1

LAB MANUAL FOR BIOCHEMSRTY FOR PTU

STUDENTS

1. Preparation of standard buffers (citrate, phosphate and carbonate) and measurement of

pH.

2. Titration curve for amino acids.

3. Separation of amino acids by two dimensional paper chromatography and gel

electrophoresis.

4. Separation of lipids by TLC.

5. Separation of serum proteins by electrophoresis on cellulose acetate.

6. Quantitative estimation of amino acids.

7. Quantitative estimation of proteins.

8. Determination of glucose by means of the enzyme glucose oxidase.

9. Enzymatic hydrolysis of glycogen by alpha- and beta- amylases.

10. Isolation and determination of RNA and DNA.

11. Effect of temperature on the activity of alpha-amylase.

12. Estimation of SGOT, SGPT, Alkaline phosphotase and Bilirubinu in the serum.

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Experiment – 1

Aim : To prepare standard buffer and measure its pH

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 263-266.

Requirement

Apparatus : pH meter

Chemical : Standard buffer tablet, potassium dihydrogen phosphate, disodium hydrogen

phosphate

Theory : Buffers are solutions of weak acids (or bases) together with their conjugate

bases (or acids) which diminish the change in pH which would otherwise occur from

addition of acid or base.

pH = pKa + log [salt] / [acid]

Procedure :

Preparation of standard buffer solution : Stock solutions: (i) Dissolve 14.2g Na2HPO4 in

one litre water (0.1mol/L). (ii) Dissolve 13.6g KH2PO4 in one litre water (0.1mol/L).

Mix the volumes of two solutions given below for 10 ml phosphate buffers of different

pH values.

Na2HPO4 (ml) (0.1mol/L) KH2PO4(ml) (0.1mol/L) pH at 20º C

0.25 9.75 5.29

0.50 9.50 5.59

1.00 9.00 5.91

2.00 8.00 6.24

3.00 7.00 6.47

4.00 6.00 6.64

5.00 5.00 6.81

6.00 4.00 6.98

7.00 3.00 7.17

8.00 2.00 7.38

9.00 1.00 7.73

9.50 0.50 8.04

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Measurement of pH :

pH meter is put on and allowed to warm.

The pointer is adjusted to 0 mV or 7.0 pH position by zero control.

Standard buffer solution is taken in beaker. Its temperature is noted and electrodes

are dipped in solution.

The meter will show the pH of buffer. Set the pointer at exact value of buffer.

Bring selector switch again to zero and clean electrodes with water.

Now take the pH of unknown solution.

Prepare an appropriate buffer solution by dissolving buffer tablet in distilled water. Place

the temperature probe into standard buffer solution. Insert electrode assembly in it and

adjust the buffer until the meter reading agrees with known pH of buffer. Remove the

electrode assembly, rinse and place it in second buffer solution. Then remove the

electrode and rinse with distilled water and place again in first solution to confirm the

calibration.

Result : pH is noted from pH meter.

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Experiment – 2

Aim : To perform identification test for a given sample of carbohydrate.

Reference : Satyanarayan U., Chakrapani U., “Biochemistry”, Uppola , 3rd

edition, 16-

17.

Requirement

Apparatus : Test tube, water bath, holder.

Chemical : Molisch’ reagent, Fehling’s solution, Benedict’s solution, conc. sulphuric

acid, picric acid, sodium carbonate, phenyl hydrazone, sodium acetate, Barford’s

solution, sample solution.

Theory :

Molisch's Test (named after Austrian botanist Hans Molisch) is a sensitive chemical test

for the presence of carbohydrates, based on the dehydration of the carbohydrate by

sulfuric acid to produce an aldehyde, which condenses with two molecules of phenol

(usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also give colored

products) resulting in a red- or purple-colored compound.

The Iodine test is used to test for the presence of starch. Iodine solution — iodine

dissolved in an aqueous solution of potassium iodide — reacts with starch producing a

purple black color.

