Ex.

no:
Date:

Estimation of protein efficiency ratio (PER) using

albino rats

Aim:
To study the protein quality of the given formulated food protein in
comparison with milk protein, casein by PER using albino rats
Procedure:
1. Choice of the animal species for experimentation:
The rat has physiological symptoms similar to that of man also there
are no problems in housing them individually weanling male rats (20-23
days) are used for the study.
2. Growing of rats:
The animals are weighed and based on the weight they are classified
into two groups (10 rats in each group).The first group was fed with
formulated diet (control) and the second group fed with test diet containing
the health mix. The cages were cleaned for feeding the rats. Bottles were
used for water from which the rats could take fresh water throughout the
days
3. Assay period:

4 week period

The formula should contain the following nutrients.

Nutrients
Amount %
Carbohydrates
60
Protein
20
Vitamin(pre-mix)
5
Mineral
5
Fat
10
The ingredients were mixed and this is in the experimental diet.
[Vitamin pre-mix]
Vitamin
Vitamin-A
Vitamin-D
Vitamin-E
Vitamin-K

Amount
2000IU
2000IU
10IU
0.5mg

Thiamin
Riboflavin
Vitamin B6
Pantotenate
Niacin
Inocitol
PABA
Biotin
Folic acid
Vitamin B12
Choline chloride

0.5mg
0.8mg
0.5mg
4mg
10mg
10mg
10mg
40mg
0.2mg
3 Mcg
200 mg

[Mineral pre-mix]
Mineral
Calcium carbonate
Cobalt chloride
Cupric sulphate
Ferrus suphate
Magnesium sulphate
Potassium Iodide
Potassium phosphate

Amount
38.14 g
0.023 g
0.047 g
0.4 g
0.4
0.079 g
38.9 g

Dry and grind to a find powder grind in a motor a portion of Nacl2 with
potassium iodine. Grind the remaining of Nacl2 and the other salt. Finally add
potassium iodine sodium chloride mixture and store in a cool dry place.
4. Feeding the rats:
The rats were feed for 30 days with the control and experimental diets to
control and other groups respectively.
PER = Weight gain / Mean Protein intake

Composition of the rat food

Diet

Rice(g)

Protein(g)

Oil(g)
250

Vitamin
tablet(g)
125

Mineral(g
)
125

Control(actua
l)
% content
Experiment
% content

1500

500

60
1500
60

20
500
20

10
250
10

5
125
5

5
1255
5

Nutrient content of rat food

Group

CHO(g)

Protein(g)

Fat(g)

Control
Experimen
t

46.92
34.18

20
28.64

10.3
13.9

Vitamin (no.
of tablet)
5
5

Mineral (no.
of tablet)
5
5

Data recorded: Control

Rat
Number
C1
C2
C3

Food
intake(g
)
340.1
220.5
216.2

Protein
Initial
Final
Gained
content(g weight(g weight(g weight(g
)
)
)
)
68.02
35.8
107
71.2
44.1
40.4
114.5
74.1
43.24
39.7
108
68.3
Data recorded: Experiment

Rat
Number
E1
E2
E3

Food
intake(g
)
280.1
289.9
252.8

Protein
Initial
Final
Gained
content(g weight(g weight(g weight(g
)
)
)
)
56.02
36.4
85.8
49.4
57.98
51.9
96
44.1
50.56
32.2
75.4
43.2

Mean value of PER study

Group
Control
Experiment

Weight gain(g)
71.2
45.6

Protein intake(g)
51.8
54.9

PER
1.38
0.830

Ex. No:
Date:
Estimation of Vitamin-A in liver
Aim
To estimate the amount of vitamin-A in goat liver
Principle
The colour produced by vitamin-A, its acetate or palmitate with trichloro
acetate is proportional intensity to its concentration property is used for
spectrophotometric estimation.
Reagents
All reagents are prepared fresh. Exposure of sample and reagent to light are
avoided all times.
1. Alcohol KOH
1g of KOH dissolved in 10ml of alcohol.
2. Chloroform TCA Reagent
15g of TCA crystals are dissolved in 25ml of alcohol free chloroform
and stored in dark.
3. Petroleum Ether
4. Stock Standard
1.5 g vitamin A palmitate in 100ml of chloroform.
5. Working Standard
1 in 10 dilution, i.e., 10 ml of the stock is made up to 100ml with
chloroform.
Procedure
Extraction of vitamin-A from goat liver:
Incubated liver slices were homogenized to 1ml of homogenate and added
1ml of saponification mixture (2N KOH in 90% alcohol). Then the tubes were
gently reflexed for 20 minutes at 60C. The tubes were cooled at room
temperature. Added 20 ml of water and mixed well.
Vitamin-A was extracted from liver with 10ml portion of petroleum ether
(42-60C), pooled the extract and washed thoroughly with water. Separate the

layers using the separating funnel, added sodium sulphate (anhydrous) to remove
the moisture. 1.0 ml of ether extract was then taken and evaporated to dryness at
60C. The dry tissue was dissolved in 1ml of chloroform.
Assay:
Aliquots of standard were pipette out into a series of clean dry test tubes in
the concentration range of 20-100g. The volumes in all test tubes are made up to
1ml with chloroform. From the fast delivering pipette added 2ml of TCA reagent
rapidly mixing with the contents of the tube. The procedure was repeated for the
sample and it is read against blank at 620nm.

