Biochemistry DPHP1054 Lab Manual

GALGOTIAS UNIVERSITY
SCHOOL OF MEDICAL AND ALLIED SCIENCES
DEPARTMENT OF PHARMACY
LABORATORY
MANUAL
BIOCHEMISTRY
PRACTICAL
SUBJECT CODE: DPHP1054
Ist YEAR
Prepared By
Mr. Debashish Paramanick
School of Medical and Allied Sciences, Galgotias University

LIST OF EXPERIMENTS
1. To perform qualitative analysis of Glucose and fructose.
2. To perform qualitative analysis of Lactose and Maltose.
3. To perform qualitative analysis of Sucrose and Starch.
4. To perform the identification test for Protein albumin
5. To perform the identification test for Protein.
6. To perform the identification test for Lipids.
7. To perform thin layer chromatography for amino acids.
8. To perform paper chromatography for amino acids.
9. To identify the normal inorganic constituents in the urine.
10. To identify the normal organic constituents in the urine.
11. To perform the qualitative test of abnormal constituents of Urine.
12. To analyze the sample of saliva.
13. To determine the effect of Different Temperatures on the Activity of
Salivary Amylase on Starch.
14. To determine the effect of different pH on the Activity of Salivary Amylase
on Starch.
15. To prepare acid buffer solution and measurement of pH
16.To prepare basic buffer solution and measurement of Ph
17.To determine sugar level in blood.
18. To determine the creatinine level in blood

EXPERIMENT NO.-1
OBJECT: - To perform qualitative analysis of Glucose and fructose.
REFERENCES: -
1) Malthora Varun practical bio-chemistry for student, 4th Edition published by Japee
brother’s daryganj, Delhi.
2) Kale S.K and Kale R.R ‘Practical biochemistry and clinical pathology’publised by Nirali
prakashan, Page no.20-26.
3) Chaudhari M.R and Gokhale S.B ‘Biochemistry and clinical pathology’ published by Nirali
prakashan, Page no.199.
CHEMICAL REQUIRED: - Conc. H2SO4, carbohydrate solution, iodine, NaOH solution,

phenylhydrazene, acetate solution,
APPARATUS REQUIRED: - Test tube, test tube holder and stand and water bath.
PROCEDURE:-
S.NO. TEST OBSERVATION INFERENCE
1. MOLISH TEST- Violet colour was Carbohydrate
2ml solution + 2 drop of molish observed at the confirmed
reagent mix it and then add 2 ml of interface.
conc. H2SO4 and rotate gently betwee
the palms.
2. FEHLING TEST- Red precipitate is Reducing sugar
1ml of Fehling solution A and 1 ml formed. present.
of Fehling solution B + add 2ml of
carbohydrate solution mixed it
properly and then boil it.
3. BENEDICT’S TEST- Red ppt was Reducing sugar
2 ml of benedict’s reagent + 8 drops formed. present.
of carbohydrate solution keep on
water bath for 2 min and then cooled
it.
4. BARFOED’S TEST- Brick red ppt. was Marked presence

2 ml of barfoed’s reagent + 2ml of formed. of
carbohydrate solution keep on monosaccharide.
boiling water bath for 3 min.
5. OSAZONE TEST- Needle shaped Glucose and
0.2 gm of sugar + 0.4 gm crystals fructose Present
phenylhydrazene + 0.6 ml solution
of acetate + 4 ml of water and heat
the solution on water bath for 3 min.
then cooled and observed crystal
under microscope.

6. SELWINOFF’S TEST- Cherry red color May be glucose
3 ml of selwinoff’s reagent + 3 drop not formed present.
of carbohydrate solution + heat for
30 sec and then cool it.
CONFIRMATORY TEST FOR GLUCOSE:-

1. 3ml water+ 2 drop of methylene Blue colour Glucose confirmed.
blue + 1 ml NaOH and the disappeared.
boiled it.
2. 3 ml of sample carbohydrate Red colour was Glucose confirmed.
solution + 1 ml of picric acid obtained
solution + 1 ml of NaOH and
the boiled it.
PREPARATION OF THE REAGENTS:-
1) Benedict’s reagent:-
Solution A: - Dissolved173 gm of Sod. citrate and 100gm of anhydrous sod. Carbonate
in about 800ml water with heating.
Solution B: - Dissolve separately 17.3 gm of crystalline copper sulphate in 100 ml of
water.
Cool the solution; add solution (B) to solution (A) with constant stirring adjust the volume to
100ml with distilled water.
2) Barfoed’s reagent: - Dissolve 13.3 gm of crystalline copper acetate in 200ml water and
the filter it. Add 1.8 ml of glacial acetic acid.
3) Fehling’s solution (Pot.cupric tartarate solution)

Solution –A (copper solution):-
Dissolve 34.66 gm of carefully selected small crystals of cupric sulphate, showing no trace of
efflorescence or of adhering moisture in sufficient water to produce 500 ml.store this solution
in small, well stoppered bottles.
Solution –B (Alkaline tartrate solution):-

Dissolve 76gm of sod.pot.tartrate and 77gm of sod. hydroxide in sufficient water to produce
500 ml.
Then mix equal volumes of the solution A and solution B immediately

before use.
4. Selwinoff’s reagent: - Dissolve 50 mg of resorcinol in 33 ml of conc. HCl dilute to 100 ml
with water.
RESULT: - The given unknown sample of carbohydrate was found to be ---------- --.

