Prepared By

Dr. Showkat Ahmad Bhat,

Assistant Professor,
Department of Biochemistry, Govt. Medical College Doda

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – I
Clinical Laboratory

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Responsibilities of health care personnel
 Laboratory hazards – Physical, Chemical and Biological.
 Laboratory safety measures – safety regulations and first aid in laboratory

Clinical Laboratory
A clinical laboratory or medical laboratory is a laboratory where tests are carried out on
clinical specimens to obtain information about the health of a patient to aid in diagnosis,
treatment, and prevention of disease or it means a facility where microbiological,
Biochemical, serological, hematological, immunohematological, immunological,
toxicological, cytogenetical, cytological, histological, pathological or other examinations
are performed on material derived from the human body, for the purpose of diagnosis,
prevention of disease or treatment of patients.

Different branches of clinical laboratory

 Clinical microbiology: This encompasses several different sciences, including
bacteriology, virology, parasitology, immunology and mycology.
 Clinical chemistry: This area typically includes automated analysis of blood
specimens, including tests related to enzymology, toxicology and endocrinology.
 Hematology: This area includes automated and manual analysis of blood cells. It
also often includes coagulation.
 Blood bank involves the testing of blood specimens in order to provide blood
transfusion and related services.
 Molecular diagnostics DNA testing may be done here, along with a subspecialty
known as cytogenetics.
 Reproductive biology testing is available in some laboratories, including Semen
analysis, Sperm bank and assisted reproductive technology.

Responsibilities of health care personnel

As a healthcare support worker, he should be responsible for assisting and caring for
patients in a fast-paced and dynamic clinical environment.
Responsibilities and Duties:
 Assist patients with basic hygiene activities
 Administer medication to patients
 Take vital signs and report findings to superiors
 Collect, store, and label biological specimens
 Sterilize medical equipment

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Keep hospital supplies properly stocked and organized
 Work closely with other healthcare professionals such as nurses, physicians, and
therapists in order to provide patients with exceptional care
 Dispose of waste and hazardous materials
 Comply with all hospital rules, regulations, and procedures
 Collect and enter patient data into electronic medical record portal
 Contact hospitalists and specialty providers to arrange consultations and
admissions
 Manage clinical staff scheduling
 Maintain adequate medical supply inventory through restocking and reordering

Laboratory hazards – Physical, Chemical and Biological.

The modern clinical laboratory is a workplace where many hazardous chemicals,
complex instrumentation, and potential pathogens are encountered on a daily basis.
However, the laboratory can be a safe place to work and learn if possible hazards are
identified and safety and infection control protocols are followed. Laboratories are
commonly used for scientific disciplines ranging from biology, chemistry, biochemistry,
physics, botany and zoology to medicine, psychology, dentistry, engineering,
agriculture and veterinary science.
We all have the responsibility to maintain a constant concern for safety in the
laboratory. Good personal habits, housekeeping practices, and laboratory technique can
all help ensure that the laboratory is a safe place to learn and work.

List of specimen, their type and Applications in laboratory

Specimen Specific type Characteristics Applications
(example)
Light latex, Disposable latex Powdered or Working with
vinyl or nitrile gloves Unpowered biological hazards
gloves (known or potentially
known infectious
materials including
work with animals)

Disposable nitrile Puncture, abrasion Working with

gloves resistant, protection biological hazards and
from splash hazards chemical splash
hazards

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Disposable vinyl Economical, durable, Working with
gloves similar to latex biological hazards

Light Natural rubber latex Chemical resistant, Working with small

chemical liquid-proof volumes of corrosive
resistant liquids, organic
gloves solvents, flammable
organic compounds

Light to Nitrile gloves Chemical resistant, Apparatus under

heavy good puncture, cut, pressure, air or water
chemical and abrasion reactive chemicals
resistant resistance
gloves
Heavy Butyl gloves High permeation Large volumes of
chemical resistance to most organic solvents, small
resistant chemicals to large volumes of
dangerous solvents,
gloves
acutely toxic or
hazardous materials
Viton® II gloves High permeation Same as butyl gloves,
resistance to most plus hazardous
chemicals material spills

Applicable Specific type Characteristics Applications

PPE (example)
(Personal
protective
equipment)
Heavy Butyl/Silver Shield Extra chemical and Same as butyl and
chemical gloves and apron mechanical Viton II gloves, added

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
resistant protection mechanical protection,
gloves hazardous material
spills

Insulated Cryogen gloves Water resistant or Cryogenic liquids

gloves water proof, handling
protection against
ultra-cold
temperatures
Applicable Specific type Characteristics Applications
PPE (example)
Lab Coats Knee length lab coat Protects skin and General use; Chemical,
clothing from dirt, Biological, Radiation,
inks, non hazardous and Physical Hazards
chemicals,
biohazards without
aerosol exposure
Flame resistant lab Flame resistant (e.g. Working with water or
coat Nomex or flame- air reactive chemicals,
resistant cotton) large volumes of
organic solvents,
potentially explosive
chemicals
Gowns Disposable gowns Clothing and skin Working with
protection biohazards

Tyvek gowns High tear resistance, Working with

protection from biohazards with
particulates potential for exposure
to airborne
transmissible disease
Cap Bouffant caps Economical Working with
protection for biohazards, especially
hygienic work in animal facilities
environments;
protection from dirt,

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 dust

Shoe Cover Disposable shoe Protection from dirt, Working with

covers dust; maintenance of biohazards, especially
hygienic work in animal facilities
environments.
Adjustable fit, non-
skid soles

Applicable Specific type Characteristics Applications

PPE (example)
Safety glasses Polycarbonate lens, Working with chemical,
side shields for eye biological, radiation,
protection; meets physical hazards;
laboratory work
ANSI and OSHA
specifications
Applicable Specific type Characteristics Applications
PPE (example)
Respirators Surgical masks Used for bacterial Working with live
filtration animals; working with
infectious material with
potential aerosol
exposure

N-95 Protects against Working with live

dusts, fumes, mists, animals or infectious
microorganisms materials with known
airborne transmissible
disease; dusty
environments

Full face Same as half- face, Working with live

but with greater animals or infectious
protection factor, and materials with known

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 greater protection of airborne transmissible
ey es and face; disease; dusty
depends on filter environments; chemical
cartridge used vapors; particulates

SYMBOLS
Flammable Poison Explosive Radioactive Corrosive Compressed
Gas

Explosive Oxidizer Low Level Flammable Corrosive Severe

Hazard Chronic
Hazard

Health Hazard (Blue)

Danger May be fatal on short exposure. Specialized
protective equipment required
Warning Corrosive or toxic. Avoid skin contact or
inhalation
Warning May be harmful if inhaled or absorbed
Caution May be irritating
No unusual hazard
Flammability (Red)
Danger Flammable gas or extremely flammable liquid
Warning Combustible liquid flash point below 100 °F
Caution Combustible liquid flash point of 100° to 200 °F
Combustible if heated
Not combustible

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Reactivity (Yellow)
Danger Explosive material at room temperature
Danger May be explosive if shocked, heated under
confinement or mixed with water
Warning Unstable or may react violently if mixed with
water
Caution May react if heated or mixed with water but
not violently
Stable Not reactive when mixed with water
Special Notice (White)
Water Reactive
OX Oxidizing Agent

The most common hazards and risks of the modern laboratory.

1. Chemical Hazards
2. Electrical Hazards
3. Biological Hazards
4. Physical Hazards

1. Chemical Hazards: Handling chemicals is a typical part of the day-to-day routine

for many lab workers, but the risks and hazards remain the same. Many organic and
inorganic chemicals are corrosive to the skin and to the eyes and can be toxic. Full
safety wear should be provided to any members of the team handling chemicals,
and provisions to treat any exposure or clean spillages should be present in the
laboratory.
It‘s not only direct contact which may be hazardous, chemical reactions which
generate heat can lead to thermal burns. This further demonstrates the importance
of ensuring the surface of the skin is protected from the potential for burns and
exposure.
Similarly, incorrect venting within the laboratory could be hazardous. Without full
and correct ventilation, a distillation or chemical reaction could lead to an explosion
in the lab. Depending upon the size of the explosion and the materials affected, this
could be hugely dangerous for the team and for the lab.
Inhalation of certain chemicals can be dangerous, with many of the most
common solvents proving to be extremely toxic. These dangers can be immediate or
slowly manifest over time – making it important that the research team are protected
from the fumes produced by these hazardous chemicals.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
2. Electrical Hazards: Even the most experienced research professional can overlook
basic safety principles when working with electricity – so it is vital that protective
measures are employed throughout the laboratory. Particularly hazardous are
electrical units which are positioned close to liquid, so these should be fitted with
ground-fault circuit interrupters to break the circuit should any current flow to
ground.
Ingesting chemicals is a huge risk in many laboratories, due to contamination on
hands, food and drink. This demonstrates the importance of safe and secure storage
for all food and drink items, away from chemical exposure. Furthermore,
comprehensive hand-washing and sanitation provisions should be accessible for all
members of the research team exposed to hazardous chemicals.

Prevention: When it comes to chemical hazards, effective prevention is the best way
to manage the risks of working with these dangerous substances. Practising proper
chemical segregation is essential in all labs, as some substances can react with each
other to create chemical reactions, fires and even explosions. Protective clothing and
good housekeeping are also important for protecting your team from chemical
hazards.
Watch the video below for more information on how to minimise chemical hazards
in your lab and what to do in an emergency.
Electrical fires are another common laboratory hazard, which can occur when
incorrect or unsafe cords and plugs are used. Any electrical appliances used in the
laboratory should be fit for purpose, up-to-date and correspond to connected
devices before they are implemented. Any electrical apparatus, from adaptors to
cables, not safety-tested could compromise the safety of the lab and research team.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Prevention: Electrical hazards can easily be overlooked in labs, which tend to have
more health and safety risks than in other workplaces. Electrical hazards are
potentially life threatening, however, so minimizing their risk is vital. All power
outlets that could be exposed to wet conditions should be equipped with ground-
fault circuit interrupters. Flexible extension cords should also be well maintained
and never used as a substitute for permanent wiring. Electrical pendants can be used
where possible to keep cords out of the way.
3. Biological Hazards: The use of bacteria, viruses, blood, tissue and/or bodily fluids
in the lab can lead to potential biological hazards. These materials can all carry
disease or hazardous allergens which could put the lab team at risk. The effects of
the diseases and allergens can be immediate or take significant time to manifest,
demonstrating the importance that all members of the lab team are given sufficient
protection, even if the dangers are not yet known.
Diseases carried by humans and animals used in research can be transmitted by
the team, who then may become carriers. This means that biological hazards could
prove to be a massive risk for not only the lab professionals working with the
materials, but anyone they come into contact with outside of work. Sometimes
incredibly infectious, biological hazards (biohazards) can be amongst the biggest
risks of the modern research lab, so every consideration must be made to ensure the
team and the wider public are protected against contagious materials.

Prevention: Proper storage and protection is key to preventing a biological

emergency in your lab. Wearing appropriate protective clothing and keeping
biological agents contained in the correct areas are essential for minimising exposure
to risk. Systems and procedures for safe use, handling, storage and transport of
biological hazards should all be in place. Appropriate housekeeping, such as
disinfecting work surfaces and properly disposing of waste, are also vital for
minimising biological risk.
4. Physical Hazards: And with so many unique risks at play in the modern lab, it can
be easy to overlook the more commonplace, physical risks. Trip hazards and
mishandling mistakes are rife in busy, bustling labs.
Handling is one of the major concerns for all lab managers, with members of the
research team susceptible to injury if not following safe handling requirements. Hot,
heavy and sharp apparatus can all compromise the health and welfare of members

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 of the research team. This makes it important that full and correct handling
equipment such as safety gloves is provided. Furthermore, training for correct lifting
should be conducted so the whole team can lift and carry without fear of injury.
Slips, trips and falls are more likely to occur in the laboratory than many other
workplaces due to the amount of time researchers spend on their feet and the
volume of different materials present. Due care and diligence must be paid by every
member of the team to reduce the presence of slip and trip hazards – to protect
themselves and other members of the team. All essential and non-essential items
which are stored in the laboratory must have a sufficient storage space, keeping
them well out of the team‘s way.
And finally, perhaps the most common of all hazards and risks in the science lab
is the humble glass tube. Many an experienced lab professional has cut their finger
or hand when forcing a rubber stopper into a glass tube. Whilst this will, perhaps,
always occur – the risk can be reduced with continued encouragement of correct
stopper replacement, using gentle pressure whilst rotating the glass tube.

Prevention: Preventing physical risk to your team in the lab can often be achieved
by effective training and good housekeeping. Staff should be trained in the proper
procedures for lifting, pulling and pushing, as well as the dangers of repetitive
movements, and the handling requirements for different equipment. Proper
housekeeping is essential for preventing slips, trips and falls in the lab, so any
potential hazards should be quickly disposed of or tidied away. Also, a safety policy
can help identify and protect your research team from any kind of potential hazard.

Laboratory safety measures – safety regulations and first aid in

laboratory
Safe Laboratory Practices & Procedures
Students and laboratory staff working in any medical institute or hospital depending
upon the nature of their work they perform are exposed to diverse real or potential
hazards. Common hazards in the medical laboratory include biological, chemical,

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
physical, and radiological, flammable liquids, toxic vapors, compressed gases, electric
shock, corrosive substances, mechanical trauma, poisons, and the intrinsic risks of
handling biological materials. Work force of laboratory/medical institution must be
―safety conscious‖ at all times.
Safety guidelines should be properly followed in order to make laboratory a safe
working place and prevent accidents and mishaps.

Laboratory Safety Guidelines

To begin, with the recognition of hazards, safety is achieved via the application of
common sense. A safety-focused attitude, good personal behavior, good housekeeping
in all laboratory work and storage areas, above all, the continual practice of good
laboratory technique.
If anyone is splashed by any of these materials, use running water from an eyewash
station or emergency shower for at least 15 minutes or until emergency assistance
arrives and provides with different instructions.
a) Report to supervisor/lab. incharge if any any accident, injury, or uncontrolled
release of potentially hazardous materials occurs, no matter how trivial the
accident, injury, or release may appear.
b) Attend all required laboratory safety training prior to the start of
practical/research assignment.
c) Read all procedures and associated safety information prior to the start of lab.
experiment.
d) Perform only those experiments authorized by lab. Incharge/supervisor.
e) Follow all written and verbal instructions. Ask for assistance if you need guidance
or help.
f) Work under direct supervision at all times. Never work alone in the laboratory.
g) Know the locations and operating procedures for all safety equipment. This
includes the eyewash station and safety shower.
h) Know the locations of the nearest fire alarms and at least two ways out of the
building. Never use an elevator in emergencies.
i) Be alert and proceed with caution at all times in the laboratory. Immediately
notify the supervisor of any unsafe conditions.
j) Know the proper emergency response procedures for accidents or injuries in the
laboratory.

Prevent potential exposure by safe laboratory practices are as follows

a) Conduct yourself in a responsible and professional manner at all times. No
pranks. No practical jokes.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 b) Dress for work in the laboratory. Wear clothing and shoes that cover exposed
skin and protect from potential splashes. Tie back long hair, jewelry, or
anything that may catch in equipment.
c) Never eat food, drink beverages, chew gum, apply cosmetics (including lip
balm), or handle contact lenses in the laboratory.
d) Use a chemical fume hood or biosafety cabinet, as directed by supervisor.
e) Observe good housekeeping - keep aisles clear.
f) Report damaged electrical equipment to the supervisor. Do not use damaged
electrical equipment.
g) Do not leave active experiments unattended. Never leave anything that is
being heated or is visibly reacting unattended.
h) Practice good personal hygiene. Wash the hands after removing gloves, before
leaving the laboratory, and after handling a potentially hazardous material.
i) While working in the laboratory, wear personal protective equipment - eye
protection, gloves and laboratory coat.
j) Properly segregate and dispose of all laboratory waste.
k) Before beginning any new or modified procedures determine the potential
physical, chemical and biological hazards and their appropriate safety
precautions.
l) Familiarize with the emergency procedures, alarms and evacuation routes.
m) Location of emergency phone, emergency eye wash, safety showers and fire
extinguishers with proper operating procedures should be known.
n) Do not smoke, apply makeup and consume food or beverages in laboratories.
Never store food or drink in laboratory refrigerators.
o) Wear protective clothing and gloves that are not permeable to the chemicals
being used.
p) Long hair and loose clothing should be confined when in the laboratory. Shoes
must be worn at all times.
q) Sandals or open toe shoes must not be worn in the laboratory.
r) All containers of chemicals should be correctly and clearly labelled.
s) The label should provide hazard and safety information about the chemicals.
t) All chemical wastes should be disposed of appropriately to the corresponding
waste containers.
u) Mouth pipette of chemicals must not be allowed. A pipette bulb, aspirator for
pipetting chemicals should be used.
v) Exposure to gases vapors and aerosols should be minimized. Appropriate
safety equipment in conjunction with fume cupboard should be used
whenever such exposure is expected.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 w) Cell phones should not be operated at any time within a laboratory. They
might cause disturbance to other laboratory users, and also cause signal
interference.
x) The warning placard in lab includes a list of emergency contact persons. In
case of any mishap, assistance from the people on that list may be informed.
y) Students need to abide by these regulations. This is necessary to keep order in
the laboratory

Common Hazards faced during working with Acids & bases

Acids and bases can provoke skin, mucous membrane and eye burns. While
using these viz. (NaOH, KOH, H2SO4, HNO3, trichloroacetic acid etc), following rules
should be adhered to:
a) Toxic materials should be clearly labeled with special tape when used in
reagents and stored in separate containers.
b) In case of spillage, wash all exposed human tissue (including eyes) generously
with water and laboratory supervisor should be informed for proper reporting
of the incident.
c) Do not taste or ingest any laboratory chemical.
d) Avoid touching chemicals with bare hands and always thoroughly wash hands
after their use.
e) Work in a fume hood when carrying out reactions that give off objectionable
gases. If there is need to smell vapor, nose should not be directly above a flask,
beaker, or other vessel containing chemicals, instead, hold the vessel at least
one foot away and cautiously fan the vapors toward nose.
f) While pipetting acids, use mechanical pipettors, pipette aids or rubber bulbs
provided in the laboratory.
g) Always add acids to water (its alphabetical!); never add water to acids.
Combining acid and water frequently generates heat; addition of the acid to the
water reduces the amount of heat generated at the point of mixing and
provides more water to disperse the heat.
h) Label all flasks, beakers, test tubes, and other vessels containing chemicals
according to their contents. This facilitates both identifying chemicals during
an experiment and following proper waste disposal procedures.
i) Laboratory wastes and residues are to be disposed of in an approved manner
as per standard disposal procedures.

Common Hazards faced during working with Electric equipment and apparatus

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Be careful when working with electric equipment to avoid an electric shock. Following
rules should be pursued:
(a) Do not touch the naked electric wires.
(b) Do not work with unearthed apparatus.
(c) Do not pull out an electric wire, during switch off an electric apparatus
from the electric network. Do it only with an electric plug.
(d) Do not touch a water pipe, a tap, and a heating radiator during working
with electric apparatus.
(e) Extension cord use is prohibited.
(f) All equipment has to be properly grounded.
(g) Never operate electrical equipment with wet hands or if spilled with fluid.
(h) Never use plugs with exposed or frayed wires.
(i) If there are unusual sparks or smoke shut down the instrument and
inform the lab staff.
(j) Electrical equipment that is not working properly should not be used.
(k) If a person is shocked by electricity, shut off the current or break contact
with the live wire immediately and don‘t touch the victim while he is in
contact with the source unless you are completely insulated against shock.

Common Hazards faced during working with compressed gases

a. In the laboratory, gas containers should be limited to the number of
containers in use at any time.
b. Low pressure gases shall also be limited to the smallest size container.
c. Containers shall be firmly strapped, chained or secured in a cylinder stand
so they cannot fall.
d. Oxidizing gases should be separated from the flammable ones.

Fire Extinguishers
Fire is mainly a chemical reaction involving the rapid oxidation of fuel or any
other combustible material wherein heat and light are liberated. Elements necessary for
the initiation of fire are already present in any clinical laboratory which includes oxygen
(air), fuel and heat or ignition source. Recent research has suggested the presence of
fourth factor that has been classified as a chain reaction in which burning is continued
or accelerated & is caused by the breakdown and recombination of the molecules from
the material burning with the oxygen in the atmosphere. This has resulted in the
modification of the previous fire triangle into a pyramid known as ―Fire tetrahedron‖
which does not abolish the established procedures in dealing with fire, instead

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
additional ways to prevent or extinguish fire have been provided by this. A fire will
extinguish by the removal of any three basic elements (heat, air or fuel).

Classification of Fires
Depending on the nature of the combustible material and requirements for
extinguishing fires have been classified into:
 Class A: Ordinary combustible solid materials like- paper, wood, plastic, fabric
 Class B: Flammable liquids/gases & combustible petroleum products
 Class C: Energized electrical equipment (table BI 11.1.A)
 Class D: Combustible/reactive metals like- magnesium, sodium, potassium

Like fires, fire extinguishers are classified depending on the type of fire to be
extinguished. Use of correct fire extinguisher is very important. e.g water should not be
used on burning liquids or electrical equipment. Pressurized-water extinguishers, as
well as foam and multipurpose dry-chemical types, are used for Class A fires.
Multipurpose dry-chemical and carbon dioxide extinguishers are used for Class B and
C fires. Halogenated hydrocarbon extinguishers are particularly recommended for use
with computer equipment. Class D fires present special problems, and extinguishment
is left to trained firefighters using special dry-chemical extinguishers.
In the event of a fire, all personnel, patients, and students who are in immediate
danger have to be evacuated first and then activate the fire alarm, report the fire, and
attempt to extinguish the fire, if possible. Personnel should work as a team to carry out
emergency procedures. Fire drills must be conducted regularly and with appropriate
documentation.

Table BI 11.1.A: Class of Fires and Remedies

Class of Fire Type of Extinguisher Operation
Class A Fires A /ABC Pull pin
Pressurized water/Dry chemical
Class B Fires ABC/BC Aim nozzle
Dry chemical/Carbon dioxide
Class C Fires BC/Halon/ABC Squeeze trigger
Carbon dioxide/Halon/Dry chemical Sweep nozzle
Class D Fires Metal X Cover burning material with
extinguishing agent (scoop,
sprinkle)
General lab safety rules
The following are rules that relate to almost every laboratory and should be included in
most safety policies. They cover what you should know in the event of an emergency,

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
proper signage, safety equipment, safely using laboratory equipment, and basic
common-sense rules.
1. Be sure to read all fire alarm and safety signs and follow the instructions in the
event of an accident or emergency.
2. Ensure you are fully aware of your facility's/building's evacuation procedures.
3. Make sure you know where your lab's safety equipment—including first aid
kit(s), fire extinguishers, eye wash stations, and safety showers—is located and
how to properly use it.
4. Know emergency phone numbers to use to call for help in case of an emergency.
5. Lab areas containing carcinogens, radioisotopes, biohazards, and lasers should
be properly marked with the appropriate warning signs.
6. Open flames should never be used in the laboratory unless you have permission
from a qualified supervisor.
7. Make sure you are aware of where your lab's exits and fire alarms are located.
8. An area of 36" diameter must be kept clear at all times around all fire sprinkler
heads.
9. If there is a fire drill, be sure to turn off all electrical equipment and close all
containers.
10. Always work in properly-ventilated areas.
11. Do not chew gum, drink, or eat while working in the lab.
12. Laboratory glassware should never be utilized as food or beverage containers.
13. Each time you use glassware, be sure to check it for chips and cracks. Notify your
lab supervisor of any damaged glassware so it can be properly disposed of.
14. Never use lab equipment that you are not approved or trained by your
supervisor to operate.
15. If an instrument or piece of equipment fails during use, or isn't operating
properly, report the issue to a technician right away. Never try to repair an
equipment problem on your own.
16. If you are the last person to leave the lab, make sure to lock all the doors and turn
off all ignition sources.
17. Do not work alone in the lab.
18. Never leave an ongoing experiment unattended.
19. Never lift any glassware, solutions, or other types of apparatus above eye level.
20. Never smell or taste chemicals.
21. Do not pipette by mouth.
22. Make sure you always follow the proper procedures for disposing lab waste.
23. Report all injuries, accidents, and broken equipment or glass right away, even if
the incident seems small or unimportant.
24. If you have been injured, yell out immediately and as loud as you can to ensure
you get help.
25. In the event of a chemical splashing into your eye(s) or on your skin,
immediately flush the affected area(s) with running water for at least 20 minutes.
26. If you notice any unsafe conditions in the lab, let your supervisor know as soon
as possible.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – II
Laboratory apparatus: Different types, use and maintenance

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Glass ware in laboratory – significance of boro-silicate glass.
 Plastic ware in laboratory, cleaning of glass and plastic ware.
 Pipettes – glass and automated
 Burettes, beakers, Petri dishes, porocelain dish
 Flasks – different types (volumetric, round bottomed, Erlenmeyer, conical etc.
 Funnels – different types (conical, Buchner etc)
 Bottles – reagent, wash bottles
 Measuring cylinders, reagent dispensers
 Tubes – test tube, centrifuge tube, folin-wu tube
 Cuvettes and its use in measurements, Cuvettes for visible and UV range
 Racks – bottle, testube, pipette and draining racks
 Tripod stand, wire gauze, Bunsen burner, desiccators, stop watch, timers

Glass ware in laboratory

Commonly used glass ware in laboratory
Name Use Glass ware/Apparatus/
equipments
Beaker Beaker are used to hold, mix, and
heat liquids

Buret Burets are used for dispensing an

accurate volume of a liquid.

Conical Flask Conical Flasks are used to hold

and mix chemicals. The small neck
is to facilitate mixing without
spilling.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Graduated Graduated Cylinders are used to
Cylinder measure a precise volume of a
liquid.

Mortar and Pestle Mortar and Pestle is used to crush

and grind materials.

Test Tube Test Tubes are used to hold and

mix liquids.

Decicator Desiccators are sealable enclosures

containing desiccants used for
preserving moisture-sensitive
items such as cobalt chloride paper
for another use. A common use for
desiccators is to protect chemicals
which are hygroscopic or which
react with water from humidity.

Volumetric Flask Used to prepare solutions to an

accurate volume

Volumetric Pipet Used to measure small amounts of

liquid very accurately. Never pipet
by mouth! Use pipetting aids.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Wash Bottle Used to rinse pieces of glassware
and to add small quantities of
water.

Watch Glass Used to hold solids while they are

being weighed or to cover a
beaker.

Glass slide and Glass slide and cover slips are

cover slips used in microscopy, serology, etc.
as the solid backing on which test
samples are taken

Petridish Petridishs are used for preparation

of culture media and the culture of
organisms they are in

Pasteur pipette/ Pasteur pipette are used for

Droppers aspiration and addition of reagents

Column Column Chromatography is a

Chromatography preparative technique used to
purify compounds depending on
their polarity or hydrophobicity. In
column chromatography, a
mixture of molecules is separated
based on their differentials
partitioning between a mobile
phase and a stationary phase

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Paper Paper chromatography is used as a
chromatography qualitative analytical chemistry
technique for identifying and
separating colored mixtures like
pigments. It is used in scientific
studies to identify unknown
organic and inorganic compounds
from a mixture.

Urinometer The urinometer, a type of

hydrometer, was used for
measuring the specific gravity of
urine. 'Specific gravity' is a
function of the number, density
and weight of the solute particles
present in the urine, and is used as
a measure of the concentrating
power of the kidney
Double Double glass distillation unit
distillation water consists of flak with heating
plant elements which has been
(glass/steel) embedded in fine glass. This
Double distillation is used for
carrying out reactions under
stirred conditions along with the
furnishing for reflux distillation.

Significance of boro-silicate glass.

Borosilicate glass is a type of glass that contains boron trioxide which allows for a very
low coefficient of thermal expansion. This means it will not crack under extreme
temperature changes like regular glass. Its durability has made the glass of choice for
high-end laboratories.
Borosilicate glass is made up of about 15% boron trioxide, which is that magical
ingredient that completely changes the behavior of glass and makes it thermal shock
resistant. This allows the glass to resist extreme changes in temperature and is
measured by the ―Coefficient of Thermal Expansion,‖ the rate at which the glass
expands when it is exposed to heat. Thanks to this, borosilicate glass has the ability to
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
go straight from a freezer to an oven rack without cracking. For you, this means you can
pour boiling hot water into borosilicate glass if you wanted to say, steep tea or coffee,
without worrying about shattering or cracking the glass.

Applications of Borosilicate Glass

 Laboratory glassware
 Scientific lenses and hot mirrors
 Bake ware and cookware
 Thermal insulation
 High-intensity lighting products
 Sight glass
 Aircraft exterior lenses
 Aquarium heaters
 Electronics (distillation water plant)
 Rapid prototyping

The high dimensional stability and ability to tolerate exposure to different

temperatures at the same time make borosilicate glass a natural material choice from
which to create laboratory glassware, also called lab ware. Petri dishes, microscope
slides, bottles, beakers, flasks, test tubes, funnels, and measuring instruments such as
graduated cylinders are all common examples. Besides the favorable thermal
properties, borosilicate glass is very resistant and non-reactive to most chemicals.

Plastic ware in laboratory, cleaning of glass and plastic ware.

Commonly used plastic ware in laboratory
Name Use Glass ware/Apparatus/
equipments
Beaker Beaker are used to hold, mix,
and heat liquids

Conical Flask Conical Flasks are used to hold

and mix chemicals. The small
neck is to facilitate mixing
without spilling.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Graduated Graduated Cylinders are used to
Cylinder measure a precise volume of a
liquid.

Spatula Spatulas are used to transfer

solids.

Test Tube Rack Test Tube Racks are used to hold

several test tubes at one time.

Volumetric Used to prepare solutions to an

Flask accurate volume

Wash Bottle Used to rinse pieces of glassware

and to add small quantities of
water.

Pasteur Pasteur pipette are used for

pipette/ aspiration and addition of
Droppers reagents

Cleaning laboratory glassware is important because contaminated or dirty glassware

can lead to inaccurate results in the lab. A good way to confirm that the glassware is
clean is to make sure that distilled water uniformly wets the surface because the
surfaces are having dust, grease and other contaminants that could alter the volume
being measured or introduce impurities into the glass ware which can give wrong
results. Laboratory cleaning procedures require exact methods to insure excellent lab

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
results. In all instances labware should be physically clean, including both chemical
residue free and grease free and in many cases even be sterile. Grease or residues will
not only contaminate the reaction and test results but will also alter the measurement of
the liquids used during test procedure.

Proper Procedure for Cleaning Laboratory Glassware

One basic procedure is to start with the gentlest methods, scraping off any solids and
then using brushes and normal soaps and detergents. If this doesn't get the job done,
move on to longer soaks and harsher cleaners. Finally, when the glassware is fully
clean, rinse it thoroughly and allow it to dry.

General Precautions
1. Careful handling and storage should be used to avoid damaging glassware.
2. Inspect the glassware before each use and discard if scratched on inner surfaces,
chipped, cracked or damaged in any way.
3. Use only plastic core brushes that have soft non-abrasive bristles or soft, clean
sponges/rags. Use brushes to clean inside of deep glassware.
4. Do not reach inside of glassware while cleaning to prevent cuts should the
glassware break.
5. Rubber sink and counter mats can help reduce the chance of breakage and
resultant injury.
6. Do not overload sinks or soaking bins.
7. Do not place metal or other hard objects, such as spatulas, glass stirring rods, or
brushes with metal parts, inside the glassware. This will scratch the glass and
cause eventual breakage and injury.
8. Never use strong alkaline products and hydrofluoric acid as cleaning agents.
These materials dissolve glass, leading to damage and eventual breakage.
9. Do not use any abrasive cleansers, including soft cleansers (e.g., Ajax, Comet,
Old Dutch, Soft Scrub, etc.), as these will scratch the glass and cause eventual
breakage and possible injury. Scotch Brite and similar scouring pads will scratch
glass and should not be used.
10. Do not use heat as a method to remove carbon residues. Heating glassware to
temperatures >800°F will cause permanent stresses in the glass and eventual
breakage.
11. Use proper drying racks for fully cleaned glassware.
12. Tongs, a dust pan, and a broom are the best tools for cleaning up broken glass. If
hands are used to pick up glass, only handle large pieces of glass and wear heavy
leather gloves to protect the hands. Broken glass must be packaged in labeled,
rigid, and sealed containers before disposal.
13. Proper instruction should be provided in the use of glass equipment designed for
specialized tasks, which can represent unusual risks for the first-time user.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Cleaning laboratory plastic ware depends on the type and properties of the plastic.

Temperature in the form of extreme heat or cold affects flexibility and strength.
Chemicals such as lubricants and oil cause cracking, and prolonged use of oxidizing
agents cause brittleness and breakage. Laboratory ware made with glass, quartz,
polyethylene and polypropylene are subject to interaction between container and
sample, or with reagents and standards and can give incorrect results. However,
polyolefins and fluorinated hydrocarbons have excellent resistance to high
temperatures and chemical attack. They have wettable surfaces and are easy to clean.

Cleaning methods
Acid bath: Immerse plastic ware into a 1M nitric acid and allow soaking overnight for
mild contamination. Keep in bath for about one week for heavy contamination. The
cleaned plastic ware is then taken out off the bath and rinsed with distilled water and
put to dry. A rinse with acetone or placement in glassware dryer at low temperature
can be used.
AUTOCLAVE
Certain chemical contaminants on plastic ware can be baked onto the plastic at
autoclave temperatures. Rinse thoroughly with distilled water before autoclaving.
Autoclave within the tolerated temperature range of the plastic being sterilized.
Remove any stoppers, caps or fittings before autoclaving.
Plastic containers such as vials, sample tubes and bottles should be autoclaved with
their closures disengaged to avoid deformation.
Note: Nylon, polyurethane, polystyrene, polyvinyl chloride (PVC), Acrylic, LPDE and
HPDE must not be autoclaved under any condition.

Pipettes (glass and automated)

A Pipette and micropipette (sometimes spelled pipet) is a laboratory tool commonly
used in different laboratories to measure and deliver accurate volumes of liquid. The
difference between the two is that micropipettes measure a much smaller volume,
starting at 1 microliter, while pipettes generally start at 1 milliliter.
Glass pipettes are excellent in chemical resistance. Since dry heat sterilization is
possible, it can be used repeatedly by washing and sterilizing depending on the
contamination. Pipettes are generally used for moving small amounts of liquid or when
measuring and dispensing liquid in mL units. When measuring less than 1 mL,
Micropipettes are more accurate and user-friendly type. Plastic pipettes such as
polystyrene are basically disposable, so there is no cleaning time involved and pre-
sterilized items are convenient in preventing

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Pipette-aides or pipettors are suction devices that are used to either suck liquids into or
expel liquids out of pipettes. For some types of measurements it may be necessary to
expel, or blow-out, the total liquid volume from the pipette using the pipette-aid like
teets, triple valve Electronic pipette aid that allows fine control and ease of use.

Serological or 'blow-out' pipettes are calibrated so that the last drop of liquid needs to
be blown-out of the tip to deliver the full volume of the pipette.

A pipette is calibrated with a series of graduation lines to allow the measurement of

more than one volume. Measure using the bottom of the concave surface of the liquid in
pipette. This figure illustrates how to read the meniscus on a measuring pipette.

The Procedure for glass Pipette:

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 1. Check that the pipette is calibrated and is it a 'blow-out' or blow in pipette. Also
make sure that the tip is not cracked.
2. Fill the pipette a bit above the capacity line desired and then slowly lower the
meniscus to required capacity line by pipette aid especially for acids.
3. Aseptically move the pipette to the receiving tube or any other glass ware and
deliver the contents, blow out the volume if blow out pipette is in use.
4. Remove the pipette aseptically and discard it into an appropriate discard
container.

The bands at the top indicate that this pipette is to be

"blown-out."

Mohr pipettes are another type of pipette. Mohr

pipettes are not 'blow-out' type, nor are the tips
part of the measurement. These types of
pipettes are only used to measure using a point-
to-point delivery system.
Mohr pipettes aren't commonly used in
microbiology applications.

Micropipettes & Autopipettes

Micropipettes are the standard laboratory equipment used to measure and transfer
small volumes of liquids.
A. Parts of a micropipette
 Plunger button
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Tip ejector button
 Volume adjustment dial
 Digital volume indicator
 Shaft
 Attachment point for disposable

Figure BI 11.19.11: Semi Automatic and Fully Automatic analyzers

B. Three sizes of micropipettes

The micropipettes in laboratories are of three different sizes each of which measures a
different range of volumes. The three sizes are P20, P200 and P1000. These sizes are
noted on the top of the plunger button.
S. Size Micropipette Range of volumes measured
No
1. P20 0.5-20µl
2. P200 20-200µl
3. P1000 100-1000µl

C. Adjusting Volume on micropipettes

The volume adjustment dial near the top of the micropipette allows adjusting the
volume that is measured. It can be dialed to the left or right to increase or decrease the
volume. The digital readout shows the volume that will be measured. By turning the

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
volume adjustment dial, the numbers in the digital readout will change. On each of the
three sizes of micropipettes (P20, P200,
 P1000) the digital readout has three numbers. These three numbers correspond to
different volumes on the different size pipettes.
 In a P100, the top number refers to 1000‘s of µl, the middle number refers to
100‘sµl and the bottom number refers to 10‘s of µl‘s.
 In a P200, the top number refers to 100‘s of µl, the middle number refers to 10‘s µl
and the bottom number refers to µ‘ls.
 In a P20, the top number refers to 10‘s of µl, the middle number refers to µl‘s and
the bottom number refers to 1/10ths of µl.

D. Pipette Tips
Liquids are never drawn directly into the shaft of the pipette. Instead, disposable plastic
tips are attached to the shaft. There are two sizes of tips. The larger blue tips are used
for the P1000 and the smaller clear tips are used for the P20 and P200.
The tips are racked in plastic boxes with covers. After receiving a box, it will be sterile.
Please be careful when touching box or tips not to contaminate them. The box should be
closed when not in use to prevent air born contamination.
Inserting the Tip
1. Select the correct size tips.
2. Open the box without touching the tips with the hands.
3. Insert the micropipette shaft into the tip and press down firmly. This will attach
the tip to the shaft.
4. Remove the micropipettor with the tip attached.
5. Close the box without touching the tips with the hands.

