Chem3pracmanual 16-02-17

School of Chemistry
CHEM30015
A DVANCED P RACTICAL C HEMISTRY
Laboratory Manual
2017
Contents
List of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Course Organisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Safety in the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
List of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Appendix A - Techniques for Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Appendix B - Crystallography and Instrumentation . . . . . . . . . . . . . . . . . . 309
Appendix C - Error Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Appendix D - Introduction to pro Fit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Appendix E - How to Prepare a Sample for Analysis by NMR Spectroscopy . . . . 345
Student Feedback
We welcome suggestions for improvements to the style and content of this manual. Please pass your
suggestions on to A/Prof Craig Hutton. In addition, it would be very helpful if you could tell the
demonstrators of any errors or omissions that you discover within the manual, so that future versions of
the manual can be corrected.
Copyright : c Prepared by members of the Staff of the School of Chemistry, The University of Mel-
bourne, 2010-2017. No part of this publication may be reproduced in any form without the permission
of the publisher.
Subject coordinator: A/Prof Craig Hutton.
1
Course Organisation
1 The Course
This subject is designed to build on the experience gained in second year practical chemistry
(CHEM20019) and complete students training in all practical aspects of chemical laboratory experi-
mentation through a comprehensive programme of advanced practical exercises. Students will develop
skills in the synthesis of different classes of complex compounds, the acquisition and analysis of ad-
vanced spectroscopic and physical data and the investigation of chemical systems through computational
techniques. Students will have the opportunity to obtain expertise in the operation and interpretation of
modern spectroscopic techniques (including chromatography, magnetic resonance, mass spectrometry,
laser spectroscopy etc.). Knowledge of various aspects of chemical safety, reporting of experimental res-
ults, data and error analysis and the use of chemical databases will be strengthened. Other components
of the course include elements of small group work, peer assessment, oral presentation, and experiment
design and testing.
The course initially concentrates on identified core skills of Chemistry (in the broad sense), where
practical exercises are grouped under ten such core areas. Each student will complete one experiment
from each of the ten core groupings. You will then undertake one of the integrated themed experi-
ments, which are designed to increase proficiency in standard techniques and provide advanced labor-
atory skills and the necessary background for further experimental studies within that theme. The third
component comprises small group work, and provides an introduction into research-based chemistry.
The final stage of the course provides an opportunity for experimental design and implementation, build-
ing on the themed experiments to develop an extension through further investigation. Assessment of this
final component of the course will include a brief oral presentation.
2 This Manual
This manual provides an outline of the structure of the Advanced Practical Chemistry course, the
requirements of the students, and detailed experimental procedures for each experiment included in the
course. It also contains important information on Laboratory Safety, Report Writing, etc. as detailed in
the Table of Contents. Please see the CHEM30015 LMS webpage [1] for any additional information or
resources, announcements or amendments.
3 Course Structure and Organisation of the Laboratory
This course relies on more independent learning than previous years, and does require a high level
of time-management. As discussed below, you are responsible for booking your prac. sessions, keep-
ing up to date with report submission, preparing and planning your work, seeking assistance from the
appropriate person immediately if you are experiencing problems, etc.
The course runs for all 12 weeks of semester 1 in 2017, according to the broad outline provided
below. Further details as appropriate will be provided on the CHEM30015 LMS website and on
the notice board outside the second level laboratory multimedia room (room 296).
3
University of Melbourne 2017
C OURSE O RGANISATION CHEM30015: Advanced Practical Chemistry
Laboratory hours are 10 am - 1.30 pm & 1.30 - 5.00 pm on Tuesdays (2 sessions), 1.30 - 5.00
pm on Wednesdays and 1.30 - 5.00 pm on Thursdays. The labs may also be open at other times
for students who need the time to tidy up their experiments, run/analyse spectra etc. but no new
experiments can be booked for these sessions. Availability will depend on the lab. staff.
Please start each session on time - starting later will limit your chances of completing the practical
work within the session, and if someone else has the equipment booked for a later session, they
will take precedence.
Students are expected to perform, on average, 7 hrs per week of laboratory work. This will
involve two sessions (or parts of several sessions) per week, each session being of up to 3.5 hours
duration. However, some flexibility is required since some experiments are not conducive to being
completed within a formal 3.5 hour session - e.g. a given experiment might require 1 hour in one
session to get it underway and more time in other sessions to complete.
Students will be required to book their preferred experiments for a given pair of sessions
per week, and the experiment they are carrying out in a given session, using the web-based prac.
allocation system [4]. Details of this system are provided below.
Practical experiments will be held in one of two locations. Certain instrument-based experiments
will be performed in the Level 2 teaching laboratory (room 294) while more synthesis-based ex-
periments will be performed in the Level 3 teaching laboratory (room 383), so please check the
schedule carefully prior to each practical session. These locations are also marked on the booking
system, LMS, and notice boards. Computational work can be performed on the computers avail-
able in the multi-media room on Level 2 (room 296), which will be available for use at any time
during normal working hours throughout the week.
The rst 8 weeks (16 sessions) of semester will comprise 10 core experiments. This part of the
course includes a computational chemistry exercise. Within each core block up to four experi-
ments may be available for you to select and complete at the times allocated (see booking system).
Each student will complete one experiment (performed individually in almost every case) from
each of the ten core groupings. Some exercises are allocated 1 session each, while others are
allocated 2 sessions. Some experiments will require work over several partial sessions equivalent
to one (or two) sessions. The ten core block themes and their abbreviations are detailed in Table
1.
In weeks 1-4 you will complete the organic synthesis experiments on level 3 and half of the
physical experiments on level 2. In weeks 5-8 you will complete the metal-based synthesis
experiments on level 3 and the remainder of the level 2 experiments.
Please double-check in which laboratory you will be performing the given experiment (see below).
Please also check your University email account for conrmations (and possible cancellations or
changes) to the bookings made.
An academic staff member will act as a senior assistant in the laboratories. Laboratory staff and
assistants will also be present to assist you during your experimentation, but you will be expected
to work somewhat independently. You should seek assistance from the appropriate staff member
or course coordinator as soon as possible if you are experiencing problems with the preparation
of your reports.
Each practical exercise has an academic staff member assigned to it who is responsible for issues
relating to that exercise, and marks the submitted reports. Some staff expect you to discuss the
prac. with them prior to you undertaking the laboratory exercise, and others encourage you to
discuss the style and content of the report. It is your responsibility to make contact with the
appropriate staff member whenever necessary. If you experience problems with concepts, data
4 16th February 2017

CHEM30015: Advanced Practical Chemistry C OURSE O RGANISATION
analysis or interpretation, writing a report, please make contact with the staff member allocated to
that exercise as soon as possible.
Assistance with library related matters can be obtained from the University Librarians who may be
available for consultations, research assistance, book & serial requests, scholarly literacy support
and other assistance.
1. Synthesis & Characterisation 1 (SC1)

2. Synthesis & Characterisation 2 (SC2)
3. Extended Synthesis & Characterisation (ESC)
4. Computational Chemistry (CC)
5. Physical Characterisation (PC)
6. Electrochemical processes (EC)
7. Kinetics (K)
8. Light/matter interactions (LMI)
9. Metal-Based Compounds: Synthesis & Characterisation 1 (M1)
10. Metal-Based Compounds: Synthesis & Characterisation 2 (M2)
Table 1: The core block themes and their abbreviations.
In weeks 9-11 students will undertake one of the integrated theme experiments in groups of
3. The core part of each theme experiment is undertaken individually (three sessions). Three
extension experiments are then to be undertaken by the group, with each group required to perform
the three modules extending the selected theme experiment. Each group member must participate
in performing at least two of these extension modules. At the completion of these experiments
the group will combine their findings enabling the preparation of a group-based oral presentation
encompassing all aspects of the entire theme experiment. Oral presentations from all groups will
be undertaken over 2 afternoon sessions in week 12.
The group presentations, in addition to describing results from the theme experiment undertaken,
will also incorporate an experimental design component. Each group will propose, developing and
explain a modication or extension to one or more of the parts of the theme experiment undertaken.
This will entail brief discussion/s with colleagues, lab assistants and/or staff to develop a practical
experimental approach that you could potentially test in the lab, if feasible.
Peer assessment will also form part of this course. Each student will critically review anothers
report, according to guidelines to be provided separately.
4 Laboratory Attendance Book
Attendance at the laboratory is compulsory. Students must fill in a log book in the laboratory stating
when they commence and finish work on each day of attendance. Your entries in the logbook must be
initialled by a member of the laboratory staff. Please also sign the log book if you change laborator-
ies during a session. Signing the laboratory book is primarily for safety purposes should there be an
evacuation, but may also be used to monitor attendance.
16th February 2017 5

5 Laboratory Practice
5.1 Prac. Server
A central file storage server has been created for this course. Most laboratory instruments and
the computers in the Multimedia room are connected to this server directly. A separate folder, STU-
DENT FILES 30015, to which students have write access, should be used to save data and files to onto
the Prac share (e.g. to/from instruments, for example). You can also create subfolders within this folder.
This server is accessible (from School of Chemistry computers only, as it is behind a firewall) using
the following methods:
MAC: Use command-K ( -K) or WindowConnect to server.

In the server address type: smb://pv-chem-file/PRAC
WINDOWS: Use My Network Places. If the prac server is not listed, add a network place using
pv-chem-file\PRAC.
Any files that you want to use in the virtual Windows environment on the multimedia room iMacs
should be accessed via the server share in Windows rather than attempting to find the files on the Mac
somehow. There should be an icon on the Windows desktop for the Prac share. Similarly, if you have
any files that you want to save from within Windows then they should be put back onto the Prac share;
they can then be accessed from the Mac again. (Think of the Prac share as being like a tunnel between
the Mac and the Windows sides in this scenario). The eChem software for example runs in the virtual
Windows environment.
Some data files are unrecognised by the Mac OS and so double-clicking them will result in a error.
Instead, it is best if you open the required files from within the relevant application or drag & drop the
file onto the application icon in the Dock.
All data should be saved directly from the lab/instrument to the CHEM30015 STUDENT FILES 30015
folder, rather than to the instruments computer directly. Data analysis etc. can then be completed using
the computers in the multimedia room (room 296) accessing the server.
The server should only be used for the transfer and short-term storage of data - do not leave data
stored for long periods. It is your responsibility to back up your data and remember that this server
(and therefore any data stored on it) is open to all within the School. Files actually stored on the com-
puters in room 296 get removed automatically each evening, but if stored on the PRAC server they are
not.
5.2 Other Requirements
Experimental results and observations should be recorded directly into a laboratory notebook.
Your notebook should be available for perusal by lab. staff at any time in the laboratory.
A completed risk assessment form, co-signed by a member of the lab staff before you start the
experiment, must also be available for perusal at any time.
If any apparatus is required to be left running overnight, a completed apparatus overnight form,
co-signed by the laboratory staff member, must be affixed to the apparatus. These forms are available
on the LMS site.
Prepare solutions away from instrumentation and make sure hands are clean and dry before operating
instruments or using computers. Remove gloves prior to using instruments.
Students will be assigned their own lockers in the Level 3 laboratory for the duration of the course.
Name tags will also be issued and should be worn in the laboratories.

6 Assessment
To gain credit for this unit (12.5 credit points), students are required to attend, on average, 2 labor-
atory sessions (of 3.5 hours duration each) every week for the 12 weeks of 1st semester, complete and
submit assessment tasks for each experiment (as detailed below), make adequate contributions to the
peer review process, and participate in a group-based oral presentation related to the Theme experi-
ment.
Satisfactory performance includes:
Completion of experimental aspects, and reports submitted on time, for all required experiments.
An overall grade in excess of 50% overall, calculated according to the allocation of marks outlined
below.
A maximum of 3 sessions of medically certified absences is permitted. More than 3 absences

deemed (by the course coordinator) to be appropriate, will be considered as grounds for inadequate
completion of the course and you will not receive a pass grade.
Assessment of students technical competence, background knowledge, reporting and interpretative

skills will be undertaken for each of the core experiments by one of three methods. The assessment
method to be used is outlined at the start of each experiment and will comprise one of the following;
1. A written report. Reports (6 pages) will be submitted electronically and in hard copy, and will
follow templates provided on LMS.
2. A viva (oral exam). Vivas of 5-10 minutes will be held with the marker at a mutually agreed time.
Topics to be covered in the viva will be provided.
3. A short oral presentation. Each student will present a brief (5 minute) slide presentation to the
marker, with other students present.
Assessment of the Theme Experiment will be by a group-based oral presentation of 15-20 minutes.
Each group of 3 students will present the outcomes of the theme experiment core and the theme exper-
iment extensions. Each group will also design and present a further extension or improvement to the
theme experiment.
The remainder of the assessment will be based on students contributions to a peer review exercise.
A breakdown of the assessment components is provided in Table 2:
Allocation of marks:
2 session pracs (3 x 11 %) 33%
1 session pracs (7 x 6 %) 42%
Themed Experiment Core 20%
Peer assessment 5%
TOTAL 100%
Table 2: Marking scheme for the course.

Requirements for all assessments types:
A safety risk assessment form must have been filled in prior to the commencement of each exper-
iment. This must be co-signed by a member of the lab staff before the experiment is started and
a copy of this assessment must be included with your report or provided to the marker of your
viva/oral presentation.
Your laboratory notebook used to record all experimental results and observations should be avail-
able for perusal at any time and should be provided to the marker during your viva/oral presenta-
tion.
Regardless of the assessment type, you will be assessed on your ability to explain the concepts, as
well as on the competence with which you completed the experiment and analysed the data.
At the 3rd year level you are expected to consider the background and context of each experiment
you are conducting. Familiarity with the mechanism/key theoretical concept for each experiment
is required.
It is most important that you do not get bogged down with a problem! If you are experiencing
difficulty with a concept, spectral interpretation, reaction mechanism, etc., or if a written report is
taking an inordinate amount of time to complete, you should seek assistance from the appropriate
person (course coordinator or person responsible for that prac.) immediately.
Over the first 8 weeks you will complete 10 individual experiments, and therefore need to undertake
more than one assessment task per week. Written reports should be submitted within 1-2 weeks follow-
ing completion of that experiment. Please make every effort to not fall behind in report submission -
late reports may have marks deducted. For experiments assessed by viva/oral presentation you should
discuss potential assessment times with the marker before starting or while undertaking the experiment.
The marker may not be available for the whole semester so do not leave it to the last minute to make
contact!
After week 4 you should have completed the organic synthesis experiments on level 3 and 2 of
the physical experiments on level 2. As such, after week 6 you are required to have submitted all
written reports for these experiments and either completed any vivas/oral presentations or at least booked
times with the appropriate markers. Similarly, after week 8 you should have completed the metal-
based synthesis experiments on level 3 and the remaining experiments on level 2. Written reports and
completed or booked vivas/oral presentations are therefore required for such experiments by the end of
week 10. Please note that online prac bookings will be dependent on a satisfactory rate of assessment
completion. Failure to maintain this will mean you cannot book another experiment until you are up to
date, which will restrict which experiments you can complete, and therefore inhibit satisfactory progress.
7 Written Reports
A laboratory report should contain all of the necessary information for another scientist reading it to
understand what you have done experimentally, repeat the experiments and their analysis, interpret the
ndings similarly and be convinced that your conclusions are supported by your results.
Each report should be completed individually and independently, even if results are shared.
Reports should be presented clearly and neatly, in a format reecting that of the relevant scientic
literature, wherever possible using word processing (e.g. MS Word, Latex etc.), chemical drawing
(e.g. ChemSketch, ChemDraw etc.), plotting (e.g. ProFit, MS Excel, Igor, MatLab etc.), equation
writing (e.g. Equation Editor, LaTeXiT etc.) software. A range of appropriate software is available

on the computers in the multi-media room (room 296), and sources of suitable freeware can also
be provided.
Students should use the report template(s) provided on the LMS, based largely on the format of a
journal article. You should also discuss report preparation with the senior lab assistants and other
staff members, especially early in the semester.
Reports must be accompanied with a signed coversheet (available on LMS) conrming that the
Universitys anti-plagiarism policy has been adhered to.
Some experiments may refer to additional information such as results sheets (which will be avail-
able on the LMS site), but such material should be used as a guide to what information should be
included in your report, and how it might be presented. Results sheets themselves should not be
submitted as a report.
Submit a hardcopy of your report in the shoot provided near the Enquiries ofce (near the entrance
to rooms 290-293). The report will be date stamped by the laboratory staff acknowledging receipt.
Marked reports will be returned as soon as possible to the pigeon holes next to the submission
shoot.
Also, an electronic version of the report must be submitted via the Turnitin facility [2] available
through the LMS webpage for this subject. See [3] for directions on how to submit electronically.
8 Vivas
Vivas will in general involve the marker asking questions to probe you understanding of the ex-
periment you have undertaken. You should be able to explain the aims of the experiment, how it was
conducted, interpretation of results/spectra/data/mechanisms, what conclusions can be drawn from the
results, any problems encountered and how these were overcome or how they affected the results.
9 Oral presentations
Oral presentations should be brief descriptions of the experiment using, for example, Powerpoint. As
described above for reports and vivas, your presentation should detail the aims of the experiment, how it
was conducted, interpretation of results/spectra/data/mechanisms, what conclusions can be drawn from
the results, any problems encountered and how these were overcome or how they affected the results.
10 Sample Submission
Under no circumstances should any chemical samples be removed from the laboratory. Label your
samples clearly with your name, sample name, yield & melting point (where applicable) and place them
in the designated tray in the laboratory. They will be checked for consistency and form part of the
assessment for certain experiments.
11 Prac. booking system
A web-based booking system is used to self-allocate which prac., from each of the blocks of experi-
ments, you prefer to complete and when. This system is accessed through [4]. It will take discipline and
planning on your part to ensure that you complete each of the the required components at an appropriate

time, as pracs are only available at particular times of the semester. Pracs should be booked by 4 pm
on the Friday of the week preceding the prac/session in question. You will receive an acknowledgement
of successful prac booking. An email may also be sent (to your University email account) if for some
reason your booking needs to be changed, so please check your ugrad email account. Please do not
attempt to book more than two pracs at a time as booking is dependent on satisfactory report submission
rates. For pracs that require multiple sessions, you will need to book both part 1 and part 2, with
part 1 before part 2. If for some reason you are unable to attend a session for which you are booked,
you will need to amend your booking in advance of the session, otherwise you will be deemed to have
completed that prac. and a later booking will not be possible.
12 Peer Review
Peer review, which may be an unfamiliar concept to you, is an important part of scientic reporting
and an element of this is included as part of this course. You will be required to prepare a short (up
to 500 words) critical analysis of a submitted report resulting from one of your peers work (from an
experiment that you did not participate in). Your report will be prepared according to the guidelines
made available via LMS, providing suggested improvements or questioning unsupported or unclear
conclusions. Similarly, another peer will assess one of your reports. There will be an opportunity to
resubmit your report after having taken into account the comments resulting from the peer assessment.
The identities of the Reviewers and authors will be kept anonymous. It is expected that this process will
be conducted rigorously and the quality and academic rigour of the review will be assessed.
We will use the PRAZE facility for the Peer Review. A link to PRAZE is provided on the
CHEM30015 LMS site under Assessment. More details will be provided on the subjects LMS page.
References
[1] http://app.lms.unimelb.edu.au/
[2] http://academichonesty.unimelb.edu.au/turnitin/
[3] http://www.lms.unimelb.edu.au/animations/Turnitin_download_papers.htm
[4] http://pracalloc.chemistry.unimelb.edu.au/
[5] http://pubs.acs.org/
[6] A Guide to Scientific Writing, D.R. Lindsay, 2nd ed., Longman-Cheshire, Melbourne, 1995
[7] Successful Laboratory Reports: A Manual for Science Students, C.S. Lobban and M. Scheefter,
Cambridge University Press, 1992
[8] The Chemists English: with Say It in English, Please! R. Schoenfeld, 3rd rev. ed., 1989

week # 1 2 3 4 5 6 7 8 9 10 11 12
Week starting 27-Feb-17 06-Mar-17 13-Mar-17 20-Mar-17 27-Mar-17 03-Apr-17 10-Apr-17 17-Apr-17 24-Apr-17 01-May-17 08-May-17 15-May-17 22-May-17
level 2 level 3 level 2 level 3 level 2 level 3 level 2 level 3 level 2 level 3 level 2 level 3 level 2 level 3 Easter level 2 level 3
STUDENT/SESSION# Easter
1 PC1 SC1.1 PC1 SC1.1 PC1 SC1.4 PC1 ESC1 PC1 M1.1 PC1 M1.1 PC1 M1.1 Easter PC1 M1.1 TE: Sulfa Drugs T1.0 Sulfa Drugs themed
16th February 2017

2 PC1 SC1.1 PC1 SC1.1 PC1 SC1.4 PC1 ESC1 PC1 M1.1 PC1 M1.1 PC1 M1.1 Easter PC1 M1.1 TE: Sulfa Drugs T1.0 group experiments
3 PC2 SC1.1 PC2 SC1.1 PC2 SC2.1 PC2 ESC1 PC2 M1.1 PC2 M1.1 PC2 M1.1 Easter PC2 M1.1 TE: Sulfa Drugs T1.0 T1.1,1.2 &1.3
Functional Organic Dye
4 PC2 SC1.1 PC2 SC1.2 PC2 SC2.1 PC2 ESC1 PC2 M1.2 PC2 M1.2 PC2 M1.2 Easter PC2 M1.2 TE: Functional Organic Dyes T3.0 themed group experiments
5 EC1 SC1.1 EC1 SC1.2 EC1 SC2.2 EC1 ESC2 EC1 M1.2 EC1 M1.2 EC1 M1.2 Easter EC1 M1.2 TE: Functional Organic Dyes T3.0 T3.1,3.2 &3.3
6 EC1 SC1.2 EC1 SC1.2 EC1 SC2.2 EC1 ESC2 EC1 M1.2 EC1 M1.2 EC1 M1.2 Easter EC1 M1.2 TE: Solar cell T2.0
7 EC2 SC1.2 EC2 SC1.3 EC2 SC2.2 EC2 ESC2 EC2 M2.1 EC2 M2.1 EC2 M2.1 Easter EC2 M2.1 TE: Solar cell T2.0 Solar Cell themed
8 EC2 SC1.2 EC2 SC1.3 EC2 SC2.3 EC2 ESC3 EC2 M2.1 EC2 M2.1 EC2 M2.1 Easter EC2 M2.1 TE: Solar cell T2.0 group experiments
9 LM1 SC1.2 LM1 SC1.3 LM1 SC2.3 LM1 ESC3 LM1 M2.1 LM1 M2.1 LM1 M2.1 Easter LM1 M2.1 TE: Solar cell T2.0 T2.1,2.2 & 2.3
10 LM2 SC1.3 LM2 SC1.4 LM2 SC2.3 LM2 ESC3 LM2 M2.2 LM2 M2.2 LM2 M2.2 Easter LM2 M2.2 TE: Solar cell T2.0
11 LM3 SC1.3 LM3 SC1.4 LM3 SC2.4 LM3 ESC4 LM3 M2.2 LM3 M2.2 LM3 M2.2 Easter LM3 M2.2 TE: Sulfa Drugs T1.0 Sulfa Drugs themed
12 LM3 SC1.3 LM3 SC2.1 LM3 SC2.4 LM3 ESC4 LM3 M2.2 LM3 M2.2 LM3 M2.2 Easter LM3 M2.2 TE: Sulfa Drugs T1.0 group experiments
13 K1 SC1.3 K1 SC2.1 K1 ESC1 K1 ESC4 K1 M2.3 K1 M2.3 K1 M2.3 Easter K1 M2.3 TE: Sulfa Drugs T1.0 T1.1,1.2 &1.3
14 K1 SC1.3 K1 SC2.1 K1 ESC1 K1 K1 M2.3 K1 M2.3 K1 M2.3 Easter K1 M2.3 TE: Functional Organic Dyes T3.0 themed group experiments
15 K2 SC1.4 K2 SC2.2 K2 ESC1 K2 K2 M2.3 K2 M2.3 K2 M2.3 Easter K2 M2.3 TE: Functional Organic Dyes T3.0 T3.1,3.2 &3.3
CHEM30015: Advanced Practical Chemistry
16 K2 SC1.4 K2 SC2.2 K2 ESC1 K2 K2 K2 K2 Easter K2 TE: Solar cell T2.0

17 K3 SC2.1 K3 SC2.2 K3 ESC2 K3 K3 K3 K3 Easter K3 TE: Solar cell T2.0 Solar Cell themed
18 K3 SC2.1 K3 SC2.3 K3 ESC2 K3 K3 K3 K3 K3 TE: Solar cell T2.0 group experiments
19 SC2.1 SC2.3 ESC2 CC TE: Solar cell T2.0 T2.1,2.2 & 2.3 Final date for
Labs open submission of
20 SC2.1 SC2.3 ESC3 CC TE: Solar cell T2.0
Wed and Thurs
21 SC2.2 SC2.4 ESC3 CC TE: Sulfa Drugs T1.0 Sulfa Drugs themed lab reports;
10-5pm as
22 SC2.2 SC2.4 ESC3 CC TE: Sulfa Drugs T1.0 group experiments Friday, 26th May
Tues is Anzac
23 SC2.2 SC2.4 ESC4 CC TE: Sulfa Drugs T1.0 T1.1,1.2 &1.3 2017
24 SC2.2 ESC3 ESC4 CC TE: Functional Organic Dyes T3.0 themed group experiments
25 SC2.3 ESC3 ESC4 CC TE: Functional Organic Dyes T3.0 T3.1,3.2 &3.3
26 SC2.3 CC TE: Solar cell T2.0

27 SC2.3 CC TE: Solar cell T2.0 Solar Cell themed
28 SC2.3 CC TE: Solar cell T2.0 group experiments
29 SC2.4 CC TE: Solar cell T2.0 T2.1,2.2 & 2.3
30 SC2.4 CC TE: Solar cell T2.0
31 SC2.4 CC TE: Sulfa Drugs T1.0 Sulfa Drugs themed
Figure 1: Schedule of experiments for 2017.

32 SC2.4 CC TE: Sulfa Drugs T1.0 group experiments
33 CC TE: Sulfa Drugs T1.0 T1.1,1.2 &1.3
34 CC TE: Functional Organic Dyes T3.0 themed group experiments
35 CC TE: Functional Organic Dyes T3.0 T3.1,3.2 &3.3
36 CC TE: Solar cell T2.0
37 CC TE: Solar cell T2.0 Solar Cell themed
38 CC TE: Solar cell T2.0 group experiments
39 TE: Solar cell T2.0 T2.1,2.2 & 2.3
40 TE: Solar cell T2.0
41
C OURSE O RGANISATION
11
Safety in the Laboratory
Read this section carefully
The University of Melbourne has adopted the internationally recognised systems: Safety MAP and
Environmental Management System (ISO14001), to ensure a safe and environment-friendly workplace
for all staff, students and visitors. As a student of the University you are responsible for adopting safe
work and study practices and you are required to comply with all relevant University and School of
Chemistry rules and procedures.
Sensible Laboratory Dress: Participants in laboratory practical classes are reminded to wear sensible
dress appropriate for the tasks being performed on the day. When the experiments involve highly cor-
rosive or toxic substances, the wearing of very short pants and skirts is discouraged. Participation in any
practical classes also requires students to wear a lab coat, safety glasses and fully enclosed shoes at all
times. The wearing of contact lenses in the lab is strongly discouraged. If their use is unavoidable, then
splash resistant chemical goggles (not just safety glasses) are mandatory. A chemical splash to the eye
while wearing contact lenses can trap chemicals between the lens and the cornea, making eye washing
more difficult.
Chemical Hygiene: Lab users are reminded to practice proper chemical hygiene at all times. Gloves
are often used to protect the skin from chemical exposure. Users must avoid touching other surfaces
(door handles, taps, pens, phones, faces, etc) while wearing gloves in order to prevent the spread of
chemical residues. Failure to practice safe chemical hygiene defeats the purpose of wearing protective
gloves.
Detailed information on University policy and procedures is provided in the Environment, Health
and Safety Manual [1].
The Laboratory Rules and Safe Work Procedures set out in this practical manual must be adhered
to at all times and the direction of School staff and demonstrators must be followed. If you have any
concerns about the safety or environmental impact of any activity in School of Chemistry practical
classes, please raise them with the staff members in charge of the class. Report all injuries, accidents or
incidents immediately to a staff member.
A completed Risk Assessment must be submitted before the beginning of each Experiment and a
copy of this should be attached to your report. For assistance with information required to complete the
risk assessment visit the Subject Website. Material Safety Data Sheets (MSDS) and other information
about chemicals can be sourced through the ChemFFX facility [2]. This is a password-restricted location
so you will need to provide your University username/email password to access it.
If you have an allergy or medical condition that you think may be affected by the chemicals, materials
or procedures to be used in these practical classes, please fill-in a Medical Status-Notification for
Laboratory Classes Form (available on the LMS under Resources & forms) and give it to your
demonstrator (for transferral to Ms. Volaric), so that any risk can be assessed and the work procedures
modified accordingly.
You should ensure that you are familiar with the location and operation of safety apparatus such as
fire blankets, fire extinguishers, safety showers etc., and laboratory evacuation procedures (see Evacu-
ation procedure below).
The following rules apply to all laboratories in the School of Chemistry:
13
S AFETY IN THE L ABORATORY CHEM30015: Advanced Practical Chemistry
Safety glasses with side-shields conforming to Australian Standard (AS) 1337 must be worn at all
times in the laboratory. Prescription spectacles with polycarbonate lenses are acceptable provided
they are fitted with side shields (available from optometrists). If you wear contact lenses, safety
goggles, which provide a total seal around the eyes, must be substituted for safety glasses.
A long-sleeved, knee length laboratory coat (available from the Chemistry Store, the Union, and
other commercial protective clothing suppliers) and shoes which enclose the feet must be worn
in the laboratory. Thongs, sandals and open style shoes are prohibited.
Do not roll up the sleeves of your laboratory coat. The sleeves are there to protect your arms.
A range of disposable gloves will be provided (e.g. nitrile, Latex). Only certain types of gloves
are appropriate for certain circumstances. Please ensure you wear the appropriate gloves for
the situation. This should be specified for your practical exercise, otherwise please consult a
demonstrator. If you have a Latex or powder allergy, please speak to a demonstrator. Also ensure
that you use long armed gloves when advised to do so.
Laboratory coats and gloves must be removed before entering the multi-media room. Gloves
should also be removed before using the laboratory computers or instrumentation, or personal
calculators, phones etc.
Take care when using hot plates (or similar) - avoid contact with your body, clothing or electrical
leads.
Tongs should be used where appropriate, when handling hot vessels.
Listening to portable media players (iPods, etc.) is not permitted in the laboratory.
Long hair must be fastened securely.
Eating, drinking, smoking or chewing of gum is not permitted in the laboratory.
Pipetting by mouth is prohibited. Pipette fillers are provided and must always be used.
In any chemical laboratory there is always a potential danger from accidental splashing or spillage
of chemicals, cuts from broken glass, burns from touching hot apparatus or splashing hot liquids and
fire. Part of your training in practical chemistry is to learn the procedures, which minimise these dangers
and allow safe working conditions. Specific safety precautions relating to particular experiments are
detailed in this laboratory manual. All laboratory glassware must be handled with due care. Hot objects
should be allowed to cool before handling. If it is essential to handle a hot object (e.g., pouring a hot
liquid from a beaker) use a cloth, an insulated glove or tongs to hold the object. In the case of a major
chemical spillage or fire, evacuation of the laboratory may be required.
Accidents and First Aid
Chemicals
All chemicals must be treated with respect. Some (e.g., concentrated acids and alkalis) are corrosive
(to skin and clothing), some (e.g., cyanides) are poisonous and many, particularly organic chemicals,
(e.g., phenols and aromatic amines) are toxic by skin absorption or breathing of vapours. Organic
solvents (e.g., hydrocarbons, ether and alcohols) are volatile and highly flammable and must not be used
in the presence of an ignition source (e.g., an electric hotplate or flame.) When a chemical substance is
used for the first time you should ask a staff member about its properties, or consult a reference book. In
this course you will find specific safety precautions and procedures detailed in the notes. The following
general precautions always apply:

CHEM30015: Advanced Practical Chemistry S AFETY IN THE L ABORATORY
Chemicals should never be touched, tasted or smelled. Always handle toxic or foul smelling
chemicals in a fume cupboard.
Spillage on the skin: If any chemical comes in contact with the skin immediately wash with
running water from a tap, shower hose or shower. Organic chemicals (e.g., toluene, phenols and
aromatic amines) are readily absorbed through the skin. After washing with cold running water for
several minutes, wash the exposed area with warm water and soap. Clothing contaminated with
a chemical should be removed immediately, placed in a plastic bag and later washed separately
from other clothing.
Chemicals in the eyes: The use of approved safety glasses or goggles will greatly reduce the risk
of injury to the eyes. An accident involving a chemical splash into the eyes must be regarded
as serious. The immediate treatment is to wash the eye with cold running water at the eye-wash
station. Then report to the demonstrator, who may advise seeking medical attention.
In the event of a chemical splash to the head or upper torso immediately remove contaminated
clothing and wash the affected area under a safety shower. Report to the demonstrator who may
advise seeking medical attention.
Fire
Many chemicals are flammable and must not be used when an ignition source (e.g., an electric hot
plate or flame) is present. You are required to know the location of the nearest fire extinguisher, fire
blanket and safety shower. If a persons hair or clothing catches fire, try to smother the flames with a fire
blanket or laboratory coat, rolling the person on the floor if necessary. The safety shower can also be
used. Do not allow the person to remain standing to prevent the flames from reaching the head. Report
to the demonstrator, who may advise seeking medical attention for burns.
The following general precautions always apply:
Do not use any type of chemical fire extinguisher on a person.
Never heat an organic liquid in an open vessel (e.g., a beaker or flask) on an electric hotplate or
over a flame.
Never distil a liquid to dryness (the vessel may crack) and always use anti-bumping granules
during a distillation or reflux operation.
Evacuation procedure
When the alarm sounds:
Stop what you are doing and turn off electricity and any gas taps.
Listen to evacuation instructions.
Move quickly from the laboratory using the nearest exit, taking only personal belongings.
In the passageway, the fire wardens will direct you to the building exit and to the assembly point
well clear of the building.
You may only re-enter the building when the chief fire warden gives the all clear.

S AFETY IN THE L ABORATORY CHEM30015: Advanced Practical Chemistry
Glassware
Glass is a very hard material but brittle and breaks readily under stress or strain. Handle all laboratory
glassware carefully. Do not use chipped or cracked glassware. In the case of breakage of laboratory
glassware, which results in a cut, any particles or splinters of glass in the wound must be removed. All
cuts must be reported to the demonstrator, who will inspect the wound and may advise seeking medical
attention. Broken glass should be carefully cleaned-up using a dust pan and brush and disposed of
in the glass bin provided. If you have any concerns relating to to cleaning up broken glassware, please
consult a demonstrator or lab staff. Replacement glassware may be obtained by asking at the service
desk.
Waste materials
All chemical waste should be disposed of in a safe and environmentally responsible manner. Chem-
ical waste, other than non-hazardous aqueous solutions, which have been neutralised, must not be
washed down the sink. Particular care must be taken in disposing of some chemical reaction residues.
Follow the specific instructions given in the laboratory manual. Waste chemical bottles will be provided
in the laboratory. Ensure that each waste bottle is used for only the type of chemical waste noted on the
label.
Care of benches and apparatus
Each student is responsible for the section of laboratory bench allotted to him/her. Any chemicals
or water spilled on the bench must be cleaned-up immediately. Concentrated acid spills should first be
neutralised with sodium bicarbonate and then washed away with cold water. Your working area and the
communal areas (e.g. reagent benches and fume cupboards) must be kept clean and tidy at all times.
Untidy work areas invite accidents! Large chemical spills (>500 mL) must be contained immediately
using vermiculite or sand. The contaminated waste should then be placed in a sealed labelled bag for
proper disposal and the floor mopped. Broken glass must be swept up using a brush and dustpan.
Mops, brooms, brushes and dustpans are available from the Preparation Room and from the side of the
fumehoods.
If your bench is left in an unacceptable state at the end of the laboratory practical session, marks
may be deducted from your assessment.
Pre-lab preparation and general considerations
It is essential when undertaking the experimental course that you be prepared before entering the
laboratory to commence your experiments. Experiments at the third year level require time management
in order to successfully complete all required sections within the allocated time. In order for your exper-
ience in the laboratory to be an enjoyable one, it is essential you have good planning and organisation.
Below are listed some points that you should keep in mind before commencing any day in the laboratory.
For safety reasons you may not enter the laboratory areas without PPE (lab coat & safety glasses).
This means that you cannot leave your PPE in your lab locker - there are coat hooks at the north
end of the Level 3 lab for safe storage of these items (during the day & between classes).
Identify the location and operation of all safety related features including safety showers, fire
blankets and extinguishers, evacuation paths/assembly points etc..

CHEM30015: Advanced Practical Chemistry S AFETY IN THE L ABORATORY
Plan your day. It is essential you plan what you are to complete in any laboratory session - this
includes thoroughly reading through the experiment you have scheduled before arriving to the lab.
Attempt all pre-lab questions relating to your scheduled experiment. There is not sufficient
time in each laboratory session for pre-lab questions to be completed on the day. Unless
a serious attempt has been made you may be refused entry to the laboratory to complete
your experiment. It is also advisable for you to have a list of any questions you may have for
demonstrators to help clarify areas you are uncertain about before commencing.
Typically you will be expected to have a time-line, flow-chart and equipment / chemical list ready
for your given experiment. You are also required to complete a risk assessment for your prac and
submit it to a demonstrator prior to starting any experimental work.
Key references are provided for each experiment - it is expected that you will consult these ref-
erences and additional sources for your reports. Further reading is necessary to provide adequate
background and current insight for the writing of reports. The background provided as an intro-
duction for each experiment is generally not sufficient for your reports.
References
[1] http://safety.chemistry.unimelb.edu.au/ehs-procedures-and-processes/
[2] http://safety.unimelb.edu.au/hazard-topics/chemical-management/
goldffx-chemical-management-system

List of Experiments
List of Experiments
Experiments in Synthesis and Characterisation 1
SC1.1 - Preparation of N-Benzylphthalimide . . . . . . . . . . . . . . . . . . . . . . . 27
SC1.2 - 2-Phenyl-4-benzylidene-5-oxazolone: Azlactone formation . . . . . . . . 29
SC1.3 - Synthesis of a para-Substituted Cinnamic Acid Using a Palladium-Catalysed

Heck Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
SC1.4 - Flow Synthesis of a Bicyclic Lactone . . . . . . . . . . . . . . . . . . . . . . . 35
Experiments in Synthesis and Characterisation 2
SC2.1 - Synthesis of 5,5-Diphenylhydantoin (DPH) . . . . . . . . . . . . . . . . . . . 41
SC2.2 - Synthesis of 3-Carbethoxycoumarin . . . . . . . . . . . . . . . . . . . . . . . 43
SC2.3 - Thiamine-Catalysed Synthesis of Benzoin . . . . . . . . . . . . . . . . . . . . 45
SC2.4 - Synthesis of 4-Phenylbenzoic acid by Palladium-catalysed Cross Coupling 47
Extended Synthesis and Characterisation of Organic Com-

pounds
ESC1 - Preparation of a Bicyclo[2.2.1]heptene by Cycloaddition . . . . . . . . . . . 51
ESC2 - Grignard Synthesis of Triphenylmethanol . . . . . . . . . . . . . . . . . . . . 53
ESC3 - Synthesis of Methyl 4-Nitrocinnamate Using the Wittig Reaction . . . . . . 57
ESC4 - Synthesis of (3-Ethyl-pentane-3-ol) . . . . . . . . . . . . . . . . . . . . . . . . 61
Kinetics
19
LIST OF EXPERIMENTS CHEM30015: Advanced Practical Chemistry
K1 - Excited State Reaction Kinetics: Pyrene Excimer Formation . . . . . . . . . . 65
K2 - Flash Photolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
K3 - Kinetics of the Acid Catalysed Aquation of Fe(II)-Tris(chelates) . . . . . . . . 83
Physical Characterisation
PC1 - Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
PC - Introduction to Investigations into Surface Tension . . . . . . . . . . . . . . . . 97
PC2 - Surface Tension Determination Pendant Drop . . . . . . . . . . . . . . . . . . 99
Electrochemical Processes
EC1 - Coupled Chemical and Electrochemical Reactions . . . . . . . . . . . . . . . 107
EC2 - Electrochemical Kinetics of Redox Reactions . . . . . . . . . . . . . . . . . . . 117
Light/Matter Interactions
LM1 - Photolysis of Ethanal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
LM2 - Fluorescence Polarisation Measurements of Molecular Motion . . . . . . . 135
LM3 - Principles of Fluorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Computational Chemistry
CC - Computational Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Metal-Based Compounds: Synthesis & Characterisation 1
M1.1 - Oxo-Molybdenum Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
M1.2 - Reactions of Coordinated Ligands . . . . . . . . . . . . . . . . . . . . . . . . . 173
Metal-Based Compounds: Synthesis & Characterisation 2
M2.1 - Copper Bypyridine Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
M2.2 - Electronic Spectroscopy of Coordination Compounds . . . . . . . . . . . . . 183
M2.3 - Geometrical Isomers of Mo(CO)4 (PPh3 )2 . . . . . . . . . . . . . . . . . . . . 191
Themed Experiments

CHEM30015: Advanced Practical Chemistry LIST OF EXPERIMENTS
Theme based Experiments and Group Modules . . . . . . . . . . . . . . . . . . . . . 197
Theme 1 - Sulfa Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
T1.0 - Synthesis and Characterisation of Sulfanilamide . . . . . . . . . . . . . . . . 201
T1.1 - Synthesis of Prontosil & Carbonic Anhydrase Binding Studies . . . . . . . . 207
T1.2 - Removal of Zn(II) From Carbonic Anhydrase - A Kinetics Experiment . . 213
T1.3 - Three Dimensional Analysis of the Carbonic Anhydrase Active Site . . . . . 219
Theme 2 - Dye-Sensitised Solar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
T2.0 - Construction and Characterisation of a Dye-Sensitised Solar Cell . . . . . . 235
T2.1 Determination of the Surface Area of Powders by Gas Adsorption . . . . . . 249
T2.2 Particle Size Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
T2.3 Photophysical study of Natural Dyes . . . . . . . . . . . . . . . . . . . . . . . . . 261
Theme 3 - Flow Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
T3.0 - Synthesis of Coumarin Dyes in Continuous Flow . . . . . . . . . . . . . . . . 269
T3.1 - Translation of Synthesis into Flow . . . . . . . . . . . . . . . . . . . . . . . . . 273
T3.2 - Photophysical Properties of Coumarin Derivatives . . . . . . . . . . . . . . . 275
T3.3 - Computational Study of the Photophysical Properties of Coumarin Derivat-

ives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

S ECTION 1: I NDIVIDUAL C ORE
E XPERIMENTS
Experiments in Synthesis and
Characterisation 1
In the following pages outline the preparations for all experiments offered in the Synthesis and
Characterisation 1 section of the course.
You must complete one experiment from the offered 3 in the SC1 block.
NMR and mass spectra of your samples may be recorded (see Ms Volaric) or typical spectra will be
provided on the LMS for analysis, where required.
SC1.1 - Preparation of
N-Benzylphthalimide
A/Prof. Craig Hutton

This experiment will be assessed by viva. Please see the LMS for details.
1 Introduction
Protecting groups are often required in synthetic sequences to prevent unwanted side reactions. For
example, amines are frequently converted to amides, imides or carbamates to prevent their reactivity as
nucleophiles or bases. Conversion of primary amines to phthalimides is a common protection strategy as
phthalimides are stable with the phthaloyl protecting group removable only under specific conditions.
Five- and six- membered cyclic imides can be formed by treating a primary amine with the corresponding
cyclic anhydride, followed by heating of the intermediate amide-acid.
2 Experimental Procedure
**Benzylamine is a corrosive lachrymator and phthalic anhydride is an irritant. Make sure

you have completed your Risk Assessment and had it signed by a demonstrator before commencing
the laboratory session.
This experiment will require several hours in one session, followed by isolation and recrys-
tallisation of the product in a subsequent session. You should consult your demonstrator
about conducting other assigned experiments during this session in parallel with this pro-
cedure.
Place phthalic anhydride (3.7 g) and acetic acid (30 mL) in a 100 mL round bottom flask. Add a
magnetic stirrer bar, and fit the flask with a condenser. Cautiously add benzylamine (2.5 mL) portion
wise with a Pasteur pipette down the condenser over a period of 10 min to the stirred mixture. (care: the
reaction is exothermic). After the addition is complete, heat the mixture at reflux for 1 h.
Carefully pour the hot solution into 125 mL of water in a 500 mL conical flask, and quickly bring
the mixture to the boil on a hot plate. Allow the mixture to cool overnight. Filter off the crude N-
benzylphthalimide and recrystallise the damp product from ethanol.
27
SC1.1 CHEM30015: Advanced Practical Chemistry
Record the yield of your product and ensure all characterisation data required is collected.
This should include the 1 H NMR, IR and mass spectra, and the melting point. Acquire the
1 H NMR spectrum using the new 43 MHz Magritek Ultra Compact Spectrometer (instruc-
tions available on the LMS). Process the spectrum either on the spectrometer computer or
using iNMR in the multimedia room. Include an analysis of your spectrum in your report
(peak assignments, multiplicity if applicable, interpretation of impurities). Also provide
an analysis of the 13 C NMR spectrum (available on the LMS).

SC1.2 -
2-Phenyl-4-benzylidene-5-oxazolone:
Azlactone formation
A/Prof. Craig Hutton

This experiment will be assessed by written report.
1 Introduction
Oxazolones are easily made from N-acyl--amino acids. Enolisable oxazolones condense readily
with aryl aldehydes to form azlactones (an old term for oxazolones). Alkyl aldehydes tend to self-
condense rather than form azlactones. 4-Subsituted-5-oxazolones have use as synthetic intermediates,
for example in the synthesis of aromatic amino acid derivatives.
**Caution: Acetic anhydride is a vesicant and a lachrymator
This experiment involves an initial short set-up and reaction time, followed by isolation
and recrystallisation of the product after standing your reaction overnight. You should
consult your demonstrator about conducting other assigned experiments during this ses-
sion in parallel with this procedure.
Preparation of 2-Phenyl-4-benzylidene-5-oxazolone
Place hippuric acid (N-benzoylglycine; 3 g), benzaldehyde (2.2 g) and anhydrous potassium acetate
(1 g, very hygroscopic) in a 50 mL round-bottomed flask. Add a stirrer bar and acetic anhydride (10
mL), then cover the flask with aluminium foil. Stir the mixture as vigorously as possible without any
splashing; be careful not to get any of the solids adhering to the wall of the flask above the liquid. The
reaction is mildly exothermic and as the azlactone begins to form the mixture becomes a paste. Continue
stirring all parts of the paste until it becomes so thick that the stirrer bar stops (or for 1/2 hour, whatever
is sooner) then leave the mixture to stand overnight.
To hydrolyse the excess acetic anhydride and complete the separation of the azlactone, mix in 25
mL of water and stir the suspension until it forms a thick slurry, free from lumps (you may have to use
a glass rod to aid this process, which can take up to 30 min).
29
Filter off the solid, wash it well with water, then press and drain. Wash the compact filter cake first
with 10 mL of an ice-cold 1:1 mixture of methanol and water, then with 10 mL of ether. Recrystallise
the dry azlactone from ethyl acetate.
3 Questions
1. Provide a detailed mechanism for this reaction.
2. Azlactone formation is a common problem during peptide synthesis, as it leads to epimerisa-

tion of the C-terminal amino acid residue. Discuss the reasons why azlactones are particularly
susceptible to epimerisation/racemisation

SC1.3 - Synthesis of a para-Substituted
Cinnamic Acid Using a
Palladium-Catalysed Heck Reaction
Prof. Mark Rizzacasa

1 Introduction
In the early 1970s R. F. Heck and T. Mizoroki independently discovered that aryl, benzyl and vinyl
halides react with alkenes in the presence of a palladium catalyst and hindered amine base to form
substituted olefins (Figure 1). Since its discovery, the so called Heck reaction has become one of the
most widely used catalytic carbon-carbon bond forming cross coupling reactions in organic synthesis.
C-C bond formed R'

R = aryl,vinyl, benzyl
Pd0
+ R via
R X R' R' Pd(II)
HX R
X = Cl, Br, I
sp2-sp2 CC bond formed
Br 1 mol% Pd(OAc)2
CO2Me 2 mol% PPh3 CO2Me
+
NEt3 (base)

Figure 1: Scheme 1
The Heck reaction is best applied for the preparation of disubstituted alkenes and the electronic
nature of the substituents on the olefin only has limited influence on the outcome of the reaction. Elec-
tron poor olefins usually give higher yields and the reaction conditions tolerate a wide range of func-
tional groups on the olefin component such esters, ethers, alcohols, carboxylic acids, nitriles, phenols
and dienes. Thus, when compared to traditional C-C bond forming reactions using sensitive and react or-
ganometallic reagents, the Heck reaction has many advantages. The reaction rate is strongly influenced
by the degree of substitution of the olefin and usually the more substituted olefin undergoes a slower
Heck reaction and terminal alkenes predominantly undergo substitution at the least substituted olefinic
carbon. The nature of the X group on the aryl or vinyl component is very important and the reaction
rates change in the following order: I >Br OTf Cl.
Interestingly, the Heck reaction is not sensitive to water, and the solvents need not be thoroughly
deoxygenated. In addition, the reaction is stereospecific as the migratory insertion of the palladium
complex into the olefin and the -hydride elimination both proceed with syn stereochemistry. There
are a couple of drawbacks of the Heck reaction: 1) the substrates cannot have hydrogen atoms on their
-carbons, because their corresponding organopalladium derivatives tend to undergo rapid -hydride
elimination to give olefins; and 2) aryl chlorides are not always good substrates because they react very
slowly.
31
I cat. Pd(OAc)2 CO2H

CO2H
+ NEt3, MeCN
Br reflux Br

In a 10 mL round bottom flask place para-bromoiodobenzene (560 mg), palladium(II) acetate (5

mg), acetonitrile (2 mL), triethylamine (0.70 mL) and acrylic acid (0.170 mL). Add a boiling chip,
equip the flask with a condenser (a B19/B14 adaptor will be required) and heat on a steam bath for 1
hour.
Cool the solution and pour into 50 mL of 2 M HCl in a 100 mL beaker, rinsing the flask with
portions of water to complete the transfer. Mix the precipitated solid and carefully break up any large
lumps. Collect the solid by vacuum filtration and wash with several portions of cold water. Recrystallise
the crude product from ethanol/water and collect the purified product by vacuum filtration. A second
crop of product may be obtained from the filtrate.
Determine the melting point of your product and obtain 1 H-NMR (d6 -DMSO as solvent) and IR
spectra.
3 Report
Your discussion should provide a concise analysis of the spectroscopic data (IR, mass spectrum
and NMR) and use this information to determine the structure of the product formed. In particular,
briefly describe how the various spectroscopic techniques provide evidence for the regiochemical and
stereochemical outcome of the reaction.
4 Questions
1. Provide a detailed mechanism for the Heck reaction. In particular, show how the oxidation states
of the palladium catalyst change in the catalytic cycle.
2. The Heck reaction can be undertaken using both aryl iodides and aryl bromides. How is the reac-
tion between para-bromoiodobenzene and acrylic acid an example of a chemoselective reaction?
3. Cinnamic acids, such as the one prepared is this experiment, may also be synthesised using a
Wittig reaction. What advantages are there to using the Heck reaction in comparison to the Wittig
reaction?
4. One geometric isomer is formed preferably in the Heck reaction to give a disubstituted alkene.
Explain the origin of this selectivity.
References
[1] William B. Martin and Laura J. Kateley, J. Chem. Ed. 2000, 77, 757-759.
[2] Belestskaya, I. P.; Cheprakov, A. V. Chem. Rev. 2000, 100, 30093066
[3] de Meijere, A.; Meyer, F. E. Angew. Chem., Int. Ed. Engl. 1994, 33, 237924.

CHEM30015: Advanced Practical Chemistry SC1.3
[4] Brase, S.; de Meijere, A. In Metal-catalyzed Cross-coupling Reactions, Diederich, F., and Stang, P.
J., Eds.; Wiley-VCH: New York, 1998, pp. 99166.
[5] Cabri, W.; Candiani, I. Acc. Chem. Res. 1995, 28, 27.

SC1.4 - Flow Synthesis of a Bicyclic
Lactone
Dr Anastasios Polyzos and Dr Wallace W. H. Wong

This experiment will be assessed by oral presentation (see below for details).
1 Introduction
This experiment illustrates a Diels-Alder cycloaddition[1] under continuous flow synthesis condi-
tions. You will compare the outcome of the flow reaction with conventional synthesis setup.
This experiment involves training on the use of the flow synthesis equipment. Please
ensure that you arrive at your allocated practical session on time as there will be ONE
training time per practical session. Note that you will take turns running the flow equip-
ment. You can prepare and start the reaction using conventional equipment while waiting
for your turn.
Preparation of 5-methyl-3-oxo-1,3,3a,4,5,7a-hexahydroisobenzofuran-4-carboxylic acid
2.1 For flow synthesis:
35
2.2 Prepare the reagent reservoirs:
1. In a 50-mL capacity bottle, combine the E,E-2,4-hexadien-1-ol (1 g), maleic anhydride (1 g), and
tetrahydrofuran (12 mL). Label this bottle reagent.
2. Swirl the contents of the flask to ensure adequate mixing of the reagents.
3. In a second 100 mL capacity bottle place 80 mL of tetrahydrofuran (THF). Label this bottle
solvent.
4. Setting up the flow machine: This part will be performed by the demonstrator and shown to you
at the training session.
5. Equip the flow unit with a 10-mL capacity PFE reactor coil.
6. Select a blue pump for performing the reaction.
7. Ensure tubing is connected as shown below.
1. Fully open the back-pressure regulator.
2. Place the exit line in a 50-mL bottle and label as waste.
3. Place both the solvent line and the reagent line for the blue pump into the bottle labelled solvent.
4. Turn on the flow unit and prime the solvent and reagent lines for the blue pump with THF from
the solvent.

5. Carefully move the reagent line from the bottle labelled solvent to that labelled reagent.
6. Ensure the pump is set back to solvent on the screen.
7. Pass solvent through the reactor coil at a flow rate of 3.0 mL/min until it is filled.
8. Once the reactor coil is filled, reduce the flow rate to 1.0 mL/min.
9. Adjust the back-pressure-regulator carefully to a pressure of 7 bar.
10. Set the temperature of the reactor coil to 120 C.
11. Once the desired temperature is reached, and the pressure is stable, the unit is ready to run the
reaction.
2.3 Synthesise the bicyclic lactone:
1. Switch the pump from solvent to reagent.
2. Set the exit stream to go to collect.
3. When the contents of the reagent bottle are almost all loaded into the reactor, switch the pump
from reagent back to solvent - It is important to perform this step when there is still a small
quantity of liquid remaining in the reagent bottle or else there is the possibility of allowing air
into the reactor.
4. Continue collecting for another 15 min.
5. Once all the reagents have exited the flow unit, turn off the heating to the reactor by pressing the
on / off button on the reactor quadrant of the screen.
6. When the temperature is below 50 C, press the Stop All button.
2.4 Purify the product:
1. While the reaction is running set up a vacuum filtration system.
2. When the reaction is complete, transfer the contents of the product bottle to a round bottomed
flask. Rinse the product bottle with THF (5 mL) and add the wash to the flask.
3. Remove the THF solvent under reduced pressure until a white solid is obtained.
4. Rinse the solid with diethyl ether (10 mL) to remove any yellow colouration from the solid.
5. Collect solid by filtration and wash with small amounts of diethyl ether.
6. Allow the product to air dry on the filter funnel for several minutes.
7. Allow the product to air dry until a constant weight is reached.
8. Calculate the yield and collect characterisation data (1H NMR, IR, melting point). The 13C NMR
and mass spectrometry data are provided on LMS. Include analysis of all the characterisation data
in your report.

2.5 For conventional synthesis:
In THF: Dispense 1 ml of E,E-2,4-hexadien-1-ol (0.61 M in THF) in an appropriate size glass con-

tainer. Add maleic anhydride, 60 mg to the glass vial. Heat the mixture until it refluxes (70 C) in a Ratek
heating block. The reaction progress is monitored by thin layer chromatography for 35-40 minutes using
ethyl acetate as the developing solvent. A sample of the Diels Alder Bicyclo Lactone product can be
obtained from your demonstrator for comparison. Based on the TLC results use your judgement to see
if you need to work-up the reaction.
In toluene: Dispense 1 ml of E,E-2,4-hexadien-1-ol (0.61 M in toluene) in an appropriate size glass
container, Add maleic anhydride, 60 mg to the glass container. Heat the mixture until it refluxes
(120 C) in a preheated sandbath. The reaction progress is monitored by thin layer chromatography for
35-40 minutes using ethyl acetate as the solvent. A sample of the Diels Alder Bicyclo Lactone product
can be obtained from your demonstrator for comparison.
When no starting materials remain, the solution is then allowed to cool slowly to room temperature,
during which time a white crystalline material deposits on the sides of reaction vessel. The reaction
mixture is cooled in an ice/water bath for 10 min to ensure complete crystallisation of the product. The
product is isolated by vacuum filtration, rinsed once with a small amount of diethyl ether, and allowed
to air dry, leaving a white crystalline solid. Use an eppendorf vial to collect the product. Compare the
outcome of the conventional reaction with the flow reaction.
Compare the outcome of the conventional reaction with the flow reaction.
3 Assessment
This practical will be assessed by a short oral presentation. Please prepare a brief (5 minute)
Powerpoint slide presentation to the marker. The audience will include other students. The presentation
should be around 5-7 slides in the following order:
i. Aims
ii. Results:
(a) Yields in batch and flow;
(b) Characterisation data (1H NMR, IR, melting point). Comment on product quality and purity.
iii. Questions: Provide answers to the following (1 slide each)
(a) Propose a mechanism of the reaction.
(b) Draw the chemical structure of the product clearly indicating the stereo outcome of the reac-
tion.
(c) Compare the reactions in the flow and conventional experiments. Summarize the advantages
of using flow processing.
iv. Conclusions
4 Questions
(i) Provide the mechanism of the reaction.

