SUNANDAN DIVATIA SCHOOL OF SCIENCE

Bachelors
In
Biomedical Science

Practical Journal
Semester II
2023-2024

Name of Student: Anushka Gupta

Roll No.: A012

1
 DECLARATION BY THE STUDENT

“I confirm that I will follow the rules for working in the laboratory, and implement all the
rules and regulations regularly”.

Student name: Anushka Gupta Signature

2
 CERTIFICATE

This is to certify that Ms. Anushka Gupta of bachelor’s in biomedical science

course has satisfactorily completed and documented the practical work in
Practical 1 during the Second Semester of the year 2023-2024 in the Journal.

Signature of Teacher In-charge

Signature of Dean

Date: College Stamp/Seal

3
 Semester II
Practical 2
INDEX

Expt. Expt. Title Page Date Sign.

No. No.

1. Preparation of normal, molar and percent solutions 5 04/01/24

2. Preparation of Buffer Solution 11 11/01/24

3. Using absorbance to determine the concentration of 10 11/01/24

CuSO4

4. Titration of Glycine Curve. 16 18/01/24

5. Qualitative tests for Carbohydrates 18 24/01/24

6. Qualitative tests for Lipids 30 22/02/24

7. Qualitative tests for Amino acids & Proteins, 23 01/02/24

9. Preparation of casein from milk 37 07/03/24

10. Titrimetric analysis of Vitamin C. 43 23/03/24

11. Estimation of total carbohydrates 47 23/03/24

12. Estimation of glucose by GOD POD method 51 27/03/24

4
 Date: 04/01/24
Expt. No: 1

PREPARATION OF NORMAL, MOLAR AND PERCENT SOLUTIONS

Aim: To learn to calculate preparation of normal, molar and percent solutions

Introduction:
Amount of the solute may be defined as the mass (in grams or milligrams) of the solute of interest
present in a mixture or solution.
Concentration on the other hand is a term used to describe a solution. It essentially is the amount
of the solute of interest per unit of the solution. The concentration of a solute may be described in
many different ways, which may be classified into 2 categories.
1. Concentration in terms of amount of solute per unit volume of solution:

a. Molarity: Molarity of a solution is defined as number of moles of solute per unit

volume of solution in litres.

Since,

Therefore,

b. Normality: Normality of a solution is defined as the number of equivalents of solute

per unit volume of solution in litres.

Where,

And,

Therefore,

5
 Relating M and N

c. g%: The weight of the solute in grams per 100 ml of solution is defined as the
concentration of the solution in g%

d. mg%: the weight of solute in milligrams per 100 ml of solution is defined as the
concentration of the solution in mg%

e. v/v%: The volume of solute in millilitres per 100 ml of solution is defined as the
concentration of the solution in v/v%

f. Osmolarity: is the number of moles of solute particles per unit volume of solution taken
in litres.

Where n = number of ions generated by one solute molecule

2. Concentration in terms of amount of solute per unit mass of solution:

a. Molality: Molality of a solution is defined as the number of moles of solute per unit
mass of solvent taken in kilograms

b. Mole fraction: The term is generally used to define a multicomponent solution, i.e. a
solution with more than one solute. Mole fraction is the ratio of number of moles of
desired solute to the number of moles of solute in the solution.

Where,
n1, n2 and n3 are the number of moles of solutes 1, 2 and 3 respectively and X1 is the
mole fraction of solute 1.
c. w/w%: The weight of solute in grams per 100 grams of solution is defined as the
concentration of the solution in w/w%.

Exercise: Preparation and standardization of reagents:

Solve the following problems:

6
Problem Solution
1. A. How many grams of solid NaOH are (0.8 g)
required to prepare 500 ml of 0.04 M
solution?
B. Express the concentration of this solution
in terms of
i. Normality (0.04N
ii. Grams/litre 1.6 g/L
iii. %w/v 0.16%
iv. mg% 160 mg%
v. osmolarity 0.08 osmoles)
2. How many ml of 5M H2SO4 are required (0.6 ml)
to make 1.5 L of 0.002 M H2SO4
solution?
3. Describe the preparation of 2 L of 0.4M (90.7 ml stock diluted to 2 L with DW)
HCl solution starting with a concentrated
HCl solution (28%w/v HCl, SG = 1.15)
4. How many mg/ml is a 10% w/v NaOH (100 mg/ml)
solution?
5. How much NaCl will you weigh in order (42 gm)
to make 350 ml of 12% w/v NaCl?
6. What is the molarity of 10%w/v NaCl (1.71 M)
solution?
7. How will you make 200 µl of 25 mg/ml (50 µl stock diluted to 200 µl)
ampicillin working solution from a 100
mg/ml stock?
8. How will you make the following (Add 1 volume ethanol to 1 volume water,
dilutions of ethanol in distilled water? DF = 2)
Mention the dilution factor in each case (Add 1 volume ethanol to 4 volume water,
i. 1:1 dilution DF = 5)
ii. 1:5 dilution (Add 1 volume ethanol to 3 volume water,
iii. 1:3::ethanol:water DF = 4)
iv. 70% v/v ethanol (70 ml ethanol in 30 ml DW)
9. How many millimoles of solute are (5.52 millimoles)
present in 2L of 2.76 x 10-3 M KMnO4
solution?
10. A solution is prepared by dissolving 376 (0.00152M)
mg of K3[Fe(CN)6] (M.W 329.2) In (0.00456M)
sufficient amount of water to give 750 ml (0.00152M)
of solution. Calculate the following (0.05%W/v)
i. Molarity of K3[Fe(CN)6] (0.00152m)
ii. Molarity of K+ ions assuming complete
ionization of the solute
iii. Molar concentration of [Fe(CN)6]- ions
assuming complete ionization of the
solute
iv. %W/V of solution

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 v. molality

11. (1.66 x 10-24 g)

i. What is the weight of 1 Hydrogen (3.32 x 10-24 g)
atom?
ii. What is the weight of 1 Hydrogen
molecule?

12. (3.72 x 10-23 dm3)

i. What is the volume of one molecule of (3.72 x 10-4 m3)
oxygen? Given 1 mole of a gas (0.021 dm3)
occupies 24.2 dm3 at STP
1m3 = 103 dm3
ii. What is the radius of this oxygen
molecule assuming it to be spHerical
13. How many water molecules are present in (1.67 x 10-21)
1 drop of water assuming that one drop of
water has a volume of 0.05 ml?
14. Concentration of commercial NH3 (liquor (15.51M)
ammonia) is 29%W/W. Its density is 0.9 (24.02m)
g/L. Calculate: (781.6 ml of stock diluted to 2L)
i. Molarity of this solution
ii. Molality of the solution
iii. What volume of liquor ammonia is
required to prepare 2L of 6M solution
15. Commercially available HCl is 37% pure. (164.3 ml of stock diluted to 20 L)
Its Specific Gravity = 1.2. How much
volume of the concentrated acid is
required to prepare 20 L of 0.1M HCl
solution?

Interpretation:
A solution of 70% Ethanol was successfully prepared from a stock of 95% Ethanol by adding
73.68mL sock to 26.32mL distilled water/diluent.

A solution of 1N NaOH by adding 40 gm of NaOH pellets in 1L of water.

A solution of 1M KOH Solution by adding 56gm of pure stock in 1L of water.

A solution of 1M K2HPO4 was made by adding 17.42g of pure stock in 1L of water.

