QUALITATIVE REACTIONS OF CARBOHYDRATES

Ex. No. 1

Date: …………..

Experiment Observation Inference

1. Molisch’s Test

Add two mL of
Molisch’s reagent (5%
α-napthol in alcohol) to about
2 mL of given carbohydrate
solution and mix well.

Incline the tube and

add about 1 mL of
concentrated sulphuric acid
along the sides of the tube.

Observe the colour at

the junction of the two
liquids.

2. Iodine Test

Add a few drops of

iodine solution to about
1 mL of the given test
solution.

3. Fehling’s Test

a) To 1 mL of Fehling’s
solution A, add 1 mL of
Fehling’s solution B and a
few drops of the test
solution containing
glucose, fructose or
maltose. Boil for few
minutes.

1
b) Perform the test with
sucrose solution.

4. Benedict’s Test

a) To 2 mL of Benedict’s
reagent, add five drops of
the test solution
containing any one of the
reducing sugars. Boil for 5
minutes in a water bath.
Cool the solution.

b) Perform the test with

sucrose solution.

5. Barfoed’s Test

To 1 mL of the test solution

add about 2 mL of Barfoed’s
reagent. Boil it for one minute
and allow to stand for a few
minutes.

6. Seliwanoff’s Test

To 2 mL of Seliwanoff’s
reagent (Resorcinol in HCl)
add two drops of the test
solution and heat the mixture
to just boiling.

2
7. Bial’s Test

To 5 mL of Bial’s reagent
(orcinol in HCl) add 2-3 mL of
sugar solution and warm it
gently. When bubbles rise to
the surface, cool under the
tap.

8. Test for sucrose

a) Perform Benedict’s test

with the given sucrose
solution

b) Add 5 drops of
concentrated HCl to 5
mL of sucrose solution in
another test tube. Heat
for 5 min in a boiling
water bath.
Add 10% sodium
hydroxide solution to give
a slightly alkaline solution
(test with litmus paper).
Now perform Benedict’s
test with this hydrolyzed
sucrose solution.

3
THINGS TO LEARN
1. Mark (+) sign wherever you get positive reaction and (-) sign wherever the reaction is
negative.

S. Carbohydrate Molisch’s Iodine Barfoed’s Fehling’s Benedict’s Bial’s Seliwanoff’s

No. solution test test test test test test test

1. Glucose

2. Fructose

3. Xylose

4. Sucrose

a. Before hydrolysis

b. After hydrolysis

5. Maltose / Lactose

6. Starch

2. Why is sucrose not able to reduce Benedict’s reagent before hydrolysis?

3. Did you get deep red color when you performed Seliwanoff’s test with sucrose solution? Explain the
theory behind it?

4
 ESTIMATION OF STARCH BY ANTHRONE METHOD
Ex.No. 2

Date: …………..

Starch is an important polysaccharide. It is the storage form of carbohydrate in plants

abundantly found in roots, tubers, stems, fruits and cereals. Starch, which is composed of several
glucose molecules, is a mixture of two types of components, namely amylose and amylopectin. Starch is
hydrolyzed into simple sugars by dilute acids and the quantity of simple sugars is measured,
colorimetrically.

AIM

To estimate the amount of starch present in the given sample.

PRINCIPLE

The sample is treated with 80 per cent alcohol to remove sugars and then starch is extracted
with perchloric acid. In hot acidic medium starch is hydrolyzed to glucose and dehydrated to
hydroxymethyl furfural. This compound forms green colored product with anthrone.

REAGENTS
 Anthrone Reagent: Dissolve 200 mg anthrone in 5mL distilled ethanol and make up to 100 mL
with ice cold 95 percent sulfuric acid.
 80 % ethanol
 52 % perchloric acid

5
  Stock standard glucose: 100 mg glucose in 100 mL water.
 Working standard: 10 mL of stock solution diluted to 100 mL with water.
SAMPLE PREPARATION:

Homogenize 0.1 to 0.5 g of the sample in hot 80 per cent ethanol to extract sugars. Centrifuge
and retain the residue. Wash the residue repeatedly with hot 80 percent ethanol until the washings do
not give color with anthrone reagent. Dry the residue well over a water bath. Add 5.0 mL of water and

6.5 mL of 52 per cent perchloric acid to the residue. Extract at 0 °C for 20 minutes. Centrifuge and save
the supernatant. Repeat the extraction using fresh perchloric acid. Centrifuge and pool the supernatants
and make up to 100 mL and use it as sample.

PROCEDURE
1. Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the working standard.
2. Make up the volume to 1.0 mL in each tube with water.
3. Pipette out 0.1 or 0.2 mL of the sample and makeup the volume to 1.0 mL with water.
4. 1.0 mL of water separately serves as blank.
5. Place all the tubes in ice and add 4 mL of anthrone reagent from a burette slowly drop by drop.
6. Heat for eight minutes in a boiling water bath.
7. Remove the tubes, cool rapidly under running tap water and read the absorbance of the green
colored solution at 630 nm.
8. Construct a standard graph by plotting concentration of standard on X-axis vs OD value along
the Y-axis

CALCULATION

Determine the starch content of the sample by multiplying the amount of glucose with the
factor 0.9.

RESULT

The starch content in the given sample =

THINGS TO LEARN

6
1. Differentiate between starch and cellulose.

2. Name the enzymes responsible for starch digestion.

ESTIMATION OF AMYLOSE

7
Ex.No. 3

Date: …………..

Starch is composed of amylose and amylopectin. Amylose has a molecular weight in the range
1,50,000 - 10,00,000 depending on its biological origin. It is now recognized that it has some elements
of non-linearity. The ratio of amylose and amylopectin plays an important role in the cooking
characteristics of cereal grains, especially rice.

AIM

To estimate the amount of amylose present in the given sample and unknown solution.

