Lab Manual KC 31001 Laboratory 6: Semester 2 2016/2017

UNIVERSITI MALAYSIA SABAH
SCHOOL OF ENGINEERING & INFORMATION TECHNOLOGY

CHEMICAL ENGINEERING PROGRAMME
LAB MANUAL
KC 31001 LABORATORY 6
Semester 2 2016/2017
Venue:
Makmal Tindakbalas, Makmal Unit Operasi, Makmal Kawalan,
Makmal Bioproses
Lecturer:
Hafeza Abu Bakar
Lab. Assistants:
En. Freddy, En. Razis, Ms. NorAemi, Ms. Chen Moi Fong
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Contents
No. Titles Pages

1. Laboratory safety 3
2. Laboratory reports and assessment 5
3. Experiments
BP1 Preparation of culture media and isolation of pure culture 8
BP2 Characterization of bacteria and establishing pure cultures 12
BP3 Quantification of cell number 15
CRE1 Kinetic studies in batch reactor 20
CRE2 Isothermal continuous stirred tank reactor (iCSTR) 24
SP1 Determination of adsorption isotherm for citric acid 30
SP2 Solid-liquid extraction (SLE) 36
SP3 Batch distillation on a binary mixture 41
PC1 Liquid/water flow control (WF) 49
PC2 Liquid/water level flow control (WLF) 60
2

Laboratory Safety in General
These guidelines are meant for safety awareness in the laboratory. However, specialized
laboratory may require specific safety rules. Good management of laboratory is important to protect
laboratory personnel/users against hazards at work.
1. Laboratory Safety awareness

- Everyone is responsible for his or her own safety and the safety of others while working in the
laboratory.
- Before working with a chemical, assessment must be made of its hazards and risk.
- Be familiar with appropriate protection measure when you are working with the following:
Flammable substances
Corrosive and toxic chemicals
Biohazards
Radioactive materials
Compresses gases
- Laboratory should be adequately ventilated.
- Chemical storage areas should be cool, dry, well ventilated and away from sunlight.
- Eating, drinking, and smoking are strictly prohibited in laboratories, as well as stores and
workshops.
2. Personal Protection
- Laboratory coat, safety goggles and gloves (if needed) should be worn all the time in the
laboratory.
- Always assure that you wash your hands before leaving the laboratory.
- Short skirts, shorts and open-toed shoes/sandals should not be worn in the laboratory to avoid
skin exposure.
3. Fire Hazards and hazardous chemicals

- Always store flammable liquids in appropriate safety cabinets/cans.
- Do not store incompatible reagents together, e.g. flammables and acids. Segregate acids and
bases, and corrosive materials from organic and flammable materials.
- Wear appropriate protective equipment such as laboratory coat, apron, gloves and eye protection
gear when you are working with flammable, corrosive and toxic chemicals.
- Ensure that no ignition sources present nearby while working with flammable chemicals.
- All electrical cords should always be in good condition. Electrical outlets should be grounded.
- Do not store ethers for long periods to avoid formation explosive peroxides.
- Corrosive chemicals can burn and irritate tissue. If inhaled or ingested, it may affect lung and
stomach tissue.
- Avoid mixing oxidizing chemicals with other chemicals during disposal.
- Careful when dealing with carcinogens (cancer causing agent). Suspected carcinogens (please
check for a full list): chloroform, benzidine, benzene, methylchloromethyl ether, vinyl chloride,
acrylonitrile, formaldehyde, etc.
- Never use lubricants on valve regulators of compressed gases.
4. Laboratory Housekeeping
- All equipment should be inspected carefully before used.
- Equipment and work bench must be cleaned after use.
- Use non-chromate cleaning solution if possible. Make sure cleaning is done in the fume hood if
sulphuric acid glass cleaner is used.
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- Keep laboratory floor dry at all times. Any spills must be immediately attended to.
5. After Hours/Long Hours Experiment

- Avoid experimental work in an unoccupied space/building if possible.
- Always place a note should any unattended experiments must be carried out, stating the
experiment, name of researcher/student and contact number.
- Always check that flames and compressed gas supplies are shut off when not in use and end of
day.
6. Emergency Procedures
- All laboratory personnel/users must be familiar with the location and uses of the safety devices in
and around the laboratory, for example:
Safety shower
Fume hood
Fire extinguisher
First aid kit
Eye wash station
Fire alarm
- Contact the laboratory personnel or University safety officer immediately when emergency
happens.
- Upon ingestion of chemicals, if victim is awake, give water. If nauseated, do not give fluids. If
unconscious, CPR might be needed.
- Upon inhalation of hazardous chemicals, move the victim out for fresh air. Perform artificial
respiration if victim is not breathing.
- For chemical spills: (i) acid spills apply neutralizer or add water if necessary and check mixture
with indicator for neutralization; (ii) solvent spills apply activated charcoal and mix thoroughly
until material is dry. Transfer mixture into plastic bag, tie up and label, place in fume hood.
Contact laboratory personnel for disposal.
4

Laboratory Reports and Assessment
General Guidelines
1. Please BE PREPARED mentally and physically and go through the manual before entering the
lab. Students should have studying the theory of the experiments and also preparing the required
tables for noting down the observations.
2. Get your laboratory observations and calculation recorded in a logbook. The raw data and
preliminary analysis counter signed by the instructor.
3. Students are encouraged to be on time or earlier. Some of the groups will perform the same
experiment on the same day. However, if the group does not have sufficient time to finish the
experiment, they should reschedule the time with the lab technician and please inform me on the
date.
4. All experiments are carried out in groups. Attendance is compulsory. Whoever unable to
attend or run the experiment with his/her group (with reasonable excuse and proof of absence)
at scheduled time, he/she should join other group to complete the experiment. Please inform the
lecturer/ instructor about the matter as soon as possible.
5. Every member of the group has to submit a short technical report for all five experiments.
The short report should emphasize more on abstract, result, discussion and questions given.
6. Lab report is due in a week for each completed experiment. The lab report should be submitted
to my room (Bilik No 56, Block A). Late report submission without reasonable reasons will cause
mark deductions.
7. As for the mini project, every group should submit only one full report. The report is due in 2
weeks. Late submission will affect your total marks. There will be a presentation on the project
after the report is submitted. The date will be notified later.
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Guidelines for Report Writing
Laboratory report should contain the followings:
1. Title page
Please provide the essential information at the front cover of the report such as:
Group number
Name & Matric number of the student
Group members names
Name of experiment
Date of experiment
Name of the instructor/ lecturer
Name of the lab assisstant
Venue
2. Abstract
This section summarizes the whole report in one, concise paragraph of about 100-200 words. It
should be a precise and specific summary. It might be useful to think in terms of writing one
sentence to summarize each of the traditional report divisions: objective, method, discussion,
conclusions. Emphasize the objective (which states the problem) and the analysis of the results
(including recommendations). Avoid the temptation to copy a whole paragraph from elsewhere in
your report and make it do double duty.
3. Introduction
The introduction of a technical report identifies the subject, the objective, and the plan of
development of the report. The subject is the what, the purpose is the why, and the plan is
the how. Together these acquaint the reader with the problem you are setting out to solve.
It also should provide the reader with any background information which the reader will need
before you can launch into the body of your paper. You may have to define the terms used in
stating the subject and provide background such as theory or history of the subject.
4. Experimental procedure
This section describes the process in chronological order. Using clear paragraph structure, explain
all steps in the order they actually happened, not as they were supposed to happen. Please show
the process flow diagram in the report for better undertanding of the experiment
5. Result and discussion

Generally, results are dominated with calculations, graphs, tables and figures. Nevertheless, the
significant results should be in verbal/ write in sentences form. Graphics need to be clear, easily
read, and well labeled. A sample calculation is sufficient in the report and let the remainder in the
appendix.
Discussion is the prominent section in a report. It shows that you really understand the
experiment that you completed. You should be able explain, analyse and interpret results that
you obtained from the experiment. You can use the explanation below to help you write the
discussion part.
Analysis: What do the results indicate clearly? What have you found? Explain what you
know with certainty based on your results and draw conclusions.
Interpretation: What is the significance of the results? What ambiguities exist? What
questions might we raise? Find logical explanations for problems in the data.
Compare expected results with those obtained.
Analyze experimental error: Was it avoidable? Was it a result of equipment? If an
experiment was within the tolerances, you can still account for the difference from the
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ideal. If the flaws result from the experimental design explain how the design might be
improved.
Analyze the strengths and limitations of your experimental design
6. Questions
Answer all questions from the manual and insert them in a section of your report before
conclusion part.
7. Conclusion
The conclusion states the knowledge comes out of the report and experiment result. It can be a
short and concise conclusion.
8. Reference
In engineering, the two most common referencing standards are:
Author-Date style (Chicago style, APA 6th, etc)
IEEE style
Please provide any resources that you refer to complete the report such as laboratory manual or
any other outside reading.
9. Appendices
Appendices may include raw data, calculations, graphs, and other quantitative materials that
were part of the research.
Evaluation System
Assessment Method Marks

Laboratory reports 50%
Laboratory Log Book 5%
Attendance / TeamWork 5%
Mini Project and Presentation 40%
Overall Total 100%
7

BIOPROCESS PRINCIPLES LABORATORY EXPERIMENT 1
PREPARATION OF CULTURE MEDIA AND ISOLATION OF PURE

CULTURE

1.0 INTRODUCTION
Cultivation is an important technique used to isolate culture and determine

microorganisms in microbiology. Handling method is designed to keep the culture pure
and to prevent pollution. Any medium for cultivation of bacteria must provide basic
nutritional requirements.
There are two types of medium: defined or synthetic medium and complex
medium. In both cases, pH of a medium may need to be adjusted to an optimum value
to permit microbial growth (referring to the number of cells not the size of the cells).
Most bacteria can grow well at pH of 7. A broth medium is one in which the components
are dissolved in water. Addition of agar results in a solid medium.
Normally, bacteria occur in nature as mixed populations. To study individual
bacteria, various microorganisms need to be isolated into pure cultures consisting of a
single type of species. Most methods for obtaining pure cultures rely on dilution
technique. The most useful method is the streak plate method, in which a mixed culture
is spread or streaked over the medium surface so that individual cells are separated
from one another. Another method to produce pure cultures involves diluting a fraction
of a mixed culture through a series of cooled liquid agar such that fewer cells exist. The
liquid agar is then poured into sterile petri dish and is allowed to solidify. Single cells will
be entrapped and grow into colonies.
In microbiology laboratory, autoclave is usually used for sterilization. Generally, a
temperature of 121oC for a period of time (approximately 20 minutes) is used to heat-
sterilize bacteriological media.
1.1 THEORIES AND EXPLANATIONS
1.1.1 Aseptic technique
Aseptic is important while working with microorganism to prevent contamination

of culture and spread of microorganisms into the environment. Generally, normal
transferring a culture from one vessel to another involves: (i) sterilizing inoculating loop
by flaming, (ii) remove the closure and briefly flame the lip of culture tube before
inserting sterile loop, (iii) Insert inoculating loop into culture tube and remove loopful of
ture, (iv) flame the lip of tube and replace closure.
NOTE: Keep all culture covered with caps or lids. Do not lay caps or lids on table top to
avoid exposing to contamination. Do not allow tube closures or Petri dishes lids to touch
anything. When transferring colonies from Petri dish, use the lid as a shield by slightly
raising it enough so the loop can be inserted but agar surface still protected from
contaminants.
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1.1.2 Streak plating
The streak plating technique isolates individual bacterial cells (colony-forming

units) on the surface of an agar plate using a wire loop. The streaking patterns shown in
the figure below result in continuous dilution of the inoculum to give well separated
surface colonies. Once again, the idea is to obtain isolated colonies after incubation of
the plate.
The following rules are important for consistent results: (i) Allow the loop to cool
before using it for inoculation (Note: Hot loop may kill the microorganism), (ii) Allow
the loop to touch the agar surface lightly and slide gently over the surface. (Note: Do
not dig the loop into agar! Hold the loop parallel to the agar surface to minimize gouging
(see figure 1.2 below)), (iii) Sterilize the loop after inoculating each section of plate, (iv)
Use petri dish cover to prevent contamination into the media and (v) when incubating,
invert the plates to prevent water condensate from spreading bacteria over agar surface.
Figure 1.1 Three times streaking method.

