Unit 1: Fundamentals of Laboratory Techniques

Assignment Brief

Student Name/ID Eshana Sandaruwani Bandara Gunasekara

Number HSC-B09-027

Unit Number and Title Unit 1: Fundamentals of Laboratory Techniques

Academic Year
Unit Tutor Ms. Buddhika Dilrukshi

Assignment Title Individual Assignment – Lab Report Portfolio

Issue Date
Formative Submission -Task 01 Please refer to the Given
Submission Date
Assessment plan
Formative Submission - Task 02 Please refer to the Given
Assessment plan
Formative Submission - Task 03 Please refer to the Given
Assessment plan
Summative Submission Please refer to the Given
Assessment plan
Submission Format

The submission is in the form of collated scientific reports (a portfolio) for the practical
work in the assignment, developed in MS Word format. You are required to follow the
standard lab report format for each experimental report (Annex 1 of the Assignment Brief).
You may use any diagrams, images or illustrations as appropriate. All work must be
supported with research and in-text citations should be provided using Harvard referencing
system as required. Bibliography should be provided using Harvard referencing system at
the end of each report. The recommended word limit per each lab report is 1500–2000
words, although you will not be penalized for exceeding the total word limit.

Unit Learning Outcomes (Assessed by this assessment)

LO1 Carry out qualitative and quantitative analysis

LO2 Carry out synthetic chemistry techniques
LO3 Demonstrate use of microscopy and aseptic technique
LO4 Demonstrate good practice with respect to reporting, health and safety, and laboratory
organization
Transferable skills and competencies developed

 Building Competencies
 Analytical Reasoning
 Critical Thinking
 Communication skills
 Relationship Building

Vocational scenario

You have recently been recruited to the position of laboratory demonstrator in a college
educational laboratory. In this laboratory, a range of chemistry and biology related
experiments are being conducted as a part of the degree programmes conducted in the
college. As part of the probationary period for the role, the college laboratory management
require you to produce a portfolio whereby you can demonstrate profound knowledge
regarding all aspects of laboratory techniques. In order to retain your employment, you are
required to successfully complete the trials of all the laboratory experiments to be
conducted and generate a portfolio of reports for the experiments undertaken. Also, you are
required to have a good knowledge of health, safety and risk assessment.

Assignment activity and guidance

Prepare a portfolio of lab reports for the following experiments (You should take the
required data from the experiments you have done in the lab). Your portfolio must have
three sections, and under each section the required experiments and explanations are as
follows:

Task 01

Experiment 1: Preparation of Paracetamol

Experiment 2: Preparation of Ethyl acetate

*Note: You should explain the reasons for carrying out the steps in the syntheses. You
must also evaluate the success of the syntheses.

Task 02
Experiment 3: Determination of the Ascorbic acid concentration in Vitamin C

Experiment 4: Determination of Cu2+ concentration in an unknown copper (II)

sulphate solution using colorimetry. (Beer-Lambart law application)

Experiment 5: Qualitative separation of amino acids using thin layer

chromatography (TLC).

*Note: You should explain the theories underpinning each technique and should evaluate
the validity of the results obtained.

Task 03

Experiment 6: Observation of Onion cells under the Light Microscope

Experiment 7: Observation of Cheek cells under the Light Microscope

*Note: You should draw the properly labelled diagrams of your observations.

Explain and justify the actions that you can take in order to improve the level of your skills
related to microscopy.

Experiment 8: Aseptic technique (Microbiology)

Part I: Streaking is a technique for the isolation into a pure culture of the organisms (mostly
bacteria), from a mixed population.

a. Briefly explain how streaking is done and the aseptic techniques you have to follow
in relation to streaking.

b. Explain the purposes of those aseptic techniques that you mentioned under the
above point.

c. Explain and justify the actions that you can take in order to improve the level of
your aseptic techniques.

Provide following details in each lab report:

a. Explain how you carry out a risk assessment for each technique undertaken.

b. Explain calibration procedures for instruments/glassware you had to use in the

above experiments.
 c. Evaluate the quality of the practical work carried out with respect to good
laboratory practice, health and safety and laboratory organisation.

d. Explain and justify the actions that you can take in order to improve the level of
your skills related to risk assessment and compliance with standards of good
practice in the laboratory.

Recommended Resources
Please note that the resources listed are examples for you to use as a starting point in
your research – the list is not definitive.

Textbooks
AHMED, N., GLENCROSS, H., WANG, Q. (Eds.) (2016) Biomedical Science Practice:
Experimental and Professional Skills. 2nd ed. Oxford: Oxford University Press.
BOYLE, J., RAMSAY, S. (2017) Writing for Science Students. London: Palgrave.
CHRISTIAN, G.D., DASGUPTA, P.K., SCHUG, K.A. (2013) Analytical Chemistry. 7th
ed. Hoboken, NJ: John Wiley & Sons Inc.
CRANWELL, P.B., HARWOOD, L.M., MOODY, C.J. (2017) Experimental Organic
Chemistry. Chichester: John Wiley & Sons Ltd.
DEAN, J.R., JONES, A.M., HOLMES, D., REED, R., WEYERS, J., JONES, A. (2017)
Practical Skills in Chemistry. 3rd ed. Harlow: Pearson Education Ltd.
EVANS, E.H., FOULKES, M.E. (2018) Analytical Chemistry: A Practical Approach.
Oxford: Oxford University Press.
GOLDMAN, E., GREEN, L.H. (Eds.) (2015) Practical Handbook of Microbiology. 3rd ed.
Boca Raton, FL: CRC Press.
HARRIS, D.C., CHUCK, L. (2015) Quantitative Chemical Analysis. 9th ed. New York:
W. H. Freeman and Co. Ltd.
JOHNSON, S., SCOTT, J. (2019) Study and Communication Skills for the Biosciences. 3rd
ed. Oxford: Oxford University Press.
JONES, A., REED, R., WYERS, J. (2016) Practical Skills in Biology. 6th ed. Harlow:
Pearson Education Ltd.
MCPHERSON, P. (2015) Practical Volumetric Analysis. Cambridge: The Royal Society of
Chemistry.
OVERTON, T., JOHNSON, S., SCOTT, J. (2019) Study and Communication Skills for the
Chemical Sciences. 3rd ed. Oxford: Oxford University Press.
ROBBINS, S. (2009) Science Study Skills. Basingstoke: Palgrave Macmillan.
SASTRY, A.S., BHAT, K.S. (2018) Essentials of Practical Microbiology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd.
SKOOG, D.A., WEST, D.M., HOLLER, F.J., CROUCH, S.R. (2014) Fundamentals of
Analytical Chemistry. 9th ed. Belmont: Brooks/Cole.

Web
sdbs.db.aist.go.jp National Institute for Advanced Industrial Science and
Technology (AIST)
Spectral Database for Organic Compounds, SDBS
(General reference)
rsc.org Royal Society of Chemistry
Learn Chemistry
(General reference)
Learning Outcomes and Assessment Criteria
 STUDENT ASSESSMENT SUBMISSION AND DECLARATION
When submitting evidence for assessment, each student must sign a declaration confirming
that the work is their own.
Student name: Assessor name:

Issue date: Submission date: Submitted on:

Programme:

Unit:
Assignment number and title:

Plagiarism
Plagiarism is a particular form of cheating. Plagiarism must be avoided at all costs and
students who break the rules, however innocently, may be penalised. It is your responsibility
to ensure that you understand correct referencing practices. As a university level student, you
are expected to use appropriate references throughout and keep carefully detailed notes of all
your sources of materials for material you have used in your work, including any material
downloaded from the Internet. Please consult the relevant unit lecturer or your course tutor if
you need any further advice.

Student Declaration
I certify that the assignment submission is entirely my own work and I fully understand the
consequences of plagiarism. I understand that making a false declaration is a form of
malpractice.

Student signature: Date:

Higher Nationals - Summative Assignment Feedback Form

Student Name/ID
Unit Title Fundamentals of Laboratory Techniques
Assignment Assessor
Number
Date Received
Submission Date 1st submission
Date Received 2nd
Re-submission submission
Date
Assessor Feedback:

Grade: Assessor Signature: Date:

Resubmission Feedback:
*Please note resubmission feedback is focussed only on the resubmitted work

Grade: Assessor Signature: Date:

Internal Verifier’s Comments:
 Signature & Date:

Please note that grade decisions are provisional. They are only confirmed once internal and
external moderation has taken place and grades decisions have been agreed at the assessment
board.

Guidelines for the assignment/task

 Reference List for each lab report. And provide a separate reference list for section 3-
All references and citation shall use Harvard Referencing Style.
 In-text citations where necessary; including for figures and diagrams
• Use proper report formatting techniques (standard lab report format is attached at
the end of the assignment brief) for the portfolio as follows;
– Each report should be typed using 1.5 spaces between lines in 12 Times New
Roman point-font.
– Margins
– Justifying and aligning
– Page numbers
– Footer/ header
– Bullets and numbering
*University rules will be strictly applied in the event of plagiarism and late submission.

Late submission of the assessment will result in a late penalty mark. Penalties for late
submission: Up to one week late, maximum mark of 50%. Over one week late, 0%. Only the
Extenuating Circumstances Panel may approve a change to submission dates.
Annex 1

Lab Report Format

TITLE PAGE OF LAB REPORT

Title of the Experiment

Program Name:

Semester and Year:

Name of Student: Name of Lecturer / Lab Demonstrator:

Date of Experiment Performed: Date of Report Submitted:

OBJECTIVES

 State the objective of the experiment clearly in a complete sentence.

 The objective of the experiment is what you’re trying to prove or accomplish.

INTRODUCTION

 Describe why the topic is of interest.

 You can use 1-2 small paragraphs.
 Start with the problem or the issue you are trying to solve then provide some
background information about it.
 It should be direct, concise and easy to understand.
 You may have to use different resource materials.
 Use proper citations.

THEORY

 Explain relevant concepts, chemical equations, and provide any appropriate

definitions.
 Relevant equations should be derived in a clear, logical manner.
 Define all variables used in the equations.
 Use proper citations.

MATERIALS

 List chemicals, apparatus, and special equipment used for the experiment.
 Ideally, you want this section to be sufficiently detailed another person could repeat
the experiment.

EXPERIMENTAL PROCEDURE

 Provide a detailed description in your own words of how you accomplished the
experimentation.
 Use sketches, diagrams or photos, to describe the experimental set-up. Label the main
components.
 The experimental procedure should be written in past passive form.
 This can be a single paragraph or one or more pages.

RESULTS

 Present the results/data in the easiest way for your reader to understand: graphs,
tables, figures, etc.
 Your results/data should have the correct number of significant figures and
appropriate units.
  If your results/data is represented by graphical methods, make sure your graph has
been appropriately labeled with the correct axis, units, and scale.
 All figures and tables must have numbers and captions.
 Table captions should be placed over the table, figure captions should be placed
below the figure.

CALCULATIONS (If needed)

 Write down equations being used to do calculations using the results/data you
obtained.
 The calculations should be clear and readable.
 Show and label calculations in complete detail for any quantity.
 If you are repeating a calculation several times, you only need to show one sample
calculation.

DISCUSSION

 Explain the results of the experiment, comment on the results you obtained, compare
obtained results with expected results,
 Give probable reasons for discrepancies from the correct results.
 Answer any questions outlined in the instructions and solve any problems that may
have been presented.
 Implementation errors should be discussed here.

CONCLUSIONS

 Describe your conclusions clearly.

 State your discoveries, judgments and opinions from the results of this experiment.
 Make recommendations for further study.
 Suggest ways to improve the results of this experiment.

REFERENCES

 Cite any resources; books, publication or websites that you used.

 Provide authors, publisher, date of publication, page number, etc.
Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab Demonstrator:

Eshana Gunasekara Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01
Practical name - observation onion cells under microscope
Objective - To observe onion cells using a light microscope at under X4 X10 X40 X100
magnifications

Introduction –

Head- The head of a microscope is a metallic cylinder that houses the eyepiece lens on one end
and connects to the nosepiece at the other. Also known as an eyepiece tube or body tube, it
serves to link the objective lens with its counterpart. The light emitted by objectives passes
through this tube and undergoes bending thereafter. In binocular microscopes, they are
customizable for maximum visualization pleasure during use by viewers. (prashant, 2024)

Arm - The arm of the microscope serves as a linkage between its base, head and eyepiece tube.
Apart from carrying the weight of the head, it also facilitates transportation. Superior
microscopes are fitted with an articulated or flexible arm that has multiple joints to maximize
viewing precision by allowing considerable movement range for microscopic observation.

Base- The base of the microscope is located at its bottom and acts as a support structure. It
stabilizes the whole instrument, while also housing illuminators, light switches, and electrical
wiring systems. (prashant, 2024)
Eyepiece - The ocular lens, also referred to as the eyepiece, is positioned at the top of the
microscope and in closest proximity to the viewer's eye. This component serves its purpose by
providing a means for examining specimens and comes equipped with lenses that offer varying
degrees of magnification ranging from 5X up to 30X; however common versions usually feature
either 10x or 15x magnification capabilities. Consequently, these lenses are utilized for
augmenting images a second time over their initial enlargement through objective lenses.

eyepiece tube- this is the component that holds the eyepiece in place, positioned just above the
objective lens. Some microscope models, such as binoculars, have a flexible eyepiece tube that
can be rotated to provide optimal visual clarity at varying distances. However, monocular
microscopes do not offer this feature and their tubes are fixed. (prashant, 2024)

Diopter Adjustment - The control knob known as Diopter Adjustment is exclusively found in the
binocular microscope, and it serves to adjust focus on a single eyepiece. Its purpose is to rectify
any visual disparities and equalize any variances between an observer's two eyes. (prashant,
2024)

Nose piece - A revolving turret, also known as a nose piece, contains all of the objective lenses
and is situated above the stage on top of the body tube. By rotating it either clockwise or
counterclockwise, users can modify magnification levels by switching between different
objective lenses housed within this circular structure. (prashant, 2024)

objective lense- The objective lens is positioned closest to the specimen and secured on the
nosepiece. A typical microscope entails 3-4 lenses with differing magnification powers
including: 4X,10X,40x,and 100X. These lenses initially captures light beams from specimens
and resize images for viewing purposes.The size of anobjective lends vary depending om it's
power; smallest being low-powered while longest having highest capacity.At higher magnitude
such as at "40x" or "100x",the retractable features allow one to push towards its end.In most
modern optical microscopes,the oil immersion type ensures high-quality vision especially on
those magnified over a hundred times".(prashant, 2024)

The control knobs utilized to focus the microscope on the specimen are called Adjustment
Knobs. There exist two types of these knobs, namely:

The Adjustment knobs - The Fine Adjustment Knob is utilized to make precise adjustments. This
smaller knob gradually moves the stage up or down for small increments, resulting in a sharper
image. The distance covered by the stage with each turn of this knob is minimal, making it ideal
for use while viewing under high power settings. Its primary function is to fine-tune and sharpen
images viewed through microscopes. (prashant, 2024)

The Coarse Adjustment Knob enables the swift movement of the stage when focusing images at
a low magnification level. This knob is relatively large compared to others and easily raises or
lowers the stage with rapid precision. (prashant, 2024)

stage - The stage is where the specimen is positioned for observation. It features stage clips that
secure the specimen slides in place. The mechanical variety of this apparatus, commonly used,
permits manipulation of slides via mechanical knobs on the stage instead of manual positioning.

Stage Control Knobs- which are the knobs responsible for mechanical movement of the stage,
come in two variants. One knob enables left and right movements while the other facilitates
forward and backward motions resulting in adjustment of slide positioning within field of view.

Aperture- The aperture on the microscope stage allows for transmitted light from the source to
reach it through a hole. (prashant, 2024)

A microscopic illuminator – this serves as a source of light in various types of microscopes.

Some compound microscopes make use of mirrors to reflect external light onto samples, while
other optical microscopes utilize low voltage electric bulbs for continuous illumination, such as
tungsten-halogen lamps or 75-150W Xenon lamps and mercury vapor lamps among others.
Selections are made based on the required intensity and wavelength needed for proper lighting.
condenser- The condenser is a set of lenses located beneath the microscope stage, next to the
diaphragm. Its purpose is to gather and direct light from the illuminator onto the specimen in
order to generate clear and crisp images at magnifications above 400X. The effectiveness of this
component increases with greater magnification power, such as those found on more advanced
microscopes featuring Abbe condensers capable of up to 1000X magnification for optimal
clarity. (prashant, 2024)

The diaphragm- otherwise referred to as the iris, is situated beneath the microscope stage and
serves a crucial purpose in regulating light exposure on samples. By functioning as an adjustable
instrument for managing both intensity and beam size of illumination directed toward specimens;
optimal conditions are achieved. As a component included in top-tier microscopes alongside
Abbe condensers, controlling focus quality along with lighting strength becomes feasible.

The Condenser Focus Knob – this is responsible for adjusting the position of the condenser,
thereby regulating light focus on a specimen. (prashant, 2024)

The Abbe Condenser – this is intended for use with top-of-the-line microscopes and has a
unique design that enables mobility while also allowing magnification above 400X. Typically,
high-end microscopes possess superior numerical apertures compared to the objective lenses
they feature. (prashant, 2024)

The the rack stop- this is to regulate the extent to which stages can move, thereby preventing
potential damage to specimens caused by overly close proximity between objective lens and
specimen slide. It effectively halts excessive ascent of the latter, avoiding collision with the
former. (prashant, 2024)

light switch- The Light Switch is a device used to control electricity, specifically for turning on
or off the illuminator. (prashant, 2024)

brightness adjustment-The system that regulates the intensity of a light bulb is called brightness
adjustment. This function manages the voltage supplied to said bulb, thereby controlling its
brightness level. (prashant, 2024)
Theory
The light microscope is an apparatus or device used in biological laboratories that employs
visible light to detect and amplify minute objects, increasing their size.Lenses are utilized to
concentrate light onto the specimen, which enlarges it and generates an image. The microscopic
lens is typically located in close proximity to the specimen.The degree of microscopic
magnification can fluctuate substantially based on the composition and quantity of lenses
comprising the microscope. Two types of microscopes exist contingent upon lens count: an
elementary light microscope (characterized by low magnification due to its sole reliance on a
solitary lens) and a compound light microscope (featuring greater magnifying capabilities than
its simple counterpart as it incorporates two or more sets of lenses- including an objective one, in
addition to an eyepiece). The dispositioning of these multiple-grade lenses is such that they
effectively refract incoming light for optimal image expansion.The light microscope operates by
using its capacity to concentrate a beam of light through an almost invisible transparent specimen
and create an image. This picture is then heightened for viewing as it goes through one or two
lenses. The clearness of the sample enables quick acceptance of illumination, thus
accommodating various microscopic particles such as cells, bacteria, and other microbial forms.
(makobi, 2024)
Visualizing an image in light microscopes involves using a glass lens, where magnification is
based on the lens's capacity to bend and focus light onto the specimen forming an image.
Refraction occurs when a ray of light passes through different mediums with bending determined
by the refractive index measuring how much substance slows down speed of light. The direction
and extent of this refraction depend on both medium's refractive indexes at their interface.
(makobi, 2024)
Magnification is the act of visually enlarging something without changing its physical size. The
degree of enlargement can be expressed as a number called "magnification." It is important to
distinguish magnification from resolution, which refers to an imaging system's ability to reveal
details in the imaged object. While high magnification alone may enable observation of minute
microorganisms, it does not facilitate differentiation between them or their structures. Therefore,
microbiologists rely more heavily on resolution for this purpose. However, achieving appropriate
levels of magnification remains necessary before resolving differences between two objects
using microscopy techniques becomes possible. (libretexts, n.d.)
The optical resolution of an imaging system is influenced by the distance between two
discernible radiating points. A microscopic setup incorporates various components like lenses,
recording and display apparatus that all add to its overall performance. The imaging environment
also plays a crucial role in determining the quality of output generated. Due to practical
complications involved, actual optical systems are intricate; consequently increasing the gap
between detectable point sources becomes more challenging. (libretexts, n.d.)
Materials
1. Microscope
2. Microscope slide
3. Cover clip
4. Sample of onion peel
5. Forceps
6. Paper towel
7. Methylene blue solution
8. Glycerin
9. Dropper

Methodology
Using forceps and a new blade, a thin layer of onion epidermis was carefully removed. The
specimen was then immersed in water droplets before securely attaching the coverslip to it. Next,
we obtained an onion bulb and sliced off a thin piece which we placed on the glass slide. A few
drops of methyl blue dye were evenly distributed over the cells followed by gently placing
another cover slip without causing air bubbles formation. After ensuring that everything is
prepared with extreme caution so as not to damage or distort image quality under examination
through microscopic lens, starting from low-power objective lenses first used ensure proper
location finding for each cell; high power objectives later enable closer inspection when finally
found along horizontally-done adjustments made possible by fine-tuning lighting source settings
depending upon surrounding conditions at time images captured - focused clearly enough such
that distinctive features like nuclei-wall components identified too able capture observations
illustrations showed downstream uses/references done accordingly.
Results
Under 4x magnification
Elongated rectangles form the clusters of onion cells and their entire composition is visible.