E. Punger Settings
The plunger will stop at two different positions when it is depressed. The first of these
stopping points is the point of initial resistance and is the level of depression that will
result in the desired volume of solution being transferred. The second stopping point is

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
when the plunger is depressed beyond the initial resistance until it is in contact with the
body of the pipettor. At this point, the plunger cannot be depressed further. This second
stopping point is only used for the complete discharging of solutions from the plastic
tip.

F. Measuring and transferring a volume of liquid

Before measuring and transferring liquid:
 Choose the appropriate size micropipettor
 Adjust to the correct volume Insert tip on the shaft.
Measuring and transferring liquid
 Depress the thumb knob to the first stop.
 Immerse the tip approximately 3 mm into the sample solution.
 Slowly release the thumb knob to the initial position. Watch as the solution is
drawn up slowly into the tip. Do not release the plunger too quickly. Rapid
release might draw bubbles in the solution and might splash solution on the non-
sterile shaft.
 Withdraw the tip from the sample solution. Place the tip against the side wall of
the receiving container.
 Smoothly depress the thumb knob to the first stop, pause and then depress the
knob to the second stop.
 Remove the tip from the receiving container, and return knob to the initial
position. Do not let the knob snap back.
 Remove the disposable tip by firmly depressing the tip ejector knob.
 Add as new tip and continue.

Burettes
A burette (also buret) is made of glass or plastic and is a straight graduated glass tube
with a tap (stopcock) at one end, for delivering known volumes of a liquid, especially in
titrations. It is a long, graduated glass tube, with a stopcock at its lower end and a
tapered capillary tube at the stopcock's outlet. The flow of liquid from the tube to the
burette tip is controlled by the stopcock valve. There are two main types of burette; the
volumetric burette and Piston burette or Digital burette. Burettes are manufactured
for specific tolerances, designated as class A or B and this also is etched on the glass.
In order to measure the amount of solution added in or drained out, the burette
must be observed at eye level straight to the bottom of the meniscus. The liquid in the
burette should be completely free of bubbles to ensure accurate measurements. The
difference in volume can be calculated by taking the difference of the final and initial
recorded volume. Using the burette with a colorless solution may make it difficult to

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
observe the bottom of the meniscus, so the black strip technique can make it easier to
accurately observe and record measurements.

Use of a Burette
a) Rinse the burette with the standard solution to be used, and align burette tube
vertically.
b) Fill the burette slightly above the zero mark. To prime the stopcock, drain the
burette no further than the nominal capacity.
c) Refill the burette with titrant free of air bubbles to approx. 5 mm above the zero
mark.
d) Drain liquid to set the zero point accurately. Important: Meniscus must be read at
eye level (parallax-free level). Automatic burettes: Fill to approximately 5 mm
above the zero mark, this is adjusted automatically after air release.
e) Wipe off any drops adhering to the discharge tip.
f) Open the stopcock and slowly add titrant to the sample (containing the
indicator). The discharge tip must not touch the wall of the vessel. Keep swirling
the sample vessel lightly while adding titrant, or place it on a magnetic stirrer.
g) Read the discharged volume at eye level.
h) Any drops remaining on the tip of the stopcock should be wiped against the
vessel wall and rinsed down. It is part of the titrated volume.

Flasks
In laboratory and other scientific settings these are usually referred to simply as flasks.
Flasks come in a number of shapes and a wide range of sizes, but a common
distinguishing aspect in their shapes is a wider vessel "body" and one (or sometimes
more) narrower tubular sections at the top called necks which have an opening at the
top. Laboratory flask sizes are specified by the volume they can hold, typically in metric
units such as milliliters (mL or ml) or liters (L or l). Laboratory flasks have traditionally
been made of glass, but can also be made of plastic.
At the opening at top of the neck of some glass flasks such as round-bottom
flasks, retorts, or sometimes volumetric flasks, there are outer (or female) tapered
(conical) ground glass joints. Some flasks, especially volumetric flasks, come with a

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
laboratory rubber stopper, bung, or cap for capping the opening at the top of the neck.
Such stoppers can be made of glass or plastic. Glass stoppers typically have a matching
tapered inner (or male) ground glass joint surface, but often only of stopper quality.
Flasks which do not come with such stoppers or caps included may be capped with a
rubber bung or cork stopper.
Use of flasks
Flasks are used for making solutions or for holding, containing, collecting, or sometimes
volumetrically measuring chemicals, samples, solutions, etc. for chemical reactions or
other processes such as mixing, heating, cooling, dissolving, precipitation, boiling (as in
distillation), or analysis.

Different types of flasks

Volumetric flasks: These are used primarily in the preparation of standard solutions.
To create a solution of a specific concentration, we need to know the volume of the
solution; the narrow neck of the volumetric flask will have a thin graduation to show
where a specific volume is reached.
a) Round-bottomed flasks and Florence flasks: These look very similar, but there
is a slight difference between the two. Both have round bottoms, designed to
spread out heat evenly when they are heated. They are frequently used by
chemists for reactions and in rotary evaporators. Whereas round-bottomed flasks
will usually have a ground glass joint on their neck, to allow connection to other
apparatus, Florence flasks, supposedly named after Florence in Italy, tend to
merely have a lip. They can also come with either a flat bottom so they are free-
standing, or a rounded bottom, and have longer necks.
b) The Kjeldahl flask: These are having an even longer neck and were developed
for use in the Kjeldahl method, which is used to determine the nitrogen content
in a substance.
c) Pear-shaped flask: These are usually rather small flasks, used for small-scale
distillations. Their shape allows recovery of more material than the round-
bottomed flasks. The rather odd-looking retort flasks are used in distillations,
though their use was primarily before the advent of condensers. Today, they are
very rarely used.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 d) Schlenk flask and the Straus flask: These are another two that look fairly
similar. Schlenk flasks are commonly used in air-sensitive chemistry, as the side
arm allows an inert gas such as nitrogen to be pumped into the vessel. The Straus
flask, on the other hand, is used to store dried solvents. The main neck is actually
filled in halfway up, and connected to a plugged smaller neck; this main neck
can be connected to other apparatus, and allows the solvent to be extracted when
the plug is slightly withdrawn or removed entirely.
e) Claisen flask: These are designed by chemist Ludwig Claisen, is designed for
vacuum distillation; distillation under vacuum produces problematic amounts of
bubbles when solutions are boiled. Claisen‘s flask includes a capillary tube that
inserts small bubbles into the liquid, easing the ferocity of boiling, whilst the
branched portion of the flask hosts a thermometer. Today, Claisen‘s flask is less
commonly used.

Funnels
A funnel is a tube or pipe that is wide at the top and narrow at the bottom, used for
guiding liquid or powder into a small opening. These are usually made of stainless
steel, aluminium, glass or plastic. The material used in its construction should be sturdy
enough to withstand the weight of the substance being transferred and it should not
react with the substance. For this reason, stainless steel or glass are useful in
transferring reagents etc. Dropper funnels, also called dropping funnels or tap funnels,
have a tap to allow the controlled release of a liquid. A flat funnel, made of
polypropylene, utilizes living hinges and flexible walls to fold flat.

Use of Funnels
Laboratory funnels are used to channel liquids or fine-grained chemicals (powders) into
lab. ware with a narrow neck or opening. Often, they are made of plastic or glass, such
as polypropylene or borosilicate 3.3 glass.

Different types of funnels

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 a) Conical funnel: This funnel is with wide mouth and narrow stem used in
laboratories. They are used to transfer liquids carefully without spillage in to
containers. They are used to prepared solutions (for transferring weighed
samples into standard flasks
b) Buchner funnel: This is a piece of laboratory equipment used in filtration. It is
traditionally made of porcelain, but glass and plastic funnels are also available. On top
of the funnel-shaped part there is a cylinder with a fritted glass disc/perforated plate
separating it from the funnel.
c) Hirsch funnel: This has a similar design; it is used similarly, but for smaller quantities of
material. The main difference is that the plate of a Hirsch funnel is much smaller, and
the walls of the funnel angle outward instead of being vertical.

Bottles (wash bottles &) reagent

Wash bottles
A wash bottle is a squeeze bottle with a nozzle and sealed with a screw-top lid, used to
rinse various pieces of laboratory glassware, such as test tubes and round bottom flasks.
When hand pressure is applied to the bottle, the liquid inside becomes pressurized and
is forced out of the nozzle into a narrow stream of liquid.
Most wash bottles are made up of polyethylene, which is a flexible solvent-resistant
petroleum-based plastic. Most bottles contain an internal dip tube allowing upright use.
Wash bottles may be filled with a range of common laboratory solvents and reagents,
according to the work to be undertaken. These include deionized water, detergent
solutions and rinse solvents such as acetone, isopropanol or ethanol. In biological labs it
is common to keep sodium hypochlorite solution in a wash bottle to disinfect unneeded
cultures.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Reagent bottles
Reagent bottles also known as graduated bottles, are containers made of glass, plastic,
borosilicate or related substances, and topped by special caps or stoppers. They are
intended to contain chemicals in liquid or powder form for laboratories and storage of
chemicals. Some reagent bottles are tinted amber (actinic) brown or red to protect light-
sensitive chemical compounds from visible light, ultraviolet and infrared radiation
which may alter them; other bottles are tinted blue (cobalt glass) or uranium green for
decorative purposes.
The bottles are called "graduated" when they have marks on the sides indicating
the approximate amount of liquid at a given level within the container. A reagent bottle
is a type of laboratory glassware. The term "reagent" refers to a substance that is part of
a chemical reaction (or an ingredient of which), and "media" is the plural form of
"medium" which refers to the liquid or gas which a reaction happens within, or is a
processing chemical tool such as (for example) a flux. Common bottle sizes include 100
ml, 250 ml, 500 ml, 1000 ml (1 liter) and 2000 ml (2 liter).
The selection of caps and stoppers that reagent bottles are closed with are as
important as the material the bottles are made of, and the decision as to which cap to
use is dependent on the material stored in the container, and the amount of heat which
the cap can be subject to. Common cap sizes include 33-430 (33mm), 38-430 (38mm),
and GL 45 (45mm). Caps range in size from narrow mouthed to wide mouthed and
often a glass or plastic funnel is needed to properly fill a reagent bottle from a larger or
equal sized container's mouth. Reagent bottle caps are commonly said to be
"autoclavable".

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Measuring cylinders & reagent dispensers
Measuring cylinders
A Measuring Cylinder (Graduated Cylinder) is a type of measuring tools and common
moderately accurate laboratory consumable used for measuring the volume of liquid.
The graduated cylinder is widely used in various hospital laboratories, school
laboratories, pharmaceuticals, food and beverage, petrochemical, aquatic products
testing, cosmetics, coatings, quality supervision departments, biological and other
enterprises‘ laboratories. The Measuring Cylinder is a common and essential measuring
device used in the laboratory, mainly made of glass and plastic. The common glass
types for measuring cylinder are Quartz Glass and Borosilicate Glass. Quartz Glass
Silica/SiO2 content is greater than 99.5%, low thermal expansion coefficient, high
temperature resistance, good chemical stability; Borosilicate Glass with Silica/SiO₂ and
Boron Trioxide/B2O3 as the main components, it has good heat resistance and chemical
stability. Plastic measuring cylinder made of PP plastic (Polypropylene) can also be
used for kitchen purpose to measuring water or other liquid.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Volume of measuring cylinders: The specifications are expressed in terms of the
maximum capacity (ml) of the measuring cylinder, and commonly used volumes
are 10ml, 25ml, 50ml, 100ml, 250ml, 500ml, 1000ml, 2000ml, etc.
 Graduation: The outer wall Graduation /scale are in ml, the 10ml graduated
cylinder represents 0.2ml per small scale, and the 50ml graduated cylinder
represents 1ml per small scale. It can be seen that the larger the cylinder, the thicker
the pipe diameter, the smaller the accuracy, and the larger the reading error caused
by the deviation of the line of sight. Therefore, in the experiment, according to the
volume of the liquid taken, try to use the smallest gauge cylinder that can be
measured at one time. Fractional metering can also cause errors. If you take 70ml of
liquid, you should use a 100mL measuring cylinder.
 Structure: The measuring cylinder has a long cylindrical shape with a mouth on
one side for easy dumping. The lower part has wide feet for stability. The cylinder
wall is engraved with a volumetric range for the user to read the volume. The
maximum measured volume is from a few milliliters to a few liters. The cylinder
wall is printed with scales from the bottom up. When observing the reading, the
user should pay attention to the line of sight that needs to be level with the lowest
point of the liquid surface of the liquid (or the highest point of the liquid surface).
 Measuring Cylinder Uses and Cautions:
i. When injecting liquid into the measuring cylinder, use your left hand to hold
the measuring cylinder, tilt the measuring cylinder slightly, and take the

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 reagent bottle in the right hand so that the bottle mouth is close to the
measuring cylinder mouth to let the liquid slowly flows in.
ii. When the amount to be injected is slightly less than the amount required,
place the cylinder on the table and use a plastic dropper to add the required
amount.
iii. The graduated cylinder does not have a ―0‖ scale, and the general starting
scale is 1/10 of the total volume. Many experimental pictures on the
chemical book, the scale surface of the measuring cylinder is at the opposite
side, which is very inconvenient to read.
iv. The view sight is going through two layers of glass and liquid, if the liquid is
turbid, the scale is even less visible, and the scale figures are not pleasing to
the eye. So the scale should face the user.
v. After injecting the liquid, wait for 1 to 2 minutes to allow the adhering liquid
to flow down the inner wall, and then read the scale value. Otherwise, the
value read is smaller than actual value.
vi. Place the graduated cylinder on a flat surface first and then view the height
of the liquid in the cylinder with your eyes directly.
vii. The meniscus is the U-shapes ―the upper surface of a liquid in the measuring
tube‖ Read the measuring volume in a graduated cylinder at the bottom of
the meniscus. Always look straight from the side of the meniscus at the
graduated cylinder to measure and never try to read from above or below.
Otherwise, the value will be higher or lower than the actual value.
viii. The scale of the cylinder refers to the number of volumes at a temperature of
20 °C. As the temperature rises, the cylinder expands and the volume
increases. So, unlike the glass test tubes, measuring cylinder cannot be
heated, nor can it be used to measure the superheated liquid, and it is
impossible to carry out chemical reaction or formulate the solution in the
measuring cylinder.

Cautions
 Never use the measuring cylinder as a reaction container
 Cannot be heated
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Never dilute concentrated acid, concentrated alkali with the cylinder
 Do not store pharmacy
 Do not use it to take hot solution
 Never clean it with decontamination powder to avoid scratching

Tubes – test tube, centrifuge tube, folin-wu tube

Test tube
A test tube, also known as a sample tube, is a common piece of laboratory glassware
consisting of a finger-like length of glass or clear plastic tubing, open at the top and
closed at the bottom.
Test tubes are usually placed in special-purpose racks.
Test tubes intended for general chemical work are usually made of glass, for its relative
resistance to heat. Tubes made from expansion-resistant glasses, mostly borosilicate
glass or fused quartz, can withstand high temperatures up to several hundred degrees
Celsius.
Chemistry tubes are available in a multitude of lengths and widths, typically
from 10 to 20 mm wide and 50 to 200 mm long. The top often features a flared lip to aid
pouring out the contents.
A chemistry test tube typically has a flat bottom, a round bottom, or a conical
bottom. Some test tubes are made to accept a ground glass stopper or a screw cap. They
are often provided with a small ground glass or white glaze area near the top for
labelling with a pencil.
Test tubes are widely used by chemists to handle chemicals, especially for
qualitative experiments and assays. Their spherical bottom and vertical sides reduce
mass loss when pouring, make them easier to wash out, and allow convenient
monitoring of the contents. The long, narrow neck of test tube slows down the
spreading of gases to the environment.
Test tubes are convenient containers for heating small amounts of liquids or
solids with a Bunsen burner or alcohol burner. The tube is usually held by its neck with
a clamp or tongs. By tilting the tube, the bottom can be heated to hundreds of degrees in
the flame, while the neck remains relatively cool, possibly allowing vapours to
condense on its walls. A boiling tube is a large test tube intended specifically for boiling
liquids.
A test tube filled with water and upturned into a water-filled beaker is often
used to capture gases, e.g. in electrolysis demonstrations.
A test tube with a stopper is often used for temporary storage of chemical or biological
samples.

These tubes are used in lab centrifuges that are used to separate solids and liquid
chemicals. This machine spins the sample to achieve this goal. Centrifuge tubes look
like miniature test tubes that have tapered tips. These can be made of the glass and
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
plastic. It has different taper designs that depend on the type of solids you need to
separate in the chemical solution. Centrifuge tubes are set inside the centrifuge machine
and then it is spun for a specific amount of time at a specific velocity. Lab technician or
scientist takes the tube out after the process is complete and pours the separated liquid
into a container

Test tube is mostly used just to contain a liquid substance. It's the most common type of
all tubes with thin walls and cylindrical shape. They are usually about the same size
and shape as a finger. Scientists also mix and heat chemicals in test tubes for
experiments. Its bottom is rounded so it can't stand on its own. A test tube stand is
usually used to hold them straight. Test tubes are most commonly made with glass, but
it's also made of metal, plastic, and ceramic. You will find these tubes in chemical,
bioscience, and clinical medicine labs.

It is used in the Folin Wu method of estimation of glucose. It is a non enzymatic

estimation of glucose. It has a constricted neck to prevent reoxidation of Cu 2O with
atmospheric O2.

Cuvettes and its use in measurements, cuvettes for visible and UV range:
A cuvette (sample cell, absorption cell) is, in its basic level, fundamentally a test tube
designed for use with optical analysis. Standard cuvettes are generally square or
rectangular in cross section to avoid refraction artefacts. Depending on what part of the
spectrum is under consideration, they may be made of quartz or optical glass although
plastic cuvettes do exist for less demanding measurements.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 A liquid sample is placed in the cuvette carefully to ensure that it is not over
filled and the outside of the cuvette is kept clean. Then the cuvette is inserted into the
instrument such as a Spectrometer or Fluorometer. The beams inside of the instrument
can pass through the quartz/glass walls of the cuvette due to their optical purity and
'read' the sample. The sample's data is then analysed against know standards of
materials.
Historically, reusable quartz cuvettes were required for measurements in the
ultraviolet range, because glass and most plastics absorb ultraviolet light, creating
interference. Today there are disposable plastic cuvettes made of specialized plastics
that are transparent to ultraviolet light. Glass, plastic and quartz cuvettes are all suitable
for measurements made at longer wavelengths, such as in the visible light range.
"Tandem cuvettes" have a glass barrier medium that extends two-thirds of the
way up in the middle, so that measurements can be taken with two solutions separated
and again when they are mixed.

A disposable plastic cuvette

Plastic: Plastic cuvettes are often used in fast spectroscopic assays, where high speed is
more important than high accuracy. Plastic cuvettes with a usable wavelength range of
380–780 nm (the visible spectrum) may be disposed of after use, preventing
contamination from re-use. They are cheap to manufacture and purchase. Disposable
cuvettes can be used in some laboratories where the beam light is not high enough to
affect the absorption tolerance and consistency of the value.

Glass: Crown glass has an optimal wavelength range of 340–2500 nm. Glass cuvettes
are typically for use in the wavelength range of visible light, whereas fused quartz tends
to be used for ultraviolet applications.
Historically, reusable quartz cuvettes were required for measurements in the
ultraviolet range, because glass and most plastics absorb ultraviolet light, creating
interference.
Glass, plastic and quartz cuvettes are all suitable for measurements made at
longer wavelengths, such as in the visible light range.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Quartz: Quartz cells provide more durability than plastic or glass. Quartz excels at
transmitting UV light, and can be used for wavelengths ranging from 190 to 2500 nm.
Glass absorbs strongly in UV region and its application is not recommended for
wavelengths below 340 nm.

What is difference between quartz Cuvettes and glass Cuvettes

This is the absolute best way to determine what material an unknown cuvette is made
out of. Other differences between quartz and glass cuvettes include the following:
 Transmission properties – as you can see from the information above quartz has
a bigger transmission range than glass.
 Thermal Properties – A quartz material has a much higher melting point than
glass.
 Chemical Compatibility – The chemical structure of quartz is stronger than glass
making it able to handle a bigger range of chemicals that would melt or damage
a glass cuvette.
 Modifications – Here is where glass cuvettes really shine. A pyrex cuvette is
super easy to modify and make attachments to. Quartz cuvettes can be modified
but is a much bigger process.

Physical characteristics of cuvette:

1. High mechanical strength, strong adaptability to temperature changes, very
strong bonding part, pressure resistance to several atmospheric pressures.
2. Extremely precise optical processing technology, the optical performance of the
light-transmitting surface is excellent, and the grouping error is ≤0.3%.
3. Use high-quality quartz glass and optical glass to ensure no bubbles and no
stripes. The quartz cuvette is greater than 80% at a wavelength of 200nm, and the
glass cuvette is greater than 80% at a wavelength of 340nm.
It has recently been found that the inability to properly measure or cause large errors
due to improper selection or use of cuvettes often occurs in experiments, and this
problem is easily overlooked by the experimenter. A brief description of the correct
choice of cuvettes is now available.

1. Common cuvettes are divided into quartz and glass.

2. Only 200-400 nm in the ultraviolet region can be used with quartz cuvettes. A
glass cuvette or a quartz cuvette can be used in the visible light region of 400-
1100 nm.
3. The standard Q and S are generally quartz, the standard G is generally glass. If
there is no mark or the mark is unclear, the instrument can be adjusted to the
ultraviolet region of about 200 nm, and the T% mode is selected. After the air is
zeroed, the display shows 100%T, and the clean cuvettes are inserted into the
sample cell holder. (Double-beam UV can only be used in the sample cell.) If the

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 transmittance is between 60% and 90% T, it is a quartz cuvette. If the
transmission is below 1%, it is a glass cuvette.
4. The cuvettes should be paired and used. The transmittance of the two cuvettes is
measured by the method of 3, and the difference is less than 0.5%.

Racks – bottle, testube, pipette and drying racks

Test tube racks are laboratory equipment used to hold upright multiple test tubes at the
same time. They are most commonly used when various different solutions are needed
to work with simultaneously, for safety reasons, for safe storage of test tubes, and to
ease the transport of multiple tubes. Test tube racks also ease the organization of test
tubes and provide support for the test tubes being worked with.

A pipette stand is a holder designed to provide safe and convenient storage for manual
and electronic pipettes. In particular, the stands for electronic pipettes can recharge the
battery of the pipettes, typically by means of charging pins or connectors. The design of
the pipette stand can be:
 wall mount holder
 linear stand
 carousel stand
The main purpose of the use of pipette stand is to reduce the cross contamination risk
between pipettes or from benchtop to your pipettes.

Laboratory drying rack is a pegboard for hanging and draining glassware in

a laboratory. It is available in different varieties and sizes. It can be used for different
materials of glassware in the laboratory room such as funnels, pipettes, mixing balls,
slides, bottle stoppers, tubing and so on. In addition to that, the pegs on the drying rack
are easily removable and replaceable in order to maintain the cleaning of the lab racks
to avoid contamination with other apparatus used on the same rack. Any common
laboratory needs to have at least two or three drying racks per lab.

Laboratory drying rack can be mainly categorized into three major types
including stainless steel laboratory drying racks, epoxy laboratory drying racks,
and acrylic laboratory drying racks.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Stainless steel laboratory drying rack
Stainless steel laboratory drying rack, which is also known as a 'Mod-Rack' pegboard, is
the drying rack made of stainless steel that uses to drain laboratory accessories. The
examples of stainless steel laboratory drying rack are flask holders, soap dispensers,
paper towel dispensers, glove box holders, drain shelves. Stainless steel pegboard
installation is very easy and quick to set up with basic hand equipment's, and it does
not damage the wall as mounting brackets and hardware are being used.

Epoxy laboratory drying rack

Epoxy laboratory drying racks are the most common type of drying rack that are used
among university labs and science classrooms in many high schools. Epoxy drying
racks are mounted directly to a wall or other solid structures which can be set up with
basic hand tools and power tools. They are easily installed by using wall anchors and
other strong fasteners due to their small weight. Typical installation is to drill holes, one
at each corner, and to use the mounting points in order to fix it to the wall.

Acrylic laboratory drying rack

Acrylic laboratory drying racks give a unique feature that other pegboards cannot do.
The clear acrylic is transparent, which means that it allows the light to pass through as
well as brightening the working area. Acrylic pegboards are mostly in the place where
there are no lights, or to be done in dim areas. Like epoxy pegboards, acrylic laboratory
pegboards are also installed with basic tools and power tools in the same way.
However, acrylic pegboards are made up of plastic, so it can be easily scratched as
compared to the epoxy and the stainless steel drying rack.

Tripod stand
A laboratory tripod is a three-legged platform used to support flasks and beakers.
Tripods are usually made of stainless steel or aluminium and lightly built for portability
within the lab. Often a wire gauze is placed on top of the tripod to provide a flat base
for glassware. Tripods are generally tall enough for a bunsen burner to be placed
underneath.
There are several different designs. The top is commonly triangular or circular,
and sometimes the three legs can be removed and adjusted depending on the
preferences of the user.
A laboratory tripod is most commonly used in middle and high schools for basic
heating experiments. However, tripods and bunsen burners have been made obsolete
by hot plates, which are considered to be safer since there is no direct contact with the
flame.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Wire gauze
Wire gauze is popular equipment in Laboratory Apparatus. it is a thin metal
sheet having net or web-like patterns. this pattern is also known as metal mesh. the
major wire gauze function is that it often placed on a supporting ring and attached to
the retort stand between the bunsen burner and flasks, beakers, etc to facilitate heat
transfer. High-quality wire gauze always made from stainless steel having ceramic
centers for general heating applications in common laboratories.
A wire gauze is a sheet of thin metal that has net-like patterns or a wire mesh.
Wire gauze is placed on the support ring that is attached to the retort stand between
the Bunsen burner and the glassware to support the beakers, flasks, or other glassware
during heating.[1][2] Wire gauze is an important piece of supporting equipment in
a laboratory as glassware cannot be heated directly with the flame of a Bunsen burner,
and requires the use of a wire gauze to diffuse the heat, helping to protect the
glassware. Glassware has to be flat-bottomed to stay on the wire gauze.
Some wire gauze has a ceramic centre. Plain wire gauze can transmit heat
efficiently, but gauze with a ceramic center will also allow the heat to be dispersed more
evenly. The ceramic at the centre of the wire gauze is enmeshed at high pressure to
prevent it from peeling.
Wire gauze may be woven from metals such as iron, steel, copper, or nichrome.
Nichrome alloy provides long life expectancy and tear resistance. The edges of the wire
gauze are turned inward to help prevent fraying, improve handling, and reduce the
danger from sharp protruding wire ends.
Ceramic-centered wire gauze is commonly available in the United States in squares of 4
inches (100 mm), 5 inches (130 mm), and 6 inches (150 mm) to accommodate different
sizes of glassware.
Wire gauze is used as a support for different containers when they are placed
across a support ring above the bunsen burner to spread the heat of the burner flame. it
also supports flasks and beakers. when the bunsen burner flame is beneath it with
a tripod, it helps to spread the flame out evenly over the container.
dealing with gauze is a fire-based work, so we need to follow some cautions and safety
rules in the laboratory So that we can save ourselves and others from severe
consequences and hazards. it also uses in many industries, arts and crafts, and many
home improvements.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Different Sizes, Varieties, and Types
It is a net-like mesh that is interconnected strands of different wires. they are made up
of different gauges of wire and various spacing patterns on the metal mesh. it is made
with different metals like iron, steel, nichrome, and copper, etc. the ceramic center wire
gauze is often used in the laboratory which has the following available sizes:
 6 x 6 inches (15 x 15 cm) (150 mm)
 5-inch (125 mm)
 5 x 5 inches (13 x 13 cm) (125 mm) with ceramic center
 4 x 4 inches (10 x 10 cm), (100 mm)
 5 inches (130 mm)

Bunsen burner
A Bunsen burner, named after Robert Bunsen, is a kind of gas burner used
as laboratory equipment; it produces a single open gas flame, and is used for heating,
sterilization, and combustion.
The gas can be natural gas (which is mainly methane) or a liquefied petroleum gas, such
as propane, butane, or a mixture.

A Bunsen burner is a type of gas burner commonly used as a heat source in laboratory
experiments. The burner consists of a flat base with a straight tube extending vertically,
known as the barrel or chimney. Natural gas (predominantly methane) or a liquified
petroleum gas such as propane or butane is supplied at the bottom of the chimney.
Bunsen burners are normally fitted with a hose barb at the base of the chimney to allow
rubber tubing to supply the gas from a gas nozzle on the laboratory bench. There may
also be a gas value on the Bunsen burner. The other critical component of a Bunsen
burner is the air hole. This is located near the bottom of the chimney, just above the gas
inlet. The air hole allows pre-mixing of air and gas before combustion occurs at the top
of the chimney. A collar around the base of the chimney, with a hole that aligns with the
air hole, acts as an air regulator, allowing the air in the pre-mixture to be adjusted.

Operation
The device in use today safely burns a continuous stream of a flammable gas such
as natural gas (which is principally methane) or a liquefied petroleum gas such
as propane, butane, or a mixture of both.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
The hose barb is connected to a gas nozzle on the laboratory bench with rubber tubing.
Most laboratory benches are equipped with multiple gas nozzles connected to a central
gas source, as well as vacuum, nitrogen, and steam nozzles. The gas then flows up
through the base through a small hole at the bottom of the barrel and is directed
upward. There are open slots in the side of the tube bottom to admit air into the stream
using the Venturi effect, and the gas burns at the top of the tube once ignited by a flame
or spark. The most common methods of lighting the burner are using a match or a spark
lighter.
The amount of air mixed with the gas stream affects the completeness of
the combustion reaction. Less air yields an incomplete and thus cooler reaction, while a
gas stream well mixed with air provides oxygen in a stoichiometric amount and thus a
complete and hotter reaction. The air flow can be controlled by opening or closing the
slot openings at the base of the barrel, similar in function to the choke in a carburettor.
If the collar at the bottom of the tube is adjusted so more air can mix with the gas before
combustion, the flame will burn hotter, appearing blue as a result. If the holes are
closed, the gas will only mix with ambient air at the point of combustion, that is, only
after it has exited the tube at the top. This reduced mixing produces an incomplete
reaction, producing a cooler but brighter yellow, which is often called the "safety flame"
or "luminous flame". The yellow flame is luminous due to small soot particles in the
flame, which are heated to incandescence. The yellow flame is considered "dirty"
because it leaves a layer of carbon on whatever it is heating. When the burner is
regulated to produce a hot, blue flame, it can be nearly invisible against some
backgrounds. The hottest part of the flame is the tip of the inner flame, while the coolest
is the whole inner flame. Increasing the amount of fuel gas flow through the tube by
opening the needle valve will increase the size of the flame. However, unless the airflow
is adjusted as well, the flame temperature will decrease because an increased amount of
gas is now mixed with the same amount of air, starving the flame of oxygen.
Generally, the burner is placed underneath a laboratory tripod, which supports
a beaker or other container. The burner will often be placed on a suitable heatproof
mat to protect the laboratory bench surface.
A Bunsen burner is also used in microbiology laboratories to sterilise pieces of
equipment and to produce an updraft that forces airborne contaminants away from the
working area.

Variants
Other burners based on the same principle exist. The most important alternatives to the
Bunsen burner are:
 Teclu burner – The lower part of its tube is conical, with a round screw nut below its
base. The gap, set by the distance between the nut and the end of the tube, regulates
the influx of the air in a way similar to the open slots of the Bunsen burner. The
Teclu burner provides better mixing of air and fuel and can achieve higher flame
temperatures than the Bunsen burner.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Meker burner – The lower part of its tube has more openings with larger total cross-
section, admitting more air and facilitating better mixing of air and gas. The tube is
wider and its top is covered with a wire grid. The grid separates the flame into an
array of smaller flames with a common external envelope, and also
prevents flashback to the bottom of the tube, which is a risk at high air-to-fuel ratios
and limits the maximum rate of air intake in a conventional Bunsen burner. Flame
temperatures of up to 1,100–1,200 °C (2,000–2,200 °F) are achievable if properly
used. The flame also burns without noise, unlike the Bunsen or Teclu burners.[11]
 Tirrill burner – The base of the burner has a needle valve which allows the
regulation of gas intake directly from the Burner, rather than from the gas source.
Maximum temperature of flame can reach 1560 °C

Desiccators
Desiccators are sealable enclosures containing desiccants used for preserving moisture-
sensitive items such as cobalt chloride paper for another use. A common use for
desiccators is to protect chemicals which are hygroscopic or which react with water
from humidity.
The contents of desiccators are exposed to atmospheric moisture whenever the
desiccators are opened. It also requires some time to achieve a low humidity. Hence
they are not appropriate for storing chemicals which react quickly or violently with
atmospheric moisture such as the alkali metals; a glovebox or Schlenk-type apparatus
may be more suitable for these purposes.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Constituents
The lower compartment of the desiccator contains lumps of silica gel,
freshly calcined quicklime, Drierite or (not as effective) anhydrous calcium chloride to
absorb water vapor. The substance needing desiccation is put in the upper
compartment, usually on a glazed, perforated ceramic plate. The ground-glass rim of
the desiccator lid must be greased with a thin layer of vacuum grease, petroleum jelly or
other lubricant to ensure an airtight seal.
In order to prevent damage to a desiccator the lid should be carefully slid on and off
instead of being directly placed onto the base.

Operation
In laboratory use, the most common desiccators are circular and made of heavy glass.
There is usually a removable platform on which the items to be stored are placed. The
desiccant, usually an otherwise-inert solid such as silica gel, fills the space under the
platform. Colour changing silica may be used to indicate when it should be refreshed.
Indication gels typically change from blue to pink as they absorb moisture but other
colours may be used.
A stopcock may be included to permit the desiccator to be evacuated. Such models are
usually known as vacuum desiccators. When a vacuum is to be applied, it is a common
practice to criss-cross the vacuum desiccator with tape, or to place it behind a screen to
minimize damage or injury caused by an implosion. To maintain a good seal,
vacuum grease is usually applied to the flanges.

A laboratory desiccator is a round shaped closed vessel made of heavy glass which is a
common laboratory glassware item and has multiple uses, such as:
• Storage of standards under dry environment
• Storage of materials for weighing to constant weight
• Prolonged storage of hygroscopic materials

• Determination of loss on drying of manufactured products such as

pharmaceuticals
The base of the desiccator supports a perforated plate below which the desiccant drying
agent is kept. Commonly used drying agents are anhydrous calcium chloride, drierite

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
(calcium sulphate) and silica. Such desiccants can be regenerated by heating. A small
amount of cobalt indicator compound is generally added which turns pink from blue on
absorption of moisture. This lets you know when to regenerate the desiccant.
The desiccator is effective only when sealed to prevent contact of contents with outside
air. Some desiccators are provided with vacuum fittings for evacuation of inside air for
long-term storage of moisture sensitive materials.

Types of Desiccators
There are four types of desiccators:
1. Standard: which are used as desiccant cartridges and need to be changed often,
depending on how often they are used. The standard laboratory desiccator
requires monitoring but is quite economical.
2. Vacuum: The vacuum desiccator requires a pump to remove any air or moisture
from the chamber. The length of the vacuum process depends on the model.
3. Automatic: This desiccator is fitted with electrical fans and heaters, and they are
continuously used to regenerate a dry atmosphere. It requires minimal
monitoring and it‘s highly reliable. But, they can be costly.
4. Gas Purge: The drying agent used in the desiccator is nitrogen and argon. They
maintain a dry atmosphere within the desiccator, at a faster rate.

Benefits of a Desiccator
 This laboratory device can absorb moisture in humid conditions and keep the
items dry and dust-free
 They also prevent any hygroscopic materials from reacting with moisture that is
caused by humid conditions.
 It‘s not used to dry material, but to maintain a dry atmosphere and keep the
material dry. Some laboratory desiccators are used for cooling a heated object or
material.
 In any regulated environment, such as a laboratory, there is a need to control the
environment because it determines the completion of various tasks. Many factors
cause a hindrance like fluctuating temperatures, dust particles, humidity, and so
on; hence, these elements have to be controlled and regulated.

Some of the top benefits of each desiccator:

a. Standard Desiccator: This unit is operated and monitored manually. It will take
in the moisture, and once saturated, you will have to replace or heat it to
regenerate the desiccant. These units provide flexibility, are affordable, and quite
convenient.
b. Automatic Desiccator: These require minimal monitoring and make use of
heaters/electric fans to prevent saturation. The humidity is maintained at a
constant low and the units are programmable; they are also set to operate on a
preset schedule. They provide control over humidity and use silica gel beads that

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 last for a long time, this also reduces the need to replace the desiccation material,
constantly.
c. Gas Purge Desiccator: If you want fast removal of humidity, the best bet is to opt
for a gas purge desiccator. They use a slow and steady flow of inert gas to extract
moisture. The gas used is usually dry nitrogen, which is available easily. These
units create a dust-free environment and are great for clean rooms.
d. Vacuum Desiccator: If you are stuck in a situation where you need to remove air
and humidity, go for a vacuum desiccator. These units remove air and moisture
with a vacuum pump. They are great at collecting dust and can be used as
standard desiccators as well.

