Diploma in Pharmacy 2nd year Practical Manual Biochemistry & Clinical Pathology

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 Practical / Experiment Page No
Practical 1 : To perform qualitative analysis of 4
carbohydrates
Practical 2 : To perform qualitative analysis of 8
proteins and amino acids
Practical 3 : To perform qualitative analysis of lipids 12
Practical 4 : To perform qualitative analysis of normal 15
and abnormal constituent in given sample of urine
Practical 5A : To detect the glucose in given sample 24
of urine by qualitative test
Practical 5B : To detect the creatinine in given 28
sample of urine by qualitative test
Practical 5C : To detect the chlorides in given sample 31
of urine by qualitative test
Practical 6A : To determine the creatinine in blood 35
/serum
Practical 6B : To determine the glucose in blood 40
/serum
Practical 6C : To determine the cholesterol in 48
blood/serum
Practical 6D : To determine the calcium in blood 52
/serum
Practical 6E : To determine the urea in blood /serum 55
Practical 6F : To determine the SGOT/SGPT in 59
blood/serum
Practical 7A : To study the hydrolysis of starch from 63
salivary amylase enzyme
Practical 7B : To study the hydrolysis of starch from 67
acid
 To perform qualitative analysis of carbohydrates.

Aim:
To perform qualitative analysis of carbohydrates.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 1 – 4

Materials Required
Fehling's solution A and B, Molish reagent, phenylhydrazine
hydrochloride, beaker, glass rod, measuring cylinder, funnel and test
tube.

Theory :
1) Fehling's Test : This test serves as the carbohydrate reduction test.
The blue alkaline cupric hydroxide solution in Fehling's solution
reduces to yellow or red cuprous oxide and precipitates when heated
with reducing sugars. OH
As a result, the test solution's ability to precipitate a yellow or
brownish-red coloured substance aids in the identification of reducing
sugars.

2) Molisch's Test : This test is a common test for all carbohydrates

greater than tetroses. The test is based on the principle that pentoses
and hexoses are dehydrated by concentrated sulphuric acid to yield
furfural and hydroxymethylfurfural, respectively. These products
condense into a purple condensation product when combined with a-
naphthol.
 3) Osazone Test : In this test, Phenylhydrazine combines with the
ketoses and aldoses to form a phenylhydrazone, which then reacts with
two more molecules of phenylhydrazine to produce osazone.
Lactosazone produces mushroom shape crystals, whereas glucose,
fructose, and mannose produce needle-shaped yellow osazone crystals.
Different osazones will exhibit crystals in a various shapes. Maltose
produces flower- shaped crystals.

Procedure
Common tests which are carried out for identification of carbohydrates
are given below:

1) Fehling's Solution Test : In this test, 1-2ml each of the Fehling's

solution A and B should be added to few drops of the test solution and
boiled for a few minutes. A yellowish-red colour appears that confirms
the presence of a reducing sugar.
i) Fehling's Solution A : This reagent should be prepared by
dissolving 34.65gm copper sulphate in distilled water and making
the volume up to 500ml.
ii) Fehling's Solution B: This reagent should be prepared by
dissolving 125gm potassium hydroxide and 173gm Rochelle salt
(potassium sodium tartrate) in distilled water and making the
volume up to 500ml.
2) Molisch Test : All members of carbohydrates give a positive result
for this test. In this test, 1-2 drops of Molisch's reagent (5% of 1-
naphthol in alcohol) should be mixed with 1-2ml of test solution. About
Iml of concentrated sulphuric acid should be added along the side of
the tube.
 A colour develops at the junction of the two liquids due to the reaction
between a-naphthol and furfural and/or due to the derivatives formed
by dehydration of sugars with concentrated sulphuric acid.

3) Osazone Formation : A sugar should be heated with

phenylhydrazine hydrochloride, sodium acetate, and acetic acid to
form yellow crystals of osazone.

Result :
Qualitative analysis of Carbohydrates was Performed.
 To perform qualitative analysis of proteins and amino acids.

Aim:
To perform qualitative analysis of proteins and amino acids.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 5 – 9

Materials Required
Stirrer, dropper, water bath, distilled water, bunsen burner, test tube holder,
clean and dried test tubes, food sample, nitric acid, ninhydrin reagent, etc

Theory :
Amino acids are the building blocks of all proteins. There are 20 amino
acids commonly found in protein. These 20 amino acids are linked
together through peptide bond, forming peptide chains and proteins.
The chains containing less than 50 amino acids are called peptides,
while those containing greater than 50 amino acids are called proteins.
An amino acid chain with more than 25 (10-50) amino acids is called a
polypeptide, e.g., insulin, hormones, etc. A protein consists of a long
polypeptide chain or several polypeptide subunits, e.g., interferon,
clotting factors, antibodies, enzymes, etc.

The word Amino Acid itself is made of two words amino + acid. This means
the chemical structure of amino acids contains two important functional
groups:
1) Amine group (-NH2 group), and

2) Acid group or carboxylic acid group (-COOH group).

For the identification of proteins following tests are performed :

1) Ninhydrin Test : Like amino acids and peptides, proteins also give
blue to violet colour with ninhydrin solution
2) Xanthoproteic Test : This test is given by proteins and peptides
which have phenylalanine or tyrosine or tryptophan (aromatic amino
acids) in their molecules. On treatment with concentrated nitric acid,
these amino acids undergo nitration in the aromatic ring.
These nitro compounds (particularly nitrophenols) are yellow in
colour. This is the same reaction which occurs when skin comes in
contact with concentrated nitric acid in the laboratories.
3) Millon's Test : When proteins or peptides are treated with Millon's
reagent (a solution of mercuric and mercurous nitrate in nitric acid), a
white precipitate is formed. When this is heated, a red coloured
precipitate is formed. This test is given only by proteins containing
tyrosine in their molecules, forming red mercury complexes with the
nitrophenol formed. The test is also given by other phenolic
compounds and is therefore not specific to proteins.
Procedure
1) Ninhydrin Test : A clean and dry test tube should be taken. The food
samples and 1-2 ml of ninhydrin solution should be added into the test
tubes and it should be shaken. The mixture should be boiled and the
change should be observed. The presence of protein is confirmed if
deep blue or purple color appears.
 2) Xanthoproteic Test : A clean and dry test tube should be taken. The
food samples and few drops of concentrated sulphuric acid should be
added to test tube and it should be shaken. Then the test tube should
be heated gently on a Bunsen burner. The presence of protein is
confirmed if there is a formation of yellow precipitate.