Benedict's reagent is used as a test for the presence of reducing sugars. This includes all

monosaccharides and the disaccharides, lactose and maltose. Even more generally,

Benedict's test will detect the presence of aldehydes (except aromatic ones), and alpha-

hydroxy-ketones, including those that occur in certain ketoses. Thus, although the ketose

fructose is not.

Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose

sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If

the sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is

an aldose. This test is based on the fact that, when heated, ketoses are more rapidly

dehydrated than aldoses. In concentrated HCl, ketoses undergo dehydration to yield

furfural derivatives more rapidly than do aldoses. These derivatives form complexes with

resorcinol to yield deep red colour.

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Procedure :

Iodine solution: Add a few crystals of iodine to 2% potassium iodide solution till the

colour becomes deep yellow.

Fehling’s reagent A: Dissolve 34.65 g copper sulphate in distilled water and make up to

500 mL.

Fehling’s reagent B: Dissolve 125 g potassium hydroxide and 173 g Rochelle salt

(potassium sodium tartrate) in distilled water and make up to 500 mL.

Benedict’s qualitative reagent: Dissolve 173 g sodium citrate and 100 g sodium

carbonate in about 500 mL water. Heat to dissolve the salts and filter, if necessary.

Dissolve 17.3 g copper sulphate in about 100 mL water and add it to the above solution

with stirring and make up the volume to 1 L with water.

Barfoed’s reagent: Dissolve 24 g copper acetate in 450 mL boiling water. Immediately

add 25 mL of 8.5% lactic acid to the hot solution. Mix well, Cool and dilute to 500 mL.

Seliwanoff’s reagent: Dissolve 0.05 g resorcinol in 100 mL dilute (1:2) hydrochloric

acid.

Bial’s Test: It is specific for pentoses. They get converted to furfural. In the presence of ferric

ion orcinol and furfural condense to yield a coloured product.

The reactions of carbohydrates are given in Table:

Experiment Observation Remarks

Molisch’s Test

Add two drops of Molisch’s

reagent (5% 1-naphthol in

alcohol) to about 2 mL of test

solution and mix well.

Incline the tube and add about

1 ml of concentrated sulphuric

acid along the sides of the

tube.

A red-cum-violet ring appears

at the junction of the two

liquids.

The colour formed is due to

the reaction of alpha-naphthol

with furfural and/or its

derivatives formed by the

dehydration of sugars by

concentrated sulphuric acid.

All carbohydrates react

positively with this reagent.

Iodine Test

Add a few drops of iodine

solution to about 1 mL of the

test solution.

Appearance of deep blue

colour

This indicates the presence of

starch in the solution. The blue

colour is due to the formation

of starch-iodine complex.

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Fehling’s Test

To 1 mL of Fehling’s solution

‘A’, add 1 mL of Fehling’s

solution ‘B’ and a few drops

of the test solution. Boil for a

few minutes.

Formation of yellow or

brownish-red precipitate.

The blue alkaline cupric

hydroxide present in Fehling’s

solution, when heated in the

presence of reducing sugars,

gets reduced to yellow or red

cuprous oxide and it gets

precipitated. Hence, formation

of the coloured precipitate

indicates the presence of

reducing sugars in the test

solution.

Benedict’s Test

To 2 mL of Benedict’s reagent

add five drops of the test

solution. Boil for five minutes

in a water bath. Cool the

solution.

Formation of red, yellow or

green colour/precipitate.

Depending on the sugar

concentration yellow to green

colour is developed.

Barfoed’s Test

To 1 mL of the test solution

add about 2 mL of Barfoed’s

reagent.

Boil it for one minute and

allow to stand for a few

minutes.

Formation of brick-red

precipitate.

Only monosaccharides answer

this test. Since Barfoed’s

reagent is weakly acidic, it is

reduced only by

monosaccharides.

Seliwanoff’s Test

To 2 mL of Seliwanoff’s

reagent add two drops of test

solution and heat the mixture

to just boiling.