Stop watch
A stopwatch is a handheld timepiece designed to measure the amount of time that
elapses between its activation and deactivation.
A large digital version of a stopwatch designed for viewing at a distance, as in a
sports stadium, is called a stop clock. In manual timing, the clock is started and stopped
by a person pressing a button. In fully automatic time, both starting and stopping are
triggered automatically, by sensors. The timing functions are traditionally controlled by
two buttons on the case. Pressing the top button starts the timer running, and pressing
the button a second time stops it, leaving the elapsed time displayed. A press of the
second button then resets the stopwatch to zero. The second button is also used to
record split times or lap times. When the split time button is pressed while the watch is
running it allows the elapsed time to that point to be read, but the watch mechanism
continues running to record total elapsed time. Pressing the split button a second time
allows the watch to resume display of total time.
Mechanical stopwatches are powered by a mainspring, which must be wound up by
turning the knurled knob at the top of the stopwatch.
Digital electronic stopwatches are available which, due to their crystal
oscillator timing element, are much more accurate than mechanical timepieces. Because
they contain a microchip, they often include date and time-of-day functions as well.
Some may have a connector for external sensors, allowing the stopwatch to be triggered
by external events, thus measuring elapsed time far more accurately than is possible by
pressing the buttons with one's finger. Stopwatches that count by 1/100 of a second are
commonly mistaken as counting milliseconds, rather than centiseconds. The first digital
timer used in organized sports was the Digitimer, developed by Cox Electronic
Systems, Inc. of Salt Lake City Utah (1962). It utilized a Nixie-tube readout and
provided a resolution of 1/1000 second. Its first use was in ski racing but was later used
by the World University Games in Moscow, Russia, the U.S. NCAA, and in the Olympic
trials.
The device is used when time periods must be measured precisely and with a
minimum of complications. Laboratory experiments and sporting events like sprints are
good examples.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 The stopwatch function is also present as an additional function of many
electronic devices such as wristwatches, cell phones, portable music players, and
computers.

Timers
A timer is a specialized type of clock used for measuring specific time intervals. Timers
can be categorized into two main types. A timer which counts upwards from zero for
measuring elapsed time is often called a stopwatch, while a device which counts down
from a specified time interval is more usually called a timer. A simple example of this
type is an hourglass. Working method timers have two main groups: Hardware and
Software timers.
Most timers give an indication that the time interval that had been set has
expired.

Mechanical timers
Mechanical timers use clockwork to measure time.[1] Manual timers are typically set by
turning a dial to the time interval desired; turning the dial stores energy in
a mainspring to run the mechanism. They function similarly to a mechanical alarm
clock; the energy in the mainspring causes a balance wheel to rotate back and
forth.[1] Each swing of the wheel releases the gear train to move forward by a small
fixed amount, causing the dial to move steadily backward until it reaches zero when a
lever arm strikes a bell. The mechanical kitchen timer was invented in 1926 by Thomas
Norman Hicks.[1] Some less accurate, cheaper mechanisms use a flat paddle called a fan
fly that spins against air resistance; low-precision mechanical egg-timers are sometimes
of this type.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
The simplest and oldest type of mechanical timer is the hourglass, which is also known
as the glass of the hour most people use these because they want to but not a lot of
people know that that was its first name. in which a fixed amount of sand drains
through a narrow opening from one chamber to another to measure a time interval.

Electromechanical timers

Short-period bimetallic electromechanical timers use a thermal mechanism, with a

metal finger made of strips of two metals with different rates of thermal
expansion sandwiched together; steel and bronze are common. An electric
current flowing through this finger causes heating of the metals, one side expands less
than the other, and an electrical contact on the end of the finger moves away from or
towards an electrical switch contact. The most common use of this type is in the
"flasher" units that flash turn signals in automobiles, and sometimes in Christmas lights.
This is a non-electronic type of multivibrator.
An electromechanical cam timer uses a small synchronous AC motor turning
a cam against a comb of switch contacts. The AC motor is turned at an accurate rate by
the alternating current, which power companies carefully regulate. Gears drive a shaft
at the desired rate, and turn the cam. The most common application of this timer now is
in washers, driers and dishwashers. This type of timer often has a friction clutch
between the gear train and the cam, so that the cam can be turned to reset the time.
Electromechanical timers survive in these applications because mechanical switch
contacts may still be less expensive than the semiconductor devices needed to control
powerful lights, motors and heaters.
In the past, these electromechanical timers were often combined with
electrical relays to create electro-mechanical controllers. Electromechanical timers
reached a high state of development in the 1950s and 1960s because of their extensive
use in aerospace and weapons systems. Programmable electromechanical timers
controlled launch sequence events in early rockets and ballistic missiles. As digital
electronics has progressed and dropped in price, electronic timers have become more
advantageous.
Electronic timers

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
A simple digital timer. The internal components—including the circuit board with
control chip and LED display, a battery, and a buzzer—are visible. Electronic timers are
essentially quartz clocks with special electronics, and can achieve higher precision than
mechanical timers. Electronic timers have digital electronics, but may have
an analog or digital display. Integrated circuits have made digital logic so inexpensive
that an electronic timer is now less expensive than many mechanical and
electromechanical timers. Individual timers are implemented as a simple single-
chip computer system, similar to a watch and usually using the same, mass-produced,
technology.
Many timers are now implemented in software. Modern controllers use
a programmable logic controller (PLC) rather than a box full of electromechanical parts.
The logic is usually designed as if it were relays, using a special computer language
called ladder logic. In PLCs, timers are usually simulated by the software built into the
controller. Each timer is just an entry in a table maintained by the software.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – III
Instruments: use, care and maintenance

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Water bath
 Oven & incubator
 Distillation apparatus- water distillation plant and water deionizers
 Reflux condenser
 Cyclomixers
 Magnetic stirrer
 Shakers
 Refrigerators: deep freezers, cold box
 Centrifuges: principal, Svedberg unit, centrifugal force, centrifugal field, rpm,
conversion of G to rpm and vice versa, components and working
 Different types of centrifuges
 Laboratory balances: physical and analytical. Mono & double pan, electronic balances
 Weighing different types of chemicals, liquids, hygroscopic compounds
 Precautionary measures while handling
 Photometry – colorimeter : principal, limitations of beer lamberts law, components,
working
 PH meter: principal, components PH measuring electrodes, Working, precautions taken
while handling

Water bath
Water bath is a device that is used in the laboratory where the water sample is kept at a
constant temperature. The temperature can be controlled using a dial or a once set that
cause the water bath to cycles to an on and off constancy of temperature. Some water
bath has an additional mechanism that allows the water bath to shake or stir that can be
set at a varying speed.

Proper use of hot water bath.

1. Fill the water bath before switching on.
2. Accurate bath fluid used: suitable liquid with its suitable temperature must be
followed. Alcohol with a low boiling point can cause safety hazard as it can give
off harmful fumes (example ethylene glycol).
3. Electrical supply must be in the correct voltage needed for the equipment.
4. Optimal condition of hot water bath, safe position.
5. Accurate water levels: water level must be above 2 inch of the heater and below 1
inch above the top of the water bath.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 6. The use of base tray for a flat-bottomed vessel or object to avoid damage to the
heater under the tank.
7. Turn on the water bath.
8. Set the temperature to its required temperature using the temperature control
and indicator.
9. The light indicator will be turn on to indicate the heater is on.
10. For a temperature above 60˚C, the lid provided must be used to cover the water
bath; this is to avoid excessive evaporation of water.
11. Place the specimen or solution needed.
12. After use, drain the water and clean the equipment.

Maintenance and care

Water bath should be drained, cleaned and re-filled weekly to avoid contamination and
build up of salt.
Use of oxygenated water to avoid rust.
Regular heating at a temperature of >60˚C for 30 minutes for biological application.
Avoid running the water bath to dry.

Cleaning and servicing

Use of mild household or moderate laboratory detergent can be used.
Scouring powder, steel wool or abrasive pads should be avoided.
Small spots due to small ferrous particles can be cleaned by standard stainless steel
cleaner and plastic scour.
Clean outside of the water bath with a damn cloth of water solution only.
Regular cleaning will ensure the water bath to maintain its optimal condition and
provide long years of service.
Service the water bath according to the protocal provided (this is different with each
company and product thus it is advisable to contact the manufacturer and as for
advice).

Oven
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Laboratory ovens are standard equipment found in most clinical, forensic, electronics,
material processing, and, research laboratories. Laboratory ovens provide uniform
temperature and precise temperature control for heating, baking, evaporating,
sterilizing and other industrial laboratory functions.
Principle
The principle of Hot Air Oven functioning depends on a precise pressure of air
convection in a power heated inner chamber. The double-walled design of the unit and
an automated control system ensure the homogenous heat and the precise sequence of
operations as well as quick recovery time after door opening. Hot Air Oven is best fitted
for the mild process of heating and drying of basic substances/instruments.
The working principle is based on fine gravity air convection in an electrically
heated chamber. The machine is equipped with various components to ensure the
uniform heating throughout the chamber. Two jacket design, automatic control unit,
PID controlled, PT sensors, temperature preset, etc. allows the machine to work
efficiently and return to normal temperature when the test process is over. It is
designed to simplify the process of dry sterilization and pre-treatment of different
materials like rubber, plastic, etc.
Inside the chamber the air flows in a forced circulation manner, this allows the
appropriate heat distribution inside the chamber. As the air inside the chamber becomes
hot, it becomes lighter and moves in the upward direction. As it reaches the top, the fan
inside the chamber pushes it back to the bottom. This creates a circular motion inside
the cabinet and makes a consistent circular flow of the air. With this process, eventually,
the optimum temperature is achieved.
It is always to be taken care that articles kept for sterilization should not be taken
out immediately. Let the oven comes to a normal temperature and then the articles are
taken out.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure BI 11.19.8: Hot Air Oven
Applications of Hot Air Oven
Items that are sterilized in a hot air oven include:
 Glassware (petri dishes, flasks, pipettes, and test tubes)
 Powders (starch, zinc oxide, and sulfadiazine)
 Materials that contain oils
 Metal equipment (scalpels, scissors, and blades)
 Glass test tubes can be sterilized using a hot air oven
 Glass test tubes can be sterilized using a hot air oven
 Hot air ovens use extremely high temperatures over several hours to destroy
microorganisms and bacterial spores. The ovens use conduction to sterilize
items by heating the outside surfaces of the item, which then absorbs the heat
and moves it towards the center of the item.

Note: Items that are not sterilized in a hot air oven are surgical dressings, rubber items,
or plastic material.

Incubators
An incubator is a device used to grow and maintain microbiological cultures or cell
cultures. The incubator maintains optimal temperature, humidity and other conditions
such as the CO2 and oxygen content of the atmosphere inside. Incubators are essential
for much experimental work in cell biology, microbiology and molecular biology and
are used to culture both bacterial and eukaryotic cells.
Louis Pasteur used the small opening underneath his staircase as an incubator.
Incubators are also used in the poultry industry to act as a substitute for hens. This
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
often results in higher hatch rates due to the ability to control both temperature and
humidity. Various brands of incubators are commercially available to breeders.
The simplest incubators are insulated boxes with an adjustable heater, typically
going up to 60 to 65 °C (140 to 150 °F), though some can go slightly higher (generally to
no more than 100 °C). The most commonly used temperature both for bacteria such as
the frequently used E. coli as well as for mammalian cells is approximately 37 °C (99 °F),
as these organisms grow well under such conditions. For other organisms used in
biological experiments, such as the budding yeast Saccharomyces cerevisiae, a growth
temperature of 30 °C (86 °F) is optimal.
More elaborate incubators can also include the ability to lower the temperature
(via refrigeration), or the ability to control humidity or CO2 levels. This is important in
the cultivation of mammalian cells, where the relative humidity is typically >80% to
prevent evaporation and a slightly acidic pH mis achieved by maintaining a CO2 level
of 5%.

Principle/ Working of Incubator

 An incubator is based on the principle that microorganisms require a particular set
of parameters for their growth and development.
 All incubators are based on the concept that when organisms are provided with
the optimal condition of temperature, humidity, oxygen, and carbon dioxide
levels, they grow and divide to form more organisms.
 In an incubator, the thermostat maintains a constant temperature that can be read
from the outside via the thermometer.
 The temperature is maintained by utilizing the heating and no-heating cycles.
 During the heating cycle, the thermostat heats the incubator, and during the no-
heating period, the heating is stopped, and the incubator is cooled by radiating
heat to the surrounding.
 Insulation from the outside creates an isolated condition inside the cabinet, which
allows the microbes to grow effectively.
 Similarly, other parameters like humidity and airflow are also maintained through
different mechanisms that create an environment similar to the natural
environment of the organisms.
 Similarly, they are provided with adjustments for maintaining the concentration of
CO2 to balance the pH and humidity required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows
for the continuous movement of the culture required for cell aeration and
solubility studies.

On the basis of the presence of a particular parameter or the purpose of the incubator,
incubators are divided into the following types:

Benchtop incubators
 This is the most common type of incubator used in most of the laboratories.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  These incubators are the basic types of incubators with temperature control and
insulation.

CO2 incubators
 CO2 incubators are the special kinds of incubators that are provided with
automatic control of CO2 and humidity.
 This type of incubator is used for the growth of the cultivation of different bacteria
requiring 5-10% of CO2 concentration.
 For humidity control, water is kept underneath the cabinet of the incubator.

Cooled incubators
 For incubation at temperatures below the ambient, incubators are fitted with
modified refrigeration systems with heating and cooling controls.
 This type of incubator is called the cooling incubator.
 In the cooling incubator, the heating and cooling controls should be appropriately
balanced.

Shaker incubator
 A thermostatically controlled shaker incubator is another piece of apparatus used
to cultivate microorganisms.
 Its advantage is that it provides a rapid and uniform transfer of heat to the culture
vessel, and its agitation provides increased aeration, resulting in acceleration of
growth.
 This incubator, however, can only be used for broth or liquid culture media.

Portable incubator
 Portable incubators are smaller in size and are used in fieldwork, e.g.
environmental microbiology and water examination.

Uses of Incubator
Incubators have a wide range of applications in various areas including cell culture,
pharmaceutical studies, hematological studies, and biochemical studies.
Some of the uses of incubators are given below:
1. Incubators are used to grow microbial culture or cell cultures.
2. Incubators can also be used to maintain the culture of organisms to be used later.
3. Some incubators are used to increase the growth rate of organisms, having a
prolonged growth rate in the natural environment.
4. Specific incubators are used for the reproduction of microbial colonies and
subsequent determination of biochemical oxygen demand.
5. These are also used for breeding of insects and hatching of eggs in zoology.
6. Incubators also provide a controlled condition for sample storage before they can
be processed in the laboratories.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
The following precautions are to be followed while running an incubator:
1. As microorganisms are susceptible to temperature change, the fluctuations in
temperature of the cabinet by repeatedly opening the door should be avoided.
2. The required parameters growth of the organism should be met before the culture
plates are placed inside the cabinet.
3. The plates should be placed upside down with the lid at the bottom to prevent the
condensation of water on to the media.
4. The inside of the incubators should be cleaned regularly to prevent the organisms
from settling on the shelves or the corners of the incubator.
5. While running the incubator for an extended period of time, sterile water should
be placed underneath the shelves to prevent the culture media from drying out.

Distillation apparatus: water and steel

Distillation is the process of separating the components or substances from a

liquid mixture by using selective boiling and condensation. Distillation may result in
essentially complete separation (nearly pure components), or it may be a partial
separation that increases the concentration of selected components in the mixture. In
either case, the process exploits differences in the relative volatility of the mixture's
components. In industrial applications, distillation is a unit operation of practically
universal importance, but it is a physical separation process, not a chemical reaction.
Distillation has many applications. For example:
 The distillation of fermented products produces distilled beverages with a
high alcohol content, or separates other fermentation products of commercial value.
 Distillation is an effective and traditional method of desalination.
 In the petroleum industry, oil stabilization is a form of partial distillation that
reduces the vapor pressure of crude oil, thereby making it safe for storage and
transport as well as reducing the atmospheric emissions of volatile hydrocarbons. In
midstream operations at oil refineries, fractional distillation is a major class
of operation for transforming crude oil into fuels and chemical feed stocks.
 Cryogenic distillation leads to the separation of air into its components –
notably oxygen, nitrogen, and argon – for industrial use.
 In the chemical industry, large amounts of crude liquid products of chemical
synthesis are distilled to separate them, either from other products, from impurities,
or from unreacted starting materials.

The method of distillation has a considerable history, dating back to 3000 BC. Evidence
suggests that the distillation of alcohol was developed as far back as the 9th century.
Some important applications of distillation are listed below.
 Distillation plays an important role in many water purification techniques. Many
desalination plants incorporate this method in order to obtain drinking water
from seawater.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Distilled water has numerous applications, such as in lead-acid batteries and
low-volume humidifiers.
 Many fermented products such as alcoholic beverages are purified with the help
of this method.
 Many perfumes and food flavorings are obtained from herbs and plants via
distillation.
 Oil stabilization is an important type of distillation that reduces the vapor
pressure of the crude oil, enabling safe storage and transportation.
 Air can be separated into nitrogen, oxygen, and argon by employing the process
of cryogenic distillation.
 Distillation is also employed on an industrial scale to purify the liquid products
obtained from chemical synthesis.

Water deionizers

Water Deionizer Systems: Deionization is a process done when there is an immediate

need of purified water distribution. It is imperative that deionization is performed
when the water is close to being utilized since extremely high water purity degrades
quickly. Deionization systems work by replacing negative and positive molecules in the
water with hydrogen (positive) and hydroxyl (negative) molecules. In effect, organic
substances are removed through filtration which improves the quality of the water and
prevents the formation of scale deposits forming. For this reason, deionized water is one
of the most preferred options of use in factories and manufacturing facilities.

Reflux condenser
A Reflux Condenser is a machine that uses a distillation technique known as reflux to
liquefy the vapor that returns to its condensate form to the system from which it
originated. In this technique, large scale distillation columns as well as fractionators are

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
utilized. Reflux condensers are used in industrial as well as laboratory distillation
processes.
Unlike distillation, the reflux process is used in chemistry to accelerate a specific
reaction thermally. This is done by conducting it in a controlled high temperature. The
role of a condenser here is to cool down the generated vapours and convert them back
into the liquid form. The liquid component is then sent back to the boiler. So the reflux
condenser purpose is to stop the loss of solvent, thereby increasing the reaction time
over which the flask can be heated.

Cyclomixers
Variable speed mixer to eliminate hand mixing. Holding tube against vibrating rubber
cup does rapid mixing of contents. Speed regulator controls the degree of vibration. A
unique touch feature operates the unit when the tube is pressed on the rubber cup.
Interchangeable adapters greatly enhance the use of Cyclo Mixer for mixing in different
applications.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Features of Cyclomixer
 Designed for mixing liquids in Schools, Laboratories & Factories
 Touch/ Continuous Operation mode Selection through bi-directional Switch
 Speed Regulation through knob provided on the control panel
 Interchangeable mixing heads for use with a variety of tubes
 Supplied with all interchangeable mixing heads

Magnetic stirrer
A magnetic stirrer or magnetic mixer is a laboratory device that employs a rotating
magnetic field to cause a stir bar immersed in a liquid to spin very quickly, thus stirring
it. The rotating field may be created either by a rotating magnet or a set of stationary
electromagnets, placed beneath the vessel with the liquid. Since glass does not affect a
magnetic field appreciably, and most chemical reactions take place in glass vessels,
magnetic stir bars work well in glass vessels. On the other hand, the limited size of the
bar means that magnetic stirrers can only be used for relatively small experiments. They
also have difficulty dealing with viscous liquids or thick suspensions. For larger
volumes or more viscous liquids, some sort of mechanical stirring is typically needed.
Magnetic stirrers are often used in chemistry and biology. They are preferred over gear-
driven motorized stirrers because they are quieter, more efficient, and have no moving
external parts to break or wear out. Because of its small size, a stirring bar is more easily
cleaned and sterilized than other stirring devices. They do not require lubricants which
could contaminate the reaction vessel and the product. They can be used inside
hermetically closed vessels or systems, without the need for complicated rotary seals.
Magnetic stirrers may also include a hot plate or some other means for heating the
liquid.
Magnetic stirrers use a rotating magnetic field to move a stir bar around in liquid
samples. The movement of this stir bar mixes the sample thoroughly with rapid
movement and agitation. The speed of the magnetic field is controlled by the user, so it

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
can be customized to the specific sample that‘s being stirred. These stirrers should be
used with glass or other non-metal beakers to prevent interference with the magnetic
field.
Magnetic stirring equipment permits variable speed stirring depending on the
requirement. It is advisable to use a gentle stirring instead of a violent one, bearing in
mind not only the stirring of the liquid culture medium and expose the cells to the gas
on the surface, but also to avoid damage to the cell.

Shakers
A shaker is a piece of laboratory equipment used to mix, blend, or agitate substances in
a tube or flask by shaking them. It is mainly used in the fields of chemistry and biology.
A shaker contains an oscillating board that is used to place the flasks, beakers, or test
tubes. Although the magnetic stirrer has lately come to replace the shaker, it is still the
preferred choice of equipment when dealing with large volume substances or when
simultaneous agitation is required
Laboratory mixers and shakers are instruments that help to form a homogenous
mixture from more than one ingredient. Used in many types of industry, such as food
and beverage, cosmetic, pharmaceutical, and electronics, as well as labs that deal with
life sciences, wastewater treatment, and biotech, laboratory mixers and shakers are an
important part of many labs.
Laboratory shakers do just that—-shake the mixtures that are placed on them.
Plates vibrate back and forth or circularly to mix the components. Some laboratory
shakers have plates that tilt up and down as well as side to side for an added mixing
element. Laboratory mixers, on the other hand, have a tool, such as a paddle or blade,
that goes into the sample to be mixed and mechanically stirs it.

Types of shakers

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Vortex shaker: Invented by Jack A. Kraft and Harold D. Kraft in 1962, a vortex shaker is
a usually small device used to shake or mix small vials of liquid substance. Its most
standout characteristic is that it works by the user putting a vial on the shaking
platform and turning it on; thus, the vial is shaken along with the platform. A vortex
shaker is very variable in terms of speed adjustment, for the shaking speed can be
continuously changed while shaking by turning a switch.

Platform shaker: A platform shaker has a table board that oscillates horizontally. The
liquids to be stirred are held in beakers, jars, or erlenmeyer flasks that are placed over
the table or, sometimes, in test tubes or vials that are nested into holes in the
plate. Platform shakers can also be combined with other systems like rotating mixers for
small systems and have been designed to be manufactured in laboratories themselves
with open source scientific equipment.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Orbital shaker: An orbital shaker has a circular shaking motion with a slow speed (25-
500 rpm). It is suitable for culturing microbes, washing blots, and general mixing. Some
of its characteristics are that it does not create vibrations, and it produces low heat
compared to other kinds of shakers, which makes it ideal for culturing microbes.
Moreover, it can be modified by placing it in an incubator to create an incubator shaker
due to its low temperature and vibrations.

Incubator shaker: An incubator shaker (or thermal shaker) can be considered a mix of
an incubator and a shaker. It has an ability to shake while maintaining optimal
conditions for incubating microbes or DNA replications. This equipment is very useful
since, in order for a cell to grow, it needs oxygen and nutrients that require shaking so
that they can be distributed evenly around the culture.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
A vortex shaker in use.
Anyone employing an incubator shaker (thermal shaker) to grow yeast or bacteria in
the laboratory needs to beware that under the usual conditions encountered in the lab,
the rate at which oxygen diffuses from the gaseous phase into the shaken liquid phase is
too slow to keep up with the rate at which the oxygen is consumed by for example E
coli dividing every half hour or S cerevisiae dividing every hour. If the investigator
measure the oxygen in the shake flask on the shaker -- polarographically, for example --
at mid-exponential phase of growth, the dissolved oxygen concentration will turn out to
be zero.

Refrigerators

A laboratory refrigerator is a common laboratory appliance that consists of a thermally

insulated compartment and a heat pump (mechanical, electronic, or chemical) which
transfers heat from the inside of the unit to its external environment so that the inside is
cooled to a temperature below the ambient temperature of the room.
It is important that you know how a laboratory refrigerator is composed, in this
way, you will be able to identify each of its functions, parts and elements, getting you to
fully familiarize yourself with its use.
The laboratory refrigerator is constituted by an evaporator, you can also observe
a conservation cabinet where you can place each of the substances or samples and,
finally, the thermostat that allows you to control the temperature and the cooling
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
system. Usually, the evaporator can be found on the top of the refrigerator cabinet, to
know that the refrigerator is in use, it determines that the evaporator is below 0 ° C,
even if you are not alarmed if it reaches temperatures of 5 ° C to 30 ° C.
In another sense, the cabinet has a space under the evaporator, where you can
place shelves for the storage of samples or substances. Generally, the laboratory
refrigerator has an appearance similar to that of a domestic one, therefore, it will not be
so complex to identify it and know what its function is.
Although it can be simple to identify, the laboratory refrigerator has the main
function of properly conserving any type of substance, either liquid or homogeneous, so
that, when performing the analyzes, they generate the results correct.
These laboratory elements, which contribute greatly to the management and
conservation of substances, must be taken as essential and vital in any laboratory,
however small it may be.

Deep freezers
Deep freezers are the testing equipment that are used to preserve and store food
products, medical equipment, blood samples, medicines and injections, etc. for a long
period of time. Deep Freezers are used for industrial purposes as well as for household
purposes. Moreover, deep freezers are also used in restaurants and supermarkets to
preserve the raw food for a long period of time. There are numerous types of deep
freezers such as blood bank refrigerators, freezer drier, ultra-low deep freezer, deep
freezer vertical and many more. These devices are available in different sizes and
shapes sometimes it is designed with compact designs and sometimes with regular
designs. The specifications and functions of the instruments varies as per the
requirements of the test application.

Ultra-low deep freezer is highly sophisticated testing machine which designed

and fitted with a cooling compressors and CFC free refrigerants. These freezers are
installed to create highly effective cooling consistently inside the cabinet. The air
cooling compressors of the freezer are designed with aerodynamic fans and washable
condense filters which keep the internal environment free from dirt and dust. The
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
instrument helps to look into the mechanical behavior and characteristics of the rubber
and polymer and other medical or industrial products at low temperatures. The initial
stages of the test procedure of the device involve a satisfactory technique for low
temperature testing. Simple temperature effect includes the glass transition,
crystallization, effects of solubility and many more.

Cold box
Cold boxes are (pressure) vessels that hold a gas or liquid at a very low temperature.
The distinctive feature of cold boxes is the double-wall construction, which allows the
insulation to be fitted between the inner and outer walls

Centrifuges: principal, Svedberg unit, centrifugal force, centrifugal field,

rpm, conversion of G to rpm and vice versa, components and working,
Different types of centrifuges

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Principal
Centrifuges are used for many separation processes in the laboratory (Figure BI
11.19.9).The centrifuge works on the sedimentation principle: substances are
separated according to their density under the influence of g-force. Depending on
the sample type, there are several centrifugation methods available. Examples
include isopycnic, ultrafiltration, density gradient, phase separation, and pelleting.
Centrifugation is a technique used for the separation of particles using a
centrifugal field. The particles are suspended in liquid medium and placed in a
centrifuge tube. The tube is then placed in a rotor and spun at a definitive speed.
Rotation of the rotor about a central axis generates a centrifugal force upon the particles
in the suspension.

Two forces counteract the centrifugal force acting on the suspended particles:
 Buoyant force: This is the force with which the particles must displace the liquid
media into which they sediment.
 Frictional force: This is the force generated by the particles as they migrate
through the solution.
Particles move away from the axis of rotation in a centrifugal field only when the
centrifugal force exceeds the counteracting buoyant and frictional forces resulting in
sedimentation of the particles at a constant rate. Particles which differ in density, size or
shape sediment at different rates. The rate of sedimentation depends upon:
 The applied centrifugal field
 Density and radius of the particle.
 Density and viscosity of the suspending medium.
Angular velocity = w radians / second;
since one revolution = 360 = 2p radians,

(r = radial distance of the particle from the axis of rotation)

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
As the centrifugal field acting on the particle is much greater than the Earth's
gravitational field, CF is generally expressed relative to the Earth's gravitational field as
multiples of g, the acceleration due to gravity (g= 980 cm/s2)

This expression relates relative centrifugal field (RCF) to the speed of the centrifuge
(rpm) and and the radius of the rotor (r). For example, if a rotor with an average radius
of 7 cm revolves at a speed of 20,000 rpm, a centrifugal field of 31,300 g is created.
The sedimentation rate of velocity (v) of a particle can be expressed in terms of its
sedimentation rate per unit centrifugal field. This is termed as sedimentation coefficient
(s). The sedimentation rate is proportional to w2 r, the centrifugal field,

Sedimentation velocity depends upon the mass of the particle, its density, shape and
also on the density and viscosity of the medium in which the particle is suspended.

Figure BI 11.19.9: Centrifuge

Applications of Centrifuge
In summary, Centrifugation is the process of using centrifugal force to separate the
lighter portion of solution, mixture or suspension from the heavier portions. In
laboratory centrifuge is used to:

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Remove cellular debris from blood to separate cell free plasma or serum
 Concentrate cellular elements and other components for microscopic analysis or
chemical analysis.
 Separate protein bound or antibody bound ligand from free ligand in
immunological assay.
 Extract solutes from aqueous or organic solvents.
 Separate lipid components like chylomicrons from other components of plasma
 To separate two miscible substances
 To analyze the hydrodynamic properties of macromolecules
 Purification of mammalian cells
 Fractionation of sub-cellular organelles (including membranes / membrane
fractions) Fractionation of membrane vesicles
 Separating chalk powder from water
 Removing fat from milk to produce skimmed milk
 Separating particles from an air-flow using cyclonic separation
 The clarification and stabilization of wine
 Separation of urine components and blood components in forensic and research
laboratories
 Aids in separation of proteins using purification techniques such as salting out,
e.g. ammonium sulfate precipitation.

Svedberg
It is a unit of time amounting to 10−13 second that is used to measure the
sedimentation velocity of a colloidal solution (as of a protein) in an ultracentrifuge
and to determine molecular weight by substitution in an equation
A Svedberg unit (symbol S, sometimes Sv) is a non-SI metric unit for
sedimentation coefficients. The Svedberg unit offers a measure of a particle's size based
on its sedimentation rate under acceleration (i.e. how fast a particle of given size and
shape settles to the bottom of a solution). The Svedberg is a measure of time, defined as
exactly 10−13 seconds (100 fs).

Relative Centrifugal Force

The relative centrifugal force (RCF) or the g force is the radial force generated by the
spinning rotor as expressed relative to the earth's gravitational force. The g force acting
on particles is exponential to the speed of rotation defined as revolutions per minute
(RPM).

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Doubling the speed of rotation increases the centrifugal force by a factor of four. The
centrifugal force also increases with the distance from the axis of rotation. These two
parameters are of considerable significance when selecting the appropriate centrifuge.

Relative Centrifugal Force (RCF) Formula

Using the G Force Formula
RCF or g force is dependent on the speed of rotation in RPM and the distance of the
particles from the center of rotation. When the speed of rotation is given in RPM and
the distance (r) is expressed in centimeters, RCF can be calculated by using the
following formula.
G Force Formula
g Force (RCF) = (RPM)2 × 1.118 × 10-5 × r
When the RPM is unknown and you are given a g force and radius (r), you can calculate
RPM using the following formula:
RPM Formula
RPM = √[RCF/(r × 1.118)] × 1 × 105
RCF (g force) = relative centrifugal force
r = rotational radius (cm)

Centrifuge Rotors
Rotors in centrifuges are the motor devices that house the tubes with the samples.
Centrifuge rotors are designed to generate rotation speed that can bring about the
separation of components in a sample. There are three main types of rotors used in a
centrifuge, which are:
Fixed angle rotors
 These rotors hold the sample tubes at an angle of 45° in relation to the axis of the
rotor.
 In this type of rotor, the particles strike the opposite side of the tube where the
particles finally slide down and are collected at the bottom.
 These are faster than other types of rotors as the pathlength of the tubes increases.
 However, as the direction of the force is different from the position of the tube,
some particles might remain at the sides of the tubes.

Swinging bucket rotors/ Horizontal rotors

 Swinging bucket rotors hold the tubes at an angle of 90° as the rotor swings as the
process is started.
 In this rotor, the tubes are suspended in the racks that allow the tubes to be moved
enough to acquire the horizontal position.
 In this type of rotors, the particles are present along the direction or the path of the
force that allows the particles to be moved away from the rotor towards the
bottom of the tubes.
 Because the tubes remain horizontal, the supernatant remains as a flat surface
allowing the deposited particles to be separated from the supernatant.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Vertical rotors
 Vertical rotors provide the shortest pathlength, fastest run time, and the highest
resolution of all the rotors.
 In vertical rotors, the tubes are vertical during the operation of the centrifuge.
 The yield of the rotor is not as ideal as the position of the tube doesn‘t align with
the direction of the centrifugal force.
 As a result, instead of settling down, particles tend o spread towards the outer wall
of the tubes.
 These are commonly used in isopycnic and density gradient centrifugation.

Types of centrifuges
Benchtop centrifuge
 Benchtop centrifuge is a compact centrifuge that is commonly used in clinical and
research laboratories.
 It is driven by an electric motor where the tubes are rotated about a fixed axis,
resulting in force perpendicular to the tubes.
 Because these are very compact, they are useful in smaller laboratories with
smaller spaces.
 Different variations of benchtop centrifuges are available in the market for various
purposes.
 A benchtop centrifuge has a rotor with racks for the sample tubes and a lid that
closes the working unit of the centrifuge.

Continuous flow centrifuge

 Continuous flow centrifuge is a rapid centrifuge that allows the centrifugation of
large volumes of samples without affecting the sedimentation rates.
 This type of centrifuge allows the separation of a large volume of samples at high
centrifugal force, thus removing the tedious part of emptying and filling the tubes
with each cycle.
 They have a shorter pathlength which facilitates the process of pelleting out the
solid part out of the supernatant, thus maintaining the speed of the process.
 They also have larger capacities which saves time as the sample doesn‘t have to be
load and unloaded over and over again like in traditional centrifuges.
 Up to 1 liter of samples can be centrifuged by this centrifuge at a time period of 4
hours or less.

Gas centrifuge
 Hematocrit centrifuges are specialized centrifuges used for the determination of
volume fraction of erythrocytes (RBCs) in a given blood sample.
 This centrifuge provides hematocrit values that can be used for testing in
biochemistry, immunity, blood test, and other general clinical tests.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Hematocrit centrifuges may be used to help diagnose blood loss, polycythemia (an
elevation of the erythrocyte count to above-normal levels), anemia, bone marrow
failure, leukemia, and multiple myeloma.
 The microhematocrit centrifuge quickly attains speeds of 11,000 rpm and RCFs of
up to 15,000 g to spin tube samples.
 The components of a hematocrit centrifuge are similar to that of the benchtop
centrifuge, but this centrifuge is specialized for the use of blood samples.

High-speed centrifuge
 High-speed centrifuge, as the name suggests, is the centrifuge that can be operated
at somewhat larger speeds.
 The speed of the high-speed centrifuge can range from 15,000 to 30,000 rpm.
 The high-speed centrifuge is commonly used in more sophisticated laboratories
with the biochemical application and requires a high speed of operations.
 High-speed centrifuges are provided with a system for controlling the speed and
temperature of the process, which is necessary for the analysis of sensitive
biological molecules.
 The high-speed centrifuges come with different adapters to accommodate the
sample tubes of various sizes and volumes.
 All three types of rotors can be used for the centrifugation process in these
centrifuges.

Low-speed centrifuge
 Low-speed centrifuges are the traditional centrifuges that are commonly used in
laboratories for the routine separation of particles.
 These centrifuges operate at the maximum speed of 4000-5000 rpm.
 These are usually operated under room temperature as they are not provided with
a system for controlling the speed or temperature of the operation.
 Swinging bucket and fixed angle type of rotors can be used in these centrifuges.
 These are easy and compact centrifuges that are ideal for the analysis of blood
samples and other biological samples.
 The low-speed centrifuge works on the same principle as all other centrifuges, but
the application is limited to the separation of simpler solutions.

Micro-centrifuge
 Microcentrifuges are the centrifuges used for the separation of samples with
smaller volumes ranging from 0.5 to 2 µl.
 Microcentrifuges are usually operated at a speed of about 12,000-13,000 rpm.
 This is used for the molecular separation of cell organelles like nuclei and DNA
and phenol extraction.
 Microcentrifuges, also termed, microfuge, use sample tubes that are smaller in size
when compared to the standard test tubes used in larger centrifuges.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Some microcentrifuges come with adapters that facilitate the use of larger tubes
along with the smaller ones.
 Microcentrifuges with temperature controls are available for the operation of
temperature-sensitive samples.

Refrigerated centrifuges
 Refrigerated centrifuges are the centrifuges that are provided with temperature
control ranging from -20°C to -30°C.
 A different variation of centrifuges is available that has the system of temperature
control which is essential for various processes requiring lower temperatures.
 Refrigerated centrifuges have a temperature control unit in addition to the rotors
and racks for the sample tubes.
 These centrifuges provide the RCF of up to 60,000 xg that is ideal for the separation
of various biological molecules.
 These are typically used for collecting substances that separate rapidly like yeast
cells, chloroplasts, and erythrocytes.
 The chamber of refrigerated centrifuge is sealed off from the outside to meet the
conditions of the operations.

Ultracentrifuges
 Ultracentrifuges are the centrifuges that operate at extremely high speeds that
allow the separation of much smaller molecules like ribosomes, proteins, and
viruses.
 It is the most sophisticated type of centrifuge that allows the separation of
molecules that cannot be separated with other centrifuges.
 Refrigeration systems are present in such centrifuges that help to balance the heat
produced due to the intense spinning.
 The speed of these centrifuges can reach as high as 150,000 rpm.
 It can be used for both preparative and analytical works.
 Ultracentrifuges can separate molecules in large batches and in a continuous flow
system.
 In addition to separation, ultracentrifuges can also be used for the determination of
properties of macromolecules like the size, shape, and density.

Vacuum centrifuge/ Concentrators

 Vacuum centrifuge utilizes the centrifugal force, vacuum and heat to speed up the
laboratory evaporation of samples.
 These centrifuges are capable of processing a large number of samples (up to 148
samples at a time).
 This type of centrifuge is used in chemical and biological laboratories for the
effective evaporation of solvents present in samples, thus concentrating the
samples.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  These are commonly used in high throughput laboratories for samples that might
have a large number of solvents.
 A rotary evaporator is used to remove the unnecessary solvents and eliminate
solvent bumping.
 The centrifuge works by lowering the pressure of the chamber, which also
decreases the boiling point of the samples.
 This causes the solvents to be evaporated, concentrating the particles to be
separated.

