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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 ph osphotase and Bilirubinu in the serum.
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Experiment – Experiment – 1 1 Aim : To prepare standard buffer and measure its pH Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 Na 2HPO4 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. bu ffer. 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 standard buffer solution. Insert Insert electrode assembly in it 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 – Experiment – 2 2 Aim : To perform identification test for a given sample of carbohydrate. rd
Reference : Satyanarayan U., Chakrapani U., “Biochemistry”, Uppola , 3 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 botanist Hans Molisch) is Molisch) is a sensitive chemical sensitive chemical test
for the presence of carbohydrates, 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, α-naphthol, though other phenols (e.g. resorcinol, thymol) 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. starch. Iodine solution — iodine
dissolved in an aqueous solution of potassium of potassium iodide — iodide — reacts reacts with with starch producing a purple black color. reducing sugars. This sugars. This includes all Benedict's reagent is used as a test for the presence of reducing monosaccharides and the disaccharides, lactose and maltose. maltose. Even more generally, Benedict's test will detect the presence of aldehydes (except aromatic ones), and and alphahydroxy-ketones, including 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 sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde 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.
5 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 m L. 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: 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
A red-cum-violet ring appears
The colour formed is due to
Add two drops of Molisch’s
at the junction of the two
the reaction of alpha-naphthol
reagent (5% 1-naphthol in
liquids.
with
furfural
and/or
formed
its
alcohol) to about 2 mL of test
derivatives
solution and mix well.
dehydration
Incline the tube and add about
concentrated sulphuric acid.
1 ml of concentrated sulphuric
All
acid along the sides of the
positively with this reagent.
of
by
the
sugars
by
carbohydrates
react
tube. I odine Tes Test
Appearance
Add a few drops of iodine
colour
of
deep
blue
This indicates the presence of starch in the solution. The blue
solution to about 1 mL of the
colour is due to the formation
test solution.
of starch-iodine complex.
6 Fehling’s Test
Formation of yellow or
The
blue
alkaline
cupric
To 1 mL of Fehling’s solution brownish-red precipitate. precipitate.
hydroxide present hydroxide present in Fehling’s
‘A’, add 1 mL of Fehling’s
solution, when heated in the
solution ‘B’ and a few drops
presence of reducing sugars,
of the test solution. Boil for a
gets reduced to yellow or red
few minutes.
cuprous oxide and it gets precipitated. precipitated. Hence, formation of the coloured precipitate indicates
the
presence
of
reducing sugars in the test solution. Benedict’s Test
Formation of red, yellow or
Depending
To 2 mL of Benedict’s reagent
green colour/precipitate.
concentration yellow to green
add five drops of the test
on
the
sugar
colour is developed.
solution. Boil for five minutes in a water bath. Cool the solution. Barfoed’s Test
Formation of brick-red
Only monosaccharides answer
To 1 mL of the test solution precipitate. precipitate.
this
test.
Since
Barfoed’s
add about 2 mL of Barfoed’s
reagent is weakly acidic, it is
reagent.
reduced
Boil it for one minute and
monosaccharides.
only
by
allow to stand for a few minutes. Seliwanoff’s Test
Appearance of deep red colour
To 2 mL of Seliwanoff’s
It is a timed colour reaction specific for ketoses.
reagent add two drops of test solution and heat the mixture to just boiling. Bial’s Test
Appearance of green colour or
It is specific for pentoses.
To 5 mL of Bial’s reagent add add
precipitate. precipitate.
They get converted to furfural.
2-3 mL of solution and warm
In the presence of ferric ion
gently. When bubbles rise to
orcinol and furfural condense
7 the surface cool under the tap.
to yield a coloured product.
