BBCCL 102

Indira Gandhi National Open University
School of Sciences BBCCL-102
Molecules of Life
Lab
BBCCL-102
Indira Gandhi National MOLECULES OF LIFE
Open University
School of Sciences Lab
MOLECULES OF LIFE
Exercise 1
Cleaning and Maintenance of Glass Ware, Safety
Measures in Laboratories 5
Exercise 2
Preparation of Normal, Molar and Percent Solutions 11
Exercise 3
Measuring pH, Calibration of pH Meter and Preparation
of Buffers 16
Experiment 4
Determination of pKa of Glycine 23
Experiment 5
Qualitative Tests for Carbohydrates 27
Experiment 6
Qualitative Tests for Amino Acids and Proteins 39
Experiment 7
Qualitative Tests for Lipids 53
Experiment 8
Determination of Nucleic Acid Purity by UV
Absorption Method 60
Experiment 9
Estimation of Vitamin C 65
Course Design Committee
Prof. Bechan Sharma Prof. Ranjit K. Mishra Prof. Vijayshri
Dept. of Biochemistry Dept. of Biochemistry Former Director,
University of Allahabad University of Lucknow SOS, IGNOU
Prof. Reena Gupta Prof. Sanjeev Puri Dr. Parvesh Bubber

Dept. of Biotechnology UIET, Panjab University Associate Professor
H.P. University, Shimla SOS, IGNOU
Prof. Seemi Farhat Basir
Prof. D. V. Devaraju Dept. of Bio Sciences Dr. M. Abdul Kareem
Dept. of Biochemistry Jamia Milia Islamia Assistant Professor
Bangalore University SOS, IGNOU
Prof. K. Vali Pasha Dr. Arvind Kumar Shakya

Dept. of Biochemistry Assistant Professor
Yogi Vemana University, SOS, IGNOU
Andhra Pradesh
Dr. Maneesha Pandey
Dr. Suneeta Joshi Assistant Professor
Dept. of Biochemistry SOS, IGNOU
Daulat Ram College
Univ. of Delhi Dr. Seema Kalra
Assistant Professor
SOS, IGNOU
Block Preparation Team

Prof. K. Vali Pasha Dr. M. Abdul Kareem
(Content Editor) (Content Writer for Experiments 1 to 9)
Dean, Faculty of Science Assistant Professor
Department of Biochemistry SOS, IGNOU.
Yogi Vemana University
Kadapa-516003 Andhra Pradesh
Course Coordinator: Dr. M. Abdul Kareem

Print Production Cover Page
Sh. Sunil Kumar Dr. M. Abdul Kareem
Assistant Registrar (Pub.) Assistant Professor,
SOS, IGNOU SOS, IGNOU.
Acknowledgement: Mr. Sumit Verma for Graphics and word processing.

July, 2020
© Indira Gandhi National Open University, 2020
ISBN:
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All rights reserved. No part of this work may be reproduced in any form, by mimeograph
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Further information on the Indira Gandhi National Open University courses may be
obtained from the University’s office at Maidan Garhi, New Delhi-110 068 or the official
website of IGNOU at www.ignou.ac.in.
Printed and published on behalf of Indira Gandhi National Open University, New Delhi
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Printed at :
BBCCL-102 MOLECULES OF LIFE
This laboratory course is of 2 credits. In this lab course, you will be introduced
to the basic requirements of biochemistry lab like cleaning, maintenance and
safety measures of the lab. Apart from these fundamentals you will also learnt
about concepts of various qualitative and quantitative tests routinely performed
in a biochemistry lab.
You are aware that this lab course is designed in connection with theory
course Molecules of life (BBCCT-101) where we have described structure,
classification and functions of biomolecules. Hence, in this lab course we
have developed experiments that will give you an opportunity to understand the
biochemical properties of biomolecules discussed in BBCCT-101.
In this course we have included 3 exercises & 6 experiments, among which

first 3 exercises are helpful in understanding the basic needs and
requirements of a biochemistry lab. Whereas, the next 6 experiments based
on the physical and chemical properties of the biomolecules. This course will
help you in acquiring essential skills and concepts in performing qualitative
and quantitative tests. Reference books provided at the end of the exercises
may be referred for more information.
may be referred for more information.
Expected Learning Outcomes:
The broad objectives of this course are to enable you to:
l identify the routinely used glass ware and apparatus;
l impart maintenance and cleaning of biochemistry lab;
l preparation of normal, molar and percent solutions;
l perform titrations and plot graphs;
l know the basic principles behind qualitative and quantitative tests;
l distinguish different types of biomolecules in a given test solution; and
l determine the purity of a given nucleic acid in a solution.
Study Guide
We advise you to go through respective blocks of BBCCT-101 before you
come to attend the practical sessions. This will enable you to easily
understand the purpose of doing experiments and their applications. You
should also read the principles of each experiment of this course along with
procedure before you start performing the experiment. It is always good to
prepare all the reagents freshly and store them under prearranged storage
conditions. Adhere to all the safety measures and follow the safety instructions
while handling the reagents.
One of the good laboratory practices is to maintain your log books up-to-date
i.e., enter the observations made while performing the experiments. Carry this
laboratory manual and your log book during lab sessions.
Like all other IGNOU laboratory courses this is an intensive residential

exercise requiring one week to complete it. Everyday there will be two
laboratory sessions of 4 hours each. So there will be a total of 14 sessions.
The first session will be introductory and the remaining 2 nd to 13th sessions will
be based on the exercises given in to course. A schedule for laboratory
exercises will be given to you in the first session. Sessions 1 to 13 will have
guided exercises under the supervision of the academic counsellor. The last
two sessions i.e., 13th and 14th will be unguided sessions that is the term end
examination. In each session you will perform exercises for 3 hours and in the
remaining 1 hour you will complete your practical note book.
You are aware that there is a time constraint as you will have limited access to
laboratory work, therefore, you are required not to miss any of the laboratory
sessions.
Assessment of the experiments will be graded and you will have to appear for
the viva-voce at the end of the practical session. At the end of the laboratory
session you should perform the assigned experiment, which will be graded
and final assessment will be made based on the continuous performance
during the laboratory sessions, maintenance of log books and records
followed by viva-voce.
For the better understanding of how to use laboratory apparatus few video
links have been provided where ever available. There might be a slight
difference in the steps or procedure being explained in the video when
compared to the procedure provided in this self-instructional; aterial. However,
the principles and reagents remain same. Hence, there is no need to worry
about slight modifications adopted in the procedure.You may also refer the books
listed at the end of each exercise.
We wish you best in this endeavor!!
IMPORTANT
 Attendance is compulsory in the Laboratory Course work held

generally at the Study Centre.
 The Laboratory Course is worth 2 credits to be completed over

7 days duration.
 6 days of Guided Laboratory work
 1 day for the Unguided Laboratory work
 To successfully complete the laboratory course you will have to

pass (at least 35% marks) in the Guided and Unguided
components separately.
Laboratory setup
Exercise 1
CLEANING AND
MAINTENANCE OF GLASS
WARE, SAFETY MEASURES IN
LABORATORIES
Structure
1.1 Introduction 1.4 Laboratory Hazards
Expected Learning Outcomes Self Assessment Questions
1.2 Cleaning and Maintenance of 1.5 Summary
Glassware
1.6 Further Readings
1.3 Safety Measures in Laboratory
“As a rule, never allow
1.1 INTRODUCTION any biological material
to dry in any of the
We are all aware that general cleanliness plays an important role in glassware. This, if
maintaining our health. Similarly cleanliness of lab, lab materials and its followed rigorously, will
automatically improve
apparatus has a significant role in good laboratory practices (GLP). To carry
the quality of glassware
out experiments in Biochemistry lab we use various types of glass ware. In washing”
order to achieve error free results it is very important to maintain glass ware or
any apparatus in clean and hygiene condition. The best practice includes
“never apply undue pressure or strain to any piece of glass ware”. We must
ensure that the glass ware we are going to use is free from traces of reagents
or chemicals. This is because remains of any such chemicals will influence
the results of the biochemical experiment.
The second part of this unit will give us an opportunity to explore some of the
common laboratory hazards and various safety measures to be followed.
There are many potentially dangerous chemicals that are carcinogenic, toxic
and inflammable. Hence it is essential to have awareness about maintenance
of glass ware and safety measures to be followed in the laboratory.
Expected Learning Outcomes

After going through this exercise, you should be able to:
 describe the laboratory procedures involved in cleaning the glass ware;
 distinguish various types of hazards; and
 select and use specific measures to avoid laboratory accidents.
5
BBCCL-102 Molecules of Life
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1.2 CLEANING AND MAINTENANCE OF

GLASSWARE
Since this is the first exercise to all our practical courses, we intend to begin
with basic rules to be followed in the laboratory. One of the basic and essential
rule or habit to be followed and implemented by all of us is to know how to
clean and maintain the glass ware (Fig. 1.1) we use for laboratory
experiments. In addition to this correct and safe handling of laboratory glass
ware and apparatus also shows a significant impact on the final results of the
experiments and on the laboratory expenditure.
Points to be remembered:
 the glass ware must be washed immediately after use

The above picture shows a
 for normal washing tap water can be used
cleaning area in a lab with
drying rack and wash bottle.  0.5% Labolene solution is used for regular cleaning of glassware
 soap solution, detergent or cleaning powder can be used for removing

any non-sticky substances
 organic solvents like acetone or alcohol can be used for removing water
insoluble substances from glassware
 chromic acid or mixture of alcohol with solid potassium hydroxide can be

used for dirty glassware
 usage of cleaning scrubber or abrasive can be minimized as they may

scratch the glass
 it is essential to remove the traces of soap or detergent from glassware,

Figure 1.1 shows some
of the commonly used to achieve this rinse the glassware with tap water followed by distilled
glass ware. water
 glassware often needs drying before its usage and can be done in oven at
600 C (except graduated glassware like pipettes as it may cause changes
in graduations)
 avoid usage of cracked, scratched or chipped glassware
1.3 SAFETY MEASURES IN LABORATORY

So far we have discussed how to clean and maintain the laboratory
glassware, learn let us about the Standard Operating Procedures (SOP) for
laboratory safety. All science students should remember that, they need to be
“attentive and aware of the surroundings”, when present in the laboratory. The
judicious practice of these SOP’s in laboratory will protect learners from
possible laboratory hazards. These SOP’s broadly covers issues related to
process, procedure, hazardous material, disposal, etc.
Following are the set of guidelines that are commonly practiced as SOPs in
the laboratory.
6
BBCCL-102 Cleaning and Maintenance of Glass Ware, Safety Measures in Laboratories
..........................................................................................................................................................................
Be familiar with hazard symbols (Figure.1.2) and aware of the chemicals
available in laboratory (location and chemical grade). FIRST AID KIT: The
following should be
 Many accidents are caused by failure to seek advice or information. present in a box that is
accessible to all:
 Never attempt to use equipment that you do not fully understand.
Dettol, Absorbent
 Procure and prepare all chemicals and reagents before you start cotton, Gauze, Roller
experiment, to avoid last minute chaos. bandage, Adhesive
tape and Scissors, 5 %
 Poisonous and dangerous chemicals should be stored separately and in
aqueous sodium
a locked cupboard. Explosive chemicals are kept in a lead lined carbonate, 2 %
rectangular box. aqueous sodium
bicarbonate in a
 Do not perform experiments or procedures that are not authorized by
dropper bottle, 5 %
supervisor and Institutional Ethical Committee. acetic acid, Saturated
solution of boric acid in
 It is always better to have the knowledge of the lab design, location of first
a dropper bottle,
aid kit and the safety measures available. Keep record of fire extinguisher Glycerine, Tincture of
like their refilling and expiry dates. Keep records of all chemicals, Iodine and Grease or
reagents, equipment’s and recurring materials up to date. Vaseline.
 All bottles and containers should be carefully and distinctly labeled so that
no confusion or possible error can arise when several containers are
being filled with different chemicals.
 Bottles should never be carried by the neck and should be carried in trays
or in buckets.
 Bottles containing liquids can be kept on the lower shelves whilst solids
are kept on the higher racks.
 Caution should be taken while working with flammable chemicals, The above picture
maintain sufficient distance from naked flame. To minimize explosions shows reagent rack and
use safety hood or a fume hood. bottles with glass
stoppers.
 Close the containers immediately after use.
 Smoking, eating and drinking of any kind of beverages is strictly prohibited

in the laboratory.
 Poisonous and corrosive reagents should never be pipetted by mouth; It

is always better to use pro-pipette or an auto-dispenser for dispensing.
Do not directly smell, sniff or taste any chemical.
 Care and cleanliness must be practiced by all working in the laboratory at

all times.
 Wore lab coats or aprons while handling laboratory reagents and toxic,
corrosive chemicals. Safety goggles and gloves should be used, while
handling inflammable and strong chemicals.
 Wore lab shoes, working on bare legs should be avoided.
 Cut short your finger nails and maintain hygiene before and after using
chemicals and reagents.
7
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 Electric switches and connections should be repeatedly inspected and kept
free from corrosion, so that no danger shall arise from short circuiting or
due to the exposure of naked wire from which the insulation has been lost.
 There should be proper disposal of waste. The floor space should be kept
perfectly free from spillage, broken glass etc. Broken glass waste, syringe
needles and other discarded objects must be placed into ‘puncture-proof’
labeled containers.
 When dealing with volatile highly inflammable liquids no flames/heaters
should be allowed in the vicinity. Care should be taken to avoid inhalation
of vapours.
 If corrosive or toxic liquids are being thrown down the sink, they should be
accompanied by generous supply of water to ensure that by the time they
reach the main drain they will be too dilute to be dangerous.
 When pouring liquids from one container to another keep both vessels
away from body so that any spillage shall not fall upon.
 When corrosive chemicals (acids) involved, carry out the transfer over a
sink so that any spillage can be easily flushed away.
 When diluting concentrated acids always add the acid slowly to the water
kept in ice and not the opposite.
 When NaOH is dissolved in water lot of heat is generated. Use cold water
and add NaOH to it. Do not handle NaOH with unprotected hands.
 While handling animal samples (or) microorganisms follow proper
protecting measures and use hand glows, face mark and head cap.
In addition to all the above said points, designated procedures need to be
followed while handling laboratory wastes (example: acrylamide gels, expired
chemicals, microbial and animal biological cultures, biological specimen and
animal carcass etc.). These practices will protect your health and
environment.
1.4 LABORATORY HAZARDS