PRINCIPLE
Amylose forms a helical coil like structure in solution in which 5 to 6 glucose residues are
present in each coil. Iodine forms an absorption complex with amylose to yield a blue color. The
absorbance of the blue color is measured at 600 nm.

REAGENTS
 1 N Sodium hydroxide
 Phenolphthalein indicator
 0.1 N HCl
 Iodine reagent: Dissolve 1 g iodine in 10 g KI in water and then make up to 500 mL.
 Standard amylose: Dissolve 50 mg amylose in little amount of distilled water and make up the
volume to 50 mL in a standard flask.
 Working standard: Pipette out 10 ml of the stock standard and make up the volume to 50 ml
with distilled water. One ml of this contains 200µg of amylose.

SAMPLE PREPARATION:

Weigh 50 mg rice flour, transfer to a boiling tube and add few drops of alcohol followed by 5 mL
of 1 N NaOH. Boil for 15 minutes in a water bath. Cool and transfer the contents to a 50 mL standard
flask and make up the volume with water.

8
PROCEDURE
1. Pipette out 0.5 to 2.5 mL of working standard amylose solution into a series of test tubes.
2. Pipette out 0.5 mL aliquot of the sample into a separate test tube.
3. Make up the volume to 2.5 mL with distilled water in all the tubes.
4. Add 2 drops of phenolphthalein. Add with shaking 0.1 N HCl till the pink color just disappears.
5. Add 0.1 mL of the iodine reagent and make up the final volume to 10 mL with water.
6. Measure the absorbance at 600 nm.
7. Prepare a standard graph with 100 - 500 µg of amylose.
8. From the standard graph, determine the amount of amylose present in the sample.

RESULT
The amount of amylose present in the given sample =

THINGS TO LEARN

1. Differentiate amylose and amylopectin

9
 ESTIMATION OF REDUCING SUGARS
Ex. No. 4

Date: …………..

All monosaccharides and many disaccharides act as reducing agents in the presence of dilute
alkali. They exhibit this reducing property due to the presence of free or potentially free aldehyde or

keto group. The keto group or aldehyde group under alkaline conditions reduces metal ions like Cu +

+and Ag+ to Cu+and Ag. During this process, reducing sugars get oxidized to lower acids. Therefore,

quantitative estimation of reducing sugars becomes possible with any one of the reduction processes.

AIM
To determine the amount of a reducing sugar present in the given unknown solution.
PRINCIPLE
Glucose is oxidized to gluconic acid by iodine under alkaline conditions.

C6H12O6+ 3NaOH + I2 C5 H11O5COONa + 2 NaI + 2 H2O

A known amount of sugar is allowed to react with an excess of iodine solution and the excess
iodine is back titrated against standard sodium thiosulphate solution.

REAGENTS

 0.1N Iodine solution

 0.1N Sodium thiosulphate
 0.1N Sodium hydroxide
 10 N HCl (Concentrated HCl)
 1% Starch
 Standard glucose: Dissolve 100 mg of glucose in little amount of distilled water and make up the
volume to 100 mL in a standard flask

PROCEDURE
Pipette out 10 mL of the working standard solution containing 10 mg glucose into an iodine
flask. Add 10 mL of 0.1N iodine solution followed by careful slow addition of 10 mL of 0.1N NaOH. The
alkali should be added drop by drop during a period of four minutes. During the addition of NaOH, red
colour of iodine changes to yellow and then to colourless solution. Rinse the sides of the flask with

10
distilled water and add about 50 mL of distilled water. Stopper the flask and leave it for 15 minutes at
room temperature.

Acidify the solution in the flask with 3 mL of 10N HCl, when the red colour of iodine is
regenerated. Titrate the excess free iodine against standard thiosulphate until red colour of the solution
in the iodine flask changes to straw yellow. Then add a few drops of starch solution to the flask when a
blue colour is formed. Continue the addition of sodium thiosulphate drop by drop till the blue colour
just disappears. Note the burette reading. This is titre value (V1 mL).

Make up the given unknown solution to a known volume (50 mL). Find out the amount of
reducing sugar present in the given unknown solution by titrating following the procedure as above

(V2 mL). For blank, pipette out 10 mL of water and titrate after adding all the reagents mentioned above

(V3 mL). Calculate the amount of reducing sugar present in 50 mL of the unknown solution as given

below.

CALCULATION

Volume of 0.1 N thiosulphate = 0.1 N iodine consumed

by 10 mL (10 mg glucose)
} V3-V1 mL = x mL

Volume of 0.1N thiosulphate = 0.1 N iodine consumed

by glucose in 10 mL of unknown solution
} V3 – V2 mL = y mL

Amount of glucose in 10 mL of unknown solution = (10 mg / x mL) X y mL = z mg

(z X 100)
Amount of glucose in 100 mL of unknown solution = mg
10

RESULT
100 mL of the given unknown solution contains ..................... mg reducing sugar.

11
THINGS TO LEARN

1. Name few reducing sugars and their sources. Write down their structures.

2. Can we estimate the non-reducing sugars by this method? If yes, can you suggest the
modifications required in this procedure?

3. How will you prepare 100 mL of 1N NaOH?

12
 REACTIONS OF PROTEINS

Ex. No. 5

Date: …………..

AIM: To test the reactions of protein and to know the theory behind the reactions.
Experiment Observation Remarks

I Precipitation reactions
1. Heat coagulation test

Heat 2 mL of protein solution

in a test tube.

2. Heller’s test

Take 2 mL of concentrated
HNO3 in a test tube. Add equal volume
of protein solution along the sides of
the tube. Do not mix the two
solutions.

3. Precipitation by heavy metals

Take 2 mL of the protein
solution in a test tube. Add lead
acetate solution until precipitation
occurs.

4. Precipitation by organic solvents

Add 95% alcohol or acetone
drop by drop to 1 mL of protein
solution.