Laboratory balances: physical and analytical. Mono and double pan,

electronic balances. Weighing different types of chemicals, liquids,
hydroscopic compounds etc. precautionary measures while handling
(diagram).

Physical Balance is the general type of balance with a horizontal beam and two arms to
hold and measure weight. Such balances have been in use for centuries. A typical
physical balance consists of two weight pans each suspended by two side arms and a
pivoted horizontal beam. The functioning of this balance is very simple. The unknown
mass is kept in one pan, usually right, and standard reference weight is added on the
other pan until the horizontal beam comes close to equilibrium.
The physical balance is a measuring instrument useful at various places. The
balances may be availed in various customized designs. Some designs include a
protective case for keeping the balances safely. Each of the manufactured physical
balances has different weight measuring capacity for measuring weights of different
objects.

Analytical balances are highly sensitive lab instruments designed to accurately

measure mass. Their readability has a range between 0.1mg - 0.01mg. Analytical
balances have a draft shield or weighing chamber to prevent the very small samples
from being affected by air currents. They're meant to detect very fine increments, so the
slightest vibrations or breeze can impact the results. As such, analytical balances should
be used in a dedicated room with as few disturbances as possible. Analytical balances
need to be monitored carefully and calibrated frequently. Most analytical balances have
both automatic internal motorized calibration and calibration with external weights.
Analytical balances usually come with many features and functions. Most of
them have counting and check counting applications, for example. These can be useful
when counting pills, ingredients or very small pieces. Dynamic weighing is also very
useful, as it allows lab professionals to weigh unstable samples such as liquids, small
animals, or insects. Percentage weighing allows for quicker, more efficient formulation,
and is quite handy for chemistry and pharmaceutical applications. Accumulation
ensures you can have large results despite the balance's small capacity; instead of
having all the material on the balance at one time, you can accumulate the results of
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 doses or packaging to receive a total without putting all the product on the balance at
the same time.
A double-pan balance is a scale which has 2 pans that are balanced against each other.
The scale functions like a see-saw, with each of the 2 pans attached to a beam over a
centered pivot point.
Usage:
The object to be weighed is placed on 1 pan. The other pan is gradually loaded with
small weights until the scale balances, as shown by a measurement gauge reading "0."
The weights are added up to get the weight of the target object.

Mono pan balances: It is basically used for weighing things accurately in chemical
laboratories. Further, this Direct Reading Mono Pan Balance is widely appreciated for
its long term high accuracy and pocket friendly rates.

Features:
 The performance and reliability are high enough for use in chemical laboratories
 The synthetic ruby knife edge and synthetic sapphire bearing plate minimize the
nonuniform waring out of the edge and ensure long-term high accuracy.
 The arrestment system ensures high stability of the projection scale.
 Easy operation :
 The knob, dials and display are arranged at the top
 The Pan is as large as 90 mm in diameter. The space above the pan is as high as 175
mm, so that a large sample or a large container can be put on the pan.
 The floor/ Base of the balance case is made of glass
 Weight values are all displayed in digits, which are arranged at uniform intervals for
easy reading.

Electronic balances
Principle
The electronic balances in this modern era operate on magnetic force restoration
principle. The current required for opposing a weight‘s downward motion in the
magnetic area is measured by the detector. Checking about the accuracy includes
performing testing for linearity, cornerload, reproducibility, and calibration. Precision
measuring is a requirement in every kind of laboratories (Figure BI 11.19.6). The
accuracy expanding from one part per million is normal region for the range of weights
from one gram to one kilogram. Even for reagents preparation the need of high precise
measuring and also analysis has been reduced. For accurate periodic verifying of
mechanical pipettors, several laboratories utilize precision balances.

The operational principle of Laboratory Balances: A modern laboratory balance

working principle handles some of the similarity with its predecessor that is equal arm
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
parity. Older device reflects not known weight torque exerted on one pivot side to
adjusting known measurement on other side. So, when pointer returns the similar
areas, there should be equal torques and measurement is identified by the moving
weights position. This modern equivalence is known as magnetic force restoration. The
substance exerting force that is being measured is handled by the electromagnet. The
usual mechanism comprises of a wire coil that is suspended in the magnetic area. Since
the magnetic field is normally adjusted relative with the coil. At every point, current
flow direction and magnetic field direction are perpendicular to each other.
Therefore, force exerted is in axis direction of the coil. This wire coil will be
sustained by springs of precision which enables it in moving in its axis direction. The
coil position is detected by an optical sensor and offers a signal feedback to an electronic
amplifier. An electronic amplifier immediately regulates in maintaining coil position at
a reference point. Quantity of force that is exerted by wire coil is directly proportional to
current flowing amount in it. Thus, by weighing that current, force exerted can be
calculated. Equal arm balance will reflect two torques that are counteracting and leads
to the needle deflection for making it returning to reference position. While the force
restoration framework of electromagnetic restricts direct power applied by obscure
against customizable. And realized straight power applied by loop at an explicit spatial
point.

Figure BI 11.19.6: Single pan electronic balance

Applications of electronic balances

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  An electronic balance is is used very commonly in laborites for weighing
chemicals to ensure a precise measurement of those chemicals which are used in
various experiments.
 Electronic balances may also be used to weigh food and other grocery items
from home.
 Electronic balances provide their results digitally, making them an easy tool for
chemists to use.
 There are many different types of electronic balances available for purchase,
from small devices perfect for measuring foods or packages for postage at home
to larger, more expensive versions used in labs or government agencies.

Weighing different types of chemicals, liquids, hydroscopic compounds

etc. precautionary measures while handling (diagram).

So, instead of weighing materials directly on the balance pan, always weigh the
chemicals in or on something--a weighing dish, a beaker, or a piece of folded paper.
When you do, remember to weigh the container first or adjust for its weight. Be careful
not to spill any chemicals on or around the balance.

Weighing Techniques
1. Toploader
o Direct Weighing
1. With nothing on the pan, set to zero by pressing the "on" button.
2. Place weighing bottle, beaker, or vial on balance and set to zero again.
3. Use a clean scoopula to transfer sample into container slowly, until you reach the
desired mass.
o Indirect weighing (Weighing by difference)
1. Place enough of the sample in a weighing bottle, put the lid on, and place on the
scale. Record the mass.
2. Take some out and place it in a different container (whatever you will be using
for the experiment). Record the new mass.The difference in mass is the mass of
the sample transferred.
3. Continue this procedure until you have as much sample as you need.
4. It is best to transfer small amounts at a time, so you do not take more than you
need. You should not put excess sample back into the weighing bottle.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure 1. A toploading balance

Figure 2. An analytical balance

1. Analytical Balance
o Use the same procedure as with a toploader, remembering these additional points:
1. Close all the doors before taking measurements.
2. Remember the number of significant figures. It is higher than on a regular
toploader.
Make sure the sample is completely cooled when weighing. If a sample is still warm, it
will weigh less because of buoyancy due to upward circulation of hot air.

For example, a 50 mL beaker 3 minutes after removal from a 110 degree oven weighs
27.0271 g. At room temperature, it weighs 27.0410 g.

Photometry-colorimeter: principal, limitations of beer-lambert,s

law, components, working.
Photometry
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Photometry is the science of the measurement of light, in terms of its perceived
brightness to the human eye. It is distinct from radiometry, which is the science of
measurement of radiant energy (including light) in terms of absolute power. In modern
photometry, the radiant power at each wavelength is weighted by a luminosity
function that models human brightness sensitivity. Typically, this weighting function is
the photopic sensitivity function, although the scotopic function or other functions may
also be applied in the same way.
Calorimetry is the field of science that deals with the measurement of the state of a
body with respect to the thermal aspects in order to examine its physical and chemical
changes. The changes could be physical such as melting, evaporation or could also be
chemical such as burning, acid-base neutralization etc.
 A calorimeter is what is used to measure the thermal changes of a body.
 Calorimetry is applied extensively in the fields of thermochemistry in calculating
the enthalpy, stability, heat capacity etc.

Calorimeter
A calorimeter is a device used for heat measurements necessary for calorimetry. It
mainly consists of a metallic vessel made of materials which are good conductors of
electricity such as copper and aluminium etc. There is also a facility for stirring the
contents of the vessel. This metallic vessel with a stirrer is kept in an insulating jacket to
prevent heat loss to the environment. There is just one opening through which a
thermometer can be inserted to measure the change in thermal properties inside. Let us
discuss how exactly heat measurements are made. In the previous article, we discussed
the specific heat capacity of substances.

The basic instrument consists (Figure BI 11.18.2)

1. Light source
2. Filter
3. Monochromator (Wavelength sector to transmit selected wavelength)
4. Dispersive element (Collimator for straight light beam transmission)
5. Cuvette to place a sample.
6. Photoelectric detector
7. Recorder in dgital form

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure BI 11.18.2: Basic instrumentation of colorimeter

Principle
The working of colorimeters is mainly based on the Beer-Lambert‘s Law. This law states
that the light absorption when passes through a medium are directly proportional to the
concentration of the medium. When a colorimeter is used, there is a ray of light with a
certain wavelength is directed towards a solution. Before reaching the solution the ray
of light passes through a series of different lenses. These lenses are used for navigation
of the colored light in the colorimeter. The colorimeter analyzes the reflected light and
compares with a predetermined standard. Then a microprocessor installed in the device
is used for calculation of the absorbance of the light by the solution. If the absorption of
the solution is higher than there will be more light absorbed by the solution and if the
concentration of the solution is low then more lights will be transmitted through the
solution.
Note: There are many models of colorimeter available in market (Figure BI 11.19.4)

Figure BI 11.19.4: Colorimeter

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Colorimeter is based on the photometric technique which states that When a beam of
incident light of intensity I0 passes through a solution, a part of the incident light is
reflected (Ir), a part is absorbed (Ia) and rest of the light is transmitted (It)
Thus,
I0 = Ir + Ia + It
In colorimeter, (Ir) is eliminated because the measurement of (I0) and It is sufficient to
determine the (Ia). For this purpose, the amount of light reflected (I r) is kept constant by
using cells that have identical properties. (I0) & (It) is then measured.
The mathematical relationship between the amount of light absorbed and the
concentration of the substance can be shown by the two fundamental laws of
photometry on which the colorimeter is based.

Beer’s Law
This law states that the amount of light absorbed is directly proportional to the
concentration of the solute in the solution.
Log10 I0/It = asc
where,
as = Absorbency index
c = Concentration of Solution

Lambert’s Law
The Lambert‘s law state that the amount of light absorbed is directly proportional to the
length and thickness of the solution under analysis.
A = log10 I0/It = asb
Where,
A = Absorbance of test
as= Absorbance of standard
b = length / thickness of the solution
The mathematical representation of the combined form of Beer-Lambert‘s law is as
follows:
Log10 I0 / It = asbcIf b is kept constant by applying Cuvette or standard cell then,
Log10 I0/It = asc
The absorbency index as is defined as
as = A/cl
Where,
c = concentration of the absorbing material (in gm/liter).
l = distance in traveled by the light in solution (in cm).

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
In simplified form,
The working principle of the colorimeter is based on Beer-Lambert‘s law which states
that the amount of light absorbed by a color solution is directly proportional to the
concentration of the solution and the length of a light path through the solution.
A ∝ cl
Where,
A = Absorbance / Optical density of solution
c = Concentration of solution
l = Path length
A = ∈cl
∈ = Absorption coefficient

Measurement in a photoelectric colorimeter: In a photoelectric colorimeter, absorbance

of a substance is found out by measuring the percentage of incident light that is
transmitted by the solution
% Transmission = Intensity of emergent light/ Intensity of incident light x 100
Absorbance = log (1/T) = - log (T)
A more satisfying way of expressing % Transmittance is by optical density (OD)
OD = - log T
The emergent light rays are passed through a photocell which will convert the light
energy into electrical impulses. The current thus generated is measured by
galvanometer. Beer‘s law states that optical density is directly proportional to the
concentration. Thus if a graph is plotted with concentration on X axis and optical
density on Y axis a straight line will be obtained and the concentration could be directly
read. When beer‘s law is obeyed:

Concentration of Unknown = OD of Unknown

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Concentration of Standard OD of Standard
Therefore,

Concentration of Unknown = OD of Unknown X Conc. of Standard

Concentration of Standard OD of Standard

This is the basic principle of all colorimetric reactions

To measure the concentration of substance in test solution 3 solutions
have to be prepared:
 Test solution which is to be analyzed.
 Standard solution prepared from known quantity of the substance to be
estimated.
 Reagent blank containing all the reagents but without the substance to be
estimated.

Applications of colorimeter
 The colorimeter is commonly used for the determination of the concentration of
colored compounds by measuring the optical density or its absorbance.
 It is also used for the determination of the course of the reaction by measuring the
rate of formation and disappearance of the light absorbing compound in the
range of the visible region of electromagnetic spectrum.
 It is used in compound identified by determining the absorption spectrum in the
visible region of the light spectrum of the electromagnetic spectrum.
 It is widely used in hospital, laboratories for estimation of biochemical samples
like serum, plasma, CSF AND urine.
 Photoelectric Colorimeter is not just used for the lab purposes in the field of
biochemistry. The quality of water can also be measured with the help of this
device. Different chemicals and their qualities are tested simply by using this
amazing measuring device.

Difference between spectrophotometer & colorimeter

S. No Colorimeter Spectrophotometer
Light Source LED, Fixed Wavelength Lamp (Tungsten, Xenon, Deuterium),
Wavelength Range
Wavelength Color Filter, Fixed Monochromator, Wavelength Range
Selector Wavelength
Portability Stationary parts, light weight, Moving parts, heavier, good for bench
good for field use use. The DR 1900 can be used in the field.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 It is lighter and battery operated, but still
has some moving parts.
Parameters Single or Limited Number of Numerous Parameters determined by the
Parameters determined by Wavelength Range.
Fixed Wavelengths

Ph meter: principle, components, PH measuring electrodes, working,

precautions taken while handling.

PH meter is an electric tool used to measure hydrogen-ion activity (acidity or alkalinity)

in solution. Basically, a pH meter consists of a voltmeter attached to a pH-responsive
electrode and a reference (unvarying) electrode.
When one metal is brought in contact with another, a voltage difference occurs
due to their differences in electron mobility. When a metal is brought in contact with a
solution of salts or acids, a similar electric potential is caused, which has led to the
invention of batteries. Similarly, an electric potential develops when one liquid is
brought in contact with another one, but a membrane is needed to keep such liquids
apart.
A pH meter measures essentially the electro-chemical potential between a known
liquid inside the glass electrode (membrane) and an unknown liquid outside. Because
the thin glass bulb allows mainly the agile and small hydrogen ions to interact with the
glass, the glass electrode measures the electro-chemical potential of hydrogen ions or
the potential of hydrogen. To complete the electrical circuit, also a reference electrode is
needed. Note that the instrument does not measure a current but only an electrical
voltage, yet a small leakage of ions from the reference electrode is needed, forming a
conducting bridge to the glass electrode. A pH meter must thus not be used in moving
liquids of low conductivity (thus measuring inside small containers is preferable).
The pH meter measures the electrcal potential (follow the drawing clock-wise
from the meter) between the mercuric chloride ofthe reference electrode and its
potassium chloride liquid, the unknown liquid, the solution insie the glass electrode,
and the potential between that solution and the silver electrode. But only the potential
between the unknown liquid and the solution inside the glass electrode change from
sample to sample.
The calomel reference electrode consists of a glass tube with a potassium chloride
(KCl) electrolyte which is in intimate contact with a mercuric chloride element at the
end of a KCL element. It is a fragile construction, joined by a liquid junction tip made of
porous ceramic or similar material. This kind of electrode is not easily 'poisoned' by
heavy metals and sodium.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 The glass electrode consists of a sturdy glass tube with a thin glass bulb welded
to it. Inside is a known solution of potassium chloride (KCl) buffered at a pH of 7.0. A
silver electrode with a silver chloride tip makes contact with the inside solution. To
minimise electronic interference, the probe is shielded by a foil shield, often found
inside the glass electrode.
Most modern pH meters also have a thermistor temperature probe which allows
for automatic temperature correction, since pH varies somewhat with temperature
(Figure BI 11.19.1).

Figure BI 11.19.1: PH meter

Applications of PH meter
The pH meter is used in many applications ranging from laboratory
1. The rate and outcome of chemical reactions taking place in water often
depends on the acidity of the water, and it is therefore useful to know the
acidity of the water, typically measured by means of a pH meter.
2. Knowledge of pH is useful or critical in many situations, including chemical
laboratory analyses.
3. pH meters are used for soil measurements in agriculture, water quality for
municipal water supplies, swimming pools, environmental remediation;
brewing of wine or beer; manufacturing, healthcare and clinical applications
such as blood chemistry; and many other applications.
4. Advances in the instrumentation and in detection have expanded the number
of applications in which pH measurements can be conducted. The devices have
been miniaturized, enabling direct measurement of pH inside of living cells.
5. In addition to measuring the pH of liquids, specially designed electrodes are
available to measure the pH of semi-solid substances, such as foods. These
have tips suitable for piercing semi-solids; have electrode materials compatible
with ingredients in food, and are resistant to clogging.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 pH meter: A pH meter is an electronic device used for measuring the pH (acidity
or alkalinity) of a liquid (though special probes are sometimes used to measure
the pH of semi-solid substances). A typical pH meter consists of a special
measuring probe (a glass electrode) connected to an electronic meter that
measures and displays the pH reading Figure BI 11.2.4.
 The probe: The pH probe measures pH as the activity of the hydrogen cations
surrounding a thin-walled glass bulb at its tip. The probe produces a small
voltage (about 0.06 volt per pH unit) that is measured and displayed as pH units
by the meter.
 Calibration and use.
For very precise work the pH meter should be calibrated before each
measurement. For normal use calibration should be performed at the beginning
of each day. The reason for this is that the glass electrode does not give a
reproducible e.m.f. over longer periods of time. Calibration should be performed
with at least two standard buffer solutions that span the range of pH values to be
measured. For general purposes buffers at pH 4 and pH 10 are acceptable. The
pH meter has one control (calibrate) to set the meter reading equal to the value of
the first standard buffer and a second control (slope) which is used to adjust the
meter reading to the value of the second buffer. A third control allows the
temperature to be set. Standard buffer sachets, which can be obtained from a
variety of suppliers, usually state how the buffer value changes with
temperature. For more precise measurements, a three buffer solution calibration
is preferred. As pH 7 is essentially, a "zero point" calibration (akin to zeroing or
taring a scale or balance), calibrating at pH 7 first, calibrating at the pH closest to
the point of interest ( e.g. either 4 or 10) second and checking the third point will
provide a more linear accuracy to what is essentially a non-linear problem. Some
meters will allow a three point calibration and that is the preferred scheme for
the most accurate work. Higher quality meters will have a provision to account
for temperature coefficient correction, and high-end pH probes have temperature
probes built in. The calibration process correlates the voltage produced by the
probe (approximately 0.06 volts per pH unit) with the pH scale. After each single
measurement, the probe is rinsed with distilled water or deionized water to
remove any traces of the solution being measured, blotted with a scientific wipe
to absorb any remaining water which could dilute the sample and thus alter the
reading, and then quickly immersed in another solution.
 Storage conditions of the glass probes

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 When not in use, the glass probe tip must be kept wet at all times to avoid the pH
sensing membrane dehydration and the subsequent dysfunction of the electrode.
A glass electrode alone (i.e., without combined reference electrode) is typically
stored immersed in an acidic solution of around pH 3.0. In an emergency,
acidified tap water can be used, but distilled or deionised water must never be
used for longer-term probe storage as the relatively ionless water "sucks" ions
out of the probe membrane through diffusion, which degrades it. Combined
electrodes (glass membrane + reference electrode) are better stored immersed in
the bridge electrolyte (often KCl 3 M) to avoid the diffusion of the electrolyte
(KCl) out of the liquid junction.
 Cleaning and troubleshooting of the glass probes
Occasionally, the probe may be cleaned using pH-electrode cleaning solution;
generally a 0.1 M solution of hydrochloric acid (HCl) is used, having a pH of one.
In case of strong degradation of the glass membrane performance due to
membrane poisoning, diluted hydrofluoric acid (HF < 2 %) can be used to
quickly etch (< 1 minute) a thin damaged film of glass. Alternatively a dilute
solution of ammonium fluoride (NH4F) can be used. To avoid unexpected
problems, the best practice is however to always referring to the electrode
manufacturer recommendations or to a classical textbook of analytical chemistry.
 Types of pH meters
pH meters range from simple and inexpensive pen-like devices to complex and
expensive laboratory instruments with computer interfaces and several inputs
for indicator and temperature measurements to be entered to adjust for the slight
variation in pH caused by temperature. Specialty meters and probes are available
for use in special applications, harsh environments, etc.
 How a pH meter works
When one metal is brought in contact with another, a voltage difference occurs
due to their differences in electron mobility. When a metal is brought in contact
with a solution of salts or acids, a similar electric potential is caused, which has
led to the invention of batteries. Similarly, an electric potential develops when
one liquid is brought in contact with another one, but a membrane is needed to
keep such liquids apart.
A pH meter measures essentially the electro-chemical potential between a
known liquid inside the glass electrode (membrane) and an unknown liquid
outside. Because the thin glass bulb allows mainly the agile and small hydrogen
ions to interact with the glass, the glass electrode measures the electro-chemical
potential of hydrogen ions or the potential of hydrogen. To complete the

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 electrical circuit, also a reference electrode is needed. Note that the instrument
does not measure a current but only an electrical voltage, yet a small leakage of
ions from the reference electrode is needed, forming a conducting bridge to the
glass electrode. A pH meter must thus not be used in moving liquids of low
conductivity.

Figure BI 11.2.4: PH meter electrodes

The pH meter measures the electrical potential (follow the drawing clock-wise
from the meter) between the mercuric chloride of the reference electrode and its
potassium chloride liquid, the unknown liquid, the solution inside the glass
electrode, and the potential between that solution and the silver electrode. But
only the potential between the unknown liquid and the solution inside the glass
electrode change from sample to sample. So all other potentials can be calibrated
out of the equation.
The calomel reference electrode consists of a glass tube with a potassium
chloride (KCl) electrolyte which is in intimate contact with a mercuric chloride
element at the end of a KCL element. It is a fragile construction, joined by a
liquid junction tip made of porous ceramic or similar material. This kind of
electrode is not easily 'poisoned' by heavy metals and sodium.
The glass electrode consists of a sturdy glass tube with a thin glass bulb
welded to it. Inside is a known solution of potassium chloride (KCl) buffered at a
pH of 7.0. A silver electrode with a silver chloride tip makes contact with the
inside solution. To minimize electronic interference, the probe is shielded by a
foil shield, often found inside the glass electrode. Most modern pH meters also
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 have a thermostat temperature probe which allows for automatic temperature
correction, since pH varies somewhat with temperature.
Caring for a pH meter depends on the types of electrode in use. Study the
manufacturer's recommendations. When used frequently, it is better to keep the
electrode moist, since moisturizing a dry electrode takes a long time,
accompanied by signal drift. However, modern pH meters do not mind their
electrodes drying out provided they have been rinsed thoroughly in tap water or
potassium chloride. When on expedition, measuring sea water, the pH meter can
be left moist with sea water. However for prolonged periods, it is recommended
to moist it with a solution of potassium chloride at pH=4 or in the pH=4.01 acidic
calibration buffer. pH meters do not like to be left in distilled water.
Note that a pH probe kept moist in an acidic solution can influence results
when not rinsed before inserting it into the test vial. Remember that a liquid of
pH=4 has 10,000 more hydrogen ions than a liquid of pH=8. Thus a single drop
of pH=4 in a vial measuring 400 drops of pH=8 really upsets measurements!
Remember also that the calibration solutions consist of chemical buffers that 'try'
to keep pH levels constant, so contamination of the test vial with a buffer is really
serious.
Note: Note that the pH scale is logarithmic and that each next value contains
ten times less hydrogen ions. A pH=0 contains the most, and is highly acidic

 Determination of pH of acids and bases

Strong acids or bases are completely ionized in solution, so that the concentration
of free H+ or OH is the same as the concentration of the acid or base.

Strong acid; HNO3  H+ + NO-3

HCl  H+ + Cl-
Strong base NaOH  Na+ + OH-
The pH of such solutions can, therefore, be very easily calculated:
a) 0.01 mol (liter HCl, pH = - log 10 (10-2) = 2
b) 0.01 mol / liter NaOH, H+  = Kw = 10-14 = 10-12
[OH-] 10-2
pH = -log 10 (10-12) = 12
Weak acids or bases dissociate only to a limited extent and the concentration of
free H+ and OH- depends on the value of their dissociation constants.

Weak acid (formic acid);

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 a) HCOOH H+ + HCOO-
Weak base (aniline);
b) C6H5NH2 + H+ C6H5NH3+

As weak acids (and or bases) are only slightly ionized in solution and a true equilibrium
is established between the acid and the conjugate base, the pH determination is carried
out using
Henderson – Hasselbalch equation.

If HA represents a weak acid, then:

HA H+ + A-

The dissociation constant may be given as;

[H+] [A-]
Ka = ______________
[HA]

Ka [HA]
and [H+] = ___________

[A-]

Taking negative logarithms,

[HA]
-log10 [H+] = - log 10 Ka + (- log 10 __________ )
[A-]

[A-]
pH = pKa + log10 ______
[HA]

In general,
[Conjugate base]
pH = pKa log 10 ___________________

[Acid]
The activities of A-
and HA are not always known, so it is convenient to express A- and
HA as concentration terms. Thus:
CA - fA
pH = pKa + log _____ + log _____

CHA fHA
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
(fA- and fHA are the activity coefficient of A- and HA respectively)

Since log (fA- / fHA) is constant for a given acid, there activity coefficient can be
incorporated into the pKa term leading to another apparent dissociation constant pKa.

CA- [Salt]
 pH = pKa + log ____ = pKa + log _______

CHA [Acid]

This relationship is known as the Henderson-Hasselbalch equation, If the

concentrations of the acid and its conjugate base are equal;

Then pH = pKa + log 1

PH = pKa

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – IV
Units of measurement

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Metric System, common laboratory measurements, prefixes in metric system

A metric system is a system of measurement that succeeded the decimalized system

based on the metre introduced in France in the 1790s. The historical development of
these systems culminated in the definition of the International System of Units (SI),
under the oversight of an international standards body.
The metric system is a system of measurement that uses the meter, liter, and
gram as base units of length (distance), capacity (volume), and weight (mass)
respectively.

To measure smaller or larger quantities, we use units derived from the metric units

 The given figure shows the arrangement of the metric units, which are smaller or
bigger than the base unit.
 The units to the right of the base unit are smaller than the base unit. As we move to
the right, each unit is 10 times smaller or one-tenth of the unit to its left. So, a ‗deci‘
means one-tenth of the base unit, ‗centi‘ is one-tenth of ‗deci‘ or one-hundredth of the
base unit and ‗milli‘ is one-tenth of ‗centi‘ or one-thousandth of the base unit.
 The units to the left of the base unit are bigger than the base unit. As we move to the
left, each unit is 10 times greater than the unit to its right. So, a ‗deca‘ means ten
times of the base unit, ‗hecto‘ is ten times of ‗deca‘ or hundred times of the base unit
and ‗killo‘ is ten times of ‗hecto‘ or thousand times of the base unit.

Kilo Hecto Deca Base Unit Deci Centi Milli

1000 100 10 1 1/10 1/100 1/1000
So, the units for length, weight (mass) and capacity(volume) in the metric system are:
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Length: Millimeter (mm), Decimeter (dm), Centimeter (cm), Meter (m), and Kilometer
(km) are used to measure how long or wide or tall an object is.
Examples include measuring the thickness or length of debit card, length of
cloth, or distance between two cities.

Kilometer Hectometer Decameter Meter Decimeter Centimeter Millimeter

(km) (hm) (dam) (m) (dm) (cm) (mm)
1000 100 10 1 1/10 1/100 1/1000

Weight: Gram (g) and Kilogram(kg) are used to measure how heavy an object, using
instruments.
Examples include measuring weight of fruits or, our own body weight.

Kilogram Hectogram Decagram Gram Decigram Centigram Milligram

(kg) (hg) (dag) (g) (dg) (cg) (mg)
1000 100 10 1 1/10 1/100 1/1000

Capacity: Milliliter (ml) and Liter (l) are used to measure how much quantity of liquid
an object can hold.
Examples include measuring the amount of juice in a juice can, or amount of water of in
a water tank.

Kiloliter Hectoliter Decaliter Liter Deciliter Centiliter Milliliter

(kl) (hl) (dal) (l) (dl) (cl) (ml)
1000 100 10 1 1/10 1/100 1/1000

Time: Second is the base unit for time. The other metric units of time are:

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Metric Conversions: Meters, grams and liters are considered the base units of length,
weight and volume, respectively.

Here‘s how we can multiply or divide for making metric conversions. To convert a
bigger unit to the smaller unit, we move left to write, we multiple by 10. Moving right
to left, from smaller unit to bigger, we divide by 10.

Let us look at some examples of converting from one unit to another.

Example 1: Convert 5 km to m.
As 1 km = 1000 m
Therefore, 5 km = 5 × 1000 = 5000 m

Example 2: Convert 250 kg to milligrams.

We know, 1 g = 1000 mg and 1 kg = 1000 g
So, we first convert the kg to g as:
1 kg = 1000 g
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Therefore, 250 kg = 250 × 1000 g = 250,000 g
Now, converting g to mg:
1 g = 1000 mg, therefore: 250,000 g = 250,000 × 1000 mg = 250,000,000 mg

Example 3: Convert 250 ml to liters.

1 liter = 1000 ml
Therefore, 450 ml = 450 ÷ 1000 = 0.45 liter

The US Standard Units or the Customary System uses customary units.

This system measures:
 Length or distance in inches, feet, yards, and miles.
 Capacity or volume in fluid ounces, cups, pints, quarts or gallons.
 Weight or mass in ounces, pounds and tons.

International system of units, SI units, definition, classification,

conversion of conventional and SI units

The international system of units is the most widely used system of measurement, and
is the modern form of the metric system. At its core, this system is built on seven
primary units. This system specifies twenty prefixes to the unit symbols and name to
state multiples and fractions of each unit.
The international system of units was introduced to the public in 1960 as a result
to earlier research that began in 1948. SI is based on the meter-kilogram second system
of units (MKS). SI was created to evolve as time progressed forward. It was intended
that units and prefixes were to be created and unit definitions to be modified, on an
international level, as the technology of the measuring tools we use on a daily basis
continues to become more advanced and more precise. An excellent example of this
evolving system would be the 24th and 25th General Conferences on Weights and
Measures (CGPM) that took place in 2011 and 2014 where a proposal was introduced to
alter the definition of ―kilogram‖. This was brought up because some began to believe
that a kilogram was an invariant of nature rather than a measurement of mass.
The International System of Units (abbreviated SI from systeme internationale, the
French version of the name) is a scientific method of expressing the magnitudes or
quantities of important natural phenomena. There are seven base units in the system,
from which other units are derived. This system was formerly called the meter-
kilogram-second (MKS) system.
All SI units can be expressed in terms of standard multiple or fractional
quantities, as well as directly. Multiple and fractional SI units are defined by prefix
multipliers according to powers of 10 ranging from 10 -24 to 10 24 .
SI base units:
The meter (abbreviation, m) is the SI unit of displacement or length. One meter is the
distance traveled by a ray of electromagnetic (EM) energy through a vacuum in
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
1/299,792,458 (3.33564095 x 10-9) second. The meter was originally defined as one ten-
millionth (0.0000001 or 107) of the distance, as measured over the earth's surface in a
great circle passing through Paris, France, from the geographic north pole to the
equator.
The kilogram (abbreviation, kg) is the SI unit of mass. It is defined as the mass of a
particular international prototype made of platinum-iridium and kept at the
International Bureau of Weights and Measures. It was originally defined as the mass of
one liter (10 -3 cubic meter) of pure water.
The second (abbreviation, s or sec) is the SI unit of time. One second is the time that
elapses during 9.192631770 x 10 9 cycles of the radiation produced by the transition
between two levels of Cesium 133. It is also the time required for an EM field to
propagate 299,792,458 (2.99792458 x 10 8 ) meters through a vacuum.
The kelvin (abbreviation K), also called the degree Kelvin (abbreviation, o K), is the SI
unit of temperature. One Kelvin is 1/273.16 (3.6609 x 10 -3 ) of the thermodynamic
temperature of the triple point of pure water (H 2 O).
The ampere (abbreviation, A) is the SI unit of electric current. One ampere is the
current that would produce a force of 0.0000002 (2 x 10 -7 ) newton between two
straight, parallel, perfectly conducting wires having infinite length and zero diameter,
separated by one meter in a vacuum. One ampere represents 6.24 x 10 18 unit electric
charge carriers, such as electrons, passing a specified fixed point in one second.
The candela (abbreviation, cd) is the SI unit of luminous intensity. It is the
electromagnetic radiation, in a specified direction, that has an intensity of 1/683 (1.46 x
10 -3 ) watt per steradian at a frequency of 540 terahertz (5.40 x 10 14 hertz).
The mole (abbreviation, mol) is the SI unit of material quantity. One mole is the
number of atoms in 0.012 kilogram of the most common isotope of elemental carbon (C-
12). This is approximately 6.022169 x 10 23 , and is also called the Avogadro constant.

SI derived units include the hertz , the newton , the pascal (unit of pressure or stress) ,
the ohm , the farad , the joule , the coulomb , the tesla , the lumen , the becquerel ,
the siemen , the volt , and the watt .

Base Units
Amount Unit Unit Definition
name symbol
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Length Meter M The distance traveled by light in a vacuum in
1/299792458 second.
Weight Kilogram Kg This is the unit for weight. The mass of the
international prototype kilogram.
Time Second S The duration of 9192631770 periods of the
radiation corresponding to the transition
between the two hyperfine levels of the
ground state of the cesium 133 atom.
Current Ampere A The constant current which, if maintained in
two straight parallel conductors of infinite
length, of negligible circular cross-section,
and placed 1 m apart in vacuum, would
produce between these conductors a force
equal to 2×10-7 newtons per meter of length.
Thermodynamic Kelvin K 1/273.16 of the thermodynamic temperature
temperature of the triple point of water.
Substance Mole mol The amount of substance of a system that
amount contains as many elementary entities as there
are atoms in 0.012 kilogram of carbon 12.
(Limited to objects with clarified
composition.) Elementary entities are
subatomic particles that compose matter and
energy.
Luminosity Candela cd The luminous intensity, in a given direction,
of a source that emits monochromatic
radiation of frequency 540×1012 hertz and
that has a radiant intensity in that direction of
1/683 watt per steradian.

Supplementary Units
Amount Unit Unit Definition
name symbol
Plane Radian Rad Radian describes the plane angle subtended by an
angle arc of a circle with the same length as the radius of
that circle corresponds to an angle of 1 radian.
Solid Steradian Sr A steradian is a solid angle at the center of a sphere
angle subtending a section on the surface equal in area to
the square of the radius of the sphere.

Derived Units
Derived units are a combination of base units and supplementary units and the
mathematical symbols of multiplication and division.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Amount Unit name Unit symbol
Area Square meter m2
Volume Cubic meter m3
Speed Meter per second m/s
Acceleration Meter per second squared m/s2
Wavenumber Reciprocal meter m-1
Density Kilogram per cubic meter kg/m3
Current density Ampere per square meter A/m2
Magnetic field strength Ampere per meter A/m
Concentration (of amount of substance) Mole per cubic meter mol/m3
Specific volume Cubic meter per kilogram m3/kg
Luminance Candela per square meter cd/m2

Some derived units are given unique names.

Amount Unit name Unit Composition
symbol
Frequency Hertz Hz 1Hz=1s-1
Force Newton N 1N=1kg･
m/s2
Pressure, stress Pascal Pa 1Pa=1N/m2
Energy, work, amount of heat Joule J 1J=1N･m
Power, radiant flux Watt W W=1J/s
Electric charge, amount of electricity Coulomb C 1C=1A･s
Electric potential/electric potential difference, Volt V 1V=1J/C
voltage, electromotive force
Resistance (electrical) Ohm Ω 1Ω=1V/A
Conductance (electrical) Siemens S 1S=1Ω-1
Magnetic Weber Wb 1Wb=1V･s
Magnetic flux density, magnetic induction Tesla T 1T=1Wb/m2
Inductance Henry H 1H=1Wb/A
Celsius temperature Degree ℃ 1t=T-To
Celsius
Luminous flux Lumen lm 1lm=1cd･sr
Illuminance Lux lx 1lx=1lm/m2

Reference Information
SI unit prefixes indicating integer powers of ten
Factor Prefix Symbol Factor Prefix Symbol
1018 exa E 10-1 deci D
1015 peta P 10-2 centi C
10 12 tera T 10-3 milli M

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
109 giga G 10-6 micro µ
106 mega M 10-9 nano N
103 kilo K 10-12 pico P
102 hecto H 10-15 femto F
10 deka Da 10-18 atto a

Non-SI units
Amount Unit name Unit symbol Definition
Time Minute Min 1min=60s
Hour H 1h=60min
Day D 1d=24h
Plane angle Degree ° 1°= (π/180) rad
Minute ′ 1′= (1/60) °
Second ″ 1″= (1/60) ′
Volume Liter l, L 1l=1dm3
Weight Metric ton T 1t=103kg

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – V
Introduction to general Bio-molecules

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Chemistry of carbohydrates: Classification: (structures of monosaccharide), Functions of
carbohydrates
 Chemistry of amino acides: Classifcation-based on structures and nutritional requirement,
Occurrence, Functions of amino acids.
 Chemistry of lipids: Classification of lipids and fatty acids. Functions of lipids
 Chemistry of nucleotides: Purine and Pyrimidine bases. Composition of nucleosides and
nucleotides. Occurrence of bases.