Cell wall

Cytoplasm

figure – onion cell 4x magnification

Under 10x magnification

As the visibility of onion cells increases, each individual cell can be easily distinguished.

Since the staining procedure is performed correctly, the nucleus should be visible. Additionally,
the cell walls are thick and well-defined.

cytoplasm

Cell wall

vacuole
Figure – 10x magnification
 Nucleus

Nucleus

vacuole

cytoplasm

Figure – 100x magnification

While the cytoplasm and plasma membrane can be differentiated, more intricate specifics remain
imperceptible at this stage.

Discussion
While examining samples under a microscope, I discovered intricate details invisible to the
naked eye. The cellular structures such as nuclei and mitochondria within biological specimens
became distinctly visible thus helping me understand how cells function and interact with each
other on a deeper level. Similarly in material science examinations, magnification helped identify
even the tiniest of defects or weaknesses present in their crystalline composition providing
valuable insight into them. The resolution capabilities of this microscopic technology allowed for
remarkable clarity revealing even minute features while staining techniques increased contrast
making differentiation among cell components easier during biological specimen examination.
This accurate analysis method was effective enough to evaluate both sample condition and
structure thoroughly resulting benefiting overall research process greatly. (microscopeclub,
2020)
Despite the high resolution, there were several obvious limitations. For instance, a limited depth
of field presented challenges in achieving optimal focus on thicker samples resulting in blurred
images of out-of-plane sections at times. Additionally, light microscopy itself posed an inherent
limitation as structures under 200 nanometers could not be observed due to shortcomings in
wavelength-based resolution capacity.

To enhance the accuracy of microscopic observations, several actions can be implemented.

Utilizing high-quality lenses and advanced imaging techniques such as confocal microscopy are
efficient ways to achieve better resolution and depth perception. By adjusting lighting conditions
with appropriate filters, clarity and contrast may be improved further. Additionally, adhering to
regular calibration practices along with meticulous upkeep facilitates optimal performance in
minimizing artifacts while promoting elevated image quality standards.
Microscopy calibration
Precise measurements of samples with a light microscope require calibration, which involves
several steps. Firstly, an eyepiece reticle is inserted into the eyepiece - this small ruler is etched
onto a glass disc. Next, a slide called stage micrometer must be positioned on the platform and
accurately centered and magnified under the objective lens. The level of magnification used at
this point will be recorded before both scales are aligned by counting divisions in the eyepiece
that correspond to identical lengths marked on-stage micrometers; ensuring accurate calibrations
across all possible micro-magnifications through repeating these alignments for each available
objective lenses. Once calibrated, measuring microscopic samples' features can map out units of
measurement correctly based on counts obtained from aligned Eyepieces' Reticles after dividing
known distances present via stage micrometers using calculated factors efficiently and
effectively creating certainty in precision standards post-calibration procedures completion
indefinitely throughout its use period recommended during regular recalibrations as standard
practice to ensure accuracy remains intact over time. (www.astm.org, n.d.)
(www.laddresearch.com, n.d.)

Risk assessment
When mounting onion cells, it is vital to handle the samples correctly. Either water or a standard
saline solution should be used and direct contact with other biological materials must be avoided.
Unclean, gloved hands also pose a risk and discarding discarded samples after the experiment
needs careful attention. To maintain accuracy in results, only proper usage of the microscope
should take place since improper handling could damage equipment or lead to inaccurate
readings due to human error. Following manufacturer instructions for upkeep and storage will
help avoid mishaps entirely while those using this instrument need prior training on its accurate
use along with holding it steady by utilizing both their hands. Another factor which may cause
trouble during these experiments involves shoddy electrical connections leading up electric
shocks especially if wires are exposed; thus microscopes having undamaged parts ought always
to get usages towards keeping away liquids too besides constantly validating power cables as
well as plugs regularly A tidy lab surface helps prevent trips that cords lying around might
induce posing sustainability hazards requiring better organization across workstations themselves
whereby allergenic reactions can happen among individuals sensitive/allergic toward onions'
constituent elements -it would assist participants previously knowing about allergies/ sensitivity
staying mindful concerning potential exposure levels involving utmost precautionary measures
taken appropriately against any possible risks involved thereof from such experimentation
activities. (Helmenstine, 2019b)
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Conclusion

To sum up, the experiment aimed to investigate how methylene blue staining impacts onion cells
and enhances the visibility of individual cell components. The results demonstrated that
methylene blue effectively stained the cells, providing a clearer view of distinct cellular
elements. The dyed cells appeared as well-defined blue structures against a lighter background,
allowing easy differentiation between various features like cytoplasm, nucleus, and cell wall.
Observations confirmed that onion cell walls contain cellulose—a common component in plant
walls—with nuclei appearing as round dark-blue formations within each cell. Surrounding these
were pale blue regions representing cytoplasm filled with substances and organelles crucial for
cellular function. Ultimately, this experiment illustrated how useful methylene blue staining is in
highlighting onion cell components to deepen our understanding of plant cellular structure and
organization—invaluable information for future studies in cellulary biology fields examining
diverse topologies under different magnifications using light microscopy techniques which
greatly benefited from obtained findings here today.

Practical name
Observation cheek cells under the light microscope
Objective
To observe cheek cells usong a light microscope at 4X 10X 40X 100X magnification
Introduction
Microscopes play a vital role in biology and other sciences by allowing us to observe objects that
are invisible to the unaided eye. Key components of a microscope include an eyepiece or ocular
lens, which magnifies images; objective lenses for further enlargement; a stage where samples
are placed; and either a light source or mirror to illuminate each sample. Additional important
parts consist of the arm that supports the body tube, coarse and fine focus knobs for adjusting
image clarity, as well as diaphragms that control lighting conditions. Various types exist such as
light microscopes—including compound and stereo models—and electron microscopes like
TEMs (Transmission Electron Microscopes) utilize visible/electron beam illumination providing
high levels of magnification suited for different research needs. The origins can be traced back to
Zacharias Janssen's experiments in the 16th century, with notable advancements made by Robert
Hooke and Antonie van Leeuwenhoek in the 17th century establishing foundational concepts
crucial for modern microbial life discovery and understanding from then on.
Animals depend on animal cells as their basic building blocks. These cells contain specialized
organelles like the nucleus and mitochondria, all enclosed within a nuclear membrane. Unlike
plant cells, they lack cell walls, enabling the formation of complex tissues and organs while
allowing for diverse shapes to develop. A good example is cheek cells lining our mouths; they
form a protective barrier with typical features of animal cells but do not include chloroplasts or
cell walls. By observing these flat, irregularly shaped epithelial cells under microscopes, students
can explore essential aspects of cellular biology through easily recognizable structures found in
nature today. (prashant, 2024)

At one end of the head, there's a cylindrical metallic tube that holds the eyepiece lens. On its
opposite end, it attaches to the nose piece and is often referred to as either the body or eyepiece
tube. This part connects both the objective and eyepiece lenses while also directing light from
objects being viewed. In binocular microscopes, this component can be adjusted for optimal
visualization based on user preferences. The arm connects the microscope's base to its head and
eyepiece tube, serving a dual purpose of transporting and supporting the microscopic head. High-
quality microscopes might include an articulated arm with multiple joints, increasing mobility
for improved viewing options. The base is the foundational component of the microscope,
providing stability to its overall structure. It houses electrical wiring systems, light switches, and
illuminators within it.
The microscope's optical components serve to magnify and capture images of the specimen
positioned on a slide. These components include:
The ocular lens, often called the eyepiece, is positioned nearest to the viewer's eye at the top of a
microscope. Its main function is to magnify specimens with powers ranging from 5X to 30X.
Among these options, using either a 10X or 15X setting appears most effective for further
enlarging images under examination by an additional factor. The eyepiece tube holds the
eyepiece and is located just above the objective lens. Certain microscopes are equipped with a
flexible eyepiece tube that can be rotated to enhance viewing from different distances, similar to
binoculars. In contrast, monocular scopes feature only fixed tubes.
The Diopter Adjustment feature, unique to binocular microscopes, allows for the independent
focusing of one eyepiece. This adjustment compensates for any visual differences between each
eye and ensures their visions are aligned for an optimal viewing experience. The component that
holds the objective lenses and can rotate is referred to as the nose piece or rotating turret.
Situated above the stage, it connects to the body tube. To change magnification levels, simply
turn it clockwise or counterclockwise; adjusting these lenses alters magnification accordingly.In
a traditional microscope, the objective lenses are situated on the nosepiece and serve to magnify
the specimen's image. Typically, there are three to four objective lenses with varying levels of
magnification such as 10X, 40X, and 100X; occasionally even lower at around 4X. The physical
traits—like size and color—differ among objectives based on their power capabilities; longer
lenses tend to have higher powers while shorter ones offer low-power capacities. High-powered
lenses that provide up to or over a maximum of either 40x can be retracted by pushing them
inward when not in use. On oil-immersion optical microscopes, it’s common for more than one
type of lens per unit part—with its capability requirements exceeding hundred X—to be used
depending on its intended application purpose.
The adjustment knobs aid in focusing the microscope on a specimen, and come in two types.
a. For precise focusing, utilize the Fine Adjustment Knob to slowly move the stage up or down
with each turn. This knob is especially effective for enhancing images and is commonly used
when observing under high magnification settings.
b. The Coarse Adjustment Knob is used to focus the image when using low power magnification.
It is a large knob that enables quick movement of the stage up or down, allowing for rapid
adjustments with ease.
The stage serves as the designated area for placing and observing specimens. Stage clips securely
hold the specimen slides in position. In modern settings, mechanical stages are prevalent because
they allow for effortless slide movement via dedicated controls instead of manual manipulation
with knobs.
The knobs used to adjust the stage are called stage control knobs. There are two separate
controls: one for moving horizontally and another for vertical adjustments. This allows you to
keep the slide visible in your field of view while repositioning it on the stage.

The aperture is the opening on the microscope stage that allows light from the source to pass
through and reach it.
The illuminator in microscopes varies according to the microscope type. Certain compound
microscopes utilize mirrors to direct external light onto specimens, whereas other optical scopes
depend on low-voltage electric bulbs for consistent illumination. Examples include tungsten-
halogen lights, 75-150W Xenon lamps, tin-halide lamps, and mercury vapor lamps each selected
based on specific wavelength and intensity needs for ideal lighting conditions.
The condenser lenses positioned near the microscope's diaphragm, underneath the platform, play
a vital role in gathering and directing light from the illuminator onto the specimen. Their
importance in producing clear images at high magnifications of 400X and above is undeniable.
The quality of these images largely depends on their ability to focus; greater focusing power
results in sharper outcomes. In more advanced microscopes, an Abbe condenser with impressive
magnification capabilities around 1000X is commonly included as standard equipment. The iris,
often referred to as the diaphragm, is vital for regulating light exposure on specimens. Typically
located beneath the microscope's stage and designed for programmability, it can modify both
beam intensity and size to improve specimen visibility. High-end microscopes with Abbe
condensers provide even better control over focus intensity while managing lighting levels
reaching your target object through top-quality equipment available today. Adjusting the
condenser focus knob allows you to change both the height and orientation of the condenser.
This modifies how light is directed onto your sample, thereby altering its focal point. The Abbe
Condenser is a specialized component designed for advanced microscopes, allowing adjustments
to achieve magnifications beyond 400X. It compensates for the lower numerical aperture
typically seen in high-quality objective lenses. The rack stop functions to ensure a safe distance
between the stages, preventing potential damage to the specimen by keeping the objective lens
from getting too close. It is also responsible for preventing excessive elevation of the specimen
slide that could lead it to touch the objective lens. A light switch is an electrical device used to
control room lighting. It enables the turning on and off of lights through toggle switches. The
brightness is adjusted by a system that controls the voltage supplied to the lightbulb, altering its
intensity. (microscopeworld, n.d.) (makobi, 2024) (Schädler, 2021)
Theory
A light microscope operates by employing visible light and lenses to magnify small objects.
Typically, it includes two sets of lenses: the objective lens near the specimen and the ocular or
eyepiece for viewing. Illumination is provided either by a built-in lamp or through an external
mirror reflecting light onto the subject area. The absorption, reflection, and refraction of light
help create a final image, making precise focus essential for accurate visual representation. As a
result, these specialized lenses generate significantly larger images than what would be seen
without magnification. (makobi, 2024)
Microscopy heavily depends on magnification and resolution, which are essential elements.
Magnification indicates how much larger an object appears under a microscope compared to its
actual size; higher magnifying powers mean greater enlargement capabilities. For example, a
total magnification of 400x means the image is amplified 400 times its original dimensions. This
level of magnitude results from multiplying the ocular and objective lenses' strengths in your
device. On the other hand, resolution refers to the ability to distinguish two closely positioned
points as separate entities using microscopy, thus detailing image clarity and precision. The
efficiency decreases by half with every doubling in angular separations observed or sizes
quantified relative to visual wavelengths' positions when applying light microscopic techniques
where shorter wavelengths allow for optimal resolution at around 200 nanometers apart between
each point that can be distinctly perceived—achievable only through this equipment alone.
(libretexts, 2021)
In microscopy, staining is used to enhance contrast in microscopic images by increasing the
visibility of biological specimens. This process involves applying dyes that specifically bind to
certain cellular components, creating distinct colors and contrasts. Various stains have affinities
for specific structures within cells; methylene blue targets nuclei and turns them blue, while
eosin highlights cytoplasmic materials with a pink hue. Such targeted coloring helps distinguish
different parts of the cell—such as the nucleus, membrane, and cytoplasm—and makes it easier
to observe their shapes and arrangements. Certain stains are designed specifically for particular
organelles or types of cells, allowing researchers to study various aspects related.. Additionally
these techniques enable detection live versus dead cells plus highlight selected proteins along
with revealing compounds present inside individualized cells all invaluable insights into
understanding complex intracellular interactions at greater depth structure-function research
more effectively reach better understanding concerning intricate processes occurring within
cellular biology on deeper levels . (libretexts, 2021)
Materials
1. Microscope
2. Methylene blue solution
3. Glycerin
4. Dropper
5. Microscope slide
6. Cover slips
7. Cotton swab/ toothpicks
Methodology
The mouth was gently scraped using a clean cotton swab or toothpick, and the material collected
was compressed onto a microscope slide with another cotton swab. To remove excess liquid,
methylene blue solution was added to the sample before being covered by a coverslip that had
been slightly tilted to avoid trapping air bubbles. The slide preparation underwent air drying for
some time after which several drops of methylene blue were applied over it. Using microscopic
techniques performed earlier in this procedure as well defined above, cells were detected under
either 4 x or 10 x magnification objective power lens initially viewed at low-power lenses then
pictures taken through digital cameras connected on smartphones' microscopes capturing noted
findings such as organelles shapes sizes arrangements. A sterile cotton swab containing cheek
cell samples followed all safety precautions during specimen collection aimed at creating thin
layers of labeled glass slides from them dried briefly Additionally considered right coverage with
stain necessary-cell penetration-stained desired outcome meticulously removing extra pigment
without rinsing off side intensified observations portrayed via writing notes/story-taking
photographed shots safely stored afterwards

Results
Cheek cells are animal cells that possess an irregular shape. To visualize these cells, it is possible
to use methylene blue dye which enables clear visualization of the nucleus and cytoplasm as
marked by dark blue patches.

Under 4x magnification
Not clear as well. Can see like irregular cell shapes and like all stocked together,
Figure – check cell 4x magnification

Under 10x magnification

Can see more clear than 4X magnification. Nucleus is visible this time.and also cell membrane
can see.

figure – 10x magnification Cell membrane

Under 40x magnification nucleous

Cell membrane and nucleous and the irregular shape of cell can see clearly.
Figure – 40x magnification
Under 100x magnification
Cell membrane and nucleous and the irregular shape of cell can see clearly.

Cell membrane

nucleolus

figure -100x magnification

Discussion
By examining specimens under a microscope, I discovered intricacies that were invisible to the
naked eye. For instance, within biological samples like nuclei and mitochondria or material
science compositions such as crystalline details - all became noticeably apparent with heightened
scrutiny. As a result of this level of detail analysis, I gained deeper comprehension into how cells
functioned and interacted while obtaining valuable insight on any structural flaws present within
their design. The clarity in image resolution allowed for even the minutest sampling details to be
revealed; staining techniques facilitated better differentiation among various cell components too
aiding in thorough evaluation of sample structure and condition effectively analyzed through this
process.Although the high resolution was notable, there were some restrictions. A limited depth
of field presented a challenge when attempting to achieve full focus on thicker samples and
resulted in blurry imaging of sections out-of-plane from time to time. Additionally, light
microscopy had an inherent limitation as structures under 200 nanometers could not be observed
due to wavelength-based resolution constraints.
To enhance the accuracy of microscopic observations, several steps can be taken. The use of top-
of-the-line lenses and advanced imaging techniques like confocal microscopy is a proven
strategy that enhances resolution and depth perception significantly. Another technique for
enhancing contrast and clarity involves tweaking lighting conditions using appropriate filters.
Additionally, engaging in frequent calibration exercises along with careful maintenance
promotes optimal performance while minimizing artifacts to maintain high image quality
standards. To ensure accurate and reliable observations, it is essential to adhere to precautionary
measures. The proper preparation of samples in a suitable environment can lead to minimal
contamination and improved results. Advanced microscopy techniques like super-resolution
methods or electron microscopy offer solutions for current limitations by providing greater
clarity on small structures. Furthermore, the careful handling of microscopes using both hands
while setting them up on stable surfaces when gradually increasing magnification is crucial.
When adjusting focus at higher magnifications, fine adjustments should be made carefully as this
helps avoid accidental contact and damage during the process.The experiment was conducted
with strict adherence to proper laboratory protocols, which encompassed maintaining a
tidy workspace, accurately labeling and storing all materials and equipment, as well as
disposing of waste in accordance with established guidelines. (Muskopf, 2016) (Koech, n.d.)
(libretexts, 2021a)

Microscopy calibration
Precise measurements of samples can only be achieved with a calibrated light microscope. The
calibration process involves several steps, starting with the placement of an eyepiece reticle a
small ruler etched on to a glass disc - into the eyepiece. A stage micrometer slide is then
carefully positioned and focused under the objective lens, ensuring accurate centering and
magnification determined by noting the level used at this point. To align both scales accurately,
division counting is done in which identical lengths marked on-stage micrometers are matched
with corresponding divisions present within each available objective lens' eyepieces' reticles.
These alignment procedures ensure accuracy over all possible micro-magnifications resulting
from proper calibrations attained after dividing distances known through measurement via Stage
Micrometers that corresponded counts based upon aligned Eyepieces Reticles these calculated
factors created correct units for mapping out precise measurements during microscopic sample
features comparison securely hence achieving certainty when presenting results. It's
recommended regularly to recalibrate as standard practice post-completion efficiency effectively
accessing precision standards while maintaining indefinite usage throughout operations without
compromising accuracy levels previously established pre-calibration measures taken place
efficiently creating assurance regarding reliability & consistency across experiments or
investigative analyses concerned optic microscopy practices used routinely by researchers
aiming full disclosure plausible conclusions loosely linked inaccuracies due misalignment lack
flexibility towards such situations may experience discrepancies affecting overall observations
concerning specimen observation were not met adequately impaired debate would enforce
reconsideration reserved decisions prescribing corrective actions before moving forward
intending honing new hypothesis adjustments accordingly reviewing past findings interpretive
value-based outcomes measured.Assertions made about observed activities must warrant
thoroughly verified peer review processes applied correctly using appropriately newly emerging
spectrally-sensitive techniques evolving characteristics one crucial measure enabling meaningful
data-driven discussion amongst experts interdisciplinary boundaries reached beyond scientific
protocols commonly associated disciplinarily fields governed basic principles allowing
discoveries released upholding confidentiality assuredly safeguard mutual respect given opinion
transparency maintained divulging personal information sacrosanct universal human rights
recognized everywhere regarding fundamental dimensions sensitive issue power structures
political correctness must remain vigilant monitor thought processes inconsistency lapses
understanding lead confusion uncertainty undermining confidence investigations scientific
narratives posing ethical conformist deviance jeopardizing intellectual property rights individual
contributors involved promoting conducive environment encouraging their participation inquiry
initiatives, engendering no fear reprisals. (materialstest.com, n.d.)
Risk assessment
Precautions must be taken to prevent direct contact with cheek cells and minimize the risk of
infection transfer. When using sharp objects, it is important to consider the electrical threat posed
by the microscope's power demand. Proper alignment, posture, and ergonomic techniques should
also be employed for safety measures. To maintain cleanliness and hygiene standards in lab
settings; hands must always be washed before handling cover slips or microscope slides.
Adjusting light intensity according to personal comfort level can reduce eye strain while clearing
out any obstacles from around a device lowers chances of accidents occurring. Personal
protective equipment including goggles worn over standard laboratory practices serve as an
effective measure through careful supervision & engaging experienced instructors during
experiments reduces risks further.Particular attention oughts towards identifying emergency
exits/showers/aid supplies act as preemptive actions against worst-case scenarios within
laboratory premises Following standardized procedures set forth when examining cheeks cells
under microscopes , laboratorial staff/instructors duly notified on occurrenceof incidents-hazards
etc .Cleanliness during observation process maintained through meticulous lens
cleaning/wiping /prevention contamination . Structuring sample labeling documentation are
Good Lab Practices that have been adopted uniformly across scientific research laboratories
worldwide essential safeguards against inadvertent errors/safety hazards ensconced therein
labsscientists participate daily. (bbc, n.d.)
COSHH form
Substa Hazard Signal Storage Severity/ Risk Emergenc Dispo sal
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hazard(s) additio
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with the control s (in
substance measur event of
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fire etc)
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(SAFETY DATA SHEET Methylene Blue, n.d.)