EXPERIMENT NO.-2
OBJECT: - To perform qualitative analysis of Lactose and Maltose.
REFERENCES: -

PROCEDURE:-

2ml solution + 2 drop of molish observed at the confirmed
reagent mix it and then add 2 ml of interface.
conc. H2SO4 and rotate gently
between the palms.
2. FEHLING TEST- Red precipitate is Reducing sugar

1ml of Fehling solution A and 1 ml formed. present.
of Fehling solution B + add 2ml of
carbohydrate solution mixed it
properly and then boil it.
3. BENEDICT’S TEST- Red ppt was Reducing sugar

2 ml of benedict’s reagent + 8 formed. present.
drops of carbohydrate solution
keep on water bath for 2 min and
then cooled it.
4. BARFOED’S TEST- Brick red ppt. was Lactose and

2 ml of barfoed’s reagent + 2ml of not formed. Maltose present.
carbohydrate solution keep on
boiling water bath for 3 min.
5. OSAZONE TEST- Mushroom Lactose Present
0.2 gm of sugar + 0.4 gm shaped crystals
phenylhydrazene + 0.6 ml solution Flower-shaped Maltose Present
of acetate + 4 ml of water and heat crystals
the solution on water bath for 3
min. then cooled and observed
crystal under microscope.
water.


500 ml.

before use.

EXPERIMENT NO.-3
OBJECT: - To perform qualitative analysis of Sucrose and Starch.
REFERENCES: -

PROCEDURE:-

2ml solution + 2 drop of observed at the confirmed
molish reagent mix it and then interface.
add 2 ml of conc. H2SO4 and
rotate gently between the
palms.
2. FEHLING TEST- Red precipitate not Non-Reducing
1ml of Fehling solution A and formed. sugar present.
1 ml of Fehling solution B +
add 2ml of carbohydrate
solution mixed it properly and
then boil it.
3. IODINE TEST- Blue colour was Starch present.
2ml of carbohydrate solution + appeared.
2 drop of iodine.
4. OSAZONE TEST- Greenish yellow Starch confirmed.
0.2 gm of sugar + 0.4 gm needle shaped crystal
phenylhydrazene + 0.6 ml arranged.
solution of acetate + 4 ml of
water and heat the solution on
water bath for 3 min. then
cooled and observed crystal
under microscope.
5. SELIWANOFF TEST A cherry red colored Sucrose
To 3ml of of Seliwanoff’s precipitate within 5 confirmed (a
reagent, add 1ml of the test minutes is obtained. positive
solution. Boil in water bath for ketohexose test)
2 minutes.

water.


500 ml.

before use.
4. Selwinoff’s reagent: - Dissolve 50 mg of resorcinol in 33 ml of conc. HCl dilute to 100 ml
with water.

EXPERIMENT NO: - 4
OBJECT: - To perform the identification test for Protein albumin
REFERENCES: -
CHEMICAL REQUIRED: - 5% NaOH solution, copper sulphate, copper sulphate, 40%

NaOH solution, lead acetate, nin- hydrine solution.
APPARATUS REQUIRED: - Beaker, measuring cylinder, test tube stands and holder.
PROCEDURE:-
1. BIURETTE TEST- Violet colour was Protein confirmed
1ml solution of protein formed
sample + 1-2 drop of 5%
NaOH solution and then
add -2 drop of 1% copper
sulphate.
2. NINHYDRIN TEST- Slowly Protein confirmed
1ml of protein sample + development of
add 2 drop of nin- hydrine blue colour
solution then heat it and
allowed to cool.
3. HEAT COAGULATION Cloudness at top Albumin confirmed
TEST part of the solution
Place about 5 ml of egg
albumin solution in a test
tube and heat the top part
of the solution only
1) Ninhydrin reagent:-
Dissolve 4 gm of ninhydrin in 100 ml of 2- methoxyethanol shake gently with 1 gm of a
cation exchange resin (hydrogen form) and filter. Add this solution to 100 ml o a 0.16 % w/v
solution of stannous chloride in acetate buffer pH 5.5, immediately before use.
RESULT: - The given unknown sample was found to be ………………

EXPERIMENT NO: - 5
OBJECT: - - To perform the identification test for proteins.
REFERENCES: -
1) Malthora Varun practical bio-chemistry for student, 4th Edition published by Jaypee
CHEMICAL REQUIRED: - 5% NaOH solution, copper sulphate, 40% NaOH solution,

lead acetate, FeCl3, tannic acid solution(10%), strong ammonia solution.
PROCEDURE:-
1. 3 ml of solution + FeCl3 First turbidity Protein present
solution(0.5%) dropwise appears and on
addition of excess
FeCl3 it redissolves.
2. 3 ml of solution + 1 ml lead White ppt. Protein present
acetate solution
3. 3 ml of solution + 5 drops of Brown ppt. Protein present
tannic acid solution(10%).
4. 3 ml of solution + 1 ml CuSO4 White ppt. Protein present
soluton.
5. BIURET TEST- Pink or Violet colour Protein present
1ml solution of amino acid was formed at the
sample + 1-2 drop of 5% NaOH junction
solution and then add -2 drop of
copper sulphate.
6. SULPHUR TEST- Brownish black ppt. Cystine present
1 ml off amino acid sample + was formed
equal volume of 40% NaOH
solution the boil it for 1 min.,cool
it and then acidify it by the
addition of dil. HCl + 2-3 drop of
lead acetate.
7. 3 ml of solution + 1 ml conc. An orange ring Protein present
HNO3 and boil for 1 min. + cool (tyrosine,
+ strong ammonia solution to phenylalanine,
form a layer on the surface. tryptophan
residues)

