Laboratory Manual

DEPARTMENT OF CHEMICAL ENGINEERING

PROGRAM – B.TECH (CHE)

Instrumentation and Material Characterization Lab (22CHC13)

SEMESTER-IV

R-22 Regulation

Prepared by: Dr. Rupam Sinha Verified by: Prof. M. Mukunda Vani & Dr. M. Mallaiah

CHAITANYA BHARATHI INSTITUTE OF

TECHNOLOGY
(An Autonomous Institution, Affiliated to Osmania University, Approved by AICTE,
Accredited by NAAC with A++ Grade and Programs Accredited by NBA)
Chaitanya Bharathi Post, Gandipet, Kokapet (Vill.), Hyderabad, Ranga Reddy - 500 075,
Telangana
www.cbit.ac.in

i
 CHAITANYA BHARATHI INSTITUTE OF
TECHNOLOGY
(An Autonomous Institution, Affiliated to Osmania University, Approved by AICTE,
Accredited by NAAC with A++ Grade and Programs Accredited by NBA)
Chaitanya Bharathi Post, Gandipet, Kokapet (Vill.), Hyderabad, Ranga Reddy - 500 075,
Telangana
www.cbit.ac.in
DEPARTMENT OF CHEMICAL ENGINEERING
Name of the Lab Course:
Instrumentation and Process Control Laboratory (22CHC13)

ii
 INDEX

S.no Item Page No

1. Institute Vision, Institute Mission, Quality Policy, Department Vision, iv
Department Mission and PEO's
2. Program Outcomes v
3. Program Specific Outcomes vi
4. Syllabus vii
5. Course Introduction ix
6. Assessment Procedure x
7. Laboratory Instructions (Do’s & Don’ts) and Safety Measures xiii

INDEX FOR THE EXPERIMENTS

Exp no Name of the experiment CO BT Page
L no

1 Calibration of flow measuring Instrument-Rotameter 1 2 1

2 Calibration of Temperature measuring Instrument 1 1
Mercury in Glass thermometer

3 Determination of pH 2 2

4 Determination of Electrical Conductivity 2 2

5 Determination of Alkalinity 2 1

6 Quantitative analysis of dilute solution using UV-

visible spectrophotometer
7 Microscopic Visualization of Human Buccal Epithelial
cells.
8 Calibration Of Refractometer

9 Study The Characteristics Of Control Valve

10

iii
 CHAITANYA BHARATHI INSTITUTE OF
TECHNOLOGY
(An Autonomous Institution, Affiliated to Osmania University, Approved by AICTE,
Accredited by NAAC with A++ Grade and Programs Accredited by NBA)
Chaitanya Bharathi Post, Gandipet, Kokapet (Vill.), Hyderabad, Ranga Reddy - 500 075,
Telangana
www.cbit.ac.in

Institute
To be a centre of excellence in technical education and research.
Vision
Institute
To address the emerging needs through quality technical education and advanced
Mission
research.

Quality Chaitanya Bharathi Institute Of Technology (CBIT) Imparts Value Based Technical
Policy Education And Training To Meet The Requirements Of Students, Industry, Trade /
Profession, Research And Development Organization For Self-Sustained Growth Of
Society.

Department To become the most sought center of excellence engaged in training and shaping
Vision students as professionals for higher education and process industries both in India and
abroad and allow the students to do R & D projects and publish same in the reputed
journals.

Imparting contemporary technical education and training manpower to create a skilled

Department human resource talent pool to serve, manage the process industries globally with a sense
Mission of responsibility towards society and the environment.

To train the students for identifying problems relevant to design and general practice of
PEO 1
chemical engineering field.

iv
 To provide experience in the three significant design areas of equipment, process and
PEO 2
plant operation of chemical industries.

To educate the students in understanding the multifaceted aspects of chemical

PEO 3
engineering and in applying the various computational methods studied, for problem
analysis and solution.

To prepare the students to pursue post graduate studies or to succeed in industry /

PEO 4
technical profession through global technical education.

Program Outcomes

1. Engineering Knowledge Apply the knowledge of mathematics, science, engineering

fundamentals, and an engineering specialization for the solution
of complex engineering problems

2. Problem Analysis Identify, formulate, research literature, and analyse complex

engineering problems reaching substantiated conclusions using
first principles of mathematics, natural sciences, and
engineering sciences.

3. Design/Development of Design solutions for complex engineering problems and design

Solutions system components or processes that meet the specified needs
with appropriate consideration for public health and safety, and
cultural, societal, and environmental considerations.
4. Conduct Investigations of Use research-based knowledge and research methods including
Complex Problems design of experiments, analysis and interpretation of data, and
synthesis of the information to provide valid conclusions.

5. Modern Tool Usage Create, select, and apply appropriate techniques, resources, and
modern engineering and IT tools, including prediction and
modelling to complex engineering activities, with an
understanding of the limitations.

6. The Engineer and Society Apply reasoning informed by the contextual knowledge to
assess societal, health, safety, legal, and cultural issues and the
consequent responsibilities relevant to the professional
engineering practice.

7. Environment and Understand the impact of the professional engineering solutions

Sustainability in societal and environmental contexts, and demonstrate the
knowledge of, and need for sustainable development.

v
8. Ethics Apply ethical principles and commit to professional ethics and
responsibilities and norms of the engineering practice.

9. Individual and Teamwork Function effectively as an individual, and as a member or leader

in diverse teams, and in multidisciplinary settings.

10. Communication Communicate effectively on complex engineering activities

with the engineering community and with the society at large,
such as, being able to comprehend and write effective reports
and design documentation, make effective presentations, and
give and receive clear instructions.
11. Project Management and Demonstrate knowledge and understanding of the engineering
Finance and management principles and apply these to one’s own work,
as a member and leader in a team, to manage projects and in
multidisciplinary environments.
12. Life-long Learning Recognize the need for and have the preparation and ability to
engage in independent and life-long learning in the broadest
context of technological
Change.

Program Specific Outcomes

Undertake research activities in the area of heat & mass transfer, separation
PSO 1
processes, Reaction engineering, related to Green Chemical Engineering.

Undertake real life projects in process industries and allied fields.

PSO 2

vi
 22CHC13 Instrumentation and material characterization lab

Instruction 3P Hours per week

Duration of SEE 3 Hours
SEE 50 Marks
CIE 50 Marks
Credits 1.5

Prerequisites: Engineering physics, Engineering chemistry

Course Objectives: This course helps the students to understand
1. The principles of different process instruments
2. The working principle of microscopes
3. The working principles and analysis processes of spectroscopic techniques
4. The working principles and analysis processes of characterization processes related to rheology and
interfacial tension
5. The working principles and analysis processes of Chromatographic techniques

Course Outcomes: After completing this course, students will be able to:
1. Calibrate different process instruments
2. Estimate material concentrations in solutions and evaluate the pH, alkalinity and electrical
Conductivity of solutions
3. Analyze and calculate the dimensions of microparticle
4. Identify functional groups and the composition of the materials
5. Determine viscosity and surface tension of liquids

CO-PO-PSO Matrix
PO1 PO2 PO3 PO4 PO5 PO6 PO PO8 PO9 PO10 PO1 PO12 PSO PSO2
7 1 1
CO 3 2 - - 3 1 - - 2 2 1 1 3 2
1
CO 3 2 - 3 3 1 - - 2 2 1 1 2 2
2
CO 3 2 - 3 3 1 - - 2 2 1 1 2 3
3
CO 2 2 - 2 3 1 - - 2 2 1 1 2 3
4
CO 3 2 - 3 3 1 - - 2 2 1 1 2 2
5

List of Experiments
(Minimum of 8 Experiments in the list are to be performed)
1. Calibration of flow measuring instrument-Rotameter
2. Calibration of temperature measuring instrument-Mercury in glass thermometer
3. Determination of pH
4. Determination of Electrical Conductivity
5. Determination of Alkalinity
6. Estimation of the dimension of microparticles using Optical microscopy
7. Calculation of Dye concentration using UV-Vis spectroscopy
8. Calculation of Dye concentration using Fluorescence spectroscopy.
9. Identification of functional groups using FTIR Spectroscopy.