Laboratory hazards (threats) varies from lab to lab based on the kind of the work
they involved. Hence these hazards could be of Biological, Chemical and
Physical.
Biological Hazards
Outbreak of COVID-19
in January 2020 is one The material causing biological hazard majorly comprises of microbial
of the examples how cultures of bacteria, virus, fungi, protozoans and other communicable disease
infectious diseases causing organisms. It is well known that microbes can easily enter into our
spread from one to vital systems like respiratory tract system through air, gastro intestinal tract
another.
through water or food and also cause skin diseases through contact. Hence,
specimen samples of microbial origin, experimental animals and human
diagnostic samples (urine, blood and stool etc) need to be handled with care
and to be discarded following SOP’s. Infectious agents are a major hazard in
any laboratory. Convenient hand washing facilities, germicidal hand- scrubs
8
may be provided to prevent spreading of infection.
BBCCL-102 Cleaning and Maintenance of Glass Ware, Safety Measures in Laboratories
..........................................................................................................................................................................
Chemical Hazards
Figure 1.2 shows various hazardous symbols used to represent types of

Methyl Isocyanate
hazards. Chemicals can be grouped into toxic, flammable, oxidizing and (MIC) gas leakage
corrosive based the nature of the hazard they cause. The adverse effects of tragedy in December
these chemicals will be by direct exposure like contact or inhalation. Hence it 1984 at United Carbide
is advised to check the labels of the chemicals carefully before handling them. India Limited (UCIL) a
pesticide plant in
Disposal and Storage systems to handle chemical, solvent waste should be
Bhopal, Madhya
established to minimize the chemical hazards. In addition to this follow SOP’s Pradesh, India lead to
while storing and transferring chemicals. the death of
approximately 3780
Physical Hazards individuals and many
suffered with respiratory
Physical hazards are the once that occur due to exposure to factors like disorders.
radiation, temperature or fire, force or pressure and electricity. Majority of the
equipment’s used in the research laboratory generate either ionizing
(isotopes) or non-ionizing (LASER, U.V. light) radiation, that may cause
damage to the smooth tissues and lead to generations of tumors and or
mutations. Coming to the next factor i.e., temperature, we use several heating You might have
or boiling procedures to perform our experiments. Uncontrolled or excess observed that, while
usage of flame or temperature than the suggested lead to generation of fire taking X-ray, all other
parts of the body are
that may cause irreparable damage. Another factor is pressure, we apply
covered except the
pressure in certain experiments, it is advised to use control valves and check portion of the body to
them regularly to avoid damage. Last but not least is electricity, it is hard to be exposed to X-rays.
imagine any laboratory without electricity. Hence, while working in laboratory, Such measures are
it’s responsibility of everyone to turn off the power supply once the work is essential to prevent
unwanted health
done and also it is advised to have maintenance inspections of all the
hazards. It is
apparatus at regular time intervals. scientifically proven
that, frequent or
Note: all these hazards can be avoided if we stay alert and responsible while
prolonged exposure to
working in laboratory. Refer the books listed at the end of this exercise to X-rays may increase the
know more about SOP's and their management. risk of cancer.
FLAMMABLE GAS RADIOACTIVE POISON
CORROSIVE BIOHAZARD IRRITANT
WHEN
OXIDIZER DANGEROUS WET FLAMMABLE SOLID
Fig. 1.2: Symbols used to represent various types of Hazards 9

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1.5 SUMMARY
 Cleaning and maintenance of the glass ware is one of the simple and
crucial aspect to be taken care in the laboratory since it has larger
influence on the results.
 Knowing and following SOP’s will help to achieve a quality work and also
minimize the wastage of the resources that will directly help the
organization and protect our environment.
 Laboratory hazards can be avoided through careful handling of chemicals

and apparatus.
Self-Assessment Questions
1. Enlist the contents of a first aid kit?
2. What are precautions to be taken while preparing NaOH solution?
3. Expand the acronym “SOP”.
4. Name any two organic solvents used in cleaning the glassware?
5. Identify the symbol used for biohazards from Fig. 1.2?
1.6 FURTHER READINGS

1. Experimental Biochemistry: A student Companion. BeeduSashidhar Rao
and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International Pvt. Ltd.
2. Practical Biochemistry: for medical, dental and allied courses. 2 nd edition,

Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1, Ahuja publishing
house.
3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III.ISBN 81-

7263-333-5, Published by Indira Gandhi National Open University, 1993
(Reprint December-2006).
10
Exercise 2
PREPARATION OF
MOLAR, NORMAL AND
PERCENT SOLUTIONS
Structure
2.1 Introduction 2.4 Percent Solutions
2.2 Molar Solutions 2.5 Summary
2.3 Normal Solutions 2.6 Further Readings
2.1 INTRODUCTION
In the first Unit of course BBCCT-101 you have learnt about various units
used for the expression of weights and volumes of substances and
solutions used in biochemistry experiments. In connection to that here in
this exercise you’ll be exposed to some units that are useful and play a
crucial role in the expression of solute (analyte) concentration per Unit
volume. Among such expression the widely used ones are Molarity (M),
Normality (N) and Percent (%) solutions. This exercise is mainly focused
on the definition and sample examples how to prepare a solution with
specific concentration. Though this is smaller exercise by content but it
plays key role in your present and future research work.

 define molarity and normality;
 distinguish between molar, normal and percent solutions; and
 perform the preparation of molar, normal and percent solutions.
2.2 MOLAR SOLUTIONS

For better understanding let us start with a simple a known example, we
all know how to prepare a sugar solution; preparation starts by taking a
table spoon of sugar and ends by dissolving it in a glass of normal water. 11
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simple experiment water is known as solvent and sugar as solute. But it is
tough to say the concentration of the sugar solution? And whether it is a
molar or normal or percent solution! To overcome such issues scientific
community has come up with a concept of expressing solute (analyte)
concentration per unit volume using the terms Molar, Normal and Percent
solutions. Now let’s discuss one by one.
A molar solution consists of “gram molecular weight” (formula weight) of

a solute or substance in grams. Gram Molecular weight (GMW) is obtained
by combining the atomic weights of all atoms present in that particular
solute molecule.
Molarity is defined as “the number of moles of solute per litre (L) of

solution”,and is expressed as
Weight of solute in grams

Moles =
Gram Molecular Weight
It’s essential to remember that, the term mole represents the amount of
substance (in grams), irrespective of the volume where the substance is
dissolved.
Example:
40
No. of moles of sodium hydroxide (NaOH) present in 40 gm is = =1
40
Molecular weight of NaOH is Na: 22.99; O: 16; H:1 i.e., 39.99 = 40
Let us understand this with a simple excercise of How to prepare 1 L of

1M solution of NaOH?
Since molecular weight of NaOH is 40, dissolve 40 grams of NaOH in 1

liter of water to make a 1M NaOH solution per 1 L.
Similarly one mole of sodium chloride (NaCl) is 58.45 (MW 58.45)
The formula to calculate how much weight of solute or substance is

needed to prepare a specific solution is:
Weight in grams = desired molarity x volume needed in litres x
GMW
Ex: How much weight of NaCl is required to prepare 500mL of 1
M NaCl solution
Weight in grams= 1M x 500 mL x 58.45 GMW
Weight in grams= 29.225 grams of NaCl to be dissolved in 500 mL
of water to make it 1M NaCl solution
2.3 NORMAL SOLUTIONS

Normality (N) is also another way of expressing the concentration of solute
in the solution. This is partially similar to Molarity but uses gram equivalent
12 weight (Eq.Wt) rather than gram molecular weight (GMW) of solute per
BBCCL-102 Preparation of Normal, Molar and Percent Solutions
..........................................................................................................................................................................
litre. In detail it is explained as”1N solution contains 1 gram-equivalent
weight of solute per liter of solution” it is also defined as “number of gram It should be noted that a
equivalents of solute per litre of the solution”. To obtain gram equivalent given solute in solution
weight one should know the no of hydrogen atoms that can be added or may have more than one
removed from the given substance. When you divide the GMW of the normality value,
substance with no of replaceable hydrogens you’ll get EW. depending upon the
number of electrons lost
GMW or gained (oxidation-
= Eq. Wt (EW) reduction status) in the
number of replaceble hydrogen atoms
reaction. Thus, normality
For example: Molecular weight of NaOH is Na: 22.99; O: 16; H:1 i.e., is not a good expression
39.99 = 40; NaOH possess 1 hydrogen atom that can be replaced , hence of concentration, as
Eq.Wt of NaOH is: compared to molarity,
wherein only one
40 molecular mass exists
= 40
1 for a given substance.
Let us understand this with another example of How to prepare 500 mL of Normality= Molarity x N
0.5N solution of NaOH.
Where N is equal to no
(Atomic weight of NaOH is Na: 22.99; O: 16; H:1 i.e., 39.99 = 40 ) of replaceable hydrogen
ions
desired normality x volume required in litres x GMW Reference 1: Experimental
Weight in grams = Biochemistry: A student
Valence
Companion
0.5 N  500 mL  40 GMW

Weight in grams = = 10 g of NaOH
1 EW
10 grams of NaOH is needed to prepare 500mL of 0.5N solution of NaOH.
2.4 PERCENT SOLUTIONS

This is another way to express the solute concentrations in a given solution.
The following are the two major well-known expressions used for this purpose.
i. weight/volume (wt/vol): Used to express weight (grams) of specific

substance in 100 mL solvent.
Example: 1% glucose solution consists of 1 gram glucose in 100 mL of water.
This can be further expressed as g% solution and mg% solution depending

on the weight of the solute in 100 mL of solvent.
ii. volume/volume (vol/vol): Used to express volume (mL) in 100 ml of

solution
Example: Prepare 100 mL of 60% (v/v) ethanol from 95% ethanol.
% of stock solution you have x unknown volume to be taken (X)= 60 % of

working ethanol solution you want to prepare x Volume wanted (mL final
volume)
60  100
= (x) 63.15 mL of ethanol
95 13
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Hence, to prepare 100 mL of 60% ethanol, from 95% ethanol stock-take
63.15 mL of and make up to 100 mL with distilled water, i.e., add 36.85 mL
of distilled water to 63.15 mL of ethanol.
Apart from the above two expressions sometimes we use a third type for
expressing the concentration of a substance in a mixture.
iii. weight/weight (wt/wt): This is used to express the percentage of a

particular substance in a mixture of substances.
Example: 1.If you notice a commercial advertisement of chocolate saying

50 % DARK, indicates that half of its weight (50%) made up of cocoa.
2. Protein content in soya bean is 40 % (i.e., in 100 grams of soya bean

by weight contains 40 grams of protein).
1. Calculate the weight of NaOH required for preparing 1 L of 0.5 M
NaOH solution?
2. How to prepare 20% glucose solution?
3. Calculate the amount of NaCl is required to prepare 1000 mL of 1 N

NaCl solution?
4. How to prepare 70% ethanol?
2.5 SUMMARY
All the above discussed expressions are widely used in biochemistry lab
for preparing various reagents and diluting the stock solutions.
 Molarity is used to express the mole concentration of a solute in a

solvent
 Normality is used to express the gram equivalent weight of solute in a

solvent
 Percentage solutions are of two types like wt/vol and vol/vol.
 The other expression used for substance in mixture is wt/wt.