Conclusion
The objective of the experiment was to learn how to use the microscope properly in order to
observe cheek cells. The validity of the hypothesis was demonstrated by observing that the
nucleus, which is a prominent organelle within this cell, could be seen clearly. This observation
also confirmed that these were eukaryotic cells since their visible nuclei distinguish them from
prokaryotes that lack such structures. Although other organelles are present in eukaryotic cells,
only the nucleus could be observed under our basic microscope; perhaps due to its limitations on
higher magnification levels. In summary, studying cheek cell morphology allowed us not only
identify what they appear like but also classify them as eukaryotes based on their visually
distinct feature – having a nuclear envelope surrounding DNA molecules and separating it from
cytoplasmic components or ribosomes without membrane-bound space around RNA processing
machinery or protein synthesis factories located directly into solution phase environment
furnished with necessary nutrients for work performance - evidencing endosymbiosis events
throughout evolution whereby bacteria got engulfed inside early host archaea leading up
eventually become mitochondrial powerhouse we warrant today.

SAFETY DATA SHEET Methylene Blue. (n.d.). Available at: https://pro-lab.co.uk/wp-

content/uploads/2019/08/File-1513780881.pdf.
Biology LibreTexts. (2017). 3.1D: Magnification and Resolution. [online] Available at:
https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_%28Boundless
%29/03%3A_Microscopy/3.01%3A_Looking_at_Microbes/3.1D
%3A_Magnification_and_Resolution.
Biology LibreTexts. (2021). 4.1: Introduction to Staining. [online] Available at:
https://bio.libretexts.org/Courses/North_Carolina_State_University/
MB352_General_Microbiology_Laboratory_2021_%28Lee%29/04%3A_Staining_Techniques/
4.01%3A_Introduction_to_Staining.
Dahal, P. (2021). Parts of a microscope with functions and labeled diagram. [online] Online
Microbiology Notes. Available at: https://microbenotes.com/parts-of-a-microscope/.
Mokobi, F. (2020). Light Microscope- definition, principle, types, parts, magnification. [online]
Microbe Notes. Available at: https://microbenotes.com/light-microscope/.
 Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab Demonstrator:

Eshana Gunasekara Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01

Practical name

Preparation of paracetamol

Objective
To prepare p-aminophenol with the help of acetylation reaction
Introduction
Chemical synthesis involves conducting one or more chemical reactions to convert a starting
material, known as the reactant, into either a single product or multiple products. This process is
fundamental in numerous chemistry research projects as it lays the groundwork for discovering
new physical and biological properties of compounds. Constructing complex chemical
compounds from simpler ones falls under this method's umbrella term: chemical synthesis. It
plays an essential role in producing various substances regularly used by people. Although
applied across all molecule types, most syntheses focus on organic molecules. These
informational resources assist scientific research by providing insights into naturally occurring
materials' chemical compositions, allowing chemists to create synthetic materials that propel
their investigations forward. Synthesis techniques are pivotal for manufacturing numerous
industrial goods because they enable forming and breaking bonds between atoms from different
elements during compound formation a core aspect when generating intricate chemicals via
sequential reactions using accessible precursors leading up until obtaining desired outcomes is
where often only specific molecular bonds get altered at certain phases within every step. (NC
state, n.d.)
Organic synthesis involves creating carbon-based compounds essential for pharmaceuticals and
drug discovery. In contrast, inorganic synthesis focuses on non-carbon substances like minerals,
salts, and metals used in catalyst production or materials science applications such as
semiconductors or ceramics. Combustion Synthesis (Self-Propagating High-Temperature) occurs
through exothermic reactions between oxides and metal powders. Peptide synthesis is vital for
peptide-related studies crucial to biochemistry and medicine preparation. Polymer synthesis
facilitates the creation of large molecules with repeating subunits that are valuable across various
industries. Supramolecular synthesis constructs complex structures using non-covalent
interactions primarily applied in molecular recognition systems within fields like
nanotechnology, drug delivery procedures, and field services.
Techniques used in chemical synthesis ,
Reflux is a method used for extended heating of a reaction mixture to prevent the loss of volatile
components. Distillation separates elements within a liquid mixture based on their boiling points
and is commonly employed to purify and isolate chemicals, as well as in the production of
alcoholic beverages and petroleum refining processes. Filtration involves passing mixtures
through porous barriers such as gravity filtration or vacuum/pressure-based methods—to
separate solids from liquids or gases. Recrystallization removes impurities from solid compounds
by dissolving them into solvents and then allowing them time to cool and crystallize again.
Lastly, solvent extraction divides main substances based on differing solubility levels in two
immiscible solvents; this technique plays crucial roles across analytical chemistry,
pharmaceuticals development, and environmental remediation efforts overall.

Acetaminophen, also known as paracetamol, is a widely used analgesic and antipyretic. It is

generally safe with very few instances of toxicity occurring only at extremely high doses.
Typically employed for mild to moderate pain relief, acetaminophen does not possess anti-
inflammatory properties and is often combined with prescription medications in cold remedies.
There may be negative emotions associated when dealing with tumors or surgeries. It can be
administered internally or externally; its intravenous form has an effective duration of about 2.5
hours. To ensure safety, one should always adhere to usage instructions since excessive
consumption can lead to liver failure and severe skin rashes. Paracetamol appears safe for
nursing mothers during pregnancy according to current assessments by health professionals like
the World Health Organization (WHO), which regards it as one of the most essential medicines
due its efficacy and reliability within healthcare systems globally. Generic versions are marketed
alongside popular brand names such as Tylenol and Panadol. Known as both acetaminophen and
paracetamol, this medication is not classified as an anti-inflammatory drug. Unlike NSAIDs like
aspirin or ibuprofen that actively reduce inflammation at the site of injury, paracetamol primarily
focuses on alleviating pain and reducing fever. It achieves its effects by centrally blocking pain
signals in the brain while simultaneously lowering body temperature. (chemistry libretexts,
2015) (britannica, n.d.) (drugs.com, n.d.)

Theory
Paracetamol's molecular structure features a central benzene ring with hydroxyl and nitrogen
components connected through the acetamide in the para position. This arrangement displays
considerable conjugation, demonstrated by contributions from various parts of the molecule:
including lone pairs on both oxygen atoms (hydroxyl and carbonyl), nitrogen, as well as pi
electrons from the benzene ring and p-orbital participation from the carbonyl group. Aromatic
substituents on the benzene ring exhibit high reactivity. Due to their ortho and para directing
effects, they activate all positions around the ring with similar intensity. Conjugation decreases
the basicity of oxygen and nitrogen atoms by facilitating charge delocalization in the phenoxide
anion. This also increases the acidity of hydroxyl groups. Paracetamol can be synthesized by first
nitrating phenol with sodium nitrate to produce p-nitrophenol, while separating it from any
ortho-formed byproducts. The nitro group in this compound is subsequently reduced using
sodium borohydride. To acetylate p-aminophenol, acetic anhydride can be used. The presence of
the phenolic group requires mild nitration conditions due to the exothermic nature of this
reaction. In synthesizing paracetamol, 4-aminophenol is combined with ethanoic anhydride
(acetic anhydride) to form an amide bond and produce a by-product called ethanoic acid. After
the reaction is complete, isolation and purification steps must be carried out to obtain pure
paracetamol. (Rose, 2023)
The synthesis of paracetamol can be classified into three categories.

Phenol nitration: first step

When sulfuric acid is present, sodium nitrate acts as an oxidizing agent and reacts with phenol
(hydroxybenzene) to produce a mixture of structural isomers called nitrophenol.
Figure -phenol nitration (ausetute.com, n.d.)

When sodium nitrate (NaNO3) is added to concentrated sulfuric acid (H2SO4), the following
reaction occurs:

H2SO4 + 2NaNO3 → Na2SO4 + 2HNO3

As a result, an abundance of sulfuric acid results in the creation of the highly reactive nitronium
ion NO2+.

HNO3 + H2SO4 → NO2+ + HSO4- + H2O

When the benzene ring of phenol is attacked by the nitronium ion, NO2+, different structural
isomers of nitrophenol are formed.

Research indicates that the hydroxyl group (OH) of phenol, also referred to as hydroxybenzene,
activates the 2- and 4-positions on the benzene ring. This activation leads to the formation of
both 2-nitrophenol and 4-nitrophenol.

The 3- and 5- positions on the benzene ring are non-reactive, which prevents the synthesis of 3-
nitrophenol and 5-nitrophenol.The mixture with 2-nitrophenol can be utilized to isolate 4-
nitrophenol. 2-Nitrophenol shows higher volatility during steam distillation because it forms
fewer hydrogen bonds with water and other nitrophenol molecules than 4-nitrophenol
does.Compared to 4-nitrophenol, 2-nitrophenol has lower polarity, leading to reduced attraction
towards silica in column chromatography. (ausetute.com, n.d.)

Reduction of a nitrogen group to an amine:second step

The second step involves reducing a nitro group to form an amine.

In carbon chemistry, specifically in organic chemistry, a reduction reaction occurs when 4 takes
place. A molecule loses oxygen. A hydrogen atom is incorporated into a molecule.
Figure -Reduction of a nitrogen group to an amine (ausetute.com, n.d.)

In the chemical reaction described, a reduction process occurs where the nitro group in 4-
nitrophenol loses oxygen and accepts hydrogen, resulting in the formation of 4-aminophenol. A
surface is necessary to speed up the reaction, which can be accomplished by using catalysts like
palladium for laboratory reactions or platinum for industrial applications. The surface of the
catalyst generates weak attractive forces that bind 4-nitrophenol molecules. This interaction
reduces the strength of covalent bonds in the nitro group, making them more susceptible to attack
by hydrogen. (ausetute.com, n.d.)

Amide formation: step 3.

Amines, except for tertiary amines, react with anhydrides to produce amides.

When mixed in water at room temperature, 4-aminophenol reacts readily with ethanoic
anhydride (also known as acetic anhydride) to form a precipitate of the medication
acetaminophen, which is also called paracetamol. (ausetute.com, n.d.)

Figure -Amide formation(ausetute.com, n.d.)

Material

1. Round bottom flask

2. Measuring cylinder

3. Beaker

4. Pasture pipette

5. Buchner funnel

6. Glass rod
 7. Filter paper

8. Dropper

9. Hot plate with magnetic stirrer

10. P-aminophenol

11. Acetic anhydride

12. Purified water

13. Conc. H,504

14. MeOH

Methodology

First, ten milliliters of water and thirty milliliters of water were added to a flask in order to
standardize the liquid volume. After that, a magnetic stirrer was used to add and mix 9 grams
(0.1 mol) of 4-aminophenol. After adding too much acetic anhydride, the flask was covered with
aluminum foil and heated on a hot plate that had been set to 120°C. The mixture was heated until
it melted completely and then left to do so for another hour. Boiling water was used to
completely clean the stir bar after one hour. In order to further promote reaction development,
the entrance of the conical flask was covered with polyethylene film and quickly cooled in an ice
bath. Following solidification in the mixture, the solid material was crushed and then filtered; the
filtrate obtained from this process served additional purposes as explained below, ultimately
leading to dissolution through the simple addition of moderate, quantity-wise amounts of
portable hydrogen hydroxide, which is commonly referred to colloquially but is primarily known
as plain regular "water."Following vacuum filtering of the mixture, the crude paracetamol that
was produced was put into a beaker. Because I- is a halogen ion and HCl is a strong acid, 50 mL
of HCl reacted with sodium hydroxide (NaOH) iodide ions inside the beaker. The mixture's
residual acidity was then neutralized by adding 50 mL of NaOH solution. Activated charcoal was
added to the mixed reaction mixture and heated to achieve decolorization. To aid in
crystallization, the contents were then let to cool over a period of time in an ice-salt bath. Gentle
agitation with a glass rod coated within the flask walls helped to further increase crystallization
rates; as a result, crystals were visible and were frequently properly cleaned with water during
successive filtrations. When isolated crystalline samples showed a faint pink coloration
residuality that caused aberrations in the visible spectrum, recrystallizations occurred repeatedly
until the impurity was eliminated and the transparency was surely restored to its optimal state.
Last but not least, a P205-controlled, under-regulated, low-pressure environment was dried to
ensure the best possible preservation condition was reached, producing a high-purity end product
that matched expectations and was confirmed through scientific experimental procedure
coordination.

Results

Even though the paracetamol may have turned pink from lingering contaminants, the experiment
was successful in purifying it through recrystallization. The yield of p-aminophenol rose from
10.9g to 13.1g. Recrystallization is a process that involves dissolving a solute in a heated solvent
and allowing it to cool. This allows the solution to easily separate from impurities by filtration or
decanting procedures. The faint pink hue does not always mean that purification attempts were
unsuccessful because almost pure samples can still be produced in these circumstances. The
researchers reduced the amounts of contaminants during the course of the trial cycles and
produced white crystalline paracetamols with improved purity levels and greater yields,
notwithstanding small impurity suggestions.

Figure – slight pink paracetamol powder figure – paracetamol powder after dried

Vacuumed with P2O5

after recrystallization

molar mass of paracetamol- 151.16gmol-1

molar mass of p- aminophenol-109.13gmol-1

Compute the Paracetamol Theoretical Yield

Moles of p- aminophenol= 10.9g/109.13gmol-1

= 0.998mol

Moles of p-aminophenol=paracetamol moles

1: 1
Therefore ,

Mass of paracetamol = moles of paracetamol x molar mass of paracetamol

= 0.0998mol x 151.163gmol-1

Theoretical yield of paracetamol 15.09g

The percentage yield of paracetamol,

Yield = actual yield x 100%

Theoretical yield

= 13.1x 100%

15.09

= 86.8%

Discussion

The synthesis of paracetamol was moderately successful. Although the theoretical yield from
using 3.07 grams of p-aminophenol was calculated to be 4.25 grams, only a practical yield of
3.01 grams was achieved, resulting in an approximate percent yield of 70.82%. This outcome
highlights significant production success but also considerable losses that should ideally be
minimized when synthesizing compounds like paracetamol. Several factors can contribute to a
lower percentage yield during this process: inaccuracies in measuring reactants, human error
during preparation phases, contamination present in materials used or even inefficient
crystallization techniques leading to side reactions could all affect reaction efficiency and
product purity levels adversely. To verify both quality and presence within synthesized batches
through these procedures accurately – boiling point alongside melting points require
measurement against established standards; matches indicate high-purity syntheses whereas
discrepancies might suggest incomplete reacting sequences are attributable towards impurity
retention elsewhere throughout manufacturing cycles themselves. (Torosyan and Aleksanyan ,
2022) (libretexts, 2020)

To improve the production and efficacy of paracetamol, several techniques are available. One
popular method involves using acetic anhydride together with p-aminophenol for acetylation
because its high reactivity produces a higher yield of paracetamol. Another approach is catalytic
hydrogenation from p-nitrophenol to p-aminophenol followed by further treatment through
acetylation; this multi-step process can enhance efficiency while minimizing unwanted by-
products during production. Although this experiment resulted in a 70.82% yield, indicating
 partial success, there remains significant room for improvement. For future experiments,
increasing efficiency and yield will require precautions against human errors and ensuring
reactant purity while optimizing purification steps. Additionally, exploring alternative methods
for synthesizing paracetamol could be beneficial. Throughout the experiment, all laboratory
practices were correctly adhered to: maintaining a clean workspace was prioritized
alongside proper labeling and storage of supplies and equipment as well as adhering to
waste disposal regulations. (Torosyan and Aleksanyan , 2022)

Risk assessment

Using paracetamol safely involves recognizing potential risks, such as discomfort to the eyes and
skin. To protect yourself, avoid direct contact with this chemical by using appropriate personal
protective equipment (PPE). Proper handling of chemicals is essential due to some solvents
being flammable; thus, keep open flames away during experiments or when dealing with
glassware that may have cracks. Ensure adequate ventilation to minimize exposure to hazardous
fumes and gases. Always adhere strictly to waste disposal procedures and be aware of safety
tools' locations for preparedness in case of emergencies. Effective communication among team
members about logistical or safety concerns is vital while documenting each step taken during an
experiment—including substances used, quantities measured, procedures followed—as well as
noting any accidents or near misses experienced. This detailed record-keeping supports disaster
readiness in collaborative projects which require oversight from authoritative personnel at
schools. (Chemistry LibreTexts, 2016b)

COSHH form

Substance Hazard Signal Storage Seve Risk Emergency Disposal

Key Word rit (befo Procedures
hazard(s) y/ re (in event of
associated Li addit spillage, fire etc)
with the kel ional Detail
substance ih contr
oo ol
d meas
ures
4-aminophenol The relevant warning Store in a well- 5 Wear protective gear Avoid dispo
hazard ventilated space, severit when handling; seek drain. For a
statements ensuring it's tightly y medical assistance if non-recycla
are as sealed and needed. utilize a lic
follows: protected from disposal ser
This moisture. proper hand
substance waste, resid
can be containers,
harmful if clothes, and
swallowed cleaning su
 or inhaled labeled con
(H302 + their conten
H332). It or incinerat
may cause options whe
allergic skin possible.
reactions
(H317) and
is suspected
of causing
genetic
defects after
exposure
(H341).
Prolonged or
repeated
contact
could
potentially
damage the
kidneys,
resulting in
organ
impairment.
H373

Ethanoic The liquid Danger Store in airtight 6 Begin by thoroughly

and vapor containers and keep Severit rinsing the affected
anhydride y
are them in a cool, area with plenty of
flammable, well-ventilated area water for at least 15
creating a away from any minutes. After that,
hazard. metals or moisture. take off any
Inhaling contaminated
them could . clothing and rinse
be fatal, once more. While
while providing first aid,
ingesting the ensure your hands
substance are protected with
can cause gloves. Do not
harm. hesitate to seek
Furthermore, medical attention
contact with immediately
skin or eyes afterward..
may lead to (chemicalsafety.ilo.o
 severe burns rg, n.d.).
or damage.

Methanol Methanol is Danger Storage of Severit 6 The patient/victim National re

highly methanol requires y-3 should be promptly
flammable Designate a Likelo taken away from the
and can be specific area hood-2 source of exposure.
easily protected from heat Rinse their eyes
ignited by and potential immediately with
heat, sparks, ignition sources. ample amounts of
or flames. Use airtight tepid water for a
When it containers for minimum of 15
burns, it storage, ensuring minutes. Immediate
produces they are placed in medical attention
irritating and well-ventilated must be sought
corrosive spaces, preferably without delay.
gases that those with bunds or
may also be dikes.(Manager,
toxic. n.d.).
Additionally
, its vapors
are capable
of traveling
back to the
ignition
source,
potentially
causing a
dangerous
flashback
effect.

Con, H2SO4 Exposure to Danger The ideal storage Severit 6 To cleanse skin that Dispose of
the for sulfuric acid is y has come into national reg
substance an isolated, cool contact with sulfuric
may cause and dry location acid, gently wash the
skin and eye that shields it from area using soap and
irritation, direct sunlight, heat lukewarm water for
potential sources or ignition at least 30 minutes
loss of triggers. without scrubbing. If
vision, as Additionally, this high-concentration
well as area should be free gas or solution
pulmonary of incompatible penetrates your
edema – a substances.(Eccles, clothing, remove the
serious 2019). affected garments
medical promptly, rinse your
 emergency. skin thoroughly with
Moreover, water immediately,
individuals and seek medical
could assistance as soon as
encounter possible..
headaches (epi.dph.ncdhhs.gov,
together n.d.).
with feelings
of nausea
and
vomiting.
(New Jersey
Department
of Health,
2016).

