School of Chemistry
INTERMEDIATE CHEMISTRY
LABORATORY
2024
CHEM2X23
Chemistry of Biology Molecules
NAME:
• Safety glasses and laboratory coats must be worn at all times in the
main part of the laboratory and wherever experimental work is carried
out.
• Eating and drinking are not permitted in any part of the laboratories
(including computer rooms). Smoking is not permitted anywhere in
the Chemistry Building.
• The use of cell phone in the laboratory is only permitted for use as
outlined in this lab manual.
• Important notices are placed on the laboratory noticeboards. Keep an eye on
them.
Additional information about the lab course is available on the CANVAS e- Learning site for this unit.
The education team for this course
Second Year Coordinator:
Prof. Cameron Kepert, [email protected]
Second Year Lab Director:
Dr Reyne Pullen, Rm 354, [email protected]
Education Support Team: [email protected]
Misconduct in Laboratory Classes (Discipline of Students, University of
Sydney, Calendar 1999, By-Laws Section57, page 45). Where, in the
opinion of a member of academic staff, the behaviour of a student in the
member’s class or during other work supervised by the member amounts
to misconduct or there is an imminent threat of misconduct by a student in
the member’s class or during other work supervised by the member, the
member may, for the purpose of halting or preventing misconduct, suspend
the student from attending the member’s classes or other supervised work
for a period not exceeding 1 week.
Students are reminded that continuous assessment is subject to the
University By-Laws concerning misconduct in examinations (Discipline of
Students, University of Sydney, Calendar 1999, By-Laws Section 59, page
45). All experimental results and reports presented for examination should
be the student’s own work. If results or information are obtained from other
sources, these sources should be acknowledged in the report.
PERIODIC TABLE OF THE ELEMENTS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1
HYDROGEN
H
1.008
2
HELIUM
He
4.003
3 4
5 6 7 8 9 10
LITHIUM BERYLLIUM BORON CARBON NITROGEN OXYGEN FLUORINE NEON
Li Be B C N O F Ne
6.941 9.012 10.81 12.01 14.01 16.00 19.00 20.18
11 12 13 14 15 16 17 18
SODIUM MAGNESIUM ALUMINIUM SILICON PHOSPHORUS SULFUR CHLORINE ARGON
Na Mg Al Si P S Cl Ar
22.99 24.31 26.98 28.09 30.97 32.07 35.45 39.95
19
POTASSIUM
20
CALCIUM
21
SCANDIUM
22
TITANIUM
23
VANADIUM
24
CHROMIUM
25
MANGANESE
26
IRON
27
COBALT
28
NICKEL
29
COPPER
30
ZINC
31
GALLIUM
32
GERMANIUM
33
ARSENIC
34
SELENIUM
35
BROMINE
36
KRYPTON
K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr
39.10 40.08 44.96 47.88 50.94 52.00 54.94 55.85 58.93 58.69 63.55 65.39 69.72 72.59 74.92 78.96 79.90 83.80
37
RUBIDIUM
38
STRONTIUM
39
YTTRIUM
40
ZIRCONIUM
41
NIOBIUM
42
MOLYBDENUM
43
TECHNETIUM
44
RUTHENIUM
45
RHODIUM
46
PALLADIUM
47
SILVER
48
CADMIUM
49
INDIUM
50
TIN
51
ANTIMONY
52
TELLURIUM
53
IODINE
54
XENON
Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
85.47 87.62 88.91 91.22 92.91 95.94 [98.91] 101.07 102.91 106.4 107.87 112.40 114.82 118.69 121.75 127.60 126.90 131.30
55
CAESIUM
56
BARIUM
57-71 72
HAFNIUM
73
TANTALUM
74
TUNGSTEN
75
RHENIUM
76
OSMIUM
77
IRIDIUM
78
PLATINUM
79
GOLD
80
MERCURY
81
THALLIUM
82
LEAD
83
BISMUTH
84
POLONIUM
85
ASTATINE
86
RADON
Cs Ba
Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
132.91 137.34
178.49 180.95 183.85 186.2 190.2 192.22 195.09 196.97 200.59 204.37 207.2 208.98 [210.0] [210.0] [222.0]
87
FRANCIUM
88
RADIUM
89- 103
104
RUTHERFORDIU M
105
DUBNIUM
106
SEABORGIUM
107
BOHRIUM
108
HASSIUM
109
MEITNERIUM
110
DARMSTADTIUM
111
ROENTGENIUM
112
COPERNICIUM
113
NIHONIUM
114
FLEROVIUM
115
MOSCOVIUM
116
LIVERMORIUM
117
TENNESSINE
118
OGANESSON
Fr Ra
Rf Db Sg Bh Hs Mt Ds Rg Cn Nh Fl Mc Lv Ts Og
[223.0] [226.0]
[267] [268] [269] [270] [269] [278] [281] [266] [285] [286] [289] [289] [293] [294] [294]
LANTHANIDES
ACTINIDES
57
LANTHANUM
La
138.91
58
CERIUM
Ce
140.12
59
PRASEODYMIUM
Pr
140.91
60
NEODYMIUM
Nd
144.24
61
PROMETHIUM
Pm
[144.9]
62
SAMARIUM
Sm
150.4
63
EUROPIUM
Eu
151.96
64
GADOLINIUM
Gd
157.25
65
TERBIUM
Tb
158.93
66
DYSPROSIUM
Dy
162.50
67
HOLMIUM
Ho
164.93
68
ERBIUM
Er
167.26
69
THULIUM
Tm
168.93
70
YTTERBIUM
Yb
173.04
71
LUTETIUM
Lu
174.97
89
ACTINIUM
90
THORIUM
91
PROTACTINIUM
92
URANIUM
93
NEPTUNIUM
94
PLUTONIUM
95
AMERICIUM
96
CURIUM
97
BERKELLIUM
98
CALIFORNIUM
99
EINSTEINIUM
100
FERMIUM
101
MENDELEVIUM
102
NOBELIUM
103
LAWRENCIUM
Ac Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr
[227.0] 232.04 [231.0] 238.03 [237.0] [239.1] [243.1] [247.1] [247.1] [252.1] [252.1] [257.1] [256.1] [259.1] [260.1]
Table of Contents
1. INTRODUCTION
1.1 The laboratory program
1.2 Laboratory organisation
1.3 Sign in / sign out
1.4 Prework
1.5 Sample re-issue
1.6 Sample submission at the end of the practical
1.7 Special consideration
1.8 Learning outcomes
2. SAFETY
2.1 Introduction
2.2 General precautions
2.3 Laboratory rules
2.4 Safety map
3. EXPERIMENTS
Experiment 1: Synthesis and characterisation of a self-assembling peptide derived
from silkworm protein
Experiment 2: Analysis of analgesics
Experiment 3: Enzymes in organic synthesis
4. APPENDICES
Appendix 1: Report Writing
Appendix 2: Referencing
Appendix 3: Spectroscopic data
Appendix 4: Melting point determination
Appendix 5: Thin layer chromatography
Appendix 6: Synthetic Techniques
Appendix 7: Volumetric analysis
Appendix 8: Using a Micropipette
Plagiarism Policy
Academic Honesty and Prohibition on
Plagiarism
The role of The University of Sydney is to create, preserve, transmit and apply
knowledge through teaching, research, creative works and other forms of
scholarship. The University is committed to academic excellence and high standards
of ethical behaviour as the cornerstones of scholastic achievement and quality
assurance. The University requires all students to act honestly, ethically and with
integrity in their dealings with the University, its employees, members of the public
and other students. The University of Sydney is opposed to and will not tolerate
plagiarism.
Plagiarism means presenting another person’s work as your own work by
presenting, copying or reproducing it without acknowledgement of the source. The
University of Sydney has a comprehensive policy on plagiarism (which also covers
forms of student misconduct related to assessment), and students found to be in
breach of the policy are subject to substantial penalties, which can range from
receiving a mark of zero for the individual piece of work, to expulsion from the
University. The full policy can be found at:
https://sydney.edu.au/policies/default.aspx?mode=glossary&word=Academ ic+honesty
The following would be considered breaches of the policy:
• Copying someone else's lab report (with or without their permission)
• Allowing another student access to your completed lab report
• Copying someone else's quiz answers
• Allowing another student access to your quiz answers
• Cheating in an exam
It is the responsibility of all students to:
1. ensure that they do not commit, collude with or allow another person to
commit plagiarism;
2. report possible instances of plagiarism; and
3. comply with this Policy and Procedure.
The University will treat all identified cases of student plagiarism seriously, in
accordance with this Policy and Procedure, and with Chapter 8 of The University of
Sydney By-Law 1999 (as amended) which deals with student discipline.
CHEM2X23 Practical Manual 2024 Introduction
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1. INTRODUCTION
Welcome to the Second Year Chemistry Laboratory! This manual contains
information about the organisation of the 2nd year laboratory program, course
requirements, safety information and notes for most of the practical exercises you
will be carrying out this semester. Before beginning your experimental work, please
read through the Introductory and Safety sections. You must also watch the
introductory video on Canvas, and complete the quiz below the video, before you
can access any of the resources on Canvas, including the pre-work quizzes. During
your first laboratory session, you must complete the relevant safety and
equipment map and have it checked by a demonstrator before you
commence any experimental work.
The 2nd year laboratories are on Level 4, rooms 424 and 439, Chemistry Building,
F11. The CHEM2 Laboratory Director is Dr Reyne Pullen (Room 354,
1.1 The laboratory Program
The CHEM2X23 laboratory program consists of 6 sessions in total and 4 different
experiments. Please check CANVAS regularly in case there are any alterations to
the laboratory sessions such as hot weather restrictions. Experiments will not
necessarily be conducted in numerical order but will follow an individual timetable
that is accessible on the Canvas HOME page. Your personalised timetable will
indicate which day you must attend CHEM2X23 laboratory classes.
Students are required to attend the class times indicated on their
personalised timetable. It is not generally possible to vary lab days for an
individual week; however, you may request a change by contacting the laboratory
director at least one week in advance.
ATTENDANCE AT ALL OF YOUR SCHEDULED LABORATORY SESSIONS IS
ESSENTIAL. You must attend at least 80% of the lab sessions to pass the course.
Students who miss a laboratory session due to illness or misadventure must apply
for Special Consideration. Students who miss more than two laboratory sessions
altogether (even with Special Consideration) may be given an “Incomplete” grade
for the course.
1.2 Laboratory Organisation
The laboratories (Room 424 and 439, Level 4) are open between the hours of 9:00
and 1:00 pm only. Students are not permitted to perform laboratory work outside
these times. Students should arrive ready to begin by 9:00 am with their laboratory
manual, lab coat and safety glasses on and dressed in the appropriate clothing.
As
a number of the experiments require the full 4 hours, those students who arrive
late may be excluded from the lab.