8
Conclusion:
We successfully made solutions of different Molarities , Normalities and Percentages of the
following compounds:-
● K2HPO4
● KOH
● NaOH
● Ethanol

Tables:

1.K2HPO4

Label Amount Volume of Dilution Total Volume

Stock 17.42g Pure pellets 1L

Dilution 1L 1M

2.KOH

Label Amount Volume of Dilution Total Volume

Stock 56g Pure pellets 1L

Dilution 1L 1M

3.NaOH

Label Amount Volume of Dilution Total Volume

Stock 40g Pure pellets 1L

Dilution 1L 1N

4.Ethanol

Label Amount Volume of Dilution Total Volume

Stock 73.68ml 95% 100ml

Dilution 26.32ml 75%

9
 Date:
Expr No.:2

USING ABSORBANCE TO DETERMINE THE CONCENTRATION OF CuSO4

Aim: Estimate the λmax of Copper SulpHate (CuSO4).

Principle:
As per Beer- Lambert’s law, the absorbance of a solution is directly proportional to its
concentration as well as the
path length.
A is directly proportional to l
A is directly proportional to C
(Where l=path length, A=absorbance, C=Concentration)
By adding proportionality constant ε,
A=εCl
Generally, l=1cm i.e., the internal diameter of the cuvettes.
λmax is the maximum absorbance of a chemical solution at a particular wavelength.
λmax of proteins is 280nm and for nucleic acids it is 260nm.

Requirements: 1% solution of Copper SulpHate, Distilled Water, Beakers, Glass Rod,

Colorimeter, Glass Cuvettes.

Procedure:
1. Fill the cuvettes with 1% solution of copper sulpHate

2. Take down the absorbance at 410nm, 430nm, 470nm, 490nm, 520nm, 540nm, 580nm,
610nm, 640nm
3.Plot a wavelength vs absorbance on a grapH paper.

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Observation:
Wavelength Absorbance
Blank(B) CuSO4© (C-B)
400 0.49 0.67 0.18
420 0.07 0.11 0.14
470 0.15 0.23 0.08
500 0.58 0.60 0.02
530 0.42 0.48 0.06
620 0.31 0.53 0.22
660 0.40 0.76 0.36
700 0.69 1.69 1.00

Interpretation:
The maximum absorbance of 1% CuSO4 solution is 10 which is seen at 700mm, Thus, the λmax
of the 1% CuSO4 solution is 700 mm as measured using a colorimeter within the wavelength
range 400-700mm.

11
 Date:18/01/24
Expt. No:2
TITRATION CURVE OF GLYCINE

Aim: To determine the titration curve of Glycine

Introduction: A titration curve is a grapH of the pH as a function of the amount of titrant (acid or
base) added. This curve empirically defines several characteristics. The precise number of each
characteristic depends on the nature of the acid being titrated:
1) the number of ionizing groups, 2) the pKa of the ionizing group(s), 3) the buffer region(s).

Principle: Amino Acids are ampHoteric molecules and can be titrated with both acid and alkali.
They have an acidic group (COOH) and a basic group (NH2) attached to the α-carbon. The α-
carbon is also attached to ionizable groups that act as weak acids or bases, giving off or taking on
protons when the pH is altered. At neutral pH amino acids are present as zwitterions. When an
amino acid is dissolved in water it exists predominantly in the isoelectric form. The isoelectric
point, pI, is the pH of an aqueous solution of an amino acid at which the molecules have no net
charge. When this dissolved amino acid is titrated with acid, it acts as a base, and with base, it
acts as an acid which makes them an ampHoteric molecule.

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These ionization follow the Henderson-Hasselbalch equation:
When the concentration of the unprotonated form equals that of the protonated form, the ratio of
their concentrations equals 1, and log 1=0. Hence, pKa can be defined as the pH at which the
concentrations of the protonated and unprotonated groups of the amino acids are equal. The pKa
also equals the pH at which the ionizable group is at its best buffering capacity; that is the
solution can effeciently resists changes in pH.

While titration the pK is the pH at the midpoint of the buffering region (where the pH changes
only slightly upon addition of either acid or base). The pK is the pH corresponding to the
inflection point in the titration curve. The end point of a titration curve represents the observed
end of the titration.For acidic amino acids, the pI is given by ½(pK1 + pK2) and for basic amino
acids it’s given by ½(pK2 + pK3).

In this experiment we are finding out the titration curve of the amino acid Glycine.

Glycine is a diprotic amino acid which means that it has two dissociable Protons, one on the α-
amino group and the other on the carboxyl group. In the case of Glycine,the R group does not
contribute a dissociable Proton.

13
The dissociation of proton proceeds in a certain order which depends on the acidity of the proton:
the one which is most acidic and having a lower pKa will dissociate first. So, the H+ on the α-
COOH group (pKa1) will dissociate before that on the α-NH3 group (pKa2).
Requirements:
Glasswares:
1. Burette -2
2. Beaker
3. Stirrer
4. pH Meter

Reagents:
1. 0.1M Hydrochloric acid
2. 0.1M Sodium Hydroxide
3. 0.1M Glycine
4. Standard Buffer of pH=4, pH= 7, pH=10

Procedure:
1. Pipette out 20ml of the amino acid solution into a 100ml beaker.
2. Standardize the pH meter using the standard buffer solutions.
3. Determine the pH of the amino acid solution.
4. Add 0.3ml of 0.1M HCl from the burette and record the pH after each addition.
5. Continue adding the acid until the pH falls to 1.6
6. Wash thoroughly the pH electrode in distilled water.
7. Take 20 ml of amino acid solution in another beaker and check its pH.
8. Now titrate the amino acid solution by adding 0.3ml of 0.1M NaOH until the pH reaches 12.5.
Plot the titration curve using the values recorded and find the pKa values.

Observation table:
Volume of HCl added pH Volume of NaOH added pH
(mL) (mL)

1 4.75 1 8.01

1 4.5 2 8.4

1 4.3 3 9.23

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 2 4.15 4 9.52

2 4.00 4 9.78

5 9.88

6 10.02

6ml 25ml

GrapH:

Chart Title
12

10

0
0 5 10 15 20 25 30 35

15
Result:
12.5 ml of O.1M NaOH (12) was required to increase this pH of 0 .1M glycine from 4.75 to
10.02. 6 ml of 0.1 HCl (PH1) is required to decrease the pH of 0.1M Glycine from 4.75 to pH
10.05.

Conclusion:
The graph indicates that 50% of the titration was completed at pH 8.01.

16
 Date:18/01/24
Expt. No:3
Preparation of Buffer Solution

Aim: Preparation of Buffer Solution

Principle: The preparation of a buffer solution involves creating a solution that resists change
in pH when small amounts of acid or base are added. This is achieved by mixing a weak acid
with its conjugate base or a weak base or a weak base with its conjugate acid in specific
proportions to maintain a stable pH within a desired range. Buffers are essential in various
biological, chemical and industrial processes where maintaining a constant pH is crucial.

Requirements: 1M phosphate buffer/ reagents( KH2Po4, K2HPo4), acid 1N HCl, base 1N

Naoh.

Procedure: Reagent 1 KH2Po4 / Reagent 2 K2HPo4(1M buffer) ——> pH observed 6.2 (1)
(1) + base 1N Naoh ————> pH observed is 7.4

Observation: Both the reagents were added in equal quantities in a beaker and mixed. The
initial pH was observed to be 6.2. In order to change the pH of the solution to the basic side, a
strong base i.e. 1N NaOh was added to the solution and the pH was observed to be 7.4.

Interpretation: A buffer was made successfully and change in pH was observed by addition
of a strong acid or base.

17
 Date:24/01/24
Expt. No:3

QUALITATIVE TEST FOR CARBOHYDRATES

Aim: To qualitatively analysis the presence of carbohydrates present in an unknown solution

on the basis of various chemical assays.

Introduction: Carbohydrates are polyhydroxy aldehydes and/or ketones.

Carbohydrates are mainly divided into monosaccharides, disaccharides and polysaccharides.

The commonly occurring monosaccharides includes glucose, fructose, galactose, ribose, etc.
Two monosaccharides combine together to form disaccharides which include sucrose, lactose
and maltose. Starch and cellulose fall into the category of polysaccharides, which consist of
many monosaccharide residues.