Appearance of deep red colour It is a timed colour reaction

specific for ketoses.

Bial’s Test

To 5 mL of Bial’s reagent add

2-3 mL of solution and warm

gently. When bubbles rise to

Appearance of green colour or

precipitate.

It is specific for pentoses.

They get converted to furfural.

In the presence of ferric ion

orcinol and furfural condense

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the surface cool under the tap. to yield a coloured

product.

Osazone Test

To 0.5 g of phenylhydrazine

hydrochloride add 0.1 g of

sodium acetate and 10 drops

of glacial acetic acid. To this

mixture add 5 mL of test

solution and heat on a boiling

water bath for about half an

hour. Allow the tube to cool

slowly and examine the

crystals under a microscope.

Glucose, fructose and

mannose produce needle-

shaped yellow osazone

crystals, whereas lactosazone

is mushroom shaped. Maltose

produces flower-shaped

crystals.

The ketoses and aldoses react

with phenylhydrazine to

produce a phenylhydrazone

which in turn reacts with

another two molecules of

phenylhydrazine to form the

osazone.

Observation : Note the various changes occurring after completion of reaction.

Result : According to observation write down the result whether test is positive or

negative.

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Experiment –3

Aim : To perform identification test for a given sample of protein.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 316.

Requirements : Test tube, dropper, pipette, beaker

Chemicals : Conc. nitric acid, chlorophenol, acetic acid.

Theory :

Heller's test: Proteins get denatured when acid is added and this forms a white coagulum

which is slightly yellow in colour because of nitro- derivatives of proteins given by

aromatic amino acids.

Heat coagulation test: Coagulation is a result of irreversible denaturation of proteins like

albumin and globulins.

Procedure :

Heller's test:

In 3ml conc. Nitric acid add 3 ml of protein solution.

White ppt. appears at junction of two fluids indicating that protein is present.

Heat coagulation test:

Fill 2/3 of test tube with protein solution. Add 4-5 drops of chlorophenol red and

mix.

A purple colour develops. Add 1% acetic acid until colour changes to faint pink.

Incline the test tube and heat the upper part and a dense coagulum is formed.

Observation: Note the various changes occurring after completion of reaction.

Result : According to observation write down the result whether test is positive or

negative.

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Experiment –4

Aim : To separate amino acids by thin layer chromatography.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 270-273, 275.

Requirements : Glass slide , beaker

Chemicals : Amino acids, n-propanol, Butanol, glacial acetic acid, ninhydrin reagent,

silica gel.

Solvent system : n-butanol : glacial acetic acid : water :: 12 : 5 : 3

Ninhydrin Solution : 250 mg ninhydrin in 100 ml acetone.

Theory : Chromatography is the collective term for a set of laboratory techniques for the

separation of mixtures. It involves passing a mixture dissolved in a "mobile phase"

through a stationary phase, which separates the analyte to be measured from other

molecules in the mixture based on differential partitioning between the mobile and

stationary phases.

Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil,

which is coated with a thin layer of adsorbent material, usually silica gel, aluminium

oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary

phase. After the sample has been applied on the plate, a solvent or solvent mixture

(known as the mobile phase) is drawn up the plate via capillary action. Because different

analytes ascend the TLC plate at different rates, separation is achieved.

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Procedure

1.Prepare slurry of stationary phase and apply on glass plate.

2. Activate the air dried plates by keeping in oven at 110o C for 1 hr.

3.Apply the sample using micropipette. Saturate the TLC chamber and place the glass

plate into it and the solvent is allowed to run.

4. Plate is air dried and spray ninhydrin solution.

5. Heat the plates in oven at 110o C for 10 min.and calculate the Rf value.

Calculation :

Rf = Distance travelled by analyte from origin

Distance traveled by solvent front from origin

Result : Separate different amino acids according to their Rf values.

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Experiment – 5

Aim : To separate amino acids by paper chromatography.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 270-273, 275.