Experimental work must be completed by
12:45 pm each day, allowing at least 15 minutes to ensure that your laboratory
space is left clean and tidy.
CHEM2X23 Practical Manual 2024 Introduction
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1.3 Sign in / Sign out
It is a legal requirement that we know exactly who is in the lab at any given time,
in case of an emergency. Each time you walk in or out of the laboratory area,
you must log this on the computer next to the entrance to the labs. You can
sign in and out by scanning the barcode on your student card or by typing in your
student number. You must sign out, even if you are just stepping outside briefly.
Your initial sign-in will be recorded as an indication of your attendance, but no other
data will be retained after the laboratory session.
1.4 Prework
Each student will be assigned to a specific lab group (note that each group has a
different experiment schedule as listed in your personalised experiment schedule
on CANVAS). For each laboratory session, it is essential that you have read the
relevant section in the lab manual, planned your experiment and thought about the
safety aspects involved. This will be assessed by online prework, which can be
found in the Assignments section of CANVAS for each Experiment.
Prework must be completed and 100% correct before attending each
laboratory session and will contribute to 10% of the final grade for the lab course.
You may attempt the prework as many times as you like. Students who have not
completed the prework will be asked to leave the lab and will receive a mark of 0
for the experiment.
1.5 Sample re-issue
If, during the laboratory session, you realise you’ve made a mistake and would like
to start again, starting materials or samples can be re-issued to you. For all re- issues, you must discuss your case (what you did wrong, and what you can
do to avoid this mistake) with the academic supervisor, who will then
authorise a reissue. Without this authorisation, the service room will not reissue
any samples to you.
1.6 Sample submissions at the end of the practical
You must upload at least TWO photos to CANVAS of the samples you are submitting
for marking. All vial(s) must be properly labelled and the information clearly legible.
The photos must be of high quality and capture
the contents of the vial and all of the
information on the sample label. You must then
submit these sample vials to your demonstrator
before the end of the practical session.
1.7 Special Consideration
If, due to illness or misadventure, you miss a laboratory session or cannot submit
a report on time, you must fill in an online request for Special Consideration within
3 working days of the laboratory date or report due date, respectively.
For more information, consult the “Special Consideration” page:
https://www.sydney.edu.au/students/special-consideration.html
CHEM2X23 Practical Manual 2024 Introduction
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1.8 Learning Outcomes
The laboratory program aims to enable you to gain confidence in working in a
laboratory, to gain essential chemical laboratory skills, and to apply your
understanding to make decisions in the laboratory. This is achieved by a
combination of demonstrations (from your demonstrator) and your individual,
hands-on experience. Specific learning outcomes for each experiment are listed at
the end of the experiment introductions. General learning outcomes for the entire
laboratory course are below.
After completing this laboratory course, you will be able to:
- Follow the proper procedures and regulations for safe handling, use and
disposal of chemicals
- Confidently use common laboratory glassware and analytical instruments
- Use Microsoft Excel (or other graphing software) to plot data, perform
simple calculations and regression analysis, and insert these graphs into a
Word document
- Record observations and results in a laboratory notebook
- Articulate results clearly in a written report with recognition of the context of
the experiment, and reflection on the results obtained
- Perform calculations relating to yield, concentration and molecular mass
- Report the appropriate number of significant figures for any quantity
- Work responsibly and independently in the laboratory, managing time
appropriately
CHEM2X23 Practical Manual 2024 Safety
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2. SAFETY
2.1 Introduction
Safety in the laboratory is a matter of good organisation and adherence to the
guidelines, regulations and prescribed safety practices. It is the responsibility of
all individuals to ensure that they work safely. There are both state and federal
legislations which cover laboratory safety. The following regulations have not been
written from a legal point of view and are not intended to be a comprehensive
compilation of safety practices and techniques but rather a general guide designed
to prevent accidents.
2.2 General Precautions
i. Responsibilities
a) All members of the School of Chemistry, including undergraduates, are
required to:
• Follow the safety regulations and procedures set out in this booklet.
Safety regulations are in place to protect all members of the School
and will be strictly enforced by the Head of School.
• Familiarise themselves with the location and operation of safety devices
(fire extinguishers, fire blankets, safety showers, first aid equipment
and fire exits) in the area in which they work.
b) If in doubt about any matter likely to affect safety, a Safety Officer must be
consulted. They will be prepared to advise or act on any such matter at any
time.
ii. Discipline
a) Running, riding bicycles or skateboards, or throwing any object within the
building is NOT ALLOWED.
b) Walking on seats, desks or benches in laboratories is hazardous and is NOT
PERMITTED.
c) Fire exits, corridors, aisles and doorways must be kept clear at all times.
Stairways must not be obstructed, nor used for seating.
d) No eating or drinking is allowed in laboratories.
e) University regulations prohibit smoking in and around the Chemistry and
other University buildings.
f) Cell phones cannot be used in the laboratory except in special circumstances
(e.g. taking a picture for your report or in an emergency).
2.3 Laboratory rules
i. Clothing and PPE
All students who work in the laboratories must be wearing the mandated
personal protective equipment (PPE) outlined below before entering:
1. Lab-approved Safety Glasses (or pre-approved prescription glasses with
side-shields)
2. Clean, white cotton lab coat
3. Fully enclosed shoes
CHEM2X23 Practical Manual 2024 Safety
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4. Long thick fabric pants or long skirt that covers the ankles (preferably
100% cotton or similar).
Leggings and tights are NOT recommended due
to the high synthetic content which can melt onto the skin in case of fire.
In addition, the close contact these garments have with the skin can
exacerbate chemical transfer if chemicals are spilt on the clothing).
Wearing the correct PPE is mandatory and non-negotiable. Students who wish
to enter the lab without the above PPE will be turned away.
Note: Lab coats should NOT be worn outside of the laboratory (eg. in
toilets, the computer room, or other public spaces). Hooks are provided to
temporarily hang your lab coats if you need to leave the lab during your session.
Bring a dedicated bag to carry your lab coat to and from the University to avoid
any possible chemical contamination of your bag and contents.
Lockers are available to store your bag and any other items during your lab
session.
As these are shared lockers, they are only available to you during your
lab session. Lockers will be cleared after hours and any items found in lockers
will be disposed of.
iii.
Masks
Face masks are not mandatory in the labs however, students may wear their
own. Students are not permitted to leave labs with a face mask, as they will be
contaminated with chemicals. In line with the University policy, please stay at
home if you are feeling unwell or are showing symptoms of COVID-19.
iv. Chemical handling
Students are not allowed to handle any chemicals within the laboratory unless
all the following conditions are met:
a) Correct PPE (as described above) is worn.
b) An assessment of all possible precautions to be considered before any
dangerous or potentially dangerous experiment is performed.
c) Suitable gloves are worn when handling corrosive or toxic substances. If you
wear gloves while handling these materials, you must never touch any item
that a person not wearing gloves could. For instance, DO NOT touch
laboratory equipment or even lab benches with your gloves on. While you
are unaffected by this action, any contaminants on your gloves will be
transferred to the hand of the next person who touches these with an
ungloved hand. Likewise, remove your gloves if you are using a computer,
keyboard or a pen that might also be used later by yourself or another person
not wearing gloves. Also, do not touch your face, hair, mobile phone, etc.
while wearing protective gloves.
d) A member of the academic staff must be present to supervise any
undergraduate student work in the laboratory.
Other conditions or techniques that must be observed or undertaken when
working in the laboratory.
e) All reagents and products must be clearly labelled to show the contents,
owner, and date.
CHEM2X23 Practical Manual 2024 Safety
6
f) Spilt chemicals must be cleaned up immediately. A dustpan and broom are
available from the service room. Cleaning brushes are attached to the
balances.
g) Operations involving noxious fumes must be carried out in an operating fume
hood.
h) Boiling chips are to be added to every liquid that is to be boiled, except
aqueous solutions for quantitative analysis. These promote steady boiling of
the liquid and eliminate the danger of superheating. If for any reason boiling
is interrupted, fresh chips must be added before heating is resumed. Never
add boiling chips to a hot liquid - allow it to cool first.
i) Do not evaporate large volumes of solvent (except water) into the
atmosphere, even in a fume hood. Use a distillation apparatus and dispose
of the solvent in the appropriate organic solvent container.
j) Never pipette any chemical by mouth. Use a pipette filler.
v. Conduct during laboratory experiments and laboratory cleanliness.
The labs run each day throughout the week, and glassware and equipment are
therefore very heavily used. It is therefore your responsibility to ensure that all
glassware is clean and dry and that all equipment is in place, for the following
day.
If you don’t leave your workspace clean and tidy at the end of a session, you
risk a 10% penalty on your mark for that session.
Cleanliness extends to avoiding the chemical contamination of your devices,
such as mobile phones. The use of mobile phones in the lab is strictly forbidden.
You may use your phone to take pictures of your experiments, samples, and the
like. A sealable zip-lock bag can be collected from the Service Room if required.
Demonstrators and supervisors will hand out 10%-mark penalties for
any inappropriate use of cell phones in the lab (e.g. checking email,
TikTok, Facebook, Twitter etc..).
All students who work in the laboratories must
1. Conduct their work in a clean and tidy manner.
2. Exercise care with all glassware. Do not exert undue pressure on glassware
as this can lead to breakage and serious injury.
3. Inspect glassware which will be vacuumed before applying a vacuum to
minimise the risk of implosion. This includes vacuum distillation apparatus,
vacuum desiccators and rotary evaporators.
4. Clean up and immediately remove any broken glass and/or paper from all
sinks.
5. Leave their work area clean and tidy after they have finished.
6. Clean all the glassware they have used to the required standard.
7. Return any equipment and/or chemicals to the correct location.
CHEM2X23 Practical Manual 2024 Safety
7
vi. Waste Disposal
Students are responsible for appropriately disposing of all chemicals generated
during experiments. In Chem2 labs, there are five designated waste containers
available to all students:
1. Halogenated waste → Exclusively for all organic compounds containing
halogens (F2, Cl2, Br2, and I2, DCM, Chloroform)
2. Non-halogenated waste → Meant for all other generic organic compounds
such as solvents (ethanol, hexane, toluene).
3. Heavy Metal Aqueous waste → Intended for compounds containing; Zn,
Fe, Mn, Co, Cu, Cd, Pb, Ag etc.
4. Strongly acidic or alkaline solutions should be neutralized and then
washed down the sink with large volumes of water.
5. Glass and Contaminated waste → Specifically for all chemically
contaminated consumables such as paper towels, filter papers etc. and
broken glass.
Students must adhere to the proper disposal methods, with failure to do so will
result in affecting lab safety marks.
vii. Equipment handling
a) A demonstrator must be consulted before using any electrical equipment or
instrument for the first time.
b) Faulty equipment of any kind must not be used.