Classification, Functions of carbohydrates and structures of

monosaccharide

Carbohydrates
Carbohydrates are amongst the most important organic compounds found in almost all
the living organisms and one of the four major macromolecules. Carbohydrates are also
called saccharide which is a Greek word and it means sugar because almost all the
carbohydrates have a sweet taste. Glucose, fructose, various sugars, starch etc. are some
common carbohydrates,
Carbohydrates Formula: The general formula for carbohydrates is Cx(H2O)y. By using
this formula we can find the molecular formula for glucose (C6H12O6).
Carbohydrate Chemistry: Chemically, carbohydrates are defined as optically active
polyhydroxy aldehydes or ketones or the compounds which produce units of such type
on hydrolysis.
Sources of Carbohydrates: We know carbohydrates are an important part of any
human‘s diet. Some common sources of carbohydrates are
 Potatoes
 Maize
 Milk
 Popcorn
 Bread

Classification of Carbohydrates: The carbohydrates can be classified on the basis of their

behavior on hydrolysis. They are mainly classified into three groups:
 Monosaccharides
 Disaccharides

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Polysaccharides

Fig. 3.1: Types of carbohydrates

Monosaccharides are those carbohydrates that cannot be hydrolyzed further to give

simpler units of polyhydroxy aldehyde or ketone. If a monosaccharide contains an
aldehyde group then it is called aldose and on the other hand, if it contains keto group
then it is called a ketose.
Depending upon the number of carbon atoms they possess monosaccharides
may be subdivided into different classes such as trioses, tetroses, pentoses, hexoses or
heptoses; Based upon whether the aldehyde or ketone groups are present they are
termed aldoses or ketoses,. Examples are:
Cabohyrdate Aldoses Ketoses
Trioses (C3H6O3) Glyceraldehyde Dihydroxyacetone
Tetroses (C4H8O4) Erythrose Erythrulose
Pentoses (C5H10O5) Ribose Ribulose
Hexoses (C6H12O6) Glucose Fructose

Structure of Glucose
One of the most important monosaccharides is glucose.
Glucose is also called aldohexose and dextrose and is abundant on earth.

Fig. 3.2: Structure of glucose

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Glucose is named as D (+)-glucose, D represents the configuration whereas (+)
represents the dextrorotatory nature of the molecule.
The ring structure of glucose can explain many properties of glucose which cannot be
figured by open chain structure.
The two cyclic structures differ in the configuration of the hydroxyl group at C1 called
as anomeric carbon. Such isomers i.e. α and β form are known as anomers. The cyclic
structure is also called pyranose structure due to its analogy with pyran. The cyclic
structure of glucose is given below:

Fig. 3.3: Cyclic structure of glucose

Structure of fructose
It is an important ketohexose. The molecular formula of fructose is C6H12O6 and
contains ketonic functional group at carbon number 2 and has six carbon atoms in a
straight chain. The ring member of fructose is in analogy to the compound Furan and is
named as furanose. The cyclic structure of fructose is shown below:

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Fig. 3.4: Cyclic structure of Fructose
Derived Monosaccharides:
Monosaccharides are modified variously to form a number of different substances. The
important derivatives are:
(i) Deoxy Sugar: Deoxygenation of ribose produces deoxyribose. The latter is a
constituent of deoxyribotides found in DNA.
(ii) Amino Sugars: The monosaccharides have an amino group—(NH2). Glucosamine
forms chitin, fungus cellulose, hyaluronic acid and chondriotin sulphate. Galactosamine
is similarly a component of chondriotin sulphate.
(iii) Sugar Acid: Ascorbic acid is a sugar acid. Glucuronic acid and galacturonic acid
occur in mucopolysaccharides.
(iv) Sugar Alcohol: Glycerol is involved in lipid synthesis; Mannitol is storage alcohol
in some fruits and brown algae.

Disaccharides
On hydrolysis, disaccharides yield two molecules of either same or different
monosaccharide. The two monosaccharide units are joined by oxide linkage which is
formed by the loss of water molecule and this linkage is called glycosidic linkage.
Sucrose is one of the most common disaccharides which on hydrolysis gives glucose
and fructose.
Maltose and Lactose (also known as milk sugar) are other two important
disaccharides. In maltose, there are two α-D-glucose and in lactose, there are two β-D-
glucose which are connected by oxide bond. Sucrose is popularly known as table sugar.
Sucrose is found in all photosynthetic plants. The chemical structure of sucrose
comprises of α form of glucose and β form of fructose

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Polysaccharides
A polysaccharide is a large molecule made of many smaller monosaccharides.
Monosaccharides are simple sugars, like glucose. Special enzymes bind these small
monomers together creating large sugar polymers, or polysaccharides. A
polysaccharide is also called a glycan. A polysaccharide can be a homopolysaccharide,
in which all the monosaccharides are the same, or a heteropolysaccharide in which the
monosaccharides vary. Depending on which monosaccharides are connected, and
which carbons in the monosaccharides connects, polysaccharides take on a variety of
forms. A molecule with a straight chain of monosaccharides is called a linear
polysaccharide, while a chain that has arms and turns are known as a branched
polysaccharide.
Polysaccharides are long chains of monosaccharides linked by glycosidic bonds.
Three important polysaccharides, starch, glycogen, and cellulose, are composed of
glucose. Starch and glycogen serve as short-term energy stores in plants and animals,
respectively. The glucose monomers are linked by α glycosidic bonds.

Homopolysaccharides
Homopolysaccharides are chemical compounds that are composed of a single type of
monomer. These monomers are monosaccharides. Therefore, the chemical structure of a
homopolysaccharide has the same repeating unit.
A polysaccharide is made out of monomers that are covalently bonded to each
other via glycosidic bonds. There can be two types of glycosidic bonds as 1-4 glycosidic
bonds and 1-6 glycosidic bonds, depending on the carbon atoms that are bonded to
each other (via an oxygen atom). The 1-4 glycosidic bonds cause the formation of a
linear homopolysaccharide whereas 1-6 glycosidic bonds result in branched structures.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Cellulose is a good example for homopolysaccharides. It is a linear homopolysaccharide
with 1-4 glycosidic bonds. The monomer of cellulose is glucose. Glucose is a
monosaccharide. Starch is another homopolysaccharide. It has two main components:
amylose and amylopectin. Amylose is a linear structure whereas amylopectin is a
branched structure. Cellulose and starch can be found in plants. There are
homopolysaccharides in animal bodies as well. For example, glycogen is a
homopolysaccharide of glucose monomers. Chitin is another homopolysaccharide
which has N-acetylglucosamine as the monomer. It is the main structural component of
insects.

Heteropolysaccharides
Heteropolysaccharides are polysaccharides that are made out of two or more different
monosaccharides. These are polymers of monosaccharides. The polymeric structure of
the heteropolysaccharide has different repeating units.
Heteropolysaccharides are complex structures. The arrangement of repeating
units decides the chemical and physical properties of the heteropolysaccharide. There
are many well-known heteropolysaccharides. These compounds have various
applications in biological systems and in industries as well.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
For example, Hyaluronic acid is a structural component that can only be found in
animal tissues. It is a heteropolysaccharide of D-glucuronic acid and N-acetyl-D-
glucosamine. Pectin can be found in plant tissues. It is also a heteropolysaccharide. This
compound is made out of a D-galacturonic acid backbone that is bonded to different
side chains.

Functions of carbohydrates
 Carbohydrates are the energy stores in living beings. Starch and glycogen,
respectively in plants and animals, are stored carbohydrates from which glucose
can be mobilized for further catabolism. Glucose can supply energy both fueling
ATP synthesis (ATP, the cell‘s energy currency, has inside a phosphorylated sugar)
and in the form of reducing power as NADPH.
 Glucose, used as energy source, ―burns‖ without yielding metabolic wastes, being
turned in CO2 and water, and of course releasing energy. Monosaccharides supply
3.74 kcal/g, disaccharides 3.95 kcal/g, while starch 4.18 kcal/g; on average it is
approached to 4 kcal/g. Glucose is indispensable for the maintenance of the
integrity of nervous tissue ,some central nervous system areas and red blood cells
are able to use only glucose as fuel.
 Their presence is necessary for the normal lipid metabolism. More than 100 years
ago Pasteur said: “Fats burn in the fire of carbohydrates“. This idea continues to
receive confirmations from the recent scientific studies. Moreover, excess
carbohydrates may be converted in fatty acids and triglycerides (processes that
occur mostly in the liver).
 Two sugars, ribose and deoxyribose are constituents of ribonucleotide building
blocks of RNA and deoxy ribonucleotide building blocks of DNA, respectively.
 They take part in detoxifying processes. For example, at hepatic level in a process
called glucuronidation, glucuronic acid derived from glucose combines with
endogenous substances such as , hormones, bilirubin etc., and exogenous
substances such as chemicals, bacterial toxins or drugs , increasing their solubility
and allowing their elimination.
 Two homopolysaccharides, cellulose (the most abundant polysaccharide in nature)
and chitin (probably, next to cellulose, the second most abundant polysaccharide in
nature), serve as structural elements, respectively, in plant cell walls and
exoskeletons of nearly a million species of arthropods (e.g. insects, lobsters, and
crabs).
 Heteropolysaccharides provide extracellular support for organisms of all
kingdoms: in bacteria, the rigid layer of the cell wall is composed in part of a
heteropolysaccharide contained two alternating monosaccharide units while in
animals the extracellular space is occupied by several types of
heteropolysaccharides, which form a matrix with numerous functions, as hold
individual cells together and provide protection, support, and shape to cells,
tissues, and organs.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Chemistry of amino acids: Classification-based on structures and
nutritional requirement, Occurrence, Functions of amino acids.

Amino acids are small organic molecules that play several important roles in living
organisms:
i) They are the principle building blocks of proteins, Nature‘s most functionally
diverse biomolecules.
ii) They serve as precursors for many biologically active molecules, such as
neurotransmitters (e.g. dopamine, serotonin, GABA, epinephrine), local mediators
(e.g. the allergy mediator histamine), energy-related metabolites (e.g. creatine,
citrulline, carnitine), the oxygen-binding molecule ‗heme‗, and DNA bases called
purines. For details, see section 5 below.
iii) They serve as an energy source during prolonged fasting, diabetes, and when the
diet is rich in proteins.
iv) Some act as regulators of gene expression and cellular signalling. This affects
multiple physiological processes that are related to growth, maintenance,
reproduction and immunity.

General structure of amino acids

As their names suggests, all amino acids contain both amino and carboxylic acid
groups. There are roughly 300 types of amino acids in nature, only 20 of which serve as
building blocks of proteins. Others function as metabolites, messengers and regulators
of biological processes.
All amino acids that appear in proteins (‗α-amino acids) possess a structure
which includes a central carbon atom called Cα, surrounded by four substituents: a
hydrogen atom, an amino group (α-amino), a carboxyl group (α-carboxyl), and a fourth
group referred to as side-chain:

Fig. 5.1: The general structure of an amino acid

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  The α-carboxyl group has a low pKa (~2), and is therefore deprotonated and
negatively charged at physiological pH (7).
 The α-amino group has a high pKa (9–10), and is therefore protonated and
positively charged at physiological pH.
 The side-chain is chemically different in each of the amino acids. They
determine the uniqueness of the 20 natural amino acids found in proteins.
 Amino acids in proteins almost exclusively possess an L configuration. Amino
acids with D configuration can be found in microorganisms (e.g. in the bacterial
cell wall and in antibiotic peptides) and in certain animals (e.g. the frog skin
peptide ‗dermorphin‘)

Fig. 5.2: Short hand symbols for amino acids

Amino acid groups

It is customary to group the 20 natural amino acids found in proteins into 4 types,
according to the polarity of their side chains. Amino acids that have polar side chains
are hydrophilic. That is, they tend to appear on the surface of water-soluble proteins
where they can interact favorably with the surrounding water. Amino acids that have

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
nonpolar side chains are hydrophobic. That is, they tend to be buried inside proteins,
where they are away from water and can interact favorably with each other.
Different methods of protein classification have been proposed. Below, some examples
based on chemical composition, structure, functions, and solubility in different solvents.

Protein classification based on chemical composition

Simple proteins
Also known as homoproteins, they are made up of only amino acids. Examples are
plasma albumin, collagen, and keratin.
Conjugated proteins

Fig. 5.3: Human Fibronectin

Sometimes also called heteroproteins, they contain in their structure a non-protein
portion. Three examples are glycoproteins, chromoproteins, and phosphoproteins.
Glycoproteins
They are proteins that covalently bind one or more carbohydrate units to the
polypeptide backbone.
Typically, the branches consist of not more than 15-20 carbohydrate units as arabinose,
fucose (6-deoxygalactose), galactose, glucose, mannose, N-acetylglucosamine (GlcNAc,
or NAG), and N-acetylneuraminic acid (Neu5Ac or NANA).
Examples of glycoproteins are:
glycophorin, the best known among erythrocyte membrane glycoproteins; fibronectin,
that anchors cells to the extracellular matrix through interactions on one side with
collagen or other fibrous proteins, while on the other side with cell membranes; all
blood plasma proteins, except albumin; immunoglobulins or antibodies.
Chromoproteins
They are proteins that contain colored prosthetic groups.
Examples:
Hemoglobin and myoglobin, which bind, respectively, one and four heme groups;
chlorophylls, which bind a porphyrin ring with a magnesium atom at its centre;
rhodopsins, which bind retinal.

Phosphoproteins

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
They are proteins that bind phosphoric acid to serine and threonine residues Generally,
they have a structural function, such as tooth dentin, or reserve function, such as milk
caseins (alpha, beta, gamma and delta), and egg yolk phosvitin.

Protein classification based on shape

On the basis of their shape, proteins may be divided into two classes: fibrous and
globular.
Fibrous proteins

Fig. 5.4: Collagen

They have primarily mechanical and structural functions, providing support to the
cells as well as the whole organism. These proteins are insoluble in water as they
contain, both internally and on their surface, many hydrophobic amino acids. The
presence on their surface of hydrophobic amino acids facilitates their packaging into
very complex supramolecular structures. In this regard, it should be noted that their
polypeptide chains form long filaments or sheets, where in most cases only one type of
secondary structure, that repeats itself, is found. In vertebrates, these proteins provide
external protection, support and shape; in fact, thanks to their structural properties,
they ensure flexibility and/or strength. Some fibrous proteins, such as α-keratins, are
only partially hydrolyzed in the intestine. Here are some examples.

Fibroin: It is produced by spiders and insects. An example is that produced by the

silkworm, Bombyx mori.

Collagen: The term ―collagen‖ indicates not a single protein but a family of structurally
related proteins (at least 29 different types), which constitute the main protein
component of connective tissue, and more generally, the extracellular scaffolding of
multicellular organisms. In vertebrates, they represent about 25-30% of all proteins.
They are found in different tissues and organs, such as tendons and the organic matrix
of bone, where they are present in very high percentages, but also in cartilage and in the
cornea of the eye. In the different tissues, they form different structures, each capable of
satisfying a particular need. For example, in the cornea, the molecules are arranged in
an almost crystalline array, so that they are virtually transparent, while in the skin they
form fibers not very intertwined and directed in all directions, which ensure the tensile

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
strength of the skin itself. Note: the different types of collagen have low nutritional
value as deficient in several amino acids (in fact, they contain no tryptophan and low
amount of the other essential amino acids). The gelatin used in food preparation is a
derivative of collagen.
α-Keratins: They constitute almost the entire dry weight of nails, claws, beak, hooves,
horns, hair, wool, and a large part of the outer layer of the skin.
The different stiffness and flexibility of these structures is a consequence of the number
of disulfide bonds that contribute, together with other binding forces, to stabilize the
proteinstructure. And this is the reason why wool keratins, which have a low number of
disulfide bonds, are flexible, soft and extensible, unlike claw and beak keratins that are
rich in disulfide bonds.
Elastin: This protein provides elasticity to the skin and blood vessels, a consequence of
its random coiled structure, that differs from the structures of the α-keratins and
collagens.
Globular proteins: Most of the proteins belong to this class

Fig. 5.5: Haemoglobin

They have a compact and more or less spherical structure, more complex than
fibrous proteins. In this regard, motifs, domains, tertiary and quaternary structures are
found, in addition to the secondary structures. They are generally soluble in water but
can also be found inserted into biological membranes (transmembrane proteins), thus in
a hydrophobic environment. Unlike fibrous proteins, that have structural and
mechanical functions, they act as: enzymes; hormones; membrane transporters and
receptors; transporters of triglycerides, fatty acids and oxygen in the blood;
immunoglobulins or antibodies; grain and legume storage proteins.
Examples of globular proteins are myoglobin, hemoglobin, and cytochrome c. At
the intestinal level, most of the globular proteins of animal origin are hydrolyzed almost
entirely to amino acids.

Protein classification based on biological functions

The multitude of functions that proteins perform is the consequence of both the folding
of the polypeptide chain, therefore of their three-dimensional structure, and the
presence of many different functional groups in the amino acid side chains, such as
thiols, alcohols, thioethers, carboxamides, carboxylic acids and different basic groups.
From the functional point of view, they may be divided into several groups.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Enzymes (biochemical catalysts).
In living organisms, almost all reactions are catalyzed by specific proteins called
enzymes. They have a high catalytic power, increasing the rate of the reaction in which
they are involved at least by factor 106. Therefore, life as we know could not exist
without their ―facilitating action‖. Almost all known enzymes, and in the human body
they are thousand, are proteins (except some catalytic RNA molecules called ribozymes,
that is, ribonucleic acid enzymes).
Transport proteins
Many small molecules, organic and inorganic, are transported in the bloodstream and
extracellular fluids, across the cell membranes, and inside the cells from one
compartment to another, by specific proteins.
Examples are:
hemoglobin, that carries oxygen from the alveolar blood vessels to tissue capillaries;
transferrin, which carries iron in the blood; membrane carriers; fatty acid binding
proteins (FABP), that is, the proteinsinvolved in the intracellular transport of fatty acids;
proteins of plasma lipoproteins, macromolecular complexes of proteins and lipids
responsible for the transport of triglycerides, which are otherwise insoluble in water;
albumin, that carries free fatty acids, bilirubin, thyroid hormones, and certain
medications such as aspirin and penicillin, in the blood. Many of these proteins also
play a protective role, since the bound molecules, such as fatty acids, may be harmful
for the organism when present in free form.
Storage proteins
Examples are: ferritin, that stores iron intracellularly in a non-toxic form; milk caseins,
that act as a reserve of amino acids for the milk; egg yolk phosvitin, that contains high
amounts of phosphorus; prolamins and glutelins, the storage proteins of cereals.
Mechanical support
Proteins have a pivotal role in the stabilization of many structures. Examples are α-
keratins, collagen and elastin. The same cytoskeletal system, the scaffold of the cell, is
made of proteins.
They generate movement.
They are responsible, among others, for: the contraction of the muscle fibers (of which
myosin is the main component); the propulsion of spermatozoa and microorganisms
with flagella; the separation of chromosomes during mitosis. They are involved in nerve
transmission. An example is the receptor for acetylcholine at synapses. They control
development and differentiation. Some proteins are involved in the regulation of gene
expression. An example is the nerve growth factor (NGF), discovered by Rita Levi-
Montalcini, that plays a leading role in the formation of neural networks.
Hormones
Many hormones are proteins. They are regulatory molecules involved in the control of
many cellular functions, from metabolism to reproduction. Examples are insulin,
glucagon, and thyroid-stimulating hormone (TSH).
Protection against harmful agents.

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Medical College Doda
The antibodies or immunoglobulins are glycoproteins that recognize antigens expressed
on the surface of viruses, bacteria and other infectious agents. Interferon, fibrinogen,
and factors of blood coagulation are other members of this group.
Storage of energy.
Proteins, and in particular the amino acids that constitute them, act as energy storage,
second in size only to the adipose tissue, that in particular conditions, such as
prolonged fasting, may become essential for survival. However, their reduction of more
than 30% leads to a decrease of the contraction capacity of respiratory muscle, immune
function, and organ function, that are not compatible with life. Therefore, proteins are
an extremely valuable fuel.

Protein classification based on solubility

The different globular proteins can be classified based on their solubility in different
solvents, such as water, salt and alcohol.

I. Nonpolar amino acids

These include the following
 Glycine (gly, G) – has a single hydrogen atom as a side chain.
 Alanine (Ala, A) – has a methyl group (CH3) as a side chain.
 Valine (val, V), leucine (leu, L) and isoleucine (ile, I) – have a branched aliphatic
side chain.
 Methionine (met, M) – has a sulfur-containing linear aliphatic side chain.
 Proline (pro, P) – has an aliphatic side chain which is covalently attached to
the α-amino group
In water-soluble proteins, nonpolar amino acids reside mainly in the hydrophobic core.
There, their interactions with each other (the ‗hydrophobic effect‗) is what holds
together the 3-dimensional fold of the protein. In membrane-bound proteins, nonpolar
amino acids reside on the surface, where they can interact favorably with membrane
lipids. Finally, nonpolar interactions involving these amino acids serve as a driving
force for the binding of ligands and substrates to the protein.
Here are some interesting facts on some of the nonpolar amino acids:
 Methionine, despite being overall nonpolar, is still able to interact weakly with
polar species, such as metals. This is thanks to the non-bonding electrons of
methionine‘s sulfur atom, which are only weakly held by the nucleus.
 Glycine, having only hydrogen as side chain, confers flexibility to the protein
areas in which it is present. Proline does the opposite (confers rigidity) due to its
side chain being fused to the rest of the amino acid.
 The branched amino acids (valine, leucine, isoleucine) are an important source
of energy in muscles and signal cells to synthesize proteins.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Fig. 5.6: Nonpolar amino acids

II. Polar-uncharged amino acids

These include the following
 Serine (ser, S) and threonine (thr, T) – have a hydroxyl group in their side chain.
 Cysteine (cys, C) – has a thiol (sulfhydril) group in its side chain.
 Glutamine (gln, Q) and asparagine (asn, N) – have an amide group in their side
chains
In proteins, polar-uncharged amino acids form hydrogen bonds with each other and
with the protein‘s ligand/substrate. This confers specificity to protein-ligand
interactions, in contrast to the hydrophobic effect which confers stability and serves as a
driving force for the interactions. Some of the polar-uncharged amino acids also
function as nucleophiles in enzymatic catalysis.
Here are interesting facts on some polar-uncharged amino acids:
 Serine and threonine are often phosphorylated in proteins that participate in
cell-cell communication. This modification is reversible and serves as a
chemcial, signal transduction tag. The two amino acids may also function as
nucleophiles in enzymatic catalysis, thanks to the hydroxyl group in their side
chain.

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Medical College Doda
  Serine and threonine on the surface of membrane/secreted proteins may also be
glycosylated (attached with sugar groups). This protects the protein and
increases its water solubility. In some cases, the attached sugar groups serve also
as a recognition code for other extra-cellular elements.
 Cysteine, despite being polar, tends to appear in core of water-soluble proteins.
Under oxidizing conditions, its thiol side chain deprotonates and tends to form a
(covalent) disulfide bond with a thiol group of a neighboring cysteine. These
bonds are important for stabilizing proteins that are secreted from cells
(hormones, antibodies, some enzymes). Cysteine also serves as a nucleophile
and an electron-transfer agent in enzymatic catalysis. Both are the result of the
non-bonding electrons of cysteine‘s sulfur atom, conferring it high chemical
reactivity and the ability to exist in different oxidation states.
 The carboxamide group of glutamine and asparagines can serve as both
hydrogen bond donor and acceptor. As a result, these amino acids are commonly
involved in hydrogen bond networks within proteins. This group in asparagine
is also glycosylated in membrane/secreted proteins (see above for the benfits of
glycosylation).

Fig. 5.7: Polar-uncharged amino acids

III. Polar-charged amino acids

Polar acidic (-ve charged) amino acids: 2 amino acids are polar acidic: asp and glu
Polar basic (+ve charged) amino acids: 3 amino acids are polar basic: lys, arg, his.

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Medical College Doda
Aspartate (asp, D) and glutamate (glu, E) – have a carboxyl group in their side chains.
As explained above, this group has a low pKa and therefore tends to become
deprotonated and negatively charged at physiological pH. For this reason, aspartate
and glutamate are referred to as ‗acidic‗. In proteins, they tend to interact
electrostatically with positively charged groups in other amino acids or in the protein‘s
ligand/substrate.
 Lysine (lys, K) and arginine (arg, R) – have nitrogen-containing groups in their
side chains (amino group in lysine and guanidine group in arginine). These
groups have high pKa and therefore tend to become protonated and positively
charged at physiological pH. For this reason, lysine and arginine are referred to
as ‗basic‗. In proteins, they tend to interact electrostatically with negatively
charged groups in other amino acids or in the protein‘s ligand/substrate.
 Histidine (his, H) – has an imidazole group in its side chain. This group has a
pKa of ~6, and therefore has a 50% chance of being protonated (positively
charged) or deprotonated (neutral) at physiological pH. This allows histidine to
function in hydrogen-transfer enzymatic catalysis, where it may functions as the
hydrogen donor, acceptor, or both.

Fig. 5.8: Polar amino acids

Here are interesting facts on some polar-uncharged amino acids:

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Carboxylation of glutamate on its γ carbon allows it to bindefficiently cations,
such as divalent calcium. This is very useful in clotting proteins, such as
prothrombin, the activity of which is regulated by blood calcium levels.
 The amino group of lysine‘s side chain is able to form Schiff base with
aldehydes. This helps some proteins bind aldehyde-containing prosthetic groups
(e.g. the pyridoxal phosphate coenzyme of aminotranferase enzyme).
 Oxidation of lysine‘s side chain in structureal proteins like collagen allows it to
participate in cross-linking reactions that stabilize these proteins.
 As explained above, the imidazole side chain of histidine can serve as both acid
and base thanks to its pKa, which is close to the physiological pH. This is
important, for example, for the catalytic mechanism of the enzyme acetylcholine
esterase, which inactivates the neurotransmitter acetylcholine. The inactivation
is important for preventing our nervous system from going into paralysis and
death, which is what happens when the enzyme is attacked and inactivated by
toxic nerve agents. Acetylcholine is an ester, and it is hydrolyzed to choline and
acetate by the enzyme, via nucleophilic attack of a catalytic serine residue on the
ester bond. To do that, the serine must deprotonate, and this is made possible by
a nearby histidine which acts as a base, abstracting serine‘s proton:

IV. Aromatic amino acids

These includes the following
 Phenylalanine (phe, F) – has a phenyl group in its side chain.
 Tyrosine (tyr, Y) – has a phenol group in its side chain.
 Tryptophan (trp, W) – has an indole group in its side chain.

Fig. 5.9: Aromatic amino acids

In contrast to the other amino acids groups, this one is considered not according the
polarity of the side chain but rather on its aromatic nature (in the chemical sense, not
the olfactory one). Aromatic groups contain delocalized π electrons which can interact
with similar electrons in other aromatic groups, as well as with positively-charged

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Medical College Doda
groups. Indeed, all of these interactions are observed in proteins between aromatic side
chains. The aromatic amino acids are important in forming closed scaffolds within
proteins, especially binding sites for ligands and substrates.
Here are interesting facts on some aromatic amino acids:
 Like serine and threonine, tyrosine may also become phosphorylated on
proteins involved in cellular communications. A well-known example is the
membrane-bound receptors which respond to growth factors. Binding of the
latter to these receptors results in their phosphorylation, this in turn conveys the
signal into the cell and results in cellular division. Genetic defects that allows for
hormone-independent phosphoryaltion of these proteins often lead to cancer.
 The phenol group of tyrosine also participates in differnt mechanisms of
enzymatic catalysis (e.g. nucleophilic attack, acid-base catalysis and
stabilization of reaction intermediates).
 The indole group in tryptophan‘s side chain is capable of participating in
different polar and nonpolar non-covalent interactions with other chemical
fspecies. Therefore, it is common in protein binding sites. It also participates in
enzymatic catalysis and electron transfer.
 Tryptophan‘s side chain fluoresce when absorbing UV light. This allows
biochemists to identify proteins or study changes in their structure by UV-
irradiating them and then recording the fluorescence of their tryptophan amino
acids.
The 20 naturally-occurring amino acids can be clustered according to their physical-
chemical properties, as shown in the following Venn diagram:

The 20 types of amino acids clustered by their physical-chemical properties (taken from
Esquivel et al. (2013)

Nutritional classification:
On the basis of nutritional requirement amino acids are of three types Essential,
Nonessential, and Conditional essential.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Essential – must be consumed in the diet
Nonessential –can be synthesized in body
Conditionally essential – cannot be synthesized due to illness or lack of necessary
precursors. Premature infants lack sufficient enzymes needed to create arginine

Fig. 5.11: Essential & non essential amino acids

Chemistry of lipids: Classification of lipids and fatty acids. Functions of

lipids

Lipid
A lipid is chemically defined as a substance that is insoluble in water and soluble in
alcohol, ether, and chloroform. Lipids are an important component of living cells.
Together with carbohydrates and proteins, lipids are the main constituents of plant and
animal cells. Bloor (1943) has proposed the following classification of lipids based on
their chemical composition.

A. Simple lipids or Homolipids. These are esters of fatty acid with various alcohols.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
1. Fats and oils (triglycerides, triacylglycerols).
These are esters of fatty acids with a trihydroxy alcohol, glycerol. A fat is solid at
ordinary room temperature wheras an oil is liquid.
2. Waxes. These are esters of fatty acids with high molecular weight monohydroxy
alcohols.

B. Compound lipids or Heterolipids.

These are esters of fatty acids with alcohol and possess additional group(s) also.
1. Phospholipids (phosphatids): These are compounds containing, in addition to fatty
acids and glycerol, a phosphoric acid, nitrogen bases and other substituents.
2. Glycolipids (cerebrosides): These are the compounds of fatty acids with
carbohydrates and contain nitrogen but no phosphoric acid. The glycolipids also
include certain structurally-related compounds comprising the groups, gangliosides,
sulfolipids and sulfatids.

C. Derived lipids.
These are the substances derived from simple and compound lipids by hydrolysis.
These include fatty acids, alcohols, mono- and diglycerides, steroids, terpenes and
carotenoids.

Fatty acids (structure, composition, classification and use)

Fatty acids (FA) consist of carbon, hydrogen, and oxygen, arranged as a linear carbon
chain skeleton of variable length, generally with an even number of atoms, with a
carboxyl group at one end. Fatty acids from 2 to 30 carbons or more occur, but the most
common and important ones contain between 12 and 22 carbon atoms and are found in
many different animal and plant fats.
They are rarely free in nature and are the main components of:
 Triacylglycerols (or triglycerides)
 Diacylglycerols
 Monoacylglycerols (the last two families of compounds are often added to processed
foods)
 Phospholipids of cell membranes
 Sterol esters.
Far from only being a convenient unit for energy storage, they are also essential for
metabolic and structural activities. In this regard fatty acids, unlike proteins and
nucleic acids, have the singular ability to be incorporated into tissues intact, thereby
altering tissue acyl compositions. Although most of them have unbranched structure,
there are many that have a branched chain. Some of these, like phytanic acid, occur
frequently but in small amounts in animal fats, waxes, and marine oils. They are rare in
plant lipids, while being major components of the lipids of gram-positive bacteria.

Nomenclature of fatty acids

System Example Explanation
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Trivial Palmitoleic acid Trivial names (or common names) are non-
nomenclature systematic historical names, which are the
most frequent naming system used. Most
common fatty acids have trivial names in
addition to their systematic names These
names frequently do not follow any pattern,
but they are concise and often unambiguous.
Systematic (9Z)-octadecenoic Systematic names (or IUPAC names) derive
nomenclature acid from the standard IUPAC Rules for the
Nomenclature of Organic Chemistry, published
in 1979, along with a recommendation
published specifically for lipids in 1977.
Counting begins from the carboxylic acid
end. Double bonds are labelled with cis-trans
isomerism-/trans- notation or E-/Z- notation,
where appropriate. This notation is generally
more verbose than common nomenclature,
but has the advantage of being more
technically clear and descriptive.
Δxnomenclature cis,cis In Δx (or delta-x) nomenclature, each double
Δ9,Δ12octadecadienoi bond is indicated by Δx, where the double
c acid bond is located on the xth carbon–carbon
bond, counting from the carboxylic acid end.
Each double bond is preceded by a cis- or
trans- prefix, indicating the conformation of
the molecule around the bond. For example,
linoleic acid is designated "cis-Δ9, cis-Δ12
octadecadienoic acid". This nomenclature
has the advantage of being less verbose than
systematic nomenclature, but is no more
technically clear or descriptive.
n−x n−3 n−x (n minus x; also ω−x or omega-x)
nomenclature nomenclature both provide names for
individual compounds and classifies them
by their likely biosynthetic properties in
animals. A double bond is located on the xth
carbon–carbon bond, counting from the
terminal methyl carbon (designated as n or
ω) toward the carbonyl carbon. For example,
α-Linolenic acid is classified as a n−3 or
omega-3 fatty acid, and so it is likely to share
a biosynthetic pathway with other
compounds of this type. The ω−x, omega-x,
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 or "omega" notation is common in popular
nutritional literature, but IUPAC has
deprecated it in favor of n−x notation in
technical documents.
Lipid numbers 18:3 Lipid numbers take the form C:D, where C
18:3, n−6 is the number of carbon atoms in the fatty
18:3,cis,cis,cis-Δ9, acid and D is the number of double bonds in
Δ12, Δ15 the fatty acid. This notation can be
ambiguous, as some different fatty acids can
have the same numbers. Consequently,
when ambiguity exists this notation is
usually paired with either a Δx or n−x term.

Classification of fatty acids

Depending on their degree of saturation/unsaturation in the carbon chain, they can be
divided into two classes:

1: Saturated fatty acids (SFA): Saturated fatty are simple forms, having no double
bonds. They are made up of two small molecules which are fatty acids and
monoglyceride. Saturated fatty acids contain long, unbranched chains of carbon atoms.

These are abundantly found in animal fat, coconut oil, palm oil, whole milk, butter.
These are solid at room temperature. The over consumption of the saturated fatty acids
is harmful to the health as it affects the blood cholesterol by increasing the level of it.

2: Unsaturated FA: Unsaturated fatty acids contain both single as well double bonds.
But this type is known by the presence of at least one double bonds in their chain. These
are mostly found in vegetable oil, plants, avocado, fish oil. By the double bonds (C=C),
they are divided into two categories, which are – monounsaturated fatty acids (MUFA)
and polyunsaturated fatty acids (PUFA).

Such fatty acids which contain only one double bond (C=C) in their chain is called as
monounsaturated fatty acids (MUFA), on the other hand, such fatty acids which
contain two or more double bonds (C=C).
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Moreover on the basis of the absence/presence of double/triple bonds they can be
grouped into two broad classes:
 monounsaturated fatty acids (MUFA), if only one double bond is present;

 Polyunsaturated fatty acids (PUFA), if two or more double bonds are present.

Table 4.1: Comparison between Saturated fatty acids and Unsaturated fatty acids
Basis for Saturated fatty acids Unsaturated fatty acids
comparison
Double Bonds Saturated fatty acids contain Unsaturated fatty acids
single chain of carbon atoms contain carbon chains with
with no double bond. one or more double bond.
(C=C).
Physical appearance Solid at room temperature. Liquid at room temperature.
Type of chain Straight chain. Bend chains at double bond.
Melting point Relatively higher. Relatively lower.
Sources to obtain Animal fats, palm oil, coconut Plant and vegetable oil,
oil. avocado, sunflower oil,
walnuts, flax, canola oil and
fish oil.
Solubility in Soluble in vitamins. Insoluble in vitamins.
vitamins
Shelf life They get spoilt quickly and are They do not get spoilt quickly.
long-lasting.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Fig. 4.1: Fatty acid classification

On the basis of essentiality

On the basis of the ability or not to synthesize them de novo from endogenous
precursors by animals, and whose deficiency can be reversed by dietary addition, they
can be classified as:
 Essential
 Non essential
Essential fatty acids or EFA are fatty acids which cannot be synthesized de novo by
animals, but by plants and microorganisms, such as bacteria, fungi and molds, and
whose deficiency can be reversed by dietary addition.
There are two essential fatty acids: linoleic acid or LA (18:2n-6) and α-linolenic
acid or ALA (18:3n-3), polyunsaturated fatty acids(PUFAs) with 18 carbon atoms,
belonging to omega-6 and omega-3families, respectively.
Animals cannot synthesize these two fatty acids because they lack desaturases that
introduce double bonds beyond the Δ9 position (carbon atoms numbered from the
methyl end), namely:
 Δ12-desaturase (E.C. 1.14.19.6), which catalyzes the synthesis of LA from oleic acid;
 Δ15-desaturase (EC 1.14.19.25), present also in phytoplankton, which catalyzes the
synthesis of ALA from linoleic acid.
Instead, animals have the enzymes needed to elongate and desaturate, though with low
efficiency, the two EFA to form PUFAs with 20, 22, or 24 carbon atoms and up to 6
double bonds, such as for example dihomo-gamma-linolenic acid or DGLA (20:3n6),
arachidonic acid or AA (20:4n6), eicosapentaenoic acid (EPA, 20:5n3),
and docosahexaenoic acid or DHA (22:6n3). If diet is deficient in EFA, also fatty acids
synthesized from them become essential. For this reason they may be
termed conditionally essential fatty acids.
It should be noted that all essential fatty acids are polyunsaturated molecules but not all
polyunsaturated fatty acids are essential, such as those belonging to the omega-7 and
omega-9 families.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Non essential
Non essential amino acids are amino acids that can be produced in our body. Their
uses and functions in our body are equally as important as the limiting amino acids.
The difference is that those kind of amino acids can be found in our food.
Functions of non-essential amino acids
The following list includse the 12 non-essential amino acids. Included is a some of the
functions and benefits and side effects (if any) of the amino acids.
Alanine: Removes toxic substances released from breakdown of muscle protein during
intensive exercise. Side effects: Excessive alanine level in the body is associated with
chronic fatigue.
Cysteine: Component of protein type abundant in nails, skin and hair. It acts as
antioxidant (free radical scavenger), and has synergetic effect when taken with other
antioxidants such as vitamin E and selenium.
Cystine: The same as cysteine, it aids in removal of toxins and formation of skin.
Glutamine: Promotes healthy brain function. It is also necessary for the synthesis of
RNA and DNA molecules.
Glutathione: Is antioxidant and has anti-aging effect. It is useful in removal of toxins.
Glycine: Component of skin and is beneficial for wound healing. It acts as
neurotransmitter. The side effect of high level glycine in the body is that it may cause
fatigue.
Histidine: Important for the synthesis of red and white blood cells. It is a precursor for
histamine which is good for sexual arousal. Improve blood flow. Side effects of high
dosage of histidine include stress and anxiety.
Serine: Constituent of brain proteins and aids in the synthesis of immune system
proteins. It is also good for muscle growth.
Taurine: Necessary for proper brain function and synthesis of amino acids. It is
important in the assimilation of mineral nutrients such as magnesium, calcium and
potassium.
Threonine: Balances protein level in the body. It promotes immune system. It is also
beneficial for the synthesis of tooth enamel and collagen.
Asparagine: It helps promote equilibrium in the central nervous system—aids in
balancing state of emotion.
Apartic acid: Enhances stamina, aids in removal of toxins and ammonia from the body,
and beneficial in the synthesis of proteins involved in the immune system.
Proline: plays role in intracellular signalling.
L-arginine: plays role in blood vessel relaxation, stimulating and maintaining erection
in men, production of ejaculate, and removal of excess ammonia from the body.