7|Page
 10. Calculation of heavy metal concentration using Atomic Absorption spectroscopy
11. Determination of viscosity using Viscometer/Rheometer
12. Determination of surface tension using Tensiometer
13. Estimation of gas composition using Gas chromatography
14. Calculation of alcohol concentration using High Pressure Liquid Chromatography
15. Estimation of Contact angle using contact angle goniometer

Text Books:
1. Characterization of Materials, 2 Volume Set by Elton N. Kaufmann -Wiley-Interscience 2003.
2. Principles of Instrumental Analysis by D.A. Skoog, F.J. Holler, and T.A. Nieman, 7th edition, Cengage
Learning, 2018.
3. Principles of industrial instrumentation, D. Patranabis, 2nd ed., TataMcGraw Hill Edu. (India) Pvt.Ltd.,
New Delhi, 2013.

8|Page
 Course Introduction

In the Instrumentation and Material Characterization Lab course, students will delve into a diverse
range of experiments aimed at understanding fundamental principles and techniques used in the
analysis of materials. From calibrating flow and temperature measuring instruments to determining
pH, electrical conductivity, and alkalinity, students will gain hands-on experience in essential
laboratory practices. Furthermore, they will explore advanced techniques such as optical microscopy
for particle dimension estimation, UV-Vis and fluorescence spectroscopy for dye concentration
calculation, FTIR spectroscopy for functional group identification, and atomic absorption
spectroscopy for heavy metal concentration determination. This comprehensive curriculum equips
students with the skills necessary for precise material analysis and characterization.

Applications: The experiments conducted in the Instrumentation and Material Characterization Lab
hold diverse applications across industries and scientific disciplines. From calibrating instruments for
precise data collection to analyzing pH, conductivity, and alkalinity for water quality, the applications
are vast. Techniques like microscopy aid in particle analysis, while spectroscopy methods support
research in pharmaceuticals and materials science. These experiments have widespread applications in
pharmaceuticals, environmental monitoring, and industrial processes, providing essential insights into
material properties and composition.

9|Page
 Assessment Procedure And Award Of CIE Marks

Following is the subdivision for the internal marks (50) of the Lab:

(i) 20 marks for the lab internal tests

Two tests are to be conducted i.e one test after 1st cycle of experiments and
second test after the second cycle. Average of two tests marks put together should
be consider (20 maximum)

(ii) 30 marks for CIE

For the CIE 30 marks will be awarded based on the rubrics provided below.

This Rubrics are general guideline. Based on the lab type (programming or hardware) and
complexity of the course Rubrics can be customized by the department in tune to program
and course offered. Performance Indicators of the Rubrics also can be changed by the
department/Program based on the need.
RUBRICS
S. Descriptors and Score
N Parameter
o Outstanding Good Fair Poor Very Poor
1 Pre- Well prepared prepared for Adequately Minimal Lacks
Experiment for the the prepared for preparation preparation
Preparation experimentati experimentat the and and without
work on with a ion with experimentat without clear
5M clear clear ion without clear specifications
specifications, specification clear specificatio and
plan/design s and specification ns and plan/design (1
and plan/design s and plan/desig M)
additional (4M) plan/design n (2M)
information (3M)
(5M)
2 Experimenta Student Student Student Student Student fails to
tion conducts solves the solves the solves the solve the
(Problem experiment problem and problem and problem problem (2M)
Solving, with all conducts conducts with few
Methodolog possible test experiments experiment test cases
y of cases in an with all with few with
Conduction) optimized possible test possible test complexity
10 M fashion.(10M) cases (8M) cases with (4M)
complexity

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 S. Descriptors and Score
N Parameter
Outstanding Good Fair Poor Very Poor
o
(6M)
3 Post Demonstrates Demonstrate Demonstrate Demonstrat Failed to
Experiment the s results and s results es Partial Demonstrates
Analysis simulation/ inference; and results and results and
[Viva, findings Able to inference; inference; inference;
Inference] /Hardware answer Few Unable to Unable to Unable to
5M results Infers Questions answer the answer the answer the
and answer posed by Questions Questions Questions
all the Instructor posed by posed by posed by
Questions (4M) Instructor. Instructor Instructor (1M)
posed by (3M) (2M)
Instructor
(5M)
4 Report Report meets Report with Report is Report is Report is
Writing all well- complete complete, incomplete,
5M requirements organized and poor unclear, poor
and it is content, adequate grammar grammar and
prepared in visuals, with poor and inadequate
original and graphics, grammar. inadequate (1M)
creative way citations and (3M) and failed
to engage references to organize
readers (5M) (4M) thoughts
(2M)
5 Conduct Excellent team Follows the Follows Followed Does not
(Ethics, spirit, strictly safety safety minimum Follows safety
Safety, Team follows ethics precautions, precautions safety precautions
Work) 5M and safety practices and ethical precautions and ethical
precautions ethics and practices and and ethical practices and
with good poor team failed to practices failed to
team work work (4M) exhibit and failed exhibit team
(5M) teamwork. to exhibit work. (1M)
(3M) team work.
(2M)
TOTAL SCORE

Assessment Procedure And Award Of SEE Marks

S. Descriptors and Score

No Parameter
. Outstanding Good Fair Poor Very Poor
1 Record The content of The content The content The content of The content
5M all the of most of of the most the few of the all the
experiments is the of experiments is experiments
well-organized, experiments experiments not well- is not well-

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 S. Descriptors and Score
No Parameter
Outstanding Good Fair Poor Very Poor
.
recorded the are well- are well- organized, organized,
tables and organized, organized, recorded the recorded the
neatly drawn recorded the recorded the tables, neatly tables, neatly
graphs. Results tables, tables, drawn graphs. drawn
and discussions neatly neatly Results and graphs.
are well drawn drawn discussion are Results and
presented. (5M) graphs. graphs. not presented discussion
Results and Results and (2M) are not
discussion discussion presented
are are not (1M)
presented. presented
(4M) (3M)
2 Write up Presentation of Presentation Presentation Presentation Presentation
about the the given of the given of the given of the given of the given
experime experiment/pr experiment/ experiment/ experiment/p experiment/
nt ogram is very program is program is rogram is program is
10M well organized organized organized minimal with minimum
with the with the and is clear without clear
required required without specifications specifications
content, content, clear and and
specifications, specification specification plan/design. plan/design.
plan/procedure s, s and (4M) (2M)
of the conduct plan/proced plan/design
and all the ure of the .
required conduct. (6M)
additional (8M)
information.
(10M)
3 Conducti Conducts Conducts Conducts Conducts Failed to
on of the experiment / experiment experiment experiment / Conducts
experime Simulate the / Simulate / Simulate Simulate the experiment /
nt and problem with the problem the problem problem with Simulate the
observati proper with with proper improper problem with
ons connections / connections connections connections / improper
15M all possible test / with most / less test less test cases. connections /
cases. All the of the test cases. (6M) less test cases.
possible cases. Failed (9M) (3M)
observations to note all
are noted. observations
(15M) (12M)
4 Results & Demonstrates Demonstrate Demonstrat Demonstrates Failed to
Analysis the s the es the the partial Demonstrate
10M experimental experimenta experimenta experimental the results
results with l results l results, results with and least
adequate with failed to least analysis /
analysis / required maximum analysis / simulation/
simulation/ analysis / analysis / simulation/ findings.