Estimation of Vitamin A in liver

Volume
of
Solution
(ml)
Blank
Standard

Sample

Volume of
working
standard
(ml)
0.2
0.4
0.6
0.8
1.0
1.0

Con.
(g)
30
60
90
120
150
-

Volume of
Volume of
O.D.
chloroform TCA reagent Value
(ml)
(ml)
(620nm)
1.0
0.8
0.6
0.4
0.2
0.0
-

CALCULATION
1ml of homogenate corresponds to
g.
Therefore 10ml of homogenate corresponds to ______10
=
g
i.e., 1.0g of liver contains
=
g.
RESULT
The amount of vitamin-A was found to be = ________ g.

2
2
2
2
2
2
2

Ex.No:
Date:

Introducing thiamine deficiency in pigeon

Aim:
1. To introduce thiamine deficiency in pigeons
2. To observe the clinical manifestations of the deficiency
3. To see the recovery by injecting by B1 hydrochloride solution.
Theory:
The prime role that thiamine plays in the body is its co-enzyme function in the
release of energy during carbohydrate metabolism. Thiamine is known to be a part
of the co-enzyme thiamine pyrophosphate made in the tissue cells by the
combination of B1 with phosphate group. Experimental work with pigeon had
revealed that in addition to its functions in general metabolism thiamine may also
have a specific role in the functioning of peripheral nerves.
In the experimental animals, B1 deficiency symptoms appear earlier (36days) after withholding the vitamin. All the deficient animals show poor growth.
Nerves symptoms and death occurs in severe cases. Pigeons and fowls developed a
severe and characteristic head retraction, vomiting and dizziness. Rats show
reduced growth, convulsions, slowing of heart beat and loss of appetite. Thiamine
is necessary for normal fertility and lactation in experimental animals. It is
interesting to know that the recovery of the deficiency can occur within few
minutes after intramuscular administration of the vitamin. If untreated the bird
might die. The most common method to induce beriberi in pigeons is to feed
double polished rice, although other food stuffs or mixture of related substances
are available for that purpose.
Procedure:
Young healthy pigeons were caged in separate cages, one as the control
and the other as the experimental bird. They were placed in an area free of
infections. Pigeons were chosen because B1deficiency could be readily induced in
these birds. The control bird was fed with the combination of cereals and pulses.
The experimental bird was fed only with double polish rice. The polished rice was
washed in water several times till the water was clear. Then it was dried and stored
for feeding. Both the control and experimental pigeons were weighed once in a
week and water and food were provided daily for the pigeon adlibtium.

Maintaining record:
The pigeons were weighed once in a week and the weights were
recorded in a tabular column. The pigeons were carefully observed and a
descriptive record was maintained. The experimental bird showed symptoms of
deficiency.
Observation:
To introduce thiamine deficiency in pigeons, the experimental and control pigeons
were fed for 28 days. The control bird was fed with bajra and the experimental bird
was fed with double washed, dried polished raw rice. The experimental pigeon
developed the symptoms of thiamine deficiency such as shivering, restlessness,
and even showed a slight head retraction towards the end of the study. In the
process of the study the experimental bird developed diarrheal symptoms also. The
bird developed fatique and weakness towards the 20th day of study.

Ex.no:
Date:

Paper Chromatography of Aminoacid

Aim
To determine the RF value of the known amino acid and to identify the amino acid
in given mixture.
Principle
Chromatography is a simple technique used for the separation of closely
related compounds in a mixture. The separation is effected by differences in the
equilibrium distribution of the components between two immiscible phases (i.e.)
the stationery and the mobile phase. These differences in the equilibrium
distribution are the result of the nature and the degree of interaction of optic
components with these two phases. The stationery phase is a porous medium to
which the sample mixture percolates under the influence of a moving solvent
(mobile phase).
Reagents
1. Solvent Mixture:
N-butanol 80ml, acidic acid 20ml, distilled water 100ml. these are
poured into a separating funnel and left side for layer separation. When the 2 layers
are separated it can be used.
2.0.1% Ninhydrin:
Weigh accurately 0.1g of Ninhydrin and dissolve in 100ml of acetone.
Procedure
The chromatographic paper used for this experiment is Wattman No.1 filter
paper. The solvent mixture ( CH3COOH, N-butanol) is put in a separating funnel
for phase separation. The upper layer is used to irrigate the chromatography paper
and the lover layer to saturate the chamber. The chromatographic chamber consists
of a glass trough and a bell jar. The lower phase is kept in the trough with in the
chamber to saturate it. The filter paper is measured according to the size of the bell
jar. The length of the filter paper is taken as 2cm greater than the circumference of
the bell jar. The height or the width is taken as 2cm greater than height of the bell
jar. A line is marked 1.5cm above the lower edge of the filter paper at a distance of
3cm; 10l of the known amino acid is spotted on the paper. The last 2 spots will be
that of the amino acid mixture. The filter paper is rolled in to a cylinder and the