Principle: Carbohydrates are also known as sugars. They can be reducing or non-reducing
sugars based on the functional groups available for the reducing reaction. Carbohydrates
though having polyhydroxy aldehydes/ ketones differ amongst themselves in the arrangement/
or confirmation of the hydroxy group into glucose, galactose, maltose etc. Presence of ketone
group instead of an aldehyde makes it fructose. Identification of these sugars and
differentiating them into reducing and non-reducing sugars; monosaccharides can be done by
various tests. The principle reaction of the tests are as follows:

a. Molisch’s Test: This is a common test for all carbohydrates dehydrated by conc. H2SO4. The

reaction is based on the fact that conc. H2SO4 catalyses the dehydration of sugars to form

furfural (from pentoses) or hydroxymethyl furfural (from hexoses). These furfurals

18
 then condense with sulfonated alpHa-napHthol to give a purple or violet coloured product.
Polysaccharides and glycoproteins also give a +ve reaction. In the event of the carbohydrate
being a poly- or disaccharide, the acid first hydrolyses it into component monosaccharides,
which then get dehydrated to form furfural or its derivatives.

b. Fehling’s Test: This forms the reduction test of carbohydrates. Fehling’s solution contains blue

alkaline cupric hydroxide solution, heated with reducing sugars gets reduced to yellow or red

cuprous oxide and is precipitated. Hence, formation of the yellow or brownish-red coloured

precipitate helps in the detection of reducing sugars in the test solution.

c. Benedict’s Test: As in Fehling’s test, free aldehyde or keto group in the reducing sugars reduce

cupric hydroxide in alkaline medium to red coloured cuprous oxide. Depending on the

concentration of sugars, yellow to green colour is developed. All monosaccharides are reducing

sugars as they all have a free reactive carbonyl group. Some disaccharides, like maltose, have

exposed carbonyl groups and are also reducing sugars, but less reactive than monosaccharides.

d. Barfoed’s Test: Barfoed's test is used to detect the presence of monosaccharide (reducing) sugars

in solution. Barfoed's reagent, a mixture of ethanoic (acetic) acid and copper(II) acetate, is

combined with the test solution and boiled. A red copper(II) oxide precipitate is formed will

19
 indicates the presence of reducing sugar. The reaction will be negative in the presence of

disaccharide sugars because they are weaker reducing agents. This test is specific for

monosaccharides. Due to the weakly acidic nature of Barfoed's reagent, it is reduced only by

monosaccharides.

e. Seliwanoff’s Test: It is a color reaction specific for ketoses. When conc. HCl is added. Ketoses

undergo dehydration to yield furfural derivatives more rapidly than aldoses. These derivatives form

complexes with resorcinol to yield deep red colour. The test reagent causes the dehydration of

ketohexoses to form 5-hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol

present in the test reagent to produce a red product within two minutes (reaction not shown).

Aldohexoses reacts so more slowly to form the same product

f. Bial’s Test: Bial’s test is used to distinguish between pentoses and hexoses. They react with Bial’s

reagent and are converted to furfural. Orcinol and furfural condense in the presence of ferric ion

to form a coloured product. Appearance of green colour or precipitate indicates the presence of

pentoses and formation of muddy brown precipitate shows the presence of hexoses.

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g. Iodine Test: This test is used for the detection of starch in the solution. The blue-black colour is due

to the formation of starch-iodine complex. Starch contain polymer of α-amylose and amylopectin

which forms a complex with iodine to give the blue black colour.

h. Osazone Test: The ketoses and aldoses react with pHenylhydrazine to produce a

pHenylhydrazone which further reacts with another two molecules of pHenylhydrazine to yield

osazone. Needle-shaped yellow osazone crystals are produced by glucose, fructose and mannose,

whereas lactosazone produces mushroom shaped crystals. Crystals of different shapes will be

shown by different osazones. Flower-shaped crystals are produced by maltose.

Requirements:
Glasswares:
1. Test tubes.
2. Test tube holder.
3. Water bath
4. Spatula
5. Dropper

Reagents:
1. Molisch’s Reagent
2. Iodine solution
3. Fehling’s reagent A & B
4. Benedict’s qualitative reagent
5. PHenylhydrazine hydrochloride
6. Sodium acetate
7. Glacial acetic acid
8. Glucose, Fructose, Maltose, Sucrose.

Procedure:

No. Test Observation Inference

1 Molisch’s Test A deep violet Presence of
coloration is carbohydrates
2-3 drops of molisch reagent is added produced at the
to 2ml of the test solution. Very gently junction of the two
layers

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 add 1ml of conc. H2SO4 along the side
of the test tube.

2 Iodine test Blue colour is Presence of

4-5 drops of iodine solution are added observed polysaccharides
to 1ml of the test solution and
contents are mixed gently.
3 Fehling's test A red precipitate is Presence of reducing
About 2 ml of sugar solution is added formed sugar
to about 2 ml of Fehling’s solution
taken in a test-tube. It is then boiled
for 10 min
4 Benedict’s test Formation of a green, Presence of reducing
To 5 ml of Benedict's solution, add red, or yellow sugars
1ml of the test solution shake the precipitate
tube. Place the tube in a boiling water
bath and heat for 3 minutes. Remove
the tubes from the heat and allow
them to cool.
5 Barfoed’s test A deep blue colour is Presence of reducing
To 2 ml of the solution to be tested formed with a red sugars. Appearance
added 2 ml of freshly prepared ppt. settling down at of a red ppt as a thin
Barfoed's reagent. Place test tubes the bottom or sides of film at the bottom of
into a boiling water bath and heat for the test tube. the test tube within 3-
3 minutes. Allow to cool. 5 min. is indicative
of reducing mono-
saccharide. If the ppt
formation takes more
time, then it is a
reducing
disaccharide.
6 Seliwanoff test A cherry red colored Presence of ketoses
To 3ml of of Seliwanoff’s reagent, precipitate within 5 [Sucrose gives a
add 1ml of the test solution. Boil in minutes is obtained. positive ketohexose
water bath for 2 minutes A faint red colour test ] Presence of
produced aldoses
7 Bial's test A blue-green product Presence of pentoses.
Add 3ml of Bial’s reagent to 0.2ml of A muddy brown to Presence of hexoses
the test solution. Heat the solution in gray product
a boiling water bath for 2 minutes
8 Osazone Test Formation of Glucose/fructose
Two ml of the test solution, add 3ml beautiful yellow Presence of lactose
of pHenyl hydrazine hydrochloride crystals of osazone Presence of maltose
solution and mix. Keep in a boiling Needle shaped
water bath for 30mts. Cool the crystals Hedgehog
solution and observe the crystals crystals Sunflower
under microscope. shaped crystal

22
Observation

Sample Qualitative analysis Remark

Molisch Fehling’s Iodine
1. Glucose(stock) purple ring (+) Blue Negative has carbs and
reducing sugars
1% Fructose dark. Blue Negative has carbs, but no
purple ring (+) reducing sugar
1). Sucrose purple ring (+) Brownish Red Negative has carbs, but no
(sugarcane juice) reducing sugar

1% Starch light Brownish Red Blue colour Has Starch but

purple ring (+) nothing else
-ve blank no ring (-) Blue Negative no carbs and reducing
sugar

Results:
We successfully performed quality test of carbohydrates by following method.
o Molisch Method
o Fehling’s Method
o Iodine test
And resulted in Glucose being carbohydrate and reducing sugar.
Sucrose is reducing sugar and Starch gave positive Molisch test.

Conclusion:
We conclude the presence of carbohydrate, reducing sugar and starch for various compounds.