Report any faulty
equipment to demonstrators or Service Room personnel immediately.
Repairs to faulty equipment, particularly electrical equipment, should not be
attempted except by suitably trained and qualified personnel.
viii. Emergencies
If the evacuation klaxons (sirens) sound, then:
a) cease all activity
b) turn off all non-vital equipment
c) secure all dangerous substances
d) evacuate the building following the instructions of the fire wardens
e) assemble in front of the New Law building.
In the event of an emergency:
a) a member of staff, service room personnel or the front office (Rm 207,
phone 14504) should be notified immediately. The appropriate Safety
Officer must then be notified as soon as possible.
b) University Security can be reached on 13333. If necessary, Fire, Police
or Ambulance can be called by dialling 0 (for an outside line), then 000,
and asking to be connected to the appropriate service. Other emergency
numbers are listed on the inside cover of the University telephone
directory.
In the case of fire in the laboratory:
a) Do not hesitate to use a fire extinguisher if it appears necessary. Fire
blankets can be more efficient if a small fire occurs in a fume hood.
Do not take personal risks. When a fire extinguisher has been
used, however briefly, the use of that particular fire extinguisher must
be reported immediately to a Divisional Safety Officer so that it may be
CHEM2X23 Practical Manual 2024 Safety
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refilled.
All accidents, floods, fires, etc, (even if trivial) are potentially dangerous
situations and must be reported immediately to demonstrators or Service
Room personnel.
Any person found damaging or improperly using any safety equipment, or
defacing safety signs and instructions will be liable to prosecution.
ix. Accidents
a) In the event of an accident, get first aid attention in the service room
immediately. The academic in charge should be informed of the
circumstances as soon as possible and an accident report form completed.
b) Minor burns, where the skin is unbroken, should be treated immediately
under cold running water for at least 15 minutes.
c) Do not allow any chemical to come into contact with the skin and take care
to avoid the inhalation of any vapours. It should be borne in mind that toxic
effects may be cumulative. If a chemical is spilt onto the skin, it should
immediately be washed off with an excess of water, or soap and water, and
the lecturer in charge is informed. Students should study the safety notices
outside the service rooms.
• Safety glasses and clean, white laboratory coats must be
worn at all times in the main part of the laboratory and
wherever experimental work is carried out.
• Long, thick fabric pants or long skirt that covers the ankles and
fully-enclosed shoes must be worn in the laboratory.
• Eating and drinking (even water) are not permitted in any
part of the laboratories.
• All water sources in the lab deliver NON-POTABLE water
unless otherwise indicated.
• Smoking is not permitted anywhere in or around the
Chemistry Building.
• Mobile phones may not be used in the laboratories except to
take a photograph to record results.
CHEM2X23 Practical Manual 2024 Safety
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2.4 Safety and Equipment Map
Below is a map of the Level 4 Undergraduate Laboratories. Mark the locations
of the following items on the map:
a) fire escapes (or exits) f) fume cupboards
b) fire blanket Waste containers for
c) fire extinguishers g) general waste
d) eye wash stations h) broken glass
e) safety showers i) chemical waste residue bottles
Your map must be checked and initiated by a demonstrator before you
start your first practical.
Ask a demonstrator to log a safety record after you have
completed your safety map.
CHEM2X23 Practical Manual 2024 Experiments
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3. EXPERIMENTS
Experiment 1 – Synthesis and
characterisation of a self-assembling
peptide derived from silkworm protein
1. INTRODUCTION
Proteins can play diverse roles in nature. Some proteins have structural
roles, such as the silk fibroin protein. This protein is a key component of
silk, a biomaterial made by silkworms.
The silk fibroin protein from the Bombyx mori silkworm contains many
glycine-alanine-glycine-alanine-glycine-serine (GAGAGS) peptide motifs
within its amino acid sequence. Multiple copies of this repetitive motif can
self-assemble through intermolecular hydrogen-bonding interactions with
each other, forming extensive anti-parallel beta sheets that give silk its
strength.
In this experiment, you will synthesise a short version of the silk fibroin
motif – the tetrapeptide GAGA (Scheme 1). This peptide can self-assemble
into beta sheet fibrils in organic solvents, resulting in a change in state from
soluble peptide to a gel.
Researchers are interested in using naturally-inspired fibrillar assemblies
that form gels for many useful applications, such as scaffolds for cell/tissue
regeneration, biocompatible materials for drug release, and as biomaterials
that can change state in response to a given stimulus.
The key stages of this multi-week experiment are to:
• Conduct Fmoc solid-phase peptide synthesis (Fmoc SPPS) to make
GAGA on resin
• Acylate the N-terminus with hexanoic acid
• Cleave the N-hexyl-GAGA from resin and isolate the peptide by
precipitation
• Acquire FTIR spectra before and after gel formation
CHEM2X23 Practical Manual 2024 Experiments
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Scheme 1. Synthesis of N-hexyl-GAGA peptide.
2. LEARNING OUTCOMES
After undertaking this experiment, you will have the following laboratory
skills:
• Solid-phase peptide synthesis, including the use of:
o an Fmoc protecting group strategy
o peptide coupling reagents to achieve amide bond formation
o acid-mediated cleavage of peptide from resin
o peptide isolation by ether precipitation and centrifugation
• FTIR-ATR operation
CHEM2X23 Practical Manual 2024 Experiments
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• Gel formation by solvent-induced self-assembly
After undertaking this experiment and the associated analysis, you will
understand:
• How self-assembly can emerge from simple chemical motifs in nature
• The iterative process of solid-phase peptide synthesis
• The mechanisms for peptide coupling and Fmoc protecting group
removal
• Hydrogen bonding interactions that drive peptide and protein
secondary structure
• Interpretation of FTIR spectra
• Interpretation of LCMS chromatographic data
CHEM2X23 Practical Manual 2024 Experiments
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3. EXPERIMENTAL SECTION
3.1. Safety
Chemical
Hazards
N,N- Dimethylformamide
(DMF)
Flammable liquid and vapour. Harmful in
contact with skin or if inhaled. Causes
serious eye irritation. May damage fertility
or the unborn child.
Dichloromethane
(DCM)
Causes skin irritation. Causes serious eye
irritation. May cause drowsiness or
dizziness. Suspected of causing cancer.
20% (v/v) Piperidine
in DMF
Highly flammable liquid and vapor. Harmful
if swallowed. Toxic in contact with skin or if
inhaled. Causes severe skin burns and eye
damage.
25% (v/v) N,N- Diisopropylethylamine
(DIPEA) in DMF
Highly flammable liquid and vapor. Harmful
if swallowed. Causes serious eye damage.
Toxic if inhaled. May cause respiratory
irritation. Toxic to aquatic life with long
lasting effects.
O-(Benzotriazol-1-yl)- N,N,N′,N′-tetramethyl
uronium
hexafluorophosphate
(HBTU)
Causes skin irritation. May cause an
allergic skin reaction. May cause
respiratory irritation.
WEAR LONG CUFFED NITRILE GLOVES
Hexanoic acid
Harmful if swallowed. Toxic in contact with
skin. Causes severe skin burns and eye
damage.
1,1,1,3,3,3- Hexafluoroisopropanol
(HFIP)
Harmful if swallowed. Harmful in contact
with skin. Causes severe skin burns and
eye damage. Causes serious eye damage.
Harmful if inhaled.
Diethyl ether
Extremely flammable liquid and vapor.
Harmful if swallowed. May cause
drowsiness or dizziness.
Fmoc-Ala-2-CTC resin
(100-200 mesh, ~0.82
mmol/gram)
N/A
Fmoc-Gly-OH Not considered hazardous
Fmoc-Ala-OH Not considered hazardous
Tetrahydrofuran (THF)
Highly flammable liquid and vapor. Harmful
if swallowed. Causes serious eye irritation.
May cause respiratory irritation. May cause
drowsiness or dizziness. Suspected of
causing cancer.
CHEM2X23 Practical Manual 2024 Experiments
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3.2. Procedure
3.2.1. General protocol for using a syringe and washing the
resin
The reaction vessel you will be using is shown below. It consists of a standard
syringe barrel, with a frit in the bottom. At the end of the syringe, either a blunt
needle or a syringe cap can be attached.
The blunt needle is used for drawing up liquids. The syringe cap is used for sealing
the vessel during reactions.
The syringe will be pre-loaded with ~250 mg of 2-chlorotrityl chloride resin (2- CTC) with a Fmoc-protected alanine already attached. The resin loading is ~0.8
mmol/g.
Note: The plunger can be quite hard to move up and down. Be persistent but slow
in moving the plunger to avoid spraying solvent out.
Always keep needle tips and pipette tips clean – do not touch them on the sides of
any containers or waste drums as this would lead to contamination.
Add solvent to the syringe. Immerse the tip of the blunt needle into the wash
solvent and carefully pull up on the plunger until the required volume has been
reached. Then, lift the tip out of the solvent and pull the plunger up to the ~11 mL
mark to draw up some air, forming an airlock so that none of the solvent remains
in the needle.
Wash: Turn the syringe upside-down (plunger pointing down), replace the needle
cover, cap the syringe and swirl gently for 1 min.
Expel solvent: Remove the cap and replace with the blunt needle, turn the
syringe back upright (needle pointing down) and gently expel the solvent into a
waste container by pushing down slowly on the plunger. Do not squash the beads
– always leave a cushion of air between the beads and the plunger.
3.2.2. Day one – Synthesis of Fmoc-GAGA on resin
I. Swelling the resin
a) Remove the syringe cap and keep it aside for later use.
b) Attach a blunt 18G needle to the end of the syringe, then remove the
needle cover and keep it aside for later use.
c) Draw 5 mL of dichloromethane (DCM) into the syringe, then immediately
point the needle end into ‘Halogenated’ waste container and expel the
DCM.
d) Draw up another 5 mL of DCM, invert the syringe pull an airlock in the
Frit
Syringe cap
Syringe top end
(Put name here)
2-CTC resin
(pre-loaded with Fmoc-Ala)
Blunt needle
with needle
cover
Frit
Plunger
CHEM2X23 Practical Manual 2024 Experiments
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syringe, remove the needle, and cap the syringe.
e) Wash resin for 1 min as previously outlined.
After 1 min, replace the cap
with the blunt syringe and expel the DCM into the ‘Halogenated’ waste
container.
f) Repeat two more washings with 5 mL DCM each time.
I. Fmoc deprotection
g) Wash the resin three times with 5 mL of DMF. Expel DMF into the ‘Non- Halogenated’ waste.
h) Draw up 5 mL of 20% (v/v) piperidine in DMF into the syringe with an
airlock, cap the needle, and soak for 5 min with swirling. Expel solvent
into ‘Non-Halogenated’ waste after 5 minutes. Repeat once more.
i) Wash the resin 3 times with DMF (5 mL each time) to remove the
residual piperidine reagent.