Figure 1.2 Correct method to hold the loop.
1.2 OBJECTIVE
In this experiment, you will learn to:

1. Prepare a broth medium and agar medium
2. Use autoclave to sterilize the prepared medium and required labwares
3. Transfer culture using aseptic technique without contaminating the culture or
exposing yourself to them
4. Isolate single colony for your next experiment by doing streaking on agar plates
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1.2 MATERIAL AND APPARATUS
Nutrient Apparatus Equipment

Peptone Microscope slides PH meter
Ethanol 95% Conical flasks (250 ml) Balance
MRS Broth Graduated cylinder (1L) Autoclave
MRS Agar Beaker (500 ml) Incubator
Gram stain Crystal Loop Oven
Violet Beaker (250 ml) Spectrophotometer
Gram stain iodine Pipette (100 microlitre)
Gram stain Safranin Pipette (1000 microlitre)
Pipette tips (100 microlitre)
Pipette tips (1000 microlitre)
Microcentrifuge tubes 1.5 ml
Cotton
Aluminium foil
Sterile petri dish
Bunsen burner
1.4 PROCEDURE
1.4.1 Preparation of medium and sterilization
1. Nutrient broth and agar will be prepared in advance by lab demonstrators.
1.4.2 Sampling analysis:
1. Insert the wire of inoculating loop into Bunsen flame until it is red and
glowing. Allow the wire to cooled for a few seconds (Do not wave the
loop in air).
2. Use your free hand to pick up the tube containing the culture. Gently
shake it to disperse the culture. Remove the tube cap and flame the lip in
Bunsen burner flame.
3. Insert the sterile loop and remove a loopful of culture. Try not to touch
the sides of tube.
4. Flame the tube lip again and replace the tube cap.
5. Place the loop containing the inoculum into the tube. Remove the loop
and cross streak the agar surface three to four times and continue
streaking into a second adjacent area.
6. Cover the agar plate with its lid.
7. Flame the inoculating loop.
8. Place the streaked plate into a 37 C incubator and incubate for 48 hours.
9. Examine the plates and describe its appearance.
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10. Keep the plates in the fridge for next experiment.
1.5 EXPERIMENTAL RESULTS
Observe plates. Describe the morphology, size and color of representative colonies from
the streaking results.
1.6 QUESTIONS
1. What can you notice in the control plate?

2. Did you obtain isolated colonies on the agar plates which were streaked? If
you did not obtain isolated colonies, what changes should you make in your
technique to ensure isolated colonies?
3. What is subculturing?
4. When and why do you need to apply isolation of pure culture? Support your
reason(s) with three examples.
5. What is the difference between streak plate and spread plate method?

11

CHARACTERIZATION OF BACTERIA AND ESTABLISHING PURE

CULTURES

2.0 INTRODUCTION
When grown on a variety of media, microorganisms will exhibit differences in the

macroscopic appearance of their growth. These differences, called cultural
characteristics, are used as the basis for separating microorganisms into taxonomic
groups.
In 1883 Hans Christian Gram invented an important differential staining method
called the Gram Stain. This staining procedure differentiates microbes into two basic
groups: Gram positive microbes and Gram negative microbes. Differential stains render
one type of microbe one color and other types of microbes another color. In the Gram
stain, Gram positive organisms retain the crystal violet-iodine and appear blue/purple
whereas Gram negative cells loose the primary dye complex during the challenge rinse
(acetone or alcohol) and are stained by the safranin which makes them pink.
In most microbiological staining procedures, the bacteria are first fixed to the
slide by the heat fixed smear. In this procedure living, potentially pathogenic bacteria
are smeared on the glass slide and allowed to dry before the slide is heated so that the
bacteria are killed and stuck (coagulation of surface proteins) to the slide. This renders
the slide safe and keeps the bacteria stuck to the slide so that simple or complex
staining and washing procedures can be carried out without washing them off of the
slide.
(source: http://www2.fiu.edu/~makemson/MCB3020Lab/Ex2GramStain.pdf)
Most bacteria can be divided into two groups based on the composition of their cell wall:
1. Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma
membrane. Characteristic polymers called teichoic and lipoteichoic acids stick
out above the peptidoglycan and it is because of their negative charge that the
cell wall is overall negative. These acids are also very important in the bodys
ability to recognize foreign bacteria. Gram-positive cell walls stain blue/purple
with the Gram stain.
2. Gram-negative cell walls are more complex. They have a thin peptidoglycan
layer and an outer membrane beyond the plasma membrane. The space
between the plasma membrane and the outer membrane is called the
periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that
is important in triggering the bodys immune response and contributing to the
overall negative charge of the cell. Spanning the outer membrane are porin
proteins that enable the passage of small molecules. Lipoproteins join the
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outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.
Table 2.1 Gram stain

CELL COLOUR
REAGENT
Gram Positive Gram Negative
Crystal violet PURPLE PURPLE
Iodine PURPLE PURPLE
Alcohol PURPLE COLOURLESS
Safranin PURPLE RED
2.2 OBJECTIVE

1. Prepare the bacterial smears for the microscopic visualization of bacteria
2. Characterize bacteria using gram staining and microscope
3. Become familiar with the chemical basis of the Gram stain and the
performance of the procedure for differentiating between the two principal
groups of bacteria (gram-positive and gram-negative).
2.3 MATERIAL
Refer to Bioprocess principles laboratory experiment 1.3.
2.4 METHODOLOGY OF OPEN REFLUX METHOD
2.4.1 Identification of bacteria
1. Observe the growth of colonies on the agar plates.

2. Smear preparation
i. On the underside of slide, label one end of slide with microorganism to
be stained.
ii. Prepare one smear for each microorganism. For agar culture, place a
small drop of water at the top center of slide. Pick up a single colony
using the aseptic technique to the slide and mix with water. Spread the
suspension over an area of circle.
iii. Allow the smear to heat dry by passing the slide through a bunsen
burner flame intermittently. Do not overheat the slide, touch the slide 2
seconds after heating, it should be warm but not hot enough to burn.
Note: Remember to hold the clean slides by their edges. Smears

require only a small amount of the bacterial culture. Do not blow on
slide or wave it in the air.
3. Place the slides on the staining rack and flood each smear with crystal violet.
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4. After staining for 1 minute, wash the crystal violet from each slide with
distilled water and drain off excess water.
5. Flood each smear with iodine.
6. After 1 minute, wash the iodine from each slide with distilled water.
7. Tilt each slide and decolorize with ethanol until alcohol draining from the
slide appears colourless, about 15 seconds.
8. Wash the slide briefly and drain off the excess.
9. Counterstain the smear with safranin for 20 to 30 seconds.
10. Wash the slide briefly with distilled water and blot dry.
11. Examine the slides. Describe the color of the smear.
12. View the slides under microscope to observe the cell morphology.
1.4.2 Establishing pure culture
1. Using aseptic technique, pick up a loopful of single colony from the agar
plate and inoculate into the prepared broth medium.
2. Swirl the medium to make sure the colony is well dispersed.
3. Incubate the broth medium in an incubator shaker at 37 C and 150 RPM for
24 hours.
4. Store the culture in fridge for next experiment.
Show and describe your Gram stains.
2.6 QUESTIONS
1. Describe the growth of each culture on the streak plates.

2. What is a pure culture? How do you prove a culture is pure?
3. Why is it important of obtaining pure cultures?
4. What is a differential staining? What are the advantages of differential
staining procedures over the simple staining technique?
5. What color does your specimen appear under microscope? Is it gram positive
or negative?
6. What features of your Gram stain exemplify the Gram stain theory? If there
are features of the Gram stain that are not as expected, what possible
sources or error may be responsible?
7. Cite the purpose of each of the reagents in a differential staining procedure.
(Define the followings: primary stain, counterstain, decolorizing agent,
mordant.)
8. Which is the most crucial step in the performance of the Gram staining
procedures? Justify your answer.
9. Why must fresh bacterial cultures be used in a Gram stain?

14

QUANTIFICATION OF CELL NUMBER

3.0 INTRODUCTION
In the study of microbiology, there are numerous occasions when it is necessary

to either estimate or determine the number of bacterial cells in a broth culture or liquid
medium (a.k.a bacterial enumeration). Determination of cell numbers can be
accomplished by a number of direct or indirect methods. The methods include standard
plate counts, turbidimetric measurements, visual comparison of turbidity with a known
standard, direct microscopic counts, cell mass determination, and measurement of
cellular activity. For accurate enumeration of bacterial numbers, including injured or
starved cells, it is important to use culture-independent techniques. The culture-
independent techniques are important as it has been established that the bacterial
diversity in any given environment is severely underestimated when assessed by means
of culture-based technique.
3.1.1 Serial dilution from stock solution
Quantification of the cell numbers is important in researches. This can be done

by series of dilution. Usually the dilution factor at each step is constant, resulting in a
geometric progression of the concentration in a logarithmic fashion. A ten-fold serial
dilution could be 1 M, 0.1 M, 0.01 M, and 0.001 M and so on. Since measuring small
volumes of solution is prone to error, a series of dilutions are performed in order to
gradually reduce the concentration of the solution from that of the stock solution.
Figure 3.1 Preparation of dilutions utilizing sterile nutrient broth blanks.
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3.1.2 Standard plate count
A viable cell is defined as a cell which is able to divide and form a population (or
colony). A viable cell count is usually done by diluting the original sample, plating
aliquots of the dilutions onto an appropriate culture medium, then incubating the plates
under proper conditions so that colonies are formed. After incubation, the colonies are
counted and, from the knowledge of the dilution used, the original number of viable
cells can be calculated. For accurate determination of the total number of viable cells, it
is critical that each colony comes from only one cell, so chains and clumps of cells must
be broken apart. However, since one is never sure that all such groups have been
broken apart, the total number of viable cells is usually reported as colony-forming units
(CFUs) rather than cell numbers. This method of enumeration is relatively easy to
perform and is much more sensitive than turbidimetric measurement. A major
disadvantage, however, is the time necessary for dilutions, platings and incubations, as
well as the time needed for media preparation.
The viable titer is determined by and counting colonies (expressed as Colony
Forming Units or CFUs) per volume plated and multiplying by the dilution factor. This
method only counts living cells because dead cells do not reproduce to form colonies.
Viable titer = (CFU/volume plated) x Dilution factor (3.1)
Later, isolated colonies will be examined for the types of cells and one will be
restreaked to obtain a pure culture. A pure culture is defined as the progeny from one
cell. Actually an axenic culture will be made from a clone (colony). Assuming that one
cell could have given rise to the colony, we call these pure cultures even though we
have no technical proof of that. Proof of pure culture involves showing that all the
colonies on the restreak are identical and Gram staining these to demonstrate all the
cells in the resulting colonies are identical and the same as those on the original plate.
3.2 OBJECTIVE

1. quantify cell viability using plate count method
2. determine the optical density of broth medium containing cell suspension
3. determine the dry cell weight
4. Use Hemacytometer to determine the total cell
3.3 MATERIAL
Refer to Bioprocess principles laboratory experiment 1.3.
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3.4 PROCEDURE
3.4.1 Determination of viable cells: Plate count method
1. Cautiously remove 8 sterile microcentrifuge tubes and fill them with 0.9 ml of
peptone water in each of the tubes. Label the tubes from 1 to 8.
2. Withdraw 0.1 ml of sample from the broth medium and pipette into tube no.1.
Shake well and remove 0.1 ml of sample from no. 1 and pipette into tube no.
2. Repeat this process up to tube no. 8.
3. Remove 0.1 ml of sample from tube no. 1 and pipette onto the surface of
agar. Sterilize a spreader with ethanol and flame and cool it at the side of
agar before spreading the pool of liquid sample.
4. Invert the plate and label.
5. Repeat the spreading process for tube no. 2 to tube no.8.
6. Incubate the agar plates at 37 C for 48 hours before counting.
3.4.2 Determination of optical density
1. Switch on the spectrophotometer and adjust the wavelength to 600 nm.

Allow it to warm-up for 15 minutes.
2. Fill an empty cuvette with 3ml of distilled water. Use it as a reference.
3. Fill an empty cuvette with 2.7 ml of distilled water. Remove 0.3 ml of sample
from broth medium and pipette into the cuvette to make up 3 ml.
4. Cover the opening with para-film and gently shake to mix.
5. Load the reference cuvette into the sample chamber and set to zero.
6. Load the cuvette containing sample and wait until a stable reading has been
achieved.
7. Record the reading.
3.4.3 Determination of dry cell weight
1. Weight a dry filter paper.

2. Load the filter paper into the filtration unit.
3. Pass 10 ml of broth medium through the filter.
4. Carefully remove the filter paper which contains the cell paste and dry in an
oven at 95 C for 24 hours.
5. Determine the dry cell weight of cells.
3.4.4 Determination of total cell
1. Total cell count can be determined by using a Hemacytometer.
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3.5 EXERIMENTAL RESULTS
Calculate the viable titer for each plating medium.
Dilution number Number of colony Cell number
3.6 QUESTIONS
1. Are all the bacteria counted by this procedure? Is this an overestimate or

underestimate of the actual number of viable bacteria in soil? What are
possible reasons that the CFU underestimates the number of bacteria?
2. Why can a standard curve be used only to estimate cell concentration of a
fresh culture of the same species grown in the same media? For instance
why cant it be used for other species, or same species grown in other media?
Why cant it be used on older cultures?
3. If the stock culture you were given was old, would you expect the
heterotrophic plate count to be higher or lower? Why?
4. Which method would you use (heterotrophic plate count or
spectrophotometer) if you had enumerate 200 different samples? Why?
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Appendix 01: Colonial Morphology

Both colonial and cellular morphology are characteristic of each species of bacteria and are
sometimes useful in the identification of an unknown microorganism. When a bacterium grows on a
solid agar surface, the number of cells increase until a visible mass of cells, called a colony, appears.
It is usually inferred that each colony arises from the division of a single cell. The most useful
culture characteristics are morphology, size and pigmentation of the colony. The figures presented
below illustrate some of the morphological characteristics of bacterial colonies and provide helpful
terminology for the description of colony morphology.