Requirements : Whatman filter paper no.1, beaker

Chemicals : Amino acids, n-propanol, Butanol, glacial acetic acid, ninhydrin reagent,

silica gel.

Solvent system : n-butanol : glacial acetic acid : water :: 12 : 5 : 3

Ninhydrin Solution : 250 mg ninhydrin in 100 ml acetone.

Theory : Paper chromatography is an analytical chemistry technique for separating and

identifying mixtures that are or can be colored, especially pigments. This can also be used

in secondary or primary colors in ink experiments. This method has been largely replaced

by thin layer chromatography, however it is still a powerful teaching tool. Two-way

paper chromatography, also called two-dimensional chromatography, involves using two

solvents and rotating the paper 90° in between. This is useful for separating complex

mixtures of similar compounds, for example, amino acids.

Procedure :

1.Apply the sample using micropipette. Saturate the chamber and place the paper into it

and the solvent is allowed to run.

2. Paper is air dried and spray ninhydrin solution and calculate the Rf value.

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Calculation :

Rf = Distance travelled by analyte from origin

Distance traveled by solvent front from origin

Result : Separate different amino acids according to their Rf values.

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Experiment – 6

Aim : To separate lipids by thin layer chromatography.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 278-279.

Requirements : Glass slide , beaker

Chemicals : Petroleum ether, diethyl ether, glacial acetic acid, silica gel, sulphuric acid

(50%v/v)

Solvent system : Petroleum ether : diethyl ether : glacial acetic acid :: 80 : 20 : 1

Theory : Chromatography is the collective term for a set of laboratory techniques for the

separation of mixtures. It involves passing a mixture dissolved in a "mobile phase"

through a stationary phase, which separates the analyte to be measured from other

molecules in the mixture based on differential partitioning between the mobile and

stationary phases.

Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil,

which is coated with a thin layer of adsorbent material, usually silica gel, aluminium

oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary

phase. After the sample has been applied on the plate, a solvent or solvent mixture

(known as the mobile phase) is drawn up the plate via capillary action. Because different

analytes ascend the TLC plate at different rates, separation is achieved.

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Procedure:

1. Prepare slurry of stationary phase and apply on glass plate.

2. Activate the air dried plates by keeping in oven at 110o C for 1 hr.

3. Apply the sample using micropipette. Saturate TLC chamber and place the glass

plate into it and the solvent is allowed to run.

4. Plate is air dried and spray sulphuric acid solution.

5. Heat the plates in oven at 110o C for 10 min.and calculate the Rf value.

Calculation :

Rf = Distance travelled by analyte from origin

Distance traveled by solvent front from origin

Result : Separate different amino acids according to their Rf values.

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Experiment -7

Aim : To determine casein in the milk.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 309,311.

Requirements : Flask, beaker

Chemicals : Acetic acid 10%, KOH 0.1N, HCl 0.1N, Phenolphthalein indicator

Theory : Casein (from Latin caseus, "cheese") is the name for a family of related

phosphoprotein proteins (αS1, αS2, β, κ). These proteins are commonly found in

mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65%

of the proteins in human milk. Casein has a wide variety of uses, from being a major

component of cheese, to use as a food additive, to a binder for safety matches. As a food

source, casein supplies essential amino acids, carbohydrates and two inorganic elements,

calcium and phosphorus.

Procedure:

1. Take 10 ml milk in 200 ml flask and add 75 ml of distilled water and 1.0-1.5 ml of

10% acetic acid.

2. Mix content vigorously. Filter the ppt. and wash with cold water to remove acetic acid.

3. Transfer ppt. and paper in flask, add 75-80 ml of neutral water, 10 ml of 0.1N KOH

and a few drops of phenolphthalein.

4. Stopper the flask and shake vigorously and titrate alkaline casein solution with 0.1N

HCl until all red colour disappears.

Calculation : Substract the corrected acid value from 10ml of alkali used to give % of

casein in milk.