23
 Date:01/02/24
Expt. No:5

QUALITATIVE TEST FOR AMINO ACIDS/PROTEINS

Aim: To qualitatively analysis the presence of Amino acids/Proteins present in an unknown

solution on the basis of various chemical assays.
Introduction: Amino acids are building blocks of all proteins, and are linked by peptide bond
(-CONH-) to form the primary structure of a protein. Amino acids comprises of an amine
group, a carboxylic acid group and a varying side chain that differs between different amino
acids. Based on the side chains, there are 20 different amino acid present in nature. Amino
acids are ampHoteric in nature, i.e. they possess an amino group as well as an carboxylic group
that make amino acids ampHoteric. It also bears an Isoelectric point (IEP). This point is the pH
at which the amino acids has a zero net charge. IEP of amino acids is one of its key features.
Every amino acid has a distinguished IEP.

Principle: Amino acids respond to all typical chemical reactions associated with compounds
that contain carboxylic acid and amino groups, usually under conditions where the zwitter ions
form is present in only small quantities. All amino acids (except glycine) exhibit optical activity
due to the presence of an asymmetric α – Carbon atom. Amino acids with an L – configuration
are present in all naturally occurring proteins, whereas those with D – forms are found in
antibiotics and in bacterial cell walls. Amino acids can be identified by performing the
following tests.
a. Ninhydrin test: In the pH range of 4-8, all α- amino acids react with ninhydrin
(triketohydrindene hydrate), a powerful oxidizing agent to give a purple colored product
(diketohydrin) termed Rhuemann’s purple. All primary amines and ammonia react

24
 similarly but without the liberation of carbon dioxide. The imino acids proline and
hydroxyproline also react with ninhydrin, but they give a yellow coloured complex instead
of a purple one. Besides amino acids, other complex structures such as peptides, peptones
and proteins also react positively when subjected to the ninhydrin reaction.
b. Millon's test: PHenolic amino acids such as Tyrosine and its derivatives respond to this
test. Compounds with a hydroxybenzene radical react with Millon’s reagent to form a red
colored complex. Millon’s reagent is a solution of mercuric sulpHate in sulpHuric acid.
c. Xanthoproteic acid test: Aromatic amino acids, such as PHenyl alanine, tyrosine and
tryptopHan, respond to this test. In the presence of concentrated nitric acid, the aromatic
pHenyl ring is nitrated to give yellow colored nitro-derivatives. At alkaline pH, the color
changes to orange due to the ionization of the pHenolic group.
d. Aldehyde Test: The test indicates presence of indole group containing amino acid. The
oxidizing agents are HgSO4 and H2SO4 which oxidize indole nucleus of tryptopHan. This
oxidized indole ring reacts with formaldehyde to give the violet colored ring at the junction
of two liquids.
e. Pauly's diazo Test: This test is specific for the detection of TryptopHan or Histidine. The
reagent used for this test contains sulpHanilic acid dissolved in hydrochloric acid.
SulpHanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid
results in the formation a diazonium salt. The diazonium salt formed couples with either
tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye).
f. Hopkins cole test: This test is specific test for detecting tryptopHan. The indole moiety of
tryptopHan reacts with glyoxilic acid in the presence of concentrated sulpHuric acid to give
a purple colored product. Glyoxilic acid is prepared from glacial acetic acid by being
exposed to sunlight.
g. Sakaguchi test: Under alkaline condition, α- napHthol (1-hydroxy napHthalene) reacts
with a mono-substituted guanidine compound like arginine, which upon treatment with
hypobromite or hypochlorite, produces a characteristic red color.
h. Lead sulpHide test: SulpHur containing amino acids, such as cysteine and cystine. upon
boiling with sodium hydroxide (hot alkali), yield sodium sulpHide. This reaction is due to
partial conversion of the organic sulpHur to inorganic sulpHide, which can detected by
precipitating it to lead sulpHide, using lead acetate solution.

25
i. Biuret test for proteins: In alkaline medium copper from CuSO4 will form a coordination
complex with the peptide bond. A minimum of 3 peptides are required to answer this test.
Biuret test is not performed on urine because of presence of peptide like link.

Requirements:
Glassware:
1. Test tubes
2. Test tube holder
3. Water bath
4. Spatula
5. Dropper
6. Bunsen burner
7. Ice box
8. Vortex mixer
9. Filter paper
10. Hair drier

Reagents:
1. Ninhydrin reagent: Ninhydrin (2%W/V) in acetone
2. Sodium hydroxide (40%W/V)
3. Conc. Nitric acid
4. Hydrochloric acid (6N)
5. SulpHanilic acid (1%w/v) in 1N HCl
6. Sodium nitrite (5%w/v) in distilled water ( to be freshly prepared)
7. Sodium carbonate (10%w/v) in distilled water
8. Millon’s reagent ( 15%w/v mercuric sulpHate in 6N sulpHuric acid)
9. Sodium nitrite solution (5%) [To be freshly prepared]
10. Bromine (5%) in acetic acid(33%) solution
11. Amonium carbonate (5%)
12. Acetic acid – Glyoxilic acid reagent – Glacial acetic acid is exposed to sun light ( for
5 – 6 hours) for the formation of small amounts of glyoxilic acid) Con. SulpHuric acid
13. Sodium hydroxide (10%w/v)
14. α NapHthol reagent (1%w/v in ethyl alcohol)
15. Hypobromite solution (To be freshly prepared): -Take 100 of 5%(W/V) sodium
hydroxide solution in a glass reagent bottle and add 1ml of pre chilled liquid bromine,
using a pro pipette. Shake the contents till bromine dissolves)
16. Urea solution 5% (w/v)
17. Lead acetate solution (10% w/v)
18. 5N NaOH
19. 1% Glycine solution
20. 10% Sodium nitroprusside solution

26
Procedure:
NO. Experiment Observation Inference
1. Ninhydrin Test: To 1 ml of Purple Color will The given solution may
given unknown solution add form be an Amino acids
1ml of Ninhydrin reagent and
boil the contents slightly for
few minutes.

2. Xanthoproteic test: To 2ml of A yellow colour Indicates the presence of

given solution add 1ml of conc.
HNO3 carefully and boil the
content. Cool and add 40%
NaOH till the solution become
alkaline.
formed in acid “Aromatic Amino
solution will turn to Acids”. (Nitration of
orange color aromatic ring to give
precipitates with the nitro substituent which
addition of alkaline. reacts with NaOH to
give an orange color)

3. Millon’s test: To 1ml of given Yellow color Confirms the presence or

solution add few drops of 10% precipitate will be Tyrosine
H2SO4. Add equal volume of observed at
10% of mercuric sulpHate & beginning which
boil the contents cool & add will turn to red
few drops color after the
of 10% Sodium nitrate addition of Sodium
(NaNO2) and warm the content Nitrate.
again

4. Aldehyde Test: Add 1 drop of
10% mercuric sulpHate in
10%H2SO4 to 1 ml of protein
solution and 1 drop of
formaline. Mix well. Add 2 ml
sides of test tube.
A violet ring will Indicates the presence of
form at the junction “TryptopHan”
of the two liquids. (Specific for Indole’
group)

5. Sakaguchi Test: To 2 ml of Red color solution Indicates the

presence
given solution add 1ml of will form of “Arginine”
(Specific

27
 40% NaOH and few drops for “Guanidonium
of alcoholic alpHa- Napthol group”)
and then add two drops of
freshly prepared sodium hypo
bromide.

6. Biuret Test: To 2ml of given A Purple Violet Indicates the

presence of
solution add 3ml of NaOH. or “Protein” (Specific
for
Mix. Add copper sulpHate Pinkish Violet “Peptide bond”)
solution dropwise, mixing after color will form
addition. Two or three drops of
copper sulpHate solution are
sufficient to obtain good
results.