1. Experimental Biochemistry: A student Companion. Beedu Sashidhar
Rao and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International
Pvt. Ltd.
14
BBCCL-102 Preparation of Normal, Molar and Percent Solutions
..........................................................................................................................................................................
2. Practical Biochemistry: for medical, dental and allied courses.
2nd edition, Dr. G. Rajagopal and Dr.B.D. Toora.ISBN 81-901769-5-1,
Ahuja publishing house.
3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III. ISBN

81-7263-333-5, Published by Indira Gandhi National Open University,
1993 (Reprint December-2006).
4. Laboratory manual of Microbiology and Biotechnology (second edition),

K.R. Aneja. ISBN 978-93-87025-49-3. MEDTECH a division of Scientific
international (Pvt. Ltd).
15
3
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Exercise
MEASURING pH,
CALIBRATION OF pH METER
AND PREPARATION OF
BUFFERS
Structure
3.1 Introduction 3.4 Precautions

3.2 Measuring pH 3.5 Summary

Calibration of pH meter 3.6 Further readings
Measuring pH of a Biological Test sample
3.3 Preparation of Buffers

How to Prepare a Buffer Solution
3.1 INTRODUCTION
In the previous exercise you have studied about how to prepare solutions
with different concentrations. The major aim of this exercise is to learn
about how to measure pH, in addition you’ll know about calibration of pH
meter along with preparation of various buffers. However in the second Unit
(water) of course BBCCT-101 you have learnt about what is pH and its
biological significance. For better understanding of the current exercise it
would be better if you recollect the concepts studied in the unit water.
In your lower classes you might have studied that various parts of our body
have different ranges of pH according to their functions (Table: 3.1). Do
you know that pH plays a significant role in many biochemical reactions
especially in enzyme catalysed reactions (Refer BBCCT-107 enzyme
catalysis). It is essential to measure the pH for conducting biochemical
experiments. Buffers play an important role in maintaining pH of the body
and regulate biochemical reactions. Hence, in this exercise we’ll be
studying about preparation of buffers and their compositions. There are
three major ways to measure pH like using pH paper strips, voltage gated
pH meter and digital pH meter. In this exercise we’ll be discussing about
16 digital pH meter that is widely used.
BBCCL-102 Measuring pH, Calibration of pH Meter and Preparation of Buffers
..........................................................................................................................................................................
 define pH;
 operate pH meter;
 determine the pH of a solution; and
 perform the preparation of different types of buffers.
3.2 MEASURING pH
pH is defined as negative logarithm of H+ ion concentration. pH meter is a
electric device and routinely used in biochemistry laboratory. We all know
that pH scale is used to measure concentration of hydrogen (H+) ions and
tells us whether the given solution is acidic, alkaline or neutral in its nature.
Principle: pH meter designed in such a way that, it can measure the
effective concentration of H+ ions in solution. A typical pH meter can
measure the potential difference i.e., Electro Motive Force (EMF) which is
developed between selective glass electrode and test solution containing H+
ions. The magnitude of this EMF varies with the varying temperature of the
solution. The output potential is in millivolts [mv] and it is recorded
glavanometrically or digitally on a scale graduated in pH units. This
relationship is defined by the following equation.
E o  2.303 RT
V   pH
F
A glass electode
Where, V= Voltage of the completed circuit [observed EMF] The pH meter having
combined electrode
E0= Potential of reference Electrode
that is reference
R= the gas constant [8.314 J/mole/oK] electrode and pH
sensitive glass
T= the absolute temperature in oK [25oC= 298 oK] electrode connected
F=the Faraday’s constant (964846 Coulombs/ equivalent weight or 9.64846 by potassium chloride
bridge present in
x 104 Coulombs mol-1).
(Source: reference 1: Experimental Biochemistry) single glass tube.
3.2.1 Calibration of pH Meter Source: Course

BCHCL-134
So for we have studied the principle behind pH meter. Let us explore how of B.Sc. Gen.
to calibrate pH meter.
Materials:
pH meter, Thermometer, Beakers, Wash bottle, Distilled water, Commercial
buffer capsules and Tissue Paper.
Test Sample:
We can use any of the following liquids as test sample: Normal tap water,
bottle water, diluted acid or base and Urine sample.
Procedure:
pH meter standardization: Prior to start using the pH meter,immerse the
electrode in electrode storage solution provided by the manufacturer or dip
in 100 mL of pH 7.0 buffer with 0.5 grams of potassium chloride (KCl) 17
..........................................................................................................................................................................
added. Later connect the electrode to the pH meter. Prepare three different
buffer solutions of pH 7.0, pH 4.0 and pH 9.0 that represent neutral, acidic
and alkaline pH respectively. For this dissolve respective commercial buffer
capsule into100 mL of deionized water separately. Then wash the electrode
with deionized water using a wash bottle, later gently wipe the electrode
with tissue paper. Now, immerse the electrode in pH 7.0 buffer solution and
use a glass rod to stir the buffer with moderate speed. Wait for 1-2
minutes, if pH meter does not display the value of buffer automatically, set
the value of buffer manually to pH 7.0 (Fig. 3.1). Remove the electrode
from pH 7 buffer solution and wash with deionized water, later wipe the
electrode with tissue paper without touching the bottom membrane. Repeat
the standardization procedure with already prepared pH 4.0 followed by pH
9.0 buffer solutions. Now the pH meter has been standardized, and is
ready for measuring the pH of given/test sample. Do not change any
settings before measuring the unknown samples.
Fig. 3.1: pH meter
3.2.2 Measuring pH of a Biological Test Sample

Once you calibrate the pH meter remove the electrode from buffer solution
and wash with deionized water and wipe the electrode with tissue paper.
Then take the given test solution into a beaker and note the temperature
value using a thermomete. Now immerse the electrode into the test
solution. Note the values displayed on LED window of the pH meter.
Results: The pH of the given test sample is .................... .
Discussion:
The pH value of a solution indicates the concentration of hydrogen ions in
a solution, indicates the relative abundance of H+ ions in it. Living cells
require either acidic or alkaline pH. Enzymes being significant biocatalysts
require optimum conditions for their activity. Alterations in the levels
of pH and temperature will affect the activity of cells, in gastric acidity the pH
value of gastric juice raises above pH 6 (alkaline) many gastric enzymes
will work in the range of (pH 1-3). Here it is important to remember
that, the pH values are not same in all parts of our body (Table 3.1).
Note: pH value depends on the role and functional status of the cell.
18
..........................................................................................................................................................................
Table 3.1: pH values of different biological fluids
Biological Fluid pH
Urine 6 to 7.6
Serum 7.8
Bile 7.8 to 8.6
Pancreatic Juice 8.0
Saliva 6.3 to 6.5
Blood 7.4
Gastric Juice 1.6 to 3.0
3.3 PREPARATION OF BUFFERS

So far we have discussed about calibration of pH meter and measuring
pH. Before we start discussing on how to prepare a buffer solution, it is
better to go through the concept of buffers, Henderson-Hasselbalch
equation and the biological importance explained in the second Unit (water)
of course BBCCT-101. A buffer is defined as a solution (chemical system)
that can resist change in pH upon the addition of small amounts of acid or
alkali. Buffer solution has a significant role in maintaining constant pH while
performing a biochemical reaction. In general, buffers are mixtures of a
conjugate acid and a conjugate base (refer Unit-2 of BBCCT-101). A buffer
has its maximum buffering capacity at its pKa value (refer table 3.2).
Table 3.2: Routinely used buffers and their buffering range
Buffer pKavalue Buffering pH range
Phosphate 6.86 6.5-7.5
Carboxylic acid
(a) Acetate 4.76 3.0 - 6.0
(b) Citrate 4.74
Borate 9.24 8.5 - 10.0
Amino acid & peptide

(a) Glycine 9.6 (p K a2 ) 2.0 - 3.0 & 9.5 - 10.0
(b) Histidine 6.0 (pKa2 ) 5.5 - 6.0
(c) Glycylglycine 8.4 (pKa2 ) 8.0 – 9.0
Zwitterionic*
(Good‘s Buffer)
(a) Tris 8.10 7.5 – 9.0
(b) MOPS 7.20 6.5 -7.9
(c) HEPES 7.55 7.0 – 8.0
19
..........................................................................................................................................................................
*Tris – Tri (hydroxymethyl) aminomethane; MOPS- 3-(N-morpholino)-
propane sulphonic acid; HEPES-N-2-hydroxyethyl piperazine-N’-2-ethane
sulphonic acid.(Source: Experimental Biochemistry: A student
Companion.BeeduSashidhar Rao and Vijay Deshpande.
ISBN 81-88237-41-8, I.K. International Pvt. Ltd.)
3.3.1 How to Prepare a Buffer Solution

While studying Unit-2 of BBCCT-101 we came to know that, buffer is
combination of conjugate acid and its conjugate base. Hence to start
preparation of specific buffer solution i1t is advised to prepare stock
solutions of respective acid and base salt. Table 3.3 and 3.4 will show how
to prepare acetate and carbonate-bicarbonate buffers with diverse range of
pH.
Example 1. Preparation of Acetate Buffer
To begin with prepare the stock solution of acid (A) and stock solution of
base (B) as following
Acetic acid stock solution A: Prepare 0.2 mol/litre solution of acetic acid
(to obtain this take 11.55 mL of acetic acid and makeup to the one litre
using distilled water).
Sodium acetate stock solution B: Prepare 0.2 mol/litre solution of sodium

acetate (to prepare this dissolve 16.4 grams of sodium acetate in one litre
of distilled water).
Now you can mix the respective volumes of these two stock solutions and
makeup to 100mL with distilled water as mentioned in the table 3.3.
Table 3.3: Shows the volume of stock solutions and distilled water
to be taken to prepare acetate buffer with specific pH range.
pH Volume of Volume of Volume of Final

Solution A Solution B distilled water (mL) volume (mL)
3.6 46.3 3.7 50 100

3.8 44.0 6.0 50 100
4.0 41.0 9.0 50 100
4.2 36.8 13.2 50 100
4.4 30.5 19.5 50 100
4.6 25.5 24.5 50 100
4.8 20.0 30.0 50 100
5.0 14.8 35.2 50 100
5.2 10.5 39.5 50 100
5.4 8.8 41.2 50 100
5.6 4.8 45.2 50 100
20
..........................................................................................................................................................................
Example 2. Preparation of Carbonate-bicarbonate buffer
Sodium carbonate stock solution A: Prepare 0.2 M solution of sodium

carbonate (anhydrous) (dissolve 21.2 grams of sodium carbonate in 1000
mL of distilled water).
Sodium bicarbonate stock solution B: Prepare 0.2 M solution of sodium

bicarbonate (dissolve 16.8 grams of sodium carbonate in 1000 mL of
distilled water).
Now you can mix the respective volumes of these two stock solutions and
makeup to 100mL with distilled water as mentioned in the table 3.4.
Table 3.4: Shows the volume of stock solutions and distilled water
to be taken to prepare Carbonate-bicarbonate buffer with
specific pH range.
pH Volume of Volume of Volume of Final

Solution A Solution B distilled water (ml) volume(ml)
9.2 4.0 46.0 50 100
9.3 7.5 42.5 50 100
9.4 9.5 40.5 50 100
9.5 13.0 37.0 50 100
9.6 16.0 34.0 50 100
9.7 19.5 30.5 50 100
9.8 22.0 28.0 50 100
10.9 25.0 25.0 50 100
10.0 27.5 22.5 50 100
10.1 30.0 20.0 50 100
10.2 33.0 17.0 50 100
10.3 35.5 14.5 50 100
10.4 38.5 11.5 50 100
10.5 40.5 9.5 50 100
10.6 42.5 7.5 50 100
10.7 45.0 5.0 50 100
3.4 PRECAUTION
 Do not touch the lower sensitive bulb of the electrode by naked hand
or using any other rough material always use cotton or tissue paper
for wiping the electrode.
21
..........................................................................................................................................................................
 Always cover the tip of electrode with storage solution (if not in use
for long time).
 Allow all of the buffers to reach the same temperature, since pH
readings are temperature dependent.
 Use separate beaker to wash/rinse the electrode.
 Note that, the electrode tip and junction are fully immersed in the
buffer, and stir the buffer at a moderate, uniform rate.
1. Define pH?
2. Enlist the pH of saliva, urine and blood?
3. What is the importance of buffer solution?
4. Calculate the volume of sodium carbonate solution and sodium
bicarbonate solution required to prepare 200 mL of carbonate-
bicarbonate buffer with pH9.6. (Refer table 3.4)?
3.5 SUMMARY
 pH is negative logarithm of hydrogen ion concentration.
 Electro Motive Force(EMF) plays a crucial role in measuring the pH.
 Calibration of pH meter is essential before we start using it.
 Buffer is the solution that resists change in pH of a solution upon
addition of small amounts of acid or base.
 Buffer solution is a combination of conjugate acid and its conjugate base.
 Cleaning, maintenance and handling of electrode is essential to obtain
error free values.

1. Experimental Biochemistry: A student Companion. Beedu Sashidhar Rao
2. Practical Biochemistry: for medical, dental and allied courses. 2 nd
edition, Dr. G. Rajagopal and Dr.B.D. Toora.ISBN 81-901769-5-1, Ahuja
publishing house.
4. Segel, I.H. Biochemical Calculations. 2nd ed. John Wiley & Sons. Inc.
NewYork (1976).
K.R. Aneja. ISBN 978-93-87025-49-3.MEDTECH a division of Scientific
22 international (Pvt. Ltd).
Experiment 4
DETERMINATION OF
pK a OF GLYCINE
Structure
4.1 Introduction 4.4 Procedure
4.2 Principle 4.5 Summary
4.3 Reagents and Equipments 4.6 Further readings
4.1 INTRODUCTION
In the previous experiment we have studied about how to prepare buffer
solutions and measure pH of unknown solution. In this experiment we shall
perform the titration of amino acid to know the effect of change in pH on the
ionisable groups of amino acid. Before you proceed further, it is advised to
refer the section 3.3.2 of Unit-3 from BBCCT-101 to recall the concepts of
amino acid titration. This will help you in correlating the core objective of
performing this experiment. The point to be remembered in this experiment
is amino acid tend to donate its protons (H+ ions) in alkaline pH and
becomes negatively (-ve) charged. Similarly, acidic pH amino acid
accepts protons to get positively (+ve) charged. The core aim of this
experiment is to study the behavior of ionizable groups of amino acid pH
with respect to pH changed. This experiment shall give us information
about pKa/b of the ionizable groups, isoelectric point and buffering range of
specific amino acid. Before going to start this experiment you’re advised to
watch a video tutorial available at the YouTube links given below:
https://youtu.be/MpvA75l5oPw ; https://youtu.be/MgUfgozUqFI

After going through this experiment, you should be able to:
 explain the principle behind the titration curves of amino acid;
 determine the pKa/b, isoelectric point and buffering capacity of

amino acid; and
 illustrate the significance of pKa and pKb .