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 S. Descriptors and Score
No Parameter
Outstanding Good Fair Poor Very Poor
.
findings simulation/ simulation/ findings. (2M)
/obtained findings findings. (4M)
results /obtained (6M)
/plotting the results
graphs. (10M) /plotting
the graphs.
(8M)
5 Viva-Voce Answers most Answers Answers Answers only Failed to
10M of the questions most of the only few of few of the answer
with good questions the questions with questions.
analytical with good questions nominal (2M)
explanation. explanation. with good explanation.
(10M) (8M) explanation. (4M)
(6M)
TOTAL SCORE

Laboratory Instructions (Do’s &Don’ts)

S.No Do’s S.No Don’ts

1. Students must wear their identity 1. Do not operate the machines without the
cards and specified uniform permission of the staff
2. Keep all the belongings at the place 2. Do not lay on the equipment
suggested by the lab instructor
3. Bring the calculator, graph paper, 3. Do not use mobile phones during
and other accessories while coming laboratory hours
to the lab
4. Bring the observation book and record 4. Never handle the electric cables
without fail with wet skin
5. Before performing the 5. Do not use any pen drive
experiments, read the instrument
manual carefully
6. Keep the experimental area clean 6. Do not eat or drink in the lab

7. Maintain silence in the lab

Safety Instructions
1. All safety rules related to Dos and Don’ts are displayed.
2. Safety shoes and Aprons are mandatory for students.

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 3. Insulated Rubber mats are provided near UPS
4. Fire extinguishers and water hose line are available near the lab, in corridor.
5. All are instructed to switch off the power supply after completion of experiment.
6. Lab has proper ventilation with exhaust fans.
7. First aid kit is available in department.

EXPERIMENT- 1
Calibration of flow measuring Instrument
ROTAMETER
Aim: To calibrate the given Rotameter and draw its calibration curve.
Apparatus: The apparatus consists of a fluid circuit which includes sump tank, monoblock
pump, rotameter and measuring tank connected in series with the help of pipeline to form a
fluid flow circuit. Further, control valves are provided to regulate the flow of water (or any
liquid). A stop watch is provided in order to measure the time taken for filling the tank with
water up to a specific desired level.
Theory: Rotameter is an instrument used for fluid flow measurement. Rotameter is a variable
area flow meter. In head flow meters the restriction size remains constant, due to which the
differential pressure across it varies with the differential flow rate through it. But in variable
area flow meters the restriction size or flow area of restriction is allowed to vary with the
fluid flow rate so as to maintain the differential pressure across it constant. Thus any change
in the fluid flow rate can be measured in terms of change of flow area, hence the name
variable-area flow meter.

Construction: Rotameter consists of a tapered glass tube mounted vertically with smaller end on
lower side. The glass tubes are used for metering low temperature and pressure fluids, but for high
temperature and pressure service metal tubes are used. A float is installed in the tube after the meter is
mounted in the flow line. Floats are usually made of corrosion resistant metals like aluminum, bronze,
monel, nickel, stainless steel etc. Usually a series of slanting notches are cut in the underside of the

14 | P a g e
 float rim that gives rotation to float so as to reduce the friction. Float material decides the flow-range
of the rotameter. Float may have different float shapes. Flow scale is marked on the glass-tube or it is
mounted close to the metering tube. Rotameter is installed in the pipe line by means of flanges or
threads along with the inlet and outlet piping supports in brackets. The meter must be installed
vertically within about 2 geometrical degrees so as to centre the float in the fluid stream.

Procedure:
1. Start the pump.
2. Operate the valve for flow of fluid through rotameter apparatus and keep it slightly open
3. Slowly adjust the valve so that the flow of fluid through rotameter is sufficient enough so
that the float shows displacement.
4. Measure the flowrate of fluid and corresponding float position in rotameter.
5. The flowrate can be calculated by knowing the time taken for filling the tank for a known
level. Hence measure the time taken for filling of tank upto a particular level.
6. Increase the flowrate by opening the valve further.
7. Take the reading for different flow rates.
8. Plot a graph of float position vs. flowrate.

Observation Table:

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 S.No. Rotameter Time reqd for Actual Error (Lph) Accuracy %
reading 1.5 liters of discharge
(Lph) water (Sec.) (Lph)

1
2

Calculations:
1. Actual discharge = (1.5X3600)/(Time required for 1.5 liters of water )
2. Error = Rotameter reading – Actual discharge.
Rotameter reading - Error
3. Accuracy = x 100
Rotameter

Sample Calculation:

Graphs:

1. Plot the graph of accuracy versus Rotameter reading

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 Sample Graph:

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 Result:
Calibration curve of Rotameter

EXPERIMENT- 2
Calibration of Temperature measuring Instrument
Mercury in Glass thermometer
Aim: To Calibrate given thermometer (mercury in glass thermometer) by immersing them in
different equilibrium systems (ice/water, normal water and boiling point of water) of known
temperatures
Apparatus: heat source, ice, water, mercury in glass thermometer.
Theory:
Thermometers are devices that measure temperature using a variety of different principles.
Most of these rely on measuring some physical property of a working material that varies
with temperature.
One of the most common devices for measuring temperature is the glass thermometer. The
mercury-in-glass or mercury thermometer is based upon the difference between the thermal
expansion coefficients of liquid mercury (about 1.8x10 -4 K -1) and glass (about 0.2x10-4 K -1 ).
The glass vessel of a mercury thermometer consists of a bulb (i.e. mercury container) and a
capillary of uniform diameter.
Temperature increase causes the fluid to expand, so the temperature can be determined by
measuring the volume of the fluid. The capillary is usually marked at two points (say at 0 °C
and 100 °C ) and graduated uniformly in between, on an assumption that the volume of a
fixed mass of mercury is a linear function of temperature. The measuring interval of a
common mercury thermometer is between -39 °C and +350 °C. Note that the European
Union bans mercury from certain electrical, electronic and non-electrical measuring devices,
such as thermometers and barometers. The alcohol thermometer is an alternative to the
mercury-in-glass thermometer. Unlike the mercury thermometer, the contents of an alcohol
thermometer (pure ethanol, toluene, kerosene etc.) are less toxic.
Thermometers are not affected by vapour pressure above the capillary column. It is only
necessary that the liquid be clearly distinguishable from the volume above the liquid. The
glass capillary magnifies the column, and can be shaped to increase the magnification. A gas
thermometer measures temperature by the variation in volume or pressure of a gas. The
constant volume thermometer consists of a bulb connected by a capillary tube to a
manometer.
A bimetallic thermometer consists of two strips of different metals (usually steel and copper)
which expand to a different degree as they are heated. The strips are joined together
throughout their length. The different expansions force the flat strip to bend if 3 temperature
changes. The metal with the higher coefficient of thermal expansion is on the outer side of
the curve when the strip is heated.