EXPERIMENT NO: - 6
OBJECT: - - To perform the identification test for Lipids.
REFERENCES: -
CHEMICAL REQUIRED: - oil, phenolphthalein, alkali solution

PROCEDURE:-
1. GREASE SPOT TEST- greasy spot Lipid present
Take a small amount of oil penetrating the
on a piece of paper paper will be
formed
2. FREE FATTY ACIDS Colour disappeared Lipid present

TEST
Take a few drops of
phenolphthalein solution in
a test tube and add to it one
or two drops of very dilute
alkali solution, just
sufficient to give the
solution a pink colour.
Now add a few drops of
the oil and shake
3. SAPONIFICATION no oil Lipids present
TEST drops (Appearance
Take 1 ml of the oil in a of some oil drops
test tube and add an equal will indicate the
amount of alcoholic KOH incomplete saponi-
solution, mix them ficatio)
thoroughly and keep the
mixture during the course
of warming and shake up
gently with a little distilled
water
4. EMULSIFICATION Water, oil is Lipid present
Take 2 clean and dry test broken in small
tubes, in one test tube pieces and floats
added 2 ml water and in on the surface;
other 2ml dilute bile salt where as in the bile
solution. Now to each tube salt (emulsifying
added 2 drops of mustard agent) solution, the
oil and shaken vigorously oil can be seen in
for about one minute. minute droplets
Allow the tubes to stands suspended in the
for two minutes liquid (permanent
emulsification).

EXPERIMENT NO. 7
OBJECT: - To perform thin layer chromatography for amino acids.
REFERENCES: -
1) Sawhney S.K and Singh Randhir “Introduction practical biochemistry”Narosa Publication
Page no.199
2) Beckett A.H, Stanlet J.B, “Practical pharmaceutical chemistry” part 2, CVS publication.
CHEMICAL REQUIRED: - Ninhydrin spray, Solvent, Amino acid sample, silica gel G.
APPARATUS REQUIRED: - Glass plate, Chromatographic Chamber, Filter paper.
PROCEDURE:-
1. Thoroughly clean glass plate free from any greasy spot or finger marks are taken. Put a base
line on opposite side of glass plate at 2 cm from one edge.
2. Prepare slurry of stationary phase (silica gel G) in water.
3. Spread a uniform layer of 250 micrometer with the help of spreader.
4. Activate the plate by keeping them at 105 C for 30 minutes.
5. Allowed the plate to cool in desiccators before use.
6. Gently put marks on the lower base line at a distance of 2 cm from lower end of plate.
7. Carefully applied the amino acid sample on the base line marks.
8. Place the plate in chromatographic chamber which has already been saturated with solvent.
9. Close the chromatographic chamber with an air tight plate .allow the solvent to ascend along
the plate.
10. Remove the plate from the chromatographic chamber and dry it at room temperature.
11. Spray the ninhydrin reagent and dry it again in oven for 10 minutes.
12. Spots are developed measure their height and calculate their Rf value.
1) Ninhydrin reagent:-
Dissolve 4gm of ninhydrin in 100 ml of 2- methoxyethanol shake gently with 1 gm of a
cation exchange resin (hydrogen form) and filter. add this solution to 100 ml o a 0.16 % w/v
solution of stannous chloride in acetate buffer pH 5.5,immediately before use.
RESULT: - The given unknown sample was found to be ……………

EXPERIMENT NO. 8
OBJECT: - To perform paper chromatography for amino acids

APPARATUS REQUIRED Square whatman filter paper, petri dish, cotton, fine capillary
CHEMICALS REQUIRED Glycine, valine, butanol, acetic acid, distilled water,
ninhydrin reagent.
Solvent system is butanol: acetic acid: water
The spraying reagent is Ninhydrin reagent.
THEORY: This is a type of partition chromatography which involves paper placed in a jar
containing a shallow layer of solvent and sealed. A small dot of sample solution is placed
onto a strip of chromatography paper. As the solvent rises through the paper by capillary
action, it meets the sample mixture which starts to travel up the paper with the solvent.
Components of the mixture are carried along with the solvent up the paper to varying
degrees, depending on the compound’s performance to be adsorbed onto the paper v/s
being carried along with the solvent. This paper is made of cellulose to which polar water
molecules are adsorbed while the solvent is less polar usually consisting of a mixture of
water and an organic liquid. The compounds within the mixture travel farther if they are
non-polar. More polar substances bond with the cellulose paper quickly and therefore, do
not travel so far. The further the circular band from the starting spot, the higher the affinity
of the amino acids for the mobile phase and faster is its migration. The relative distances
are measured in terms of retention factor (Rf).
PROCEDURE:
1. Take a whatmann filter paper and mark the centre by drawing two perpendicular
diameters.
2. Make a wick of cotton and insert it through the hole of the chromatogram.
3. In a petri dish, place the solvent mixture (40-50 mL) and cover it with another petri dish.
4. Remove the wick from the chromatogram and apply the given mixture of amino acids
using a fine capillary to spot at the centre of centre of the chromatogram.
5. Repeat the spotting two times and insert the wick back when the spot has dried.
6. Introduce the system of chromatogram and wick into the chromatography chamber
(containing the solvent system).