7. Pauly’s Test: Red color solution Indicates the

To 2ml of given presence of
solution add 2% SulpHonic will form “Tyrosine” &
acid
in 10% HCL and cool in Ice “Histidine”
add 4ml of 1% Sodium
Nitrate and again cool in Ice.
Make the solution with
Alkaline with 10% Na2CO3
and then add 5ml of Ethanol.

8. Hopkin’s Coal Test A reddish violet ring Indicates the

Take 2 ml of the protein will form at junction presence of
solution and add 2 ml of of the two fluids. ‘’TryptopHan’’
Hopkin’s-Cole reagent. Mix, (The indole group of
incline the test tube, and add 3- TryptopHan reacts
4 ml of conc. SulpHuric acid with glyoxylate
along the wall of the test tube. released by the
Mix the test tube upright and action of H2SO4 on
rotate between palms. Acetic Acid to give
a purple color.)
9. Lead Acetate: To 1ml of the Precipitate will be Indicates presence
of
amino acid solution taken in a formed “Cysteine”
test tube, add few drops of
sodium hydroxide (40%) and
boil the contents for 5 – 10min

28
 over a bunsen burner. Cool the
contents and add few drops of
10% Lead acetate solution and
observe.

Conclusion:

According to the ninhydrin test, all the samples hare amino acids since the test was the for all
samples.
The result was a dark purple colour for 1%. glycine and 1y alanine.
Ninhydrin turned a dark orange with 1) cysteine. This is not unusual since cysteine forms a
red colour upon reacting with ninhydrin due to its thiol (sulfhydryl) group.

Result:

Amino Acid Colour

Colour

1% glycine dark purple

1%. alanine dark purple

1% cysteine dark orange

29
 Date:22/02/24
Expt. No:4

QUALITATIVE ANALYSIS OF LIPIDS

Aim: To qualitatively analysis the presence of Lipids in an unknown solution on the basis of
various chemical assays.

Introduction: Lipids are one of the major constituents of foods, and are important in our diet
for a number of reasons. They are a major source of energy and provide essential lipid nutrients.
Nevertheless, over-consumption of certain lipid components can be detrimental to our health,
e.g. cholesterol and saturated fats. In many foods the lipid component plays a major role in
determining the overall pHysical characteristics, such as flavor, texture, mouth feel and
appearance.

Principle: The term lipid is used to define naturally compounds that are saponifiable esters
of long chain fatty acids. Lipids are insoluble in water but soluble in fat solvents such as
acetone, alcohol, chloroform etc. Lipid are generally classified into simple lipids comprising
of Glycerides and Waxes; and Compound lipids which include pHospHolipids, spHingolipids
and Lipoproteins. Following are the test which will aid in qualitative analysis of lipids.

Requirements:

Glasswares:
1. Test tubes
2. Test tube holder
3. Spatula
4. Dropper
5. Conical flask
6. Round bottom flask
7. Reflux condenser
8. Heating mantle
9. Burettes
10. Burette stands

Reagents:
1. Fat or Oil
2. 0.1N KOH
3. 0.5 N alcoholic potassium hydroxide ( alcoholic KOH) ( prepared by dissolving 30 g
potassium hydroxide in 20 mL of water and make the final volume to 1 L using 95 %

30
 ethanol. Leave the solution to stand for 24 h before decanting and filtering the
solution.
4. 0.5 N Hydrochloric acid
5. PHenolpHthalein.indicator
6. Oil or fat
7. Hanus solution ( it’s prepared by dissolving 18.2 g of iodine in 1L of glacial acetic acid
and then add 3 ml of bromine water for increasing the halogen content.
8. 15% potassium iodide solution
9. 1% starch solution
10. 0.1 N Sodium thiosulfate solution

Procedure:
1. Solubility Test
Principle: The oil will float on water because of lesser specific gravity.
Procedure: Take 3ml of solvents in each test tube and add 5 drops of sample. For water
and ethanol, it is insoluble and for chloroform and ether, it is soluble and hence the
given sample is lipid.
2. Transparency Test:
Principle: All the lipids are greasy in nature. Therefore, the test may be taken as
group test for lipids. The oil does not wet the paper.
Procedure: Take 3ml of ether in a test tube and dissolve 5 drops of oil in tit. Put a drop
of the solution on the filter paper and let it dry. A translucent spot on the filter paper
was observed and this indicates the greasy character of the lipid.
3. Test for unsaturation:
Principle: The unsaturated fatty acids absorb iodine at the double bonds until all the double
bonds are saturated with iodine. Hence the amount of iodine required to impart its color to
the solution is a measure of the degree of the fatty acids.
Procedure Take 1ml of of chloroform and add a drop of methanol and one drop of oil.
to this add 1drop of iodine. Chloroform dissolve sample give red color which
decolorizes the iodine giving brown color. This indicates the presence of fatty acids
4. Acid value (Acid Number)
Principle: The acid value (AV) is the number that expresses, in milligrams the quantity of
potassium hydroxide required to neutralize the free acids present in 1 g of the substance.
The acid value may be overestimated if other acid components are present in the system,
e.g. amino acids or acid pHospHates. The acid value is often a good measure of the break

31
 down of the triacylglycrols into free fatty acids, which has an adverse effect on the quality
of many lipids.

Acid value is the measure of hydrolytic rancidity. In general, it gives an indication about
edibility of the lipid.
- Edible oil contain > 1%; PHarmaceutical oil must not have any acidity.

Procedure
1. Place 5.0 g of fat or oil in a dried conical flask.
2. Add 25 ml of absolute ethanol and add ( 2-3) drops of pHenolpHthalein
3. Heat with shaking in water bath (65%) for 10 minutes ,then cool Titrate the solution
against 0.1 N KOH until pink color appears (end point).
4. Record your observations.
5. Calculate the acid value (AV) and free fatty acid (%FFA) using above laws.

5. Saponification Number
Principle: The saponification value is the number of mg of potassium hydroxide required
to neutralize the free acids and to saponify the esters in 1 g of the substance. The
saponification number is a measure of the average molecular weight of the triacylglycerols
in a sample. Saponification is the process of breaking down a neutral fat into glycerol and
fatty acids by treatment with alkali. The smaller the saponification number the larger the
average molecular weight of the triacylglycerols present i.e. Saponification value is
inversely proportional to the mean molecular weight of fatty acids (or chain length).

Procedure
1. Weigh approximately 2 g of the fat or oil into a 250 mL conical flask.
2. Add 25 mL of alcoholic potassium hydroxide solution ( 0.5 N).
3. Attach a reflux condenser and heat the flask contents on a boiling water bath for 1 hour
with occasional shaking.
4. While the solution is still hot, add 3-5 drops of pHenolpHthalein indicator and titrate the
excess potassium hydroxide with the 0.5 N hydrochloric acid (Vml of hydrochloric acid at
end point (disappearance of pink colour). Mark this point as S

32
5. Do same above procedure but without sample (Vml of hydrochloric acid at end point
represents B).
6. Calculate the saponification number by using the following law:

6. Ester Value
Principle: The ester value is defined as the mg of KOH required to react with glycerin
(glycerol / or glycerin) after saponify one gram of fat. It is calculated from the
saponification Value (SV) and the acid Value (AV):