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 There are some important sources of error in measuring with mercury (or alcohol)
thermometers.
1. Achieving thermal equilibrium needs a good while. Therefore when measuring with
mercury thermometer must wait at least 10 mins before recording temperature values.
2. Glass, not being a crystalline material, cannot adjust its volume promptly to an abrupt
drop in temperature. Consequently when you transfer a thermometer from boiling water
into an ice/water bath, mercury thermometers tend to show lower than 0 °C for the
melting point of ice. This type of error is called zero point depression.

3.

The glass capillary that contains the mercury (or any other liquid) thread is never quite even in diameter

t=T e-Tm
where Te is exact (correct) temperature established by well-known phase equilibria
and Tm is the measured one
4.

The value of t can be either negative or positive number. Measuring the temperatures of some equilibriu

5.

Stem error and correction. Thermometers are normally scaled on the assumption that when the reading i

) using the equation

stem

ΔTstem ktm - tstem 

where tm (in ˚C) is the temperature read in the thermometer, t stem (in ˚C) is the mean
temperature of the stem measured by an another (auxiliary) thermometer in the middle of
the stem (or estimated by another way), and l is the length of the stem outside the vessel
(in cm). Constant k depends on the difference of the thermal expansion coefficients of
thermometer liquid and glass. In case mercury-in-glass thermometers its value is about
0.00016 ˚C -1 .
It can be easily seen from the above mentioned errors that for precise measurements the
thermometer should be calibrated and stem correction should be used. With knowledge
of both corrections, the true temperature can be calculated by the equation:
te  tm Δt  Δt stem
Procedure: Mercury-in-glass-thermometer (3 point calibration)
1. To determine the calibration, check your thermometers with the following 3 systems:

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 i. ice/water system
ii. Normal water
iii. boiling water / water vapour at actual atmospheric pressure
2. Keep both test and standard thermometers in the Ice bath. Wait about 10 mins to reach
thermal equilibrium between the system and thermometer. After that, record 5-6
equilibrium temperature values.
3. Shift the thermometers from ice bath to hot water bath and note the reading.
4. Repeat above step with the normal water.

Observation Table:

S.No System Correct value of measured temperature t =Te-Tm

temperature (Te) (Tm)
1 Ice
2 Water
3 Steam

Calculations:
1. Calculate the boiling point of water at the actual atmospheric pressure using vapour
pressure table or equation according to ITS–90 (tb).
2. Calculate the correction values of thermometers at the three secondary standard
temperatures
Δt1  0.00C – tm1,
Δt2  32.38C-tm2,
Δt3 tb - tm3
3. Plot corrections (Δt) vs. measured temperature (tm) for the mercury thermometer
4. Connect measuring points by straight lines. Give the results in form of a table

Graphs:

1. Plot the graph of corrections versus measured thermometer reading(Tm).

2. Plot the graph of corrected temperature (Te) versus thermometer reading (Tm)

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 Sample Graphs

Result:

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 Calibration graph for the mercury thermometer.

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 EXPERIMENT 3:

Determination of pH
Aim: To determine the pH of a given sample using pH meter.
Apparatus: Digital pH meter, pH electrode, Stand, Beakers, Wash bottle, Glass rod
and Tissue Paper.
Reagents required: Capsules for pH 4.0, 7.0 and 9.2, distilled water or Buffer
solutions for pH .4.0 & 7.0.
Sample: Tap water and Domestic wastewater.
Principle: The electro-chemical potential developed between a known liquid inside
glass electrode and unknown liquid outside gives the corresponding pH value of the
unknown liquid i.e., sample.

Theory:
pH is referred as a potential of hydrogen ion concentration. pH is a measure of
positive ion concentration. pH describes the degree of acidity or alkalinity of a
solution The pH scale is used to express the concentration of hydrogen ions in a
liquid. The pH scale ranges from 1 to 14, i.e, most acid to most alkaline. If the H +
concentration is greater than OH- , then the sample is acidic where the pH value is
less than 7. If the OH- ion concentration is greater than the H+ ion concentration the
sample is alkaline where the pH value is greater than 7. If equal amount of OH- , H+
ions are present, the sample is neutral, with a pH of 7. The meaning of pH is potential
of Hydrogen (H) which means the strength of Hydronium ions in the given solution.
However since the concentrations are very minute, pH is taken as the negative
logarithm of the Hydrogen ion concentration expressed in grams per litre.
pH = - log [ H+]……………………(1)
The hydrogen ion concentration is an important parameter in determining the quality
of water and wastewater. The pH of the water/wastewater sample is measured by pH
meter. A pH meter consists of a measuring probe connected to electronic meter
measures and displays the pH reading.
Acids and bases have free hydrogen and hydroxyl ions respectively. Since the
relationship between hydrogen ions and hydroxyl ions in a given solution is constant
for a given set of

conditions, either one can be determined by knowing the other. Thus pH is a

measurement of both acidity and alkalinity even though by definition it is a selective
measurement of hydrogen ion activity.
An ion is a charged particle, created by an atom or molecule which has either gained or
lost electron(s). The presence of ions in solution allows electrical energy to pass
through the solution as a conductor. Different compounds form ions in solution in
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 different amounts, depending on the ability of the atoms to gain or lose electrons. They
will dissociate (or ionize) in solution to form hydrogen (H+) or hydroxyl (OH-) ions in
the solution. Molecules that dissociate easily will form strong acids or bases when in
aqueous solution. In an aqueous solution, hydrogen ions normally combine with the
water solvent to form the hydronium ion concentration.
Normally, the terms ‘hydronium ion’ and ‘hydrogen ion’ are used interchangeably
in pH measurement applications.
Procedure:
Take 20ml of ready-to-use pH 4 and pH 7 buffer solutions in two separate beakers.
Or
Preparation of buffer solutions:
1. Dissolve 1 buffer pH 4 tablet/ capsule in 50 ml double distilled water taken
in a glass beaker.
2. Make up the volume of volumetric flask to 100 ml with distilled water.
3. Repeat the procedure with pH 7 and pH 9 capsules in different beakers to
obtain 3 different buffer solutions.
Calibration of instrument

1. Switch ON the pH meter 10 minutes before start of the experiment allowing it to

warm up.
2. Press and Hold ‘CAL’ key for 5 seconds and adjust the indicator to ‘pH’ mode.
3. Scroll over prefixed pH values you desire to calibrate i.e pH 4, pH7, pH 9.2
depending on the respective buffer solution you immerse the pH electrode.
4. The reading on the display should be the same as the pH value of the buffer solution.
Calculation of pH value
1. Filter the water sample if there is any visible turbidity or precipitate.
2. Put the pH electrode in sample taken in a beaker & press ‘READ’.
3. Note the pH value of the sample.
4. Repeat the process for 3 times and Note down the average of all 3 values.
Observation and Calculations:

S. Sample Reading Average

No
.
1
2
3

Results:

24 | P a g e
 The pH of the given sample = ………..

Precautions

1. For best results the temperature of the buffer solution should be set at the
temperature knob during experiment.

2. In general, the glass electrode is not subject to solution interference like

color, high salinity, colloidal matter, oxidants, turbidity or reductants.

3. Oil and grease, if present in the electrode layer, should be removed by

gentle wiping or detergent washing, followed by rinsing with distilled
water, because it could impair the electrode response.

4. Electrodes used in the pH meter are highly fragile, hence handle it

carefully.