3) Millions Test : A clean and dry test tube should be taken. The food
samples and 2-3 drops of Millon's reagent should be added into the test
tubes and it should be shaken. The presence of protein is confirmed if
there is the formation of white precipitate or if the sample changes to
brick red on heating

Result :
Qualitative analysis of proteins and amino acids was performed.
 To perform qualitative analysis of lipids.

Aim:
To perform qualitative analysis of lipids.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 10 – 14

Materials Required
Filter paper, phenolphthalein solution, dilute alkali solution, test tube,
water, etc

Theory :
1. Test for Free Fatty Acids: Titrating oil against KOH in the presence of
phenolphthalein indicator yields an estimate of the amount of free
fatty acids in the oil. The amount of KOH needed to neutralise the free
fatty acids in 1gm of sample is known as the acid number.

2. Emulsification : When oil or liquid fat is dispersed in water and

shaken, it becomes finely divided to form an emulsion; this process is
emulsification which is permanent and complete in the presence of
emulsifying agents (e.g., bile salts, proteins, soaps, mono- and
diglycerides). Emulsification aids in the processes of fat digestion in
intestines. Emulsifying agents lower the liquid surface tension.

3. Saponification Test : Hydrolysis of esters by alkali yields the parent

alcohol and salt. The salt yielded by long chain fatty acids is soap; this
 process is saponification. Since oils and fats contain long chain fatty
acids, they are the starting materials for soap preparation.
Procedure
The following qualitative tests are performed for identification of lipids :
1. Test for Free Fatty Acids: A few drops of phenolphthalein solution
should be taken in a test tube and 1-2 drops of very dilute alkali
solution should be added to it to develop a pink colour. This solution
should be shaken with a few drops of oil. The pink colour disappears
due to neutralisation of the alkali by the free fatty acids present in the
oil.
2. Emulsification: In a clean and dry test tube, 2ml water should be
added, and in another test tube, 2ml dilute bile salt solution should be
added. Each test tube should be added with 2 drops of mustard oil and
vigorously shaken for a minute. On leaving the tubes undisturbed for 2
minutes, the oil in the first tube breaks down in small pieces and floats
on the water surface; while the oil in the second tube suspends in the
bile salt liquid in the form of minute droplets (i.e., permanent
emulsification
3. Saponification Test: Iml of oil should be thoroughly mixed with an
equal amount of alcoholic KOH solution in a test tube. The mixture
should be kept during the course of warming and shaken gently with
small amount of distilled water. Some oil drops appear indicating
incomplete saponification; while no oil drops indicate complete
saponification.

Result :
Qualitative analysis of lipids was performed.
 To perform qualitative analysis of normal and abnormal
constituent in given sample of urine.

Aim:
To perform qualitative analysis of normal and abnormal constituent in
given sample of urine.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 15 – 19

Materials Required
Dilute HCl, dilute H,SO conc. HNO, AgNO, solution, ammonium
molybdate solution, BaCl2, NaOH, phenolphthalein indicator,
ammonium oxalate solution, 1% acetic acid, sodium hyprobromate
solution (NaOBr), urea powder, benedict uric acid reagent, anhydrous
Na₂CO., sodium nitropruside, picric acid, Fehling's reagent,
sulphosalicyclic acid, chlorophenol red, benedict's reagent, solid
(NH),SO, strong ammonia solution, conc, nitric acid, benzidine
powder, red litmus paper beaker, glass rod, measuring cylinder,
funnel, test tubes, test tube holder

Theory :
Urine is a sterile liquid, by-product of the body, formed by the kidneys
by a process known as urination. It is excreted through the urethra by a
process known as micturition. Analysis of urine is performed to
diagnose various pathological conditions. Urine analysis is based on
either physical or chemical characteristics of urine which help in
assessment of processes occurring within the body.
 Normal Constituents of Urine: Urine is an aqueous solution, having
more than 95% water and 5% of other constituents mentioned below,
with their decreasing concentrations
1) Urea (9.3gm/lt),
2) Chloride 187gm/lt).
3) Sodium 117gm/11),
4) Potassium 0.750gm/11).
5) Creatinine 0.670gm/lt), and
6) Other dissolved ions, inorganic and organic compounds (proteins,
hormones, and metabolites).
In the liver, ammonia and carbon dioxide undergo ornithine cycle to
synthesise urea, which is non-toxic to mammals (unlike ammonia). The
processed form of ammonia is urea which is non-toxic to mammals as
ammonia is highly toxic in nature. Urine is sterile until it reaches the
urethra, where epithelial cells lining the orethra have facultative
anaerobic, gram-negative rods, and cocci.
Physical features of urine are its colour, turbidity or transparency, odour,
pH (acidity or alkalinity), and density. Many of these features can be
identifiable physically while for some laboratory testing is performed:
1) Colour: Normally yellow amber in colour, but colour may depend on
recent diet and the concentration of urine Concentration of urine
reduces due to drinking of more water and it causes lighter colour of
urine, whereas dark coloured urine indicates dehydration. Red urine
shows the presence of RBCs in urine due to kidney damage and
disease.
2) Odour: Its smell indicates various pathological conditions, for
example, urine of diabetic patients has sweet or fruity smell due to the
presence of ketones (organic molecules of a particular structure) or
glucose. Commonly, fresh urine has a mild odour while aged urine
smells stronger just like ammonia.
 3) pH: The pH of normal urine ranges between 4.6-8 (average 6.0), which
depends on the diet of an individual. For example, high protein diets
make urine acidic while vegetarian diets result in alkaline urine.
4) Density: It is the ratio of the weight of a volume of a substance
compared with the weight of the same volume of distilled water; and is
also known as specific gravity. The density of normal urine ranges
between 0.001-0.035.
5) Turbidity: The turbidity of the urine sample is evaluated from person
to person and is reported as clear, slightly cloudy, cloudy, opaque, or
flocculent. Generally, fresh urine is either clear or very slightly cloudy.
Excess turbidity is observed in the presence of suspended particles in
the urine that can be determined through the examination of
microscopic urine sediment. Turbidity of the urine generally increases
due to urinary tract infections or obstructions.
Abnormal Constituents of Urine
Urine has the following constituents, whose abnormality indicates a
pathological condition.
1) Proteins : Presence of abnormal concentration of albumin and
globulin in the urine is known as proteinuria (albuminuria), which may
be of two types:
i) Physiologic Proteinuria: Less than 0.5% protein is observed in
urine due to severe exercise, after a high protein meal, and during
pregnancy.
ii) Pathologic Proteinuria: Amount of proteins increases in case of
glomerulonephritis and nephrotic syndrome Proteinuria may also
occur due to poisoning of renal tubules by heavy metals (like
mercury, arsenic, or bismuth)
2) Glucose : Presence of glucose in urine is known as glycosuria which
occurs due to diabetes mellitus and endocrinal disorders such as
hyperpituitarism. hyperthyroidism, Cushing's syndrome, and
 pheochromocytoma Transient glycosuria may be observed rarely due
to emotional stress (like in exciting athletic contest).
3) Other Sugars : Presence of fructose sugar in urine due to disturbed
fructose metabolism is known as fructosuria. Similarly, gallactosuria
and lactosuria occur rarely in infants, pregnant women, and in
lactating mother Consumption of food rich in pentose sugars (like
grapes, cherries and plums) causes pentosuria The condition of
pentosuria also occurs in inherited diseases in which pentoses are not
metabolised.
4) Ketone Bodies : Under normal conditions, less than Img of ketone
bodies are excreted through urine in 24 hours. Excretion of ketone
bodies increases in many conditions like starvation, diabetes mellitus,
pregnancy, ether anaesthesia, and in certain types of alkalosis.
5) Bilirubin and Bile Salts : In case of obstructive or hepatic jaundice,
bilirubin is excreted in urine. A condition in which bile salts are
excreted in urine is known as bilirubinuria In certain stages of liver
disease, bile salts are excreted without bile pigments in urine. Traces of
bilirubin without bile saits are excreted in urine during excessive
haemolysis. Bilirubin is absent in urine of an adult healthy person. It is
a waste product, produced by the liver from the haemoglobin of RBCs
that are broken down and removed from circulation. It is utilised in the
synthesis of bile (a fluid involved in food digestion). In some liver
diseases like biliary obstruction or hepatitis, excess amount of bilirubin
is formed which is eliminated through urine. Presence of bilirubin in
urine indicates liver disease (like development of jaundice).
6) Blood: Blood is excreted in urine due to lesions of kidney, urinary tract
infection, and in case of nephritis Free haemoglobin molecules may
also be detected after quick haemolysis, e.g., black water fever ( a
consequence of malaria) and severe burns.
Procedure
Result :
Qualitative analysis of normal and abnormal constituent in given
sample of urine was performed.
To detect the glucose in given sample of urine by qualitative test.

