet colored complexes in presence of H2SO4. Addition of Sodium nitrite intensify and
stabilize colour.
Millon’s reagent
Reagent:
Millon's reagent:
Dissolve 10 gm of mercuric sulphate(HgSO4) +100ml DI water + 7 ml Conc.H2SO4
1% sodium nitrite:1 gm in 100 ml DI water
Principle
Tyrosine has hydroxyphenyl(Phenol) group. The hydrophobic group is in the core of protein. The
protein is denatured by mercuric sulphate in boiling water exposing hydroxyphenyl group.
Sodium nitrite reacts with sulfuric acid to form nitrous acid. The exposed hydroxyphenyl groups
react with nitrous acid. The compound formed chelates Hg2+ & give red colour precipitates.
Sakaguchi’s Test
This test is for Guanido group Which is the R-group of arginine.
Reagent:
1%w/v α-Napthol: Dissolve 1 gm α-Napthol in 100 ml of methanol
10%w/v NaOH: Dissolve 10gm of NaOH & make it upto 100ml with DI water.

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Alkaline hypochloride : Make 100 ml 10 % NaOH & add 8 ml 5-6 % Analytical grade Sodium
hypochloride.
Principle
In an alkaline medium, alpha-Napthol combines with guanidino group of arginine to form a
complex, which is oxidized by bromine/chlorine.
Sulphur Test (Lead acetate test):
Reagent:
2% Lead acetate in 10% NaOH: add 20 gm lead acetate, 100 gm NaOH in 1 liter of water. There
is no need to make exactly up to 1 liter. Above solution will be more than 1 liter in volume.
Principle:
When protein containing cysteine & cystine is boiled with strong alkali, organic
sulphur(R-SH) is converted to sulphide (Na2S]. Addition of lead acetate to this solution causes
precipitation of insoluble lead sulphide (PbS), which is black-gray in colour. Methionine does
not give this test due to the presence of thioether linkage (H3C-S-CH2-R) which does not allow
the release of sulphur in this reaction.
Heat coagulation test:
Reagent:
1% acetic acid: 1 ml acetic acid up to 100 ml with DI water.
Principle
Proteins have net zero charge at their iso-electric pH (pI). So, at pI, protein molecules have
minimum repelling force. Thus proteins are easily precipitated at pI. When proteins are heated,
weak bonds like hydrogen-bonds, salt bonds and van-der-wal forces are broken. Proteins are said
to be denatured.
Core hydrophobic regions of denatured Albumin can form intermolecular associations
and cause precipitation.Thus, in order to precipitate proteins like albumin, two conditions are
required. 1) Bring albumin to its pI(5.4) by adding few drops of 1% acetic acid. 2)Heat the
solution
Half & Full Saturation Test:
Reagent:
Saturated ammonium sulphate [(NH4)2SO4]: Add ammonium sulphate in 500 ml DI water till it
stops dissolving.
Ammonium sulphate [(NH4)2SO4] power
Principle

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When ammonium sulphate is added to protein solution, water concentration decreases. This
removes shell of water from outer surface of protein molecules, favoring formation of hydrogen
bonds among protein molecules and causing their precipitation. While proteins like globulin,
gelatin and casein are precipitated in half-saturated ammonium sulphate solutions, albumin is
precipitated in full-saturated ammonium sulphate solution.
Protein molecules contain both hydrophilic & hydrophobic aminoacids. In aqueous medium,
hydrophobic amino acids form protected areas while hydrophilic amino acids form hydrogen
bonds with surrounding water molecules (solvation layer).When proteins are present in salt
solutions (e.g.ammonium sulfate), some of the water molecules in the solvation layer are attracted
by salt ions. When salt concentration gradually increases, the number of water molecules in the
solvation layer gradually decreases until protein molecules coagulate forming a precipitate; this is
known as “salting out”.
For example,albumin requires higher salt concentration for precipitation than casein or
gelatin.Albumin particals are smaller in size & so have larger surface area,so they hold more
water molecules around them.so a higher concentration of Ammonium sulphate is required.The
salt concentration used is described as 'half saturation'(for casein,gelatin,globulin) or ' full
saturation' (for albumin).

33
Sr.
no.
Procedure Observation Inference
1 Biuret test:
Main test: Protein solution treated
with copper sulphate solution in
presence of alkali (NaOH or KOH),
Control test: with copper sulphate
solution in presence of alkali (NaOH or
KOH),
More intense violet colour
Obtained in main test
solution than control
peptide bond present
2 Ninhydrin test
Take 2 ml of the sample in a test tube
and add 3-4 drops of Ninhydrin
solution and boil the contents.
an intense blue coloured
complex is formed
protein present with
free –NH2 group
3 Xanthoproteic test:
Take about 2 ml of the sample in a test
tube and add few drops of conc.
HNO3 into it and heat the test tube.
a yellow coloured substance
is formed due to
xanthoproteic acid which is
formed by the nitration of
certain amino acids
with amino acid with
aromatic group may
present in protein such
as tyrosine and
tryptophan