Chemistry of nucleotides: Purine and Pyrimidine bases. Composition of

nucleosides and nucleotides. Occurrence of bases. Structures mandatory

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Medical College Doda
Nucleic acid, naturally occurring chemical compound that is capable of being broken
down to yield phosphoric acid, sugars, and a mixture of organic bases (purines and
pyrimidines). Nucleic acids are the main information-carrying molecules of the cell,
and, by directing the process of protein synthesis, they determine the inherited
characteristics of every living thing. The two main classes of nucleic acids are
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the master blueprint
for life and constitutes the genetic material in all free-living organisms and most
viruses. RNA is the genetic material of certain viruses, but it is also found in all living
cells, where it plays an important role in certain processes such as the making of
proteins.

Nucleic Acids- Nucleosides and Nucleotides

 Nucleotide is any member of the class of organic compounds in which the
molecular structure comprises a nitrogen-containing unit (base) linked to a sugar
and a phosphate group.
 They are monomeric units of nucleic acids and also serve as sources of chemical
energy (ATP, GTP), participate in cellular signalling (cAMP, cGMP) and function
as important cofactors of enzymatic reactions (coA, FAD, FMN, NAD+).
 The molecule without the phosphate group of nucleotides is called as nucleoside.
 Nucleosides are glycosylamines consisting simply of a nitrogenous base and a five-
carbon sugar (either ribose or deoxyribose).

Structure of Nucleotides
A single nucleotide is made up of three components: a nitrogen-containing base, a five-
carbon sugar (pentose), and at least one phosphate group With all three joined, a
nucleotide is also termed a ―nucleoside phosphate‖.
Individual phosphate molecules repetitively connect the sugar-ring molecules in
two adjacent nucleotide monomers, thereby connecting the nucleotide monomers of a
nucleic acid end-to-end into a long chain.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unlike in nucleic acid nucleotides, singular cyclic nucleotides are formed when
the phosphate group is bound twice to the same sugar molecule, i.e., at the corners of
the sugar hydroxyl groups

Nitrogenous bases
 The nitrogenous base is either a purine or a pyrimidine.
 There are five major bases found in cells. The derivatives of purine are called
adenine and guanine, and the derivatives of pyrimidine are called thymine,
cytosine and uracil.
 Purines include adenine and guanine and have two rings.
 Adenine has an ammonia group on its rings, whereas guanine has a ketone group.
 Pyrimidines include cytosine, thiamine, and uracil and have one ring.
 Thymine (found in DNA) and uracil (found in RNA) are similar in that they both
have ketone groups, but thymine has an extra methyl group on its ring.
 Bonds between guanine and cytosine (three hydrogen bonds) are stronger than
bonds between adenine and thymine (two hydrogen bonds).
Pentose Sugar
 The five-carbon sugar is either a ribose (in RNA) or a deoxyribose (in DNA)
molecule.
 In nucleotides, both types of pentose sugars are in their beta-furanose (closed five-
membered ring) form.

Structure of Nucleosides

 While a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or

more phosphate groups, a nucleoside has only a nitrogenous base and a five-
carbon sugar.
 In a nucleoside, the base is bound to either ribose or deoxyribose via a beta-
glycosidic linkage at 1‘ position.
 Examples of nucleosides include cytidine, uridine, adenosine, guanosine,
thymidine and inosine.

Properties of Nucleotides

Properties of purine bases

 Sparingly soluble in water
 Absorb light in UV region at 260 nm. (detection & quantitation of nucleotides)
 Capable of forming hydrogen bond
 Aromatic base atoms numbered 1 to 9
 Purine ring is formed by fusion of pyrimidine ring with imidazole ring.
 Numbering is anticlockwise.
Adenine : Chemically it is 6-aminopurine
Guanine : Chemically it is 2-amino,6-oxy purine
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Medical College Doda
Can be present as lactam & lactim form

Properties of pyrimidine bases

 Soluble at body pH
 Also absorb UV light at 260 nm
 Capable of forming hydrogen bond
 Aromatic base atoms are numbered 1 to 6 for pyrimidine.
 Atoms or group attached to base atoms have same number as the ring atom to
which they are bonded.
Cytosine: Chemically is 2-oxy ,4-amino pyrimidine
Exist both lactam or lactim form
Thymine: Chemically is 2,4 dioxy ,5-methyl pyrimidine
Occurs only in DNA
Uracil: Chemically is 2,4 dioxy pyrimidine
Found only in RNA

Properties of Pentose Sugars

 A pentose is a monosaccharide with five carbon atoms.

 Ribose is the most common pentose with one oxygen atom attached to each carbon
atom.
 Deoxyribose sugar is derived from the sugar ribose by loss of an oxygen atom.
 The aldehyde functional group in the carbohydrates react with
neighbouring hydroxyl functional groups to form intramolecular hemiacetals.
 The resulting ring structure is related to furan, and is termed a furanose.
 The ring spontaneously opens and closes, allowing rotation to occur about the
bond between the carbonyl group and the neighboring carbon atom yielding two
distinct configurations (α and β). This process is termed mutarotation.

Classification of Nucleotides
On the basis of the type of sugar present, nucleotides may be:
1. Ribonucleotides if the sugar is ribose.
2. Deoxyribonucleotides if the sugar is deoxyribose.

Classification of Nucleosides
On the basis of type of nitrogenous bases present, nucleoside derivatives may be also
grouped as following:
1. Adenosine nucleotides: ATP, ADP, AMP, Cyclic AMP
2. Guanosine nucleotides: GTP, GDP, GMP, Cyclic GMP
3. Cytidine nucleotides: CTP, CDP, CMP and certain deoxy CDP derivatives of
glucose, choline and ethanolamine
4. Uridine nucleotides: UDP

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Medical College Doda
5. Miscellaneous : PAPS (active sulphate), SAM (active methionine), certain
coenzymes like NAD+, FAD, FMN, Cobamide coenzyme, CoA

Functions of Nucleotides
 The nucleotides are of great importance to living organisms, as they are the
building blocks of nucleic acids, the substances that control all hereditary
characteristics.
 Polynucleotides consist of nucleosides joined by 3′,5′-phosphodiester bridges. The
genetic message resides in the sequence of bases along the polynucleotide chain.
 Nucleotides have a variety of roles in cellular metabolism. They are the energy
currency in metabolic transactions.
 They act as essential chemical links in the response of cells to hormones and other
extracellular stimuli.
 They are the structural components of an array of enzyme cofactors and metabolic
intermediates.
 The structure of every protein, and ultimately of every biomolecule and cellular
component, is a product of information programmed into the nucleotide sequence
of a cell‘s nucleic acids.
 Serving as energy stores for future use in phosphate transfer reactions. These
reactions are predominantly carried out by ATP.
 Forming a portion of several important coenzymes such as NAD+, NADP+, FAD
and coenzyme A.
 Serving as mediators of numerous important cellular processes such as second
messengers in signal transduction events. The predominant second messenger is
cyclic-AMP (cAMP), a cyclic derivative of AMP formed from ATP.
 Serving as neurotransmitters and as signal receptor ligands. Adenosine can
function as an inhibitory neurotransmitter, while ATP also affects synaptic
neurotransmission throughout the central and peripheral nervous systems. ADP is
an important activator of platelet functions resulting in control of blood
coagulation.
 Controlling numerous enzymatic reactions through allosteric effects on enzyme
activity.
 Serving as activated intermediates in numerous biosynthetic reactions. These
activated intermediates include S-adenosylmethionine (S-AdoMet or
SAM) involved in methyl transfer reactions as well as the many sugar coupled
nucleotides involved in glycogen and glycoprotein synthesis.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – VI
Fundamental Chemistry

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Valency, Molecular weight & Equivalent weight of elements and compounds.
Normality, Molarity, Molality. Valency

Molecular Weight
Molecular weight is a measure of the sum of the atomic weightvalues of the atoms in
amolecule. Molecular weight is used in chemistry to determine stoichiometry in
chemical reactions and equations. Molecular weight is commonly abbreviated by M.W.
or MW. Molecular weight is either unit less or expressed in terms of atomic mass units
(amu) or Daltons (Da).
Both atomic weight and molecular weight are defined relative to the mass of the
isotope carbon-12, which is assigned a value of 12 amu. The reason the atomic weight of
carbon isnotprecisely 12 is because it is a mixture of isotopes of carbon.
Sample Molecular Weight Calculation
The calculation for molecular weight is based on the molecular formula of a
compound (i.e., not the simplest formula, which only includes the ratio of types of
atoms and not the number). The number of each type of atom is multiplied by its atomic
weight and then added to the weights of the other atoms.
For example, the molecular formula of hexane is C6H14. The subscripts indicate
the number of each type of atom, so there are 6 carbon atoms and 14 hydrogen atoms in
each hexane molecule. The atomic weight of carbon and hydrogen may be found ona
periodic table.

Atomic weight of carbon: 12.01

Atomic weight of hydrogen: 1.01

Molecular weight = (number of carbon atoms) (C atomic weight) + (number of H atoms)

(H atomic weight)
Molecular weight = (6 x 12.01) + (14 x 1.01)
Molecular weight of hexane = 72.06 + 14.14
Molecular weight of hexane = 86.20 amu

Molecular Weight Versus Molecular Mass

Molecular weight is often used interchangeably with molecular mass in chemistry,
although technically there is a difference between the two. Molecular mass is a measure
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Medical College Doda
of mass and molecular weight is a measure of force acting on the molecular mass. A
more correct term for both molecular weight and molecular mass, as they are used in
chemistry, would be "relative molecular mass".

Equivalent weight
Elements combine with other elements to from compounds. Each element combines
with another element with a defininte weight. This combining weight of element to
another metal is called the equivalnet weight of the element. If we take certain weight of
any element as standard, we can find equivalent weight of corresponding other element
on regard of the standard element.
Equivalent weight is defined as ―The number of parts by weight of any element
that combine with (or displace) directly or indirectly, 1.008 parts by weight of
Hydrogen or 8 parts by weight of oxygen or 35.5 parts by weight of chlorine‖.

Some examples:

C+ O2→ CO2

As the definition says, the weight of element that combines with 8 parts by weight of
oxygen is the equivalent weight of that element. So let‘s find the weight of carbon that
combine with 8 parts by weight of oxygen in the above reaction.
In the above reaction, one mole of carbon reacts with one mole of oxygen to give one
mole of carbon dioxide.
i.e. 32 parts by weight of oxygen is combining with 12 parts by weight of carbon.
1 part by weight of oxygen is combining with (12/32) parts by weight of carbon.
8 parts by weight of oxygen is combining with (12/32) x 8 parts by weight of
carbon
=3

Therefore equivalent weight of carbon is 3 gm.

2Na + 2HCL →2NaCl + H2

Here, 2 mole of Na reacts with 2 moles of HCl to give 2 moles of NaCl and one mole of
H2 .

In this reaction, Na has displaced hydrogen from HCl.

2 Parts by weight of hydrogen is replaced by (23 x 2) 46 parts by weight of Na.

1 part by weight of hydrogen is replaced by 46/2 parts by weight of Na.

=23
Therefore, equivalent weight of Na is 23.

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Medical College Doda
 Equivalent weight of Acid, Base, salt and oxidizing/Reducing agents:
1. Equivalent weight of Acid:
For an acid, equivalent weight is the molecular weight divided by its basicity.
i.e. Equivalent weight of acid = Molecular weight of the acid/basicity
Basicity of an acid is the number of replaceable hydrogen of an acid. In HCl there
is one replaceable hydrogen, so its basicity is one. In H2SO4 there is 2 replaceable
hydrogen so its basicity is two

Calculate equivalent weight of HCL and H2SO4

For HCL;
The molecular formula is HCL is (1.008 + 35.6) =36.451 gm.
Basicity=1
Equivalent weight =Molecular weight / Basicity
= 36.451/1
= 36.451 gm
For H2SO4
The molecular formula is (2+ 32 + 4 x 16) = 98gm.
Basicity =2
Equivalent weight = 98gm/2= 49 gm.

2. Equivalent weight of Base:

Equivalent weight of base = Molecular formula /Acidity
Acidity of base is the number of replaceable hydroxyl ion which is generated when
dissolved in water. The number of replaceable hydroxyl ion for NaOH is 1, so its acidity
is 1. For Ca(OH)2, there is two replaceable hydroxyl ions, so its acidity is 2.

Calculate equivalent weight of NaOH:

Molecular formula of NaOH is (23+ 16+ 1.008)= 40gm.
Acidity=1
Equivalent weight= 40/1
= 40 gm.

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Medical College Doda
3. Equivalent weight of Salt:
For a salt, equivalent weight = Molecular formula / number of the charge on basic
radical
For eg: Equivalent weight of NaCl= (23+35.5)/1
=1
Equivalent weight of Na2CO3 = (2 x 23+ 12+ 3 x 16)/2
= 106/2
= 53
4. Equivalent weight of Oxidant and Reductant:
Equivalent weight of oxidant or reductant = molecular weight/ no. of change in
oxidation no of Oxidant and reductant.
For example,
When KMnO4changes to MnSO4, the oxidation number of Mn changes from +7 to +2
state. The total change in oxidation number is 5. So,
Equivalent weight of KMnO4in acidic medium = molecular weight of KMnO4/5
= 161/5
= 32.2 gm

Equivalent Mass
i. Equivalent mass of an element is the mass of the element which combines with
or displaces 1.008 parts by mass of hydrogen or 8 parts by mass of oxygen or 35.5
parts by mass of chlorine.
Atomic wt. of the element
Eq. Mass of an element = _____________________________

Valence of the element

Molecular weight of the acid

ii. Equivalent mass of an acid = ___________________________________

Basicity of the acid

Basicity is the number of displaceable H+ ions from one molecule of the acid.

Molecular weight of the base

iii. Equivalent mass of a base = ___________________________________

Acidity of the base

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Medical College Doda
 Acidity is the number of displaceable OH – ions from one molecule of the base.

Molecular weight of the salt

iv. Equivalent mass of a salt = __________________________________________

Total positive valency of the metal atoms

Formula weight of the ion

v. Equivalent mass of an ion = ________________________________

Change weight of the ion

vi. Eq. mass of an oxidizing / reducing agen t = Mol. Wt. or atomic wt.
__________________________________

No. of electron lost or gained by one molecule

of the substance

Relationship between normality and molarity of a solution

Mol. Mass
Normality of a solution = Molarity × _____________
Eq. Mass

For an acid, M = basicity and for a base M = acidity

Hence Normality of an acid = Molarity x Basicity

Normality of a base = Molarity x Acidity

For monobasic acids e.g., HCl, HNO3 and monoacidic bases eg. NaOH, KOH, the
molecular weight and equivalent weight are the same

Mol. Wt.
Eq. Wt = ____________ or Eq = Mol Wt
1

Hence N = M i.e. Normal solution is equal to Molar solution.

For dibasic acids eg. H2SO4, H2CO3, H2C2O4 and diacidic bases eg. Na2CO3, Ca(OH)2,
Ba(OH)2, the molecular weight is equal to times that of equivalent weight.

Mol. Wt.
Eq. Wt. = _______________ or 2E=M
2

Normality

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Medical College Doda
Normality: The normality of a solution is the gram equivalent weight of a solute per
liter of solution. A gram equivalent weight or equivalent is a measure of the reactive
capacity of a given chemical species (ion, molecule, etc.). Normality is the only
concentration unit that is reaction dependent.

Important Formulas
No. of geq of solute
1. (Normality) N = _______________________________

Volume of the solution in liters

Wt. of solute per litre of solution

2. N = -------------------------------------------
geq wt. of solute

Wt. of solute 1000

3. N = ------------------------ × --------------------------
g eq. Wt. of solute Vol. of solution in ml.

No. of milli eq. of solute

4. N = ---------------------------
Vol. of soln. in ml.

Percent of solute × 10
5. N = --------------------------------
geq wt. of solute

Strength in gm/lt of solution

6. N = --------------------------------------
geq Wt of solute

10 ×sp. gr. of the soln. × wt. % of the solute

7. M= --------------------------------------------------------
g eq. Wt of solute.

8. N1 V1 =N2 V2 (Dilution Law)

9. N1V1/n1 =N2V2/n2
10. If two solutions of the same solute are mixed then normality of the resulting
solution
N= V1N1 + V2 N2/V1+V2
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Medical College Doda
11. Volume of water added to get a solution of normality N2 from V1 ml of normality
N1 is
V2-V1= (N1-N2/N2) ×V1

12. If ‗a‘ gram of solute is present in υ ml of a given solution,

a 1000
N= __________________________ x _________
Eq. Mass of the solute υ

Thus a solution of sulfuric acid having 0.49 gram of it dissolved in 250 ml of solution
will have its normality,

0.49 1000
N= ______ x _______ = 0.04 (Eq. Mass of sulfuric acid = 49)
49 250

 Normal solution: One gram equivalent weight of a substance (solute) present in

one liter of solution is called normal solution and denoted by (N).
Example 1: Since hydrochloric acid is a strong acid that dissociates completely in
water, a 1 N solution of HCl would also be 1 N for H+ or Cl- ions for acid-base
reactions.
 Semi-normal solution: A solution, one liter of which contains ½ gram equivalent
of a solute is called semi-normal solution and denoted by N/2.
 Deci-normal solution: 1/10 gram equivalent of the substance present in one liter
of a solution is called deci-normal solution and denoted by (N/10).

Molarity
When we are interested in the actual concentration of molecules of a chemical in
solution, it is better to have a universal measurement that works regardless of how the
chemical is supplied. As long as the molecular weight (sometimes called formula
weight) is known, we can describe a solution in the form of moles per liter, or simply
molar (M). Molarity of a solution is defined as the numbers of moles of the solute
dissolved per liter of solution. It is denoted by M.

Important Formulas
No. of moles of solute
1. (Morality) M = ___________________________________

Volume of the solution in liters

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 M can be calculated from the strength as below:

Strength in grams per liter

M= _____________________________

Molecular mass of solute

If ‗a‘ grams of the solute is present in υ ml of a given solution, then

a 1000
M= x
______________ ________

Mol. Mass υ

Thus a solution of sulfuric acid having 4.9 g of it dissolved in 500 ml of solution will
have its molarity,

4.9 1000
M= ________ x ________ = 0.1
98 500
Wt. of solute per litre of solution
2. M =
Mol. Wt. of solute

Wt. of solute 1000

3. M = ×
Mol. Wt. of solute Vol. of solution in ml.

No. of milli moles of solute

4. M =
Vol. of soln. in ml.

Percent of solute × 10
5. M =
Mol. wt. of solute

Strength in gm/lt. of solution

6. M =
Mol. wt. of solute

10 × Sp. gr. of the soln. × wt. % of the solute

7. M=
Mol Wt of solute

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Medical College Doda
8. M1 V1 = M2 V2 (Dilution Law)
9. M1V1/n1 =M2V2/n2
10. If two solutions of the same solute are mixed then molarity of the resulting solution
M= V1M1 + V2 M2/V1+V2
11. Volume of water added to get a solution of molarity M2 from V1 ml of molarity M1
is
V2-V1= (M1-M2/M2) ×V1

Calculating Morality from Percent Solutions

To determine the morality of a mass percent solution, proceed as follows:
a. Determine the mass of solution by multiplying the volume of the solution by the
density of the solution.
b. Determine concentration in percent by mass of the solute in solution.
c. Calculate the molar mass of the compound, MM.
d. Multiply mass by mass % and divide by molecular mass to find the number of
moles present in the whole solution.
e. Divide the number of moles by the volume in liters of the solution to find the
molarity of the solution.

Example: Determine molarity of 37.2% hydrochloric acid (density 1.19 g/ml).

1. Mass of solution = 1,000 ml x 1.19 g/ml = 1,190 g
2. Mass % = 37.2 % = 0.372
3. Molar mass of hydrochloric acid = 36.4 g/mol
bvgf
4. mass x mass % 1,190 g x 0.372
—————— = —————— = 12.1 moles
MM HCl 36.4 g/mol

5. Molarity = moles/liters = 12.1 moles/1 liter = 12.1 M

Molality:
The molal unit is not used nearly as frequently as the molar unit. A molality is the
number of moles of solute dissolved in one kilogram of solvent. Be careful not to
confuse molality and molarity. Molality is represented by a small "m" whereas molarity
is represented by an upper case "M." Note that the solvent must be weighed unless it is
water. One liter of water has a specific gravity of 1.0 and weighs one kilogram; so one
can measure out one liter of water and add the solute to it. Most other solvents have a
specific gravity greater than or less than one. Therefore, one liter of anything other than

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Medical College Doda
water is not likely to occupy a liter of space. To make a one molal aqueous (water)
solution of sodium chloride (NaCl), measure out one kilogram of water and add one
mole of the solute, NaCl to it. The atomic weight of sodium is 23 and the atomic weight
of chlorine is 35. Therefore the formula weight for NaCl is 58, and 58 grams of NaCl
dissolved in 1kg water would result in a 1 molal solution of NaCl.
Percent Solutions Mathematically,

Number of moles of the solute

m= X 1000
_________________________________

Weight of the solvent in grams

‗m‘ can be calculated from the strength as below:

Strength per 1000 grams of solvent

m= ___________________________________

Molecular Mass of solute

If ‗a‘ gram of the solute is dissolved in ‗b‘ gram of the solvent then,

a 1000
m= _________________________ x ______

Mol. Mass of the solute b

Thus a solution of anhydrous sodium carbonate (Mol. mass = 106) having 1.325 grams
of it dissolved in 250 grams of water will have its molality.

1.325 1000
m= ________ x ______ = 0.05
106 250

 Molal solution: A molal solution is that solution which contains 1 gm molecule

of solute dissolved in 1000 gm of solvent.
Example: Determine molality of the sugar solution.
Molality = molsolute / msolvent
Molality = 0.0117 mol / 0.341 kg = 0.034 mol/kg
The molality of the sugar solution is 0.034 mol / kg.

Weight in volume: Prepare 2 liters 0.85% sodium chloride

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Medical College Doda
With 1% defined as 1 gram per 100 ml, 0.85% is 0.85 grams per 100 ml. Since two liters is
20x the volume of 100 ml, we need 20 x 0.85 grams which is 17 grams NaCl. For this
quantity we can use a top loading balance or even a trip balance.
A typical electronic balance is accurate to one hundredth of a gram, which is
sufficiently accurate for weighing out 17 grams. First we "tare" the instrument by
placing a weigh boat onto the pan and setting it to ―zero.‖ We don't want to
contaminate our chemical stocks, so we either clean the spatula or spoon before dipping
it into the container or we simply shake the chemical out onto the boat.
Suppose that we tap out 16.97 grams of NaCl. Should we go to the trouble to get
that last 0.03 gram? No. Consider that if it was necessary to be more accurate, we would
describe the formula as something like 0.846% NaCl, or maybe 0.8495%. If there is some
advantage to being precise then we should exercise precision, otherwise trying to be too
precise just wastes time.

Percent by weight: To make up a solution based on percentage by weight, one would

simply determine what percentage was desired (for example, a 20% by weight aqueous
solution of sodium chloride) and the total quantity to be prepared.
If the total quantity needed is 1 kg, then it would simply be a matter of calculating 20%
of 1 kg which, of course is: 0.20 NaCl 1000 g/kg = 200 g NaCl/kg.
In order to bring the total quantity to 1 kg, it would be necessary to add 800g water.

Percent by volume: Solutions based on percent by volume are calculated the same as
for percent by weight, except that calculations are based on volume. Thus one would
simply determine what percentage was desired (for example, a 20% by volume aqueous
solution of sodium chloride) and the total quantity to be prepared.
If the total quantity needed is 1 liter, then it would simply be a matter of
calculating 20% of 1 liter which, of course is: 0.20 NaCl 1000 ml/l = 200 ml NaCl/l.
Percentages are used more in the technological fields of chemistry (such as
environmental technologies) than they are in pure chemistry.

 Mass percent solutions: Mass percent solutions are defined based on the grams
of solute per 100 grams of solution.
Example: 20 g of sodium chloride in 100 g of solution is a 20% by mass
solution. Volume percent solutions are defined as milliliters of solute per
100 mL of solution.
Example: 10 mL of ethyl alcohol plus 90 mL of H2O (making approx. 100
mL of solution) is a 10% by volume solution. Mass-volume percent

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Medical College Doda
 solutions, indicated by w/v% are defined as the grams of solute per 100
milliliters of solution.
Example: 1 g of phenolphthalein in 100 mL of 95% ethyl alcohol is a 1
w/v% solution.
 Conversion between Percent Solutions: To convert mass percent to volume
percent or vice versa proceed as below:
Example: A 10% by mass solution of ethyl alcohol in water contains 10 g
of ethyl alcohol and 90 g of water.
(a) Determine the volume of the component (ethyl alcohol in our
example):
Mass of ethyl alcohol = 10 g (given)
Density of ethyl alcohol = 0.794 g / ml

10 g
Volume of ethyl alcohol = ———— = 12.6 ml
0.794 g/ml

(b) Determine the volume of the total solution by dividing the mass of the
solution by the density of the solution.
Mass of solution = 100 g (given)
Density of solution (10% ethyl alcohol) = 0.983 g/ml

100 g
Volume of solution = ————— = 101.8 ml
0.983 g/ml

(c) Determine the percent by volume by dividing the volume of the

component by the volume of the solution.
Volume of ethyl alcohol 12.6
Volume Percent = —————————— = ——— = 12.4%
Total volume of solution 101.8

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Medical College Doda
 Unit – VII
Solutions

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Definition, use, classification where appropriate, preparation and storage of
solutions

A solution is a homogeneous mixture composed of only one phase. In such a mixture, a

solute is a substance dissolved in another substance, known as a solvent. The solvent
does the dissolving. The solution more or less takes on the characteristics of the solvent
including its phase and the solvent is commonly the major fraction of the mixture. The
concentration of a solute in a solution is a measure of how much of that solute is
dissolved in the solvent

 Characteristics of solution
 A solution is a homogenous mixture.
 The particles of solute in solution cannot be seen by naked eye.
 The solution does not allow beam of light to scatter.
 A solution is stable.
 The solute from the solution cannot be separated by filtration (or physically).

Stock and working solutions.

Stock solutions are solutions of known concentration that are prepared by the
pharmacist or technician for convenience in dispensing. They are usually strong
solutions from which weaker ones may be made conveniently. Stock solutions usually
are prepared on a weight-in-volume basis, and their concentration is expressed as a
ratio strength or less frequently as a percentage strength. Stock solutions can save a lot
of time, conserve materials, reduce needed storage space, and improve the accuracy
with which we prepare solutions and reagents. Here are several illustrated types of
applications using stock solutions.
We typically refer to the strength of a stock solution by a number followed by the times
symbol x. For example, a stock solution that is concentrated by a factor of 10 is called a
10 times concentrated stock, a 10x concentrate, a solution of 10x strength, or simply a
10x solution. A normal working solution is a 1x, or normal strength solution.

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Medical College Doda
Molar and Normal solutions of compounds and acids. (Nacl, NaOH,
HCL, H2SO4, H3PO4, CH3COOH)
Molarity is another standard expression of solution concentration. Molar solutions use
the gram molecular weight of a solute in calculating molar concentration in a liter (L) of
solution.
The gram molecular weight (GMW) of a substance (sometimes called the "formula
weight") is the sum of the combined atomic weights of all atoms in the molecule
expressed in grams. For example, the GMW of NaCl is equal to the atomic weight (these
atomic weights may be found on a periodic table or as a formula weight on the bottle of
substance) of Na (22.99) and the atomic weight of Cl (35.45) for a total of 58.44 g.
A 1 molar (M) solution will contain 1.0 GMW of a substance dissolved in water to make
1 liter of final solution. Hence, a 1M solution of NaCl contains 58.44 g.
Example:

1: Molar solution of Nacl

The molecular weight of a sodium chloride molecule (NaCl) is 58.44, so one gram-
molecular mass (=1 mole) is 58.44 g.
If you dissolve 58.44g of NaCl in a final volume of 1 liter, you have made a 1M
NaCl solution, a 1 molar solution

2: Molar solution of NaOH

Molarity = no. of moles of solute / 1 liter . * one moles of sodium hydroxide = 40 gm of
sodium hydroxide. so we can said ; if want prepare 1 molar NaOH solution then we
need 40 gm NaOH dissolve in one liter of water so it became one 1 molar NaOH
solution.

3: Normal solution of HCL

4: Normal solution of H2SO4

5: Normal solution of H3PO4

6: Normal solution of CH3COOH

Preparation of percent solutions – w/w, v/v, w/v (solids, liquids and

acids), Conversion of a percent solution into a molar solution

Concentration: Concentration is defined as the abundance of a constituent divided by

the total volume of a mixture. Furthermore, in chemistry, four types of mathematical
description can be distinguished: mass concentration, molar concentration, number

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Medical College Doda
concentration, and concentration. The term concentration can be applied to any kind of
chemical mixture, but most frequently it refers to solutes in solutions.
There are five units of concentration that are particularly used. The first three:
molality, molarity and normality are dependent upon the mole unit. The last two:
percent by volume and percent by weight have nothing to do with mole, only weight or
volume of the solute or substance to be diluted, versus the weight or volume of the
solvent or substance in which the solute is diluted. Percentages can also be determined
for solids within solid. These formulas all have one thing in common. A quantity of
solute is measured out, mixed with solvent, and the volume is brought to some final
quantity after the solute is completely dissolved. That is, solutions are typically
prepared volumetrically. Because solutes add volume to a quantity of solvent, this
method of preparation of solutions is necessary to ensure that an exact desired
concentration is obtained.

Weight/weight (w/w) solutions

The weight of the solute relative to the weight of the final solution is described as a
percentage. For example, suppose you have a dye that is soluble in alcohol. Rather than
write the instructions, ―take 3 grams dye and mix with 97 grams absolute alcohol,‖ you
can describe the solutions simply as 3% dye in absolute alcohol. The formula applies to
any volume of solution that might be required. Three grams dye plus 97 grams alcohol
will have final weight of 100 grams, so the dye winds up being 3% of the final weight.
Note that the final weight is not necessarily equal to the final volume.
Aqueous weight-in-weight solutions are the easiest to prepare. Since 1 milliliter
of water weighs one gram, we can measure a volume instead of weighing the solvent. A
very common use of w/w formulas is with media for the culture of bacteria. Such
media come in granular or powdered form, often contain agar, and often require heat in
order to dissolve the components. Using a w/w formula the media and water can be
mixed, heated, and then sterilized, all in a single container.

Weight-in-volume (w/v) solutions

When we describe a concentration as a percentage without specifying the type of
formula, we imply that the solution is to be made using the weight-in-volume (w/v)
method. As with w/w, weight-in-volume is a simple type of formula for describing the
preparation of a solution of solid material in a liquid solvent. This method can be used
to describe any solution, but is commonly used for simple saline solutions and when the
formula weight of the solute is unknown, variable, or irrelevant, which is often the case
with complex dyes, enzymes or other proteins. Solutions that require materials from

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Medical College Doda
natural sources are often prepared w/v because the molecular formula of the substance
is unknown and/or because the substance cannot be described by a single formula.
A one percent solution is defined as 1 gram of solute per 100 milliliters final
volume. For example, 1 gram of sodium chloride, brought to a final volume of 100 ml
with distilled water, is a 1% NaCl solution. To help recall the definition of a 1% solution,
remember that one gram is the mass of one milliliter of water. The mass of a solute that
is needed in order to make a 1% solution is 1% of the mass of pure water of the desired
final volume. Examples of 100% solutions are 1000 grams in 1000 milliliters or 1 gram in
1 milliliter.

Volume / volume (v/v) solutions

Volume-in-volume is another rather simple way of describing a solution. We simply
describe the percent total volume contributed by the liquid solute, in case we assume
that the solvent is water unless some other solvent is specified. V/v is often used to
describe alcohol solutions that are used for histology or for working with proteins and
nucleic acids. For example, 70% ethanol is simply 70 parts pure ethanol mixed with
water to make 100 parts total.

Saturated and supersaturated solutions

Introduction
When solid solute (substance or particles) and liquid solvent are mixed, the only
possible reactions are dissolution and crystallization.
 Dissolution is the dissolving process of the solid solute.
 Crystallization is the opposite, causing the solid solute to remain undissolved.

Types of Saturation
Kinds of Saturation Definition
Saturated Solution A solution with solute that dissolves until it is unable to
dissolve anymore, leaving the undissolved substances at the
bottom.
Unsaturated Solution A solution (with less solute than the saturated solution) that
completely dissolves, leaving no remaining substances.
Supersaturated A solution (with more solute than the saturated solution) that
Solution contains more undissolved solute than the saturated
solution because of its tendency to crystallize and
precipitate.
Example 1: Saturated Solution

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Medical College Doda
Example 1: Above is illustrated an example of a saturated solution. In Figure 1.1-1.3,
there is a constant amount of water in all the beakers. Figure 1.1 shows the start of the
saturation process, in which the solid solute begins to dissolve (represented by red
arrows). In the next beaker, Figure 1.2, much of the solid solute has dissolved, but not
completely, because the process of crystallization (represented by blue arrows) has
begun. In the last beaker, Figure 1.3, only a small amount of the solute solvent
remains undissolved. In this process, the rate of the crystallization is faster than the
rate of dissolution, causing the amount of dissolved to be less than the amount
crystallized.
Example 2: Unsaturated Solution

Example 2: Next, an unsaturated solution is considered. In Figure 2.1-2.3, there is a

constant amount of water in all the beakers. Figure 2.1 shows the start of the process,
in which solid solute is beginning to dissolve (represented by red arrows). In the next
beaker, shown in Figure 2.2, a large amount of solute has dissolved. The size of the
red arrows is much larger than those of the blue arrows, which means that the rate of
dissolution is much greater than rate of crystallization. In the last beaker, shown in
Figure 2.3, the solute solvent has completely dissolved in the liquid solvent.
Example 3: Supersaturated Solution

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Medical College Doda
 Example 3: This is an example of a supersaturated solution. In Figure 3.1-3.3, there is a
constant amount of water in all the beakers. Figure 3.1 shows a beaker with more solid
solute than in the saturated solution (Figure 1.1) dissolving. In Figure 3.2, solid begins
to crystallize as it slowly decreases the rate of dissolution. In the last picture, Figure
3.3, the solids become a crystallized form which begins to harden.

Factors Affecting Saturation

 The solubilities of ionic solutions increase with an increase in temperature, with
the exceptions of compounds containing anions.
 Finely divided solids have greater solubilities.
 In contrast to the solubility rate, which depends primarily on temperature, the
rate of crystallization depends on the concentration of the solute at the crystal
surface.
 In a still solution, concentration builds at the solute surface causing higher
crystallization; therefore, stirring the solution prevents the buildup, maximizing
the net dissolving rate.
 The net dissolving rate is defined as the dissolving rate minus the crystallization
rate.
 If the rates of solubility and crystallization are the same, the solution is saturated,
and dynamic equilibrium is reached.
 Le Chatelier's principle predicts the responses when an equilibrium system is
subjected to change in temperature, pressure or concentration. This principle
states the following:
o For an increase of temperature, solubility increases which causes an
endothermic reactions.
o For an decrease of temperature, solubility decreases which causes an
exothermic reactions.
o Adding an inert gas to a constant-volume equilibrium mixture has no
effect on the equilibrium.
o An increase in the external pressure causes a decrease in reaction volume
and shifts equilibrium to the right.

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Medical College Doda
Standard solutions. Technique for preparation of standard solutions and
Storage. E.g: glucose, albumin etc.

Standard solutions are solutions that contain a known and accurate amount (i.e.
concentration) of a substance or element. These solutions are commonly used to help
identify and determine the concentration of a substance whose concentration is
unknown. When working to analyze, let's say an unknown substance, you want to be as
accurate as possible with all results. This is where standard solutions step in. Because
these solutions contain accurate concentrations of a chemical component, they will
increase confidence regarding the determination of substances with unknown
concentrations.

Standard solution of Glucose

Standard solutions in general contain a known amount of substance dissolved in a
known quantity of another substance. Usually, a standard glucose solution refers to a 1-
percent glucose solution. Preparing a 1-percent standard glucose solution involves
dissolving 1 g of glucose in 100 ml of water. Glucose standard solutions are used to
create calibration curves against which unknown solutions are measured. These curves
then help determine the concentration of the unknown solution.
Intravenous sugar solution, also known as dextrose solution, is a mixture of dextrose
(glucose) and water. It is used to treat low blood sugar or water loss without electrolyte
loss. Intravenous sugar solutions are in the crystalloid family of medications.

Standard solution of Albumin

Dilutions- Diluting Normal, Molar and percent solutions. Preparing

working standard from stock standard.
Diluted solution: that solution in which there is a relatively small amount of solute
dissolved in the solution.
Concentrated solution: that solution which contains a relatively large amount of solute.
Dilution: it is the process of reducing the concentration of a solute in solution, usually
simply by mixing with more solvent. A dilution is a solution made by adding more
solvent to a more concentrated solution (stock solution), which reduces the
concentration of the solute. An example of a dilute solution is tap water, which is
mostly water (solvent), with a small amount of dissolved minerals and gasses (solutes).
An example of a concentrated solution is 98% sulfuric acid (~18 M).