Aim:
To detect the glucose in given sample of urine by qualitative test.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 20 – 24

Materials Required
Urine sample, Benedict's solution, Fehling's solution A. Fehling's
solution B. Burner, test tube, test tube holder and measuring cylinders.

Theory :
A test for glucose in urine measures the level of glucose in urine (pee).
Glucose is type of sugar and serves as body's main source of energy for
the cells. Glucose is delivered to cells via blood. Generally, there is
little to no glucose in urine. However. if blood sugar level is too high,
kidneys will excrete some of it through urine Therefore, a high urine
glucose level may indicate that blood glucose is also high, which could
be an indication of diabetes. The doctor will generally order blood
glucose test in case of high urine glucose level in order to help in the
diagnosis.
Procedure
Benedict's Test
1) A clean and dry test tube should be taken.
2) 2ml of given urine sample should be measured using the measuring
cylinder.
3) The measured urine sample should be poured into test tube.
4) 5ml of Benedict's reagent should be added into the test tube containing
urine sample.
5) The test tube holder should now be fixed and the test tube should be
brought near the bunsen burner and allowed to heat for 2 minutes.
6) The tube should be stirred continuously while heating.
7) The changes should be observed.

Fehling's Test
1) A clean and dry test tube should be taken
2) 2ml of given urine sample should be measured using the measuring
cylinder
3) The measured urine sample should be poured into test tube.
4) 2ml of Fehling's solution A should be added into the test tube
containing urine sample and shaken well.
5) 2ml of Fehling's solution B should be added into the same test tube
and all the solution should be mixed slowly.
6) The test tube holder should now be fixed and the test tube should be
then brought near bunsen burner and allowed to heat for 2 minutes.
7) The tube should be stirred continuously while heating.
8) The changes should be noticed.
Observation and Conclusion
1) Benedict's Test: A yellow precipitate gradually forms in the test tube
after the sample is heated, indicating the presence of sugar in the urine
sample.
2) Fehling's Test: A green precipitate progressively forms in the test tube
when the sample is heated, indicating the presence of sugar in the
urine sample.

Result :
The given sample of urine contains glucose.
 To detect the creatinine in given sample of urine by qualitative
test.

Aim:
To detect the creatinine in given sample of urine by qualitative test.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 25 – 29

Materials Required
Sodium nitropruside, 10% NaOH, picric acid, anhydrous Na,CO,,
beaker, glass rod, measuring cylinder, funnel, test tubes and test tube
holder.

Theory
By applying a photoelectric method, the Folin modified method
estimates creatinine. It represents the waste product of creatinine
metabolism. The presence of creatinine in urine is known as
creatinuria. In cases of fever, starvation, or diabetes, its excretion
increases.
Procedure
1) (Weyl's Test): 5 ml of urine should be treated with 5 drops of sodium
nitropruside and 2 ml of 10% NaOH. Formation of yellow colour from
ruby red colour confirms the presence of creatinine.
2) Jaffe's Test: 5 ml urine should be treated with I ml of standard
solution of picne acid. To this solution 3 gm of anhydrous Na₂CO,
 should be added and mixed well by shaking. A deep orange colour
confirms the presence of creatinine.

Result :
The given sample of urine contains creatinine.
 To detect the chlorides in given sample of urine by qualitative
test.

Aim:
To detect the chlorides in given sample of urine by qualitative test.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 30 – 33

Materials Required
Concentrated nitric acid, acetic acid, Robert's solution, sulphosalicylic
acid or a solution containing 13% salicylic acid and 20% sulphuric
acid, test tube stand, test tube holder, test tubes, graduated pipette (5
ml. capacity), and spirit lamp.

Principle
Albumin precipitates due to nitric acid. Albumin coagulates when
heated or exposed to sulphosalicylic acid.