34
4 Millon’s test:
Take 1-2 ml of the sample in a test tube
and add 2 drops of Millon’s reagent.
white coloured precipitate
which then changes to brick
red on boiling.
egg albumin may be
present
5 Millon’s test:
Take 1-2 ml of the sample in a test tube
and add 2 drops of Millon’s reagent.
No change in colour or ppt
formed
Gelatin may be
present
6 Hopkins-Cole Test:
Place 2 to 3 ml of protein solution add
an equal volume of Hopkins-Cole
reagent in a test tube and mix. Add
permit 5 to 6 ml of conc.H2SO4 to flow
slowly down the side of the tube, thus
forming a sharp layer of acid beneath
the amino acid solution.
reddish-violet color forms at
junction of contact of the
two fluids
presence of the indoly
group
(tryptophan)may
present in protein
7 Sakaguchi test Formation of a red color indicates presence of
guanidine group. This
is a very sensitive and
specific test
Mix 1 ml of NaOH with 3 ml of test
solution and add 2 drops of alpha
napthol. Mix thoroughly, add 4 to 5
drops of bromine water.
8 Nitroprusside test an intensely red but
somewhat unstable colour
observed .
Protein may contains
sulphahydryl groups in
its structure
Mix 0.5 ml of a fresh solution of
sodium nitropruside with 2 ml of the
test solution and add 0.5 ml of
ammonium hydroxide
9 Folin test:
Add 5ml of the alkaline solution to 1
ml of the test solution. Mix thoroughly
and allow to stand at room temperature
for 10 mins. Add 0.5 ml diluted Folin-
Ciocalteau reagent rapidly with
immediate mixing. indicates presence
of indolyl or phenol group.
Observe for development of
color after 30 mins.
Development of
characteristic blue color
Indolyl or phenol
group containing
amino acid may
present iin protein.
10 Ehrlich Test:
0.5 mL of the amino acid solution to a
test tube. Add 2 mL Ehrlich reagent
Observe colour change Aromatic amines may
be part of protein
structure
11 Protein precipitation reaction:
Add solid ammonium sulfate to about 5
mL of protein solution in a test tube the
(salt should be added in quantities of
approximately 1 g at a time)
Agitate the solution gently after each
addition to dissolve the ammonium
sulfate
proteins can aggregate and
form precipitates from the
solution
Protein confirmed

35
12 salts of Heavy Metals: Heavy metal
salts usually contain Hg2+, Pb2+,
Ag1+, Tl1+, Cd2+ and other metals
with high atomic weights. Since salts
are ionic, they disrupt salt bridges in
proteins. The reaction of a heavy metal
salt with a protein usually leads to an
insoluble metal protein salt.
- Treat 3 mL of the protein solution
provided with a few drops of mercuric
nitrate.
A white precipitate
formation should be
observed.
Protein confirmed
13 By Acid Reagents
The precipitation of a protein in the
presence of acid reagents is probably
due to the formation of insoluble salts
between the acid anions and the
positively charged protein particles.
These precipitants are only effective in
acid solutions.
Treat 3 mL of protein solution provided
with a few drops of trichloroacetic acid
solution.
A white precipitate
formation should be
observed.
Protein confirmed
Result: Different tests used in Qualitative analysis of proteins were introduced.

36
Experiment no 9
Aim: To perform Qualitative analysis of given unknown sample of protein (albumin)
REFERENCE: -
2 Conc. HNO3 LR 2ml 40 ml 120ml
3 Conc.H2SO4 LR 2ml 40 ml 120ml
4 1% Ninhydrin solution LR 2ml 40 ml 120ml
5 NaOH solution
10%,20% 40%
LR 2ml 40 ml 120ml
6 CuSO4 solution LR 2ml 40 ml 120ml
7 Formalin solution LR 2ml 40 ml 120ml
8 Millon’s reagent LR 2ml 40 ml 120ml
9 1% Alpha naphthol LR 2ml 40 ml 120ml
10 Sodium nitrite LR 1gm 20 gm 60gm
11 Chlorophennol red LR 2ml 40 ml 120ml
12 Ammonium Sulphate LR 10gm 20 ml 60ml
13 Albumin LR 1gm 20 gm 60gm
1 Test tubes 5 20