Osazone Test
Glucose, fructose and
The ketoses and aldoses react
To 0.5 g of phenylhydrazine
mannose produce needle-
with phenylhydrazine to
hydrochloride add 0.1 g of
shaped yellow osazone
produce a phenylhydrazone phenylhydrazone
sodium acetate and 10 drops
crystals, whereas lactosazone
which in turn reacts with
of glacial acetic acid. To this
is mushroom shaped. Maltose
another two molecules of
mixture add 5 mL of test
produces flower-shaped flower-shaped
phenylhydrazine phenylhydrazine to form the
solution and heat on a boiling
crystals.
osazone.
water bath for about half an hour. Allow the tube to cool slowly and examine the crystals under a microscope.
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 – Experiment – 3 Aim : To perform identification test for a given sa mple of protein. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 – Experiment – 4 Aim : To separate amino acids by thin layer chromatography. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 edition, 2006, 270-273, 275. Requirements : Glass slide , beaker
Butanol, glacial acetic acid, ninhydrin reagent, Chemicals : Amino acids, n-propanol, Butanol, 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 laboratory techniques for the
separation of mixtures. mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, 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). paper). This layer of adsorbent is known as the stationary phase. phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile the mobile phase) is phase) is drawn up the plate via capillary via capillary action. Because action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
10 Procedure
1.Prepare slurry of stationary phase and appl y on glass plate. o
2. Activate the air dried plates by keeping in oven at 110 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 spra y ninhydrin solution. o
5. Heat the plates in oven at 110 C for 10 min.and calculate the R f value. Calculation :
R f =
Distance travelled by analyte from origin Distance traveled by solvent front from origin
Result : Separate different amino acids according to their R f values.
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Experiment – Experiment – 5 5 Aim : To separate amino acids by paper chromatography. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 edition, 2006, 270-273, 275. Requirements : Whatman filter paper no.1, beaker
Butanol, glacial acetic acid, ninhydrin reagent, Chemicals : Amino acids, n-propanol, Butanol, 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 an analytical chemistry technique for separating and
identifying mixtures that are or can be colored, especially pigments. especially pigments. This This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional called two-dimensional chromatography, involves 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 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 sp ray ninhydrin solution and calculate the R f value.
12 Calculation :
R f =
Distance travelled by analyte from origin Distance traveled by solvent front from origin
Result : Separate different amino acids according to their R f values.
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Experiment – Experiment – 6 6 la yer chromatography. Aim : To separate lipids by thin layer Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 Petroleum ether : diethyl ether : glacial acetic acid :: 80 : 20 : 1 laboratory techniques for the Theory : Chromatography is the collective term for a set of laboratory separation of mixtures. mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, 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). paper). This layer of adsorbent is known as the stationary phase. phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile the mobile phase) is phase) is drawn up the plate via capillary via capillary action. Because action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
14 Procedure:
1. Prepare slurry of stationary phase and appl y on glass plate. o
2. Activate the air dried plates by keeping in oven at 110 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 spra y sulphuric acid solution. o
5. Heat the plates in oven at 110 C for 10 min.and calculate the R f value. Calculation :
R f =
Distance travelled by analyte from origin Distance traveled by solvent front from origin
Result : Separate different amino acids according to their R f values.
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Experiment -7 Aim : To determine casein in the milk. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 commonly found in mammalian milk, making up 80% of the proteins in cow in cow milk 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 essential amino acids, carbohydrates acids, carbohydrates and two inorganic elements, calcium and phosphorus. 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 – 10 – (actual (actual value + check value) Check value = 0.2 - 0.3 Result : Write down the % casein present in milk.
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Experiment – Experiment – 8 8 b lood serum. Aim : To estimate level of glucose in blood Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 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
-
-
0
Mix them and incubate at 37 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.
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Experiment – Experiment – 9 9 Aim : To perform the quantitative estimation of proteins in serum u sing Biuret method.
P.C., “Pharmaceutical Biochemistry”, Vallabh Reference : Sharma P.K., Dandiya P.C., st
Prakashan, 1 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 : 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
-
-
0
Mix them and incubate at 37 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.