23
..........................................................................................................................................................................
4.2 PRINCIPLE
Amino acids owing to the presence of amino and acidic groups tend to
accept and donate protons in acidic and alkaline conditions. In this process
amino acid attains +ve charge (cation) in acidic pH and –ve charge (anion)
in alkaline pH. However amino acids exists as Zwitter ion (possess both
+ve and –ve charges) at neutral pH (Equation 4.1). This property of amino
acid enables to determine the following:
pKa: Dissociation constant in acidic pH (pK1)
pKb: Dissociation constant in alkaline pH (pK2)
pI: Isoelectric point.
Equation:
4.3 REAGENTS AND EQUIPMENTS

Preparation of Reagents
i) Standardized sodium hydroxide (0.1 N): dissolve 4 g of NaOH in 1L
distilled water
ii) Standardized hydrochloric acid (0.1 N): Pipette out 8.33 mL of
concentrated HCl into 1L volumetric flask and make the volume to
1000 mL with distilled water.
iii) Glycine solution (0.1 N): Dissolve 7.5 g of glycine powder in 1000mL
distilled water
Glassware: conical flask, pipette, burette, wash bottle, beakers and
measuring cylinders
Equipment: pH meter.
4.4 PROCEDURE
Prepare all the reagents freshly prior to start the titration.
i) Take 20 mL of 0.1 N, glycine solution in a 50 mL beaker and measure
the pH and note it in the log book. Take 50 mL burette and fill it upto
“0” (zero) mark with 0.1 N HCl and slowly start titrating against glycine
in the beaker. Record the change in pH value for every 1 mL addition
of acid (Table4.1). Repeat the procedure of amino acid titration with
0.1N NaOH and record the change in pH value for every 1 mL addition
of base (Table 4.2).
Plotting the titration Curve:
ii)Plot the curve by taking amount of acid or alkali added on x-axis, against change
24 in the values of pH on the y-axis (Fig. 4.1).
BBCCL-102 Determination of pKa of Glycine
..........................................................................................................................................................................
The point where half of the volume of acid or alkali consumed in the
titration will give pKaand pKb value.
pI value of glycine can be calculated by using the following equation,
(pI)=1/2 (pKa + pKb)
Observation Table 4.1:

Volume of 0.1N HCl added (mL) Observed pH
1.0
2.0
3.0
4.0
5.0
6.0
Continue up to 20 mL
Observation Table 4.2:

Volume of 0.1N NaOH added (mL) Observed pH
0
1.0
2.0
3.0
4.0
5.0
6.0
Continue up to 20 mL
Resuls : The pKa and pKb values obtained by plotting the titration curve are
________ and pI value is ______________.
25
..........................................................................................................................................................................
1. Define the terms pKa and pKb?
2. What is isoelectric point of an amino acid?
3. Write the equation to calculate pI?
PRECAUTIONS:
1. Rinse pH electrode with distilled water while changing form acid to
alkaline solutions.
2. Note the change in pH regularly after addition of 1 mL of acid/alkali.
3. Immerse the electrode properly in glycine solution and avoid the

touching of the electrode bulb with walls and bottom of the beaker.
4. Use electrode clamp to hold and avoid erratic values.
4.5 SUMMARY
 In this experiment we have learnt about the behavior of ionizable
groups in amino acid with respect to pH change.
 This experiment gave us information about pKa/b of the ionizable

groups, isoelectric point and buffering range of specific amino acid.
 Plotting the titration curves and determining the dissociation constants

pKa (acid, pK1), pkb (base, pK2) of the ionizable groups will give us the
value of isoelectric point (pI) of specific amino acids.
 pKa is the point where amino acid is half protonated and pKb is the
point where amino acid is half deprotonated and pI is the midpoint of
both these points.

Rao and Vijay Deshpande. ISBN 81-88237-41-8, I.K. International Pvt.
Ltd.
2. Maniatis T et al. Molecular cloning- a laboratory manual, 1982.
3. Sambrook, J., & Green, R. M. (2012). Molecular cloning a laboratory

Manual. (4th ed.). New York: Cold Springer Harbor Laboratory Press.
26
Experiment 5
QUALITATIVE TESTS FOR

CARBOHYDRATES
Structure
5.1 Introduction 5.4 Summary
5.2 Materials and Principles
5.3 Procedure for Qualitative

Analysis of Carbohydrates
Self Assessment Questions
5.1 INTRODUCTION
In the previous experiments we have studied about cleaning and
maintenance of glassware, measuring of pH, preparation of buffers and
significance of pKa value. In this experiment we will know about various
qualitative tests routinely used to identify the presence or absence of
carbohydrates in a given test solution.
These tests are specific to the functional group present in that particular
sugar molecule. As we all know that carbohydrates are polyhyroxy
aldehydes or ketones that occur in nature as monosaccharides,
disaccharides, oligosaccharides and polysaccharides (Refer to Unit-5 of
BBCCT-101). Due to the presence of free hydroxyl group at anomeric
carbon atom they exhibit reducing property. Hence, we call them
reducing sugars. However there are few carbohydrates that do not show
reducing property (example: starch and glycogen). These tests are
widely used in clinical biochemistry for the diagnosis of diseases like
diabetes, glycosurias etc. These sugars undergo oxidation and react with
phenylhydrazine to form osazones. The shape of osazone crystals acts
as final confirmatory test. Apart from learning how to do various tests,
we’ll be discussing the principles and preparation of reagents used for
identification of carbohydrates in a given test sample.
For better understanding of this experiment, it is advised to go through

the physical and chemical properties of major biomolecules discussed in
BBCCT-101. Before performing the experiment watch the video available
at the given YouTube link : https://youtu.be/Ewe7i1D9lSQ. 27
..........................................................................................................................................................................
After performing this experiment, you should be able to:
 explain the principle behind the specific test;
 identify specific carbohydrate in an unknown solution;
 distinguish between identification and confirmation tests; and
 enlist various tests used for identification of carbohydrates.
5.2 MATERIALS AND PRINCIPLES

Materials Required
i. Chemicals required
Distilled water, Conc. hydrochloric acid, Sodium hydroxide (10 N).

Conc. Sulphuric acid, Iodine solution, Phenylhydrazine hydrochloride,
Sodium acetate, Glacial acetic acid,concentrated nitric acid.
Phenylhydrazine hydrochloride, Sodium acetate, Glacial acetic acid.
ii. Glassware: 10 mL boiling test tubes, pipettes (1-10 mL), dropper,

glass rod, microscope, glass slides and watch glass, test tube holder,
spatula and blotting paper.
iii. Minor Equipment & other requirements: water bath, light

microscope.
iv. Preparation of carbohydrate (sugar) solution [1% (w/v) in distilled

water] (1g in 100 mL : Prepare individual sugar solutions of glucose,
fructose, ribose, lactose, galactose, mannose and starch respectively.
vi. Preparation of Reagents:
Molisch’s [α-naphthol reagent (5% w/v in ethyl alcohol)]: Dissolve 5 g of

α-naphthol crystals in 100 mL ethyl alcohol..
Iodine Solution: Prepare a 3% (w/v) solution of potassium iodide in

distilled water. Add few crystals of iodine until the solution becomes deep
yellow in colour.
Fehling’s reagent:
Reagent A: Dissolve 6.92 grams of cupric sulphate in distilled water and

make up the volume to 100 mL in a volumetric flask. Store this reagent in
a reagent bottle.
Reagent B: Dissolve 34.6 grams of sodium potassium tartrate and 25

grams of potassium hydroxide in distilled water and make up the volume to
100 mL with distilled water and store in a reagent bottle.
Benedict’s qualitative reagent: Dissolve 17.3 grams of sodium citrate

and 10 grams of sodium carbonate in 50 mL of distilled water and heat the
28 solution in a hot water bath, with constant stirring. Separately, prepare
BBCCL-102 Qualitative Tests for Carbohydrates
..........................................................................................................................................................................
cupric sulphate solution by dissolving 1.73 grams of this salt in 20 mL of
distilled water. Mix the cupric sulphate solution with sodium citrate-
carbonate solution and make up the volume to 100 mL.
Barfoed’s reagent: Dissolve, 13.3 grams of cupric acetate in 80 mL of

distilled water. Add 2 mL of glacial acetic acid and make up the volume to
100 mL with distilled water.
Seliwanoff’sReagent (0.05% (w/v): Dissolve 0.05g of resorcinol in 100 mL

of 4 N hydrochloric acid.
Bial’s Reagent: Prepare 0.3% orcinol in concentrated hydrochloric acid

and add 0.25 mL of 10% (w/v) ferric chloride solution.
Principles In this section we will be studying about principles of various

qualitative tests used for carbohydrate analysis and preparation of reagents.
Molisch’s test: This is a general test for the identification of

carbohydrates.
Principle: In the presence of sulphuric acid, the sugars undergo

dehydration to furfural or hydroxymethyl furfural, which condenses with
α-naphthol (1-hydroxy naphthalene), resulting in the formation of a purple
coloured ring. The sulphuric acid also brings about the hydrolysis of
glycosidic bonds of oligo and polysaccharides.
Reaction showing
formation of purple colour
Reaction showing formation purple colour
Iodine test: This is a general test for the identification of polysaccharides.
Principle: Molecular iodine reacts with polysaccharides forming coloured

adsorption complexes. Variations in the colour of the polysaccharide-iodine
complex is observed (starch gives blue colour, while glycogen forms a red-
brown colours).
29
..........................................................................................................................................................................
Tests for reducing sugars: This is a general test for the identification of
sugars with free hydroxyl group at anomeric carbon atom.
Principle: The cupric (Cu2+) present in the alkaline copper sulphate

solution is reduced to cuprous (Cu+) hydroxide by the reducing sugar
which undergoes spontaneous dehydration to cupric oxide, appearing as
a coloured precipitate (Fig. 5.1). The colour of precipitate varies from
green to red (red, brick-red, orange, green) based on the concentration of
reducing sugar.
Fig. 5.1: Benedicts test
This is the basic principle for the following biochemical tests (i) Fehling’s
(ii) Benedict’s, and (iii) Barfoed’s. However in Barfoed’s test, the reduction
of cupric ion is under mild acidic condition and the test is more rapid for
monosaccharides than disaccharides.
For the better understanding of the principle working behind each reaction
we are providing the following reaction mechanisms.
Benedict’s test:
Barfoed’s test:
30 Reaction showing formation of red precipitate

..........................................................................................................................................................................
Seliwanoff’s test:
Principle: Ketosugars (ex. Fructose) when exposed to acid medium
undergo dehydration forming hydroxymethyl furfural more rapidly than
aldohexoses. This furfural condenses with resorcinol (m-dihydroxy
benzene) to produce a deep pink colour molecule. This test is helpful to
distinguish between aldoses and ketoses.
Reaction showing formation of pink color.
Bial’s test:
Principle: Pentose sugars like ribose or xylose undergo dehydration in acid

medium to form furfural derivative, which condenses with orcinol (3,5-
dihydroxy toluene) to produce a green coloured complex. However,
presence of hexose sugar produce a brown color complex.
Reaction showing formation of brown color complex.
31
..........................................................................................................................................................................
Mucic acid test:
Principle: Monosaccharides like galactose, (galactose containing sugars
such as lactose gives positive response to this reaction). The presence
of strong acids like nitric acid, produce saccharic acids. The saccharic
acid formed is insoluble and form clear crystals. This acid derivative is
known as galactaric or meso-galactaric acid (mucic acid), thus the name
for the test.
Heat
Reaction showing formation of mucic acid crystals.
Test for sucrose:
Principle: Sucrose being disaccharide does not give positive results

to reducing tests. However, upon acid hydrolysis (by adding few
drops of diluted hydrochloric acid) yields reducing sugars glucose
and fructose. Later, these sugars give positive results to reducing
test (Benedict’s and seliwanoff’s test).
Note: Before testing the solution by Benedict’s reagent, neutralize

the acid added during hydrolysis by adding few drops of 10N
NaOH.
Osazone test: This is the final and confirmatory test for qualitative
analysis of carbohydrates
Principle: Reducing sugars upon reaction with Phenylhydrazine

produces osazones, which are the characteristic derivatives of
carbohydrates. These osazone derivatives have definite crystalline
shape. These crystals made it easy and possible to confirm the type
of carbohydrate.
Sugars lacking free anomeric hydroxyl group (non-reducing sugars)

do not respond to this test. Whereas glucose, fructose and mannose
produces similar type of osazone, i.e., glucosazone. An osazone
crystal differs from the other with respect to time of crystallization,
crystal shape and melting point.
32
..........................................................................................................................................................................
Reaction showing formation of osazone crystals.
5.3 PROCEDURE FOR QUALITATIVE ANALYSIS