7. Iodine Value (I.V)

Principle: The iodine value (IV) gives a measure of the average degree of unsaturation of a
lipid: the higher the iodine value, the greater the number of C=C double bonds. By definition
the iodine value is expressed as the grams of iodine absorbed per 100g of lipid. Iodine value
(I.V.) is directly proportional to the degree of unsaturation (No of double bonds.) and inversely
proportional to the melting point (M.P.) of lipid. An increase in I.V.indicates high susceptibility
of lipid to oxidative rancidity due to high degree of unsaturation. One of the most commonly
used methods for determining the iodine value of lipids is "Hanus method". The lipid to be
analyzed is weighed and dissolved in a suitable organic solvent, to which a known excess of
iodine chloride is added. Some of the IBr reacts with the double bonds in the unsaturated lipids,
while the rest remains:
R-CH=CH-R + IBrexcess R-CHI-CHBr-R + IBrremaining
The amount of IBr that has reacted is determined by measuring the amount of IBr remaining
after the reaction has gone to completion (IBrreacted =IBrexcess - IBrremaining). The
amount of IBr remaining is determined by adding excess potassium iodide to the solution to
liberate iodine, and then titrating with a sodium thiosulfate (Na2S2O3) solution in the
presence of starch to determine the concentration of iodine released:
IBrremaining + 2KI KBr + KI + I2
I2 + starch + 2Na2S2O3 (blue) 2NaI + starch + Na2S4O6 (colorless)

33
 TYPICAL IODINE NUMBERS
Coconut oil 8 - 10

Butter 25 - 40

Palm oil 37 - 54

Olive oil 75 - 95

Peanut oil 85 - 100

Corn oil 115 - 130

Fish oils 120 - 180

Soybean oil 125 - 140

Safflower oil 130 - 140

Sunflower oil 130 - 145

Procedure
1. Weigh approximately 0.25 g of the fat or oil into a 250 mL conical flask.
2. Add 10 ml of chloroform.
3. Add 30 ml of Hanus solution and close the flask completely by Para film, then leave the
solution for 30 minutes with shaking continuously.
4. Add 10 ml of 15% potassium iodide solution and then shake.
5. Add 100 ml of distilled water (DW).
6. Titrate the iodine solution against 0.1 N Sodium thiosulfate solution till yellow color
formed , then add 2-3 drops of starch solution where blue solution formed and then
continue with titration till the blue color is disappeared (Volume (ml) of Na2S2O3 at end
point represents S)
7. Do same above procedure but without sample (Volume (ml) of Na2S2O3 at end point
represents B).
8. Calculate the iodine number by using the following law:

B: V ml of Na2S2O3 volume for blank

S: V ml of Na2S2O3 volume for sample

34
Results:
1)
Solvents Observation
Hair oil vegetable oil Ghee
Water Floats Floats Floats
Ether dissolves dissolves Did not dissolve
Iodine test dissolves dissolves Floats
Ethanol sinks sinks Floats

2) Transparency test
Both, the hair oil a edible all showed a translucent spot on the filter paper. The spot for hair
oil, however, was much yellower than that of vegetable oil's.

(3) Unsaturation test

The reaction system turned from red to light brown after a few minutes. Ghee was used.

Interpretation:
1)Solubility test
Since oil has a lower specific gravity than water it floats, However, it has a higher specific
gravity than methanol and Both were insoluble in water & methanol. But both oil were
soluble in ether and chloroform.

2) Transparency test
The presence of translucent spot for both oils swept that they are Both greasy in nature.
However, since the hair oil was originally deep yellow in colour, it formed a yellow-coloured
translucent spot.
Vegetable oil formed a white transeunt spot.

3) Unsaturation test
Using Ghee resulted in formation of a light brown colour because it has a high in unsaturated
C=C) Content. So I2 would react with unsaturated part and change from red to light blown.

35
 Date:07/03/24
Expt. No:7

PREPARATION OF CASEIN FROM MILK and Estimation of protein

content by Folin Lowry Method

Aim: To perform the isoelectric precipitation of casein present in milk

Introduction: Milk is a mixture of proteins, most of them present in very small amounts. Milk
proteins are classified into three main groups of caseins (80%), whey proteins and minor
proteins. Casein is a heterogeneous pHospHorous containing protein, is present in milk as
calcium salt and calcium caseinate. At some pH value, called the isoelectric point (IEP), all the
positive charges and all the negative charges on the [casein] protein will be in balance, so that
the net charge on the protein will be zero. At this point casein from milk can be precipitated
out by titrating it with an acid.
Principle: Casein is a heterogeneous mixture of protein. Mainly comprises of a mixture of
alpHa, beta and kappa caseins to form a cluster called micelle which gives milk its white
opaqueness. Casein, like any other proteins, is composed of amino acids. As we know amino
acids are ampHoteric in nature. At the isoelectric point, all the positive charges and negative
charges on the amino acid will be equal and the net charge will be zero. The same pHenomenon
applied on proteins. Thus proteins also have an isoelectric point; a pH at which the protein net
charge is zero. For casein, the IEP is approximately 4.6 and it is the pH value at which acid
casein is precipitated. Most proteins show minimum solubility at their isoelectric point and this
principle is used to isolate the casein by adjusting the PH of milk to (4.5-4.8) its isoelectric
point. Casein is also insoluble in ethanol and this property is used to remove unwanted fat from
the preparation. In milk, which has a pH of about 6.6, the casein micelles have a net negative
charge and are quite stable. During the addition of acid to milk, the negative charges on the
outer surface of the micelle are neutralized (the pHospHate groups are protonated), and the
neutral protein precipitates.
The same principle applies when milk is fermented to curd. The lactic acid bacillus produces
lactic acid as the major metabolic end-product of carbohydrate fermentation. The lactic acid
production lowers the the pH of milk to the IEP of casein.

36
The Lowry method is based on the reaction of Cu+, produced by the oxidation of peptide bonds,
with Folin–Ciocalteu reagent (a mixture of pHospHotungstic acid and pHospHomolybdic acid
in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves
reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly
tryptopHan, also tyrosine). Experiments have shown that cysteine is also reactive to the
reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen
in the Lowry assay.The result of this reaction is an intense blue molecule known as
heteropolymolybdenum Blue. The concentration of the reduced Folin reagent
(heteropolymolybdenum Blue) is measured by absorbance at 660 nm. As a result, the total
concentration of protein in the sample can be deduced from the concentration of tryptopHan
and tyrosine residues that reduce the Folin–Ciocalteu reagent.

Requirements:
Glasswares:
1. Burette -1
2. Beaker
3. Stirrer
4. pH Meter
5. Whatman No 1 filter paper strip (Size 25×50mm) - 2 no.
Reagents:
1. Raw milk - 100ml
2. 0.2N HCl - 50ml
3. Diethyl ether - 50ml
4. 50% Ethanol - 50ml
5. 10% Acetic acid

Fo lin Lowry Chemical Reagents:

1] Buffer ‘K’, pH 7.4

2] BSA - 0.2 mg/ml

3] Solution A: 20 g of Na2CO3 in 1l of 0.1 N NaOH

4] Solution B: 50 g/l CuSO4 in 10 g of NaK tartarate

5] Solution C: 50 ml of solution A + 1 ml of solution B

6] Folin - Ciocalteau reagent (1:1 dil with distilled water)

7] casein sample

8) Colorimeter - Cuvettes, tissues roll,

37
Procedure:

1. Measure 100ml of milk in a measuring cylinder and transfer 25ml of milk to four
Oakridge centrifuge tubes each.
2. Centrifuge the milk in a centrifuge at 4000rpm at room temperature (25- 30o C) for 20
minutes. This is done to remove the fats and lipids from the mixture.
3. After centrifugation, carefully remove the fats and lipids from the surface of the milk
with a spatula.
4. Then transfer the milk from all the tubes into a beaker and add equal volume of distilled
water and stir well. Now check the pH.
5. Start adding 0.2N HCl drop by drop into the milk mixture and stir well.
6. Note the PH at which precipitation (white curdy substances) appears. The pH should be
4.6.
7. Take the curdy precipitate and allow it to sediment.
8. Now decant the supernatant using a filter paper and funnel and wash the precipitate with
distilled water to remove the salts, then wash with diethyl ether and ethanol.
9. Dry the precipitate and take the weight of the casein and record it.
10. Prepare the series of tubes for the BSA std curve and use casein and whey samples for
protein estimation.