(ii) Draw the chemical structure of the product clearly indicating the stereo outcome of the reaction.
(iii) Compare the reactions in the flow and conventional experiments. Summarise the advantages of
using flow processing.

References
[1] Diels, O.; Alder, K. Liebigs Ann. Chem., 1928, 460, 98.
[2] Kurti, L.; Czako, B. Strategic Applications of Named Reactions in Organic Synthesis. Elsevier
Academic Press: Burlington, MA, 2005.

Experiments in Synthesis and
Characterisation 2
In the following pages outline the preparations for all experiments offered in the Synthesis and
Characterisation 2 section of the course.
You must complete one experiment from the offered 3 in the SC2 block.
NMR and mass spectra of your samples may be recorded (see Ms Volaric) or typical spectra will be
provided on the LMS for analysis, where required.
SC2.1 - Synthesis of
5,5-Diphenylhydantoin (DPH)
A/Prof. Uta Wille

This experiment will be assessed by a viva.
1 Introduction
The synthesis of 5,5-diphenylhydantoin (DPH) can be performed by the base-catalysed reaction of

urea with benzil, which is a variation of the classical benzilic acid rearrangement. The sodium salt of
DPH is known as phentoin sodium (marketed in Australia as Dilantin), which is a commonly used
antiepileptic that acts by suppressing the abnormal brain activity seen in seizures. The synthesis of
5,5-diphenylhydantoin is accompanied by formation of the by-product diphenylglycouril.
Place benzil (2.10 g, 10.0 mmol), urea (1.00 g, 16.7 mmol), 3 mL of water and 20 mL of ethanol in
a 100 mL round bottom flask. Fit the flask with a regular condenser and heat the mixture strongly in a
heat-on block. When the mixture is boiling and homogeneous (e.g. all solids are dissolved), add 5 mL
of a 10% aqueous sodium hydroxide solution via the condenser. Mix well, add a boiling chip and let the
mixture reflux for 15 to 20 minutes.
During this reflux period a white precipitate forms, which is the by-product diphenylglycouril. In
order to remove the by-product, dilute the hot suspension with 50 mL of water, cool and separate the
solid by suction filtration. (Dry weigh this product and submit it with your DPH sample and report. Do
not determine the melting point as it is rather high).
The desired product is obtained by slightly acidifying (pH <7) the filtrate with acetic acid. After
this, heat the resulting suspension strongly in a heating mantle for 15 minutes, then allow to cool to
room temperature. Isolate the solid by filtration and wash it with small portions of cold water. Purify
the crude product by recrystallization from ethanol.
Determine the yield and the melting point and run and interpret an infrared spectrum of your product.
41
Record the yield of your product and ensure all characterisation data required is collec-
ted. This should include the 1 H and 13 C NMR, IR and mass spectra, and the melting point.
Acquire the 1 H NMR spectrum using the new 43 MHz Magritek Ultra Compact Spectro-
meter (instructions available on the LMS).Use d6-dmso for this experiment. Process the
spectrum either on the spectrometer computer or using iNMR in the multimedia room.
Include an analysis of your spectrum in your report (peak assignments, multiplicity if ap-
plicable, interpretation of impurities). Also provide an analysis of the 13 C NMR spectrum
(available on the LMS). Analysis and peak assignments are to be included in your report.
3 Questions
1. The benzilic acid rearrangement is the rearrangement of benzil to benzilic acid in the presence of
base (sodium or potassium hydroxide). Give a mechanism for this rearrangement.
2. Draw a detailed mechanism for formation of 5,5-diphenylhydantoin from benzil and urea.
3. Give a mechanism for the formation of the by-product diphenylglycouril.
4. Provide the IUPAC name for the heterocycle found in 5,5-diphenylhydantoin.

SC2.2 - Synthesis of
3-Carbethoxycoumarin
Prof. Richard OHair

1 Introduction
Coumarin and its derivatives are widely distributed in plants. Coumarin itself occurs in many essen-
tial oils including lavender oil, woodruff and tonka beans, and is in part responsible for the characteristic
odour of new-mown hay. A number of 3-substituted-4-hydroxycoumarins are powerful anticoagulants
and are used to control blood clotting, and as rodenticides.
In this experiment 3-cabethoxycoumarin is prepared via a Knoevenagel reaction, followed by lac-
tonisation.
In a dry 25 mL round bottom flask place salicylaldehyde (1g), diethyl malonate (1.4g), piperidine
(0.1 mL), acetic acid (1 drop) and absolute ethanol (5 mL). Add a boiling chip, equip the flask with a
regular condenser and a drying tube (to protect the mixture from atmospheric moisture), then boil the
mixture gently on a steam bath for 2 hours.
Pour the yellow reaction mixture into a 50 mL conical flask, then use portions of water totalling
about 17 mL to rinse out the reaction flask and so complete the transfer. Mix the contents of the conical
flask thoroughly then chill the suspension in ice (for about 15 minutes). Collect the solid by suction
filtration then wash it with several small portions of cold water. Recrystallise the crude product from
ethanol/water using charcoal if necessary. Determine the melting point and run an infrared spectrum of
your product.
43
3 Questions
1. The Knoevenagel reaction is a special case of an aldol reaction, which always leads to condensa-
tion (e.g. formation of the , -unsaturated product). Explain.
2. In contrast to aldol reactions, which require strong bases, Knoevenagel reactions can be performed
using weak bases, like piperidine. Explain.
3. The -ketoester immediately below undergoes decarboxylation upon base hydrolysis followed by
neutralisation with acid. Give a mechanism for this transformation.
4. Similar treatment of 3-carboethoxycoumarin gives the carboxylic acid (below), which is stable to
decarboxylation. Explain why decarboxylation does not occur.

SC2.3 - Thiamine-Catalysed Synthesis of
Benzoin
Prof. Jonathan White

1 Introduction
Thiamine, commonly known as vitamin B1, is a co-enzyme, which acts in concert with appropri-
ate enzymes in living systems to bring about a range of organic reactions essential to metabolism [1].
Examples include oxidative and non-oxidative decarboxylation of -keto acids, which are central to
aerobic and anaerobic breakdown of pyruvic acid. Thiamine is also involved in the transformation of
aldehyde containing precursors into acyloins (-hydroxy ketones), which is the basis of this practical.
The mechanism of action of thiamine is essentially the same in all the above reactions and provides an
early example of nucleophilic catalysis [2].
Dissolve thiamine hydrochloride 1.3 g (3.85 mmol) in 4 mL water in a 100 mL B19 quick-fit conical
flask. Then add 15 mL of ethanol to the reaction flask and cool in an ice-bath for 5 minutes. Add
2.5 mL of 3M NaOH dropwise with stirring/swirling over a period of 3 minutes while maintaining the
temperature in the range of 10-15 C.
To the above yellow solution add 7.5 mL (73.9 mmol) of pure benzaldehyde and place the reaction
mixture in a 70 C thermoregulated water bath for 15-20 minutes. (The internal temperature of the
reaction flask will be 69-65 C). Stopper the flask and leave it overnight at room temperature.
45
After 30 hours, filter the yellow needle-like crystals of benzoin were off on a 4.25/5 cm Buchner
funnel, then wash with 10 mL ethanol and dry at the vacuum pump for 10-15 minutes. Determine the
yield and melting point of the yellow solid. If possible obtain a second crop of benzoin from the filtrate.
Determine the infrared and proton nuclear magnetic resonance spectra, and the mass spectrum of
your product. Assign these spectra (see demonstrator for assistance). The proton NMR spectrum of
your products is to be run on the desktop NMR spectrometer in room 380 (the balance room). NMR
tubes and deuteriochloroform (CDCl3 ) can be obtained from your demonstrator.
3 Questions
1. Thiamine contains two heterocyclic ring systems, circle and name these heterocycles.
2. Identify the catalytic site of thiamine in this reaction (information required to answer this question
can be obtained from references below) and propose a mechanism for the thiamine catalysed
conversion of benzaldehyde into the product benzoin. Highlight the role of thiamine at each step
of this mechanism.
References
[1] McMurry 5th Edition or later: Chapter 29 The Organic Chemistry of Metabolic Pathways
[2] D. Enders, Nucleophilic Carbenes in Assymmetric Organocatalysis, Accounts of Chemical Re-

search, 2004, 534-541.

SC2.4 - Synthesis of 4-Phenylbenzoic acid
by Palladium-catalysed Cross Coupling
Dr. Wallace W. H. Wong

1 Introduction
Palladium-catalysed carbon-carbon bond formation has become an indispensable reaction in the tool
kit of synthetic chemists. Richard Heck, Ei-ichi Negushi and Akira Suzuki shared the Nobel Prize in
Chemistry in 2010 for their development of this important class of reactions (Figure 1). Suzuki-Miyaura
cross coupling[1] is arguably the most widely applied and is used in the synthesis of natural products,
pharmaceuticals, agrochemicals, liquid crystals and polymers. A building block commonly constructed
using Suzuki-Miyaura cross coupling is the biphenyl unit (Figure 2).
Figure 1: Reagents involved in Heck, Negishi and Suzuki cross-coupling reactions. Substituent, R, can
be aryl, vinyl or alkyl and leaving group, X, is typically a halide
Figure 2: Synthesis of biphenyl by Suzuki-Miyaura cross coupling
This experiment involves a relative short setup and reaction time, followed by isolation and
recrystallisation of the product. Other assigned experiments can be conducted in parallel with
this procedure.
47
2.1 Preparation of 4-phenylbenzoic acid
Place 4-bromobenzoic acid (0.5 g) and phenylboronic acid (0.37 g) in a 100 mL Erlenmeyer flask.
Add an aqueous solution of sodium carbonate (1 M, 15 mL),swirl the reagent mixture carefully until the
reactants dissolve. Begin heating the reaction on a steam bath and add the palladium catalyst solution[2]
(0.25mM, 0.01 mol%). The product should precipitate directly from the solution during the reaction
time of 30 min. Allow the reaction to cool to room temperature and acidify the reaction by carefully
adding HCl (1 M). Collect the crude product by filtration and recrystallise from HCl (1 M, 4 mL) and
ethanol (15 mL). Collect crystals of the product by vacuum filtration and dry the crystals by drawing air
over it.
This should include the 1H NMR, IR and mass spectra, and the melting point. Acquire the
1H NMR spectrum using the new 43 MHz Magritek Ultra Compact Spectrometer (instruc-
an analysis of the 13C NMR spectrum (available on the LMS).
3 Questions
1. Provide the mechanism of the reaction. This includes the catalytic cycle and the key steps of the
cycle should be labelled. Assume an active Pd(0) catalyst species is present in your reaction.
2. Inert atmosphere is normally used for Suzuki-Miyaura cross coupling reactions. Explain why this
is normally required.
3. Apart from the fact that the reaction was done in air, only water was used as the reaction solvent.
Comment on the green chemistry aspect of this experiment. Can the product be obtained using
Negishi cross coupling? Explain.
References
[1] Miyaura, N.; Suzuki, A., Chem. Rev. 1995, 95, 24572483.
[2] Chalker, J. M.; Wood, C. S. C.; Davis, B. G., J. Am. Chem. Soc. 2009, 131, 16346-16347.

Extended Synthesis and Characterisation of
Organic Compounds
In the following pages the preparations for all experiments offered for the Extended Synthesis and
Characterisation of Organic Compounds component of the course are included.
You must complete one experiment from those offered.
The experiments in this section require two full sessions (or parts thereof) to complete. You must
be prepared to start work immediately when you enter the laboratory and work efficiently if you are to
complete the assigned tasks within the time allocated.
Appropriate spectra can either be recorded (See Ms Volaric) or may be made available on the LMS.
Be sure to check the available files before completing your final laboratory session for the experi-
ment.
ESC1 - Preparation of a
Bicyclo[2.2.1]heptene by Cycloaddition
Dr. Wallace Wong

1 Introduction
This preparation illustrates an example of a cycloaddition reaction: the Diels-Alder reaction between
phencyclone and norbornadiene. A 1 H NMR spectrum will be provided which you should use to ascer-
tain the stereochemistry of the adduct produced in this transformation.
2.1 Preparation of Phencyclone
9,10-Phenanthrenequinone (0.75 g, 3.6 mmol), 1,3-diphenyl-2-propanone (0.80 g, 3.8 mmol) and

methanol (45 mL) are placed in a 100 mL round bottom flask equipped with a watercooled condenser
and a magnetic stirrer bar. The mixture is stirred while heating in a heat-on block. When the methanol
begins to reflux, a solution of methanolic potassium hydroxide (0.2 g KOH in 0.8 mL dry methanol) is
added dropwise (pipette) through the top of the condenser over a period of 1-2 min. When the addition
is complete, the mixture is stirred at reflux for an additional 15 min. After cooling in an ice bath (10-15
minutes) the black crystals are collected by suction filtration, washed with three portions of ice-cold
methanol, and dried in air. Determine the yield of phencyclone.
51
ESC1 CHEM30015: Advanced Practical Chemistry
2.2 Reaction of Phencyclone with Norbornadiene
Phencyclone (0.5 g, 1.3 mmol), norbornadiene (0.3 g, 3.25 mmol) and toluene (5 mL) are added to
a 25 mL round bottom flask equipped with a reflux condenser. A boiling chip is added and the mixture
heated at reflux using a heating mantle as the heat source. The progress of the reaction is monitored
by TLC (1:1 dichloromethane/ light petroleum) every 15 minutes and the heating is discontinued when
the TLC indicates the absence of phencyclone (approximately 1-1.5 hours). The reaction is cooled to
room temperature, and the product collected by vacuum filtration. Evaporation of the toluene (rotary
evaporator) yields additional product. The combined solids are recrystallised from dichloromethane /
methanol and dried to afford a white crystalline solid. Read Appendix A for mixed-solvent recrystal-
lisation technique. Determine the melting point and infrared spectrum of your product.
3 Questions
1. Obtain the 1 H NMR spectrum of the cycloadduct (the bicyclo[2.2.1]heptene) from your demon-
strator.
a. Analyze the 1 H NMR spectrum of the bicyclo[2.2.1]heptene cycloadduct. You do not need to
provide a full analysis of the 1 H and 13 C NMR spectra; rather focus on analysing the parts of
the spectrum that are relevant to parts b) and c) below.
b. Propose a structure that shows the stereochemistry of the bicyclo[2.2.1]heptene cycloadduct.
c. Discuss how the 300 MHz 1 H NMR spectrum supports the structure of the cycloadduct.
d. Provide a mechanism for the Diels-Alder reaction that explains how the diene and dienophile
approach one another to afford a product with the proposed stereochemistry.
2. The Diels-Alder reaction is one of the most useful synthetic methods to make six-membered rings.
It requires an alkene (dienophile) and a 1,3-diene. Predict the product of each of these reactions:
3. Identify the starting materials that can be used to produce the compounds shown using a Diels-
Alder reaction.

ESC2 - Grignard Synthesis of
Triphenylmethanol

1 Introduction
Organohalides (RX) react readily with magnesium to form Grignard reagents, so named after their
discoverer Victor Grignard. Grignard reagents are both bases and nucleophiles. The nucleophilic reac-
tion of Grignard reagents and carbonyl compounds constitutes an effective means to construct carbon-
carbon bonds.
As bases Grignard reagents react with any acidic protons such as water, alcohols, amines, thiols, etc.
to generate the corresponding alkane and destroying the reagent. The formation of a Grignard reagent
is therefore highly sensitive to the presence of water, and great care should be taken to ensure your
glassware is properly dried prior to commencing either part of this experiment.
Grignard Synthesis of Triphenylmethanol
In this experiment you will prepare the tertiary alcohol triphenylmethanol from an aryl halide, bro-
mobenzene, and a ketone (benzophenone).
2 Pre-lab Exercises
1. In the first part of this experiment, the formation of the Grignard reagent, you will use 1 g of
magnesium and 4 mL of bromobenzene. Calculate the molar amounts used for each of these
substances.
2. Determine which reagent is the limiting reagent in the first step. Calculate the amount of benzo-
phenone you will require to perform the second step of the experiment (Part 2). You will need
these calculations prior to commencing the experiment. Check your results with the demonstrator
before you start.
53
3 Experimental Procedures
3.1 Part 1 - Generation of the Grignard Reagent (1 hour)
NOTE: All glassware for Parts 1 and 2 should be pre-dried in an oven and then stored in a
dessicator.
Assemble the oven-dried glassware for the reaction according to the diagram in Figure 1 below using
a 100 mL round bottom flask, reflux condenser and calcium chloride drying tube. Ensure the glassware
is assembled quickly as moisture from the atmosphere will react with the Grignard reagent.
Figure 1: Equipment setup for the Grignard reaction
Carefully weigh 1 g of magnesium and gently crush in a pestle and mortar to expose the fresh metal
surface from the oxide coating. Add the metal to the reaction flask along with 10 mL of anhydrous THF.
Carefully add one small crystal of iodine (I2 ) to the mixture.
In dry vessel with an appropriate seal such as a rubber septum, syringe 4 mL of bromobenzene and
15 mL of anhydrous THF.
*NOTE: BEFORE COMMENCING THE REACTION HAVE AN ICE-WATER BATH
ON HAND. THE FORMATION OF THE GRIGNARD REAGENT IS EXOTHERMIC.
IF AT ANY POINT THE REACTION BECOMES TOO VIGOROUS IT MAY NEED
TO BE COOLED IN THE ICE-WATER BATH. LET YOUR DEMONSTRATOR KNOW
BEFORE YOU COMMENCE THE GRIGNARD REACTION.

CHEM30015: Advanced Practical Chemistry ESC2
To commence the reaction, slowly (!) add 5 mL of the bromobenzene solution drop-wise (via syr-
inge) through the septum into the round bottom flask containing the magnesium and THF. Gently warm
the flask with a heating block. The THF may start to boil. Formation of small cloudy spots on the surface
of the metal will indicate commencement of the reaction, another indicator that the reaction has began
will be the disappearance of the purple colour of the iodine.
Keep the reaction at a gentle reflux, carefully balancing heating and cooling by raising the reaction
flask off the heating block. If the reaction is progressing too vigorously, raise the reaction flask out of
the heating mantle and place in the ice-water bath to cool and allow the reaction to subside. You may
need to add an additional 5 mL of THF if your solvent volume becomes too low.
Continue to add the remainder of the bromobenzene solution drop-wise to your reaction mixture. Be
sure to maintain the reaction at a moderate reflux, as previously, raising the reaction vessel out of the
water bath as required. Be careful not to cool the reaction so far as to cause it to stop entirely.
Once all the bromobenzene has been added and the reaction mixture ceases refluxing spontaneously,
gently heat the reaction to reflux for 30 minutes to ensure complete formation of your Grignard reagent.
3.2 Part 2 - Addition of the Carbonyl Compound (1 hour)
While you are waiting for the Grignard reaction to complete, prepare a solution of benzophenone
(weight as determined in your pre-lab section) in 15 mL of anhydrous tetrahydrofuran (THF) in an
oven-dry 50 mL round bottom flask stoppered with a rubber septum.
When the Grignard formation is complete, allow the reaction mixture to cool to room temperature.
Once the reaction mixture has cooled, slowly add 5 mL of your benzophenone solution dropwise through
the septum, using the same syringe you used in the previous step. Once you have added 5 mL of the
ketone solution, place your reaction flask back in the heating block and warm the reaction. Continue the
dropwise addition of the benzophenone solution until you have added the entire solution and rinse the
flask with an additional 5 mL of dry THF and add this to the reaction.
Reflux your mixture for a further 15 mins.
3.2.1 Quenching the Reaction
Allow your reaction mixture to cool to room temperature before proceeding.

In a 250 mL beaker place 25 g of ice and 25 mL of 1 M sulfuric acid. Carefully pour your reaction
mixture into the 250 mL beaker containing the ice-cold aqueous acid solution. Rinse your reaction
vessel with 10 mL of additional 1 M sulfuric acid, followed by CH2 Cl2 (20 mL) and add to the beaker.
Stir the mixture with a glass rod until the salts and unreacted magnesium have completely dissolved.
This is a good point to stop if you are running short on time. Label your beaker and cover
with a watchglass or aluminium foil. Check with your demonstrator where you should leave your
beaker. If you do this you will need to add 20 mL of CH2 Cl2 before the next step.
3.2.2 Isolation of the crude triphenylmethanol product
Your mixture will be contaminated with a fine particulate matter that is difficult to remove by fil-
tration. To assist with filtration, add Hyflo medium filter aid to a Buchner funnel. Filter the mixture
through the Buchner funnel and rinse with 3 x 20 mL CH2 Cl2 . Add the filtrate to a 250 mL separating
funnel and use another 25 mL of CH2 Cl2 to rinse out the Buchner flask. Shake the mixture thoroughly
in the separating funnel, being careful to vent regularly. Allow the layers to separate and collect each
layer in a separate conical flask. Extract the aqueous layer with 10 mL of CH2 Cl2 (twice) and combine

the organic layers. Wash the combined organic fraction by shaking successively with 25 mL of water,
25 mL saturated aqueous sodium bicarbonate and 25 mL saturated aqueous sodium chloride solution.
Dry the organic fraction with anhydrous sodium sulfate (a few grams will be required) and filter into
a clean conical flask. Evaporate the majority of the organic solvent on a steam bath until rapid bubbling
slows, then add 20 mL of hexane to the oily residue. Continue heating until crystallisation occurs, then
remove the flask from the hotplate and cool to room temperature before placing on an ice-bath. Wash
well with 3 x 20 mL ice-cold hexane.
Recrystallise the triphenylmethanol from hot CH2 Cl2 and hexane (you will need 4 times the
volume of CH2 Cl2 in hexane).
**Note: the use of excessive volumes of solvent will reduce your overall yield - if your yield seems
low take a second crop from your mother liquor.
4 Questions
1. The alkyl moiety in Grignard reagents can be considered to have a negative polarity, i.e.
R -Mg+ -Hal . Explain.
2. If you hadnt bothered to dry your glassware or used a drying tube, what by-product would have
formed in this reaction?
3. What would be the product of phenylmagnesium bromide and carbon dioxide? Provide a detailed
mechanism.
4. What would be the product of the reaction of excess phenylmagnesium bromide and methyl ben-
zoate? Provide a detailed mechanism.
5. How might the addition of iodine help initiate formation of the Grignard reagent?
6. Ethers are typically used as solvents in Grignard reactions. Why?

ESC3 - Synthesis of Methyl
4-Nitrocinnamate Using the Wittig
Reaction
A/Prof. Uta Wille

1 Introduction
The Wittig reaction was discovered in 1954 by Georg Wittig (Heidelberg, Germany), for which he
was awarded the Nobel prize in Chemistry in 1979. This reaction is a highly versatile and very powerful
method for the synthesis of alkenes from readily available alkyl halides and carbonyl compounds, e.g.
aldehydes and ketones. In the first step, the Wittig reagent (ylide) is prepared through deprotonation of
a phosphonium salt, which in turn is obtained by reaction of an alkyl halide with triphenyl phosphine.
In a second step, the ylide is reacted with the carbonyl compound to generate an alkene. Ylides, which
can be described as either zwitterionic species with a carbon-phosphorous single bond, or as neutral
compounds with a carbon-phosphorous double bond (this resonance form is called ylene) possess a
highly nucleophilic carbon centre and are therefore very reactive. Thus, phosphorous ylides usually need
to be prepared under exclusion of moisture and oxygen, but in this reaction the ylide is stabilized and
can be prepared in aqueous solution using sodium hydroxide as the base. One major shortcoming of the
Wittig reaction is, however, the production of large quantities of triphenylphosphine oxide as a reaction
by-product, which can be difficult to separate from the desired product of the reaction. Here we will use
silica gel chromatography to purify a small amount of the product. In your report you should consider
reasons for the production of the observed double-bond stereochemistry in the alkene.
2.1 Preparation of the Ylide
2.1.1 Preparation of Carbomethoxymethyltriphenylphosphonium bromide
In a 50 mL round-bottom flask equipped with a magnetic stirrer bar triphenyl phosphine (2.62 g,
12.5 mmol) is dissolved in toluene (15 mL). Methyl bromoacetate (1.5 g, 10 mmol) is added, and
the reaction mixture is stirred at room temperature overnight. The crystalline material is collected by
vacuum filtration, washed at the pump with cold toluene, followed by petrol and dried (you could place
the product on a watch glass an let it dry in air). Typical yield: 3.3 g (79%).
Run an infrared spectrum of this material.
2.1.2 Preparation of Carbomethoxymethyltriphenylphosphorane
In a round bottom flask equipped with a magnetic stirrer bar the phosphonium salt (3.2 g, 7.7 mmol)
prepared in the previous step is dissolved in water (80 mL). Aqueous sodium hydroxide solution (2.5 M,
57
ca. 4 mL) is added to this mixture. The white precipitate that forms on addition of the base is collected
by vacuum filtration and washed at the pump with water.
Drying of the product: The remaining water is removed water by dissolving the solid in dichloro-
methane and washing with saturated aqueous sodium chloride solution, followed by drying the organic
layer over magnesium sulphate. The solvent is then removed by rotary evaporation to afford the product
as a white crystalline material, which is purified by recrystallisation from ethyl acetate. Typical yield:
2.1 g (82%).
Run an infrared spectrum of this material.
2.2 Synthesis of Methyl 4-Nitrocinnamate
In a 50 mL round bottom flask equipped with a magnetic stirrer bar, the previously prepared ylide
(1.0 g, 3.0 mmol) is dissolved under nitrogen in dichloromethane (25 mL). p-Nitrobenzaldehyde (450
mg, 2.98 mmol) is added, and the reaction mixture is stirred under nitrogen at room temperature
overnight. The reaction is quenched with water (20 mL) and extracted with dichloromethane (3 x 20
mL). The combined organic fractions are washed with water (20 mL) and saturated aqueous sodium
chloride (20 mL). After drying (MgSO4 ) the solvent is removed on the rotary evaporator.
A small quantity of the residual solid is purified by chromatography through a small plug of flash-
grade silica gel as described below (ask a demonstrator for assistance, if you have not yet been taught
how to set up a silica gel column).
Flash-grade silica gel (20 g) is suspended in 10% ethyl acetate/n-hexane and poured carefully into
a filter column. The excess solvent is released until the silica gel has set (dont let the column dry
out). The surface of the silica is covered with a filter paper and the product of the reaction (200 mg),
dissolved in the minimum amount of dichloromethane (ca. 2 mL), is pipetted onto the surface of the
filter paper. The compound is allowed to adsorb onto the silica before a total of 100 mL of 10% ethyl
acetate/n-hexane is carefully added in two 50 mL portions. The eluent is collected and checked by TLC
to ensure that the entire product has been eluted from the column. If the product has not been eluted
(or elution is not yet finished), add an additional 50 mL portion of 20% ethyl acetate/n-hexane, and
then 30% ethyl acetate/n-hexane until elution of the product is complete. The solvent is removed on the
rotary evaporator to afford a white crystalline solid.

Determine the melting point of the product and record a TLC of the product in a solvent system
that provides an Rf value for your product of approximately 0.5. Develop your chromatogram using the
phosphomolybdic acid dip provided (consult a demonstrator).
Run an infrared spectrum of the chromatographed material.
(peak assignments, multiplicity if applicable, interpretation of impurities). Also provide an
analysis of the 13 C NMR spectrum (available on the LMS). Analysis and peak assignments
are to be included in your report.
3 Questions
1. From the 1 H NMR spectrum obtained from your demonstrators, assign the recorded signals to the
protons in your product. Comment on the configuration of the alkene C=C double bond in your
product. Your report must contain a detailed analysis of the 1 H NMR spectrum and a mechanism
that accounts for formation of the observed alkene isomer.
2. As mentioned in the introduction, phosphorous ylides are generally quite unstable and cannot be
isolated. Suggest a reason for the special stability of the ylide used in this experiment.
3. Less reactive ylides, such as the one used in this reaction, react readily with aldehydes, but usually
fail to react with ketones. Suggest a reason for this finding.
4. Aside from the desired alkene formed in this reaction, triphenylphosphine oxide is also produced.
With consideration of the relative bond energies of the bonds broken and formed during this
reaction, suggest a driving force for the Wittig reaction.

ESC4 - Synthesis of (3-Ethyl-pentane-3-ol)
Prof. Mark Rizzacasa

1 Introduction
Organohalides (RX) react readily with magnesium to form Grignard reagents, so named after their
discoverer Victor Grignard.
Acting as a base Grignard reagents will react with any protons that are more acidic than those found
on alkenes and alkanes, thereby readily reacting with water, alcohols, amines, thiols, etc. to generate the
corresponding alkane.
The formation of the Grignard reagent is highly water sensitive, and great care should be taken to
ensure your glassware is properly dried prior to commencing either part of this experiment.
In this experiment you will prepare the tertiary alcohol 3-ethyl-pentane-3-ol using a prepared Grignard
reagent ethyl magnesium bromide according to the scheme below.
Pre-lab Question:
You are told to use 0.02 mol of diethyl carbonate in the preparation outlined below. The Grignard
reagent you will use is prepared as a 3.0 M solution in diethyl ether. Calculate the volume of
Grignard reagent required. You will require this value prior to commencing the experiment below.
Be sure to check your result with a demonstrator.
For this reaction you will use a prepared Grignard reagent ethyl magnesium bromide. It is provided
as a prepared solution in diethyl ether (obtained from the prep room).
You will require a 100 mL round bottom flask and suitably sized magnetic stirrer bar, a condenser and
drying tube packed with calcium chloride. Your glassware must be dry (check with your demonstrator
before collecting glassware from the oven). Place the hot glassware into a dessicator directly from the
oven, unless you plan to use it immediately.
Assemble the glassware according to Figure 1 and attach the drying tube to the apparatus.
Place X mL (as determined in your pre-lab exercise) of the Grignard solution into the round bottom
flask and ensure the mixture is stirring. Dissolve 0.02 mol of diethyl carbonate in an equal volume of
61
Figure 1: Equipment setup for the Grignard reaction
anhydrous diethyl ether. Perform the next step very carefully as the reaction is exothermic . Using
a syringe, carefully add the diethyl carbonate solution dropwise into the Grignard solution. Once the
addition is complete, heat the reaction at gentle reflux for 2 hours. Cool the reaction mixture to room
temperature before carefully hydrolysing the mixture by slowly adding 50 g of crushed ice. Acidify the
reaction mixture with saturated aqueous ammonium chloride solution, until the precipitated magnesium
hydroxide just dissolves. Separate the aqueous and organic phases using a separatory funnel and extract
the aqueous layer with diethyl ether (2 times). Combine the organic fractions and wash successively
with saturated aqueous sodium bisulfite, saturated aqueous sodium bicarbonate and small amounts of
water. Dry the organic fraction over anhydrous MgSO4 , filter the solution and remove the solvent using
a rotary evaporator.
Your product should be a liquid and will require purification.
Purify your crude product by distillation, referring to the additional information available on the Web
site for distillation set up. Have your distillation set up checked by a demonstrator before commencing
the distillation.
Be sure to make note of the boiling point of your product and obtain the refractive index of your
purified product.
Record the yield of your product and ensure all required characterisation data are collec-
ted. This should include the mass spectrum, IR, refractive index and boiling point. NMR
and mass spectra may be recorded (see Ms Volaric) or made available on the LMS. Peak
assignments are to be included in your report.
3 Questions
1. What type of solvent can be used in Grignard reactions and why?

2. Why would you use ammonium chloride to acidify the reaction mixture instead of using concen-
trated sulfuric acid?
3. Write the mechanism for formation of the Grignard reagent RMgBr by reaction of RBr with Mg.
4. The structure of Grignard reagents can be described by the Schlenk equilibrium:
2 RMgBr
)

*
R2 Mg + MgBr2
The position of the equilibrium is influenced by solvent, temperature and the nature of the sub-
stituent R. Explain the role of the solvent in this equilibrium.

Kinetics
In the following pages the preparations for all experiments offered for the Kinetics component of the
course are included.
You must complete one experiment from the offered 3.
It is your responsibility to complete your experimental work within the time prescribed. At third
year level this will require you to be well prepared and ready to start as soon as you enter the laboratory.
You must work efficiently if you are to complete your work within the specified time.
K1 - Excited State Reaction Kinetics:
Pyrene Excimer Formation
Prof. Ken Ghiggino

Learning Goals from this Experiment
1. To learn about the kinetics of excimer formation in an aromatic molecule.
2. To calculate the rate constants for excimer formation and decay.
3. To learn how to handle sensitive laser photolysis instrumentation.
4. To learn about the use of LabVIEW for data acquisition.
5. To learn about complex kinetics and data analysis.
1 Introduction
An exciplex is an excited state complex formed by the association of two different species, one in its
excited and the other in its ground state. An excimer is an excited state dimer, i.e., an exciplex involving
the ground state and excited state of the same species.
The kinetic processes involved in excimer formation and decay can be summarised by the following
scheme:
Figure 1: Kinetic scheme of the excimer formation process
Excitation of a solution of light absorbing molecules by a very short pulse of light produces an
initial concentration of excited molecules [1 M ]. At very low concentrations, these excited molecules
will decay back to their ground state with a rate constant, kM , (=1/lifetime of the excited state), possibly
with the emission of light (fluorescence). At higher concentrations, interactions can occur between the
excited-state molecules and molecules in their ground state, within the lifetime of the excited state, to
form excimers. These excimers may either lose their excess energy and break apart (again accompanied
65
K1 CHEM30015: Advanced Practical Chemistry
by the emission of light) to form two ground-state molecules, or simply dissociate back to the excited
and ground state molecules.
Since the monomer and excimer emit in different spectral regions, we can monitor the emission from
the monomer or excited dimer species. Kinetic analysis of the fluorescence decay curves, obtained by
collecting the emission as a function of time after excitation, yields the rate constants associated with
the decay of the monomer population and the rate constants for the formation and dissociation of the
excimer.
The monomer (1 M ) fluorescence response function
The emission intensity vs time response of excited pyrene monomer in solution will have the form
given by equation 1.
" #
[1 M ] (2 X)
iM (t) = kFM 1 = kFM [exp(1t) + A exp(2t)] (1)
[ M ]0 (2 1 )
where X = kM + kDM [1 M], A = X 1

2 X
and 1 , 2 and A are fitting parameters which are (rather complic-
atedly) related to the rate constants shown in Figure 1.
The dimer (1 D ) fluorescence response function

The emission intensity versus time response of the pyrene excimer in solution will have the form
given by equation 2.
" #
[1 D ] kFD kDM [1 M]
iD (t) = kFD kDM 1 = [exp(1t) exp(2t)] (2)
[ M ]0 (2 1 )
From these equations and the experimental decay profiles, the rate constants for excimer formation,
dissociation and decay can be calculated.[1]
A few useful, simplifying relationships are:
(i) as [1 M] 0 : 1 kM and 2 kD + kMD
(ii) as [1 M] : 1 kD
(1 +2 )
(iii) at all [1 M] : [1 M]
= kDM
Pre-lab Questions
1. What is meant by concentration quenching in fluorescence?[1]
2. Explain the difference between excimers and exciplexes?[1]
3. What is the unit of a second order rate constant?
4. Why is it necessary to remove oxygen from the pyrene solutions by bubbling nitrogen gas through
it?
5. Write down any safety precautions that need to be considered when:
(i) Handling your pyrene solutions

(ii) Using the Nd:YAG laser
6. Calculate the mass of pyrene needed to prepare the stock solution. What volumes are required of
the stock solution to prepare the 3 more dilute solutions?

CHEM30015: Advanced Practical Chemistry K1
2 Experimental section
Apparatus
Figure 2 shows a schematic diagram of the instrumentation. A laser beam of 355 nm is incident into
the sample compartment where the sample, contained in a quartz cuvette, fluoresces. The intensity of
the fluorescence is detected by the photomultiplier via an interference wedge which allows a specific
detection wavelength to be selected. This wavelength selection is controlled by a computer using a
LabVIEW program (see below). The photomultiplier tube is connected to a digital oscilloscope, which
gives a waveform representing the decay of the pyrene fluorescence. The data are fed into the computer
using another LabVIEW program, and can be saved to a file for analysis.
Sample cuve7e
355 nm laser
Tuneable wavelength
transmission lter
Neutral
PMT power density
supply & lter/s
stepper Laser
driver power
supply
Photomul?plier tube
Computer Q-switch
Digital storage trigger
oscilloscope (50
termina?on)
Figure 2: Schematic diagram of the apparatus set up using a laser and oscilloscope to obtain fluorescence
decay curves.
2.1 Monochromator Control
The monochromator is controlled by the LabVIEW virtual instrument fluorimetercontrol.vi

(see Figure 3). It should be opened along with CROdownload.vi at the beginning of the session. Setting
up the fluorimeter and changing wavelength are straightforward.
1. When the instrument is first switched on it needs to be initialised. This is done by clicking the
mouse on the run arrow while the switch on the left side of the control panel is in the initialise
position (as shown in the figure above). The stepper motor then scans the variable filter to the end
position and then back to the desired wavelength which is set in the top numeric control panel
(labelled Desired Wavelength (nm)). The wavelength must be in the 368-699 nm range.
2. Once initialisation is complete the run/initialisation switch automatically flicks to the run position.
3. Once initialised, a new desired wavelength in the 368-699 nm range can be set in the top numeric
control panel (labelled Desired Wavelength (nm)) before clicking the mouse on the run arrow.

Figure 3: Front panel of fluorimetercontrol.vi
The fluorimeter will move to the desired position 1 nm. When the desired wavelength is reached
the Actual Wavelength (nm) indicator will display the new wavelength.
Note: Occasionally the GPIB control box freezes up (the stepper motor does not click happily
away when a new wavelength is entered on the control panel). If this happens the GPIB control box
needs to be reset by turning it off and on. After doing this flick the switch on the left side of the
monochromatorcontrol.vi control panel to the initialise position and rerun the program.
Experimental Procedure
You will be provided with a Eppendorf Reference auto-pipette to assist you in preparing your
samples. See the operating instructions before you attempt to use this device.
2.2 Pyrene Solutions
Prepare a 1.0 102 M stock solution of pyrene in cyclohexane in a 10 mL standard flask. (Note:
make sure the pyrene has fully dissolved in the cyclohexane before proceeding). In three 10 cm3 volu-
metric flasks use your stock solution to prepare solutions of 5 103 M, 5 104 M and 5 106 M
pyrene in cyclohexane, these will be used to conduct the experiments described below.
2.3 Fluorescence Spectrum of Pyrene
1. Measure the emission spectrum of your most dilute solution prior to degassing. If there is any
evidence of excimer emission under these conditions, this solution should be diluted further until
no sign of any excimer emission is present. Only then degas the solution as below. The actual
concentration of this solution is not required in the calculations, but that of the 5103 M solution
is.
2. Purge the solutions in a cuvette for 15-20 min. with nitrogen and seal the cuvette with a stopper.
Record emission spectra and emission decay curves as soon as possible after degassing.
3. Use the Hitachi F-4500 fluorimeter or the Cary Eclipse (Varian) fluorimeter to record the fluores-
cence spectra of the 4 solutions. See notes that have been provided on a separate sheet of paper
kept adjacent to the respective instrument, which will guide you through the procedure for re-
cording the spectra. If you encounter any difficulties, see the demonstrator. Set the excitation
wavelength to 350 nm and record the fluorescence spectra between 355 - 600 nm.

2.4 Decay of Excited States of Pyrene
You will only record time-resolved emission traces of the 5 106 M and 5 103 M solutions.
1. Place your nitrogen-purged sample into the cell compartment of the time-resolved emission ap-
paratus and shut the lid of the sample compartment and the enclosure.
2. Use your fluorescence spectra of the 5 106 solution to select an appropriate wavelength to
follow the decay kinetics of the monomer emission. Set the wavelength on the detector of the
apparatus using LabVIEW - see below. Record the decay kinetics.
(i) Double click fluorimetercontrol.vi, which appears on the desktop. [This opens up the
LabVIEW program]. See section 2.1 for more details.
(ii) Click on the Initialize switch and then click . This will initialize the monochromator.
Once this is completed, the switch will automatically flip back to Run.
(iii) Enter the desired wavelength in the appropriate box and then click . The monochromator
will be set at this selected wavelength.
(iv) Close fluorimetercontrol.vi.
3. Open CRO-download.vi, which appears on the desktop in the Launcher folder.
4. After you have read the laser safety instructions below, start the laser using the operation instruc-
tions below. Note that the safety interlocks must be set to allow operation of the laser. Cau-
tion: Do not open the sample compartment since the invisible laser beam is of high intensity
and may cause eye damage - opening the enclosure or sample compartment will deactivate
laser operation.
5. Turn on the power supply to the photomultiplier (PM) tube (high voltage (HV) 670V). Caution:
Never open the sample compartment when the power supply is on. The excess room light
will damage the PM tube.
6. Turn on the Tektronix CRO .
7. The signal (and trigger, if used) cables should be terminated into 50 at the oscilloscope input.
8. The oscilloscope can be triggered either by the Q-switch as the external trigger (positive slope
+1 V) or internally directly off the input signal (negative signal (negative slope, trigger level
something like -20 mV)). Internally triggering is the best option to start with. If required
externally trigger the CRO following these instructions:
Use the Q-Switch output from the laser power supply as the external trigger to the CRO. Use
a 50 terminator at the CRO external input as well as the PMT signal input.
Ensure in the trigger menu on the CRO that it is set to trigger from the EXT source with a
positive slope and a level of 0.5-1 V, Edge trigger.
On the Horizontal menu (timebase) use Delayed trigger only (rather than Main only), with
delayed runs 162 ns after main.
The Delayed timebase (as distinct from the Main timebase) can be set using the Sec/div knob
(e.g. 25 ns/div).
The trace can also be positioned with the trigger position % in the timebase.
9. Activate both the flashlamp and Q-switch on the laser remote controller, and ensure the laser head
shutter is open.

10. Make adjustments on the CRO to obtain a steady peak that maximises the decay trace on the
screen. [You may need your demonstrator to help with this part]
11. It may be necessary to reduce the laser pulse energy to get an uncorrupted kinetic trace (the decay
should be free of noise and spurious oscillations. Ask the demonstrator if this is necessary, and if
so how to do this (a laser flashlamp energy of 1.6 J should be sufficient - this is set on the laser
remote controller). A neutral density filter in front of the detector may also be required to obtain
an uncorrupted trace.
12. The signal-to-noise can be improved by acquiring in Signal averaging mode under Acquire
(e.g. 32 averages).
13. Click on the button to transfer the decay curve onto the computer, only after you have a
satisfactory (i.e. noise free) emission trace on the CRO. Save your data in an appropriately named
file. [pyrenexx/yy/zz appears as a prompt - change this to your own file name and save this in the
student folder. Make sure the switch icon on the screen reads yes and the ns/division selected on
the computer is the same as the time base scale used on the Tektronix CRO]. Take note of
the time base on the CRO.
14. Place your nitrogen-purged second (high concentration) sample into the cell compartment. Using
the same detection wavelength as above, collect a monomer fluorescence decay profile of the
5 103 M solution.
15. Use your fluorescence spectra of the 5 103 M solution to select an appropriate emission wave-
length to follow the growth and decay kinetics of the dimer emission in this concentrated solution.
Change the detection wavelength (using the fluorimetercontrol.vi). You should select an
appropriate time-scale to ensure adequate data in both the rise and decay of the curve. Ensure you
save these curves for analysis.
2.5 Data analysis
All calculations and raw data manipulations should be carried out by transferring the saved data files
of your measured emission-time curves into pro Fit.
1. Load the file containing data for the 5 106 M pyrene solution at the monomer detection
wavelength into pro Fit. [When you open the file you will need to select Without titles in
the import options window]. Take the maximum value in Column 2 and delete all values before
this, so the maximum value matches with zero in Column 1. Plot these points (time in x-axis
and intensity in y-axis) and fit a curve. The emission from the monomer region should be fit by
a single exponential function. To do this, you will need to create a single exponential analysis
function of the form: y = A exp(1 x) in pro Fit. [Note: the in-built Exp function in the pro
Fit FUNC menu is of the form y = A exp((x x0)/t0) + const. If you use this you will need to
deselect x0 and probably const in the Use for fitting check box. You will also have to convert
t0 to a rate constant.] In the parameters window (select Parameters from the WINDOW menu)
enter initial guesses for the values for A (i.e. the intensity at t=0) and k (or t0). Then select Fit
from the CALC menu, ensuring that the X Column is Column 1 and the Y Column is Column 2.
2. From the low concentration fit obtain 1 (= kM ). This value will be used in subsequent steps to
evaluate the other rate constants in Figure 1.
3. Load the file containing data for the 5103 M pyrene solution at the dimer detection wavelength
into pro Fit. Identify the point where the onset of dimer formation occurs. NB: This is not the
point of maximum intensity (as was the case in 1. above, it is the first non-baseline point in the
curve. Delete all values before this point, so the first dimer formation value matches with zero in
Column 1. Plot these points (time in x-axis and intensity in y-axis) and fit a curve.

4. Since pro Fit does not have a default bi-exponential function, you will need to upload an external
function. This function is provided as a file (Excimer.func) on the server. The function looks
something like:
y := a[1] (exp((x a[5]) a[2]) exp((x a[5]) a[3])) + a[4];
5. Click the To menu button to have it appear in the Function Menu. Pro Fit will use the parameters
a[1], a[2], a[3] and a[4] to fit to your data to the User function. N.B. Suggested initial guesses for
the parameters are; a[1]=roughly the maximum signal intensity; a[2]=0.03; a[3]=0.0015. Enter
these values in the Parameters window. If adequate fitting cannot be achieved, try varying these
initial guesses.
6. From the high concentration fit from the dimer region obtain 1 (1 =a[2]) and 2 (2 =a[3]) as
defined in Equation 2.
Using A = 0.1, calculate X = (A2 + 1 )/(A + 1)
7. Use X = kM + kDM [1 M] to get a value for kDM . Then use the following relationships to obtain the
remaining rate constants in Figure 1:
Y = 2 + 1 - X,
kMD = (X - 1 )(2 - X)/kDM [1 M]
kD = Y - kMD
8. Summarise in table form the four reactions and their corresponding rate constants (i.e. kM , kDM ,
kD , and kMD ). Show the units associated with the rate constants.
9. Compare your rate constants with those that can be calculated from the data in Figure 10 in the
Birks reference [1] (use the simplifying relationships discussed in Birks paper and outlined
above). Make a table showing your values along with those calculated from the reference.
10. Compare the decays obtained from the monomer region at the high and low concentrations. What
differences are observed in the decays from this region? Does the monomer emission still decay
exponentially at high concentrations?
11. Calculate kdi f f , the diffusion controlled rate constant using kdi f f = 8000RT /3. R = gas constant
(J mol1 K1 ), T = temperature (assume 293 K) and = viscosity of the medium. (Viscosity has
the units, kg m1 s1 . See reference [2] below. N.B. the 8000 term in this expression takes the
conversion to dm3 mol1 s1 into account).
12. Compare the value between the rate constant of dimer formation (kDM ) with kdi f f . Discuss any
expected similarities between these values and account for any discrepancy.
13. Comment on how the presence of O2 may affect the value of the rate constant.
14. Comment on the affect of temperature on the excimer formation and decay processes.
In your report you will need to show the fluorescence spectra of the 3 diluted pyrene solutions, the
monomer decay curves for the 5 106 M and concentrated pyrene solutions fitted with single expo-
nential functions, and the dimer emission curve for the 5 103 M pyrene solution fitted with a double
exponential function. Also include all calculations.
Laser Operation
The laser used in this experiment (a Quantel Ultra20) is based on a crystal of yttrium aluminium
garnet; Y3 Al5 O12 , (YAG)) that is doped with a small amount (1%) of neodymium3+ ions. This crystal

is excited by a flashlamp. It is commonly known as a Nd:YAG laser, and produces fundamental output
at 1064 nm. This infrared radiation can be converted to other wavelengths through a non-linear optical
process. The Nd:YAG laser used in this experiment is Q-switched which means that it operates in
a pulsed mode that can produce high energy, short pulses of light at low pulse repetition rates. This
laser is frequency tripled (wavelength/3) to produce 355 nm (ultraviolet) light. In this frequency
conversion process, 532 nm light is also produced which is separated inside the laser. The 355 nm
radiation is produced in pulses of 10 nanoseconds in duration with 4 milliJoules/pulse (mJ/pulse) at
50 pulses/sec (Hz). It is therefore quite a powerful laser, and should be operated with great caution (see
operating and safety instructions below).
To operate this laser:
Turn on keyswitch on the power supply unit, if not already on. Water flow should occur to cool
the laser head - check that this is the case by observing bubbles flowing in the cooling water lines.
Ensure that the laser is positioned so that the correct laser wavelength output (355 nm) is switched
in and entering the sample compartment. This is selected using the rotatable device on the front
of the laser head, but you should not have to alter this (check with Alf or demonstrator).
Ensure that all cabinet covers are in place, which will deactivate the safety interlocks (check the
control module that interlocks are not active).
Ensure that the internal beam block is activated (left side of laser head - small metal pin should
point to ).
Push the Flashlamp start button - this will activate the flash lamp that excites the Nd:YAG crystal.
The Start light will flash.
Push the Q-switch button - this will activate the Q-switch device in the laser that makes it operate.
The Q-switch light will flash. Only have the Q-switch on when actually taking measurements.
Turning the Q-switch off will stop the laser lasing and so the sample will not be being irradiated.
In case of emergency, hit the red button on the end of the control module (if this is activated it
needs to be twisted clockwise to deactivate - see p32 of Users Manual).
When measurements are finished, press the Q-switch STOP button and the flash lamp STOP
button.
Laser Safety
Due to the potential power of this laser, it is extremely important that all safety guidelines for the
use of this laser are followed absolutely.
The laser (Q-switch and Flashlamp) should always be deactivated before the enclosure or sample
compartment is opened.
Do not attempt to defeat the interlocks on the enclosure or sample compartment.
Laser glasses will be provided and should be used whenever necessary.
Be aware that 532 nm radiation might also be present.
Do not allow items to interfere with the laser beam as this can cause unwanted and dangerous
scattered laser beams.
A beam stop should be used to intercept any transmitted laser light that exits the sample.
Whenever the laser is not being used to acquire data, turn off the Q-switch and activate the manual
beam stop on the side of the laser head.

References
[1] J.B. Birks, Excimers, Rep. Prog. Phys., 1975, 38, 903-974. (A copy of this article is on the LMS).
[2] CRC Handbook of Chemistry and Physics, 69th ed, Weast ed, CRC Press 1988.

K2 - Flash Photolysis
Prof. Muthupandian Ashokkumar

Experiment Aims
1. Learn about the technique of flash photolysis.
2. Study the phosphorescent behaviour of a chromophore embedded in a solid matrix.
3. Appreciate the importance of elementary photochemical processes.
4. Learn about the reactivity of the benzophenone ketyl radical anion in solution.
5. Become familiar with the use of the LabVIEW Program for data acquisition.
1 Introduction
1.1 Phosphorescence Decay of the Triplet State
Flash irradiation of a chromophore rapidly excites it from the ground singlet state, S0 , to the first
excited singlet state, S1 . In many molecules the triplet state, T1 , is then rapidly populated by S1 T1
intersystem crossing (ISC).
The triplet state can decay by both phosphorescence (radiative decay) and non-radiative processes,
mainly T1 S0 ISC (see Figure 1). Oxygen, which has a triplet ground state, facilitates this last process
with high efficiency. This means that in the presence of O2 , ISC is very rapid and the triplet lifetime is
extremely short. Phosphorescence is therefore often difficult to detect.
However, by embedding the chromophore in a polymer through which oxygen can only diffuse
slowly, radiative decay of the triplet state becomes the dominant decay process and the triplet lifetime is
significantly extended[1] making it possible to detect this form of emission.
Figure 1: Energy levels and some possible transitions of a chromophore.
75
1.2 The Decay of the Benzophenone Ketyl Radical Anion
Irradiation of a solution of benzophenone initially leads to the excited singlet state (S1 ). Rapid
ISC subsequently results in the population of the T1 level with almost unit efficiency. If the ketone is
irradiated in water or benzene, no reaction occurs and the ground state is re-populated mainly by ISC.
If, on the other hand, photolysis is carried out in a 50% (v/v) mixture of propan-2-ol and water then all
of the triplet undergoes reaction instead of returning to the benzophenone ground singlet state (S0 ).
In the triplet state the excitation is localised on the carbonyl group and this reactive group abstracts
the -hydrogen atom from propan-2-ol to form the protonated ketyl radical, (C6 H5 )2 C OH. At pHs near
its pKa , this species is appreciably dissociated into the ketyl radical anion, (C6 H5 )2 C O :
(C6 H5 )2 C OH )

*
(C6 H5 )2 C O + H
+
(Ka = 6 1010 M)
In the pH range 10-13, the ketyl radicals react with ketyl radical anions to give the alkoxide ion of
benzpinacol:
k
(C6 H5 )2 C OH + (C6 H5 )2 C O
(C6 H5 )2 C(OH)C(O )(C6 H5 )2
2 Pre-Lab Questions and Exercises
Before entering the lab, endeavour to:
(i) Construct a flow sheet of the procedures for the experiment.
(ii) Answer the prelab questions.
You must include a flow sheet and answers to prelab questions in your report.
Pre-Lab Questions
1. What will be the final pH of the solution of benzophenone in NaOH (see Section 5.1)?
2. How is absorbance defined in terms of incident and transmitted light intensities? [2]
3. What are the units of (i) a first-order and (ii) a second-order rate constant? [2]
4. (a) Write the integrated forms of the rate equations, which relate concentration to time, for:
(i) a first-order, and

(ii) a second-order process [2]
(b) In each case, how would you plot a set of concentration-time data to evaluate the rate constant?
(c) What will be the effect on the slope of each plot if a quantity proportional to concentration (e.g.
absorbance) is plotted instead of concentration?
5. In one part of this experiment, a chromophore is excited by a flash of light and the excited state
subsequently decays by phosphorescence. The time-dependent phosphorescence intensity is used
to derive kinetic parameters. Answer the following questions:
What is the reactant in this situation?

What is the reaction?

How is the intensity of emitted light related to the concentration of the reactant?
6. Show that the decay of the ketyl radical is second order with respect to the ketyl radical anion with
a rate constant kobs = k[H+ ]/Ka .
7. A photomultiplier (PM) tube [3] produces an output voltage that is proportional to the intensity of
light incident upon it. The PM tube output is sent to a computer running the LabVIEW program,
which displays a plot of the voltage versus time. Sketch the expected trace on the computer screen
when the following experiments are executed:
(i) the flash produces a phosphorescent species,

(ii) the flash produces a short-lived intermediate species which absorbs light.
8. When a light beam is passing through a spectrophotometer cell filled with water into the PM tube,
the voltage output registered is 500 mV. If the cell is then filled with an absorbing solution the
voltage registered is 200 mV. Calculate the absorbance of the solution.
3 Apparatus
The flash photolysis apparatus is drawn schematically in Fig. 2; it has the following features:
a commercial photographic flash gun which delivers a flash of 100 J in a pulse lasting 50 ms;
a photomultiplier (PM) tube;
a 12 volt quartz-iodine lamp for monitoring the absorbance/transmission of the sample;
a variable interference filter to select the wavelength to be monitored; the position of the filter can
be set using the calibration graph provided;
a variable offset potential which is used to set appropriate baseline values; and
a silica flash photolysis cell of 10 cm pathlength.
Figure 2: Schematic diagram of the flash photolysis apparatus.
The various electronic and computer control units are:

the flash photolysis apparatus - this contains
(i) a low voltage power supply for the flash,

(ii) a high voltage power supply for the PM tube,
(iii) a variable, stabilised, ripple-free DC supply for the light source, and
(iv) a variable zero offset potential.
a USB analogue-to-digital (A/D) interface unit
a computer
Details of data collection and plotting programs are given in Section 4.
4 Using LabVIEW for Data Acquisition
1. Launch the Flash Photolysis program by double-clicking on the Flash Photolysis.vi icon on the
Desktop.
2. The file opens with a front panel window as shown in the figure below.
The front panel window shows a Waveform Graph (X-axis: Time in seconds; Y-axis: Light in-
tensity in arbitrary units) and 3 numeric controls (milliseconds to wait (Delay time), scan length
which controls the duration of acquisition (X-axis limits) and points per second which controls
the number of data points to be acquired per second.
Note: Normally, the delay should be set at 100 ms. The total number of points during the period
of any scan length can be fixed to 1000. e.g. if the scan length is 2 seconds then the number of
points per second is 500 if the scan length is 4 seconds then the number of points per second is
250, etc.