7. Ensure that the chromatogram rests on the rim of the petri dish and does not dip into it.
The chromatogram should remain in a stretched state and now cover it with another petri
dish.
8. Leave the system undisturbed to allow the development of chromatogram.
9. When the solvent front has almost reached three-fourth of the chromatogram, remove it
and immediately mark the solvent front with a pencil.
10. Spray the chromatogram with spraying reagent (ninhydrin reagent) to visualize the
separated components. Calculate Rf value of each components at four different points.
CALCULATIONS: Calculation of Rf value of amino acid
Rf = distance travelled by amino acid from application point /distance travelled by solvent
from application point
RESULT
Rf value of amino acid was………
PRECAUTIONS
1. The chromatogram should remain in the stretched position on the rim of the petri dish.
2. Do not disturb the system while the chromatogram develops.
3. The chromatogram should be dried properly before introducing it in the
chromatography chamber.
4. The solvent mark should be marked immediately with a pencil after removing it from
the chromatography chamber.

EXPERIMENT NO. 9
OBJECT: - To identify the normal inorganic constituents in the urine.
REFERENCES: - 1) Kale S. R & Kale R. R “Practical Biochemistry and clinical

pathology” 10th edition 2004, Nirali publication, page no-23.
APPARATUS REQUIRED: - Beaker, test tubes, test tubes stands, holder and urine
sample etc.
CHEMICAL REQUIRED:-
S.N. NAME OF CHEMICAL EACH STUDENT QUANTITY REQUIRED FOR

TOTAL STUDENTS (50)
1. Ag NO3 solution 1 ml 50 ml
2. Sodium nitroprusside 3 ml 150 ml
3. 10% NaOH 2 ml 100 ml
4. Picric Acid solution 1 ml 50 ml
5. Anhydrous Na2Co3 3 gm 150 gm
INORGANIC CONSTITUENTS IN THE URINE:-
S.No. TEST OBSERVATION INFERENCE
1. TEST FOR BICARBONATE- Effervescence of CO2 Bicarbonate present

Test for bicarbonate-3-ml urine + gas
dil HCl
2. TEST FOR CHLORIDE- White curdy pt of silver Chloride present
5-ml urine sample + 1 ml Conc. chloride soluble in
HNO3 + 1-ml silver nitrate ammonium hydroxide
solution.
3. TEST FOR PHOSPHATE- Yellow ppt was formed Phosphate present
3-ml urine sample +3-ml conc.
nitric acid + 3-ml ammonium
molybdate solution. Heat to boil
4. TEST FOR SULPHATE- An opaque milkiness or Sulphate present
5-ml urine + 1-ml conc. HCL + 2- a thick white ppt of
ml Barium chloride solution. Barium sulphate
insoluble in conc. HCL
5. TEST FOR AMMONIA- The Red litmus paper Ammonia present
5-ml urine sample 2-ml 40% turns blue.
NaOH, boil hold a red litmus
paper in vapour.
6. TEST FOR CALCIUM- White ppt of Calcium Calcium present
5-ml urine sample added few oxalate.
drops of sodium hydroxide + 1%
acetic acid + 2-ml of ammonium .
oxalate solution
PRECAUTIONS: - Reagents should be freshly prepared.

Urine sample should be taken early in morning.
Na2 Co3 used should be anhydrous.
RESULT: - The normal inorganic constituent of urine in the sample was found to be-----

EXPERIMENT NO. 10
OBJECT: - To identify the normal organic constituents in the urine.
REFERENCES: - 1) Kale S. R & Kale R. R “Practical Biochemistry and clinical

pathology” 10th edition 2004, Nirali publication, page no-23.
sample etc.
CHEMICAL REQUIRED:-
S.N. NAME OF CHEMICAL EACH STUDENT QUANTITY REQUIRED

FOR TOTAL STUDENTS
(50)
1. AgNO3 solution 1 ml 50 ml
2. Sodium nitroprusside 3 ml 150 ml
3. 10% NaOH 2 ml 100 ml
4. Picric Acid solution 1 ml 50 ml
5. Anhydrous Na2CO3 3 gm 150 gm
ORGANIC CONSTITUENTS IN THE URINE–
1. SCHIFF’S TEST- Black or yellow

Moisten a strip of paper brown stain form Uric acid present
with AgNO3 Soln + a drop
of Urine
2. WEYL’S TEST- Ruby red colour is
Urine + drops of sodium formed and soon Creatinine is present
nitroprusside + 10% change to yellow
NaOH
3. JAFFER’S TEST- A deep arrange colour
Urine + saturated soln. of is formed Creatinine is present
Picric acid + anhydrous
Na2 CO3 mix well by
shaking
PRECAUTIONS: - Reagents should be freshly prepared.