37
4 Beaker 2 10
5 Glass rod 5 20
Theory:
Proteins (also known as polypeptides) are organic compounds made of amino acids
arranged in a linear chain. The amino acids in a polymer are joined together by the peptide
bonds between the carboxyl and the amino groups of adjacent amino acid residues.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins
are essential parts of organisms and participate in virtually every process within cells. Proteins
are important in:
Preparation of Protein solutions:
Egg albumin solution (1:21): Mix 50 ml of egg(both white and yellow) in 1 liter
of tap water. Use only for 24 hours
Sr.
no.
METHOD OBSERVATION INFERENCE
1 BIURET TEST
10% NaOH (2 ml ) +1% CuSO4 (2
ml)
divide above mixture in two parts of 2
mlpart 1: add 2 ml OS
part 2: add 2 ml H2O
Pink or Violet Colour
develops in part 1. No
such color develop in
part 2
Two or more peptide
linkages present.
Protein present
2 XANTHO-PROTEIC TEST
OS (0.5 ml) + HNO3con (1 ml) Mix it.
(Solution turns yellow) + 40%NaOH (1
ml) in above mixture.
Solution turns orange
Note: Use Fresh(tightly packed)
conc.HNO3otherwise test come negative.
Yellow-Orange colour
develops.
Aromatic Amino Acids
Tyrosine and
Tryptophan present in
protein.
3 NINHYDRIN TEST
OS (1 ml) + 1% Ninhydrine (2
drops)Mix, Boil (1 min).
Cool.
Blue or Purple colour
develops.
Alpha Amino groups of
proteins at N-terminal
are responsible for
positive test with
proteins.
4 Aldehyde Test
1 ml Protein Solution + 1 drop of
1:500
formalin. Mix.
Violet color is formed. Indole group present in
protein. Tryptophan
present in the protein.

38
Slant the test tube and slowly add 1
ml of conc. H2SO4 . Mix.
Add 1 drop of 1% sodium nitrite
solution in Test tube. Mix.
Use Fresh(tightly packed)
conc.H2SO4 &1:500formaline
otherwise test come negative.
5 MILLION’S TEST
0.5 ml protein sol. +50 ul sodium
nitrate
sol.n
+100 ul Millon’s reagent. mix
well &Heat
Red coloured
precipitate 0bserved.
Hydroxyphenyl group
present in protein.
Tyrosine present in
protein.
6 SAKAGUCHI TEST
1 ml Protein sol.n
+ 2 drops of alpha
Napthol +1 ml Alkaline sodium
Hypoochloride
Carmine Red colour
observed.
Guanidino
gro
up present in protein.
Arginine present in
protein.
7 SULPHER TEST (Lead acetate test)
0.5 ml OS + 0.5 ml Lead acetate
reagentBoil for 1 minute
Black- Grey colour
seen.
Sulfhydryl group
(-SH)
present in protein.
Cysteine &
Cystine
present in protein
8 HEAT COAGULATION TEST
5 ml Protein solution + 1- 2 drops of
chlorophenol red.
If faint pink colour appears, add 1%
aceticacid drop wise till you get faint
pink colour with yellowish tinge (pH
5.4 is obtained)
If yellow colour appears, add 2%
Na2CO3 solution drop wise till you
get faint pink colour with yellowish
tinge (pH 5.4 is
obtained)
White precipitates seen
in upper part of
solution, as compared
to clear lower part of
solution
Albumin is
precipitated when
denatured at its
pI~5.4
9 HALF SATURATION TEST
2 ml of the protein sol.n
+ 2 ml of
saturated sol.n
of (NH4)2 SO4 (Thus,
saturated (NH4)2 SO4 is half diluted)
No White precipitate
formed.
Casein, Gelatin
and
Globulin
are
precipitated at
half
saturation with
(NH4)2
SO4

39
10 FULL SATURATION TEST
5 ml. Of protein sol.n
+ a
pinch ofAmmonium Sulphate powder,
Shake
Repeat above steps till some
undissolved
(NH4)2 SO4 remains at the bottom of
the testtube.
White precipitate
formed
Albumin precipitates
at full saturation with
(NH4)2 SO4
11 MOLISCH’S TEST
1ml OS + 2 drops of α-napthol
solution,mix
Add 2 ml. of conc. Sulphuric
acidcarefullythrough the side of the test
tube without shaking.
Purple ring is formed at
the junction of acid and
solution.
Proteins
conta
inCarbohydrates
Result: Qualitative analysis of given protein sample was performed and it was found to be
Albumin.

40
Experiment no 10
Aim: To perform Qualitative analysis of given unknown sample of protein (Casein)
REFERENCE: -
2 Conc. HNO3 LR 2ml 40 ml 120ml
3 Conc.H2SO4 LR 2ml 40 ml 120ml
4 1% Ninhydrin solution LR 2ml 40 ml 120ml
5 NaOH solution
10%,20% 40%
LR 2ml 40 ml 120ml
6 CuSO4 solution LR 2ml 40 ml 120ml
7 Formalin solution LR 2ml 40 ml 120ml
8 Millon’s reagent LR 2ml 40 ml 120ml
9 1% Alpha naphthol LR 2ml 40 ml 120ml
10 Sodium nitrite LR 1gm 20 gm 60gm
11 Chlorophennol red LR 2ml 40 ml 120ml
12 Ammonium Sulphate LR 10gm 20 ml 60ml
13 Casein LR 1gm 20 gm 60gm