3. Data acquisition is started by clicking on the button. When the data collection is completed,
an option will appear on the screen to save the file. If the data is useable, save it with a file name
of your choice in the appropriate folder on the computer. Otherwise quit this option by clicking
on the cancel window. Ensure that each file is readable before proceeding (check with any text
editor such as NotePad).
4. When the data collection is completed, close the Flash.vi file without saving any modification
that you made during the data collection. Then quit the LabView SE program.
5 Experimental Section
5.1 Preparation of the Benzophenone Solution
(Complete this section before answering Questions)
1. Prepare a 25 cm3 solution of 0.010 M benzophenone in propan-2-ol.
2. Dilute 20 cm3 of this solution with an equal volume of 0.1 M NaOH (supplied).
3. Rinse the silica photolysis cell with a little of the mixture and then fill it to 90% of its capacity.
4. Outgas the mixture in the cell by passing a small capillary tube into one of the filling ports and
all the way to the other side of the cell, and gently bubbling N2 through the mixture. During
outgassing, place a dark cloth or a piece of aluminium foil over the cell in order to reduce photo-
decomposition. Continue outgassing the mixture until it is needed for photolysis.
5.2 Phosphorescence Decay of the Triplet State
A PMMA cylinder containing a dissolved chromophore (naphthalene) is provided.
1. Place the PMMA cylinder containing the naphthalene in the sample compartment and set the
monochromator to the wavelength of its phosphorescence [4]. Note: Cover the sample compart-
ment with a black cloth prior to turning on the Photomultiplier Tube (PMT) power supply.
2. Turn ON:
(i) the flash power supply,

(ii) Set the PM high voltage switch to position 8. Cover the apparatus with the black cloth and
switch on the PM tube power supply.
3. Follow the procedure given in the section 4 for data collection. First record the output signal in the
absence of the flash. This is the baseline signal that you need to subtract from the phosphorescent
decay kinetics. Turn on the flash (the set delay after the flash should be 100 ms) and record
the phosphorescence decay. It is recommended that suitable time scales for the phosphorescence
experiment are 4 sec and 8 sec (Scan length). Collect 6 decay curves (3 + 3). Calculate the rate
constants as explained in section 5.4.
WARNING: Each time when you need to remove the black cloth, make sure to TURN OFF the PMT
power supply.

5.3 Decay of the Benzophenone Ketyl Radical Anion
1. Turn off the PM tube, then remove the PMMA cylinder from the apparatus. Insert the silica cell
containing the outgassed benzophenone solution, and set the wavelength of the monochromator to
630 nm (this is the wavelength of maximum absorption of the benzophenone ketyl radical). Note:
Cover the sample compartment with a black cloth prior to turning on the PMT power supply.
2. Turn the PM tube back on.
3. Record and save the LAMP OFF baseline (both lamp and flash must be turned off).
4. Turn ON the monitoring lamp (flash should still be turned off), allow sufficient time for it to
stabilise, and record the LAMP ON baseline.
As there may be slight fluctuations in the lamp intensity over time, you should record a
LAMP ON baseline immediately before each flash photolysis experiment. In particular, if
the high voltage to the PM tube is altered, or the lamp intensity is changed then another
LAMP ON baseline must be recorded.
5. Turn on the flash (the set delay after the flash should be 100 ms) and record the phosphorescence
decay. Follow the procedure given in the section 4 for data collection. A typical decay curve,
along with the lamp- off and on baselines, is shown in Figure 3. It is recommended that suitable
time scales for this experiment are 2 sec and 4 sec (Scan length). Collect 6 decay curves (3 + 3).
Calculate the rate constant values as explained in section 5.4.
Figure 3: Schematic diagram of transient absorption of the benzophenone ketyl radical.
5.4 Using pro Fit for Data Analysis
5.4.1 Naphthalene Phosphorescence Data Analysis
1. Open the program pro Fit by double clicking on the icon.
2. Open the decay data file (LabView data file) by selecting OPEN from the FILE menu and selecting
the appropriate file.

3. Column 1 is the x-axis data (Time, ms) and Column 2 is the y-axis-data (Intensity, mV). Label
these appropriately.
4. Create 2 more columns by clicking the Data window size button (this is the button with two
double-headed arrows crossing each other in the top left-hand corner of the data window) and
entering a value of 4 into the Number of columns.
5. Open the baseline file and determine the average intensity by highlighting the entire column and
selecting Statistics from the CALC menu. Using the formula entry method, subtract this av-
eraged baseline intensity value from the (decay) intensity data into Column 3. (If you are not
familiar with the procedure of formula entry, consult with your demonstrator.)
6. To Column 4, insert a formula to obtain the natural log of the background-subtracted intensity
values in Column 4 i.e. ln(Int).
7. A plot of ln(int) vs time should give a straight line. Fit the data to a linear regression. The slope is
the first-order rate constant.
5.4.2 Benzophenone Ketyl Radical Data Analysis
1. Open the data file (LabView data file) from the FILE menu.
2. Column 1 is the x-axis data (Time, ms) and Column 2 is the y-axis-data (Intensity, mV). Label
these appropriately.
3. Create 2 more columns by clicking the Data window size button and entering a value of 4 into
the Number of columns.
4. Determine the I0 value by subtracting the averaged lamp off baseline value from the averaged
lamp on baseline value.
5. Determine the I values by subtracting the averaged lamp off baseline value from the kinetic data
into Column 3.
6. Using the Formula Entry method, get absorbance = log (I0 /I) values and convert the intensity
values accordingly to Column 4.
7. A plot of 1/abs vs Time should give a straight line. From the slope and intercept the second-order
rate constant and initial concentration can be calculated.
6 Calculations And Questions
1. Determine the order of reaction for the triplet decay observed during the phosphorescence of the
species contained in the PMMA sample.
2. Determine the rate constant for the triplet decay observed. Compare your value of the rate constant
(averaged) with that obtained from the literature[4] and suggest reasons for any discrepancy.
3. Confirm the order of reaction for the benzophenone ketyl radical decay.
4. The (molar extinction coefficient) of the ketyl radical is 5000 M-1 cm-1 and the optical path
length of the cell is 10 cm. Using your plots, calculate the concentration of the ketyl radical
produced by the flash at t=0.

5. Calculate the value of the observed rate constant, kobs, with appropriate units. Compare your
value of kobs with that given in the literature[4] at a similar pH (see Prelab Question 1) and suggest
reasons for any discrepancy. Deduce the value of k (see Q6 of Prelab questions).
6. In the study of the benzophenone ketyl radical, it is important to exclude oxygen from the solution
to prevent the reaction between the radical and oxygen from contributing to the radicals decay.
The second order rate constant for the reaction of ketyl and oxygen is in the vicinity of 1010 M-1
sec-1 . If, after an inadequate degassing, the residual oxygen concentration is 2 10-5 M, estimate
roughly the expected half-life of the radical (assume that the oxygen is in excess so that you can
treat the reaction as a pseudo-first order one). How would this small concentration of oxygen
effect your measured rate constant?
7. Why is that you do not use a lamp to follow the kinetics in the naphthalene experiment whereas
in the case of benzophenone experiment, you do use a lamp.
7 Report
Your report should include, (i) a flow sheet, (ii) answers to the prelab questions, (iii) results and
discussion (answers to question 1 and 2) and (iv) answers to questions 3-7. Use of proper units and error
limits is important. Graphs/traces should be properly labelled.
In addition to the above, your report should also include sample calculations for transforming data,
and presentation of all the results as a summary / table. The logic of presentation and setting-out of
report / ease of reading are also considered when marking a report.
A copy of the marking scheme for K2 is available on LMS. Your report should include all sections
mentioned in the marking scheme.
References
[1] 3rd year Photomolecular Sciences notes, (see LMS)
[2] P.W. Atkins, Physical Chemistry, 6th edition, Oxford University Press; Sections 16.2,17.3, & 25.3
[3] R. P. Wayne, Photochemistry, Butterworths, London, 1970, pp.201, 205; for more detail, see J. F.
Rabek, Experimental Methods in Photochemistry and Photophysics, Wiley-Interscience, Chichester,
1982, Part I, pp.459-501
[4] D. M. Goodall, P. W. Harrison and J. H. M. Wedderburn, J. Chem. Ed., 49, 669, (1972) (A copy of
this paper is available near the experimental setup in the lab)

K3 - Kinetics of the Acid Catalysed
Aquation of Fe(II)-Tris(chelates)
Dr. Stephen Best

Preamble
Before entering the laboratory you should have planned the preparation of your solutions (i.e. know
the masses of reactants and the strategy you will use for conducting your successive dilutions. Also
note that you are expected to maintain a laboratory notebook and use this to record your calculations,
observations and to record details of the samples and the output files generated during the experiment.
You MUST bring your laboratory note book to the viva for the experiment as this will help you explain
how you did the experiment and the strategy you employed to interpret your results. The emphasis of
this element of the reporting is to put an emphasis on your development of the technical and recording
skills that underpin experimental chemistry. You should NOT rewrite your notes for the purpose of the
report, I just need to be convinced that these notes are sufficient for you to be able to identify what you
have done in the experiment, where your files are kept and any observations relevant to the chemistry or
analysis.
Please note that if you plan to do this experiment you can prepare your solutions while doing one of
the other experiments in the level 2 laboratory. Inform the level 2 technical officer of your intention to
do this so that your solutions can be stored safely until you are ready to continue the experiment.
measure the effect of pH on the observed rate constants for aquation of homoleptic 2,2-bipyridyl
(bipy) and 1,10 phenanthroline (phen) complexes of iron(II),
use the shape of the pH prole to determine the relative importance of the contributing kinetic
pathways to the aquation of tris(bipy)iron(II),
rationalise the differences in kinetic behaviour of the bipy and phen complexes in terms of the
properties of the two ligands,
use the reported pressure dependence of k for these systems to calculate the activation volume for
the reactions at low pH and to discuss this property in terms of the mechanisms of aquation,
to explore the concept of reaction mechanism in the context of coordination compounds.
1 Introduction
In this experiment, the aquation kinetics of two cations, tris(2,2-bipyridyl)iron(II) and tris(1,10-
phenanthroline)iron(II), will be measured and compared. The rate of dissociation of a number of com-
plexes of the type M(AA)3 show a complicated acid dependence. The pH dependence for the aquation
83
of the tris(bipy)iron(II) complex, and can be rationalised in terms of a mechanism such as that given by
Figure 1.
Figure 1: Kinetic scheme for acid-catalysed aquation.
The aquation reaction of transition metal complexes will typically be first order in the concentration
of the complex, where the observed rate constant, kobs , can have a more complicated pH dependence.
Application of the steady state approximation to the mechanism shown in Figure 1 can be shown to
give the following expression for kobs :
k1 (k3 + k4 [H + ])
kobs =
k2 + k3 + k4 [H + ]
The rate constant will be approximately independent of pH at low or high values of [H+ ], changing
over from one regime to the other at intermediate pH values. These reactions can be followed using a
recording spectrophotometer monitoring the absorbance changes at the appropriate wavelength with the
solutions thermostatted at 45 -50 C, when the reactions have half lives around 5 to 30 minutes. The
aquation of the [Fe(phen)3 ] 2+ , however, has a different acid dependence and this will be quantified by
your measurements.
2.1 Operation of the kinetics spectrophotometer
The sample compartments of the four Novaspec spectrometers are temperature controlled. Note the
temperature and the temperature stability over the course of your measurements. The analogue outputs
of the four spectrometers have been interfaced to a PowerLab data recording system. In this way the time
dependence of the absorbance change of four independent reactions can be recorded simultaneously. For
each of the reactions the rate constant for the chemical reaction is obtained by fitting the time dependence
of the absorbance of the solution to an exponential function.
Details of the method of setting up the PowerLab data acquisition software are outlined in the notes
by the instrument. If you are uncertain about the operation of the equipment, consult the demonstrator
before beginning your kinetic runs.

2.2 Reactant solution preparation for the [Fe(bipy)3 ]2+ study
Prepare a fresh solution of 100 mL of 2.0 103 M FeSO4 7 H2 O. Carefully weigh out 6 105
moles of 2,2-bipyridyl in a small beaker, use a pipette to add 10 mL of the FeSO4 solution to this; the
red [Fe(bipy)3 ]2+ complex forms in solution. Quantitatively transfer this solution to a volumetric flask
and dilute to 100 mL using water.
From the stock solution of HCl, 12 M prepare 25 mL each of HCl at 5.0 M, 2.0 M, 1.0 M, 0.5 M,
0.2 M, 0.1 M, 0.05 M, and 0.02 M by successive dilution.
2.2.1 Measurement of the aquation kinetics of [Fe(bipy)3 ]2+
Adjust the wavelength of each of the Novaspec spectrometers to 530 nm. If not already turned on
start the chart program (double click the chart icon button). Note that this program MUST be left running
until you have recorded your full set of kinetics data.
Care: Avoid spillage of acidic (corrosive) solution in the sample compartment of the spectro-
meter.
1. The set of four Novaspec spectrometers used for the kinetic experiments are located in Lab 293.
You will need 60 minutes to complete all the kinetic runs. Organise your time so as to be able to
complete your measurements within the 1 hour time period so that other students can complete
their measurements without unnecessary delays.
2. Pour approximately 30-40 mL of [Fe(bipy)3 ]2+ solution into the large test-tube (35 15 mm)
included in the blue kit. Clamp the test-tube in the 50 C thermostatted bath and allow the solution
to equilibrate for 10 minutes.
3. Use the Auto-Pipetter to deliver exactly 1.50 mL of your acid solutions into clean, dry, plastic
cuvettes. Use a fresh, dry, blue pipette tip for each acid addition. Place the cuvettes into the four
Novaspec spectrometers (labelled Channel 1, 2, 3, 4) which are also equilibrated near to 50 C.
Note the temperature of the sample block at the start and end of your measurements. Your results
will depend on the accurate dilution of acid and the 1:1 mixing of the solutions of the acid and
the [Fe(bipy)3 ]2+ complex. The syringe pipette can very accurately deliver solution volumes if
used properly, check with a demonstrator if you are not clear on the proper procedure to use the
Auto-Pipetter.
4. Wait for 10 minutes for the temperature of the acids to equilibrate in temperature. Keep track of
which run (i.e. acid concentration) corresponds to which Novaspec as this will seriously affect
your analysis of the results. Carefully record the details in your lab book and update the details
as you conduct the experiment (these notes need to be submitted with your experimental report).
Note that you can add comments to the PowerLab chart trace.
5. Open the folder on the desktop called <Master-K3Initialised.adicht>. This will open the Chart
software and configure the four recorder channels with the correct settings for data collection.
6. Zero the spectrometers before starting a new run (the 1.5 mL of acid solution in the cuvette should
be sufficient to allow a valid zero of the spectrometer).
7. Just before you make the first addition of the metal complex start recording the spectrometer
output by clicking the start button at the bottom right side of the Chart window.
8. Pipette 1.5 mL of the [Fe(bipy)3 ]2+ into the acid solution in the cuvette in the Novaspec spectro-
meter. Stir quickly with a dry glass rod to mix the solution then lower the lid of the spectrometer.
Repeat for each of the four solutions. [Tip: start with the solution with the lowest acid concentra-
tion, this reacts more slowly and will give you a chance to refine your technique.]

9. Record the change in absorbance for the different solutions until a stable reading is obtained (the
trace should take the form of an exponential decay or rise) or until you judge the reaction to be
ca 80% complete. Depending on the acid concentrations, the reaction time will vary from 5 to 20
minutes. To measure the acid dependence of the aquation kinetics of [Fe(bipy)3 ]2+ you will need
data for eight different acid concentrations.
10. Save your data on the prac server (rather than on the instrument computer) as a Chart Data file
(*.adicht) and Chart text file (*.txt) include your name and date in the filename e.g. MyName-
HiconcFebpyDate (I would recommend giving the date in the form YYMMDD).
11. Repeat Steps 2-10 for the other kinetic runs.
2.3 Measurement of the aquation kinetics of [Fe(phen)3 ]2+
Important: Change the wavelength to 510 nm for the [Fe(phen)3 ]2+ complex.
Using an analogous procedure to that outlined for [Fe(bipy)3 ]2+ , measure the aquation kinetics of
tris(1,10-phenanthroline)iron(II). Use the same iron(II) complex reactant concentration, but it is only
necessary to determine the rate at four acid concentrations: 1.0 M, 0.5 M, 0.1 M and 0.05 M. Monitor
the reaction at 510 nm; the reactions are slower so allow around 20 minutes for these runs.
2.4 Treatment of the kinetic data
All of these complexes should aquate according to first order kinetics with a rate constant that de-
pends on the acidity of the solution - however it is unlikely that you will have followed all reactions
to completion. In this case the rate constant can be obtained from a fit of the time dependence of the
absorbance data to an exponential function (this method for the determination of the first order rate con-
stant does not require an equilibrium value of absorbance to determine values of kobs ). The data file
should be saved using the CHART software and the file closed.
Data processing for determination of the rate constants and the pH profile analysis for aquation of
[Fe(bipy)3 ]2+ and [Fe(phen)3 ]2+ takes the form of (1) fitting the kinetic scans to an exponential function
to extract the first-order rate constant, kobs , for each measurement. Make sure you make an assessment
of the error of your reported rate constant. (2) Using the rate constants obtained for measurements at
different pH to extract the rate constants shown in Fig. 1. The analysis can be conducted using routines
available in proFit (e.g. the proFit function pHProfile) or Igor (ExptK3Calc k.pxp) which are on the
computers in the Multimedia room but it is also possible to use Excel to complete the analysis. A
spreadsheet setup to conduct the analysis will be available on the LMS.
3 Report
The report should consist of a short introduction (1-2 sentences) that put the purpose of the exper-
iment into context. This is followed by sections describing the experimental procedure (as conducted
by you) and a summary of your results (in tabular form). The analysis will be concerned with the
deduction of the rate constants (or ratio of the rate constants) shown in Fig.1. The discussion will be
concerned with the interpretation of the results you have obtained, together with measurements of the
activation volumes for these reactions which are given in reference 3, in terms of the reaction mechan-
ism. The conclusion will be a short summary (2-3 sentences) of how the experiment went and what, in
broad terms, was learnt in completing the experiment. The report should be brief and succinct and be
no more than 1-2 pages of writing.

4 Assessment
Your mark for the experiment will be based on your ability to record and communicate what you did
in the experiment where this will be determined during the viva (40%) together with a component based
on the quality of your experimental results (20%). The report and interpretation mark will be based on
your submitted report (20%) and the discussion of the experimental analysis in the viva (20%).
You must submit the report within 1 week of completing the experiment and the viva will be con-
ducted at the time of, or shortly after, your submission of your report. Please note that most of the
marks awarded are based on how you prepare for, conduct and engage with the experiment. The report
should be brief, experiment focussed and provide you with an opportunity to think about the chemical
significance of the observations.
For the viva, make sure you understand what you have done and why you have done it. If things have
gone wrong be able to explain what you think is unreliable (and why) and how this limits the reliability
of your conclusions. I would expect you to know what is meant by the steady state approximation, be
able to show how kobs relates to the proposed mechanism and what one means by the A, Ia , I, Id and
D reaction pathways (discussed in the CHEM20018 subject and in the Inorganic Chemistry textbooks).
The dependence of the rate constant with pressure from reference [3] (reproduced in Table 1) may be
helpful in discussing the reaction pathway. Mostly the viva gives you an opportunity to demonstrate
your engagement with the conduct and analysis of the experiment.
Aquation of Fe(bipy)2+ +
3 ; temp = 25 C; [H ] = 2.5 M
pressure (bar) 1 34 344 626 1020 1293
kobs (min-1 ) 0.0464 0.0452 0.0387 0.0365 0.0302 0.0272
Aquation of Fe(phen)2+ +
3 ; temp = 35 C; [H ] = 1.0 M
pressure (bar) 1 690 1380
-1
kobs (min ) 0.0220 0.0147 0.0097
Table 1: High pressure data from reference 3
References
[1] R.G. Wilkins, Kinetics and Mechanism of Reactions of Transition Metal Complexes, 2nd ed. VCH,
1991 (UniM ERC 546.6 WILK); M.T.Weller et. al. , Inorganic Chemistry, 6th ed. OUP, 2014 (UniM
ERC 546 WELL); C.E. Housecroft and A.G. Sharpe, Inorganic Chemistry, 4th ed. Pearson, 2012
(UniM ERC 546 HOUS).
[2] F. Basolo, J.C. Hayes and H.M. Neuman, J. Amer. Chem. Soc. 1954, 76, 3807.
[3] J.M. Lucie, D.R. Stranks and J. Burgess, J. Chem. Soc. Dalton Trans. 1975, 245.

Physical Characterisation
In the following pages the preparations for the experiments offered for the Physical Characterisation
component of the course are included.
You must complete one experiment from those offered.
PC1 - Dynamic Light Scattering
Dr. Georgina Such

to learn about the interaction of light with scattering materials,
to investigate the effect of pH and/or ionic strength on the size of a nanoparticle properties,
to investigate the effect of polymer composition on nanoparticle behaviour.
1 Introduction to Light Scattering
Light is scattered from most materials to some extent. Objects that we see are visible due to light
scattering from their surfaces. Scattering of light depends on the wavelength or frequency of the light
being scattered, the angular distribution of the scattered light, and certain physical properties of the
material scattering the light. Analysis of the scattered light from a sample can reveal important inform-
ation about the structure and dynamics of the material being examined. A study of the scattered light
intensity as a function of scattering angle gives information about the structure, spatial configuration, or
morphology of the scattering medium.
There are various types of light scattering; elastic and inelastic. Examples of elastic light scattering
are Rayleigh, Mie and Tyndall scattering and an example of inelastic scattering is Raman scattering,
which can give vibrational frequency information complementary to infra red absorption spectroscopy.
The Rayleigh Theory can predict the light scattering for small particles and molecules whose dia-
meters are less than 1/10th that of the laser wavelength. This equates to particle sizes of less than about
60 nanometres for the helium neon laser used in the Zetasizer Nano which has a wavelength of 632.8
nanometres. For particles which are this small, the scattering produced is isotropic - that is, equal in all
directions. The intensity of light they produce is proportional to the particle diameter to the sixth power
i.e. I d 6 .
Mie theory is an exact description of how spherical particles of all sizes and optical properties scatter
light. When particles become larger than one tenth of the laser wavelength, the scattering changes from
being isotropic to a distortion in the forward scattering direction. When the size of the particles becomes
equivalent to or greater than the wavelength of the laser, the scattering becomes a complex function with
maxima and minima with respect to angle. This is known as non-isotropic or Mie scattering. Mie theory
correctly explains the maxima and minima in the plot of intensity with angle. The Malvern Zetasizer
Nano uses Mie theory by default to convert the intensity size distribution into volume and number for
all sizes of particles.
Light scattering techniques are reasonably easy to perform and are very useful in studies about the
structure of macromolecules and molecular assemblies. But particle size analysis is a more complex
subject than first appears because the problem is how to describe a 3-dimensional object with one num-
ber. Most techniques use an equivalent spherical diameter. This is convenient since the size of a sphere
89
PC1 CHEM30015: Advanced Practical Chemistry
can be described with a single number. All particle size analysis techniques measure some property
of a particle and report results as the equivalent spherical diameter based on this measured parameter.
Different measurement techniques often give different sizes for the same sample.
There are two main approaches in elastic light scattering; static and dynamic. In static light scatter-
ing, the experimental variable is the time-average intensity of scattered light, whereas in dynamic light
scattering it is the fluctuations in light intensity that are studied. Static light scattering can be used to de-
termine the molecular weight of polymers. Dynamic light scattering (DLS) analyses are routinely used
in chemical and biology laboratories to detect aggregates in macromolecular solutions, to determine the
size of proteins, nucleic acids, and complexes or to monitor the binding of ligands.
1.1 Dynamic Light Scattering and Brownian Motion
Dynamic light scattering is a non-invasive technique for measuring the size of particles and mo-
lecules in suspension Brownian motion is the random movement of particles due to the bombardment
by the solvent molecules that surround them. The technique of dynamic light scattering measures the
speed of particles undergoing Brownian motion.
The speed of the Brownian motion is influenced by:
Particle size
Sample viscosity
Temperature
The importance of the viscosity in determining the speed of Brownian motion means that the temper-
ature must be accurately known - this is automatically read back by the software. The temperature also
needs to be stable during a measurement otherwise convection currents in the sample will cause non-
random movements which will ruin correct size interpretation. The smaller the particle is, the more rapid
the Brownian motion becomes. The larger the particle is, the slower the Brownian motion becomes. The
higher the temperature the more rapid the Brownian motion.
Velocity of the Brownian motion is defined by the translational diffusion coefficient (D). The trans-
lational diffusion coefficient can be converted into a particle size using the Stokes-Einstein equation
(Equation 1).
kT
dH = (1)
3D
The equation contains k, the Boltzmann constant, T the absolute temperature and , the viscosity.
The equation shows there is a direct relationship between the hydrodynamic diameter obtained and the
viscosity value defined. Therefore, the accuracy of the viscosity value entered into the software cannot
be overemphasised.
1.1.1 Definition of Hydrodynamic Diameter (dH ):
The diameter of a hard sphere that diffuses at the same speed as the particle or molecule being
measured. The hydrodynamic diameter will depend not only on the size of the particle core, but also
on any surface structure, as well as the type and concentration of any ions in the medium.

CHEM30015: Advanced Practical Chemistry PC1
1.1.2 Effect of Ionic Strength
The ions in the medium and the total ionic concentration may affect the particle diffusion speed by
changing the thickness of the electric double layer called the Debye length (1 ). A low concentration
ionic medium will produce an extended double layer of ions around the particle, reducing the diffusion
speed and resulting in a larger, apparent hydrodynamic diameter. Higher ionic concentration media
(>10mM) will compress the electrical double layer and reduce the measured hydrodynamic diameter.
1.1.3 Effect of Surface Structure
Any change to the surface of a particle that affects the diffusion speed will correspondingly change
the apparent size of the particle. An adsorbed polymer layer projecting out into the medium will reduce
the diffusion speed more than if the polymer is lying flat on the surface. The nature of the surface and the
polymer, as well as the ionic concentration of the medium can affect the polymer conformation, which
in turn can change the apparent size by several nanometres.
1.1.4 Effect of non-spherical particles
The sphere is the only object whose size can be unambiguously described by a single number. For
a non-spherical particle, DLS will give the diameter of a sphere that has the same average translational
diffusion coefficient as the particle being measured.
1.2 Correlation in Dynamic Light Scattering:
In this experiment, you will use a commercial Dynamic Light Scattering instrument (Malvern ZetaS-
izer Nano-S90). A schematic diagram of this apparatus is shown in Figure 1. It consists of a He-
lium:Neon laser (632.8 nm), temperature controlled sample chamber, single photon counting detector
and an in-built correlator.
Cuvette
containing Laser
sample
Small Large
Laser Particles Particles
Intensity
Intensity
Photon
counting
device Time Time
Figure 1: Dynamic light scattering apparatus.
Correlation in DLS is a technique for extracting the time dependence of a signal in the presence
of noise. In a DLS measurement, the time analysis is carried out with a correlator which constructs
the time autocorrelation function, G, of the scattered intensity according to Equation 2. Note from this
equation that intensities are multiplied together i.e. each channel of the correlator works by multiplying
together light intensities separated by its delay time. The product of intensities at short delay time is
high i.e. the degree of correlation is high. At long delay times, when there is no correlation, the product
of the two intensities will be equal to the square of the average light intensity.


I(t) I(t + )
G () = (2)
I(t)2
where I = intensity, t is the time and = the delay time.

For a DLS measurement, the intensity autocorrelation function is normalised such that G tau ap-
proaches 1 as tau approaches infinity. An example correlation function is shown in Fig. 2 in which the y
axis on the correlogram plot is labelled as g(2) minus 1. The small g indicating that the data plotted have
been normalised. The 2 indicating that it is a second order correlation function because it involves in-
tensities which are the squares of electric fields. The x axis, labelled as time, represents the delay times,
or taus, of the correlator. For a DLS measurement, the intensity autocorrelation function is normalised
such that G() 1 as . From an analysis of the autocorrelation function, a range of physical
parameters can be derived, often based on various assumptions.
Figure 2: Example correlation function.
Visual observation of the correlation function can provide us with a lot of information about our
sample and data quality (Fig. 3): The intercept indicates signal-to-noise (i.e. how easily the sample is
being measured). Time when decay starts indicates the average size (the longer the correlation persists,
the larger the average size). The gradient of the decay indicates the width of the distribution (a steep
gradient indicates a narrow distribution, a shallow gradient indicates broad distribution). The baseline
will indicate if there are any large sedimenting particles present (which may be too large to accurately
measure by DLS).
Time when decay starts

indicates mean size
Intercept
Gradient indicates
the polydispersity
of sample
Baseline
Figure 3: Interpreting the correlation function.

The correlation function can be modelled with an exponential expression such as:
2 Dr
G() = B + A e2q (3)

where B = baseline at infinite time, A = amplitude (or intercept), q = scattering vector = 4n
0 sin( 2 )
where n = dispersant refractive index, 0 = laser wavelength and = detection angle (usually 90 for
DLS), D = diffusion coefficient and is the correlator delay time. It is clearly critically important to
know the refractive index of the system (solvent).
The correlation function contains the diffusion coefficient information required to be entered into
the Stokes-Einstein equation. These diffusion coefficients are obtained by fitting the correlation function
with a suitable algorithm. Two methods of analysis are used:
Cumulants analysis:
Determines a mean size and polydispersity index
Distribution analysis:
Determines actual size distribution from suitable data
2 Introduction to Polymer Particle Sizing
The use of nanotechnology to deliver therapeutics has generated significant research interest due to
the potential of nanoparticles to release drugs specifically in a target site and thus limit negative side
effects. Encapsulation is also important for the delivery of biological therapeutics e.g., DNA, which
degrade non-specifically in the body if not protected. Polymeric nanoparticles are of particular interest
as drug delivery systems as they are simple to synthesise and can be tuned to incorporate a range of
functionality. However, to deliver drugs to a specific site it is important to engineer the nanoparticles
with stimuli-responsive properties, so they are stable in the blood stream and only deliver their cargo
specifically in a target site. A range of stimulus has been used in the literature, including variation in pH,
redox potential, temperature and light. The use of pH as a stimulus is particularly relevant for delivery
of a drugs inside a cell as there is a change in the pH from the blood stream (pH 7.4) to endosomal
vesicles in the cell (pH 6.5-5) where nanoparticles are internalised. This pH variation can be harnessed
to achieve disassembly of the carrier and drug release. To design pH responsive nanoparticles a useful
polymer is poly[2-(diethylamino)ethyl methacrylate) (PDEAEMA) as it undergoes a transition from
hydrophobic to hydrophilic at approximately pH 7 due to the protonation of the amino groups in the
structure. The pH of this transition can be tuned by changing the substituents on the nitrogen e.g.,
poly(2-(diisopropylamino)ethyl methacrylate (PDPAEMA) has a transition at pH 6.4.
There are many techniques to synthesis polymer nanoparticles; one of the most well documented
approaches is self-assembly to form structures like polymersomes or micelles. These structures are typ-
ically formed from an amphiphilic copolymer with both hydrophobic and hydrophilic domains. These
polymers will self-assemble in an aqueous environment to form spherical nanoparticles. A micelle is a
nanoparticle with a hydrophobic core where hydrophobic drugs can be loaded and a hydrophilic shell.
(Figure 4). Micelles that are being used for drug delivery typically contain a hydrophilic segment with
low-fouling properties such as poly(ethylene glycol) (PEG) to minimise non-specific biological interac-
tions.
In this experiment two pH responsive micelles will be synthesised using PEG-b-PDEAEMA and
PEG-b-(PDEAEMA-r-PDPAEMA) (see Figure 5). The polymers have similar molecular weight, (30,000
Da), but differ in the composition of their pH responsive block, PEG block is 2000 Da. The disassembly
behaviour of the nanoparticles in simulated biological conditions will be investigated using dynamic
light scattering (DLS). The DLS analysis will be used to understand the potential of these materials as
drug delivery systems.

Figure 4: Amphiphilic polymer micelle formation.
Figure 5: Polymers under investigation.
3.1 Operation of the Malvern DLS instrument
You will use the Malvern ZetaSizer Nano-S90 instrument. One of the most crucial aspects of DLS
measurements is the cleanliness and homogeneity of the sample.
3.2 Procedure
1. Make up some phosphate buffered saline (PBS). To make up 1 L we need to dissolve 80 g NaCl,
2g KCl, 14.4g Na2 HPO4 , 2.4g KH2 PO4 in 1 L of MilliQ water. Stir until dissolved.
2. Adjust pH to 6.5 using a pH meter and 0.1M HCl and 0.1M NaOH. Note: Please rinse the pH
probe will MilliQ water before adjusting the pH.
3. Polymer 1: PEG-b-(PDPAEMA-r-PDEAEMA) solution will be provided to at a concentration of

2.5 mg/mL at pH 6.5 in PBS.
4. Divide this solution into five vials and adjust the solutions to be pH 6.5 (initial), pH 6.8, pH 7.2,
pH 7.6 and pH 8.
5. Polymer 2: Make up 2.5 mg/mL PEG-b-PDEAEMA solutions in 25 mL PBS. The PEG-b-

PDEAEMA will be provided for you. Note: polymer is fluffy powder so weighing needs to
be done with care.
6. Divide this solution into five vials and adjust the solutions to pH 6.5, pH 6.8, pH 7.2, pH 7.6 and
pH 8 as above.
7. Filter particle solution using a 0.45 m PES membrane

8. Incubate solutions (1 mL) in the Thermomixer Comfort (incubator) at 37 C for 5 minutes (300
rpm).
9. The DLS will be conducted using a Malvern Zetasizer Nano S90 operating at a scattering angle
of 90 .
3.3 DLS Procedure:
1. Turn on computer and instrument
2. Open up Zetasizer software
3. Browse SOPs, press green arrow and then choose test1 Nach(temperature set at 37 C)
4. Place your sample in the instrument (1 mL of sample each time), name the sample and then press
the green arrow again.
5. Write down intensity mean size and polydispersity both given in the intensity PSD tab. What do
these values tell you? While you are measuring your sample draw the typical correlation function
you observe. Explain what this graph represents?
6. Take note of count rate of the measurement, what is this telling you?
7. Take at least three measurements for each pH (5 pH points for each polymer) and report the
average value.
8. Measure a control sample just PBS) without any particles at pH 6.5 to assess the background
signal obtained using a buffer. What does this signal tell you about some of your measurements
for polymer 2? Once the solutions at all five pH values are measured, graph the change in size
with pH for both particles. Error bars should be used based on the three measurements taken.
4 Assessment/Discussion Points
The assessment will be a combination of a five minute talk and a two page handout with the table
of particle sizes and the two disassembly graphs (submitted at start of presentation). Assessment will be
determined 50% on results obtained and 50% on the clarity of the presentation. The points that should
be covered in this presentation include:
1. How does DLS work?
2. Why are nanoparticles useful for therapeutic delivery?
3. Which particle you investigated has more potential for intracellular drug delivery and why (refer-
ence to graphs)?
4. What are the limitations of DLS for particle analysis?
5. What other instruments could be used to verify our results?

References
[1] Paul Barrett, ATA Scientific, Malvern Instruments, Dynamic Light Scattering Training - Achieving
reliable nano particle sizing.
[2] Xinpeng Ma, Yiguang Wang, Tian Zhao, Yang Li. Lee-Chun Su, Zhaohui Wang, Gang Huang,
Baran D. Sumer, Jinming Gao, Utra-pH-Sensitive Nanoprobe Library with Broad pH Tunability
and Fluorescence Emissions, Journal of American Chemical Society 2014, 136, 11085-11092.
[3] Georgina Such, Yan Yan, Angus Johnston, Sylvia Gunawan, Frank Caruso, Interfacing Materials
Science and Biology for Drug Carrier Design Advanced Materials 2015, 27, 2278-2297.

PC - Introduction to Investigations into
Surface Tension
In a liquid, the shape of the liquid mimics the shape of the container, but at the upper surface of the
liquid the phenomenon of surface tension operates and this defines the surface boundary.
The surface tension of a liquid, , is the force per unit length on the surface that opposes the ex-
pansion of surface tension. That is molecules located in a bulk solution are generally subjected to equal
forces of attraction in all directions, whereas molecules residing at an interface experience unbalanced
forces of attraction resulting in a net inward pull (Figure 1).
Figure 1: For water molecules at an air-water interface the attractive forces result in a net inward pull.
The addition of surface-active reagents, such as a surfactant and alcohol molecules invariably consist
of a hydrophilic (i.e. water loving) and hydrophobic (i.e. water-fearing) part (Figure 2).
Figure 2: Schematic representation of the hydrophilic and hydrophobic portions of alcohols and surfact-
ants.
97
PC-I NTRODUCTION TO S URFACE C HEMISTRY CHEM30015: Advanced Practical Chemistry
This structure enables the molecules to orient themselves at the surface and interfaces so that there is
a minimisation of the overall interfacial energy of the system. Considering a water-air interface again, the
hydrophilic group will orient itself toward the water while the hydrophobic portion will protrude into the
air. The interface is now between the hydrophilic group and the water on one side and the hydrophobic
portion and the air on the other side. The forces of attraction are now much more balanced and the net
inward pull has been significantly reduced when compared to the water-air interface (Figure 3).
Figure 3: The surface tension has been greatly reduced in the presence of surface active reagents.
A further consequence of the hydrophilic/hydrophobic portions of the surfactant molecules, is the

formation of micelles. These aggregates generally consist of 50 to 100 surfactant molecules. Micelle
formation occurs at a particular surfactant concentration known as the critical micelle concentration
(cmc).
To measure the surface tension of a liquid a number of methods are available. The three most
common methods are:
Capillary action
Du Nouy Ring
Wilhelmy Plate
For the purpose of these experiments you will be using one of either the Wilhelmy Plate or the
Pendant drop method. Be sure to confirm before your scheduled Laboratory time which experiment you
are to perform.

PC2 - Surface Tension Determination
Pendant Drop
Prof. Paul Mulvaney

1. To demonstrate the relationship of surface tension and surface excess.
2. To determine the Critical Micelle Concentration (CMC) of sodium dodecyl sulfate (SDS).
3. To demonstrate the principles and use of an OCA 20 Tensiometer.
4. To develop analytical skills.
1 Introduction
As mentioned previously there are a number of methods available for measuring the surface tension
of a liquid. In this experiment the Pendant Drop method will be employed. In the pendant drop method
the profile of a drop hanging (pendant) from a syringe needle is measured. Very small drops look
spherical, but as a hanging drop becomes larger, it becomes elongated due to the action of gravity. The
shape of the drop is then determined by the balance of the surface tension of the fluid and the pull of
gravity.
The Young-Laplace equation describes the shape of the drop exactly, so by measuring the pendant
drop profile along with the density of the fluid, the surface tension can be determined.
The Young-Laplace Equation is:
P = (1/R1 + 1/R2 ) (1)
where: P = pressure difference inside and outside the drop

R1 , R2 = principle radii of curvature
= surface tension
To describe the curvature at any point on the profile, z, Eq. 1 becomes:
(1/R1 + 1/R2 ) = g z + 2/b (2)
where: = the change in density inside and outside the drop

b = local radius or curvature at the point z
g = gravity constant
99
Eq. 2 is the starting point for the drop shape analysis software. In Eq. 1 P is in units of pressure
(N/m2 ) and R1 and R2 are in units of distance (m), is in units of N/m.
Once the surface tension is known the surface excess, , can be calculated from equation 3 below.
d = i i di (3)
Which for a solute that does not dissociate in water, e.g. 1-pentanol, becomes:
= (1/kT)d/d ln C (4)
where: i = chemical potential of component i

k = Boltzmann constant
T = temperature
C = concentration of the additive
2 Pre-Lab Questions
1. Derive the surface excess function for SDS in water.
2. Explain why the Gibbs-Duhem equation does not hold for surfactant concentrations above the cmc
[This is very important and needs to be taken into consideration when processing the experimental
results later.]
3. Calculate all weights and volumes needed to make up the solutions required in this experiment.
Tabulate these and include them as a table in your report.
Before entering the laboratory you should know what mass of SDS is required, and what aliquots
are to be dispensed (see Pre-Lab. Questions).
3.1 Solution Preparation
NOTE: Do not shake solutions containing SDS as this will cause frothing which will result
in inaccurate results. Gentle swirling of the solutions will provide adequate mixing. You will be
provided with an Eppendorf Multipette Plus auto-pipette. If in doubt, be sure to read through the
operating instructions provided before using the pipette.
Using a 250 cm3 volumetric flask, weigh out enough SDS to make a stock solution of 25 mM.
Use pipettes and 25 cm3 volumetric flasks to accurately prepare SDS solutions of 0, 0.5, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15 and 20 mM concentrations.
3.2 Cleaning
Cleanliness is a major contributor in obtaining accurate and reproducible results. To clean the syr-
inge, the following steps should be followed:
1. Rinse through with distilled water (three times)
2. Rinse through with ethanol (three times)

3. Rinse through with the next solution to be tested (three times)
4. Dry with fresh Kimwipes
5. Make sure disposable gloves are used when handling cleaned glassware.
Note: Once this has been done for the initial measurements with pure water, proceed from step
3 onwards with the solutions in increasing concentration of the surfactant in the second part of
the prac.
3.3 Procedure
3.3.1 Measurement of the surface tension of pure water
1. Turn on the tensiometer. Be sure to remove the lens cap from the camera.
2. Turn the computer on.
3. Remove the syringe from the holder and clean it thoroughly, using Milli-Q water. Fill the syringe
with Milli-Q water and carefully place it back on the holder. Take care when positioning the
needle - ensure it is sitting perfectly vertical in its holder so the camera is viewing it at 90 (seek
help from a demonstrator).
4. Run the Prac6.ref, located on the Computer Desktop. DO NOT load software BEFORE turn-
ing on the tensiometer!
5. A Results Collection Window should appear. Before continuing check the following settings:
Select System. Make sure that the following parameters are set:
System Phases
Drop Phase: Water
Surrounding Phase: Air
Phase Densities
Drop Phase: 0.9982 g/cm3
Surrounding Phase: 0.0013 g/cm3
Select M-Info and make sure that the following parameters are set:
Ref Size: 0.72mm
Mag [pixel/mm]: 115-120
Select C-Info and make sure the following parameters are set:
-Default Method-
Aspect: 1.0035
Acc. Of gravity (g): 9.80180
DO NOT CLOSE THIS RESULTS COLLECTION WINDOW, AS THIS WILL ALTER

THE ABOVE MENTIONED PARAMETERS.
6. Select Drop Window (by minimising the Result Collection Window OR click onto white
background of Drop 1 window).
7. Select Image > Start Grabbing (alternatively, you can hit the video camera icon on the menu
bar).
8. Make sure the needle appears on the screen as shown in the diagram below. Use the micrometer
to drive the syringe in order to produce the largest possible droplet size.

9. Make sure that the magnification dial on the camera is set to 0.7 and adjust the focus of the camera
(using the dial at the lens-end of the camera). The lamp brightness should be set to 50 on the
scale located next to the power switch. Do not turn the lamp up to full brightness, as this will
saturate the camera.
10. Adjust the reference (red) lines in the Drop Image window (there are three in total) such that the
rightmost one crosses the far left-hand-side of the drop, and the middle one crosses the needle.
11. Wait approximately 30 seconds then hit the following icons (on the icon bar) in the following
order:
Snap
Profile extraction
Fit to Laplace-Young Eq.
12. The surface tension value will appear on the top-left corner of the screen. Note this value in
your results sheet. Repeat the surface tension measurements by producing new droplets in order
to get an average. [For pure water, the surface tension value should be 73 mN/m. If your value is
substantially different from this you will need to adjust the magnification until you get 73 mN/m
for Milli-Q water].
3.3.2 Measurement of surfactant adsorption rate
N.B. Keep the needle in place when undertaking the following procedure.
1. Remove the syringe from the holder. Fill the syringe with 2 mM SDS solution and carefully place
it back on the holder. Take care when positioning the needle.
2. Repeat steps 8-10 of section 3.3.1.
3. Every 30 seconds for the next 10 minutes (a total of 20 images), hit the following icons (on the
icon bar), in the following order, simultaneously noting the surface tension values that appear on
the top-left corner of the screen, before taking the next image.
Snap
Profile extraction
3.3.3 Determination of CMC of SDS
1. Remove the syringe form the holder. Clean it thoroughly using Milli-Q water. Fill the syringe
with Milli-Q water and carefully place it back on the holder.
2. Repeat steps 8, 10 and 11 of section 3.3.1.

3. Remove the syringe from the holder. Fill syringe with the lowest concentration SDS solution and
carefully place it back on the holder.
4. Repeat steps 8 and 10 of section 3.3.1.
5. Wait about 5 minutes then hit the following icons (on the icon bar), in the following order:
Snap
Profile extraction
6. Repeat steps 3-5 of section 3.3.3 for all your solutions, working through form the lowest concen-
tration of SDS to the highest. Record the surface tension for each solution.
7. At the end of the experiment, be sure to quit from Prac6-ref software. Do NOT save the changes
to the file.
8. Turn the tensiometer off and replace the lens cap.
NOTES:
Wear gloves!
Remember, cleaning the syringe is important (failure to do so will introduce unnecessary skewing
to your results).
If errors persist, check to see if you have the correct drop-type selected (right-click the mouse
button, and from the resulting menu select Drop-Type, followed by the image of the drop beading
form the left to the right).
It is essential that the droplet remain as stationary as possible. The use of fluid cells and covering
shields may limit interference from vibrations and air currents, but they are not infallible measures.
Try not to lean on the bench or tap your feet, as this may upset the instrument.
If, in plotting surface tension vs. time, you get two curves, it is likely that vibrations have disturbed
your experiment. Look around and see what might be causing them.
If all else fails, consult your demonstrator.
4 Data Manipulation
1. Plot the surface tension vs. time for the 2 mM SDS solution (Section 3.3.2).
Relating to Section 3.3.3:

2. Tabulate the average surface tension values determined for each concentration of SDS (see the
results sheet illustrating the required format for this information) and then plot these results, i.e.
graph vs. [SDS].
3. Identify the cmc from this graph.
4. Plot the surface tension vs. the natural log of each concentration of SDS used below the cmc. Fit
a polynomial to these points using the curve fit menu, of the form
y = m0 + m1 x + m2 x2
where x = loge [SDS].
5. The slope of each point on the above graph will give d()/d(ln[SDS]). Therefore,
dy
= m1 + 2m2 x = slope
dx
It then follows that the surface excess () can be determined from the equation:
slope 1 d
= 2RT or = 2RT d ln[SDS] for SDS solutions (see pre-lab questions).
6. Complete the rest of the table in your report with the information shown on the results sheet.
7. Plot vs. [SDS].
5 Questions
1. After what interval of time does droplet surface tension reach equilibrium?
1. What is the maximum and at what concentration of SDS does it occur?
2. How does your value compare with the literature values? [Be sure to quote the literature value
and a reference in your report].
3. Determine the cmc and compare the value with that given in the literature [quote a value and the
reference source].
4. What would you expect would happen to the cmc of SDS if 0.1 M butanol were added? Explain.
5. What would you expect would happen to the cmc of SDS if 0.1 M NaCl were added? Explain.
6. Why is there virtually no change in above the cmc?

References
[1] Surface Chemistry Lecture Notes (See LMS).
[2] D.J. Shaw, Introduction to Colloid & Surface Chemistry, Butterworth-Heinemann Publishers,
1992.
[3] P.C. Hiemenz, Principles of Colloid & Surface Chemistry, Marcel Dekker Publishers, 1986.
[4] D. Myers, Surfaces, Interfaces and Colloids, VCH Publishers, 1991.
[5] M.J. Rosen, Surfactants and Interfacial Phenomena, Wiley-Interscience Publishers, 1978.
[6] A.W. Adamson and A.P. Gast, Physical Chemistry of Surfaces, 6th ed., Wiley-Interscience Pub-
lishers, 1997.

Electrochemical Processes
In the following pages the preparations for all experiments offered for the Electrochemical Processes
EC1 - Coupled Chemical and
Electrochemical Reactions
Dr. Chris Ritchie

This experiment will be assessed by a Mini Presentation and Viva (see below).
1 Introduction
A feature common to catalytic systems that underpin biology (enzymes), industry and chemical syn-
thesis is their exploitation of changes in chemical reactivity with a change in redox. This is no better
illustrated than by transition metal complexes where a one-unit change in oxidation state can change
the acidity of coordinated ligands by 6 orders of magnitude (e.g. [Fe(OH2 )6 ] 2+/3+ ) or the rate of ligand
substitution by more than 12 orders of magnitude (e.g. Cr II /Cr III ) [1]. In some cases the change in reac-
tions/reactivity is directed by the metal but there are important examples where the characteristics of the
ligand can be made to dominate the chemistry as is often observed in enzymes. Characterisation of the
thermodynamic and kinetic properties of catalytic systems is of practical and fundamental importance.
A broad range of electrochemical techniques have been developed which may be used to gain insight
into the redox behaviour and dynamics of electro-active species [2, 3]. Cyclic voltammetry is one of
the most useful of these techniques, the results being sensitive both to the reduction potential and the
reactions of the molecule in oxidised and reduced form. In this experiment we examine the cyclic
voltammetry of compounds that undergo coupled chemical-electrochemical reactions with the aims of
(i) understanding the voltammetric signatures that are obtained and (ii) relating the differing chemical
behaviour to the molecular interactions within the compound.
2 Aims
Examine the UV-Vis and NMR spectroscopy of the Co III N6 complexes.
Measure cyclic voltammetry of the related Co III N6 complexes: [Co(en)3 ]I3 and [Co(diNOsar)]Br3
and to contrast the differing reversibility of their reduction chemistry.
Explore the reversibility of the redox chemistry of another redox system (to be allocated by SPB
prior to commencing the experiment).
3 Background
The macrocyclic ligand diNOsar (abbreviation for its common name, dinitro sarcophagene, more
correctly this is 8-dinitro-3,6,10,13,16,19-hexaazabicyclo-[6.6.6]icosane) was discovered by Alan Sargeson
[4, 5, 6, 7] at the ANU in the early 1970s being prepared by a template synthesis involving the condens-
ation of [Co(en)3 ] 3+ with formaldehyde (HCHO) and nitromethane (CH3 NO) to give the cage complex,
[Co(diNOsar)] 3+ . The condensation reaction that forms [Co(diNOsar)] 3+ from [Co(en)3 ] 3+ is believed
to occur through a number of condensation reactions as shown in Figure 1.
107
EC1 CHEM30015: Advanced Practical Chemistry
Figure 1: The condensation reaction that forms [Co(diNOsar)] 3+ from [Co(en)3 ] 3+ .
It is interesting to note that while each of the condensations at the nitrogen atoms could in principle
give different enantiomers the product is predominantly a racemic mixture of isomers with all six nitro-
gen atoms having the same absolute configuration (a higher energy form with , , , , , absolute
configurations of the N atoms has also been isolated). While the preparation of Co(diNOsar)]Br3 is
relatively straightforward, it is time consuming and samples will be provided.
4 Pre-Lab Exercises
(Hint: read the notes on cyclic voltammetry [8, 9] first! (available on LMS)
1. Analyse the cyclic voltammogram shown in Figure 2 to estimate the following parameters: E pa ,
E pc , E1/2 , i pa , i pc , E.
2. The inset in the voltammogram depicts regions very close (A), intermediate (B) and far (C) from
the working electrode. Complete Table 1 to indicate whether the predominant species is the ox-
idised (ox) or reduced (red) component of the couple or if similar concentrations of the oxidised
and reduced species (ox/red) are expected.
Cathodic scan Anodic scan

Region
-1.0 -1.38 -1.6 -1.35 -1.3 -1.1
A ox
B ox
C ox
Table 1: To be completed for pre-lab question 2.

CHEM30015: Advanced Practical Chemistry EC1
Figure 2: Cyclic voltammogram for pre-lab question 1.
3. Why are CV experiments conducted using unstirred solutions?
5.1 UV-Visible Spectra
Samples of [Co(en)3 ]I3 and [Co(diNOsar)]Br3 will be provided for the UV/Vis and electrochemical
experiments. Note: ensure the solid samples are completely dissolved prior to commencing any
analysis.
Note that spectra collected under basic conditions must be collected immediately after sample dis-
solution.
Prepare separate aqueous solutions (25 mL) of accurately known concentration of 5 mM [Co(en)3 ]I3
and [Co(diNOsar)]Br3 in 0.1 M HCl and 0.1 M NaOH (separately) and record the visible spectra (300-
700 nm) of these four solutions using one of the UV/Vis spectrometers (e.g. the Perkin Elmer lambda 2,
Shimadzu UV-2401 PC or Varian Bio50). Make appropriate dilutions to bring the absorbance to about
1 OD unit. Retain the acidic solutions for the electrochemical experiments. Save the spectra as a text
file so you can plot it in your report. (Note: one of the spectra will go off range, use an autopipette
to dilute your solution by a factor of 5 and immediately the re-run spectrum). Remember to record the
colours of the acidic and basic solutions in your laboratory note book and also which of the solutions it
was necessary to dilute. Note that absorbance readings larger than 2 (i.e. less than 1 % of the incident
light reaches the detector) are likely to be unreliable.
5.2 Electrochemical Measurements
Before commencing the electrochemical measurements you should be familiar with the instructions
for the use of the PowerLab electrochemical instrument and EChem software and introductory notes on
cyclic voltammetry [8, 9] are available from the LMS page.
Please note: the eChem software is also available on the Multimedia room computers using the Vir-
tual Windows environment. You can analyse your data on those computers and paste diagrams directly
into your report.

5.2.1 Experimental Procedure
The electrochemical measurements should take about 1 hour to complete.

Electrochemical measurements will be conducted on the following deaerated solutions (remember
to run CVs at different scan rates for the reversible processes - this is adjusted in the Staircase Cyclic
Voltammetry window:
i. K3 [Fe(CN)6 ] (1 mM in 1.0 M KCl) - supplied. Initial potential = +500 mV; Upper potential = +500
mV; Lower potential = 0 mV. If the distance between the redox peaks is larger than 59-63 mV, clean
the glassy electrode (see below) and repeat the experiment.
ii. [Co(en)3 ]I3 (5 mM in 0.1 M HCl) - use your sample prepared earlier for UV-Vis analysis. Clean the
electrode. Initial potential = 0 mV; Upper potential = 0 mV; Lower potential = -900 mV.
iii. [Co(diNOsar)]Br3 (5mM in 0.1 M HCl) - use your sample prepared earlier for UV-Vis analysis.
Clean the electrode. N.B. the voltage range for steps 2 and 3 varies i.e. different ranges will need to
be used for the two experiments. Initial potential = 0 mV; Upper potential = 0 mV; Lower potential
= -1100 mV.
In each case 5-10 mL of solution will be needed for these measurements. Degas solution for 2-3
mins.
The electrochemical measurements will be conducted using a standard three-electrode geometry,
with glassy carbon working, platinum foil counter (or auxiliary) and Ag/AgCl reference electrodes. The
reference electrode should be stored with the junction (sintered glass frit) in contact with a solution of
3 M KCl. Before placing in the electrochemical cell the electrodes should be rinsed with distilled water
and lightly dried using a clean tissue.
The measurements conducted on K3 [Fe(CN)6 ] are used to verify that the electrochemical cell is cor-
rectly set up and can be used to test and calibrate the potential of the reference electrode (note that if the
tip of the reference electrode dries up crystallisation of the supporting electrolyte can occur and blocks
the sintered glass frit). This measurement also provides an example of a reversible, diffusion controlled
electron transfer process (actually, the Fe III /Fe II couple of ferricyanide is only strictly quasi-reversible
at a glassy carbon working electrode). The metal-based reduction of [Co(en)3 ] 3+ and [Co(diNOsar)] 3+
should occur at similar potentials and with similar peak currents, although the chemical and electrochem-
ical reversibility of the two processes is very different. You will be asked to consider the reversibility of
these two processes. Note also that one of the complexes has two reduction processes within the solvent
window, one metal-based the other ligand-based. Be careful to examine only the reversible process for
the different scan speeds (it is also advisable to clean the tip of the working electrode before doing those
scans).
For the reversible (or quasi-reversible) redox processes (i.e. where you see a well-defined return
wave) record the cyclic voltammogram for scan rates of 10, 50, 100 and 500 mV/s. Determine E p ,
i pc /i pa and plot the value of the peak current of the anodic and cathodic scans against the square root of
the scan speed. In light of these values comment on the reversibility of the electrochemical process.
5.2.2 Instructions for Use of the Adi Potentiostat and Powerlab Interface for Electrochemistry
Please note that the electrodes used for the electrochemical are expensive and fragile. If you are
unsure about the handling of the electrodes please consult a demonstrator.
This sub-section describes the operating procedure. Apply these instructions to Section 5.2.1.
1. Turn on the potentiostat and the PowerLab interface (switch at the back of the unit). This should
be done before turning on the computer.