5. Dip the electrode in distilled water after conducting experiment.

6. Wash the glassware with tap water and then with distilled water
before and after the experiment.

7. To dry the electrode, dab it with tissue paper but DO NOT WIPE
THE ELECTRODE.

EXPERIMENT 4:

25 | P a g e
 Determination of Electrical Conductivity
Aim: To determine the electrical conductivity of the given sample using Conductivity
meter

Apparatus: Conductivity meter, beakers, tissue paper

Principle
Conductivity is determined using the distance between the electrodes and their
surface area. According to Ohm’s Law, the current through a conductor between
two points is directly proportional to the potential difference across the two points.
Conductivity is measured with a probe and a meter. A voltage is applied between
the two electrodes in the probe immersed in the sample water. The drop in voltage
caused by the resistance of the water is used to calculate the conductivity per meter
and displays the result for the user. According to Ohm’s law, Conductivity (G),
the inverse of resistivity (R) is determined from the voltage and current values.
R=V/I then, G=1/R=I/V.

Theory
Electrical Conductivity: The electrical conductivity (EC) is a measure of the
capacity of a substance or solution to carry an electrical current. Conductivity of a
substance is defined as the ability or power to conduct or transmit heat, electricity
or sound. When an electrical potential difference is placed across a conductor, its
movable charges flow, giving rise to an electric current. This property is called
conductivity. The conductivity is represented by reciprocal value of electrical
resistance in ohms relative to cubic centimeter of water at 25ºC. The measured EC
value is used as an alternate method to estimate the total dissolved solids (TDS)
concentration of the sample. The EC is represented by the symbol ’k’ and its units
are millisiemens per meter (mS/cm) or micromhos per centimeter
(µmho/cm).Since the charge on ions in solution facilitates the conductance of
electrical current, the conductivity of a solution is proportional to its ion
concentration. The conductivity of water is directly linked to the concentration of
the ions and their mobility. The ions in water act as electrolytes and conduct the
electricity.
The conductivity also depends on the value of the pH, on the temperature of
measurement and on the amount of CO2 which has been dissolved in the water to
form ions. The conductivity is also affected by the concentration of ions already
present in the water such as chloride, sodium and ammonium. Chemical
composition of water determines its conductivity. Hence this becomes the most
widely used measure of the purity of water.

Procedure

26 | P a g e
 1. Switch on the instrument.
2. The instrument shows 0 value for both EC and TDS as soon as the instrument
is switched on.
3. Insert the electrode in the sample till the reading displays a non fluctuating value.
4. To note down the TDS reading press “Range” and it will display the value of
TDS in ppm.
5. Press “Range” again to display the value of EC in micro Seimens.
6. Also, note down the temperature of the sample which is displayed on the meter.
7. After reading, take the probe out of the solution and dab it with tissue paper
but do not wipe the probe
8. Now take the required sample to be tested in another beaker and dip the probe in
it
9. Note down the reading on the display. Take 3 average readings of each sample.
Observations

S. No Temperature, oC Conductivity TDS

1
2
3

Calculations

Results
The Electrical Conductivity of the given sample =..........mS
TDS of the given sample =.............mg/l

Precautions / Instructions
1. Wash the glassware with tap water and then with distilled water before and after the
experiment
2. When not in use, always dip the electrode in distilled water and do not expose it to
air.
3. As conductance is influenced by temperature, always use a conductivity meter
with temperature control.
4. Always prepare the calibration solution freshly before the start of the experiment.
5. As it involves instruments for analyzing do not forget to calibrate the instrument.
6. Switch on the conductivity meter for atleast 30 minutes before starting the
experiment so that the instrument gets stabilizes.

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 EXPERIMENT 5:
Determination of Alkalinity
Aim: To determine the amount of the following types of alkalinity present in the
given water sample: a. Hydroxide alkalinity b. Carbonate alkalinity c.
Bicarbonate alkalinity.
Apparatus required: Cylindrical beaker, Measuring jar, Burette
Reagents required: Standard Sulphuric Acid (0.02 N), Phenolphthalein
Indicator, Methyl Orange Indicator
Theory
Alkalinity of water is a measure of its capacity to neutralize acids. It is primarily
due to salts of weak acids, although weak or strong bases may also contribute.
Alkalinity is significant in many uses and treatments of natural waters and
wastewaters. Because the alkalinity of many surface waters is primarily a
function of carbonate, bicarbonate, and hydroxide content, it is taken as an
indication of the concentration of these constituents. It is expressed in terms of
CaCO3 equivalent to hydrogen ions neutralized. The major portion of alkalinity in
natural water is caused by carbonates, bicarbonates and hydroxides which may be
ranked in order of their association with high pH values. Highly alkaline water
leads to caustic embrittlement and causes deposition of precipitates and boiler
tubes. Bicarbonates of calcium and magnesium include temporary hardness to
water. Boiler water always contains carbonates and hydroxide alkalinity,
chemically treated water (limeor lime soda ash softening water) will be alkaline
due to the presence of carbonates and excess hydroxide. High alkalinity in
natural water will favour the growth of algae and phytoplankton.

Principle

Hydroxyl ions present in a sample as a result of dissociation or hydrolysis of

solutes react with standard acid added. Alkalinity thus depends on the end-point
pHused. Alkalinity is usually imparted by bicarbonate, carbonate and hydroxide. It
is measured volumetrically by titration with 0.02 N sulphuric acid and is reported
in terms of CaCO3 equivalent. For samples whose initial pH is above 8.3, the
titration is conducted in two steps. In the first step, the titration is conducted until
the pH is lowered to 8.2, the point atwhich phenolphthalein indicator turns from
pink to colourless. This value corresponds to the points for conversion of
carbonate to bicarbonate ion. The second phase of titration is conducted until the
pH is lowered to 4.5, corresponds to methyl orange end point, which corresponds
to the equivalence points for the conversion of bicarbonate ion to carbonic acid.
Titration to decolourisation of phenolphthalein indicator will indicate complete
neutralization of OH- and 1/2 of CO3- while sharp change from yellow to orange of
methyl orange indicator indicates total alkalinity (complete neutralization of OH-,
CO3- and HCO3). The equation in its simplest form is as follows:

28 | P a g e
 CO32- + H+ = HCO3 (pH 8.3)

From pH 8.3 to 3.7 the following reaction may occur:

HCO3- + H+ = H2CO3

Sampling and storage: Collect samples in polyethylene or borosilicate glass

bottles and store at a low temperature. Fill bottles completely and cap tightly. As
wastewater samples may be subject to microbial action and to loss or gain of CO2
or other gases when exposed to air, analyze samples without delay, preferably
within one day. If biological activity is suspected analyze within 6 hours. Avoid
sample agitation and prolonged exposure to air.
Procedure
Phenolphthalein Alkalinity
1. Take 30 ml of sample in a conical flask.
2. Add 3-4 drops of phenolphthalein indicator. If the pH of sample is above 8.3,
sample turns pink.
3. Titrate with 0.02 N H2SO4 in a burette till the color disappears.
4. Note down the volume of H2SO4 added (V1).

Total Alkalinity

1. Take 30 ml of sample in a conical flask.

2. Add 3-4 drops of Methyl Orange Indicator. The sample turns yellow.

3. Titrate it against 0.02 N H2SO4 till the colour of the sample turns orange.

4. Note down the total volume of H2SO4 added (V2).

29 | P a g e
 Fig. 1 Titration set up.

Observations

Burette reading Volume of

S.N Volume of H2SO4
o Sample
(V1) (VH2SO4, ml)
Initial Final
Phenolphthalein
Alkalinity
1
2
3
Total Alkalinity
1
2
3

Calculations:
Let V1 = Amount of H2SO4 used (volume)

1.
Phenolphthalein Alkalinity of the sample = V H2SO4 × NH2SO4× 1000/ V1
(ml) × 50(0.6 x0.02 x 1000 x 50)/30 = 20 mg/L as CaCO3

2.
Total Alkalinity of the sample = V H2SO4 × NH2SO4× 1000/ V1 (ml) ×
50110 mg/L asCaCO3 Where, V = mL of H2SO4 titrant consumedN =
Normality of H2SO4

The types of alkalinities present in the samples are calculated using the
equations given in the following table and the results are tabulated.