Urine sample should be taken early in morning.
Na2 Co3 used should be anhydrous.
RESULT: - The normal organic constituents of urine in the sample was found to be-------

EXPERIMENT NO. 11
OBJECT: - To perform the qualitative test of abnormal constituents of Urine.
REFERENCES: - 1) Kale S. R & Kale R. R “Practical Biochemistry & Clinical

Pathology” 10th edition 2004, Nirali publication page no. 21, 22.
sample etc.
CHEMICAL REQUIRED: -
S.N. NAME OF CHEMICAL EACH QUANTITY REQUIRED FOR

STUDENT TOTAL STUDENT
1. Conc. HNO3 3 ml 150 ml
2. Benedict’s solution 5 ml 250 ml
3. Fehling solution A & B 2 ml each 200 ml
4. Ammonium Sulphate 1 gm 50 gm
5. Sodium nitroprusside 0.12 ml 6.0 ml
6. Strong ammonia soln 2 ml 100 ml
7. Benzedine powder 0.5 gm 25 gm
8. Glacial acetic acid 1 ml 50 ml
9. H2O2 Solution 2.4 ml 120 ml
ABNORMAL CONSTITUENTS IN THE URINE:-
1. HELLER’S NITRIC ACID White ring at the Protein is present

TEST- junction of two
Concn HNO3 + add urine fluids.
drop wise
2. BENEDICT’S ETST- Brick and color Sugar is present
Urine + Benedicts soln boil changes to yellow
& cool
3. Fehling test Red yellow ppt. Sugar is present
Fehling A & B + add 2 ml
of urine
4. Test for ketone Permanent Red Ketone is present
Urine+Ammonium colour developed
sulphate +sodium
nitroprusside+ ammonia
soln from the side of the
test tube wait for 10
minutes.
5. Test for blood Green blue colour Blood is present
Benzedine powder + due to Iron
glacial acetic acid shake Benzedine
formation
for 1 minute + urine +
drops of H2O2
6. Test for bile pigments Various colors are Bile pigments are present
5ml Urine + 1 ml or 2 ml formed in filter
dil. HCl and filter it, allow paper
filter to dry followed by
adding a drop of conc.
HNO3.
PRECAUTIONS: - Urine sample should be taken early in the morning

Reagents should be freshly prepared.
Complex reagents should properly prepare.
1 Benedict’s reagent:-
Solution A: - Dissolved173 gm of Sod. Citrate and 100gm of anhydrous sod.
Carbonate in about 800ml water with heating.
Solution B: - Dissolve separately 17.3 gm of crystalline copper sulphate in 100
ml of water.
Cool the solution; add solution (B) to solution (A) with constant stirring adjust the
volume to 100ml with distilled water.

Dissolve 34.66 gm of carefully selected small crystals of cupric sulphate, showing no
trace of efflorescence or of adhering moisture in sufficient water to produce 500 ml.store
this solution in small, well stoppered bottles.

Dissolve 76gm of sod.pot.tartrate and 77gm of sod. Hydroxide in sufficient water to
produce 500 ml.
Then mix equal volumes of the solution A and solution B

immediately before use.
RESULT: - The abnormal constituents of urine in sample was found to be -----------------

EXPERIMENT NO. 12
OBJECT: - To analyze the sample of saliva.
REFERENCES:-. Tortora derrickson-Pincipal of anatomy and physiology 11th edition

published by john Wiley and sons Page no. 904.
CHEMICAL REQUIRED: - Phosphate buffer, NaCl solution, dil. Iodide solution, 1%

Acetic acid, Potassium oxalate solution, Conc. HNO3, Ammonium molybdate.
sample etc.

1. TEST FOR AMYLASE- Gradual disappears Amylase present
2.5 ml starch solution + of blue colour
1ml phosphate buffer + 1
ml NaCl + 1ml saliva
solution(for 1 min) + add 1
drop of mixture dil. Iodine
solution
2. TEST FOR MUCIN- Thread like Mucin present
2ml of saliva + 1% acetic precipitate was
acid drop wise formed
3. TEST FOR CALCIUM- White precipitate Calcium present
2ml of saliva + 5 drops of was formed
1% acetic acid + 5 ml of
potassium oxalate solution.
4. TEST FOR Yellow colour ppt Inorganic phosphorous
INORGANIC was formed present
PHOSPHOROUS-
2ml of saliva + few drop of
conc. HNO3
+ add ammonium
molybdate + heat
PRECAUTIONS: - Saliva sample should be taken early in the morning.

Reagents should be freshly prepared.
RESULT: - The constituent of saliva in the sample was found to be ------------------ --.

