SATHYABAMA UNIVERISY
DEPT.OF BIOINFORMATICS
SBIX4001 - Biochemistry Lab Manual
List of Experiments
1. Estimation of Glycine.
2. Estimation of Ascorbic Acid.
3. Estimation of Protein by Biuret Method.
4. Estimation of Protein by Lowry’s Method.
5. Estimation of DNA by Diphenylamine Method.
6. Colorimetric Assay for Salivary Amylase Activity.
7. Colorimetric Assay for Catalase Activity.
8. Estimation of Glucose by Anthrone Method.
9. Qualitative Analysis of Sugars.
10. Qualitative Analysis of Amino Acids.
Experiment 1
Estimation of glycine by Sorenson’s Formol Titration
AIM:
To estimate the amount of amino acid present in the given solution.
PRINCIPLE:
The acid group present in the glycine can be titrated with NaOH. It is not
easy in this case because the amino group present will interfere at the end
point. To prevent i t e x c e s s o f f o r m a l d e h y d e i s u s e d b y
w h i c h t h e a m i n o g r o u p i s b l o c k e d b y t h e formation of
methylene glycine. Then it is titrated with NaOH using
phenolphthaleinindicator.
REAGENTS REQUIRED:
(i)
(ii)
(iii)
(iv)
(v)
Standard oxalic acid 0.1 N
Link solution NaoH
Formaldehyde
Phenophthalein indicator
Amino acid solution
PROCEDURE:
Standardisation of NaOH
Rinse a clean burette first with distilled water and then with the
link solution of Na O H. R i n s e a c l e a n 2 0 ml pipette first with
distilled water and then with the givenstandard oxalic acid. Pipette
out 20ml of oxalic acid into a clean conical flask. Add a few drops of
phenolphthalein indicator and titrate against NaOH taken in the burette.
Thee n d p o i n t i s th e a p p e a r a n c e o f p e r ma n e n t p a l e p i n k
c o l o u r . R e p e a t t h e t i t r a t i o n f o r concordant values.
Estimation of Amino acid
Make up the given amino acid solution with distilled
w a t e r t o 1 0 0 m l i n a volumetric flask, observing the usual
precautions. Shake the solution well for uniformconcentration. Pipette
out 20ml of amino acid solution into a clean conical flask. Add5 ml of
HCHO and keep it for 2 minutes. Then titrate it against the NaOH solution
takenin the burette. Phenolphthalein is used as the indicator. The end point is
the appearanceof permanent pale pink colour. Repeat the titration for
concordant values
Blank titration
Pipette out 20ml of distilled water in a clean conical flask. Add 5ml
of HCHOand keep it aside for a few minutes. Add 1-2 drops of
phenolphthalein indicator. Titrateit against the NaOH solution taken in
the burette. The end point is the appearance of permanent pale pink
colour. Repeat the titration for concordant values.
Result:
The amount of amino acid present in the whole of the given solution =
_______ g
EXPERIMENT 2
Estimation of Ascorbic acid
AIM:
To estimate the amount of Ascorbic acid present in the given solution.
PRINCIPLE:
Ascorbic acid (Vitamin C) is a strong reducing agent. Ascorbic acid can
readily lose two of its hydrogen atoms from the hydroxyl groups on the
double bonded enediolcarbons.The titration method for estimation of
ascorbic acid is based on an oxidation reduction reaction,in which it reduces
the dye 2,6-dichlorophenol indophenols and In turn ,It gets oxidized to
dehydro ascorbic acid by 2,6 dichlorophenol indophenol dye. The dye
originally blue in colour, gets reduced to a colourless form,and at the end of
the titration ,when no more of ascorbic acid is available for the reduction of
the dye, the excess oxidized dye in the acidic solution imparts a faint pink
colour to the titration mixture.
REAGENTS REQUIRED:
Standard Indophenol solution
42mg of sodium bicarbonate and 52mg of 2, 6 dichlorophenol
indophenol were dissolved in 50ml of water and diluted to
200ml. The solution was filtered and stored.
Standard Ascorbic acid
o

50mg ascorbic acid was dissolved and made up to 250ml using
0.6% oxalic acid.