Result of Hydroxide Alkalinity Carbonate Bicarbonate

titration asCaCO3 Alkalinityas Alkalinity as
CaCO3 CaCO3
P=0 0 0 T
P < T/2 0 2P T-2P

30 | P a g e
 P = T/2 0 2P 0
P > T/2 2P-T 2(T-P) 0
P=T T 0 0

Result:
1.
Phenolphthalein alkalinity in mg/L as CaCO3 = ………………………
2.
Total alkalinity in mg/L as CaCO3 = ………………………………….
3.
Hydroxide Alkalinity (mg/lt) = ……………………………………….
4.
Carbonate Alkalinity (mg/lt) = ………………………………………..
5.
Bicarbonate Alkalinity (mg/lt) = ………………………………………

Conclusion:

31 | P a g e
 EXPERIMENT NUMBER: 6

Quantitative analysis of dilute solution using UV-Visible

Spectrophotometer
Aim: To determine the solute concentration of dilute solution.
Theory
Properties of Light: Light is a type of energy that travels as a wave-particle. The
wavelength of light is the distances between peaks in the waves as light travels.
Wavelengths are measured in nanometers (nm) and different wavelengths of light
represent differing colors. White light is a mixture of the visible light spectrum.
Light of long wavelengths (infra-red) and very short wavelengths (ultraviolet) are
invisible to humans but can be observed by other organisms. As the wavelength
decreases, the energy of the light is increased.

Figure. Electromagnetic spectrum and associated peak wavelength emitted. [credit:

Diagram of the Electromagnetic Spectrum. NASA (2010). Public domain.]
Spectrophotometry: Spectrophotometers (spectro-image/color; photo-light; meter-
measure) are used for chemical analysis of solutions based on properties of
absorption or transmission. Figure 8.3 shows a schematic of a spectrophotometer.

32 | P a g e
 Figure. A spectrophotometer splits light through a monochromatic prism, reducing
the light from the light bulb to a single wavelength. A small portion of the single
wavelength light passes through the aperture, which is a hole that captures the single
wavelength light. The aperture focuses the light to go through the solution contained
in the cuvette. [credit: Center for Online and Continuing Education in partnership
with Ozlem Yavuz-Petrowski and the College of Science, Florida Atlantic
University. (2020).

The Absorbance of a Solution: For each wavelength of light passing

through the spectrometer, the intensity of the light passing through the reference cell
is measured. This is usually referred to as I0 - that's I for Intensity. Transmittance
refers to the amount of light that passes through the solution.

The transmittance of a light source through a cuvette. The intensity of light, I0,
decreases as it passes through the solution. The light detected by the sensor, I,
reflects the transmittance of the solution.

Figure. Light absorbed by sample in a cuvette with length l.

The intensity of the light passing through the sample cell is also measured for that
wavelength - given the symbol, I. If 𝐼 is less than I0, then the sample has absorbed
some of the light (neglecting reflection of light off the cuvette surface). A simple bit
of math is then done in the computer to convert this into something called the
absorbance of the sample - given the symbol, 𝐴. If the light is being absorbed by
chemicals in the solution, this results in a lower transmission. Absorbance is
therefore inversely related to transmittance as expressed by the equation:

33 | P a g e
 The absorbance of a transition depends on two external assumptions: 1. The
absorbance is directly proportional to the concentration (𝑐) of the solution of the
sample used in the experiment. 2. The absorbance is directly proportional to the
length of the light path (𝑙), which is equal to the width of the cuvette.
Assumption one relates the absorbance to concentration and can be expressed as, 𝐴
∝ 𝑐. The absorbance (𝐴) is defined via the incident intensity I0 and transmitted
intensity I by

Assumption two can be expressed as, 𝐴 ∝ 𝑙. Combining equations from assumption

one and two gives, 𝐴 ∝ 𝑐𝑙. This proportionality can be converted into an equality by
including a proportionality constant (𝜖).
𝐴 = 𝜖𝑐𝑙
This formula is the common form of the Beer-Lambert Law, although it can be also
written in terms of intensities:

The constant ϵ is called molar absorptivity or molar extinction coefficient and is a

measure of the probability of the electronic transition. On most of the diagrams you
will come across, the absorbance ranges from 0 to 1, but it can go higher than that.
An absorbance of 0 at some wavelength means that no light of that particular
wavelength has been absorbed. The intensities of the sample and reference beam are
both the same, so the ratio I0/I is 1 and the log10 of 1 is zero.
The Beer-Lambert Law: You will find that various different symbols are given for
some of the terms in the equation, particularly for the concentration and the solution
length. The Beer-lambert law is expressed as follows,

The Greek letter epsilon, 𝜖, in these equations is called the molar absorptivity - or
sometimes the molar absorption coefficient. The larger the molar absorptivity, the

34 | P a g e
 more probable the electronic transition. In UV spectroscopy, the concentration of
the sample solution is measured in mol L-1 and the length of the light path in cm.
Thus, given that absorbance is unitless, the units of molar absorptivity are L mol -1
cm-1. However, since the units of molar absorptivity is always the above, it is
customarily reported without units.

Procedure:
Methodology:
1. Step-1 (Scanning): To find out λmax
Sample is scanned to determine the maximum absorbance (λmax) of light passing through the
sample.
2. Step-2: Calibration
Standard curve or reference curve and its equation are determined by analyzing at least
four samples having different concentrations. The absorbance of these four samples is
measured in MEASUREMENT program and absorbance vs. concentration plot is obtained.
For dilute solutions this plot is a straight line and equation of standard curve is obtained
using excel software.
3. Step-3: Analysis of unknown sample
The unknown sample is placed in sample chamber and its absorbance is measured in
MEASUREMENT program. Absorbance value is put in equation or graph of standard
curve and concentration is found out.

Steps for analysis :

For Step-1
1. Switch on spectrometer and computer.
2. Wait for at least 10 minutes for the stabilization of spectrometer.
3. The spectrometer is taken in SCAN mode.
4. Required parameters are given in INSTRUMENT mode.
5. Required parameters are given in SAMPLE mode.
6. Cuvettes (two) filled with pure solvent are placed in respective chambers.
7. Base line correction is done.
8. Sample cuvette is placed in sample chamber for scanning of sample to determine
maximum absorbance of light (λmax) passing through the sample.

For Step-2&3
1. The instrument is taken in MEASUREMENT program.
2. Required parameters are given in INSTRUMENT mode.
3. Required parameters are given in SAMPLE mode.
4. Four samples of known concentration are placed in sample chamber one after another for
their absorbance values and standard curve is determined.
5. The unknown sample absorbance is measured in same way and concentration of that is
found out from standard curve.

35 | P a g e
 Result and Analysis
1. Instrument name(Make and Model:
2. Sample name :
3. Sample amount:

For step-1 (scanning):

Table 1: Observed data
Concentration, ppm Peaks, nm % absorbance

(a) λmax. for ( sample name ) is .

(b) Plot of standard curve of (sample name)
(c) Absorbance of sample :
(d) Concentration of sample :

Precautions to be taken:
4. Spectrometer is stabilized for at least 10 min. after switch on.
5. Sample chamber should be closed during analysis.
6. Sample should be clear enough (nearly free of materials except solvent and solute).
7. Sample should be so diluted that absorbance should not exceed a
maximum limit (3.0% absorbance). Otherwise sample should be diluted
with solvent only.