13
 Experiment Observation Remarks

5. Precipitation by salts

To few mL of a protein
solution add saturated
ammonium sulphate solution
drop by drop.

II. Colour reactions

6. Xanthoproteic reaction
To about 1mL of
protein solution add equal
volume of concentrated nitric
acid. Then add few drops of
NaOH to make the solution
alkaline. Cool and observe the
colour change.

7. Glyoxylic reaction for

tryptophan Hopkins Cole
Test
Add 2 mL of glacial
acetic acid to 2 mL of the test
solution. Then pour about
2 mL of concentrated H2SO4
carefully down the sides of the
test tube. Observe the colour
change at the junction of the
two liquids.

14
 8. Biuret test

Add five drops of copper

sulphate solution to 2mL of
the test solution followed by 2
mL of NaOH. Mix thoroughly
and note the colour produced.

THINGS TO LEARN

1. You would have noticed that when you handle nitric acid that part of your fingers which is in
direct contact with nitric acid turn yellow. Can you explain the reaction behind it?

2. When hen’s egg is boiled, it gets solidified.

a. Can you explain this phenomenon with your knowledge on proteins?

15
 b. Is it possible to rescramble the egg and get the original form? If not, why?

3. When we cut our hair and nail, we don’t feel pain. Why ?

4. Differentiate denaturation and renaturation.

5. Why does your palm turn purple when it comes in contact with ninhydrin?

16
 SORENSON’S FORMAL TITRATION OF AMINO ACIDS

Ex. No. 6

Date: …………..

Amino acids are the basic building blocks of proteins. They carry both amino and carboxyl
groups on the same molecule. In nature, amino acids mostly occur as proteins as well as in free form in
many tissues. Estimation of free amino acids is important in agricultural biochemistry for nutrients
content information.

AIM
To estimate the amount of amino acid present in the given solution by Sorenson’s method.

PRINCIPLE
A solution containing amino acid is neutral in reaction. The carboxyl group of amino acids cannot
be accurately titrated in aqueous medium with alkali because the carboxyl group and the basic amino
group of the amino acid react to form zwitter ions, which are not dissociated completely at the end
point of alkaline indicator, phenolphthalein. In the presence of formaldehyde, amino acids form the
dimethylol derivatives.

NH3+ H

R – C – COO- + 2 HCHO R – C – COO- + H+

H N – CH2OH

CH2OH
Zwitter ion Dimethylol amino acid

The presence of formaldehyde, hence, protects the basic amino group from forming zwitterion
and permits the carboxyl group to react freely (maximum acidity). The acidity may be titrated against
standard NaOH, using phenolphthalein as indicator. This reaction forms the basis for Sorensen’s formal
titration method of estimating amino acids.

REAGENTS
 Formaldehyde
 0.02 N oxalic acid
 Approximately 0.02 N NaOH

17
  0.1% phenolphthalein

PROCEDURE
1. Pipette out 10 mL of standard oxalic acid solution into a conical flask; add few drops of
phenolphthalein and titrate against sodium hydroxide. The end point is the appearance of pale
permanent pink color.
2. Repeat the titration for concordant values. Calculate the normality of sodium hydroxide
solution using the formula
V x N = V xN
oxalic acid oxalic acid NaOH NaOH

3. Pipette out 10 mL of the given unknown solution into a conical flask.

4. Add 5 mL of formaldehyde and a few drops of indicator.
5. Titrate against the standard sodium hydroxide solution (x mL). Repeat for concordant values.
6. For blank, take 5.0 mL of formaldehyde and 10 mL of water and titrate against the alkali after
adding the indicator (y mL). Repeat for concordant values.

CALCULATION
The difference in the titre values (x-y mL) gives the volume of alkali required for neutralizing the
amino acids present.

Calculate the strength of amino acid using the formula,

N =V N
V1 1 2 2

where,

V1 = 10 mL (volume of amino acid solution); V2 = (x-y) mL (volume of NaOH)

N1 = Normality of amino acid solution; N2 = Normality of NaOH

Find out the concentration of amino acid by the formula,

Normality x Equivalent weight = weight (g/l)

Equivalent weight of the simplest amino acid (glycine) is 75.

18
RESULT

The strength of amino acid solution........... N

The concentration of amino acid is ............ g in 100 mL of the given solution.

THINGS TO LEARN

1. What is the function of formaldehyde in Sorensen’s formal titration?

2. Name the amino acids, which are deficient in rice proteins?

3. Name the amino acid that is deficient in pulse protein?

4. Name few plant sources that are rich in sulphur containing amino acids.

19
 ESTIMATION OF TOTAL FREE AMINO ACIDS
Ex. No.7

Date: …………..

Amino acids are colourless ionic compounds that form the basic building blocks of proteins.
Apart from being bound as proteins, amino acids do exist in the free form in many tissues and are
known as free amino acids. They are mostly water soluble in nature. Very often in plants during disease
conditions, the free amino acid composition exhibits a change and hence, the measurement of the total
free amino acids gives the physiological and health status of the plants.

AIM
To estimate the amount of total free amino acids present in the given sample.

PRINCIPLE
Ninhydrin, a powerful oxidizing agent, decarboxylates the alpha-amino acids and yields an
intensely bluish-purple (Rheumann’s purple) coloured product which is colorimetrically measured at
570nm.

Ninhydrin + alpha-amino acid Hydrindantin + Decarboxylated Amino acid + CO2 + NH3

Hydrindantin + Ninhydrin + NH3 Rheumann’s purple product + H2O

REAGENTS
 Stock: 25 mg leucine in 25 mL water.
 Working standard : 1 mL of stock diluted to 10 mL (100 µg / mL)
 Citrate buffer- pH 5.5 M: Dissolve 1.44 g citric acid and 5.34 g sodium citrate in 100 mL
water
 Ninhydrin reagent: Dissolve 1 g ninhydrin in 100 mL of 0.5 M citrate buffer, pH 5.5
 Ninhydrin-citrate-glycerol reagent: Mix 25 mL of ninhydrin solution, 60 mL glycerol (60%)
and 10mL of 0.5M citrate buffer. Adjust the final pH of the reagent to 5.5.