2. Turn on the Computer.
3. Once the Computer has booted, click on the EChem icon located on the desktop.
4. While the program is booting, set up the electrochemical cell as follows:
Place 8-10 mL of the reference electrolyte solution (1.0 M KCl) in the electrochemical cell.
Insert the glassy carbon working and Pt wire counter electrodes and a stopper (in place of
the Ag/AgCl reference electrode) into the holes at the top of the cell.
5. Nitrogen is supplied from the Broen valve linked to a manifold. Connect one of the outputs from
the manifold to the electrochemical cell, using the remaining quickfit joint. Turn on the N2 line
and purge the solution for 5 mins under gentle bubbling. Remove the stopper and insert the
reference electrode.
6. In the EChem program, click on the Technique heading on the screen and pull down the cursor
with the mouse until Cyclic Voltammetry is reached (Fig. 3).
Figure 3:
7. Then release the mouse button. The screen (Fig. 4) should appear.
Figure 4:

You will return to the Staircase Cyclic Voltammetry window many times. Therefore you should
note the following:
i. The scan rate is varied in the Rate box. For most runs this will be 100 mV/s as shown.
For the [Co(diNOsar)] 3+ complex it will be necessary to adjust this value when examining
the reversible couple (see below);
ii. As the scan rate increases so too does the peak current. You should be aware of this and adjust
the current range accordingly (as shown in point 11 below);
iii. Steps should be more than 1000 but must not exceed 2500. If it does increase the Width in
lots of 10 ms until Steps is less than 2500.
iv. Height can only take integer values and must be greater than, or equal to, 1.
v. The Sampling period (S period) must be equal to, or shorter than, the Width.
vi. A graphical summary of the data collection parameters can be obtained by selecting view.
8. Change the Initial Potential to 0 mV, the Upper Potential to 1000 mV and the Lower Potential to
-1,200 mV, then click on OK. If you wish to see what the excitation profile (Potential vs Time
response) looks like before leaving this box, click on the view button.
9. Collect the de-gassed solution from the main laboratory. Remember to turn off the nitrogen supply.
10. Connect the three wires from the potentiostat to the three electrodes (the alligator clips are appro-
priately labelled).
11. Click on the Start (sample) button on the bottom right of the screen to run the CV. If the response
runs off the scale then you will need to adjust the Y-scale by clicking on the voltage range button

on the right of the screen. The default value is 100 mA, this will be too high for many experiments.
For the [Fe(CN)6 ]3 scan, the current range should be ca. 20 A. For the more concentrated cobalt
solutions you should choose a more appropriate current range.
12. The background electrolyte should give a straight line (20 A current range) if the solution is
properly degassed. If any peaks are observed in the response, apart from curvature at the end
of the scan, the glassy carbon electrode needs to be polished in an alumina slurry. Consult a
demonstrator if this needs to be done.
13. The convention for plotting voltammograms is for the voltage to increase from left to right and for
negative currents to indicate reductions. In some cases the software loads with positive currents
corresponding to reductions. If this is the case select potentiostat and the following window will
appear. Check the box labelled invert and close the box (OK) and re-run the voltammogram.
14. Before plotting the CV, you should change over the solutions so that the next one is being degassed
while you are plotting. Remove the vial top (with the electrodes attached). Rinse the electrodes

with a wash bottle and wipe them clean with a tissue. Insert the electrodes into the next solution
to be measured and commence degassing the solution.
15. Go to the File heading and then Save As... Save the document with the same name as already
in the list of files, e.g. MyName E3. Make sure that you use an appropriate file name for your
experiment, since this name that will be printed out on your plots. Note also that the EChem
software saves all CVs in the one file. The different CVs are represented as different pages of
the overall experiment.
16. Return to the File heading and go to Page Set-up. In the Orientation box make sure that you make
a landscape (sideways) plot. This command only needs to be performed once, unless you turn the
computer off. Click on the OK button.
17. Return to the File heading again and then Print. You may prefer to save the plot as a file for
inclusion in your report.
For the [Co(diNOsar)] 3+ solution, you should perform two experiments, one over the entire range
and a second experiment where you stop the scan just after the first current response. The peak
potentials and currents for the responses can be read off the screen plot by moving the cursor with
the mouse. The current and potential are shown on the top right hand of the screen.
18. After you have finished, turn off the computer and potentiostat, turn off the nitrogen supply and
make sure that the reference electrode is returned to its 3 M KCl reservoir. If the reference elec-
trode dries out it could be destroyed. Consult with a demonstrator to make sure that you have done
everything correctly.
Remember to clean the glassy carbon electrode carefully and check the cleanliness by running a scan
of a KCl(aq) solution. To clean the glassy carbon electrode, place a small amount of alumina powder
(about the tip of a spatula) into the Petri dish, add a drop of water, then move the electrode tip firmly
through this slurry in figure of eight patterns for about a minute.
6 EChem Software
If you prefer, you can process your data using the EChem software (EChem 2.1.16) from home/your
computer. The link to the software is: http://www/edaq.com/software-dowloads, and the licence code
is: http://www/edaq.com/software-dowloads. Remember, you will need to save the data from the
prac. server to a USB stick.
7 Report
A report and question sheet is available on LMS. This will provide you with a guide to the material
that you should include in your report and how it might be presented.
8 Assessment: Mini Presentation and Viva
Examination of EC1 will be conducted by presentation of your findings and interpretation of your
results in the form of a 5-10 minute presentation. Following the presentation you will be questioned on
details of the experimental setup, the techniques used and your understanding of the chemical processes
investigated.

References
[1] General Inorganic Chemistry texts such as: Shriver and Atkins, Inorganic Chemistry, Cotton and
Wilkinson, Advanced Inorganic Chemistry.
[2] A.J. Bard and L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley,
New York, 2001.
[3] Southampton Electrochemistry group, Instrumental Methods in Electrochemistry, Ellis Horwood,

Chichester, 1985.
[4] A.M. Sargeson, Chem. Brit., 1979, 15, 23.
[5] A.M. Sargeson, Pure Appl. Chem.. 1984, 56, 1603.
[6] R.J. Geue, T.W. Hambley, J. Harrowfield, A.M. Sargeson and M.R. Snow J. Am. Chem. Soc. 1984,
106, 5478.
[7] A.M. Bond, G.A. Lawrence, P.A. Lay, and A.M. Sargeson,. Inorg. Chem. 1983, 22, 2010.
[8] P.T. Kissinger and W.R. Heineman, J. Chem. Educ. 1983, 60, 702-6.
[9] G.A. Mabbott, J. Chem. Educ. 1983, 60, 697-702.

EC2 - Electrochemical Kinetics of Redox
Reactions
Prof. Paul Mulvaney

1 Experiment Aims
To understand the relationship between cell current and EMF of electrochemical cells
To measure electrode potentials at various currents using a three-electrode cell.
To understand how diffusion affects the rate of electron transfer when high potentials are applied
to the working electrode.
2 Background
2.1 Introduction and theory of electron transfer
Redox reactions involve the transfer of an electron. In an electrochemical cell, these reactions (ox-
idative and reductive) occur at an electrode surface. The dependence of the current on the potential
difference between the anode and the cathode are characteristic of the redox reaction and the conditions
used.
In the first year and second year we examined static systems, i.e. we measured and analysed the
potentials of cells in the absence of any current flow. The cell potential was found to be given by the
Nernst equation, but differs because of three kinetic factors:
1. The rate of electron transfer at electrode-solution interfaces, i.e. the rate of oxidation or reduction
of electroactive species is finite.
2. The approach of electroactive species from the bulk of the solution to positions close enough
(<1 nm) to an electrode surface to allow electron transfer is limited by diffusion in the solution.
3. Conductance of the cell electrolyte. Cells with high solution resistance have a voltage drop across
the solution of V = iRs . This factor can be reduced by adding a high concentration of an electrolyte
or salt to the solution. The conductances of the electrolyte that you will use are sufficiently great
that currents are not limited by electrolytic conduction.
In these experiments, we will learn about the kinetics of charge transfer at electrode surfaces, and
about ion migration in solution. Because electrochemical reactions involve the consumption of elec-
trons, the reaction can be monitored by measuring the electric current. The unit of current is Ampere
(symbol A):
117
1A = 1 Coulomb of electrons per second = 1Cs-1

1 Coulomb = 6.21018 electrons
Conversely we can measure the amount of material in solution that is consumed per unit time
(mol s-1 ). To convert between these two units, we use Faradays constant, F.
1mol = 96487 C, i.e. F = 96487 C mol-1
For an anodic reaction (Oxidation of a solution species) we define the current, ia , to be positive, and
cathodic currents ic are defined to be negative. Both anodic and cathodic reactions take place simultan-
eously and independently at an electrode surface. Consider reaction 1 given below:
O + ne
)

*
R (1)
It is observed experimentally that when we apply a voltage to an electrode containing a redox couple,
the current depends exponentially on the voltage or potential applied to the cell. It has been shown that
the cathodic and anodic currents obey the following equations:

nFE (1 )nFE
ic = ic exp and ia = ia exp (2)
RT RT
Here is called the transfer coefficient, and 0 < < 1. It is determined experimentally. At the
Nernst potential, Eeq , there is no NET current flow, and the system is at equilibrium. It is convenient to
measure the current flow as a function of the potential using the Nernst potential as a reference point.

(1 )nF nF
i = ic + ia = io exp exp (3)
RT RT
The first term is the rate of oxidation of R, the second is the rate of reduction of O. is the overpo-
tential with respect to the equilibrium or Nernst value, Eeq . i.e.
= E Eeq (4)
Equation 3 can be simplified under some conditions. For large positive potentials, the second term
is very small and:
(1 )nF
ln i = ln io + (5)
RT
Similarly if << 0.12 V then the oxidation current becomes negligible and the total current
becomes almost equal to the reduction current, ic , and equation 2 can be written as:
nF
ln(i) = ln io (6)
RT
Equations 5 and 6 are two versions of the Tafel equation and are used in the estimation of and io .
The Tafel equation deals only with the role that electron transfer plays in controlling current (Factor 1)
and ignores the part played by mass transport. According to the Tafel equation, we can get any current
we want by simply dialling up a big enough potential. However, when a reaction becomes very rapid,
the chemical species that is reacting at the electrode surface (the electroactive species), is depleted
from the electrode surface region. The rate of reaction is then no longer controlled by the Butler-Voler
or the reduced Tafel form; instead it is the rate at which the electroactive material can be brought to the
surface by diffusion (concentration gradient set up by its depletion), or convection (stirring).

Figure 1: Concentrations of reacting species in solution near electrode.
This means there is a limiting current that can be achieved (see Fig. 1).
Diffusion is important because as the reactant is used up near the surface, its concentration decreases.
New reactant must diffuse from the bulk solution up to the electrode. The rate of diffusion is propor-
tional to the concentration gradient. At sufficiently high reaction rates, the surface concentration of the
electroactive species will be reduced to almost zero and at this point (Fig. 1, curve (c)), the current is
limited by diffusion and will no longer increase with increasing overpotential.
By stirring the solution, we also create convection currents, which augment the flow due to diffusion.
A well-stirred solution can be achieved by spinning the surface where the electron transfer is occurring.
If the solution is well stirred, a steady-state is achieved with the electrode potential and current density,
I, related by:
RT (id,a i)
Eapp = E1/2 + ln (7)
nF (i id,c )
where id,a , id,c , are the anodic and cathodic limiting currents, which are proportional to [R and [O]
respectively, and E1/2 is the half-wave potential which is often close to Eeq [1] (see Fig. 2).
Levich has shown that for a rotating disk electrode, which we will be using, the limiting current due
to diffusion and convection obeys equation 8:[1]
id = 0.620nFA D2/3 1/2 1/6 [Fe(CN)64 ] (8)
where n = 1, A is the electrode area, D is the diffusion coefficient (m2 s1 ), = 2 f (s1 ) and f
is the rotation rate of the RDE, is the kinematic viscosity of water (= 8.9 107 m2 s1 ) and the
ion concentration is in mol m3 (NOTE UNITS!!!). A plot of limiting current vs 1/2 yields the ions
diffusion coefficient, D.
A less rigorous approach to the diffusion limit is often used, which relates the rate to the concentra-
tion gradient, which is [Fe(CN)64 ]/. This gives equation 9.
id = nFAmo [Fe(CN)64 ] (9)
where:
mo = D/ (10)
This form is used to estimate the thickness of the depleted region next to the electrode.

Figure 2: Typical current-voltage curve for a solution containing both Fe(CN)64 and Fe(CN)63 at Pt
RDE using equation 7 with n = 1 and E1/2 taken to be 0.40V vs an arbitrary electrode.
2.2 Experimental Considerations
In voltammetry, the cell current and the potential of one electrode, the working electrode, are mon-
itored. Because current must pass through a counter electrode to complete the circuit, the potential of
the counter electrode may not remain constant (especially at high current densities). Consequently, a
separate reference electrode must be included in the cell. A negligible current passes between work-
ing and reference electrodes to ensure the Ere f remains constant and independent of the current passing
between the working and counter electrode. In your measurements correction of the ohmic potential
drops in solution is not necessary because the reference and working electrodes are close together and
electrolyte solutions used are of high conductance.
The electrochemical measurements will be carried out on a simple, one-electron, reversible system
3/4
Fe(CN)6 using a rotating disk electrode (RDE, radius of 2.7 mm) made of platinum, which is inert.
No side reactions of platinum need be considered. In order to produce the stable diffusion layer necessary
for steady-state currents the working electrode will be a rotating disk electrode (RDE). The reference
electrode is Ag/AgCl in saturated KCl (Ere f = 0.196 V vs. SHE) and the counter electrode is a platinum
flag electrode. The electrolyte around the counter electrode is separated from the rest of the cell by
a sintered-glass frit to minimise migration of species formed at the counter electrode into the vicinity
of the working electrode. It is necessary to ensure there is electrolyte solution around the electrode
otherwise there is no circuit and the apparatus will behave erratically.
An essential part of voltammtery is an electrical device called a potentiostat which controls and
stabilises the potential of a working electrode. For example, if a working electrode potential is set at
0.2 V relative to the reference electrode, the potentiostat maintains this potential while the cell current,
and any redox equilibrium, changes to match this potential. A potentiostat can be used to vary the
electrode potential continuously, swept, between chosen upper and lower limits at a selected rate
(V s1 ) while recording the current passing through the working electrode at each potential.
To simplify the data manipulation, the potentiostat is connected to a MacLab 2e which is basically
an analogue to digital converter (ADC), which allows the voltage and current to be converted directly
into numbers suitable for manipulation by computer. The MacLab has its own software Echem, which
can be used to both program the potentiostat and to record on a computerised chart recorder the resultant
current-voltage curve. We will be carrying out linear Sweep Voltammetry with RDE on a reversible, one

electron redox couple.
3 Pre-Lab Questions
1. What would you expect the potential (relative to the SHE) of a platinum electrode to be in
0.005M Fe[CN]64 / 0.01M Fe[CN]63 solution at very low measuring currents, if E 0 = 0.33 V
vs. Ag/AgCl?
2. What is the area of a circle of radius r cm? Hence determine the area of a Pt disk of radius 2.7 mm.
3. If a current of 1.2 mA passes through a 1k resistor, what will be the potential difference between
the two ends of the resistor?
4. Why do the solutions used in this experiment contain 1 M background electrolyte concentration?
Why are salts like KCl and KNO3 used as background electrolytes and not ZnCl2 or KI?
5. Why is nitrogen bubbled through your solutions before measurements are made?
You should determine what masses are required before you enter the laboratory.
Make up quantitatively each of the following solutions. You should know what masses are required
before entering the laboratory. You will be provided with a Brand Transferpette autopipette. For operat-
ing instructions refer to the manual in the lab.
Please note: the eChem software is also available on the Multimedia room computers using the Vir-
tual Windows environment. You can analyse your data on those computers and paste diagrams directly
into your report.
i. 25 mL of 0.30 M K3 Fe(CN)6 in milliQ water (accurately)
ii. 25 mL of 0.30 M K4 Fe(CN)6 3 H2 O in milliQ water (accurately)
iii. 250 mL of 1 M KCl (18.52 g)
To reduce the time spent dissolving the lumps, use the provided mortar and pestle to crush sufficient
material for all solutions. Place the weighed amount into a volumetric flask, fill to the mark and then
add a magnetic stirring bar. The salts dissolve readily once stirred.
Read the instructions provided with the instrument before proceeding.
Apparatus set up and equilibration
Rinse the cell, electrodes and counter electrode compartment with a small amount of solution (iii)
then fill the cell with exactly 150 g of KCl solution (do this on a balance).
Pick up the Perspex lid and insert the bubbling frit into the Perspex lid from underneath, holding
it in pace with a rubber O-ring. Carefully place the reference electrode in the slot right next to the
main hole in the centre of the Perspex lid, replace the lid on top of the cell and then move the cell
under the Pt disk and carefully lower the disk into the cell until it is immersed.
Place the counter electrode into the solution,

It will be easiest if you make sure the final slot for adding solution is at the front for easy access.
If it is not, carefully rotate the cell lid around. Carefully connect the gas tubing to the bubbling
frit, and bubble slowly for at least 15 minutes (2 bubbles per second. Consult your demonstrator
about use of the gas line).
Finally raise the frit so that it sits over the solution, and leave it on so that it provides a blanket of
inert gas over the solution.
Once the apparatus is set up, turn on all the equipment. On the computer screen you will see several
icons. Double click on Echem. Now connect the potentiostat. The green wire is the working electrode.
It is connected onto the main RDE marked disk (NOT ring). The yellow is attached to the reference,
and the red to the counter electrode.
Now you are ready to measure a CV.
Set the motor speed with the dial on the front to approximately 5 Hz. Careful, it is sensitive. From
the menu Technique select Cyclic Voltammetry.
** NOTE: A sheet of Typical data for experiments 1-6 is provided for comparison and should be
on the bench or in the drawer.
4.1 Experiment 1 - Solvent Reactions
Select the upper and lower voltages to be 1200 mV and -1200 mV. Make the start potential 0 mV,
and adjust the scan rate to 100 mVs1 . Click OK.
You will now see a chart in front of you with the current on the Y-axis and potential on the X- axis.
On the right there is a range setting. Set it to 2 mA. The potential you have set is not applied until you
actually start. Now press start and watch what happens. The screen records the current and the potential
for you, and you can see in the top right hand corner the actual values as they are measured. You should
see that the current is fairly low, but rises sharply at both ends of the scan.
Create a folder on the PRAC server with the label being the day of the experiment. Now go to the
File menu and select Save As. Select the folder on the hard drive for storage, choose the data format
as TEXT and give the curve a name, e.g. EXP1.txt. Now select the file on the bottom of the screen.
It is number 1. Choose CUT from the menu. This deletes the file.
FOR ALL DATA IT IS ESSENTIAL YOU STORE THE FILE IMMEDIATELY AFTER THE
SCAN AS A TEXT FILE AND DELETE IT FROM THE RECORD.
4.2 Experiment 2 - Oxidation of Fe(CN)64
To avoid these solvent reactions we work over a much smaller potential range. Return to CV and
reset the lower and upper potentials to be +800 mV and -400 mV. Starting at -400 mV, choose a scan
rate of 50 mVs-1 with a step width of 40 ms. Disk rotation rate should be about 5 Hz. Press start.
Record a blank as before on a current range of 500 A and ensure there is no evidence for oxygen
reduction.
DELETE THE SCAN USING CUT BEFORE CONTINUING ON
Carefully add in, using the auto pipette, 1 mL of 0.30 M Fe(CN)64 . What is the ferrocyanide ion
concentration?
Lower the bubble for 2 minutes to aid mixing, then raise it again so it just covers the headspace.
Record the CV using 500 A scale. You should see a small current at potentials more positive than
+0.1V. If not reduce the range to 200 A and rescan. Now using the cursor, measure the current at a

potential where there is no current and at a point where the current plateaus at its highest value. Write
down the concentration and maximum current in your notebooks. SAVE the scan e.g. as Expt2.1.txt.
Repeat this procedure for 4 aliquots of ferrocyanide up to a total added volume of 4 mL, saving the
curves as TEXT files after each scan. Make sure the volumes are added accurately, and do not adjust
the rotation rate or scan rate. Adjust the current range if the current goes off scale using the Range
control on the right hand side of the screen. Keep the nitrogen gas flowing over the solutions during the
measurements, but do not directly bubble the solution with gas.
4.3 Experiment 3 - The Effect of Rotation Rate
The actual limiting current depends on the convection and diffusion to the RDE. As the electrode
is rotated faster, species in solution will be drawn more quickly to the surface. The Levich equation
predicts the limiting current increases with 1/2 . We will now try to confirm this. Repeat your scans
at 50 mVs-1 , over the same potential range but varying the RDE rotation rate from about 1 Hz up to
about 12 Hz (at least 4 points). Use a 40 ms step size. After each scan, use the cursor to measure the
maximum, limiting current. Record the speed and current in your books.
BE SURE TO DELETE ALL SCANS ONCE YOU HAVE DONE THIS
4.4 Experiment 4 - Effect of Scan Rate
If we raise the potential too quickly, a proper diffusion layer cannot be formed. Instead, as we
increase the potential we get a peak in the current. To see this effect, fix the rotation rate at around 3
Hz. Now run scans at 10, 50, 100, 200 and 400 mVs-1 . After completing each scan SAVE you file (e.g.
exp4.txt) and delete the file using CUT as per the previous experiments.
4.5 Experiment 5 - The Tafel Equation
Now we want to investigate the regime where the Tafel equation applies. It predicts that the current
depends exponentially on potential. Choose Linear Sweep Voltammetry (LSV) from the menu. It
simply does half a scan, i.e. in one direction only. Set the potential limits to be +400 mV to 0 mV with
a rotation rate 5 Hz and a scan rate < 50 mVs-1 .
Why?
Use a 40 ms step width. Your current range should be around 2 mA. Adjust this to ensure you get
good resolution. You want to examine the region where the current is just rising up form zero. In LSV,
the potential is only swept in one direction.
Press START. Save your scan as EXP5.1.txt and cut to delete.
4.6 Experiment 6 - Both O and R present
So far we have worked with just the reduced form of the redox couple. Now add 4 mL of the
Fe(CN)63 solution. You now have equal amounts of oxidant and reductant in the solution. Bubble for
at least 5 minutes with nitrogen. Record the CV from 600 mV to -600 mV starting around 0 mV. Use a
slow scan rate of 50 mVs-1 and a rotation rate of 5 Hz. Record the limiting current at a high positive and
negative potentials and the potential where there is no current. SAVE the file.

5 Data Processing
The text files can all be opened directly in pro Fit and replotted or manipulated. To open a file in
pro Fit, start the program on one of the iMacs available in the computer lab, select OPEN from the
FILE menu, and then select the desired file. The first window you will see is the Text file import
options window. Make sure that without titles is selected, then press OK. You will see two columns
of numbers. To label these columns simply click on the column title (e.g. column 1) and type the new
column title (e.g. Current). To plot the data, select scatter plot from the DRAW menu, ensuring that
the New Window check-box is selected.
5.1 Experiment 1
Plot the CV out with a label showing:
Rotation state
Scan Rate
Gas present
Solution composition (1M KCl)
Label the plot with the reactions occurring at large anodic and cathodic potentials.
5.2 Experiment 2
Plot out the CVs on the same graph showing the effects of [Fe(II)] on the CV. (To put them on the
same graph, open up the required sets of data. For the first data set, select Scatter Plot form the
DRAW menu and ensure that the New Window check-box is selected. For all the subsequent
data sets to be plotted on the same graph plot the data as normal but ensure that the Plot into
current graph check-box is selected.)
Plot Max Current vs. [FE(II)].
Determine the slope of your plot.
Using equation 8, calculate a value for mo .
5.3 Experiment 3
Plot the limiting current vs. 1/2 .
Does the Levich equation work? Explain.
From the slope calculate D (m2 s1 ) using equation 7.
From mo find , the diffusion layer thickness, using equation 9. Make sure that your units corres-
pond.
5.4 Experiment 4
Plot graphs of the CVs at 3 Hz, showing the effects of increasing scan rate.
Explain the origins of the peaks.[1, 2, 3, 4, 5]
If there is no Fe(III) present, why is there a cathodic current at high scan rates?

5.5 Experiment 5
Open the data collected using LSV (Linear sweep voltammetry). We need to calculate In(Current).
Firstly, we cannot calculate the log of a negative number or zero, so scan through your values and delete
any non-positive values. Secondly, we need to create more columns in our data set. To do this, select
the Data window size button (this is the button with two double-headed arrows crossing each other
found in the top left-hand corner of the data window) and enter the desired number of columns i.e.
3. Next, to enter formulas into a column select Data Transform from the CALC menu. In the Data
Transformations window, firstly select the FORMULA check box and into the space provided enter
ln(c1). Secondly, direct the output for this formula by setting Y to column 3, then click OK. Column 3
should now contain the required values. (If the current you recorded is in mA the formula will need to
include the conversion to A. Correct the formula to ln(c1/1000)).
Plot ln i vs. E for the LSV. You should be able to see a reasonably linear portion between approx-
imately +0.1 and +0.25 V.
Go to your data and highlight the data points (current and potential) that are in your linear regime,
and re-plot this selection in a new graph.
Fit this selected data to a linear fit by selecting fit from the CALC menu and select Linear
Regression form the algorithm list. (Make sure that the Selected Rows Only check-box is
selected so as to fit the linear part of your data).
Print out your curves of current vs. potential and ln(current) vs. potential with the fit. Calculate
the transfer coefficient, , and io from equation (4). Record the values on your results sheet. Note
0 < < 1.
5.6 Experiment 6
Plot out the curve for the solution of both Fe(CN)64 and Fe(CN)63 .
Record the potential at which there is zero current. This is the Nernst potential for the ferricyanide
couple.
6 EChem Software
If you prefer, you can process your data using the EChem software (EChem 2.1.16) from home/your
computer. The link to the software is: http://www/edaq.com/software-dowloads, and the licence
code is: RWAW-U9F7-UEGJ-HU. Remember, you will need to save the data from the prac. server to a
USB stick.
7 Report
With your report you must hand in the following:
1. All relevant plots clearly labelled.
2. Answers to Pre-Lab questions should be incorporated into an appropriate section in your report.
3. Include any working involved in the calculation of your answers.

References
[1] A.J. Bard and L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, John
Wiley, New York, 1980, (a) pp. 100-108; (b) pp. 123-127; (c) pp. 22-26; (d) pp. 283-298; (e)
pp. 158-164.
[2] J. O. M. Bockris and A.K.N. Reddy, Modern Electrochemistry, Vol. 2, Plenum Press, New York,
1973, pp. 1265-1347; ch 9.3, pp.1036-1080.
[3] J. Koryta and J. Dvorak, Principles of Electrochemistry, Wiley, Chichester, England, 1987.
[4] J. Albery, Electrode Kinetics, Clarendon Press, Oxford, 1973.
[5] D. Hibbert, Introduction to Electrochemistry, MacMillan, Sydney, 1993.

Light/Matter Interactions
In the following pages the preparations for all experiments offered for the Light/Matter Interactions
The interaction with matter is fundamental to many processes in everyday life. Interactions include
the scattering of incident light, the transmission or absorption of light, the production of light follow-
ing electronic energy absorption, chemical reaction, biomolecular reactions, sonochemically produced
species etc. Light can be used to initiate chemical processes (photochemistry) or study properties of
chemical species through a range of spectroscopic methods.
In this component of the course you will investigate some of the chemical and spectral changes that
can be induced by the absorption of light.
LM1 - Photolysis of Ethanal
Dr. Alessandro Soncini

To understand the photochemical decomposition of small molecules.
To determine the rate influencing parameters for a gas-phase chain reaction.
To develop expertise in the use of high vacuum equipment.
1 Introduction
The photochemical decomposition of ethanal proceeds by a chain mechanism of overall stoichiometry:

h
CH3 CHO CH4 + CO
The photolysis is induced by radiation from a medium pressure, mercury arc lamp.
2 Using LabVIEW for Data Acquisition
Launch LabVIEW by double-clicking on the LabVIEW SE icon.
Close the Untitled window by selecting from the File menu.
Open the file Ethanal.vi by selecting from the File menu.
The file opens with a front panel window. The following control buttons will appear at the top of
the window.
The front panel window also shows a Chart (Title: Photolysis of Ethanal; X-axis: Time in
Seconds; Y-axis: Pressure in Torr.
Data acquisition is started by clicking on the button.
(When the data collection is completed, close the Ethanal.vi file without saving any modification
that you made during the data acquisition. Then quit the LabVIEW SE program.)
129
LM1 CHEM30015: Advanced Practical Chemistry
Risk assessments for the safe use of glassware under vacuum,[1] and the use, handling and clean
up of mercury[2] must be reviewed before the laboratory session. Instructions for operating a
vacuum line are provided in Appendix B. If in doubt, consult the laboratory manager. When
using the vacuum line, safety glasses must be worn.
1. Switch on all the equipment and let it warm up, adjusting the thermostat to give the required
furnace temperature.
Degas the ethanal as follows:
(a) isolate the bulb of ethanal from the vacuum system;

(b) freeze the ethanal in the bulb using a Dewar of liquid nitrogen;
(c) open the bulb to the vacuum system and pump out the air;
(d) isolate the bulb from the vacuum system and allow to thaw;
(e) repeat the procedure (b) to (d) ensuring throughout that the tap to the pumping system is
closed when the ethanal is in the liquid state.
The pressure in the reaction vessel is measured with a strain gauge that is attached to and operated
by a flexible diaphragm in contact with the gas in the reaction vessel. The readout from the strain
gauge is shown on a digital meter, which, in turn is connected to a computer.
2. With the mercury lamp on and the shutter between it and the reaction vessel open, allow the
furnace temperature to stabilise at about 90 C. Then, with the reaction vessel isolated from the
rest of the line, close the shutter and let in about 7 mmHg of ethanal. After the sample has come
to thermal equilibrium, expose the sample to the mercury arc lamp simultaneously clicking the
LabVIEW button. The reaction will be followed for 20 minutes. Time in seconds along with
corresponding pressure readings will be recorded (a total number of 1200 data points (1 point /sec)
will be acquired for each run). When the data collection is completed, a file dialogue menu will
appear on the screen to save the data. DO NOT CANCEL THIS MENU. You must choose
a file name to save your data. IF YOU MISS THIS YOU WILL LOSE YOUR DATA AND
WILL HAVE TO REPEAT THE EXPERIMENT. Save your data files.
3. Repeat the experiment with initial pressures of 7, 3 and 1 mmHg at 130-140 C.
4. Repeat with 7 mmHg pressure of ethanal (130-140 C) but with the metal gauze in front of the Hg
lamp. Measure the percentage transmission of the gauze on a UV/Vis spectrophotometer at 313
nm.
4 Mechanism
4.1 Background
Before proceeding it is advisable to consider carefully the nature of the reaction. The primary
photochemical step is:
h
CH3 CHO CH3 + CHO
followed by:

CHEM30015: Advanced Practical Chemistry LM1
CHO CO + H H + CH3 CHO CH3 CO + H2 CH3 CO CH3 + CO
All the foregoing reactions occur very quickly and together these can be regarded as the initiation of
the subsequent chain reaction. The nett effect of the above four steps is:
2 CH3 CHO + h (intensity I) H2 + 2 CO + 2 CH3 (1)
Small amounts of H2 and CO are produced in this initial step but these amounts are negligible
compared to the amounts produced in the subsequent chain reaction. (NOTE: It may be assumed that
at the pressures used here, reaction (1) is dependent on light intensity only).
In the ensuing chain reaction the chain carrier is the CH3 radical:
k
CH3 + CH3 CHO
2
CH4 + CH3 CO (2)
k3
CH3 + CO
CH3 CO (3)
k
2 CH3
4
C2 H6 (chain termination) (4)
The nett effect of (2) and (3) is the conversion of one mole of CH3 CHO to one mole of both CH4
and CO and the regeneration of the CH3 .
Question a) Consider the reactions (1) to (4) and apply a steady-state approximation to the CH3 and
CH3 CO radical concentrations and determine the rate expression.
Question b) Also show that the pressure of CH3 CHO at any time is equivalent to 2P0 - PT where PT
is the experimentally measured total pressure.
As the temperature is raised the rate constant for reaction 2 will increase while the rate constants of
3 and 4 will stay approximately constant. (These latter two reactions have very low activation energies).
Thus the chain length increases at higher temperature.
The experimental results should enable you to confirm the predictions of your steady-state analysis.
In the first instance (i.e. from Question a) you should be able to confirm the apparent order of the reaction
with respect to ethanal and then determine the composite rate constant.
4.2 Data Analysis
4.2.1 Using pro Fit for Data Analysis
Open the program pro Fit by double clicking on the icon.
Open the data file (LabVIEW data file) by selecting Open from the FILE menu.
Column 1 is the x-axis data (Time, sec) and Column 2 is the y-axis-data (Pressure, Torr). Label
appropriately.
Create 1 more column by clicking the Data window size button (this is the button with two
double-headed arrows crossing each other in the top left-hand corner of the data window) and
entering a value of 3 into the Number of columns.
Using the Formula Entry method, the y-axis values can be converted as required using appro-
priate formula. (If you are not familiar with the procedure of formula entry, consult with your
demonstrator.)

4.2.2 Calculations
1. Assuming the reaction to be first order with respect to ethanal, plot the appropriate integrated form
of the rate equation, using data from the pressure-time curve, and determine an accurate value of
the observed rate constant for each separate run.
The following steps may be used to determine the apparent rate constant value:
(i) Double-click on the column number of Column 3. In the Column format window, select
NEW calculation and enter the formula, ln((2P0 )-C2) in the space provided. Then press OK.
(Column 2 = Total pressure; P0 =initial pressure; Note that you have to enter the actual (2P0 )
value in the formula - eg., if the initial pressure is 3 Torr, then the formula to be entered is
ln(6-C2) ).
(ii) Plot ln(2P0 - Pt ) vs Time.
(iii) Fit this plot to a straight line. Slope = apparent rate constant.
[HINT: If a number of points do not lie along the linear fit remove these points and re-plot
before taking the slope and obtaining the rate constant.]
Question c) Does the rate constant vary with the initial ethanal pressure, P0 ? Does this correlate
with your rate expression derived in Questions a?
[Hint: The rate constant of reaction (4) would be expected to increase with increasing total
pressure (third body effect).]
2. For the experiment at lower light intensity, assume the order with respect to ethanal is unchanged
and examine the variation in apparent rate constant. From the equation:
dP
rate = = kI m [ethanal]
dt
where I = incident light intensity and k is independent of I, it follows that:
kobs = kI m
Question d) Using values of kobs at the two light intensities, deduce the value of m. How does
this compare to the theoretically derived value of m, obtained in Question a? Why may there be a
difference?
3. From the two experiments with P0 = 7 mmHg at different temperatures, determine the apparent
activation energy.
Question e) How does this value compare with a literature value (e.g. Atkins)?
In your report tabulate the rate constants for all runs, stating units, temperature and pressure
conditions.
5 Questions and Derivations
In addition to the tabulated results, ensure that your report includes the following:
Introductory questions:
Question a) Using equations (1)-(4), determine the rate expression for the decomposition of ethanal,
d[CH3 CHO]
i.e., dt .
Question b) Show why the pressure of CH3 CHO at any time is equal to 2P0 - PT , where P0 is the
initial pressure of ethanal and PT is the total pressure experimentally measured.

Comparative Questions:
Question c) Comment on the variation in kobs with P0 and comment on any trend observed. Explain.
Question d) Compare your experimentally determined value of m with your theoretically derived
value from (a) above, and comment on any difference between the two.
Question e) Comment on the magnitude of the activation energy obtained in relation to a literature
value.
Additional Hints:
Please write the detailed report in accordance to the Report Writing Instructions given in this manual,
and follwing the report template provided on LMS.
Note: A results sheet is provided (see LMS) which should be treated as a guide as to the information
that should be included in the Report
References
[1] http://safety.chemistry.unimelb.edu.au/pdf/Use of Glassware Under Vacuum RA v2.1.pdf
[2] http://safety.chemistry.unimelb.edu.au/pdf/Use, handling & clean up of mercury v1.2.pdf
[3] Atkins, P.W., Physical Chemistry, (6th ed.), OUP, Ch. 25 and 26.
(Information in Atkins treats the decomposition of ethanal by pyrolysis rather than by light. Keep
this in mind when consulting the appropriate data).

LM2 - Fluorescence Polarisation
Measurements of Molecular Motion
A/Prof. Trevor Smith

To learn about fluorescence polarisation and its applications.
To learn about the dynamics of molecular motion, particularly of a biomolecule.
To use the Perrin Equation to determine the limiting anisotropy of a fluorescent probe, and the
molar volume and rotational correlation time of a dye-polymer conjugate.
To learn how to use fluorescence instrumentation.
To learn about data acquisition and analysis.
1 Introduction
The emission of light (e.g. fluorescence) following the absorption of visible or ultraviolet radiation
provides a very sensitive method of monitoring a variety of photophysical phenomena. The study of the
polarised nature of light provides additional information in such studies.
Figure 1: Electric and magnetic fields of plane-polarised light.
If light of a particular electric vector orientation (plane polarised light, Figure 1) impinges on
a sample, only those molecules that are oriented such that a component of their absorption transition
moment lies parallel to this electric vector can absorb the light. Specifically, the probability of the
absorption of light is proportional to the cosine squared of the angle between the plane of polarisation
of the exciting light and the absorption transition dipole (cos2 ). Hence, when we excite an ensemble of
randomly oriented fluorophores with plane-polarised light we are performing a photo-selection process,
135
creating a population of excited molecules which nominally have their excited dipoles lined up with the
polarisation direction of the excitation (Figure 2).
Figure 2: Photo-selection.
The term fluorescence anisotropy is used to characterise the degree of depolarisation of fluores-
cence following photo-selection when the incident light is linearly polarised. It is defined as:
Ik I
r= (1)
Ik + 2I
where Ik and I are the intensities measured with a linear emission polariser set to analyse emission
components parallel and perpendicular, respectively, to the electric vector of linearly polarised incident
light. The excitation light is usually polarised vertically relative to the sample frame of reference, and
the emission is therefore analysed for its components in the vertical and horizontal planes. The degree
of polarisation of fluorescence can therefore be calculated by measuring the emission intensity under
two polarisation conditions with Ik corresponding to the excitation/emission polariser combination, IVV
and I corresponding to IV H (V: vertical; H: horizontal; the first subscript corresponds to the orientation
of the excitation polariser and the second subscript to the emission polariser). The quantity Ik + 2I is
proportional to the total fluorescence intensity I, and is therefore related to the fluorescence quantum
yield.
The term fundamental emission anisotropy describes the situation in which no depolarising events
occur subsequent to the initial formation of the emitting state, such as those caused by rotational diffu-
sion or electronic energy transfer. It also assumes that there is no overlap between differently polarised
transitions. The (theoretical) value of the fundamental emission anisotropy depends on the angle, ,
between the absorption and emission transition moments in the following way:
3hcos2 i 1
r0 = (2)
2
where hi denotes an average over the orientations of the photoselected molecules. r0 can take on
values ranging from -0.2 for = 90 (perpendicular transition moments) to 0.4 for = 0 (parallel
transition moments).
However, if molecular rotation occurs during the lifetime of the excited state, then further depolarisa-
tion of the emission will result. Polarisation measurements can therefore be used to determine properties
of the rotating molecule such as its size and hence its rotational dynamics.
r can be determined with a spectrofluorimeter equipped with polarisers. It is not sufficient to keep the
excitation polariser vertical and to rotate the emission polariser because the transmission efficiency of a
monochromator depends on the polarisation of the light. The determination of the emission anisotropy
requires four intensity measurements: IVV , IV H , IHV , IHH . For horizontally polarised exciting light,

the vertical component (IHV ) and the horizontal component (IHH ) of the fluorescence detected through
the emission monochromator are different, although these components are identical before entering the
monochromator. The so-called G factor, G = IHV /IHH , measured through the monochromator, is
different from unity and can be used as a correcting factor for the ratio Ik /I because it represents the
ratio of the sensitivities of the detection system for vertically and horizontally polarised light:
IVV Ik
=G (3)
IV H I
The emission anisotropy is thus given by:
IVV G IV H
r= (4)
IVV + 2G IV H
For the sake of simplicity, this relation is often written as:
Ik G I
r= (5)
Ik + 2G I
The Perrin equation [4, 5] can be written as:
1 1 f 1 3 f
= 1+ = 1+ (6)
r r0 rot r0
where ro is the limiting emission anisotropy (i.e. the anisotropy in the absence of fluorophore ro-
tation) and f and rot are the fluorescence lifetime and rotational correlation time of the fluorophore,
respectively. The rotational correlation time is the time taken for the initial anisotropy to fall to 1/e of
its original value due to molecular rotation. The alternative expression substitutes the Debye rotational
relaxation time for the rotational correlation time ( = 3rot ) which is the time for a given orientation to
rotate through an angle given by the arccos e1 (68.42 ).
Experimental determination of the anisotropy can lead to an estimation of rot , thereby providing
information on the rotational mobility of the emitter. For a protein with an attached fluorophore, the
value of rot can reflect the ease of rotation either for the macromolecule as a whole or for an internal
rotation of its parts.
The rotational correlation time can be determined directly from time-resolved fluorescence meas-
urements or from the Stokes-Einstein equation:
V
rot = (7)
RT
where V is the volume of the rotating dye-polymer conjugate assuming its shape to be spherical, is the
viscosity of the solution, R is the ideal gas constant, and T is the absolute temperature.
From equations 6 and 7, the Perrin equation can be rewritten as:
1 1 f RT
= 1+ (8)
r r0 V
Serum albumins are blood serum proteins of considerable interest since they rapidly form a covering
layer on synthetic polymers and play a supporting role in blood clotting. In this experiment you will use
fluorescence anisotropy measurements and the Perrin equation to determine information relating to the
rotational motion of a protein macromolecule, bovine serum albumin, (BSA).
This will be achieved using a non-covalently bound fluorescent probe, 8-anilino-1-naphthalene sulf-
onic acid (ANS, Figure 3). ANS is a fluorescent probe whose fluorescence decay kinetics depend on the

probes microenvironment such that, in a polar microenvironment, the fluorescence is rapidly and effi-
ciently quenched. ANS is an extraordinarily sensitive fluorescent probe for determining the microscopic
viscosity and polarity of chemical and biological systems. It is well known that under aqueous condi-
tions, ANS is virtually non-fluorescent (the quantum yield, f = 0.004) due to an efficient fluorescence
quenching mechanism.[6] A decrease in polarity of the ANS environment leads to both an increase in
the fluorescence lifetime (and a concomitant increase in fluorescence quantum yield, f = 0.98) and a
slightly blue shifted fluorescence maximum.[6] ANS associates with BSA non-covalently by partition-
ing into hydrophobic pockets within the proteins tertiary structure. You will determine the limiting
anisotropy of ANS, the molar volume of the protein and the rotational correlation time for ANS-labelled
BSA as a function of temperature.
Figure 3: 8-anilino-1-naphthalene sulfonic acid (ANS).
1. What is meant by photo-selection in fluorescence?
2. What affect would you expect the viscosity of the solution to have on the fluorescence anisotropy?
3. Describe, in terms of what the detector sees, how IHH and IHV can give the G-factor
4. What would you expect the effect on these measurements to be if the concentration of ANS is too
high or too low?
3 Apparatus
In this exercise you will use a steady-state fluorescence polarisation spectrometer and (possibly) a
time-resolved fluorescence instrument. In each case the sample is contained in a standard 1 cm path
length fluorescence cuvette. These are delicate and should be handled with great care. Keep fingers off
the faces of the cuvette.
The steady-state instrument is based on a Hitachi 4500 spectrofluorimeter equipped with excitation
and emission polarisers. The polarisers are not easily cleaned and so do not touch the polarising
material with fingers. The excitation lamp of the fluorimeter produces unpolarised light over a broad
wavelength range. The desired excitation wavelength is chosen by the excitation monchromator and an
excitation polariser must be used to transmit plane-polarised light to photo-excite the sample. For most
measurements vertically polarised excitation light will be used. The emission is collected at right angles
to the excitation beam with the desired emission wavelength selected by the emission monochromator.
The polarisation of the emission is analysed by the setting of the emission polariser. All four excita-
tion/emission polarisation combinations, IVV , IV H , IHV and IHH must be determined at one temperature,
and subsequently only IVV and IV H , need to be collected.
The technique that you may use to record the time-resolved fluorescence behaviour is called time-
correlated single photon counting (TCSPC) [2, 3, 7] or single photon timing. As the name suggests,
this is an extremely sensitive method whereby a histogram is generated corresponding to the times

between when a short light pulse excites the fluorophore and a single photon of emission is detected.
The sample is excited with short pulses of light at high repetition rate, and the detected emission is
reduced to the level that the probability of detecting a single photon of emission per excitation pulse is
less than unity.
A separate description on this instrument and operating instructions on its use will be provided
if it is necessary for you to use this photon counting apparatus.
4.1 Preparation of ANS/BSA solutions
Use only Milli-Q water for the preparation of the following solutions. Prepare a dilute solution of
the protein ( 0.4 g L-1 BSA). A solution of 8.0 106 M ANS is already prepared for you. Prepare
a mixture of 0.4 g L-1 BSA in the presence of 8.0 106 M ANS. Assume that the viscosity of
this solution is the same as that of water. The ANS is non-fluorescent in water due to an efficient
emission quenching process, however, upon non-covalent attachment to BSA on mixing, it becomes
highly fluorescent through incorporation into hydrophobic pockets within the protein structure. Thus
any unbound ANS should not interfere with the fluorescence anisotropy measurements.
4.2 Unpolarised Fluorescence Spectra of Solutions
1. Remove the excitation and emission polarisers from the Hitachi F-4500 fluorimeter.
2. Set the excitation wavelength to 370 nm.
3. Record the fluorescence spectra of the water, then the ANS, BSA and ANS-BSA complex solu-
tions between 400-600 nm. Comment on these spectra. See the notes that have been provided on
a separate sheet of paper kept adjacent to the instrument, which will guide you through the pro-
cedure for recording the spectrum. If you encounter any difficulties, see the demonstrator. Ensure
that there is sufficient emission intensity from ANS-BSA complex solution before continuing.
4.3 Polarised Fluorescence Spectra of Solutions
1. Install the excitation and emission polarisers in the appropriate holders in the Hitachi F-4500
fluorimeter.
2. Set the temperature controller to 20 C and allow sufficient time for thermal equilibration of the
BSA-ANS solution. This will require at least 5 minutes after each temperature change.
3. Set the excitation polariser to horizontal and record the two spectra corresponding to IHV and
IHH by changing the emission polariser. These combinations, IHV and IHH , need only be collected
once, and will be used to calculate the correction term, G.
4. Save these and subsequent spectra as text files under appropriate filenames.
5. Set both the excitation and the emission polarisers to vertical.
6. Record the emission spectrum of the BSA-ANS solution between 400-600 nm. This will corres-
pond to IVV .
7. Set the emission polariser to horizontal and repeat the measurement of (4). This will correspond
to IVH .

8. Increase the bath temperature by 5 C and allow sufficient time for the solution to thermally
reequilibrate.
9. Repeat steps 5-8 at this temperature, and at further 5 C intervals up to 60 C.
5 Calculations and Questions
All calculations and raw data manipulations should be carried out by transferring the saved data files
of your measured emission-time curves into a spreadsheet program such as pro Fit or MS Excel.
1. Load the files containing the spectrum data for IHV and IHH into the analysis program. You should
manipulate the data so you have three columns; one for wavelength, one for the emission intensity
IHV and one for IHH .
2. Calculate the G-factor as the correction term (G = IIHH

HV
) to be used in subsequent calculations.
Calculate G across the emission range recorded as a new column of data.
3. Load the files containing the spectral data for IVV , IVH , recorded at 20 C into the analysis program.
You should again manipulate the data so you have two additional columns; one for the emission
intensity IVV and one for IVH corresponding to the same wavelength values as for the G-factor.
4. Create a new column of data containing the anisotropy values for each wavelength (Equation 4)
including the G-factor.
5. Calculate the average anisotropy value from several data points around the peak in the emission
band.
6. Repeat the procedure of steps 3-5 for the other temperatures. Use the same G-factor data in each
case.
7. Create a new data file containing the temperature (in Kelvin) and corresponding average aniso-
tropy values.
8. Determine the values of the viscosity of water at the different temperatures used.[8]
9. Calculate 1/r and T/ for each temperature and tabulate your data.
10. Plot the 1/r values versus T/ (N.B. use the correct units for the viscosity term).
11. Determine the value of the limiting anisotropy, r0 , from the intercept of the graph.
12. Calculate the value of the molar volume, V, of the dye-polymer conjugate from the slope of
the graph, using the fluorescence decay time determined by you experimentally (or provided by
A/Prof. Smith).
13. Use Equation 2 to calculate the angle between the absorption and emission transition moments for
ANS.
14. Calculate the volume of one molecule of the conjugate.
15. Calculate the value for rot , assuming the protein adopts a spherical shape.
16. Compare the value you have determined for the molar volume with the value determined from X-
ray scattering experiments.[9] Discuss possible reasons for any discrepancies between the values
obtained from the two methods.

17. Comment on the observed temperature dependence on the rotational correlation time calculated
using the steady-state data.
18. Comment on any observed (or expected) temperature dependence on the fluorescence decay time.
19. What other phenomena would be expected to alter the rotational motion of macromolecules in
solution?
In your report you will need to show the plot of the 1/r values versus T/ and a pair of typical IVH and
IVV spectra. Also include a plot of the G-factor vs wavelength. Include a table of all relevant data. You
should also make some attempt at determining the error associated with the values you have calculated.
References
[1] Bigger SW, Craig RA, Ghiggino KP, Scheirs J, Fluorescence Anisotropy Measurements in Under-
graduate Teaching. J. Chem. Ed., 1993. 70: p. A234-A239 (N.B. there is a slight discrepancy in the
definition of the Perrin equation in this reference)
[2] Lakowicz JR (1999) Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Pub-

lishers, New York
[3] Valeur B (2002) Molecular Fluorescence. Wiley-VCH, Brisbane
[4] Perrin FJ, J. Phys., 1926. 7: p. 390-401
[5] Perrin FJ, Phys. Radium, 1936. VII.7: p. 1-11
[6] Hlady V, Andrade JD, Fluorescence Emission from Adsorbed Bovine Serum Albumin and
Albumin-bound 1-Anilinonaphthalene-8-sulfonate Studied by TIRF. Coll. & Surf., 1988. 32: p.
359-369
[7] OConnor DV, Phillips D (1983) Time-Correlated Single Photon Counting. Academic Press
[8] Weast RC, CRC Handbook of Chemistry and Physics. CRC Press, Boca Raton, USA
[9] McClure RJ, Craven B, J. Mol. Biol., 1974. 83: p. 551-555

LM3 - Principles of Fluorimetry
Prof. Ken Ghiggino

To learn about the fluorescent behaviour of a chemical species.
To understand the Stern-Volmer equation.
To learn about fluorescence quenching.
To obtain fluorescence data for several complexes.
1 Introduction
The fluorescence emission spectrum and fluorescence quantum yield are characteristics of the excited
singlet state(s) of a chemical species. Fluorescence measurements also provide information about the
photochemistry of molecules. In practice, the fluorescence emission spectra can be obtained from many
commercial spectrofluorimeters [1]. Fluorescence quenching refers to any process that decreases the
intensity of fluorescence and occurs as a result of the interaction of the fluorophore with another species.
In this experiment, you will use a commercial spectrofluorimeter to obtain fluorescence emission
spectra of:
(i) quinine bisulfate (QBS), a fluorescence standard, and
(ii) Leucophor PAF, a commercial optical brightener [2] whose structure appears in Figure 1.
Figure 1: Structure of Leucophor PAF.
You will also investigate the phenomenon of fluorescence quenching and the applicability of the
Stern-Volmer equation by:
143
(i) measuring the fluorescence of quinine bisulfate (QBS) as a function of electrolyte concentration,
and
(ii) determining the the applicability of the Stern Volmer equation for describing your experimental
data.
2 Prelab Questions and Exercises
Your answers to the following questions are designed to help you prepare for the prac. and to give
you some guidance as to what should be included in your report. You should incorporate your answers
to these questions into your report. Do not hand them in as an attachment.
1. What is the definition of absorbance (in terms of transmitted and incident light intensities)?
2. What is the difference between a fluorescence emission spectrum and a fluorescence excitation
spectrum [4]?
3. Show that the ratio of intensities of light absorbed by two solutions is given by the expression:
I1abs q1 (1 10A1 )
= =
I2abs q2 (1 10A2 )
where Iabs is the intensity of light absorbed, q is the number of photons absorbed by each solution
and A is the absorbance of the solution.
4. What are the units for a second-order rate constant?
5. What features of the absorption spectrum of a substance would help you choose an appropriate
excitation wavelength for recording its fluorescence emission spectrum?
6. What is meant by the term collisional quenching?
3 Theory
3.1 Fluorescence Quantum Yield
The fluorescence quantum yield[4], f , of a molecule is defined as the number of fluorescence

photons emitted per photon absorbed, i.e.:
qem
f = (1)
qabs
You can determine the relative fluorescence quantum yields of two solutions experimentally using
[4]:
f1 (1 10A2 )n21 1
= (2)
f2 (1 10A1 )n22 2
where A is the absorbance of the solution measured at the excitation wavelength, n is the refractive
index, and is the area under the fluorescence emission spectrum.
The factors in parentheses take account of the fraction of light absorbed by each solution, so that the
terms correspond to the qem terms in equation (1) and the (1-10A ) terms to the qabs terms. This equa-
tion is important because, if you know the absolute fluorescence quantum yield of one of the compounds,
the other can easily be calculated.

3.2 Fluorescence Quenching
The term fluorescence quenching refers to the observed decrease in the fluorescence from a molecule
caused by interaction with another species. Collisional quenching occurs when collisions between the
fluorescence molecule and another species deactivate the singlet excited state from which fluorescence
occurs. This process can be quantitatively modelled by the Stern-Volmer equation, which can be derived
from kinetic equations[1] for the quenching process. This equation relates the decrease in fluorescence
quantum yield to the concentration of the quencher:
0f
= 1 + kq 0 [Q] (3)
f
where 0f and f are the fluorescence quantum yields in the absence and presence of quencher re-
spectively, kq is the second-order rate constant for the quenching process, 0 is the fluorescence lifetime
of the fluorescent species in the absence of quencher, and [Q] is the molar concentration of the quencher.
0
A plot of ff versus [Q] will be a straight line with a slope equal to kq 0 . If the value of 0 is known,
then the value of kq can be calculated. This means that you can obtain the rate constant for the quenching
process from steady-state fluorescence measurements and a Stern-Volmer plot without having to make
any time-dependent measurements.
q
The ratio q0,em
em
is often assumed to be equal to II0 , where I0 and I are the maximum intensities of fluor-
escence emission in the absence and presence of quencher respectively. This approximation assumes
that the area, , under a fluorescence emission spectrum is proportional to the maximum fluorescence
intensity, I.
0 q .q
Since ff = q0,em abs
0,abs .qem
, then combining the approximation for the emission terms with the ratio of
absorbance terms (see Question 1) yields:
0f (1 10A )I0 I00

= = 0 (4)
f (1 10A0 )I I
where I is the corrected maximum fluorescence intensity:

I
I0 = (5)
(1 10A )
4 Apparatus
You will be using a UV-Visible absorption spectrophotometer (Shimadzu or Varian) to record the
absorption spectra and a fluorimeter (Hitachi or Varian) to record the fluorescence spectra of your solu-
tions. See notes that have been provided on a separate sheet of paper kept adjacent to the respective
instrument, which will guide you through the procedure for recording the spectrum. If you encounter
any difficulties, see the demonstrator. You will also be provided with a Brand Transferpette autopipette
to assist you in preparing your samples. For operating instructions see manual in the teaching laboratory.
5.1 Fluorescence quantum yield of Leucophor PAF
NOTE: As far as practicable, keep solutions of Leucophor PAF shielded from light to avoid
degradation due to photo-isomerisation.

1. To a 10 cm3 volumetric flask add 4 cm3 of the stock solution of QBS in 2 M H2 SO4 and add to
it an equal quantity of distilled water. Fill the fluorescence cell with this solution and measure its
absorption spectrum using distilled water as the reference. (The absorbance above 350 nm should
be approximately 0.5 - dilute if necessary). Record this value.
2. Measure the absorption spectrum of the concentrated Leucophor PAF solution that is supplied.
Place an appropriate quantity of this solution in a 10 cm3 volumetric flask and adjust its concen-
tration with distilled water until its absorbance at the excitation wavelength to be used (for both
solutions) is close to 0.5. Record this optical density and the wavelength you have chosen to excite
your sample for the fluorescence measurements.
3. Set the fluorimeter excitation wavelength to this wavelength.
4. Measure the fluorescence emission spectrum of your solvent. This is a blank measurement
that you will subtract from your raw spectra to eliminate spectral noise. If the solvent exhibits a
significant amount of fluorescence, an alternative source of clean solvent should be used.
5. Place the QBS solution in the cell compartment of the fluorimeter.
6. Record the fluorescence emission spectrum over the wavelength range 380 to 600 nm. If the
spectrum is worthy of analysis, save it as a .txt file using a suitable file name. If not, adjust the
instrument settings to obtain a suitable spectrum.
7. Record the fluorescence emission spectrum of the adjusted Leucophor PAF solution with the same
excitation wavelength as that used for QBS, and the same instrument settings (slit width, PMT
voltage etc.)
8. Record fluorescence excitation spectra of both solutions. Comment on the comparison of the
excitation spectra with the corresponding absorption spectra.
9. If using the Varian Eclipse collect emission spectra twice: once with correction turned off and
once with correction turned on. What is the effect of correcting emission spectra?
10. Save all your spectra in data files for the data analysis outlined in Section 6.
5.2 Fluorescence quenching: Stern-Volmer plot
1. To each of four 10 cm3 volumetric flasks add 4.0 cm3 of the QBS stock solution. Add to the flasks
0.5, 1.5, 2.5 and 4.0 cm3 of the 0.10 M NaCl stock solution. Use the digital pipette to make up
each flask to a total volume of 8 cm3 with distilled water.
2. For each solution:
(i) measure accurately its absorbance at the excitation wavelength you chose, and
(ii) record its fluorescence emission spectrum over the wavelength range 380 to 600 nm.
(iii) save each of your spectra in data files for analysis in Section 6.
6 Calculations and Questions
All calculations and data manipulations should be carried out by transporting the saved data files of
your measured spectra into an appropriate program (Excel, pro Fit, etc.).
1. Plot the spectra of (i) the QBS standard (f = 0.546) and (ii) the Leucophor PAF and determine the
area under each spectrum. You can then calculate a value for the quantum yield of fluorescence
of Leucophor PAF using equation 2.