Example analysis :
Aim: Determination of concentration of dilute amino acid solution.

Result: Sample name : Amino acid

Sample amount: Minimum 3 ml.

For step-1 (scanning):

Table 1: Observed data of aqueous amino acid solution

Concentration, ppm Peaks, nm % absorbance

25 225 0.260
50 225.5 0.520
75 225.5 0.781
100 226 1.038

So, the λmax. is 225.5 nm for this amino acid.

36 | P a g e
 For step- 2&3 (measurement):

Fig-1: Standard curve of

amino acid

If the absorbance of an unknown sample is 0.8234, the concentration of that sample should
be, from standard curve equation, x=( 0.8234/0.0104) =79.17 or 79 ppm.

37 | P a g e
 Experiment Number: 7
Microscopic Visualization of Human Buccal Epithelial cells.
Aim: To prepare a temporary slide of Human Buccal epithelial cells and visualize the
sample using Inverted Microscope.

Material Required: Human cheek epithelial cells, sterile tooth picks, glass slide, coverslip,
droppers, tweezers and 0.1% w/v Methylene blue solution and Inverted Microscope (Model:
CKX53).

Principle:
An inverted microscope is a microscope with its light source and condenser on the top,
above the stage pointing down, while the objectives and turret are below the stage pointing
up. It was invented in 1850 by J. Lawrence Smith. It is useful for observing living cells or
organisms at the bottom of a large container (e.g., a tissue culture flask) under more natural
conditions than on a glass slide, as is the case with a conventional microscope. Inverted
microscopes are also used in micromanipulation applications. These microscopes may also
be fitted with accessories for fitting still and video cameras, fluorescence illumination,
confocal scanning and many other applications.

Animal cells are typical of the eukaryotic cell type, enclosed by a plasma membrane and
containing a membrane-bound nucleus and organelles. The cell is the structural and
functional unit of life. The animal cells lie within the size range of 10-100 µm. When we
visualize a typical animal cell under a microscope it is clearly visible that they lack a rigid
cell wall. This has allowed to develop a greater diversity of cell types and tissue types within
animals. The specialized tissues like nerve tissue and muscle tissue associate to form organs
which has enabled movement of animals. most animal tissues are bound together in an
extracellular matrix proteins like collagen. The animal consists of various internal organelles
which are as follows:

 Centrioles - Centrioles are self-replicating organelles made up of nine bundles of

microtubules and are found only in animal cells. They appear to help in organizing cell
division, but aren't essential to the process.
 Cilia and Flagella - For single-celled eukaryotes, cilia and flagella are essential for the
locomotion of individual organisms. In multicellular organisms, cilia function to move fluid
or materials past an immobile cell as well as moving a cell or group of cells.
 Endoplasmic Reticulum - The endoplasmic reticulum is a network of sacs that
manufactures, processes, and transports chemical compounds for use inside and outside of
the cell. It is connected to the double-layered nuclear envelope, providing a pipeline between
the nucleus and the cytoplasm.
 Endosomes and Endocytosis - Endosomes are membrane-bound vesicles, formed via a
complex family of processes collectively known as endocytosis, and found in the cytoplasm
of virtually every animal cell. The basic mechanism of endocytosis is the reverse of what
occurs during exocytosis or cellular secretion. It involves the invagination (folding
inward) of a cell's plasma membrane to surround macromolecules or other matter diffusing
through the extracellular fluid.
 Golgi Apparatus - The Golgi apparatus is the distribution and shipping department for the
cell's chemical products. It modifies proteins and fats built in the endoplasmic reticulum and
prepares them for export to the outside of the cell.

38 | P a g e
 Intermediate Filaments - Intermediate filaments are a very broad class of fibrous proteins
that play an important role as both structural and functional elements of the cytoskeleton.
Ranging in size from 8 to 12 nanometres, intermediate filaments function as tension- bearing
elements to help maintain cell shape and rigidity.
 Lysosomes - The main function of these microbodies is digestion. Lysosomes break down
cellular waste products and debris from outside the cell into simple compounds, which are
transferred to the cytoplasm as new cell-building materials.
 Microfilaments - Microfilaments are solid rods made of globular proteins called actin.
These filaments are primarily structural in function and are an important component of the
cytoskeleton.
 Microtubules - These straight, hollow cylinders are found throughout the cytoplasm of all
eukaryotic cells (prokaryotes don't have them) and carry out a variety of functions, ranging
from transport to structural support.
 Mitochondria - Mitochondria are oblong shaped organelles that are found in the cytoplasm
of every eukaryotic cell. In the animal cell, they are the main power generators, converting
oxygen and nutrients into energy.
 Nucleus - The nucleus is a highly specialized organelle that serves as the information
processing and administrative centre of the cell. This organelle has two major functions: it
stores the cell's hereditary material, or DNA, and it coordinates the cell's activities, which
include growth, intermediary metabolism, protein synthesis, and reproduction (cell division).
 Peroxisomes - Microbodies are a diverse group of organelles that are found in the
cytoplasm, roughly spherical and bound by a single membrane. There are several types of
microbodies but peroxisomes are the most common.
 Plasma Membrane - All living cells have a plasma membrane that encloses their contents.
In prokaryotes, the membrane is the inner layer of protection surrounded by a rigid cell wall.
Eukaryotic animal cells have only the membrane to contain and protect their contents. These
membranes also regulate the passage of molecules in and out of the cells.
 Ribosomes - All living cells contain ribosomes, tiny organelles composed of approximately
60 percent RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands
of RNA. In prokaryotes, they consist of three strands of RNA.

Procedure:
1. Take a tooth pick and gently scrape the inner wall of your cheek.
2. Place the scrapings on clean glass slide, add a drop of methylene blue/Safranin.
3. Allow the cells to take up the stain for a period of 2-3 minutes.
4. Carefully blot the excess of methylene blue/safranin stain present on the glass slide.
5. A drop of a mountant like glycerin can be added; Carefully place a cover slip on the cells
avoiding the entrapment of any kind of air bubbles.
6. Place the slide under the microscope to observe the cells.
7. Set the main switch of the microscope (ON) and turn the light intensity control knob to
obtain appropriate brightness.
8. Set the light path at 100% for binocular observation.
9. Turn the revolving nosepiece to bring the 10X objective into the light path. Be sure to turn
the revolving nosepiece until it clicks.

39 | P a g e
10. Adjust the interpupillary distance of the eyepieces
11. Bring the required objective into the light path and focus on the specimen.
12. Under 20X objective provided with the correction collar, set the scale on the correction
collar according to the thickness of the vessel bottom.
13. The morphology of the cells was Observed and recorded.

Observation:
Results:

40 | P a g e
 Block Diagram of Inverted Microscope CKX53

41 | P a g e
Diagram a typical animal cell

42 | P a g e
 Experiment No. 08
Calibration Of Refractometer
Aim
To get the calibration curve for the Mettler Toledo refractometer for ethanol – water
mixtures at different temperatures.

Requirements

Mettler Toledo refractometer, heating plate, thermometer, Ethanol, Distilled water, 50 ml

beakers (# 10), pipette, cleaning tissues.