Figure A.1 Morphological characteristics of bacterial colonies.
19

CHEMICAL REACTION ENGINEERING LABORATORY EXPERIMENT 1
KINETIC STUDIES IN BATCH REACTOR

(Saponification of Ethyl Acetate)
1.0 INTRODUCTION
The design of a chemical reactor calls for prior information regarding (a)
the order of reaction and (b) the value of the reaction rate constant which gives
the reaction rate equation. This information is to be obtained invariantly through
laboratory- scale experiments conducted at constant temperature. The
equipment used is generally of batch type in case of homogeneous reaction, and
of flow type in case of the homogenous or heterogeneous reactions. The present
experiment is very typical of the kinetic method that is normally used for simple
homogeneous reaction.
This experiment can be divided into two parts: (i) to find the reaction
order and the rate constant at constant temperature, (ii) to determine the
activation energy for saponification of ethyl acetate. Reaction rates depend on
the composition and the temperature of the reaction mixture. Every chemical
reaction occurs at finite rate and, therefore, can potentially serve as the basis for
a chemical kinetic method of analysis. On the other hand, activation energy is
the energy barrier that has to be overcome for a reaction to happen.
The saponification id ethyl acetate in dilute aqueous solution can be

written as:
CH3COOC2H5 (l) + NaOH (l) = CH3COONa (l) + C2H5OH (l)

(1.1)
Because that the reaction saponification of Ethyl Acetate is a single

reaction, let A be NaOH and B be CH3COOC2H5, the rate equation can be defined
as
rA = k CA.CB
= k f (CA)
= k CAn
(1.2)
when one mole of A reacts with one mole of B (refer to eq. 1.1)
20

In determining the rate constant of the reaction, we can use the
differential method. The differential method of analysis deals with the differential
rate equation to be tested, evaluating all terms in the equation including the
derivative dCi/dt, and testing the goodness of fit of the equation with the
experiment. By plotting the graph concentration CA versus time, t, we can find
the slope or tangent at point for the suitably selected concentration values by
using mirror method. The slope of these points dCA/dt is the rates of reaction at
certain compositions. Then, plot the graph of log [dCA/dt] vs log [CA ].
rA = k CAn
log [-dCA/dt] = n log [CA ] + log k (1.3)
The graph of log [dCA/dt] vs log [CA] is a straight line. The rate constant,
k, and the order of the reaction, n can be directly obtained by finding the
intercepts and the slope from the graph of log [dCA/dt] versus log [CA].
The Arrhenius equation:
K=AeE/RT
(1.4)
where k = rate constant,
A = frequency factor
E = activation energy
R = Ideal gas constant
T = temperature
Rearranging the Arrhenius equation,
! !
ln =
! !
ln (1.5)
1.2 OBJECTIVE
1. To verify that the saponification of ethyl acetate in dilute aqueous

solution is a second order reaction.
2. To report the value of the reaction rate constant.
3. To find the activation energy of the reaction of saponification of ethyl
acetate in dilute aqueous solution
1.3 MATERIAL
1. Ethyl Acetate, pure 10ml

2. NaOH(aqueous,0.05M) 1 litre
3. HCL (aqueous, 0.1M) 500ml
4. Phenolphthalein indicator
5. Batch reactor (1 litre beaker)
6. a dozen conical flaks(250ml)
21

7. measuring pipette(5ml) and burettes 10ml and 25ml
8. pipettes
9. stop-watch
1.4 METHODOLOGY
1.4.1 Ascertain the order of reaction:
1. Place 1 litre of NaOH solution in a clean 1 litre beaker, which serves

as the reactor.
2. Transfer 10ml of 0.1M HCl solution into each of twelve conical
flasks, which are serially labeled.
3. Place the reactor on a magnetic stirrer and start stirring vigorously.
Keep a clean and dry 10ml pipette handy.
4. By means of a clean and dry measuring pipette, transfer exactly
10ml of ethyl acetate into the reactor and start the stop watch
simultaneously. The ethyl acetate forms a 0.1 M solution in the
reactor itself and saponification reaction is now on.
5. At the reaction times 1,2,3,4,5,6,7,8,9,10,11,12 minutes, pipette
10ml of the reaction solution mixture into each one of the labeled
conical flasks. Note: The stopwatch must not stop until all the
twelve samples are transfer.
6. Back-titrate the contents of twelve conical flasks with aqueous
NaOH from the burette using phenolphthalein as indicator
(appearance of first shade of pink color is the end point of titration).
7. The experiment should be carried out at room temperature.
Explanation: Note that when a sample of reaction mixture is

transferred into the conical flask containing HCl, the alkali in the
reaction mixture gets neutralized and so the saponification reaction
stops. Back-titration of the contents of this flask therefore helps in
determining the amount of unreacted NaOH present in the reaction
mixture at the time of its removal from the reactor. In other words,
the experiment helps to find the alkali concentration (CA) in the
reactor as a function of time (t).
1.4.2 Determining the activation energy of saponification:
Repeat steps 1-6 in section 1.4.1. In this experiment conduct the

experiment at 40 C and 50 C instead of the room temperature.
22

Record the following at (i) room temperature, (ii) 40 C, and (iii) 50 C.
Saponification reaction conducted at T = ____ C..

Readings of Burette Volume of
Time,t
(mL) NaOH used, V,
(min)
Vinitial Vfinal mL
1.6 QUESTIONS
1. Find the order of reaction and rate constant using

a. Differential method
b. Integral method
Compare the result of the two methods.
2. Evaluate the rate constants when temperature changes.
3. Using Arrhenius equation, calculate activation energy, E and frequency
or pre-exponential factor, k0. Justify the differences found in
comparison.
4. Perform the mass balance.
5. For each case, calculate the conversion. Draw the conversion vs time
graph.
23

CHEMICAL REACTION ENGINEERING LABORATORY EXPERIMENT 2
ISOTHERMAL CONTINUOUS STIRRED TANK REACTOR
2.0 INTRODUCTION
Continuous stirred tank reactors (CSTR) may consist of one or more

cylindrical tanks. Normally the tanks are arranged with their axes vertical,
although this is not essential. The stirring of the contents of each tank is affected
by an agitator which is mounted on a shaft inserted through the vessel lid. In
addition, the tank is fitted with the auxiliary equipment necessary to maintain the
desired reaction temperature and pressure conditions.
The well-stirred tank reactor is used almost exclusively for liquid phase
reactions, although instances of gaseous reactions have been reported. In
normal operation, a steady continuous feed of reactants is pumped into the
vessel and since there is usually negligible density change on reaction, an equal
volume of the reactor contents is displaced through an overflow pipe situated
near the top of the vessel.
Figure 2.1 Single continuous stirred tank reactor.
The stoichiometric equation of the saponification of sodium hydroxide (A)

and the ethyl acetate (E):
CH3COOC2H5 + NaOH CH3COONa + C2H5OH

(2.1)
The reactants, ethyl acetate and sodium hydroxide are introduced at the
respective volumetric feed rate of vA and vE.
24

The flow rate for input, vin = vA + vE
(2.2)
The ratio of the reactor volume, V, over the total inlet volume flow rate, v,
is called space time
Residence time, = V / v
(2.3)
And vin = vout
(2.4)
v
Volumetric flow rate of sodium hydroxide = A
time
(2.5)
v
Volumetric flow rate of Ethyl Acetate = E
time
(2.6)
v + vA
Total volumetric flow rate = E
time
(2.7)
Volume of the liquid in the reaction vessel is constant at V.
At the beginning of the experiment, moles of Ester and Alkali changes

with respect to time, the equation is given by:
d [n e ] d [C E ]
=V = vin (CE) vout (CE) VkCECA
dt dt
(2.8)
The CE and CA at the exit stream is taken as those exiting in the vessel
using the result from the calculation of the equation 2.3, 2.4, and 2.8 we get,
d CE (CE )in (CE ) out

= KC E C A
dt
(2.9)
dCA (C A )in (CE ) out
= KCE C A
dt
(2.10a)
d [C E ] d [C A ]
= =0
dt dt
(2.11)
25

After a certain passage of time CA and CE reaches constant value, CA and
CE.
CE = (CE)in - k (CE)
(2.12)
CA = (CA)in - k (CE )(CA)
(2.12a)
Eliminating (CA) from (2.12) and (2.12a),

k (CE )2 + (CE) {1 k [CE,in CA,in]} (CE)in = 0
(2.13)
For the special case (CA)in = (CE)in
k (CE)2 + (CE) (CE)in = 0

(2.14a)
OR k (CA)2 + (CA) (CA)in = 0 (2.14b)
The conversion of the ethyl acetate and sodium hydroxide can be

evaluated from equation (2.14a) or the equation (2.14b) to determine the
conversion if rate constant , k, value is known or to determine the k value if
experimental conversion is known.
We can write the equation (2.14b) to
k (CA) 2 + (CA) (CAo) = 0

(2.14c)
where CA is the concentration of the Sodium Hydroxide which had reacted

with the ethyl acetate in the continuous stirred tank reactor.
CAO is the concentration of the Sodium Hydroxide which before
reacted with the Ethyl Acetate.
is the space-time of experiment.
Also, for CSTR,

! !! (!!! !!! )/!!! (!!! !!! )
! !!
=
!!!
=
!!!!
therefore = !!!!
(2.15)
Before using equation (2.14c) to find the rate constant, k, we need to find
the CA, CAo, and the .
Vmixture = Vtotal VNaOH(exact) VHCl (2.16)
!"#$%& !" !"#$%&! !!
= =
!!! !"!#$ !"#$%&'()* !"#$ !"#$ !! !!!
(2.3a)
26

!"#$%&'( !" !"#$ !" !""# (!"#$%&'()* !"#$ !"#$ !" !"#$) ! !! ! !
!! =
!"#$% !"#$%&'()* !"#$ !"#$
=
!! !!!
(2.17)
!!" !!!"#$ !"#$% (!!!! )!"#$

! = =
!!"#$%&' !!"#$%&'
(2.18)
Substitute the equation (2.3), (2.17) and (2.18) into the equation (2.14c).
Then we can find the rate constant, k, by solving this equation. We find the
average of the CAs which we find before, then substitute that value of the
average of CA into the equation (2.14c).
2.2 OBJECTIVE
To determine the rate constant for the given reaction at ambient temperature.
2.3 MATERIAL AND APPARATUS
1. Sodium hydroxide, NaOH (aqueous, 0.05M, reactor)

2. Ethyl acetate (0.05 M, reactor)
3. Sodium Hydroxide, NaOH (aqueous, 0.1M, titration)
4. Hydrochloride acid, HCl (aqueous, 0.1 M, titration)
5. Distilled water (H2O)
6. Phenolphthalein indicator
7. Isothermal Continuous Stirred Tank Reactor
8. 7 conical flasks ( 250 ml)
9. Measuring pipette ( 5ml )
10. Burettes 50 cm
11. Water vessel
12. Digital stop watch
13. Magnetic stirrer
14. Measuring calibrated cylinder (1000mL)
2.4 METHODOLOGY OF OPEN REFLUX METHOD
1. Fill the reservoir with the 0.1 M of Sodium Hydroxide, NaOH, and 0.1
M strength of Ethyl Acetate.
2. Start the stirrer.
3. Adjust the flow rates of the Ethyl Acetate and the Sodium Hydroxide to
the required level, 2.5L/hr.
4. Once the flow rates are adjusted to the known levels, introduce the
streams into the vessel.
27

5. Collect the sample for the time, t = 0, 1, 2, 4, 6, 8, 10, 12, 16 and 30th
minutes by pipette each of them out into the prepared and labeled
conical flasks.
Note: Do not stop the stop-clock until all the twelve samples are
transferred.
6. The contents which in the vessel are stirred and the reaction is allowed
to complete until the Ethyl Acetate has reacted.
7. Once a constant value of concentration is reached, stop the
experiment.
8. Backtitrate the contents of the seven conical flasks with aqueous
NaOH from the burette by using phenolphthalein as indicator.
Note: The end point of the titration is indicated by the appearance of

the first shade of pink colour.
Find the values of (CA) and tabulate the observations.
Volumetric flow rate of Sodium Hydroxide, vA= _____ L/hr

Volumetric flow rate of Ethyl Acetate, vE = _____ L/hr
Total volumetric flow rate, (vA + vE) = _____ L/hr
Total reactor volume, Vr = _____ mL
Time, t VNaOH, initial VNaOH, final

V(exact) (ml) CA (mol/L)
(min) (ml) (ml)
2.5 QUESTIONS
1. What is the rate constant for CSTR?
28

2. Perform mass balance for the saponification of ethyl acetate and
sodium hydroxide in isothermal CSTR system.
3. Calculate the substances conversion. Draw the conversion vs time
graph.
4. Compare the conversion in a batch reactor with the conversion in the
CSTRs for the same reaction time. (show the graph)
5. In terms of conversion, why and when will you choose to use CSTR for
your production?
6. Formulate a model for the system of CSTRs which should describe the
change in concentration due to changes in inlet flow rate.
29

SEPARATION PROCESS LABORATORY EXPERIMENT 1
DETERMINATION OF ADSORPTION ISOTHERM FOR CITRIC ACID
1.0 INTRODUCTION
In this experiment adsorption isotherms operation is introduced. It is the

equilibrium relationship between concentration in liquid phase and concentration
in substance particles adsorption in certain temperature.
The term adsorption is used to describe the attachment of gases or
dissolved substances to the surface of a solid. The quantity of absorbed
substances is a function of the type of system investigated and the partial
pressure and the concentration of the substance in lab at a constant temperature.
1.1.1 Equilibrium relation for adsorbents: adsorption isotherms
Attractive forces between the exposed particles cause gases or dissolved

substances B attach themselves (absorbate) reversibly to the surfaces of solid
phases A (adsorbing agents) (adsorption).
A + B ABads (1.1)
Depending on the strength of the interactions one can differentiate

physiosorption (weak Van der Waals forces, very low adsorption enthalpies, a
number of superimposed adsorption layers) and chemisorptions (chemical bonds,
substantial adsorption enthalpies, monolayer of the adsorbed substance).
The extent of adsorption is expressed by the quality of adsorbent nB,ads
referred to the surfaces area of or the mass mA of the adsorbing agent.
n
= B ,ads , where =adsorption molality.
mA
(1.2)
In cases of monolayer formation, the quotient of the real ( ) and the
maximum adsorption molality is defined as degree of coverage .