OF CARBOHYDRATES
We have explained the procedure under three phases. Phase-I focuses
on identification of the sample as mono or disaccharide or polysaccharide.
Phase-II is helpful in identifying the sample as Aldose or Ketose sugar.
Finally Phase-III is confirmatory using osazone crystals. Learners need to
note their observations in the tabular format (Table 5.1) while performing
this experiment at practical session
33
..........................................................................................................................................................................
Table 5.1: Qualitative analysis of carbohydrates
Sl. Test Observation Inference

No.
1. Solubility Test: A 1. Soluble in cold 1. Presence of

pinch of sugar water monosaccharide
(carbohydrate sample
2. Insoluble in cold 2. Presence of
being tested) is
dissolved in 2 mL of water Polysaccharides
distilled water.
2. Molisch’s Test: Add A purple ring Presence of

few drops of α- appears at the Carbohydrates
naphthol reagent to 1 interphase of
mL of sugar solution sugar solution and
taken in a test tube, the conc. sulphuric
and vortex the acid.
contents. Carefully, add
conc. H2SO4 along the
side of the test tube,
keeping the tube in an
inclined position (do
not shake the test
tube, while adding the
acid).
3. Iodine test: Add few 1. Appearance of 1. Presence of

drops of iodine reagent blue color Starch
to 2 mL of the sugar 2. Appearance of 2. Presence of
solution and mix. red color dextrin
3. Appearance of 3. Presence of
brown color glycogen
4. No change in 4. Absence of
color polysaccharides
4. Test for Reducing

Sugars:
1. Fehling’s Test: Mix Appearance of Presence of

1 mL each of orange to brick red reducing sugar
reagent A and B in a color
test tube. Add few
drops of sugar
solution/test sample
and mix. Heat the
tube in a boiling
water bath for 5 to
34 10 min.
..........................................................................................................................................................................
2. Benedict’s Test: Appearance of Confirms the presence
Add few drops of green/yellow/ of reducing sugar
sugar solution to 2 orange/ red color
mL of Benedict’s (the development of
reagent taken in a color depends on
test tube, and place the concentration of
the tube in a boiling sugar present in
water for 5 to 10 the solution)
minutes.
3. Barfoed’s Test: To Presence of reducing

2 mL of the reagent Appearance of monosachharide.
taken in a test tube, red precipitate at
Note: If the time taken
add few drops of the the bottom of the
for the formation of
sugar solution and tube within 5
the precipitate is
heat the contents in minutes
more, it is suggestive
a boiling water bath. of a reducing
disaccharide.
Seliwanoff’s test: Add Appearance of Presence of keto-

5.
few drops of sugar pink or cherry red sugar
solution to 2 mL of the color
Seliwanoff’s reagent
taken in a test tube,
and place the tube in
a boiling water bath for
5 minutes.
Bial’s test: Add few Appearance of

6. Presence of pentose
drops of sugar
green color sugar
solution to 2 mL of the
Bial’s reagent, taken in
a test tube, and heat
in a boiling water bath
for 5-10 minutes.
7. Mucic acid test: To

5 mL of the sugar Appearance of Presence of galactose
solution in a 50 mL gritty crystals of
glass beaker add 2 mL mucic acid
of conc. Nitric acid.
Concentrate the
contents to a small
volume (2-3 mL) by
heating (over a flame
or on steam bath).
After concentration,
cool the solution
gradually to room
temperature.
35
..........................................................................................................................................................................
8. Osazone test: Take Microscopic

3 mL of the sugar examination of the
solution in a test osazones reveals
tube, add 0.5 g of the following shapes
phenylhydrazinereagent, of crystals (Fig. 5.3):
add 0.1 g of sodium
acetate and few i)n eedle shaped i)
drops of acetic acid. crystals arranged
The contents are singly or in groups
(Feathery).
mixed well and
placed in a boiling a ) glucose is
a) Crystals
water bath for 15 confirmed
formed after 10
b) fructose is
minutes. Cool the minutesb)
confirmed
solution to room b) Crystals formed
temperature and within 5-10
observe the shape of minutes
the crystals formed,
under a light ii) Long fine needles. ii) Xylose is
microscope. confirmed
(Fig. 5.2) iii) Sunflower shaped iii) Maltose is
crystals. confirmed
iv) Puff shaped iv) Lactose is

crystals. confirmed
v) Broad glass piece v) Presence of

shaped crystals galactose
formed within 30
minutes
vi) White colored vi) Presence of

irregular shaped Mannose
crystals are
formed after 45
minutes
36 Fig. 5.2 : Preparation of osazone crystal slide.

..........................................................................................................................................................................
Fig. 5.3: Osazone crystals.
Note:
1. The reducing ability of carbohydrates is due to the presence of

free hydroxyl group at anomeric carbon atom (glucose). However,
in certain sugars (sucrose) this hydroxyl group is involved in
glycosidic bond formation and in some anomeric hydroxyl group of
sugar blocked by either alkylation (α- or β- methyl glycosides).
Hence, they do not respond to reducing tests. Interestingly,
maltose and lactose being disaccharides possess a free anomeric
hydroxyl group enabling it positive to reducing tests.
2. It is better practice to cross check the inference of all tests and

compare with the results obtained in osazone test before finalising
your results.
3. For microscopic examination decant the upper liquid portion from

the test tube and collect a small precipitate of ozone on a glass
slide. Gently spread the precipitate and observe under light
microscope.
Results:
The given test sample shows positive response to the following tests
______ , ______, and the osazone crystals observed are ________ shape
and hence it is confirmed that the given carbohydrate is
____________________.
SELF ASSESSMENT QUESTIONS
1. Give two differences between Mono and Disaccharides?
2. Write the composition of sucrose and lactose?
3. What is the significance of Iodine test? 37

..........................................................................................................................................................................
4. Justify the non-reducing property of sucrose.
5. Draw the structure of Maltosazone?
6. Identify the test used to detect Honey sugar?
7. List the chemicals used in Osazone test?
5.4 PRECAUTIONS
1. Ensure adequate distance from burner while boiling the reagents
2. Always add reagents with the help of pipette or dropper
3. Use separate pipettes or dropper to add reagents (avoid contamination)
4. Use clean and dry test tubes for individual test
5. Avoid contact of microscope lens with osazone crystals.
5.5 SUMMARY
 In this experiment we have studied about various tests and their
principle involved in identifying the presence of carbohydrates in a
given test sample (sugar solution).
 Reducing tests are specific to the sugars containing free anomeric
hydroxyl group (reducing sugars). However in certain sugars this
hydroxyl group is involved in glycosidic bond formation. Hence such
sugars do not show positive response towards reducing test and
known as non-reducing sugars.
 Iodine test is specific for polysaccharides like starch, dextrin and
glycogen and selwinoff’s test is for ketose sugars.
 Osazone crystals formed are very useful for confirmation of
carbohydrates. However reducing sugars show positive response to
this test and individual sugars have distinct osazone crystals.

1. Experimental Biochemistry: A student Companion. BeeduSashidhar Rao
2nd edition, Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1,
3. Life Sciences Protocol Manual January 2018. Department of
Biotechnology, Ministry of Science and Technology, Govt. of India.
New York (1976).
K.R. Aneja. ISBN 978-93-87025-49-3. MEDTECH a division of Scientific
38
Experiment 6

AMINO ACIDS AND
PROTEINS
Structure
6.1 Introduction 6.4 Procedure for
Expected Learning Outcomes identification of Proteins
6.2 Materials and Principles Self Assessment Questions
6.3 Procedure for Qualitative 6.5 Summary

analysis of amino acids
6.1 INTRODUCTION
In the previous experiment you have learnt about various qualitative tests
routinely used to identify carbohydrates. As we all know that amino acids
are building blocks of proteins, these tests will help us in understanding the
chemical nature of proteins with special emphasis to amino acid
composition. However, in this experiment we’ll be focusing on the principles,
preparation of reagents and procedures of qualitative tests used for
identification of amino acids (Fig. 6.1) and proteins in a given test sample.
These tests are specific to the functional group present in that amino acid
(Refer to Unit-3 of BBCCT-101). In general amino acids are like aliphatic,
aromatic, sulphur containing and imino acids. Coming to qualitative analysis
(Table 6.1) of proteins, it has a significant contribution in understanding the
kidney function. For a better understanding of this experiment, it is advised
to go through the physical and chemical properties of amino acids
discussed in BBCCT-101. Before performing the experiment watch the
video available at the given YouTube link: https://youtu.be/EyGVlgZoHaQ.

 explain the principle behind the specific qualitative test;
 identify specific amino acid in an unknown solution;
 distinguish between identification and confirmation tests; and
 enlist various tests used for qualitative identification of amino acids. 39
..........................................................................................................................................................................
6.2 MATERIALS AND PRINCIPLES

Materials Required:
i.G lassware: 10 mL. boiling test tubes, pipettes (1-10 mL.), dropper,
glass rod, microscopic glass slides and watch glass.
ii. Minor Equipment and accessories: water bath, test tube holder,
spatula and blotting paper.
iii. Amino acid solution:Prepare 0.1% (w/v) of the individual amino acid
(standard amino acid samples are commercially available) in distilled
water.
iv. Preparation of Reagents:
Ninhydrin reagent (2% w/v): Prepare by dissolving 2 g of Ninhydrin

powder in 100 mL acetone.
α-naphthol reagent (1% w/v in ethyl alcohol): Dissolve α-naphthol 1 g

of α-naphthol crystals in 100 mL ethyl alcohol.
Sodium hydroxide solution (40% w/v): Prepare by dissolving 40 g of

sodium hydroxide pellets in 100 mL of distilled water.
Lead acetate solution (10% w/v): Prepare by dissolving 10 g of Lead

acetate powder in 100 mL of distilled water
Sodium nitroprusside (10% w/v) reagent: Prepare by dissolving 10 g of

Sodium nitroprusside powder in 100 mL of distilled water.
Glycine (2% w/v) solution: Prepare by dissolving 2 g of glycine powder

in 100 mL distilled water.
Urea solution 5% (w/v): Prepare by dissolving 5 g of Urea powder in 100

mL distilled water.
Hypobromite reagent (to be prepared freshly): This is prepared by

adding bromine water to NaOH solution. Dissolve 5 g of NaOH in 50 mL of
distilled water and add one commercially available elemental bromine vial.
(Perform this this under fume hood with adequate care and guidance)
Isatin reagent (1% w/v): Dissolve 1 g of isatin powder in 100 mL

acetic acid solution.
Sulphalinic acid (1% w/v): Dissolve 50 mg of Sulphalinic acid in a mixture

of 0.1 mL of hydrochloric acid and 10 mL of water and add 0.1 mL of
sodium nitrite solution.
Sodium carbonate (10% w/v): Dissolve 10 g of sodium carbonate in 100

mL distilled water,
Million’s reagent (15% w/v): Dissolve 15g of mercuric sulphate in 100 mL

of 6 N sulphuric acid.
40
BBCCL-102 Qualitative Tests for Amino Acids and Proteins
..........................................................................................................................................................................
Sodium nitrite solution (5% w/v): Dissolve 5 g of sodium nitrite powder
in 100 mL of distilled water. (To be freshly prepared),
Nitrosonaphthol reagent (0.1% w/v): Dissolve 0.1g of nitrosonaphthol in
100 mL of ethanol.
In addition to the above reagents few commercially available ready to use
acids, alkalis and solvents like glacial acetic acid, conc. sulphuric acid,
conc. nitric acid, hydrochloric acid and liquor ammonia, are also needed to
perform the qualitative analysis of amino acids.
PRINCIPLES:
Ninhydrin test: This test is a identification test for the presence of amino
acids.
Principle: In this reaction amino acids react with ninhydrin (a powerful
oxidizing agent) reagent to give a purple coloured complex (Ruhemann’s
purple) while, imino acids (proline and hydroxyproline) react with ninhydrin
to produce yellow colour.
Reaction showing formation of Rheumann’s purple.
Xanthoproteic test: This test is a identification test for the presence of

aromatic amino acids.
Principle: In this reaction aromatic amino acids (tyrosine or tryptophan or

proteins with aromatic amino acids) undergo nitration in the presence of
nitric acid to produce nitro-derivatives that are yellow in colour. At the end
of the reaction addition of NaOH (alkaline pH), the colour changes to
orange due to the ionization of the phenolic group.
Reaction showing formation of yellow colour.
Ehrlich’s test: This is a identification test for the presence of amino acid
tryptophan. 41
..........................................................................................................................................................................
Principle: In this reaction indole ring of tryptophan reacts with para-
dimethylamino benzaldehyde under acidic conditions to give a purple
colour.
Hopkins-Cole test: This test is a confirmatory test for the presence of

amino acid tryptophan.
Principle: In this reaction indole moiety of tryptophan condenses with

aldehydes under acidic environment to yield purple or violet coloured
compounds.
Condences / acidic conditions