edges are loosely stitched without touching each other. The filter paper is placed in
the petridish containing the upper layer of the solvent mixer. The solvent is allowed
to run until the solvent front reaches 0.5cm below the upper edge of the filter
paper. The filter paper is air dried at room temperature by suspending it from the
line with the clip. When it is completely dried it is sprayed with the 0.1% solution
of
Ninhydrin in acetone and dried in a hot air oven at 100 0c for few
minutes. Violet coloured spots that developed are marked at their centre and the
distance travelled by each amino acid is noted. Also the distance travelled by the
solvent is noted.
RF value of the amino acid =
Distance travelled by AA (cms)
Distance travelled by solvent (cm)
The amino acids in the mixture are identified with the RF value of the known
amino acid.
Determination of Unknown Amino Acid
Distance
Distance
S.n Amino acid
travelled by
travelled by
RF Values
Standard
o
solvent (cm)
Amino acid(cm)
Values
1.

Arginine

2.

Cysteine

3.

Tyrosine

4.

tryptophan

The unknown amino acids may be arginine, tyrosine, tryptophan, and cysteine
because their RF values are close to the known amino acids.

Ex.no:
Date:

Electrophoretic separation of serum

protein by electrophoresis

Aim
To separate the serum proteins by paper electrophoresis.
Principle
When a protein molecule is placed in a solution at a P H alkaline to its isoelectric
print. The protein molecule acquires a net charge. When a mixture of protein is
placed in a buffer solution that protein species with isoelectric point farthest from
the PH of buffer until acquire greatest charge until the molecular size and shape are
the contributing factors.
In a mixture of protein similar in size and shapes the species with the greatest
net charge will have fast and therefore, will be farthest in the electric field. Serum
proteins are usually analyzed at PH 8.6 and at this PH all the serum protein are
negatively charged. Albumin has the greatest magnitude of charge and it therefore
moves the greatest distance in a given length of time. It is followed in order by 1,
2, , globulin.
Reagents
Buffer: ( PH 8.6, Ion strength 0.05).
1.84g of barbitone and 10.3g of sodium barbitone in 1 litre of distilled
water. Check the PH with PH paper.
Dye (Bromophenol blue):
0.1g of the dye and 1g of mercuric chloride are dissolved in 100ml of 2%
acetic acid. Wash the solution in 5% acetic acid.
Fixative Solution:
5% CH3COOH containing 0.3% sodium acetate.
Procedure
Wattman no.1 filter paper was cut into strips of 439 cms. Moistened with
buffer and removed the excess buffer by blotting between the filter papers.

Poured the buffer into compartments of the tank to a uniform level. The paper
strips were kept in a position on the track with 2 inches dipping into the buffer.
Sealed the tank and switched on the current and allowed and equilibrate for 15
minutes with a current of 1.5amp per strip. Turn of the current serum (0.6) was
applied on the paper; using pen sealed the tank, turned off the current and allowed
to run for 5 hours. The current was turned off, removed the strip and air dried
them. The strips were then stained with bromphenol blue dye for 1 hour and
washed off the excess dye with 5 % acetic acid and finally put into fixative
solution for 6minutes, air dried and finally dried for 15 minutes at 1100c.
Electron:
Cut the bonds and placed them in a different marked tubes. A similar blank
strip was cut and placed in a sixth tube. Added 5ml of NaOH solution to each and
shook till the colour was removed from the strip. Left for half an hour. Read at
540nm.
Result
The ratio of albumin: globulin is =

Ex.No:
Date:

High Performance Liquid Chromatography

(HPLC) instrument name: Waters

Introduction
Chromatography is the term used to describe a separationtechnique in which a
mobile phase carrying a mixture is caused to move in contact with a selectively
absorbent stationary phase. There are a number of different kinds of
chromatography, which differ in the mobile and the stationary phase used.
High Performance Liquid Chromatography (HPLC) is one mode of
chromatography, one of the most used analytical techniques. Chromatographic
process can be defined as separation technique involving mass-transfer between
stationary and mobile phase. HPLC utilizes a liquid mobile phase to separate the
components of a mixture. The stationary phase can be a liquid or a solid phase.
These components are first dissolved in a solvent, and then forced to flow through
a chromatographic column under a high pressure. In the column, the mixture
separates into its components. The amount of resolution is important, and is
dependent upon the extent of interaction between the solute components and the
stationary phase. The stationary phase is defined as the immobile packing material
in the column. The interaction of the solute with mobile and stationary phases can
be manipulated through different choices of both solvents and stationary phases. As
a result, HPLC acquires a high degree of versatility not found in other
chromatographic systems and it has the ability to easily separate a wide variety of
chemical mixtures.
Principle
HPLC separates mixture of compounds on the basis of polarity.
Polarity refers to: the greater the difference in electron affinity i.e. electro
negativity between atoms in a covalent bond, the more polar the bond. Partial
negative charges are found on the most electronegative atoms, the others are
partially positive. The molecular electrostatic potential energy of a hydrogen ion at
a particular location near a molecule. Negative electrostatic potential corresponds
to: a partial negative charge, Positive electrostatic potential corresponds to: partial
positive charges.