=
max
(1.3)
The correlation between the adsorption molality and the free equilibrium
concentration of B at constant temperature is described by great numbers of
adsorption isotherms of limited validity.
30

dC B
From kinetic consideration of the adsorption rate ,
dt
dC B
= k ads c B (1 ) k des
dt
(1.4)
where C B = concentration of B in the solution
k ads, k des = rate constant of the adsorption and desorption,
respectively.
dC B
For the case of an established adsorption, ( = 0 ) with definition (1.3),
dt
Langmuirs adsorption isotherm follows:
max KC B ,eq
=
1 + KC B ,eq
(1.5)
Where C B = equilibrium concentration of B in solution.
k
K= ads = constant.
k des
This simplified for very low equilibrium concentrations ( KC B ,eq < 1 ) to:-
= max KC B ,eq = K ' C B ,eq
(1.5a)
Thus, the adsorption molality should be linearly correlated with C B ,eq
(Henrys isotherm).
In contrast, the adsorption molality tends toward a concentration in
depending limiting value max at very high equilibrium concentrations ( KC B , eq >1)
= max
(1.5b)
In the region of validity of the isotherm (4) the correlation between
adsorption molality and equilibrium concentration can be linearized by simple
rearranging.
1 1 1 1
= + (1.5c)
MAX K C B,eq max
1
Consequently, the graph plots of as a function of the reciprocal

1 1
equilibrium concentration gives a straight line with slope and the
C B,eq max k
Freundlichs empirically determined adsorption isotherm for many systems
= C B,eq
(1.6)
where , = system-dependent constants
31

whose logarithmic expression is as followed:
lg = lg C B ,eq + lg
(1.6a)
This form allows a simple graphic evaluation and thus the determination
of the constants and .
1
Therefore, plot the expressions of the adsorption molality ( , , lg ),

which correspond to the relationships (1.5a), (1.5c) and (1.6a), as functions of
1
the corresponding equilibrium concentrations ( C B,eq , , lg(C B,eq ) ). In the
C B,eq
present case, all results are straight line.
The initial ( C B ,O ) and equilibrium concentrations ( C B ,eq ), which are
required for the evaluation, can be calculated from the volume of the volumetric
flack Vk=250ml and the volumes of NaOH of known concentration, CNaOH,1 of
citric acid solution when the tribacity of the absorbed material (citric acids) is
considered by applying the following relationships.
c V V
cB,O = NaOH NaOH ,0 st (1.7a)
3V0VK
c NaOHVNaOH ,1
c B ,eq = (1.7b)
3V1
From this, the adsorption molality can be determined from following
correlation, which is derived from equation (1.2).
n (C B ,0 C B ,eq ) Vs
= B ,ads = (1.8)
mA mA
Where Vk = 250mL=0.25L
VS = 100mL, system volume = 0.1L
MA=3 mass of adsorbing agent = 0.003kg
1.2 OBJECTIVE
1. To determine whether isotherm adsorption occur for liquid-gases

adsorption systems.
2. To estimate the equilibrium concentration balance of citric acid after
stirring the solution with different starting concentration and activated
carbon.
3. To investigate which isotherm rule (Henry, Freundlich or Langmuir
isotherm adsorption) that this adsorption system is valid.
1.3 MATERIAL
32

No. Chemicals and apparatus Units
1 Retort stand 1
2 Burette clamp 1
3 Magnetic stirrer with stirrer bar 3
4 Laboratory balance 1
5 Burette, 50 ml 1
6 Volumetric flask, 500 ml 4
7 Volumetric flask, 250 ml 6
8 Volumetric pipette, 10 ml 1
12 Graduated pipette, 50 ml 1
13 Erlenmeyer flask, 250 ml 6
14 Pipettor 1
15 Beaker, 100 ml 1
16 Funnel 1
17 Circular filter paper 1
18 Stopper 6
19 Citric acid, 250g
20 NaOH, 1M
21 Phenolphthalein indicator, 100 ml
22 Activated carbon, granulated,
500 g
23 Distilled water, 5 litres
1.4 PROCEDURE
1. Prepare 2 sets of 0.5 M citric acid with 52.54 g dissolved in distilled

water. Fill them into the 500 mL volumetric flasks and top up with
distilled water until calibration mark.
2. Prepare 0.5 M NaOH and fill it into another 500 ml volumetric flask.
Top up with distilled water until calibration mark.
3. Pipette each volume of the 0.5 M citric acid (Vst) listed in table 1 into a
separate clean 250 ml volumetric flask and top off to calibration mark.
4. Add 3.00g activated carbon into a clean Erlenmeyer flask. Repeat for
other five Erlenmeyer flask.
5. Pour 100 ml each of the six concentration series solution into separate
250 ml Erlenmeyer flasks containing the activated carbon. After that,
seal the flask mount and set onto the magnetic stirrer.
33

6. Stir the content of the flasks vigorously for 30 minutes and filter the
suspensions stirring.
7. Add the filtrate solution with 3 drops of phenolphthalein.
8. Titrate the respective sample volumes (V1) given in the table 1 with
0.5 M NaOH and in similar manner, repeat the titration process with 10
ml of the 0.5 M solution of citric acid (Vo).
9. Repeat the titration process.
Table 1.1: Volumes of stock solution for the preparation of the

concentration series (Vst) and the sample volumes after filtration (V1)
for the determination of the equilibrium concentration of citric acid.
Vst (ml) V1 (ml)

250.0 10.0
187.5 10.0
125.0 25.0
62.5 25.0
25.0 50.0
12.5 50.0
Record the following. Include any relevant observation.
Vo = __________ ml
VNaOH,o= ________ ml
Burette Readings (ml) Volume of

Vst (ml) V1 (ml)
Initial Final NaOH
250.0 10.0
187.5 10.0
125.0 25.0
62.5 25.0
25.0 50.0
12.5 50.0
1.6 QUESTIONS
1. Find CB,0, CB,eq and . Then find the respective logarithms and
reciprocals for CB,eq and . Tabulate the data.
34

2. Plot the correlation between the of the equilibrium concentration CB,eq
and the adsorption molatity using (i) Henry, (ii) Freundlich, and (iii)
Langmuir isotherm adsorption theory.
3. Determine the slope of the regression lines of (i), (ii) and (iii).
4. Determine the adsorption constant (k) and the maximal adsorbed
substance amount of citric acid per gram of activated carbon.
5. What is the major difference in Henry, Freundlich and Langmuir
isotherm adsorption? Under what circumstances that they are
applicable/not applicable?
6. Discuss and conclude your findings.
35

SOLID-LIQUID EXTRACTION
2.0 INTRODUCTION
Solid-liquid extraction (leaching) is the process of removing a solute or

solutes from a solid by using of liquid solvent. Leaching is widely used in
chemical industries where mechanical and thermal methods of separations are
not possible or practical. Extraction of sugar from sugar beets, oil from oil
bearing seeds, production of a concentrated solution of a valuable solid material
are typical industrial examples of leaching. Leaching process consists of three
parts: (i) diffusion of the solvent through the pores of the solid, (ii) the diffused
solvent dissolves the solutes (i.e. transfer the solute to the liquid phase), (iii)
transfer of the solution from porous solid to the main bulk of the solution.
The extraction of the corn/soybean/nut oil from the maize/soybean/nuts
using the extracting solvent, hexane, is carried out in this experiment. At first 2
kg of maize (corn)/soybean/nuts is poured into extraction bag, followed by
putting the bag into the extraction chamber. The solvent hexane is poured into
the solvent charge. The experiment started once the extractor is stabilized. Along
the experiment, samples weight from valve 2 and 3 will be taken.
Solid-liquid extraction by percolation of solvent downward through a fixed

bed of solid is a common method of operation. It can be used in both simple
batch system or in a semi-continuous system such as the Bollman extractor. In
this experiment, solvent and solid are fixed and it is left to determine how the
extraction proceeds with time.
The efficiency of extraction varies with the contact between solid and
solvent. For cellular solids case, sufficient contact would occur to allow
penetration of solvent into the cells and diffusion of solute out again. At very
high flow rate, contact will be limited and extraction efficiency will be impaired.
At very long contact times, Murphee efficiency will be high as the system will
have the opportunity to approach equilibrium, but the overall operation of the
plant would be uneconomic as the throughput would be very low. An optimum
can occur between these two extremes.
The rate of extraction depends on many factors such as type of solvents,
physical state of solids, and the nature contact between solvent and solid
(contact time, etc). In the case of a free-draining solid, i.e. no flooding of the
extraction chamber, the contact time should be independent of the flow rate
36

since it will always be just the time taken for solvent to percolate through the
column. Therefore, reducing the flow rate (i.e. the rate of solvent input), it is not
expect to alter the efficiency, but is expected to reduce throughput (i.e. the
amount extracted per unit time).
For this experiment, the composition of the effluent can be calculated by
using equation (2.1);
1 ( / p s ) (2.1)
x=
( / o ) 1
Where =density of mixture
s=density of hexane
0=density of soya bean
As for amount of solute content in effluent, we can get it by plotting the

graph of density of effluent vs time. Then we calculate the area under the curve.
The value of the area will be multiply by the solvent flow rate to get the amount
of solvent.
It is expected the profile of extraction to be exponential with time but the
practical systems has to be done to confirm the model. The amount of effluent
should be inversely proportional to the reboiler.
2.2 OBJECTIVE
1. To demonstrate the characteristics of an extraction by downward

percolation through a fixed bed at two solvents flow rates.
2. To compare methods for estimating the amount of material extracted (to
compare the amount of oil that is extracted from the corns).
2.3 EQUIPMENT AND MATERIAL
1. QVF Teaching system: CTS 6 Solid Liquid Extractor

2. Maize (Corn) / Soy bean / nuts
3. Hexane
4. Erlenmeyer flasks
5. Weighting machine
6. Stop watch
37

2.6 PROCEDURE
CAUTION: Read all operating instructions FIRST to ensure that you fully
understand all safety precautions and operating instructions. DO NOT
start up without the permission from your supervisor/lab assistants.
1. Before the extraction is started, weight 2 kg of blended corn and pour it

into the extraction bag.
2. Place the extraction bag into the extraction chamber.
3. Fill the solvent, approximately 28 litre of hexane, into the solvent
charge. Take down the volume of the hexane.
4. 15 minutes after solvent has begun to pass through the product cooler,
measure the flow rate, FI1. Adjust the heat input to give a flow rate of
approximately 500 ml/min.
5. When a steady value for FI 1 has been determined, record its value and
the values indicated by TI 1 and PI 4 [steam heating].
6. Carefully take a sample from the reboiler.
7. Close valve V.5 and begin the solvent transfer to the extraction vessel.
8. Take a sample of the first effluent.
9. Record the collection of this sample as T=0.
10. Sample reboiler contents and effluent every 2 minutes for the first 10
minutes. Then every 5 minutes until 30 minutes have elapsed then at
T= 45, 60, 90 minutes.
11. Record the final temperature indicated by TI 1.
12. Lastly, shut down the extractor and leave it to cool.
13. Record the final volume in the reboiler.
14. Determine the density for each of the samples taken.
Record the following data.
Weight of solid charge = ____ kg

Solvent charge = ____ L
FI 1 = ____ ml/min
TI.1 (Initial) = ____ 0C
TI 1 (Final) = ____ 0C
TI.2 (Initial) = ____ 0C
TI 2 (Final) = ____ 0C
PI 4 = ____ bar
FI 2 = ____ L/min
FI 3 = ____ L/min
Final volume in reboiler= ____ L (at ____ 0C)
38

Weight of density bottle (empty) = _____ g
Volume of the sample = ____ cm3
Time Weight of sample for Weight of sample for

(min) sample point V2 (g) sample point V3 (g)
0
2
4
6
8
10
15
20
25
30
45
60
90
120
2.6 QUESTIONS
1. Draw a labeled diagram of the equipment.