% Casein = 10 – (actual value + check value)

Check value = 0.2 - 0.3

Result : Write down the % casein present in milk.

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Experiment – 8

Aim : To estimate level of glucose in blood serum.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 294-295.

Requirements : Beaker, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : Blood sample, glucose estimation kit.

Theory : Enzymatic method yields maximum specificity for glucose estimation. Glucose

can be measured by its reaction with glucose oxidase, in which gluconic acid and

hydrogen peroxide are formed. Hydrogen peroxide than reacts with an oxygen acceptor,

such as ortho-dianisidine, phenylamine-phenazone or any other chromogenic oxygen

acceptors, in a reaction catalysed by peroxidase to form a colour.

Procedure

Mix them and incubate at 370

C for 20 min. Read the absorbance of standard and test

against blank at 540 nm.

Calculation :

Plasma glucose (mg/dl) = reading of test x 100

reading of standard

Result : Write down the concentration of glucose in blood.

Sr.No. Reagents Blank Standard Test tube

1. Glucose reagent 1 ml 1 ml I ml

2. Glucose standard - 0.01 ml -

3. Serum sample - - 0.01 ml

4. Distilled water 0.01 ml - -

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Experiment – 9

Aim : To perform the quantitative estimation of proteins in serum using Biuret method.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 296.

Requirements : Beaker, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : Blood sample, protein estimation kit.

Theory : Copper in alkaline solution reacts with peptide linkages of amino acids in

protein producing a violet colour which is measured colorimetrically.

Procedure :

Mix them and incubate at 370

C for 10 min. Read the absorbance of standard and test

against blank at 540 nm.

Calculation :

Serum Total Protein (g/dl) = reading of test x 6

reading of standard

Result : Write down the concentration of proteins in serum.

Sr. No. Reagents Blank Standard Test tube

1. Biuret reagent 1 ml 1 ml I ml

2. Protein standard - 0.01 ml -

3. Serum sample - - 0.01 ml

4. Distilled water 0.01 ml - -

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Experiment – 10

Aim : To estimate SGOT level in serum.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 297.

Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : SGOT estimation kit

Theory : Serum aspartate aminotransferase (AST) also known as serum glutamic

oxalacetic transaminase (SGOT) is a tissue enzyme that catalyzes the exchange of amino

and keto groups between alpha amino acids and alpha-keto acids. AST is widely

distributed in tissue principally cardiac hepatic muscle and kidney. Injury to these tissues

results in the release of the AST (SGOT) enzyme to general circulation.

AST catalyzes the following reaction.

L-Aspartate + 2-Oxoglutarate ----- Oxalacetate + L-Glutamate

In the present method a diazonium salt is used which selectively reacts with the

oxalacetate to produce a color complex that is measured with colourimeter..

Procedure :

Reagent Blank Standard control Test

Substrate

reagent

0.5 ml 0.5 ml 0.5 ml 0.5 ml

Deionised

water

0.1 ml - - -

Sample - - - 0.1 ml

Standard - 0.1 ml - -

Mix and incubate at 370C for 60 min.

SGOT

colour

reagent

0.5 ml 0.5ml 0.5ml 0.5ml

Sample - - 0.1ml -

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Mix and incubate at 370C for 20 min.

Add 3ml of alkali reagent to each test tube. Read the absorbance against distilled water at

505 nm.

Calculation :

SGOT (U/L) = Absorbance of test - Absorbance of std x Conc. of std (160 U/L)

Absorbance of std - Absorbance of blank

Result : Write down the SGOT level in serum.

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Experiment -11

Aim : To estimate SGPT level in serum.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 297.

Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : SGPT estimation kit

Theory : Alanine transaminase or ALT is a transaminase enzyme. It is also called serum

glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT). ALT is

found in serum and in various bodily tissues, but is most commonly associated with the

liver. It catalyzes the two parts of the alanine cycle.