2. Print, on the same axes, the spectrum of the QBS solution prepared in Section 5.1 together with
the blank spectrum recorded in Section 5.1 and the four spectra recorded in Section 5.2.
3. Using the data from Section 5.2, plot I0 /I v. [NaCl]. Calculate a value of kq , the rate constant
for the quenching process (assuming collisional quenching), given that the excited-state lifetime
of QBS at 20 C is 20.68 ns.[7]
Issues you should address when writing up your report are:
1. Fluorescent whitening agents are commonly used to whiten textiles and paper. With reference to
your experimental results (absorption and emission spectra for Leucophor PAF), describe briefly
how this is achieved.[6]
2. Comparison of the value of kq determined in (3) above with the rate constant kdiff , for diffusion-
controlled collisions using the following form of the Debye-Smoluchowski equation:
8000RT
kdi f f = 3 .
[In this equation, R is the ideal gas constant (J mol-1 K-1 ), T is the absolute temperature (K) and
is the viscosity of the medium (kg m-1 s-1 );[8] the constant includes a factor of 1000 so that kdiff
is in units of dm3 mol-1 s-1 .] You should address any assumptions made in your calculation and
how this affects the value of kdiff .
3. Comment on any differences between kq determined in (3) with the rate constant kdiff and what
this implies about the process of quenching experienced by QBS.
7 Report
You should discuss what is expected for your report with Dr. Yuning Hong before commencing your
write up. Your report should take the form of a scientific paper and contain an abstract, introduction,
methods section, results, discussion and conclusions. It should be properly referenced. All figures/plots
should contain captions. Graphs should be properly scaled and axes should be properly labelled. Use of
proper units and error limits is also important. Your calculations should be included in the appropriate
section. You should incorporate the answers to the prelab questions into your report.
Show plots of the spectra recorded using the most concise way. The logic of presentation, data
interpretation and setting-out of report / ease of reading are also considered when marking this report.
References
[1] C. A. Parker, Photoluminescence of Solutions, Elsevier, 1968, pp.252-258.

[2] K. J. Smit and K. P. Ghiggino, Dyes and Pigments, 1987, 8, 83.
[3] any appropriate 1st year resource
[4] 3rd year Photochemistry Lecture Notes (see LMS)
[5] See e.g. R. Livingston, Physico Chemical Experiments, MacMillan, New York, 1943, pp.43-44;
P.R. Bevington, Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New
York, 1969, pp.265-270; I. Estermann, ed., Methods of Experimental Physics, Vol. I, Academic,
New York, 1959, pp.58-59
[6] R.P. Wayne, Photochemistry, Butterworths, London, 1970, pp.251-3 or any other photochemistry
book.

[7] S.R. Meech and D. Phillips, J. Photochem., 1983, 23, 193.
[8] CRC Handbook of Chemistry and Physics, 69th ed., R.C. Weast ed., CRC Press, Boca Raton,
1988, p. F-40 (use conversion tables on pp. F-38-39).

Computational Chemistry
In the following pages the procedure to follow for the Computational Chemistry component of the
course is included.
In this component of the course you will investigate some of the methods used in determining chem-
ical information using computational methods.
This is a compulsory exercise. It can be done in the Multimedia Room in the assigned session/s with
demonstrator assistance, or it can be done independently on any computer at your leisure.
CC - Computational Chemistry
Dr. Lars Goerigk

BEFORE YOU BEGIN:
You will need your WEBMO login data from CHEM20019 to carry out this experiment.
You may want to re-familiarise yourself with the topic Computational Chemistry (see CHEM20019
lectures and related experiment).
Please bring a calculator to the lab sessions.
Please read these instructions carefully to avoid unnecessary mistakes.
This experiment is assessed through a report. You can find a template and the marking scheme on
the LMS. Please also read the appendix for more information on how to write this report.
If you need to re-access WEBMO from off-campus to finish your report, you have to establish a
VPN connection first (see UOM IT website on how to do this).
Aim
This experiment develops the concepts and methods introduced in CHEM20019. By the end of
this experiment you should have an enhanced appreciation of the benefits and power of Computational
Chemistry.
Introduction
Many molecular properties are not directly measureable but can be determined by solving the
Schrodinger equation. Generally, this cannot be done exactly for molecules, and resort must be made to
computer-based methods, usually involving approximating the molecular wavefunction through molecu-
lar orbitals (MOs). These MOs are mathematically described by a linear combination (a sum) of atomic
orbitals (AOs). These AOs are also called basis functions and a collection of AOs is called basis set.
The most appropriate combination of basis functions is the one that minimises the energy of the mo-
lecule (Variational Method). The more basis functions used to approximate the molecular wavefunction,
the more accurate the result, but only at the expense of increased computing time. In this exercise a
balance is struck; small to medium sized basis sets are used in most calculations for reasonable results.
All calculations in this experiment are performed using the Gaussian09 computer package accessed
through a web interface called WebMO. WebMO connects with Gaussian09 in a user-friendly manner
making it simple to perform complex calculations.
In this experiment you will:
Revise the construction of molecules in WebMO/Gaussian09.
151
CC CHEM30015: Advanced Practical Chemistry
Compare structural and energetic differences between different forms of a molecule.
Learn how to create a potential energy curve along a reaction coordinate and develop a greater
understanding of transition states and reaction barriers.
Discover how to calculate partial charges and molecular orbitals, and appreciate how these are
connected with familiar chemical notions (electrophilic substitution and effect of -electrons).
Determine the relative reactivity of several Diels-Alder systems.
Compare the relative shifts in 13 C NMR spectra of several similar molecules.
1 Optimisation of Ethylene and Fluoroethylene
Gaussian 03 in conjunction with WebMO will be used to determine the preferred states and shapes of
molecules (minimum energy structures). In this exercise, you will examine a series of simple molecules
and see how bond lengths, angles, and relative energies develop and change as ethylene is modified
through substitution of hydrogen atoms by fluorine atoms.
1.1 Ethylene
1. Open a web-browser (e.g. Firefox, Safari).
2. Type in the URL: http://webmo.chemistry.unimelb.edu.au/
3. Click the Login to WebMO to get to the login screen.
4. Enter your user name and password and click return.
5. You should be into the WebMO system and see a screen like this:
6. Click on the New Job menu and Create New Job. You should get a drawing palette that can be
used to create the molecule you wish to study.
7. On the left side of the drawing palette is a series of tools. As you pass the mouse over the tools an
explanation for each should appear.

CHEM30015: Advanced Practical Chemistry CC
8. To create ethylene, select the Periodic Table Tool from the side menu and then click on
carbon. Now you can click anywhere in the palette window to create a carbon atom. Try this.
Then create another carbon atom nearby, dragging the mouse back to the first carbon to form a
single bond. If you make a mistake, you can always Undo from the Edit menu. Below is a picture
showing the C-C bond if this is done correctly.
9. The ethylene backbone requires a C=C bond, so click on one carbon and drag the mouse as if
you were forming another bond, and let go when you reach the other carbon. This should form a
double bond. Triple bonds are formed in a similar manner.
10. Now click the Comprehensive Cleanup button resembling a brush from the side menu. This
button cleans up the molecule, adding hydrogens, charges and changing bond angles and lengths
to approximately appropriate values. This process is sometimes erratic and shouldnt always be
trusted for complex molecules, but for simple ones such as ethylene it works fine. You have now
created ethylene! Click the bottom right arrow to continue to the next screen.
11. The next screen should resemble this:

12. Select Gaussian and click on the arrow again to continue onwards to the Configure Gaussian Job
Options screen. This Choose Computational Engine screen may not always appear however, in
which case you should be taken straight to step 13.
13. In the Configure Gaussian Job Options screen, you will be presented with the formula of your
created molecule (C2 H4 ) and a series of drop down menus.
14. The Calculation menu allows you to choose the type of computation. Choose Optimize+Vib
Freq. This will result in determination of equilibrium bond-lengths and vibrational frequencies.
15. The Theory menu allows you to choose the method. You can leave this as Hartree-Fock for the
time being.
16. The Basis Set menu gives you a choice between small, medium and large basis sets. Large basis
sets give more accurate results but slower calculations. Set this to 6-31G(d), a modest basis set.
17. The Charge box lets you set the molecular charge. You want 0. If you were calculating a cation
you would enter a positive number and if you were calculating an anion you would enter a negative
one.
18. The Multiplicity box allows you to specify the multiplicity (related to the spin-pairing in the
molecule). You want Singlet.
19. Your screen should resemble the following image:
20. Click on the rightmost arrow to initiate the calculation. Progress of the job can be monitored and
controlled in the WebMO Job Manager. Once it is completed (after 12 s), click on either the
job name or magnifying glass on the far right of the screen to view the results.
21. The View Job screen should show the optimised structure of the molecule as well as calculated
energies and frequencies (scroll down). You can also find bond lengths and angles using the Select
tool (the one resembling a mouse cursor ). After clicking this icon, you can select a single atom

in the molecule by left clicking. To select multiple atoms, hold down shift and click. If 2 adjacent
atoms are selected, you will see at the bottom of the molecule viewer the bond length between
those atoms. If you select 3 atoms, you get the bond angle, whereas 4 atoms gives the dihedral
angle. The order you select atoms is important for these last two angle measurements.
22. Now, open up your worksheet and paste an image of your molecule into it. This can easily be
done by clicking the Print icon in the left-hand side menu, right clicking on the image that
pops up, selecting Copy Image, and then pasting the image into the area provided. Resizing the
image is recommended.
23. Next, fill out the table in your worksheet. All the required information is available on the View
Job page. As we are looking at ethylene, in the case of C-R bond length, enter the C-H length,
and for the C=C-R angle, determine any of the C=C-H angles. To find the angle, make sure you
select the C atom furthest from the H atom first, then the other C atom and then the H atom. Note
that angles in your report must be rounded to one decimal place and bond lengths in A to three.
Total energies in Hartree (Eh ) must be stated with six decimal places; anything less leads to errors
when relative energies are converted to chemically more useful units, such as kJ/mol.
24. To return to the Job Manager screen, click on the Job Manager link on the left side
of the screen.
1.2 Fluoroethylene and 1,2-difluoroethylene
1. Start a new job and repeat steps 8-9.
2. Now return to the Periodic Table Tool but this time select fluorine. Create a C-F bond from one
of the carbons so that the molecule resembles the following:
3. Now perform a Comprehensive Cleanup (click the brush ). Note that the C atom already
attached to the F atom receives only one H atom this time in order to satisfy its valence demands.
4. Now start the same Optimise + Vib Freq job as you did for ethylene. The point here is to compare
the properties of the two molecules. The calculation can take up to 20 s.
5. Repeat steps 21-23 to work out the C=C and C-F bond lengths, the C=C-F angle. Enter the values
in the table provided in the worksheet.
6. Repeat this process with cis-1,2-difluoroethylene and trans-1,2-difluoroethylene. To do this it is

essential to press the Comprehensive Mechanics Cleanup button (click the wrench ) located
just under the brush, instead of the usual Cleanup. This form of cleanup takes into consideration
on which side of the carbon atom you have placed the fluorines, whereas the normal cleanup does
not discern between cis and trans. These calculations take 30 s each.
7. Complete the table provided in your worksheet.
8. All calculations so far were carried out with the moderately-sized 6-31G(d) AO basis set. While
smaller basis sets may already provide reasonable structures, energetic properties usually require
a larger basis set. A common strategy is to carry out geometry optimisations such as the calcu-
lations that you just did with a small basis set and to use the resulting structure for an energy

calculation with a larger basis set without readjusting the geometry. Such an energy calculation
is also called single point calculation. Next, you will carry out such single point calculations
for the two difluoroethylenes with the larger 6-311+G(2d,p) basis set. For this purpose, you
re- open the output of the respective geometry-optimisation calculation. Directly under the mo-
lecular viewer, you can find the button New Job Using This Geometry. This will open a new
molecular-editor window with your optimised structure. Do NOT use the Comprehensive Clean
Up button, but proceed to the Configure Gaussian Job Options screen (remember, we want
to keep the already optimised structure). Prepare a HF calculation with the 6-311+G(2d,p) basis
set and the same charge and multiplicity as before. In the Calculation menu, choose Molecular
Energy. Start the calculation and add the resulting RHF Energy to your table.
9. In addition to the images that you added to your worksheet and to the table that you filled out,
please address the following questions.
Question 1.1: What are the general trends in bond lengths and angles as you move from ethylene
to fluoroethylene, ethylene to the cis and trans isomers, and as you move from cis to trans? Find an
explanation for these trends.
Question 1.2: a) Find out what the cis-effect in difluoroethylenes is. b) Which out of the cis or
trans isomers is the most stable in terms of RHF energy for the 6-31G(d) basis set? Report the energy
difference in kJ/mol. The appendix at the end of this document has a conversion table for Hartrees to
kJ/mol. What is the energy difference between cis and trans for the larger basis set? Discuss the results
in the context of the cis effect. What do you observe and how do you interpret the obvious qualitative
difference in your results?
2 SN 2 Reactivity and Transition States
All SN 2 reactions follow the basic mechanism:
Where Nu is the nucleophile, L is the leaving group and X, Y and Z are substituents. If the nucleophile
is stronger than the leaving group then it bonds preferentially to the carbon, resulting in the detachment
of the leaving group.
In this exercise you will investigate the reactants, transition states and products of the symmetric
SN 2 reaction:
F + CH3 F FCH3 + F
The potential energy profile of the reaction is shown below.

Using HF calculations, you will determine the structures and energies of the reactants, the products,
the transition state and the entrance and exit channel complexes in order to draw the energy diagram.
To begin, well calculate the energies of the reactants and products.
2.1 Reactants and Products
1. Create a new job in WebMO.
2. Go to the Periodic Table Tool , select Fluorine and click once on the page. Do not perform
either form of cleanup as it will assume we want to attach a hydrogen whereas we require the
anion.
3. Go to the next page. Choose Optimise+Vib Freq for Calculation, Hartree-Fock for Theory and
Basic: 3-21G for the Basis set. For consistent results the same basis set should be used for all
molecules in the exercise at hand, and as transition states can be computationally draining, it is
best to use a smaller set to cut down on time. To make this fluorine an anion, set the Charge to
-1, and change multiplicity to Singlet. Start the job.
4. Once completed (the job should take 5 s), note down the RHF energy.
5. Create a new job and this time build CH3 F. To do this quickly, create a carbon atom and bond it
to a fluorine atom, then click the brush to add the 3 hydrogen atoms to the carbon.
6. Initiate the job as before, making sure that the charge is 0 and multiplicity is singlet. Make sure the
basis set is Basic: 3-21G. Again, note down the RHF energy once the job is completed (10 s).
The total energy of the reactants (or products) is the sum of the RHF energies of F and CH3 F.
Question 2.1: The reactants and products are essentially the same in this reaction. Does this suggest
that the reaction is endothermic, exothermic or thermoneutral? What does this tell you about the reactant
and product energies?
2.2 Calculating the Minimum Energy Structure
1. Create a new job.
2. Build a C atom and bond it to an F atom, cleaning up to make CH3 F as you did previously.
3. Now bond the methyl group to another fluorine, and use the Comprehensive Mechanics Cleanup
to create our molecule.

4. Note the charge that now appears on the C atom. This is a result of the cleanup balancing charges
and trying to make the product neutral. We want CH3 F2 to have a charge of -1, so click the Select
tool and right click (control click) the C atom.
5. Go down and click on Charge... and in the adjust charge box type 0.
6. Continue on to the job options page and set the settings to the same as those for the fluoride anion:
Optimise+Vib Freq, Hartree-Fock, Basic: 3-21G, Charge = -1, Singlet.
7. Start the job. Once completed (28 s), copy and paste the molecule into your worksheet using
Print to create an image file.
8. Note down the RHF energy. As a way to ensure youve found the minimum energy structure,
examine the frequencies of the vibrational modes, available in the Vibrational Modes table. If
the structure is indeed a minimum, they should all be positive.
Mathematical Aside: The vibrational frequencies depend on the second derivative of the potential
energy with respect to the displacements. A minimum is associated with all positive vibrational frequen-
cies. In contrast, the transition state is associated with one imaginary vibrational frequency associated
with a negative second derivative of the potential energy with respect to the displacements.
2.3 Calculating the Transition State Structure
1. The transition state and the minimum energy structure have the same chemical formula, so repeat
steps 1-5. Note: You may be tempted to use the New Job Using This Geometry button on the
results page of the previous structure, but do not do this. This structure has already been optimised
by Gaussian to give a lower energy state than the transition state, and therefore the structure you
end up with for the transition state will be incorrect.
2. On the job options page, instead of the usual Optimise+Vib Freq, select Transition State Op-
timisation. Remember to change the basis set, charge and multiplicity to the values mentioned in
Step 6.
3. Searching for the transition state can take some time (30 s). Copy and paste the structure into
your worksheet and note down the RHF energy.
4. VERY IMPORTANT: A good computational chemist does not accept everything the computer
spits out. It is important that one always uses ones own chemical expertise to determine if a result
is reasonable or wrong. A bad input , for instance, usually also results in a wrong output (Rubbish
in = rubbish out). Verify that the optimised transition state of the Sn2 reaction looks according
to what you know about Sn2 reactions. If that is not the case, you may need to modify the input
structure by selecting four atoms (shift+click) and changing the related dihedral angle (menu
option Adjust followed by dihedral angle). If you are unsure about what a dihedral angle is,
please have a look at your previous course material and re-familiarise yourself with its definition
(e.g. the first CHEM20019 lecture on Computational Chemistry). Sometimes it takes several
attempts to find the correct transition state.
Question 2.2: Using the reactants and products RHF energies as a zero point (E = 0), label the
reactants, products, transition state and minimum energy structures on the energy diagram in your work-
sheet. In the last two labels make sure to include the energy difference (in kJ/mol) between the reactants
and both the transition state and minimum energy structures.

3 Partial Charges, Electron Withdrawing and Donating Groups in Sub-

stituted Benzenes
Electron Withdrawing Groups (EWGs) and Electron Donating Groups (EDGs) are common sub-
stituents that respectively, donate electrons to or withdraw electrons from a ring system. Donation or
withdrawal changes the polarity of the molecule and opens up certain atoms in the ring to electrophilic
attack.
Electron donating groups are:
Activating (increase reaction rate) due to extra electrons donated to the ring.
Ortho/para directing: They direct the second substituent to the ortho or para positions.
Have a formal negative charge or a charge.
Examples of EDGs are: OH, OR, NH2 , alkyl groups.

Electron withdrawing groups are:
Deactivating (decrease the rate of reaction) due to the rings withdrawn electrons.
Meta directing: They direct the second substituent to the meta position.
Have a formal positive charge or a + charge.
Examples of EWGs are: CN, NO2 , CO2 H, CHO, C(O)R

Halogens are exceptions to both groups, being ortho/para directing as well as deactivating.
Partial charges are a way of determining how negative an atom is in a molecule compared to the
formal charges of Lewis structures. With partial charges, which most of the time can be found down
near the bottom of the results page of a job, you can graphically detect how positive (blue) or negative
(red) an atom is. In the following exercise you will perform a partial charge calculation to help explain
the ortho-, para- or meta-directing nature of EWGs and EDGs.
1. Create a new job and build the molecule anisole (methoxybenzene). The easiest way to do this
is to click on the Build drop-down menu located at the top of the screen, select Fragment... and
then Phenyl from the Fragment menu. Click OK to select the phenyl group and now click on
the screen to create a Phenyl group. If the molecule is hard to see, zoom out by clicking the
magnifying glass in the side menu, then holding the left mouse button and pulling the cursor
down.
2. Now you need to bond the phenyl ring to an O atom (make sure you bond to the carbon without
a hydrogen attached), and then bond another C atom to that O atom. As you may have noticed
when selecting the phenyl ring, in the Build drop-down menu there are shortcuts to the common
atoms C, H, O and N. Use these to create the required bonds.
3. Once you have done this, perform a Comprehensive Cleanup by clicking the brush and con-
tinue on to the next screen.
4. In the Job Options page change the Calculation to Geometry Optimization, the basis set to Basic:
3-21G basis set, Charge=0 and Multiplicity=singlet.
5. Run the job.

6. Once the job is finished (35 s), open the results and scroll down to the Partial Charges Table.
Here the atomic charges are listed and numbered as on the molecule viewer. To make things easier
to visualise, click on the magnifying glass on the right of the Partial Charges Table. This creates
a new picture of the molecule with atoms coloured in red and blue to show an accumulation or
deficiency of negative charge, respectively, and the charge for each atom.
Question 3.1: Present your results in the worksheet. Examining the partial charges, which positions
on the benzene ring would you say are most susceptible to electrophilic attack? Is this what you would
expect?
Question 3.2: Why is the methoxy group in the plane of the benzene ring? What is the C-O-C bond
angle? What does this say about the hybridisation of the oxygen atom? Note that sp2 atoms have a 120
bond. Remember that the order of atom selection is important when finding the bond angle.
4 Molecular Orbitals for Benzene
We will now extend the notions of the previous exercise, investigating and interpreting molecular
orbitals for the benzene molecule. By the end of this exercise you should be able to find the HOMO and
LUMO orbitals for a molecule including their energies and location of nodes.
1. Create a new job. Go to Build, Fragment... but this time change the Category to Rings, then
select Benzene from the Fragment section and click ok. Click anywhere on the molecule viewer
once more to create a benzene ring.
2. This ring is already completed and does not require cleanup, so continue on to the job options
screen.
3. In the Job Options page change the Calculation to Geometry Optimization and the basis set to
Basic: 3-21G basis set.
4. Run the job.
5. Once the job is finished (10 s) you can view the output and use the optimised geometry for a
calculation of the molecular orbitals. To do this, choose New Job Using This Geometry.
6. In the Job Options page change the Calculation to Molecular Orbitals, leaving the rest of the
menus as they are.
7. Once the job is completed (2 s), view the results and scroll down to the Molecular Orbitals
section. Going across the table, we have the orbital number, the symmetry, occupancy and energy.
8. At some point in the table, the occupancy changes from 2 to 0. The last orbital to contain 2 (i.e.
the highest occupied orbital) is the HOMO of benzene, and the first to have 0 (i.e. the lowest
unoccupied orbital) is the LUMO. Some general terminology: HOMO-1 refers to the orbital just
below the HOMO (in this case this would be orbital 20), HOMO-2 is two below the HOMO
(orbital 19), etc.
9. Clicking the magnifying glasses on the right of the table shows the specific molecular orbital.
Different colors for the MO signify different signs for the wave function (for occupied orbitals
blue and red; for unoccupied orbitals green and yellow). Look at the HOMO and the next 4 filled
MOs (i.e. HOMO-1, HOMO-2, HOMO-3, HOMO-4) either by clicking the magnifying glass in
the table each time or, after doing it once and opening up the MO Viewer, clicking on the table on
the left-hand side of the viewer.

10. Right clicking (Option clicking) the orbitals will allow you to select the transparency of the orbitals
through the Opacity menu. The default setting is Solid, but the other options allow you to see the
molecule as well as the orbitals, making it easier to locate which atoms have a positive, negative
or neutral effect.
Question 4.1: The following question relates to the HOMO, HOMO-1, HOMO-2, HOMO-3 and
HOMO-4 orbitals. Which of these HOMO-n orbitals correspond to the three filled MOs in benzene?
Examining the MOs, can you see any connection between the number of nodes in the orbitals and the
energy values in the Molecular Orbitals table? Present the relevant orbitals and their energies in the
report.
5 Diels-Alder Reactions
In a Diels-Alder reaction, a diene (hydrocarbon containing 2 alkene groups) reacts with a dienophile
(a substituted alkene) to produce a ring system. The Diels-Alder reaction is considered by chemists to
be a good way to create rings due to its low energy requirement. In this section you will focus on the
Diels-Alder reaction, discovering why it makes ring systems easy to create.
Eq.1:
Frontier Molecular Orbital (FMO) theory tells us that the molecular orbitals of any two reactants
that are closest in energy to each other are generally the most important in determining reactivity. This
is because the strength of the electronic interactions depends on the overlap and energy difference of
orbitals.
Therefore, in Diels-Alder reactions, the most important aspect in deciding reactivity between similar
types of dienes and dienophiles is the energy gap between the diene HOMO and dienophile LUMO, or
conversely the gap between the diene LUMO and dienophile HOMO. A small energy gap is usually
associated with a reactive system. As an example, ethylene and butadiene is a sterically favourable
Diels-Alder reaction, but has a very small yield due to a large HOMO-LUMO energy gap. In this
exercise you will examine this unfavourable reaction and two other reactions to determine trends in
relative reactivity.
Eq.2:
Eq.3:

To start, well study the reaction of ethylene with butadiene (Eq.1).
1. Create a new job and build ethylene as in the first exercise. Remember to use a Comprehensive
Mechanics Cleanup before continuing on to the job options page.
2. In the Job Options page change the Calculation to Geometry Optimization and the basis set to
Basic: 3-21G basis set. For consistent results, all of the Diels-Alder jobs should be run using the
same basis set, so make sure you do this every time.
3. Run the job.
4. Once the job is finished (7 s) you can view the output. VERY IMPORTANT: You have learned
in your Organic Chemistry lectures that the diene participating in a Diels-Alder reaction must be
in its s-cis conformation, as also shown in Eqns. 1-3. If your resulting structure is in the s-trans
conformation, instead, you have to adjust the relevant dihedral angle and repeat the geometry
optimisation.
5. Use the optimised geometry for a calculation of the molecular orbitals. To do this, choose New
Job Using This Geometry.
6. In the Job Options page change the Calculation to Molecular Orbitals, leaving the rest of the
menus as they are.
7. Once the job is completed (1 s for ethylene), open the results page and scroll down to the
Molecular Orbitals table. Here we will be calculating the gap between the HOMO of the diene
and the LUMO of the dienophile. As ethylene is a dienophile, for this first example write down
the LUMO energy. It may be helpful to write down which reactants are dienes and which are
dienophiles.
8. Run the same job for the other reactant, which in the first example means finding the HOMO
energy of butadiene.
9. Repeat the previous steps for Eqns. 2 and 3, ensuring to perform the correct cleanup when building
the molecule. Keep in mind that when building trans, trans-1,4-diphenylbutadiene, use the Com-
prehensive Cleanup brush tool . For all other reactants, use the Comprehensive Mechanics
Cleanup . Do not build the products of the reactions.
10. Calculate the HOMO-LUMO energy differences for all 3 reactions (subtract the HOMO energy
from the LUMO energy).
Question 5.1: Present the relevant HOMO and LUMO energies and briefly discuss the relevant
HOMO-LUMO differences for the three reactions. Are they consistent with the fact that the reaction of
ethylene with butadiene is unfavourable whereas reactions 2 and 3 occur readily?

6 NMR and Shielding
This exercise focuses upon calculations of the relative shifts in 13 C isotropic shielding in butane,
2-butene and 2-butyne.
A nucleus is shielded from an applied magnetic field by being surrounded by a cloud of electrons.
When electrons move in an applied magnetic field (B0 ), an induced magnetic field (Bi ) is generated in a
direction opposite to B0 . The net result is that there is an effective magnetic field (Be f f ) that the nucleus
actually feels, which is less than the applied magnetic field. Effectively, nuclei are shielded from the
applied magnetic field by the electron cloud. The degree of shielding depends on the atom itself and its
location within a molecule. Gaussian09 can be used to calculate the shielding and the NMR chemical
shift (usually with tetramethylsilane (TMS) as a reference value).
In this exercise the molecular geometry is first optimised using a Density Functional Theory (DFT)
approximation and then the NMR shielding is calculated using Hartree-Fock theory (which is a wave-
function theory). For the DFT geometry optimisation youll employ the hybrid functional B3LYP
Becke-style 3-Parameter Density Functional Theory (using the Lee-Yang-Parr correlation functional).
To find out more about NMR shielding calculations and associated background theory, you may wish to
look at [1] or [2].
1. Start a new job, build butane and perform the usual Comprehensive Cleanup before continuing
on to the Job Options page.
2. Before you can initiate the NMR calculation, you need to optimise the molecules geometry using
the B3LYP/6-31(d) method and basis set. Go to the Theory drop-down menu and select B3LYP.
The Routine: 6-31G(d) basis set is default so should already be selected. Finally, set the Calcu-
lation to Geometry Optimisation. VERY IMPORTANT: Verify that all molecules are in their
lowest conformation. Sometimes, you may have to re-adjust the methyl groups accordingly and
repeat the optimisations.
3. This job can take some time (40 s), as the DFT B3LYP method is slower than Hartree-Fock.
When completed, open up the results page and select the New Job Using This Geometry button.
4. The molecule is already in the molecule viewer, so continue on to the Job Options page.
5. This time, change the Calculation menu to NMR, and the Theory to Hartree-Fock. Leave the
basis set as Routine: 6-31G(d). This is the minimum recommended level for performing these
calculations.
6. The NMR calculation should be fast. Once completed, open up the Job Results page and scroll
down to the Relative NMR Shifts table. Here you can read the relative isotropic and anisotropic
shift values, as well as look at the 1 H and 13 C NMR spectra. Note the isotropic values for the first
2 carbon atoms in the table, i.e. the NMR shift of one outer sp3 carbon (C1) and one inner sp3
carbon (C2).

7. Repeat this process for 2-butene and 2-butyne.
Question 6.1: Calculate the shifts in 13 C isotropic shielding for butane, 2-butene and 2-butyne,
using the 2 inner carbons of butane as a reference value. Fill out the tables in your worksheet, focusing
only on the first 2 carbons (labelled C1 and C2) for butane, 2-butene and 2-butyne. Express the results
to only one decimal place.
Do your calculated values agree with the experimental ones?
Question 6.2: A positive value indicates that there is less shielding than in the reference model,
and a negative value indicates there is more. What is the trend as you move from alkane to alkene, and
alkane to alkyne, specifically regarding the inner (C2) carbon atoms? Try to explain this trend.
Appendix - Units of Energy
The usual unit of energy in quantum chemical calculations is the Hartree. A unit conversion table is
given below.
Hartree kJ/mol eV kcal/mol cm1

1 2625.5 27.21 627.51 219474
Appendix - Report
Your report can be based on the template uploaded to the LMS. Even though it is tempting to answer
each question with bullet points, please write proper answers (1 to 2 paragraphs per question, depending
on the context) in academic English. Also, please interpret your results within the chemical context of
each exercise.
References
[1] Cheeseman, J. R.; G. W. Trucks; T.A. Keith; M. J. Frisch. A Comparison of Models for Calculating
Nuclear Magnetic Resonance Shielding Tensors, J. Chem. Phys., 1996 104, 5497-5509.
http://www.chemia.uj.edu.pl/ migda/Literatura/pdf/JCP05497.pdf
[2] http://bouman.chem.georgetown.edu/nmr/interaction/chemshf.htm
[3] Clauss, A. D.; S. F. Nelsen. Integrating Computational Molecular Modeling into the Undergraduate
Organic Chemistry Curriculum. J. Chem. Ed., 2009 86(8), 955-8.
http://pubs.acs.org.ezp.lib.unimelb.edu.au/doi/pdf/10.1021/ed086p955
[4] de Dios, A. C. The NMR Chemical Shift. Georgetown University, 2008.

http://bouman.chem.georgetown.edu/nmr/interaction/chemshf.htm
[5] Foresman, James B.; leen Frisch (1996). Exploring Chemistry with Electronic Structure Methods.
Pittsburgh, PA: Gaussian Inc.. pp. 6, 9, 21-22, 29-30, 40-5, 208-210

Metal-Based Compounds: Synthesis &
Characterisation
In the following pages the preparations for all experiments offered for the Synthesis and Character-
isation of Metal Based compounds component of the course are included.
You must complete one experiment from those offered in the M1 and one from those in the M2
sections.
The experiments in this section require two full sessions to complete. You must be prepared to start
work immediately when you enter the laboratory, good time management and efficiency are required if
you are to complete the assigned tasks within the time allocated.
Additional resources for these experiments such as NMR spectra and visualisation aids may be
required and can be found on the LMS.
Be sure to check the available files before completing your final laboratory session for the experi-
ment.
M1.1 - Oxo-Molybdenum Chemistry
A/Prof. Colette Boskovic

1 Introduction
Metal oxides are important industrial catalysts and certain biological systems, notably those contain-
ing Mn, Fe and Mo, exploit oxo-metal active sites for a variety of catalytic reactions. The high-valent
chemistry of the early transition metals is, in particular, dominated by oxo complexes. The oxo ligand,
formally O 2 , stabilises high-valent complexes by both and interactions (Fig. 1).
Figure 1: The and bonding interactions of the filled orbitals of O 2 with empty metal d orbitals.
Oxo complexes are susceptible to both nucleophilic and electrophilic attack, exemplified for Mo
chemistry by equations 1 and 2, respectively.
Mo VI O + : PIII R3 Mo IV + OP V R3 (1)
Mo VI O + H+ [Mo VI OH]+ + H+ [Mo VI ] 2+ + H2 O (2)
Equation 1 is an oxygen atom transfer reaction which is accompanied by a change in the oxidation
states of the reactants (related but reversed reactions are possible when suitable oxygen atom donors
react with Mo IV ). Equation 2 represents a simple two-step protonation which is not accompanied by a
redox change. In the absence of other ligands, such protonations lead to polyoxomolybdate formation. In
the presence of suitable ligands various complexes are formed. This reaction may be used to selectively
replace oxo ligands by anionic ligands.
These reactions are exploited in the synthesis of innumerable Mo complexes, account for much of
the chemistry of such complexes, and are important in catalytic oxygen atom transfer reactions such
167
M1.1 CHEM30015: Advanced Practical Chemistry
as those mediated by industrial oxidation catalysts and the molybdoenzymes sulfite oxidase, xanthine
oxidase and nitrate reductase.
In this experiment various oxo-Mo complexes containing the N,N-diethyldithiocarbamate ligand
(Fig. 2) are prepared and studied by a variety of techniques.
Figure 2: Structure of the N,N-diethyldithiocarbamate ligand.
The synthesis of the dioxo-Mo(VI) complex cis-MoO2 (S2 CNEt2 )2 (Figure 3) is central to the exper-
iment: from it a number of colourful oxo-molybdenum complexes are prepared and these are charac-
terised by a variety of analytical and spectroscopic techniques. Their structures and role in oxygen and
sulfur atom transfer reactions are also explored.
Figure 3: Molecular structure of cis-MoO2 (S2 CNEt2 )2 .
This experiment highlights:
A number of synthetic strategies based on equations 1 and 2.
The study of complexes with a variety of coordination numbers, geometries and nuclearities.
Analysis of infrared and 1H NMR spectra of the complexes.
An examination of stoichiometric and catalytic oxygen atom transfer reactions.
Comparison of the course of a simple oxygen atom transfer and sulfur atom transfer reactions.
2 Prelaboratory Exercises
(To be submitted to Dr Boskovic prior to commencing the experiment)
1. Write balanced equations, including molybdenum oxidation states, for the two steps involved in
the formation of cis-MoO2 (S2 CNEt2 )2 .
2. Why doesnt the synthetic reaction for the formation of cis-MoO2 (S2 CNEt2 )2 begin until the
HCl(aq) is added?
3. Use the elemental analysis data provided in the oxo-Mo file for cis-MoO2 (S2 CNEt2 )2 to calcu-
late the atomic proportions (C:H:N:S:Cl) and formula of the sample analysed. Comment on the
presence of chlorine in the sample, noting that this sample was recrystallised from dichlorometh-
ane solution by addition of ether.

CHEM30015: Advanced Practical Chemistry M1.1
4. Comment on the structural information provided by the bands in the (CN) and (Mo=O)regions
and other informative regions of the IR spectrum of cis-MoO2 (S2 CNEt2 )2 (Figure 2 in the oxo-
Mo file).
5. Determine the symmetry point group of cis-MoO2 (S2 CNEt2 )2 (hint: a model is helpful).
3 Experimental
The dispensing of malodorous (smelly!) or toxic substances such as HNEt2 , CS2 , concentrated
HCl and chlorinated solvents should only be done in a fume hood.
3.1 Preparations
3.1.1 cis-MoO2 (S2 CNEt2 )2
Success depends on vigorous magnetic stirring during the addition of hydrochloric acid - so use
a large (5 cm) stir-bar and an effective stirrer unit. Diethylamine (2.4 mL - use a 5 mL pipette and
bulb) and sodium hydroxide (0.9 g) are added to water (50 mL) in a 250 mL Erlenmeyer flask. After
stirring for 5 min, the mixture is treated with carbon disulfide (1.4 mL), a watchglass is placed over
the top of the flask and the solution is stirred for a further 10 mins. Sodium molybdate(VI) dihydrate
(3.5 g) is added to the mixture, which is then treated dropwise (about 3-4 drops/sec from a dropping
funnel, over about a 10 min period) with a solution of 6 mL of concentrated hydrochloric acid (12 M) in
water (100 mL). Vigorous stirring is required during the dropwise addition as the dense yellow-brown
product precipitates. The solid is isolated by vacuum filtration on a glass fritted funnel, washed with
water (60 mL), ethanol (60 mL) then ether (60 mL) and dried at the pump. In each washing remove
the filter funnel from the Buchner flask, add the solvent to the solid and use a spatula to waft the solid
around in the washing solvent, then replace the funnel on the flask and suck the solvent off the solid.
The crude material may be employed in the syntheses that follow. The remainder of the sample can be
recrystallised later by dissolving it in dichloromethane (15 mL/g), filtering, and adding ether (20 mL/g)
to the clear filtrate.
3.1.2 The Red Compound (R)
This compound is moderately air-sensitive and all work should be performed quickly and efficiently.
In a small round bottom flask connected with a reflux condenser, a mixture of cis-MoO2 (S2 CNEt2 )2 (1.0
g) and triphenylphosphine (1.0 g) in 1,2-dichloroethane (bp 82 C, 10 mL) is refluxed for 15 mins. Make
sure that reflux (in a pre-heated bath) is commenced immediately after adding the solvent to the starting
materials. Upon completion of the reflux pour the reaction mixture, with swirling, into ice-cold ethanol
(50 mL) contained in a 100 mL Erlenmeyer flask. Filter the crystals and wash with ethanol, then ether
and vacuum dry (see Fig. 4). Record an infrared spectrum as soon as possible after the compound has
dried. Perform the test tube experiments (following page) with a fresh sample, R is air-sensitive so do
the tests and synthesis of B straight away.
3.1.3 The Blue Compound (B)
In a small round bottom flask, a solution of the Red Compound (0.25 g) and sulfur (0.1 g) in 1,2-
dichloroethane (5 mL) is refluxed for 30 minutes. After cooling, ca. 1 mL of the reaction mixture is
loaded onto an analytical column. The column is prepared by placing a plug of cotton wool in the
bottom of a 10 mL burette and adding a slurry of silica gel (3 g, 70-230 mesh) in CH2 Cl2 (10-15 mL).
Swirl the solution before each addition so as to transfer the silica gel into the burette. Excess solvent

Figure 4: Vacuum drying on a filter frit.
may be drained from the column by opening the tap of the burette. Be careful not to allow the solvent
level to drop below that of the top of the silica gel since air bubbles will be trapped in the column leading
to poor separation of the compounds. Elute the column with dichloromethane (fume hood) and collect
the blue fraction. Evaporate the blue fraction to dryness on a rotary evaporator and triturate with ethanol
(5 mL). The solid is collected by vacuum filtration, washed with ether (20 mL) and dried at the pump.
3.1.4 The Purple Compound (P)
A solution of cis-MoO2 (S2 CNEt2 )2 (0.5 g) in dichloromethane (5 mL) is filtered through a fluted
filter paper, then the filtrate is treated with a solution of triphenylphosphine (0.16 g) in methanol (10
mL). The mixture is swirled for a few seconds then left to stand for 15 minutes. The purple solid formed
is vacuum filtered, washed with methanol and dried at the pump. Crush a crystal of the sample on a
piece of white paper to determine its colour.
3.1.5 The Yellow Compound (Y)
A solution of crude cis-MoO2 (S2 CNEt2 )2 (0.5 g) in acetone (35 mL) is filtered through a fluted filter
paper to remove any impurities. The filtrate is then treated with 2.5 mL of concentrated hydrochloric
acid and the mixture stirred for 20 mins. The product is isolated by filtration, washed with 10 mL of
acetone and dried at the pump. If you have time, large crystals of the product can be obtained by allowing
the reaction mixture to stand undisturbed for 2-3 h (or overnight) following the initial mixing of the acid
into the cis-MoO2 (S2 CNEt2 )2 solution.
3.1.6 Oxygen and Sulfur Atom Transfer Chemistry
You have already observed the reactions which take place when cis-MoO2 (S2 CNEt2 )2 is reacted
with one-half mole equivalent and excess PPh3 (the preparations of P and R, respectively). Try the
following test tube experiments to reveal more about these and related reactions. Record and account
for the colour changes observed - write appropriate chemical equations. The masses used need only
approximate those given below.
1. Make up separate solutions of cis-MoO2 (S2 CNEt2 )2 and R (10 mg/5 mL) in dichloromethane.
Observe the colours of the solutions and mix them together with swirling.
2. Allow a solution of R (5 mg/5 mL) in dichloromethane to stand in air (leave overnight - but
observe at intermediate stages).

3. Add about five drops of hydrogen peroxide solution to a solution of R (20 mg/5 mL) in dichloro-
methane, swirl for a minute. Dissolve an excess of PPh3 (ca 0.1 g) in the solution then allow it to
stand. Swirl to again mix in the H2 O2 phase.
4. To a solution of B (10 mg/5 mL) in dichloromethane add 0.1 g of PPh3 and swirl.
5. One final observation you should be aware of and explain: When a solution of cis-MoO2 (S2 CNEt2 )2
and a fifty-fold molar excess of PPh3 is monitored in air by 31 P NMR, the initial spectrum reveal-
ing only the presence of PPh3 is slowly replaced by a spectrum consistent with the presence of
only OPPh3 . A similar observation results when a solution of cis-MoO2 (S2 CNEt2 )2 and a fifty-
fold molar excess of both PPh3 and dimethylsulfoxide (Me2 SO) is monitored in a sealed tube
by 31 P NMR. Blank experiments show that in the absence of cis-MoO2 (S2 CNEt2 )2 , PPh3 is not
converted to OPPh3 even in the presence of oxygen or Me2 SO.
3.2 Analytical and Spectral Data
Some Supplementary data and spectra are provided in the oxo-Mo file on the LMS, together with
tips that will assist you in characterising the unknowns.
Elemental Analyses: Elemental analyses for all compounds are given in the oxo-Mo file. The
analytical data will assist you in determining the formulae of the compounds. Notes on the use of
analytical data are given in the Appendices.
Mass Spectra: Mass spectra for compounds R, B, P and Y are provided in the oxo-Mo file.
Infrared Spectra: Record the infrared spectra of compounds R, B, P and Y as pressed KBr disks.
The spectrum of cis-MoO2 (S2 CNEt2 )2 is provided in the oxo-Mo file. Note that compound Y also
displays two (Mo-Cl) stretches at 294 and 220 cm1 , which are outside the operating frequency of our
instrument.
Nuclear Magnetic Resonance Spectra: The 1 H NMR spectra of compounds R and B are provided
in the oxo-Mo file file. The spectrum of compound Y consists of a broad triplet (due to two very
closely coincident triplet resonances and a multiplet due to overlapping diastereotopic CH2 groups).
Notes on the NMR spectra of two inequivalent spin 12 nuclei are available in the Appendices.
4 Report
The experiment consists of two parts: experimental and deductive. Extra data and tips are provided
in the oxo-Mo file to help with the deductions. You may use the results sheet available on the LMS as
guide.
Your report should include:
A full write-up of the synthetic procedure in the experimental section, including yields (mass and
percentage) for the synthesised compounds;
The atomic proportions (C:H:N:S:Cl) in compounds R, B, P and Y calculated on the basis of

elemental analysis and mass spectral data and the m/z value for the most intense component of the
isotope pattern;
Simulations of the isotope patterns present in the mass spectra of compounds R, B, P and Y
using the programme available in the laboratory or via Sheffield Chemputer:
(http://winter.group.shef.ac.uk/chemputer/);
Balanced equations for the formation of compounds R, B, P and Y, indicating the oxidation
states of the molybdenum at each stage. Two steps exist for compound P;

The recorded IR spectra of compounds compounds P, R and Y;
Comment on the structural information provided by the bands in the (CN) and (Mo=O) regions
and other informative regions of the IR spectra and deduce the number and types of ligands present
in each of compounds R, B, P and Y;
Assignments of the NMR spectra of compounds R and B, accounting especially for the complex
patterns in the methylene region. An explanation for the complex patterns in the methylene region
of the spectrum for compound Y should also be provided;
Structural diagrams for compounds R, B, P and Y consistent with the spectral data. Seven co-
ordinate structures based on the pentagonal bipyramid only should be considered for compounds
B and Y;
The symmetry point groups of compounds R, B, P and Y in solution;
Oxygen Atom Transfer. Account for the observations of the test tube experiments in terms of
oxygen atom transfer reactions and explain the process leading to the catalytic oxidation of PPh3
by O2 revealed by the 31 P NMR experiment. Write balanced equations or a balanced catalytic
cycle for each experiment.
References
[1] X.F. Yan and C.G. Young, Aust. J. Chem, 1991, 44, 361-7.
[2] C.G. Young et al, J. Chem. Soc. Dalton Trans., 1983, 2135-8.
The following are more general references about the significance of this field of chemistry:
[3] C.G. Young, Molybdenum: MPT-Containing Enzymes, in Encyclopedia of Inorganic Chemistry

2, Pergamon, 2005.
[4] C.G. Young, Molybdenum, in Comprehensive Coordination Chemistry II, Pergamon-Elsevier,

2004, 415.

M1.2 - Reactions of Coordinated Ligands
Dr. David Jones

1 Background
The oxidation of 1,10-phenanthroline (phen) to 1,10-phenanthroline-5,6-quinone (quin) will be ac-

complished through the formation of the tris(phenanthroline)cobalt(III) complex followed by oxidation
of the coordinated ligand.
Although the complex is optically active, racemic [Co(phen)3 ] 2+/3+ complexes are isolated in this
experiment. In cases where optically resolved complexes are used it is found that the following steps
proceed WITH RETENTION OF OPTICAL PURITY (and absolute configuration).[1, 2]
D(+)[Co(phen)3 ] 2+ + 21 Cl2 D(+)[Co(phen)3 ] 3+ + Cl

D(+)[Co(phen)3 ] 3+ + 9 HNO3 D(+)[Co(quin)3 ] 3+ + 9 HNO2 + 3 H2 O
The second reaction is conducted in a refluxing solution of concentrated H2 SO4 and HNO3 and the
retention of the optical activity gives an impressive demonstration of the inertness of cobalt(III) ammines
to ligand substitution (which in this case would lead to racemisation).
In the final stage of the experiment the [Co(quin)3 ] 3+ complex is decomposed, yielding 1,10- phen-
anthroline-5,6-quinone. The utility of this series of reactions is reflected by their use, in a study [3]
of the intercalation of ruthenium phenanthroline complexes with DNA, to prepare 1,10-phenanthroline-
5,6-quinone.
The reactivity of an organic molecule is often changed markedly when it is co-ordinated to a metal.
This may take the form of either more facile reaction or completely different forms of reaction. Further-
more the regio- or stereo-chemical course of the reaction may be altered decisively. An example of this
is provided by the enhanced reactivity of 1,10-phenanthroline when co-ordinated to cobalt(III). In this
case stoichiometric oxidation to the quinone can be accomplished using a concentrated nitric-sulfuric
acid mixture.
Examination of the structures of tris chelate octahedral complexes reveal that they exist in enantio-
meric forms. In cases where the central metal ion is inert to substitution the enantiomers retain their
optical activity in solution, however optical activity is lost when one end of the chelate detaches itself
from the metal since reformation of the co-ordinate bond is not generally stereoretentive and this results
in racemisation. Therefore reactions involving optically resolved tris chelates which proceed without
173
loss of optical activity necessarily proceed without dissociation (whole or partial) of the chelate. Stud-
ies of the racemisation of tris chelate complexes have been used to elucidate the rates and mechanisms of
certain substitution and electron transfer reactions. A particularly elegant example of the use of optically
activity to examine electron transfer reactions was provided by F.P. Dwyer where the following electron
transfer reaction can be studied by the loss of optical activity [4, 5, 6]:
(+)[Os(bipy)3 ] 3+ + ()[Os(bipy)3 ] 2+ ()[Os(bipy)3 ] 3+ + ()[Os(bipy)3 ] 2+
kelectron transfer > 5 10 4 M 1 s 1
In this case the rate of loss of optical activity is many orders of magnitude faster than the rate of
racemisation of either (+)[Os(bipy)3 ] 3+ or ()[Os(bipy)3 ] 2+ on their own, where in fact, there is no
loss of optical activity of solutions of either [Os(bipy)3 ] 2+ or [Os(bipy)3 ] 3+ on standing for several days.
This strongly suggests that racemisation occurs by an outer-sphere electron transfer mechanism:
(+)[Os(bipy)3 ] 3+ + ()[Os(bipy)3 ] 2+ (+)[Os(bipy)3 ] 2+ + ()[Os(bipy)3 ] 3+
2 Pre-Lab Questions
1. What is the typical colour of Co III N6 (where N represents a N-donor ligand) complexes?
2. What are typical rates of ligand substitution reactions of Co II and Co III amine complexes?1
3. By what mechanisms may octahedral complexes undergo racemisation?
4. Estimate the relative rates of racemisation of [Co(phen)3 ] 2+ and [Co(phen)3 ] 3+ ?
As mentioned in the introduction to this experiment, synthesis of 1,10-phenanthroline-5,6-quinone

can be achieved from the starting material 1,10-phenanthroline. Due to time constraints, the synthesis
you will perform in this experiment begins at the second step in the synthetic method: the preparation of
tris(1,10-phenanthroline)cobalt(III) tetrafluoroborate from tris(1,10-phenanthroline)cobalt(II) bromide.
3.1 Preparation of tris(1,10-phenanthroline)cobalt(III) tetrafluoroborate
Rubber gloves should be used when handling BROMINE. All operations involving the use
of bromine MUST be carried out in a fume hood.
Prepare a solution of saturated bromine water by adding 30 mL of water to 9-12 drops of liquid
bromine in a 50 mL quickfit flask (care!, excess bromine may result in isolation of the bromide salt).
Stopper the flask and shake vigorously to aid dissolution of the bromine.
Place a mixture of 1.5 g (1.97 mmol) of [Co(phen)3 ]Br2 in 30 mL of freshly prepared bromine water
in a 100 mL B19 rb flask equipped with a magnetic stirring bar. Add 15 mL of water to the flask and
attach a water condenser. Heat the solution under mild reflux for 25 minutes (fume hood). Initially the
reaction mixture will contain a large amount of undissolved solid, but as the reaction proceeds the solid
the rate of the water self exchange reaction as a guide to the reactivity of the Co II metal ions. See, M.L. Tobe in
1 Use
Comprehensive Coordination Chemistry, Vol. 1 Ch. 7.

should dissolve. If a precipitate is still evident after 20 minutes, remove the condenser and boil until a
clear solution is obtained.
Remove the solution from the hotplate then add 7.5 mL of 48% aqueous HBF4 (rinse out your
measuring cylinder immediately after dispensing the HBF4 as this acid also acts as a source of F , which
attacks the glass) to the solution. Allow the solution to cool slowly to room temperature (whereupon
most of the product ought to have crystallised from solution) then cool the flask to 0 C in an ice-salt bath
to ensure complete precipitation. The yellow product is collected by suction filtration using a Buchner
funnel, rinsed with 2-3 mL of ice-cold water and air dried for 15 minutes. Record the yield and obtain
an IR spectrum (KBr disc) of your product. Identify the peaks due to vibrations of the BF4 anion, and
note the differences in the vibrational bands due to complexed and uncomplexed phenanthroline.
3.2 Preparation of tris(1,10-phenanthroline-5,6-quinone)cobalt(III) hexafluoro-phosphate
The following reaction involves the use of concentrated acid, with BROMINE generated
by the action of concentrated acid on NaBr. This reaction MUST be completed in a fume
hood. Strong acids MUST be placed in the appropriate waste container.
Add 1.0 g (1.2 mmol) of [Co(phen)3 ](BF4 )3 , 0.6 g (5.8 mmol) of NaBr and 10.0 mL of H2 SO4
(18 M) to a 50-mL quickfit round-bottomed flask equipped with a magnetic stirring bar and a water
condenser (at this stage the water condenser is used to prevent loss of the bromine from the reaction
flask). Cool the mixture in an ice-bath and add 5.0 mL of HNO3 (15 M) to the ice-cold mixture. Reflux
with stirring for 30 minutes using a Heat-On block attached to a stirring hotplate. (if unavailable a
preheated oil-bath set 120 C may be used).
Caution: Do NOT use oil baths if they have been contaminated with water. The water will boil,
sometimes explosively, and splash hot oil over a large area.
Prepare a solution of 2.68 g of KPF6 (14.4 mmol) in 20-25 mL of water in a 100 mL beaker. Cool
the hot reaction mixture in ice and add SLOWLY to the KPF6 solution with stirring (CAUTION, you
are adding concentrated acid to water in this step and the buildup of heat will result in decomposition of
the PF6 counterion). A heavy precipitation of the pale yellow reaction product should form immediately
on adding the reaction mixture to the KPF6 solution. Allow to stand for 10 minutes. Collect the crystals
by suction filtration using a 5.5 cm Buchner funnel (make sure that you use acid resistant hardened filter
paper e.g. Whatman no. 541) and wash with 10 mL of warm 1 M nitric acid. Transfer the product
to a plastic petri dish, label it with your name and bench number. The product from this reaction dries
very slowly; dry the sample using the vacuum oven (40-50 C). Record the yield and IR spectrum and
compare the IR spectrum with those obtained of the previous products.
Transfer the product to a plastic petri dish, label it with your name and bench number.
(Option: Ask (nicely) the laboratory staff to dry it in the vacuum oven for you in the morning if you
are running out of time).
3.3 Isolation of 1,10-phenanthroline-5,6-quinone
CAUTION: this compound is a severe irritant to the throat and nasal membrane. Chlo-
roform is poisonous and carcinogenic, ALL manipulations using chloroform must be con-
ducted in a fume hood.
Suspend 0.60 g of tris(1,10-phenanthroline-5,6-quinone)cobalt(III) hexafluorophosphate from part b
and 0.60 g of Na2 H2 EDTA in 20 mL of warm water in a 50 mL B19 conical or rb flask. The suspension
is stirred magnetically and, using a hand-held pH meter, carefully adjust the pH to 5.0 - 5.5 by the
dropwise addition of a saturated solution of NaHCO3 . Attach a water condenser and reflux, with stirring
for 30 mins. or until a red solution is obtained (possibly with a yellow precipitate). The mixture is

cooled to room temperature and the bright yellow product is extracted into chloroform (30 + 15 mL).
Use a separating funnel to isolate the non- aqueous phase. Dry the chloroform extract over anhydrous
magnesium sulphate (1 g) and filter the solution into a 250 mL quickfit round bottom flask. Remove
the solvent by rotary evaporation and recrystallise the impure quinone from the minimum volume of hot
methanol. Record the yield, IR spectrum and melting point2 of the purified product.
4 Report
Complete the report for this experiment using the report sheets as a guide for the questions and data
that must be included in your formal report. The report sheets will be available from the LMS site. NMR
and IR spectra of appropriate compounds may be provided.
References
[1] R.D. Gillard, R.E.E. Hill, and R. Maskill, J. Chem. Soc. A, 1970, 1447.
[2] H.R. Hunt, J. Chem. Educ., 1977, 54, 710.
[3] K.J. Black, H. Huang, S. High, L. Starks, M. Olson and M.E. McGuire, Inorg. Chem., 1993, 32,
3391.
[4] F.P. Dwyer and E.C. Gyarfas, Nature, 1950, 166, 1181.
[5] M.W. Dietrich and A.C. Wahl, J. Chem. Phys., 1963, 38, 1591.
[6] F.P. Dwyer and E.C. Gyarfas, J. Am. Chem. Soc., 1951, 73, 2322.
General references:
[7] J.E. Huhey, E.A. Keiter and R.L. Keiter, Inorganic Chemistry; Principles of Structure and Reactiv-
ity, Harper Collins, 1993, 548-555.
[8] F.A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, 5th ed., Wiley, 1988, Ch.29.
[9] D.F. Shriver, P.W. Atkins and C.H. Langford, Inorganic Chemistry, 2nd ed., Oxford University Press,
1994, Ch.15.
[10] Z. Szafran, R.M. Pike, and M.M. Singh, Microscale Inorganic Chemistry, Wiley, New York, 1991.
[11] Socrates, G., IR and Raman Characteristic Group Frequencies, Tables and Charts, 3rd ed., 2001.
2 Beilstein Supp. series IV, 24, 1741.