EXPERIMENT NO. 13
Aim: To determine the effect of Different Temperatures on the Activity of Salivary

Amylase on Starch.
Theory
All living beings need energy to survive. It is from the food we consume that we get our
energy. We know that the energy we are getting is by the process of digestion that breaks
down the complex substance of starch into simpler molecules of glucose, which are
further metabolized into CO2 and water through the process of glycolysis. The human
digestive tract starts at the mouth and ends at the anus.
In the Beginning
The digestion of the food starts as soon as we put food in our mouth. Our teeth cut the
food into small pieces and the salivary glands secrete saliva that mixes with these food
materials. The saliva contains an enzyme called salivary amylase which hydrolyses starch
into maltose. The complete digestion of starch occurs only in the small intestine by the
action of pancreatic amylase.
The activity of enzymes is strongly affected by several factors, such as temperature and
pH.
Effect of Temperature
All enzymes are proteinaceous in nature. At a lower temperature, the enzyme salivary
amylase is deactivated and at the higher temperature, the enzyme is denaturated.
Therefore, more time will be taken by an enzyme to digest the starch at lower and higher
temperatures. Optimum temperature for the enzymatic activity of salivary amylase ranges
from 32 °C to 37 °C. The optimum temperature means that the temperature at which the
enzyme shows the maximum activity. At this optimum temperature, the enzyme is most
active and hence, takes less time to digest the starch.
Effect of pH
The optimum pH for the enzymatic activity of salivary amylase ranges from 6 to 7.
Above and below this range, the reaction rate reduces as enzymes get denaturated. The
enzyme salivary amylase is most active at pH 6.8. Our stomach has high level of acidity
which causes the salivary amylase to denature and change its shape. So the salivary
amylase does not function once it enters the stomach.
How to test it?
The effect of temperature and pH on the activity of salivary amylase on starch can be
studied by using the Iodine test. If we add saliva on starch, the salivary amylase present
in saliva gradually acts on starch and converts it into maltose. Starch keeps on giving
blue colour with iodine till it is completely digested into maltose. At this point, no blue
colour is formed. This is the end point or achromic point.
Learning Outcomes:
 Students understand the process of digestion of starch by salivary amylase.
 Students understand the effect of temperature and pH on the activity of salivary
amylase on starch.
 Students do the experiment better in the real lab having gone through the
animation and simulation
Materials Required
Three series of test tubes having iodine solution in each, test tubes, ice cubes, water, 15
ml 1% starch solution + 3 ml 1% NaCl, saliva solution, droppers, thermometer, Bunsen
burner and wire gauze.
Procedure
 Take beaker containing 15 ml of 1% starch solution + 3 ml of 1% NaCl solution.
 Divide and pour this solution into three test tubes and mark them as A, B and C.
 Maintain the temperature of the beaker containing ice cubes at 5°C.
 Take beaker containing ice cubes and keep it on the table.
 Take another two beakers containing water and heat over the Bunsen burner.
 Now transfer experimental tube A into a beaker containing ice.
 Transfer the second experimental tube B into water bath set at 37°C and third
experimental tube C into the beaker maintained at 50°C.
 Using a dropper, take 1 ml saliva solution and transfer the solution into test tube
A.
 Similarly, add 1 ml saliva solution into test tube B and test tube C.
 Immediately, using a dropper, take few drops from experimental tube A and
transfer this into first series of test tubes having iodine solution.
 Similarly, using fresh droppers, do the same procedure for test tube B and test
tube C and transfer the solution into second and third series of test tubes having
iodine solution.
 Note this time as zero minute reading.
 After an interval of 2 minutes, again take a few drops from each tube and add to
the iodine tubes and note the change in colour of iodine.
 Keep on repeating the experiment at an interval of every 2 minutes till colour of
iodine does not change.
Results
It takes less time to reach achromic point at 37°C, as the enzyme is maximum active at
this temperature, while at higher and lower temperatures more time is taken to reach the
achromic point.
Conclusion
All enzymes are proteinaceous in nature. At lower temperatures, the enzyme salivary
amylase is deactivated and at higher temperatures, the enzyme is denaturated. Therefore,
more time will be taken by enzyme to digest the starch at lower and higher temperatures.
At 37° C, the enzyme is most active, hence, takes less time to digest the starch.
URL: http://amrita.olabs.edu.in/?sub=79&brch=18&sim=236&cnt=705

EXPERIMENT NO.14
Aim: To determine the effect of different pH on the Activity of Salivary Amylase on

Starch.
Materials Required
Three series of test tubes having iodine solution in each, test tubes, pH tablets of 5, 6.8
and 8, beaker containing water with thermometer, 15 ml 1% starch solution + 3 ml 1%
NaCl, saliva solution, droppers, Bunsen burner and wire gauze.
Procedure
 Take a beaker containing 15 ml of 1% starch solution + 3 ml of 1% NaCl
solution.
 Divide and pour this solution into three test tubes and mark them as A, B and C.
 Add pH tablet 5 into test tube A, pH tablet 6.8 into test tube B and pH tablet 8
into test tube C.
 Now transfer experimental tube A, B and C into a beaker containing water and a
thermometer for recording temperature. Temperature of this beaker is to be
maintained at 37°C.
 Using a dropper, take 3 ml saliva solution and add 1 ml of solution to each of the
three test tubes.
 Immediately using a dropper, take few drops from experimental tube A and
transfer this into the first series of test tubes having iodine solution.
 Similarly, do the same procedure for test tube B and test tube C and transfer the
solution into second and third series of test tubes having iodine solution.
 Note this time as zero minute reading.
 After an interval of 2 minutes, again take a drop from each tube and add to the
iodine tubes and note the change in colour of iodine.
 Keep on repeating the experiment at an interval of every 2 minutes till colour of
iodine does not change.
Results
pH 5 is acidic and pH 8 is alkaline, therefore salivary amylase did not act in these tubes.
Whereas, the enzyme acted in the tube with pH 6.8 (i.e., slightly acidic) and digested the
starch.
OBJECT: - To determine serum total cholesterol