Theory
Along with sodium, potassium, and bicarbonate, chloride is an anion
(negatively charged ion) that helps in controlling the amount of body
fluids and the acid-base balance. It is present in all body fluids but is
more concentrated outside of cells. Chloride enters the body through
foods, and the kidneys remove extra chloride by excreting it in the
urine Urinary Chloride Test is prescribed whenever the doctor
suspects any disturbance with acid base balance, changes in the levels
 of body fluids or electrolytes (rons present im body fluids), or in a
condition known as renal tubular acidosis.

Procedure
1) Nitric Acid Ring Test:
i) 5 ml of conc nitric acid should be taken in a test tube.
ii) The test tube should be inclined and the urine sample should be
added to it with a dropper so that it slowly drips down the side
and forms a distinct layer.
iii) At the point where the two liquids meet, a white ring forms,
indicating the presence of albumin in the urine sample.
Or
i) 5 ml. of Robert's solution should be taken in a test tube.
ii) The test tube should now be inclined and 2-3ml of the given
sample of urine should be added to it with a dropper along the
inner side of test tube so that it forms a layer over the Robert's
solution.
iii) The presence of albumin in the sample is indicated by the
presence of white ring at the junction of two layers.
2) Heat Coagulation Test:
i) 6 to 8ml of urine should be taken in a test tube.
ii) The test tube should now be inclined at an angle and the upper
one-third of test tube should be heated at a low flame.
iii) The heated part of the urine becomes turbid.
iv) 1% acetic acid should be added drop by drop and boiled or a
drop of 33% acetic acide should be added.
v) Appearance of turbidity confirms the presence of albumin in the
urine sample whereas disappearance of turbidity confirms the
presence of phosphate.
3) Sulphosalicylic Acid Test:
i) 3mL of urine should be taken in a test tube.
ii) Few drops of sulphosalicyclic acid should be added and heated
gently.
 iii) The presence of albumin in the urine sample is indicated by a
white, hazy, or turbid precipitate (coagulation) in the solution.

Observation
Less than 250 mg (in a 24-hour urine sample) of protein are present in
normal urine Albumin is found in urine at levels above normal when
pathogenic conditions, such as albuminuria, are present. This level is so
small that it cannot be detected by any easy tests. Albumin levels in urine
are significantly high in case of renal disruption and high blood pressure

Result :
The given sample of urine contains chlorides.
 To determine the creatinine in blood /serum.

Aim:
To determine the creatinine in blood /serum.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 34 – 38

Materials Required
Picric acid, sodium hydroxide, stock creatinine, standard creatinine,
flasks, graduated pipettes and photoelectric colorimeter.

Theory
By employing a photoelectric colorimeter and the modified Folin
method, creatinine in blood is assessed Protein free blood filtrate, ie,
folin Wu is used. The unknown creatinine found in the filtrate is
treated with picric acid to create red-colored creatinine picrate in an
alkaline medium. The optical density of this red-coloured creatinine is
compared to that of standard solution after being converted by picric
acid to creatinine picrate The concentration of creatinine in given
blood sample can be calculated using colorimetry principle.
Clinical Significance
1) When creatine phosphate spontaneously degrades in the body,
creatinine, the waste product of creatine metabolism, is produced. It
is a non-threshold substance. The glomeruli often filter it. Changes
in its excretion can be a sign of specific metabolic disorders because
its excretion is not related with food protein.
2) Its excretion rises during fevers, starvation, consumption of a diet
low in carbohydrates, and in diabetes mellitus.
3) It may rise as a result of excessive tissue damage that releases
creatine or as a result of improper phosphorylation of creatine.
4) Therefore, creatinine excretion should be used as a measure of
endogenous protein metabolism since it is independent of food
proteins.
5) An adult of average size normally has an endogenous creatinine
clearance that varies from 100 to 130 ml/minute, which is a rough
measure of glomerular filtration rate.
6) Reduced glomerular filtration rate is indicated by values below 90
ml/minute.
7) Normal value is 0.7 to 2.0 mg/100 ml blood.

Procedure
1) Preparation of Unknown Sample:
i) 5 ml of folin wu filtrate should be pipetted out in a flask labeled
as "S".
ii) 2 ml of 1% picric acid should be added and mixed well.
iii) 10% of 0.5 ml of sodium hydroxide solution should be added.
iv) Then, the solution should be kept for 15 minutes and the optical
density should be measured using green filter (530 M𝜇). It
should be noted as "Es"
 2) Preparation of Standard Sample:
i) 5 ml of standard creatinine solution should be pipetted out in a
flask labeled as "S"
ii) 2 ml of 1% picric acid should be added and mixed well.
iii) 10% of 0.5 ml of sodium hydroxide solution should be added.
iv) Then, the solution should be kept for 15 minutes and the optical
density should be measured using photoelectric colorimeter
with green filter (530 m𝜇). It should be noted as "Es".
3) Preparation of Blank Sample:
i) 5 ml of distilled water should be pipetted out in a flask labeled
as "B"
ii) 2 ml of 1% picric acid and 0.5 ml of 10% sodium hydroxide
solution should be added to it.
iii) Then, the solution should be kept for 15 minutes and compared
in a calorimeter using green filter (530 m𝜇).
Calculations
The optical density of the blank should be subtracted from the optical
density of the unknown and the standard by using the principle of
photoelectric calorimetry.

Optical density of
Concentration of (Eu) unknown sample Concentration of
creatinine in unknown = × standard creatinine (Cs)
blood sample CU Optical density of
standard sample "Es"

Standard creatinine solution is prepared by diluting Iml of stock solution (1

mg) to 500 ml.

Here,
1 ml of standard creatinine solution = 0.002 mg creatinine.
5 ml of standard creatinine = 0.01 mg creatinine.
Now, 5 ml filtrate = 0.5 ml blood
Therefore
Optical density of
Concentration of unknown sample "(Eu)"
creatinine in unknown = × 0.01 mg creatinine
blood sample CU Optical density of
= “X” mg (suppose) standard sample "Es"
Now "x" mg of creatinine is present in 5 ml of folin wu filtrate.
But 1 ml of folin wu filtrate = 0.1 ml of blood
5 ml of folin wu filtrate = 0.5 ml of blood
"x" mg of creatinine is present in 0.5 ml of blood.