M2.1 - Copper Bypyridine Complexes
A/Prof. Brendan Abrahams

1 Background and Objectives
One of the pleasures in making transition metal complexes is the aesthetic appeal of their colour and
exquisite crystalline form; in this exercise we synthesise two such visually attractive materials.
The central aim of this exercise is to prepare and investigate the bis-2,2-bipyridine complex of
copper(II) chloride. The complex is analysed for copper by electrodeposition and for chloride gravi-
metrically. The bis-bipyridine complex of copper(II) tetrafluoroborate is also prepared (for the purposes
of this exercise BF4 can be regarded as a non-coordinating anion). The sort of information that can
be obtained about complex species in solution from conductometric measurements is then illustrated
by conductometric studies in nitromethane solution on the chloride and tetrafluoroborate derivatives,
together with a conductometric titration of the tetrafluoroborate with chloride ion. These investigations
make it possible to determine if counteranions are bound to the metal centre or are uncoordinated. The
usefulness of d d spectra is illustrated by comparing the visible spectrum of the bis-bipyridine copper
chloride complex (in nitromethane) with spectra provided of selected copper(II) complexes known to
have tetragonal ligand fields. Molecular models are constructed to provide some insight into factors
influencing stereochemistry in these systems. Finally, you can propose a unifying explanation for their
various observations.
1.1 The electronic spectra of distorted copper(II) complexes, d d spectra
Cupric complexes often adopt, if the ligands permit it, a tetragonal geometry with four ligand atoms
at short distances approximately at the corners of a square (say the xy plane) and with two other ligands
at longer distances on the axis (the z axis) at right angles to that plane. The order of the energies of the
d-orbitals expected on this basis is dx2 y2 > dz2 > dxy > dxz = dyz .
In the ground state of a d9 CuII complex with this geometry, we expect all the levels to be filled
except for dx2 y2 which has a single vacancy. According to this simple picture then, we expect three
possible electronic transitions to this vacancy, namely, dxz,yz dx2 y2 , dxy dx2 y2 and dz2 dx2 y2 .
The blue or green colours of most cupric complexes are associated with a broad, usually asymmetric,
absorption band, generally in the range 600-900 nm, which is made up of at least two and possibly
more overlapping components. These component bands are taken to have basically the d d transition
origins described above, although definitive assignments are difficult to make.
The d d electronic spectra of [Cu(H2 O)6 ] 2+ , [Cu(gly)2 (H2 O)2 ] (gly = glycinate ion) and
[Cu(en)2 (H2 O)2 ] 2+ (en = ethylenediamine) in aqueous solution each show a broad, asymmetric band
with maxima at 820, 630 and 540 nm respectively. Assume that the cupric species that are present in
each of these three cases are tetragonally distorted with two extended axial CuH2 O bonds.
177
2 Pre-Lab Questions
You are required to prepare written answers to these questions before commencing the experiment.
You might be asked to show these answers to a demonstrator during the experiment.
1. What is the formula of basic copper carbonate?
2. (a) Draw an energy level diagram showing the d orbital splitting in an octahedral complex and in
a tetragonally distorted octahedral complex (with two trans ligands further from the metal ion
than the other four).
(b) Comment on the trend in max observed for the three complexes [Cu(H2 O)6 ] 2+ ,
[Cu(gly)2 (H2 O)2 ] and [Cu(en)2 (H2 O)2 ] 2+ .
3 Experimental
3.1 Preparation of Cu(bipy)2 Cl2 hydrate
Add a solution of CuCl2 2 H2 O (0.70 g) in hot water (14.0 mL) to a boiling solution of bipyridine
(1.40 g) in acetone (40 mL). Note that boiling acetone presents a fire risk. Heat the solution on a water
bath in a fumehood. Filter the hot solution through a fluted filter paper and maintain the filtrate at the
boiling point on the steam bath. Add 60 mL boiling acetone to the boiling filtrate. Allow to cool.
Whilst waiting for the crystals to grow start the preparation (below) of Cu(bipy)2 (BF4 )2 . Collect the
blue crystals, wash with a mixture of acetone (25 mL) and water (3 mL) and allow the crystals to dry.
Record your yield.
3.2 Preparation of Cu(bipy)2 (BF4 )2
Transfer by pipette 1.5 mL of the 40% aqueous HBF4 solution provided to a DRY (it is very
important the flask is dry) 25 mL conical flask. Add about 1 g of basic copper carbonate followed
by 8 mL ethanol. Gently simmer for 10 min. on the steam bath. Considerable solid should remain
undissolved at this stage; if this is not so add a further 0.2 - 0.3 g basic copper carbonate and continue
gentle heating for a further 5 min. Place 8 mL of acetone in a small DRY conical flask and heat to
boiling on a steambath (fumehood) and add immediately to your solution. Remove the excess copper
carbonate by hot filtration under gravity through a preheated fluted filter paper.
Add the filtrate to a solution of 1.2 g bipyridine in 30 ml acetone. Blue crystals should start to
separate after a minute or two. To prevent evaporation stopper the flask either with a quickfit stopper
(if you have used a quickfit flask) or with a cork or rubber stopper wrapped in clean aluminium foil.
Standing for 1 - 2 hrs. is usually sufficient to give a good crop of product. Collect the crystals on a
Hirsch funnel, wash with ice-cold acetone and dry in a stream of air. Record your yield.
3.3 Analysis: Determination of copper in Cu(bipy)2 Cl2 hydrate by electrodeposition
3.3.1 Preparation of the electrode
If the cathode (the larger of the two electrodes) is covered in copper (either the usual copper colour
or a black deposit) follow the procedure in part 3.3.5 for the clean-up of the electrodes. Avoid touching
the clean cathode with fingers. Weigh the cathode on a zeroed 4 decimal place balance. Record the
weight and store in a clean dry beaker until ready to use.

3.3.2 Dissolution of the sample
Weigh out accurately approximately 0.25 g complex in a clean dry 50 mL conical flask. Add 2-
3 mL conc. sulphuric acid (18 M) and gently heat to the boiling point on a gauze over a bunsen for
approximately 10 minutes. Warning, do not leave the solution unattended while it is being heated. The
purpose of this step is to remove all chloride from the system as gaseous HCl prior to the electrolysis.
Allow the mixture to cool. Transfer the product very carefully to a 100 mL beaker containing 10 mL of
distilled water - care! - incautious mixing of water and H2 SO4 can be dangerous and also lead to a
loss of material by spattering. Make sure the copper complex is transferred completely by washing in
thoroughly with distilled water but keep the total volume below approximately 40 mL. Add 2 mL conc.
nitric acid (14 M) and approximately 0.5 g sulphamic acid (to destroy any nitrite ion contaminant of the
nitric acid).
3.3.3 Deposition of copper
The electrodeposition apparatus is located in a dedicated fumehood in the laboratory. Attach the
electrodes to the power supply; the stems of the electrodes fit into the holes in the plastic block and are
clamped in position, anode inside cathode, so that there is maximum separation between the two. Check
at this point that you have recorded the mass of the cathode. Direct contact between the electrodes
MUST be avoided.
Introduce the electrode assembly into the solution for analysis and allow sufficient space below the
electrodes for the magnetic stirrer bar to rotate. Add water until approximately 10% of the cathode
surface is above the liquid. Switch on the current and adjust to approximately 2 A. Deposition should
be quite rapid.
After 20-30 minutes, or when the solution appears colourless, add distilled water so that half the
previously unsubmerged part of the electrode becomes submerged. If after a further 15 minutes no
further copper is detected on the newly submerged surface, the electrolysis is complete.
With the potential still applied stop the stirrer and gradually lower the beaker (or raise the electrode
assembly) whilst continuously spraying the electrodes with distilled water from a wash bottle. Switch
off. Avoiding knocking the cathode, submerge it in distilled water in a beaker. Finally wash with acetone
and dry in a clean dry beaker with a stream of nitrogen. Accurately weigh the cathode with deposited
copper and record the weight.
3.3.4 Calculation
Calculate the weight of copper deposited and the percentage copper in your compound. Compare
this with the theoretical percentages for various integral values x in Cu(bipy)2 Cl2 (H2 O)x .
3.3.5 Clean-up of electrodes
Dissolve the copper off the platinum electrodes by immersing in 7 M aqueous nitric acid. When all
the copper has dissolved wash thoroughly with distilled water and rinse with acetone. After the acetone
has evaporated heat to bright red using a bunsen burner. Allow to cool in a clean, dry beaker.
3.4 Determination of Chloride in Cu(bipy)2 Cl2 hydrate by Gravimetric Analysis
3.4.1 Dissolution
Weigh accurately about 0.15 g of Cu(bipy)2 Cl2 (H2 O)x in a small, clean, dry beaker. Tip the solid
into a clean 250 mL beaker making sure that the solid either ends up in the 250 mL beaker or remains

adhered to the beaker. Reweigh the small beaker so as to determine the mass of solid transferred to the
250 mL beaker. Dissolve in nitric acid (100 mL, 1 M). Add 20 mL approx. 0.1 M silver nitrate solution
to precipitate AgCl. Heat the mixture on a steam bath for approximately 10 minutes with occasional
stirring with a glass rod fitted with a rubber policeman or sleeve (the rubber protected end of the rod
is used to agitate the mixture and to detach solid from the walls of the beaker without scratching). This
converts the precipitate into a form more suitable for filtration. Be careful at this stage that none of the
mixture is lost from the beaker, e.g. do not remove the stirrer rod with adhering solid from the beaker
and put it down on the bench. Be equally careful that no foreign material gets into the beaker. Cover the
beaker with a watch glass and allow to cool for at least an hour.
3.4.2 Collection of the silver chloride precipitate
Dry a sintered glass crucible by heating in a microwave oven for 2-3 minutes (medium heat). Cool
for 15 minutes in a desiccator then weigh, record the weight. It is very important that the crucible is
weighed to at least four decimal places. Use the same balance for subsequent weighing. Attach the
crucible to a suction filter flask and collect the precipitate. Wash all the precipitate into the crucible
using 0.01 M HNO3 (use a plastic pipette). Use a rubber policeman attached to a glass stirring rod
to remove solid adhering to the walls of the beaker. Wash the precipitate with two 10 mL aliquots of
acetone. Suck air through the precipitate for a few minutes to partially dry it then dry in a microwave
oven for 2-3 minutes (medium heat). Cool for 15 minutes in a desiccator, then again weigh (to at least
four decimal places).
3.4.3 Calculation
Calculate the percentage of chloride in the complex. Compare your result with calculated chloride
content for various integral values of x in Cu(bipy)2 Cl2 (H2 O)x .
3.5 Conductance Measurements
Some notes on conductance measurements are available in the Appendices and general information
regarding conductometric titrations is available in reference [1].
In solution the possibility arises that solvent molecules or anions may be coordinated to the copper in
addition to the bipyridine. Coordination of anions will result in a change in the charge of the species in
solution and the number of charged species. Conductance measurements provide an extremely elegant
means of examining reactions of this sort, the interpretation relying on the observation that, for almost all
electrolytes of a given valency type {i.e. whether 1:1 (e.g. Li+ F ), 2:1 (e.g. Mg 2+ 2 Cl or 2 Na+ SO42 )
or 3:1 (e.g. Fe 3+ 3 Cl )} at a given molar concentration and temperature, molar conductances fall in
predictable ranges. The ranges are solvent dependent, e.g. in aqueous solution the ranges for 103 M
solutions are:
1:1 96-115 S cm2 mol1

2:1 225-270
3:1 380-432
whereas in nitromethane, the solvent used in this experiment, the ranges for 103 M solutions are:
1:1 70-100 S cm2 mol1

2:1 170-200

Nitromethane is toxic and flammable: avoid swallowing, contact with skin and inhalation.
Prepare a 10 mL solution of Cu(bipy)2 (BF4 )2 in nitromethane of accurately known concentration

close to 5 103 M (range 4.8 5.2 103 M) by carefully determining the mass of sample transferred
to a clean and dry 10 ml volumetric flask. This is best achieved by weighing a vial containing the sample
and transferring the sample via a funnel to the volumetric flask. The mass of the empty sample tube is
then measured and the mass transferred to the volumetric flask is determined by difference. Carefully
pipette 1.0 mL of the nitromethane solution to the clean, dry, sample tube to be used as the conductomet-
ric titration cell containing a clean dry magnetic stirrer bar. Carefully add 4.0 mL of nitromethane. Insert
the conductance electrode and measure the conductance of the solution (the conductance electrode must
be clean and dry before placing in the solution - this is achieved by rinsing the electrode with acetone
and drying with a hairdryer). The electrode assembly has already been calibrated using an aqueous KCl
solution of known conductivity so that the readout (vertical scale) represents the specific conductivity,
, in units of S cm1 (consult notes from the LMS regarding the definition and significance of and
the molar conductivity, ). Use this solution to perform the conductometric titration described below.
3.5.1 Calculation
Use the initial specific conductivity measurement (t = 0) to calculate the molar conductivity of
Cu(bipy)2 (BF4 )2 in nitromethane. From this value deduce the form of the complex ion in nitromethane
in the two cases (i.e. whether Cu(bipy)2 (BF4 )2 in nitromethane behaves as a 1:1, 2:1 or 3:1 electrolyte).
Remember that the solution is diluted by a factor of 5 when the 4.0 mL of nitromethane is added.
3.6 Conductometric Titration of Cu(bipy)2 (BF4 )2 complex with Cl ion in nitromethane
Instructions for the use of the Conductiometric Titration Apparatus is available in Room 380. (This
titration takes about 20 minutes to complete)
You will be provided with the titrant, 2.5 102 M solution of benzyl triphenylphosphonium chlor-
ide ((C6 H5 )3 P(CH2 C6 H5 )+ Cl ) in nitromethane. Using a micropipette deliver 20 L aliquots of the
((C6 H5 )3 P(CH2 C6 H5 )+ Cl solution to the nitromethane solution containing Cu(bipy)2 (BF4 )2 . During
the titration the solution should be stirred with the magnetic stirrer bar. After the addition of each aliquot
wait for 10 s to allow the mixing of the solution and then record the specific conductivity of the solution.
Continue the titration until approximatley 400 L of the ((C6 H5 )3 P(CH2 C6 H5 )+ Cl solution has been
added. At the end of the experiment , the electrode assembly should be washed with acetone and air
dried.
3.6.1 Graphs and Calculation
Plot the specific conductivity, , (vertical scale) versus volume (L of Cl added) and determine
the number of moles of Cl needed to reach the endpoint. What is the likely formula of the copper
complex at the end point in the titration of Cu(bipy)2 (BF4 )2 complex with Cl ion?
A plot of specific conductivity, , (on the vertical scale) of a 1 mM solution of Cu(bipy)2 Cl2 xH2 O in
nitromethane as [(C6 H5 )3 P(CH2 C6 H5 ]+ Cl is added is available on the LMS. Use at t = 0 to calculate
and determine whether Cu(bipy)2 Cl2 xH2 O is a 1:1, 2:1 or 3:1 electrolyte.
3.7 Electronic spectral investigations
Record the absorption spectrum in a 1 cm glass cuvette (dont forget the reference cell) of a solution
of accurately known concentration of approx. 0.012 g Cu(bipy)2 Cl2 hydrate in 5 mL nitromethane in
the range 450-1100 nm.

3.7.1 Calculation
Calculate the molar extinction coefficient(s) (max ) at the wavelength(s) of maximal absorbance
(max ) - note: there should be more than one local maximum. Discard the nitromethane in the water-
insoluble waste contained in the appropriate fume hood.
3.8 Molecular Models
Models of [Cu(en)2 ] 2+ and [Cu(bipy)2 ] 2+ each with a square planar CuN4 geometry are available
from the prep. room. Please return these intact when you have finished with them. Consider whether
bipyridine and ethylenediamine are equally able to provide the square planar N4 component of the
tetragonal ligand arrangement normally preferred by cupric ion (especially bear in mind the non bonded
repulsive interactions between hydrogen atoms - van der Waals radius approximately 1.2 A). Structures
of a range of related copper complexes are available on the molecular graphics web page (see LMS). It
is essential that students carefully consider these models.
4 Report
You need to bring to the viva:
The appropriate pages from your duplicate notebook showing the raw data and calculations;
Your risk assessment;
The completed proforma sheet (available on the LMS);
In the viva you should be able to provide answers to the following questions:
1. Why was the tetrafluoroborate anion introduced as the acid (HBF4 ) rather than as a simple
metal salt (e.g. NaBF4 )? Refer to an equation in your answer. (Hint: Think about what
happens when acid is added to anions like carbonate and hydroxide.)
2. In the titration of Cu(bipy)2 (BF4 )2 with Cl what is the likely formula of the copper complex
at the end point?
3. Given that bipyridine is a stronger field ligand than ethylenediamine, what conclusion can
you draw from a comparison of the max values of the tetragonally distorted octahedral com-
plexes, [Cu(en)2 (H2 O)2 ] 2+ , [Cu(H2 O)6 ] 2+ and [Cu(gly)2 (H2 O)2 ] with that of your
Cu(bipy)2 Cl2 solution?
4. What is meant by the term a poorly coordinating anion? Would you expect Cl or BF4 to
be the more strongly coordinating anion?
5. Which experimental technique might allow you to confirm the proposed structures for the
complex ions in the solid state?
References
[1] For gravimetric analysis and conductometric titrations: A. I. Vogel et al, Vogels Textbook of Quant-
itative Inorganic Analysis
[2] For copper chemistry: C. M. Harris, T. N. Lockyer, H. Waterman, Nature 1961, 192, 424

M2.2 - Electronic Spectroscopy of
Coordination Compounds
Dr. Stephen Best

1 Learning Goals from this Experiment
Synthesis of chromium(III) complexes by simple substitution at elevated temperature and by redox

catalysis.
Application of UV-Vis spectroscopy to (i) determine the relative positions of ligands in the spec-
trochemical series, (ii) quantitate the molar decadic extinction coefficient and (iii) determine the
metal coordination environment using the rule of average environment.
Distinguish between the inner- and outer-sphere pathways of a redox reaction based on analysis
of the products.
2 Introduction
Your earlier studies in Chemistry will have introduced the concepts of crystal-field theory, which
readily explains the splitting of the energies of the d orbitals of a metal ion in an octahedral complex
(giving the t2g and eg orbitals) and the relationship between this and the colour of transition metal
complexes. The spectrochemical series, the sequence of ligands arranged according to their ability to
cause a large splitting of the energies of the d-orbital, allowed a discussion of magnetism and spin state
applicable to both simple metal complexes and metal centres in metalloproteins such as myoglobin.
Ligand-field theory provided a molecular-orbital based explanation of the factors responsible for the
ordering of ligands in the spectrochemical series. This explains, for example, why weak-field ligands
are donors and strong-field ligands are acceptors.
The placement of ligands into a spectrochemical such as that given below is best achieved through
consideration of homoleptic octahedral complexes (homoleptic in this context means that only one type
of ligand is bound to the metal). A ligand frequently omitted from the spectrochemical series is acet-
ylacetonato. In the experiment you will prepare the homoleptic octahedral complexes of acetylacetonato
and ethylenediamine, measure the UV-Vis spectra and from this and other measurements you will make
will be able to place the acetylacetonato ligand in the spectrochemical series.
halides<OH <C2 O42 <H2 O<NCS <py<NH3 <en<phen<NO2 <CN , CO
In general, the determination of the energy difference between the t2g to the eg orbitals of an oc-
tahedral complex (0 ) from the electronic spectra is only straightforward for the d1 and d9 cases. The
spectrum shows only one d-d band, with energy 0 . In the other cases (d2 to d8 ) interactions between
the d electrons modify the transition energy and more than one absorption band is observed and there is
a less direct relationship between the band energies and 0 .
In addition to 0 the excited state energies also depend on electron-electron repulsion of the metal
d-electrons (most commonly designated by the Racah parameter, B). Tanabe and Sugano were the first
183
to realise that general maps of the excited states energies of transition metal complexes of a given d-
electron count could be obtained by plotting the energy of the excited state divided by B against the
field strength (0 ), once again divided by B. The so-called Tanabe-Sugano diagram for the d3 case is
shown in Fig. 1. By convention the lowest energy term (often, though not correctly, called the ground
state) is plotted as the horizontal axis and the excited terms are plotted relative to the ground term. For
chromium(III) compounds, the lowest energy (or ground) term is labelled 4 A2g (pronounced quartet-
A-two-g), corresponding to a (t2g )3 electronic configuration. The only transitions which occur with
significant probability are between terms of the same spin multiplicity (i.e. terms with the same left
superscript). At room temperatures only the ground term is populated so only three d-d transitions are
expected ((viz 4 A2g 4 T2g , 4 A2g 4 Tlg and 4 A2g 4 Tlg in order of increasing energy) and these
give rise to the bands observed in the absorption spectrum. For the d3 case it can be shown that 0 is
approximately equal to the energy of the 4 A2g 4 T2g transition. A slightly more sophisticated approach
is to use the ratio of the energies of the transitions to determine the values of the Racah parameter, B
(related to the inter-electron repulsion) and 0 .
60
4
T1g
2
A1g
50
4
T1g
40 2
T2g
4
T2g
E/B
30
2
T1g
2
G 20 2
Eg
4
P
10
4
4 A2g
F 0
0 5 10 15 20 25 30 35
D O /B
Figure 1: Tanabe-Sugano Diagram for the d3 electron configuration.
The preceding discussion deals with the case of homoleptic complexes, but in most cases more
than one type of ligand is bound to the metal. In this case the complexes are still treated as being
octahedral, but the value of 0 takes a value that is averaged over all the ligands bound to the metal. The
validity of this approach can be assessed by experiment, where the additivity of the effect of the ligands
on the splitting of the energies of the metal d-orbitals is tested by application of the rule of average
environment. This states that the value of 0 (the energy splitting of the d-orbitals for a particular
geometry) for a mixed ligand complex, MAa Bn-a , where n is the co-ordination number of the complex,
can be derived from those of the pure ligand complexes (MAn , MBn ). This is given by the following
equation:
RULE OF AVERAGE ENVIRONMENT

a (MAn ) (n a) (MBn )
(MAa Bna ) = +
n n

For chromium(III) complexes with a coordination number of six (i.e. n = 6) this gives:
a (CrA6 ) (6 a) (CrB6 )
(CrAa B6a ) = +
6 6
An important characteristic of the chemistry of metal complexes is the dependence of the lability of
metal complexes on the electron configuration of the metal. This is described in terms of the crystal field
activation energy. For particular electron counts there can be a dramatic change (more than 12 orders
of magnitude) in the lability of complexes as a result of a change in oxidation state of the metal. This
behaviour can be used to great effect in synthetic studies, where redox-catalysed substitution can be
used either to speed up the reaction or change the reaction products. In order to understand the reaction
products it is necessary to identify the mechanism of the redox reaction. In this experiment you will use
the rule of average environment to identify the kinetic product of a redox-catalysed substitution reaction
of chromium(III) and from that deduce the intimate mechanism for the redox reaction.
3 Overview
By the examination of the UV-Vis spectra a series of octahedral or pseudo-octahedral complexes of

chromium(III) the crystal field parameter 0 will be calculated. This will be used to deduce a spectro-
chemical series for chromium(III). In order to include Cl in the range of ligands considered it will be
necessary to collect spectra from CrCl3 (anhydrous) in the solid state. By the use of the rule of aver-
age environments the co-ordination sphere of the chromium(III) product of the chromium(II) catalysed
dissolution of anhydrous CrCl3 will be deduced. The complexes, [Cr(en)3 ]Cl3 and Cr(acac) will be
synthesised and the extinction coefficients at the maximum absorption (max ) of their lowest energy d-d
transition measured and this will be used to assess their purity.
4 Questions
(the concepts associated with these questions underpins the experiment, make sure know the answers
to these questions BEFORE starting the experiment)
(a) The complexes MA6 and MB6 have lowest wavenumber absorption bands of 16000 and 13000 cm1
respectively. Use the rule of average environment to calculate the wavenumbers of the mixed-ligand
complexes, MAa B6a .

(b) Describe the co-ordination environment of Cr 3+ in anhydrous CrCl3 and chrome alum,
KCr(SO4 )2 12 H2 O.1 Knowledge of these structures, together with experimental observations made
in this experiment will allow the structure of CrCl3 6 H2 O to be deduced.
(c) What are the rate constants for water exchange for hexaaquachromium(II) and hexaaquachromi-
um(III)? Estimate the half-lives of ligand exchange at these metal centres.
Your risk assessment must be completed before attempting any part of the experiment. Note that the
isolation of a good sample of [Cr(en)3 ]Cl3 3 H2 O is tricky. Check the tips document before commencing
your experimental work.
All manipulations involving methanol, toluene and acetylacetone must be completed in a fume
hood. The ZnHg amalgam must be deposited in the mercury waste container immediately after
use.
5.1 Preparation of [Cr(en)3 ]Cl3 3 H2 O
Place one piece granular zinc (briefly washed with 6 M HCl to remove any surface ZnO, then rinsed
with methanol) and 2.66 g (10.0 mmol) of CrCl3 6 H2 O in a dry 50 mL round-bottom flask. Add 10
mL (0.15 mol) of ethylenediamine, followed by 10 mL of methanol. Attach a water condenser fitted
with a drying tube containing CaCl2 (to prevent ingress of water vapour). Place the assembly on a steam
bath and heat the mixture to reflux, with occasional swirling, for 1 hour. (Make sure that the inside of
the condenser is dry before you assemble that apparatus). Note that this preparation will fail if your
glassware is damp!
5.1.1 Isolation of Product
Cool the solution to room temperature. Collect the grey-green crystalline product by suction filtra-
tion using a Buchner funnel. Remove any unreacted zinc using tweezers. Wash the filter cake with three
5 mL portions of 10% ethylenediamine in methanol by which time the washings should be colourless.
Purify by dissolving the crude yellow product in a minimum quantity (no more than 20 mL) of warm,
weakly acidic, water (distilled water to which 1 drop of conc. HCl has been added). Filter carefully so
as to remove all traces of zinc powder and slowly add ethanol (to a maximum of 100 mL) until a heavy
yellow precipitate is formed. A highly crystalline sample can be obtained by warming the suspension
until the precipitate just redissolves then cool the solution in an ice bath. Filter the bright yellow crystals,
wash with 210 mL of cold ethanol and air dry. A second crop of crystals may be obtained from the
filtrate by the addition of more ethanol and cooling. Dry in a desiccator. Record the melting point and
yield.
5.2 Preparation of Cr(acac)3
In a 100 mL conical flask fitted with a watch glass cover place 20.0 mL of distilled water (graduated
cylinder) and 1.30 g (4.9 mmol) of chromium(III) chloride hexahydrate. When the chromium complex
has dissolved, add 2 to 3 drops of concentrated ammonia, 1.0 g (83 mmol) of urea and 4.0 mL (38 mmol)
of acetylacetone (acacH). A large excess of acacH is used, as it helps the reaction go to completion.
1 Chrome alum is isostructural with CsCr(SO ) 12 H O, consult Fig. 1 of S.P. Best and J.B. Forsyth, J. Chem. Soc. Dalton
4 2 2
Trans., 1991, 1721. Note Fig. 1. RasMol structures of CsCr(SO4 )2 12 H2 O and anhydrous CrCl3 are available from the LMS
(M2.2/resources) folder.

NOTE: The acetylacetone must be dispensed in the fumehood.
Add 1 mL of concentrated ammonia to the reaction mixture immediately before placing on the
steam bath. This ensures a good yield of the product. Heat the mixture, on a steam bath with occasional
stirring, for 1 hour. As the urea releases ammonia and the solution becomes more strongly basic, deep
maroon crystals will begin to form. These may form as a crust at the surface of the reaction mixture.
5.2.1 Isolation of Product
Cool the reaction flask to room temperature. Collect the crystalline product by suction filtration
using a Buchner funnel. A second crop of crystals can be obtained by adding 5 mL of concentrated
ammonia to the filtrate and then heating on a steam bath for a further 20 minutes. Wash the crystals
with three 2 mL portions of distilled water and air dry. Recrystallise half of your product by dissolving
the crude Cr(acac)3 in a minimum volume of acetone, filter and add an equal volume of distilled water.
The crystalline product is filtered from the ice-cooled solution, washed with a small quantity of ice-cold
acetone:water (1:1, ca. 2 mL) and air dried. Dry in a desiccator then record the melting point and yield.
5.3 Characterisation of the Products
Prepare solutions of [Cr(en)3 ]Cl3 3 H2 O in water and Cr(acac)3 in toluene (note: use glass cuvettes)
and measure their absorption spectra using either the Perkin Elmer Lambda 2 or the Shimidazu UV-2401
PC UV-Vis spectrometers. The concentration of the solutions must be known accurately and should give
an absorbance of approximately 0.5 absorbance units. The required concentration may be estimated by
assuming that the molar absorbance coefficient at the band maximum (max ) for all the compounds is
50 cm1 M1 . Use your spectra to calculate the molar extinction coefficient for each clearly resolved
band.
Note:
A = cl
where A = absorbance, l = path length (in cm), c = molar concentration and = molar absorbance
coefficient* in cm1 M1 .
(* also known as the extinction coefficient or, more correctly, the molar decadic extinction coeffi-
cient).
5.3.1 Electronic Spectral Investigations
Chromium(III) salts are susceptible to photochemical decomposition. Therefore solutions of

chromium(III) complexes should not be exposed to light for prolonged periods.
(i) Solution spectra:

In addition to the two compounds above electronic spectra should be recorded for solutions of the
following compounds in water slightly acidified with nitric acid (ca. 0.01 M):
(a) CrCl3 6 H2 O
(b) Chrome alum, KCr(SO4 )2 12 H2 O.
It is not necessary to know the concentration of these solutions, however the spectra should be
recorded soon after preparation. Spectra of a solution obtained immediately following dissolution
of K3 [Cr(CN)6 ] in water and again after exposure to light for ca. 15 minutes are available on
the Web. You will be required to use the spectra of these five compounds in the report for this
experiment.

Figure 2: UV-Vis. spectra recorded from a paraffin dispersion of hydrated CrCl3 6 H2 O.
(ii) Solid State Spectra:

The spectrum of [CrCl6 ]3 may be obtained from anhydrous chromic chloride in the solid-state (in
aqueous solution [CrCl6 ]3 is unstable as one or more of the chloride ligands are rapidly aquated).
So as to compare the solid state and solution spectra, solid state spectra are also measured for
chrome alum and CrCl36 H2 O. This set of three solid-state spectra will be used to deduce, using the
rule of average environments, the composition of the inner co-ordination sphere about chromium
in the CrCl36 H2 O salt.
A convenient method for collecting the solid-state spectra is to disperse the solid in paraffin and to
press this dispersion between glass plates (in much the same manner as used to prepare a nujol mull
for infrared spectroscopy). A particular characteristic of the crystal structure of CrCl3 simplifies
the collection of spectra using this method. In this case it is not necessary to finely grind the
sample. This is not generally the case, since light scattering by the sample is most serious when
using shorter wavelength radiation (why is this not such a problem when collecting IR spectra?).
The spectrum of CrCl3 6 H2 O, obtained as a paraffin dispersion, is given in Figure 2. The solid-
state spectra of anhydrous chromic chloride (paraffin dispersion) and of chrome alum (as a single
crystal) are available on the Web. If you wish you may collect your own spectra of these samples
(consult SPB).
(iii) Chromium(II) catalysed dissolution of anhydrous CrCl3

The sample of anhydrous CrCl3 provided will not dissolve in water at an appreciable rate. This
observation is understood in terms of the kinetic inertness of chromium(III). Dissolution of the
solid in water may, however, be catalysed by traces of chromium(II). Take ca. 0.1 g of anhydrous
chromic chloride and place it in a test tube with 10 mL of 0.1 M sulphuric acid. Bubble nitrogen
through the solution for 2-3 minutes to displace dissolved oxygen then add a piece of amalgamated
zinc, stopper the tube and observe the reaction which takes place over the next 5 minutes. Note
that amalgamated zinc contains mercury (toxic and volatile). Handle the amalgamated zinc with
care and dispose of properly. It is better not to shake the tube because this will increase the
likelihood of ingress of O2 which will rapidly oxidise the chromium(II) catalyst formed by zinc
amalgam reduction of chromium(III). When most of the solid has dissolved filter the solution and
obtain the UV-Vis. spectrum of the filtrate. Heat the remaining solution on a steam bath for at
least 45 minutes and record the UV-Vis spectrum of that solution, this will be used to identify the
thermodynamic product of the reaction. Place the remaining amalgamated zinc in the mercury

residues container (located in one of the fume hoods).
6 Determination of the Racah interelectron repulsion parameter, B.
In those cases where more than one d-d transition is observed in the electronic spectra of metal
complexes it is possible to extract values for both 0 and B. This can be done by using the ratio of the
energies of the two lowest spin-allowed d-d bands to determine the value of 0 /B that applies for the
complex. This task is simplified by use of a tabular version of the Tanabe-Sugano diagram giving the
ratio of the energies of the transitions against 0 /B. A spreadsheet with this information is available on
the LMS. Once the value of 0 /B is known the energies of the transitions can be expressed as a function
of B. Since the energies of the transition are known the value of B can be evaluated. For those complexes
that present two spin-allowed d-d bands use the method described above to determine both 0 and B.
Does the trend in B values for the different ligands follow the spectrochemical series?
7 Report
Complete the report sheets are available from the Lab WEB page. Assessment will be based on your
experimental results and calculations (50%) and your performance in a viva discussion with Dr Best.
Your viva mark will be based on your ability communicate what you did in the experiment (30%) you
should use the observations noted in your lab note book for this part and a discussion of the interpret-
ation of the results (20%). The report and viva must be completed within 2 weeks of commencing the
experiment.
The following relationship gives the conversion between wavelength and the frequency unit of re-
ciprocal centimetres (cm-1 ).
1 107
= ( in nm, in cm1 ), also... 1 cm1 = 1.24 104 eV = 0.01196 kJ mol1

References
[1] Gillard, R. D., Mitchell, P. R., Inorg. Synth. 1972, 13, 184.
[2] Weller et. al., Inorganic Chemistry, Oxford University Press, 6th ed., 2014, Ch. 20 (UniM ERC 546).
[3] Housecroft and Sharpe, Inorganic Chemistry, Pearson, 4nd ed. 2012 (UniM ERC 546).
[4] Gispert, Coordination Chemistry, Wiley-VCH, 2008 (UniM ERC 541.2242).
[5] Kettle, S. F. A. Physical Inorganic Chemistry, University Science Books, 1996.

M2.3 - Geometrical Isomers of
Mo(CO)4(PPh3)2
A/Prof. Colette Boskovic

1 Introduction
1.1 Isomerism
The study of the numbers and types of isomers has been central to the development of coordina-
tion chemistry. Today a number of spectroscopic methods are available which allow us to differentiate
between isomers, and to determine their structures accurately. One of the most simple, but effective, is
infrared spectroscopy. The energies of vibrational transitions fall in the range 10-4000 cm1 , the precise
value being dependent upon the masses of the atoms involved and the strength of the bond (force con-
stant). For a vibration to be excited by direct absorption of an appropriate quantum of energy (i.e. for
that vibration to be infrared active), then there must be a change in dipole moment during the course of
the vibration. Hence in the simple case of CO2 , no change in the dipole moment occurs for the symmet-
ric stretch, and thus this vibration is infrared inactive. For the asymmetric vibration however, the dipole
fluctuates along the internuclear axis (z-axis), and hence this is seen in the infrared spectrum.
1.2 Metal carbonyl complexes
The chemistry of metal carbonyl complexes is a vast and still rapidly expanding area. Carbonyls are
important at the fundamental level and have many industrial and other applications. Carbon monoxide is
an extremely useful ligand in transition metal chemistry, stabilising metal centres in low oxidation states.
The infrared spectra of these carbonyl derivatives are particularly informative since the CO stretching
band is very intense, and also very sensitive to small steric and electronic changes in the molecule [1].
In addition these bands are often very easily observed and identified because they appear in a region of
the IR spectrum generally devoid of bands of other origin.
One of the most common reactions of transition metal carbonyl complexes, is the substitution of
carbon monoxide for other ligands, for example phosphines. This experiment is concerned with some
substitution chemistry of molybdenum hexacarbonyl, Mo(CO)6 . Substitution of one of the carbonyls
191
simply gives Mo(CO)5 L which can exist only as a single isomer. Replacement of a second carbonyl
gives rise to the possibility of geometrical isomers, namely the cis and trans isomers of Mo(CO)4 L2 .
The identity of the isomer(s) can easily be ascertained by interpretation of infrared spectra. Isolating the
cis and trans isomers of a variety of Mo(CO)4 L2 derivatives has not always been simple. For example,
if sufficient brute force (sufficiently high temperature) were used to effect di-substitution of Mo(CO)6 to
give Mo(CO)4 L2 the thermodynamically more stable trans isomer was often the only product obtained
because of relatively rapid thermal isomerisation under these forcing conditions. The method used here
is a particularly convenient way of obtaining a number of cis Mo(CO)4 L2 derivatives in good yield. The
procedure is based on the ready conversion of Mo(CO)6 to cis Mo(CO)4 (piperidine)2 (where piperidine
is the cyclic secondary amine NHC5 H10 ) followed by the very facile displacement of the amine ligands
by phosphine, arsine and related ligands.
2 Pre-Laboratory Exercises
(To be submitted to Dr Boskovic prior to commencing the experiment.)
1. What is meant by the inert gas rule (also known as the effective atomic number rule and the 18
electron rule)? Show how the rule applies to the mononuclear carbonyls of Cr, Fe and Ni and to
the binuclear Fe2 (CO)9 .
2. Describe in terms of orbital overlap considerations the nature of the multiple so-called synergic
bond formed between CO and various metal centres in low oxidation states; why is it important
the metal should be in a low oxidation state?
3. Describe in terms of orbital overlap considerations the nature of the bond formed between low
valent metal centres and various trivalent phosphorus derivatives such as phosphines, PR3 , and
phosphites, P(OR)3 .
4. Using appropriate internal coordinates as a basis set, determine the number and symmetry species
of (CO) bands that might be expected in the infrared spectra of octahedral complexes of general
formula cis-M(CO)4 L2 and trans-M(CO)4 L2 . Note that an outline on the use of group theory to
analyse the IR spectra of metal carbonyl species is given in the Appendices.
3 Experimental
Molybdenum hexacarbonyl has a small, but finite, vapour pressure at room temperature and
the vapour is quite poisonous, like all metal carbonyls. Thus, always work with metal car-
bonyls in an efficient fume hood and do not handle the solids. NB: Avoid multiple handling
of solids when using the balances and weigh by difference to get the exact amount of solid
[Mo(CO)6 ] used.
3.1 Preparation of cis-Mo(CO)4 (NHC5 H10 )2
Carry out this preparation in the fume hood. Exercise care when manipulating all the chemicals
involved. Mop up all spillages and dispose of them in the appropriate manner.
Place the sample of Mo(CO)6 provided (2.0-2.1 g pre-weighed), 5.0 mL piperidine, 5.0 mL THF
and 12.5 mL diglyme (diethylene glycol dimethylether, CH3 OCH2 CH2 OCH2 CH2 OCH3 ) and 2-5 dark-
coloured boiling chips in a 100 mL quickfit B19 round bottom flask. Attach a quickfit B19 water
condenser and place the glass vessel in a heat-on block and reflux (temperature set 150 C). Ensure

good thermal contact between the flask and the heat-on block. It is crucial that no water be allowed
into the reaction mixture: make sure all glassware is scrupulously dry. Allow to reflux for at least 60
minutes (start timing from the start of reflux) after which time a considerable quantity of yellow crystals
should have precipitated out of a now brown solution. Allow the reaction mixture to cool. Collect the
solid on a small Buchner funnel or, better, a small Hirsch funnel, using water pump suction. Wash with
n-heptane (2 5 mL portions) and air dry on the filter. Collect the crystals in a ziplock sample bag and
record the yield. Record the IR spectrum of the sample in KBr disc over the normal full range (400-4000
cm1 transmittance mode). Wrap the sample in Al foil to exclude light.
3.2 Preparation of cis-Mo(CO)4 (PPh3 )2
Place approximately 1 g of your cis-Mo(CO)4 (NHC5 H10 )2 , 1.5 g of triphenylphosphine, and 20 mL

of dichloromethane in a 50 or 100 mL quickfit B19 round-bottom flask. Allow the mixture to reflux for
20-30 minutes on a steambath, with frequent agitation /stirring. Try to avoid steam condensing on the
outside of your condenser whereupon water may run down to the joint and creep through by capillary
action with undesirable consequences. Make sure the joint is seated properly but be careful not to jam
the two components together and be prepared to dry off the external condensed water frequently with a
tissue. Allow the reaction mixture to cool to room temperature and filter under gravity through a fluted
filter paper. Add 30 mL of methanol to the filtrate and cool the mixture in ice. Allow approx. 30 min. for
the solid to separate. Collect the solid product on a Hirsch funnel, wash with methanol (2 10 mL) and
air dry. Record the yield of your crude product. Set aside approximately 0.5 g for the next reaction and
recrystallise the remainder of your product (whose mass also should be recorded) by dissolving in the
minimum of chloroform then adding methanol whilst swirling the liquid until crystallisation just starts,
then cooling in ice. Additional product can be recovered, if necessary, by adding further methanol.
Collect the crystals under suction on a Hirsch funnel and wash with a little methanol (2 5 mL). Dry the
solid in the funnel by drawing air over it. Record the yield of recrystallised product and its IR spectrum
(400-4000 cm1 transmittance mode) in KBr disc.
3.3 Conversion of cis- to trans-Mo(CO)4 (PPh3 )2
Place approx. 0.5 g (accurate mass recorded) of crude cis-Mo(CO)4 (PPh3 )2 in a 100 mL round bot-
tom flask together with 10 mL of toluene. Reflux for 30 min using a heat-on block. While the reaction
mixture is still warm, remove the solvent on a rotary evaporator. Dissolve the brown residue in 30 mL
chloroform and filter by gravity through a filter paper. To the filtered solution add 60 mL of methanol
and cool in an ice bath. Collect the crude product on a Hirsch funnel, wash with methanol (2 10 mL)
and air dry. Record the mass of crude product. Recrystallise the product from chloroform/methanol as
for the cis isomer above. Record the yield of recrystallised product and its IR spectrum (400-4000 cm1
transmittance mode) in KBr disc.
3.4 Infrared Spectroscopy
CHLORINATED HYDROCARBON SOLVENTS are cancer suspect agents, and DI-

CHLOROMETHANE (CH2 Cl2 ) vapour is extremely irritating to the eyes and can cause
conjunctivitis. Prolonged inhalation can lead to liver and kidney damage. Thus, only use
CHLORINATED hydrocarbons in a FUME HOOD and place all residues in the chlorinated
waste bottles.
Infrared spectra of your products should be obtained as solutions in dichloromethane. Instructions
on the use of the solution IR cells are available in the instrument room. Plot your spectra in absorbance
mode over the range 1800-2200 cm1 (the two recrystallised phosphine products) or 1700-2200 cm1
(the piperidine product). A small quantity of solid (about 10 mg) in 1 mL of solvent should give you

a sufficiently concentrated solution for the collection of a well-defined IR spectrum. Spectra should be
acquired as soon as possible after dissolution of the samples. It might be necessary to first filter your
solution through tissue paper in a Pasteur pipette prior to collecting your spectrum.
4 Report
Your report should include:
A full write-up of the synthetic procedure in the experimental section, including yields (mass and
percentage) for the synthesised compounds (you may use the results sheet available on the LMS
as guide);
A table of the frequencies (from the solution spectra) of the IR absorptions for
cis-Mo(CO)4 (PPh3 )2 , trans-Mo(CO)4 (PPh3 )2 and cis-Mo(CO)4 (NHC5 H10 )2 in the region 1700-
2200 cm1 ;
Schematic diagrams illustrating the CO stretching vibrations for cis-M(CO)4 L2 and

trans-M(CO)4 L2 .
Comments on the IR solution spectra obtained for cis-Mo(CO)4 (PPh3 )2 and

trans-Mo(CO)4 (PPh3 )2 with reference to the IR activity and/or equivalence of the stretching vi-
brations determined in the prelaboratory exercises.
Comments on the similarities and/or differences in the carbonyl regions of the IR spectra for
cis-Mo(CO)4 (NHC5 H10 )2 and cis-Mo(CO)4 (PPh3 )2 .
Answers to the following questions:
1. At what temperature is cis-Mo(CO)4 (PPh3 )2 formed?

2. At what temperature is trans-Mo(CO)4 (PPh3 )2 formed?
3. Why isnt trans-Mo(CO)4 (PPh3 )2 formed in the preparation of cis-Mo(CO)4 (PPh3 )2 ?
4. Infrared spectra of solutions have the disadvantage that solvent bands may overlap with
important absorption bands. Are there any advantages in solution IR over solid (e.g. KBr
disc) IR measurements?
References
[1] F.A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, 5th ed., 1034-1040.
[2] A. Vincent, Molecular Symmetry and Group Theory, 2nd ed.
[3] D. J. Darensbourg, Inorg. Chem., 1979, 18, 14.
[4] M. Y. Darensbourg, D. J. Darensbourg, J. Chem. Ed., 1970, 47, 33.

S ECTION 2: T HEMED E XPERIMENTS
Theme based Experiments and Group
Modules
1 Introduction
The aim of this section of experiments is to provide experience in a collaborative research environ-
ment, while expanding on the concepts introduced in the initial theme experiments. You are required to
complete the initial experiment individually before commencing the group exercises.
For the group exercises you will work in groups of two or three students to complete each of the
modules specified for your topic. Each group will need to perform the three modules extending one
of the selected themed experiments performed previously. How you divide the workload is up to you,
however everyone must participate in at least one module. You have two laboratory sessions to complete
all the modules within your group. After completion of the modules you will need to organise yourselves
and collate all your data from each experiment and discuss your results. At the completion of these
experiments the group will combine their findings enabling the preparation of a report encompassing all
aspects of the entire problem.
The three topics offered for this year are listed in table 1 along with each of the corresponding group
modules.
Medicinal Chemistry: Synthesis and Activity of Sulfonamides

T1.0 Synthesis and Characterisation of Sulfanilamide
T1.1 Synthesis of Prontosil & Carbonic Anhydrase Binding Studies
T1.2 Removal of Zinc from Carbonic Anhydrase: A Kinetic Study
T1.3 Structural Investigations of ligand binding: Sulfanilamide & Carbonic Anhydrase: a case study
Materials Chemistry and Devices: Dye-Sensitised Solar Cells
T2.0 Construction & Characterisation of Dye-Sensitised Solar Cells
T2.1 Determination of Surface Area of Powders by Gas Adsorption
T2.2 Particle Size Determination from Turbidity Measurements
T2.3 Photophysical Studies of Dyes and Pigments for DSSCs
Flow Chemistry
T3.0 Synthesis of Coumarin Dyes in Continuous Flow
T3.1 Translation of Synthesis into Flow
T3.2 Photophysical Properties of Coumarin Derivatives
T3.3 Computational Study of the Photophysical Properties of Coumarin Derivatives
Table 1: Themed Experiments.
197
Theme 1 - Sulfa Drugs
1 Medicinal Chemistry: Synthesis and Activity of Sulfonamides
Background
The term sulfa drugs is a collective term used to describe an extensive series of sulphonamide
compounds used therapeutically both historically and in current medical treatment. These compounds
were the first class of compounds to be widely used as antibiotics and have been attributed with sav-
ing thousands of lives in the First World War. Sulfa drugs have been used to treat a wide variety of
infections including pneumonia (sulfapyridine in 1938), urinary tract infections (Sulfacetamide, 1941)
and gastrointestinal tract infections (succionylsulfathiazole, since 1942). Although the high incidence
of sulfa allergies among patients and the effectiveness of penicillin has resulted in a reduction in the pre-
scription of sulfa drugs, this group of compounds is still widely used today for the treatment of bacterial
infections, particularly those that exhibit resistance to penicillin based pharmaceuticals.
Sulfa drugs work as anti-metabolites in bacterial cells. The parent structure of sulfanilamide is
conserved throughout a wide array of sulfa drug analogues (Figure 1).
Figure 1: A selection of sulfanilamide derivatives produced for the current pharmaceutical market.
The success of these compounds as antibiotics can be attributed to their similarity in structure to
p-aminobenzoic acid, a key component in the bacterial synthesis of folic acid (Figures 2 and 3). Folic
acid is essential for the synthesis of DNA and cannot pass through the bacterial membrane. Inhibition
of folic acid synthesis will kill the microorganism.
It should be noted that although sulfanilamide has been proven to be a potent antibiotic, it was
never widely used in humans due to its high toxicity (low solubility often resulted in the compound
crystallising out within the kidneys) when compared to its many derivatives. Over the years over 5000
analogues of sulfanilamide have been developed to treat diseases such as dysentery, meningitis, and
urinary infection to name a few.
199
T HEMED E XPERIMENTS - T HEME 1 CHEM30015: Advanced Practical Chemistry
Figure 2: p-aminobenzoic acid and sulfanilamide
Figure 3: Folic acid
More recently sulfonamides have been found to inhibit Carbonic Anhydrase a crucial enzyme in-
volved in cellular respiration. Carbonic anhydrase is a Zinc metalloenzyme that catalyses the hydration
of CO2 to bicarbonate. Current research in this area has linked this inhibition to potential chemotherapies
targeted at treating a variety of diseases including glioma and a number of cancers.
During this module you will perform the synthesis of sulfanilamide from nitrobenzene as outlined
in the scheme below (Figure 4).
Figure 4: Reaction scheme for the synthesis of sulfanilamide from nitrobenzene.
As a group you will investigate the synthesis of a sulfanilamide analogue, prontosil, and its inhibi-
tion of the enzyme carbonic anhydrase. You will also investigate the importance of Zinc in the catalytic
activity of the enzyme through a series of kinetics experiments. The final module based around mo-
lecular visualisation and structure will investigate the active site of carbonic anhydrase using computer
visualisation exercise and illustrate the techniques involved in X-ray crystal structure determination of
small molecules (in this instance sulfanilamide).

T1.0 - Synthesis and Characterisation of
Sulfanilamide

1 Introduction
1.1 Aims:
1. To gain experience in multi-step synthesis and the use of protecting groups in chemical synthesis.
2. To gain experience in standard synthetic techniques and purification methods.
3. To characterise the final compound and the synthetic intermediates using spectroscopic techniques
including mass spectrometry.
**NOTE: It is essential that you complete a risk assessment and hazard analysis for each step in the
synthetic pathway and have it signed off by a demonstrator prior to commencing the lab.
Session 1
2.1 Step 1: Synthesis of Acetanilide
Acetylation of the primary amino group of aniline introduces a protecting group preventing further
reaction of the amine functionality in the subsequent synthetic steps. Although conversion of the amine
through to the amide reduces the overall activation of the benzene ring toward substitution, the remaining
ortho/para directing influence is beneficial to the synthesis of the desired product, sulfanilamide.
!
Figure 1: Acetylation of the primary amino group of aniline
Procedure:
201
T1.0 CHEM30015: Advanced Practical Chemistry
Pour 100 mL 0.4 M hydrochloric acid into a 250 mL Erlenmeyer flask equipped with a magnetic
stirrer bar. To this solution add 3.6 mL of aniline and warm the mixture to 50 C with stirring.
While the first solution is warmed, prepare a separate solution of sodium acetate by dissolving 6.0 g
of sodium acetate trihydrate in 20 mL of water.
Measure out 4.4 mL of acetic anhydride into a clean DRY beaker and set to one side.
With all three solutions prepared add the acetic anhydride to the warm solution of the aniline hy-
drochloride salt ensuring the mixture is vigorously stirred at all times. Immediately after adding the
acetic anhydride, pour the sodium acetate solution into the reaction flask. Continue to stir the reaction
mixture and allow it to cool to room temperature before placing the flask in an ice bath (with stirring)
until precipitation is complete.
Collect the crystalline product by vacuum filtration, washing the filter cake with a small amount
of ice-cold water. Air dry the product on the filter funnel before collecting. Your sample must by
completely dry before commencing the next step in the synthesis. Collect your sample in a glass vial
and check with your demonstrator to have it placed in a desiccator until your next session.
your product. Assign these spectra (see demonstrator for assistance). The proton NMR spectrum of your
products is to be run on the desktop NMR spectrometer in room 380 (the balance room). NMR tubes
and deuteriochloroform (CDCl3 ) (or D6 -DMSO if required) can be obtained from your demonstrator.
** Be sure to label your compound with your name and your lab group.
Record the % yield of your product and prepare samples for characterisation before moving
on to the following step.
Questions:
1. With reference to your synthetic procedure and the compound structures explain how the unre-
acted aniline and acetic anhydride are removed from your acetanilide product.
2. With reference to their structure explain why aniline is soluble in aqueous hydrochloric acid and
acetanilide is not.
3. Provide mechanisms of formation of acetanilide.
2.2 Step 2: Synthesis of 4-Acetamidobenzenesulfonyl Chloride
Session 2
Steps 2 and 3 must be completed one after the other in one session. Be sure to plan your time
accordingly. Once the sulfonyl chloride is prepared it cannot be stored so it is to be used immediately in
the synthesis of 4-acetamidobenzenesulfonamide.
Prelab Question:
1. Chlorosulfonic acid reacts violently with water; provide the equation for this reaction and com-
ment on the hazards of the resultant products.
Read through the experimental procedure for both steps (2 & 3) before commencing the exper-
iment.
THIS PROCEDURE IS TO BE CARRIED OUT IN THE FUMEHOOD. CHLOROSULF-
ONIC ACID IS HAZARDOUS, ENSURE YOU HAVE READ THE MSDS FORM, FILLED OUT
YOUR HAZARD ASSESSMENTS AND ANSWERED THE PRELAB QUESTIONS BEFORE
COMMENCING THIS PART OF THE EXPERIMENT.

CHEM30015: Advanced Practical Chemistry T1.0
!
Figure 2: Reaction scheme for synthesis of 4-acetamidobenzenesulfonyl chloride.
USE THE RUBBER GLOVES PROVIDED
Experimental Procedure:
** It will be necessary for you to carefully grease the joints of your glassware for this experi-
ment. You must be careful not to over grease the joints and ensure they are seated properly.
Into a clean, dry 100 mL B19 round bottom flask add acetanilide (2.7 g) and an appropriately sized
magnetic stirrer bar, attach a Claisen adaptor and pressure equalising dropping funnel to the flask as per
Figure 3). Attach a vacuum distillation adaptor on the side arm and connect this to the water aspirator.
There is a gas trap in the line between your apparatus and the water aspirator line, which will remove
the HCl vapour evolved during the reaction.
Figure 3: Assembly of glassware for the synthesis of 4-acetamidobenzenesulfonyl chloride.

Caution: Make sure that the tap of the pressure equalising funnel is closed before you pour 8
mL of chlorosulfonic acid into the dropping funnel.
Set the reaction flask up in a cool water bath (not an ice bath) so that the temperature is maintained
at 15 to 20 C when you add chlorosulfonic acid from the pressure equalising funnel. Ensure that the
reaction mixture is stirred vigorously and the temperature is maintained below 20 C.
Once the acetanilide has dissolved and the exothermic reaction has subsided, warm the reaction to
70-80 C and stir at this temperature for 20 mins.
Remove the reaction from the heat and allow it to cool to room temperature before placing the vessel
in an ice bath.
Place 150 g of crushed ice in a beaker while the reaction is cooling, then very carefully and slowly
pour the reaction mixture over the ice.
(CAUTION! THE REACTION MIXTURE MUST BE POURED SLOWLY TO AVOID SPLAT-
TERING).
Stir the ice slurry with a glass-stirring rod to prevent the formation of large lumps as the product
precipitates. If large lumps form, carefully break them apart with the stirring rod.
Collect the product using vacuum filtration and wash repeatedly with 15 mL portions of cold water
until the filtrate tests neutral to pH paper. Make sure the product is washed thoroughly as all the excess
acid must be removed prior to the next synthetic step.
Air dry the product on your filter funnel for at least 10 mins. The sulfonyl chloride is susceptible to
hydrolysis and must be used immediately in the following reaction.
Move on immediately to the synthesis of 4-acetamidobenzene sulfonamide. It is not necessary
to prepare characterisation samples of this material or record a precise yield for this step.
2.3 Step 3: Synthesis of 4-acetamidobenzenesulfonamide
TO BE CARRIED OUT IN THE FUME HOOD
!
Figure 4: Reaction scheme for synthesis of 4-acetamidobenzenesulfonamide.
Place the crude 4-acetamidobenzenesulfonyl chloride obtained in step 2 into a 125 mL Erlenmeyer
flask and slowly add 15 mL of concentrated (28%) aqueous ammonia. (Caution! When adding the am-
monia a vigorous reaction may occur if the crude material was not washed sufficiently to remove
strong acid impurities!)
Following the addition of the ammonia solution the reaction mixture should be a thick suspension.
Stir the suspension and make sure to break up any lumps.
Heat the suspension to 70-80 C for 30 mins.
Cool the mixture to room temperature then place the flask on an ice bath to complete precipitation.