41
Preparation of protein solution:
Casein solution(0.5%) : Dissolve 5 gm of Casein powder in 20 ml of 40%
NaOH & make upto 1 Liter with water
Sr. no. METHOD OBSERVATION INFERENCE
1 BIURET TEST
 10% NaOH (2 ml ) +1% CuSO4 (2
ml) divide above mixture in two
parts of 2 ml
 part 1: add 2 ml OS
part 2: add 2 ml H2O
Pink or Violet Colour
develops in part 1. No
such color develop in
part 2
Two or more peptide
linkages present.
Protein present
2  XANTHO-PROTEIC TEST
 OS (0.5 ml) + HNO3con (1 ml)
Mix it. (Solution turns yellow) +
40%NaOH (1 ml) in above
mixture.
 Solution turns orange
Note: Use Fresh(tightly packed)
conc.HNO3 otherwise test come
negative.
Yellow-Orange colour
develops.
Aromatic Amino Acids
Tyrosine and
Tryptophan present in
protein.
3  NINHYDRIN TEST
 OS (1 ml) + 1% Ninhydrine (2
drops)
 Mix, Boil (1 min).
 Cool.
Blue or Purple colour
develops.
Alpha Amino groups of
proteins at N-terminal
are responsible for
positive test with
proteins.
1 Test tubes 5 20
4 Beaker 2 10
5 Glass rod 5 20

42
4  Aldehyde Test
 1 ml Protein Solution + 1 drop of
1:500 formalin. Mix.
 Slant the test tube and slowly add 1
ml of conc. H2SO4 . Mix.
 Add 1 drop of 1% sodium nitrite
solution in Test tube. Mix.
 Use Fresh(tightly packed)
Violet color is formed. Indole group present in
protein. Tryptophan
present in the protein.

43
conc.H2SO4 &1:500formaline
otherwise test come negative.
5  MILLION’S TEST
 0.5 ml protein sol. +50 ul sodium
nitrate sol.n
+100 ul Millon’s
reagent. mix well & Heat
Red coloured
precipitate 0bserved.
Hydroxyphenyl group
present in protein.
Tyrosine present in
protein.
6 SAKAGUCHI TEST
1 ml Protein sol.n
+ 2 drops of alpha
Napthol + 1 ml Alkaline sodium
Hypoochloride
Carmine Red colour
observed.
Guanidino group
present in protein.
Arginine present in
protein.
7  SULPHER TEST (Lead acetate
test)
 0.5 ml OS + 0.5 ml Lead acetate
reagent
 Boil for 1 minute
Black- Grey colour
seen.
Sulfhydryl group (-
SH) present in protein.
Cysteine & Cystine
present in protein
8 HEAT COAGULATION TEST
5 ml Protein solution + 1- 2 drops of
chlorophenol red.
 If faint pink colour appears, add 1%
acetic acid drop wise till you get
faint pink colour with yellowish
tinge (pH 5.4 is obtained)
 If yellow colour appears, add 2%
Na2CO3 solution drop wise till you
get faint pink colour with yellowish
tinge (pH 5.4 is obtained)
White precipitates seen
in upper part of
solution, as compared
to clear lower part of
solution
Albumin is precipitated
when denatured at its
pI~5.4
9 HALF SATURATION TEST
2 ml of the protein sol.n
+ 2 ml of
saturated sol.n
of (NH4)2 SO4 (Thus,
saturated (NH4)2 SO4 is half diluted)
No White precipitate
formed.
Casein, Gelatin and
Globulin are
precipitated at half
saturation with (NH4)2
SO4
10 FULL SATURATION TEST
5 ml. Of protein sol.n
+ a pinch of
Ammonium Sulphate powder, Shake
Repeat above steps till some
undissolved (NH4)2 SO4 remains at the
bottom of the test tube.
White precipitate
formed
Albumin precipitates at
full saturation with
(NH4)2 SO4
11  MOLISCH’S TEST
 1ml OS + 2 drops of α-napthol
solution, mix
 Add 2 ml. of conc. Sulphuric acid
carefully through the side of the test
tube without shaking.
Purple ring is formed at
the junction of acid and
solution.
Proteins contain
Carbohydrates

1
Result: Qualitative analysis of given protein sample was performed
and it was found to becasein

2
Experiment no 11
Aim: To perform Quantitative analysis of given unknown sample of protein (Albumin)
REFERENCE: -
1. Kale S.R.,Kale R.R., “Practical Biochemistry And Clinical
Biochemistry”, Nirali Prakashan,24 th edition, 2015 page no 4 to 6
3. Rajagopal and Ramakrishna “Practical Biochemistry for Medical students” 5 th
edition .
Theory:
One commonly used method for determining the total protein in a sample
is the Biuret method. The Biuret method is based on the complexation of
Cu2+ to functional groups in the protein’s peptide bonds as shown in
Figure 1.The formation of a Cu2+-protein complex requires two peptide
bonds and produces a violet-colored chelate product which is measured by
Colourimeter 540 nm. Over a given concentra tion range,the measured
absorption at 540 nm is linear
with respect to the concentration of total protein. This relationship
allows a standard curve to be created that is used to calculate the
concentration of an unknown sample.
COLOURIMETER
Colourimeter - A colourimeter is used to measure the concentration of substance in coloured
sample by comparing the amount of light it absorbs, with that absorbed by a standard preparation
that contains a known amount of substance being tested. In colourimetric determination a
specific reagent or reagents are used which react with substance under determination to form a
coloured complex. The concentration of coloured complex is directly proportional to the
concentration of substance under determination. The depth of coloured complex is measured on
photometer or spectrophotometer. Most colourimetric analytical tests are based on Beer-