20
SAMPLE PREPARATION

Extract one g of finely ground plant material in 10 mL of 80% ethanol at 55 °C for 5min.
Centrifuge the content at 10,000 rpm for 5 min and save the supernatant in a separate container. Re-
extract the residue in 5 mL alcohol as described above.

PROCEDURE
1. Pipette out 0.2 to 1.0 mL of the working standard solution.
2. Pipette out 0.2 mL of the given unknown solution and 0.5 mL of plant extract into two different
test tubes.
3. Make up the volume to 1 mL with distilled water in all the tubes.
4. Take 1 mL water for blank.
5. Add 4 mL of ninhydrin - citrate - glycerol reagent to all the tubes and mix well.
6. Heat the tubes in a boiling water bath for 10 min.
7. Cool the tubes to room temperature.
8. Measure the absorbance at 570 nm within 1 h against the reagent blank.
9. Draw a standard curve by plotting absorbance versus concentration and find out the
concentration of the total free amino acids in the sample and express as percentage equivalent
of leucine.

RESULT
The total free amino acid content of the given sample =

The amount of leucine present in the given unknown solution =

THINGS TO LEARN

1. Can we estimate non-protein amino acids by this method?

21
2. Name one amino acid that is significantly altered under abiotic stress conditions in plant.

3. Name a non-protein amino acid in the human body.

22
 ESTIMATION OF PROTEIN BY BIURET METHOD
Ex. No. 8

Date: …………..

Proteins contain amino acids linked by peptide bonds. Differences in the number of amino acids
and the sequence of amino acids result in different types of proteins. There are more than three
methods for the estimation of proteins. A colorimetric method is described here.

AIM
To estimate the amount of protein present in the given unknown solution by Biuret method.

PRINCIPLE
Compounds containing two or more peptide bonds (eg. proteins) take up a characteristic purple
colour when treated with dilute copper sulphate in alkaline solution.

The colour is apparently caused by the formation of complex of the copper atom with four
nitrogen atoms, two from each of two peptide chains. It requires 1-20 mg protein for colour formation.

REAGENTS
 Protein standard: Dissolve 1000 mg of ovalbumin in 100 mL of 0.1 N NaOH.
 Biuret reagent: Dissolve 1.5 g CuSO 4.5H2O and 6.0 g sodium potassium tartarate in about 500
mL water. To this add 300 mL of freshly prepared 10N NaOH. Make up to 1 litre.

23
PROCEDURE
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the standard protein solution into a series of
tubes.
2. Pipette out 0.2 and 0.4 mL of the given unknown solution into two more tubes.
3. Make up the volume to 1.0 mL in all the tubes with distilled water.
4. 1 mL of distilled water serves as blank.
5. Add 4 mL of Biuret reagent to each tube and mix thoroughly.
6. After 30 minutes at room temperature read the absorbance of the solution at 550 nm
against the reagent blank.
7. Construct the standard graph and calculate the amount of protein present in 100 mL of the
given unknown solution.

RESULT

100 mL of the given unknown solution contains ............... mg protein.

THINGS TO LEARN
1. What is meant by a peptide bond?

2. Name the major proteins of maize, wheat and milk?

24
 ESTIMATION OF FREE FATTY ACIDS OF AN OIL

Ex. No.9

Date: …………..

Oils and fats are triglycerides i.e., esters of fatty acids with glycerol. A little amount of free fatty
acid is also present in oils along with the triglycerides. It increases during storage. The free fatty acid
content is also known as acid number/ acid value of oil. The acid value of an oil or fat is defined as the
number of mg of sodium/ potassium hydroxide required to neutralize the free fatty acids present in one
gram of oil or fat.

AIM
To estimate the free fatty acid content of the given oil.

PRINCIPLE

The free fatty acid in oil is estimated by calculating the amount of sodium hydroxide required to
neutralize the free fatty acid present in the oil and expressed as percent oleic acid.

REAGENTS

 0.1N Sodium hydroxide

 0.1N Oxalic acid
 0.1% Phenolphthalein
 Oil
 95% alcohol (Neutralized)

PROCEDURE
1. Pipette out 5 mL oil into a clean dry conical flask.
2. Add 25 mL neutralized alcohol using a burette.
3. Add a few drops of phenolphthalein and heat the contents to boiling.
4. Titrate while hot against standard sodium hydroxide solution (x mL).

25
 5. For standardization of sodium hydroxide, pipette out 10 mL of 0.1N oxalic acid into a clean
conical flask, add a few drops of indicator and titrate against sodium hydroxide. Repeat the
titration for concordant value (V1 mL).
6. Calculate the strength of NaOH.
7. Find the weight of 5 mL of oil and calculate the free fatty acid content in 100 g oil.

CALCULATION

I. Strength of sodium hydroxide

V NaOHX N NaOH = V oxalic acid X N oxalic acid

N NaOH= (10 X 0.1) / V NaOH

II. Free fatty acid content

x mL X N NaOH 0.0282
F.F.A content = x x 100 g
0.1 weight of oil

Where x mL = Volume of sodium hydroxide required to neutralize 5 mL (find out the weight of 5 mL of
oil).

RESULT
The free fatty acid content of the given oil is ....................% Oleic acid.

The acid value (of a product like biodiesel) is expressed as FFA ( ) % oleic acid x 1.989 .

26
THINGS TO LEARN
1. Which among the following samples will record higher free fatty acid content?
a. Fresh oil
b. Oil stored for 3 months

2. Give the difference between free fatty acid and acid value.

27
 DETERMINATION OF IODINE VALUE OF AN OIL
Ex. No. 10

Date:

The iodine value is a measure of degree of unsaturation of oil. It is constant for a particular oil or
fat. Iodine value is a useful indicator in studying oxidative rancidity of oil since greater the unsaturation
greater the possibility of the oil to go rancid.