Collect the precipitated product using vacuum filtration, washing the filter cake with ice-cold water.
Allow the solid to air dry on the filter before calculating final yields.
Record the % yield of your product and prepare samples for spectroscopic characterisation
before moving on to the final step.
tubes and deuteriochloroform (CDCl3 ) or D6 -DMSO can be obtained from your demonstrator.
Questions:
1. Propose a mechanism for the ammonolysis of 4-acetylaminobenzenesulfonyl chloride.
2.4 Step 4: Synthesis of Sulfanilamide
Session 3
HN NH3Cl NH2
HCl/H2O Na2CO3
reflux H2O
O S O O S O O S O
NH2 NH2 NH2
Figure 5: Reaction scheme for synthesis of sulfanilamide.
Weigh the 4-acetamidobenzene sulfonamide you prepared in the previous step and place it into a 50
mL QFB19l round bottom flask along with a magnetic stirrer bar.
Measure out 6 M hydrochloric acid equal to twice the weight of the 4-acetamidobenzene sulfonamide
(or a minimum volume of 2 mL) and add to the flask.
Fit the round bottom flask with a condenser and set up for reflux (check all water lines are attached
securely before starting water flow). Heat the reaction mixture to reflux with constant stirring for 1 hr.
After 1 hr allow the reaction mixture to cool to room temperature.
Once cooled, transfer the reaction mixture to a glass beaker and neutralise the reaction mixture
through slow addition of saturated aqueous Na2 CO3 until the solution tests slightly alkaline to pH paper.
After the solution has been neutralised, cool the beaker in an ice bath to promote precipitation (some
precipitate may form during the neutralisation step). If required scratch the inside of the beaker with a
glass rod to promote crystallisation.
Collect the product using vacuum filtration and wash the filter cake with a small amount of ice-cold
water then air-dry.
Calculate the crude yield of sulfanilamide and purify by recrystallisation from hot water.
Dissolve the crude material in a minimum amount of boiling water, remove from the heat and allow
the solution to cool slowly to room temperature. Transfer the flask to an ice-bath to promote crystallisa-
tion of as much product as possible before collecting the recrystallised solid using vacuum filtration.
Once your product has dried, record the weight, % yield and prepare samples for characterisation.

your product is to be run on the desktop NMR spectrometer in room 380 (the balance room). NMR
Be sure to transfer your purified sulfanilamide sample to a pre-weighed sample bag and label
clearly with your name and sample information.
Questions:
1. Provide a mechanism for the formation of sulfanilamide.
2. Calculate your overall yield for the multi-step synthesis of sulfanilamide from aniline.
3. Write acid-base chemical equations to explain the solubility of sulfanilamide in both dilute NaOH
and HCl.
4. Starting with nitrobenzene and any commercially available amine. Propose a reasonable synthetic
methodology for the preparation of the anti-malarial sulfadiazine (pictured in the background
information at the beginning of this section).
5. As discussed in the introduction to this section the antibacterial effect of sulfonamides is attrib-
utable to their ability to inhibit folic acid synthesis within bacteria. The potency of sulfonamides
as antibacterial agents was found to increase when analogues were synthesised having pKa val-
ues closer to that of PABA (6.5) when compared to sulfanilamide (pKa = 10.4). One of the
most widely used sulfa drugs, sulfisoxazole (Figure 6) has a pKa of 5.0 attributable to the res-
onance stabilisation of the sulfioxasole conjugate base. Draw out the resonance structures for the
conjugate base of sulfisoxazole.
!
Figure 6: Sulfisoxazole.
6. In addition to increased binding affinity and therefore increase efficacy the change in lowering
of pKa values in newer generation sulfa drugs has alleviated earlier solubility issues. Under bio-
logical conditions sulfanilamide precipitated in the kidneys. Explain the increased solubility ob-
served with newer generation sulfa drugs such as sulfisoxazole.
7. You should provide NMR, IR and mass spectral analyses in your report.
3 References:
1. John C. Gilbert and Stephen F. Martin, 2002, Experimental Organic Chemistry: a miniscale and
microscale approach.

T1.1 - Synthesis of Prontosil & Carbonic
Anhydrase Binding Studies
Dr. Chris Donner & A/Prof. Brendan Abrahams

This experiment will be assessed by a presentation.
1 Introduction
Prontosil is a type of azo dye, which is produced by the diazotisation of sulfanilamide (para-
aminobenzenesulfonamide), followed by an azo coupling reaction with m-phenylenediamine.
Figure 1: Reaction scheme for synthesis of sulfanilaminde.
Prontosil was historically used for bacterial staining, however in 1935, Gerhard Domagk, in a desper-
ate attempt to save his ill daughter, discovered that the oral administration of prontosil cured streptococci
infections. However, when tested in vitro it was observed that all antibacterial activity was lost. The an-
tibacterial activity observed by Domagk actually arose from the metabolite sulfanilamide; the prontosil
was being reduced within the gut, producing the biologically active sulfanilamide and the remaining azo
functionality.
The discovery by Ernest Foueneau in 1936 that prontosil is metabolized to the active drug sulfanilam-
ide led to the now commonly used term pro-drug and the concept of bioactivation. A prodrug may
be defined as a drug that is delivered in an inactive form, and is metabolised within the body into the
corresponding active compound. Strategies employed in rational drug design today employ the prodrug
concept to design structures that will potentially increase a drugs solubility, stability and activity.
Although it was determined that sulfanilamide was the active component responsible for the antibac-
terial properties of prontosil, both sulfonamides are potent inhibitors of the enzyme carbonic anhydrase.
Carbonic anhydrase catalyses the hydration of carbon dioxide to give bicarbonate ion:
The enzyme is especially active in tissues involved in respiration. One litre of mammalian blood
can contain up to 1-2 grams of this enzyme. Carbonic anhydrase is a protein of MW approx 30,000
and contains approx 260 amino acids and a tightly bound Zn 2+ ion within its catalytic site (see cartoon
below).
207
Figure 2: The active site of bovine carbonic anhydrase (determined by X-ray crystallography). Carbonic
anhydrase (CA) is a powerful catalyst with a turnover rate at 25 C for hydration of carbon dioxide of
106 s1 . In addition to catalysing the hydration of CO2 , CA can catalyse the hydration of acetaldehyde,
however the rate is 1000-fold lower for this reaction.
It is generally assumed that a proton is lost from the water bound molecule to give Zn-OH, which is
in effect a stabilised form of OH , that can exist at low pH values where OH is not normally present.
Nucleophilic attack on CO2 by the hydroxide ion results in bicarbonate formation.
Zinc plays an essential role in many enzymes including a variety of dehydrogenases, aldolases,
peptidases and phosphatases. Zn 2+ has a filled 3d shell and usually forms four bonds with tetrahedral
symmetry, often with nitrogen- or sulfur-containing ligands. A common feature in zinc metalloenzymes
(Figure 2) is the surrounding of the Zn 2+ at the active site by the three imidazole groups from adjacent
histidine residues. This leaves the fourth zinc coordination position free to interact with a substrate.
Generally Zn 2+ in metalloenzymes plays the role of a Lewis acid.
1.1 Visible Absorption Spectra
In this experiment we will use the optical properties of the free and enzyme-bound substrate to
investigate the binding properties of the carbonic anhydrase enzyme.
As a general rule, the wavelength of UV (or visible) absorptions depends on the extent of conjuga-
tion: the more (longer) the conjugation, the longer the wavelength absorbed and the larger the extinction
coefficient. This can be seen in the examples provided below.
If coplanarity of the -system is prevented (by steric interference for example), the intensity of
the transition is reduced and the wavelength of the light absorbed is consequently reduced
(hypochromic shift) with a corresponding reduction in the extinction coefficient. This can be seen for
the examples of cis and trans stilbene.

1.2 Experiment Aims:
1. To synthesise the azosulfonamide prontosil.
2. To use UV spectroscopy to study the binding properties of the enzyme carbonic anhydrase with
the sulfonamide prontosil.
3. To determine the binding ratio between prontosil and the enzyme.
2 Experimental Procedures:
2.1 Prontosil Synthesis
In a 100 mL Erlenmeyer flask dissolve 0.60 g of m-phenylenediamine in 30 mL of water and add 4.0
g of sodium acetate trihydrate to the solution. Place the mixture on an ice bath and then start to prepare
the second solution as below.
In a 50 mL Erlenmeyer flask dissolve 1.0 g of p-aminobenzenesulfonamide (this is the sulfanilamide
you prepared in experiment T1.0) in 12.0 mL of dilute aqueous HCl (1 M). Place the solution on an ice
bath to cool.
Form the diazonium salt by adding 10 mL of an ice-cold solution of 0.7 M sodium nitrite to the
acidified p-aminobenzenesulfonamide solution, with constant stirring.
Slowly, with constant stirring, add half of the cold diazonium solution to the flask containing m-
phenylenediamine.
Adjust this mixture to a pH of 8 by adding saturated aqueous sodium bicarbonate solution. Continue
adding the remainder of the diazonium solution and again adjust to pH 8 with saturated aqueous sodium
bicarbonate solution.
Stir the reaction mixture for 15 min.
Collect the precipitated material using a Buchner funnel. Wash the filter cake thoroughly with cold
water until the filtrate is neutral (pH 7). This should require approx 200 mL of water.
Dry the crude product on the filter for at least 5 min.
Collect the deep orange powder and recrystallise 0.25 g of the crude product from a minimum
volume of hot ethanol/water (50:50). Dissolve in ethanol first, then add H2 O until cloudiness appears.
Solubility in ethanol is 0.5g/100mL. Read Appendix A for two-solvent recrystallisation technique.

Remember to keep a sample for characterisation before making the solutions for the binding
studies. You will require a melting point, IR, NMR and mass spectral data, which should be
provided to all members of your group.
2.2 Enzyme assay
Part 1: Visible Absorption spectra of free and enzyme-bound prontosil

The following reagents will be provided:
A. 0.05 M AMPSO buffer, pH 9.0 (AMPSO, an acronym for 3-[(1,1-dimethyl-2-hydroxylethyl)amino]-

2- hydroxypropanesulfonic acid)
B. 1104 M Bovine Carbonic Anhydrase (BCA) in 0.05 M AMPSO buffer, pH 9.0
C. 5105 M BCA in 0.05M AMPSO buffer, pH 9.0
Keep the enzyme solution in the ice bath, adding only the amount required into each tube just
prior to taking the absorbance reading.
You will need to prepare:
D. 25 mL of a 1103 M stock solution of 2,4-diaminoazobenzene-40 -sulfonamide (prontosil) in eth-

anol
E. 2105 M prontosil working solution in 0.05 M AMPSO buffer (dilute 1 mL of solution D to 50

mL with 0.05 M AMPSO buffer)
You will be recording the visible absorption spectra of a free prontosil solution (from prontosil
prepared in your group) and a solution of enzyme-bound prontosil.
For free prontosil solution mix 3.0 mL of your 2105 M prontosil (solution E) and 1.0 mL AMPSO
buffer. For the enzyme-bound prontosil solution mix 3.0 mL of the prontosil solution (solution E) with
1.0 mL of the 1104 M BCA solution (solution B). The mixture should be mixed gently and thoroughly
by inversion, using a small square of parafilm to seal the tube using pressure from your thumb. Do not
shake vigorously as this can cause the enzyme to denature!
Record the absorption spectra for the free prontosil and enzyme-bound prontosil between 550 and
350 nm.
Plot your results for absorbance versus wavelength for the free prontosil and the enzyme-prontosil
complex as an overlay on the same page. Obtain the max for the free prontosil.
Part 2: Titration of BCA active site with prontosil
The hypochromia (reduction in absorbance) observed on formation of the prontosil-enzyme complex
can be used as a basis to titrate the enzymes binding site(s). This technique allows calculation of the
binding ratio between the enzyme and the ligand (prontosil).
First you must determine the optimal max for the titration. This is the wavelength where the dif-
ference in absorbance between free prontosil and the enzyme-prontosil complex is the greatest and will
provide the greatest sensitivity for the titration. Determine max by inspection of the plots of absorbance
versus concentration obtained in Part 1 and set the spectrophotometer to record at this wavelength.
Keep the enzyme stock solution in the ice bath, adding only the amount required into each
tube just prior to taking the absorbance reading.
Prepare a series of test tubes (numbered 1-8) containing 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mL
respectively of the 2105 M prontosil solution (solution E) prepared in Part 1. Add sufficient AMPSO

buffer (solution A) to each of the test tubes to make a final volume of 4.0 mL. When you are ready to
take your reading add 1.0 mL of the 5105 M BCA solution (solution C) to each test tube, mix the
solution by gentle inversion and record the absorbance of the solution at the predetermined max value.
3 Data analysis and report questions
1. Plot the results you obtained for the titration experiment as a function of absorbance (A at max )
versus [prontosil].
2. For the plot you have drawn what does the gradient of the line for absorbance versus [prontosil]
represent?
3. From your plot of absorbance versus molar concentration of prontosil, what can you conclude?
4. What are the molar concentrations of prontosil and BCA at the equivalence point? What can you
conclude from this?
5. If your BCA solution had been heated briefly (i.e. some denaturing of BCA had occurred), predict
what effect would this would have on your plot of absorbance versus molar concentration of
prontosil and on the resulting binding ratio?
6. Provide a mechanism for the synthesis of prontosil from sulfanilamide (remember this is a two-
step process; provide mechanisms for both steps).
References & Acknowledgements
1. This module has been adapted from the Journal of Chemical Education 66(7), 609 (1989). As-
sociate Professor Chris Brown from Griffith University is acknowledged for assistance in the
development of this experiment.
2. McMurray, Organic Chemistry, 8th Edn., pp 517 - 521
3. McMurray, Organic Chemistry, 8th Edn., pp 968 - 972
4. Norman and Coxon, Principles of Organic Synthesis, Sections 13.1 and 13.4

T1.2 - Removal of Zn(II) From Carbonic
Anhydrase - A Kinetics Experiment

1 Introduction
As outlined earlier in this section, Carbonic anhydrase (CA) catalyses the inter-conversion of carbon
dioxide and bicarbonate, an essential reaction in respiration and metabolism. While for the most part
we have discussed the role of CA inhibitors in disease treatment Carbonic anhydrase deficiency can also
result in a number of disease states including renal tubular acidosis and osteopetrosis (abnormally dense
bones, a condition opposite of osteoporosis).[1]
Carbonic anhydrase has been studied for many years,[2] and a number of investigations have fo-
cussed on the role of the Zn2+ cofactor, which is essential for catalytic activity. By removing the Zn2+
and substituting it with other metal ions such as cobalt, researchers are able to probe the structure and
function of the enzymes active site.
The Zn2+ can be removed by exchange reactions with other ligands, such as 1,10-phenanthroline,
EDTA, a common chelating agent and pyridinecarboxylates. In this experiment you will use 2,6-
pyridinedicarboxylate, a highly efficient compound for the removal of zinc.[3] While EDTA could be
used in a similar fashion for this experiment the rate of Zinc removal would be much slower.
Figure 1: 2,6-pyridinedicarboxylate (dipic).
Unlike the enzyme kinetics experiment offered in the second year lab course, this experiment studies
the rate of Zn2+ removal from the enzyme active site by the ligand, rather than an investigation of the
catalytic role of the enzyme itself. Several groups[4, 5, 6, 7, 8] have studied the CA-Zn / dipic exchange
reaction, using varying methods, the conditions outlined in this experiment most closely correspond to
those of Kidani and Hirose.[4, 5] When collating your results for your report be sure to compare your
observations with those reported in the literature.
2 Background Theory: Kinetics
According to Michaelis-Menten kinetics, when dipic is present in large excess ([dipic]/[CA] 25),
the reaction can be considered to be pseudo-first-order with respect to CA-Zn:
213
d[apoCA]
= kobs [CAZn] (1)
dt
where:
CAZn represents the active enzyme
apoCA is the inactive enzyme where the Zn2+ ion has been removed
kobs is the pseudo-first order rate constant.
The value of kobs increases as the concentration of dipicolinate increases but will level off at high
dipic concentration.
This behaviour indicates that the dipicolinate (given the symbol L) and CAZn first form a ternary
CAZnL complex, which slowly breaks apart to form the zinc-dipicolinate complex and apoCA. The
proposed mechanism is provided below:
KEML
CAZn + L CAZnL (where the back reaction is fast) (2a)
d k
CAZnL apoCA + ZnL (where kd is slow) (2b)
Once formed, the ZnL complex rapidly complexes with an additional dipicolinate to form ZnL2 .
The rate of formation of apoCA is thereby determined by step 2b and can be written as:
d[apoCA]
= kd [CAZnL] (3)
dt
According to reaction 2a:
[CAZnL]
KEML = (4)
[CAZn][L]
Since dipic is present in large excess, [L] remains at its initial value, [L]0 , and equation 4 can
therefore be rearranged to give:
[CAZnL] = KEML [CAZn][L]0 (5)
To obtain [CAZn], the concentration of apo-enzyme formed and the concentration of enzyme tied
up as CAZnL must be subtracted from the initial concentration, [CAZn]0 :
[CAZn] = [CAZn]0 [apoCA] [CAZnL] (6)
Substituting for [CAZnL] gives:
[CAZn] = [CAZn0 [apoCA] KEML [CAZn][L]0 (7)
Equation 7 can be rearranged to give:
[CAZn]0 [apoCA]
[CAZn] = (8)
1 + KEML [L]0
Combining equations 3 and 5 the reaction can be shown to be first-order with respect to CAZn
according to equation 9:

d[apoCA]
= kd kEML [L]0 [CAZn] (9)
dt
Substitution of equation 8 into equation 9 gives:
d[apoCA] [CAZn]0 [apoCA]

= kd kEML [L]0 (10)
dt 1 + KEML [L]0
Equation 10 can be integrated to give:

[CAZn]0 [apoCA] kd kEML [L]0t
ln = (11)
[CAZn]0 1 + KEML [L]0
[CAZn]0 [apoCA]
Given that [CAZn]0 is the fraction of CAZn remaining (FCAZn ), we can rewrite the expression
as:
kd kEML [L]0
ln (FCAZn ) = .t (12)
1 + KEML [L]0
Thus, a plot of ln(FCAZn ) versus time should be linear with slope (= -kobs ) given by:
kd kEML [L]0
slope = kobs = (13)
1 + KEML [L]0
To determine the value of kobs , a known amount of dipic is added to a solution of CAZn at t0 .
Aliquots are withdrawn at suitable time intervals (t), and the amount of CAZn remaining is measured.
We can then plot lnFCAZn versus t with the slope is equating to kobs .
Referring to equation 13, both sides can be inverted to give:
1 1 1 1
= . + (14)
kobs kd kEML [L]0 kd
To obtain kd and KEML , kobs is measured for several dipicolinate concentrations ([L]0 ). Plotting
1/kobs versus 1/[L]0 should result in a straight line with a slope equal to 1/kd KEML and an intercept of
1/kd .
2.1 Rate Measurements
Equation 12 is valid for any quantity proportional to [CAZn], for example, the enzymatic activity.
For a typical enzyme-catalysed reaction, at high concentration of substrate, S, the velocity, V, is inde-
pendent of [S] and given by Vmax = kcat [E], where kcat is the turnover number, and [E] is the total enzyme
concentration. Thus, Vmax is directly proportional to [E].
Although the native enzyme substrate can be used, frequently (in the case of CA this would be
CO2 ) the enzyme also catalyses other reactions, which are easier and/or less expensive to perform. For
carbonic anhydrase, a convenient assay reaction is the hydrolysis of p-nitrophenyl acetate (pNPA) as
outlined below.

The product, p-nitrophenol, absorbs strongly at 348 nm, allowing for the monitoring of its formation
using standard UV spectrometry.
The reaction velocity (which is really Vmax , because pNPA is in large excess) is proportional to
A/t, the slope of an absorbance/time plot, which canbe obtained
directly from the spectrophotometer.
A
Thus, equation 12 can be implemented by plotting: ln At (or simply ln At ) versus time.
( t )0
Because the hydrolysis of pNPA occurs to a small extent without
carbonic anhydrase, a baseline
correction will be required. This is usually determined as A
t , the assay velocity after complete
2+
removal of Zn from the enzyme.
(Note: Be careful. The t in equation 12 is the elapsed time for the reaction of dipicolinate with
CAZn. The t in dA
dt corresponds to the reaction time for the pNPA hydrolysis.)
As a group you will need to perform 5 kinetics experiments with the same concentration of Bovine
Carbonic Anhydrase (CA) and varying 2,6-Pyridinedicarboxylate (dipic) concentrations of 0.20 M, 0.10
M, 0.048 M, 0.032 M and 0.016 M, maintaining total volumes of 500 L. The following solutions will
be provided in the kit for this experiment:
A. Bovine carbonic anhydrase (ca. 3104 M in 0.125 M phosphate buffer, pH 7.4). This solution
must be kept on ice to prevent the enzyme denaturing (1.2 mL in an eppendorf vial).
B. Phosphate buffer (0.125 M, pH 7.4), (12 mL in a labelled tube).
C. 2,6-Pyridinedicarboxylate (dipic) (0.40 M in 0.125 M phosphate, pH 7.4), (1.2 mL in a labelled

eppendorf vial).
D. HEPES buffer (0.25 M, pH 8.0), (12 mL in a labelled tube).
E. p-nitrophenyl acetate (pNPA) (2103 M), (12 mL in a labelled tube) freshly prepared 30 minutes
before class. This is the substrate. The hydrolysis of pNPA will occur without the presence of the
enzyme (albeit at a much slower rate)) so you will need to work quickly in order to obtain accurate
results.
**Before commencing your kinetics runs make sure the table of sample solutions for different DIPIC
concentrations is filled out. One entry is completed for you.
Table 1: Final Dipic concentration and volumes in 500 L reaction mixture.
Final Dipic CA stock Phosphate buffer stock Dipic stock Total volume
concentration 3104 M 0.125 M, pH 7.4 0.4 M
[M] L L L L
0.016
0.032
0.048
0.100 250 125 125
0.200

Preparation of the solutions.
1. The first kinetics run will contain 0.10 M dipic, which gives a convenient rate of Zn(II) removal.
Place 250 L of the CA solution and 125 L of the pH 7.4 phosphate buffer (no dipic yet) into a
clean microfuge tube. Place the tube into a 25 C water bath to equilibrate.
2. While the CA is equilibrating, begin preparing cuvettes (10 at a time is suggested) for the assays.
To each cuvette, add 1.7 mL of DI water, 0.3 mL of HEPES buffer, and 1.0 mL of pNPA.
3. Prepare the Novaspec UV/Vis spectrophotometer for measurements at 348 nm. Absorbance read-
ings should be acquired at 5-second intervals for a total of 100 seconds. Check with your demon-
strators if you are unsure on how to do this.
Baseline Correction
4. To obtain the un-catalysed assay velocity, add 40 L of water to one of the assay cuvettes, quickly
invert it several times using a piece of Parafilm between your finger and the top, place it into the
sample compartment, and initiate the time-based absorbance readings.
5. When the measurements are complete, obtain the average A t over the 100-second observation
period (should be very small for the uncatalysed reaction), or plot A versus t and obtain A
t from
the slope.
Kinetics Runs
6. The CA should now be equilibrated. Have a timer ready. Add 125 L of the 0.40 M dipic to the
CA tube, and mix it gently. Start the timer. After 1 minute, repeat step 4 with 40 L of the reaction
mixture (rather than water). This time the absorbances should show a linear increase.
7. Using the method from step 5, obtain the average A t . Usually the plot is linear for the entire
100-second period, but if it shows signs of curvature, use only the initial linear part (e.g. 0 to 50
seconds).
8. Repeat the assays at reasonable intervals for about one hour. Suggested intervals (as total elapsed
time) are: 1 (already obtained), 4, 8, 12, 18, 24, 32, 42, and 55 minutes. If the final average A
t is
not very close to the uncatalysed velocity from step 5, perform another assay at a longer time to
obtain the At value.
9. Perform 4 additional kinetics experiments with the same concentration of CA and dipic concen-
trations of 0.20, 0.048, 0.032, and 0.016 M, maintaining total volumes of 500 L. (For example,
to obtain 0.048 M dipic, equilibrate 250 L of CA with 175 L phosphate buffer, and add 62.5
L dipic to initiate the reaction.) Prepare more assay cuvettes as needed. Use good judgment in
choosing the assay intervals, especially for the 0.20 M dipic run. If it is not
possible to obtain an
A
infinity-time assay for the slower runs (due to time constraints), the t from a faster run can
be used in the calculations.
10. When all measurements are completed, ensure you save all your data. Copy and share your results
with all the other members in the group. Clean all cuvettes and discard used pipette tips in the bin.
4 Data Analysis and Questions:
1. For each of your runs, correct the average A A

t values for the residual t at infinite time to obtain
A

t corr . Record these in a table (a suggested format may be provided) and include these data in
your report.

2. Plot A
t corr versus the reaction time.

3. Use the slopes of the lines to determine kobs .

1
4. Prepare a plot of kobs versus [L]0 . Obtain kd and KEML .
5. Compare your results with those reported in the literature (Kidani and Hirose).[5] (Note: The
authors use the symbol k2 instead of kd in their calculations.)
References
[1] Bhagavan, N.V Medicinal Biochemistry, 4th ed.; Harcourt/Academic Press: San Diego, 2001; p
890.
[2] Lindskog, S.; Malmstrm, B.G. J. Biol. Chem. 1962, 237, 1129-1137.
[3] Hunt, J.B.; Rhee, M-J.; Storm, C.B. Anal. Biochem. 1977, 79, 614-617.
[4] Kidani, Y.; Hirose, J. J. Biochem. 1976, 79, 43-51.
[5] Kidani, Y.; Hirose, J. J. Biochem. 1977, 81, 1383-1391.
[6] Pocker, Y.; Fong, C.T.O. Biochemistry 1980, 19, 2045-2050.
[7] Pocker, Y.; Fong, C.T.O. Biochemistry 1983, 22, 813-818.
[8] Hendry, P.; Lindoy, L.F.; Yellowlees, D. Inorg. Chim. Acta-Lett. 1982, 65, L237-L239.
[9] Williams,K.R.; Adhyaru, B., J. Chem. Edn., 2004 , 81, 1045-1047.

T1.3 - Three Dimensional Analysis of the
Carbonic Anhydrase Active Site

Analysis of crystallographic data for a small molecule: Sulphanilamide
Refer to Appendix B for information relating to X-ray crystallography
1 Introduction
Much of our knowledge of structural chemistry has come from the technique of single crystal X-
ray diffraction, which exploits the regular periodic nature of crystals. When exposed to radiation of an
appropriate wavelength (comparable to atomic dimensions) a crystal behaves as a 3D diffraction grating.
The technique of X-ray diffraction generally involves directing a monochromatic beam of X-rays at a
crystal (typically with dimensions less than 0.5 mm) and measuring the intensities and positions of the
scattered X-rays.
In a crystal, reflections of X-rays occur from families of parallel, regularly spaced planes that are
defined by three integers, known as Miller indices. The reflected X-rays from a particular set of planes
are labelled with the Miller indices corresponding to that family of planes. The intensity of the reflected
X-ray is related to the amount of electron density in a particular family of planes. By measuring the
intensity and position of many hundreds, often thousands of reflected X-rays we can obtain a 3D picture
of the distribution of electron density (ie. the atomic arrangements) within the repeating unit of the
crystal.
1.1 Structure Solution
In order to solve a crystal structure we need to know not just the intensity of each reflection but also
the relative phase of each reflection. The problem is that we cannot determine the phase of a reflection
from experimental measurements. This is known as the phase problem. To overcome this problem
the technique of direct methods is often employed which is a method of deriving relative phases by
consideration of relationships between Miller indices. For the purposes of this practical exercise we will
treat the direct methods approach as a black box structure solution method.
In the structure solution, peaks of electron density are identified. Generally, the height of a peak may
be related to the atomic number of the atom that the peak represents. Some peaks correspond to ghost
atoms, that is, they are not real atoms. We use our knowledge of chemistry to decide what peaks are
chemically reasonable. If the solution process is successful we would normally expect to identify the
positions of most of the non-hydrogen atoms in the structure.
The portion of the structure that we assign is known as the asymmetric unit. When the symmetry
operations associated with the space group are applied to the asymmetric unit, the contents of the unit
cell are generated. Since the crystal is made up of identical unit cells the combination of the asymmetric
unit with space group provides us with the full crystal structure.
219
1.2 Structure Refinement
The structure solution should provide the coordinates of most non-hydrogen atoms in the crystal
structure. Using these atomic positions it is possible to produce a model for the crystal structure
from which we are able to produce calculated intensity data for all the reflections. By comparing the
calculated diffraction data with the experimental diffraction we can obtain an indication of the agreement
between the two sets of data. In a least squares procedure our model is refined so that the difference
between the calculated and experimental diffraction data is minimised. The agreement between the
calculated and experimental diffraction is indicated by an agreement value, R.
This process of least-squares refinement is normally followed by the generation of a difference Four-
ier map that often reveals the position of the remaining non-hydrogen atoms. Hydrogen atoms may also
begin to appear at this stage.
Along with the coordinates, each atom is refined with an isotropic displacement parameter (also
known as thermal parameter) that provides an indication of the size of the sphere within which the
nucleus is found. The greater the vibration of the atom, the larger the displacement parameter. Typically,
displacement parameters fall in the range 0.01 to 0.08. If the identity of an atom has been incorrectly
assigned then the displacement parameter will often (but not always) have an unreasonably high or low
value. Once all the non-hydrogen atoms have been located it is common to apply anisotropic (i.e. not
the same in all directions) displacement parameters. When these are applied, the boundary of each atom
is defined by an ellipsoid rather than a sphere. Finally, hydrogen atoms are included in the model at
geometrically estimated positions. For hydrogens attached to oxygen atoms this can be difficult because
the H atom may be disordered over a number of positions.
The refinement is said to reach convergence when the parameters (coordinates and displacement
parameters) in the least squares refinement do not change.
A more in depth discussion the theory and instrumentation involve in X-ray crystallography
can be found on the LMS.
This exercise involves structural analysis using computer programs, however no prior knowledge of
computers, beyond the basics, is assumed.
2 Crystallographic Experimental Data
2.1 Analysing the Reflection Data and Determination of Space Group
On the desktop of the PC there is a folder labelled Prac ***. Open the folder for your group e.g.
Tue 2. There is a folder labelled SULFAN there is a file called sulfan.hkl which contains the reflection
data. Open this file.
Part of the file is represented here:
5 3 -1 2.69 0.96
5 3 -1 2.96 0.66
-5 -3 2 91.63 3.42
-5 -3 -2 90.28 3.94
-5 3 2 93.08 4.09
Table 1: Crystallographic data for sulfanilamide.
The first three numbers of a line represent the Miller indices of a reflection. The next value, F 2 , is
related to the intensity and the fifth number is the value (standard deviation) associated with the F 2 .

We do not need to be concerned with the final value. If the value of F 2 is not at least three times
then the reflection is considered to be either weak or absent. If the reflection is absent it may be part of
a set of systematic absences. Systematic absences arise because the symmetry in the unit cell leads to
complete destructive interference of some classes of reflection. Systematic absences can give valuable
clues as to the symmetry within the unit cell.
Scroll through the data and locate approximately ten reflections of the type 0 k l i.e. reflections
with Miller indices such as 0 1 5, 0 1 6, 0 2 3 etc. Record the F 2 and values for these reflections. There
is no need to consider reflections where k or l >10. You will find that only when k is an even number
is this class of reflection present.
The systematic absences for the 0 k l reflections can be summarised as:
0 k l: k = 2n
This means that for reflections of the type: 0 k l, only when k is an even number is the reflection
present. Now check systematic absences for reflections of the type h 0 l and h k 0. What are the
reflecting conditions for these two classes?
These three sets of systematic absences uniquely identify the space group as Pbca. This symbol
means that the unit cell is primitive (P ), and that within each unit cell there are b -glide planes, c-glide
planes and a-glide planes. A glide plane is a symmetry operation that when applied to a set atoms
results in an equivalent set of atoms in a position related to the first by a reflection and a translation. In
addition to the glide plane, there are two fold-screw axes (a 2-fold axis is a rotation of 180 followed by
a translation) and centres of inversion.
These symmetry transformations, eight in all in this space group, are listed in International Tables -
a crystallographic resource book that gives the symmetry operations and systematic absences associated
with each of the possible 230 space groups. The data for the space group, Pbca, will be provided.
2.2 Solving the Structure
In this exercise we will use a program (SHELX-97) to solve the crystal structure. The program
requires a data file (sulfan.hkl) that contains the reflection data and an instruction file that contains
information about the crystal e.g. cell dimensions, symmetry etc. A template for this file has already
been created. You will need to fill in the missing information.
Open up the file sulfan.ins. To do this click once on the file and when it is highlighted right click the
mouse and open the file using WordPad.
The first line of the file is a title. Make up a title that includes your names e.g.:
TITL Chris Tell and Ray DAtions sulfanilamide in Pbca
and the cell dimensions (in Aand

On the next line enter the wavelength of the radiation (0.71073 A)
degrees) in the order a, b, c, , , .
In an orthorhombic cell all angles (,
For this cell a = 14.752(4), b = 5.6307(14), c = 18.462(5) A.
and ) are 90 .
Your CELL line should read:
CELL 0.71073 14.752 5.6307 18.462 90 90 90

The next line beginning with ZERR provides information about the expected number of formula
units in the unit cell (Z ). As a guide it is common to use the number of symmetry operations associated
with the space group as the value of Z. The rest of the line lists the error values associated with the unit
cell dimensions and angles.
Your ZERR line should read:
ZERR 8 0.0012 0.0012 0.0008 0 0 0
LATT gives information about the type of cell. The cell is primitive and centrosymmetric which
corresponds to a value of 1. We now list the symmetry operations associated with the space group.
There are eight symmetry operations associated with Pbca but we only list three because x, y, z is
assumed by the program and the fact that it is centrosymmetric means that once four are given (including
x, y, z) the other four are implied. The symmetry operations for Pbca are from the book International
Tables. A photocopy of the relevant page is supplied.
Give one symmetry operation on each line, for example:
SYMM 1/2 - x, -y, 1/2 + z
On three separate lines list the 2nd , 3rd and 4th symmetry operations as listed in International Tables
(Pbca is space group number 61).
Different atoms scatter X-rays to different extents. On the SFAC line we indicate what elements may
be present in the crystal. (SFAC represents scattering factors).
The SFAC line should read:
SFAC C H N O S
This should be followed by the UNIT line, which provides an estimate of the unit cell contents with
respect to each type of atom listed in the SFAC line:
UNIT 48 64 16 16 8
The next line indicates what technique will be used to solve the structure. We will be using direct
methods, which will be indicated by the instruction: TREF. TREF should be followed by a number that
represents the number of direct method attempts. Try a number like 100. The solution with the best
CFOM (combined figure of merit) is the one we will use.
The next line indicates the format of the reflection data. The format type is 4. This line becomes:
HKLF 4
Finally the file ends with an END line.

Once you have completed the file, close it and indicate that changes should be saved. Sometimes
you may get a warning about possible changes in format during the save - do not worry about this, save
it anyway.
We are now ready to run the program. Double click on the Wingx32 icon on the desktop. Under
the File menu select Job paths and then select Select Work Folder . When the Select Work Folder
and Jobname window opens, select browse. Find your recently edited instruction file and click open.
Once your file has been selected, click OK to close this window. Under the SOLVE menu, select
SHELXS-97 and solve the structure.

The results of the attempt to solve the structure may be viewed using the Graphical Model Editor
which is the 3rd icon from the left on the WinGX menu bar. A PowerPoint presentation is available
to help you work through the solution and refinement of the crystal structure. After you have assigned
atoms, the refinement process can begin. In the refinement process the following procedure is normally
used.
1. Identify any ordered non-hydrogen atoms not found in the structure solution.
2. Identify disordered non-hydrogen atoms. These will normally be refined with partial site occu-
pancies.
3. Apply anisotropic displacement parameters to ordered non-hydrogen atoms.
4. Place ordered hydrogen atoms at geometrically estimated positions.
5. Check for convergence. The maximum shift/esd should be <0.01.
6. On the final refinement run include the ACTA command after the PLAN line.
The inclusion of the ACTA command results in the generation of a CIF which is an abbreviation
for Crystallographic Information File. This summarises all the important crystallographic information
in a form that many programs can read. It is also the preferred format for submission to journals and
crystallographic databases.
2.3 Analysis of Structure
Structural data such as bond distances and bond angles may be obtained using the graphical editor.
In answering the questions below it may be useful to draw one or more labelled figures of the structure.
i) Give the range of C-C bond distances and C-C-C bond angles in the molecule. Typically, C-C bonds
whereas the C=C separation in alkenes is 1.33 A.
in alkanes are 1.54 A, Comment on the C-C
bond lengths found in sulphanilamide compared to those found in alkanes and alkenes.
ii) The conformation of the molecule adopted in the crystal structure shows one of the oxygen atoms
lying close to the plane defined by the 6-membered ring. Suggest a reason why this particular
conformation is adopted.
iii) The nitrogen atom bound to the sulfur atom adopts a pyramidal shape while the nitrogen atom bound
to the 6-membered ring is trigonal. Provide an explanation for the adoption of these geometries at
the nitrogen centres.
3 Molecular visualisation of the Carbonic Anhydrase active site using on-

line tools: Deep View
3.1 Overview
During this exercise you will investigate the active site of carbonic anhydrase, it is important that you
pay particular attention to the metal binding site, nearby amino acid residues of the protein interacting
with the metal and coordination geometries of inhibitors, in particular, sulfonamides.
The images you generate can be included in your group report and should be used to illustrate key
aspects of your discussion. The software used in this exercise is public domain software and while quite
easy to use is a powerful tool for the viewing and manipulation of enzyme / protein structures. Deep

TM TM
View is available for Windows , Apple Macintosh or Linux computer operating systems. You are
encouraged to download the software for your personal computers and investigate the software and its
full capabilities outside of the prescribed workshop exercise. A full user guide and tutorials are located
at the Swiss PDB Viewer Deep View web site located at: http://spdbv.vital-it.ch/.
3.2 Prelab Questions and Exercises
1. Draw a mechanism for the carbonic anhydrase catalysed hydration of carbon dioxide to bicarbon-
ate outlined in the introduction to experiment T1.1, include the relevant amino acid side chains
and their role.
2. It is recommended that you read through the introductory section of the Deep View user guide
prior to commencing this exercise. The sections relating to the working environment (pages1-3)
and basic commands (pages 15-34) will be particularly useful.
4 Deep View Exercise: Basic Controls and Navigation
4.1 Structure Search
Before loading the Deep View software you will need to download a structure file for carbonic
anhydrase.
The RCSB Protein data bank (PDB) is a repository of experimentally determined X-ray crystal
structures and NMR structures of proteins and protein ligand complexes. It is freely available and can
be easily searched to find the 3D coordinates of each protein with the coordinate files presented in a
standard PDB format.
Load a web browser and go to: www.rcsb.org/pdb. You should come to the RCSB web site that
will look similar to Figure 1.
Each structure within the repository is assigned a unique accession code. Whenever data and or
images are generated from a structure it is essential to quote the accession code for the PDB file used.
Structure files can be located on the PDB web site using this accession code.
If the accession code for a specific structure is unknown it is also possible to search for a protein
using a standard text search.
Use the keyword search to locate structures for human carbonic anhydrase II
How many structures do you find?
Repeat this search using the search term hCAII.
How many results did you obtain for this search?
There are many structures available for carbonic anhydrase, the accession code is the 4 alpha-
numeric code displayed next to each title. Clicking on the title or accession code of any file will bring
up detailed information for the entry. The tabs along the top of the page provide access to a wealth of
information regarding the structure file Figure 2.
Select any of the structures listed in your search results and investigate the details available for the
file.

Figure 1: Screen shot of the RCSB Protein Data Bank web site front page. It should be noted the exact
layout will change from week to week as the molecule of the month and featured molecules are changed
regularly.
When you have finished investigating the information tabs return to the original search page.
In instances where your search generates a large number of hits it is possible to refine your search
parameters in a number of ways. One useful tool when looking for enzymes with bound substrates is to
add complex into your search term.
Repeat the search for human carbonic anhydrase II, this time adding the term complex into your
keyword search.
How many results did you find?
Adding the term complex into the search will provide only those structures where the human
carbonic anhydrase enzyme has a substrate bound. This type of search is particularly useful when
looking to investigate inhibitors of enzyme systems.
For this exercise we will be using the PDB file 1CA2.
Perform a search for this structure and bring up the detail page for the file.
What is the primary citation for this structure?
Download the PDB file in text format using the links in the upper right corner of the page. Save
the file to your group folder on the computer.
Locate the Deep View program (Swiss Pdb-Viewer) icon in the applications folder on your com-
puter and start the program by double clicking the icon.
An alert will appear about screen colours press ok and move on.
An open file dialog box will appear.

Figure 2: Screen shot of the detailed information page available for structure files.
Open the file you downloaded (1CA2) in the previous step. If for any reason you do not see the
open file dialog box, or you pressed cancel. The PDB file can be loaded using the load command
under the file menu in the Deep View program.
Load the PDB file for carbonic anhydrase (1CA2.pdb) located in your group folder. Once loaded
the screen should look like the screen shot provided in Figure 3.
The top four buttons on the left hand side of the screen control movement and positioning of the
molecule within the workspace. Familiarise yourself with each of the buttons and their function.
! Clicking this button positions the image at the centre of the workspace on the screen.
! Sets the movement setting so you can move the protein by clicking on the structure
and moving the mouse.
! This button sets the mouse to controlling Zoom, allowing you to zoom in or out on
specific regions of the structure within the workspace.
! Pressing this button changes the mouse settings to control rotation of the image within
the workspace.
The remaining buttons along the toolbar include tools for the measuring of atomic distances, bond
angles and labelling of selected atoms. A full explanation of these functions can be found in the Deep
View user guide. The mutate residue and torsion functions will not be used in this exercise.

!
Figure 3: Screen shot of Deep View with 1CA2 loaded into the main display.
One of the benefits of using visualisation tools is it allows us to get a feel for things in three dimen-
sions.
The default display settings of Deep View provide a rudimentary wireframe representation of the
enzyme. The display can be changed so that the enzyme appears more three-dimensional. (Later
on in this exercise you will investigate additional display modes to create coloured and shaded
representations of the enzyme system).
Go to the Display menu and select Render in 3D. Also turn on Render in Solid 3D from
the same menu.
Bring up the alignment window (Wind>Alignment). This window shows the entire amino acid
sequence of the protein and allows specific residues to be highlighted.
Look through the alignment hovering your mouse over each of the tryptophan (W) residues
as you do. Do not click on the residue just hover the mouse over the W; you will see where
in the protein the residue is located, as it will be flashing.
Go to the Control Panel (Wind>control panel) from this window the residues within the protein
can be selected and the graphical representations of the residues or the whole protein within the
workspace can be controlled.
Remove the water molecules from the display by going to the end of the file in the con-
trol panel and selecting all the HOH residues. Be careful not to select the zinc atom. With
the water molecules selected, go to the Select menu and choose Inverse Selection (Se-
lect>Inverse Selection) and press return. The water molecules remain listed in the control
panel but are no longer visible within the display.
Select the entire protein by clicking in the far left region of the control panel. Note that the
selection is highlighted in both the control panel and the alignment window. You can select

regions of residues by clicking on the left hand side of the residue that starts your selection
and dragging the mouse to select a range. Regions or domains of specific structures can also
be selected by clicking on the small s or h denoting sheets or helices.
Select the entire protein and colour the residues by their secondary structure. (Color menu
>Secondary Structure).
What colour corresponds to which structure (-helices and -Sheets)? Turning off the
side chain display may make this more evident. You can do this by selecting the minus
(-) sign above the side column in the Control Panel. Pressing the (+) sign will turn
the display back on.
With the side chains switched off you should be able to see the zinc atom present within the en-
zyme active site. Rotating the image will allow you to see the cleft formed within the protein by the
arrangement of a series of -Sheets.
4.2 Viewing controls and structural representations
There are a number of ways we can represent the structural data when using molecular modelling
packages to investigate molecules and proteins. How we display the molecule can convey a variety of
information. Deep View provides a number of common representation modes that can be utilised to
generate images. These are all accessed from the Control Panel and include:
Ball and stick: With and without side chains (these are the default representations). Side chain
visibility is controlled by the (+) and (-) symbols above the side column.
Space-filling or CPK: In this representation covalent bonds are not visible and atom representa-
tions are determined by their atomic radii. While this provides more realistic representation of
the atoms, it often obscures other parts of the model and is best used sparingly when illustrating
specific concepts. CPK representation is toggled on and off using the (+) and (-) symbols above
the :: column.
The final representation is the ribbon representation. This is a simplified cartoon representation
used to display secondary structural motifs within the protein. Neither the atoms nor the bonds are
visible with this representation, instead a flat ribbon structure traces out the shape of helices and
sheets. This display can be overlayed on top of a CPK representation or displayed independently.
Toggling of the ribbon representation is controlled by the (+) and (-) symbols above the ribn
column in the Control Panel.
Colours can be set manually using the boxes under the col heading in the Control Panel. The small
black triangle under the col heading allows you to select which structural element the colour selections
are applied to.
At any time you can save an image of the display space. To do this, go to the File menu >Save
>Image
A save image dialog box will open. Be sure to name your file in such a way that you understand
what it is and select the appropriate folder to save your images to. (You are more likely to find your files
again if you keep them all in your group folder. This folder can then be copied to a USB thumb drive so
you can use your files from home.)
Be sure to append the .jpg file extension to your file name before pressing the save button e.g.
active site.jpg.
Images saved in this fashion can be inserted into word processing and presentation documents for
your presentation and report.

4.3 Investigating the active site.
Locate the Zn(II) ion on the control panel
Change the colour to something more visible and the representation to space-filling (::).
Go to the Select menu and use Neighbours of Selected Residues to display only the neighbour-

ing residues within 3 A(this will select bonded residues nearby to the zinc atom).
List the residues displayed according to this selection.
Which atoms are coordinating to the Zn(II)? Rationalise this according to the rules for hard / soft
Lewis acids and bases.
Other than amino acid residues is there anything else bonded to the zinc?
What type of coordination geometry does the zinc atom in the active site display?
Is the active site arrangement consistent with the mechanism for CO2 hydration you outlined in
your pre-lab question?
4.4 Structural insights into enzyme inhibition.
Here, you will look at an alternative PDB file for carbonic anhydrase in which there is a sulfonamide
inhibitor complexed with the enzyme.
Return to your web browser and the RSCB protein data bank website.
Search for human carbonic anhydrase complexes.
Select the file 1AZM and download as you did for 1CA2 in the previous exercise.
Locate and download a copy of the primary citation for this structure. What disease is the sulfon-
amide complexed in 1AZM a treatment for?
What is the name and structure of the sulfonamide complexed with carbonic anhydrase in this
file?
Open the 1AZM PDB file in Deep View (a side chain error may appear, select ok and move on)
Using the techniques outlined in the previous section, deselect the water molecules and highlight
the sulfonamide ligand within the active site.
Generate images for your report as you work, remember you can control the quality of your graphics
and the colours of the representations using the preferences menu.
What atom / atoms of the sulfonamide coordinate to the zinc?
Using the measure distance and measure bond angle tools report the bond distances and angles
for the sulfonamide compound and the zinc atom.
Are the bond angles for the sulfonamide moiety consistent with those exhibited by sulfanilamide
in the crystal structure exercise?
Using Deep View calculate the molecular surface of the protein. There are three surface represent-
ations available within the Deep View software. Each of these is described in detail in the User Guide
(Pages 32 and 33 - Displaying Surfaces).

Generate a representation that shows the active site pocket with the Zn(II) highlighted and the
coordinated amino acids and sulfonamide represented using a ball and stick representation.
Be sure to label the coordinated residues. Save this representation in an orientation that allows
you to describe the critical aspects of the carbonic anhydrase active site for your report.
You may also wish to generate a representation of the enzyme substrate complex with the VDW
surface applied to the substrate. This will provide insight into the way in which the substrate fills
the binding pocket.
Using the Slab may assist you in creating a clear image. Slab view allows you to see a cross section
of the protein allowing the interior residues to be visible even with a surface applied.
As an additional exercise you may wish to investigate the effect of metal substitution on the sur-
rounding residues within the active site. The search results for human carbonic anhydrase complexes
you performed earlier will include structure files for cobalt substituted carbonic anhydrase. Download
and investigate one of these files. Pay particular attention to the bond distances and metal coordination
geometry compared to the native zinc enzyme. Remember to generate images for your report as you
work. It is easier to create them as you go than trying to recreate a particular orientation you found
useful.
References
[1] DeepView The Swiss-PdbViewer User Guide v. 3.7 available from: http://www.expasy.org/spdbv/
[2] J. A. Cowan, Inorganic Biochemistry: An Introduction (Wiley VCH, New York, 1977)
[3] A. Messerschmidt, M. Cygler, W. Bode, Eds., Handbook of Metalloproteins (John Wiley & Sons
Ltd, West Sussex, 2004).

Theme 2 - Dye-Sensitised Solar Cells
1 Introduction
This theme practical involves the construction and characterisation of dye-sensitised solar cells
(DSSCs). Besides using a commercial dye, you will need to extract naturally occurring pigments and
compare their performance in the solar cell with that of the commercial dye. In the three expansion prac-
ticals, factors affecting DSSC performance, which include the surface area and particle size of electrode
materials, and the photophysics of a potential sensitising dye, are explored.
1.1 Background
As our society becomes more focused on finding alternatives to fossil fuels and smarter energy tech-
nologies, it is important to understand the role that chemistry can play in the area. One area where
chemistry continues to have a large impact is the development and refinement of DSSCs.[1, 2, 3] Relev-
ant areas of chemistry include biological extraction, electrochemistry, electronic spectroscopy, physical
characterisation, energy and electron transfer, and the design and synthesis of organic and inorganic
photovoltaic materials. Within the School of Chemistry these are all fields of active research. Chemistry
can also play a significant role in the development of new materials that can act as transparent electrodes.
The interaction between light and matter was studied in some of the core practical exercises. Here
you will extend this knowledge to harvest light to generate electrical energy and investigate the processes
that affect the efficiency of such an approach.
1.2 Dye-sensitised solar cells
Photovoltaic devices, such as solar cells, are based on the concept of charge separation at an interface
of two materials that exhibit different electrical conduction mechanisms. In a DSSC, this interface
consists of a photo-sensitised semiconductor and an electrolyte giving a photo-electrochemical system.
There are many prerequisites for this system to make it an efficient solar cell. The aim is to harness
energy from sunlight through light absorption where an electron can be freed from a material and be
put into an external electrical circuit in order to do useful work. In a semiconductor such as TiO2 , an
electron can be promoted from the valence band to the conduction band following the absorption of light
in the ultraviolet region of the spectrum (wavelengths <400 nm). This causes problems and expenses,
e.g. high grade optical components are required and the radiation spectrum of the Sun is not maximal in
the ultraviolet region. High energy photons can also cause photochemical damage to the device. Ideally
we would like to harvest all the light within the emission spectrum of the Sun that reaches our part of
the troposphere. This can be achieved using a highly visible-light absorbing dye. The absorption of
light by the dye results in the electronic excitation of an electron in the dye from the highest occupied
molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The absorption of light
itself is insufficient for conversion of light into electrical current since optical absorption does not in
itself free the electron from the dye. However, if the first excited state of the dye is higher in energy than
the conduction band of the semiconductor to which the dye is attached, an electron could be transferred
from the LUMO of the dye to the conduction band of the semiconductor, and thus be freed to do useful
work. This is the basis of typical DSSCs. A schematic of such a solar cell is shown in Figure 1.
231
T HEMED E XPERIMENTS - T HEME 2 CHEM30015: Advanced Practical Chemistry
Figure 1: Photochemical processes in a typical dye-sensitised solar cell
1.3 Characterisation of suitable natural pigments
There are numerous natural pigments that absorb strongly in various parts of the ultraviolet and vis-
ible (UV-Vis) regions of the electromagnetic radiation spectrum, and may therefore be thought suitable
for DSSC applications. The UV-Vis absorption spectrum of each dye must therefore be determined in
order to identify likely candidate dyes. However, the energy of the lowest energy excited-state of the
dye relative to that of the conduction band of the semiconductor must also be determined in order to
find a suitable candidate dye (see Figure 2). This can be achieved through electrochemical studies of the
semiconductor and the dye coupled with UV-Vis spectroscopy.
Figure 2: Relative energy levels of the components in a dye-sensitised solar cell.
2 Objectives
In this exercise you will construct several DSSCs using natural pigments derived from berries, leaves
etc. and a commercial dye (N719) targeted specifically for the DSSC market. Organic-, inorganic- or
polymer-based solar cells can be constructed using a variety of structures but the type you will construct,
which is a typical device structure, is shown in Figure 3. Through completion of the core component
and the subsequent modules 2.1-2.3 you will investigate further aspects involved in solar cell efficiency,
characterisation and function. In particular, the spectroscopy of some of the dyes used will be carried
out. The surface area of the semiconductor will be determined as this affects the amount of dye that

CHEM30015: Advanced Practical Chemistry T HEMED E XPERIMENTS - T HEME 2
can be adsorbed on the semiconductor. Since the size of the particles of the semiconductor affects the
surface area, methods of determining the dimensions of nanometre-sized particles will be investigated.
Figure 3: A typical organic solar cell device structure.
References
[1] Gratzel, M., Recent Advances in Sensitised Mesoscopic Solar Cells, Acct. Chem. Res., 2009, 42,
1788.
[2] Gratzel, M., Photoelectrochemical Cells, Nature, 414, 15 November 2001.
[3] Gratzel, M., Solar Energy Conversion By Dye-Sensitized Photovoltaic Cells, Inorganic Chem-
istry, 2005, 44, issue 20, 6841-6851.

T2.0 - Construction and Characterisation
of a Dye-Sensitised Solar Cell
Dr. Georgina Such

1 Introduction
1.1 Aims
The goals of this practical component are:
1. To construct a number of working photovoltaic devices in the form of a dye-sensitised solar cell
(DSSC). The dyes used will be extracted from natural substances, e.g. raspberries (anthocyanin
dyes), leaves (chlorophyll) or other sources, and tested to determine their relative efficiency. You
may provide your own dye (see experimental section for possible candidates). You will also be
provided with a commercial dye (ruthenium-based DSSC dye N719) for comparison.
2. To characterise and compare the efficiency of the cells based on these low-cost and readily avail-
able natural pigments with those based on the commercial dye.
3. To learn about the various aspects of chemistry that are integral in solar energy research and
development.
2 Time management and safety aspects
Construction and characterisation of the solar cells will take three laboratory sessions. The timing of
these sessions is not very prescriptive, but it is important that you have your time planned before starting
the experiment to ensure that there will be enough time to complete each section of the experiment. You
are to work in groups of 2 or 3 students. Each group will construct several DSSCs: one anthocyanin-
sensitised cell, one chlorophyll-sensitised cell, one N719-sensitised cell, and one cell sensitised with a
naturally occurring dye of the groups own choice.
In the first laboratory session you will need to prepare the anatase titanium dioxide working elec-
trodes (anatase is one of the three semiconducting crystalline phases of TiO2 with a band gap of 3.2
eV; a fourth phase does not semiconduct[1]) and extract the pigments for the dye baths. You will also
need to run most of the UV-Vis absorption spectra of the extracted pigments. By the end of the first
session the working electrodes should be in their dye baths. The second and third sessions will involve
the preparation of the carbon counter electrode, cell assembly, and testing of your prepared cells under
different conditions.
235
2.1 Safety precautions and waste management
TiO2 can be a skin and eye irritant. The fine nanoparticles should not be inhaled.
KI3 electrolyte contains both iodine and potassium iodide. Iodine is an irritant to the eyes, skin
and respiratory system. Flush affected areas with water.
The electrodes sintered in an oven at 500 C are extremely hot. Handle with thermally-insulated
gloves and tweezers.
Perform all deposition of carbon soot for the solar cells in the fume hood. Do not remove the
candle from the fume hood. It might set off the smoke detector!
Waste disposal: Dispose of all materials in the proper containers provided.
You are to work in pairs. Each group will construct several DSSCs: one anthocyanin sensitised
cell, one chlorophyll sensitised cell, one N719 sensitised cell and one cell sensitised with a naturally
occurring pigment of the groups own choice. Possible sources of naturally occurring pigments include:
saffron, turmeric powder, red onion skins, raspberry tea, hibiscus tea, beetroot juice, Ribena, fluorescent
dyes or anything else you would like to try. You will need to bring the material in question to the first
laboratory session in order to extract it along with the other pigments.
Due to the time needed for the oven to cool down after heating at higher temperatures, there will only
be one opportunity per laboratory session for sintering the working electrodes and counter electrodes,
respectively. Your demonstrator will announce the times by which your electrodes need to be ready for
the sintering. You will need to take this into account when you plan your experiments.
3.1 Preparation of the working electrode
A TiO2 paste may be provided - ask your demonstrator. If the TiO2 paste is not provided perform
the following procedure:
Weigh out 1 g of TiO2 powder in a beaker and place in a mortar and pestle. Gradually
add 0.035 M acetic acid solution (pH 3-4) to the TiO2 in 1 mL increments. With each
addition of acetic acid grind the slurry until you have a paste that has a soupy colloidal
consistency. Approximately 7-8 mL of acetic acid will be needed.
Transfer the mixture to a small beaker or vial and sonicate for 5 minutes (in an ultrasonic
bath) to break up aggregates within the mixture. Add 1-2 drops of 0.1 M sodium
dodecyl sulphate surfactant solution and mix gently with a stirring rod. Be careful not
to agitate the solution too briskly after you have added the surfactant, otherwise
you will cause the surfactant to foam and bubbles to form in your mixture.
The quantity of the prepared paste is sufficient to make 50-70 working electrodes if ali-
quots of 60 L are dispensed. In other words, there is enough paste to practice making
working electrodes as well as making more than the required four working electrodes, if
needed. Do not throw away the paste until you have completed the entire practical.
Seal the beaker with Parafilm and label it with the name of the group, the contents, and
the date prepared.