3
Lambert's law which states that the absorbance of a solution at the appropriate wavelength is
directly proportional to its concentration and the light path through the solution. This law can be
applied for measuring the concentration of substance in an unknown (test) solution by using
formula.
Concentration of test C
Absorbance of test A,
- x Concentration of standard C Absorbance of standard As
Formerly the amount of light absorbed by a coloured solution i.e. absorbance was referred as
optical density (O.D.)
Absorbance A = 2-log%T Absorbance is zero when there is 100% transmission. In colourimetric
tests, the light path is kept constant by using optically matched cuvettes usually of 10mm light
path distance or tubes of known light path distance. Basic components of a photometer and
spectrophotometer are a) A light source b) Monochromator / filter c) Cuvette d ) Photodetector
and
e) Galvanometer. The instrument which uses a light filter for wavelength at which photometric
measurements are made is called photometer, while the instrument which uses a prism or
diffraction grating with a slit to get monochromatic light is called spectrophotometer.
The colourimeter is supplied with colour filters which ranges between wavelength 400-700 nm.
Wavelength 400-419 420-449 450-479 480-504 505-534
Filter Deep violet Violet Blue Blue green Green
Wavelength 535-564 565-589 590-639 640-689 690-700
Filter Yellow green Yellow Orange Red Deep red
Basic steps involved in use of colourimeter / spectrophotometer are 1. Put on the main switch 2.
Select a proper filter / wavelength 3. Adjust 0% without cuvette 4. Place a cuvette filled with
blank / distilled water and adjust 100%T. 5. Replace the blank / distilled water with test and
standard solution and record their absorbance
(optical density).
Reagent blank is used to correct any absorbtion of light by reagents. It contains reagents and
chemicals used for development of colour but do not contain the substance being tested.
Procedure:
The Biuret reagent was prepared by adding 3 g of CuSO4•5H2O and 9
g of sodium potassium citrate to 500 mL of 0.2 N NaOH solution,
followed by the addition of 5 g of KI. The resultingsolution was then
brought to a total volume of 1 L with 0.2 N NaOH.
Protein standards and the sample were prepared with saline solution (8.5 g/L)
according to Table

4
1. 3.0 mL of Biuret reagent was added to each standard and sample, the
solution was mixed welland incubated at room temperature for 30
minutes.
Result:
quantitative analysis of protein is performed quickly and easily using the
Biuret reagent methodand colourimeter

5
Experiment no 12
AIM:
To demonstrate estimation of quantity of creatinine in given sample of urine by Alkaline
picrate method (Jaffe's reaction)
REFERENCE: -
3. Rajagopal and Ramakrishna “Practical Biochemistry for Medical students” 5 th edition
APPARATUS:
Glassware Conical flask, pipette, test tubes, volumetric flask, cuvettes, photo electric colorimeter
/ spectrophotometer. Chemicals 24 hours urine sample, picric acid (0.9%w/v), Sodium hydroxide
(3%w/v), Distilled water Creatinine stock (1mg/ml) 100mg creatinine in 100ml of 0.1N HCI,
Creatinine working standard (0.01mg/ml) Dilute 1.0 ml of stock creatinine solution to 100ml
with distilled water in volumetric flask.
To estimate quantity of creatitine in given sample of urine
Theory :
Urine It is chief excretory fluid eliminated through kidney. Waste products are eliminated
through urine. Normal Constituents of urine Urine Contains organic constituents urea, uric acid,
creatinine and inorganic constituents chlorides, phosphates, sulphates, bicarbonates, ammonia and
calcium etc.
1: Creatinine Creatinine is end product of creatine metabolism. It is an
anhydride of creatine.
Proposition 2 : Creatine Creatine is present in muscles, brain and blood in free form as well as in
the form of creatine phosphate. Creatine phosphate gives supply of readily available energy to
muscles. Concept structure:
Creatine phosphate + ADP
ATP + Creatine
(in muscles) During severe exercise creatine phosphate stored in muscles is converted to
ATP
Proposition 3: Excretion of Creatinine Creatinine is formed in body from spontaneous