It is used to analyze density, purify and quantify compounds. It has a mobile

phase, a stationary phase, and detector. The mobile phase is continuously pumped
at a fixed flow rate through the system and mixed by the pump. The injector is
used to introduce a plug of a sample into the mobile phase without having to stop
the mobile phase flow, & without introducing air into the system. The mixture of
components is carried in a narrow band to the top of the column. Some compounds
in the sample mixture will have greater preference for stationary phase than the
mobile phase and will be retained in the column longer.

STANDARD OPERATING PROCDURE FOR HPLC

Empower project

Configuration system

Project

New Project

Next

Next

Next

Next

Project name

Finish

Browse project

Ok

Run samples

INSTRUMENT METHOD
Edit in toolbar

Instrument method

W 600

Flow

Solvents

W717

Temperature

W2489

Lamp on

Save as

Close

Edit

New method set

Yes

Selected created an instrument method

Next

Next

Finish

Close

Give the samples name and their profiles

Select your project from instrument method

Monitor

Check the straight base line

Run

Processing method

Click on the particular chromatogram

Review

Processing method wizard

Click creating a new in default

Process type--LC

Integration algorithm--Traditional

Use processing method

Ok

3. PROJECT

Injection or channels

Right click

Review


Processing method wizard

Ok

Processing type--LC

Integration algorithm --Traditional

Ok

Peak width

Next

Threshold

Next

Start end of next

Selected the peak bottom

Next

Next

Next

Yes or no

Enter the peak name

Method name

Finish

File

Save as


Processing method

Project name

Ok

File

Save

All

Update

Reporting method

Select required line

Preview publisher

Use the following report method

Ok
Application
HPLC is used for chemistry and biochemistry research analyzing complex
mixtures, purifying chemical compounds, developing processes for synthesizing
chemical compounds, isolating natural products, or predicting physical properties.
It is also used in quality control to ensure the purity of raw materials, to control and
improve process yields, to quantify assays of final products, or to evaluate product
stability and monitor degradation. In addition, it is used for analyzing air and water
pollutants, for monitoring materials that may jeopardize occupational safety or
health, and for monitoring pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug products, for identifying
confiscated narcotics or to check for adherence to label claims.

Ex. No:
Date:

Gas Chromatography with mass

Spectrophotometer (GCMS)
Instrument Model: shimadzu gcms-qp2010

Introduction
In the simplest terms the GC/MS instrument represents a device that
separates chemical mixtures (the GC component) and a very sensitive detector
(the MS component) with a data collector (the computer component).
Working Principle
Both gas chromatography (GC) and mass spectrometry (MS) bring
something to their union. GC can separate volatile and semi volatile compounds
with great resolution, but it cannot identify them. MS can provide detailed
structural information on most compounds such that they can be exactly identified,
but it cannot readily separate them. Therefore, it was not surprising that the
combination of the two techniques was suggested shortly after the development of
GC. It is one of the most accurate tools for analyzing environmental samples. The
GC works on the principle that a mixture will separate into individual substances
when heated. The heated gases are carried through a column with an inert gas
(such as helium). As the separated substances emerge from the column opening,
they flow into the MS. Mass spectrometry identifies compounds by the mass of the
analyst molecule. A library of known mass spectra, covering several thousand
compounds, is stored on a computer. Mass spectrometry is considered the only
definitive analytical detector.

STANDARD OPERATING PROCEDURE FOR GCMSANALYSIS

He gas cylinder open

Check the gas leakage

Switch on
GC on
MS on
PC-on

Go to Real time analyzer

Click to instrument in menu bar

Click to vacuum control

Click to Auto start

Wait for 20 minutes for vacuum pump ready - completed

Close

GC&MS Ready

Go to file

Select open method file

Select desired method file

Go to Acquisition

Download initial parameter

Wait about 3 hours for GC&MS Ready

Go to tuning

Go to peak view monitor

Check water

Filament on

Check the peak ratio is must be 18>28> 32

Filament off

Check Leak 69

PFTBA on

Filament on

Check the peak ratio is must be 69>18>28

Filament off

PFTBA off

Go to start auto tuning

Wait 2 minutes for auto tuning completion

Check the peak profile and spectrum

Spectrum base peak is must be a 69

Save in tuning file as from file

Close tuning software


Go to sample log in from assistant bar

Fill up the sample profile

Ok

Standby

Wait for GC&MS Ready

Sample inject exactly 1 micro liter

Click to Start

Wait for analyzing chromatogram

Go to GCMS Post run analysis after completed a programmer time and got a
Chromatogram