Preparation of test solution
o Given solution was made up to 100ml with 0.6% oxalic acid.
o

PROCEDURE:

Titration I: Standardisation of dye
The burette was rinsed and filled up to the convenient mark with dye
solution. 10ml of std ascorbic acid solution was pipetted out into a clean
conical flask. It was titrated against the dye taken in the burette. The end
point was the appearance of permanent pale pink colour which remains for
10 minutes. The titrations were repeated for concordant value. The dye
equivalent of std ascorbic acid was calculated.

Titration II: Estimation of Ascorbic acid
10ml of made up solution was pipetted out into a clean conical flask. It was
titrated against the dye taken in the burette, till the permanent pale pink
colour was obtained. The titrations were repeated for concordant value.
Using the dye equivalent of ascorbic acid, the amount of ascorbic acid in the
given solution was calculated.
RESULT: - The amount of ascorbic acid present in 100ml of given
solution=_______mg
EXPERIMENT 3
Estimation of Protein by Biuret method
Aim:
To estimate the protein using Biuret method.
Principle:
The –CO-NH- bond (peptide) in polypeptide chain reacts with copper
sulphate in an alkaline medium to give a purple which can be measured at
540 nm.
Reagents Required:
1. Biuret Reagent:
Dissolve 3 g of copper sulphate (CuSO4.5H2O) and 9g of sodium
potassium tartarate in 500 ml of 0.2 mol/lttre sodium hydroxide; add 5 g of
potassium iodide and make up to 1 litre with 0.2 mol/litre sodium hydroxide.
ProteinStandard:5 mg BSA/ml.
Apparatus and Glass wares required:Test tubes, Pipettes, Colorimeter, etc.,
Procedure:
Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of
labeled test tubes. Pipette out 1 ml of the given sample in another test tube.
Make up the volume to 1 ml in all the test tubes. Atube with 1 ml of distilled
water serves as the blank. Now add 3 ml of Biuret reagent to all the test
tubes including the test tubes labeled 'blank' and unknown'. Mix the contents
of the tubes by vortexing / shaking the tubes and warm at 37 ºC for 10 min.
Now cool the contents and record the absorbance at 540 nm against blank.
Then plot the standard curve by taking concentration of protein along X-axis
and absorbance at 540 nm along Y-axis.
Then from this standard curve calculate the concentration of protein in the
given sample.
Result:
The given unknown sample contains ----μ g protein/ml
S.No
Contents
Blank
S1
Volume of Working Standard
1 Solution (ml)
----Concentration of working standard
2 (µg)
----3 Volume of Unknown Solution (ml)
4 Volume of Distilled Water (ml)
3
Volume of Biuret solution
5 (ml)
7 Optical density at 540nm
0.2
Standards
S2 S3 S4
0.4
0.6
0.8
Test
Solution
T1 T2
S5
1 ----- -----
50 100 150 200 250
1
0.8
3
0.6
3
0.4
3
0.2 ---3
3
0.5
1
0.5
---3
3
EXPERIMENT 4
ESTIMATION OF PROTEIN BY LOWRY’S METHOD.
AIM:
To estimate the amount of protein present in the given unknown solution by
Lowry‟s method.
PRINCIPLE:
The aromatic amino acids tyrosine and tryptophan present in the protein
sample reacts with folin‟s phenol reagent to give a blue color complex. The
color intensity was measured colorimetrically at 620nm.
REAGENTS REQIRED:
1. Stock solution: 100mg of BSA (Bovine Serum Albumin) is made up to
100ml with distilled water in SMF.
2. Working standard solution: 10ml of stock standard is made up to 100ml
with distilled water in SMF.
3. Alkaline copper reagent:
Solution A: 2% Sodium carbonate in 0.1N Sodium hydroxide
Solution B: 0.1% Copper sulphate in 1% Sodium potassium tatrate
4. Mix 50ml of solution A with 1ml of solution B prior to use.
5. Folin‟s phenol reagent: Dilute the reagent with water in 1:2 ratio.
PROCEDURE:
0.5, 1.0, 1.5, 2.0 and 2.5ml of the working standard solution of protein is
pipetted out into a clean dry test tube marked as S1 to S5 respectively. The
given unknown solution is diluted to 100ml using distilled water, from that
take 0.5 and 1.0ml into the test tube marked as T1 and T2. The final volume
is made up to 2.5ml with distilled water. 2.5ml of distilled water serves as
blank. Add 4.5ml of Alkaline copper reagent and 0.5ml of folin‟s reagent to
all the tubes. The contents are mixed well and kept it in a room temperature
for 30min‟.The intensity of color developed is read colorimetrically at
620nm.