2. Tabulate the density (kg dm-3) and composition (kg solute/kg solvent)
of V2 and V3 at their respective time.
3. Plot a calibration curve of density vs. solute composition.
4. Plot a graph of
a. density of reboiler content vs time
b. density of effluent vs time
5. Use the calibration graph to convert the data for density of effluent to
effluent composition [kg solute/kg solvent]. Alternatively calculate
each composition.
6. Use the transformed data to plot a graph of effluent composition vs
time.
7. Use this graph to estimate the amount of solute extracted during the
course of the experiment.
8. Using the final density of the reboiler contents and the corrected final
volume, calculate the amount of solute in the reboiler.
9. Plot a graph of loge [composition] vs time for the effluent.
10. Why is the effluent composition not zero at time zero?
39

11. Explain briefly why the effluent density increased during the course of
the experiment while the density of the reboiler contents decreased.
12. Explain why the change in density for the reboiler contents appears to
be less in rate and magnitude than for the effluent.
13. Comment on the agreement or otherwise between the estimates of the
amount of oil extracted obtained:
a. by composition-time curve or reboiler composition.
b. between the two extractions.
14. Comment on the shape of the composition versus time cuves.
15. Comment on the shape of the loge [Composition] versus time graphs.
16. Suggest the efficiency of the extraction.
17. What are the effects of contact time on the extraction of corn oil?
18. Carry out mass balance analysis.
40

BATCH DISTILLATION ON A BINARY MIXTURE

3.0 INTRODUCTION
Distillation is the main process involved in petroleum refining. The products of oil
processing are usually grouped into three categories: light distillates (liquefied-
petroleum gas, gasoline, naphtha), middle distillates (kerosene and diesel), and heavy
distillates or residuum category (fuel oil, lubricating oils, wax and tar).
The fractioning column of LS-32205 consists of equally-spaced plates using a
liquid downcomer which is important in determining the stability and transfer efficiency
of the system. As the downcomer area increases, the area available for gas dispersers
(bubble cap, perforations) decreases.
The most common gas disperser for cross-flow plates has been the bubble-cap.
This device has a built-in seal which prevents liquid drainage at low gas flow rates. The
plate-column capacity is determined by the maximum allowable capacity to avoid
increasing pressure drop which leads to flooding and on the other hand, the minimum
allowable capacity to determine the effectiveness of dispersion and contacting of the
phases.
3.1.1 Batch distillation
Batch distillation is the process of separating a specific quantity of a liquid

mixture into products, which is extensively used in the laboratory and in small
production units. A batch of liquid is charged to the reboiler and the system is first
brought to uniform operation under total reflux. Then a portion of the overhead product
is continuously withdrawn in accordance with the established reflux policy. The column
operates as an enriching section. The progress of batch distillation can be controlled
either by (i) constant reflux, varying overhead composition, or (ii) constant overhead
composition, varying reflux.
3.1.2 Useful calculations for binary distillation
3.1.2.1 Convert data to mole fraction
!"
Number of mol of component, = (3.1)
!"
where V is the volume of component (cm3), is the density of component

(g/cm3), Mw is the molecular weight of the component (g/mol).
41

Mole fraction is the percentage of component X exits in the mixture in unit mol.
!
% = 100% (3.2)
! + !
Taking the example of 10 V/V% ethanol in ethanol-water mixture,
!"!"! !.!"#!/!"!
Number of mol of 10 ml ethanol, = = 0.1715 mol
!"!/!"#
!"!"! !!/!"!
Number of mol of 90 ml distilled water = = 5 mol
!"!/!"#
!.!"!#!"#
Mol fraction of ethanol in mixture, !"!!"#$ = 100% = 3.312%
!.!"!#!"#!!!"#
3.1.2.2 McCabe-Thiele method (example calculation steps)
For a given set of results as followed,

Determine the ethanol composition with the refractive index
Composition (mol)
Feed (XF) 0.2792
Distillate (XD) 0.45
Bottom (XB) 0.163
Reflux ratio,

= (3.3)

Ratio value of q obtained to determine the q line equation which required to
draw the q line on the McCabe-Thiele diagram.
!,!"# + ! (3.4)
=
!
where CP is the heat capacity of the mixture (J/mol.K), T is the temperature
difference between dew point of feed at feed input ( C), F is the latent heat of
vaporization of the substance.
Given the heat capacity of ethanol and water,

CP,water=158.8 KJ/kmol. C; CP,ethanol=75.4 KJ/kmol. C
Heat capacity of mixture is,
158.8 74.5
!,!"# = 0.2792 + 0.7208 = 98.685/

From T-x-y diagram the feed dew point is 82.5 C for a feed of xF=0.24 (mol
fraction). Feed input is given at T=80 C, hence,
= 82.5 80 = 2.5
Latent heat of water is 40.656 KJ/mol, while the latent heat of ethanol is 38.58
KJ/mol. Hence the latent heat of feed is,
KJ KJ
! = 0.279238.58 + 0.720840.656 = 40.0710! /
kmol kmol
Therefore,
98.685
2.5 + 40.0710! /
= = 7.157
40.0710! /
42

The q-line equation is given as
1 (3.5)
=
1 1 !
Top operating line (TOL) equation is given as
1
= + (3.6)
+1 +1 !
3.1.3 Chemical safety and precaution
Ethanol is volatile and will evaporate quickly. Avoid exposing this chemical to the
atmosphere for extended period of time. Work in a well-ventilated area. Dispose all
unused chemicals in an appropriate manner after the experiment. Under no
circumstances should the chemicals be allowed to flow into the main drains. For details
refer to the MSDS of each chemical.
3.2 OBJECTIVES AND AIM
Objectives:
1. To perform batch distillation on a binary mixture using a bubble cap
distillation column.
2. To investigate the effects of a constant reflux ratio on product composition
at top and bottom.
Aim:
1. To calibrate a refractive index graph for binary mixture
2. To draw temperature profile for the column
3. To calculate tray efficiency using McCabe Thieles graphical method
4. To study a bubble cap tray in action
3.3 EQUIPMENT AND MATERIAL
1. LS-32205 Bubble Cap Distillation Unit

2. binary mixture (ethanol and water)
3. Refractometer
4. 200 ml ethanol (for refractive index calibration)
5. Distilled water (for refractive index calibration)
6. Syringe
7. Stir rod
8. Beakers
43

Figure 3.1 The schematic diagram for bubble cap distillation unit.
3.4 PROCEDURE
3.4.1 Obtaining refractive index calibration data
1. Using a syringe, draw 10 ml component A (distilled water) and pour into a

100 ml beaker (A).
2. Switch on the refractometer.
3. Drop a few drops of distilled water onto the sampling area such that the
refractometer lens is well immersed.
4. Measure the refractive index of component A and jolt down the refractive
index shown on the refractometer.
5. Pour away the component A (distilled water) on the refractometer. Use a
clean dry cloth, wipe the sampling area.
44

6. Using a clean syringe, draw 10 ml component B (ethanol) into previous
beaker (A). Stir the mixture using a stir rod.
7. Drop a few drops of mixture from beaker (A) onto the sampling area such
that the refractometer lens is well immersed. Measure and record the
refractive index.
8. Pour away the sample. Use a clean dry cloth, wipe the sampling area.
9. Repeat 6-8 for another 9 times until the mixture contain up to 90 ml of
component B (ethanol).
10. Upon completing step 9, repeat step 1-9 by switching to 10 ml of component
B (ethanol) in beaker (B) and mix with up to 90 ml component A (distilled
water) intermittently to get the refractive index.
11. Record the refractive index according to the concentrations as shown in
Table 3.1, section 3.5.
3.4.2 Experimental set up and checking
1. Identify the components of LS-32205 bubble cap distillation unit:

a. Bubble cap distillation column
b. Reflux unit (Reflux divider and reflux valve)
c. Reboiler/evaporator and two condenser units
d. Sampling ports
e. Feed tank and feed valves
f. Dosing pump
g. Top and bottom product tank
h. Water jet vacuum with pressure gauge
i. Temperature sensors and digital temperature display
j. Control Panel
2. Check that the binary mixture to be distillated has been poured into the
reboiler.
3. Attach the inlet hose from the condenser unit to a cold water supply and the
outlet hose to a drain.
4. Ensure that all hand valves on the unit are closed except the one on the
water inlet valve V9.
5. Connect the unit to a 415 VAC 3 phase 50 Hz power supply and switch on
the power supply.
3.4.3 Constant reflux
1. Collect 10 ml sample in the reboiler to test the refractive index.

2. Switch ON the main power switches on the unit.
3. Close all the valves on this unit.
4. Open valves V8 and V11 if initial vacuum suction is required. Keep an eye on
the pressure gauge, close V8 and V11 when the vacuum pressure reaches
0.2 bar.
5. Open valve V9. Start the cooling water supply.
NOTE: Do not open valves v8 and v11 when the experiment is running.
45

6. Turn on the reboiler heater ON/OFF switch located on the Control Panel.
7. Set the power of the reboiler to maximum using the power regulator.
8. Set the temperature of reboiler, T15 to 95 C by pressing UP and DOWN
button.
NOTE: High power setting is required in order to supply sufficient energy to

warm the system up in the initial startup, for around 20 minutes.
9. Turn on the reflux switch and set the reflux to 100%. The reflux controller
indicates percentage of condensate returning to the column. Open valve V12
on the Top product tank.
10. Set the feed flow to zero since this is a batch process.
11. Wait for the vapor to condense in the glass condenser.
NOTE: Please take note of the temperature change on T11, when T11 reaches
about 80 C, it will begin to show condensate collection.
12. Once the top product appears in the divider vessel, leave the column at total
reflux (100%) for another 10 minutes to allow the system to stabilize.
13. Record all the temperatures as the initial experiment point.
14. Change the reflux control to a desired value (80%).
15. At every intervals of 10 minutes, record all the temperatures and draw 10 ml
sample each from the top product tank (DV3) and the reboiler tank (DV1).
Measure and note down the refractive index of each sample at constant room
temperature. To quickly cool down the reboilers sample, place it in a beaker
filled with cold water.
16. Take 6 set of readings. Then, empty the top product vessel and measure its
volume to get the product output rate of the system.
17. Tabulate all the data as shown in table 3.2 in section 3.5.
18. All alcohol samples can be reused after the experiment, as long as they are
uncontaminated.
3.5 EXERIMENTAL RESULTS
3.5.1 Refractive index calibration
Record the refractive index from section 3.4.1 into the table below.
Table 3.1 Refractive index calibration table

A (ml) B (ml) Mol fraction A Mol fraction B Refractive index
0 10
10 0
10 10
20 10
30 10
40 10
50 10
46

60 10
70 10
80 10
90 10
10 20
10 30
10 40
10 50
10 60
10 70
10 80
10 90
Plot the refractive index curve for ethanol (graph of refractive index against mol
fraction ethanol). The curve obtained is the ethanol water refractive index
calibration.
3.5.2 Effects of a constant reflux ratio on product composition
Tabulate all data for batch distillation (constant reflux) as shown in the table below.
Operating condition:
Binary mixture of ________ / _________ (____V/V %)
Feeds refractive index = ___________@_______ C
Heater Set temperature = __________ C
Heater Power = 2kW
Table 3.2 Batch distillation (constant reflux) result tabulate table

Reflux (%) 100% Constant reflux: _____%
T1 ( C)
T2 ( C)
T3 ( C)
T4 ( C)
T5 ( C)
T6 ( C)
T7 ( C)
T8 ( C)
T9 ( C)
T10 ( C)
T11 ( C)
T12 ( C)
T13 ( C)
T14 ( C)
T15 ( C)
47

Top product
Refractive index
Composition(mol%)
Bottom product
Refractive index
Composition(mol%)
i. Plot the mol percent product against time graph for top and bottom products.
ii. Plot the T-x-y diagram of ethanol water for the temperature composition
relationship.
iii. Plot the VLE data for ethanol water binary mixture using McCabe Thiele
diagram. You can refer the calculation steps in section 3.1.2. Calculate the
number of steps from top product to reach the bottom product composition
which indicates the number of equilibrium stages.
Discuss your findings.
3.6 QUESTIONS
1. Discuss the difference of a bubble cap distillation column compared to sieve

plate and valve plate columns.
2. When should/shouldnt batch distillation been applied?
3. What is the definition of an equilibrium stage?
4. Where in the column is the temperature highest?
5. What are the assumptions behind McCabe-Thieles graphical method?
Determine the overall column efficiency.
6. How does a constant reflux ratio affect the top and bottom product
composition? How (do you think) would the reboiler heating and feed
condition influent the degree of separation?
7. Can you perform the energy balance and find the heat loss for the distillation
column?
8. What is (are) affecting the performance of the distillation column? Suggest
ways to improve the performance of a distillation column.
48

PROCESS CONTROL LABORATORY EXPERIMENT 1
LIQUID/WATER FLOW (WF) CONTROL
1.0 INTRODUCTION
In this experiment, we use water to simulate a liquid phase flow process.