6
breakdown of creatine phosphate. It is a nonthreshold substance. It is filtered by glomerulli.
It's excretion is not related with food protein. It is remarkably constant. It's variation in
excretion indicates metabolic disorder. It's excretion in urine increases in fever, starvation, on
carbohydrate free diet and in diabetes mellitus.
Proposition 4 : Alkaline Picrate Method Creatinine reacts with picric acid in alkaline medium
to form reddish yellow coloured complex creatinine picrate. The intensity of colour
developed is directly proportional to the amount of creatinine present in urine and is
compared with that of standard creatinine solution similarly treated against reagent blank at
green filter / 520nm using colourimeter or spectrophotometer respectively. Estimation of
quantity of creatinine in urine is useful in calculation of creatinine clearance.
Concept Structure:
Creatinine in
urine colourless
Picric acid in
alkaline media
Creatinine plcrate
Reddish yellow
coloured complex
1. Ability to add proper amount of urine, creatinine working standard, picric acid, sodium
hydroxide solution and distilled water in blank, standard and test solution respectively.
2. Ability to operate colourimeter.
3. Ability to measure % Transmittance and Absorbance.
PROCEDURE:
1. Prepare creatinine working standard by diluting 1.0ml of stock creatinine solution to 100ml
with distilled water in volumetric flask. So the concentration of creatinine will be 0.01mg/ml.
Dilute 1.0ml of given urine sample to 100ml in volumetric flask. Prepare blank standard, test
solution as mentioned in following table
Standard
Test
Blank 5.0 ml
5.0 ml
Distilled water Creatinine working standard (0.01mg/ml) Diluted urine Picric acid (0.9%w/v) |
Sodium hydroxide (3%w/v)
1.0ml 1.Oml
1.Oml 1.Oml
5.0ml 1.Oml 1.Oml
WONO

7
Allow it to stand for 15 minutes. Set photoelectric colorimeter at green filter / spectrophotometer
at 520 nm Adjust 0% T (transmission) Adjust 100% T (transmission) with distilled water.
Measure Absorbance or calculate Absorbance for blank standard and test
OBSERVATION : Absorbance of blank Ag = Absorbance of standard As= Absorbance of Test
A. =
CALCULATIONS :
A
Concentration of creatinine in test solution C
A.-A Where Cs = 0.05mg of creatinine
A Concentration of creatinine in test solution C, 98 x 0.05 - Ag-Ą,
Now as 1.0ml of urine is diluted to 100ml and 5.0 ml is taken in test solution concentration of
creatinine in 0.05ml undiluted urine = C,
Hence concentration of creatinine in 100ml urine
C,x100 0.05
A -AB
x 0.05 x100
As -Ą,
0.05
Ą, -A As-A
-
X 100
Hence mg of creatinine in 100ml sample of urine
x100 mg/100ml
- ...... - ........
xmg/100ml ............ mg/100ml
G of creatinine in 1000 ml sample of urine
–
– - 1000
X
10
g/lit
x 10 g/lit
1000

8
Yg/lit | .......... g/lit
Volume of 24 hours urine sample in liters x Y
hence amount of creatinine in 24 hours urine sample
X.......
1 ....... g/lit
RESULT:
Amount of creatinine in given sample of urine .. Normal range is 1.5 to 3.0 g/24 hrs.
........ g / 24 hrs
QUESTIONS:
Name the method used for estimation of creatinine in urine?
Which colour developing agent is used for estimation of creatinine in urine by colorimeter?
What is colour of creatinine picrate?
State the wavelength and filter at which estimation of creatinine in urine is done by colorimeter
If patient's urine report 3.5g creatinine in 24 hours, Name the diseases he is suffering from?
State the condition in which creatinine excretion is increased in urine.
Name the standard used for estimation of creatinine in urine by colorimeter
How creatinine is formed in body? State the factors by which creatinine excretion in urine is
affected. What is normal value of excretion of creatinine in urine?

9
Experiment no 13
AIM : To demonstrate estimation of quantity of glucose in given sample of serum. (GOD / POD
method ).
REFERENCE: -
APPARATUS: Glassware : Test tubes, Beakers, Test tube stand, Glass rod, Photoelectric
Colorimeter, Centrifuge, Incubator etc. Chemicals: 1.
(Buffer Enzyme) This reagent is prepared by mixing following constituents in a 100 ml of
phosphate buffer (M/10, pH 7).
Glucose Oxidase : 650 unit 2. Peroxidase
: 500 unit 3. 4 amino phenozone : 20 mg
4. Sodium azide : 30 mg 2. Phenol reagent
: 100 mg/100 ml 3. Glucose Standard : 100 mg/100 ml
Theory:
Diabetes Mellitus Increase in sugar (glucose) level in blood than normal level is known as
diabetes mellitus. Proposition 2: Hypoglycemia Decrease in blood glucose level than normal
level is known as hypoglycemia. Preposition 3: GOD / POD method Glucose oxidase (GOD )
oxidises glucose to gluconic acid and hydrogen peroxide. Glucose + 0, +H,0 Glucose oxidase
Gluconic Acid + H, O, In presene of enzyme peroxidase. hydrogen peroxide reacts with phenol
and 4-amino antipyrine (4 AAP)to form red coloured Quinoneimine dye absorbance of coloured
dye is measured at 510 nm and directly proportional to glucose concentration in sample. H,O, +
Phenol + 4 Amino antipyrine_Peroxidase Q uinoneimine dye + H2O
PROCEDURE:
Preparation of glucose reagent.
Mix 2 parts of buffer enzyme reagent and one part of phenol reagent to gate glucose reagent. 2.
Preparation of test, standard and blank solutions as follows.
Blank 3.0 ml
Standard
3.0 ml
Sr. No.Reagent