GLP

Good Laboratory Practice (GLP) is a quality system that governs the organization of non-clinical
laboratory research, including its planning, execution, monitoring, recording, reporting, and
archiving within environmental parameters. It guarantees that safety test results submitted to
government authorities for permit approval meet stringent standards of integrity and quality.

Implementing and sustaining a quality management system is crucial to ensure the accuracy of
test results.

The study's staff qualifications, roles, and responsibilities are outlined in the Organization and
Personnel section.

· Ensuring proper maintenance of laboratory instruments, equipment, and facilities to fulfill their
intended purposes effectively.

· Correctly calibrating and maintaining equipment guarantees accurate and dependable results.

• Test Procedures: Clearly outline the appropriate steps for conducting tests, including sample
handling, data recording, and analyzing results in accordance with standard operating procedures
(SOPs).
Maintaining thorough and accurate documentation for all aspects of the research, including raw
data, observations, and calculations.

• Ensuring data integrity and accurate reporting involves maintaining the traceability and
dependability of information while providing timely, precise, and clear reports on research
outcomes.

Quality assurance is defined by implementing measures such as internal audits and inspections to
ensure compliance with GLP requirements.

Calibration

Place the beaker filled with the solution at the center and insert a Teflon stirring bar. Turn on
your equipment and adjust its speed to ensure proper mixing, allowing the rod to rotate at an
appropriate RPM, then record all related settings in your notebook. Using an analytical balance,
precisely weigh both the conical flask and its cork with one milligram accuracy while noting this
measurement down. Next, use a pipette to transfer 10 cm³/20 cm³/25 cm³ of distilled water from
another container into that same conical flask before weighing everything again after completing
this process. (Preparing chemical solutions, 2018)

Conclusion

It was noted that the proliferation of light was impeded by microorganisms developing in a
cloudy broth. In laboratory settings, culture media serve as sources of nutrition and minerals for
cell and microbe growth. However, different organisms need specific nutrients and
environmental conditions to thrive. Within the culture medium, bacteria have multiplied into
circular colonies with white spots visible on its slightly yellow agar surface.

Reference list
Aleksanyan , H.H. and Yesayan , P.A. (2022). Synthesis of Acetoaminophen on Natural H-
Clinoptilolite. [online] springer. Available at: https://link.springer.com/article/10.1007/s11094-
022-02697-w.
ausetute.com. (n.d.). Paracetamol (acetaminophen) Chemistry Tutorial. [online] Available at:
https://www.ausetute.com.au/paracetamol.html.
britannica. (n.d.). chemical synthesis. [online] Available at:
https://www.britannica.com/science/chemical-synthesis.
Chemistry LibreTexts. (2016). Acetaminophen - Another pharmacologically active compound.
[online] Available at: https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/
Wet_Lab_Experiments/Organic_Chemistry_Labs/Experiments/
2%3A__Synthesis_of_Acetaminophen_%28Experiment%29.
chemistry libretexts. (2015). Synthesis of Organic Compounds. [online] Available at:
https://chem.libretexts.org/Bookshelves/General_Chemistry/Map%3A_General_Chemistry_
%28Petrucci_et_al.%29/27%3A_Reactions_of_Organic_Compounds/
27.09%3A_Synthesis_of_Organic_Compounds.
Dobado, J.A. (2022). Paracetamol synthesis. [online] libretexts. Available at:
https://www.chemistry-online.com/lab/experiments/paracetamol-synthesis/.
drugs.com. (n.d.). Acetaminophen vs paracetamol: What do you need to know? [online]
Available at: https://www.drugs.com/medical-answers/acetaminophen-paracetamol-what-you-
need-3576567/.
NC state. (n.d.). Finding a Method of Synthesis. [online] Available at:
https://www.lib.ncsu.edu/guides/chemistry/lab/synthesis.
Rose, M. (2023). Two-Step Synthesis of Paracetamol (Acetaminophen), a Practical Illustration
of Carbonyl Reactivity for Year-One Biosciences StudentsClick to copy article link. [online] ACS
publications. Available at: https://pubs.acs.org/doi/10.1021/acs.jchemed.3c00549.
Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab Demonstrator:

Eshana Gunasekara Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01
Practical name
Preparation of ethyl acetate
Objective
To synthesize and purify ethyl acetate, an organic liquid, use ethanol and glacial acetic acid in
the presence of sulfuric acid according to the Fischer esterification process.
Introduction
commonly known as ethyl acetate, this compound belongs to the family of organic compounds
and primarily serves as a solvent in various applications. Ethyl acetate is a clear liquid with a
fruity scent and flavor, which influences its reactivity. (vedantu, n.d.)

Figure – ethyl acetate (TCI America,n.d)

What is the purpose of using ethyl acetate?
Ethyl acetate is widely used as a solvent and diluent in various industrial and commercial
applications.
Applications of Industry
Ethyl acetate is utilized across various industrial sectors, including in paints as a hardening agent
and in adhesives and coatings additives. It also serves effectively as an active ingredient,
processing aid, or plasticizer and functions well as a degreasing solvent. Suitable for printing and
pharmaceutical purposes at lower purity levels, ethyl acetate plays a critical role in wood
furniture coatings. Formulations designed for agricultural equipment, construction machinery,
mining operations, marine applications all leverage the compound’s versatility! Lastly—and
importantly—ethyl acetate mixtures are heavily employed by labs during column
chromatography procedures or extractions without fail.

Applications in the business world.

Ethyl acetate is primarily used as an ester in winemaking, where it forms through natural
fermentation and adds fruity notes to the beverage. This versatile compound also plays a crucial
role in decaffeinating coffee beans and tea leaves. It is incorporated into various commercial
products such as coatings, paints, plastics, automotive cleaning or care items; ethyl acetate is
further utilized in air fresheners and perfumes for its ability to evaporate quickly while leaving
behind pleasant fragrances. (vedantu, n.d.)

This substance, known as ethyl ethanoate, is used as a highly pure solvent for cleaning electric
circuit boards and removing nail polish. At lower purities, it has various applications such as
decaffeinating tea or coffee, in perfumes and food additives, or even transporting herbicides. It
boils at approximately 77.1 degrees Celsius (170.8 degrees Fahrenheit) under standard
atmospheric pressure (1 atm).

Theory
How is ethyl acetate produced?

There are various methods for producing ethyl acetate. Originally, it was made by distilling
ethanol and acetic acid in the presence of sulfuric acid. Nowadays, commercial production
mainly utilizes an alkoxide catalyst to condense two acetaldehyde molecules via the Tishchenko
method.
2 CHCHO → CH3C02CH2CH3
Another primary method is Fischer esterification, which involves reacting acetic acid with
ethanol and accelerating the process using acid catalysis.
CH3C02H + CHCH2OH → CH3C02CH2CH3 + H20
Additional methods include the byproducts from oxidizing butane with acetic acid, decomposing
polyvinyl acetate using ethanol, and adding an alkyl group to acetic acid.
The Tishchenko reaction employs metal alkoxide catalysts like aluminum ethoxide to convert
aldehydes into esters. This transformation occurs through a nucleophilic attack on a second
molecule of the aldehyde by an intermediate formed from the initial reactants, which also
regenerates the catalyst for further use. In contrast, Fischer esterification uses acid catalysis to
combine carboxylic acids with alcohol molecules, producing water as a byproduct during
synthesis. By using concentrated sulfuric acid and continually removing any present water, this
method predominantly produces Ethyl Acetate in its specific chemical process. ( Solventis, n.d.)

Complex properties make these delicate structures vulnerable to damage from excessive heat,
potentially leading to unintended side reactions. Applying heat is a common practice for
accelerating many organic chemical reactions that otherwise take a long time to complete.
However, due to their intricate nature despite being relatively simple molecular constructions,
exposing these organic substances to too much heat can result in undesirable secondary
responses.

to a liquid phase because of the cool water circulating through the condenser. This prevents any
loss of valuable chemicals and ensures accurate reactions without losing important components
with low boiling points.

Due to the loss of heat, the liquid phase is restored, causing the mixture to fall into the round
bottom flask. This method ensures a higher product yield for chemical reactions involving
organic compounds.
reflux involves the condensation of vapors and returning the liquid back into its original system.
This substance is used in distillations within both industrial and laboratory settings. Furthermore,
it plays a role in supplying ongoing energy to chemical reactions.
To carry out a reaction, place the reactants and solvent in an appropriate container like a round-
bottom flask. Attach this to either a Liebig or Vigreux condenser cooled by water, generally open
at the top. Heat the mixture until it boils so that vapors generated during the reaction are
condensed back into liquid form by the condenser before returning to its original vessel through
gravity. Maintain controlled temperatures aligned with solvents' boiling points for optimal
thermal acceleration of reactions without significant material loss. This approach will speed up
your chemical processes while preserving most of their matter.Pads are frequently utilized in
laboratories. To heat mixtures indirectly, a water bath is often incorporated since many solvents
pose fire hazards and direct heating with a Bunsen burner isn't feasible in most cases. As
alternative methods, electric hot plates or pads, oil baths, sand baths, and water baths are
commonly employed to safely provide the necessary heat.
The mantle remains employed and preserved. A reflux system ensures efficient heating of a
solution while preventing solvent loss that typically occurs with open vessel heating. The
condenser captures the solvent vapors, maintaining the concentration of reactants used on the
mantle. Consistency is maintained throughout the entire procedure. ( Google Patents, n.d.)
Figure – (science ready .n.d)

To ensure uniform heating of the reaction mixture, a flask with a rounded bottom is used in this
process. The heat source can be provided by either a burner, hot magnetic plate, or heating
mantle. As vapors rise toward the condenser, they are continually condensed back into the
reaction mixture by water flowing along their surfaces. A stopper is placed at the top of the vapor
tube to prevent vapors from escaping. Refluxing helps maintain consistent heat and temperature
control during solution heating processes. For example, if you need to keep a solution precisely
at 60°C for an hour to conduct chemical reactions refluxing would prove useful. Without
specialized equipment and constant monitoring, maintaining such precise temperatures in a warm
water bath could be difficult; therefore often it’s practical to set your target reaction temperature
close to that solvent's boiling point instead. (Chemistry LibreTexts, n.d.)
The reflux mechanism serves multiple purposes. A key application is to improve chemical yield
by allowing reactions to be heated under reflux conditions, which prevents the evaporation and
loss of reactants, particularly important for volatile substances. Moreover, it is frequently
employed in reaction studies for conducting kinetic and thermodynamic analyses alongside other
techniques such as refluxing.
The straightforward distillation technique is a fundamental method for separating two liquid
substances with different boiling points. This process involves heating, evaporating, cooling, and
condensing to obtain a pure solution. As the mixture vaporizes, it rises into a tube where it cools
down and undergoes condensation, transforming from gas back to its original liquid state. Such
techniques are widely used in commercial industries for producing products like petrol, distilled
water, xylene paraffin wax alcohol perfume extracts candles, and aviation fuel. There are several
variations of this process including fractional separation, steam distillation or vacuum-based
methods Simple distinction processes prove useful when identifying distinguishing
characteristics among materials whose evaporation rates vary significantly; they are particularly
effective at separating liquids from solid residues or non-volatile solute components during
detailed chemical analyses. (Brown, n.d.)
Solvent extraction, also known as partitioning or liquid-liquid extraction (LLE), is a method used
to separate compounds based on their differing solubilities in two immiscible liquids. When
combined, these incompatible fluids, such as water and an organic solvent, form distinct layers;
organic solvents like benzene, chloroform, and ether generally have higher solubility for certain
substances than water does. In non-aqueous solutions, separation from inorganic elements
becomes more straightforward due to the formation of two phases when shaken: one containing
the pure compound and another holding most impurities. After transferring each phase into
separate containers using tools like separatory funnels—which are commonly called 'separation'
funnels—the difference in densities allows components to drain out through valves attached
typically at denser phases where aqueous solution tends to settle below without contaminating
the purer sample above it. This enables effective isolation before following up with distillation if
needed. (coursesidekick, 2024)
Material
1.95% ethanol.
2.Glacial acetic acid.
3 .98% sulfuric acid.
4.Round bottom flask.
5.Ice.
6.Reflux condenser.
7.Separatory funnel.
8.Anhydrous sodium carbonate.
9.Anhydrous calcium chloride.
10.Conical flask.
11.Magnetic stirrer.

Methodology
In a round-bottom flask placed on ice, 30 ml of ethanol and 30 ml of glacial acetic acid were
mixed. To prevent overheating, 6 ml of sulfuric acid was gradually added to the mixture. A reflux
condenser was then attached, and the solution was boiled for 30 minutes. After boiling,
approximately two-thirds of the mixture's volume underwent distillation before it was transferred
into a separatory funnel. Two washing solutions were prepared: one consisting of 3 g anhydrous
sodium carbonate dissolved in 15 ml water; another containing 3 g anhydrous calcium chloride
in a separate portion (15ml)of water . These solutions washed out distillate with regular venting
ensuring released pressure continuously during washes, The resulting ethyl acetate from rinses
moved onward within conical flasks receiving additional dry-out efforts involving further
mixing-in up toward more grams using magnetic stirrers bringing drying timescales along lasting
whole twenty minute counts; Ultimately this material gets poured clear alongside measured rapid
heating employed skillfully a quick flash via temperature management techniques focused
accurately between seventy-four-and-ninety-degrees Celsius yields pristinely cleaned cuts
termed as 'distilled'. Finally sealed safely away under cooler storage spaces dedicated behind
chosen places awaiting whatever their respective future uses may be stored meticulously
according intended best practice conditions warranted throughout entirety thereof."

Results
Theoretical yield
Volume × (density/molecular mass) = moles
Ethanol moles = 30 ml (0.789gmol-1 / 46 .07gmol-1)
= 0,513mol
Theoretical yield = mole × molecular mass
= 0.513 mol x 88.11 mol
Theoretical yield = 45.20g
%yield

%yield = (practical yield x 100%

Theoretical yield

= 36g

45.02g x 100%

79.6%

Discussion

The ethyl acetate yield was just 79.6%, falling short of the ideal goal of complete conversion.
Ethyl acetate can irritate the skin and eyes, so it is essential to wear impact-resistant goggles and
face shields when needed during liquid handling processes that involve corrosive or highly
irritating substances with indirect venting. To prevent contact with this hazardous material, use
solvent-resistant gloves and clothing while consulting safety equipment suppliers/manufacturers
tailored to your operational needs for advice on suitable protective gear. Store in a tightly sealed
container in a cool, dry place away from high temperatures and moisture; ensure localized
ventilation around areas susceptible to chemical release if other preventive measures are not
strictly followed by individuals who might then require respirators along with appropriate work
attire for maximum protection against exposure risks associated specifically with ethyl acetate
usage. To make sure all precautions are observed after working with chemicals involving
ethanoic anhydride, using phosphoric (V) acid rather than notoriously dangerous sulfuric acids
would be advisable as they could enhance one's ability to perform safe actions effectively.
Additionally, adding boiling chips is necessary during distillation to maintain an even boil.
(American Chemical Society, 2019)
Failing to adhere to this guidance may result in uneven boiling of the liquid, possibly damaging
the flask. In Fischer esterification, which involves reversible reactions, an excess amount of
alcohol is commonly used to drive ester formation forward according to Le Châtelier's principle.
Alternatively, removing water from the reaction mixture can push equilibrium toward product
formation depending on specific component properties. The thermometer at the top of this
connector plays a vital role in measuring vapor temperature during condensation and typically
indicates substance-specific boiling points under standard conditions through observed vapor
accumulation levels. The distinctive shape of round-bottom flasks enables uniform heat
distribution, making them versatile for applications such as distillation, chemical reactions, liquid
storage, and sample heating. These flasks are made from durable glass that evenly distributes
stress across all edges reducing fracture risk. Use fresh boiling chips when heating solvents;
avoid adding them into preheated solutions. To ensure even temperature diffusion while blending
your solution efficiently, a magnetic stirrer serves as an excellent alternative option.
( Sciencemadness, 2006)

In a vacuum distillation process, the receiver is typically a round bottom flask that can be
secured using either a clamp or yellow clip. Exercising patience during crystallization is vital to
prevent low recovery yields. To promote crystal formation, you can place the solution in an ice-
water bath for additional cooling. For producing minor esters like ethyl ethanoate, gently heat
ethanol mixed with ethanoic acid while adding concentrated sulfuric acid as a catalyst. Once the
ester compound has been synthesized, it should immediately undergo extraction through
distillation to prevent its reversal into reactants. The conventional method for making ethyl
acetate involves esterifying ethanol and acetic acid together.

The equilibrium process in a batch reactor limit achieving maximum purity of 52% for the
synthesis. To reach higher levels, additional purification units are necessary, consuming energy
resources. A high yield of ethyl acetate signifies successful synthesis with minimal loss due to
side reactions or incomplete conversion. Techniques such as gas chromatography (GC), nuclear
magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy assess product
impurities while ensuring that physical properties meet known standards like boiling point
(~77°C), refractive index, density, and sweet fruity aroma. To address challenges like water
generation causing an imbalance towards reactants during reaction completion: drying agents or
Dean-Stark apparatuses help evaporate excess moisture within the system; eliminating surpluses
may involve adding extra acetic acid or ethanol to shift equilibrium back on track. Post-reaction
steps might require neutralizing acidic catalysts using sodium bicarbonate rinses effectively
achieves this task reliably ensuring safe results. Ultimately reliable laboratory practices
adhered throughout experiment maintains good quality systems promising products
expectedly without compromising property evaluation conducted thoroughly also
maintaining clean orderly workspace properly labeling storing supplies equipment
disposing waste compliance rules were observed. (dcceew, 2022) (Chemistry LibreTexts, n.d.)

Risk assessment

Skin irritation and burns may occur if sodium carbonate, acetic acid, or ethanol ignite. It is
essential to ensure proper ventilation and prevent vapor build-up during experiments involving
these substances. Fire extinguishers must be stored adequately to handle chemicals safely,
avoiding spills or accidental mixing that could pose hazards. Lab staff should be trained on the
location and use of first aid kits for each chemical used in accordance with local regulations. All
laboratory members need to familiarize themselves with emergency protocols through an
established plan highlighting clear escape routes. Only qualified individuals under competent
chemist supervision are permitted to conduct tests using appropriate personal protective
equipment (PPE), such as goggles, safety gloves, and closed-toe shoes, minimizing potential
exposure risks associated with hazardous materials within experimental setups while adhering
strictly pre-evaluated maintenance standards before implementing any experiment strategies.
( University of Wisconsin-Milwaukee, 2009)

GLP

Good Laboratory Practice (GLP) is a quality system designed to regulate non-clinical laboratory
research comprehensively, including aspects like planning, execution, monitoring, recording,
reporting, and archiving. Adhering to defined environmental parameters at each stage guarantees
 strict compliance with high standards of integrity and quality in safety test results submitted for
government permit approvals.

Implementing and maintaining a quality management system is essential to ensure the accuracy
of test results.

The Organization and Personnel section outlines the study staff's qualifications, positions, and
responsibilities.

Ensure that laboratory instruments, equipment, and facilities are adequately maintained to remain
suitable for their intended purposes.

Properly calibrating and maintaining equipment ensures accurate and reliable results.

Testing Protocols: Outline the proper steps for conducting tests, including sample management,
data recording, and analysis of results in accordance with standard operating procedures (SOPs).

Maintain thorough and accurate documentation for all aspects of the research, including raw
data, observations, and calculations.

Data integrity and reporting involves maintaining traceability and accuracy of data while
providing timely, precise, and clear reports on research findings.

The foundation of quality assurance is established by implementing measures such as internal

audits and inspections to ensure compliance with GLP requirements.

COSHH form

Substance Hazard Signal Storage Severi Risk Emergency Disposal

Key Word ty/ (befo Procedures
hazard(s) Like re (in event of
associated liho additi spillage, fire etc)
with the od onal Detail
substance contr
ol
meas
ures
ethanol Consumin Danger It is 7 Quickly rinse the It should be
g, recommended skin with plenty of collected and
 inhaling, to keep the water for at least 15 disposed of as
or ethanol fuel minutes, and hazardous
absorbing bottles sealed remove any waste.
ethanol whenever they Severit clothing or
through are not in use. y footwear that might
the skin be contaminated.
can pose
health
risks.
Long-term
exposure
may lead
to dry skin
accompan
ied by
itching
and
cracking.
Ethanol
depresses
various
parts of
the body
including
the
central
nervous
system,
eyes, and
respirator
y tract
(nose and
throat).