II. Glycine coupling
WEAR LONG CUFFED NITRILE GLOVES FOR THE FOLLOWING
SECTION
j) Into a 20 mL glass vial containing pre-weighed Fmoc-Gly-OH (0.267 g,
0.898 mmol) and HBTU (0.304 g, 0.802 mmol), add 1.8 mL of 25%
DIPEA in DMF using a P1000 pipettor with a filter tip.
k) Cap the vial and swirl gently to mix until the HBTU is completely
dissolved.
l) Once dissolved, immediately draw the solution into the syringe, with a
small airlock, then replace the needle with the syringe cap, and label the
syringe with your name.
m) Place your syringe on the rocker and leave it to react for 30 min.
n) After 30 min, replace the syringe cap with the needle and expel the
reaction solution (‘Non-Halogenated’ waste). Wash the resin three times
with 5 mL DMF each time.
III. Fmoc deprotection
o) Repeat steps h) and i), which are the two rounds of piperidine treatment
followed by three washes with DMF.
IV. Alanine coupling
p) Repeat coupling step II) (steps j to n) but this time with Fmoc-Ala-OH
(0.280 g, 0.899 mmol) in place of Fmoc-Gly-OH.
V. Fmoc deprotection
q) Repeat steps h) and i) only, which are the two rounds of piperidine
treatment followed by three washes with DMF.
VI. Glycine coupling
r) Repeat Glycine coupling II), (steps j to n) only.
CHEM2X23 Practical Manual 2024 Experiments
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VII. Resin storage
s) Wash the resin three times with 5 mL DCM each time, expelling solvent
into ‘Halogenated’ waste.
t) Cap the blunt needle, remove and dispose of it in the ‘Sharps’ bin.
Cap
the syringe cap for storing the dry resin. Label your syringe with your lab
day and the date, alongside your name. Place in the rack for storage at 4
°C until the next lab session.
3.2.3. Day two – N-termincal acylation and cleavage from resin
I. Swelling the resin and Fmoc deprotection
u) Repeat steps a) to i).
II. N-terminal acylation with hexanoic acid
WEAR LONG CUFFED NITRILE GLOVES FOR THE FOLLOWING
SECTION
v) Into a 20 mL glass vial containing pre-weighed HBTU (0.304 g, 0.802
mmol), add 1.8 mL of 25% DIPEA in DMF using a P1000 pipette.
w) Add 0.100 mL of hexanoic acid (0.860 mmol) using a P200 pipette with a
filter tip. Cap the vial and gently swirl until the HBTU is completely
dissolved.
WEAR LONG CUFFED NITRILE GLOVES WHEN HANDLING HBTU
x) Once dissolved, immediately draw the solution into the syringe, with a
small airlock, and close the end with the syringe cap. Place your syringe
on the rocker and leave to react for 30 min.
y) After 30 min, expel the reaction solution then wash the resin three times
with 5 mL DMF each time.
z) Then wash the resin three times with 5 mL DCM each time.
III. Cleavage from resin
aa) Draw 5 mL of 30% HFIP:DCM into the syringe with an airlock. Close the
end with the syringe cap, then place the syringe on the rocker and leave
to react for 60 min.
bb) After 60 min, the 30% HFIP:DCM solution contains your peptide. Expel
this solution into a clean 15 mL centrifuge tube. Label this tube with your
name. DO NOT THROW THE SOLUTION AWAY.
cc) To ensure complete recovery of the peptide from resin, draw 4 mL of
30% HFIP:DCM into the syringe, let resin soak for 4 min with occasional
swirling, then add this extra solvent into the same 15 mL centrifuge tube.
See your demonstrator for the next step (evaporation using a nitrogen
manifold).
IV. Peptide precipitation
dd) Once only about 2 mL of solvent remains after evaporation, remove and
chill the tube on ice.
ee) Add 8 mL of cold diethyl ether, cap the tube and gently invert to mix.
CHEM2X23 Practical Manual 2024 Experiments
17
ff) Set the tube in the rack for centrifugation (5000 rpm for 5 min).
gg) After centrifugation, remove the ether with a plastic pipette, being careful
not to disturb the pelleted precipitate. Add another 4 mL of cold ether,
carefully resuspend the pellet, and place in the rack for centrifugation
(5000 rpm for 5 min).
hh) While waiting, separately weigh two empty 1.5 mL microfuge tubes and
record the weight. Label tubes with your name.
ii) After the second centrifugation, carefully remove 2 mL of the ether with a
plastic pipette. Resuspend the solid pellet in the ether remaining in the
tube, then transfer the slurry evenly into the two weighed 1.5 mL
microfuge tubes using a small metal spatula. Centrifuge the two
microfuge tubes in the small centrifuge for 2 min (see demonstrator).
jj) Carefully remove all the remaining ether with a P200 pipette, then place
your two labelled centrifuge tubes with pellets in the designated rack for
drying overnight.
3.2.4. Day three – Characterisation and gel formation
I. Yield
DO NOT wear gloves during this process.
Weigh the tube containing the dried residue on the same balance that you weighed
the tube on before and record the mass.
Calculate the percentage yield.
Do your best to minimise air exposure as the
peptide tends to absorb moisture from the air.
II. ATR-FTIR spectroscopy
Take an IR spectrum of the dried peptide (before assembly) and of your xerogel.
Save both spectra to analyse and turn in with your report.
III. Self-assembly of gel and FTIR analysis
Weigh ~5 mg of the peptide into a clean 20 mL glass vial, then add 0.5 mL
tetrahydrofuran (THF). Cap the vial and sonicate the suspension for 5 min.
Remove the cap, then heat the vial gently on a hotplate until either the peptide
dissolves or the solvent begins to bubble lightly.
Remove the vial from the hotplate, then transfer the solution into a clean 1.5 mL
microfuge tube. Close the tube and let this solution cool slowly to room
temperature (~10 min). Do not disturb the sample during this gel formation step.
When cool, invert the closed tube to see if a gel has formed. Carefully decant off
any solution that did not gel. If the entire sample is still liquid, repeat the gel
formation process. You may also need to add more peptide if this still doesn’t work.
Use your phone to take a photo of before and after with regard for the
next step – this will be used for your sample submission. With the
‘organogel’ in your tube, remove the residual THF solvent under a high vacuum to
make a ‘xerogel’ – see your demonstrator to do this (takes ~30 min).
Finally, take an ATR-FTIR spectrum of this dried ‘xerogel’ powder and compare the
spectrum against your earlier spectrum for the product before gel formation.
CHEM2X23 Practical Manual 2024 Experiments
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IV. Mass spectrometry
Please collect a mass spectrum for two samples from your demonstrator. You will
analyse and discuss these as part of your oral presentation.
4. SAMPLE SUBMISSION AND ASSESSMENT
Before leaving the laboratory for the day, check that you have kept copies of
your FTIR spectra exported as “.txt” with you. You will need them for writing your
oral presentation.
PLEASE CHECK CANVAS FOR ALL ASSESSMENT INFORMATION
CHEM2X23 Practical Manual 2024 Experiments
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Experiment 2 – Analysis of analgesics
1. INTRODUCTION
Analgesic drugs are substances that relieve pain. They fall into 5 main categories
based on their mode of action:
a) Opioids - morphine and related drugs
b) Non-steroidal anti-inflammatory agents (NSAIDs) and related drugs e.g.
aspirin and paracetamol
c) Local anaesthetics
d) Centrally acting (i.e. central nervous system) non-opioid drugs
e) Drugs used in specific painful conditions e.g. ergotamine for migraines
In this experiment, several commonly used analgesics which can be purchased
over the counter in tablet form will be analysed for their contents. Compounds
commonly used in over-the-counter preparations are aspirin (group ii above) and
paracetamol (group ii above).
aspirin
paracetamol
m.p. 138-140 °C
m.p. 169-172 °C
Some of these compounds have additional properties: e.g. aspirin is anti- inflammatory and paracetamol is anti-pyretic (i.e. reduces temperature).
Tablets containing any of the above compounds - aspirin or paracetamol. In
this experiment, you will:
a) Determine the key component present in the tablet by FTIR analysis
b) Isolate the key component.
2. LEARNING OBJECTIVES
After undertaking this experiment, you will have the following laboratory skills:
• FTIR-ATR operation
• Extraction of active components from drug formulations
• Liquid-liquid extraction
After undertaking this experiment and the associated analysis, you will
understand:
• Interpretation of FTIR spectra
• Separation of organic compounds according to their acidity/basicity
• Use of FTIR to determine component identity
• Use of Melting Point Device to determine the purity of extracted components
CHEM2X23 Practical Manual 2024 Experiments
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3. EXPERIMENTAL SECTION
3.1. Safety
Chemical
Location Hazards
Asprin (acetylsalicylic
acid)
Bench Harmful if swallowed
Paracetamol (4- acetamidophenol)
Bench
Harmful if swallowed. Harmful to
aquatic life with long lasting effects
Ethyl Acetate F5
Highly flammable liquid and vapour.
Causes serious eyes irritation. May
cause drowsiness or dizziness
Hydrochloric acid
(3M)
C1
May be corrosive to metals. Causes
severe skin burns and eye damage.
May cause respiratory irritation
Sodium sulphate V5 Non-hazardous
Sodium carbonate
(10%)
C1
Causes serious eye damage. May
cause respiratory irritation
3.2. Procedure
3.2.1. Determining key components in an unknown tablet
1. Crush all tablets to a fine powder using a mortar and pestle and transfer the
powder to a pre-weighed 50 mL beaker.
2. Using a small amount of tablet powder (enough to cover the FTIR diamond),
obtain an FTIR spectrum of your unknown. Compare your spectrum of the
unknown tablet to the reference spectra (provided within this laboratory
manual at the end of this experiment) to determine the major component in
your tablet (aspirin or paracetamol).
3. Weigh the amount of powder you have after the FTIR scan.
3.2.2. Separation of compounds in unknown tablet
I. Extraction of paracetamol from an unknown tablet
1. Slowly add 3 M hydrochloric acid (3 mL) to the powder in the beaker and stir
the mixture carefully with a Teflon rod for 3 minutes.
2. Using a funnel, decant the suspension into a 100 mL separating funnel,
leaving the insoluble tablet powder behind.
Rinse the beaker with 5 mL of
ethyl acetate and carefully decant it into the separating funnel (without the
insoluble tablet powder).
3. Thoroughly mix the contents of the separating funnel (hydrochloric acid +
ethyl acetate) by inverting and rapidly swirling, releasing the pressure
every few seconds, and allowing the layers to separate. Failure to release
the pressure may cause the separating funnel to burst.