The aim of the experiment is to control the flow rate by using a PID single loop
flow control. This experiment is to observe and obtain the response of the
controlled variable, PV when (i) the process is run under an open loop condition
by changing the manipulated variable values (% of valve opening), (ii) the set
point is increased and decreased using a step function at a fixed PID in a closed
loop. The next objective is to observe the change of output when a different
value of PID is used for the same set point input.
1.1.1 PID Theories
The Proportional Integral (PI) control

!
! (1.1)
= ! + + !
! !
where KC = gain, I = integral time, and PS = constant.
Taking Laplace transform and rearranging, we get the following transfer

function,
() 1 (1.2)
= ! (1 + )
() !
The Proportional Derivative (PD) control
(1.3)
= ! + ! ! + !

where KC = gain, D = detention time, and pS = constant.

function,
(1.4)
49

()
= ! (1 + ! )
()
The Proportional Integral Derivative (PID) control

!
! (1.5)
= ! + + ! ! + !
! !
Taking Laplace transform and rearranging,
() 1 (1.6)
= ! (1 + + ! )
() !
In proportional mode, a smooth, linear relationship exists between the

controller output and the error. For the two-position mode had the
controller output of either 100% or 0%, over some range of errors about
the setpoint, each value of error has a unique value of controller output in
one-to-one correspondence. The range of error to cover 0% to 100%
controller output is called the proportional band, because the one-to-one
correspondence exists only for errors in this range. The proportional band
(PB) of a proportional controller depends on the inverse of the gain.
100 (1.7)
=
!
1.1.2 Model WF922 description
The process consists of two pipelines of 1 each, circulating water around

a tank T21 as follows:-
NORMAL OPERATION
1. Pipeline Controlled Flow (CF): P21-FT21-CF1-FCV21-T21

- Water is pumped from tank T21 by pump P21 via a Vortex
Flowmeter FT21, a manual valve CF1, a Control Valve FCV21 and
back into tank T21.
- Manual valves CF2 and WF2 are shut.
- The interconnecting manual valve MVX and the pump P20
discharge manual discharge valve MF are shut.
- This is the Controlled Flow (CF) stream.
2. Pipeline Wild Flow (WF): P22A/B-FI22-FE22/FT22-WF1-T21

- Water is pumped from tank T21 by one or two pumps P22A, P22B,
via a Variable Area Flowmeter (Rotameter) FI22, an orifice plate
50

FE22 with a Differential Pressure (DP) Flow Transmitter FT22, a
manual valve WF1 and back into tank T21.
- Manual valves CF2 and WF2, the interconnecting manual valve MVX
and the manual discharge valve MF of pump P20 are all shut.
- This is the Wild Flow (WF) stream.
- NOTE THAT PUMP P20 IS OFF AND NOT USED.
For Ratio Control, Wild Flow (WF) x R = Controlled Flow (CF)

R is the instrument ratio factor
there must be a Control Valve at the Controlled Flow (CF) pipeline.
there must be flowmeters with linearised signal transmission (e.g. 4-
20 mA representing the flowrates expressed in the same
engineering units) at both the WF and the CF pipelines.
the instantaneous WF is multiplied by the instrument ratio factor R
(i.e. R x WF) and this signal becomes the instantaneous remote
setpoint (SV=R x WF) cascaded to the PID flow controller controlling
flow at the CF pipeline. The CF pipeline must have a closed loop in
flow control, with its PV, SV and MV. In this case the PV is the
variable to be controlled i.e. PV = CF, SV is R x WF and MV is the
controller output to the control valve. One WF pipeline can set more
than one CF pipeline but each CF pipeline must have its own flow
closed loop with its own PID flow controller and control valve. There
is no PID controller for the WF pipeline.
INTERCHANGE OPERATION
If manual valves CF2/WF2 are opened but CF1/WF1 are shut, the flow
pipeline circuits are interchanged and become:-
i. Pipeline Controlled Flow (CF): P22A/B-FI22-FE22/FT22-CF2-FCV21-

T21
ii. Pipeline Wild Flow (WF): P21-FT21-WF2-T21
However, note that the flowmeter signals to the PID flow controller
(FIC21) must also be interchanged so that FE22/FT22 becomes the CF
which is the process measurement (PV) signal to the PID flow controller
(FIC21), whilst FT21 becomes the WF to be multiplied by the
instrument ratio factor R, setting the setpoint (SV) of FIC21.
The Control Valve FCV21 is always at the CF pipeline, so that there is a
closed flow loop around the CF pipeline.
To interchange the flowmeter signals to FIC21, an external selector
switch is provided at the panel and its two position are:
51

1: FT21=PV=CF
2: FT22=PV=CF
For FT22 to be the CF, select Position 2: FT22=PV=CF.
The NORMAL OPERATION is for FT21 to be the Controlled Flow CF. Thus
CF1/WF1 Open, CF2/WF2 Shut
Controller Selector Switch Position 1: FT21=PV=CF
The recorder FR21 displays: Red pen/Channel 1 : FT21
Green pen/Channel 2 : FT22
The CF pipeline must have a closed loop in flow control, with its PV, SV
and MV. In this case the PV is the variable to be controlled i.e. PV = CF,
SV is R x WF and MV is the controller output to the control valve. One WF
pipeline can set more than one CF pipeline but each CF pipeline must
have its own flow closed loop with its own PID flow controller and control
valve. There is no PID controller for the WF pipeline. Controller FIC21 is
configured for Ratio control using one PID (PID1).
NOTE: Since only ONE PID is used i.e. PID1, the PV, SV and MV
are understood to mean PV1, SV1 and MV1 throughout.

1.2 OBJECTIVE AND AIM
Objective: To control water flow using PID controller.
Aim: Learn how to
1. Adjust Manipulated Variable (MV) and Set Point Variable (SV).

2. Move between Manual (M) and Auto (A) control mode.
3. Set the PID values of proportional band (PB), integral time (I) and
detention time (D).
4. Operate and read the Chart Recorder.
1.3 EQUIPMENT
Model WF922 flow ratio process control training unit
Control System and Instrumentation
The processes can be operated and controlled from the control panel or the DCS
(Distributed Control System), if a DCS is connected. Otherwise, operation and
control is only from the control panel. For panel operation and control, the PANEL,
SCADA/DDC selector switch should be at the PANEL, SCADA position. This
52

selector switch is at the front cubicle beneath the Instrument control panel. Note
that for SCADA operation, a DCS with a SCADA configuration must be connected.
All instruments and controls are Tag-named as follows:-

the first letter describes the process variables such as F for flow, L for
level, P for pressure and T for temperature.
the subsequent letters described the functions, E for the measuring

element, T for transmitter of the measurement sensor, I for indicator, R
for recorder, C for control (ON/OFF or PID), S for switch (ON/OFF), A for
Alarm and V for valve. H and L refer to High and Low alarm (annunciator)
limits when used with A (alarm).
CV is for control valve pneumatically operated and SV is for electrically

operated ON/OFF solenoid valve. PID throttling control valve requires an
IP converter (CY) which converts a 4-20 mA signal proportionately into a
3-15 psi signal. The letters CY represent such a control accessory. If a
pneumatic Positioner is used at the control valve, it is further represented
as PP. If the Positioner is integral with the IP converter, the integral unit is
represented as EP i.e. IP + PP.
the Tag ends in digits differentiating different instruments of the same

process variables and functions.
For more specific details, please refer to your instructor on Typical

Notation/ Symbols.
Field-Mount Instruments: Transmitters, Control Valve, Gauges, etc .
The instruments mounted at the field (plant) are asterisked (*) in (c) and further
detailed below. For more details, please refer to a separate manual for the
instrument specifications etc.
Instrument Description Label

Variable Area This flowmeter is for local indication only FI22
Flowmeter or and has no transmitting output.
Rotameter.
Orifice plate It is a flow element for 1 nominal pipe. FE22
Differential It measures the pressure drop (h) across FT22

Pressure (DP) the tapping points of the orifice for a
Transmitter 3
calibrated flow range of 0-6 m /Hr.
53

Vortex Flowmeter Calibrated Range: 0-6 m3/Hr FT21
Control Valve Control Valve, 1. Equal % characteristics, FCV21
Air to Open (ATO).
Pneumatic Pneumatic Positioner for FCV21 with By- PP
Positioner pass.
Current-to-Air Converts 4-20 mA to 3-15 psig FCY21
Converter proportionately.
Temperature Measure temperature TG21

Gauge
Pressure Gauges Measuring pressure drop across the control PG21,
valve FCV21. Liquid filled and complete PG22
with snubbers to minimise pressure
pulsation.
Solenoid valve Normally open (NO) SV21
Pressure Relief Safety instruments to vent excess pressure PRV20,
Valves. in the system. PRV21,
PRV22A/B
Panel-Mount Instruments
Instrument Description Label

Single Loop Configured for Ratio control using only one FIC21
Multifunction PID (FIC21, PID1).
Controller
Chart Recorder Two analog inputs with pen/bar graph and FR21
selective display in engineering units. If the
selector switch is at Position 1, then:
Red pen (Channel 1): FT21,
Calibrated Range 0-6 m3/Hr
Green pen (Channel 2): FE22/FT22,
Calibrated Range 0-6 m3/Hr
* Note that the Red pen (Channel 1)

is always for CF and the Green pen
(Channel 2) is always for WF.
CF is either FT21 (during NORMAL

OPERATION, at Position 1) or FT22 (during
INTERCHANGE OPERATION, at Position 2).
Analog display in the form of horizontal

coloured bar or pen- chart paper are to be
read as follows:
54

Analog display in % x Maximum
(calibrated range), engineering units
= Actual readings, engineering units
The chart drive is set for fast speed (500

mm/Hr). The recorder chart drive is started
by pressing ON the RCD button with the
front swing cover opened. The chart speed
is regularly printed on the chart paper.
Distributed Control System (DCS)
The *Yokogawa CENTUM CS1000 is a Distributed Control System (DCS) used in

Direct Digital Control (DDC) mode. The same DCS can also be configured with
the panel controllers to become a SCADA (Supervisory Control and Data
Acquisition) system, if additional communication hardware modules and software
are preconfigured in a DCS and the panel controller.
The panel control systems can be by-passed and be taken over by the DCS (in
DDC) mode by switching over the hard-wiring so that all measurement signals
from the field are connected directly to the DCS and all control outputs to the
field are directly from the DCS. A PANEL, SCADA/DDC selector switch is provided
at the front cubicle for this switch-over. Any of our process plants can be
operated and controlled from either the panel control centre OR the DCS control
centre, by switching this selector switch accordingly.
The DCS is configured in Direct Digital Control (DDC) mode, in the sense that the
DCS receives signals directly from the plant and it controls (PID, ON/OFF) the
plant directly. This selector switch must be in the DDC position.
For SCADA operation, the control functions (PID or ON/OFF) are still done at the
panel controllers but the DCS Operator Stations are used for the operators
interface with the process. This selector switch must be in the PANEL, SCADA
position for SCADA operation.
* IF OTHER MAKE OF DCS IS USED INSTEAD, THE GENERAL FUNCTIONS AND

FEATURES DESCRIBED HERE CAN ALSO BE ENGINEERED INTO THE TOTAL
TRAINING SYSTEMS. HOWEVER, THE DCS ENGINEERING AND CONFIGURATION
OF SUCH MAKE OF DCS WILL BE OUTSIDE OUR CURRENT SCOPE OF WORK.
55

Instrument Control Panel
Note the following:

Controller, Recorder, Annunciators.
A 1-2 position selector switch for 1: FT21 = PV = CF, NORMAL
OPERATION.
2: FT22 = PV = CF,
INTERCHANGE
Instrument wiring termination and power supply and wiring:

The Instrument panel can be opened from the back to access the
Instrument terminations.
The front cubicle below the Instrument panel can be opened from the
front.
Annunciators
The Annunciators are:-
FAH(CF) : Flow of the Controlled Flow stream (CF) exceeds the preset High
flow limit, PH1, set at the PID1 page or panel.
FAL(CF) : Flow of the Controlled Flow stream (CF) drops below the preset
Low flow limit, PLI, set at the PID1 page or panel.
A buzzer will come on and the respective alarm window [FAH(CF), FAL(CF)] will
lit up when the above abnormal or alarm conditions occur. Pressing the
Acknowledge button will silence the buzzer sound. The dedicated alarm window
remains lit as long as its process variable is still in the alarm condition. The alarm
window light will go off when the process variable is restored to normal.
The Test button is to test if the Annunciator alarm window lights are working.
Front Cubicle
Beneath the Instrument control panel is another cubicle with the following at the
front of the cubicle:-
Switch for incoming mains 415V/50Hz/3 phase.
PANEL, SCADA/DDC selector switch
Pumps Start-Stop pushbuttons for P20, P21, P22A, P22B
56

1.4 PROCEDURE
1.4.1 Getting started
1. Tank T21 should be filled with water up to almost the level of its lower
overflow pipe.
2. Quickly check the various manual valves as follows:-

All pumps (P20, P21, P22A, P22B) suction, discharge and by-pass valves
are fully opened. The pump by-pass valves B20, B21, B22A, B22B can
be shut later after the respective pump has started pumping.
The manual by-pass valve around the control valve FCV21 should be
always shut but its two adjacent manual valves should be always
opened.
The bottom manual drain valve of tank T21 is always shut.
Model WF922 is connected to a common drain system for draining
outside the building. Trace the piping system from the tank overflow
pipe to its final discharge.
The interconnecting manual valve MVX is fully shut.
The manual discharge valve MF at the discharge of pump P20 is fully
shut.
2. Instrument air supply (IAS) is required to operate the control valve system.
FCY21/PP/FCV21. Check that the pressure is in accordance to the pressure
indicated at the air pressure regulator (IAS). Once set, it need not be
readjusted frequently.
3. Main power supply (415V/50Hz/3 phase) is preconnected to the Model plant.