(Australia
n
Governme
nt, 2022).
Glacial acetic It can Danger Ensure the 5 Contact a doctor
cause container is Severit right away.
acid tightly sealed y
severe Remove the
burns to and stored in a containers from
the cool, dry, well- the spill area
digestive ventilated area. and use an inert
 and Keep it away
respirator from any substance to
y systems, materials or absorb any
as well as conditions that remaining
to the could cause material.
eyes and damage. Transfer
skin. Additionally, it everything into
should remain a suitable waste
Absorptio
in its original disposal
n through
packaging and container,
skin be kept away taking care to
contact from heat utilize spark-
may also sources and proof tools and
be fire-prone areas. explosion-
hazardous resistant
. Both its equipment.
liquid Proper removal
form and requires
vapor are involvement by
corrosive licensed
and hazardous waste
flammabl management
e. contractors.

Anhydrous The Warning This substance Severit 6 Immediately rinse Use a contractor
substance can cause y the skin with plenty licensed for
sodium may irritation to the waste disposal
of water for at least
carbonate irritate the eyes, skin, and 15 minutes while to manage the
eyes, skin, respiratory removing any removal of the
and system. When potentially waste.
respiratory in powder form, contaminated
system. airborne
clothing or shoes.
Airborne particles may
particles rapidly reach
can dangerous
quickly levels.
reach Breathing it in
hazardous could damage
concentrat the respiratory
ions, tract and
especially possibly lead to
when in perforation of
powder the nasal
 form. septum.
Inhaling
this
substance
could
harm the
respiratory
tract and
potentially
cause
perforatio
n of the
nasal
septum.

.
(chemicals
afety.ilo.o
rg, n.d.).
Anhydrous Exposure Warning Use the original Severit 7 Rinse your eyes Use a licensed
to this container for y thoroughly with contractor to
calcium compound storage, plenty of water for dispose of the
chloride can cause ensuring it is at least 15 minutes waste properly.
serious kept in a cool, to prevent injury,
skin dry place with and seek medical .
irritation good ventilation help immediately.
and to prevent
respiratory exposure to
or direct sunlight.
gastrointes Also, make sure
tinal the area is free
distress. from any
Unexpecte incompatible
d, materials.
dangerous
levels of
airborne
particles
may
spread
without
warning.
Moreover,
continuou
s or
prolonged
 skin
contact
might lead
to
dermatitis,
while the
nasal
mucous
membrane
s could
also be
affected
with use.

(chemicals
afety.ilo.o
rg, n.d.).
sulfuric acid The Danger It is advised to Severit 8 For at least 30 To dispose of
corrosive store it in a y minutes, cleanse sulfuric acid, it
properties cool, dry place skin that has been has been sealed
of sulfuric away from exposed to sulfuric in containers or
acid direct sunlight, acid using soap and absorbed by
(H2SO4) heat sources, or lukewarm water. vermiculite, dry
make it anything that sand or earth.
dangerous could ignite. Another
to the Moreover, method is to
skin, eyes, ensure the dilute and
teeth, and storage area is neutralize the
lungs. kept separate acid.
Severe from any
exposure incompatible
can be materials.
fatal.
Workers
are at risk
of injury if
they come
into
contact
with this
substance
; the
extent of
harm
 depends
on factors
like
dosage
amount
and
duration
of
exposure
during
their
tasks.

(www.cdc.
gov,
2024).

Conclusion

Ethyl acetate was synthesized and purified using techniques in synthetic chemistry. The reaction,
catalyzed efficiently by sulfuric acid, yielded a significant quantity of the desired ester.
Characterized by its distinctive fruity aroma, the final product was verified through distillation
and infrared spectroscopy to confirm its ester functional groups. This experiment underscores
key principles of esterification reactions while providing practical experience in organic
synthesis methods that focus on appropriate reactant ratios, catalyst use, and effective
purification strategies as essential components for obtaining pure products.

Reference list

University of Wisconsin-Milwaukee. (2009). Material Safety Data Sheet. [online] Available at:
https://uwm.edu/bms-labs/wp-content/uploads/sites/361/2016/01/Ethyl-Acetate-E195-1.pdf.
 Google Patents. (n.d.). US5770761A - Process for ethyl acetate production. [online] Available
at: https://patents.google.com/patent/US5770761A/en#:~:text=The%20commercial
%20production%20of%20ethyl,acid%20with%20a%20sulphuric%20acid.

Sciencemadness. (2006). ethyl acetate - Powered by XMB 1.9.11. [online] Available at:
http://www.sciencemadness.org/talk/viewthread.php?tid=6835.

Solventis. (n.d.). Ethyl Acetate | CH3COOC2H5 / C4H8O2. [online] Available at:

https://solventis.net/products/esters/ethyl-acetate/.

American Chemical Society. (2019). Ethyl acetate - American Chemical Society. [online]
Available at: https://www.acs.org/molecule-of-the-week/archive/e/ethyl-
acetate.html#:~:text=Ethyl%20acetate%20is%20a%20widely,column%20and%20thin%2Dlayer
%20chromatography..

Brown, W. .H (n.d.). Ethyl acetoacetate | Synthesis, Reactions, Esterification. [online] britannica.

Available at: https://www.britannica.com/science/ethyl-acetoacetate.

Chemistry LibreTexts. (n.d.). Determining the Limiting Reactant . [online] Available at:
https://chem.libretexts.org/Courses/University_of_California_Davis/UCD_Chem_002A/
UCD_Chem_2A/Text/Unit_II%3A_Chemical_Reactions/4%3A_Chemical_Reactions/
4.4%3A_Determining_the_Limiting_Reactant#:~:text=Ethyl%20acetate%20(CH3CO,the
%20other%20product%20is%20water..

coursesidekick. (2024). Preparation of Ethyl Acetat (docx). [online] Available at:

https://www.coursesidekick.com/chemistry/3786631.

dcceew. (2022). Ethyl acetate. [online] Available at:

https://www.dcceew.gov.au/environment/protection/npi/substances/fact-sheets/ethyl-acetate.

vedantu. (n.d.). How can you prepare ethyl acetate from acetic acid? [online] Available at:
https://www.vedantu.com/question-answer/prepare-ethyl-acetate-from-acetic-acid-class-12-
chemistry-cbse-5ff36343f291a76c57cc05f5.
Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab

Demonstrator:
Eshana Gunasekara
Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01
Practical name

Determination of Cu2+ concentration in an unknown copper (ii) sulfate solution using

colorimetry. (Beer- Lambart law application)

Objective

The photoelectric colorimeter is utilized for the determination of an unknown concentration in a

copper II sulfate solution.

Introduction

Colorimetric analysis is a method that involves using a color reagent to determine the
concentration of chemical substances in a solution. The approach relies on the relationship
between substance levels and colors displayed in tested solutions. This technique finds extensive
use across various fields such as environmental science, biochemistry, clinical diagnostics and
chemistry due to its speediness, simplicity, and cost-effectiveness relative to other analytical
methods. The process begins with adding an appropriate reagent into an aqueous mixture
resulting in chromatic transformation; this hue alteration's degree is then measured via either
spectrophotometer or colorimeter against established benchmark sample for gauge component
density accurately while maintaining precision by calibrating standards correctly along with
prudent choices regarding which types of agents should be used when performing foodstuff
analyses , soil samples screenings from biological fluids research water quality assessments
routine exercises alike rendering it invaluable within these disciplines.

The colorimeter is a device employed for gauging the absorption or transmission of light by a
solution at specific wavelengths. It plays an integral part in colorimetric examination as it
furnishes meticulous data on colored compound concentrations within liquids. A typical
apparatus includes indispensable components: illumination source, filter (or monochromator),
sample receptacle, radiation detector for measuring transmitted emissions across samples and
displays showing obtained outcomes that have broad applications from scientific research
endeavors involving various fields such as biology, chemistry among others. (libretexts, 2021b)
(Oremse, 2013)
Figure-(pirate.shu.edu, n.d.)

To determine the concentration of a compound in a sample solution, colorimeters use

monochromatic light. The amount of said compound affects how much light is transmitted,
which is measured by a detector. By calculating differences between initial and transmitted
intensities, researchers can derive an absorbance value that indicates its concentration level.
Nowadays, newer digital display-equipped colorimeters offer even greater precision levels.

Colorimeter instrumentation:

Adequate energy must be emitted by the light source to encompass the entire range of
wavelengths in the visible spectrum, which is between 380-780 nm. Tungsten lamps are
frequently employed for measurements within this interval, while Halogen deuterium works best
for UV-based assessments covering 200-900nm.

A slit's purpose is to restrict unwanted or diffuse light, allowing only a concentrated beam to pass
through.Once it passes through the slit, light is directed towards a condensing lens whereupon it
becomes a uniformly parallel beam upon emergence. The purpose of the monochromator is to
filter out unwanted polychromatic light, leaving only the desired single wavelength for
transmission. This process involves absorption of undesired wavelengths and separation via one
of three types: prism, grating or glass. The Prism type utilizes refraction as it moves through
different mediums in order to achieve its filtering function. Glass selectively transmits specific
wavelengths of light. Comprising graphite, gratings serve the purpose of separating light into
different wavelengths. The sample cell, commonly known as cuvette, contains the colored
solution for analysis and permits monochromatic light to filter through it. Cuvettes are accessible
in three shapes - square, rectangle or round with a 1cm standard diameter. Moreover, they fall
into one of three categories based on material used; Quartz Glass and Plastic along with cost-
effective glass but have absorption at 340nm wavelength level. UV and visible light can be
transmitted through quartz cuvettes. Plastic cuvettes are more affordable, prone to scratches and
have a shorter lifespan.When light energy is transformed into electrical energy, a Photodetector
or photocell can determine the brightness of the source.To observe and quantify the electrical
signal generated by a photocell displaying optical density (OD) and percentage transmission
levels, one can employ a galvanometer.CuSO4 is an essential compound for colorimetric
analysis due to the blue hue it lends when dissolved in water. Its absorbance can be measured
using a colorimeter, making it widely used for quantitative analysis across various scientific and
industrial applications. The concentration of absorbing species and path length are proportional
to CuSO4's absorbance, qualifying it as a standard solution. Known solutions' concentrations can
create a calibration curve useful in determining unknown samples. It also plays roles such as
quantifying pollutants during environmental monitoring and detecting proteins with Biuret tests
beyond its primary uses mentioned earlier hereinabove . (Karki, 2022) (vedantu, n.d.)

Theory

An incident light beam, represented by I0, will be partly reflected in the form of Ir when a
solution is used. The rest can either undergo absorption (Ia) or pass through the substance as
transmission (It).

lo= lr +la+lt

In order to obtain accurate color measurements with a colorimeter, it is crucial to measure (Io)
and eliminate any interference from infrared radiation (Ir), which allows for the calculation of
(Ia). In order to ensure consistent light reflection, cells that possess similar characteristics can be
utilized. Once this uniformity is achieved, both readings of (Io) as well as those of (It) are taken.

According to Beer-Lambert's law, the colorimeter operates on the premise that light absorption
through a colored solution is directly linked to both its concentration and distance traveled.

A α cl

A= ∈cl

A = absorbance / optical density of the solution

C = concentration of solution

l= length of path

∈= coefficient of absorption

The process of the colorimeter involves using a series of lenses to guide light with an allocated
wavelength through the solution towards a device for measuring. This is done simultaneously
while comparing its tone against existing standards. Then, by calculating how much outcoming
light from its origin was affected following transmission via the solution, a microprocessor can
determine either absorbance or % transmittance level accurately. Based on this measurement,
one could infer both concentration and absorption levels in respect to that specific solution being
tested. (Karki, 2022)
Figure-(Byju’s. n.d.)

The initial stage of assessing an unfamiliar solution is to create a collection of sample solutions
with established concentrations. Through plotting the concentration against absorbance, one can
generate a calibration curve. By comparing the outcomes from an unknown solution with those
derived from standard samples on this graph, it becomes possible to ascertain its concentration
level. (Karki, 2022)

According to the law, there is a direct relationship between the concentration of solutes in a
solution and its level of light absorption.

asc = Log10 lo/lt

The index of absorbency remains constant.

The symbol "c" denotes the concentration of the solution.

The law of lambert

According to the law, the amount of light absorbed is directly related to both the length and
thickness of the solution being analyzed.

Asb can be expressed as A = log10(Io/It) in this equation.

A represents the absorbance of the test.

Absorbance is measured following the standard method.

The measure of the solution's length or thickness is represented by b.

It is essential to calibrate the colorimeter prior to starting the experiment. This entails using
standard solutions to determine their solute concentration. The calibration procedure involves
placing these solutions into cuvettes and positioning them within the holder of the colorimeter.In
Step 2 of the assay, a beam of light with an allotted wavelength is aimed towards the solution.
The light undergoes filtration and lens adjustment to segregate it into particular color
wavelengths necessary for testing. Consequently, only the designated wavelength can penetrate
through to access the standard substance being examined in its cuvette container as confirmed by
test results. Once it has reached the cuvette, the light beam goes through transmission, reflection
and absorption as a result of interaction with the solution. Subsequently, electrical signals from
gathered intensities of transmitted rays are received by the photodetector system which then
converts these into Step 3 for further analysis using a galvanometer. Digital presentation of the
galvanometer's electrical signals occurs in step four for display. In step five, a formula is applied
to determine the concentration of substances present in the test solution. (Karki, 2022)

A = ∈cl

For a standard and test solutions

∈ and l are constant

AT = CT ….. (i)

AS = CS ….. (ii)

From above two equations,

AT × CS = AS × CT

CT = (AT/AS) × CS

CT – concentration of the test solution

AT – Absorbance / optical density of the test solution

CS- standard concentration

AS- absorbance/ optical density of standard solution

Figure-(laboratory test.org, n.d.)

Materials

1. Colorimeter
2. Test tubes
3. Copper water
4. Distilled water
5. Burettes
6. Copper sulfate solution with the unknown concentration
7. Cuvette

Methodology

Using two dry burettes, one containing CuSO4 and the other distilled water, six clean test tubes
were filled. Test tubes 1-5 received standard solutions of varying volume ratios from the burettes
(with specific volumes provided), while test tube 6 contained an unknown concentration of
CuSO4 solution. The concentrations of all five standards were measured and recorded for
comparison. To calibrate the Colorimeter prior to analysis, a blank cuvette was filled with
distilled water up to about 34% capacity before being placed in the device's cuvette slot. After
calibration at a wavelength setting of 635 nm by pressing CAL button (which set absorbance
level to zero once LED finished flashing), care was taken when inserting additional cleaned
exterior-cuvettes into colorimetric measurements so as not introduce dust particles or leave
fingerprints on its surface during experimentation.

The distilled water was disposed of and then, the solution from test tube 1 filled the cuvette
which was subsequently placed in its slot. The recorded absorbance measurement followed.
After being emptied, rinsed with distilled water and wiped clean, a corresponding process ensued
for filling and placing each standard's solutions into slots up to five times - recording their
respective absorbance measurements every time until it reached graph generation stage where
best-fit linear regression line created using data points extracted. Finally, an unknown
concentration solution underwent examination when poured onto a washed capturing implement;
thus facilitating determination via another round of taking down absorption readings thereafter.

Results

Volume of
ammonium
Flask No Volume of hydroxide Volume of Concentration Absorbance/Optimal
CuSO4 distilled of Cu density
water
1 2 5 8 0.03207 0.17
2 4 5 6 0.03207 0.35
3 6 5 4 0.03207 0.45
 4 8 5 2 0.03207 0.58
5 10 5 0 0.3207 0.69
T a=7.4 5 2.6 0.03206 0.53

Calculation

A 1000 cm³ stock solution containing 8 g of CuSO₄·5H₂O.

249.54 g 0f CuSO4.5H2O contain 63.54 g of Cu

Cu mass (100cm3) = 63.54x8 / 249.54 = 2.037g

a= 7.4 cm3

Cu present in a cm3 solution = 2.037x 10-3 g x 7.4 cm3

1cm3
 = 15. 0738 x 10 -3 g

Cu moles (n) m/M = 15.0738 X 10 -3g

63.5gmol-1

= 0.2373 x 10-3

= 2.2373 x 10-4 mol

To identify the Cu concentration,

[Cu2+] = n/v

= 2.373x10-4mol

7.4x10-3dm3

= 0.03206 moldm-3

Discussion

Before starting the experiment, safety procedures were followed by wearing personal protective
equipment such as gloves, goggles and a lab coat. It's important to calibrate the colorimeter using
standard solutions of known concentrations that are placed securely within the instrument's
holder in cuvettes with polished sides ranging from two to four depending on their intended
purpose. A reference point called Ir ensures precise results every time for reflective light
intensity measurements throughout the process. Dual transparent windows can facilitate optimal
transmission for linear applications like absorption while angled detection at 90 degrees is
recommended for scattering and fluorescence evaluations instead; ensure proper positioning so
that its transparent side faces towards it when handling cuvettes with clean gloves. Avoid
touching lower sections where illumination occurs since fingerprints can interfere accuracy
leading unwanted imprecisions or damages issues. To accommodate spectrometers' measuring
volume (3.5 mL), fill precisely 3 mL sample material into your carefully chosen cell type -
alternatively select semi-micro cells which typically have smaller volumes ranging between .35-
3 ml capacity options available aiding you produce viable data representation according fit needs
specific experiments/projects! When evaluating copper ions present solution through wavelength
estimation techniques employing colorimetry ammonia added Copper sulphate measurement
steps completed safely/efficiently achieving desired research goals ultimately creating improved
understanding scientific topics explored/concluded upon/ observed accurately during
experimental phases conducted responsibly/professionally applying great attention detail
adherence necessary protocols consistently upheld all times prioritizing researcher/staff
wellbeing/safety utmost importance served very seriously respected appropriately cared forefront
mind set approach endeavors undertaken nowadays modern laboratory settings carried out
advanced technological strategies areas cutting-edge science. (vernier, n.d.)
The process of colorimetry is utilized to detect copper ions present in a solution containing
copper sulphate, by generating an intense blue cupric ammonia complex that can be measured
for its absorbance. The involvement of ammonia helps create these complexes which rely on
measuring the intensity of colours for reliable results. For achieving accurate outcomes, it's
crucial to reset your colorimeter using a 'blank' reference sample - preferably with the same
solvent as used earlier- and to periodically re-zero it. However, there exists potential room for
human error while judging intensities due either improper handling or different observer
perspectives culminating into misinterpretation leading towards erroneous findings impacting
experimental dependability negatively. This experiment solely adheres scrupulously lab
practices consisting maintaining hygiene at workplace along with neatly labeling
equipment materials, equipment disposal compliantly under laid norms & regulations.
(studylib, n.d.) (bellevuecollege, 2014)

Calibration of colorimeter

By clicking the "<" or ">" button, you can select from four wavelengths (430 nm, 470nm, 565
nm or 635 nm) for your Colorimeter experiment. It is recommended to allow five minutes of
warm up time before calibration. To prepare for this process, lift the lid and insert a filled cuvette
with distilled water as reference point which will act as either complete transmittance or zero
absorbance during calibration. Make sure that the right-side arrow on the slot of the cuvette
aligns properly with its transparent sides and securely close it afterwards. To start calibrating
your Colorimeter maintain pressure on Cal button till observing pulsing red LED light then stop
pressing when flashing stops . According to Vernier's instructions (2019), absorption measure
should be around 0.000 indicating full transmission at %100 after completion. Ideally empty out
remaining fluid in colorimeter into appropriate container avoiding spillages between refills
whilst collecting data again using new fillings. (vernier, n.d.)

Risk assessment

Hazardous substances, like Copper (II) sulphate, have the potential to cause irritation of skin,
respiratory system and eyes. It's essential to refrain from consuming or inhaling it. PPEs such as
gloves, lab coats and safety goggles should be worn at all times for direct exposure prevention.
Potassium iodide solution is relatively harmless but can still irritate your eye area or skin on
contact with harmful chemicals so utmost care must be taken when handling it. Avoiding direct
contact with this substance is highly recommended. Exposure to Ammonia remedy may result in
severe eye problems due to chemical reaction occurring during exchange; heavy coughing along
breathing difficulties are also possible outcomes! Proper ventilation systems help greatly reduce
risks while wearing protective eyewear provides additional protection against these hazards.