Go to view

Click Data explorer

Drag the analyzing data

Right click on the chromatogram

Click to peak integrate (TIC all group)

Click Auto to give desired points

Right click on the chromatogram

Click to Show Qualitative table

Edit

Select all

Edit

Register to the spectrum compound table

Go to spectrum process

Click to similarity search

Double click on the name table

Copy marked compound name

Close

Right click to edit compound name

Copy

Ok

Go to TIC

Right click on the name table

Click to edit compound name

Paste

Ok

Close

Save

Go to print report

Double click the report format

Go to data explorer


Drag the data file

Go to print preview

Print

Ok

Get result

EX.NO:
DATE:
ESTIMATION OF SERUM BILIRUBIN
(METHOD OF HALLOY AND EVILYN, 1937)
AIM:
To estimate the serum bilirubin of the given sample.
THEORY:
Serum is diluted with water and methanol, added in an amount sufficient to
precipitate the protein, yet sufficient to ensure that all bilirubin reacts with the
diazo reagent.
REAGENTS:
1. ABSOLUTE METHANOL
2. HCL 15%:
Volume by volume with water.
3. DIAZO REAGENT SOLUTION-A:
1g of sulphanilic acid in 15ml of concentrated Hcl and make up to 1l
with water.
4. DIAZO REAGENT SOLUTION-B:
Dissolve 0.5g of sodium nitrate and water and make up to 100ml
water. 0.3ml of solution B is mixed with 100ml of solution A.
5. STOCK STANDARD:
10mg of bilirubin or 100ml of chloroform.

6. WORKING STANDARD:
1 in 5 dilutions made with methanol. I.e., 0.02mg or 20/ml.

PROCEDURE:
Two tubes are taken and into each tube-0.2ml of serum and 1.8ml of water
are added. To the unknown added 0.5ml of azo reagent and a (blank for sample)
control 0.5ml of 15% Hcl. 2.5ml of methanol is added (to the control and total
bilirubin tubes) and kept for 30min and read at 540nm against water. Subtract
reading of control- blank from that of the working test. For standards, 0.5, 1, 1.5,
2.0ml of working standard are taken and make up to 9ml of methanol. 1ml of diazo
reagent is added and read at 540nm. A blank for standard is prepared with 9ml of
methanol and 1ml of diazo reagent.
DIRECT BILIRUBIN:
In a test tube 0.2ml of serum and 1.8ml of distilled water are taken. Then
0.5ml of diazo reagent is added. Finally instead of methanol 2.5ml of water is
added. Kept for 30min and read against same control (blank for sample) at 540nm.
RESULT:
The serum bilirubin of given sample was found to be
(i)
(ii)

Direct =
Total =

mg%
mg%

ESTIMATION OF SERUM BILIRUBIN

S. Volume
No of soln
(ml)

1
2

Blank
Standard
0.5
1.0
1.5
2.0
Test
Sample
Control
(0.2)
Total
(0.2)
Direct
(0.2)

Con Volume
c
of
distilled
Water
(ml)
-

Diazo Volum Methanol Distille O.d

reage e of hcl
(ml)
d water value
nt
(ml)
(ml)
(ml)
1

9.0

10
20
30
40

1
1
1
1

8.5
8.0
7.5
7.0

1.8

0.5

2.5

1.8

0.5

2.5

1.8

0.5

0.5

Calculation:
From the graph, concentration of bilirubin (direct) =
0.2ml of serum contains
=
Therefore, 100ml of serum contains
=

g
g of bilirubin (direct)
x
x 100

0.2

1000
=

(direct)
For total bilirubin = (Total-control)
0.2ml of serum contains
Therefore, 100ml of serum contains
0.2

=
=
=

g
y

x 100

1000
=

(total)

mg% of bilirubin

mg% of bilirubin

Ex.no:
Date:

Estimation of Icetric Index

Aim:
To estimate icetric index of the given sample.
Theory:
The icetric index measures the degree of jaundice by measuring the intensity
of yellow colour of serum. The normal range is from 4-6. Clinically obvious
jaundice is present when it is above 15. It is generally 1/3 rd of serum bilirubin
expressed in m/l. Sometimes icetric index is raised without an increase in serum
bilirubin.
Reagents:
1. Stock Dichromate Standard:
0.1g of potassium dichromate is dissolved in water and 0.1ml of
concentrated H2SO4 is added and made up to 100ml. This has icetric index of 10.
2. Working Standard:
Dilute 1ml in100ml (icetric index).
3. 0.9% Saline:
Procedure:
Icetric Index
1ml of serum is diluted with working standard till the colour matches with
working standard. Both are read at 500nm against water.
Result:
The icetric index of the given sample of serum is
Calculation:
Reading of unknown =
Reading of standard =
Icetric index