Turn on the main power supply switch at the front cubicle. The panel
instruments all lit up. The PANEL, SCADA/DDC selector switch should be in the
PANEL, SCADA position.
Whenever any annunciator is activated, press the acknowledge button to silence

the buzzer. Rationalise the cause of the alarm condition. Refer to the earlier
section annunciators and rationalise the cause of the alarm condition.
4. Switch controller FIC21 to Manual (M) mode with its MV = +100% e.g. 106.3%
to open the control valve FCV21 fully.
5. Switch the selector switch to Position1: FT21 = PV = CF, for NORMAL

OPERATION. Open fully CF1/WF1, Shut fully CF2/WF2, MVX, MF.
6. Switch On the following pumps with the manual by-pass valves opened:-
Pump P21: Manual by-pass B21 open.
Pumps P22A, P22B: Manual by-pass B22A, B22B open.
7. Check that pump P20 is OFF and its manual discharge valve MF is fully shut.
57

8. Shut the pumps manual by-pass valves, B21, B22A, B22B.
9. Start the recorder FR21 by pressing its RCD button with the front swing cover
opened. Check the chart speed (500 m/Hr) which is regularly printed on the
chart. The recorder chart drive should be stopped (press RCD) when its chart
recording is not required. Stop the chart drive.
PID Controllers setup
1. Controller FIC21 is configured for Ratio control using one PID (PID1). Be
familiar with the controller as follows:-
Display FIC21 and change from Auto (A) to Manual (M) mode and vice
versa.
Change the setpoint SV to 3.6 m3/Hr. (60%)
Switch to Auto (A) mode for single loop flow control.
In Manual (M) mode, manually stroke the control valve FCV21 fully
opened with MV = +100% e.g. 106.3%. Check that the control valve
FCV21 is fully opened.
2. Access the PID tuning panel or page at PID1 and change the PID values to the
following first (I) trial PID values.
FIC21 (PID1): PB1 = 150%, TI1 = 10 secs, TD1 = 0 secs,
GW1=0.0%, GG1=1.0
3. Also note the PH1 and PL1, values.
PH1 and PL1 are used to set the High and Low flow alarm limits (in m3/Hr) of
the process variable PV, which is the controlled flow CF. PH1 should be set at
4.0 m3/Hr; PL1 at 1.2 m3/Hr. Do not change these PH1, PL1 values once they
are set at these values.
4. Access the instrument ratio factor R at the PARAMETER page or panel at

CGN1. Set CGN1 to 1.0 so that the flow ratio R = 1.0.
1.4.2 Training: PID SINGLE LOOP FLOW CONTROL (NORMAL

OPERATION): FT21-FIC21-FCY21/PP/FCV21
1. The PID flow controller is FIC21 (PID1) and the controlled variable PV is FT21.
The flow single loop is:- FT21-FIC21-FCY21/PP/FCV21
2. Restore the pipelines and control to NORMAL OPERATION position i.e.

Open fully CF1/WF1, Shut fully CF2/WF2.
Shut the interconnecting manual valve MVX.
Shut the manual discharge valve MF, of pump P20.
Open manual discharge valves of the pumps P21, P22A, P22B.
Controller selector switch is in Position 1: FT21=PV=CF.
58

Check that the adjacent upstream/downstream manual valves of FCV21
is fully Opened but its manual by-pass valve is fully shut.
The Positioner (PP) at FCV21 must be CONNECTED i.e. ON, and not By-
passed.
3. Switch ON only pump P21 and shut its manual by-pass valve B21.
4. With the controller FIC21 still in Manual (M) mode at MV=100%, set its first (I)
Trial Linear PID values as follows:-
FIC21(PID1) : PB1 = 150%, TI1 = 10 secs, TD1 = 0 sec
: GW1 = 0.0%, GG1 = 1.0
5. Set the setpoint SV = 1.8 m3/Hr.
6. Manually adjust MV so that PV approaches SV. Switch FIC21 to Auto (A) Mode.
7. The recorder FR21 chart drive should be at fast speed (500 mm/Hr) with RCD
ON. Check the chart speed which is regularly printed out on the chart.
8. Observe the response of FT21 at the red pen until it is almost Steady at the
setpoint to within 0.02 m3/hr, or continues to Oscillate even after 3 cycles.
9. Write down the setpoint (sv) and pid values on the recorder Chart paper
besides its response. These chart recordings constitute the results of your
experiment and should be kept for your report.
Attach the recorder chart paper(s) with clearly marked details.
59

PROCESS CONTROL LABORATORY EXPERIMENT 2
LIQUID/WATER LEVEL FLOW (WLF) CONTROL
2.0 INTRODUCTION
Level control is very important in chemical industry. It is usually applied in

reaction process control and storage tank control. The purpose of this control is
to control the level of the tank at its desired value. Failure to control the level of
a system may cause the tank to overflow or dry up, which may eventually lead to
unwanted incident or explosion.
The model WLF922 uses water to simulate a liquid phase level and flow
process. The liquid flow is measured by the differential pressure across an orifice,
and the water level of the tank is measured through the differential pressure (DP)
level transmitter. Level conductivity probes are used to detect water level.
2.1.1 PID Theories
The Proportional Integral (PI) control

!
! (1.1)
= ! + + !
! !
where KC = gain, I = integral time, and PS = constant.

function,
() 1 (1.2)
= ! (1 + )
() !
The Proportional Derivative (PD) control
(1.3)
= ! + ! ! + !

where KC = gain, D = detention time, and pS = constant.
60

function,
() (1.4)
= ! (1 + ! )
()
The Proportional Integral Derivative (PID) control

!
! (1.5)
= ! + + ! ! + !
! !
Taking Laplace transform and rearranging,
() 1 (1.6)
= ! (1 + + ! )
() !
In proportional mode, a smooth, linear relationship exists between the

controller output and the error. For the two-position mode had the
controller output of either 100% or 0%, over some range of errors about
the setpoint, each value of error has a unique value of controller output in
one-to-one correspondence. The range of error to cover 0% to 100%
controller output is called the proportional band, because the one-to-one
correspondence exists only for errors in this range. The proportional band
(PB) of a proportional controller depends on the inverse of the gain.
100 (1.7)
=
!
2.1.2 Open Tank Flow and Level Measurement
The most common industrial flowmeter is the orifice plate with a DP

transmitter. This works on the fluid mechanics principle that the liquid
volumetric flowrate (F) is proportional to the square root of the pressure
drop (h) across an orifice flow element, i.e.
!
= = if the density is assumed constant (2.1)
!
This equation is only true if the flowrate is high enough to be called

turbulent flow.
On the other hand, a DP transmitter is also the most common level
measuring device in industrial process plant. It works on the principle
that the hydrostatic pressure at any point in a column of the liquid
depends on the level of the liquid column above this point, multiplied by
the liquid density. If the tank liquid density is constant, the hydrostatic
pressure is a measure of the level. An open tank system requires one wet
61

leg connecting the tank bottom to the high pressure chamber of the
differential pressure transmitter. The low pressure chamber is opened to
the atmosphere, cancelling the pressure at the top space of the open tank.
The DP transmitter therefore measures H (tank level) D (density of tank
liquid) g (gravity). If the density (D) of the tank liquid is constant, the
measured DP is a measure of the tank level H.
2.1.3 Model WLF922 description
The process consists of a level tank T31 and a collection tank T32
connected with the appropriate pumps and piping system for the study
of:-
Measurement and Control of Flow into T31 from T32
Water is pumped by pump P32 from tank T32 into T31 and is referred to
as the INFLOW. The PID single inflow control loop is: FE31/FT31-
FIC31-LCY31/PP/LCV31.
Measurement and Control of Level in tank T31
The PID single level control loop is: LT31-LIC31-LCY31/PP/LCV31.
T31 as an Open or Closed Tank
The top vent (V) and overflow drain (D) manual valves are opened to
operate T31 as an Open tank (so that the top space of T31 is at
atmospheric pressure), and vice versa operating T31 as a Closed tank.
The top space of T31 is pressurised with air from the pressure regulator
AR31 to about 2.5 to 3.0 psig, as monitored by the pressure gauge PG31.
Excessive pressure will cause the pressure relief valve PRV31 to vent.
Note that a pressure of 2.5 psig is equivalent to about 1760 mm Water
level at ambient temperature.
The level transmitter LT31 is installed to measure the tank level,

using the two wet legs technique. However, note that when T31 is
pressurised as a Closed tank, the inflow rate will be slightly reduced but
its outflow will be increased slightly.
Level Process as Self Regulating (SR) or Non-Self Regulating (NSR)
The first thing to note in controlling a level process (or any process) is to
check whether it is Self Regulating (SR) or Non-Self Regulating (NSR). For
Self Regulating Single Capacity Level Process, the outflow of T31 is by
62

gravity from the tank bottom discharge pipe. Two outflow gravity pipes
are provided each with its own manual valve. Only one pipe is to be
operated as the outflow. The second manual valve remains shut.
The Self Regulating process is able to control its own level and
unlikely to overflow or run dry. When the inflow increases, the level also
increases which therefore increases the outflow. Similarly, when the
inflow decreases, the outflow also decreases. The outflow is dependent
on the level. Such self regulating mechanism makes the control of a Self
Regulating process easier to control.
Panel-Mount Instruments
The panel-mount instruments are listed below.
Switch the PANEL, SCADA/DCS selector switch at the front cubicle to the
PANEL, SCADA position for Panel operation.
LIC31/FIC31: Single Loop Programmable Controller, PID. Configured

for:-
LIC31-Loop 1, PID1, Level PID Controller
FIC31-Loop 2, PID2, Flow PID Controller
In Cascade mode, LIC31 is the master or primary,
FIC31 is the slave or secondary.
Three control strategies can be operated as

described earlier in (c).
LIC31, Level Single Loop, PID1, Loop 1:

LT31-LIC31-LCY31/PP/LCV31
Selector switch in Position 1: LIC31.
LIC31 in Auto (A) or Manual (M) mode.
FIC31, Flow Single Loop, PID2, Loop 2:
FE31/FT31-FIC31-LCY31/PP/LCV31
Selector switch in Position 2: CASCADE LIC31-
FIC31.
FIC31 in Auto (A) or Manual (M) mode.
LIC31/FIC31, Level (primary) - Flow (secondary)
Cascade:
LT31-LIC31-FIC31-LCY31/PP/LCV31 (FE31/FT31)
Selector switch in Position 2: CASCADE LIC31-
FIC31. FIC31 in Cascade (C) mode.
63

LIC31 in Auto (A) mode. Control output of LIC31
(MV1) becomes the remote setpoint (SV2) of FIC31.
LFR31 : chart recorder, two analog inputs with pen/bar graph and
selective display in engineering units.
Red pen (Channel 1): Level, Calibrated Range 0-800 mm WG
3
Green pen (Channel 2): Flow, Calibrated Range 0-3 m /Hr
Analog display in % x Maximum (calibrated range),
engineering units = Actual reading, engineering units. The
chart drive is set for fast speed (500 mm/Hr). The recorder
chart drive is started by pressing ON the RCD button with the
front swing cover opened.
FIELD-MOUNT INSTRUMENTS: TRANSMITTERS, CONTROL VALVE,

GAUGES, ETC.
The instrumentation mounted at the field (plant) are further

detailed below:
LT31 Differential Pressure (DP) Level Transmitter

Measures level of tank T31, Open (to atmospheric
pressure) or Closed (pressurised).
Calibrated Range = -800 to 0mm Water Gauge (WG)
Transmitted to LIC31.
FE31 1 Orifice plate, radius *ID-D taps connected to FT31.
Orifice d = 11.076 mm for 0-3.0 m3/Hr.
FT31 Differential Pressure (DP) Flow Transmitter
Calibrated for 0-3.0 m3/Hr, with square root function
Transmitted to FIC31
LIC31 PID controller (PID1, Selector: Position 1) configured in
the panel-mount Single
Loop Programmable Controller LIC31/FIC31
FIC31 PID controller (PID2, Selector: Position 2) configured into
the panel-mount Single Loop Programmable Controller
LIC31/FIC31.
Note that only FIC31 can be Cascaded (i.e. press C) and
the Selector switch must be in Position 2.
LCV31 1 Control Valve with Equal % characteristics, Air-to-
PP Open (ATO)
Pneumatic Positioner (PP) for LCV31, with By-pass.
64

LCY31 Current-to-Air Converter
Converts controller output from 4-20 mA into a
proportional pneumatic signal of 3-15 psig to stroke the
control valve LCV31.
LS32 Conductivity Level Switch
PG31, PG32 Pressure Gauges
SV31 Solenoid valve, normally open (NO).
PRV31, Pressure Relief Valves.
PRV32,
PRV33
LFR31 Chart recorder, two analog inputs with pen/bar graph
and selective display in engineering units.
Red pen (Channel 1): Level, Calibrated Range 0-800
mmWG
Green pen (Channel 2): Flow, Calibrated Range 0-3
m3/Hr
Analog display in % x Maximum (calibrated range),
engineering units = Actual reading, engineering units.
The chart drive is set for fast speed (500 mm/Hr).
LAH31, The annunciators
LAH32, LAH31: Tank level at T31 measured by LT31 exceeds the
FAL31 preset High level alarm limit. The High alarm limit is set
at PH1 at the PID1 page.
FAL31: Inflow to T31 measured by FE31/FT31 is below
the preset Low alarm limit. The Low alarm limit is set at
PL2 at the PID2 page.
LAH32: Tank level at T32 exceeds the High level limit
(shortest probe) of the Level Switch LS32.
REMARKS: The PV, SV in the main display pages (faceplates) of LIC31

and FIC31 become PV1,SV1 and PV2,SV2 respectively when the selector
switch is in Position 2.
2.2 OBJECTIVE
Objective: To control water level using PID controller.
Aim: Learn how to
1. Adjust Manipulated Variable (MV) and Set Point Variable (SV).