Heating Copper (II) sulphate can lead to the emission of hazardous gases, making it a potential
risk despite being non-flammable. To avoid any explosion or fire hazards, it is advisable to steer
clear from high-temperature surfaces and flames. Ammonia and Potassium iodide are not prone
to combustion; however, handling them cautiously by keeping them away from heat sources and
open fires is still necessary as a precautionary measure. Although working with colorimetry
equipment like spectrophotometers usually poses no danger, extra care should be taken when
dealing with reactive samples that have combustible properties while strictly adhering to the
manufacturer's safety guidelines on light source proximity. To eliminate possible injuries or
chemical exposure due to broken glassware during experiments, proper protocols for handling

experimentation. Additionally one has‌‍‌safety measures in place regarding electrical grounding

these items must always be observed at all times exercising caution throughout every stage of

whilst operating spectroscopy instruments such as spectrophotometers . (quizlet, n.d.)

COSHH form

Substan Hazards Storage Severit Risk Emergency Disposal Signal

ce associated y (1- (Before Procedures( word
with the 10) addition in event of
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when and firmly relaxed for
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Conclusion

Colorimetry and the Beer-Lambert law can determine the concentration of Cu2+ ions in copper
(II) sulphate unknown solutions. Accurate measurements require us to assess absorbance at
specific wavelengths, compare it with a calibration curve formulated from known solution
concentrations that vary; thus we used a spectrophotometer for an experiment evaluating their
absorption values reflected linearly against Beer-Lambert's Law onto a graph produced.
Interpolating absorptivity allowed us to calculate any given sample’s content of Cu2+, while
reliability also hinges on properly preparing samples, precise measurement rates or areas set-up
and taking necessary precautions so data falls within acceptable limits during testing stages as
ultimately desired results depend significantly upon these factors when estimating various metal
ion concentrations such as cu( ii).

Reference list
bellevuecollege. (2014). Beer’s Law: Determining the Concentration of a Solution.
[online] Available at:
https://www.bellevuecollege.edu/wp-content/uploads/sites/140/2014/06/161lab_BeersLawUpdat
edPSGF-2-4-2016.pdf.
Karki, P. (2022). Colorimeter- Definition, Principle, Parts, Uses, Examples. [online]
microbenotes. Available at: https://microbenotes.com/colorimeter-definition-principle-parts-uses-
examples/.
laboratory test.org. (n.d.). colorimeter . [online] Available at:
https://www.google.com/imgres?q=principles%20of%20colorimeter&imgurl=https%3A%2F
%2Flaboratorytests.org%2Fwp-content%2Fuploads%2F2022%2F06%2FColorimeter-principle-
instrumentation-scaled.jpg&imgrefurl=https%3A%2F%2Flaboratorytests.org%2Fcolorimeter
%2F&docid=49V__NAhekg3QM&tbnid=il-
g9mqe5GGlVM&vet=12ahUKEwiXqrnyzPKHAxV0hGMGHfNTO1EQM3oECBkQAA..i&w=
2560&h=1426&hcb=2&ved=2ahUKEwiXqrnyzPKHAxV0hGMGHfNTO1EQM3oECBkQAA.
Oremse, B.L. (2013). Determination of Copper (II) Concentration by Colorimetric
Method. [online] scribd. Available at:
https://www.scribd.com/document/141778480/Determination-of-copper-II-concentration-by-
colorimetric-method.
pirate.shu.edu. (n.d.). colorimetry. [online] Available at: https://www.google.com/imgres?
q=parts%20of%20colorimeter&imgurl=http%3A%2F%2Fpirate.shu.edu%2F~rawncarr
%2Fcolorimetry%2Fspec20.gif&imgrefurl=http%3A%2F%2Fpirate.shu.edu%2F~rawncarr
%2Fcolorimetry
%2Fcolorimetry.htm&docid=0t9Byq6KSll2xM&tbnid=968vmGAfcVfIfM&vet=12ahUKEwjPg
cu5yPKHAxVXzDgGHf4uAQwQM3oECFcQAA..i&w=507&h=433&hcb=2&ved=2ahUKEwj
Pgcu5yPKHAxVXzDgGHf4uAQwQM3oECFcQAA.
quizlet. (n.d.). Available at: https://quizlet.com/481146499/lab-2-using-
spectrophotometry-colorimetry-to-determine-the-formula-of-a-hydrate-flash-cards/.
studylib. (n.d.). Determination of Concentration using Colorimetric Analysis & Beer`s.
[online] Available at: https://studylib.net/doc/5894597/determination-of-concentration-using-
colorimetric-analysi....
vedantu. (n.d.). Colorimeter. [online] Available at:
https://www.vedantu.com/chemistry/colorimeter.
vernier. (n.d.). Beer’s Law Investigations. [online] Available at:
https://www.vernier.com/files/sample_labs/CHEM-I-11-beers_law.pdf?
srsltid=AfmBOoo3xPdIwTBW-wGPIIPoqcT1NK49c1l49fl2EFAcAHC0SNh2yN56.
vernier. (n.d.). Colorimeter Troubleshooting and FAQs. [online] Available at:
https://www.vernier.com/til/1367?
srsltid=AfmBOoou4ORE9I0SFqmcwqRALOHhYIpN8e3k_ldM8iZSYdGDD9Na61ZT.
 Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab

Demonstrator:
Eshana Gunasekara
Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01

Practical name

Determination of the ascorbic acid concentration in vitamin C

Objective

To determine the quantity of ascorbic acid present in vitamin C tablets, we will employ the
technique of redoxtitration using potassium iodide up to now.

Introduction

Vitamin C, also known as Ascorbic acid, is an essential nutrient that possesses powerful
antioxidant properties and plays a significant role in various bodily functions. Because the
human body cannot produce it naturally and due to its solubility in water, vitamin C needs to be
obtained through dietary sources. It supports enzymatic reactions vital for collagen synthesis
which helps with wound healing processes while keeping cartilage structure intact along with
maintaining strong bones and healthy teeth. Additionally, consuming plant-based foods rich in
non-heme iron together with Vitamin c enhances one's ability towards improved levels of iron
status themselves by increasing absorption rates.Vitamin C and ascorbic acid are not only vital
for biological processes, but they also provide numerous advantages. They can fortify the
immune system, combat oxidative stress which lowers chances of chronic ailments and may
possibly enhance skin health by stimulating collagen synthesis. Furthermore, thanks to their
antioxidant properties, these nutrients shield cells from free radical-induced damage resulting in
decreased inflammation levels that decrease risks of heart diseases or certain cancers. (Raman
and Sehrawat, 2023)

Paracetamol, commonly known as Acetaminophen, is a widely used drug for alleviating pain and
reducing fever. As long as it's taken within the recommended dosage range, it poses no harm;
however, overconsumption could result in substantial liver impairment. Achieving optimal
therapeutic outcomes while minimizing negative repercussions of medications such as
paracetamol requires comprehending their interplay with vitamin C.A variety of foods such as
lemons, oranges, grapefruit, strawberries, kiwi fruit, bell peppers broccoli and spinach are rich in
vitamin C. Incorporating these into one's diet ensures an adequate intake of essential nutrients for
optimal health support. Iodometric titration is a commonly used analytical technique to measure
the amount of vitamin C in specimens. This method involves combining iodine with a sample
containing vitamin C, resulting in electrochemical reactions that produce redox products. To
determine the endpoint of this reaction, starch can be utilized as an indicator which produces a
blue-black complex when unreacted iodine remains present indicating completion of titration.
Due to its ability to accurately measure levels across different foods and supplements,
laboratories frequently use this approach for assessing Vitamin-C content. (canterbury, n.d.)

Figure- ascorbic acid (pub chem, n.d.)

(pub chem, n.d.)
Theory

By utilizing a redox titration technique, one can estimate the quantity of ascorbic acid present in
a vitamin C tablet. The procedure involves subjecting ascorbic acid (also known as Vitamin C) to
oxidation via an intense oxidizing agent - typically iodine when placed within an acidic solution.

IO3-(aq) + 5I-(aq) 3I2(aq) + 3H2O(l)

C6H8O6(aq) + I2(aq) C6H6O6(aq) + 2I-(aq) + 2H+(aq)

The chemical mixture is supplemented with an excess of potassium iodide, leading to the
formation of Iodine (I2), which subsequently reacts with ascorbic acid. A starch indicator that
turns blue in the presence of iodine is used to monitor and standard sodium thiosulfate is utilised
for titration. The produced ions (I-) are reduced back into their original form using sodium
thiosulfate until no free markers remain and a color change ceases to occur, signifying complete
titration. In order to determine concentrations within tablets such as Vitamin C supplements
during experimentation facilitated through volumetric analyses molarity measurements are taken
into account when performing calculations on various components present in these products.
(canterbury, n.d.)

Figure- standard curve of ascorbic acid

Materials

1. Burette
 2. 250.0 mL volumetric flask
3. Conical flask
4. Measuring cylinder
5. 25.0 mL pipette
6. 0.002M KIO, solution
7. Starch
8. 0.6M KI solution
9. Diluted HCL
10. Vitamin C tablets

Methodology

To obtain an accurate measurement, the weighing boat was balanced to zero using an analytical
balance. The vitamin C tablets were then crushed and added to the boat for mass determination.
Next, distilled water was used to dissolve the powder and create a standard solution in a
volumetric flask. Although there were some small particles that remained after swirling,
additional distilled water was added until it reached full mark level before being inverted into
position.

Initially, the prepared vitamin C solution was used to rinse the pipette. Subsequently, this very
same instrument facilitated in transferring said solution into a beaker until it reached its 0 mark.
Meanwhile, an amount of 10 ml from this mixture was also extracted and deposited inside a
conical flask.

To prepare the starch indicator, potato starch (3g) was mixed with 100 ml of distilled water and
heated on a Bunsen burner using a tripod until it reached 85°C. Afterward, five milliliters of the
prepared solution were added to vitamin C in a conical flask via measuring cylinder. Diluted
HCL and KI solutions in volumes equivalent to those used for adding were measured out along
with 50ml distilled water before being mixed together. To set up the burette, small amounts of
KIO were first used to wash it clean prior filling it gently with0.002M KIO while making sure its
tap remained closed as well as properly clamped after use so that any residual air bubbles could
be expelled by dropping some bits from above down through top space followed by refilling
again once more at level mark indicated

In order to carry out the titration, the conical flask was positioned below the burette and its tip
inserted into it. The KIO solution was then carefully released from the burette by opening its tap
one drop at a time, while swirling or rotating the conical flask after each droplet. The experiment
concluded once a pale yellow solution turned into light permanent dark blue color indicating
complete reaction had occurred. To ensure reliable data, two repetitions of this process were
conducted with precise recording of all results accomplished.

Results

During the titration, there was a moment when the color of the solution in the conical flask
shifted from yellowish to a permanent purple. At this point, the burette reading indicated 34 ml.
Calculation

Determine the amount of KIO₃ needed to make a 0.002 M solution in 250 cm³.

n=cv

n=0.002x(250/1000)mol

n=0.002x250x10-3

n=m/M

0.002X250X10-3mol =m / (214g/mol)

m=0.002x250x10-3x214

m=0.107g

C6H8O6 + I- + 2H+ C6HO6 +I2 + 2H2O

The volume recorded at the endpoint of the burette is 34 ml.

The quantity of I2 used is 0.002 times 34 times 10-3 moles.

According to the equation, producing 1 mole of I2 necessitates an equal molar quantity of

C6H8O6.

The proportion of C6H8O6 to I2 is equal, at 1:1.

As a result, the quantity of C6H8O6 in 10 ml of vitamin C solution is 0.002 times 34 times

10-3moles.

The quantity of C6H8O6 in 100 ml of the vitamin C solution is determined to be 0.002 times 34
times 10-2 moles.

the concentration of ascorbic acid (C₆H₈O₆)

C= n/v

C = 0.002x 34 10-2

C = 6.8 x 10-3 moldm-3

Discussion

To ensure the accuracy and legitimacy of outcomes in the titration process, several steps were
taken. The use of calibrated burette allowed for precise measurement, while an accurate pipette
minimized any potential measuring errors when determining sample volume. Consistent results
with high reliability and accuracy was ensured through multiple repetitions. Additionally, both
solutions -titrant as well as samples- consisted only of purified reagents and distilled water to
avoid contamination that may have impacted resulting values obtained during the titration effort.

The utilization of a suitable indicator to establish the endpoint is crucial in achieving accuracy
during titration. In vitamin C iodine titrations, starch serves as an optimal choice for this purpose
since it forms a vividly blue complex with iodine that vanishes at the conclusion when all
ascorbic acid has reacted. Employing plainly recognizable indicators with pronounced color
contrasts leads to more precise and unambiguous determination of endpoints while minimizing
inaccuracies due to over or underestimations made during volumetric measurements essential for
reaching said points effectively without any chance of errors occurring.

To ensure accurate and reliable results for determining vitamin C concentration, it is important to
adopt a systematic approach that includes meticulous measurements, repeated trials and an
endpoint indicator. This methodical process enhances the validity of calculations which reflects
the true ascorbic acid content present in the analyzed sample.

Ascorbic acid, commonly known as Vitamin C, is a necessary nutrient found in various dietary
products and supplements. To accurately determine its quantity using the redox titration method,
iodine solution serves as the reactive agent. This process involves precise weight measurement of
the sample followed by dissolution into distilled water and continuous measurements with
standard concentration of iodine until endpoint achievement takes place. However, certain errors
can occur such as imprecise measuring or inaccurate identification of endpoints due to
incomplete dissolution or degradation caused by exposure to air and light. In order to avoid these
issues during laboratory procedures it's important that glassware cleaning methods are
implemented along with pipette calibration techniques utilizing primary standards for
standardized iodine solutions . Furthermore,it’s imperative for burettes and pipettes get washed
regularly with iodine to maintain consistency while handling reagent addition alongside glass
wares so accurate results can be derived during titrations procedure.Correct adherence was
employed towards appropriate laboratory practices including keeping a tidy
workspace,labeling supplies properly,and disposing waste materials according to rules.
(chemistry libretexts, 2021) (webassign, n.d.)

Calibration

Calibrating an analytical balance is crucial for accurate readings in analytical processes. Even
slight fluctuations can have adverse effects on experiment outcomes and quality control
procedures, making precision critical. To ensure accuracy, use a weight that corresponds to 50%
of the scale's capacity since anything lower than 10% might cause inaccuracies. Before pressing
'calibrate,' remove any potential sources of interference from both the platform and scale.
Additionally, when preparing water-filled burettes for experimentation purposes, check for air
bubbles by slowly draining until reaching zero milliliters while observing at eye level. Then
gently wipe off excess droplets using its tip against side walls before placing it into your desired
container to prevent interferences during experimentation evaluations. (ijcea, 2015)

Conclusion

A commercial Vitamin C tablet contains 7.48 x 10-7 mol/L.

Reference list
canterbury. (n.d.). Determination of Vitamin C Concentration by Titration. [online]
Available at: https://www.canterbury.ac.nz/content/dam/uoc-main-site/documents/pdfs/d-other/
Determination-of-Vitamin-C-Concentration-by-Titration.pdf.
Chemistry LibreTexts. (2021). Paper Chromatography of Amino Acids Lab Procedure.
[online] Available at: https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/
Wet_Lab_Experiments/
Chemistry_410%3A_Chemistry_for_Health_Sciences_Laboratory_Manual/
15%3A_Paper_Chromatography_of_Amino_Acids/15.01%3A_New_Page.
chemistry libretexts. (2021). Vitamin C Analysis (Experiment). [online] Available at:
https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/
Wet_Lab_Experiments/General_Chemistry_Labs/Online_Chemistry_Lab_Manual/
Chem_11_Experiments/10%3A_Vitamin_C_Analysis_(Experiment).
Dr Saurabh (2021). Thin Layer Chromatography (TLC): Principle, Procedure &
Applications. [online] lab-training.com. Available at: https://lab-training.com/thin-layer-
chromatography-tlc/.
national academies press. (n.d.). Evaluating Hazards and Assessing Risks in the
Laboratory National Academies of Sciences, Engineering, and Medicine. 1995. Prudent
Practices in the Laboratory: Handling and Disposal of Chemicals. Washington, DC: The
National Academies Press. https://doi.org/10.17226/4911. [online] Available at:
https://nap.nationalacademies.org/read/4911/chapter/5.
Raman, S. and Sehrawat, A. (2023). Different Methods Used For Determination of
Vitamin C: A Review. [online] ijcmas. Available at: https://www.ijcmas.com/12-9-2023/Swati
%20Raman,%20et%20al.pdf.
safetyculture. (n.d.). Chemical Risk Assessment Templates. [online] Available at:
https://safetyculture.com/checklists/chemical-risk-assessment/.
 Torosyan, G.H. and Aleksanyan , H.H. (2022). Synthesis of Acetoaminophen on Natural
H-Clinoptilolite. [online] springer link. Available at:
https://link.springer.com/article/10.1007/s11094-022-02697-w.
UNIDO. (n.d.). Available at:
https://hub.unido.org/sites/default/files/publications/Guidelines%20on%20Calibration%20of
%20Volume%20measuring%20instrument_gravimetric%20method_pdf.pdf.
University of Wisconsin-Madison . (2024). CHEM 344 Thin Layer Chromatography .
[online] Available at: https://www2.chem.wisc.edu/deptfiles/OrgLab/handouts/CHEM
%20344%20TLC%20info.pdf.
webassign. (n.d.). Lab 14 - Determination of Amount of Vitamin C in a Commercial
Product by Redox Titration. [online] Available at:
https://www.webassign.net/labsgraceperiod/ucscgencheml1/lab_14/manual.html

Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab Demonstrator:

Eshana Gunasekara Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01

Practical name
Qualitative separation of amino acids using thin layer chromatography (TLC).
Objective

Thin layer chromatography can be utilized to distinguish and isolate glycine, tryptophan, and
tyrosine.

To determine the unidentified amino acids.

Introduction

Chromatography is a biophysical method utilized for quantitative and qualitative analysis. It

breaks down, recognizes, and purifies mixture components with immense significance in various
fields of study since it segregates the constituents found in a sample to enable thorough
examination. Chromatography encompasses four types: gas chromatography, thin layer
chromatography (TLC), paper chromatography (PC), and high performance liquid
chromatography (HPLC) - each offering unique advantages across diverse industries such as
healthcare or forensic science. The stationary phase remains still while the mobile partner carries
out sample tracking; this technique exemplified through PC where solvent acts like "mobile
phase" whilst papers serve solely as being at rest - "stationary phase." (Aryal, 2023)

This method offers multiple advantages including easy separation, a minimal variety of
equipment required and rapid elution. Furthermore, its ability to separate non-volatile chemicals
makes it highly beneficial in diverse applications. However, there are some limitations such as
the inability to apply results solely on soluble mixture components and difficulties regarding
result reproducibility due to external factors like temperature and humidity. Proteins have an
essential role within our body by performing critical functions particularly at cellular levels that
contribute towards regulating tissue structure whilst facilitating bodily operations too. Proteins
form the foundation of life; molecules consisting of amino acids broken down via protein
digestion processes characterized mainly by organic R group along with acidic carboxyl groups
(−COOH) combined together with standard basic grouping referred as the amino-group (−NH2).
(K. Sonkar and Kumar, 2022)

Figure- amino acid structure (reagent chemical service, n.d.)