Use the law of conservation of mass to perform the calculation for the dilution:

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Medical College Doda
 MdilutionVdilution = MstockVstock

Fig 1.5: Dilution Process

Dilution Example
As an example, preparation of 50 ml of a 1.0 M solution from a 2.0 M stock solution. The
first step is to calculate the volume of stock solution that is required.
MdilutionVdilution = MstockVstock
(1.0 M)(50 ml) = (2.0 M)(x ml)
x = [(1.0 M)(50 ml)]/2.0 M
x = 25 ml of stock solution
So to make theabove solution, pour 25 ml of stock solution into a 50 ml volumetric flask.
Dilute with solvent to the 50 ml line.

Specimen dilutions: Serial dilutions, Reagent dilution & dilution factors

A simple dilution is one in which a unit volume of a liquid material of interest is
combined with an appropriate volume of a solvent liquid to achieve the desired
concentration. The dilution factor is the total number of unit volumes in which your
material will be dissolved. The diluted material must then be thoroughly mixed to
achieve the true dilution. For example, a 1:5 dilution (verbalize as "1 to 5" dilution)
entails combining 1 unit volume of solute (the material to be diluted) + 4 unit volumes
of the solvent medium (hence, 1 + 4 = 5 = dilution factor). The dilution factor is
frequently expressed using exponents: 1:5 would be 5e-1; 1:100 would be 10e-2, and so
on.

Example 1:

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Medical College Doda
Frozen orange juice concentrate is usually diluted with 4 additional cans of cold water
(the dilution solvent) giving a dilution factor of 5, i.e., the orange concentrate represents
one unit volume to which you have added 4 more cans (same unit volumes) of water.
So the orange concentrate is now distributed through 5 unit volumes. This would be
called a 1:5 dilution, and the OJ is now 1/5 as concentrated as it was originally. So, in a
simple dilution add one less unit volume of solvent than the desired dilution factor
value.

Example 2: Suppose you must prepare 400 ml of a disinfectant that requires 1:8 dilution
from a concentrated stock solution with water. Divide the volume needed by the
dilution factor (400 ml / 8 = 50 ml) to determine the unit volume. The dilution is then
done as 50 ml concentrated disinfectant + 350 ml water.

Serial dilution, as the name suggests, is a series of sequential dilutions that are
performed to convert a dense solution into a more usable concentration.
 In simple words, serial dilution is the process of stepwise dilution of a solution
with an associated dilution factor.
 In biology, serial dilution is often associated with reducing the concentration of
cells in a culture to simplify the operation.

Objectives of Serial dilution

 The objective of the serial dilution method is to estimate the concentration (number
of organisms, bacteria, viruses, or colonies) of an unknown sample by enumeration
of the number of colonies cultured from serial dilutions of the sample.
 In serial dilution, the density of cells is reduced in each step so that it is easier to
calculate the concentration of the cells in the original solution by calculating the
total dilution over the entire series.
 Serial dilutions are commonly performed to avoid having to pipette very small
volumes (1-10 µl) to make a dilution of a solution.
 By diluting a sample in a controlled way, it is possible to obtain incubated culture
plates with an easily countable number of colonies (around 30–100) and calculate
the number of microbes present in the sample.

Serial dilution formula/calculations

 Serial dilution involves the process of taking a sample and diluting it through a
series of standard volumes of sterile diluent, which can either be distilled water or
0.9 % saline.
 Then, a small measured volume of each dilution is used to make a series
of pour or spread plates.
 Depending on the estimated concentration of cells/organisms in a sample, the
extent of dilution is determined. For e.g., if a water sample is taken from an

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Medical College Doda
 extremely polluted environment, the dilution factor is increased. In contrast, for a
less contaminated sample, a low dilution factor might be sufficient.
 Serial two-fold and ten-fold dilutions are commonly used to titer antibodies or
prepare diluted analytes in the laboratory.
 The dilution factor in a serial dilution can be determined either for an individual
test tube or can be calculated as a total dilution factor in the entire series.
 The dilution factor of each tube in a set:

 For a ten-fold dilution, 1 ml of sample is added to 9 ml of diluent. In this case, the

dilution factor for that test tube will be:

 After the first tube, each tube is the dilution of the previous dilution tube.
Now, for total dilution factor,

 Total dilution factor for the second tube = dilution of first tube × dilution of the
second tube.
Example:

For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)

For the second tube, dilution factor = 10-1 (1ml added to 9 ml)
Total dilution factor = previous dilution × dilution of next tube

= total dilution of 10-1 × 10-1 = 10-2

Procedure of Serial dilution

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Medical College Doda
The following is the procedure for a ten-fold dilution of a sample to a dilution factor of
10-6:
1. The sample/culture is taken in a test tube and six test tubes, each with 9 ml of
sterile diluents, which can either be distilled water or 0.9% saline, are taken.
2. A sterile pipette is taken.
3. 1 ml of properly mixed sample/culture is drawn into the pipette.
4. The sample is then added to the first tube to make the total volume of 10 ml. This
provides an initial dilution of 10-1.
5. The dilution is thoroughly mixed by emptying and filling the pipette several times.
6. The pipette tip is discarded, and a new pipette tip is attached to the pipette.
7. Now, 1 ml of mixture is taken from the 10-1 dilution and is emptied into the second
tube. The second tube now has a total dilution factor of 10-2.
8. The same process is then repeated for the remaining tube, taking 1 ml from the
previous tube and adding it to the next 9 ml diluents.
9. As six tubes are used, the final dilution for the bacteria/cells will be 10 -6 (1 in
1,000,000).

Applications/Uses
Serial dilution is performed in a number of experimental sciences like biochemistry,
pharmacology, physics, and homeopathy.

1. Serial dilution is used in microbiology to estimate the concentration or number of

cells/organisms in a sample to obtain an incubated plate with an easily countable
number of colonies.
2. In biochemistry, serial dilution is used to obtain the desired concentration of
reagents and chemicals from a higher concentration.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 3. In pharmaceutical laboratories, serial dilution is performed to receive the
necessary concentration of chemicals and compounds as this method is more
effective than individual dilutions.
4. In homeopathy, homeopathic dilutions are used where a substance is diluted in
distilled water or alcohol. It is believed than dilution increases the potency of the
diluted substance by `activating its vital energy.

Limitation/Problems
Even though serial dilution is a useful technique in laboratories, it faces some
challenges. Some of which are:

1. An error might occur during the propagation of the sample, and the transfer
inaccuracies lead to less accurate and less precise transfer. This results in the
highest dilution to have the most inaccuracies and the least accuracy.
2. Because serial dilution is performed in a stepwise manner, it requires a more
extended period of time which limits the efficiency of the method.
3. Serial dilution only allows the reduction of bacteria/cells but not the separation of
bacteria/cells like in other techniques like flow cytometry.
4. This technique also requires highly trained microbiologists and experts in aseptic
techniques.

Examples
 A simple example of serial dilution performed in our daily life is tea or coffee. In
coffee, we add a certain amount of cold press coffee and add water over it so
obtain a desired concentration of coffee.
 Another example of serial dilution is the dilution of acids and bases in chemistry to
obtain a required concentration.
 Serial dilution of culture to determine the number of bacteria in a given sample
through a plating technique is also an essential example of serial dilution.

Reagent dilution & dilution factors

Reagents – Dilution

How to dilute acids and bases.

Dilution of acids are exothermic reactions, proper precautions are to be taken while
diluting acids. Wear goggles at all times, aprons and gloves would add an extra layer of
protection.
Acids: Concentration of acids available
 HCl 12 M
 H2SO4 18 M
 HNO3 16 M
Preparation of 1 M HCl

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 To prepare 1 Liter of 1 M HCl measure out 83 ml of 12 M concentrated acid in a fume
cupboard and add it to 917 ml of distilled water. Using a magnetic stirrer to mix the
acid would be a good idea, if that is not available use a glass rod.
Preparation of 1 M H2SO4
 To prepare 1 Liter of 1 M H2SO4 measure out 56 ml of 18 M concentrated acid in a fume
cupboard and add it to 944 ml of distilled water. Using a magnetic stirrer to mix the
acid would be a good idea. The dilution of sulfuric acid is exothermic so add small
quantities of the acid to water – Extra care should be taken while diluting sulphuric
acid.
Preparing 1M HNO3
 To prepare 1 Liter of 1 M HNO3 measure out 63 ml of 16 M concentrated acid in a fume
cupboard and add it to 937 ml of distilled water. Using a magnetic stirrer to mix the
acid would be a good idea, Nitric acid is an oxidizing acid, if it comes in contact with
your skin it will turn yellow due to nitration. The skin will eventually be replaced.
Preparation of 1 M NH4OH
 Ammonium hydroxide is volatile and pungent make sure the fume cupboard exhaust is
turned on before you attempt to dilute the base.
 To prepare 1 Liter of 1 M NH4OH measure out 63 ml of 16 M ammonium hydroxide to
937 ml of distilled water. Using a magnetic stirrer to mix the base, if that is not available
use a glass rod. Put the caps on the bottles right away and transfer the prepared
solution to an appropriate glass ware and keep the lid closed.
Dilution
Dilution Factor Although dilution ratio is sometimes confused with dilution factor, the
two are not the same. A dilution factor describes the ratio of the volume of solute to the
total, final volume of the entire diluted solution. Unfortunately, like the dilution ratio, a
dilution factor also is typically expressed as two numbers separated by a colon (e.g.,
1:10). However, in this case • first number represents the number of volumes of solute
liquid. • second number represents the number of volumes of the entire solution. •
dilution factor = volumes of solute : volume of entire solution For example, a dilution
factor of 1:5 means that • one volume of solute is combined with • four volumes of
solvent, for a total of • five volumes of finished, diluted product Thus, 20% (1/5) of the
volume of the entire solution consists of the solute, and the rest is the solvent. A
dilution factor is employed in a serial dilution, the stepwise dilution of a (liquid) solute
to a desired concentration

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – VIII
Basic concepts of acids, bases, salts and indicators

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Basic concepts, determination of PH-Henderson Hasselbalch’s equation.
 Buffer solution.
 PH determination of buffers

1. Acids: Acids are those compounds that dissolve in water and generate hydrogen
ions or H+ Ions. The examples of acids include Hydrochloric acid, citric acid,
sulphuric acid, vinegar, etc. One example of the acidic reaction is shown below-
Hydrochloric acid + water → H++ Cl-

2. Bases: A base is a type of substance or a compound that produces hydroxyl ions in

solution. The bases like potassium hydroxide, calcium hydroxide, sodium
hydroxide produce OH- ions when dissolved in water.
Potassium Hydroxide + H2O → K+ + OH-

3. Salts: The substances obtained as a result of the reaction between an acid and bases
are called as Salts. The table salt or sodium chloride is one of the typical examples
of salts.
4. Oxides: The compounds which consist of one oxygen atom called as Oxides.

5. Indicator: Indicators are substances that are used to test whether a substance is
acidic or basic or neutral in nature. They change their colour when added to a
solution containing an acidic or a basic substance.
Two types of indicators are:
 Natural
Examples: Turmeric, litmus, china rose and red cabbage.
 Artificial
Examples: Gentian Violet, Methyl Orange.
Litmus: Litmus is a natural dye extracted from lichens. It is the most commonly used
natural indicator. Litmus turns acidic solutions red and basic solutions blue. Neutral
solutions do not change the color of either red or blue litmus.
Turmeric: Turmeric gives brownish red color in basic medium and yellow in acidic
medium.
China rose: A solution of china rose turns green in a basic solution, and bright pink or
magenta in an acidic solution.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Identification of Basic Substances
Soap, milk of magnesia and limewater turn red litmus paper blue. This indicates the
basic nature of these solutions. Soap, milk of magnesia and limewater turn turmeric red
and the china rose green.
Identification of Acidic Substances
Red litmus paper when dipped in lemon juice and vinegar remains red. But they turn
blue litmus paper red. This indicates the acidic nature of these solutions. Lemon juice
and vinegar turn turmeric yellow and china rose pink

Determination of pH- Henderson Hasselbalch’s equation.

Determination of pH of acids and bases

Strong acids or bases are completely ionized in solution, so that the concentration of
free H+ or OH is the same as the concentration of the acid or base.

Strong acid; HNO3  H+ + NO-3

HCl  H+ + Cl-
Strong base NaOH  Na+ + OH-
The pH of such solutions can, therefore, be very easily calculated:
c) 0.01 mol (liter HCl, pH = - log 10 (10-2) = 2
d) 0.01 mol / liter NaOH, H+  = Kw = 10-14 = 10-12
[OH-] 10-2
pH = -log 10 (10-12) = 12
Weak acids or bases dissociate only to a limited extent and the concentration of
free H+ and OH- depends on the value of their dissociation constants.

Weak acid (formic acid);

a) HCOOH H+ + HCOO-
Weak base (aniline);
b) C6H5NH2 + H+ C6H5NH3+
As weak acids (and or bases) are only slightly ionized in solution and a true equilibrium
is established between the acid and the conjugate base, the pH determination is carried
out using

Henderson – Hasselbalch equation.

If HA represents a weak acid, then:

HA H+ + A-

The dissociation constant may be given as;

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 [H+] [A-]
Ka = ______________
[HA]

Ka [HA]
and [H+] = ___________

[A-]

Taking negative logarithms,

[HA]
-log10 [H+] = - log 10 Ka + (- log 10 __________ )
[A-]
[A-]
pH = pKa + log10 ______
[HA]

In general,
[Conjugate base]
pH = pKa log 10 ___________________

[Acid]
The activities of A-
and HA are not always known, so it is convenient to express A- and
HA as concentration terms. Thus:
CA - fA
pH = pKa + log _____ + log _____

CHA fHA

(fA- and fHA are the activity coefficient of A- and HA respectively)

Since log (fA- / fHA) is constant for a given acid, there activity coefficient can be
incorporated into the pKa term leading to another apparent dissociation constant pKa.
CA- [Salt]
 pH = pKa + log ____ = pKa + log _______
CHA [Acid]

This relationship is known as the Henderson-Hasselbalch equation, If the

concentrations of the acid and its conjugate base are equal;

Then pH = pKa + log 1

PH = pKa

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Determine pH with the help of pH Paper
Litmus and pH paper contain a chemical that changes color as it makes contact with an
acid or base. The paper will turn red in acids and blue in bases. Usually a color chart is
provided with the pH paper in order for the user to determine the pH range of the
indicator Figure BI 11.2.5. Using paper to determine the pH is not as accurate as a pH
meter, which provides results with the exact pH measurement; whereas the pH paper
only results in a pH range.

Figure BI 11.2.5: pH chart for determination of PH with the help of pH Paper

Measuring with pH paper

Find the pH of a substance using pH paper.
 Dip the end of the pH strip into the chemical or substance that was to be tested.
 After a couple of seconds, remove the paper
 Compare the color of the pH strip to the color chart provided with the pH paper
kit.
 Do not re-use a pH paper to retest or test another chemical. Always use a new
pH strip.
Test pH Paper on Saliva
 Test the litmus or pH paper using your saliva to test the color changes of the pH
paper.
 Take two pieces of the pH paper (one pink and one blue).
 Place the paper in mouth and remove after a few seconds.
 Determine whether the saliva is acidic or basic.
 If the paper changed from red to blue, this indicates that the saliva is basic.
 If the paper changes from blue to red, this indicates that the saliva is acidic.
 If nothing happened, the saliva is neutral.

Buffer solution. pH determination of buffers. Blood pH. Fluid

buffers.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
In chemistry, pH is a measure of the activity of the (solvated) hydrogen ion. P [H],
which measures the hydrogen ion concentration is closely related to, and is often
written as, pH. Pure water has a pH very close to 7 at 25°C. Solutions with a pH less
than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline.
The pH scale is traceable to a set of standard solutions whose pH is established by
international agreement. Primary pH standard values are determined using a
concentration cell with transference, by measuring the potential difference between a
hydrogen electrode and a standard electrode such as the silver chloride electrode.
Measurement of pH for aqueous solutions can be done with a glass electrode and a pH
meter, or using indicators.
pH measurements are important in medicine, biology, chemistry, agriculture,
forestry, food science, environmental many other applications.
pH can be viewed as an abbreviation for power of Hydrogen or more
completely, power of the concentration of the Hydrogen ion.
The mathematical definition of pH is a bit less intuitive but in general more
useful. It says that the pH is equal to the negative logarithmic value of the Hydrogen
ion (H+) concentration,
Or
pH = -log [H+]

pH can alternatively be defined mathematically as the negative logarithmic value of the

Hydroxonium ion (H3O+) concentration. Using the Bronsted-Lowry approach

pH = -log [H3O+]

pH values are calculated in powers of 10. The hydrogen ion concentration of a solution
with pH 1.0 is 10 times larger than the hydrogen concentration in a solution with pH
2.0. The larger the hydrogen ion concentration, the smaller the pH.
 when the pH is above 7 the solution is basic (alkaline)
 when the pH is below 7 the solution is acidic

Preparation of buffer solution:

A buffer solution is one that resists pH change on the addition of acid or alkali.
Such solutions are used in many biochemical experiments where the pH needs to be
accurately controlled. The buffer solution contains appreciable amounts of both a weak
acid and its salt or a weak base and its salt e.g. a suitable concentration of acetic acid
and sodium acetate and ammonia in aqueous solution constitute buffer solutions.
Buffer solutions tend to keep the concentration of [H3O+] and [OH-] constant and
hence the pH and pOH, even when small amounts of strong acid or strong base are
added to them. The major buffering systems found in cellular fluids involve phosphate,
bicarbonate, amino acids and proteins.
A buffer solution resists change in hydrogen ion concentration on the addition of
acid or alkali. This resistance is called the buffer action. The magnitude of the buffer
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
action is called the buffer capacity (β), and is measured by the amount of strong base
required to alter the pH by one unit:
db
β = _________
d(pH)

Where, d(pH) is the increase in pH resulting from the addition (db) of base.
It is to be noted that the buffer capacity of a particular acid and its conjugate base will
be maximum when their concentrations are equal i.e. when pH = pKa of the acid. Buffer
capacity also depends upon the total concentrations of acid and salt as well as on their
relative proportions- greater the total concentration, greater the buffer capacity.
The usual concentration of acid and salt in buffer solutions is of the order of 0.05-
0.20M and generally the mixtures possess acceptable buffer capacity within the range
pH = pKa + 1.

Principles of buffering (Fig BI 11.2.1)

Fig BI 11.2.1: Principles of buffering

Buffer solutions achieve their resistance to pH change because of the presence of

an equilibrium between the acid HA and its conjugate base A-

HA H+ + A-
When some strong acid is added to an equilibrium mixture of the weak acid and
its conjugate base, the equilibrium is shifted to the left, in accordance with Le Chatelier's
principle. Because of this, the hydrogen ion concentration increases by less than the
amount expected for the quantity of strong acid added.
Similarly, if strong alkali is added to the mixture the hydrogen ion concentration
decreases by less than the amount expected for the quantity of alkali added. The effect

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
is illustrated by the simulated titration of a weak acid with pK a = 4.7. The relative
concentration of undissociated acid is shown in blue and of its conjugate base in red.
The pH changes relatively slowly in the buffer region, pH = pK a ± 1, centered at pH =
4.7 where [HA] = [A-]. The hydrogen ion concentration decreases by less than the
amount expected because most of the added hydroxide ion is consumed in the reaction

And only a little is consumed in the neutralization reaction which results in an increase
in pH.

Once the acid is more than 95% deprotonated the pH raises rapidly because most
of the added akali is consumed in the neutralization reaction.

Criteria for buffers used in biological research

i) They should possess adequate buffer capacity in the required pH range.
ii) They should be available in a high degree of purity.
iii) They should be water soluble and impermeable to biological membranes.
iv) They should be enzymatically and hydrolytically stable.
v) They should not be toxic.
vi) They should not absorb light in the visible or ultraviolet regions.
vii) They should only form complexes with cations that are soluble.
Not all buffers that are commonly used meet all these criteria.
The pH of a buffer is expressed by Henderson – Hasellbatch equation:

pH = pKa + log [ Salt]

[Acid]

From the above equation, the pH of a buffer solution depends on two factors; first on
the pKa value (pKa is the pH at which the concentrations of the acid and its conjugate
base are equal) and second on the ratio of salt to acid.

Preparation of Buffer
(a) Acetate Buffer :
(i) Take 10 ml of 0.1N acetic acid in a beaker. Measure the pH. Add 1 ml of 0.01
N HCl. Mix well, measure the pH.
(ii) Take 10 ml of 0.1 N sodium acetate solution in a beaker. Measure the pH.
Add 1 ml of 0.01N HCl. Mix and measure the pH.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 (iii) Mix equal volumes of the acetic acid and sodium acetate solution. Measure
the pH. Now add 0.1 N HCl, mix and measure the pH.
(iv) Mix acetic acid and sodium acetate as in (iii). After measuring the pH, add 1
ml of 0.01N NaOH solution and check the pH. Observe the resistance in
change in pH of acetate buffer and draw the conclusions.
(b) Phosphate Buffer:
Prepare 0.1 N solution of NaH2PO4 (monosodium hydrogen phosphate) and
Na2HPO4 (Disodium hydrogen phosphate) separately. Mix them in various
proportions as indicated below and measures the pH. Calculate, the pH using
Hasselbatch equation, and compare with the observed value (Table BI 11.2.A).

(c) Table BI 11.2.A: preparation of Phosphate Buffer:

Vol. of Vol. of [H2PO4] Log[H2O4] Calculate Measure
NaH2PO4 Na2HPO4 [HPO4] [HPO4] d pH d pH
(0.1 N) (0.1 N)
10 ml 10 ml 1000μmoles 0.0001 6.77 ?
=1
1000μmoles
9 ml 11 ml 900 = 0.818 0.0872 6.61 ―
1100
6 ml 14 ml 600 = 0.428 0.3686 6.33 ―
1400
12 ml 8 ml 1200 = 1.50 --------- 6.88 ―
800
14 ml 6 ml 1400 = 2.33 -------- 7.07 ―
600

Applications
Buffer solutions are necessary to keep the correct pH for enzymes in many organisms to
work. Many enzymes work only under very precise conditions; if the pH moves outside
of a narrow range, the enzymes slow or stop working and can denature. In many cases
denaturation can permanently disable their catalytic activity. A buffer of carbonic acid
(H2CO3) and bicarbonate (HCO3−) is present in blood plasma, to maintain a pH between
7.35 and 7.45. Industrially, buffer solutions are used in fermentation processes and in
setting the correct conditions for dyes used in colouring fabrics. They are also used in
chemical analysis and calibration of pH meters.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 The majority of biological samples that are used in research are made in buffers,
especially phosphate buffered saline (PBS) at pH 7.4.
Common Buffer Preparations as shown below table (Table BI 11.2.B)
Buffer Stoc Components Amount Conc. Final
k per Liter Stock Soln Conc.
Soln. Soln.
PBS (Phosphate 10X NaCl 80 g 1.37 M 137 mM
Buffered Saline) KCl 2g 27 mM 2.7 mM
pH ~7.3 Na2HPO4×7H2O 11.5 g 43 mM 4.3 mM
KH2PO4 2g 14 mM 1.4 mM
TAE (Tris acetate 50X Tris base 242 g 2 M (Tris 40 mM
EDTA) Acetic acid 57.1 mL acetate) (Tris
pH ~8.5 (glacial) 37.2 g 0.1 M acetate)
EDTA 2 mM
TBE 10X Tris base 108 g 0.89 M 89 mM
(Tris borate EDTA) Boric acid 55 g 0.89 M 89 mM
pH ~8.0 EDTA 40 mL 0.02 M 2 mM
(0.5 M pH
8)
TE (Tris EDTA) 1X Tris base 1.2 g 10 mM 10 mM
pH ~7.5 H2 EDTA (acid) 0.29 g 1 mM 1 mM

Fluid buffers or Human body Buffer system

A buffer has the capacity to resist the change in pH of solution after the addition of
small amount of an acid or an alkali. Buffer solutions are prepared by mixing a weak
acid with its salt of strong base (or weak bases and their conjugated acids).The body‘s
first line of defense against extreme changes in H+ concentration is the buffer systems
present in all body fluids figure BI 11.2.1.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure BI 11.2.1: Human body Buffer system
Extracellular Buffer Systems:
 Bicarbonate–carbonic acid buffer system: The major source of metabolic acid in
the body is the gas CO2, produced principally from fuel oxidation in the TCA
cycle. Under normal metabolic conditions, the body generates more than 13 moles
of CO2 per day (approximately 0.5-1 kg). CO2 dissolves in water and reacts with
water to produce carbonic acid, H2CO3, a reaction accelerated by the enzyme
carbonic anhydrase (Figure BI 11.2.2). Carbonic acid is a weak acid that partially
dissociates into H+ and bicarbonate anion, HCO3-.
Carbonic acid is both the major acid produced by the body, and its own
buffer. pKa of carbonic acid itself is only 3.8, so at the blood pH of 7.4 it is almost
completely dissociated and theoretically unable to buffer and generate
bicarbonate. However, carbonic acid can be replenished from CO2 in body fluids
and air because the concentration of dissolved CO2 in body fluids is
approximately 500 times greater than that of carbonic acid. As base is added and
H+ is removed, H2CO3 dissociates into hydrogen and bicarbonate ions, and
dissolved CO2 reacts with H2O to replenish the H2CO3 (Figure BI 11.2.2).
Dissolved CO2 is in equilibrium with the CO2 in air in the alveoli of the lungs,
and thus the availability of CO2 can be increased or decreased by an adjustment
in the rate of breathing and the amount of CO2 expired.
The pKa for the bicarbonate buffer system in the body thus combines K h
(the hydration constant for the reaction of water and CO 2 to form H2CO3) with
the chemical pKa to obtain the value of 6.1 used in the Henderson-Hasselbalch
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 equation (BI 11.2.3). To use the terms for blood components measured in the
emergency room, the dissolved CO2 is expressed as a fraction of the partial
pressure of CO2 in arterial blood, PaCO2.

Figure BI 11.2.2: carbonic acid – Bicarbonaten buffer system

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure BI 11.2.3: The Henderson-Hasselbalch equation for the bicarbonate
buffer system Bicarbonate and Hemoglobin in the Red Blood Cells

The bicarbonate buffer system and hemoglobin in red blood cells cooperate in buffering
the blood and transporting CO2 to the lungs. Most of the CO2 produced from tissue
metabolism in the TCA cycle diffuses into the interstitial fluid and the blood plasma
and then into red blood cells. Although no carbonic anhydrase can be found in blood
plasma or interstitial fluid, the red blood cells contain high amounts of this enzyme, and
CO2 is rapidly converted to carbonic acid (H2CO3) within these cells. As the carbonic
acid dissociates, the H+ released is also buffered by combination with hemoglobin. The
side chain of the amino acid histidine in hemoglobin has a pKa of 6.7 and is thus able to
accept a proton. The bicarbonate anion is transported out of the red blood cell into the
blood in exchange for chloride anion, and thus bicarbonate is relatively high in the
plasma.
As the red blood cell approaches the lungs, the direction of the equilibrium
reverses. CO2 is released from the red blood cell, causing more carbonic acid to
dissociate into CO2 and water and more hydrogen ions to combine with bicarbonate.
Hemoglobin loses some it of its hydrogen ions, a feature that allows it to bind oxygen
more readily. Thus, the bicarbonate buffer system is intimately linked to the delivery of
oxygen to tissues. The respiratory center within the hypothalamus, which controls the
rate of breathing, is sensitive to changes in pH. As the pH falls, individuals breathe
more rapidly and expire more CO2. As the pH rises, they breathe more shallowly. Thus,
the rate of breathing contributes to regulation of pH through its effects on the dissolved
CO2 content of the blood. Bicarbonate and carbonic acid, which diffuse through the
capillary wall from the blood into interstitial fluid, provide a major buffer for both
plasma and interstitial fluid. However, blood differs from interstitial fluid in that the
blood contains a high content of extracellular proteins, such as albumin, which
contribute to its buffering capacity through amino acid side chains that are able to
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
accept and release protons. The protein content of interstitial fluid is too low to serve as
an effective buffer.

 Intracellular Buffer Systems:

Phosphate anions and proteins are the major buffers involved in maintaining a
constant pH of intracellular fluids. The inorganic phosphate anion H2PO4
dissociates to generate H+ and the conjugate base, HPO4-2 with a pKa of 7.2. Thus,
phosphate anions play a major role as an intracellular buffer in the red blood cell
and in other types of cells, where their concentration is much higher than in blood
and interstitial fluid. Organic phosphate anions, such as glucose 6-phosphate and
ATP, also act as buffers. Intracellular fluid contains a high content of proteins that
contain histidine and other amino acids that can accept protons, in a fashion similar
to hemoglobin.
The transport of hydrogen ions out of the cell is also important for maintenance
of a constant intracellular pH. Metabolism produces a number of other acids in
addition to CO2. For example, the metabolic acids acetoacetic acid and β-
hydroxybutyric acid are produced from fatty acid oxidation to ketone bodies in the
liver, and lactic acid is produced by glycolysis in muscle and other tissues. The pKa
for most metabolic carboxylic acids is below 5, so these acids are completely
dissociated at the pH of blood and cellular fluid. Metabolic anions are transported
out of the cell together with H+. If the cell becomes too acidic, more H+ is
transported out in exchange for Na+ ions by a different transporter. If the cell
becomes too alkaline, more bicarbonate is transported out in exchange for Cl- ions.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – IX
Biomedical waste management

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Biomedical waste management

Safe Laboratory Practices & Procedures

Students and laboratory staff working in any medical institute or hospital depending
upon the nature of their work they perform are exposed to diverse real or potential
hazards. Common hazards in the medical laboratory include biological, chemical,
physical, and radiological, flammable liquids, toxic vapors, compressed gases, electric
shock, corrosive substances, mechanical trauma, poisons, and the intrinsic risks of
handling biological materials. Work force of laboratory/medical institution must be
―safety conscious‖ at all times.
Safety guidelines should be properly followed in order to make laboratory a safe
working place and prevent accidents and mishaps.

Laboratory Safety Guidelines

To begin, with the recognition of hazards, safety is achieved via the application of
common sense. A safety-focused attitude, good personal behavior, good housekeeping
in all laboratory work and storage areas, above all, the continual practice of good
laboratory technique.
If anyone is splashed by any of these materials, use running water from an eyewash
station or emergency shower for at least 15 minutes or until emergency assistance
arrives and provides with different instructions.
k) Report to supervisor/lab. incharge if any any accident, injury, or uncontrolled
release of potentially hazardous materials occurs, no matter how trivial the
accident, injury, or release may appear.
l) Attend all required laboratory safety training prior to the start of
practical/research assignment.
m) Read all procedures and associated safety information prior to the start of lab.
experiment.
n) Perform only those experiments authorized by lab. Incharge/supervisor.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 o) Follow all written and verbal instructions. Ask for assistance if you need guidance
or help.
p) Work under direct supervision at all times. Never work alone in the laboratory.
q) Know the locations and operating procedures for all safety equipment. This
includes the eyewash station and safety shower.
r) Know the locations of the nearest fire alarms and at least two ways out of the
building. Never use an elevator in emergencies.
s) Be alert and proceed with caution at all times in the laboratory. Immediately
notify the supervisor of any unsafe conditions.
t) Know the proper emergency response procedures for accidents or injuries in the
laboratory.

Prevent potential exposure by safe laboratory practices are as follows

z) Conduct yourself in a responsible and professional manner at all times. No
pranks. No practical jokes.
aa) Dress for work in the laboratory. Wear clothing and shoes that cover exposed
skin and protect from potential splashes. Tie back long hair, jewelry, or
anything that may catch in equipment.
bb) Never eat food, drink beverages, chew gum, apply cosmetics (including
lip balm), or handle contact lenses in the laboratory.
cc) Use a chemical fume hood or biosafety cabinet, as directed by supervisor.
dd) Observe good housekeeping - keep aisles clear.
ee) Report damaged electrical equipment to the supervisor. Do not use damaged
electrical equipment.
ff) Do not leave active experiments unattended. Never leave anything that is
being heated or is visibly reacting unattended.
gg) Practice good personal hygiene. Wash the hands after removing gloves, before
leaving the laboratory, and after handling a potentially hazardous material.
hh) While working in the laboratory, wear personal protective equipment -
eye protection, gloves and laboratory coat.
ii) Properly segregate and dispose of all laboratory waste.
jj) Before beginning any new or modified procedures determinethe potential
physical, chemical and biological hazards and their appropriate safety
precautions.
kk) Familiarize with the emergency procedures, alarms and evacuation
routes.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 ll) Location of emergency phone, emergencyeyewash, safety showers and fire
extinguishers with proper operating procedures should be known.
mm) Do not smoke, apply makeup and consume food or beverages in
laboratories. Never store food or drink in laboratory refrigerators.
nn) Wear protective clothing and gloves that are not permeable to the
chemicals being used.
oo) Long hair and loose clothing should be confined when in the laboratory. Shoes
must be worn at all times.
pp) Sandals or open toe shoes must not be worn in the laboratory.
qq) All containers of chemicals should be correctly and clearly labelled.
rr) The label should provide hazard and safety information about the chemicals.
ss) All chemical wastes should be disposed of appropriately to the corresponding
waste containers.
tt) Mouth pipette of chemicals must not be allowed. A pipette bulb, piplip or
aspirator for pipetting chemicals should be used.
uu) Exposure to gases vapors and aerosols should be minimized. Appropriate
safety equipment in conjunction with fume cupboard should be used
whenever such exposure is expected.
vv) Cell phones should not be operated at any time within a laboratory. They
might cause disturbance to other laboratory users, and also cause signal
interference.
ww) The warning placard in lab includes a list of emergency contact persons.
In case of any mishap, assistance from the people on that list may be informed.
xx) Students need to abide by these regulations. This is necessary to keep order in
the laboratory

Common Hazards faced during working with Acids & bases

Acids and bases can provoke skin, mucous membrane and eye burns. While
using these viz. (NaOH, KOH, H2SO4, HNO3, trichloroacetic acid etc), following rules
should be adhered to:
j) Toxic materials should be clearly labeled with special tape when used in
reagents and stored in separate containers.
k) In case of spillage, wash all exposed human tissue (including eyes) generously
with water and laboratory supervisor should be informed for proper reporting
of the incident.
l) Do not taste or ingest any laboratory chemical.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 m) Avoid touching chemicals with bare hands and always thoroughly wash hands
after their use.
n) Work in a fume hood when carrying out reactions that give off objectionable
gases. If there is need to smell vapor, nose should not be directly above a flask,
beaker, or other vessel containing chemicals, instead, hold the vessel at least
one foot away and cautiously fan the vapors toward nose.
o) While pipetting acids, use mechanical pipettors, pipette aids or rubber bulbs
provided in the laboratory.
p) Always add acids to water (its alphabetical!); never add water to acids.
Combining acid and water frequently generates heat; addition of the acid to the
water reduces the amount of heat generated at the point of mixing and
provides more water to disperse the heat.
q) Label all flasks, beakers, test tubes, and other vessels containing chemicals
according to their contents. This facilitates both identifying chemicals during
an experiment and following proper waste disposal procedures.
r) Laboratory wastes and residues are to be disposed of in an approved manner
as per standard disposal procedures.

Common Hazards faced during working with Electric equipment and apparatus
Be careful when working with electric equipment to avoid an electric shock. Following
rules should be pursued:
(l) Do not touch the naked electric wires.
(m) Do not work with unearthed apparatus.
(n) Do not pull out an electric wire, during switch off an electric apparatus
from the electric network. Do it only with an electric plug.
(o) Do not touch a water pipe, a tap, and a heating radiator during working
with electric apparatus.
(p) Extension cord use is prohibited.
(q) All equipment has to be properly grounded.
(r) Never operate electrical equipment with wet hands or if spilled with fluid.
(s) Never use plugs with exposed or frayed wires.
(t) If there are unusual sparks or smoke shut down the instrument and
inform the lab staff.
(u) Electrical equipment that is not working properly should not be used.
(v) If a person is shocked by electricity, shut off the current or break contact
with the live wire immediately and don‘t touch the victim while he is in
contact with the source unless you are completely insulated against shock.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Common Hazards faced during working with compressed gases
e. In the laboratory, gas containers should be limited to the number of
containers in use at any time.
f. Low pressure gases shall also be limited to the smallest size container.
g. Containers shall be firmly strapped, chained or secured in a cylinder stand
so they cannot fall.
h. Oxidizing gases should be separated from the flammable ones.

Fire Extinguishers
Fire is mainly a chemical reaction involving the rapid oxidation of fuel or any
other combustible material wherein heat and light are liberated. Elements necessary for
the initiation of fire are already present in any clinical laboratory which includes oxygen
(air), fuel and heat or ignition source. Recent research has suggested the presence of
fourth factor that has been classified as a chain reaction in which burning is continued
or accelerated & is caused by the breakdown and recombination of the molecules from
the material burning with the oxygen in the atmosphere. This has resulted in the
modification of the previous fire triangle into a pyramid known as ―Fire tetrahedron‖
which does not abolish the established procedures in dealing with fire, instead
additional ways to prevent or extinguish fire have been provided by this. A fire will
extinguish by the removal of any three basic elements (heat, air or fuel).