10
Glucose reagent Serum / Plasma
Glucose standard 4. Distilled water
Test 3.0 ml 0.02 ml
0.2 ml
0.02 ml
Mix well incubate at a 370c for 15 minutes. The final colour is stable for one hour. Set the
spectrophotometer at 530 nm or photoelectric colourimeter on green filter. Adjust 100 %
Transmission and 0 absorbance with a distilled water. Read absorbance for standard test and
blank
OBSERVATION TABLE:
1. Absorbance (optical density) of Blank : 2. Absorbance ( optical density) of Standard: 3.
Absorbance ( optical density) of Test : CALCULATION :
A Ag A
X
C, =
As — Ag When Cs = 0.02 mg
C = quantity of glucose in 0.02 ml serum.
Hence quantity of glucose in a 100 ml serum
=
C X 100
- mg/100 ml 0.02 A-Ą X 0.02
- x 100 mg/100 ml As — A
0.02
A - AB
- x 100 mg/100 ml Ą A
.. -
x 100 mg/100 ml
RESULT :
Given sample of serum/plasma contains Glucose ----- ----------------- mg/100 ml Normal values :
70 - 105 mg/100 ml

11
Experiment No. 14
AIM: To detect abnormal constituents in given sample of urine by qualitative tests.
REFERENCE: -
APPARATUS:
Glassware
Test tubes, Beakers, Test tube stand, Gas, Burner, Glass rod etc.
Chemicals Concentrated HNO3, 0.1 N.Chlorophenol red solution, Ammonium sulphate powder
1 N Sulphosalicylic acid solution, Fehling A solution, Glacial Acetic acid solution, Benzidine
powder Sulphur powder Strong Ammonia., Solution, Fehling B solution, 0.1 N. HCl solution,
Hybrogen Peroxide Solution
Theory:
Pathological urine The urine which contains essential of body like glucose, proteins, ketone
bodies, bile salt, bile pigment, blood , etc.
Abnormal constituent
Sugar (Glucose) Protein Ketone bodies Bile salt Bile pigment Blood
Significante disease
Glycosuria Protinuria Ketonuria Jaundice Hepatitis Haematuria
PROCEDURE :
Table for performing test on urine sample with reagents. 1. Use urine sample as a original
solution 2. Perform following tests on given urine sample and interpret as mentioned in
following table.
Physical properties
Table for performing test on urine sample with reagents.

12
Test
Inference
Volume
Normal
Observation (a) 1000 ml-1500 ml
per day (b) More than 1500 ml
per day
(c) Less than 1000 ml/
day
(1) Polyuria may be due to more water intake, less
perspiration, high protein diet diuretic substance like alcohol, coffee, tea, diseased state like
diabetes
inspidius may be due to
1. Hard physical work 2. Fever 3. Dehydration 4. Vomitting, diarrhoea 5. Acute nephrittis.
Anuria due to acute renal insufficiency. (a) Normal due to presence of urochrome pigment.
(d) No urine (a) Pale yellow
Colour (observe within 60 minutes)
(b) Marked yellow (b) Normal due to decreased urine output. (c) Light yellow (Slightly (c) Seen
after heavy meals.
yellow than pale
yellow) (d) Yellowish green (d) Abnormal indicates condition like jaundice.
brown (e) Reddish
(e) Abnormal due to haematuria. (1) Dirty bluish
(f) Abnormal like in cholera and typhus. (g) Milky
(g) Abnormal like in pyuria or chyluria (Pus and fat
respectively) (h) Brown colour (h) Abnormalprecipitates of urate, phosphates etc. (a) Peculier,
aromatic (a) Normal. (b) Unpleasant aromatic (b) Due to various drug metabolites and microbial
decomposition. (a) 6 - 7.5
(a) Normal (b) Acidic (below 6) (b) Normally seen after high meat diet. Abnormally
seen in acidosis. (c) Alkaline (above 8) (c) Abnormally seen in alkalosis.
Odour :PH
ABNORMAL CONSTITUENTS
ANALYSIS OF URINE FOR ABNORMAL CONSTITUENTS
White ppt appears Albumin present
Test for proteins (Albumin and globulin) (i) Sulphosalicylic acid test:
3 ml clear urine + sulphosalicyclic
acid drop by drop. (ii) Hellers nitric acid ring test :
3 ml conc. HNO, + add from side of test tube dropwise urine
Albumin confirmed