A standard graph is drawn from the obtained values. From the graph the
amount of protein present in the given unknown solution is calculated.
S.No
Contents
Blank
S1
Standards
S2 S3 S4
S5
Test
Solution
T1 T2
Volume of Working Standard
1 Solution (ml)
----0.5
1 1.5
2 2.5 ----- ----Concentration of working standard
(µg)
2
----50 100 150 200 250
3 Volume of Unknown Solution (ml)
0.5
1
4 Volume of Distilled Water (ml)
2.5
2 1.5
1 0.5
2
1.5
Volume of Alkaline Copper
Solution
5 (ml)
4.5 4.5 4.5 4.5 4.5 4.5 4.5
4.5
6 Volume of Folin's Reagent (ml)
0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5
Keep all the tubes in room temperature for 30
Minutes
7 Optical density at 620nm
RESULT:
Amount of protein present in the given unknown sample is
_________mg/100ml
EXPERIMENT 5
ESTIMATION OF DNA BY DIPHENYLAMINE METHOD.
Aim:
To estimate the DNA content of the given unknown solution by
diphenylamine method.
Principle:
The Reaction is specific for deoxyribose .Deoxypentose on acid
hydrolysis forms dihydroxy linoleic acidwhich is ablue coloured complex
compound. Inthisreaction,thedeoxyribose of the purine nucleotide alone
react.The value obtained therefore represents only one halfofthe value
ofthedeoxyribose present.
Reagents Required:
1. Phosphoric acid
2. Diphenylamine
3. Standard DNA Solution: 20mg of DNA was dissolved in 100ml
of Trichloroacetic acid and was heated in a boiling waterbath for
10minutes l
Procedure:
0.4 to 2ml of working DNA solution of concentration range of 40 ug
to 200ug was pippetted out in a 5 different test tubes and marked as S1 to
S5.1.0 and 2ml of unknown DNA solution were taken in another 2 test tubes
worked as T1 and T2.The volumes of all test tubes were made up to 2.0 ml
using distilled water.Then 4ml of the diphenylamine reagent was added to
all the test tubes . A blank was simultaneously prepared using 2.0 ml of
distilled water and 4mlof diphenylamine reagent.The test tubes were treated
in a boiling waterbath for 10minutes and then cooled.
The blue colur developed was read using colorimeter at 620nm. A standard
graph was drawn with optical density on the y axis and concentration on X
axiss.Fromthisgraph,the amount of DNA graph present in unknown solution
can be determined.
Result:
The amount of DNA present in the given unknown solution is
__________mg/dl
S.No
Contents
Blank
Standards
S2
S3
S4
S1
Volume of Working Standard
1 Solution (ml)
----0.4 0.8
1.2 1.6
2 Concentration of standard (µg)
----3 Volume of Unknown Solution (ml)
4 Volume of Distilled Water (ml)
2.0
1.6 1.2
0.8 0.4
Volume of diphenyl amine reagent
4.0 4.0
4.0 4.0
5 (ml)
4.0
Keep all the tubes in boiling water bath for 10 Minutes
6 Optical density at 620nm
S5
Test
Solution
T1
T2
2.0
-----
-----
4.0
1.0
1.0
4.0
2.0
4.0
EXPERIMENT 6
EFFECT OF PH ON THE ACTIVITY OF SALIVARY AMYLASE
Aim
To determine the optimum PH for salivary amylase.
Principle
Amylase present in salivary secretion hydrolysis α1,4-linked D-Glucose units and
produce maltose as a final product.
Materials required
1. Substrate
2. Activator
3. Inhibitor
4. Buffer
Solution A
water.
Solution B
water.