AIM
To determine the iodine number of the given oil.
PRINCIPLE
Oils contain both saturated and unsaturated fatty acids. Iodine gets incorporated into the fatty
acid chain wherever the double bonds exist. Hence the measure of iodine absorbed by oil gives the
degree of unsaturation. Iodine value or Iodine number is defined as the (g) of iodine absorbed by 100 g
of the oil.

Excess amount of Hanus iodine solution (IBr) is allowed to react with the oil for a definite period
of time. IBr adds to the double bonds present in the unsaturated fatty acids. To the remaining IBr,
potassium iodide solution is added and the liberated iodine is titrated against the standard sodium
thiosulphate.

REAGENTS
 Hanus iodine reagent
 Oil
 15% potassium iodide solution
 1N sodium thiosulphate
 1% starch solution (freshly prepared)
PROCEDURE
1. Weigh about 0.25 g oil into an iodine flask and dissolve in 5 mL of chloroform.
2. Add 20 mL of Hanus iodine reagent using a pipette, draining it in a definite time.
3. Mix well and allow to stand in dark for exactly 30 min, with occasional shaking.
4. Add 10 mL of 15 percent KI solution, shake thoroughly and add 10 mL of freshly boiled and
cooled water, washing down any free iodine on the stopper.
5. Titrate against sodium thiosulphate until yellow solution turns almost colourless.

28
 6. Add few drops of starch as indicator and titrate until the blue colour disappears.
7. Towards the end of the titration, stopper the flask and shake vigorously so that any iodine
remaining in solution in chloroform is taken up by potassium iodide solution.
8. Simultaneously run a blank without the sample.

CALCULATION
The quantity of thiosulphate required for blank minus the quantity required for the sample gives
thiosulphate equivalent of iodine absorbed by the fat or oil taken for analysis.

Iodine Number = (B -S) x N x 12.60

Wt. of sample (g)

where,

B = mL thiosulphate for blank

S = mL thiosulphate for sample
N = Normality of thiosulphate
Amount of fat / oil taken should be adjusted such that the excess iodine in the added 20 mL of
Hanus iodide solution has 60 percent of excess iodine of the amount added. ie. if (B-S) is greater than
B/2, repeat with smaller amount of the sample.

RESULT
The iodine number of the given oil =

29
THINGS TO LEARN

1. Give the iodine value of coconut oil, groundnut oil, sunflower oil, corn oil, rice bran oil, gingelly
oil and olive oil. Based on the iodine values, give your inferenceon the degree of unsaturation.

30
 ESTIMATION OF ASCORBIC ACID
Ex. No. 11

Date: …………..

Ascorbic acid, commonly known as vitamin C, is abundant in fresh fruits and vegetables. It is a
water-soluble and heat labile vitamin. Ascorbic acid is synthesized from glucose in plants. Ascorbic acid
is known for its antioxidant properties.

AIM
To estimate the amount of ascorbic acid in the sample (lime juice) and the given unknown
solution by the dye method.

PRINCIPLE
Ascorbic acid reduces the dye, 2,6 dichlorophenol indophenol (a blue colored compound which
attains red color in acid solution) to a colourless compound. The ascorbic acid gets oxidized to
dehydroascorbic acid.

REAGENTS
 4% oxalic acid
 Standard ascorbic acid solution in 4% oxalic acid (0.1 mg /mL)
 Dye solution: 42 mg sodium bicarbonate and 52 mg of dye (2,6 dichlorophenol indophenol) in
200 mL distilled water.

PROCEDURE
1. Pipette out 10 mL of the working standard solution into a conical flask.
2. Add 10 mL of 4% oxalic acid and titrate against the dye (V 1 mL). End point is the appearance

of pink colour, which persists for a few minutes.

3. Repeat the titration for concordant values. The amount of the dye consumed is equivalent
to the amount of ascorbic acid present.
4. Pipette out 10 mL of the given unknown solution into a conical flask.
5. Add 10 mL of 4% oxalic acid and follow the titration as above (V2 mL).

6. Repeat the titration for concordance.

7. Pipette out 10 mL of the diluted lime juice and follow the titration as above (V3 mL).

31
 8. Repeat the titration for concordance.
9. From the titre values, calculate the amount of ascorbic acid in
i. 100 mL of the given unknown solution
ii. 100 mL of fresh lime juice.

CALCULATION

I. Amount of ascorbic acid present in 10 mL of working standard = 1.0 mg

Volume of dye solution required to oxidize 1.0 mg ascorbic acid = V1 mL

Amount of ascorbic acid required to reduce V1 mL of the dye solution = 1.0 mg.

Therefore,

Amount of ascorbic acid required to reduce 1 mL of dye solution (dye factor)

= (1.0/V1) = x mg

II. Amount of Ascorbic acid required to reduce 1 mL of dye solution = x mg ascorbic acid
Therefore, Amount of ascorbic acid required to reduce V2mL of dye solution = (x X V2) mg

= y mg ascorbic acid

10 mL of the unknown solution contains = y mg ascorbic acid

Therefore, 100 mL of unknown solution contains = y x 100/10 mg ascorbic acid

RESULT
(i) The ascorbic acid content in the given unknown solution is
(ii) The ascorbic acid content of fresh lime juice is

32
THINGS TO LEARN

1. Find out the Vitamin ‘C’ content in tomato, grapes and Indian gooseberry.

2. Name the condition occurring due to deficiency of Vitamin. C

33
 ASSAY OF AMYLASE

Ex. No. 12

Date: …………..