= Reading of unknown 10 dilution factor

Reading of standard

Dilutionfactor =Volume of serum

Volume of serum+ saline

Ex. no:
Date:

Estimation of Malondialdehyde (T BARS)

(Sadasivadu et al., 1997)

Principle:
In this method malanodialdehyde and thiobarbituric acid reactive substances
(T BARS) were measured by their reactivity with thiobarbituric acid in the acidic
condition to generate a pink colored chromophore which is read at
spectrophotometer at 530nm.
Reagents
1) Trichloroacetic acid (TCA) (40%)
40gm of TCA in 100ml of distilled water
2) Thiobarbituric acid (TBA) (0.67%)
0.67% of thiobarbituric acid in 100ml of hot or warm water. (TBA not
dissolved in cold water)
Procedure
0.5 ml of serum, 0.5 ml of 40% TCA and 1.0ml of 0.67% TBA was added
one after the other in a boiling water bath for 10 mins centrifuged at 6000 rpm for
15 mins and read the supernatant at 530nm
Note: Use double the volume of serum, TCA &TBA for reading in the
spectrophotometer otherwise the quantity may be very less and cannot read in the
spectrophotometer.
Calculation:
E= kCL
C= E/KL=. nmol/dl

K= Molar extinction coefficient (extinction offered by 1M solution), i.e., 1.5105

E= Extinction/absorbance
C= Concentration in moles/liter
L= Length of cuvette used (1cm)

Ex.No:
Date:

Estimation of Erythrocyte Glutathione (GSH)

(Beutler, 1975)

Principle
Virtually all the non protein sulphydryl groups of erythrocytes are in the
forms of reduced glutathione. 5, 5 dithiobis 2-nitrobenzoic acid (DTNB) is
disulfide chromogen that is readily reduced by sulfydryl compounds to an intensely
yellow compound. The absorbance of the reduced chromogen is measured to the
GSH concentration.
Reagents
1. Precipitating Solution:
Place in a 100 ml volumetric flask 1.67 g of glacial meta phosphoric
acid.0.20 g of disodium or di potassium ethylene di amine tetra acetic acid
(EDTA), and 30 g of sodium chloride and bring to volume with distilled water.
This solution is stable for 3 weeks at 4 C. A fine precipitate may form owing to
EDTA, but this does not interfere with the test.
2. Phosphate solution: 0.30 mol/l
In a 1 liter of volumetric flask, place 42.5 g of Na2 H Po4 and bring to volume
with distilled water. This solution is stable indefinitely at 4 C. if crystals form,
dissolve by heating.
3. DTNB Reagent: 40 mg of DTNB in 100 ml of 1% sodium citrate
4. GSH Standards (50 mg %)
Procedure
1. Place 0.2 ml of whole blood into a 10 ml of test tube and add 1.8 ml of
distilled water mix to haemolyse.
2. Promptly add 3.0 ml of precipitating solution and mix.
3. Allow to stand for 5 minutes at room temperature and then filter through
coarse grade filter paper.
4. Prepare these mixtures for each sample.
Reagents

Blank(ml)

Assay (ml)

Filtrate

2.0

Precipitating solution
Water

1.2
0.8

Phosphate solution

8.0

8.0

DTNB Reagents

1.0

1.0

5.
6.
7.
8.
9.

Invert three times to mix.

Read absorbance at 412 nm within 4 min of preparing the mixtures.
Measure the heamatocrite of the original whole blood specimen.
Assay the GSH standard, omitting the filtration step.
Graph the standard, curve determine GSH concentration of blood specimen
from the graph

10.Calculate GSH concentration GSH, mg/dl of erythrocytes

GSH Concentration (From standard curve)
Hematocrit

Ex. no:
Date:

Estimation of superoxide dismutase

(Marklund & Marklund method (1974) modified by Nandhi
et
al., 1988)

Principle
This method utilizes the inhibition of auto oxidation of pyrogallol by
superoxide dismutase.
Reagents
1) Tris Hcl buffer (0.1M)
pH adjusted to 8.6 using Hcl.
3.027g of Tris + 0.186g of EDTA + 50mM Hcl (0.05ml conc. Hcl in 10 ml
of distilled water) + make up to 500 ml with distilled water.
2) Pyrogallol (20mM concentration)
25mg of pyrogallol in 10ml of distilled water
Procedure
For control
To 2.9 ml of Tris buffer, 0.1 ml of pyrogallol solution was added and mixed;
and reading was taken at 420nm exactly after 1min 30 sec and 3 mins 30 sec. The
absorbance per 2 mins was recorded and the concentration of pyrogallol was
adjusted so that the rate of change of absorbance per min was approximately
0.020- 0.023 nm.
For sample
To 2.8 ml of Tris buffer, 0.1 ml of sample was added and mixed. The
reaction was started by adding 0.1 ml of adjusted pyrogallol solution (as per
control). Reading was taken at 420 nm exactly after 1min 30sec and 3 mins 30 sec
and the above absorbance was recorded per 2 min.