To estimate milligram percentage of creatinine

If
0.5 ml = "x" mg creatinine
100 ml xx100 0.5 mg creatinine

Substituting the value of 'x'

Then.
"cu" Eu x 1 x 100 x 10 = Eu x 2mg Es 10 15 x 2mg % creatinine
Es 100 1 5 Es
CU = Eu x 2mg % creatinine. Es
Es

Result :
Creatine concentration in blood /serum was determined.
 To determine the glucose in blood /serum

Aim:
To determine the glucose in blood /serum.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 39 – 43

Materials Required
Alkaline copper sulphate solution, phosphomolybadic acid, sodium
tungstate 10%, sulphuric acid 2/3 n, benzoic acid solution, stock
glucose solution, standard glucose solution no. 1, standard glucose
solution no. 2, fluoride oxalate solution, folins sugar tube, pepettes
graduated (2 ml, 5 ml), flasks and photoelectric calorimeter.

Theory
Folin Wu (modified) method is used in which protein-free filtrate
(also known as folin-Wu filtrate) is produced so that 10 ml of the
filtrate can make 1 ml of blood sample. By treating the blood proteins
with tungstic acid, protein-free filtrate is produced. Then an alkaline
copper sulphate solution is used to heat this protein-free filtrate that
contains glucose. Hence, glucose breaks down copper sulphate to
form an equivalent amount of cuprous oxide.

To form an equivalent amount of molybdenum blue, this cuprous

oxide is reduced with phosphomlybdic acid The intensity of the
 molybdenum blue colour depends on the amount of cuprous oxide,
which is related to the amount of glucose present in a particular
sample of "folin wu" filtrate

Figure 1: Folins Sugar Tube

Reactions
C6H12O6+CU++ CU++ Oxidation
Glucose cupric products of glucose

CU+ Na2 PO4: 12 MoO3 CU++ + 12 MoO

cuprous cupric molubdenum blue

A similar procedure and a photoelectric calorimeter are used to compare

the blue colour of the standard solution to the blue colour produced from
the test blood sample. The optical density of the test and standard are
determined and the colorimetric principle can be used to determine the
blood glucose levels.
Clinical Significance
1) Monosaccharides, like glucose, fructose, galactose, etc. are the main
end product of carbohydrate digestion absorbed in the blood, and
glucose metabolism provides:
i) Major amount of energy for body activities
ii) Reserve fat depots
iii) Tissue glycolipids
iv) Amino acids
2) Thus, it would seem that the metabolism of glucose plays a crucial
part in the metabolism of carbohydrates, which is closely linked to the
metabolism of protein and fat.
3) The hormones of the islets of Langerhans control the availability of
glucose from various dietary sources and its management and
utilisation. Following is a schematic explanation of this:

ẞ cells of Langerhans
Glucose (Excess) Glycogen (stored)
Insulin

Energy

Fat depots

Tissue repair
4) As a result, the blood sugar level is kept stable, normal, and expressed
as fasting, ie, after a meal.
5) A real symptom of diabetes mellitus is hyperglycaemia, or an increase
in blood sugar levels. Excess free glucose accumulates in the blood as
a result of inadequate insulin production or impaired insulin secretory
function.
 6) According to the severity of the condition, high fasting blood sugar
readings in diabetes mellitus range from normal to 500 mg/100 ml and
above.
7) Blood sugar levels above 500 mg/100 ml are related with an increased
risk of coma.
8) Blood sugar levels are increased by 150 mg% as a result of the thyroid,
pituitary and adrenal glands' hyperactivity, which also involves
emotional stress states.
9) Convulsions and the latter stages of numerous diseases are both
accompanied by a similar rise in blood sugar levels.
10) Hyperglycaemia, a moderate increase in blood sugar levels, can
be caused by sepsis and a number of infectious diseases.
11) Additionally, various brain illnesses, such meningitis, encephalitis,
tumours, and haemorrhages are associated with an increase in blood
sugar levels.
Preparation of Reagents
1) Sodium Tungstate: 10% w/v of 10gm of Na₂ WO, 2H₂O (sodium
tungstate) should be dissolved in 100 ml of distileed water.
2) Sulphuric Acid: 3.5gm of 2/3 N of sulphuric acid should be dissolved
in 100 ml of distilled water.
3) Alkaline Copper Sulphate Solution:
i) A solution A should be prepared by dissolving 40gms of
anhydrous sodium carbonate and 7.5gms of tartaric acid in
400ml of distill water.
ii) A solution B should be prepared by dissolving 4.5gm of copper
sulphate crystalline in 200ml of distill water.
iii) Solution B should be added to solution A with constant surring
and the final volume should be maintained with distilled water
to 1 litre.
 4) Phosphomolybdic Acid Reagent: 70gm of molybdic acid, 10gm of
sodium tungstate AR and 400ml of Sodium hydroxide 10% should be
dissolved in 400ml of distil water. It should be boiled for 30 minutes
to remove ammonia. Phosphoric acid of specific gravity 1.75 should be
added. Distilled water should be added to make the final volume of 1
litre.
5) Benzoic and Solution: 2.5gms of benzoic acid should be dissolved in
1000 ml (1 litre) of distil water and heated.
6) Stock Glucose Solution (1gm/100ml): 1gm of glucose (AR) should
be dissolved in 100 of ml benzoic acid solution.
7) Standard Glucose Solution No. 1 (10 mg/100ml): 1 ml of stock
glucose solution should be dissolved in 100 of ml benzoic acid solution
8) Standard Glucose Solution No. 2 (20 mg/100 ml): 2 ml of stock
glucose solution should be dissolved in 100 of ml benzoic acid
solution.
Procedure
1) Three folin-wu tubes should be cleaned and labelled as:
i) Unknown 'U'
ii) Standard I-Std I
iii) Standard II - Std II
2) 2ml of "Folin wu filtrate" should be taken to the folin wu tube
labelled as "U".
3) 1ml of standard sugar solution 1 (0.1mg sugar) should be taken in a
folin wu tube labelled as "Std I".
4) 1 ml of standard sugar solution II (0.2mg sugar) should be taken in a
folin wu tube labelled as "Std II".
5) 1ml of alkaline copper sulphate solution should be added to all three
tubes.
6) Tubes should be kept in boiling water bath for 6 to 8 minutes.
7) Then, the tubes should be removed from the water bath and 1ml
phosphommolyddic acid should be added to all the tubes.
 8) The tubes should be again kept in boiling water bath for 2 minutes
and thes cooled at room temperature after 2 minutes.
9) 25ml of distilled water should be added to each tube and mixed well
and recorded. The optical densities should be compared using
photoelectr colorimeter having tube filter 420 mµ.