Introduction

The Mettler Toledo refractometer is used for the simple determination of the refractive index
of liquid samples. It has the following characteristics:
1. Measures samples with a refractive index in the range 1.3200 to 1.5000.
2. Needs a minimum amount of sample (min. 0.4 ml) for measurement.
3. Keeps the temperature of the sample being measured constant between 15 to 40 °C.
4. The light source for the measurement is an LED and prism is made of sapphire,
which is strongly resistant to corrosion, very rigid and of high thermal conductivity.

Refractive index

The refractive index ‘n’ of a substance is the ratio of the velocity of a ray of light in a
vacuum to its velocity in the medium. If a ray of light at a particular angle passes from
optically less dense (air) to optically more dense (water) medium, it changes its direction
except when the incident light is vertical. According to Snell's law of refraction, the ratio of
the refractive indices of the two media is proportional to the ratio of the angle of refraction
and angle of incidence of the ray of light:

n1 sin (β)
=
n2 sin (α )

If a ray of light passes into an optically less dense medium from an optically denser
medium, it also changes its direction. If the angle of incidence α is increased, it reaches a
critical value (angle of refraction β = 90°) at which the ray of light no longer passes into
the optically less dense medium. If this "critical angle" is exceeded, total reflection
occurs. The critical angle α is used to calculate the refractive index:

n1 1
=
n2 sin (α )

43 | P a g e
 Measurement Principle
The light emitted passes through the prism and encounters the sample. It is partially refracted
(angle of incidence < critical angle) and partially reflected (angle of incidence > critical
angle). The reflected light is recorded using an optical sensor (CCD). The boundary between
the dark and light areas represents the critical angle needed to calculate the refractive index.

Procedure

1. Prepare 10 different mixture samples of the given liquids with known mole fraction.
2. Place the refractometer on a table and place one or two drops of the liquid mixture sample
on the prism.

3. Press the OK button on the meter and note down the refractive index and temperature from
the screen.
4. Clean the prism with cleaning tissues and repeat the above steps for measuring the next
liquid mixture sample.
5. Repeat steps 1 to 4 for different temperatures.

Observations

Refractive Index of Ethanol-Water mixtures

Serial Volume of ethanol Volume of water Mole fraction of Refractive

number (ml) (ml) ethanol index

44 | P a g e
 Result

These measured refractive indexes were plotted against to their mole fraction of the
mixtures.

Note:
1. Compare these refractive index data of samples at different temperatures with the
refractive index values in the literature (taking into consideration errors in
observation and measurements, and the refractometer).
2. Write the report in your own words, mention the conclusions of the experiment and
validate your conclusions by stating proper references.

45 | P a g e
 Experiment No. 09
Study The Characteristics Of Control Valve
Objective:
To determine the flow coefficient Cv of the linear control valve, equal % control valve and
quick open control valve.

Theory:
Valve is essentially a variable orifice. Control valve is a valve with a pneumatic, hydraulic,
electric (excluding solenoids) or other externally powered actuator that automatically, fully or
partially opens or closes the valve to a position dictated by signals. transmitted from
controlling instruments. Control valves are used primarily to throttle energy in a fluid system
and not for shutoff purpose. Control valves are used to control conditions such
as flow, pressure, temperature, and liquid level by fully or partially opening or closing in
response to signals received from controllers that compare a "set point" to a "process
variable" whose value is provided by sensors that monitor changes in such conditions.
Control Valve is also termed as the Final Control Element. The opening or closing of control
valves is usually done automatically by electrical, hydraulic or pneumatic actuators.
Positioners are used to control the opening or closing of the actuator based on electric, or
pneumatic signals. These control signals, traditionally based on 3-15psi (0.2-1.0bar), more
common now are 4-20mA signals for industry, 0-10V for HVAC systems.
TYPES OF CONTROL VALVE:
Depending upon the valve plug design the control valves can be divided in three categories:
i) Equal % type.
ii) Linear type.
iii) Quick opening type (On/Off type).

Equipment set up:

The present set-up consists of three nos. of pneumatic control valves. One control valve is
with equal % characteristics (air to close type), second is with linear characteristics (air to
open type) and third is quick opening characteristics (ON/OFF). Sump tank with pump is
provided for continuous water circulation. Manometer is provided at the inlet of valves to
measure pressure. Valves are given for water supply to the control valve. Valves are provided
for air supply to control valves. The air regulator and pressure gauge is given for regulating
air pressure.

The schematic is given below:

46 | P a g e
Objective-1:
To study the quick opening control valve characteristics and to calculate the gain at various
conditions.

Procedure:
1. Hand valve HV1, HV2 and HV5 are opened and HV3, HV4 and HV7 are closed.
2. Air regulator is adjusted to give 15 PSI output.
3. The unit is switched ON and pump speed is adjusted for maximum flow.
4. The hand valve HV1 is adjusted to give maximum flow through control valve.
5. Flow through the rotameter and stem position are noted.
6. Air regulator output is varied, and the corresponding flow rate is measured.
7. Step 5 and 6 are repeated for the different pressure and the readings are tabulated.
8. Gain of the control valve at various operating pressure is calculated using the formula:

Formula:

Gain= Q/Qmax
S /Smax
Where,
Q= Instantaneous flow in LPH
Qmax = Maximum flow in LPH
S = Instantaneous stem position in mm
Smax = Maximum stem position in mm

Table 1:
Sl. no Control air Stem Flow rate S/Smax Q/Qmax Gain
pressure position calculated

Objective-2:
To study the Equal Percentage control valve characteristics and to calculate the gain at
various conditions.

Procedure:

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 1. Hand valve HV1, HV3 and HV6 are opened and HV2, HV4, HV5 and HV7 are closed.
2. Air regulator is adjusted to give 15 PSI output.
3. The unit is switched ON and pump speed is adjusted for maximum flow.
4. The hand valve HV1 is adjusted to give maximum flow through control valve.
5. Flow through the rotameter and stem position are noted.
6. Air regulator output is varied, and the corresponding flow rate is measured.
7. Step 5 and 6 are repeated for the different pressure and the readings are tabulated.
8. Gain of the control valve at various operating pressure is calculated using the formula:

Formula:

Gain= Q/Qmax
S /Smax
Where,
Q= Instantaneous flow in LPH
Qmax = Maximum flow in LPH
S = Instantaneous stem position in mm
Smax = Maximum stem position in mm

Table 2:
Sl. no Control air Stem Flow rate S/Smax Q/Qmax Gain
pressure position calculated

Objective-3:
To study the Linear control valve characteristics and to calculate the gain at various
conditions.
Procedure:
1. Hand valve HV1, HV4 and HV7 are opened and HV2, HV3, HV5 and HV6 are closed.
2. Air regulator is adjusted to give 15 PSI output.
3. The unit is switched ON and pump speed is adjusted for maximum flow.
4. The hand valve HV1 is adjusted to give maximum flow through control valve.
5. Flow through the rotameter and stem position are noted.
6. Air regulator output is varied, and the corresponding flow rate is measured.
7. Step 5 and 6 are repeated for the different pressure and the readings are tabulated.
8. Gain of the control valve at various operating pressure is calculated using the formula:
Formula:
Gain= Q/Qmax
S /Smax
Where,

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 Q= Instantaneous flow in LPH
Qmax = Maximum flow in LPH
S = Instantaneous stem position in mm
Smax = Maximum stem position in mm

Table 3:
Sl. no Control air Stem Flow rate S/Smax Q/Qmax Gain
pressure position calculated

Graphs:

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 GAP ANALYSIS BY THE COURSE FACULTY /

COORDINATOR

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Faculty name & Signature with date

HoD/BoS Chairman remarks:

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HoD/BoS chairman Signature with date

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