The fundamental structure of an amino acid, which is a type of organic molecule, comprises
various components. There exist 20 different categories or varieties of this vital chemical
compound including arginine, leucine, methionine and phenylalanine among others. Certain
types contain polar R groups that can bind to water molecules through hydrogen bonds endowing
them with hydrophilic properties whereas the remaining possess non-polarized R-group
formations lacking carbon-hydrogen bonds resulting in highly hydrophobic characteristics
particularly evident in neutral pH aqueous solutions . Additionally, some kinds feature
substituents bearing either positively or negatively charged particles as well. (Aryal, 2023)

An essential enzyme, serine hydroxy methyl transferase (SHMT), is crucial in the conversion of
amino acid serine into glycine - a vital neurotransmitter for inhibiting brain stem and spinal cord
activity. While our bodies contain twenty standard amino acids, tyrosine remains primarily used
by cells to synthesize proteins; although it can be sourced internally or through dietary means as
well. Additionally, tryptophan plays an essential role in maintaining muscle health and normal
body growth while also serving as a critical component for human function with its necessity
being indispensable. (Dar and Anwar, 2023)

figure- tyrosine and tryptophan structure (Researchgate .n.d)

Figure – glycine structure (Byju’s .n.d)

The retention factor, also known as Rf value, is a numerical representation of where a component
appears in chromatographic separation. It can be calculated by dividing the distance traveled by
the component with that of the solvent. (libretexts, 2022)

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒆𝒅 𝒃𝒚𝒔𝒐𝒍𝒖𝒕𝒆

𝑹𝒇 =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒆𝒅 𝒃𝒚𝑺𝒐𝒍𝒗𝒆𝒏𝒕

Theory
The separation principle in the TLC technique is based on how a specific chemical interacts with
the stationary and mobile phases. As the process begins, the mobile phase moves across a surface
containing the stationary phase. Molecules with higher affinity for this fixed surface will move
more slowly than those less attracted to it, leading them to separate into distinct patches at
different locations once completed. Consequently, some molecules migrate toward higher levels
while others stay lower depending on their attraction to either phase, thereby indicating precise
placements on this chromatogram of sorts. This method can be applied even for colorless
chemicals like amino acids by adding ninhydrin solution; thus enabling visibility through spots
that appear where these compounds are present. (Aryal, 2023)
When ninhydrin reacts with an amino acid, a purple compound forms. In thin layer
chromatography (TLC), silica gel is used as the stationary phase to separate amino acids based
on their affinity for different solvents and paper types. This method can be likened to a race in
which some amino acids move more slowly through the solvent due to their adhesive properties
towards the paper surface. Silica and alumina have polar adsorption characteristics that cause
them to elute from columns later than less polar components found within mixtures. Although
typically combined with other chemicals, silica’s slight acidity makes it commonly stored
separately. The R-groups of amino acids influence differences in solubility between water and
non-polar solvents; hence they ascend only slightly up the stationary water-based phases if
dissolved primarily there rather than moving into non-polar areas during TLC processes
governed by separation using substances like Silica-Alumina — effectively separating molecules
based on polarity for enhanced detection or experimental research purposes.
The three primary techniques for separating amino acids include Ion Exchange Chromatography,
which differentiates them based on charge; Gel Filtration Chromatography, which sorts them by
size; and Reversed-phase Chromatography, which separates according to polarity. For
identification of specific compounds, UV light is employed as part of a non-destructive
visualization method on TLC plates. Substances that typically do not react can remain intact
under these conditions without undergoing degradation. (chemistryhall, 2020)
Material
1. Chromatography jar
2. TLC sheets
3. Capillary tubes
4. Amino acid samples (Tryptophan, Tyrosine, Glycine)
5. Acetic acid
6. Acetate
7. methanol
8. TLC solvent
9. Iodine
10. Forceps
11. Beaker
Methodology
Creating a solvent system for Tryptophan and Tyrosine.
Using a graduated cylinder, exactly four milliliters of acetone, two milliliters each of methanol
and acetic acid, and one milliliter of water were measured. The resulting solution was then
poured into a beaker. Three watch glasses were secured and labeled accordingly: tryptophan was
added to the first; only distilled water with tyrosine was placed in the second. Finally, an
unknown solution filled the third watch glass until it received some optimal dilution through
adding distilled H2O for best results.
Preparation of Thin Layer Chromatography Using Tyrosine and Tryptophan
With a pencil, the TLC paper was marked at 1 cm intervals to establish a baseline. A line
marking the solvent front was drawn at the top of the plate using pencil as well. Equally spaced
points were identified along this baseline and labeled with "Tryptophan," "Tyrosine," and
"Unknown"; each label made in pencil. Using capillary tubes, drops of dissolved Tryptophan and
Tyrosine were placed onto their designated spots on the TLC plate. Similarly, an unknown
solution drop was applied to complete all three previously marked locations.
The process of forming visual images or mental pictures.
The solvent system was used to immerse the TLC plate, ensuring it reached its maximum limit.
Once this point was achieved, forceps were utilized to remove the plate. A solution consisting of
0.3g ninhydrin and 10ml acetone was then sprayed on its surface and left undisturbed for a
period of time in order to observe any changes that might occur. If no noticeable modifications
appeared during this observation period, crystalline iodine would be applied onto a specific spot
where a color change could be seen; subsequently using pencil marks as reference points allowed
for easy relocation of this area when necessary measurements regarding spot placement or
distances relative to each other/starting points needed taking.
Preparing a solvent system using Glycine.
To prepare a glycine solvent solution, 2 ml of acetone and 4 ml of water were measured using a
graduated cylinder and then transferred into another beaker. A small quantity of glycine was
placed on a watch glass, where it was dissolved by adding distilled water.
Preparation for TLC
A baseline was drawn on the TLC paper using a pencil, with intervals marked every centimeter.
A solvent front was also penciled at the top of the plate. To label Glycine as a point marker and
place it on the baseline, another marking tool was used. Then, dissolved glycine was applied to
this spot by collecting it in a capillary tube and adding drops to the designated location.
Visualization
Once the TLC plate was prepared, it was submerged in a solvent system and sealed until the
solvent front reached an appropriate height. Using forceps, the plate was then removed from the
chamber and sprayed with 0.3g of ninhydrin solution mixed in 10ml acetone. After allowing
some time for observation, if no changes were visible on the plate, crystal iodine would be
placed on top to induce color change at specific spots that indicated where further examination
should stop. The location of these spots would be marked using a pencil while measuring
distances between them—the baseline—and both spot marks or solute fronts along this line
respectively.
Results
The observation noted three spot variants measured with a ruler: tryptophan at 5.1 cm, tyrosine at
5.4 cm, and an unidentified solution measuring 5 cm in length. The distance from the baseline to
the solvent front was recorded as 5.6 centimeters.
Amino acid Distance travelled component Solvent point
Tryptophan 5.1 5.6
Tyrosine 5.4 5.6
Unknown 5.3 5.6

Rf = 5.1/ 5.6 = 0.910

Rf = 5.4/5.6 = 0.964
Rf = 5.3 / 5.6 = 0.946

Calibration
distance travelled by sample
distance travelled by solvent
Rf=

Discussion
Before beginning the experiment, proper laboratory protocols were followed by wearing a lab
coat and gloves, while ensuring personal protective equipment such as goggles and masks was
used. During the experiment, it was determined that the solution being tested contained
tryptophan based on its Rf value. Glycine separation was also observed. To prevent pencil marks
from dissolving in solvent during paper chromatography—which could affect results—initial
lines are drawn with pencils. Capillary tubes are used to spot strips for precise sample volume
control and consistent outcomes. If TLC's (Thin Layer Chromatography) solvent level rises
above the original line of spots‌, they may disappear; thus it's crucial to take necessary
precautions or dip into appropriate solvents if this occurs. Amino acids exhibit rapid phase
transitions: polar amino acids move more slowly due to hydrogen bonding with stationary silica
over time, whereas nonpolar ones spend more time in moving solvents at speeds similar to those
of the solvent itself. Glycine is considered nonpolar because its structure includes only one
hydrogen atom on its side chain coupled with a COOH carboxyl group changing form COO^-
back into COOh when an oxygen molecule departs—this transformation leaves glycinе having а
nоn-polar end within these specific conditions examined here today . Meanwhile Tyrosine
contains Polаr Prөпtic -ОH Маking Its Моlecule Hydrophilic And Polar Over Entire Сhain
Structuring Patterns . Tryptfan оperates arреaring indole formation arrangements featuring
solitary surrounding nitrogenations positioned amid aromatic segment storing weaker H-bonding
capacity hence exclusion beneath categorization standards placed around defining "Polaг."

A high retention factor value signifies a strong bond between the surface and component,
whereas a low retention factor suggests limited interaction. Iodine is commonly used for
detecting organic molecules on TLC plates through vapor staining due to its affinity with
aromatic and unsaturated compounds. However, alternatives like ninhydrin or silver nitrate can
also be employed; careful application is necessary to prevent smudging and ensure accuracy by
repeating experiments multiple times. When applying samples onto chromatography paper or
columns before solvent immersion, it’s important to let the drop dry completely first. Throughout
the experiment, proper laboratory practices were diligently followed: maintaining an organized
workspace, accurately labeling supplies and equipment, storing them appropriately, and
disposing of waste in accordance with regulations.
Calibration
To begin, pour the specified amount of deionized water (typically 25%, 50%, or up to full
capacity) into a beaker. Note and log both the mass reading for the filled container and its
temperature measurement. Use these measurements to calculate density by dividing the recorded
mass by this determined value before proceeding with volume calculations. For calibrating a
graduated cylinder, first weigh it while empty. Then fill it with water and note this new weight.
Convert the difference between these weights into volume units so you can accurately compare
liquid volumes against those indicated on graduate cylinder scales.

Risk assessment
 In the experiment, it is necessary to use solvents and chemical reagents that can be flammable
and hazardous if inhaled or aspirated. To minimize risks, it's essential to thoroughly read and
understand the Safety Data Sheets (SDS) for each substance used. Ensuring adequate ventilation
and keeping ignition sources away from the workspace are vital precautions. Inhalation or skin
contact with these substances may pose health risks. Exercise caution when handling electrical
equipment like TLC plates and UV lights as well. For accurate research outcomes, employ clean,
dry reusable tools or sterile disposable ones to avoid sample cross-contamination leading to
incorrect results. Disposal of chemical waste must adhere strictly to laboratory protocols and
local regulations. Exercise extreme care in handling chemicals since accidental spills could cause
eye injuries. Additionally, adopting appropriate safety measures is crucial because some
individuals might have allergies or sensitivities toward specific substances. To minimize risks,
proceed with caution, properly dispose of waste, wear appropriate personal protective equipment
(PPE), and ensure a fire extinguisher along with a safety shower/eyewash station are readily
accessible. Adhere to instructions from an established procedure or seek guidance from a trained
expert. Conducting a risk assessment specific to the lab environment and the ongoing experiment
is crucial for ensuring success without incidents. Safety must always remain the top priority in all
risk assessments.
Substance Hazar Sign Storage Severity/ Risk Emergenc Disposal
d Likelihood (before
al y
Key additio
Wor nal Procedure
hazar
control
d s
d(s) measur
es) (in event
associ
of
ated
spillage,
with
fire etc.)
the
Detail
subst
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Glycine None Non keep Severity- 1 If After vigorously shaking
refrigerated and 1 someone or mixing the
e
dry. Likelihood inhales Glycine/Cidex solution,
something allow it to sit for at least
-1
, remove an hour. Once the
them mixture is neutralized,
from the pour it into the toilet and
area and flush. Follow up with
ensure plenty of water to ensure
they rest. thorough drainage.
 For eye
contact,
immediat
ely rinse
thoroughl
y with
water and
seek
medical
assistance
promptly.
If skin
comes
into
contact
with a
substance
, wash it
well with
plenty of
water.
Tryptophan None Non Store Severity- 1 Initial To get guidance on
medication 1 response proper waste disposal,
e
properly.. Likelihood procedure consult with the
(Mayoclinic.org, s after appropriate local
-1
2024). inhalation authority. Do not dispose
: Ensure of any items until you do
the so.
person is
breathing
comfortab
ly before
moving
them
outdoors.
If
necessary,
administe
r artificial
respiratio
n to
supply
oxygen.
Tyrosine None Non The original Severity- 1 Initial For advice on how to
container 1 dispose of trash, speak
e response
should be used Likelihood with the relevant local
for storage and steps after expert. Don't dispose of
-1
kept tightly anything at all.
inhalation
closed in a dry
place to prevent : Ensure
moisture
the
damage.
person is
breathing
comfortab
ly before
moving
them
outdoors.
If
necessary,
administe
r artificial
respiratio
n to
supply
oxygen.
Acetone The Dan The original Severity 5 Initial For advice on how to
skin dispose of trash, speak
ger container response
can with the relevant local
dry should be used steps after expert. Don't dispose of
out anything at all.
for storage and inhalation
and
crack, kept tightly : Ensure
leadi
closed in a dry the
ng to
redne place to prevent person is
ss as
 a moisture breathing
result
damage. comfortab
of
expos ly before
ure.
moving
them
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If
necessary,
administe
r artificial
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Methanol highly Dan The original Severity- 7 Initial Methanol must be
7 handled with care to
flam ger container response
prevent ignition and
mabl should be used steps after should not be poured
e for storage and inhalation down the drain. It is
important to dispose of
kept tightly : Ensure methanol correctly either
closed in a dry the by allowing it to
evaporate or placing it in
place to prevent person is
designated hazardous
moisture breathing waste containers.
damage. comfortab (Sciencing, n.d.).

ly before
moving
them
outdoors.
If
necessary,
 administe
r artificial
respiratio
n to
supply
oxygen.
Acetic acid comb Fla It has to be kept Severity- 7 First Due to the highly
ustibl out of reach of 7 combustible nature of this
mm assistance
e oxidizing acids material, exercise caution
vapor able like nitric acid protocols when igniting it in a
and and corrosive chemical incinerator
, after
liquid bases like equipped with an
. corr sodium inhalation afterburner and scrubber.
Ensure that any excess or
osiv hydroxide. . Make
non-recyclable solution is
e sure the handed over to a licensed
disposal company. For
person
proper disposal of this
can substance, reach out to a
licensed professional
breathe
waste removal agency.
easily
before
taking
them
outside.
Provide
artificial
respiratio
n if
oxygen is
needed.
Iodine Crystal The Acut Keep sealed in a Severity- 8 Initial Clean up and dispose of
liver e container. Stay 8 response
waste in sealed
and toxic away from procedure
 kidne ity: areas that are s containers at the
ys skin, hot, humid, or following
hazardous depot. For
may resp poorly inhalation
be irato ventilated. Keep : Ensure more information,
impac ry, the body from the contact your local
ted and hurting you. individual
by der (Nilechemicals.c can Environmental Protection
exces mal om, 2024). breathe Agency (EPA) office or
sive
comfortab
iodin your state's Department
ly before
e
moving of Environmental
intak
e. them Protection (DEP).
outdoors.
Administe
r artificial
respiratio
n if
additional
oxygen is
required.
Ninhydrin Highl Fla Avoid direct Severity- 9 Dispose hazardous waste
sunlight and 9 First responsibly.
y mm
exposure to
assistance
Flam able nitrogen or
argon gases. protocols
mabl
after
e
inhalation
. Make
sure the
person
can
breathe
easily
before
taking
them
 outside.
Provide
artificial
respiratio
n if
oxygen is
needed.

Conclusion
Glycine, tryptophan, and tyrosine can be identified and separated using thin-layer
chromatography (TLC). For effective separation of glycine, a solvent system consisting of
distilled water and acetone is suitable. To separate tryptophan and tyrosine, methanol or acetic
acid combined with distilled water may also be utilized. These solvents aid in the separation
process by allowing each compound to travel at different rates on the TLC plate. This technique
further enables the detection of unknown amino acids by comparing their migration distances
against those of known standards. When applying samples onto the TLC plate, it is crucial to
ensure that they remain distinct without being washed away before adequate resolution occurs;
this involves maintaining an appropriate distance from both the starting point horizontally as
well as ensuring sufficient height above initial solvent levels during processing until clear
identification is achieved. As an illustrative example: employing this method could possibly
identify suspected tryptophans in an unidentified solution based upon comparisons with
movement patterns observed for standard compounds.

Reference list
Aryal, S. (2023). Thin Layer Chromatography: Principle, Parts, Steps, Uses. [online]
microbenotes. Available at: https://microbenotes.com/thin-layer-chromatography/.
chemistryhall. (2020). Thin Layer Chromatography: A Complete Guide to TLC. [online]
Available at: https://chemistryhall.com/thin-layer-chromatography/.
K. Sonkar, P. and Kumar, N. (2022). Analysis of Amino Acids Using Thin Layer
Chromatography. [online] researchgate. Available at:
https://www.researchgate.net/publication/362644470_Analysis_of_Amino_Acids_Using_Thin_L
ayer_Chromatography.
libretexts. (2022). Thin Layer Chromatography - Chemistry LibreTexts. [online] Available at:
https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/
General_Lab_Techniques/Thin_Layer_Chromatography.
Dar, A. and Anwar, J. (2023). Separation of Amino Acids, Dyes, and Pigments Using Novel
Pressurized Circular TLC Assembly for Secure Medical Imaging Applications. [online] national
library of medicine. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10115537/.
ijcea. (2015). Development of Calibration and Standard Addition Polarographic Determination
of Ascorbic Acid. [online] Available at: https://www.ijcea.org/vol6/457-A1001.pdf.
libretexts. (2020). Acetaminophen - Another pharmacologically active compound. [online]
Available at: https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/
Wet_Lab_Experiments/Organic_Chemistry_Labs/Experiments/
2%3A__Synthesis_of_Acetaminophen_%28Experiment%29.

Program Name:
HND-Batch 10

Semester and Year:

Second Semester 2024

Name of Student: Name of Lecturer / Lab Demonstrator:

Eshana Gunasekara Mis Buddika Dilrukshi
HSC-B09-027

Date of Experiment Performed: Date of Report Submitted:

2024.09.24 2024.10.01
 Practical name
Aseptic Transfer of bacterial from stock culture to a new culture medium
Objective
To streaking new culture media and transfer of bacterial by using aseptic techniques.
Introduction
Bacteria are microorganisms made up of single cells with prokaryotic structures. These
cells do not have organelles or a true nucleus, making them simpler than their eukaryotic
counterparts. When discussing the domain Bacteria (with an uppercase "B"), it is recognized as
one of the three domains that encompass all living organisms; the other two domains are Archaea
—organisms similar to bacteria in having prokaryotic characteristics—and Eukarya. Bacteria
exist on Earth in staggering abundance and outnumber plants and animals together due to their
high global biomass levels.

In microbiology, studying bacterial growth is crucial for scientists to understand bacteria's

behavior, physiology, and genetics. Factors influencing this growth include temperature, pH
levels, oxygen availability, and nutrient access. In laboratory settings, culture media such as
selective media are used to regulate environmental conditions while supplying necessary
nutrients for bacterial proliferation. Streak plating remains a key method for isolating pure
colonies by spreading small samples across sterile agar plates with loops. Techniques like
spread-pour-plating quantify bacterial populations by accurately counting both surface-level
metabolic activity and within-agar colony formation. Continuous-culture systems called
hemostats maintain cultures in the exponential growth phase consistently providing cells for
experiments or industrial applications uniformly needed fresh supplies of growing cells.. By
carefully controlling these factors researchers can analyze different types & strains offering
insights into medicine-research essential biological processes impacting various fields--
applications globally throughout this domain . (openstax.org, n.d.) (BD Editors, 2019)
Streak plate
When working with organisms that grow on agar plates, the streak plate method is preferred for
obtaining a pure culture. This technique helps isolate bacteria (mainly) from a mixed population
and develop them into distinct colonies. The procedure involves spreading an inoculum over the
agar surface to effectively reduce bacterial density.

In microbiology, obtaining pure bacterial cultures is crucial, and the streak plate method
facilitates this process effectively. This technique separates individual cells on an agar surface,
allowing them to grow into isolated colonies for easier identification and study of specific
microorganisms within a mixed culture. It is indispensable in clinical microbiology, research
settings, as well as industrial applications. Selecting appropriate media during streaking plays a
vital role in determining the success of these methods. Nutrient agars support non-fastidious
organisms while blood agars contain mammalian blood that aids in hemolytic activity and helps
identify pathogenic elements. Selective and differential mediums isolate particular microbes
from others to aid swift identification processes. The correct selection between media types
along with employing proper methodologies like The Streak Plate Method are fundamental steps
towards better understanding microbial life—ultimately providing increased potential for
advancements across medical fields or industries engaged in research work—and offering
insights into how these tiny creatures impact our lives every day. (Thiel, 1999)
Culture media preparation
Culture media, or growth media, are gels or liquids that supply the nutrients needed for
cultivating microorganisms and bacteria. They are customized to meet specific cell type
requirements, with nutrient broths and agar plates frequently employed for microbial
development. Some bacterial strains may require specialized culture media designed specifically
to accommodate their unique needs.

Laboratory culture media are nutrient solutions essential for supporting the growth of
microorganisms. These media, available in solid or liquid forms, contain vital nutrients such as
proteins, vitamins, carbohydrates, and minerals. The specific composition of each medium is
tailored to environmental conditions like pH levels and temperature to ensure optimal microbial
growth based on strain requirements. In various microbiological applications—such as antibiotic
production research or pathogen detection—the reproducibility of experimental results is crucial
for accuracy while maintaining safety standards against contamination risks. To ensure
successful cultivation efforts, it’s important to use proper aseptic techniques by regularly
sterilizing equipment and applying flame sterilization when using inoculation loops and needles.
Additionally, minimizing airborne contamination during the handling of culture vessels
contributes positively toward professional integrity within laboratory environments by enhancing
both effectiveness and safety practices universally recognized in this field.
Theory
In laboratory settings, bacterial cultures can be composed of either a single pure type or various
types. To maintain their growth and purity, subculturing techniques are utilized. Aseptic methods
must be employed to prevent contamination from surface- and air-borne sources; this demands
skillful execution and careful judgement. Using pre-sterilized tools like metal wires or
inoculating loops helps ensure that the environment remains free from contaminants while
handling bacterial culture samples.