Calculations
By using photoelectric calorimetry principle
Optical density of unknown
blood glucose sample Eu Concentration of
Concentration of glucose in
= × std glucose (Cs)
given blood sample (Cu) Optical density of standard
glucose Es
Now as 1 ml of standard sugar solution is used for experiment it is fact that:
1 ml of standard sugar solution = 0.1 mg of sugar.
ie. Cs concentration of standard glucose = 01 mg of sugar.

Therefore,
Eu
Concentration of glucose in given blood sample CU = × Cs
ES
Eu
Eu
= ×.1mgES= “ x ” (suppose)
ES

Now "x" mg of sugar is present in 1 ml of folin wu filtrate of given blood

sample, ie, in 0.1 ml blood.

Note: (1 ml 0 folin wu filtrate = I ml of blood)

To determine mg percentage of sugar

If 0.1 ml blood = "x" mg of sugar

100 ml blood = 100xx mg 0.1 ml blood.

On substituting the value of 'x'

Then,
100 Eu
Cu= × ES ×.1
0.1
 Eu
Concentration of unknown glucose in given blood sample = ES × 100 mg
percent sugar.
Optical density of unknown
Concentration of glucose in blood glucose sample Eu
= × 100
given blood sample (Cu) Optical density of standard
sugar solution

Result :
Glucose concentration in blood /serum was determined.
 To determine the cholesterol in blood/serum.

Aim:
To determine the cholesterol in blood/serum.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 44 – 48

Materials Required
Absolute alcohol, Diethyl ether, Chloroform, Acetic anhydride,
Standard solution of cholesterol (100 mg%), test tube and stir rod.

Theory :
The blood contains both esters and free cholesterol Normally, the ester
fraction forms 70% of the total cholesterol while free cholesterol forms
roughly 30% An alcohol-ether mixture is used to extract the cholesterol
and cholesterol esters from the serum (Alcohol precipitates the
proteins or either solubilises the cholesterol part). The contents are
centrifuged. The protein free extract is dried using evaporation. Using
the Liebermann-Burchard procedure, the cholesterol residue is
dissolved in chloroform and colorimetrically quantified.
Reagent Preparation
100 mg of cholesterol should be dissolved in some amount of alcohol. It
should be slightly warmed (in water bath), if needed. The volume
should be maintained to 100 ml. with alcohol.

Procedure
1) Test:
i. 8mL of alcohol and 2ml. of ether should be taken in a centrifuge
tube and then mixed.
ii. 0.2mL of blood should be added and mixed. It should be kept in
slanting position for half an hour and centrifuged
iii. The supernatant should be collected (which contains
cholesterol) in another tube. This test tube should be kept in
boiling water for the evaporation of solvent and the residue, ie,
leaving behind a residue of cholesterol that adheres to the
bottom of the flask.
iv. The best quality chloroform, acetic anhydride, and sulphuric
acid should be used. Chloroform needs to be extremely
anhydrous. The colour produced by regular chloroform or
outdated stock will be weak and unpredictable.
2) Standard:
i. 0.2, 0.4, 0.6 and 0.8ml. of standard cholesterol solution should be
added to the labelled tubes, 1.e., S1, S2, S3, and S4
ii. All test tubes should be stored in a hot water bath to allow the
solvent to evaporate and leave any residue behind.
iii. The blank test tube should be cleaned and dried.
iv. The following reagents should now be added in test, different
standard, and blank.
 v. It should be mixed well and kept in dark for 10 minutes.
vi. The optical density should be read at 610nm.

Calculations
Optical density of test =
1) Optical density of standard S₁ = concentration = 0.02 mg
2) Optical density of standard S₁ = concentration = 0.04 mg
3) Optical density of standard S₁ = concentration = 006 mg
4) Optical density of standard S, concenuation = 0.08 mg

Optical density of test Conc of std.

The concentration of = × × 100
cholesterol Optical density of std. Vol. of blood/serum used

Interpretation
1) Normal serum cholesterol level is 150-250 mg/dl.
2) The normal level of serum cholesterol is 150-250 mg/dl.
3) Following conditions are caused due to hypercholesterolemia:
i) Diabetes mellitus
ii) Obstructive jaundice
iii) Nephrotic syndrome.
iv) Cirrhosis of liver
v) Hypoparathyroidism.
vi) Xanthomatosis.
4) Following conditions are caused due to hypocholesterolemia:
i) Hyperthyroidism
ii) Pernicious anaemia
iii) Haemolytic anaemia
iv) Malabsorption syndrome

Result :
Cholesterol concentration in blood /serum was determined.
 To determine the calcium in blood /serum

Aim:
To determine the calcium in blood /serum

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 49 – 52

Materials Required
Ammonium oxalate, 4% solution, ammonia, 2% solution, 0.01 N
potassium permanganate, 2N H,SO, solution, test tube and stir rod.

Theory :
In plasma and serum, calcium can be found in three different forms:
1) Protein-bound
2) Jonised part
3) Coupled with other compounds like citrate
Unlike the other two, calcium that is attached to proteins is not diffusible
When it comes to extremely young children, the upper limit seems to be a
little higher During pregnancy, serum calcium levels tend to reduce slightly.
One of the reasons of tetany is a decrease in serum calcium that affects the
ionised calcium.
Calcium precipitates out of the serum as oxalate. The precipitate is first
washed. and then it is dissolved in acid and titrated with KMnO, solution.
Procedure
1) 2ml serum should be dissolved in a test tube containing 2ml distilled
water.
2) 4% of Iml ammonium oxalate should be added to the above mixture
and allowed to stand for 30 minutes and then centrifuged.
3) The supernatant fluid should be removed without disturbing the
precipitate.
4) The test tube should be allowed to dry for 5 minutes by standing the
tube inverted on a filter paper.
5) 3ml Ammonia solution (2%) should be added and mixed well.
6) It should be centrifuged again and the supernatant fluid should be
poured off.
7) The precipitate should be mixed with 2ml H2SO4 (2N) and warmed by
placing the beaker of almost boiling water to complete solution of the
oxalate.
8) The mixture should be removed and kept at 60° to 70°C.
9) The mixture should be titrated with 0.01 KMnO 4 and a faint pink
colour is obtained which lasts for a minute.
10) Calcium solution should be treated with 2ml H2SO4 (2N)
solution at 60-70°C and titrated with 0.01N KMnO4 solution and
results in faint pink colour.
11) 2ml of H2SO4 (2N) solution should be titrated to the same end-point
as a blank.
Calculations
Each Iml 0.01N KMnO4 = 0.2 mg Ca"
Hence since 2ml of serum is used
Conc. of Calcium = (Titration of Test - Titration of Blank) X 0.2X
Conc. (mg%)=(Titration of Test-Titration of Blank) X 10.
Expected Values
9-11 mg/dl

Result :
Calcium concentration in blood /serum was determined.
 To determine the urea in blood /serum.