:1% Starch solution
:1% Sodium chloride
:2N Sodium hydroxide
:Phosphate buffer
: 0.2m Disodium hydrogen phosphate-28.392g/1000ml distilled
: 0.2m Sodium dihydrogen phosphate-31.202g/1000ml distilled
S.No
Solution A(ml)
Solution B(ml)
PH
1.
6.5
93.5
5.7
2.
18.5
81.5
6.2
3.
49.0
51.0
6.8
4.
84.0
16.0
7.5
5.
94.7
5.3
8.0
5. Dinitro salicylate:30g of sodium potassium tatrate, 1g of 3,5- Dinitro salicylate
and 1.6g of sodium hydroxide is weighed and made upto 100ml
6. Enzyme source
:1ml of saliva is diluted to 20ml.
Procedure
A set of testtubes marked as ET and EB are taken. Add 2.5ml of buffer (prepared at
various PH ), 2.5ml of substrate followed by 1ml of activator to all the tubes, the tubes
are then preincubated at 37’ C for 10min. Add 0.5ml of enzyme to tubes marked as ET
and incubated at 37’ C for 15min, then the reaction is arrested by adding 0.5ml of sodium
hydroxide, then add 0.5ml of enzyme to the test tube marked as EB, then 0.5ml of DNS is
added to all the test tubes. The colour developed is read at 540nm
RESULT
The optimum PH for salivary amylase is _____________
S.No Contents
B
5.7
1.
6.8
7.5
8.6
Volume of buffer
2.5
2.5
(ml)
2.
Volume of
2.5
2.5
substrate(ml)
3.
Volume of
1.0
1.0
Activator(ml)
Preincubate all the tubes at 37’ C for 10min
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
4.
Volume of
0.5
Dis.water(ml)
5.
Volume of Enzyme
(ml)
Incubate all the tubes at 37’ C for 15min
-
-
-
-
-
-
-
0.5
-
0.5
-
0.5
-
0.5
6.
7.
8.
Volume of NaoH
(ml)
Volume of Enzyme
(ml)
Volume of DNS (ml)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
-
0.5
-
0.5
-
0.5
-
0.5
-
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Keep all the tubes in boiling water bath for 5min
9.
10.
Optical density at
540nm
Difference in OD
EXPERIMENT 7
Estimation of catalase activity (CAT) (EC 1.11.1.6)
Catalase (CAT) scavenges highly toxic H2O2 produced in several reactions in the cell, thus
preventing injury to metabolic machinery of the tissue. It detoxifies H2O2 without
consuming cellular reducing equivalents and provides cell with energy efficient mechanism
to remove H2O2. It is found abundantly in plant tissues and its activity is associated with
peroxisomes where, it removes H2O2 produced during photorespiration.
Reagents
1. Assay buffer: 0.1 M potassium phosphate buffer (pH 7.0)
2. 0.2 M H2O2
3. Dichromate reagent: Prepare 5% potassium dichromate. Mix it with glacial acetic acid in
the ratio of 1:3
Procedure
1. To 0.55 ml of 0.1 M potassium phosphate buffer (pH 7.0), add 0.4 ml of 0.2 M
H2O2 and 50 μl of enzyme extract. Mix thoroughly and incubate for one
minute.3. Add 3.0 ml of 5% potassium dichromate: acetic acid (1:3) solution
to it. . Run a control containing 0.6 ml assay buffer and 0.4 ml of 0.2 M H2O2
without enzyme extract along with the samples. Keep the tubes in boiling
water bath for 10 min, cool and record the absorbance at 570 nm using
dichromate: acetate solution as blank.
2.
Subtract the absorbance of samples from that of control and calculate the
amount of H2O2 from the standard curve. One CAT unit is defined as amount
of enzyme required to consume one μmol of H2O2 min-1 or mg-1 protein.
EXPERIMENT 8
Determination of Total Carbohydrate by Anthrone Method
PRINCIPLE
Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric
acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural.
This compound forms with anthrone a green coloured product with an
absorption maximum at 630 nm.
MATERIALS

2.5 N HCl

Anthrone reagent: Dissolve 200 mg anthrone in 100 mL of ice-cold 95%
H2SO4. Prepare fresh before use.

Standard glucose: Stock—Dissolve 100 mg in 100 mL water. Working
standard—10 mLof stock diluted to 100 mL with distilled water. Store
refrigerated after adding a fewdrops of toluene.