Ex .no:
Date:

Activity of alkaline phosphatase

Fiskes Subarrow method

Aim
To determine the activity of alkaline phosphatase by Fiskes subarrow
method.
Principle
Phosphatase is the enzymes catalyze splitting of phosphoric acid from
certain mono phosphoric acid clusters, of considerable importance in several body
processes. Two types of phosphatases are commonly estimated.
Alkaline phosphatase shows maximum activity at pH 10.The method used is
that in which enzyme catalyzes the hydrolyses of phosphate esters to phosphoric
acid and alcohol. Amount of phosphoric acid produced during hydrolyses is a
measure of enzyme activity. The liberated phosphoric acid containing inorganic
phosphate is estimated by Fiskes subarrow method.
Reagents
1. Buffered substrate 0.01M: Dissolve 1.075g of sodium betaglycerophosphate
in 500ml of alkaline buffer.
2. Alkaline buffer- pH 10
Solution A 0.2M anhydrous sodium carbonate (21.2g per litre)
Solution B 0.2M sodium bicarbonate (16.8g per litre)
Mix 27.5 ml of A and 22.5 ml of B to 200ml of distilled water.
3. TCA 10 %
4. Acid molybdate: Dissolve 5g of ammonium molybdate in 100ml of 5N
sulphuric acid.
5. ANSA: Dissolve 30g of sodium meta bisulphide, 6g of sodium sulphide and
500g of ANSA, separately in small quantities of water. Combine all solution
and made up to 250ml of water.
6. Stock standard: Dissolve 2.194g of pure kH2PO4 in water. Volume was made
up to 500ml with water.
7. Working standard: 1 in 250 dilution.
Procedure:
6ml of buffered substrate was added to test and placed in water bath for 37 c
for 2 min. 0.3ml of serum was added to it and incubate at 37c for 15min. Tubes
were removed and 1.2ml of TCA was added. 5ml of filtrate will act as test.
To control, 6ml of water, 0.3ml of serum and 2ml of 10% TCA was added
and treated above. 5ml of filtrate will act as control. Standard ranging from 4-

20g concentration was added and value was made up to 5ml with water. To all
the tubes 0.8ml of acid molybdate, 0.2ml of ANSA were added. Read the colour
developed against reagent blank at 660nm after 10min.
Result:
The activity of alkaline phosphatase in serum was found to be

Estimation of serum alkaline phosphatase

S.
No

Volume
of
solution
(ml)

Concen Volume
tration of
(g)
distilled
water
(ml)

Volume
of buffer
substrate
(ml)

Volume Volume of Volume O.D.

of TCA ammoniu of ANSA Value
(ml)
m
(ml)
Molybdat
e (ml)

1.

Blank

0.3

1.2

0.8

0.2

2.

Control
0.3ml
serum

1.2

0.8

0.2

3.

Standar
d
5.0

0.04

1.2

0.8

0.2

1.2

0.8

0.2

4.
Sample
0.3ml
serum
Calculation
mg of organic phosphorous / 100ml
O.D. of control

concentration of standard
*

O.D. of standard

* 100
volume of serum (0.2)

Concentration of standard = 8 /ml

5ml contains =
Units of alkaline phosphatase/100ml
= O.D. of experiment O.D. of control O.D. of blank
*
O.D. of standard
Concentration of standard
*3
Volume of serum

Ex.no:
Date:

units/mL.

Activity of acid phosphatase

Fiskes Subarrow method

Aim
To determine the activity of acid phosphatase by Fiskes Subbarow method.
Principle
The enzyme catalyses the hydrolysis of phosphate ester to phosphoric acid
and alcohol. The liberated phosphoric acid is the measure of enzyme activity. The
liberated phosphoric acid containing inorganic phosphate is estimated by Fiskes
Subarrow method.
Reagents
1. Citrate buffer :
Solution A: 0.1M of citrate (21.01g per litre)
Solution B: 0.1M of sodium citrate (29.41g per litre)
20.5ml of solution A and 29.5ml of solution B is diluted to 100ml.
2. Buffer substrate: 0.1 M of sodium beta glycerophosphate 2.16g dissolved
in 100ml of citrate buffer.
3. TCA 10 %
4. Acid molybdate: Dissolve 5g of ammonium molybdate in 100ml of 5N
sulphuric acid.
5. ANSA: Dissolve 30g of sodium meta bisulphide, 6g of sodium sulphide and
500g of ANSA, separately in small quantities of water. Combine all solution
and made up to 250ml of water.
6. Stock standard: Dissolve 35g of pure KH 2PO4 and add 10ml of 1N sulphuric
acid and made upto 100ml with distilled water.
7. Working standard: 1 in 10 dilution.
Procedure
To the test and control tubes, 2ml of buffered substrate was added and placed
in a water bath at 37C for 15 min. To test, 0.1ml of serum was added and
incubated for an hour. Then 1ml of 10% TCA was added to control and test
tubes. Tubes were then centrifuged; 1ml of supernatant was taken with 1ml of
molybdate and 0.4ml of ANSA. Final volume is made up to 1ml with distilled
water. In a series of test tubes standard concentration solution of 8-40g was
added and tested as above. The colour was read at 660nm after 20 minutes. The
enzyme activity was determined from standard graph drawn by plotting
standard concentration along x axis and O.D along y axis.
Result
The activity of acid phosphatase in serum was found to be

Estimation of serum acid phosphatase

S. Volume of
No solution (ml)

1.
2.