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Experiment – Experiment – 10 10 Aim : To estimate SGOT level in serum. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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) enz yme 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
0.5 ml
0.5 ml
0.5 ml
0.5 ml
0.1 ml
-
-
-
Sample
-
-
-
0.1 ml
Standard
-
0.1 ml
-
-
reagent Deionised water
0
Mix and incubate at 37 C for 60 min. SGOT
0.5 ml
0.5ml
0.5ml
0.5ml
-
-
0.1ml
-
colour reagent Sample
19 0
Mix and incubate at 37 C 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 st
Prakashan, 1 edition, 2006, 297. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : SGPT estimation kit
a transaminase enzyme. enzyme. It It is also called serum Theory : Alanine transaminase or ALT is a transaminase glutamic pyruvic transaminase (SGPT) or alanine aminotransferase (ALAT). ALT is found in serum in serum and in various bodily tissues, but is most commonly associated with the liver. It liver. It catalyzes the two parts of the alanine the alanine cycle. It catalyzes the transfer of an an amino group from alanine from alanine to a-ketoglutarate, to a-ketoglutarate, the the products of this reversible transamination reversible transamination reaction being pyruvate being pyruvate and glutamate. and glutamate. glutamate + pyruvate ⇌ α-ketoglutarate + alanine Procedure : Reagent
Blank
Standard
control
Test
Substrate
0.5 ml
0.5 ml
0.5 ml
0.5 ml
0.1 ml
-
-
-
Sample
-
-
-
0.1 ml
Standard
-
0.1 ml
-
-
reagent Deionised water
0
Mix and incubate at 37 C for 30 min. SGPT
0.5 ml
0.5ml
0.5ml
0.5ml
-
-
0.1ml
-
colour reagent Sample
0
Mix and incubate at 37 C for 20 min.
21
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.
22
Experiment – Experiment – 12 12 Aim : To estimate Alkaline Phosphatase level in serum. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 proteins, and alkaloids. alkaloids. Procedure :
Reagent
Blank
Standard
Control
Test
Working
0.5 ml
0.5ml
0.5ml
0.5ml
1.5 ml
1.5ml
1.5ml
1.5ml
buffered substrate Distilled water
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 o f test - Abs of std x 10 Abs of std Result : Write down the Alkaline Phosphatase level in serum.
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Experiment -13 Aim : To estimate Billirubin level in serum. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 edition, 2006, 298-299. Requirements : Beaker, pipettes, test tube, syringe, Centrifuge, Colorimeter. Chemicals : Billirubin estimation kit
normal heme catabolism. catabolism. Heme Heme is Theory : Bilirubin is the yellow breakdown product of normal heme found in in hemoglobin, a hemoglobin, a principal component of red red blood cells. Bilirubin cells. Bilirubin is excreted in bile and urine, and urine, and and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, bruises, the yellow color of urine (via its reduced breakdown product, urobilin), urobilin), the brown color of faeces (via its conversion to stercobilin), stercobilin), and the yellow discoloration in jaundice. in jaundice. Procedure : Reagent
Blank
Test
Total bilirubin
1 ml
1 ml
-
0.5 ml
Distilled Water
0.5 ml
-
Sample
0.5 ml
0.5 ml
reagent Sodium nitrite reagent
Mix well reagents and wait for 30 sec before next addition. 0
Then add 0.15 ml of serum sample to each test tube.mix well and incubate at 37 C 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.
24
Experiment -14 Aim : To estimate Total Serum Cholesterol. Reference : Sharma P.K., Dandiya P.C., “Pharmaceutical Biochemistry”, Vallabh st
Prakashan, 1 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 56 0nm0 Procedure :
Reagent
Blank
Standard
Test
Cholesterol
1 ml
1 ml
1 ml
Cholesterol Std
-
0.1 ml
-
Sample
-
-
0.1s ml
reagent
0
Mix well and incubate at 37 C for 5 min and note the absorbance at 560nm. Calculation :
Total Serum Cholesterol (mg/ (mg/ 100ml) = Abs of test x 200 Abs of std Result : Write down the Total Serum Cholesterol.