CHEM2X23 Practical Manual 2024 Experiments
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4. Run off the bottom acid layer into a conical flask (this layer contains residue
paracetamol) and POUR the ethyl acetate layer into a clean 50 mL conical
flask (this layer contains paracetamol).
5. Extract the acid layer with two further portions of ethyl acetate (5 mL each)
in the same manner as above, combining the ethyl acetate layers from each
extraction. Decant the acid layer into the non-halogenated waste drum.
6. Dry the ethyl acetate layer over anhydrous sodium sulfate.
Filter the
solution after several minutes through fluted filter paper into a pre-weighed
and unlabelled sample vial.
7. Remove all of the solvent using the rotary evaporator.
8. Record the mass of the compound, correctly label your vial, and submit this
sample to your demonstrator at the end of the class.
9. Confirm the identity of the compound you have extracted by FTIR analysis
(as in part (a)). Save the spectrum as “.txt” and keep it for your report
writing. Then run a melting point test on your extracted compound for
checking the purity.
II. Extraction of aspirin from an unknown tablet
1. Slowly add 10% sodium carbonate solution (10 mL) to the powder in the
beaker and stir the mixture carefully with a Teflon rod for 3 minutes.
2. Using a funnel, decant the suspension into a 100 mL separating funnel,
leaving the insoluble tablet powder behind.
3. Rinse the beaker with 5 mL of ethyl acetate and carefully decant it into the
separating funnel (without the insoluble tablet powder).
4. Thoroughly mix the contents of the separating funnel (sodium carbonate
solution + ethyl acetate) by inverting and rapidly swirling, releasing the
pressure every few seconds, and allowing the layers to separate. Failure
to release the pressure may cause the separating funnel to burst.
5. Run off the bottom basic layer into a conical flask (this layer contains
deprotonated aspirin) and decant the ethyl acetate layer into a Non- halogenated waste drum.
6. Extract the basic layer with two further portions of ethyl acetate (5 mL each)
in the same manner as above, decanting the ethyl acetate layers from each
extraction into the Non-halogenated waste drum.
7. Filter the basic layer to remove the insoluble starch and binders that were in
the tablet using a glass funnel (lined with filter paper) into a clean 50 mL
conical flask.
8. Acidify the solution using 7 mL of 3M hydrochloric acid (add slowly to avoid
excess evolution of CO2 gas), ensuring that you mix thoroughly during the
addition of acid with a Teflon rod. After the addition of the 7 mL of acid, keep
stirring until there is no more CO2 evolving.
9. Filter the solid using a Büchner funnel (lined with filter paper) at the pump,
then wash the precipitate with cold water.
10.Transfer the solid into a weighed and labelled sample vial, correctly labelled,
record the weight in your lab notebook, and submit the sample to your
demonstrator at the end of class.
CHEM2X23 Practical Manual 2024 Experiments
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11.Confirm the identity of the compound you have extracted by FTIR analysis
(as in part (a)). Save the spectrum as “.txt” and keep it for your report
writing. Then run a melting point test on your extracted compound to check
the purity.
4. SAMPLE SUBMISSION AND ASSESSMENT
Before leaving the laboratory for the day, check that you have kept copies of your
FTIR spectra exported as “.txt” with you. You will need them for writing your
report.
PLEASE CHECK CANVAS FOR ALL ASSESSMENT INFORMATION
CHEM2X23 Practical Manual 2024 Experiments
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CHEM2X23 Practical Manual 2024 Experiments
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CHEM2X23 Practical Manual 2024 Experiments
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Experiment 3 – Enzymes in organic
synthesis
1. INTRODUCTION
Amino acids, the building blocks of proteins, have the general structure shown
below. Except for glycine (R = H), which is achiral, all amino acids in proteins have
the L-configuration at the a-carbon. The D/L nomenclature is usually used for
amino acids and carbohydrates and corresponds to the (R)/(S) nomenclature for
all amino acids except cysteine (R = CH2SH), as shown below. In this experiment,
the (R)/(S) nomenclature will be used for ease of comparison with the data
reported in the references.
H3N H CO2 R H3N CO2 R H3N CO2 R H 1 2 3 4 (R
CH2SH) L-amino acid (S)-amino acid =
In the synthesis of any organic compound containing one (or more) chiral centres,
the number of stereoisomers formed in a reaction always needs to be considered.
Recall that enantiomers have identical physical properties except for the way in
which they interact with polarized light and hence are not usually separable using
standard organic purification methods. For this reason, the preparation of
compounds in high optical purity is often accomplished by the use of chiral
reagents rather than resolution methods. Diastereomers have different physical
and chemical properties and may be separated in several ways.
Enzymes may be thought of as highly efficient chiral organic reagents developed
by nature to carry out organic reactions in biological systems. In this experiment,
an enzyme will be used to selectively react with one of the enantiomers of N- acetyl-D,L-phenylalanine methyl ester. The enzyme used is Subtilisin Carlsberg, a
non-specific serine protease which specifically hydrolyses the carboxyl group of
the L-enantiomer of derivatised (i.e. N-acetylated, C-esterified) amino acids. The
reaction is monitored via TLC and the optical purity of the product will be
determined by measurement of the optical rotation of the product.
2. LEARNING OBJECTIVES
After undertaking this experiment, you will have the following laboratory skills:
• Liquid-liquid extraction
• Thin-layer chromatography (TLC)
• Distillation
• Enzyme-catalysed reactions
• pH controlled-reactions
• Use of a polarimeter to measure optical rotation
After undertaking this experiment and the associated analysis, you will
understand:
• Use of enzymes for enantioselective organic synthesis
• Determination of enantiomeric purity
CHEM2X23 Practical Manual 2024 Experiments
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3. EXPERIMENTAL SECTION
3.1. Safety
Chemical Location Hazards
Acetyl chloride Bench
Highly flammable liquid and vapor. Causes
severe skin burns and eye damage.
Dichloromethane T1
Causes skin irritation. Causes serious eye
irritation. May cause drowsiness or
dizziness. Suspected of causing cancer.
D-phenylalanine
methyl ester
hydrochloride
Bench Not hazardous
Ethanol F3
Highly flammable, serious eye irritation,
suspected carcinogenic, suspected of
damaging fertility or an unborn child
Methanol F3
Highly flammable liquid and vapour. Toxic
if swallowed, in contact with skin or if
inhaled. Causes damage to organs.
pH Indicator Bench Flammable liquid and vapour, irritant
Sodium sulfate V5 Not hazardous
Sodium Carbonate
(10%)
C1
Causes serious eye damage. May cause
respiratory irritation
Subtilisin Carlsberg
(serine protease
from Bacillus
licheniformis)
Bench Not hazardous
Triethylamine Bench
Highly flammable liquid and vapor.
Harmful if swallowed. Toxic in contact with
skin or if inhaled. Causes severe skin burns
and eye damage. May cause respiratory
irritation
CHEM2X23 Practical Manual 2024 Experiments
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3.2. Procedure
Part A.
Preparation of N-Acetylphenylalanine Methyl Ester (week 1)
H3N CO2CH3Cl H3N CO2CH3Cl CH3CONH CO2CH3 CH3CONH CO2CH3 i) Na2CO3 ii) CH3COCl, NEt3
1. Weigh 2 g of phenylalanine methyl ester hydrochloride and dissolve it in 10%
sodium carbonate solution (25 mL) in a 100 mL separating funnel.
2. Add 10 mL dichloromethane and shake vigorously (Caution! Remember to
immediately relieve the pressure!). Run off the organic layer (bottom) into a
100 mL conical flask.
3. Extract the aqueous layer with dichloromethane (2 x 5 mL), collecting the
extracts in the 100 mL conical flask.
4. Dry the dichloromethane extracts over sodium sulfate and filter off the
hydrated sodium sulfate through a fluted filter paper, into a 100 mL conical
flask.
5. Put a spot of this starting material onto a thin layer chromatography (TLC)
plate.
6. Cool the conical flask in an ice bath. Add triethylamine (2.6 mL) and then
acetyl chloride (1.0 mL) dropwise while swirling the flask.
7. Swirl the reaction for an additional 10 min.
8. To determine whether the reaction has finished, run a TLC plate with the
reaction mixture and the starting material with an elute of 10% methanol in
DCM.
9. Observe the TLC plate under UV light after running it. If the reaction has not
gone to completion, ask a demonstrator for more acetyl chloride.
10. When your TLC plate indicates that all starting material has been consumed
move to step 11.
Place this TLC plate in a ziplock bag and take it with you to
prepare your report.
11. Transfer the mixture to a 100 mL separating funnel and wash the organic
layer (bottom layer) in turn with 10% sodium carbonate solution (10 mL),
followed by 3 M hydrochloric acid (2 x 5 mL).
12. Dry the organic phase over sodium sulfate in a 100 mL conical flask, then
filter it into a pre-weighed 21 mL sample vial.
CHEM2X23 Practical Manual 2024 Experiments
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13. Dry the solvent with a rotary evaporator.
14. Weigh the vial with the crude product in it and calculate the yield of crude N- acetyl phenylalanine methyl ester.
15. Clearly label your product, including your SID and leave it in the trays
provided. The product is normally obtained as a viscous pale-yellow oil which
should crystallize upon standing over the next week.
16. Discard the collected dichloromethane in the Halogenated waste drum in
the fume-hood.
Part B.
Enzymatic Resolution of Enantiomers (week 2)
Before starting part b), obtain the Subtilisin carlsberg protease enzyme (100 L)
from your demonstrator.
1. Add water (5 mL) and universal indicator solution (1 mL) into the sample vial
containing your crude product (N-acetylphenylalanine methyl ester) from
part a.
2. Adjust the pH to ~ 7 by adding 2 mL of 10% sodium carbonate solution into
the vial.
3. Seal the vial tightly and shake the vial vigorously to mix the crude product
into water.
4. If your solids have not dissolved (it happens in cold days), consult a
demonstrator and he/she will melt the solid with a hair dryer. The pH will
drop after the vigorous shaking, add 10% sodium carbonate (in 0.25 mL
increment, follow the volume mark on plastic pipette) to adjust the pH back
to ~7.
5. Add 100 L of the enzyme and place your reaction (seal tightly) in one of the
50oC water baths provided.
Frequently shake your solution and add further
10% sodium carbonate solution dropwise to maintain the pH ~7. Continue
adding sodium carbonate solution and shaking until the pH has been
CHEM2X23 Practical Manual 2024 Experiments
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stabilised. At this stage, you should observe the formation of some insoluble
yellow flakes which indicates the enzyme reaction has been initiated.
6. Incubate the sample for 30 min at 50oC.
7. After the 30 min incubation (step 6), add 5 mL of DCM into the vial, seal the
vial tightly and shake vigorously.
8. Transfer the mixture to a 100 mL separating funnel, collect the DCM layer
and extract the solution further with dichloromethane (3 x 5 mL).