2. Move between Manual (M) and Auto (A) control mode.
65

3. Set the PID values of proportional band (PB), integral time (I) and
detention time (D).
4. Operate and read the Chart Recorder.
2.3 EQUIPMENT
Model WLF922 level flow process control training unit
CONTROL SYSTEM AND INSTRUMENTATION
The processes can be operated and controlled from the control panel or the DCS
(Distributed Control System), if a DCS is connected. Otherwise, operation and
control is only from the control panel. For panel operation and control, the PANEL,
SCADA/DCS selector switch should be at the PANEL, SCADA position. The
selector switch is at the front cubicle beneath the Instrument control panel. Note
that for SCADA operation, a DCS with a SCADA configuration must be connected.
All instruments and controls are Tag-named as follows:-

the first letter describes the process variables such as F for flow, L for
level, P for pressure and T for temperature.
the subsequent letters described the functions, E for the measuring

element, T for transmitter of the measurement sensor, I for indicator, R
for recorder, C for control (ON/OFF or PID), S for switch (ON/OFF), A for
Alarm and V for valve. H and L refer to High and Low alarm (annunciator)
limits when used with A (alarm).
CV is for control valve pneumatically operated and SV is for electrically

operated ON/OFF solenoid valve. PID throttling control valve requires an
I/P converter (CY) which converts a 4-20 mA signal proportionately into a
3-15 psig signal. The letters CY represent such a control accessory. If a
pneumatic Positioner is used at the control valve, it is further represented
as PP. If the Positioner is integral with the I/P converter, the integral unit
is represented as EP i.e. IP + PP.
the Tag ends in digits differentiating different instruments of the same

process variables and functions.
2.4 PROCEDURE
66

2.4.1 Getting started
1. Tank T32 should be filled with water up to and just below the level of the
shortest level probe (not visible) of the Level Switch LS32 which is slightly
below the tank (T32) overflow pipe outlet. Top up the water later whenever
necessary.
2. To operate Model WLF922 independently, open the by-pass manual valve
B33 of pump P33 to divert any flow back into T32. Shut the interconnecting
pipeline from Model WLF922 to Model WT922 at the appropriate manual
valves MV-T.
3. Also shut the manual valve MV-D at the discharge of pump P33, leading to
the drain. For Independent Operation, the panel-mount selector switch for
P33 must be in the Manual or OVER-RIDE P33 position.
4. Quickly check the various manual valves as follows:-
Locate the Inflow pump P32. Check that its manual suction, discharge
and by-pass valves are fully opened.
For the Outflow pump P31, open fully its manual suction valve but
shut fully its two manual parallel discharge valves.
For pump P33, open fully its manual suction and by-pass valve but
make sure its manual discharge valves MV-T and MV-D are fully shut.
Operate T31 as an Open tank with the top vent (V) and overflow
drain valves fully opened. The pressurising air inlet to T31 is isolated
at its inlet manual valve (with the valve handle at 90 to the air
supply inlet tubing), located next to the preset Air Regulator AR31.
Operate T31 as a Self Regulating process. Open only the manual

GATE valve at the gravity discharge pipe at the tank bottom. The
second manual globe valve at the second gravity discharge pipe must
remain shut, thus operating only one gravity discharge pipeline at the
bottom of tank T31. Shut the two manual discharge valves of Outflow
pump P31, one of which is at the strainer inlet. For a Self Regulating
process, pump P31 must be OFF.
The manual by-pass valve around the control valve LCV31 should be
always shut but its two adjacent manual valves should be always
opened.
The bottom manual drain valve of T32 is always shut.
Model WLF922 is connected to a common drain system for draining

outside the building. Trace the drain piping system from the tank T32
overflow pipe to its final discharge.
4. Compressed air is required to operate the control valve system

LCY31/PP/LCV31.
67

Check that the pressure is in accordance to the pressure indicated at
the air pressure regulator (IAS). There is no need to adjust this
pressure (IAS) too frequently.
It is good practice to purge the air regulator (IAS) to remove any
condensed water. Use the drain/bleed valve at the bottom of the air
regulator (IAS).
The air regulator (AR31) for pressurising tank T31 is preset to about
3.0 psig and need no adjustment. Any higher pressure will cause the
pressure relief valve PRV31 to open.
Do not pressurise T31 yet. Shut the pressurising air inlet manual
isolation valve (with the valve handle at 90 to the air supply inlet
tubing) located next to AR31 near the top of tank T31.
5a. Main power supply is pre-connected to the Model plant. Turn on the main
power supply switch at the front cubicle. The panel instruments all lit up.
Whenever any annunciator is activated, press the Acknowledge button to
silence the buzzer. Rationalise the Cause of the alarm condition.
5b. Note the following switches and pushbuttons but do not switch ON any pump
yet.
PANEL, SCADA/DC : Switch to PANEL, SCADA position for Panel Selector

switch operation.
Pump P32 : Pumping inflow from T32 to T31. To be switched ON during

operation.
Pump P31 : Do not use unless T31 is to be operated as a Non-Self
Regulating process, in which case the bottom gravity discharge pipes of
T31 must be all shut. Check that the two manual discharge valves of
pump P31 are shut.
Pump P33 : Leave it in OVER-RIDE P33 position for the time being.
PID Controller
1. Controller LIC31/FIC31 is configured with PID controller: LIC31 (PID1, Loop 1)
Switch to Position 1 (LIC31): Single Level Loop LIC31 (PID1, Loop 1).
Note PV1, SV1, MV. control output to the
control valve.
In Position 1, PV1, SV1 are displayed as
PV,SV for LIC31
PV1 is process variable i.e Level of T31.
SV1 is its setpoint and MV is manipulated
variable or control output to the control
valve.
68

2. With the 1-2 Selector switch in Position 1, display LIC31. (Press the blue push
button located at the bottom right side of the controller LIC31/FIC31, to select
the display of LIC31, if necessary.) Note the A (Auto) and M (Manual)
pushbuttons. Press M and leave LIC31 in Manual (M) mode.
3. In Manual (M) mode, manually stroke the control valve LCV31 with MV = 50%,
from LIC31
4. Change to the Self-Tuning page (STC1. The STC should be in the DISP
(Display) mode. Make sure STC is not in ON mode.
Instrumentation
1. Check the Positioner (PP) manual By-pass switch at LCV31/PP. Make sure the
Positioner (PP) is connected to LCV31 i.e. do not by-pass it, but USE THE
Positioner (PP).
2. Set the 1-2 Selector switch to Position 1 to operate LIC31 and display LIC31,
Loop 1 accordingly.
3. With LIC31 in Manual (M) mode, adjust MV = 25%. Check that LCV31 is about
25% OPENED (see the indicator scale at the valve stem).
Also note that the control valve LCV31 is Air-to-Open (ATO).
Chart recorder
1. Start the recorder, LFR31 by pressing the RCD button ON, with the front
swing cover opened. Check the recorder chart speed (500mm/Hr) which is
regularly printed out on the chart.
2. The recorder chart drive should be stopped (press RCD) when its chart
recording is not required. Switch OFF the recorder chart drive.
Operate tank T31 as a SELF REGULATING LEVEL
1. Shut the two manual discharge valves of pump P31. Do not operate P31.
2. Make sure only the manual GATE valve at the gravity discharge pipe at the
bottom of tank T31 is opened. The second gravity discharge pipeline and its
manual globe valve must remain shut.
3. Check that the controller Selector switch is in Position 1. Display LIC31 at the
controller.
With LIC31 in Manual (M) mode, adjust MV = 60%. Check that LCV31 is about
60% opened. The Positioner (PP) should be connected and used throughout.
4. Start pump P32 and then shut its manual by-pass valve B32.
69

5. Note the rising water level at tank T31 at its level sight glass.
6. Make sure the overflow drain manual valve at the top of tank T31 is fully
opened for water to overflow back into T32.
2.4.2 Training: Study level control using loop LT31-LIC31-LCY31/PP/LCV31

1. Operate tank T31 as a Self Regulating Open tank process
2. With the controller Selector switch in Position 1, display LIC31 faceplate at the
controller.
3. Set the setpoint (SV1) of LIC31 to 400 mm.
4. With LIC31 in Manual (M) mode, set MV = 75%. Check that the stem position
of control valve LCV31 is about 75%.
5. Set the first (I) trial PID values at the PID1 page or panel as follows:- LIC31
(PID1, Loop1): PB1 = 30%, TI1 = 25secs, TD1 = 0
6. Press the RCD pushbutton with the front swing cover opened.
7. Start pump P32 and watch the level (PV1) increases. Shut the pump by-pass
valve B32.
8. When the level is near to the setpoint SV1, switch LIC31 to Auto (A) mode
and observe the Level (red pen) response at the recorder LFR31.
9. Set the second first (II) trial PID values at the PID1 page or panel as
follows:- LIC31 (PID1, Loop1): PB1 = 10%, TI1 = 15secs, TD1 = 0
Repeat steps 6-8.
Attach the recorder chart paper(s) with clearly marked details.
70

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Lab Manual KC 31001 Laboratory 6: Semester 2 2016/2017

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UNIVERSITI MALAYSIA SABAH

SCHOOL OF ENGINEERING & INFORMATION TECHNOLOGY

No. Titles Pages

1. Laboratory Safety awareness

3. Fire Hazards and hazardous chemicals

5. After Hours/Long Hours Experiment

Laboratory report should contain the followings:

5. Result and discussion

Assessment Method Marks

PREPARATION OF CULTURE MEDIA AND ISOLATION OF PURE

Cultivation is an important technique used to isolate culture and determine

1.1 THEORIES AND EXPLANATIONS

1.1.1 Aseptic technique

Aseptic is important while working with microorganism to prevent contamination

The streak plating technique isolates individual bacterial cells (colony-forming

Figure 1.1 Three times streaking method.

In this experiment, you will learn to:

Nutrient Apparatus Equipment

1.4.1 Preparation of medium and sterilization

1. Nutrient broth and agar will be prepared in advance by lab demonstrators.

1.4.2 Sampling analysis:

1.5 EXPERIMENTAL RESULTS

1. What can you notice in the control plate?

CHARACTERIZATION OF BACTERIA AND ESTABLISHING PURE

When grown on a variety of media, microorganisms will exhibit differences in the

2.1 THEORIES AND EXPLANATIONS

Table 2.1 Gram stain

In this experiment, you will learn to:

Refer to Bioprocess principles laboratory experiment 1.3.

2.4 METHODOLOGY OF OPEN REFLUX METHOD

2.4.1 Identification of bacteria

1. Observe the growth of colonies on the agar plates.

Note: Remember to hold the clean slides by their edges. Smears

1.4.2 Establishing pure culture

2.5 EXPERIMENTAL RESULTS

Show and describe your Gram stains.

1. Describe the growth of each culture on the streak plates.

QUANTIFICATION OF CELL NUMBER

In the study of microbiology, there are numerous occasions when it is necessary

3.1 THEORIES AND EXPLANATIONS

3.1.1 Serial dilution from stock solution

Quantification of the cell numbers is important in researches. This can be done

Figure 3.1 Preparation of dilutions utilizing sterile nutrient broth blanks.

Viable titer = (CFU/volume plated) x Dilution factor (3.1)

In this experiment, you will learn to:

Refer to Bioprocess principles laboratory experiment 1.3.

3.4.1 Determination of viable cells: Plate count method

3.4.2 Determination of optical density

1. Switch on the spectrophotometer and adjust the wavelength to 600 nm.

3.4.3 Determination of dry cell weight

1. Weight a dry filter paper.

3.4.4 Determination of total cell

1. Total cell count can be determined by using a Hemacytometer.

Calculate the viable titer for each plating medium.

Dilution number Number of colony Cell number

1. Are all the bacteria counted by this procedure? Is this an overestimate or

KINETIC STUDIES IN BATCH REACTOR

1.1 THEORIES AND EXPLANATIONS

The saponification id ethyl acetate in dilute aqueous solution can be

CH3COOC2H5 (l) + NaOH (l) = CH3COONa (l) + C2H5OH (l)

Because that the reaction saponification of Ethyl Acetate is a single

1. To verify that the saponification of ethyl acetate in dilute aqueous

1. Ethyl Acetate, pure 10ml