Observation table:
Volume of HCl added (mL) pH

0.1N 6.5
0.1N 6.0

12N 3.5

12N 3.0

12N 1.0
Amt. of Std. Vol. of Stock Distilled Folin - Ciocalteau Absorbance at
BSA water reagent
(μ g ) 660 nm
(mL) (mL) (mL)

0.0 0 1.0 0.5 Auto Zero

0.093
0.1 40 0.9 0.5

0.2 60 0.8 0.5 0.0845

0.4 80 0.6 0.5 0.12

0.6 120 0.4 0.5 0.1255

0.8 140 0.2 0.5 0.12366667

1.0 200 0.0 0.5 0.139

1.0 UK UK 0.0 0.5 0.196

BSA amount
1.2

0.8

0.6

0.4

0.2

0
0 0.05 0.1 0.15 0.2 0.25

39
Result:
015 absorbance Corelate to 32 Mg/ml
0.06 absorbance corelates to 13 Mg/ml
0.02 absorbance corelates to 4,5 Mg/ml

Calculations:
1)Casein
→ for 0.1% sample: 32x100 = 3200Mg/ml in 10'% casein stock
→ for 0.0 %. Sample: 45x1000 = 4500 Mg/mL in 10% casein stock
10ml in 10% stock = 0.03859
Average casein conc in 10% stock 3200 + 4500 = 3850Mg/ml Therefore 10 ml of stock has
0.03859g

since 1g of curd → 0.03859 casein

20.39 of curds → 0 0385x20.3 =0.781558

2) Whey
13 x 100=1300 Mg/ml
mass of whey:
100ml milk = 103g milk • mass of whey: 103-20.whey= 82.78 whey
1ml = 1.038g
Whey 1300 Mg - 1.03 g
x → 8273
x = 0.1040 whey

Interpretation:
o The casein sample was expected to have around 2.8g (80x0.035) of piston but only
0.78155 g was detected (28% of expected weight) As for the whey sample, it was
expected to have 0.7g (20 x 0.035) a protein, but only 0.1049g was detected (~15% of
expected weight).

o The decrease in the protein mass in comparison to the expected miss. could be due to the
proton extraction method conducted. It used highly concentrated (12N) HCl, which could
have denatured it e protons by disrupting there non-covalent hydrogen bards. They
denatured the protein concentration than what was expected.

40
 Date:23/03/24
Expt. No:8

TITRIMETRIC ANALYSIS OF VITAMIN C

Aim: Titrimetric analysis of Vitamin C

Introduction: Vitamin C, known chemically as ascorbic acid, is an important component of

a healthy diet. The history of Vitamin C revolves around the history of the human disease
scurvy, probably the first human illness to be recognized as a deficiency disease. Its symptoms
include exhaustion, massive hemorrhaging of flesh and gums, general weakness and diarrhea.
Resultant death was common. Scurvy is a disease unique to guinea pigs, various primates, and
humans. All other animal species have an enzyme which catalyzes the oxidation of L-
gluconactone to L-ascorbic acid, allowing them to synthesize Vitamin C in amounts adequate
for metabolic needs. Determining the concentration of ascorbic acid present in various food
items, and tablets can be done by titrimetric analysis.

Principle: This method determines the vitamin C concentration in a solution by a redox

titration with potassium iodate in the presence of potassium iodide. Vitamin C, more properly
called ascorbic acid, is an essential antioxidant needed by the human body (see additional
notes).When iodate ions (IO3−) are added to an acidic solution containing iodide ions (I−), an
oxidation-reduction reaction occurs;

The iodate ions are reduced to form iodine

− −
IO3 + 6 H+ + 5 e → ½ I2 + 3H2O

While the iodide ions are oxidised to form iodine.

− −
2I → I2 + 2 e

Combining these half-equations demonstrates the reaction between iodate and iodide

− −
IO3 +5I +6H+→3I2+3H2O

It is the iodine formed by this reaction that oxidises the ascorbic acid to dehydroascorbic acid
as the iodine is reduced to iodide ions.

−
ascorbic acid + I2 → 2I + dehydroascorbic acid
Due to this reaction the iodine formed is immediately reduced to iodide as long as there is any
ascorbic acid present. Once all the ascorbic acid has been oxidised, the excess iodine is free to

41
react with the starch indicator, forming the blue-black starch-iodine complex. This is the
endpoint of the titration.

The method is suitable for use with Vitamin C tablets, fresh or packaged fruit juices and solid
fruits and vegetables.

Requirements:
Glass wares:
1. burette with stand
2. 100 mL volumetric flask
3. 20 mL pipette
4. 250 mL conical flasks
5. 10 mL
6. 100 mL measuring cylinders
Reagents:
1. Potassium iodate
2. Starch indicator solution
3. Potassium iodide solution
4. Dilute hydrochloric acid: (1 mol L−1)

Solution Preparation
1. Potassium iodate solution: (0.002 mol L−1). If possible, dry 1 g of potassium iodate for
several hours or overnight at 100°C. Allow to cool and accurately weigh about 0.43g
of potassium iodate and dissolve in 1 L of distilled water in a volumetric flask.
2. Starch indicator solution: (0.5%). Weigh 0.5 g of soluble starch and add it to 50 mL of
near boiling water in a 100 mL conical flask. Stir to dissolve and cool before using.
3. Potassium iodide solution: (0.6 mol L−1) Dissolve 10 g solid KI in about 50 mL of
distilled water in a 100 mL volumetric flask and dilute to 100 mL with distilled water.
Procedure:
Sample Preparation
For vitamin C tablets: Dissolve a single tablet in 200 mL of distilled water (in a volumetric
flask if possible).
Titration

42
1. Pipette 20 mL of the sample solution into a 250 mL conical flask and add about 150 mL of
distilled water, 5 mL of 0.6 mol L−1 potassium iodide, 5 mL of 1 mol L−1 hydrochloric acid
and 1 mL of starch indicator solution.
2. Titrate the sample with the 0.002 mol L−1 potassium iodate solution. The endpoint of the
titration is the first permanent trace of a dark blue-black colour due to the starch-iodine
complex.
3. Repeat the titration with further aliquots of sample solution until you obtain concordant
results (titres agreeing within 0.1 mL).
4. Repeat the titration procedure with standard Vitamin C solution of known concentration.

Interpretation:

If vitamin C is present in the solution, the triiodide is converted to the iodide ion very
quickly. However, when all the vitamin C is oxidized, iodine and triiodide will be present,
which react with starch to form a blue-black complex. The blue-black color is the endpoint of
the titration.

Conclusion:

Once all the ascorbic acid has been oxidised, the excess iodine is free to react with the starch
indicator, forming the blue-black starch-iodine complex. This is the endpoint of the titration.
The method is suitable for use with vitamin C tablets, fresh or packaged fruit juices and solid
fruits and vegetables.

43
 Date:23/03/24

Expt. No.: 9

ESTIMATION OF TOTAL CARBOHYDRATES

Aim- To estimate carbohydrate content using Anthrone method

Introduction
Carbohydrates are polyhydroxy aldehydes and ketones. Carbohydrates are the essential part of
the storage and basic materials in the plants and animals. The carbohydrates are stored away
as free sugars and polysaccharides. The Anthrone reaction is the basis of a rapid and convenient
method for the determination of carbohydrates, either free or present in polysaccharides.
Anthrone is a tricyclic sweet-smelling ketone. It is utilized for a well-known cellulose measure
and in the colorimetric determination of carbohydrates. Anthrone dissolved in sulpHuric acid
is used for the quantitative determination of different carbohydrates. Quantitative
determination is only possible where the identity of sugar components is known because colour
development varies with the different sugars. Nevertheless, the anthrone method is widely used
for the determination of total carbohydrates in plant and food materials. Extraction from these
materials can be done by ethanol, acetone, water, acid etc. I used Anthrone method to estimate
the total carbohydrate content in raisin and used Dextrose as standard. Anthrone reaction
changes with time period of incubation and also with temperature. If kept for longer time period
the oxidation of carbohydrates changes resulting change in anthrone reaction and conclusion.