3.

Blank
Standard
1.0
2.0
3.0
4.0
5.0
Sample
Test (1ml)
Control (1ml)

Concentr Volume
ation
of
(g)
distilled
water
(ml)
8.6

Volume of Volume of
Molybdate ANSA
(ml)
1.0

0.4

8
16
24
32
40

7.6
6.6
5.6
4.6
3.6

1.0
1.0
1.0
1.0
1.0

0.4
0.4
0.4
0.4
0.4

7.6
7.6

1.0
1.0

0.4
0.4

Calculation:
1ml of supernatant contains
So 3.1ml of supernatant contains
Therefore 0.1ml of serum has
100ml of serum contains
=
Enzyme activity =
Ex.No:
Date:

g of enzyme

g of enzyme

/0.1 * 100/1000
mg of enzyme
*1.77
IU/dL
Estimation of serum phospholipids
method of connerty,briggs&faton

O.D.
Value

Aim:
To estimate the amount of serum phospholipids present in a given sample.
Principle
The technique most by used for phospholipids has been to extract them in
suitable solvent for example ethanol, ether and digest an aliquotes of extract with
sulphuric acid and hydrogenproxide to oxidize the phosphorus through the
inorganic phosphate and determine this by one of the method used to determine
serum inorganic phosphate.
A different approach is that of Zilversain and Davis (1950) who precipitated
protein with TCA and digested the precipitate which contains a phospholipid with
sulphuric acid + perchloric acid mixture.
Reagents:
1.TCA
50g of TCA in a litre of water.
2. Digestion Mixture:
50ml of water, 25ml of conc.sulphuric acid and 25ml of perculoric acid
(specific gravity-1.75).
3. Sodium acetate solution:
500g of sodium acetate (anhydride) in one litre of water.
4. Ammonium molbdate solution:
25g/l of water.
5ParamethylaminePhenol) sulphate (Metol)
1g in 100ml of 3% sodiumsulphite solution.
6. Stock standard solution:
4.39g of anhydrous pottassiumdihyhydrogen phosphate (KH2PO4) dissolved IN
2ml of conc.sulphuric acid and made up to a litre.
7. Working standard solution:
Dilute 1ml of stock standard to 250ml with water.
Procedure:
Pipette out 0.2ml of serum into a clean dry centrifuge tube and add 5ml of
TCA drop by drop with shaking and centrifuge the tubes to give a tightly packed
deposit. Decant the supernatant and allowed the tubes to stand inverted over the
filter paper .Wipe around the inner side of the rim and add 1ml of digestion
mixture and heat gently until the liquid becomes colorless. Allow to cool and
carefully add 1ml of water and boil for 10 minutes to convert pyrophosphate to
orthophosphate. Add 1ml of sodium acetate and make it up to 10ml with distilled
water and then add 1ml of molybdate and 1ml of metol. Mix well and allow to

stand for 15 minutes and read at 720nm against the blank prepared by mixing 025ml of conc.sulphuric acid, 1ml of sodiumacetate, 1ml of molybtate, 1ml of metol
and 8.75ml of water.The standard take 5ml of working standard solution containing
0.25ml of conc.sulphuric acid 1ml of molybdate,1ml of sodium acetate and 1ml of
metol. Make up the final volume to 12ml by adding water. The color remains stable
for few hours.
Result:
The amount of phospholipids present in 100ml of serum =
Estimation of serum phospholipids
S.n Volume of Concentrati Volume
Volume
o
solution(ml on(mcg)
of
of sodium
)
ammoniu acetate(m
m
l)
molybdat
e I (ml)
1
Blank
1
1
2
Standard
1
8
1
1
2
16
1
1
3
24
1
1
4
32
1
1
5
40
1
1
3
Samples
2.5
_
1
1
2.5
_
1
1

Volume of
distilled(m
l)

Volume
of metol
(ml)

O.D
valu
e

0.00

8
7
6
5
4

1
1
1
1
1

6.5
6.5

1
1

Calculation:
From the graph the O.D value

corresponds to

(i.e.) 2.5ml of the filtrate contains=

=
=
of phosphorus

of phosphorus
25/2.5

25ml of filtrate is made from 2ml of serum

of phosphorus.

(i.e.) 2ml of serum contains

=
A 1 in 10 dilution was made to take the the reading as colour developed was too
dark.
Therefore 2ml of serum containing =
Therefore 100ml of serum contains =
=
mg of phosphorus.
Phospholipids content=

of phosphorus.

mg of phosphorus.