An isolated colony, composed of a pure bacterial culture without external biological

contaminants, allows for untainted observations and experimental data. A bacterial colony is
formed when a single parent cell divides to produce identical offspring. In microbiology
laboratories, the unique characteristics of microorganisms are identified by examining their
visual traits on agar plates in a process known as "colony morphology."
In bacteria, horizontal gene transfer primarily occurs through conjugation, facilitating efficient
exchange between donor and recipient species. Besides conjugation, transformation and
transduction also serve as mechanisms for genetic exchange within bacterial populations. When
transferring cultures, it is crucial to use wire inoculating needles or loops while maintaining
sterility by heating them until they glow red in a Bunsen burner flame.

To maintain hygiene in the laboratory, it's crucial to use disinfectants capable of eliminating
bacterial spores and sanitizing all work areas. Additionally, cleaning benches with alcohol or a
disinfectant solution can be beneficial. In microbiology labs, using sterile loops for closely
transferring cultures is preferred, while regularly flaming culture bottle necks helps prevent
contamination. (Ahern, 2019) (LibreTexts, 2021) (Kaiser, 2016) (Karki, 2022)
Streak plate
Microbiologists employ streaking as a technique to obtain pure strains of microorganisms,
primarily bacteria. This method involves separating individual cells onto an agar plate, allowing
them to form distinct colonies for accurate analysis and reproducibility. The goal is to isolate
uncontaminated cultures from mixed samples by using inoculum with controlled dilution that is
mechanically applied on the surface of the agar plates. Various methods like quadrant streaks or
zigzag patterns can be utilized during streaking for optimal results in microbial research. To
ensure precision and prevent contamination, aseptic techniques are crucial in this process. These
include sterilizing inoculating needles or loops before and after use, maintaining clean
workspaces, wearing sterile gloves, and minimizing contact with culture vessels—practices
essential not only for preserving the integrity of microbial cultures but also ensuring laboratory
personnel's safety. (Biology LibreTexts, 2024) (Dahal, 2024)

Figure 01 streaking agar (Microbiology learning: The "why"ology of microbial testing , 2019)
Aseptic techniques in general
Aseptic techniques, which encompass various practices and procedures, are crucial in
microbiological work to prevent the contamination from unwanted microorganisms. It is
essential to maintain these conditions throughout experiments.

Autoclaving is a technique used to sterilize equipment and media by subjecting them to

pressurized steam at 121°C for a specified period. This method effectively eliminates all forms of
microbial life, including spores.

A 70% ethanol solution is used for sanitizing equipment and surfaces. It effectively targets a
range of microorganisms, disinfecting benches, gloves, and tools both before and after handling
microbial cultures.

The flame of a Bunsen burner creates an updraft that prevents aerial pollutants from settling on
sterile surfaces in open areas. It is also used to sterilize inoculation loops and needles through
incineration.

Sealing Windows and Doors: To reduce the risk of airborne pollutants entering a sterile
environment, minimize air movement by keeping windows and doors closed.

Before beginning any task, it's crucial to gather and organize all necessary equipment and
supplies. This reduces movement requirements and minimizes the risk of contamination during
the process.

Laminar Flow Cabinets and Biosafety Cabinets play an essential role when handling hazardous
or sensitive biological materials. They utilize HEPA filters to purify the air, ensuring that only
clean, particle-free air circulates over the workspace, thus maintaining a sterile environment.
(Cleveland Clinic, n.d.) (Aryal, 2022b)

Culture media preparation

In preparing culture media, essential components like water and nutrients are included with
specific growth factors tailored for the optimal development of individual bacteria. Organic
compounds such as vitamins and carbon sources naturally found in soil can also be provided
through the culture medium. Aseptic techniques are vital in minimizing contamination by
avoiding contact with potentially infected objects or fluids at sensitive areas; this is
accomplished using methods like the Aseptic Non-Touch Technique (ANTT). Miller's Luria
Bertani Broth supports both nurturing and preserving recombinant Escherichia coli, while adding
antibiotics to LB allows for selective cultivation of antibiotic-resistant E.coli strains.
Sterilizing glass petri dishes and agar gel is essential, which can be done using an autoclave or by
opting for pre-sterilized plastic versions to eliminate unwanted bacteria. To preserve a sterile
environment, it's crucial to sanitize hands and work surfaces with 70% alcohol before handling
containers, flasks, plates, or dishes in the cell culture hood—ensuring they are wiped down
beforehand as well. Additionally, storing items inside resealable bags after cleaning offers extra
protection against airborne contaminants and outside microorganisms that could compromise
research specimens within these vessels.

Laminar flow cabinets are commonly used for aseptic transfers. Their surfaces can be sanitized
with alcohol and UV light prior to use, ensuring a sterile environment. These workstations
maintain positive pressure in the working area by utilizing sterilized filtered air, preventing
contaminants from settling on materials being handled.

In microbiology laboratories, aseptic techniques are employed to prevent contamination of both

the targeted microorganisms and their environment, including lab personnel. The biosafety level
dictates which activities or projects can be undertaken in biological labs; BSL-4 is the most
stringent containment category. It is crucial to thoroughly clean workspaces, properly adjust
Bunsen burners, incinerate according to protocols, and sterilize inoculating instruments when
transferring organisms from broth or plate cultures using methods that comply with Biosafety
Level 2 standards. (biology libertex, n.d.) (Fatima, 2022) (Biology LibreTexts, 2016)
Materials
Culture media preparation
• 70% of Ethanol
• Autoclave machine
• Incubator
• Analytical balance
• Weighing board
• Conical flask
• Distilled water
• Measuring cylinder
• Stirring rod
• Spatula
• Bunsen burner
• Petri dish
• Marker
• Cotton
• Luria Bertani Broth, Miller
• Test tubes
• Inoculum loop
• E. Coli
• Mannitol Salt Agar (MSA)
streak plate
 Bacterial culture (E.coli)
 sterile nutrient agar plates
 Inoculating loop
 Bunsen burner
 70% ethanol or disinfectant
 Printed towels
Aseptic Techniques: Transferring Bacteria from Stock Culture to a New Culture Medium
 Bacterial stock culture (e.g., Escherichia coli)
 Sterile nutrient agar plates or broth tubes
 Inoculating loop or needle
 Bunsen burner
 70% ethanol or disinfectant

Methodology
Culture media preparation
Before weighing out 27.755g of Mannitol Salt Agar (MSA) Base powder with an analytical
balance, the bench top was wiped down with 70% ethanol. Next, the MSA base was poured into
a conical flask, and 250ml of distilled water was added using a measuring cylinder. The mixture
was then stirred with a rod until well combined. Next, the agar mix was heated gently on low
heat to avoid spills, before the flask was sealed tightly with a cotton ball and removed for
autoclaving at 121°C for fifteen minutes to sterilize within machine guidelines with no room for
any exceptions or deviations from established protocols. The agar's name was written on the
bottom of the petri dish, which was then slightly opened and positioned between two Bunsen
burners. The plate was placed in a refrigerator set to 2-8 degrees Celsius after carefully pouring
sterilized solution into it in small portions. Afterwards, an analytical balance was used to weigh
3.75g of Luria Bertani Broth Miller. This was then combined with 150ml of distilled water,
which had been measured using a measuring cylinder, in a conical flask. The combination was
carefully mixed using a rod until all ingredients were completely combined before being heated
with a Bunsen burner without any spillage; It was then placed in test tubes sealed tightly with
cotton wool for storage. Samples of E.coli retrieved from cold storage were thawed in hot water
in beakers, while inoculation loops were heated over flames until red-hot, all as part of the
seamless preparation for upcoming lab experiments. Afterwards, the E.coli that was stored was
employed to inoculate an inoculum loop that was subsequently heated in a Bunsen burner. The
bacteria loop was moved into every LBB mixture in all test tubes. After they hardened, these
cultures were stored in the fridge and then transferred to an incubator for temperature regulation.
To streak samples onto culture media, a sterilized loop containing E.coli was used to transfer the
sample taken from between two lit Bunsen burners, as shown in the diagram. At last, the petri
dishes containing the cultured samples were securely closed and left to be examined the next day
after 24 hours.
Streak plate
To perform the streak plate technique, a sterile environment was created by sanitizing the work
area with 70% ethanol and igniting a Bunsen burner. Sterile nutrient agar plates were prepared in
advance. The inoculating loop was sterilized by heating in a flame until it turned red and then
allowed to cool briefly. The aseptic techniques were used to open the bacterial culture vessel and
retrieve a small amount of bacteria sample by dipping the cooled loop into it. By gently swirling
near a flame on a partially opened lid, a first streak was created on the surface with careful
handling. The identical mixing procedure is carried out with intervals of heating and cooling
before spinning begins at each stage. Once all four sections on the agar plate are isolated, they
are separated individually and allowed to grow freely without any external influence. Colonies
that have been acquired can now be assisted by being incubated upside down at the required
temperature for a period ranging between one and two days.

Aseptic Techniques: Transferring Bacteria from Stock Culture to a New Culture Medium
In order to guarantee bacteria are moved from a stock culture to a fresh medium following
aseptic methods, the workbench is thoroughly sanitized with 70% ethanol. A Bunsen burner
produces a clean space to reduce the presence of pollutants and reduce airborne dangers.
Following the combination of powder and distilled water in a conical flask, the mixture is stirred
until dissolved before being sterilized in autoclave at 121°C for fifteen minutes, then cooled to a
manageable temperature before being transferred into sterile petri dishes or test tubes. When
needed, frozen cultures are thawed by warming them in beakers of warm water; inoculating
loops are heated on gas flames to become red-hot, then cooled to prevent contamination during
transfer steps. Vessels holding media are kept closed to accurately move small quantities of
bacterial cultures between centrifuge vials, returning tools and sealing everything before
incubating plates and test tubes while closely monitoring conditions.
Results
According to our findings, the Cloudy broth showed substantial growth that blocked the passage
of light. Bacterial colonies were visible inside the petri dish. The culture medium showed an
increase in microbial numbers with a yellow agar hue and round white speckled groups on top.

Figure - Culture media preparation

Figure -E.coli growth in Luria Bertani Broth, Miller.

Figure - Bacterial Growth in Agar.

Calculation
Preparation of a mannitol salt solution

111.02 g, 1000 ml

250 ml = (111.02 × 300 ml) / 1000 ml.

33.306g,
Preparation of Luria Bertani Broth, Miller Edition

25g 1000 ml
For 150 ml,= (25 * 150) / 1000 ml.

= 3.75 g

Discussion
Culture media preparation
Our observation showed that bacterial growth in Cloudy (turbid) broth blocked the passage of
light. Before starting the experiment, the lab bench was sanitized with 70% Ethanol, which has
strong antibacterial properties and can quickly eliminate most microbes. Empirical results have
demonstrated that this concentration is the most effective for producing these results. To achieve
unique and recognizable colonies in experiments, we employed the quadrant streak technique,
which consists of slowly diluting microbial samples across the entirety of a new plate, whether it
is a broth culture or a plate/slant colony. Autoclaving is crucial for sterilization, but caution is
needed when handling heavy glassware in hot conditions to prevent injuries to lab workers.
Additionally, there may be challenges in small labs due to limited space for autoclaves, which
could lead to errors from overuse. (Katz, 2008) (Biology LibreTexts, 2017b)
It is important to take into account important factors like the placement of the autoclave in the
laboratory and consistently offer training on how to operate it safely. To avoid potential burn
hazards from elevated temperature and pressure, it is necessary to wear appropriate protective
gear while using this equipment. If reducing risk requires moving away from traditional
methods, alternative approaches such as using pre-weighed gamma-irradiated dehydrated
culture-media prep can be used. Several laboratories have implemented innovative strategies to
enhance outcomes and reduce overall risks for enhanced safety measures. Precision weighing
and measuring of ingredients are essential elements in attaining the intended composition of
culture media preparations.

Calibrated volumetric equipment and precision balances are used in labs to conduct regular
calibration checks and maintain accuracy. Air pockets may develop when agar at 4C is stored,
but they can be removed by letting the sample stand upright for one to two hours before
incubating. Bacteriologists frequently utilize Luria-Bertani broth to promote the optimal growth
of different species. Mannitol Salt Agar prevents overgrowth when streaking samples from
heavily contaminated sources by being selective.
In order to fully sterilize an inoculating loop, it is crucial to ensure the entire wire is exposed to a
consistent orange color as it is angled through a gas burner flame. This method ensures
successful burning of all impurities found on the wire. In order to prevent recontamination
following sterilization, it is important not to place the loop back down. It is recommended to
slightly loosen the covers/caps of bottles or tubes for easier removal and to avoid contamination
by airborne microbes. Using a Bunsen burner in your workspace increases the ambient air
temperature, leading to a significant decrease in the risk of accidental contaminations when
working with cultures. The experiment followed proper lab protocols, such as keeping a tidy
workspace, correctly labeling and storing tools, and disposing waste according to regulations.
(Macwilliams and Liao, 2006) (bionumbers.hms.harvard.edu, n.d.) (Katz, 2008) (Biology
LibreTexts, 2017b)
streak plate
The purpose of the steak plate analysis was to assess the microorganisms on a steak with streak
plating. Different colony formations were observed in various parts of the meat sample, which
was caused by differences in bacterial concentrations. Issues in outcomes could be caused by
mishandling procedures, contamination during inoculation, or flawed streaking methods - like
using a contaminated loop, not sterilizing it properly between streaks or not allowing enough
time for cooling which can kill off bacteria. In order to improve the accuracy of results, it is
essential to strictly follow rigorous hygienic protocols such as sterilizing instruments and work
surfaces, using fresh and properly stored media, and ensuring the correct incubation conditions
are maintained, all of which are critical in determining the quality of outcomes. Neglecting strict
aseptic procedures during experiments allows outside contaminants to enter and affect test
results, while improper sample handling can introduce foreign microbes, altering bacterial load
and impacting research conclusions adversely. It is essential to always follow Good Laboratory
Practice (GLP), which involves thorough documentation of experiments, including controlled
conditions and repeatable attempts to ensure validity and reproducibility. Risk Assessment
programs must be executed meticulously, with safety standards being the top priority to prevent
personnel exposure to hazards. Conducting risk assessment can greatly reduce risk levels,
promoting safe and enduring scientific endeavors. (Coursehero.com, 2024) (Dahal, 2023b)
(Biology LibreTexts, 2017c)

aseptic techniques for transferring bacteria from a stock culture to new culture medium
In the hands-on session focused on aseptic techniques for moving bacteria from a stock culture to
fresh culture medium, multiple results and observations were noted. The original purpose was to
uphold sterility and accuracy, and avoid contamination throughout the process. However, some
issues arose that affected the desired results, such as the accidental introduction of contaminants
during transfer, which compromised the purity of the resulting cultures. Possible reasons might
include poor sterilization procedures or improper equipment handling practices being carried out
incorrectly in work spaces.

To avoid repeat mistakes and improve results in future tests, there are several measures that can
be implemented. It is crucial to adhere strictly to aseptic techniques, which include thorough
sterilization of all tools and the work surface before and during the procedure. Proper use of a
Bunsen burner to create sterile updrafts, along with regularly flaming the mouths of culture
tubes, can help prevent airborne contaminants from contaminating the medium. Additionally,
wearing gloves while maintaining a controlled environment free of unnecessary movements or
disturbances could also decrease the chances of contamination. During the hands-on session, it
was clear that some aspects of Good Laboratory Practices (GLP) and safety protocols were not
given proper attention. While efforts were made to keep a clean environment, more
improvements are needed to fully assess risks and put in place appropriate protective measures.
This involves wearing appropriate personal protective equipment like lab coats and goggles,
while also being aware of possible dangers when working with bacterial cultures. Sanders, 2012)
(Amrita Vishwa Vidyapeetham, 2011)

Calibration of autoclave
Ensuring the reliability and effectiveness of sterilization cycles requires performing autoclave
calibration. This procedure validates if the autoclave meets specified standards and requirements,
ensuring steady performance. The process involves several important steps that examine various
aspects of the autoclave's function. Firstly, temperature sensors are carefully positioned inside the
autoclave chamber to analyze how heat is distributed. This measure ensures all parts of the
chamber remain at consistent temperatures during sterilization to prevent incomplete sterilization
caused by uneven temperatures. The sensors collect temperature readings during a standard cycle
and then compare them to set parameters to identify any deviations. The next step is to carefully
examine the pressure gauges to verify that they accurately reflect the real chamber pressure.
Proper gauge readings are extremely important because they directly affect the temperature
levels of the sterilization process. Trained calibration technicians use traceable standard pressure
equipment to double-check and adjust autoclave readings to meet standards when needed.
Validating cycle timers is also a critical part of autoclave calibration, as the precision of
sterilization cycles is essential for effectively eliminating microbes. Technicians verify
autoclave's timer with certified stopwatches to ensure cycle durations meet predetermined
specifications.

Moreover, the calibration process involves examining the autoclave's control system and
software. This measure ensures proper operation of electronic components and software
algorithms in managing critical factors like temperature, pressure, and time. Engineers perform a
series of evaluations to confirm the accuracy and reliability of the software, making adjustments
as needed to ensure the sterilization device operates at peak performance within acceptable
limits. The calibration process is conducted alongside a thorough documentation process.
Technicians thoroughly record all findings, changes, and verifications in detailed reports. The
recording procedure is essential for meeting regulations and offering support for maintenance or
issue resolution in the future. These detailed records provide a comprehensive view of the
autoclave's performance status and accuracy verification history, ensuring that traceability and
accountability standards are maintained during system reviews to uphold optimal measurement
excellence. (Consolidated Sterilizer Systems, 2015) (beyer, n.d.)
Calibration of incubator
Calibrating incubators is crucial to ensuring the precise environmental conditions needed for
various biological and medical uses. The process involves various checks and adjustments to
ensure precise monitoring and maintenance of temperature, humidity, and CO₂ levels in the
 incubator. This validation guarantees perfect conditions necessary for cultivating or storing
cultures, embryos, and other fragile samples.

The initial step in calibrating an incubator is validating temperature accuracy. Calibrated probes
are placed strategically within the unit's chamber to take actual temperature readings, which are
then compared to the interface display to achieve this. If there is a discrepancy between the
readings recorded and those displayed on the screen, it indicates that adjustments need to be
made to maintain the desired temperatures in all parts of the incubator chamber.

After confirming the temperature, the incubator's humidity levels are calibrated. It is important to
manage humidity carefully to prevent specimens from becoming too dry or too damp. Calibrated
hygrometers are used to measure the relative humidity inside the incubator and these
measurements are compared with predetermined levels set for the environment of this machine.
If any inconsistencies arise, adjustments will be made to its management system until
environmental conditions align correctly.

It is essential to calibrate the CO₂ sensor or gas analyzer in incubators that depend on precise
CO₂ levels. This procedure includes checking the carbon dioxide concentration in the incubator
and comparing it with the values shown. It is essential to ensure precise CO₂ levels in cell culture
incubators to preserve the optimal pH balance in media cultures. Any variations from
recommended settings require prompt adjustment of the control system for accurate management
purposes.

During the calibration process, the alarms and control systems in the incubator are monitored.
These systems are set up to alert those in charge in case there is a change in the surroundings
beyond the usual set conditions. Calibrators test the functionality of these alarms by purposely
creating situations that activate them. This ensures their responsiveness and reliability, improving
the safety of any samples being incubated.

To summarize, thorough documentation is essential for calibrating an incubator. The technicians

meticulously record every measurement, adjustment, and validation to produce detailed reports
which act as historical records for vital regulatory compliance and quality assurance reasons.
Adequate documentation also allows for convenient future reference for calibrations and
troubleshooting by offering detailed information about the unit's calibration background.
(biotechservdev, 2022) (Anon, n.d.)
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tinal laborator refrigerated for pathoge risk national and
into contact with
illness and y short-term, nic of international
pathogenic strains,
urinary strains). frozen for long- strains). infect regulations for
it is crucial to
tract term storage). ion if biological
promptly seek
infections. prope materials in
medical help and
r order to ensure
- adhere to
safety safety.
Laborator appropriate
meas
y strains decontamination
ures
(like E. methods.
are
coli K-12)
not
are
follo
generally To combat fire,
wed.
non- employ water spray,
pathogeni - Low alcohol-resistant
c but still to foam, dry chemical
require mode or carbon dioxide.
careful rate In case of necessity
handling risk wear protective
to avoid for gear and use a self-
contamina non- contained breathing
tion. patho apparatus (SCBA).
genic
labor
atory
strain
s.
(Low et al., 2013)

Conclusion
It was noted that the expansion of light was impeded by the growth of microorganisms in a
cloudy solution. Culture media serve as nutrients and minerals for the growth of cells and
microbes in laboratory environments. Nonetheless, different organisms need distinct nutrients
and environmental factors. In the medium, bacteria have multiplied forming circular colonies
that display white spots on the slightly yellow agar surface.