9. Collect the dichloromethane extracts (including the emulsion) in a 100 mL
conical and labe it ester.
10. Acidify the aqueous phase to pH 3 with ~2 mL of 3 M hydrochloric acid (be
aware of the carbon dioxide evolving), then extract it with ethyl acetate (4 x
5 mL). Collect the extracts in a second 100 mL conical flask. Label it acid.
11. Dry each of the extracts over sodium sulfate, by swirling the solution with a
few spatulas of the sulfate.
12. Filter the acid through a fluted filter paper into a 100 mL round bottom flask.
13. Filter the ester through a fluted filter paper into a pre-weighed and
unlabelled sample vial.
14. Evaporate the acid extract by rotary evaporation to approximately 10 mL –
consult a demonstrator.
15. Transfer to a pre-weighed and unlabelled sample vial (rinse with 1-2 mL of
ethyl acetate if needed) and use the rotary evaporator again to remove all of
the solvent. Empty the solvent from the trap into non-halogenated waste
drum.
16. Evaporate the DCM from the ester extract. Dispose of the collected
dichloromethane into the Halogenated waste drum.
17. Dispose of the waste ethyl acetate in the Non-halogenated waste drum in the
fume-hood.
18. Record the mass of the compounds, correctly label your vials and submit
these samples to the Service Room at the end of class.
19. Weigh each vial and calculate the yield for both the unhydrolysed ester and
the acid.
Determination of enantiomeric purity of products
For each of the products obtained in part (B), determine the optical rotation of the
product using the following procedure.
Using an analytical balance, accurately weigh about 0.1g of the product into a 5
mL volumetric flask and make the flask up to volume with ethanol. Transfer the
solution into a polarimetry cell (Important: Record the pathlength of the
polarimetry cell used) and measure the observed optical rotation (CONSULT A
DEMONSTRATOR FOR THIS). Calculate the specific optical rotation of your
sample from the observed optical rotation and the mass of sample used, using the
following formula;
= [] =
() ℎ ℎ () ×
(/) =
×
The optical purity of a substance is generally expressed as the enantiomeric
CHEM2X23 Practical Manual 2024 Experiments
30
excess or ee, which is defined as follows:
% = () − () () + () × 100
Therefore, a mixture which contains 90% (R)-enantiomer and 10% (S)- enantiomer would have an ee of 80%.
The optical purity of a compound can be determined from the specific optical
rotation of a sample, if the specific optical rotation of a pure enantiomer is known.
In this case the optical purity, or ee, is simply defined as:
% = []
[]
× 100
From the optical rotation value you measure, and the following values of [a]D of
the pure compounds, calculate the ee of your products.
(R)-N-acetylphenylalanine methyl ester; [] − 21.4
(S)-N-acetylphenylalanine; [] + 46.0
4. DATA ENTRY AND ASSESSMENT
PLEASE CHECK CANVAS FOR ALL ASSESSMENT INFORMATION
CHEM2X23 Practical Manual 2024 Appendices
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APPENDICES
Appendix 1: Report Writing
Each laboratory exercise will be assessed based on your written report and on how
well you have completed the experiment. Approximately 50% of the marks are
allocated to the sample: the major aim of the practical course is to train you to carry
out practical experiments, and the assessment is weighted accordingly.
Reports must be submitted electronically, via Canvas. A link to the Dropbox can be
found in the Experiment resource folders. DO NOT include your name, provide your
SID instead!
Each lab report must contain the following sections:
Introduction:
- What this prac is measuring
- The technique used in this prac
- An application of this work, or why it’s important
Experimental method:
- A brief description of the experiment
- Include any information required for someone else to repeat the experiment
- Do not include unnecessary information or overly wordy descriptions
Results and discussion:
- Include any yields, calculations, and analytical values relevant to the
experiment
- Required data will be outlined for each experiment separately in the laboratory
notes
- Answer the questions posed in the laboratory notes for the specific experiment
Note: The results and discussion marks include marks relating to both the
execution of the experiment (e.g. yield, sample purity, measurement accuracy) as
well as to the reporting and analysis of results.
Conclusions:
- What your results show
- Has the experiment achieved what it set out to do?
Writing and presentation – you are required to produce a report that is free from
grammatical errors, with the correct use of symbols and chemical names, and with
clearly presented, numbered, and labelled data tables and figures.
CHEM2X23 Practical Manual 2024 Appendices
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WRITING EXPERIMENTAL REPORTS
An example lab report can be found on Canvas.
Experimental details should be described accurately and concisely in proper English
sentences, using the past tense and passive voice.
Standard abbreviations for units should be used (g, mL, min, h, °C etc.). Note
there are no full stops after such abbreviations, and they are read as singular or
plural depending on the context. You should leave a space between the number
and the unit.
Pay careful attention to your use of significant figures throughout, as marks will
be deducted for incorrect use. To refresh yourself on the use of units and significant
figures, refer to the module on the first-year webpage,
firstyear.chem.usyd.edu.au/iChem/significant_figures.shtml. There is additional
information in the Training folder on Canvas.
Use correct chemical nomenclature for all chemicals you use, and do not capitalise
the first letter of a chemical unless it is at the beginning of the sentence.
For your long reports, you must include a narrative in your results section. This
means that the individual results (e.g. yield, tables, and figures) should not merely
be placed one after the other, but they should be accompanied by text that refers
to each result and gives overall order to the section.
Where chemical structures are required, you should use ChemDraw or similar
structure-drawing software to prepare the structure.
All graphs must be prepared using Microsoft Excel, or other appropriate graphing
software. A tutorial on Excel is available on Canvas. To access it, go to
eCommunities and register in the ExSite – Excel learning site.
CHEM2X23 Practical Manual 2024 Appendices
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Appendix 2: Referencing
Referencing should be carried out according to the American Chemical
Society (ACS) guidelines for references – more details can be found here:
http://pubs.acs.org/doi/pdf/10.1021/bk-2006-STYG.ch014)
Citing References in the Body of a Paper
Use a superscript Arabic numeral after the punctuation mark.
e.g. The synthesis of the compound has been described previously.1
References should be numbered sequentially. If a reference is repeated, do
not give it another number; rather, use the original reference number.
Creating a Reference List
Arrange the references numerically according to the numbers you used within
the text. All references end with a full stop. You should include the titles of all
journal articles and book chapters.
Journal Articles
Author, A. A; Author, B. B; Author, C. C. Title of Article. Journal Abbreviation
(italics) Year (bold), Volume (italics), Pages.
e.g. Takahashi, T. The Fate of Industrial Carbon Dioxide. Science 2004,
305, 352-353. You can use the full journal title, or use standard journal
abbreviations (found here
http://images.webofknowledge.com/WOK46/help/WOS/A_abrvjt.html).
Text Books
Author, A. A.; Author, B. B. Book Title (italics), Edition (if any); Publisher:
Place of Publication, Year; Pages.
e.g. Zumdahl, S. S. Chemical Principles, 4th ed.; Houghton Mifflin: Boston, MA,
2002; p 7.
Books with chapters written by different authors
Many books have chapters written by different authors (s), and the whole
book is edited by someone else.
Author, A. A.; Author, B. B. Chapter Title. In Book Title (italics); Editor, A. A.,
Editor, B. B., Eds.; Series Information (if any, including series number);
Publisher: Place of Publication, Year; Volume number (if any), Pages.
e.g. Lenhart, J. L.; Fischer, D. A.; Sambasivan, S.; Lin, E. K.; Soles, M. A.
Utilizing Near Edge X-ray Absorption Fine Structure to Probe Interfacial
Issues in Photolithography. In Polymers for Microelectronics and
Nanoelectronics; Lin, Q., Pearson, R. A., Hedrick, J. C., Eds.; ACS
Symposium Series 874; American Chemical Society: Washington, DC,
2004; pp 98-117.
Websites
Author, A. A. (if any). Title of Site. URL (accessed date), and other identifying
information.
e.g. ChemFinder.Com. http://chemfinder.cambridgesoft.com (accessed July 14,
2013).
CHEM2X23 Practical Manual 2024 Appendices
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Appendix 3: Spectroscopic data
Revision on NMR and IR spectroscopy:
http://scilearn.sydney.edu.au/OrganicSpectroscopy.
Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy is the most
powerful tool for identifying organic compounds available to synthetic chemists. It
provides information on the number of hydrogen atoms in a molecule, the atoms
to which the hydrogens are attached, and the atoms that are nearby.
For instance, in the following spectrum of ethanol (Fig. 1), there are three signals.
The integrals (area under the curves) are in the ratio of 1:2:3, which corresponds
to 1 hydrogen atom, 2 equivalent hydrogens, and 3 equivalent hydrogens (OH,
CH2 and CH3).
The peak with a chemical shift of 4.84 ppm corresponds to a H bonded to an
electronegative element (OH). The peak at 3.46 ppm comes from the H bonded to
a carbon that is close to an electronegative element (CH2), and the peak at 1.02
ppm is from an H that is bonded to a carbon atom that is not close to an
electronegative element (CH3). The signals are split and appear as a singlet, a
quartet, and a triplet. The n+1 rule tells us that the quartet must have 3
neighbouring H atoms on the next carbon; the triplet must have 2 neighbouring H
atoms on the next carbon. [N.B. H atoms bonded to oxygen do not normally show
splitting]. This tells us that the CH2 and CH3 must be connected.
The value of the splitting gives information on the relative conformations. This is
called the coupling constant and can be calculated by: (chemical shift 1 – chemical
shift 2) × operating frequency of the spectrometer. For the quartet, this is (3.54
– 3.39) × 45 = 6.75 Hz. For the triplet, (1.17 – 1.02) × 45 = 6.75 Hz. This tells
us that the CH2 and CH3 are connected, and this coupling constant value is typical
of a linear alkane.
Figure 1: 1H NMR spectrum of ethanol.
CHEM2X23 Practical Manual 2024 Appendices
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Other diagnostic couplings are:
Alkenes: Z-configuration = less than 10 Hz; E-configuration = greater than 10 Hz.
Aromatic rings: ortho coupling = 6–10 Hz; meta coupling = 1–3 Hz, para coupling =
0–1 Hz.