PROCEDURE
2. Weigh 100 mg of the sample into a boiling tube.Hydrolyse by keeping it
in a boiling water bath for three hours with 5 mL of 2.5 N HCl and cool
to roomtemperature.Neutralise it with solid sodium carbonate until the
effervescence ceases.Make up the volume to 100 mL and
centrifuge.Collect the supernatant and take 0.5 and 1 mL aliquots for
analysis.Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of
the working standard.'0' serves as blank.Make up the volume to 1 mL in
all the tubes including the sample tubes by adding distilled water.Then
add 4 mL of anthronereagent.Heat for eight minutes in a boiling water
bath.Cool rapidly and read the green to dark green colour at 630 nm.
Draw a standard graph by plotting concentration of the standard on the Xaxis versus absorbance on the Y-axis.From the graph calculate the
amount of carbohydrate present in the sample tube.
S.No
1
2
3
4
5
7
Contents
Blank
B
S1
Standards
S2 S3 S4
Volume of Working Standard
Solution
(ml)
----0.2 0.4 0.6 0.8
Concentration (µg)
----20 40 60 80
Volume of Unknown Solution (ml)
--------- ---- ----- ----Volume of Distilled Water (ml)
1.0
0.8 0.6 0.4 0.2
Volume of Anthrone Reagent (ml)
4.0
4.0 4.0 4.0 4.0
Keep all the tubes in boiling water bath for 10 Minutes
Optical density at 620nm
S5
Test
Solution
T1 T2
1.0
100
--------4.0
--------0.2
0.8
4.0
CALCULATION
Amount of carbohydrate present in 100 mg of the sample
= (mg of glucose / volume of test sample) X 100
NOTE:
Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.
EXPERIMENT 9
Qualitative Analysis of Carbohydrates
No.
1
Test
Molisch’s Test
2-3 drops of betanaphthol solution are
added to 2ml of the test
Observation
Inference
A deep violet
coloration is produced Presence of
at the junction of two carbohydrates.
layers.
--------0.4
0.6
4.0
solution. Very gently add
1ml of Conc. H2SO4 along
the side of the test
tube..
Iodine test
Presence of
polysaccharide.
4-5 drops of iodine
Blue colour is observed.
solution are added to 1ml
2 of the test solution
Absence of
No Blue colour is
polysaccharide
andcontents are mixed
observed.
gently.
Fehling's test
About 2 ml of sugar
A red precipitate is
solution is added to about
formed
2 ml of Fehling’s solution
taken in a test-tube. It is
No red precipitate is
then boiled for 10 min
formed
Presence of
reducing sugar
Absence of
reducing sugar
Benedict’s test
4
To 5 ml of Benedict's
solution, add 1ml of the
test solution and shake
each tube. Place the tube
in a boiling water bath and
heat for 3 minutes.
Remove the tubes from
the heat and allow them to
cool.
Formation of a green,
red, or yellow
precipitate
Presence of
reducing sugars
No formation of a
green, red, or yellow
precipitate
Absence of
reducing sugars
Barfoed’s test
5
To 2 ml of the solution to
be tested added 2 ml of
freshly prepared Barfoed's
reagent. Place test tubes
into a boiling water bath
and heat for 3 minutes.
Allow to cool.
A deep blue colour is
formed with a red ppt.
settling down at the
bottom or sides of the
test tube.
Presence of
reducing sugars.
Appearance of a
red ppt as a thin
film at the
bottom of the
test tube within
3-5 min. is
indicative of
reducing monosaccharide. If the
ppt formation
takes more time,
then it is a
reducing
disaccharide.
Seliwanoff test
To 3ml of ofSeliwanoff’s
reagent, add 1ml of the
test solution. Boil in
A cherry red colored
water bath for 2 minutes precipitate within 5
minutes is obtained.
Presence of
ketoses
[Sucrose gives a
positive
ketohexose test ]
6
No cherry red colored
solution formed
Absence of
ketoses
.
7
Bial's test
Add 3ml of Bial’s reagent A blue-green product
to 0.2ml of the test
solution. Heat the solution
in a boiling water bath for A muddy brown to gray
2 minutes.
product
Presence of
pentoses.