It catalyzes the transfer of an amino group from alanine to a-ketoglutarate, the products

of this reversible transamination reaction being pyruvate and glutamate.

glutamate + pyruvate ⇌ α-ketoglutarate + alanine

Procedure :

Reagent Blank Standard control Test

Substrate

reagent

0.5 ml 0.5 ml 0.5 ml 0.5 ml

Deionised

water

0.1 ml - - -

Sample - - - 0.1 ml

Standard - 0.1 ml - -

Mix and incubate at 370C for 30 min.

Mix and incubate at 370C for 20 min.

SGPT

colour

reagent

0.5 ml 0.5ml 0.5ml 0.5ml

Sample - - 0.1ml -

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Add 3ml of alkali reagent to each test tube. Read the absorbance against distilled water at

505 nm.

Calculation :

SGPT (U/L) = Absorbance of test - Absorbance of std x Conc. of std (160 U/L)

Absorbance of std - Absorbance of blank

Result : Write down the SGPT level in serum.

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Experiment – 12

Aim : To estimate Alkaline Phosphatase level in serum.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 299-301.

Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : Alkaline Phosphatase estimation kit

Theory : Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme responsible for

removing phosphate groups from many types of molecules, including nucleotides,

proteins, and alkaloids.

Procedure :

Reagent Blank Standard Control Test

Working

buffered

substrate

0.5 ml 0.5ml 0.5ml 0.5ml

Distilled water 1.5 ml 1.5ml 1.5ml 1.5ml

Mix and incubate at 37ºC for 3 min.

Serum - - -

0.05ml

Reagent 3 - 0.05 ml -

-

Mix well and incubate at 37ºC for 15 min.

Reagent 2 1ml 1ml 1ml 1ml

Serum - -

0.05ml -

Mix well and measure optical density of all the test tubes against distilled water at

510nm.

Calculation :

Serum Alkaline Phosphatase (KA units/ 100 ml) = Abs of test - Abs of std x 10

Abs of std

Result : Write down the Alkaline Phosphatase level in serum.

23

Experiment -13

Aim : To estimate Billirubin level in serum.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 298-299.

Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : Billirubin estimation kit

Theory : Bilirubin is the yellow breakdown product of normal heme catabolism. Heme is

found in hemoglobin, a principal component of red blood cells. Bilirubin is excreted in

bile and urine, and elevated levels may indicate certain diseases. It is responsible for the

yellow color of bruises, the yellow color of urine (via its reduced breakdown product,

urobilin), the brown color of faeces (via its conversion to stercobilin), and the yellow

discoloration in jaundice.

Procedure :

Reagent Blank Test

Total bilirubin

reagent

1 ml 1 ml

Sodium nitrite

reagent

- 0.5 ml

Distilled Water 0.5 ml -

Sample 0.5 ml 0.5 ml

Mix well reagents and wait for 30 sec before next addition.

Then add 0.15 ml of serum sample to each test tube.mix well and incubate at 370C for 5

min and note the absorbance at 540nm against distilled water.

Calculation :

Total Billirubin (mg/dl) = Abs of test - Abs blank x 10

Abs of std

Result : Write down the Total Billirubin level in serum.

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Experiment -14

Aim : To estimate Total Serum Cholesterol.

Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh

Prakashan, 1st edition, 2006, 301-302.

Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter.

Chemicals : Cholesterol estimation kit

Theory : Serum is treated with ferric chloride - acetic acid reagent to precipitate proteins.

The protein free supernatant is treated with conc. sulphuric acid. A reddish purple colour

is developed which is measured colorimetrically at 560nm0

Procedure :

Reagent Blank Standard Test

Cholesterol

reagent

1 ml 1 ml 1 ml

Cholesterol Std - 0.1 ml -

Sample - - 0.1s ml

Mix well and incubate at 370C for 5 min and note the absorbance at 560nm.

Calculation :

Total Serum Cholesterol (mg/ 100ml) = Abs of test x 200

Abs of std

Result : Write down the Total Serum Cholesterol.