13
White ring at the junction of two fluids.
Test
Observation Turbidity or precipitates
Inference Albumin confirmed
(iii) Heat co-agulation test :
3ml urine + 2 drops of chlorophenol red, adjust the pH faint pink colour by adding 1 % Na, co,
boil for two
minutes. Add 5 drops acetic acid.
Turbidity remains
Albumin confirmed
Test for sugar (Glucose) (i) Benedicts test:
5ml urine + 5ml benedicts reagent boil for two minutes and cool
(i) Green ppt. (ii) Yellow ppt. (iii) Red ppt.
Glucose present -1% Glucose present -2% Glucose present more than 2% Glucose confirmed.
Red/Yellow ppt appears
(ii) Fehlings test:
2 ml fehlings A + 2 ml fehlings B boil for few minutes, add 2-3 ml urine boil again.
Test for Ketones : (Rothera's test) 5 ml urine + solid (NH), SO, to saturate it completely + 2
drops of sodium nitropruside solution + 2 ml strong ammonia solution from sides of test tube
wait for 10 minutes.
Permanganate colour develops
Ketones like acetone present.
Test for bile salts 5 ml urine in beaker sprinkle sublimed sulphur powder on surface.
Bile salts present.
(i) Powder sinks to
bottom (ii) Powder floats on the
surface
Bile salts present.
Bile pigments present.
Test for bile pigments (a) Gmelins test (modified)
10 ml urine + 2-3 drops of dilute. HCI filter it through paper, allow it to dry put a drop of conc.
HNO, at the
apex of paper. (b) Nitric acid test :
3 ml conc. Nitric acid, add urine slowly from side of test tube.

14
Coloration on paper in following order green, blue, violet, red and yellowish red seen. Fine play
of colours.
Bile pigments present.
Test for blood (Benzidine test) Pinch of benzidine powder + 1 ml of glacial acetic acid shake for
1 minute + 2 ml urine + few drops of H,O,
Blood present.
Green/blue colour due to iron benzidine formation.

15
Experiment No. 15
AIM: To demonstrate Estimation of Cholesterol in serum by enzymatic method
REFERENCE: -
Theory:
Cholesterol is a steroid with a secondary hydroxyl group in the C position and found in blood,
bile and brain tissue. It serves as a precursor to bile acids, steroids and vitamin D. It is
synthesized in many types of tissues, but particularly in the liver and intestinal walls.
Approximately 75% of cholesterol is newly synthesized and a 25% originates from dietary
intake. Measurements of serum cholesterol levels
are important in the diagnosis and classifications of hyper-cholesteremias. Elevated cholesterol
levels may occur with hypothyroidism, nephritic syndrome, diabetes and various liver diseases.
There is a correlation between elevated serum cholesterol level and incidence of coronary artery
disease. Normal cholesterol levels are affected by stress, diet, age, gender, hormonal balance
and pregnancy. Depressed levels are associated with hyperthyroidism and severe liver diseases.
Principle:
Cholesterol analysis was first reported by Liebermann in 1885 followed by Burchard in 1889. In
Leibermann Burchard reaction, cholesterol forms a blue green dye from polymeric unsaturated
carbohydrates in an acetic acid/ acetic anhydride/ concentrated sulfuric acid medium. The Abell
and Kendall method is specific for cholesterol but is technically complex and requires corrosive
agents. In 1974, Allain and Roeschlau were able to combine cholesterol esterase and cholesterol
oxidase into a single enzymatic regent for the determination of total cholesterol. Vitro
cholesterol reagent combines the use of three enzymes with the peroxidase/phenol/4-aap system

16
of Trinder11 for the measurements of total cholesterol in human serum. The series of reaction
involved in the assay system are as follows:
1. Cholesterol esters are enzymatically hydrolysed by cholesterol esterase (CE) to cholesterol
and free fatty acids.
2. Free cholesterol is then oxidized by cholesterol oxidase (CHOD) to cholest-4-ene-3-one
and H2O2.
3. In presence of peroxidise (POD), the formed hydrogen peroxide affects the oxidative
coupling of phenol and 4-aminoantipyrine(4-AAP) to form a red coloured quinoneimine
dye. Cholesterol esters + H2O → cholesterol + fatty acids
Cholesterol+O2→ cholest4-ene-3 one +H2O2
2 H2O2+4-AAP+Phenol → quinoneimine dye+4 H2O
The intensity of the colour produced is directly proportional to cholesterol concentration. It is
determined by measuring the increase in absorbance at 500-550nm.
Reagents:
R1: Cholesterol standard 200 mg/dl
R2: Pipes buffer, pH-6.9, 90 mmol/l
Phenol 26 mmol/l
Cholesterol oxidase 500 µ/l
Cholesterol esterase 500 µ/l
Peroxidase 1250 µ/l
4-Aminoantipyirine 0.4 mmol/l
Procedure:
1. Prepare the test tubes as shown below:
Blank Standard Sample
R2 (Reagent) 1.0 ml 1.0 ml 1.0 ml
dH2O 10 µl
R1 (Standard) - 10.0 µl -
Serum - - 10.0µl
Absorbance

17
2. Mix and incubate for 5 minutes at 37ºC or 10 minutes at 20-25ºC. Measure the absorbance
of specimen and standard against reagent blank at 545. The colour is stable for 60 minutes.
Calculation: Calculate the total Cholesterol concentration by using the following formulae
Total Cholesterol concentration
= (Absorbance of specimen/Absorbance of standard) x [Standard] (Unit conversion: mg/dl x
0.0259 = ___ mmol/L)
Expected value:
Risk classification Total Cholesterol
Desirable <200 mg/dl (5.2 mmol/l)
Borderline high 200-239 mg/dl (5.2-6.2 mmol/l)
High >240 mg/dl (6.2 mmol/l)

Biochemistry lab manual II semester

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