Table 1: Typical 1H chemical shift ranges in organic compounds
type of
proton
type of compound chemical shift range,
ppm
RCH3 primary aliphatic 0.8- 1.2
R2CH2 secondary
aliphatic
1.0- 1.5
R3CH tertiary aliphatic 1.2- 1.8
C=C-H Vinylic 4.6- 5.9
C=C-H vinylic,
conjugated
5.5- 7.5
C≡C-H acetylenic 2-3.5
Ar-H aromatic 6-8.5
Ar-C-H benzylic 2.2-3
C=C-CH3 allylic 1.7
HC-OH alcohols 3.4-4
HC-OR ethers 3.3-4
RCOO-CH esters 3.7- 4.1
HC-COOR esters 2-2.2
HC-COOH acids 2-2.6
HC-C=O carbonyl
compounds
2-2.7
RCHO aldehydic 9-10
ROH hydroxylic 2-4
ArOH phenolic 4-12
C=C-OH enolic 15- 17
RCOOH carboxylic 10- 13
RNH2 amino 1-5
CHEM2X23 Practical Manual 2024 Appendices
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Table 2: Typical IR absorption frequencies for common functional groups
Frequency (cm-1) Bond Functional
group
3640-3500 (s,
sh)
O–H stretch, free hydroxyl
alcohols, phenols alcohols, phenols
3500-3200 (s, br) O–H stretch, H–bonded alcohols, phenols
3400-3250 (m) N-H stretch 1°, 2° amines, amides
3300-2500 (m) O-H stretch carboxylic acids
3330-3270 (s, n) -C≡C-H (C-H stretch) alkynes (terminal)
3100–3000 (s) C–H stretch Aromatics
3100–3000 (m) =C–H stretch Alkanes
2830–2695 (m) H–C=O: C–H stretch aldehydes
2260–2210 (v) C≡N stretch nitriles
2260–2210 (v) -C≡C- stretch alkynes
1760–1665 (s) C=O stretch carbonyls (general)
1760–1690 (s) C=O stretch carboxylic acids
1750–1735 (s) C=O stretch esters, saturated aliphatic
1740–1720 (s) C=O stretch aldehydes, saturated
aliphatic
1725-1700 (s) C=O stretch ketones, saturated
aliphatic
1710-1665 C=O stretch α,β-unsaturated
aldehydes, ketones
1680–1640 (m) –C=C– stretch alkenes
1650–1580 (m) N–H bend 1° amines
1600–1585 (m) C–C stretch (in–ring) aromatics
1550–1475 (s) N–O asymmetric stretch nitro compounds
1500–1400 (m) C–C stretch (in–ring) aromatics
1470–1450 (m) C–H bend alkanes
1370–1350 (m) C–H rock alkanes
1360–1290 (m) N–O symmetric stretch nitro compounds
1335–1250 (s) C–N stretch aromatic amines
1320–1000 (s) C–O stretch
alcohols, carboxylic acids,
esters,
ethers
1000–650 (s) =C–H bend alkenes
910–665 (s, b) N–H wag 1°, 2° amines
m = medium; w = weak; s = strong; n = narrow; br = broad; sh = sharp
CHEM2X23 Practical Manual 2024 Appendices
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Cleaning an NMR Tube
NMR tubes are intended for multiple uses, so the tubes need to be thoroughly cleaned
before reuse. Using the NMR cleaner (as shown in the photo), the tubes can be quickly
cleaned before any reagents dry on them and become difficult to remove. To do this
follow the steps below:
1. Dispose of your sample in the designated waste container located in the fume
hood (do not draw dissolved reagents through the NMR cleaner).
2. Fill the glass Büchner funnel with about 5 mL of distilled water.
3. Invert the NMR tube over the washing needle then turn on the vacuum and
gently press the NMR tube down against the Suba seal so the water is drawn
through the needle and into the filtration flask.
4. Repeat step 2 using 5 mL of acetone in the NMR cleaner instead of water.
5. Once your NMR tube is thoroughly cleaned, please place it in the “Rinsed NMR
tubes” beaker.
Washing needle
Glass Buchner Funnel
NMR tube
ü Suba seal
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Appendix 4: Melting point
determination
Melting points are useful for identifying compounds, and to obtain an
indication of the sample’s purity. Melting points should always be quoted as a
range, not as a single value. A narrow range indicates a high purity, whereas
a wide range and lower melting temperature suggest the presence of
impurities.
In some cases, a sample will not melt, but will instead decompose. The
temperature of decomposition should be reported, with a note to indicate that
it refers to decomposition rather than melting.
Melting points of organic solids are generally taken by putting the sample in a
capillary tube sealed at one end. The sample must be thoroughly crushed to
get the solid crystals as small as possible so that they will pack together tightly
and conduct heat well during the heating process.
1. Take the flat end of a spatula and crush the crystals with a pulling
motion on a piece of dry filter paper.
2. Do this repeatedly until the solid is very finely ground.
3. Take the capillary tube and push it into a small pile of the ground-up
solid until there is 2–3 mm of solid in the open end of the tube.
4. Invert the tube and drop the capillary through the “tube tapper” to
pack the crystals tightly in the bottom of the tube.
5. Do this repeatedly, until you have 2–3 mm of well-packed solid in the
tube.
6. Place the tube into a cool Melting Point machine and follow the specific
instrument instructions to determine the melting point.
Each melting point should be measured twice. The first measurement will be
rough and can use a more rapid heating rate (10-20°C/min).
For the second measurement, you should start your measurement from at
least 10°C lower than the expected melting point and heat at a slower rate
of 2°C/min. CHEM2X23 Practical Manual 2024 Appendices
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Appendix 5: A Guide to Thin Layer
Chromatography
Thin-layer chromatography (TLC) is an important technique in chemistry and can be
used to assess the course of a reaction, the purity of a sample, or to identify
compounds in an unknown mixture by comparison with standards.
Some
compounds are coloured so they are easily seen on the TLC plate with the naked
eye. (The word “chromatography” comes from the Greek word for colour,
chroma.) Many different techniques are currently used to visualise colourless
compounds and hence the term chromatography no longer refers exclusively to
coloured compounds.
TLC plates are prepared commercially and consist of an inert backing material (e.g.
aluminium) covered with a thin, even layer of adsorbent material (e.g. silica) called
the stationary phase.
The mixture to be analysed is dissolved in an appropriate
solvent and this solution is spotted onto the TLC plate using a capillary tube (Figure
5-1a).
The various components in the mixture are adsorbed onto the stationary phase.
The TLC plate is then placed into a tank containing the developing solvent, or mobile
phase (Figure 5-1b). The mobile phase travels up the plate by capillary action and
the various compounds in the mixture are carried along with the mobile phase at
different rates, depending upon the relative strengths of their attractions to the
stationary and the mobile phases.
Ideally, when the solvent front has almost reached
the top of the plate, the components should be well-separated and appear in columns
of discrete spots (Figure 5-2).
Figure 5-2 TLC plate before and after development
In Figure 5-2, analyte A is more strongly attracted to the stationary phase (relative
to the mobile phase) than analyte B. The position of the analyte spots is defined by
their retention factor, Rf.
CHEM2X23 Practical Manual 2024 Appendices
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Rf = distance analyte has travelled / distance solvent front has travelled
The silica layer also contains a small amount of a fluorescent compound
(manganese-activated zinc silicate) which allows the visualisation of some
compounds under UV-C light (254 nm). The adsorbent layer will fluoresce light- green, while spots containing compounds that absorb UV-C light (“UV-active”) will
not and will appear dark (quenching).
Some compounds will fluoresce under UV
and appear as coloured spots.
If the compound is not UV active, a variety of
staining solutions (such as iodine, and permanganate, may be used to illuminate
the components.
Preparation of the TLC plate
1. With gloved hands, and only handling it by the
edges, take a TLC plate from the container and
place it on a clean, dry surface.
2. Using a pencil and a ruler, lightly draw a line 1
cm from the edge. Press lightly with the pencil
so as not to damage the silica layer.
3. On this line, mark points at intervals as
specified in the experiment. Label the points
lightly. Include your initials at the top of the
plate.
Figure 5-3 TLC plate
preparation
Loading the TLC plate
1. Before loading the TLC plate, you can practice spotting
on a practice plate (1 cm x 1 cm) as follows.
a. Dip the capillary into one of your samples then
briefly touch the tip of the capillary onto the
plate. The solvent will evaporate in a few
seconds.
b. Briefly reapply the tip of the capillary onto the
same spot on the plate. Try to keep the spots
as small as possible (1-2mm). Do not spot
more than 3 times.
c. Check the spots under the UV light – if your
spot is too dark it will smear.
2. Spot your samples onto your prepared TLC plate as
described in your experimental procedure.
The number
of times each analyte needs to be spotted depends on its
concentration.
CHEM2X23 Practical Manual 2024 Appendices
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Developing the TLC plate
1. When the spots are dry, place the plate into the solvent tank using
forceps to grip the plate at the very top. Make sure that the solvent
level is below the baseline. Put the cover back on the tank (see Fig.5- 4).
2. Allow the plate to develop until the solvent front is about 1 cm
from the top.
3. Use forceps to grasp the plate above the level of the solvent and
remove it from the solvent tank.
Immediately mark the position of
the solvent front with a pencil.
4. Place the plate on some paper towel in the fume hood until it is dry
(about 1 minute).
Figure 5-5 Loading TLC plate into development tank and marking the solvent front
Visualising the developed TLC plate
The spots on the developed TLC plate are usually visualised by observing them
under UV light and/or by placing them into a tank with an appropriate stain.
Coloured compounds can be observed under natural light.
1. Rule a pencil line between the two indicator marks (to indicate the solvent front
Fig. 5-5).
2. If visualising colourless, but fluorescent, compounds, use large forceps and
place the TLC plate under UV light (in the UV light box).
Using a long pencil,
lightly outline the shape of the spots on the plate.
3. Sketch the developed TLC plate in your logbook. Include:
a. starting positions of all samples;
b. positions and shapes of all spots after development;
c. colours of spots under 356 nm and 254 nm UV light (or natural light);
d. calculation of all Rf values.
The position of the analyte spots is defined by their retention factor, Rf.
Rf = distance analyte has travelled / distance solvent front has travelled.
See Figure 5-2 for a diagram of how to determine the Rf measurement.
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CHEM2X23 Practical Manual 2024 Appendices
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Appendix 6: Synthetic Techniques
Reflux Set-up
Distillation set-up
Heating
mantle
Hotplate
Distillation flask
(not more than ½ full)
Clamp here
Thermometer
Still head
Water out
Water in
Condenser
Clamp here
clamp here
Heating
mantle
Hotplate
clamp here
condenser
water out
water in
Round-bottomed flask (not
more than ½ full)
CHEM2X23 Practical Manual 2024 Appendices
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Separating funnels
1. Hold the stopper firmly (in a safe and controlled manner) whenever
you invert the separating funnel.
2. Open the tap to release pressure IMMEDIATELY after you first invert
the funnel, and frequently after that.
3. Make sure to hold the funnel pointing away from your face and body,
and away from others.
4. Shake well, to ensure good mixing between layers.
5. To empty, rest the funnel in a retort ring, allow layers to separate,
then take the stopper out and turn the tap with both hands.
Filtration techniques
1. Flute the filter paper to maximise surface area. Ask a demonstrator for the best
technique.
This is carried out in a fumehood with the
funnel pointing into the fumehood and away
from yourself and others.