THEORY: Cholesterol of serum is extracted into ethyl acetate alcohol (1:1) mixture and the
extract is centrifuged. The cholesterol gets extracted in supernatant liquid and reacted with
ferric chloride-acetic acid and sulphuric acid reagents to produce a color derivative. The
intensity of color is read in a colorimeter at 550 m micron and compared with standard
PROCEDURE: Take 0.1 ml of plasma in centrifuge tubes 5 ml of ethyl acetate alcohol (1:1)
mixture is added and the contents are vigorously shaken for 2 minutes. The contents are
centrifuged after 30 minutes and 1.0 ml supernatant liquid, is used for develop color.
Two ml of ferric chloride-acetic acid reagent is added followed by 2 ml of concentrated
H2SO4. After 30 minutes, absorbance (A) is read at 560 m micron and compared with
standard.

CALCULATION: Concentration of total cholesterol in serum
= ET-EB/Es-EBx100
Normal range= 150-250mg/100ml

Practical application: Increased serum total cholesterol occurs in atherosclerosis
hypothyroidism etc. and low level causes thyrotoxicosis

EXPERIMENT NO. 15
OBJECT: - To prepare acid buffer solution and measurement of pH

THEORY: The pH meter measures at electrical potential developed by pair of electrode
pins in a solution. For measurement of pH, an electrode system sensitive to change in H+ ion
concentration of solution is taken. The electrode system consists of sequence of electrode
whose potential raise with pH (H+ concentration of the solution).
REAGENT S REQUIRED:
• Citric acid: Dissolve 2.101 gm of citric acid in 100ml distilled water.
• Sodium citrate solution 0.1 M: Dissolved 2.941gm of sodium citrate in 100ml distilled
water.
PROCEDURE:
46.5ml of citric acid with 3.5ml of sodium citrate solution and up to 100ml with distilled
water. It corresponds to 0.1 M citrate buffer and standardized with pH meter and measures
the pH of the prepared solution.
RESULT:
Citrate buffer was prepared and the pH observed was ………

EXPERIMENT NO. 16
OBJECT: - To prepare basic buffer solution and measurement of pH

THEORY: The pH meter measures at electrical potential developed by pair of electrode
pins in a solution. For measurement of pH, an electrode system sensitive to change in H+ ion
concentration of solution is taken. The electrode system consists of sequence of electrode
whose potential raise with pH (H+ concentration of the solution).
REAGENT S REQUIRED:
• Monobasic: Dissolve 2.78gm of sodium dihydrogen phosphate in 100ml of distilled
water.
• Dibasic sodium phosphate (0.2M): Dissolve 5.3gm of disodium hydrogen phosphate or
7.17 gm sodium hydrogen phosphate in 100ml distilled water.
PROCEDURE:
39 ml of dihydrogen sodium phosphate is mixed with 61 ml of disodium hydrogen
phosphate This made up to 200ml with distilled water .This gives phosphate (PO4)2 buffer
of 0.2M and standardized with pH meter with standard buffer. Washed electrode with
distilled water and introduced it into phosphate buffer prepared and measures the pH of the
prepared solution.
RESULT:
Phosphate buffer was prepared and the pH observed was ………

EXPERIMENT NO. 17
OBJECTIVE: To determine blood sugar by Hexokinase method.

PRINCIPLE: • Glucose +ATP↔Glucose 6 phosphate +ADP • Glucose 6 Phosphate +
NAD ↔ 6- Phosphogluconate + NADH+H⁺
• Conversion of NADH from NAD at 340nm ,increase in O.D. is measured at fix interval
• Increase O.D. /min is directly conc. of glucose in the specimen = Delta O.D.
PROCEDURE:
• Pipette 1.0 ml Of Glucose Reagent in Cuvette & Keep It In a Water-bath at 37 For
1min(for incubation) • Add 10 μl of sample mix well & read change in O.D /minute , up to
3 minute
• Repeat steps 1, 2 & 3 by using Standard.
CALCULATION: • Plasma glucose = Delta O.D. per min(test) /Delta O.D. per min(Std.)
× 100
Result: The blood sugar level was found to be…….