Principle
Carbohydrates are dehydrated with concentrated H2SO4 to form “Furfural”, which condenses
with anthrone to form a green color complex which can be measured by using calorimetrically
at 620nm (or) by using a red filter. Anthrone reacts with dextrins, monosaccharides,
disaccharides, polysaccharides, starch, gums and glycosides. But the yield of color varies from
carbohydrate to carbohydrate.

Fig: Structure of
Furfural

Requirements:

Sample:
1. Raisin (20 mg / 40 mg / 60 mg)
Reagents:
1. Working stock: Dextrose- 1mg/ml
2. Anthrone reagent: 0.2% (to be made in concentrated H2SO4)
3. Diluent: Distilled water
Glassware:
1. Medium size test tubes: 10ml x 9
2. Glass pipettes with dispenser
Miscellaneous
1. Micropipettes and tips: 100μl and 1000μl

44
 2. Water Bath at 100OC
3. Weighing balance
4. Vortex
5. Centrifuge
6. ELISA plate reader

Procedure:
1. 0.2% Anthrone preparation: 0.24g of Anthrone to 120 ml of H2SO4 (conc. 98%).
(Prepare the reagent in a glass beaker covered with foil to protect from light).
2. Standard Dextrose: Main stock 1mg/ml (10mg/10ml) in Distilled Water
3. Unknown Sample preparation:
A. Chop the raisins and weigh the required amount using a weighing balance.
B. Transfer the weighed chopped raisins to Kahn test-tube and add 1 ml conc. H2SO4.
C. Boil it in the water bath for 10 minutes at 100OC and then cool the tubes.
D. Centrifuge the tubes at 8000 RPM for 10 minutes at RT and separate supernatant.
E. Use 20 µl of 1:10 dilution of supernatant for carbohydrate estimation (UK1).
F. Use 20 µl of undiluted supernatant for carbohydrate estimation (UK2).

Observations:
Table: Estimation of carbohydrate using anthrone reagent
Concentration of Stock Diluent Total Anthrone Absorbance at
Dextrose (μl) (μl) Volume (0.2%) Heat the 620nm
(µg/ml) (ml) (ml) tubes in a
water
bath at Abs Abs-
100º C blk
for 10
10 50 950 1 5 0.10 0.03
mins &
100 500 500 1 5 0.76 0.69
cool the
200 1000 0 1 5 1.22 1.15
tubes
unknown sample 500 500 1 5 0.79 0.72
-ve blank 0 1000 1 5 Load 150 0.07 0.00
µl in 96
well
plate

GrapHs:
Standard curve of Dextrose concentration vs (Abs-blk)

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 Abs-Blk
1.4

1.2

0.8

0.6

0.4

0.2

0
0 50 100 150 200 250

Result:
Unknown sample calculation:
Conc. of glucose in sample = 119Mg/mL (grapH)
Since 2 dilutions were performed on original stock :
That is 50x and 2X,
Then concentration of glucose in stock is 19 x 2 x 50= 11900kg/m = = 1119 mg/mL
100mg of sugar stock has 11.9 X100
= 1190 mg = 1.19g of glucose

Conclusion:
The glucose concentration of sugar stock is 1.19g of glucose.

Interpretation:
The grapH follows a straight line co relation however it uses only a 4 pointer including the
blank ,So it is unreliable. Hence，use more concentrators to plot a standard glucose curve.
Moreover, the stock has 1.19 g of glucose (as calculated). This is impossible since exactly
1000g of sugar was added to 100 ml of glucose to make the stock.

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 Date: 27/03/2023
Expt No.:12

ESTIMATION OF GLUCOSE BY GLUCOSE OXIDASE PEROXIDASE

METHOD

Aim: Estimation of Glucose by GOD POD Method

Introduction:

Glucose is the major source of energy in the body. Glucose estimation is the most frequently
performed test in clinical laboratories, is primarily run to aid in the diagnosis of diabetes
mellitus. Elevated serum glucose levels may be associated with pancreatitis, pituitary or thyroid
dysfunction, pregnancy induced diabetes and liver disease. Hypoglycemia occurs most
frequently due to over dosage of insulin. It is also observed in other conditions like
hypothyroidism, hypopituitarism and hypoadrenocorticism.

The Glucose Oxidase-Peroxidase (GOD POD) method is a commonly used enzymatic method.
Glucose is oxidized by Glucose oxidase and produces gluconate and hydrogen peroxide. The
hydrogen peroxide is oxidatively coupled with 4- aminoantipyrine and pHenol in the presence
of peroxidise enzyme, to give a coloured complex-quinonemine. The intensity of the coloured
complex (quinoneimine) is proportional to the glucose concentration in the sample and can be
measured pHotometrically at 505nm.

GOD POD Reaction

GOD POD was performed to check the pre parandial and post parandial samples. Pre parandial
blood glucose refers to fasting blood glucose or blood glucose before a meal. The test is
generally carried out in the morning, after an overnight fasting. A fasting blood sugar test offers
information about how the body is managing the blood sugar levels. Normally, the range of
glucose in a person’s blood is between 70 to 100 mg/dl. Fasting blood sugar levels between
100 to 126 mg/dl are considered as pre-diabetic or impaired fasting glucose and blood sugar
levels of 126 mg/dl or higher are diagnosed as diabetes.

Post parandial refers to the blood sugar is a measure of your blood glucoses after you've eaten.
A common use is in relation to blood sugar (or blood glucose) levels, which are normally
measured 2 hours after eating in a postprandial glucose test. This is because blood glucose

47
levels usually rise after a meal. The normal post parandial glucose levels should be under
140mg/dL.

Requirements:

1. ERBA kit

Samples- serum samples

2. Plasticware

1.5 ml Eppendorf tubes -12

15 ml Falcon tubes – 1

3. Instrument

UV/VIS SpectropHotometer at 505 nm

Water bath 37°C

4. Miscellaneous

Syringe

1 ml and 0.01 ml Micropipette and tips

Distilled water

Rack

Procedure: (A)- PRE PARANDIAL

1. 5 ml of whole blood samples were collected from volunteers.

2. The blood samples are kept at 370C for the sometime for clotting to occur.

3. The blood samples were centrifuged at 1000rpm for 10 mins at 40C.

4. The supernatant (serum) was collected and transferred to new eppendorf tubes.

5. The reagents were added into the tubes according to the following table -

BLANK STANDARD TEST

10µL D/W 10µl of 100 mg/dL 10µL serum sample
1000µL reagent 1000µL reagent 1000µL reagent

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6. The tubes were incubated at room temperature for 15mins.

7. The O.D readings were taken at 505nm.

8. Followed the same protocol to estimate the glucose levels 2 hours postprandial.

Observations:

Test Tube Colour Change Abs Abs-Blk

Blank No 0.88 0.00

Standard Yes 1.04 0.16

Test Yes 1.09 0.21

Result and Calculations:

Conc. of standard = 1% glucose

= 1g in 100 dL = x9 in
1dL = x: 100: 10mgal
conc. of glucose
in test (mg/dL) = (Abs. test / Abs. std )X conc. of standard = 0.21/0. 16 x 10 = 13.125 mg/dL

Conclusion:

The glucose Concentration of the Serum Sample is 18.125 mg/dl.

Interpretation:

Since the expected pre-prandial blood glucose concentration is 70-100 mg/dL, 13.125mg/dl is
much below (83%) the lower limit of the range. The patient is Hypoglycaemic. This could
have been due to severe blood loss.

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