Tryptophan + aldehydes Purple colored compound
Lead sulphide test: This test is a identification test for sulphur containing
amino acids.
Principle: This test mainly depends on formation of inorganic sulphide

from organic sulphur. The sulphur containing amino acids, (cysteine and
cystine) upon boiling with sodium hydroxide (hot alkali) produce sodium
sulphide. This can be detected by precipitating inorganic sulphide to lead
sulphide(black), using lead acetate solution.
Sodium nitroprusside test:
Principle: Sodium nitroprusside reacts with the thiol group of the cysteine
under alkaline condition to yield an intense purple coloured compound,
which fades after few minutes.
Sullivan and McCarthy’s test:
Principle: Addition of sodium nitroprusside to an alkaline solution of

methionine followed by acidification of the reaction yields a red colour.
Sakaguchi test:
Principle: α-naphthol (1-hydroxy naphthalene) reacts with a guanidine

group containing amino acid like arginine under alkaline condition,, which
upon treatment with hypobromite or hypochlorite, produces a characteristic
red colour.
Isatin test:
Principle: Imino acids such as proline and hydroxyl proline condense with
isatin under acidic conditions to yield a blue coloured adduct.
Protine + Isatin ----- Blue coloured adduct
42
..........................................................................................................................................................................
Pauly’s diazo test:
Principle: This test is based on the formation of diazonium salt where,
sulphanilic acid upon diazotization in the presence of sodium nitrite and
HCl produces diazonium salt. The diazonium salt formed, can combine
with either tyrosine or histidine in alkaline medium to give a red coloured
product (azo dye).
Millon’s test:
Principle: This test is specific to phenolic group containing amino acid
such as tyrosine. Tyrosine reacts with mercuric ions in acidic condition in
the presence of sodium nitrite, to give a red colour complex (Millon’s red).
Tyrosine
M illions reagent
Reaction showing formation of red color complex.
Gerngross test
Principle: This is a confirmatory test for the presence of amino acid

tyrosine. Tyrosine reacts with nitrosonaphthol in acid medium to give a
purple-red coloured complex.

OF AMINO ACIDS
Table 6.1: Qualitative analysis of amino acids

No.
1 Ninhydrin test: Take 1. Appearance of 1. Presence of

1 mL of amino acid purple to blue α-amino acids
solution in a test color.
tube and add few 2. Presence of
2. Appearance of imino acids
drops of ninhydrin
yellow color.
reagent and mix the
contents. Place the
test tube in a boiling
water bath for 5 min.
Later remove and
cool to room
temperature.
43
..........................................................................................................................................................................
2 Xanthoproteic test: After adding the Presence of aromatic

Take 1 mL of amino alkali, the color amino acids
acid solution in a test changes to
tube and add few orange.
drops of nitric acid
and mix well. Boil the
contents over a
bunsen flame, for
few minutes. Add few
drops of alkali after
cooling the test tube
under running tap
water.
3 Ehrlich’s test: Take Appearance of a Presence of

1 mL of amino acid purple color tryptophan.
solution and add few
drops of Ehrlich’s
reagent and vortex
the contents.
4 Hopkins-Cole test: A purple- violet The formation of ring

Take 1 mL of amino ring appears at confirms the presence
acid solution and add the junction of the of tryptophan.
1 mL of acetic acid- amino acid
glyoxylic acid solution and the
reagent, in a test conc. sulphuric
tube and mix. Then acid.
carefully, add (use
pipette)conc. H2SO4
along the side of the
test tube, keeping
the tube in an
inclined position (do
not shake the test
tube, while adding
the acid).
5 Lead sulphide test: Appearance of a Presence of cysteine

Take 1 mL of amino black precipitate of or cystine.
acid solution and add lead sulphide
few drops of sodium
hydroxide (40%) to 1
mL of the amino acid
solution taken in a
test tube, and boil
the contents for 5-10
min over a
44
..........................................................................................................................................................................
Bunsenflame. Cool
the contents and add
few drops of 10%
lead acetate solution.
6 Sodium
Appearance of Presence of cysteine.
nitroprusside test:
intense purple
Take 1 mL of amino
colour
acid solution and add
few drops of sodium
nitroprusside reagent
and mix well. Add
few drops of liquor
ammonia and mix well.
7 Sullivan and Appearance of red Presence of

McCarthy’s test: colour methionine.
Take 1 mL of amino
acid solution in a test
tube, add few drops
of sodium hydroxide
(5 N), followed by
addition of few drops
of glycine (2%) and
10% sodium
nitroprusside solution
and mix well. Place
the test tube in a hot
water bath, maintained
at 40o C, for 15
minutes. Cool the
tube in ice cold
water for 5 minutes
and add 0.5 mL of 6
N HCl. Mix the
contents and allow to
stand for 15 min at
room temperature.
8 Sakaguchi test: Appearance of Presence of arginine.

Take 1 mL of amino red colour
acid solution in a test
tube and chill it in
ice box. Add few
drops of sodium
hydroxide solution,
followed by 1 mL α-
naphthol reagent and
mix well. After 2 min
add few drops of 5% 45
..........................................................................................................................................................................
urea solution,
followed by freshly
prepared hypobromite
reagent, drop wise.
9 Isatin test: Apply a Appearance of a Confirms the

drop of amino acid blue coloured spot presence of imino
solution on a on the filter paper acid.
Whatman No. 1 filter
paper strip (size 25 x
50 mm) and dry the
spot using a hair
dryer or in a hot air
oven. Later apply a
drop of isatin reagent
on to the dried spot.
Repeat the drying
procedure for few
minutes and observe.
10 Pauly’s diazo test: Appearance of a Presence of either

Take 1 mL of red color tyrosine or histidine.
sulphanilic acid
reagent in a test
tube and chill the
contents in a ice box.
Add few drops of
pre-cooled sodium
nitrite solution and
mix. Quickly add few
drops of pre-cooled
amino acid solution
and mix well. (Later
add sodium
carbonate solution
drop by drop).
11 Millon’s test: Take 1 Appearance of Confirms the presence

mL of amino acid red color of tyrosine
solution in a test
tube and add few
drops of Millon’s
reagent and mix well.
Boil the contents
over a bunsen flame
for 3-5 min. Later
cool the contents
46
..........................................................................................................................................................................
under running tap

water and add few
drops of sodium
nitrite solution.
12 Gerngross test: Appearance of a Confirms the

Take 1 mL of amino purple-red colour presence of tyrosine.
acid solution and add
few drops of
nitrosonaphthol
reagent and vortex.
Boil the contents
over a bunsen flame
for 3-5 minutes. Cool
the contents under
running tap water
and add 1-2 drops of
nitric acid.
Amino acid
solution
Fig. 6.1: Schematic representation of qualitative analysis of amino acids
Results: The given test sample shows positive response to the following tests
______ , ______, ________ and hence it is confirmed that the given solution
contains_____________ functional group and is ____________________ amino
acid. 47
..........................................................................................................................................................................
1. Name the identification test for α-amino acids.
2. Give an example for imino acid
3. Xanthoproteic test is used for the identification of ____ amino acid.
4. Example for sulphur containing amino acids.
5. Test used for identification of methionine.
6. Name confirmatory test for imino acid.
7. Millons test is used for identification of ____ amino acid.
6.4 PROCEDURE FOR IDENTIFICATION OF

PROTEINS
So far we have learnt how to perform qualitative analysis of amino acid.
Now in this section we shall be exploring the tests routinely used for
qualitative analysis of proteins in a given solution. We have studied in Unit-
3 of BBCCT-101 that, amino acids are building blocks of proteins. These
proteins are essential to perform diverse functions like structural –
muscular proteins; immune molecules – antibodies; signaling molecules –
hormones and so on. Proteins being vital in our life their clinical diagnosis
has gained a significant role in the clinical diagnosis of several diseases
and also useful for the analysis of health status. Qualitative analysis of
proteins plays vital role in the clinical diagnosis of kidney function. For
instance, appearance of abnormal levels of proteins like albumin and or
globulin in urine indicates malfunctioning of kidney (nephron) and during
pregnancy it is referred as proteinuria.
In this section we will be mainly focusing on the qualitative analysis (Biuret,

Sulphosalicylic acid and heat coagulation tests) only, however in BBCCL-
106 we will perform quantitative analysis of proteins. Before performing the
experiment watch the video available at the given YouTube link: https://
www.youtube.com/watch?v=EyGVlgZoHaQ
Materials Required:
Protein solution: To perform this experiment we need to prepare a test

sample by dissolving 0.5% (w/v) casein or albumin in 0.1 N NaOH. Or we
can use readily available Hen’s egg white dissolved in 0.1 N NaOH
solution (avoid yolk).
Reagents:
Sodium hydroxide solution (10% w/v): Dissolve 10 g of NaOH crystals in

100 mL of distilled water
48
..........................................................................................................................................................................
Cupric sulphate solution (A) (0.15% w/v): Dissolve 0.15 g of cupric
sulphate powder in 100 mL distilled water.
Sodium potassium tartrate solution (B) (0.6% w/v): Dissolve 0.6g of

sodium potaasium tartrate powder in 100 mL of distilled water.
Alkaline Copper reagent: Mix equal volume of reagent (A) and (B) before
test.
Saturated ammonium sulphate solution: Add 766.8 g of ammonium

sulphate powder to 1L distilled water.
Sulphosalicylic acid 5% (w/v) reagent: Prepare by dissolving 5g of

sulphosalicylic acid powder in 20% sodium sulphate (20 g of sodium
sulphate in 100 mL of distilled water) solution.
Acetic acid (1% v/v): Take 10 mL of acetic acid in 100 mL volumetric

flask and makeup the volume to 100 mL with distilled water.
PRINCIPLES
Biuret test:
Principle: Cupric copper reacts with the peptide bonds present in proteins
under alkaline condition to yield a violet colour complex. This colour complex
is the indicator for the presence of peptide bonds and hence the detection
of proteins in the give test sample possible.
Reaction showing the formation of violet color complex
Procedure: Take 2 mL of the protein solution add few drops of alkali (10%
NaOH) and mix well. Later to this mixture add copper reagent drop wise
with continuous mixing the contents or vortex the test tube.
Fig. 6.2: Biuret test

49
..........................................................................................................................................................................
Observation: Formation of a violet-purple colour indicates the presence of
protein.
Sulphosalicylic acid test:

Principle
Precipitation is the principle working behind this test. Here, the addition of
sulphosalicylic acid precipitates proteins present in the sample solution. In
this reaction addition of sulphosalicylic acid interacts with positively charged
amino acids and aggregates the protein. The positive test would be
visualized as white turbidity.
Procedure: Take 2 mL of the test sample in a test tube. To this add

sulphosalicylic acid reagent drop by drop and mix the contents regularly.
Fig. 6.3: Formation of precipitate
Observation: After addition of few drops of reagent you can observe the
formation of a white precipitate or turbidity.
Heat coagulation test:
Principle: This is a less sensitive method when compared to the

sulphosalicylic acid test. Moreover the principle in this test is also based
on precipitation. Proteins when exposed to heat in acidic conditions
undergo structural denaturation leading to aggregation.
Procedure: Take a clean and dry test tube and add 2 mL of test sample
followed by 0.5mL of 1% acetic acid solution along the walls of the test
tube. Heat the sides of the test tube in a slanting position over a Bunsen
burner for 1 to 2 minutes.
Fig. 6.4: Heat coagulation test

Observation: Upon mild heating, observed the formation of precipitation
indicating the presence of proteins.
50
..........................................................................................................................................................................
Results: The given test solution gives positive/negative results for the
________, _______, and _____ tests hence it is confirmed that the
presence /absence of protein in the given test sample.
1) What are the building blocks of proteins?
2) Name the important bond formed between amino acids?
3) Presence of protein in Urine sample indicates a malfunction of _____
organ?
4) Egg white is a rich source of _____ protein.
Precautions:
1. Avoid continuous heating of reagents
2. Use separate pipette to avoid contamination of reagents
6.5 SUMMARY
 In this experiment we have discussed about various tests, principles,
reagents and procedures involved in identifying the presence of amino
acids in a given test sample.
 Alpha amino acids give positive response (purple color) to ninhydrin
test, whereas imino acids like proline gives yellow color.
 Xantoproteic test indicates the presence of aromatic amino acids
 Sodium nitroprusside test confirms the presence of thiol group
containing amino acids
 Millon’s test indicates the presence of tyrosine
 Isatin test confirms the presence or absence of imino acid.
6.6 FURTHER READING

Ltd.
edition, Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1,
New York (1976).
K.R. Aneja. ISBN 978-93-87025-49-3. MEDTECH a division of
Scientific international (Pvt. Ltd).
51
7
..........................................................................................................................................................................
Experiment