Aim:
To determine the urea in blood /serum.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 53 – 58

Materials Required
Distilled water, blood serum, photometer, pipette and cuvette.

Theory :
Liver converts ammonia (primarily produced by the breakdown of
amino acids) into urea. These reactions are used in kinetic enzymatic
estimation of urea:
Urea + 2H2O ___urease _____2NH4++CO32-
2-oxoglutarate + NH4+ + NADH______glutamate dehydrogenase ___L-
glutamate + NAD++H₂O
Urea is hydrolysed by urease into ammonia. Ammonia and 2-
oxoglutarate are combined by glutamate dehydrogenase to produce
glutamate. A decrease in absorbance at 340 nm (Warburg's optical test)
is used to photometrically detect the conversion from NADH to NAD
in this reaction.
Procedure
1) The photometer should be switched on and allowed to heat for 10
minutes at 37°C.
2) The wavelength should be set at 340 nm and distilled water should be
used to make the blanking All of the absorbance that is further
described is read using distilled water as a reference.
3) 3 cuvettes are available, i.e., one for blank reaction, one for the
standard reaction and one for the sample (blood serum) reaction.
4) For Blank Reaction: 002ml of distilled water and 2ml of the reagent
(working solution) should be pipetted out into the cuvette. The stop-
watch should be pressed at this moment. The solution should be
pipetted out inside the cuvette once again up and down to ensure
proper mixing. The cuvette should be transferred immediately into the
heated photometer. The initial absorbance A should be measured
exactly 30 seconds after the working solution has been pipetted, and
the second absorbance A₂ should be measured after 1 minute.

A1 blank
A2 blank

5) 0.02ml of distilled water and 2ml of the reagent (working solution)

should be pipetted out into the cuvette. The stop-watch should be
pressed at this moment. The solution should be pipetted out inside the
cuvette once again up and down to ensure proper mixing The cuvette
should be transferred immediately into the heated photometer. The
initial absorbance A, should be measured exactly 30 seconds after the
working solution has been pipetted, and the second absorbance A,
should be measured after 1 minute.

A1 Standard
A2 Standard

6) 0.02ml of the serum sample and 2ml of reagent (working solution)

should be pipetted out. A, and A should be measured as compared to
the previous measurements.

A1 Sample
 A2 Sample

7) The differences in absorbance should be calculated.

∆A blank = A1blank - A2blank

∆A Standard = A1 Standard - A2Standard
∆A Sample = A1 Sample - A2Sample

Calculate the Concentration:

Urea (mmol/l) = ∆A Sample - ∆A blank /∆A Standard - ∆A blank. C standard
(15 mmol/l)

Result :
Urea concentration in blood /serum was determined.
 To determine the SGOT/SGPT in blood/serum.

Aim:
To determine the SGOT/SGPT in blood/serum.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 59 – 64

Materials Required
0.1 M phosphate buffer having pH 7.4, distil water, monopotassium
phosphate anhydrous, disodiumphosphate solution, mono potassium
dihydrogen solution, aspartic acid, oxoglutaric acid, NaOH,
chloroform, alanine, oxaloacetate, L partate, alpha-ketoglutarate, 2,4-
dinitrophenyl hydrazine, 2,4- dinitrophenylhydrazone, sodium
pyruvate, DNPH, pipette, small beaker, incubator and volumetric flask.
Theory :
The enzyme known as serum glutamic-oxaloacetic transaminase
(SGOT/AST) is found in the kidneys, heart, skeletal muscles, and liver cells.
When there is an injury. these enzymes are released into the tissues through
the blood stream. Heart and liver cells contain serum glutamic pyruvic
transaminase (SGPT/ALT), which is released into the blood when there is
damage. The following two methods are used:
1) Reitman and Frankel Method: L-aspartate and alpha-ketoglutarate
are produced when SGOT is transformed into glutamate and
oxaloacetate, respectively. In an alkaline medium, this oxaloacetate
reacts with 2,4-dinitrophenyl hydrazine to produce 2,4-
 dinitrophenylhydrazone. It turns brown colour by producing
hydrozone derivative and pyruvate standard, a calibration curve.
2) Calorimetric Method: DNPH is higher in oxaloacetate and pyruvate
with oxoglutarate and supports the intensity of colour limiting the
error.
Reagent Preparation
1) Disodium Hydrogen Phosphate Dihydrate (0.1M): 8.9 g of
disodium hydrogen phosphate dehydrate should be dissolved in 200 ml
water in 500 ml volumetric flask.
2) Monopotassium Phosphate Anhydrous (0.1M): 1.36 g
monopotassium phosphate anhydrous should be dissolved in 50 ml of
distilled water and the solution should be diluted to 100 ml. 420 ml
disodiumphosphate solution should be mixed with 80 ml mono
potassium dihydrogen solution.
3) Substrate for SGOT: 2.66 g DL aspartic acid and 30 mg oxoglutaric
acid should be dissolved in 20.5 ml of 1 N NaOH in a beaker by adding
drop wise to adjust pH. This solution should be then transferred into
100 ml in a volumetric flask with phosphate buffer solution. Iml of
chloroform should be added as a preservative and then it should be
refrigerated. The substance should be discarded if it becomes turbid.
4) Substrate for SGPT: 1.78 g DL alanine and 30 mg oxoglutaric acid
should be dissolved in 20 ml phosphate buffer. 1.25 ml of 0.4 N NaOH
should be taken in small beaker and the volume should be maintained
up to 100 ml with buffer. I ml chloroform should be added as a
preservative.
Procedure
 Pyruvate Standard
 Stock Standard: 220mg sodium pyruvate should be treated with 100ml
of phosphate buffer, which should be discarded after preparing the
working standard.
 Working Standard: 10ml stock standard should be added to 100ml
phosphate buffer and 2ml should be dispensed in a test tube and
refrigerated using stopper. One tube should be used for preparing the
calibration curve and the rest should be refrigerated. Remaining
solution should be discarded. 200mg DNPH should be dissolved in IN
HCI (diluting 90ml concentration with water). Distilled water should
be added to make up the volume up to 1L..
 Sodium Hydroxide Solution 1N: 40gm of sodium hydroxide should
be added in 500ml of water and the volume should be maintained up
to 1000ml the substrate should be pipetted out into a test tube, which
is then incubated in a water bath at 40°C for 10 minutes. 0.2ml of
serum should be mixed and incubated for 60 minutes (GOT) and 30
min (GPT) then the test tube should be removed. DNPH should be
added to stop the reaction for 20 minutes at room temperature 110ml of
0.4N sodium hydroxide should be added and rubber stopper should be
placed to mix by inverting (A).
Calculations
For SGPT:
(A1-A₂) X 857U/L
For SGOT: (A1-A₂) X 514 U/L