Starch degrading enzymes are universally distributed. They act on starch, glycogen and allied
polysaccharides. Alpha-amylase causes endocleavage of substrates and hydrolyzes a-1,4 linkages in a
random manner. It has the ability to bypass a-1, 6 branch points. Thus, viscosity reduction of the
substrate is fast but the production of reducing sugars is slow.

b-amylase hydrolyzes alternate bonds from the non-reducing end of the substrate. The enzyme
degrades amylose, amylopectin or glycogen in an exo- or stepwise fashion by hydrolyzing alternate
glycolysidic bonds. The end product is b maltose. b-amylase is incapable of bypassing branch points i.e.,
conversion of amylopectin to maltose. The other product is a large limit dextrin. The viscosity reduction
of the substrate due to b- amylase action is slow but the production of reducing sugars is fast.

AIM

To perform the assay of amylase and to determine the activity of the enzyme in the given
sample.

PRINCIPLE

The reducing sugars produced by the action of a- and /or b- amylase react with dinitrosalicylic
acid and reduce it to a coloured product, which is a measured at 530 nm.
MATERIALS

 Sodium acetate buffer, 0.1M pH 4.7

 Starch 1% solution : Prepare a fresh solution by dissolving 1g starch in 100mL acetate buffer,
slightly warm, if necessary.
 Dinitrosalicylic acid reagent : Dissolve by stirring 1g dinitrosalicylic acid, 200mg crystalline
phenol and 50 mg sodium sulphite in 100 mL 1% NaOH. Store at 4°C since the reagent
deteriorates due to sodium sulphite. If long storage required, sodium sulphite may be added at
the time of use.
 40% Rochelle salt solution (potassium sodium tartrate)

34
  Standard maltose solution: Dissolve 50 mg maltose in 50 mL of distilled water in a standard flask
and store in a refrigerator.
Extraction of amylase

Extract 1g of (fresh) sample material with 5-10 mL of ice-cold 10 mM CaCl 2 solution overnight at
4°C or for 3 h at room temperature. Centrifuge the extract at 5,000 rpm at 4°C for 20 min. The
supernatant is used as enzyme source.

PROCEDURE

1. Pipette out 1 mL of starch solution and 1mL of properly diluted enzyme in a test tube.
2. Incubate it at 37°C for 15 min.
3. Stop the reaction by the addition of 2 mL of dinitrosalicylic acid reagent.
4. Heat the solution in a boiling-water bath for 5 min.
5. While the tube is warm, add 1mL of 40 % sodium potassium tartrate solution.
6. Then cool it in running tap water at room temperature.
7. Make up the volume to 10 mL by addition of 6 mL distilled water.
8. Read the absorbance at 530 nm.
9. Terminate the reaction at zero time in control tubes and read as above.
10. Pipette out 0.2 to 1.0 mL of the standard maltose solution corresponding to the concentration of
200 µg - 1000 µg.
11. Make up the volume to 2.0 mL with distilled water.
12. Add 2 mL of dinitrosalicylic acid reagent and proceed as per the steps for the sample.
13. Construct the standard graph and calculate the amount of maltose produced by the enzyme
amylase in the given extract.
14. A unit of α- and/or β amylase is expressed as mg of maltose produced during 15 min incubation
with 1% starch.
RESULT

Activity of amylase in the given sample =

35
THINGS TO LEARN

1. Diabetics are advised to include cereals like ragi, wheat in their diet rather than rice. Can you find
reason behind it?

36
 ESTIMATION OF TOTAL PHENOLS

Ex. No. 13

Date : …………..

Phenols are secondary metabolites with an aromatic ring and hydroxyl groups. It is widespread
in plant kingdom, occurring in all parts of plants. Phenolic compounds are water-soluble and form
complexes with protein by hydrogen bonding. It includes simple phenols, phenolic glycosides,
flavanoids, phenolic acids or phenyl propanoids, phenolic quinines and polyphenolic compounds like
lignins, melanins and tannins. They are highly susceptible to enzyme oxidation by specific phenolase.
Phenols can be estimated by various methods. Estimation of phenols using Folin-Ciocalteau reagent is
described below.
AIM

To estimate the amount of total phenols in the given sample and unknown solution.

PRINCIPLE
Phosphomolybdic acid and phosphotungstic acid present in the Folin- Ciocalteau reagent is
reduced by phenolic hydroxyl group. Blue colour is developed in alkaline condition, which is measured
at 660 nm.

REAGENTS
 Folin-Ciocalteau reagent – commercially available
 20% Sodium carbonate
 Stock standard phenol: Dissolve 50 mg pyrocatechol in 50 mL water
 Working standard: Dilute 5 mL of this stock solution to 100 mL, which contains 50 mg/mL
SAMPLE PREPARATION

Phenols are highly soluble in water as well as in alcohol. Eighty percent (80%) ethanol is
employed for efficient extraction. Boiling alcohol prevents enzyme oxidation of phenols. 250 mg plant
material (fresh) is ground in two 5 mL portions of 80% ethanol and centrifuged. Pool the extract
(supernatant) and make up to 10 mL in a standard flask.

37
PROCEDURE

1. Pipette out 0.1, 0.2, 0.3, 0.4 and 0.5 mL of the working standard solution into a series of test
tubes.
2. Take 0.2 mL of the given unknown solution in a separate test tube.
3. Pipette out 0.1 mL aliquot of the ethanol extract in a test tube and evaporate on a water bath.
4. Make up the volume to 3.5 mL with distilled water.
5. In the blank tube add 3.5 mL distilled water.
6. Add 0.5 mL of Folin-Ciocalteau reagent to all the tubes.
7. Mix well and wait for 5 min.
8. Add 1 mL of 20 % sodium carbonate solution to all the tubes.
9. After 30 min measure the absorbance at 660 nm.
10. Draw a standard curve by plotting absorbance versus concentration and find out the
concentration of the total phenols in the sample and unknown solution. The result is expressed
as mg equivalent of pyrocatechol per 100 g fresh (or dry) plant tissue.