LIPIDS
Structure
7.1 Introduction 7.3 Procedure for Qualitative

Expected Learning Outcomes Analysis of Lipids
7.2 Materials and Principles Self Assessment Questions
7.4 Summary
7.1 INTRODUCTION
In the previous experiments (5 & 6) we have studied about various
qualitative tests routinely used to identify the presence or absence of
carbohydrates, amino acids, proteins. In this experiment we’ll know about
qualitative tests used to identify lipids in a given test sample.
Lipids being insoluble in water known as hydrophobic biomolecules, but

they are soluble in organic solvents like benzene, chloroform, hexane and
diethyl ehter.Recall the properties of lipids which we have studied in block-3
of BBCCT-101. It is well known fact that lipids are high energy compounds
and are classified as simple, conjugated and derived lipids. Apart from
acting as energy source, lipids have diverse functions ranging from plasma
membrane formation, micelle formation, hormonal action and thermal
insulator etc.
However the availability of free floating lipids in blood and their

accumulation in various vital parts of our body is one of the major causes
for life style disorders like obesity and cardiovascular diseases. However in
this experiment we will be studying about the different tests performed to
identify the chemical nature of lipids i.e., saturated or unsaturated along
with presence or absence of sterols. For better understanding of this
experiment, it is advised to go through the physical and chemical
52 properties of lipids discussed in Unit 9 of BBCCT-101.
BBCCL-102 Qualitative Tests for Lipids
..........................................................................................................................................................................
The tests conducted to identify lipids are specific to the chemical nature
and functional groups present on them. Qualitative analysis of lipids has
a significant contribution in identifying the adulteration of edible oils and
hence has vital role in industry, as well as in health sector. Before
performing the experiment watch the video available at the given YouTube
link: https://www.youtube.com/watch?v=l2QOi9mZoFc

 explain the principle behind the specific test;
 identify specific fatty acid in a given solution;
 distinguish between identification and conformation tests; and
 enlist various tests used for identification of lipids.
7.2 MATERIAL AND PRINCIPLES

Materials Required:
Glassware: 10 mL boiling test tubes, pipettes (1-10 mL), dropper, glass

rod and watch glass.
Equipment and accessories: Water bath, test tube holder, spatula and
blotting paper.
Test sample: The commercially available oil samples like Coconut oil,
Ghee, Palm oil, Butter and cholesterol can be used.
Reagents and Chemicals:
Alcoholic-KOH (2% w/v KOH in ethyl alcohol): Dissolve 2 g of

potassium hydroxide (KOH) in 100 mL of ethyl alcohol.
Dilute potassium permanganate solution (0.05% w/v): Dissolve 50 mg

of potassium permanganate in 100 mL of distilled water.
Hubl’s Iodine Solution: To prepare this (a) dissolve 2.6 g of iodine in 40

mL of ethanol (95% v/v), ( b) dissolve 6.0 g of mercuric chloride in 40 mL
of ethanol (95% v/v). Transfer both solutions ‘a’ and ‘b’ into a beaker and
make up the volume to 100 mL with same ethanol.
Potassium bisulphate (solid)
Bromine water
Acetic anhydride and Conc. sulphuric acid
Solvents: Chloroform, ether, benzene, carbon tetrachloride, hexane, 53

..........................................................................................................................................................................
PRINCIPLES
Solubility test: This is a primary step to know the chemical nature of the
given test sample
Principle: Due to hydrophobic of nature of lipids they are insoluble in water
and are soluble in organic solvents.
Saponification test:
Principle: Lipids upon alkaline hydrolysis release glycerol and fatty acids.
Later sodium (Na+) or potassium (K+) ions combines with fatty acids to
form “soap” (foam). Hence, this is known as saponification reaction.
Reaction showing formation of soap molecule.
Acrolein test:
Principle: Formation of acrolein or acrylic aldehyde that has characteristic
pungent odor is the key principle. In general lipids upon heating with
potassium bisulphate produce acrolein.
Reaction showing formation of acrolein.
Hubl’s iodine test (Tests for unsaturation):

This test is mainly helpful to assess the unsaturation in a given oil sample.
The two principles that will explain the reaction mechanism are given
below.
Principle
i) Bromine or iodine upon reacting with unsaturated fatty acids produce
di-halo adducts. Consumption of more bromine indicates the higher
percentage of unsaturation.
ii) Decolorization of alkaline potassium permanganate is also an indirect
measure of unsaturation in fatty acids, where unsaturated fatty acids
54
undergo incomplete oxidation.
..........................................................................................................................................................................
Reaction showing formation of di-halo adduct.
Salkowski test and Liebermann-Burchard test (Test for cholesterol):
Principle: Cholesterol in the presence of concentrated sulphuric acid and

acetic anhydride undergo dehydration producing colored product which is
blue-green in color.
Blue-Green color
product
(chromophore chode)
Reaction showing formation of blue-green chromophore.

OF LIPIDS
Table 7.1: Qualitative analysis of lipids

No.
1 Solubility test: To i) Completely i) Indicates

small quantities of fat/ soluble in all presence of fat or
fatty acid taken in a four solvents oil in the given
separate test tube a - d. solution
add the following
organic solvents
separately:
1. a) Chloroform,
b) ether, ii) Found small ii) Indicates

c) benzene, floating presence of fat or
droplets on oil in the given
d) hexane (3 mL/ water. solution.
test tube)
2. Distilled water
55
..........................................................................................................................................................................
2 Saponification test: Appearance of Indicates the presence

Take approximately foamy solution of oil or fat
100 mg of oil or fat in
a test tube. Add 3 mL
of alcoholic-KOH and
mix well. Place the
tube in a boiling water
bath for 15-20 min.
3 Acrolein test: Take Release of Indicates Presence of

approximately 100 mg pungent odor glycerol
of test sample in a
test tube and add a
pinch of potassium
bisulphate and mix
well. Heat the mixture
over a Bunsen burner
for 1-2 minutes.
4 Tests for i) i) Indicates

unsaturation: Decolourisation presence
of potassium unsaturated fats
i) Bromine water
permanganate or fatty acids
test: Take 1 mL of
oil or 100 mg of or bromine
ii) Indicates
fat in a test tube water is
presence of
add 2 mL of non- observed
saturated fats
polar organic ii) No and fatty acids
solvent (Refer decolorisation
solubility test) and
observed.
dissolve
completely. To this
add potassium
permanganate
solution or bromine
water drop by drop.
ii) Hubl’s iodine i) Decolourisation i) Indicates

test: Take 1 mL of of iodine presence of
oil or 100 mg of solution is unsaturated fats
fat in a test tube observed
add 2 mL of non- ii) Indicates absence
polar organic ii) No of unsaturated
solvent (Refer decolrisation of fats or oils or
solubility test) and Iodine is presence of
dissolve observed. saturated fats or
completely. Add oils.
Hubl’s reagent
drop wise mixing
56 the contents.
..........................................................................................................................................................................
5 Test for cholesterol: Observes Indicates presence of

formation of blue- cholesterol
1. Salkowski test:
green color at the
Take 1 mL of oil or
junction of two
fat sample in a
liquids
test tube and
dissolve in 1 mL
chloroform to this
add equal volume
of concentrated
sulphuric acid
along the walls of
the tube. (Do not
mix the contents).
2. Liebermann- Formation of an Confirms the presence

Burchard test: emerald green of cholesterol
Take approximately color
100 mg of fat or 1
mL of oil in a test
tube, dissolve by
adding 1 mL of
chloroform add
equal volumes of
acetic anhydride
followed by drop
wise addition
(along walls) of
concentrated
sulphuric acid.
1. List the organic solvents used for solubility test?
2. What is the importance of Iodine test?
3. Name the test used to identify the presence of cholesterol in the test
sample?
4. _______________ test principle based on soap formation.
7.4 SUMMARY
 The solubility test gives us an idea about the nature of the sample i.e.,
polar or non-polar.
 Formation of foamy solution in saponification test indicates the

presence of fat. 57
..........................................................................................................................................................................
 Hubl’s iodine test confirms the presence or absence of unsaturation
(double bonds) in the given sample.
 Salkowski test and Liebermann-Burchard test indicates and confirms

the presence of sterol ring containing molecule i.e., cholesterol.
7.5 FURTHER READING

Ltd.

edition, Dr. G. Rajagopal and Dr.B.D. Toora.ISBN 81-901769-5-1, Ahuja
publishing house.

4. Segel, I.H. Biochemical Calculations. 2nd ed. John Wiley &

Sons.Inc.New York (1976).

K.R. Aneja.ISBN 978-93-87025-49-3.MEDTECH a division of Scientific
58
Experiment 8
DETERMINATION OF PURITY
OF NUCLEIC ACID (DNA) BY
UV ABSORPTION METHOD
Structure
8.1 Introduction Self Assessment Questions
8.4 Summary
8.2 Principle and Materials
8.3 Procedure for determining the
purity of nucleic acids
8.1 INTRODUCTION
In the previous experiment we have studied about qualitative analysis of
lipids by using various colour indication tests. In this experiment we’ll
study about spectrophotometric method that is based on Ultra Violet
(U.V.) light absorption method. This is a widely used method in
molecular biology research to determine the purity of DNA.
For better understanding of this experiment it is advised to go through

Units-13 &14 of course BBCCT-101. We all know that nucleic acids act
as genetic material and are made up of nitrogenous bases like purines
and pyrimidines. Due to the presence of conjugate structures these
bases show absorption maxima at 260nm. On the other side proteins
with aromatic amino acids shows absorption maxima at 280nm. That’s
why this method has gained significance in terms of both qualitative and
quantitative determination of nucleic acids.
In this method we are not going to observe any colour change either in
the substrates or in the products. But it is the ability of
spectrophotometer that makes it possible to read the changes in the
intensity of U.V. light. However, you’ll know more about this technique in
skill enhancement course (BBCS-183) i.e., Tools and techniques in
Biochemistry. However in this experiment we’ll be focusing more on
determining the purity of DNA. 59
..........................................................................................................................................................................

 explain the principle behind the determination of purity of nucleic

acids;
 identify specific absorption maxima of nucleic acids and proteins;
 determine the purity of nucleic acid; and
 know how to operate spectrophotometer.
8.2 PRINCIPLE AND MATERIALS

Principle: Presence of conjugate structures in the DNA shows absorption
maxima at 260nm. This ability of DNA is exploited and used in determining the
purity of DNA/RNA through U.V absorption spectrophotometric method.
The ratio between absorbance values at 260 nm and 280 nm
(OD260:OD280) provides an estimate of the purity of the DNA (Fig. 8.1).
Pure preparation of DNA and RNA have (OD260:OD280) ratio value 1.8
and 2.0 respectively. If there is any protein or phenol contamination in the
sample, ratio value will be less than 1.8 and 2.0. Due to this significance,
the ratio of absorption at 260 nm and 280 nm has been used as a
measure of purity of isolated nucleic acids.
Fig. 8.1: Absorption of U.V. light by DNA and protein.
Materials Required:
Glassware: Cuvette, 100 mL volumetric conical flask, Beaker, Pipette and

graduated measuring cylinder.
Equipment’s: U.V. Spectrophotometer
Chemicals: Commercial DNA sample, Ethylene diamine tetra acetic acid

(EDTA), Sodium phosphate, Sodium chloride, Tris-HCl
Preparation of DNA stock solution (100µg/ mL): Weigh 500mg of

commercially available DNA into a 100 mL volumetric flask and dissolve in
60 little volume of 20 mM sodium phosphate, pH 7.0 or 0.1 M sodium chloride
BBCCL-102 Determination of Nucleic Acid Purity by UV Absorption Method
..........................................................................................................................................................................
and make up the volume to 100 mL with 20 mM sodium phosphate, pH 7.0
or 0.1 M sodium chloride.
Preparation of DNA working standard solution (50µg/ mL): Dilute 1mL

of the stock solution to 100 mL, with 20 mM sodium phosphate, pH 7.0 or
0.1 M sodium chloride in a 100 mL volumetric flask.
Preparation of Tris-EDTA (TE) buffer:
i) Preparation of 1 M Tris- HCl: Weigh121.1 g of Tris base and

dissolve completely in 800 mL of distilled water. Allow the solution to
reach room temperature then adjust the pH to 8 by adding
concentrated HCl.
ii) Preparation of 0.5 EDTA: Dissolve 186.1 g of disodium EDTA to

800 mL of distilled water. Completely dissolve the EDTA on a
magnetic stirrer later adjust the pH to 8.0 with alkali (NaOH).
iii) Take 10 mL of 1M Tris-HCl (pH 8.0) solution and 2 mL of 0.5 M

EDTA solution, mix both the solutions and make up the volume to
1000 mL in a graduated measuring cylinder.
Note: DNA sample isolated from biological material can also be used
as test sample.
8.3 PROCEDURE FOR DETERMINING THE

PURITY OF NUCLEIC ACIDS
Perform the following steps to measure the purity of DNA in the given
sample:
I. Standardize the UV-Visible spectrophotometer with Tris-EDTA buffer as It is advised to go

blank at both 260 and 280 nm through the instruction
manual provided along
II. Dissolve the DNA sample in appropriate volume (either 1:50 or 1:100 mL) with the
of same buffer and measure the absorbance at both 260 and 280 nm. spectrophotometer prior
to start the experiment.
III. Calculate the concentration of DNA by using following formula
Purity of DNA= OD at 260 nm / OD at 280 nm
However for routine practice in the laboratory learners can use

commercially available DNA powder and prepare the DNA sample as
discussed in the above section 8.2.
Results:
A. Absorbance values of the DNA sample at 260and 280 nm using UV-
VIS Spectrophotometer are
A260 : ______ OD; A280 : _____ OD and the ratio of absorption is
OD at 260 nm / OD at 280 nm i.e, __________ .
61
..........................................................................................................................................................................
Standard reference values for various nucleic acid solutions with absorption
at 260 nm =1, the following approximations are valid:
1 A260 unit* of double stranded DNA = 50 µg/ mL
1 A260 unit* of single stranded DNA = 37 µg/ mL
1 A260 unit* of single stranded RNA = 40 µg/ mL
1 A260 unit* of oligonucleotide = 20 to 33 µg/ mL