EXPERIMENT NO. 18
OBJECTIVE: To determine the creatinine level in blood

DRAW VOLUME: Blood: 0.6 mL in green top (lithium heparin) tube or microtainer
Alternate tube: Red top tube
PROCESSED VOLUME: Serum/plasma: 0.2 mL
PROCEDURE:
These methods are based on the Jaffé reaction, in which creatinine reacts with picr
ate in alkaline solution to form an orange‐
red complex with an absorption maximum at 490500 nm, which is measured spectroph
otometrically. The major drawback of these methods is their lack of specificity: interfe
ring substances include ketones, some drug, e.g. cephalosporins (positive) and bilirubi
n (negative). This effectively negates the use of end‐
point assays. The effects of interference have been successfully reduced by the adoptio
n of kinetic assays, in which colour generation is typically measured between 20 and 8
0 seconds after initiation of the reaction (interfering substances tend to have an immed
iate (80 sec, e.g. protein), and reaction conditions, and reagent concentrations are preci
sely controlled. Such methods are widely used in automated analyzers.
Result: The blood creatinine level was found to be…….

SCHEME FOR IDENTIFICATION OF UNKNOWN CARBOHYDRATE:-
Molish test
Carbohydrate +ve Carbohydrate –ve
Iodine test
+ve -ve
Polysaccharide +ve Mono-di-saccharide
Starch-blue
Dextrin-purple
Glycogen-reddish brown
Half saturation test Benedict’s test
+ve -ve +ve -ve

Starch Dextrin, glycogen Monosaccharide or Non-reducing
disacharide reducing disacharide
Inversion test
Barfoed’s test +ve
Sucrose
+ve -ve
Monosaccharide Reducing disaccharide
Seliwanoff’s test Osazone test
+ve -ve Sunflower Puff

shaped crystals shaped
crystals
Fructose Glucose Maltose Lactose


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GALGOTIAS UNIVERSITY

SCHOOL OF MEDICAL AND ALLIED SCIENCES

SUBJECT CODE: DPHP1054

School of Medical and Allied Sciences, Galgotias University

1. To perform qualitative analysis of Glucose and fructose.

2. To perform qualitative analysis of Lactose and Maltose.

3. To perform qualitative analysis of Sucrose and Starch.

4. To perform the identification test for Protein albumin

5. To perform the identification test for Protein.

6. To perform the identification test for Lipids.

7. To perform thin layer chromatography for amino acids.

8. To perform paper chromatography for amino acids.

9. To identify the normal inorganic constituents in the urine.

10. To identify the normal organic constituents in the urine.

11. To perform the qualitative test of abnormal constituents of Urine.

12. To analyze the sample of saliva.

13. To determine the effect of Different Temperatures on the Activity of

Salivary Amylase on Starch.

14. To determine the effect of different pH on the Activity of Salivary Amylase

15. To prepare acid buffer solution and measurement of pH

16.To prepare basic buffer solution and measurement of Ph

17.To determine sugar level in blood.

18. To determine the creatinine level in blood

School of Medical and Allied Sciences, Galgotias University

OBJECT: - To perform qualitative analysis of Glucose and fructose.

CHEMICAL REQUIRED: - Conc. H2SO4, carbohydrate solution, iodine, NaOH solution,

4. BARFOED’S TEST- Brick red ppt. was Marked presence

School of Medical and Allied Sciences, Galgotias University

S.NO. TEST OBSERVATION INFERENCE

PREPARATION OF THE REAGENTS:-

3) Fehling’s solution (Pot.cupric tartarate solution)

Solution –B (Alkaline tartrate solution):-

Then mix equal volumes of the solution A and solution B immediately

School of Medical and Allied Sciences, Galgotias University

OBJECT: - To perform qualitative analysis of Lactose and Maltose.

CHEMICAL REQUIRED: - Conc. H2SO4, carbohydrate solution, iodine, NaOH solution,

S.NO. TEST OBSERVATION INFERENCE

2. FEHLING TEST- Red precipitate is Reducing sugar

3. BENEDICT’S TEST- Red ppt was Reducing sugar

4. BARFOED’S TEST- Brick red ppt. was Lactose and

3) Fehling’s solution (Pot.cupric tartarate solution)

Solution –B (Alkaline tartrate solution):-

Then mix equal volumes of the solution A and solution B immediately

School of Medical and Allied Sciences, Galgotias University

OBJECT: - To perform qualitative analysis of Sucrose and Starch.

CHEMICAL REQUIRED: - Conc. H2SO4, carbohydrate solution, iodine, NaOH solution,

S.NO. TEST OBSERVATION INFERENCE

School of Medical and Allied Sciences, Galgotias University

3) Fehling’s solution (Pot.cupric tartarate solution)

Solution –B (Alkaline tartrate solution):-

Then mix equal volumes of the solution A and solution B immediately

School of Medical and Allied Sciences, Galgotias University

OBJECT: - To perform the identification test for Protein albumin

CHEMICAL REQUIRED: - 5% NaOH solution, copper sulphate, copper sulphate, 40%

PREPARATION OF THE REAGENTS:-

RESULT: - The given unknown sample was found to be ………………

School of Medical and Allied Sciences, Galgotias University

OBJECT: - - To perform the identification test for proteins.

CHEMICAL REQUIRED: - 5% NaOH solution, copper sulphate, 40% NaOH solution,

RESULT: - The given unknown sample was found to be ………………

School of Medical and Allied Sciences, Galgotias University

OBJECT: - - To perform the identification test for Lipids.

CHEMICAL REQUIRED: - oil, phenolphthalein, alkali solution

2. FREE FATTY ACIDS Colour disappeared Lipid present