Interpretation
When the optical density changes by more than 500 mu ml against
transaminase then it is noted and the normal value is SGOT, SGPT: 0-50
U/L, moderate range: 400-2000 U/L, high range 2000-30,000 U/L.

Result :
SGOT/SGPT in blood/serum was determined.
 To study the hydrolysis of starch from salivary amylase enzyme.

Aim:
To study the hydrolysis of starch from salivary amylase enzyme.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 65 – 70

Materials Required
Starch, phosphate buffer solution, iodine solution, test tube, flask, pipette
and stir rod.

Theory :
Starch is made up of mainly two parts:
1) Amylose: A linear polymer composed of D-glucose units that are a-1-4
bound About 20-30% of the structure is made up by this
polysaccharide.
2) Amylopectin: It is a polysaccharide with highly branched polymer
containing bonded glucose units from 0-1-6.
The enzyme amylase breaks down large alpha-linked polysaccharides like
starch and glycogen to produce maltose and dextrin. It is the most common
type of amylase present in people and other mammals. Amylase is found in
saliva. This form of amylase is also known as "ptyalin".
Maltose units are formed when the enzyme randomly affects a-1-4 bounds in
the amylose structure of starch. a-1-6 bonds that are part of the amylopectin
structure of starch are unaffected by amylase. A large branching dextrin
structure is formed in the media as a result of hydrolysis process carried out
with-amylase, in addition to the maltose and glucose units.

Optimum Conditions for a-Amylase

1) Optimum pH: 5.6-6.9
2) Human Body Temperature: 37°C
3) Presence of Certain Anions and Activators:
i) Chloride and bromide is most effective
ii) Iodide is less effective
iii) Sulphate and phosphate is least effective
Procedure
Part-A
8) Iml. of 1% starch solution and ImL. phosphate buffer solution of pH =
6.8 should be added into 4 test tubes.
9) Each test tube should be placed into water baths at 0°C, 25°C, 37°C and
95°C, respectively.
10) After a few minutes Iml. diluted (1/10) saliva should be added
into each test tube.
11) 1 drop of iodine solution should be placed on 4 watch glasses and 1
drop of this solution should be taken from each test tube to check for
any remaining starch (starch gives blue colour with iodine solution).
12) Hydrolysis time of ImL starch for each test tube should be
recorded.

Part-B
1) 7 test tubes should be taken and the following solution should be
prepared:
Reagent T1 T2 T3 T4 T5 T6 T7
1% Starch Solution 0.2mL 0.5 mL 1 mL 2 mL 3 mL 4 mL 5 mL
Distilled Water 4.8mL 4.5 mL 4mL 3 mL 2 mL 1 mL 0 mL
Phosphate Buffer 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL
Saliva (1/20 diluted) 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

2) Each test tube should be shaken thoroughly.

3) Each mixture should be examined for colour with iodine every minute.
4) Changes in colour should be recorded.

Result :
Hydrolysis of starch from salivary amylase enzyme was studied.
 To study the hydrolysis of starch from acid.

Aim:
To study the hydrolysis of starch from acid.

Reference :
‘ Dr. Gupta G.D. , Dr. Sharma Shailesh, Kaur Manpreet, “Practical
Manual of Biochemistry & Clinical Pathology” Published by Nirali
Prakashan, Page no 71 – 74

Materials Required
lodine solution, Benedict's reagent, starch paste, conc. HCI, boiling water
and six test tubes.

Theory :
1) Polysaccharides are polymers and their complete hydrolysis results in
hundreds of monosaccharide molecules.
2) The end product of completely hydrolysed starch is glucose.

Complete hydrolysis
Starch Glucose
With acid

3) Partial hydrolysis of starch results in a variety of sugars, including

dextrin, maltose, and glucose.
Complete hydrolysis
Starch Dextrin + maltose + glucose
With acid

4) A specific test for polysaccharides, oligosaccharides, and disaccharides

is hydrolysis with acid.
 5) One of the important elements in this reaction is heating time.
6) The solutions must react with Benedict's reagent to confirm that the
hydrolysis of sucrose or starch occurred. (If hydrolysis is carried out,
monosaccharides will be released and a red-colored cuprous oxide
precipitate will be produced).
7) Conc HCI should be added carefully.
8) Achromic Point: It is a point at which iodine solution fails to form any
colour. All the erythrodextrin get converted into archrodextrin and
maltose at this point.
9) Chromic Point: It begins at zero and continues until the achromic
point is reached, or it is the time needed to reach that point.

Procedure
 Two sets of test tubes each containing six test tubes should be taken.
 1 ml of iodine solution should be added in all the test tubes of first
set.
 5ml of Benedict's reagent should be taken in all test tubes of second
set.
 35ml of starch paste should be taken in a beaker.
 15 to 20 drops of conc. HCI should be added in this beaker and then
mixed. As a result, the contents will swell.
 This mixture should now be transferred into a test tube.
 This test tube should be placed in boiling water and time should be
recorded.
 Sample should be taken from the test tube for iodine and Benedict's
test.
 Sample should be taken from the bottom of the test tube, if possible.
Iodine and Benedict's test should be performed.
 The procedure should be repeated till achromic point is reached.
Interpretation
1) As starch is hydrolysed by acid in test tubes containing Benedict's
reagent. the Benedict's test will gradually turn positive.
2) The hydrolysis of starch will cause the iodine test to gradually turn
negative as any polysaccharides that are present turn into reducing
monosaccharides

Result :
The hydrolysis of starch from acid was studied.