Classification of Fires
Depending on the nature of the combustible material and requirements for
extinguishing fires have been classified into:
 Class A: Ordinary combustible solid materials like- paper, wood, plastic, fabric
 Class B: Flammable liquids/gases & combustible petroleum products
 Class C: Energized electrical equipment (table BI 11.1.A)
 Class D: Combustible/reactive metals like- magnesium, sodium, potassium

Like fires, fire extinguishers are classified depending on the type of fire to be
extinguished. Use of correct fire extinguisher is very important. e.g water should not be
used on burning liquids or electrical equipment.Pressurized-water extinguishers, as
well as foam and multipurpose dry-chemical types, are used for Class A fires.
Multipurpose dry-chemical and carbon dioxide extinguishers are used for Class B and
C fires. Halogenated hydrocarbon extinguishers are particularly recommended for use

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
with computer equipment. Class D fires present special problems, and extinguishment
is left to trained firefighters using special dry-chemical extinguishers.
In the event of a fire, all personnel, patients, and students who are in immediate
danger have to be evacuated first and then activate the fire alarm, report the fire, and
attempt to extinguish the fire, if possible. Personnel should work as a team to carry out
emergency procedures. Fire drills must be conducted regularly and with appropriate
documentation.

Table BI 11.1.A: Class of Fires and Remedies

Class of Fire Type of Extinguisher Operation
Class A Fires A /ABC Pull pin
Pressurized water/Dry chemical
Class B Fires ABC/BC Aim nozzle
Dry chemical/Carbon dioxide
Class C Fires BC/Halon/ABC Squeeze trigger
Carbon dioxide/Halon/Dry chemical Sweep nozzle
Class D Fires Metal X Cover burning material with
extinguishing agent
(scoop,sprinkle)

Describe the disposal of laboratory waste

Laboratory waste is waste that is generated from laboratories in industry and in
educational centres such as Medical Institutions, ccolleges and universities. This waste
can be broken down into a number of categories: Hazardous; Clinical; Biological;
Electrical; Laboratory. Reducing laboratory waste will have a number of benefits, saving
money and reducing disposal costs while also encouraging safety in the lab.

Various Types of Wasts Produced in Medical Institution laboratories/Research

institutions/hospitals
1. Infectious waste (Biological waste): Manage biological wastes in accordance
with the Biohazardous Waste and Sharps Disposal policy. Infectious waste
includes biomedical waste previously contaminated with pathogens possessing
enough virulence capable of infecting a susceptible host resulting in an infectious
disease which includes bacteria, viruses, parasites, or fungi capable of causing
infection or spread of infectious diseases in the community.
2. Pathological Waste: This includes anatomical waste from humans and animals
often generated from hospitals and veterinary hospitals like: blood, amputated
body parts, placenta, tissues and animal carcasses etc. They are significantly
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 dangerous with a high risk of disease and infection in a vulnerable individual who
has direct contact with them.
3. Microbiological/Clinical Waste: This is usually generated from laboratories
during the course of experiments and clinical trials like cell cultures, infectious
microorganisms, vacutainers, body fluid, drainage bags, used culture dishes, and
other materials that were previously in contact with contagious agents.
4. Sharps: Sharp is defined as any object which could readily puncture or cut the
skin of an individual; these consist of both used and unused sharp objects such as
hypodermic needles, syringes, scalpels, broken ampoules, and glassware. They are
considered highly dangerous because these can cause punctures and cuts on the
skin and also harbor dangerous pathogens.
Sharps –including, but not limited to:
Needles, syringes, knives, razor blades, lancets, capillary tubes, metal shavings,
etc. Glass or plastic pipettes and pipette tips. Any broken glass, glass slides, cover
slips, plastic, metal, pottery with sharp edges, etc. Anything that could puncture
through a garbage bag causing the bag to rupture and spill, or risking injury and
exposure to personnel.
5. Pharmaceutical Waste: This includes chemical wastes, expired drugs, drug
preparations added to an intravenous solution etc. excludingempty glass
ampoules, drugs and other metabolic products excreted by patient undergoing
therapy, empty pills bottles or strip packages from where the drug/capsules have
been previously removed. However, the unsafe disposal of such wastes may lead
to significant danger.
6. Chemical Waste: This includes chemicals that deteriorate over time and become
hazardous (e.g., ether forms explosive peroxides). They are also generated during
the production of biological preparations such as disinfectants and insecticides in
medical, dental and veterinary laboratories.
7. Radioactive Waste: Manage radioactive wastes in accordance with the Radiation
Safety Manual. This type of waste may only be removed by Environmental Health
and Safety personnel. Radioactive Waste is generated from the medical or research
use of radiology, radionuclide (e.g., during radioimmunoassay), and other
radiological procedures that emit radiation at above the level set by regulatory
authorities. Examples: nuclear medicine treatments, cancer related therapies, and
medical devices that use radioactive isotopes.
8. Cytotoxic and Genotoxic Wastes: These include unused cytotoxic drugs, solid
materials such as sharp objects, tissues, any other items which may have come into
contact with a cytotoxic drug and/ or carcinogenic matter.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 9. Non-risk Waste: This is general waste that is generated within hospitals and they
are similar to municipal waste from a normal home. They are considered non-
infectious and includes office waste, food waste, aerosols, as well as non-
infectious, non-anatomical waste from patient care areas (i.e., disposable diapers,
pads, papers, cartons, gloves, trays, catheters/bags (empty), and casts) etc.

Waste Management
Rapid population growth with concomitant increases in the demand for
healthcare services has led to new and more complex societal problems. One such
difficulty is the proper management of healthcare wastes produced during diagnosis
and treatment at healthcare institutes and facilities, research centres and laboratories.
Clinical laboratories are significant generators of infectious waste, including
microbiological materials, contaminated sharps, and pathologic wastes such as blood
specimens and blood products. Most waste produced in laboratories can be disposed of
in the general solid waste stream. However, improper management of infectious waste,
including mixing general wastes with infectious wastes and improper handling or
storage, could lead to disease transmission.
The first step toward an effective waste management process is to separate out the
different types of waste materials at the initial source of its generation. Through
segregation different types of waste can be identified, labeled and placed in different
waste containers and can be treated differently.
Generally, different wastes are categorized based on the level of risk they carry
and are assigned specific containers with color coding. For example, human anatomical
waste are collected in yellow plastic containers, microbiological waste in yellow/red,
waste sharps in plastic bag puncture-proof in translucent blue/white containers,
domestic hospital waste in green bags, etc. (Figure: BI 11.1.1.)
Hospital waste disposal methods are broadly classified under incineration and
non-incineration technologies.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Figure. BI 11.1.1: Waste Management

How to Reduce Laboratory Waste

1. Look at purchasing procedures. Buy only what is needed, reducing wastage due
to expiry.
2. Find a reliable supplier who will deliver small amounts of chemicals at short
notice. Ask if they will take back unused chemicals.
3. A centralised purchasing programme should be considered. This means that all
orders are placed with a delegated person who may be able to take advantage of
bulk pricing.
4. All chemicals and wastes in the lab should be labelled. A waste chemical has no
use. This labelling system should be standardised.
5. Separate waste into the following streams for treatment, reuse or disposal:
Sharps including scalpels and syringes; Glassware; Biological samples; General
lab waste such as wipes, gloves, tissue; Chemicals.
Cleaning and waste disposal services in a laboratory requires strict adherence to
applicable policies and procedures. It is a joint effort between laboratory personnel,
Building Services personnel and Environmental Health and Safety personnel. The goal
should be to provide cleaning and waste disposal services for laboratories, develop
procedures that everyone understands and will follow and through this process avoid
the hazards to personnel conducting these services.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Laboratory Waste Disposal Procedures
The waste materials placed in the municipal waste are suitable for this type of disposal,
especially:
 Do not place any liquids in the municipal waste.
 Do not dispose of chemical waste, including stock containers with unused
product, in the municipal waste.
 Empty or rinsed containers must be free of any hazardous residue and be
marked "empty."
 All sharps must be in an appropriate, puncture-resistant container to prevent
injuries.
 If a material can be mistaken as a hazardous, radioactive, or biological waste,
but is not, it must be identified as non-hazardous.
Building Services will dispose of glass if it is cleaned of any hazardous materials and is
properly packaged. The total weight must not exceed 40 pounds and the container must
be able to be easily and safely handled by Building Services personnel.
For all other types of waste, make sure the container is appropriately labeled and
separated from municipal waste:
Manage hazardous wastes in accordance with the Hazardous Materials Management
and Disposal Policy and Procedures manual (Table BI 11.1B). This type of waste may
only be removed by Environmental Health and Safety personnel.

Table BI 11.1B: Packaging / Containerization

S. Waste Pakaginf and Packaging considerations
No containerization
challenges
1.

Potentially sharp
when broken In most cases, anything that is
Plastic or glassware can potentially sharp should be
crack or break during treated as sharps waste,
transit rendering sharp warranting a puncture-proof,
edges, which can sealable rigid plastic container.
penetrate conventionally If volume are too high to
used bages and poor warrant the use of sharps
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 quality rigid plastic containers, then improvise by
containers risking SPILL double bagging and using lined
box sets so that the contents are
protected from breakage during
transit and the likelihood of
sharps edges poking through is
minimized, for example libels
clearly indicating the contents
of the packaging inform waste
handlers how to handle it.
2.

Packaging/container Use small containers that can

Heavy can break under load withstand relative load. Smaller
Glassware or risking SPILL waste containers are easier to lift and
liquids in high handlers can injure carry. Use good quality
volume, limbs, etc themselves trying to lift packaging that can withstand
and carry the waste from load or improvise with double
point A to point B bagging, etc.
3.

Light No additional risk posed Can use larger volume

if packaged correctly containers or bags

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
4. Liquid posses the risk of Container/packaging needs to
seeping or spilling out be leak-proof and obviosly
the container. Liquid should be liquid-proof (a
waste in high volumes cardboard box would not be
can be heavy suitable, for example, as it
would fall apart when wet
unless well-lined and protected
from liquid)
Wet
5. No additional risk posed Can use lightweight packaging
if packaged correctly or bags as no risk of leakage or
seepage

Dry

6. Waste stream in Example include long sharps

question is too large or such as trocars or ampulated
oddly shaped to fit into limbs. Select the right sized
conventional HCRW container for the waste stream.
packaging If does not fit. Or you can not
find container to fit the waste,
don‘t try and fit it in because if
Clumsy, large or the container con not close and
oddly-shaped seal properly it defeats the
object of SAFE containment and
puts handlers downstream at
risk. Improvise were necessary,
taking into account the nature
of thr waste and prescribed
colour-coding and labelling
requirements.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Unit – IX
ASSIGNMENT TOPICS

Chapter at a Glance

The Learner will be able to answer questions on following topics

 Radioactive isotopes
 Arterial Blood gases

Radioactive isotopes
In radioactive processes, particles or electromagnetic radiation are emitted from the
nucleus. The most common forms of radiation emitted have been traditionally classified
as alpha (a), beta (b), and gamma (g) radiation. Nuclear radiation occurs in other forms,
including the emission of protons or neutrons or spontaneous fission of a massive
nucleus. Of the nuclei found on Earth, the vast majority is stable. This is so because
almost all short-lived radioactive nuclei have decayed during the history of the Earth.
There are approximately 270 stable isotopes and 50 naturally occurring radioisotopes
(radioactive isotopes). Thousands of other radioisotopes have been made in the
laboratory.
The history of the use of radioisotopes for medical purposes is filled with names
of Nobel Prize winners. It IS inspiring to read how great minds attacked puzzling
phenomena, worked out the theoretical and practical implications of what they
observed, and were rewarded by the highest honor in science. For example, in 1895 a
German physicist, Wilhelm Konrad Roentgen, notice d that certain crystal s became
luminescent when they were in the vicinity of a highly evacuated electric-discharge
tube. Objects placed be - tween the tube and the crystals screened out some of the
invisible radiation that caused this effect, and he observed that the greater the densit y
of the object so placed, the greater the screening effect. He called this new radiation X
rays, because x was the standard algebraic symbol for an unknown quantity. His
discovery won him the first Nobel Prize in physics m 1901.
A French physicist, Antome Henri Becquerel, newly appointed to the chair of
physics at the Ecole Polytechnique in Pans, saw that this discovery opened up a new
field for research and set to work on some of its ramifications. One of the evident
features of the production of X rays was the fact that while they were being created, the
glass of the vacHenri Becquerel greemsh phosphorescent glow. This suggested to
several physicists that substances which become phosphorescent upon exposure to
visible light might give off X rays along with the phosphorescence

What Is Radiation?
Radiation is the propagation of radiant energy in the form of waves or particles. It
includes electromagnetic radiation ranging from radio waves, infrared heat waves,
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
visible light, ultraviolet light, and X rays to gamma rays. It may also include beams of
particles of which electrons, positrons, neutrons, protons, deuterons, and alpha particles
are the best known.

What Is Radioactivity?
It took several years following the basic discovery by Becquerel, and the work of many
investigators, to systematize the information about this phenomenon. Radioactivity IS
defined as the property, possessed by some materials, of spontaneously emitting alpha
or beta particles or gamma rays as the unstable (or radioactive) nuclei of their atoms
disintegrate.

What Are Radioisotopes?

In the 19th Century an Englishman, John Dalton, put forth his atomic theory, which
stated that all atoms of the same element were exactly alike. This remained
unchallenged for 100 years, until experiments by the British chemist, Frederick Soddy,
proved conclusively that the element neon consisted of two different kinds of atoms. All
were alike in chemical behavior but some had an atomic weight (their mass relative to
other atoms) of 20 and some a weight of 22. He coined the word isotope to describe one
of two or more atoms having the same atomic number but different atomic weights.*
Radioisotopes are isotopes that ar e unstable, or radioactive, and give off radiation
spontaneously. Many radioisotopes ar e produced by bombarding suitable targets with
neutrons now readily available inside atomic reactors. Some of them, however, ar e
more satisfactorily created by the action of protons, deuterons, or other subatomic
particles that have been given high velocities in a cyclotron or similar accelerator.
Radioactivity is a process that is practically uninfluenced by any of the factors, such as
temperature and pressure, that ar e used to control the rate of chemical reactions. The
rate of radioactive decay appears to be affected only by the structure of the unstable
(decaying) nucleus. Each radioisotope has its own half-life, which is the time it takes for
one half the number of atoms present to decay. These half-lives vary from fractions of a
second to millions of years, depending only upon the atom. We shall see that the half-
life is one factor considered in choosing a particular isotope for certain uses.
Most artificially made radioisotopes have relatively short half-lives. This makes
them useful in two ways. First, it means that very little material is needed to obtain a
significant number of disintegrations. It should be evident that, with any given number
of radioactive atoms, the number of disintegrations per second will be inversely
proportional to the half-life. Second, by the time 10 halflives have elapsed, the number
of disintegrations per sec - ond will have dwindled to V-[02i th^ original number, and
the amount of radioactive material is so small it is usually no longer significant. (Note
the decrease in the figure above.)

How Are Radioisotopes Used?

A radioisotope may be used either as a source of radiation energy (energy is always
released during decay), or as a tracer: an identifying and readily detectable marker
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
material. The location of this material during a given treatment can be determined with
a suitable instrument even though an unweighably small amount of it is present in a
mixture with other materials. On the following pages we will discuss medical use s of
individual radioisotopes — first those used as tracers and then those used for their
energy. In general, tracers ar e used for analysis and diagnosis, and radiant-energy
emitters are used for treatment (therapy). Radioisotopes offer two advantages. First,
they can be used in extremely small amounts. As little as one-billionth of a gram can be
measured with suitable apparatus. Secondly, they can be directed to various definitely
known parts of the body. For example, radioactive sodium iodide behaves in the body
just the same as normal sodium iodide found in the iodized salt used in many homes.
The iodine concentrates in the thyroid gland where it is converted to the hormone
thyroxin. Other radioactive, or "tagged", atoms can be routed to bone marrow, red
blood cells, the liver, the kidneys, or made to remain in the blood stream, where they ar
e measured using suitable instruments.* Of the three types of radiation, alpha particles
(helium nuclei) are of such low penetrating power that they cannot be used for
measurement from outside the body. Beta particles (electrons) have a moderate
penetrating power, therefore they produce useful therapeutic results in the vicinity of
their release, and they can be detected by sensitive counting devices. Gamma rays ar e
highly energetic, and they can be readily detected by counters — radiation
measurement devices—used outside the body.
For comparison, a sheet of paper stops alpha particles, a block of wood stops beta
particles, and a thick concrete wall stops gamma rays.
In one way or another, the key to the usefulness of radioisotopes lies in the
energy of the radiation. When radiation is used for treatment, the energy absorbed by
the body is used either to destroy tissue, particularly cancer, or to suppress some
function of the body. Properly calculated and applied doses of radiation can be used to
produce the desired effect with minimum side reactions. Expressed in terms of the
usual work or heat units, ergs or calories, the amount of energy associated with a
radiation dose is small. The significance lies in the fact that this energy is released in
such a way as to produce important changes in the molecular composition of individual
cells within the body.

Major Uses of Radioisotopes

Radioisotopes in Medicine
Radioactive tracers are also used in many medical applications, including both
diagnosis and treatment. They are used to measure engine wear, analyze the geological
formation around oil wells, and much more.
Radioisotopes have revolutionized medical practice, where they are used extensively.
Over 10 million nuclear medicine procedures and more than 100 million nuclear
medicine tests are performed annually in the United States.
Diagnostic Medical Applications

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Diagnostic medical applications involve testing for a disease or condition. In nuclear
medicine, this could involve using PET scans, or isotopic studies. The radiation
involved for each of these types of tools will vary in mrem or mSv amounts.
PET Scanning
Positron Emission Tomography or PET scan is a type of nuclear medicine imaging.
Depending on the area of the body being imaged, a radioactive isotope is either injected
into a vein, swallowed by mouth, or inhaled as a gas. When the radioisotope is collected
in the appropriate area of the body, the gamma ray emissions are detected by a PET
scanner (often called a gamma camera) which works together with a computer to
generate special pictures, providing details on both the structure and function of
various organs. Watch this informational video on how this technique works.
PET scans are used to:
 Detect cancer
 Determine the amount of cancer spread
 Assess the effectiveness of treatment plans
 Determine blood flow to the heart muscle
 Determine the effects of a heart attack
 Evaluate brain abnormalities such as tumors and memory disorders
 Map brain and heart function

Other Isotopic Tests

Radioisotopes have revolutionized medical practice, where they are used extensively.
Over 10 million nuclear medicine procedures and more than 100 million nuclear
medicine tests are performed annually in the United States. Four typical examples of
radioactive tracers used in medicine are technetium-99 (9943Tc)(4399Tc), thallium-201
(20181Tl)(81201Tl), iodine-131 (13153I)(53131I), and sodium-24 (2411Na)(1124Na).
Damaged tissues in the heart, liver, and lungs absorb certain compounds of technetium-
99 preferentially. After it is injected, the location of the technetium compound, and
hence the damaged tissue, can be determined by detecting the γ rays emitted by the Tc-
99 isotope. Thallium-201 (Figure 11.5.711.5.7) becomes concentrated in healthy heart
tissue, so the two isotopes, Tc-99 and Tl-201, are used together to study heart tissue.
Iodine-131 cocentrates in the thyroid gland, the liver, and some parts of the brain. It can
therefore be used to monitor goiter and treat thyroid conditions, such as Grave‘s
disease, as well as liver and brain tumors. Salt solutions containing compounds of
sodium-24 are injected into the bloodstream to help locate obstructions to the flow of
blood.
Small doses of II-131 (too small to kill cells) are used for purposes of imaging the
thyroid. Once the iodine is concentrated in the thyroid, the patient lays down on a sheet
of film and the radiation from the II-131 makes a picture of the thyroid on the film. The
half-life of iodine-131 is approximately 8 days so after a few weeks, virtually all of the
radioactive iodine is out of the patient's system. During that time, they are advised that
they will set off radiation detectors in airports and will need to get special permission to
fly on commercial flights.
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Some isotopes that are used to diagnose diseases are shown in Table 11.5.111.5.1.
All of these nuclear isotopes release one form of ionizing radiation (either/add particle
or ray). In addition, each isotopic application would involve a specific amount of
mrem/mSv radiation.

Therapeutic Radiation
There are many techniques used to treat cancer. Surgery can be used to remove
cancerous tumors inside or on the body. With chemotherapy, ingested or injected
chemicals are used to kill rapidly dividing cells (cancerous and noncancerous). Other
cancer treatment methods include immunotherapy, stem cell replacement, hormone
therapy, and targeted therapy.

Radiation therapy and Chemotherapy: Two different treatment procedures

Patients diagnosed with cancer might be required to do chemotherapy or radiation
therapy. Sometimes, both of these methods are used for a patient
Current therapeutic radiation applications involve the use of gamma, x-rays, or
protons. Recently, some research facilities are investigating the use of alpha and beta
tagged molecules to kill cancer cells. These radioisotopes will first locate a cancer
related molecule on a tumor cell. Then, the alpha or beta tagged species will inject its
radiation into the tumor. Sr-89 (beta emitter) and Ra-223 (alpha emitter) have been used
in clinical research trials of certain types of bone cancers.
Radiation Therapy is used as a treatment to control malignant cells within cancer
patients. Oncologists (specialists that deal with cancer) utilize radiation frequently to
help slow or cure the spread of cancer within individuals. Radiation is specifically
applied to malignant tumors in order to shrink them in size. Medical professionals,
mainly radiation oncologists, administer a variety of dosages to patient, contingent to
the patients current health, as well as other treatments such as chemotherapy, success of
surgery, etc.

External Beam Therapy (Photon and Proton Therapy)

External Beam Therapy (EBT) is a method of delivering a high energy beam of
radiation to the precise location of a patient's tumor. These beams can destroy cancer
cells and with careful planning, NOT kill surrounding cells. The concept is to have
several beams of radiation, each of which is sub-lethal, enter the body from different
directions. The only place in the body where the beam would be lethal is at the point
where all the beams intersect. Before the EBT process, the patient is three-dimensionally
mapped using CT scans and x-rays. The patient receives small tattoos to allow the
therapist to line up the beams exactly. Alignment lasers are used to precisely locate the
target. The radiation beam is usually generated with a linear accelerator. The video
below illustrates the basic preparation and administration of external beam therapy.

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Side Effects of Radiation Therapy
Patients receiving radiation therapy can experience a variety of side effects. For
example, sterility could occur if reproductive organs are irradiated. Skin that has been
irradiated can appear dry and feel itchy. Some patients will loose sensation in the
irradiated area. Radiation can affect the production of white and red blood cells. A
reduction of white blood cells results in immunity disorders. Red blood cell lose causes
anemia. Gastrointestinal issues such as diarrhea and nausea are common during
radiation therapy. Some patients will lose hair as well. Lastly, dry mouth and tooth
decay are prevalent during radiation treatments.

Arterial Blood gases

Blood gas test measures the amount of oxygen and carbon dioxide in the blood. It may
also be used to determine the pH of the blood, or how acidic it is. The test is commonly
known as a blood gas analysis or arterial blood gas (ABG) test. Red blood cells transport
oxygen and carbon dioxide throughout the body. These are known as blood gases. As
blood passes through the lungs, oxygen flows into the blood while carbon dioxide flows
out of the blood into the lungs. The blood gas test can determine how well the lungs are
able to move oxygen into the blood and remove carbon dioxide from the blood.
ABG) Arterial Blood Gas Analysis is used to measure the partial pressures of
oxygen (PaO2), carbon dioxide (PaCO2), and the pH of an arterial blood sample.
Oxygen content (O2CT), oxygen saturation (SaO2), and bicarbonate (HCO3-) values are
also measured. A blood sample for ABG analysis may be drawn by percutaneous
arterial puncture from an arterial line. The ABG analysis is mainly used to evaluate gas
exchange in the lungs. It is also used to assess integrity of the ventilatory control system
and to determine the acid-bas level of the blood. The ABG analysis is also used for
monitoring respiratory therapy (again by evaluating the gas exchange in the lungs).
Imbalances in the oxygen, carbon dioxide, and pH levels of blood can indicate the
presence of certain medical conditions. These may include:
 kidney failure
 heart failure
 uncontrolled diabetes
 hemorrhage
 chemical poisoning
 a drug overdose
 shock

Collecting the ABG specimen

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Arterial blood gas (ABG) sampling is a commonly performed procedure which allows
healthcare professionals to quickly obtain information on a patient‘s respiratory status
(blood oxygen and carbon dioxide levels), as well as the patient‘s acid-base balance.
Taking an arterial blood gas (ABG) involves using a needle and syringe to directly
sample blood from an artery (usually the radial artery). Below is a step by step guide
explaining how to take an arterial blood gas sample.
 Wash hands
 Introduce yourself to patient
 Confirm patient details like Name / date of birth
 Take note of whether the patient is requiring oxygen and record how much (e.g.
FiO2 concentration or flow rate)
Gather equipment
 Arterial blood gas syringe
 Needle (23G)
 Alcohol wipe – 70% isopropyl
 Gauze
 Tape
 Lidocaine – with small needle/syringe for administration
 Sharps container
 Gloves
 Apron

Local anaesthetic
The sample is routinely obtained from the radial artery and it is recognised that that the
procedure causes significant pain for the patient and that this can be markedly reduced
by the use of subcutaneous local anaesthetic. The British Thoracic Society recommends
the routine use of local anaesthetic for obtaining ABG samples except in emergencies, or
in unconscious or anaesthetised patients.

Preparation
1. Position the patient‘s arm preferably on a pillow for comfort with the wrist
extended (20-30°) (Figure BI 11.16.12)
2. Prepare all the equipment in the equipment tray using an aseptic non touch
technique
3. Palpate the radial artery on the patient‘s non-dominant hand (most pulsatile
over the lateral anterior aspect of the wrist)

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 4. Clean the site with an alcohol wipe for 30 seconds and allow to dry before
proceeding
5. Wash hands again
6. Don gloves and apron
7. Prepare and administer lidocaine subcutaneously over the planned puncture
site (aspirate to ensure you are not in a blood vessel before injecting the local
anaesthetic).
8. Allow at least 60 seconds for the local anesthetic to work
9. Attach the needle to the ABG syringe, expel the heparin and pull the syringe
plunger to the required fill level.

Figure BI 11.16.12: Position of arm & Palpate the radial artery

Taking the sample

1. Palpate the radial artery with your non-dominant hand‘s index finger around
1cm proximal to the planned puncture site (avoiding directly touching the
planned puncture site that have just cleaned)
2. Warn the patient befour to insert the needle
3. Holding the ABG syringe like a dart insert the ABG needle through the skin at an
angle of 45° over the point of maximal radial artery pulsation (which you
identified during palpation) (Figure BI 11.16.13).
4. Advance the needle into the radial artery until thre is blood flashback into
the ABG syringe
5. The syringe should then begin to self-fill in a pulsatile manner (do not pull back
the syringe plunger)
6. Once the required amount of blood has been collected remove the needle and
apply immediate firm pressure over the puncture site with some gauze

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 7. Engage the needle safety guard
8. Remove the ABG needle from the syringe and discard safely into a sharps bin
9. Place a cap onto the ABG syringe and label the sample
10. Continue to apply firm pressure for 3-5 minutes to reduce the risk of haematoma
formation

Figure BI 11.16.13: Insert the ABG needle through the skin at an angle of 45°

To complete the procedure

 Dress the puncture site
 Thank the patient
 Dispose gloves and equipment into an appropriate clinical waste bin
 Wash hands
 Take the ABG sample to be analysed as soon as possible after being taken as
delays longer than 10 minutes can affect the accuracy of results

Terms used in connection with ABG's

a. Acid-Base Balance - a homeostatic mechanism in the human body that strives to
maintain the optimal pH, so that body process may function optimally (normal
pH of arterial blood = 7.35-7.45)
b. Buffer System - combination of body systems that work to keep optimal acid-
base balance
c. Partial Pressure - the amount of pressure exerted by each gas in a mixture of
gases
d. PO2 - partial pressure of oxygen
e. PCO2 - partial pressure of carbon dioxide
f. PAO2 - partial pressure of alveolar oxygen

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 g. PaO2 - partial pressure of arterial oxygen
h. PACO2 - partial pressure of alveolar carbon dioxide
i. PaCO2 - partial pressure of arterial carbon dioxide
j. PvO2 - partial pressure of venous oxygen
k. PvCO2 - partial pressure of venous carbon dioxide
l. P50 - oxygen tension at 50% hemoglobin saturation
m. Respiratory Acidosis - condition of lowered pH (acidosis) due to decreased
respiratory rate (hypoventilation)
n. Respiratory Alkalosis - condition of increased pH (alkalosis) due to increased
respiratory rate (hyperventilation)

Acid/Base Balance
pH is the measurement used to determine acidity or alkalinity of arterial blood. pH is a
measure of an acid or base solution and the relative strength of that solution.
Below is the pH scale, 7 being the arbitrary center point indicting a neutral
solution. An example of an acid is carbonic acid. Carbonic Acid is formed when carbon
dioxide (CO2) chemically combines with water (H2O) to form carbonic acid (H2CO3).
The "H" at the beginning of a chemical formula usually designates and acid.

Neutral
4 5 6 7 8 9 10
death acidosis | | alkalosis death
7.35 7.45
normal
Analyzing the ABG
Follow the steps as indicated in order to best interpret the results.

Blood pH Bicarbonate Partial pressure Condition Common causes

of carbon dioxide
Less than 7.4 Low Low Metabolic Kidney failure, shock,
acidosis diabetic ketoacidosis
Greater than High High Metabolic Chronic vomiting, low
7.4 alkalosis blood potassium
Less than 7.4 High High Respiratory Lung diseases,
acidosis including pneumonia
or COPD
Greater than Low Low Respiratory Breathing too fast,
7.4 alkalosis pain, or anxiety

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 Part B: Following is a step-by-step account of how to analyze ABG if the prime
concern is oxygenation
Patient pH 7.45 CO2 32 identify:
HCO3 23 a. (condition) ________________________________
b. compensation YES or NO
c. name the possible diagnosis:_________________
Answers: a. resp alkalosis
b. yes because HCO3 is less than 24
c. possible hyperventilation
Possible causes Hyperventilation, respiratory stimulation, gram-negative
bacteremia.
Signs & symptoms Rapid, deep breathing, twitching, anxiety, fear

Part B: Use this guide to analyze ABG's if the patient's primary diagnosis is hypoxia
or any condition where O2 may be compromised.
step 1 if normal, go to if high, go to step 2 if low, indicates poor
Examine PO2 step 2 (patient may be oxygenation
over ventilated)
*may require immediate intervention, as in obstructed airway, COPD, or if on a
ventilator.
step 2 if normal, if low, (and O2 is if high, (with normal
Examine pH patient is either sufficient) go to step O2) go to step 2 prev
in no acute 2 prev page page
distress or is
compensating
step 3 - Examine After checking all of the above steps if they are within normal
patient limits, then the patient is either in compensation or is adequately
symptoms: ventilated. If ABG's are normal, but the patient still has
symptoms of hypoxia, then repeat ABG's in a short time. Then
the problem should be apparent.

Figure BI 11.16.14: different types of ABG analyzers

Laboratory determination of blood gas analysis –Micro method
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
Procedure:
 By using heparinized capillaries, blood can be collected from fingertips, a lobe of
ear or a heel.
 After filing with the blood capillaries are sealed by using modelling wax.
 Immediately before use blood is remixed & by cutting the sealed ends the blood
is introduced into each equilibration chamber of apparatus.
 After determining blood pH & pCO₂, the bicarbonate & base deficit (or excess)
are determined by using a Nomograms.
 Although under ordinary conditions capillary blood is similar to arterial, it can
be assumed to so in patients with poor peripheral circulations.
 For reliable results a good flow of capillary blood is essential. If this cannot be
obtained arterial blood must be used.
 In hypothermia following corrections has to be made. The measurements are
made at 38 ⁰ C. pH at T ⁰ C = p H at 38 ⁰ C+ 0.0146 (38 –T) PCO₂ at T ⁰ C =
Antilog (log PCO₂ at 38 ⁰ C – 0.021 (38-T)
 Blood must not be exposed to air in any circumstances before its analysis.

Principle of blood gas analyser works with three in-built electrodes (Figure BI
11.16.14)
1. pco₂ electrode
2. pO ₂ electrode
3. pH sensitive glass electrode

 Principle of pco₂ electrode

 Test solution –Arterial blood separated by plastic membrane permeable to
gaseous CO₂ but not permeable to dissolved ions.
 CO ₂ of blood diffuses through plastic membrane & reacts with the buffer system
to change pH.
 The pCO₂ electrode takes advantage of linear correlation between p H & log
PCO₂ over the range 11-90 mm Hg.
 The hydrogen ion concentration change due to the dissolution of CO₂ is detected
by the pH sensitive glass electrode.
 A potential difference exists between glass electrode & Reference electrode this is
measured on the meter. The meter‘s scale is usually calibrated for in semi
logarithmic fashion, since pH is inversely proportional to the log of pCO

 Principle of pO ₂ electrode
Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
  Polarographic method Reference platinum & (silver –silver chloride) electrode is
immersed in a buffer containing (phosphate & sodium chloride) .These
electrodes are separated from test solution.
 Test solution –Arterial blood separated by plastic membrane permeable to
gaseous O₂ but not permeable to dissolved ions.
 O₂ of blood diffuses through plastic membrane & 4 AgCl + 4 e⁻
 A potential difference exist between glass electrode 4 OH ⁻
 The electrons necessary for electro reduction are produced at reference electrode
(anode ) as follows- 4 Ag⁺ + 4 Cl ⁻ react with the buffer Following reactions take
place
 At platinum electrode (cathode) electro reaction occurs. O₂ + 2H ₂ O+ 4 e⁻ &
Reference electrode. This is measured on the meter. The meter‘s scale is usually
calibrated for in semi logarithmic fashion. The current through the system is
directly proportional to PO ₂ & can be recorded directly after amplification into
PO₂.
 Principle of pH sensitive glass electrode
 The measurement of pH is called potentiometric analysis. It has been reported
that a difference in electrical potential could be measured between two solutions
of different pH separated by a thin glass membrane. The potential thus produced
varies with the hydrogen ion concentration of two solutions .The glass
membrane is sensitive for H ⁺ions. It is on this principle that the glass electrode is
constructed for pH measurement.

Integral Parts of combined pH electrode:

a. Simple glass electrode
b. Calomel reference electrode with combined pH electrode

a. A simple glass electrode: Modern glass electrodes are constructed from glass
containing Lithium oxide which is soft, hygroscopic imparting low resistance.
The inner surface of glass membrane is in contact with a buffer pH. Into this
buffer dips a silver /silver chloride electrode, the internal glass electrode .The
glass electrode functions like semi permeable membrane selectively permeable
only to H⁺ ions.
b. Calomel reference electrode: consist of Mercury (Hg ⁺²) in contact with a
solution of potassium chloride saturated with Calomel (HgCl). It is surrounded
by a outer vessel holding saturated Potassium chloride which acts as a salt

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 bridge between reference and test solution. This electrode is not sensitive to Ph
with Combined pH electrode.
Only when glass electrode is coupled with a Calomel reference electrode
the potentiometric measurements are possible. The potential difference (or
electrical voltage) between two electrodes depends upon the hydrogen ion
concentration of test or standard solution. It is the logarithmic response
measured in millivolts on which pH meter is calibrated in both milliv

Laboratory determination of blood gas analysis

1. The pH of blood: heparinized whole arterial blood (or heparinized capillary
blood) is used. The pH determination is performed immediately after collection
of blood .The blood can be stored at 0-4 ⁰ C up to 2-3 hrs, without significant
change in pH.
2. PCO₂: (The respiratory parameter) plasma carbonic acid can not to be found
directly .But it is determined by measuring PCO₂. The PCO₂ of arterial blood is
usually directly proportional to the amount of carbon dioxide which is being
produced in the body & inversely proportional to the rate of alveolar ventilation
in the lungs.
3. TCO₂: Total CO₂ is mainly bicarbonate & also includes dissolved carbon dioxide.
Measurement of CO₂, carbonic acid & bicarbonate of plasma derived from blood
plasma, collected under liquid paraffin gives measure of CO₂ content. It is
reported as CO₂ per 100ml at standard conditions of temperature & pressure. If
the plasma is equilibrated with normal alveolar air (40 mm Hg) before it is
measured, the CO₂ combining power is obtained.Ordinarily the CO₂ content &
CO₂ combining power are practically identical.
4. The plasma bicarbonate: (non respiratory parameter ) can be determined by
finding out the actual bicarbonate concentration of plasma separated from blood
taken anaerobically & expressed as mill equivalents per liter .This can be
calculated from the p H & PCO₂ by using the Henderson's –Hassel Balch
equation or from total carbon dioxide & the PCO₂.
5. Standard bicarbonate: is expressed as milliequivalents per liter, is the
concentration of bicarbonate in plasma separated from whole blood taken
anaerobically which has ben equilibrated at 37⁰C a PCO₂ of 40 mm Hg with
oxygen to give full saturation to the hemoglobin. The plasma concentration
bicarbonate is influenced by change in PCO₂ & degree of oxygen saturation.
Alkali reserve is plasma bicarbonate. It is this fraction of plasma which is used to
neutralize all the acidic compounds entering the blood & tissue. Plasma

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda
 bicarbonate can also be calculated (in terms of mequ /lt) by dividing CO₂
combing power by 2.24.
6. PO₂: determination is carried out to assess the oxygen carrying capacity of blood
hemoglobin. The increased oxygen affinity of hemoglobin is indicated by
elevated PO₂ values. The measurement of arterial PO₂ is also used in conjunction
with that of PCO₂ in assessment of respiratory disorders .A low PO₂ is a measure
of anoxia. It may also occur with a high PCO₂ when there is alveolar hypo
ventilation due depression or obstruction of respiration. A low PO₂ with low
PCO₂ may also be observed in pulmonary edema.
7. Base excess: is the amount of acid required to titrate blood to p H 7.4 at 37⁰ C &
PCO₂ at 40 mm Hg. Base deficit is the reverse concept.

Standardization:
 By measuring the p H or the blood at its actual PCO₂ & at accurately known
PCO₂ values, one higher & one lower than the normal PCO₂. This can be
achieved by equilibrating two portions of the blood with carbon dioxide &
oxygen mixture with PCO₂ values between 30 -60 mmHg respectively .This
reduces experimental error since the actual PCO₂ values are between 30-60
mmHg.
 A Nomogram is constructed by plotting log PCO₂ against p H .It is possible to
calculate standard bicarbonate & base excess (or deficit).

Prepared By Dr. Showkat Ahmad Bhat, assistant Professor, department of Biochemistry, Govt.
Medical College Doda