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CHEM2X23 Practical Manual 2024 Appendices
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Recrystallization
Recrystallisation is a method that allows you to purify your desired compound from
other impurities based on their solubility. It requires using a solvent that your
compound dissolves in at high temperatures but will then crystallise out at low
temperatures. By dissolving both impurities and a compound in an appropriate
solvent, either the desired compound or impurities can be removed from the
solution, leaving the other behind. Ideally, the impurities in your mixture will
remain soluble at low temperatures so when you filter your crystals, the impurities
stay dissolved in the solvent.
To do a recrystallization:
1. Heat a small volume of solvent on a hot plate.
2. Slowly add hot solvent to your compound in a conical flask until it all dissolves.
Try to use a minimum amount of solvent and keep it hot to maximise the
solubility of your compound. The volume of added solvent will vary depending
on the mass and solubility of the product being recrystallised. While keeping
the volume to a minimum, make sure that the solvent is not evaporated to
dryness, otherwise your soluble impurities will remain in your recrystallized
product.
3. When all your compound is dissolved, carefully move your flask to the side
and allow it to cool slowly. The slower you do your recrystallization, the larger
your crystals will be.
4. Collect and dry your product (crystals) by vacuum filtration.
TROUBLESHOOTING:
a. If there are impurities that do not dissolve at high temperatures, you may
need to do a hot filtration. This requires warming some filtration equipment
with some hot solvent and filtering the insoluble impurities, then doing
recrystallisation as normal to remove the soluble impurities.
b. If your compound is not crashing out upon cooling, you may have used too
much solvent. The excess solvent can be easily removed by placing your
dissolved product back onto the hot plate and evaporating some of the
solvents to reduce the volume. You can try to blow off some solvent with a
gentle stream of nitrogen while heating it.
c. You can try to put your flask on ice to encourage recrystallisation and
formation of the crystals. If that doesn’t work, you may need to remove all
the solvent and start again.
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Appendix 7: Volumetric analysis
Volumetric analysis involves the preparation, storage, and delivery of solutions of
known concentrations and/or volumes. It is intrinsic to this process that all
equipment and vessels that are used are thoroughly clean and have been rinsed
with the appropriate liquid, which, as outlined below, depends on the purpose of
the equipment.
1. Equipment and washing
Pipettes and burettes are designed to deliver solutions of a specific concentration.
Before use, they must be rinsed with the solution they are to contain.
Volumetric flasks and reaction vessels are designed to receive known amounts
(mass and/or volume) of reagents. They must be rinsed at least 3 times with
deionised water before use.
1.1
The pipette is a device for delivering a fixed volume of solution. Use the
pipette filler to suck up a few mL of solution. Use this solution to rinse the
entire inside surface of the pipette up to the graduation mark - hold the pipette
horizontally and rotate it through at least 360 . Drain. Repeat this rinsing
process twice more. The pipette is then carefully filled to a level well above the
graduation line. Ensure the tip of the pipette is well submerged during filling
and that there is enough solution to fill the pipette. Remove the pipette from
the filler, preventing the outflow of the liquid by placing your dry forefinger
firmly over the top. The outside is then dried by wiping i t
with absorptive
tissue or filter paper. Place the tip in contact with the side of an empty glass
beaker and gradually relax your forefinger to control the outflow, until the
bottom of the meniscus of the liquid exactly corresponds with the mark. The
contents are then allowed to run down the inside of the receiving flask. The
pipette should be held vertically, with its tip touching the glass throughout
delivery. You will need to hold the flask at an angle. When running has ceased,
hold the pipette in contact with the side of the flask for about 3 seconds to
allow for internal drainage. The drop retained inside by surface tension is not
expelled - it has been allowed for in calibration.
1.2 The burette is a device for measuring the volume of solution
delivered. It must drain cleanly and the tap must be in perfect order. Like the
pipette, before use, it must be thoroughly rinsed with the solution it is to
deliver. Remove the burette from its stand, close the tap and pour in about
5 mL of solution. Hold the burette horizontally and carefully rotate it to rinse
thoroughly the entire inside surface. Drain by opening the tap. Repeat this
rinsing process twice more. Clamp the burette in its stand with the
graduations facing the operator and with the tip 13 cm above a white tile. Fill
by pouring the reactant solution through a small funnel. Remove the funnel.
Carefully run liquid to waste, to remove any air bubbles below the tap and to
adjust the level to below 0.00 mL (read to two decimal places).
CHEM2X23 Practical Manual 2024 Appendices
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2. Standard solution
The required quantity of primary standard is weighed out precisely into a clean,
dry, covered beaker, using the difference method. The whole of this weighed-out
quantity is transferred to a clean, but not necessarily dry, volumetric flask (a fixed- volume container). About 50 mL of deionised water is added to the beaker and
stirred to hasten dissolution. All of the solid must be dissolved - the solution may
be heated if necessary. The solution is then poured down the stirring rod into a
small funnel in the neck of the flask. The beaker is then rinsed several times with
water to ensure all visible traces of solute are transferred to the volumetric flask.
The level of solution in the flask is brought nearly to the reference line by the
addition of water. The flask is stoppered and shaken by inversion and swirling.
Water is added by drops until the bottom of the meniscus corresponds exactly to
the line. The flask is stoppered and shaken by inversion and swirling for at least 1
minute to give a homogeneous solution. The solution thus obtained contains a
precisely known mass of standard reactant in a precise volume. These figures are
converted to concentration, and the flask is labelled.
3. Titration
Equipment must be clean and appropriately rinsed. The burette is rinsed with and
then filled with one reactant solution (not necessarily the standard solution).
The solution from the burette is run into the flask, which is agitated throughout the
titration so that all parts of the mixture reach the endpoint simultaneously. The left
hand opens the burette tap and the right hand holds the flask by the neck,
imparting a swirling motion. [Left-handed people will find it easier to rotate the
burette through 180 , use the right hand to control the burette tap and the left
hand for swirling the conical flask. After the titration, rotate the burette through
180
again to read the scale.] Addition is rapid at first, but, as the indicator takes
longer to revert to its original colour (with the approach of the endpoint),
progressively decreasing volumes are added. Single drops, each about 0.05 mL,
or even half-drops, are added in the last stages. As the endpoint is approached, a
wash bottle must be used to wash down half-drops from the burette tip and
solution sticking to the inside of the flask. The endpoint is now read as closely as
possible to 0.05 mL and recorded. A white paper slide with a broad line may be
attached to the burette so that the line is vertical; by using the line, and taking
care to avoid parallax error, precise readings of volume can be made. Take care to
read the scale correctly. Usually, 2 or 3 accurate titrations are performed and
should be within 0.10 mL of each other.
Note In some cases, for example when the expected volume is unknown or the
end-point unfamiliar, a rapid titration may be performed first to locate the end- point approximately.
5. Weighing by difference
When very accurate masses are required for analysis, you must weigh by
difference. This involves measuring out approximately the correct mass of the
sample onto weighing paper or a weigh boat, obtaining the exact mass of this
sample (to 4 decimal places), transferring the sample to the flask and then
reweighing the weighing paper or weigh boat. The difference in mass between the
original mass, and the final mass, will correspond to the amount of sample you
have in your flask.
You must transfer all solids from the weighing vessel to the flask – if you spill any
sample, you should start the process again.
CHEM2X23 Practical Manual 2024 Appendices
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Appendix 8: Using a Micropipette
Micropipettes are called a lot of different
names, most of which are based on the
companies which manufacture them. For
example, you might hear them called
“Gilsons”, as a large number of these
devices used in laboratories are made by
this company.
Regardless of the manufacturer,
micropipettes operate on the same
principle: a plunger is depressed by the
thumb and as it is released, the liquid is
drawn into a disposable plastic tip.
When the plunger is pressed again, the
liquid is dispensed.
The tips are an important part of the
micropipette and allow the same device to
be used for different samples (so long as
you change your tip between samples)
without washing. They come in several
different sizes and colours, depending on
the micropipette they are used with, and
the volume to be dispensed.
The most commonly used tips are: blue (200-1000 µL), yellow (2-200 µL) and
white (<2 µL)
Tips are loaded onto the end of the micropipette by pushing the end of the device
into the tip using firm pressure. Once used, tips are ejected using the tip eject
button. Never touch the tip with your fingers as this poses a contamination risk.
The plunger can sit at any one of three positions:
Position 1 is where
Position 2 is reached by
Position 3 is reached by
the pipette is at rest
pushing down on the plunger pushing down from
until resistance is met
in position 2
Each of these positions plays an important part in the proper use of the pipette 1
2
3
CHEM2X23 Practical Manual 2024 Appendices
50
To Draw Up Liquid
Hold the micropipette with the thumb resting on the plunger and the fingers
curled around the upper body.
1. Push down with the thumb until Position 2 is reached.
2. Keeping the plunger at the second position, place the tip attached to
the end of the micropipette beneath the surface of the liquid to be
drawn up. Try not to push right to the bottom (especially if you are
removing supernatant from a centrifuged pellet) but ensure that the
tip is far enough below the surface of the liquid that no air is drawn up.
3. Steadily release pressure on the plunger and allow it to return to
Position 1. Do this carefully, particularly with large volumes, as the
liquid may shoot up into the tip and the body of the micropipette. If
bubbles appear in the tip, return the liquid to the container by pushing
down to Position 3 and start again (you may need to change to a dry
tip).
4. To remove the last drop of liquid from the tip, push down to Position 3.
If delivering into a liquid, remove the tip from the liquid before
releasing the plunger.
To Dispense Liquid
1. Hold the micropipette so that the end of the tip is inside the vessel you
want to dispense into. When delivering smaller volumes into another
liquid, you may need to put the end of the tip beneath the surface of
the liquid (remember to change the tip afterwards if you do this to save
contaminating stock). For smaller volumes, you may also need to hold
the tip against the side of the container.
2. Push the plunger down to Position 2. If you wish to mix two liquids or
resuspend a centrifuged pellet, release it to Position 1 and push to
Position 2 a few times to draw up and expel the mixed liquids.
3. To remove the last drop of liquid from the tip, push down to Position 3.
If delivering into a liquid, remove the tip from the liquid before
releasing the plunger.
4. Release the plunger and allow it to return to Position 1
Changing the Volume
Some micropipettes deliver fixed volumes; however, the majority are
adjustable. Each brand uses a slightly different method to do this – Gilsons
have an adjustable wheel, others have a locking mechanism and turning the
plunger adjusts the volume. All have a readout which tells you how much is
being delivered and a range of volumes which can be dispensed. Trying to
dispense less than the lower value of the range will result in inaccurate
measurements. Trying to dispense over the upper range will fill the tip and
allow liquid to enter the body of the pipette. Do not overwind the volume
adjustment, as this affects the calibration of the micropipette.
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