RESULT

1. The amount of total phenol present in the given sample =

2. The amount of pyrocatechol present in the given unknown solution =

THINGS TO LEARN
1. Name few sources rich in polyphenols.

38
Ex.No.14 EXTRACTION AND ESTIMATION OF LYCOPENE AND CAROTENES

Date:

Lycopene is responsible for the red color of tomato fruit and the flesh of watermelon. It is a
tetraterpene having a straight chain structure. Though it has no nutritional value, its contribution to the
appearance of tomato has a role in consumer acceptance. Carotenes also belong to tetraterpene group.
Carotene is the pro-vitamin of Vitamin A.
Principle
Lycopene and carotenes are extracted in acetone and then taken up in petroleum ether.
Lycopene has absorption maximum at 473 and 503 nm. Carotenes have absorption maximum at 453
nm. The molar extinction coefficient of lycopene and carotenes are used for finding the concentration.

Reagents
 Acetone, AR grade
 Petroleum ether 40-60%
 Anhydrous sodium sulfate AR grade
 Sodium sulfate, 5% solution
Procedure
1. Take plant material (tomato or carrot), cut into small pieces and homogenize in a waring blender
or mortar and pestle to a smooth consistency.

2. Weigh 1 g of this pulp.

3. Extract the pulp repeatedly in acetone using pestle and mortar or a mixer until the residue is
colorless.

4. Transfer the acetone extract to a separating funnel containing about 20 mL of petroleum ether
and mix gently.

5. Add about 20 mL 5% sodium sulfate solution and shake gently.

6. Remove the upper petroleum ether layer and re extract the lower aqueous phase with 20 mL
more petroleum ether.

7. Pool the petroleum ether extracts and wash it once with a little distilled water.
8. Add 10g of anhydrous sodium sulfate into the petroleum ether and keep it aside for 20 min.

9. Decant the petroleum ether extract into a 25 mL volumetric flask and make up the volume with
petroleum ether.

39
 10. Measure the absorbance in a spectrophotometer at 453 nm using petroleum ether as blank.

Calculation
mole /l
E -------------- of lycopene at 503 nm = 17.2 x 10
1 cm

i.e 3. 12 µg of lycopene will have unit absorbance

Hence mg of lycopene in 100 g sample =

3.12 x Absorbance of sample x Total volume x 100

-------------------------------------------------------------------------
Wt of sample(g) x 1000

The extinction coefficient of 1 % b-carotene at 453 nm

(E)
= 0.2592
-----------
1 cm
1 µg/mL solution would give an extinction of 0.2592

Hence mg of b-carotene in 100 g sample =

= Absorbance of sample Total volume x 100

-------------------------------- x -------------------------------------
0.2592 Wt of sample (g) x 1000

RESULT

The lycopene content of the given sample =

The carotene content of the given sample =

40
THINGS TO LEARN

1. Name few sources rich in carotenoids and lycopene

2. Name few plant pigments

3. What is the biological importance of carotenoids?

41
 SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

Ex. No. 15

Date: …………..

AIM
To separate the amino acids in the given mixture by paper chromatography.

PRINCIPLE
Chromatography is a collective name for the separation processes where the separation of the
components is effected by their partition between a stationary phase with a large surface and a mobile
phase, which flows over the first. In paper chromatography, filter paper which is composed almost
entirely of cellulose fibers, act as the inert carrier for the stationary liquid phase, usually water, while the
solvent mobile phase is drawn through the paper by capillary action.

REAGENTS
 Chromatography (Whatman) paper
 Spotting lamp / hair drier
 Capillary tubes / Micropipette
 Standard amino acids 2 to 3 mg / mL
 Solvent - n-Butanol : acetic acid : water 4:1:5 v/v or phenol : water 4 : 1 v/v
 Chromatography chamber/ cabinet
 Detection reagent: 0.5% ninhydrin in 95% alcohol or methyl cellosolve

SAMPLE PREPARATION
For free amino acids, fresh biological samples are weighed and extracted in 70% alcohol. The
extract may be concentrated in a rotavapor, if necessary. A 10% homogenate will be sufficient for most
biological samples.

Proteins may be hydrolyzed, in 2.5 N HCl, prior to the determination of amino acids. The
samples containing about 10 mg protein may be taken and hydrolyzed with 5 mL of 0.5 N HCl under
vacuum at 110°C for 18 hours. HCl is then evaporated in a rotary evaporator completely, and the residue
dissolved in 70% alcohol.

42
SPOTTING

Cut Whatman (No.1) filter paper or any other chromatography paper to the required size. Draw
a pencil line about 4 cm from one edge of the paper and mark points at an interval of 4 cm. Apply the
unknown sample at the central spot and the standard amino acids on both sides as spots. About 50 µL
will be ideal for spotting. Spotting lamp or hair drier may be used for rapid drying of the sample. The
spot should be as small and uniform as possible. Handle the paper by its edges / corners.

Fold the filter paper into a cylinder and stitch it. Saturate the chromatography chamber with the
vapors of solvent mixture. Place the cylinder inside the chamber with its edge dipping in the solvent. The
sample spots should be well above the solvent level. The solvent moves through the paper by capillary
action. The different amino acids depending upon their polarity and solubility get resolved. After the
solvent moves to about 4 cm below the top edge of the paper, remove it. Mark the distance moved by
the solvent on paper and air dry it. Spray with ninhydrin reagent and then bake in an oven at 100° C for a
few minutes. The amino acid spots appear purple; mark them with a lead pencil. Measure the distance
travelled by the solvent from the sample spots. Measure the distance travelled by the solute (amino
acids), and calculate the Rf (Retention factor / Resolution factor) value.