Presence of
hexoses.
8
Osazone Test
Two two ml of the test
solution, add 3ml of
phenyl hydrazine
hydrochloride solution and
mix. Keep in a boiling
water bath for 30mts. Cool
the solution and observe
the crystals under
microscope.
Formation of beautiful
yellow crystals of
osazone
Needle shaped crystals
Hedgehog crystals
Sunflower shaped
crystals
Glucose/fructose
Presence of
lactose
Presence of
maltose
EXPERIMENT 10
Qualitative Analysis of Amino Acid
S.NO Experiment
1
Ninhydrin Test:
To 1ml of amino acid solution taken in a
test tube, add few drops of ninhydrin
reagent and vortex the contents. Place the
test tube in a boiling water bath for 5
minutes and cool to room temperature.
2
Inference
Indicates the
presence of
alpha amino
acids
The colourof the
solutionchanges to
orange
Indicates the
presence of
aromatic
amino acids
The appearance of
Indicates the
Indicates the
presences of
imino acids
Xanthoproteic acid Test:
To 1ml of the amino acid solution taken in
a test tube, add few drops of nitric acid and
vortex the contents. Boil the contents over a
Bunsen flame, using a test tube holder, for
few minutes. Cool the test tube under
running tap water and add few drops of
alkali.
3
Observation
Observe the colour of
the solution. If the
colour of thesolution
is blue,
And
if the colour
ofsolution is yellow,
Pauly'sdiazo Test:
Take 1ml of sulphanilic acid reagent in a
test tube and chill the contents in a small ice
bucket. Add few drops of prechilled sodium
nitrite solution and vortex. Add immediately
few drops of pre chilled amino acid solution
and vortex. This is followed by dropwise
addition of sodium carbonate solution until
the color appears.
4
The colour of the
solution in the test
tube changes to blue
This indicates
the presence
of Histidine
purple-violet ring
appears in the test
tube
Indicates the
presence of
tryptophan
.
Sakaguchi Test:
To 1 ml of prechilled amino acid solution
and few drops of NaOH is mixed and 2
drops of alpha naphthol is added. Mix
thoroughly and add 4-5 drops of
hypobromite reagent and observe.
8
Indicates the
presence of
tyrosine
Hopkins-Cole Test:
Mix 1 ml of the amino acid solution with 1
ml acetic acid – glyoxilic acid reagent, in a
test tube, vortex. Then carefully, add conc.
Sulphuric acid along the side of the test
tube, keeping the tube in an inclined
position (do not shake the test tube , while
adding acid)
7
The colour
of the solution in the
test tube changes to
red
Histidine Test:
To 1ml of amino acid solution, add 5%
bromine in 33% acetic acid until an yellow
color was formed. After 10 minutes, add
2ml of 5% ammonium carbonate solution
and placed in a boiling water bath for 10
minutes.
6
presence of
histidine and
tyrosine
Millon's Test:
To 1ml of the amino acid solution in a test
tube, add few drops of millon’s reagent and
vortex. Boil the contents over a Bunsen
flame for 3 – 5 minutes. Cool the contents
under running tap water and add few drops
of sodium nitrite solution.
5
red colour during the
addition of
sodumcarbonate
solution
Lead sulphide Test:
The color of the
solution in the test
tube changes to red
Indicates the
presence of
arginine
A black precipitate
appears in the test
tube
Indicates the
presence of
Cysteine
To 1ml of the amino acid solution taken in The content of the
a test tube, add few drops of sodium
test tube changes to
hydroxide (5N), followed by addition of few red colour
drops of glycine (1%) and 10% sodium
nitroprusside solution and vortex. Place the
test tube in a hot water bath, maintained at
40°C, for 15 minutes. Cool the test tube in
ice cold water for 5 minutes and add 0.5ml
of 6N HCl. Vortex the contents and allow to
stand for 15 minutes at room temperature.
Indicates the
presence of
Methionine
To 1ml of the amino acid solution taken in
a test tube, add few drops of sodium
hydroxide (40%) and boil the contents for 5
– 10min over a bunsen burner. Cool the
contents and add few drops of 10% Lead
acetate solution and observe.
9
Folin's McCarthy Sullivan Test: