21BTC201L – BIOCHEMISTRY LABORATORY

Offered to
II YEAR - B.TECH – BIOTECHNOLOGY
Specializations: Genetic Engineering

Department of
Biotechnology School of
Bioengineering
College of Engineering and Technology
SRM Institute of Science and
Technology Kattankulathur - 603203
2024
 SYLLABUS

BIOCHEMISTRY LABORATORY

Lab 1 – Introduction to commonly used instruments and laboratory safety – Video lecture
Exp. No.: 1 – Stoichiometric Calculations
Lab 2 - Preparation and measurement of pH of standard buffers
Exp. No.: 2 - Measurement of pH using pH metre
Exp. No.: 3 - Preparation of Biological Buffers
Lab 3 - Qualitative analysis of Monosaccharide in food samples
Exp. No.: 4 – Reducing hexose sugars: Aldose (Glucose) and Ketose (Fructose)
Exp. No.: 5 - Reducing pentose sugar and hexose sugar (Aldose – Ribose/Ketose
– Ribulose)
Lab 4 - Qualitative analysis of Disaccharides in food samples
Exp. No.: 6 – Reducing (Maltose/Lactose) and Non-Reducing disaccharide (Sucrose)
Lab 5 - Qualitative analysis of Polysaccharides in food samples
Exp. No.: 7 – Polysaccharide –Before/after hydrolysis with acid/enzyme (Starch)
Lab 6 - Qualitative analysis of Fatty Acids and Lipids in food samples
Exp. No.: 8 - Fatty Acids and Cooking oil
Lab 7 - Separation of amino acids on Thin Layer Chromatography
Exp. No.: 9 – Separation of amino acids by TLC
Lab 8 - Estimate blood glucose, compares normal and diabetes mellitus samples
Exp. No.: 10 – Glucose estimation by Dinitro salicylic acid method
Lab 9 - Quantitative analysis of proteins
Exp. No.: 11- Estimation of proteins by Lowry’s method
Lab 10 - Quantitative estimation of serum cholesterol
Exp. No.: 12 – Estimation of Cholesterol by Zak’s method

REFERENCE: Biochemistry Laboratory Manual

 INDEX

Exp. Teacher’s
Name of the experiment Page Number Date
No. Signature

1 Stoichiometric Calculations

2 Measurement of pH using pH metre

3 Preparation of Biological Buffers

4 Aldose (Glucose) and Ketose (Fructose)

Reducing pentose sugar (Aldose –
5
Ribose/Ketose – Ribulose)
Reducing (Maltose/Lactose) and
6
Non- Reducing (Sucrose)
disaccharide
Polysaccharide –Before/after hydrolysis
7 with acid/enzyme (Starch)
8 Fatty Acids and Cooking oil

9 Separation of amino acids by TLC

Glucose estimation by Dinitro salicylic acid

10
method
11 Protein Estimation by Lowry’s method

12 Cholesterol Estimation by Zak’s method

Each experiment carries 4 marks and will be considered for 60% weightage as CLA

End Semester (University Practical) – 40%

 EXP NO: 1
DATE:

STOICHIOMETRIC CALCULATIONS
NORMALITY:
The normality of a solution is defined as the number of gram equivalents of a substance in 1
litre of the solution. Normality is denoted by the symbol ‘N’. For example, if a solution is termed as
1N, it contains 1 gram equivalent of the substance in 1 litre of the solution.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑚
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡𝑠
𝑙𝑖𝑡𝑟𝑒

𝑔𝑖𝑣𝑒𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)

where,

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑚
𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡𝑠 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡

and
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟
𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡
𝑊𝑒𝑖𝑔ℎ𝑡 =
𝑣𝑎𝑙𝑒𝑛𝑐𝑦

MOLARITY:
The molarity of a solution is defined as the number of moles of a substance in 1 litre of
the solution. Molarity is denoted by the symbol ‘M’. For example, if a solution is termed as 1M it
contains 1 mole of the substance in 1 litre of the solution.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑚
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑠
𝑙𝑖𝑡𝑟𝑒

𝑔𝑖𝑣𝑒𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)

where,

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑚
𝑚𝑜𝑙𝑒𝑠 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡

MOLALITY:
The molality of the solution is defined as the number of moles of a substance in 1Kg of the
solvent. Molality is denoted by the symbol ‘m’.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓
𝑀𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑠
𝐾
𝑔
𝑠𝑜
𝑙𝑣
𝑒𝑛
𝑡
 𝑔𝑖𝑣𝑒𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)
where,

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓
𝑚𝑜𝑙𝑒𝑠 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡
OSMOLARITY:
The osmolarity of the solution is defined as the number of osmoles of a solute per litre of
the solution. Osmolarity is denoted by the symbol ‘Osm’ or ‘osmol’. Osmolarity is distinct from
molarity because it measures osmoles of solute particles rather than moles of solute. The distinction
arises because some compounds can dissociate in solution, whereas others cannot.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓
𝑂𝑠𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑂𝑠𝑚𝑜𝑙𝑒𝑠
𝑙𝑖𝑡𝑟𝑒

OSMOLALITY:
The osmolality of the solution is defined as the number of osmoles of a solute per kg of the solvent.
Osmolarity is denoted by the symbol ‘Osm’ or ‘osmol’.

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓
𝑂𝑠𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑂𝑠𝑚𝑜𝑙𝑒𝑠
𝐾𝑔 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

CALCULATIONS:
 MARK SPLIT UP
COMPONENTS Present but OBTAINED
Absent
Present not completed

Procedure (On the day of experiment) 1 0

Performance, Observation and Viva

2 1.5 0
(On the day of experiment)
Results and Inference with any
reference and answers for 1 0.5
questions
(On next experiment)
TOTAL MARKS 4 2.0 0.5

Signature of the Faculty with date

EXP NO: 2
DATE:

MEASUREMENT OF pH USING pH metre

AIM:
To become familiar with operating the pH metre, and to learn how to use the Henderson-
Hasselbalch equation to make buffer solutions at a desired pH value.
PRINCIPLE:
The pH metre measures the electrical potential developed across a pair of electrodes by
dipping it into a solution. For the measurement of pH, an electrode system sensitive to changes in
the hydrogen ion concentration of the solution is chosen. This electrode system consists of a
sequence of electrodes whose potential varies with the pH of the solution.

DIGITAL PH metre
REQUIREMENTS:
1. 0.1N HCl
2. 0.1N NaOH
3. Coloured Soft Drink
4. Colourless Soft Drink
5. Urine
6. Tap Water
7. Lime Juice
8. Pomegranate Juice
9. pH metre
10. Stirrer
11. Glass rod etc…

PROCEDURE:
The pH metre is cleaned using distilled water and the probe is patted dry using soft tissue.
After cleaning, the pH metre was calibrated using the standard buffers and cleaned again. The test
sample was taken in a beaker and its pH was recorded. Following this, 1 drop of 0.1N HCl was
added to the sample and the pH was recorded again. Next two drops of 0.1N HCl were added to the
sample and the pH was recorded. Finally, 5 drops of HCl was added to the sample and the pH was
recorded. The procedure was repeated using 0.1N NaOH in the place of HCl with a fresh sample
and the pH was recorded.

RESULT:
OBSERVATIONS:
Coloured Your own
Fresh Colourless
Tap Water Milk Soft samples if
citrus juice Soft Drink
Drink any
Test Sample
(As it is pH)
(+) 1 drop
HCl
(+) 2 drops
HCl
(+) 5 drops
HCl

(+) 1 drop
NaOH
(+) 2 drops
NaOH
(+) 5 drops
NaOH
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TOTAL MARKS 4 2.0 0.5

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EXP NO: 3
DATE:

PREPARATION OF BIOLOGICAL BUFFERS

AIM:
To prepare the biological buffers and to measure their pH.

PRINCIPLE:
Buffers may be mixtures of weak acids and their conjugate base or weak bases and their
conjugate acids. They resist changes in pH upon addition of small amounts of strong acids or bases.
In a physiological system, this property is vital for the maintenance of homeostasis. For eg. Most of
the body fluids contain natural buffering systems such as blood, saliva, urine, mucous etc.

1. Citrate buffer (pH 5.5):

Stock solutions:
1. 0.1M Solution of Citric acid
21.01g of citric acid is dissolved in 1000 ml of distilled water.
2. 0.1M Solution of Sodium citrate
29.41 g of sodium citrate is dissolved in 1000 ml of distilled water.

PROCEDURE:
Pipette out exactly 16.0 ml of citric acid solution into a 100 ml standard flask. To this add
exactly 34.0 ml of sodium citrate. The solution is made up to 100 ml with distilled water. The
resultant solution is 0.1M citric acid-sodium citrate buffer whose pH is measured using a pH metre.
The pH metre is first standardised using a standard buffer and the electrode is washed with
distilled water. The pH of the citrate buffer is measured as 5.5.

2. Carbonate-Bicarbonate Buffer (pH 10.0):

Stock solutions:
1. Sodium Carbonate solution (0.2M)
Dissolve 2.12 g of anhydrous sodium carbonate in 100 ml of water.
2. Sodium Bicarbonate Solution (0.2M)
 Dissolve 1.68 g of sodium bicarbonate in 100 ml of water.
PROCEDURE:
Pipette out exactly 27.5 ml of sodium carbonate solution (0.2M). To this added 22.5 ml of
sodium bicarbonate solution (0.2M). The resultant solution is 0.1M carbonate buffer whose pH is
measured using a pH metre.
The pH metre is first standardised using a standard buffer and the electrode is washed with
distilled water. The pH of the carbonate buffer was measured as 10.

3. Glycine - HCl Buffer (pH 2.5):

Stock solutions:
1. 0.2 M of Glycine
Dissolve 1.501 g of Glycine in 100 ml of water.
2. 0.2 M of HCl
PROCEDURE:
Take 50mL of 0.2 M of Glycine solution and add 32.5 ml of 0.2 M HCl solution and make
up to 100mL. The resultant solution is Glycine -HCl buffer whose pH is measured using a pH
metre.
The pH metre is first standardised using a standard buffer and the electrode is washed with
distilled water. The pH of the Glycine -HCl buffer is measured as 2.5.

4. Tris Buffer (pH 8.0):

Stock solutions:
1. 0.2 M of Tris
Dissolve 1.121 g of Tris in 100 ml of water.
2. 0.2 M of HCl

PROCEDURE:
Take 50mL of 0.2 M of Tris solution and add 26.8mL of 0.2 M HCl solution and make up to
200mL. The resultant solution is Tris - HCl buffer whose pH is measured using a pH metre.
The pH metre is first standardised using a standard buffer and the electrode is washed with
distilled water. The pH of the Tris -HCl buffer is measured as 8.0.
5. Phosphate Buffer (pH 7.0):
Stock solutions:
1. Monobasic sodium phosphate solution (0.2M)
Dissolve 2.78 g at monobasic sodium phosphate in 100ml of water
2. Dibasic sodium phosphate solution (0.2M)
Dissolve 5.365g of dibasic sodium phosphate heptahydrate and 7.179 g of dibasic
sodium phosphate deco carbonate in 100 ml of water.

PROCEDURE:
Mix 39 ml of monobasic sodium phosphate solution with 61 ml of dibasic sodium
phosphate solution. This is made up to 200 ml with distilled water. The resultant solution is 0.1 M
phosphate buffer whose pH is measured using a pH metre.

The pH metre is first standardised using a standard buffer and the electrode is washed with
distilled water. The pH of the phosphate buffer is measured as 7.

RESULT:
The buffers of the required pH were prepared.
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Procedure (On the day of experiment) 1 0

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TOTAL MARKS 4 2.0 0.5

Signature of the Faculty with date

 QUALITATIVE ANALYSIS OF SUGARS – GENERAL PROCEDURE

NAME PRINCIPLE PROCEDURE OBSERVATIONS

Molisch’s Test Molisch’s Test is a sensitive Place 2 mL of a known A red or purple-coloured

chemical test for all carbohydrate solution in a test tube, compound was observed at the
carbohydrates, and some add 1 drop of Molisch’s reagent interface.
compounds containing (10% α-naphthol in ethanol).
carbohydrates in a combined Pour 1-2 mL of conc. H2SO4 down
form, based on the dehydration the side of the test tube, so that it
of the carbohydrate by sulfuric forms a layer at the bottom of the
acid to produce an aldehyde tube.
(either furfural or a derivative), Observe the colour at the interface
which then condenses with the between two layers and compare
phenolic structure resulting in a your result with a control test.
red or purple-coloured
compound. A brown colour due to charing must
be ignored and the test should be
repeated with a more dilute sugar
solution.
Iodine Test Iodine dissolved in an aqueous A few drops of 0.01 M iodine in The immediate formation of a
solution of potassium iodide 0.12 M KI are added to a 1% vivid blue colour indicates
reacts with the starch producing solution of the carbohydrate in amylose. With starch a blue-
a purple black colour. The question. black colouration forms due to
colour can be detected visually the polyiodide complex formed.
with concentrations of iodine as
low as 0.00002M at 20°C.
However the intensity of the
colour decreases with increasing
temperature and with the
presence of water-miscible,
 organic solvents such as ethanol.
Also the test cannot be done at
very low pHs due to the
hydrolysis of the starch under
these conditions.

Fehling’s Test Fehling’s solution reacts with Fehling’s I consists of 7 g of A red precipitate of cuprous
reducing sugars on heating and hydrated copper (II) sulphate oxide forms at the end of the
reduces the Cu (II) ion to Cu (I) dissolved in 100 mL of distilled reaction.
producing a precipitate of red water.
copper oxide. Fehling’s II is made by dissolving
35 g of potassium sodium tartrate
and 10 g of sodium carbonate in
100 mL of dist. water.
Fehling's reagent: Equal volumes
of Fehling I and Fehling II are
mixed to form a deep blue solution.
To 1 mL of Fehling’s solution A
(aqueous solution of CuSO4) add 1
mL of Fehling solution B (solution
of potassium tartrate).
Add 2 mL of the sugar solution,
mix well and boil.
Barfoed’s Test Barfoed’s reagent, cupric acetate Barfoed's reagent consists of a A brick-red cuprous oxide
in acetic acid is slightly acidic 0.33 molar solution of neutral precipitate forms if reduction
and is balanced so that it can copper acetate in 1% acetic has taken place.
only be reduced by acid solution. The reagent does not
monosaccharide but not less keep well and it is therefore
powerful by reducing sugars. advisable to make it up when it is
Disaccharides may also react actually required.
with this reagent, but the To 1-2 mL of Barfoed’s reagent,
reaction is much slower when add an equal volume of sugar
compared to monosaccharides. solution.
 Boil for 5 min. in a water bath and
allow standing.

Benedict’s Test Benedict’s solution reacts with Benedict's reagent A colour change through green to
reducing sugars on heating and Solution 1 yellow, brown and finally to red
reduces the Cu(II) ion to Cu(I) Sodium citrate 86.5g indicates the presence of reducing
producing a precipitate of red Sodium carbonate sugar.
copper oxide. The resulting (anhydrous) 50g
colour change depends on the Dissolve in 400mls H2O
type and concentration of sugar, Solution 2
so this test can be used semi- Copper sulphate 5H2O 8.7g
quantitatively to indicate Dissolve in 50 ml of H2O. Add 2 to
approximate concentrations. 1 with rapid stirring then dilute to
500 ml. To 2 ml sugar solution, add
2 ml of Benedict’s reagent and
stand the test tube in boiling water
for a
few minutes.
Bial’s Test: The components include Bial’s reagent:
orcinol, hydrochloric acid, and Dissolved 1.5gms of orcinol in 50
ferric chloride. A pentose, if ml of conc. HCl and added 20 to
present, will be dehydrated to 30 drops of 10% solution of ferric
form furfural which then reacts chloride in water.
with the orcinol to generate a
Procedure:
coloured substance. The
solution will turn bluish and a Mix 5 ml of Bial’s reagent with 5
precipitate may form. ml of sugar solution and keep in a
boiling water bath.
1 mL of sugar solution with 3 mL
Seliwanoff’s reagent (0.5 g
resorcinol per litre 10% HCl) in
boiling water.
Seliwanoff’s Test Seliwanoff’s Test distinguishes Heat 1 mL of sugar solution with 3 In less than 30 seconds, a red
between aldose and ketose mL of Seliwanoff’s reagent (0.5g colour must appear for ketoses.
sugars. Ketoses are resorcinol per litre of 10% HCl) in Upon prolonged heating, glucose
distinguished from aldoses via boiling water. will also give an appreciable
their ketone/aldehyde colour.
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.
Phenylhydrazine Osazone formation involves Place 0.2 g of the unknown sample The times required for the
hydrazone formation at C-1 of in a test tube and add 0.4 g of formation of the osazones can be
test an aldose (or C-2 of a ketose) phenylhydrazine hydrochloride, 0.6 a valuable aid in distinguishing
and oxidation of C-2 (or C-1) of g of crystallised sodium acetate, and among various sugars. The
an alcohol group to a ketone (or 4 mL of distilled water. Place the following figures are the times
an aldehyde). The new carbonyl test tube in a beaker of boiling required for the osazone to
group is also converted to a water. Note the time that the test precipitate from the hot solution:
hydrazone. It has been tube was immersed and the time of fructose (2 min); glucose (4-5
suggested that the reaction stops the precipitation. After 20 min, min); xylose (7 min); arabinose
here (rather than further remove the test tube from the hot (10 min); galactose (15-19 min);
oxidation at C-3, etc.) because water bath and set it aside to cool. raffinose (60 min); lactose,
of hydrogen- bonding A small amount of the liquid and osazone soluble in hot water;
stabilisation of the osazone. solid is poured on a watch glass. maltose, osazone soluble in hot
Tip the watch glass from side to water; mannose, 0.5 min
side to spread out the crystals, and (hydrazone); sucrose, 30 min
absorb some of the mother liquid (owing to hydrolysis and
with a piece of filter paper, taking formation of glucosazone).
care not to crush or break up the
clumps of crystals. Examine the
crystals under
a low-power microscope (about 80-
100×), and compare with
 photomicrographs. The formation
of tarry products due to oxidation of
the phenylhydrazine may be
prevented by the addition of 0.5 mL
of saturated sodium bisulfite
solution. This should be done
before heating if it is desired to
isolate the osazone and determine
its melting point.

OSAZONE CRYSTALS UNDER MICROSCOPE

GLUCOSAZONE CRYSTALS XYLOSAZONE CRYSTALS

FRUCTOSAZONE CRYSTALS MALTOSAZONE CRYSTALS

GALACTOSAZONE CRYSTALS LACTOSAZONE CRYSTALS
EXP NO: 4
DATE:

QUALITATIVE ANALYSIS OF SUGARS

REDUCING MONOSACCHARIDE – ALDOSE (Glucose)

NAME PROCEDURE OBSERVATION INFERENCE

Solubility Test
1. A small amount of the substance was 1. Soluble in water
dissolved in water and observed.

2. A small amount of the substance was 2. Soluble in hot water

dissolved in hot water and observed. May be a
monosaccharide
3. A small amount of the substance was 3. Soluble in dilute HCl
dissolved in dilute HCl and observed.

4. A small amount of the substance was 4. Soluble in alcohol

dissolved in alcohol and observed.
Molisch’s Test To 2 drops of Molisch’s reagent add 2 ml of sugar
solution in a test tube and add 2 ml of conc. H2SO4 Reddish violet ring is formed Presence of
by the sides of the tube at the junction of two liquids carbohydrates.
Iodine Test 1 to 2 drops of iodine reagent is added 2 to 3 ml
of sugar solution. No colour change. Absence of
polysaccharide
Barfoed’s Test To 8 drops of sugar solution add 5 ml of
Barfoed’s reagent placed in a water bath for a
few minutes and allow to stand. Red precipitate was formed Presence of a
monosaccharide.
Benedict’s Test To 8 drops of sugar solution added 5 ml of
Benedict’s reagent in a test tube and kept in the
boiling water bath for 2 minutes. Blue colour turns orange. Presence of reducing
sugar.
Fehling’s Test To a few drops of sugar solution added 5 ml of A brownish orange precipitate Presence of reducing
Fehling’s solution and heated the mixture. was obtained sugar.

Bial’s Test: Mix 5 ml of Bial’s reagent with 5 ml of sugar No green colour was observed Absence of pentose
solution and keep in a boiling water bath. sugar.
Seliwanoff’s Test A few drops of sugar solution added 5 ml of
Seliwanoff’s reagent and kept it in a boiling No cherry red colour was Absence of ketose
water bath for a few minutes. observed sugar.
Phenylhydrazine Reagent: 20 gm of phenyl hydrazine chloride is
test dissolved in 120 ml of water, slightly warmed to Yellow precipitate is formed Presence of reducing
get a solution and a few ml of solution and 2 ml sugar.
of
glacial acetic acid is added.
Procedure: To 2 to 3 ml of sugar solution added Fine needle or feather shaped
2 to 3 drops of phenyl hydrazine, 8 drops of Presence of
crystals are formed within 10-
glacial acid and a pinch of sodium acetate and Glucosazone
heated in boiling water bath. 12 minutes
 REDUCING MONOSACCHARIDE – KETOSE (Fructose)

NAME PROCEDURE OBSERVATION INFERENCE

Solubility Test
1. A small amount of the substance was dissolved
in water and observed.

2. A small amount of the substance was dissolved

in hot water and observed.

3. A small amount of the substance was dissolved

in dilute HCl and observed.

4. A small amount of the substance was dissolved

in alcohol and observed.

Molisch’s Test To 2 drops of Molisch’s reagent add 2 ml of sugar

solution in a test tube and added 2 ml of conc.
H2SO4 by the sides of the tube
Iodine Test 1 to 2 drops of iodine reagent is added 2 to 3 ml of
sugar solution.

Barfoed’s Test To 8 drops of sugar solution add 5 ml of Barfoed’s

reagent placed in a water bath for few minutes and
allow to stand.

Benedict’s Test To 8 drops of sugar solution added 5 ml of

Benedict’s reagent in a test tube and kept in the
boiling water bath for 2 minutes.

Fehling’s Test To a few drops of sugar solution added 5 ml of

Fehling’s solution and heated the mixture.

Bial’s Test: Mix 5 ml of Bial’s reagent with 5 ml of sugar

solution and keep in a boiling water bath.

Seliwanoff’s 1 mL of sugar solution with 3 mL Seliwanoff’s

Test reagent (0.5 g resorcinol per litre 10% HCl) in
boiling water.
To few drops of sugar solution, added 5 ml of
Seliwanoff’s reagent and kept it in boiling water
bath for a few minutes.
Phenylhydrazi To 2 to 3 ml of sugar solution added 2 to 3 drops of
ne test phenyl hydrazine, 8 drops of glacial acid and a
pinch of sodium acetate and heated in boiling water
bath.
OSAZONE CRYSTALS: GLUCOSAZONE CRYSTALS OSAZONE CRYSTALS: FRUCTOSAZONE
CRYSTALS

RESULT:
The given sugars are identified to be ………………………………

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EXP NO: 5
DATE:

QUALITATIVE ANALYSIS OF SUGARS REDUCING MONOSACCHARIDE – PENTOSE

NAME

Solubility Test 1. A small amount

in water and observed.
Iodine Test 1 to 2 drops of iodine reagent is added 2 to 3 ml of
sugar solution.

Barfoed’s Test To 8 drops of sugar solution add 5 ml of Barfoed’s

reagent placed in a water bath for few minutes and
allow to stand.

Benedict’s Test To 8 drops of sugar solution added 5 ml of

Benedict’s reagent in a test tube and kept in the
boiling water bath for 2 minutes.

Fehling’s Test To a few drops of sugar solution added 5 ml of

Fehling’s solution and heated the mixture.

Bial’s Test: Mixed 5 ml of Bial’s reagent with 5 ml of sugar

solution and kept in boiling water bath.
Seliwanoff’s 1 mL of sugar solution with 3 mL Seliwanoff’s
Test reagent (0.5 g resorcinol per litre 10% HCl) in
boiling water.
To few drops of sugar solution added 5 ml of
Seliwanoff’s reagent and kept it in boiling water bath
for few minutes.
Phenylhydrazi To 2 to 3 ml of sugar solution added 2 to 3 drops of
ne test phenyl hydrazine, 8 drops of glacial acid and a pinch
of sodium acetate and heated in boiling water bath.
OSAZONE CRYSTALS: XYLOSAZONE CRYSTALS

RESULT:
The given sugar is identified to be ………………………………..

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 EXP NO: 6
DATE:

QUALITATIVE ANALYSIS OF SUGARS REDUCING DISACCHARIDE –

LACTOSE/MALTOSE

NAME

Solubility
Test 1. A small amount

Iodine Test 1 to 2 drops of iodine reagent is added 2 to 3 ml of

sugar solution.

Barfoed’s To 8 drops of sugar solution add 5 ml of Barfoed’s

Test reagent placed in a water bath for a few minutes and
allow to stand.
Benedict’s To 8 drops of sugar solution added 5 ml of Benedict’s
Test reagent in a test tube and kept in the boiling water
bath for 2 minutes.

Fehling’s Test To a few drops of sugar solution added 5 ml of

Fehling’s solution and heated the mixture.

Bial’s Test: Mixed 5 ml of Bial’s reagent with 5 ml of sugar

solution and kept in boiling water bath.

Seliwanoff’s A few drops of sugar solution added 5 ml of

Test Seliwanoff’s reagent and kept it in a boiling water
bath for a few minutes.
Phenylhydraz To 2 to 3 ml of sugar solution added 2 to 3 drops of
ine test phenyl hydrazine, 8 drops of glacial acid and a pinch
of sodium acetate and heated in boiling water bath.

OSAZONE CRYSTALS:

LACTOSAZONE CRYSTALS MALTOSAZONE CRYSTALS

 NON- REDUCING DISACCHARIDE – SUCROSE

OBSERVATION INFERENCE

NAME PROCEDURE
BEFORE ACID AFTER ACID BEFORE
AFTER ACID
HYDROLYSIS HYDROLYSIS ACID
HYDROLYSIS
HYDROLYSIS
Solubility 1.A small amount of the 1. Soluble in 1. Soluble in water
Test substance was dissolved water
in water and observed. 2. Soluble in hot water
2. Soluble in hot
2.A small amount of the water
substance was dissolved 3. Soluble in dilute HCl
in hot water and May be a sugar
observed. 3. Soluble in
dilute HCl 4. Soluble in alcohol May be a sugar
3.A small amount of the
substance was dissolved
in dilute HCl and 4. Soluble in
observed. alcohol
4.A small amount of the
substance was dissolved
in alcohol and observed.

Molisch’s To 2 drops of Molisch’s Reddish violet Reddish violet ring is Presence of

Test reagent add 2 ml of sugar ring is formed at formed at the junction carbohydrates.
solution in a test tube and the junction of of two liquids
added 2 ml of conc. two liquids
H2SO4 by the sides of the
tube
Iodine Test 1 to 2 drops of iodine No colour No colour change. Absence of Absence of
reagent is added 2 to 3 ml change. polysaccharide polysaccharide
of sugar solution.
Barfoed’s To 8 drops of sugar solution Red precipitate Red precipitate was Absence of Presence of a
Test add 5 ml of Barfoed’s was not formed formed monosaccharide monosaccharide
reagent placed in a water .
bath for a few minutes and
allow to stand.
Benedict’s To 8 drops of sugar No characteristic Blue colour turns Absence of Presence of 2-4g
Test solution added 5 ml of colour change orange. reducing sugar reducing sugar.
Benedict’s reagent in a
test tube and kept in the
boiling water bath for 2
minutes.

Fehling’s To a few drops of sugar No characteristic A brownish orange Absence of Presence of

Test solution added 5 ml of observation precipitate was reducing sugar reducing sugar.
Fehling’s solution and obtained
heated the mixture.
Bial’s Test: Mixed 5 ml of Bial’s No green colour No green colour was Absence of Absence of
reagent with 5 ml of sugar was observed. observed pentose sugar. pentose sugar.
solution and kept in a
boiling water bath.
Seliwanoff’s A few drops of sugar No cherry red Cherry red colour was Absence of Presence of
Test solution added 5 ml of colour was observed ketose sugar. ketose sugar.
Seliwanoff’s reagent and observed.
kept it in a boiling water
bath for a few minutes.
Phenylhydr To 2 to 3 ml of sugar No characteristic Yellow precipitate is Absence of Presence of
azine test solution added 2 to 3 observation. formed reducing sugar reducing sugar.
drops of phenyl Fine needle or feather
hydrazine, 8 drops of
glacial acid and a pinch of shaped crystals are Presence of
sodium acetate and heated formed within 10-12 Glucosazone
in boiling water bath.
minutes

OSAZONE CRYSTALS: GLUCOSAZONE CRYSTALS

RESULT:
The given sugars are identified to be ………………………..
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 EXP NO: 7
DATE:

QUALITATIVE ANALYSIS OF SUGARS POLYSACCHARIDE - STARCH

NAME PROCEDURE
 added 2 ml of conc. H2SO4
by the sides of the tube
Iodine Test 1 to 2 drops of iodine reagent Blue coloured No colour change. Presence of Absence of
is added 2 to 3 ml of sugar solution was polysaccharide polysaccharide
solution. obtained.
Barfoed’s To 8 drops of sugar solution No characteristic Red precipitate May be a Presence of a
Test add 5 ml of Barfoed’s colour change was was formed polysaccharide monosaccharide
reagent placed in a water observed . .
bath for a few minutes and
allow to stand.
Benedict’s To 8 drops of sugar solution No characteristic Blue colour turns Absence of Presence of 2-4g
Test added 5 ml of Benedict’s colour change was orange. reducing sugar. reducing sugar.
reagent in a test tube and observed
kept in the boiling water bath
for 2
minutes.
Fehling’s To a few drops of sugar No characteristic A brownish orange Absence of Presence of
Test solution added 5 ml of colour change was precipitate was reducing sugar. reducing sugar.
Fehling’s solution and heated observed obtained
the mixture.
Bial’s Test Mixed 5 ml of Bial’s reagent No green colour No green colour Absence of Absence of
with 5 ml of sugar solution was observed was observed pentose sugar. pentose sugar.
and kept in boiling water
bath.
Seliwanoff’s A few drops of sugar No cherry red No cherry red Absence of Absence of
Test solution added 5 ml of colour was colour was ketose sugar. ketose sugar.
Seliwanoff’s reagent and observed observed
kept it in a boiling water bath
for a few minutes.
Phenylhydr To 2 to 3 ml of sugar No yellow Yellow precipitate May be a Presence of
azine test solution added 2 to 3 drops precipitate is is formed polysaccharide. reducing sugar.
of phenyl formed.
hydrazine, 8 drops of glacial
acid and a pinch of sodium
 acetate and heated in boiling Fine needle or Presence of
water bath. feather shaped Glucosazone
crystals are formed
within 10-12
minutes

OSAZONE CRYSTALS: GLUCOSAZONE CRYSTALS

RESULT:

The given sugar is identified to be …………………………………

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EXP NO: 8
DATE:
QUALITATIVE ANALYSIS OF FATTY ACIDS AND LIPIDS

AIM: To determine the presence or absence of lipids and fatty acids in given sample

PRINCIPLE:
Lipids are insoluble in water known as hydrophobic biomolecules, but they are soluble in
organic solvents like benzene, chloroform, hexane and diethyl ether. It is a well known fact that
lipids are high energy compounds and are classified as simple, conjugated and derived lipids.
Apart from acting as energy source, lipids have diverse functions ranging from plasma
membrane formation, micelle formation, hormonal action and thermal insulator etc.
Qualitative analysis of fatty acids and lipids involves the identification of characteristic
functional groups and chemical reactions associated with these compounds.

REQUIREMENTS:
Reagents and Chemicals:
1. Alcoholic-KOH (2% w/v KOH in ethyl alcohol): Dissolve 2 g of potassium hydroxide
(KOH) in 100 mL of ethyl alcohol. Dilute potassium permanganate solution (0.05%
w/v): Dissolve 50 mg of potassium permanganate in 100 mL of distilled water.
2. Hubl’s Iodine Solution: To prepare this (a) dissolve 2.6 g of iodine in 40 mL of ethanol
(95% v/v), ( b) dissolve 6.0 g of mercuric chloride in 40 mL of ethanol (95% v/v).
Transfer both solutions ‘a’ and ‘b’ into a beaker and make up the volume to 100 mL
with the same ethanol.
3. Potassium bisulphate (solid)
4. Bromine water
5. Acetic anhydride and Conc. sulphuric acid
6. Solvents: Chloroform, ether, benzene, carbon tetrachloride, hexane,
7. Glassware, water bath, test tube holder, spatula and blotting paper.
8. Test sample
PROCEDURE:

NAME PRINCIPLE PROCEDURE OBSERVATION INFERENCE

Solubility This is a primary step Solubility test: To small

Test to know the chemical quantities of fat/ fatty
nature of the given acid taken in a separate
test sample. Due to test tube add the
the hydrophobic following organic
nature of lipids they solvents separately:
are insoluble in water i) Completely soluble in
i) Indicates presence of fat
and are soluble in 1. a) Chloroform b) ether all four solvents
or oil in the given solution
organic solvents. c) benzene d) hexane (a) - (d).
(3 mL/test tube)
ii) Indicates presence of fat
ii) Found small floating
or oil in the given solution
2. Distilled water droplets on water.

Translucent The lipid will not wet

spot test the filter paper, unlike
water. The lipids will
form a translucent Add a few drops of given Translucent spot will
spot due to their sample on dry and clean appear on the filter
greasy texture, and filter paper and observe. paper. Indicates presence of fat or
penetrate the filter oil in the given solution
paper. Unlike lipids,
the spot of water will
disappear from the
paper.
Free Fatty It is based on the 1. Few drops of The pink colour
Acid Test neutralisation reaction phenolphthalein solution disappears as the alkali
where the alkali is added to a test tube. is neutralised by the
neutralised by adding 2. One or two drops of free fatty acids present
free fatty acids, using dilute NaOH was added, in the oil.
an indicator. sufficient to give the Presence of free fatty acids
solution a pale pink
colour.
3.Add a few drops of
sample and shake.

Saponificati Lipids upon alkaline Take approximately 100 Appearance of Indicates the presence
on test: hydrolysis release mg of oil or fat in a test foamy solution of oil or fat
glycerol and fatty tube. Add 3 mL
acids. Later sodium of alcoholic-KOH and
(Na+) or potassium mix well. Place the
(K+) ions combine tube in a boiling water
with fatty acids to bath for 15-20 min.
form “soap” (foam).
Hence, this is known
as saponification
reaction.
Acrolein Formation of acrolein Take approximately 100 Release of Indicates presence of
test: or acrylic aldehyde mg pungent odour glycerol
that has characteristic of a test sample in a test
pungent odour is the tube and add a pinch of
key principle. In potassium bisulphate and
general lipids upon mix well. Heat the
heating witH mixture
potassium bisulphate over a Bunsen burner
produce acrolein. for 1-2 minutes.

TESTS FOR UNSATURATION

Bromine i) Bromine has the Take 1 mL of oil or 100 i) Decolourisation of i) Indicates presence of
Water ability to undergo an mg of fat in a test tube, potassium unsaturated fats or fatty
Test: addition reaction with add 2 mL of nonpolar permanganate or acids
unsaturated organic solvent (Refer bromine water is
compounds. The solubility test) and observed
reddish-brown colour dissolve completely. To
of bromine water this add potassium ii) No decolorization ii) Indicates presence of
disappears when it permanganate solution or observed. saturated fats and fatty acids
reacts with bromine water drop by
unsaturated drop.
compounds due to the
addition of bromine
 across the double or
triple bonds.
Consumption of more
bromine indicates the
higher percentage of
unsaturation.

ii) Decolorization of
alkaline potassium
permanganate is also
an indirect measure of
unsaturation in fatty
acids, where
unsaturated fatty
acids
undergo incomplete
oxidation

Hubl’s Huble's reagent Huble’s reagent Huble's i) Decolourisation is i) Indicates presence of

Iodine Test comprising iodine is reagent: (a mixture of 7% observed unsaturated fats or fatty
added to the lipid and mercury chloride in acids
the degree of the alcohol and 5% iodine in
unsaturation can be 96% alcohol is added in
determined by the equal proportion). Add ii) No decolorization
3.5 mL of mercury observed.
 decolorizing of the chloride (HgCl2) to a ii) Indicates absence of
reagent. final solution of 50 mL in unsaturated fats and fatty
The degree of ethanol. In a separate acids
unsaturation is beaker, add 2.5 g of
indicated by the iodine in 48 mL of
amount of iodine ethanol to a final volume
solution of 50 mL. Now mix both
accommodated in a the solutions in equal
lipid solution. The pink ratio to a final volume of
colour of the Huble's 100 mL.
reagent with
chloroform disappears Procedure: Added 3 mL
with the addition of of chloroform and add 3
fatty acids to it. The mL of Huble's reagent to
more the number of the test tube and shake
drops of fatty acids well. The solution turns
required, the less is the pink because of the
degree of unsaturation presence of free iodine.
of fatty acid. test samples added in a
dropwise manner.

TESTS FOR CHOLESTEROL

Salkowski Cholesterol in the Take 1 mL of oil or fat Formation of blue-green Indicates presence of
test: presence of sample in a test tube and colour at the cholesterol
concentrated dissolve in 1 mL junction of two liquids
sulphuric acid and chloroform to this add
acetic anhydride equal volume of
undergo dehydration concentrated sulphuric
producing coloured acid along the walls of
 products which are the tube. (Do not mix the
blue-green in colour. contents).

Liebermann Cholesterol forms a Take approximately 100 Formation of an Confirms the presence
- blue green dye from mg of fat or 1mL of oil in emerald green of cholesterol
Burchard polymeric unsaturated a test tube, dissolve by color
test: carbohydrates in an adding 1 mL of
acetic acid/ acetic chloroform and equal
anhydride/ volumes of acetic
concentrated sulfuric anhydride followed by
acid medium in the dropwise addition (along
presence of walls) of concentrated
chloroform. sulphuric acid.

RESULT:
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EXP NO: 9
DATE:

SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY

AIM:
To separate amino acids present in samples by Thin Layer Chromatography (TLC) method.

PRINCIPLE:

Chromatography is a technique used for the separation of closely related compounds in a

mixture. This separation is aided by the difference in equilibrium distribution of components
between the two phases- stationary phase and mobile phase. Thin layer chromatography is used
to obtain quantitative as well as qualitative data. A TLC plate is made up of a thin layer of silica
adhered to glass or aluminium support. The silica is the stationary phase and the solvent mixture
is the mobile phase. Separation results from partition equilibrium of the components in the
mixture. The separation depends on several factors: solubility of compounds, attraction between
the compound and silica and size of compound. An important factor used in thin layer
chromatography is the Rf value.

Separation of amino acids by TLC: Amino acids interact to different extents with the silica based
on their ‘R’ groups. The amino acids that interact strongly with silica move a small distance
while those that do not interact strongly move farther. Since amino acids are colourless
compounds, ninhydrin is used to detect them. Ninhydrin reacts with α-amino acids that result in
purple coloured spots (due to formation of complexes - Ruhemann’s purple). This method is used to
separate amino acids chiefly for preparative purposes that are to purify them rather than simply
analyse them.
MATERIALS REQUIRED:

1. Standard amino acids

2. Distilled water
3. Pre-coated TLC plate
4. TLC Chamber
5. Butanol
6. Acetic acid
7. Spraying reagent
8. Hot air oven
9. Blade and scissors
10. Pencils and rulers etc.

PREPARATION OF REAGENTS:

1. Preparation of mobile phase:

Butanol, acetic acid and water were mixed in the ratio 4:1:1 respectively, for 5 ml.

2. Preparation of ninhydrin solution:

0.3% solution of Ninhydrin in butanol containing 3 ml acetic acid

PROCEDURE:

1. The TLC plate was taken and after covering the silica side with paper, an appropriate size
was chosen and cut with the help of scissors.
2. The smooth base of the TLC plate was determined and using a pencil, a straight,
horizontal line about 0.5 cm from the base was drawn.
3. Spots were the standard and samples had to be loaded were marked at sufficient distance
apart and numbered.
4. Using capillary tubes, a spot of the standard and each of the samples were made on the
TLC plate. The plate was allowed to dry in the air.
5. The TLC chamber was filled with 5 ml of solvent (mobile phase) and the TLC plate was
lowered in an upright position into the chamber such that the solvent was only half-way
to the horizontal line drawn.
6. The chamber was left undisturbed for one hour and the mobile phase was allowed to
move up the silica by capillary action, carrying along with it, the sample molecules.
7. The plate was removed from the TLC chamber and briefly dried in the hot air oven at 60°
Celsius after marking the distance to which the solvent had travelled, with a pencil.
 8. Ninhydrin solution was sprayed on the TLC plate and it was again placed in the hot air
oven for Ruhemann’s purple spots to develop.
9. The TLC plate was removed from the hot air upon development of coloured spots/bands
and observed.

OBSERVATION:

The TLC plate sprayed with ninhydrin solution was placed inside the hot air oven and
allowed to develop. Ruhemann’s purple colour bands were observed in the plate and Rf values
were calculated.

𝑅𝑓 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑚𝑜𝑣𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

RESULT:
Rf Values for Amino acids on TLC
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EXP NO:
10 DATE:

GLUCOSE ESTIMATION BY DINITRO SALICYLIC ACID METHOD

AIM:

Estimation of reducing sugars from given sample(s) by Dinitrosalicylic Acid (DNS).

PRINCIPLE:

Reducing sugars have the property to reduce many of the reagents. One such reagent is
3,5- dinitrosalicylic acid (DNS).This method involves the oxidation of the aldehyde functional
group present in, for example, glucose functional group is aldehyde. Simultaneously, 3,5-
dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions:

oxidation
aldehyde group ----------> carboxyl group

reduction
3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid

REACTION INVOLVING DNS (estimation of reducing sugars by DNS method)

REAGENTS

Dinitrosalicylic Acid Reagent Solution, 1%

Solution "A" is prepared by dissolving 300g of sodium potassium tartrate in about 500
ml distilled water.

Solution "B" is prepared by dissolving 10 g of 3,5-dinitrosalicylic acid in 200 ml of 2N

NaOH solution.
 The dinitrosalicylate reagent is prepared by mixing solutions A and B and raising the
final volume to 1 litre with distilled water.

Standard Glucose Solution: 0.1g anhydrous glucose is dissolved in distilled water and then
raised to 100 ml with distilled water.

PROCEDURE:

1. Take 7 clean, dry test tubes.

2. Pipette out standard sugar solution in different test tubes and make up the volume of all
test tubes to 1 mL with distilled water.
3. Add 2 mL DNS reagent to all the test tubes and mix and plug the test tube with cotton or
marble and keep the test tube in a boiling water bath for 5 minutes.
4. Take the tubes and cool to room temperature.
5. Add 7 ml of water after cooling the solution completely.
6. Read extinction at 540 nm against the blank.
7. All the tubes must be cooled to room temperature before reading, since the absorbance is
sensitive to temperature.