Rf = Distance travelled by the solute

Distance travelled by the solvent

Compare the Rf values of amino acid spots in the mixture with that of standard amino acids. The amino
acids may be quantitatively estimated by measuring the intensity of the purple coloured spots (cut and
eluted in acetone) in a colorimeter at 570 nm.

RESULT
1. Rf value of the given standard amino acids are ………..

2. The given mixture is found to contain amino acids with Rf values…………..

43
THINGS TO LEARN

1. What are other solvents used for separation of amino acids?

2. Will the Rfvalue of a compound be the same in different solvent systems?

3. Why does proline give yellow spot with ninhydrin?

44
 SEPARATION OF PHENOLS THROUGH THIN LAYER CHROMATOGRAPHY

Ex. No. 16

Date: …………..

Thin Layer Chromatography (TLC) is an easy technique to adopt for separation, identification
and characterization of unknown organic compounds. A variety of small molecules like amino acids,
sugars, organic acids, lipids, etc. are separated by this technique. The greater advantage of TLC is the
speed at which separation is achieved. When volatile solvents are used the time required to effect
separation is only about 30 min. and with non-volatile solvents it is seldom longer than 90 min.
AIM

To separate the phenols in the given mixture and the sample.

PRINCIPLE
The general principle involved in TLC is similar to that of column chromatography, i.e.,
adsorption chromatography. In the adsorption process, the solute competes with the solvent for the
surface sites of the adsorbent. Of course, the partition effect in the separation is also not ruled out. The
adsorbent normally used, contains a binding agent such as calcium sulphate, which facilitates the
holding of the adsorbent to the glass plate.

REAGENTS
 Glass plate (20 x 20 or 20 x 10 cm)
 Glass tank with lid
 Spreader
 Developing solvents: Differ with group of compounds to be separated.
 Adsorbent: Silica gel G / alumina
 Sample should be extracted following the procedures indicated for each group of compounds.
For e.g., extraction with 80 per cent alcohol for phenols and sugars.
 Standards
 Spraying agent: This also differs as for the group of compounds of interest.

PROCEDURE
Place dry, clean glass plates (5 Nos. 20 x 20 cm) on the plastic base-plate over a plain surface.

45
 Prepare slurry of the adsorbent in water (sometimes buffer) in the ratio 1: 2 (W/V). Stir the
slurry thoroughly for 1 - 2 min. and pour into the applicator positioned on the head glass plate. Coat the
slurry over the glass plates at a thickness of a 0.25 mm for qualitative analysis by moving the applicator
at a uniform speed from one end to the other. Leave the plates to dry at room temperature for 15 - 30
min.

Heat the plates in an oven at 100 - 120°C for 1 - 2 h to remove the moisture and to activate the
adsorbent on the plate. The dried plates in a rack can be stored in a desiccator over silica gel to prevent
moisture absorption.

SAMPLE APPLICATION
Leave 2.5 cm from one end of the glass plate and at least one inch equal distance from the
edges. Apply the sample and standard by means of a micropipette or syringe as small spots. All spots
should be placed at equal distance from one end of the plate. See that the adsorbent does not flake off
at the sample application point. Measured volumes are applied for quantitative estimation. Sample
application can be done repeatedly for a more concentrated sample spot.

DEVELOPING THE CHROMATOGRAM

Pour the developing solvent into the tank to a depth of 1.5 cm. Allow it to stand for at least an
hour with a cover plate over the top of the tank to ensure that the atmosphere within the tank becomes
saturated with solvent vapour. This is called equilibration. After equilibration, remove the cover plate,
and place the thin layer plate (sample applied) vertically in the tank so that it stands in the solvent, with
the spotted end dipping in the solvent. Replace the cover plate. The separation of the compounds
occurs as the solvent moves upward. Develop the chromatogram at constant temperature in order to
avoid anomalous solvent running effects.

Once the solvent reaches the top of the plate, remove it from the tank. Dry and proceed for the
identification of the separated compounds.

TWO DIMESIONAL
In order to improve the resolution of a particular separation, two dimensional chromatography
may be used. Here, separation of the compounds is done in two different solvent systems. Apply the
sample as a single spot at one corner of the plate. Develop in the first solvent system, remove from the
tank and dry. Again develop in the second solvent system in the direction at right angles to the first
development.

46
CALCULATION

If the commercially available adsorbent contains a fluorescent dye, the separated compounds
will show up as blue, green or black spots when viewed under UV light. These areas can be scrapped,
eluted with a suitable solvent, and quantitative estimations of the separated compounds can be carried
out. When such a dye is not used in the adsorbent, one of the following methods of identification can
be followed.

i. Spray with 50 per cent sulfuric acid and heat. This will result in most compounds getting charred
and show up as brown spots.
ii. Examine the plates under UV light which show the locations of UV absorbing or fluorescent
compounds.
iii. Expose to iodine vapor for unsaturated compounds.
iv. Spray with Folin- Ciocalteau reagent for phenols.
v. Spray with ninhydrin for amino acids.
vi. If the compounds are radioactive, the plates may be subjected to autoradiography and can be
detected as dark spots on X-ray film. Alternatively, a radiochromatogram scanner may scan the
plate.

PREPARATIVE TLC
The TLC technique can also be used for the isolation of separated compounds and in that case, it
is called as preparative TLC. Instead of a thin layer, with a thick layer (up to 5 mm) of adsorbent coated,
a greater quantity of the sample can be applied onto the plate. After separation, the area of the
separated compound is scrapped off, eluted with a suitable solvent and recovered in a relatively pure
form.
In preparative TLC, usually the sample is applied as a streak rather than a spot, since a large
quantity can be applied. The compounds are also separated as series of bands which may be scrapped
off and eluted.

47
RESULT
1. The Rf value of the given compound is ……………..
2. The given sample was found to contain ………………..

THINGS TO LEARN

1. What is the solvent system for separation of organic acids?

2. Why is sulphuric acid used as spraying agent for identification of carbohydrates?

48
49