*
Unit definition: The concentration of nucleic acid dissolved in 1 mL
buffer (20 mM sodium phosphate, pH 7.0 or 0.1 M NaCl), which has
an absorbance of 1 (at A260). The spectrophotometric measurement is
made in a 1 cm cuvette, at 20o C. (Source, reference 1: Experimental
Biochemistry)
1. What is EDTA?
2. At _________ wavelength protein show maximum absorbance?
3. Write the formula used to measure the purity of DNA.
PRECAUTIONS
1. Wore gloves to avoid contamination
2. Use separate pipettes to avoid contamination.
8.4 SUMMARY
 In this experiment we came to know that presence of conjugate
structures in the nucleic acids shows absorption maxima at 260nm.
This property is typically made use in determining the purity of
nucleic acids through U.V absorption spectrophotometric method.
 The ratio between absorbance values at 260 nm and 280 nm

(OD260:OD280) is helpful is assessing the purity of the
nucleic acid.
 Pure preparation of DNA and RNA have ratio (OD260:OD280) value

of 1.8 and 2.0 respectively. If there is any protein or phenol
contamination in the sample ratio value will be less than
62 1.8 and 2.0.
BBCCL-102 Determination of Nucleic Acid Purity by UV Absorption Method
..........................................................................................................................................................................
8.5 FURTHER READING

1. Experimental Biochemistry: A student Companion. Beedu Sashidhar Rao
2. Maniatis T et al. Molecular cloning - a laboratory manual, 1982.
3. Sambrook, J., & Green, R. M. (2012). Molecular cloning a laboratory

Manual. (4th ed.). New York: Cold Springer Harbor Laboratory Press.
4. Hoisington, D., Khairallah, M. & Gonzalez-de-Leon, D. (1994).

Laboratory Protocols: CIMMYT Applied Biotechnology Center. (2nd
ed.). Mexico, D.F.: CIMMYT.
5. Life Sciences Protocol Manual January 2018. Department of

Biotechnology, Ministry of Science and Technology, Govt. of India.
63
Molecules of Life
9
BBCCL-102
..........................................................................................................................................................................
Experiment
ESTIMATION OF
VITAMIN-C
(ASCORBIC ACID)
Structure
9.1 Introduction Self Assessment Questions
9.4 Summary
9.2 Principle and Materials
9.3 Procedure for Quantitative
estimation of Vitamin C
9.1 INTRODUCTION
In the previous experiment we have studied about qualitative estimation of
nucleic acids using U.V absorption method. In this experiment we’ll learn
about quantitative method based on titration. This method is widely used to
quantify concentration of vitamin C (ascorbic acid) in a given test sample.
We all know that vitamins are vital amines that play significant role in
biochemical reactions. Vitamins are of two different types like water soluble
and fat soluble. The vitamin we are discussing in this experiment falls under
water soluble group. Vitamin C abundantly found in citrus fruits like lemon,
orange etc. Due to its vast biological applications this vitamin has got both
medical commercial importance.
Hence the knowledge of quantifying this vitamin in biological sample plays

an important role in both academic as well as industrial point of view. The
best part of this method is less expensive and ease of doing. Since, we
are using a dye method to detect the end point there is no need of any
external instrument to measure the intensity of colour developed or
disappeared. For better understanding of this experiment it is advised to go
through section 12.3.2 of Unit-12 of course BBCCT-101.
64
BBCCL-102 Estimation of Vitamin C
..........................................................................................................................................................................

 explain the principle behind the quantifying vitamin C;
 identify specific reagents and their roles in the experiment;
 distinguish between qualitative and quantitative tests; and
 estimate the amount of vitamin C in a biological sample.
9.2 PRINCIPLE AND MATERIALS

Principle: In this reaction reduced form of vitamin C, donates its protons
(H+ ions) to get oxidized and reduces the dye. In brief vitamin C, present in
the given test sample reduces 2,6-dichlorophenol indophenol (DCIP), a blue
coloured dye to pale pink or colourless leuco form in acidic medium. The
appearance of pink colour indicates the end point of titration. In this
reaction vitamins C acts as a reducing agent and get converted to
dehydroascorbic acid (oxidised).
Reaction showing formation of colourless DCIP.
Materials Required:
Glassware: 100 mL Volumetric conical flask, Pipette, Burette, Burette

stand and 100 mL standard flask.
Preparation of Reagents:
DCIP Dye solution: Separately weigh 26 mg of the dye and 21 mg of sodium

bicarbonate powder. Transfer both of them into a 100 mL volumetric flask, dissolve
slowly and make up the volume to 100 mL with distilled water. Filter the reagent and
use (store in dark coloured glass bottle). 65
..........................................................................................................................................................................
Oxalic acid solution 4% (w/v): Weigh 4g of oxalic acid crystals and
dissolve in 100 mL of distilled water.
Vitamin C stock solution(1 mg/mL):Take 100 mg of vitamin C (Tablet),
and dissolve it with 4% oxalic acid in 100 mL volumetric flask. Later,
make up the final volume to 100 mL with oxalic acid solution.
Vitamin C working standard solution (0.1mg/mL): Take 10 mL of vitamin
C stock solution and makeup to 100 mL, with 4% oxalic acid in a 100 mL
volumetric flask.
Preparation of Test sample: Follow the steps as shown in (Fig. 9.1), in brief
take 10g of citrus fruit, juice into a 100 mL beaker. Transfer juice into 100 mL
volumetric flask and bring the final volume to 100 mL with 4% oxalic acid
solution. Dilute the citrus solution ten times with oxalic acid solution before
titration. 5 mL of diluted juice is used for titration and the titration
(refer section 9.3) isrepeated thrice and average value is obtained (V2 ).
10 g citrus fruit
Extract Juice
Into 100 mL beaker

Filter the extract (use glass wool)
Into 100
100 mL
mL volumetric
volumetricflask
flask
Make up to 100 mL with 4%

Oxalic acid solution (stock)
Dilution 1:10
Take 1 mL and add 9 mL of

4% oxalic acid solution
(working standard)
Take 5 mL to start against

titration DCIP dye
Fig. 9.1: Estimation of vitamin C by titration.
9.3 PROCEDURE FOR QUANTITATIVE

ESTIMATION OF VITAMIN-C
Transfer 5 mL of the vitamin C working standard solution into a 100 mL
conical flask. Later, add 10 mL of oxalic acid solution and mix well. Now
titrate the contents against the DCIP solution, taken in a burette. Add dye
solution drop by drop into the conical flask (Fig. 9.2) and mix the contents
thoroughly.The appearance of a pale pink colour from blue, indicates the
endpoint (appears for few minutes). Repeat the procedure thrice to
obtain an average value of dye consumed, as shown in the Table 9.2.
66
..........................................................................................................................................................................
Figure 9.2 Titration of Vitamin-C against DCIP dye
Table 9.1: Quantitative Estimation of Vitamin C
Sl. Volume of Volume of

Sample for Volume DCIP dye consumed
No. working oxalic acid
Titration (burette, mL)
standard solution
(mL) (mL) Initial Final (Initial- final)
1. Blank (5 mL Distilled water) ——— 10
2. Vit. C Standard
5 10
Titration 1
Titration 2 5 10
Titration 3 5 10
3. Test sample 5 mLof

diluted 10
Titration 1 juice
5 mL of
Titration 2 diluted juice 10
5 mLof
Titration 3 diluted juice 10
67
..........................................................................................................................................................................
Calculation: Vitamin C content (mg/100g sample) is calculated by the
following equation:
0.5 mg V1 mL 100mL 10

Vitamin C (mg / 100 g) =    100
V 2 mL 5 mL Weight of the sample (g)
Where,
V1 = Volume of the dye consumed for standard vitamin C (mL).
V2 = Avarage Volume of dye consumed for the sample (mL).
X 10 = Dilution factor.
Note: 10 g of citrus fruit is estimated to contain 0.5-1.0 mg of vitamin C
Results: The amount of vitamin C present in 100 grams of fruit is ______ mg
Sourece: Reference 1, Experimental Biochemistry
1. List the fruits rich in vitamin C
2. Name the dye used in the quantitative estimation of vitamin-C
3. Whether we are using any indicator in the titration of vitamin-C? Justify

your answer.
Precautions
Use clean and dry glassware, No need to add any indicator as DCIP dye
acts as self-indicator.
9.4 SUMMARY
 In this experiment we came to know how the simple oxidation and
reduction reaction can be used to estimate the quantity of vitamin C
 Conversion of blue coloured 2, 6-dichlorophenol indophenol into the

pink coloured product acts as indicator of end point.
 The formula used to express the concentration of Vitamin C in 100 g

sample.
 This experiment has got academic and industrial significance.
 This can be used to check the vitamin C content present in the

pharmaceutical products.
68
..........................................................................................................................................................................
9.5 FURTHER READING

Ltd.

2nd edition, Dr. G. Rajagopal and Dr.B.D. Toora. ISBN 81-901769-5-1,
3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III. ISBN 81-

7263-333-5, Published by Indira Gandhi National Open University, 1993
(Reprint December-2006).
4. Segel, I.H. Biochemical Calculations. 2nd ed. John Wiley &

Sons.Inc.New York (1976).

K.R. Aneja. ISBN 978-93-87025-49-3.MEDTECH a division of Scientific
6. Harris. L.J & Rays, S. N., Lancet, I, 71: 462.(1935).
7. Analytical Uses of 2,6-DCIP. Product Information Bulletine, J.T. Baker

Chemical Company, Commodity # H114, New Jersey, USA (1971).
69

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Indira Gandhi National Open University

School of Sciences BBCCL-102

Prof. Reena Gupta Prof. Sanjeev Puri Dr. Parvesh Bubber

Prof. K. Vali Pasha Dr. Arvind Kumar Shakya

Block Preparation Team

Course Coordinator: Dr. M. Abdul Kareem

Acknowledgement: Mr. Sumit Verma for Graphics and word processing.

In this course we have included 3 exercises & 6 experiments, among which

The broad objectives of this course are to enable you to:

l identify the routinely used glass ware and apparatus;

l impart maintenance and cleaning of biochemistry lab;

l preparation of normal, molar and percent solutions;

l perform titrations and plot graphs;

l know the basic principles behind qualitative and quantitative tests;

l distinguish different types of biomolecules in a given test solution; and

l determine the purity of a given nucleic acid in a solution.

Like all other IGNOU laboratory courses this is an intensive residential

 Attendance is compulsory in the Laboratory Course work held

 The Laboratory Course is worth 2 credits to be completed over

 6 days of Guided Laboratory work

 1 day for the Unguided Laboratory work

 To successfully complete the laboratory course you will have to

Expected Learning Outcomes

1.2 CLEANING AND MAINTENANCE OF

 the glass ware must be washed immediately after use

 soap solution, detergent or cleaning powder can be used for removing

 chromic acid or mixture of alcohol with solid potassium hydroxide can be

 usage of cleaning scrubber or abrasive can be minimized as they may

 it is essential to remove the traces of soap or detergent from glassware,

 avoid usage of cracked, scratched or chipped glassware

1.3 SAFETY MEASURES IN LABORATORY

 Smoking, eating and drinking of any kind of beverages is strictly prohibited

 Poisonous and corrosive reagents should never be pipetted by mouth; It

 Care and cleanliness must be practiced by all working in the laboratory at

 Wore lab shoes, working on bare legs should be avoided.

1.4 LABORATORY HAZARDS

Figure 1.2 shows various hazardous symbols used to represent types of

FLAMMABLE GAS RADIOACTIVE POISON

CORROSIVE BIOHAZARD IRRITANT

Fig. 1.2: Symbols used to represent various types of Hazards 9

 Laboratory hazards can be avoided through careful handling of chemicals

2. What are precautions to be taken while preparing NaOH solution?

3. Expand the acronym “SOP”.

4. Name any two organic solvents used in cleaning the glassware?

5. Identify the symbol used for biohazards from Fig. 1.2?

1.6 FURTHER READINGS

2. Practical Biochemistry: for medical, dental and allied courses. 2 nd edition,

3. Preparative Organic Chemistry CHE-08 (L), Chemistry Lab-III.ISBN 81-

2.2 Molar Solutions 2.5 Summary

2.3 Normal Solutions 2.6 Further Readings

Expected Learning Outcomes

 distinguish between molar, normal and percent solutions; and

 perform the preparation of molar, normal and percent solutions.

2.2 MOLAR SOLUTIONS

A molar solution consists of “gram molecular weight” (formula weight) of

Molarity is defined as “the number of moles of solute per litre (L) of

Weight of solute in grams

Let us understand this with a simple excercise of How to prepare 1 L of

Since molecular weight of NaOH is 40, dissolve 40 grams of NaOH in 1