RESULT:
OBSERVATIONS

Tube Glucose Conc. of Test Water DNS Water OD at

No. Std. Glucose (ml) (ml) reagen (ml.) 540nm.
Vol. (µg/mL) t (ml.)
(ml.) Heat for
1 0.0 - - 1.0 2.0 5 min. 7.0
2 0.2 200 - 0.8 2.0 in a 7.0
3 0.4 400 - 0.6 2.0 boiling 7.0
4 0.6 600 - 0.4 2.0 water 7.0
5 0.8 800 - 0.2 2.0 bath 7.0
6 1.0 1000 - 0.0 2.0 7.0
7 - - 0.4 0.6 2.0 7.0

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EXP NO:
11 DATE:
ESTIMATION OF PROTEIN BY LOWRY'S METHOD

AIM
To estimate the amount of protein present in the given sample by Lowry's method

PRINCIPLE

The Lowry protein assay is a biochemical assay for determining the total level of protein
in a solution. The method combines the reactions of Copper ions with peptide bond under
alkaline conditions, with the oxidation of aromatic protein residues. The Lowry method is best
used with protein concentrations of 0.01-1 mg/ml, and is based on the reaction of Cu 2+ produced
by the oxidation of peptide bonds, with Folin-Ciocalteu reagent. This reaction involves reduction
of Folin's reagent and oxidation of aromatics (mainly Tryptophan and Tyrosine). In addition
tyrosine and tryptophan residues of protein possess the reduction of phosphomolybdate
and phosphotungstate components of the folin’s phenol reagent to form bluish products.
Experiments have shown that Cysteine is also reactive towards the reagent. The concentration of
the reduced Folin's reagent is measured by absorbance maximally at 750nm. As a result, the total
concentration of protein in the sample can be deduced from the concentration of Trp and Tyr
residues that reduce the reagent.
REQUIREMENTS

1. Complex-forming Reagent
Prepare fresh before use by mixing the following solutions in
100:1:1 proportion
a.
Solution A- 2% Na2CO3 in 2N NaOH
b.
Solution B- 1% CuSO4
c.
Solution C- 2% sodium potassium tartrate
2. Folin's Reagent
It is prepared in the ratio 1:2 with distilled water just before use.
3. Standard solution
Prepare the standard 1 mg/mL concentration of BSA.

PROCEDURE

1. Take a series of test tubes and add increasing volume of working standard solution to each tube.
2. Take 1ml of water as blank, and 0.5 ml and 1 ml of unknown protein solution in tubes T1 and
T2, respectively.
3. Make up the volume in all the tubes to 1 ml using distilled water.
4. Add 4.5 ml of complex-forming reagent to all the tubes, and incubate the tubes at room
temperature for 10 minutes.
5. Add 0.5 ml of Folin's reagent to each tube, and incubate at room temperature for 30 minutes.
6. After incubation, read the absorbance at 640 nm.
7. Plot a graph of OD versus protein concentration, and determine the concentration of the
unknown protein sample from the graph.

RESULT

The graph of OD vs. protein concentration is plotted, and the protein concentration of
the given sample is found to be

OBSERVATIONS

S.NO REAGENTS B S1 S2 S3 S4 S5 T1 T2
1 Volume of working - 0.2 0.4 0.6 0.8 1.0 - -
standard (ml)
2 Concentration of - 200 400 600 800 1000 - -
working standard
(µg)
3 Volume of unknown - - - - - - 0.2 0.4
solution (ml)
4 Volume of distilled 1.0 0.8 0.6 0.4 0.2 - 0.8 0.6
water (ml)
5 Volume of alkaline 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5
copper reagent (ml)
6 Volume of Folin’s 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
phenol reagent
The contents are mixed well and kept at room temperature for 20 minutes. The blue
colour developed is read at 640 nm
7 Optical density 640
nm
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EXP NO:
12 DATE:

ESTIMATION OF CHOLESTEROL BY ZAK’S METHOD

AIM:
To estimate the amount of cholesterol in the given food/serum sample.

PRINCIPLE:

Cholesterol is a steroid lipid, amphipathic in nature. It consists of a basic cyclopentano

perhydro phenanthrene (CPPP) nucleus. It is synthesised in liver from Acetyl CoA. It acts as a
precursor for steroid hormones and vitamin D.

In Zak's method, the proteins present in the sample are first precipitated by adding FeCl 3-
CH3COOH reagent. The protein free filtrate is treated with conc. H2SO4. In the presence of
excess conc. H2SO4, cholesterol present in the sample gets dehydrated to form cholesta-3,5-
diene, and by the catalytic action of Fe3+ ions a red coloured complex is formed. The intensity of
red colour is measured at 560 nm.

Figure: Structure of Cholesterol

REAGENTS REQUIRED:
1. Stock Standard Solution: About 100 mg of cholesterol is dissolved and made up to 100
ml with glacial acetic acid (concentration 1 mg / ml).
2. Working Standard: About 4 ml of stock solution was made up to 100 ml with ferric
chloride acetic acid reagent (concentration in 0.04 mg / ml).
3. Ferric chloride of 0.05% in acetic acid.
4. Concentrated sulphuric acid.
 5. Glacial acetic acid.
6. Preparation of unknown food sample: 20 ml of food sample and 40 ml of chloroform
was added and centrifuged. The supernatant was used for estimation.
7. Glassware etc…

PROCEDURE

1) 0.5 ml to 2.5 ml of working standard was pipetted out into clean test tubes.

2) 0.5 ml and 1 ml of unknown sample supernatant was taken in test tubes.

3) The volume was made up to 5.0 ml with ferric chloride and 3.0 ml of concentrated
sulphuric acid were added.

4) The test tubes were kept at room temperature for 15 minutes.

5) The absorbance was read at 540 nm.

6) Standard graph of OD vs. concentration of cholesterol was drawn for the values
obtained. From the standard graph the amount of cholesterol present in the food sample was
calculated.

RESULT:
The amount of cholesterol present in the given food sample is found to be………………
OBSERVATIONS
S. NO. REAGENTS B S1 S2 S3 S4 S5 T1 T2
1 Volume of standard cholesterol _ 0.5 1.0 1.5 2.0 2.5 _ _
(ml)
2 Concentration of cholesterol _ 20 40 60 80 100 _ _
(µg)
3 Volume of food sample _ _ _ _ _ _ 1 1
supernatant (ml)
4 Volume of 0.05% ferric chloride 5 4.5 4 3.5 3 2.5 _ _
acetic acid reagent (ml)
5 Volume of conc. sulphuric acid 3 3 3 3 3 3 3 3
(ml)
Incubate the tubes for 15 minutes at room temperature
6 OD at 540 nm

CALCULATION:
 MARK SPLIT UP
COMPONENTS Present but not OBTAINED
Absent
Present completed

Procedure (On the day of experiment) 1 0

Performance, Observation and Viva

2 1.5 0
(On the day of experiment)
Results and Inference with any reference
and answers for questions 1 0.5
(On next experiment)

TOTAL MARKS 4 2.0 0.5

Signature of the Faculty with date