The working electrodes are prepared by doctor blading the paste across a glass substrate, which have
been masked with adhesive tape. It takes some practice in order to produce thin, even, reproducible films
of the paste. The TiO2 films of state-of-the art research working electrodes are 10-20 m in thickness.
The thickness of the adhesive tape used for masking is 35-70 m. The actual thickness of the produced
film will depend on the paste consistency, but generally one layer of doctor bladed paste is sufficient. A
thinner film is better than a too thick film. A thick film will tend to crack and peel off the glass substrate,
which will make it useless for making a solar cell. Reproducibility is also important since the evenness
and thickness of the film will affect the efficiency of the solar cell and therefore the comparison of the
different dyes. The group will need to produce four working electrodes of equal quality. The best way to
achieve this is to first practice doctor blading on microscope glass slides. These can be reused as many
times as you like just by washing off the paste into the waste bucket for TiO2 using water followed by
ethanol and then drying with a tissue.
3.1.1 Practising doctor blading
1. Take two microscope glass slides. Wash them with water and ethanol and dry them with a tissue.
2. Mask out a square (22 cm2 ) on the glass slides with adhesive tape and at the same time attach
them to the glass surface of the bench. There is no need to be too thorough in the attachment,
because it then may be hard to remove the slide from the bench. The size of the square is not
critical for practising doctor blading.
3. Place a small amount of TiO2 paste on the piece of tape furthest away from you.
4. With a glass rod or the side of a Pasteur pipette, even out the paste on the tape by placing the
side of the rod in the paste and moving the rod sideways (make sure you do not get paste inside
the masked square) so that you end up with an even amount of paste on the tape across the entire
width of the square. This is action 1 in Figure 1.
5. With a steady hand and even pressure, pushing slightly down onto the microscope slide, drag the
glass rod towards you until you reach the other side of the masked square (see action 2 in Figure
1). The result should be an even, slightly transparent film of paste covering the entire masked
square. If not, try again on the second glass slide. Continue practising until you can produce two
films of equal quality. When ready, follow the instructions below for doctor blading the working
electrodes.

Figure 1: Illustration of doctor blading. Adapted from reference [2]
3.1.2 Doctor blading working electrodes
1. Wash the four solar cell glass slides by sonicating them briefly in ethanol. Remove them from
the ethanol using a pair of tweezers and let them dry by having them stand on one edge on a

tissue leaning against, for example, the side of a petri dish. Avoid touching the faces of the glass
slides-hold them using a pair of clean tweezers or by the edge of the glass.
2. Using a digital multimeter, determine the conductive side of four of the solar cell glass slides
with which you have been provided. Set the multimeter to resistance (). The conductive side of
the glass should provide a reading of 10-20 , whereas the non-conductive side will provide a
reading of infinite resistance (which may be displayed as 0 ). It is important that the conductive
side is facing upwards when you doctor blade the paste, i.e. the TiO2 working electrode is formed
on the conductive side of the glass.
3. Once you have established that the conductive side is facing upwards, mask out the surface of the
slides using adhesive tape. You should mask 1-2 mm on three edges and 4-5 mm on the fourth
edge. Unlike, when practising the doctor blading, the actual size of the masked area is important.
The size of the working electrodes affect the performance of the solar cell and you want to produce
working electrodes of equal size.
4. Follow points 3-5 of Section 3.1.1 to produce four working electrodes of equal quality.
5. Let the paste dry slightly, but not completely, before you remove the masking adhesive tape.
6. Carefully transport the working electrodes to the oven for sintering. Note down the location of
your electrodes in the oven.
3.1.3 Sintering of working electrodes
The working electrodes should be sintered in the oven at 500 C for 15 minutes. However, the
complete process of sintering the electrodes will take 1 hour due to the time required for the oven to
heat up and cool down. The sintering will be managed by the demonstrator present during the laboratory
session. Prepare the dye baths while waiting for the working electrodes to be sintered.
3.2 Preparation of dye baths
The dyes used in this experiment vary in molecular structure considerably (see Figure 3 for some
examples). The key functional groups required for sensitisation of the TiO2 surface are the carbonyl or
hydroxyl groups needed to facilitate chelation of the dye molecule to the TiIV of the TiO2 surface. The
different natural sources of pigments will require slightly different procedures and solvents in order to
extract the pigment. Below are some instructions for the preparation of anthocyanin and chlorophyll
pigments. For the fourth dye bath (the pigment of your own choice), you will need to determine the
procedure yourself.
3.2.1 Anthocyanin pigment from berries
Take 2-3 berries from the supplied raspberries and place in a small beaker. Crush the berries with a
small amount of water forming a deeply coloured solution. Wear gloves as cyanin stains hands. Decant
the liquid into the small glass jar provided. Take a small amount of the liquid and obtain a UV-Vis
absorbance spectrum (300-800 nm). The extract solution may need to be diluted and/or filtered signific-
antly in order to reach appropriate optical density (0-1 OD units). Set aside the rest of the extract until
the working electrodes are ready.

3.2.2 Chlorophyll pigment from citrus leaves
For the chlorophyll pigment it is necessary to extract the chlorophyll from fresh citrus leaves and
convert this to the corresponding catabolite, chlorophyllide. This conversion removes the phyto tail from
the chlorophyll exposing the necessary C=O and -OH for the pigment to chelate the TiO2 surface (see
Figure 2).
Take a couple of citrus leaves. Put one of them aside. Grind the remaining leaf with a mortar and
pestle along with 2-3 mL of acetone. This can take up to 30 minutes. Filter the resulting dark green
solution into a small beaker. Cut 5-6 pieces (4x4 mm2 ) from the unground leaf you saved earlier and
place these into the container with your extract. Set aside the extract until the working electrodes are
ready.
3.2.3 Pigment of choice
Prepare an extract from your pigment of choice. You will need to come up with the appropriate
method and solvent. Ask your demonstrator for advice if needed. Remember to obtain a UV-Vis absorb-
ance spectrum.
3.2.4 Commercial dye N719
The solution for the N719 dye bath with dye is already prepared for you. Transfer a small amount
to a small glass jar. Remember to obtain a UV-Vis absorbance spectrum. Do not throw away the dye
solution after you have used it, because it is expensive and can be reused.
3.3 Sensitising the working electrodes
Once the working electrodes have cooled down after the sintering, put one into each of your four dye
baths. Make sure the working electrode is covered by the liquid. If needed, dilute the extracted solutions
with the appropriate solvent until the working electrode is submerged. Seal all the containers except the
one containing the berry extract and put away into a dark cupboard for storage until the next session.
Label all containers with the name of the group, the name of the liquid contents, and the date prepared.
Check the working electrode in the anthocynanin dye bath after 5 minutes. If there are no white
patches on the TiO2 surface (if there are, put it back into the extract solution for another 5 minutes),
wash the dark purple film gently with water and then ethanol and let dry. Store your anthocyanin-
sensitised working electrode in acetic acid solution (pH 3-4) in a sealed container in the dark together
with the electrodes until the next session.
3.4 Preparation of carbon counter electrode
You will need to prepare four counter electrodes. This must be done in a fume hood.
3.4.1 Carbon deposition
1. Wash the four solar cell glass slides by sonicating them briefly in ethanol. Remove them from the
ethanol using a pair of tweezers and let them dry by having them stand on one edge on a tissue
leaning against, for example, the side of a petri dish. Avoid touching the faces of the glass slides
hold them using a pair of clean tweezers or by the edges of the glass.
2. Using a multimeter check which side of the solar cell glass slides is the conductive side. The task
is to deposit an even layer of carbon soot on the conductive side using a candle flame.

3. Using a pair of tweezers, hold one of the glass slides firmly but gently. You will need a 4 mm
uncoated edge of the glass slide. This can be achieved by making sure the pair of tweezers covers
this area of the glass slide. With the conductive side facing downwards and towards the flame,
pass it through the candle flame quickly. Continue passing it through the flame until you have
produced a thin, even layer of carbon soot on the glass slide.
4. Repeat points 2-3 for the other glass slides.
5. Carefully transport your prepared carbon counter electrodes to the oven. Note down the location
in the oven of your counter electrodes.
3.4.2 Sintering of carbon counter electrodes
The counter electrodes should be sintered at 400 C for 15 minutes. However, like for the working
electrodes, the process of sintering takes longer due to the time required for the heating up and cooling
down of the oven. The sintering will be managed by the demonstrator present during the laboratory
session. Prepare the equipment needed to test your solar cells while waiting for your counter electrodes
to be ready.
3.5 Assembly of your solar cells
Carefully remove your sensitised working electrodes from the solution they have been stored in.
Do not discard the dye baths and remember to obtain a UV-Vis spectrum of the chlorophyllide
pigment. Rinse the slide with a little solvent. If the electrode was sensitised in an aqueous solution:
rinse with water followed by some ethanol and let it dry on the bench. If the electrode was sensitised in
ethanol or acetone: rinse with fresh ethanol or acetone and let it dry on the bench.
Once your carbon counter electrodes have cooled down, bring them to your bench. Place the dry
working electrode on the bench so that the TiO2 side is facing up. The carbon counter electrode is placed
on top so that the conductive carbon side of the counter electrode faces the TiO2 surface. The two glass
plates should be offset so that the counter electrode covers all of the TiO2 and the 4 mm strips of glass,
not coated by either TiO2 or carbon are is exposed (see Figure 3). The two exposed sides of the device
will be the contact points for the negative and positive electrodes so that electricity can be extracted to
test the cell. Use the two binder clips supplied to hold the slides together loosely at the other edges.
Bring your assembled glass slides to a fume hood. Carefully place one or two drops of the KI3
(I/I3 ) electrolyte solution at the edges of the glass slides.
Alternating between the two binder clips gently
release the pressure applied by slightly opening and closing the clips. The electrolyte solution will be
drawn into the space between the electrodes by capillary action. Ensure the solution wets the entirety
of the TiO2 surface. Wipe off any electrolyte solution located on the outside of the glass slides and
transport your now completed DSSCs to your testing set-up on the bench.
3.6 Testing your solar cell
You will need two multimeters (see Figure 4, one marked I to measure current, and one marked
V to measure voltage), one potentiometer, crocodile clip leads, a torch and a retort stand with a clamp
for the torch. The first test of the solar cell should be done with ONLY a light source and a voltmeter
(Note switched to DC Volts, see International Electrical Symbols, Figure 4b) across the cell (Figure
5a). This will give you the unloaded voltage output of the solar cell, as a voltmeter is a high impedance
device and will not load the cell.
To measure the current/voltage characteristics, prepare to connect the solar cell, the multimeters and
the potentiometer according to the circuit in Figure 5b. DO NOT connect the current meter (I) until you

have wired the rest of the circuit with the variable load (potentiometer) in the fully counter clockwise
position (CCW, this is the maximum resistance position and therefore minimum load and current) and
you are ready to collect data. Turn on the light source and finally connect the current meter (switched
to mA) between points A and C. The reason this is left until last is that the current meter is a low
impedance device and therefore current will flow out of the solar cell, and the solar cell function may
degrade as soon as the current meter is connected. Monitor both voltage and current as you turn the
variable load clockwise (CW). Short circuit current is achieved when the variable load is in the fully
clockwise position. Ask a demonstrator for advice if needed.
The light from the torch should enter the solar cell through the TiO2 -coated glass slide. The negative
electrode should be the working electrode and the positive electrode should be the counter electrode.
3.6.1 Measuring current density (mA/area unit)
The current from the solar cell depends on the area of the working electrode (the area coated with
TiO2 ). To make a fair comparison of the efficiency of the different dyes, the current from the solar cell
should be given in mA per unit area of the working electrode, i.e. current density (J). The easiest way
to do this is to find the area of the working electrode by using a ruler and divide the measured current
by this area. Another method is to mask the surface of the solar cell so that only a known area of the
working electrode is exposed to light. This can be achieved by using a piece of carton or black paper
where an aperture of a known area has been created. The same mask can then be used for all four cells.
The measured current needs to be divided by the area of the aperture of the mask.
3.6.2 Obtaining a current density-voltage curve
The first step to obtain a measure of the efficiency of your solar cells is to produce a current density-
voltage (J-V) curve (see Figure 6). This is done by measuring the output current and voltage of your cell
when it is illuminated by the torch. The output current and voltage depend on the loading of the cell. By
varying the resistance (i.e. the load in ) of the potentiometer in small increments, current and voltage
data can be gathered, and a current density-voltage curve produced.
Start with connecting the solar cell to the testing set-up. The light intensity affects the solar cell
performance so try achieving the same intensity for all your cells by fixing the location of the cell and the
torch in relation to each other. Turn the knob of the potentiometer until a maximum voltage is observed
on the voltmeter. Record the voltage and the corresponding current. To obtain the next current-voltage
data point, turn the potentiometer lever until you see a slightly lower voltage value on the voltmeter.
Record the new voltage and corresponding current. Repeat the process of slowly decreasing the voltage
by turning the lever and record the data points. Continue until a maximum current is obtained. Plot the
data points with the current density as a function of the voltage (see Figure 6). Ask a demonstrator for
advice if needed. Obtain a J-V curve for each of your solar cells.
Please retain your completed solar cells, because you may require them at a later stage.
3.6.3 Characterising the performance of your solar cells
You will now use your current density-voltage (J-V) plots to calculate two important parameters, the
cell efficiency () and the fill factor (ff ), which are used to quantify the performance of the solar cell.
For a solar cell to be useful, it needs to deliver a significant amount of power. The power delivered
from a power source, such as a solar cell, is given by:
P = I V (1)
where P = power, or in the case of current density, J, the power density is:

P = J V (2)
Look at one of your J-V plots. The maximum (m) power density (Pm ) occurs somewhere between
V = 0 (short circuit) and V = VOC (open circuit) at a voltage Vm . At VOC there is no current flowing
through the solar cell, i.e. it is the voltage where the J-V curve intercepts the x-axis. Vm corresponds to
a current density Jm , which is smaller than the short-circuit current density JSC . At JSC . the voltage is
zero, i.e. it is the current density where the J-V curve intercepts the y-axis. The maximum power density
obtained from the solar cell is:
Pm = Jm Vm (3)
The maximum power density can be found by multiplying your measured voltages with their corres-
ponding current densities and plotting the power density as a function of the voltage on the same axes
as your J-V curve. The maximum of the power density curve will give you Pm , the maximum power
density. Vm is obtained by drawing a straight line parallel with the y-axis through the maximum of
the power density curve. Vm corresponds to the intercept of this line on the x-axis. Jm is obtained by
drawing a second line, this time parallel with the x-axis, from where the Vm straight line intercepts the
J-V curve to the y-axis.
The efficiency of a solar cell is defined as the maximum power density output divided by the power
density input. If the incoming light has a power density Ps , then the solar cell efficiency is:
Jm Vm
= (4)
Ps
In order to be able to calculate the efficiency of your solar cells you will need to measure the power
density of your light source, i.e. the torch that you have been using. Ask a demonstrator to help you do
this.
The fill factor, ff, gives a measure of how much of the VOC and JSC are used at the maximum power
density output from the cell. It is defined as:
Jm Vm
ff = (5)
JSC VOC
By convention, is given as a percentage, whereas the ff is given as a fractional number to two

decimal places. All four quantities JSC , VOC , ff, and are used to characterise the performance of a solar
cell. Calculate and create a table containing these quantities for all four of your solar cells.
4 Analysis and questions
The following topics and questions should be discussed and answered, respectively, in the practical
report:
4.1 Solar cell performance
Compare, using the absorbance spectra, the J-V plots and the table of the four quantities JSC , VOC , ff
and , the behaviour and efficiency of your DSSCs based on the various dyes you have used. Comment
on the most important properties of the dyes that might influence the solar cell efficiency.

4.2 Ru-based DSSC
A range of Ru(II) complex photosensitisers have been developed for DSSC applications. Some,
like the N719 dye (di-tetrabutylammonium cis-bis(isothiocyanato)bis(2,2-bipyridyl-4,4-dicarboxylato)-
ruthenium(II); see Figure 7) which you used, show solar-to-energy conversion efficiencies up to 11-12%
under appropriate conditions.[3] Ruthenium-based dyes exhibit ligand-centred charge transfer (LCCT)
transitions (- ) as well as metal-to-ligand charge transfer (MLCT) transitions (4d ). This phe-
nomenon can be observed in the UV-Vis spectrum of N719. Looking at your own UV-Vis spectrum
of N719, confirm that this is the case. The absorption bands at lower energies represent the MLCT
transitions, whereas the more energetically demanding transitions correspond to LCCT transitions.
4.3 Additional measurements of your Solar Cells
Consider any other measurements you can undertake on your devices and perform appropriate meas-
urements where possible. For example, experiment with placing a number of cells in series or parallel.
Can you measure any improvements in the cell output? You can also determine the photostability of
each dye, and the longevity of the cell. What light flux have you used? What is the emission spectrum of
the light source you used? You might also think about electrochemical measurements that might be per-
formed. In your report you should describe what experiments you performed, what results you gained,
and how you interpret these outcomes.
Investigate in the literature some of the spectral and electrochemical properties of this dye and dis-
cuss briefly. In this experiment the solvent used for the DSSC is tert-butanol:acetonitrile (1:1). The
following points should be discussed in addition to other information you can acquire.
The onset of the absorption of the N719 dye and most related dyes is in the range of 720 nm.
Confirm that this is the case. What energy (in eV) does this absorption onset correspond to?
Is this dye fluorescent or not? What are the consequences on DSSC efficiency if the dye is fluor-
escent?
What is the formal potential of the dye redox couple (D+ /D ) versus NHE?
What effect might a high iodine concentration have on the efficiency of a DSSC?
The dye injects an electron into the conduction band of the TiO2 . From what electronic state do
the electrons get injected?
4.4 Other questions
1. Why is it important that the dye bath solutions are diluted significantly in order to reach an appro-
priate optical density (0-1 OD) when measuring an UV-Vis absorbance spectrum?
2. What are the structures of the various dyes you have used in this practical?
3. Can you identify any structure-efficiency correlations? In other words, is there any trend between
the class of dye and the measured efficiency?
4. What is a typical value for the fill factor in an efficient DSSC? How does the value of the fill factor
correlate with the shape of the J-V curve? Take note of any differences in electrode materials
and sensitiser dye, and include references. Comment on the shape of your plots. Discuss any
discrepancies.
5. What is the solar spectrum that reaches our part of the troposphere and how well do the various
absorption spectra of the dyes used harness this energy?

6. What would you expect to happen if you were to blend two or more of the dyes you have used?
7. What would you expect if two or more cells were connected to each other in series or parallel?
8. There are numerous variations on this theme on the internet (e.g. YouTube). What additions or
improvements can you suggest for the development of DSSC?
Before leaving the lab at the end of your session ensure your electrodes have been stored correctly.
Please retain your completed solar cells - you may require them at a
later stage.
References
[1] Zhang, R. Y.; Elzatahry, A. A.; Al-Deyabb, S. S.; Zhao, D. Y. Nano Today 2012, 7, 344-366.
[2] Smestad, G.P.; Gratzel, M. J. Chem. Educ. 1998, 75, 752-756.
[3] Hallett, A.J., Jones, JE, Dalton Trans., 2011, 40, 3871-3876
[4] http://www.dyesol.com/n719-industry-standard-dye.html

Figure 2: Molecular structures of anthocyanin (left) and chlorophyll and its catabolite chlorophyllide
(right).
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Figure 3: Assembled solar cell showing offset glass plates, clips and electrical contact points. The
sensitised TiO2 layer is in contact with the carbon-coated conductive layer. Reproduced from reference
[2].

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''1!
2! 3!
Figure 5: a) Initial test of completed solar cell, unloaded. b) Experimental set-up for measuring the
current-voltage characteristics of the completed solar cell. Reproduced from reference [2].

Figure 6: A typical current-voltage curve for a 4 cm2 DSSC. Reproduced from reference [2].
Figure 7: Structure of dye N719[4].

T2.1 Determination of the Surface Area of
Powders by Gas Adsorption
A/Prof. Trevor Smith & Prof. Paul Mulvaney

1 Introduction
1.1 Aims
The goals of this practical component are:
1. To learn about high vacuum technology.
2. To use the measurement of the adsorption of gases onto solids to estimate the surface area of
solids.
2 Introduction
Adsorption isotherms relate the amount of gas adsorbed to a solid to the pressure, at a given temper-
ature. The isotherms of gases that adsorb physically (i.e. the gas molecules do not react chemically with
each other or with surface atoms[2]) can be represented as:
x = f(P,T)
where x is the amount of gas adsorbed per unit mass of adsorbent (mol g1 ), P is the pressure, and
T is the temperature.
By determining the adsorption isotherm, information such as the specific surface area (i.e. the sur-
face area per gram of adsorbent) can be calculated. The surface area of the substrate of a DSSC is an
important factor influencing the efficiency of the cell.
2.1 The Langmuir Isotherm
The Langmuir isotherm was proposed to describe cases of adsorption under conditions where:
there is no change in energy of adsorption with change in fraction of surface covered, and
maximum adsorption corresponds to a layer one molecule thick (monolayer).
249
If k1 and k2 are rate constants for the processes of condensation and evaporation of gas molecules at
pressure P, then the fraction of the surface covered is:[1, 2]
k1 P
= (1)
(k2 + k1 P)
If xm is the maximum amount of gas adsorbed per gram of adsorbent (mol g1 ), corresponding to a
monolayer, and x is the amount of gas adsorbed at pressure P, then = xxm and:
P k2 P
= + (2)
x k1 xm xm
A plot of P/x versus P yields the monolayer amount xm . If the area for one adsorbed gas molecule is
known then the surface area per gram of the adsorbent (i.e. the specific surface area) can be calculated.[2]
2.2 The BET or Multilayer Adsorption Isotherm[1, 2, 3, 4, 5, 8]
Unfortunately, many adsorbent-adsorbate systems do not follow the simple Langmuir isotherm but
show the formation of many layers (multilayer build-up).
Brunauer, Emmett and Teller proposed a process of the Langmuir type with evaporation-condensation
reactions for each of the many layers. The summation of all adsorption-desorption reactions produces
the multilayer. The limit of the multilayer is condensation of the gas to liquid, when the gas pressure
reaches the saturated vapour pressure P0 of the liquid.
The derivation of the BET isotherm assumes a heat H1 of adsorption for the first layer that is
different from all succeeding layers. The second layers, etc., have a heat of adsorption equal to the heat
HL of condensation of the gas. The isotherm is usually of the form:

P 1 c1 P
= + . (3)
x(P0 P) xm c xm c P0
where c is the so-called BET c constant:[2]

HL H1
c exp (4)
RT
The left-hand side of equation 3 may also be written as:
P/P0
x(1 P/P0 )
P P P X
A plot of [x(P0 P)] versus P0 {or of Y versus X, where X = P0 and Y = [(1X)x] }, if linear, leads to
values of c and xm . The linear range is usually observed between PP0 values of 0.08 and 0.35.
If the cross-sectional area Am (in A 2 ) of the adsorbing gas molecules is known, the specific surface
area S (i.e. the surface area per gram of adsorbent) can be calculated from the equation:
S = xm LAm 1020 m2 g1 (5)
where L is the Avogadro number.

Answer Questions 1 and 2, and construct a flow sheet for the experiment. Questions 3-9 should be
answered whilst the silica powder is baking out.
1. How many of each of the following units of pressure make up one atmosphere: mmHg, Pascal,
Torr, mbar?
2. Two bulbs are linked by a tap (Figure 1). Both have been evacuated by a high-vacuum pump.
With the tap closed, a gas is introduced to the left bulb until the manometer reads a pressure of 96
Torr. V1 is known to be 55 cm3 . After the tap has been opened, the manometer shows a pressure
of 40 Torr. What is the total volume of the two bulbs? What is the volume of V2 ?
!
Figure 1: Two bulbs linked by a tap.
3. Under what conditions does the BET equation reduce to the Langmuir equation[2]?
4. (a) How would you calculate H1 and S from a plot of equation 3?
(b) What is the value of HL for nitrogen[10], i.e. HL (N2 )?
5. (a) What is the volume of a sphere of radius r cm?
(b) What is the relationship between the volume of a material, its mass and its density?
(c) If you have 4.2 g of a powder of density 3.1 g cm3 and you know that it exists as spherical
particles of radius 0.15 mm, how many particles are in the sample?
6. Calculate the surface area of two samples of the same substance; the first exists as a single sphere
of volume 1.00 cm3 and the second as 1000 spheres, each of volume 0.001 cm3 .
What conclusion can you draw about the surface area:volume ratio for a finely-divided powder
compared to that for a coarse powder?
7. Calculating the specific surface area, S (m2 g1 ), from BET monolayer capacity uses equation 5.
(a) How is Am normally estimated?[3, 6]
(b) For N2 at 77 K, Am is usually put at 16.2 A 2 . Show how this number has been obtained.[3, 6]
(c) What physical restrictions does the use of the value of Am in (b) put on the type of interaction
between nitrogen and the solid surfaces?[3, 6]
8. Read through the experimental procedure for this experiment. Show that the number, n, of moles
of gas adsorbed for each step is given by:
(P1 P)V1 (P Pe )V2

n= (6)
RT1 RT2
where P1 is the pressure of gas in V1 before contact with the sample, P is the current equilibrium
pressure in V1 + V2 , Pe is the previous equilibrium pressure, and T1 and T2 are the temperatures
(Kelvin) in V1 and V2 respectively.
State any assumptions made in deriving Equation 6.

9. The BET equation can also be written as the difference between two hyperbolae:
x 1 1
=
xm (1 P/P0 ) 1 + (c 1)(P/P0 )
(a) How does the shape of the isotherm change as c is reduced from 100 (say) to 1?[7] What is
the significance, in terms of the molecular interaction between the surface and the adsorbate,
of changes in the value of c?
(b) For c = 1, can xm be calculated? If so, what is its significance? [Consider whether the
adsorption model that leads to the BET equation is likely to be valid when c = 1.]
4 Apparatus
Figure 2: Vacuum line.
The sample bulb of volume V2 is connected to a vacuum line with taps T1 , T2 , T3 , T4 and T5 , together
with manometers M1 , M2 and M3 (Figure 2). The section isolatable by T3 , T4 and T5 constitutes a gas
pipette of volume V1 in which known aliquots of nitrogen gas can be measured for exposure to the
sample.
Note that tap T1 shown in Figure 2 in fact represents an array of three taps which lead to different
sections of the pumping system. T1 is the means of isolating the line from the pumps. Where these
notes instruct you to open or close T1 , they mean open or close whichever of the 3 taps is
appropriate to isolate the vacuum line from or open it to the pumps.
NOTE: SAFETY GLASSES MUST BE WORN THROUGHOUT THIS EXPERIMENT

READ THE INSTRUCTIONS FOR THE SAFE USE OF VACUUM LINES.
In this experiment, as a model system, you will introduce known amounts of nitrogen gas to a
sample of silica powder in a glass bulb (Figure 2) and determine how much of the nitrogen is adsorbed
at equilibrium. The amount of adsorbed gas can simply be determined if the amount of nitrogen still in
the gas phase is measured. The obvious technique to use for this is measurement of the gas pressure as
long as two conditions are met:
1. The amount of nitrogen introduced to the powder is known;

2. The volume occupied by the gas is known.
These conditions can be satisfied by determining the volumes V1 and V2 ; the procedure then will be
to:
(a) introduce nitrogen into V1 ,
(b) measure the pressure,
(c) open T4 to permit the gas to adsorb on the powder and
(d) measure the pressure in V1 + V2 .
Therefore, the experiment contains the following steps:
determination of the two volumes V1 and V2 ;
cleaning of the powder by baking out under vacuum;
addition of known amounts of nitrogen through volume V1 and subsequent measurement of the
equilibrium pressure in the gas phase of both V1 and V2 ;
calculation of the number of mole of adsorbed gas;
determination of the BET parameters c and xm .
5.1 Determination of Volume V2
Measure the atmospheric pressure with the laboratory barometer and also note the ambient tem-
perature. V2 comprises two sections: the volume of the bulb and the tube between joint J and tap T4 .
Determine the volume of the bulb by measuring the increase in weight when the bulb is filled with water,
using the appropriate density. Repeat this determination.
Estimate the volume of the tube by assuming that:
any volume associated with J and T4 themselves is negligible, and
the internal diameter of the tube is 4 mm.
5.2 Determination of Volume V1
Evacuate the entire system including the bulb. Close T1 and observe the manometer reading in order
to check the line for any leaks. Close T3 and T4 . Admit air via T5 until the pressure in V1 is about 50
Torr; as measured on M2 allow at least 1 minute for the pressure reading to stabilise. Record the exact
pressure.
Open T4 and, from the new pressure, calculate V1 (Question 2 in Section 3). Repeat this determina-
tion.

5.3 Adsorption measurements
(a) Open T4 and T5 and remove the bulb. On a piece of paper, accurately weigh, about one gram of the
sample of high-area silica-gel (or TiO2 ) powder, and transfer into bulb, using a small glass funnel.
Reconnect the bulb to the line.
Note: The following procedure must be carried out carefully to prevent sudden changes
which may cause the sample powder to be ejected violently from the bulb, damaging the
vacuum line and its components.
(b) Close T4 and T5 and pump out the line through T1 and T3 . Open T4 slowly, whilst watching the
sample closely. The sample may bubble as the air around it is evacuated. When the pressure has
decreased to below 1 Torr and bubbling has ceased, close T4 again.
(c) Carefully place the furnace around the bulb and support it so that the bulb is centrally located. Insert
the themocouple into the furnace so that it is as close as possible to the bulb. Slowly open T4
again and wind up the furnace Variac to approximately 112 volts. Allow the sample to bake out
with continuous pumping until the pressure falls below 0.05 Torr (this will take 2 hours). The
temperature will reach approximately 400 C. Carefully remove the furnace and place it away from
the bulb.
(d) Let the sample cool to room temperature and then slowly immerse the bulb in a Dewar flask of liquid
nitrogen. Close T3 and T4 . Close T1 and allow approximately 500 Torr (or 40 kPa) of nitrogen,
as measured on M1 , to enter the line through T2 . Measure the reading on the manometer, M2 , and
record it as your zero pressure; all subsequent pressures will be recorded relative to this zero
value.
Note: You will have to top up the liquid nitrogen in the Dewar occasionally; it is
important that you make sure the bulb is always immersed in liquid nitrogen.
(e) Using T3 , pipette about 300 Torr of nitrogen into V1 . Measure on M2 this pressure (P1 ) and then
open T4 to admit the gas to the silica sample. [This initial aliquot should be almost totally adsorbed
if sufficient time is allowed.] When there is no further change, measure the equilibrium pressure
(P).
* Close T4. *
(f) Repeat this process with a 250 Torr aliquot of nitrogen (i.e. add sufficient nitrogen gas to make
the pressure in V1 reach about 250 Torr). Then add aliquots of approximately 200 Torr until the
equilibrium pressure reaches about 200 Torr - you should require several such aliquots.
6 Analysis of Data
You can use the pro Fit program (Excel or similar) to get the required plot of equation 3. If using
the pro Fit program follow the following steps (if using Excel or another alternative, follow a similar
procedure). Select New Data from the FILE menu. To the first 9 columns, re-label the titles as;1
Column 1P1; Col 2P; Col 3 P or dP;

Col 4(P1-P)V/T1; Col 5(dP)V2/T2; Col 6Mol Ads;
Col 7Tot Ads(x); Col 8P/P0 (X-axis); Col 9X/[(1-X)x] (Y-axis).
1 (to adjust label simply click on the column title)

Enter the experimental P1 and P values into the appropriate columns (column 1 and column 2 re-
spectively).
Select Data Transform from the CALC window. The Data Transforms window will appear.
Using the lists in the In/Out section, set X to Row Index and Y to dP, respectively. Select the
Differential/Integral check-box and from the lists select d/dx and P, then click OK.
Now, move all the data in dP (column 3) down one row by selecting the data in this column, cutting
it, selecting the 2nd row in this column and pasting the data. In the first row of this dP column enter the
value zero.
Enter the following formulae into the respective columns. To enter a formula, double-click the
number at the top of the desired column (the column number, not the column title). This will open the
Column Format window. Select NEW in the Calculation section to open the Data Transformations
window and select the Formula check-box and in the space provided enter the following formulae (in
bold);
(i) Column 4: ((c1-c2)*V1)/T1

V1= pipette volume (cm3 ); T1= room temp in K.
(ii) Column 5: (c3*V2)/77.33 V2=bulb volume (cm3 )
(iii) Column 6: ((c4-c5)*0.101325)/(P0 *8.314*M)

P0 =atm. pressure (mm Hg); M=mass of silica (g).
(iv) Column 7: To each cell of this column you will need to enter the cumulative sum of the data in
column 6. For example, to enter the cumulative sum of the first 3 cells in Column 6, select the
three data, select Statistics from the CALC menu. The sum will be listed (along with other column
statistics) in the statistics menu. Manually enter each row of Column 7. Screen shots below to
illustrate.
Figure 3: Example calculation
(v) Column 8: c2/Po Po=atm. pressure (mm Hg)
(vi) Column 9: c8/((1-c8)*c7)
Plot Column 8 (x-axis) versus Column 9 (y-axis) and fit this to a linear regression to obtain the value
of the gradient.
1. Calculate H1 and S.
(Hint: Would you expect H1 to be positive or negative? Data points may need to be removed

from graph to obtain a workable intercept value. Please explain this in your report if this is the
case.)
2. Assuming that the density of silica gel is 2.65 g cm3 and that the silica grains are spherical with
an average radius of 5 x 106 cm, calculate the estimated surface area, Sest (Question 5 in Section
3).
3. Samples A, B and C, provided on the bench, have surface areas measured by a different tech-
nique. Comparing silica gel with these samples comment on which value, S or Sest , appears more
appropriate for it (use Question 6 in Section 3).
4. How can you rationalise the value of S with your visual observation and with Sest values? [Hint:
How can the surface area of a sphere be increased?]
References
[1] Unit 610-310 Surface Chemistry Lecture Notes. (available on LMS)
[2] Shaw, D.J., Introduction to Colloid and Surface Chemistry, 3rd ed., Butterworths, London, pp. 108-
126, (1980.
[3] McClelland, A.L. and Harnsberger, H.F., J. Colloid and Interface Science, 23, 577 (1967).
[4] Genot, B., J. Colloid and Interface Science, 50, 413 (1975) [An advanced mathematical treatment
of BET theory].
[5] Gregg, S.J. and Sing, K.W., Adsorption, Surface Area and Porosity, Academic Press, New York,
1967
[6] ibid p.62
[7] ibid pp.45-6.
[8] Colston Research Society, Structure and Properties of Porous Materials, Everett, D.H. and Stone,
F.S. (eds.), pp. 68-94, 227-260, 266-294, 1958.
[9] CRC Handbook of Chemistry and Physics, 69th ed., R.C. Weast ed., CRC Press, Boca Raton, 1988,
pp.F-68-110.
[10] Aylward, G.H. and Findlay, T.J.V., SI Chemical Data, John Wiley and Sons, Sydney, 1971, p.101.
[note: be careful to distinguish whether the data given are per mole of atoms or molecules].

T2.2 Particle Size Determination

1 Introduction
Aims
To gain familiarity with particle size determination using turbidity
To evaluate the particle size distribution of TiO2 powder used in the construction of your dye-
sensitised solar cell in section T2.0.
2 Introduction
Light interacts with matter in different ways; it may be absorbed, scattered or transmitted. This
experiment is concerned with the scattered radiation. The intensity of the scattered light, I, measured at
an angle, , to the incident beam can be used to obtain the size of a particle that scatters the light, if the
particle size is less than approximately /20; where is the wavelength of the incident light. When this
condition is met the process is termed Rayleigh scattering [1].
Small particles (diameter, d <30 nm) generally satisfy this criterion for visible light [1]. The phase
of the scattered light is equal to that of the incident light under this condition, which means that the
particles scatter as point sources. The general geometry for this experiment is shown in Figure 1.
Figure 1: Schematic diagram showing the scattering of light by a particle.
When unpolarised light intensity I0 is passed through a sample containing N number of particles;
the intensity of scattered light I that reaches the detector located at a distance r and in an angle
relative to the direction of incidence is given by equation 1:
4 d 6 N m2 1

I
1 + cos2

= 2 4 (1)
I0 8r m2 + 2
where:
N is the number of particles;
257
m is the particles refractive index to that of the medium (n/nm , where n and nm are the refractive indices
of the particle and medium, respectively),
d is the particle diameter,
r is the distance from the sample to the detector,
is the wavelength of the incident light.
In this experiment you will use an absorption spectrometer to measure the transmitted light intensity
as a function of wavelength. In this case, the scattering angle, , is fixed at 0, where the scattered light
is equal to the transmitted light.
The intensity of the transmitted light decreases exponentially as it passes through the dispersion
(Equation 2);
It = I0 exp(L) (2)
where:
It is the intensity of the transmitted light,
L is the length of the cell,
is the turbidity of the dispersion, (Equation 3).
2
25 d 6 N m2 1

= (3)
34 m2 + 2
It is experimentally more meaningful to consider the weight concentration (mass of particles per unit
volume of dispersion), c, instead of N (number concentration which is the number of particles per unit
volume of dispersion). This is obtained from Equation 4:
d 3

c=N (4)
6
where is the density of the particles. Combining Equations 3 and 4 provides Equation 5:
2
44 d 3 c m2 1

= (5)
4 m2 + 2
In this experiment the optical density (O.D.) is measured as a function of wavelength using a UV-
visible spectrophotometer (Equation 6).
Intensity of incident light I0

O.D. = log10 = log10 (6)
Intensity of transmitted light It
Note that the term O.D. is used in place of absorbance because the particles used do not absorb
visible light, but rather scatter it, resulting in an apparent extinction of the transmitted light. Optical
density and absorbance are numerically equivalent.
Combining Equations 2 and 6 provides Equations 7 and 8.
It
L = ln (7)
I0
I0
L = 2.303log10 (8)
It
The optical density is related to the turbidity, (Equation 9):

2.303 (O.D.)
= (9)
L
Prepare a dispersion of colloidal silica (LUDOX AS-40, Aldrich 420840) by correctly noting down
the exact mass of colloidal silica. Use pH neutral water to dilute the dispersion until the diluted solution
shows an absorbance value <1. It is important to note down the dilution factor, so that the concentration
of the diluted dispersion, expressed in g/mL, is accurately known.
Prepare a sample of TiO2 powder, used for the construction of your solar cells, in pH 3-4 solution.
Sonicate your sample in an ultrasonic bath to break apart any clumps. This solution looks slightly milky
(red-brown when transmitting light). Be sure to accurately record the wt.% of your TiO2 solution (it
should not be more turbid than the silica solutions you prepared) in order to calculate the concentration
in g/mL.
Note: You may need to dilute this solution as well in order to obtain an absorbance value of <1. It is
again important to note down the dilution factor to calculate the concentration of the diluted dispersion
in g/mL for the calculation.
Thoroughly rinse two 1 cm optical cells with water.
Fill one cell with water and place in the spectrophotometer. Using single beam mode, take a back-
ground spectrum between 400 and 800 nm.
Place the diluted dispersion samples in a sample cell and record spectra in the same wavelength
range (400 - 800 nm).
Save all the spectra as a spreadsheet (.csv file) for analysis.
Using the mass of 1 mL of the two diluted dispersions used to obtain the spectra, calculate the
densities (g/mL) of the dispersions (these values are needed for your calculations).
Transmission electron microscopy data will be provided by the demonstrator. Take a ruler and
measure the diameters of 100 particles using the scale on the TEM micrographs.
4 Data Analysis
Plot the graph of OD (Abs) vs 1/4 .
1. Calculate the diameter (d) using the gradient of the graph OD (Abs) vs 1/4 (Rearrange Equations
5 & 9 use a properly labelled spread sheet). You need the concentrations of the diluted dispersions
in g/mL (see Experimental Procedure). Note that SI units should be used for both turbidity and
wavelength. The data presented in Table 1 provides refractive index information at 25 C.
Sample Density (g/mL) Refractive Index

Water 0.997 1.332
Polystyrene 1.05 1.59
Silica 2.20 1.45
TiO2 4.27 2.90
Table 1: Data for calculations

2. Calculate the particle diameters for all wavelengths using the spread sheet and compare the values
to that obtained in I. Is the size consistent in the wavelength range used? Calculate the average
diameter of silica particles and the standard deviation.
From the TEM data, plot the size distribution of the particles and find the average and the standard
deviation, compare these values with those obtained from your turbidity measurements.
5 Questions
The following questions should be answered as part of your report:
1. Given that the silica particles is not monodisperse, do you expect the size measured from turbidity
to be a simple average of the true particle size, or will it be biased towards smaller or larger sizes?
Explain your answer.
2. What are the sources of error in the experiment? For which concentration (high or low limits) of
the dispersions was the error smallest? Why?
3. What is meant by the terms monodisperse and polydisperse? Suggest a method that may be used
to measure the polydispersity of your sample [1].
4. In the light of results for your light scattering reference solution, discuss the behaviour of the TiO2
dispersion. What is the relevance of nano-scale particle sizes for the operation of dye sensitised
solar cells?
5. There are a number of different experimental techniques that can be used to determine the size of
a particle. List at least four different methods and discuss how each method works. How would
the average diameter of a particle, calculated using these different methods, compare [2].
Comment on the reasons for any possible differences between methods.
6 Particle sizing using a laser light scattering apparatus
A commercial dynamic light scattering (DLS) apparatus (Malvern ZetaSizer Nano-S90) is also avail-
able for use (see experiment PC2). If you have time, you can experiment with using this apparatus for
these particle sizing measurements, and compare the particle sizes you determine from DLS with those
from the method above - which is more physically believable and why might these values differ. Please
speak with a demonstrator about this.
References
[1] D. J. Shaw, Introduction to Colloid and Interface Science, Butterworth (1966).
[2] X. Renliang, Particle Characterisation: Light Scattering Methods, Dordrecht, The Netherlands, Ch
2, pp. 56-110 (2000)

T2.3 Photophysical study of Natural Dyes

1 Introduction
Aims
To explore three different types of electronic spectroscopy: absorption, fluorescence excitation

and dispersed fluorescence spectroscopy.
To investigate the type of information each provides about the sample molecule.
Obtain proficiency in the use of different spectroscopic instruments (specifically a UV-Vis spec-
trophotometer and a fluorescence spectrometer).
Measure the dispersed fluorescence spectrum of a molecule in the presence of different concen-
trations of dynamic quencher to determine the kinetic rate constant for quenching.
Fluorescence is a phenomenon that is used both in nature and in technological applications. It arises
from the radiative deactivation of the electronically excited state of a molecule via an allowed transition
between two states of the same multiplicity. This is most usually between two singlet states. Emis-
sion usually occurs to higher wavelength (higher energy) than the absorption band responsible for the
emission. Two types of fluorescence spectra can be recorded; fluorescence emission and fluorescence
excitation spectra. The former records the wavelengths of photons emitted from the sample under ap-
propriate excitation conditions, whilst the excitation spectrum records the wavelengths of the photons
absorbed that give rise to the observed emission.
2.1 Sample preparation
The fluorophore stock solution is to be obtained from the prep room. The sample solution is a 0.3
mg/100 mL solution of pyrene sulfonic acid, sodium salt in 0.0135M KCl.
You will also be provided with a solution of potassium chloride to use as the solvent for your
quencher solutions. All your solutions should be prepared form these stocks.
In this experiment you will take quenching measurements by acquiring emission spectra of solutions
containing varying concentrations of a quencher compound. In this instance the quencher to be used is
iodide (KI). The maximum concentration of iodide you require should not exceed 0.015 M .
Make 10 mL each of a series of solutions, the concentration of your fluorophore should be constant
and the concentration of the quencher varied for each sample.
Your solution series should be prepared such that the concentration of the fluorophore in each sample
is diluted by a factor of ten from the stock solution and the quencher concentrations are evenly distributed
between 0 and the maximum concentration listed above.
261
2.2 Acquisition of the Absorption Spectrum
Record the absorbance spectrum of your diluted fluorophore in the range 200-500 nm using one
of the available UV-Vis spectrometers. Use the fluorescence cuvettes in the spectrometer. Record the
spectrum using a cuvette with just the solvent to obtain the baseline. Absorbance due to the cuvette (and
solvent) will occur in the deep UV.
After collecting the spectrum note the position of any peaks in the spectrum for later use and print
both the spectrum and the peak report.
Hint: After collecting your spectrum, keep the solutions in the cuvettes for use in the fluorimeter.
You may wish to save your data as Spreadsheet Ascii (*.csv). Doing this will allow you to analyse
and plot your results with an appropriate program.
2.3 Fluorescence Excitation and Emission Spectra
Use one of the available spectrofluorimeters (Cary Eclipse, Hitachi F4500 or F4010). The instruction
guidelines for each instrument are kept with the instrument.
2.3.1 Recording the fluorescence emission spectrum
Make sure that the computer and spectrofluorimeter are on as per the respective guidelines.
Insert your cuvette containing the fluorophore solution into the cuvette holder.
Follow the operation instructions provided to collect fluorescence emission spectrum of your
sample.
If the spectrum is off scale then click on the appropriate buttons to rescale the x- or y- axis.
If the maximum measured intensity is too great (>1000) or too small (noisy), you will need to scan
the spectrum again, but this time the excitation and/or emission slits should be opened or closed as
appropriate. Slits widths can be varied between 2.5 and 20 nm. (Reducing the slit widths increases
the spectral resolution achievable at the expense of a reduction in the absolute amount of light reaching
the photomultiplier). Once you have set your slit widths (related to bandwidth), keep them the same
for the rest of the experiment). Should the emission spectrum still be off scale with the slits closed
down completely, you should change the excitation wavelength or the photomultiplier voltage. You
should seek advice before doing this. Choose a lower intensity peak in the absorption spectrum or even
a minimum between peaks to bring the emission spectrum onto scale. You may need to scan a few
times to optimise conditions. You should note these in your final report and keep them the same for the
quenching measurements in Section 2.4.
Examine the emission spectrum to see whether there is any evidence of second order diffraction -
incident or emitted light being recorded at long wavelengths. If there is, you may need to truncate the
scan range.
2.3.2 Background spectrum
To check that there is no spurious fluorescence or other light transmission from the solvent used,
record a background emission spectrum of the solvent using the same conditions as for the fluorophore
solution. You need to change Result Filename: for each run. This should appear as a flat baseline,
except possibly very near the excitation wavelength. Any second order diffraction should also be visible
in the background.

2.3.3 Recording the fluorescence excitation spectrum
Choose Setup parameters again and then choose Excitation.
In the Emission (nm): field put the wavelength corresponding to maximum intensity from your
Emission spectrum.
Set the instrument to scan the same range of wavelengths used in the absorption spectrum of 2.2,
but DO NOT let the upper end of this range be greater than 10 nm below the position of the
emission monochromator (e.g. if your emission monochromator is set at 500 nm, then scan from
200 to 490 nm)
Set Scan Speed (nm): to 300 nm/min.
Record the fluorescence excitation spectrum
Rescale the spectrum as before. If you need to remove any of the spectra select as appropriate from
the menu down the bottom and press the eraser button. (This only removes the spectrum from the current
plot and does not delete the data file). Save the data in a .txt (.csv) file to allow you to analyse your data
in the computer room.
Print the Emission, Excitation and Background spectra. Include a copy of this plot and the absorb-
ance spectrum with your report.
All data recorded by the spectrofluorimeter may be plotted using an appropriate program.
2.4 Quenching measurements
2.5 Recording the emission spectra
Record the emission spectra of your five fluorophore + quencher solutions prepared earlier, using a
different Result Filename: each time.
Click the cursors facility and use the mouse to move this cursor until it is in line with the maximum
of each spectrum. From the list at the bottom select the spectrum that you want the maximum for and
read off the value.
Plot the six spectra (fluorophore alone, plus five quencher solutions) on the same graph.
2.6 Photophysics of chlorophyll
Place your chlorophyll solution from experiment A1 in the UV lightbox. Illuminate with 365nm
light. What do you see? If you do not see any red fluorescence, see a demonstrator. Dilute your sample
in acetone such that the top 1 cm of the cell is fluorescing. Your sample is now ready for fluorescence
measurements (why is the dilution important?). What wavelength do you think is being emitted?
Using the skills learnt above, generate absorption, emission and excitation spectra for chlorophyll.
You will be judged on the quality of these spectra.
Attempt to record a fluorescence quantum yield of your sample. You will need to source a suitable
reference compound. If possible, attempt to record a fluorescence decay profile using the laser source in
the instrument laboratory. Can a fluorescence lifetime be determined, and if so, in conjunction with the
quantum yield, additional rate constants can be calculated.

3 Theory
3.1 Fluorescence Intensity and Decay Rate
The intensity of fluorescence is the number of photons being emitted by a sample in a particular
time.
This will be proportional to the number of fluorophore molecules in their excited state, [F*]. If
every absorption event results in a fluorescence photon being emitted, then we can describe the kinetics
of these processes as:
absorption: F + h F Rate = kA [F] (1)
fluorescence: F F + h0 Rate = k f [F] (2)
where F is a fluorophore molecule, [F] is the concentration of fluorophore in the ground state and
[F*] is the number of excited state fluorophores, kA is the rate constant for adsorption and kF is the
fluorescence rate constant.
In a time-resolved experiment, the excitation light is pulsed very quickly (faster than the fluorescence
decay time). Under these conditions, Eq. 1 stops (there is no more light) and the excited state population
decays via Eq. 2. This is a simple first order decay process with a half life, t1/2 = ln(2)/(kF ).
Under steady state conditions (where the existing light remains on), the steady state approximation
can be applied to the kinetic equations above, leading to:
kA
[F ] = [F] (3)
kF
3.2 Fluorescence Quenching
The fluorescence properties of a molecule (fluorophore) are influenced by the molecular environment
that it experiences. One of the most important effects of the environment is fluorescence quenching, in
which a second chemical species (the quencher) attenuates the emission by the fluorophore. Fluores-
cence quenching can also then be used as a probe for the environment of a molecule if the quenching
mechanism is understood.
There are two main mechanisms for quenching fluorescence; static and dynamic (kinetic). You will
explore one of these: dynamic quenching. In brief, in static quenching [F] is decreased by the quencher,
Q, binding to the fluorophore to make a binary complex, FQ , that is non-fluorescent (or dark). Therefore
there are fewer free fluorophores, resulting in less fluorescence. In dynamic quenching, a quencher
molecule interacts with an excited state fluorophore, facilitating a non-radiative transition to the ground
state and thereby decreasing [F*]. The amount of observed fluorescence is thereby decreased.
Under steady state conditions, the fluorescence intensity, which is proportional to [F*], is not sens-
itive to the static or dynamic mechanism.
Why?
However, a time-dependent measurement contains the information you need. The fluorescence in-
tensity as a function of time decays according to Eq. (2) in the absence of quencher. In the presence of a
dynamic quencher, the excited state molecule can now decay by a second reaction: dynamic quenching:
F + Q F + Q + Rate = kQ [Q][F ] (4)

The total rate of decay of F* is the sum of Eqs. 2 and 4:
Rate = k f [F ] + kQ [Q][F ] = (k f + kQ [Q])[F ] (5)
The concentration of Q does not change through the reaction. Therefore Eq. 5 can be written as:
Rate = k0 [F ], where k = k f + kQ [Q] (6)
Again, this is a first order rate equation. The fluorescence intensity should decay exponentially
with a rate constant, k. Knowledge of k f from the decay rate in absence of quencher, and [Q] from
the experiment, therefore yield the quenching rate constant kQ . In the presence of a static quencher,
the number of free fluorophores is decreased, however, the decay rate of the remaining ensemble of
fluorophores is unaffected. Therefore, if you can measure the time-resolved fluorescence in the presence
and absence of quencher the fluorescence rate will change for dynamic quenching, but not for static
quenching.
4 Analysis of Results
In the case of dynamic quenching, when a quencher Q is added to a fluorophore solution, it provides
another mechanism for the fluorophore to return to its ground state. This is a second order process
depending on the concentrations of quencher and excited state fluorophore as described by Eq. 4.
The fraction of molecules in a quenched solution that fluoresce is given by the ratio of the rate
constants with and without quencher, i.e.
kf
F = (7)
k f + kQ [Q]
where f is the fraction of molecules that fluoresce, compared with the number of molecules that
fluoresce in the absence of quencher. The fraction of molecules that fluoresce is proportional to the
intensity of the fluorescence spectrum. Therefore:
0f I0 kQ [Q]
= = 1+ (8)
f I kf
This is the Stern-Volmer Equation. The zero superscript and subscript imply zero quencher concen-
tration.
If a dynamic quenching mechanism is operative, then a plot of I0 /I versus [Q] is linear and has
a slope of kQ / kF and an intercept of 1. This combination of rate parameters is usually referred to
as the Stern-Volmer quenching constant, denoted KSV .kF can be determined (in principle) from a time
resolved decay curve of the unquenched solution (Eq. 6.2) and hence the true bimolecular quenching
rate constant, kQ can be determined (be wary of units when reporting these values). For the purposes of
this report, assume that the fluorescence lifetime is 100 ns (kF = 107 s1 ).
References
[1] Physical Chemistry with Applications to Biological Systems, R. Chang, (Macmillan Publishing,
New York), 1977, p. 441.
[2] Atmospheric Chemistry: Fundamentals and Applications, B. J. Finlayson-Pitts and J. N. Pitts, Jr.,
(Wiley, New York) 1986.

[3] Fluorescence and Phosphorescence Spectroscopy, Chapters 1 and 2, D. Rendell, John Wiley &Sons,
1987.
[4] Principles and Applications of Photochemistry, R. P. Wayne, OUP, 1988.
[5] Fraiji et al., J. Chem. Ed., 69, 425 (1992)

Theme 3 - Flow Synthesis
267
T3.0 - Synthesis of Coumarin Dyes in
Continuous Flow
Dr Anastasios Polyzos and Dr Wallace W. H. Wong

1 Introduction
Coumarins consist of a benzopyrone core structure and are found naturally in many plants. In science
and technology, coumarin dye derivatives have been used as gain medium in dye lasers, sensitisers in
solar cells and chromophores in fluorescence microscopy and cell biology. Some coumarin derivatives
are known anticoagulants and possess anti-cancer properties.
This experiment involves training on the use of the flow synthesis equipment. Please ensure that
you arrive at your allocated practical session on time as there will be ONE training time per practical
session. Note that you will take turns running the flow equipment.
2.1 Preparation of 3-carbethoxycoumarin (Dye 1) in continuous flow
2.1.1 Prepare the reagent reservoirs:
Combine the salicylaldehyde (1.22 g), diethyl malonate (1.60 g), acetic acid (1 drop) and ethyl
acetate (5 mL) in a container of appropriate size. Label this bottle reagent.
Swirl the contents of the flask to ensure adequate mixing of the reagents.
In a second 100-mL capacity bottle place 80 mL of ethyl acetate. Label this bottle solvent.
269
2.1.2 Setting up the flow machine:
Equip the flow unit with a 10 mL capacity PFE reactor coil.
Select a blue pump for performing the reaction.
Ensure tubing is connected as shown below.
Fully open the back-pressure regulator.
Place the exit line into a 100 mL bottle labelled waste. Prepare a separate empty container
labelled product for collected the reaction mixture.
Place both the solvent line and the reagent line for the blue pump into the bottle labelled solvent.
Turn on the flow unit and prime the solvent and reagent lines for the blue pump with ethyl acetate
from the solvent.
Carefully move the reagent line from the bottle labelled solvent to that labelled reagent.
Ensure the pump is set back to solvent on the screen.
Pass solvent through the reactor coil at a flow rate of 3.0 mL/min until it is filled.
Once the reactor coil is filled, reduce the flow rate to 1.0 mL/min.

Adjust the back-pressure-regulator carefully to a pressure of 5 bar.

Set the temperature of the reactor coil to 130 C.
Once the desired temperature is reached, and the pressure is stable, the unit is ready to run the
reaction.
2.1.3 Synthesis of 3-carbethoxycoumarin (Dye 1):
Add the piperidine (0.1 mL) to the bottle labelled reagent and mix the solution with a pipette.
Once thoroughly mixed, switch the blue pump from solvent to reagent.
When the contents of the reagent bottle is completely loaded into the reactor, switch the blue
pump from reagent back to solvent. Avoid introduction of air bubbles into the reactor.
Collect the reaction mixture when the reagents start to exit the reactor (10 min) and continue
collecting until all reagents have exited the system (10 min).
Once all the reagents have exited the flow unit, turn off the heating to the reactor by pressing the
on / off button on the reactor quadrant of the screen.
When the temperature is below 50 C, press the Stop All button.
Purify the product:
When the reaction is complete, transfer the contents of the product bottle to a 100 mL round
bottom flask.
Remove the organic solvent under reduced pressure.
Once the solvent has been removed a yellow oil should result.
Add ethanol (5 mL), mix well and place in an ice bath. Slowly add water (20 mL) and swirl the
flask frequently to ensure good mixing. Label the container and place in freezer for product to
crystallise.
The resulting white crystalline precipitate is collected by filtration and washed with small amounts
of ice cold ethanol/water (1:1).
Allow the product to air dry on the filter funnel for several minutes.
Calculate the yield and collect characterisation data (1H NMR, IR, melting point). The 13C NMR
and mass spectrometry data are provided on LMS. Include analysis of all the characterisation
data in your report.
2.2 Conventional preparation of Dye 1
In a dry 25 mL round bottom flask place salicylaldehyde (1.22 g), diethyl malonate (1.60 g), piperid-
ine (0.1 mL), acetic acid (1 drop) and absolute ethanol (5 mL). Add a boiling chip, equip the flask with
a regular condenser and a drying tube (to protect the mixture from atmospheric moisture), then boil the
mixture gently on a steam bath for 2 hours.
Pour the yellow reaction mixture into a 50 mL conical flask, then use portions of water totalling
about 17 mL to rinse out the reaction flask and so complete the transfer. Mix the contents of the conical
flask thoroughly then chill the suspension in ice (for about 15 minutes). Collect the solid by suction
filtration then wash it with several small portions of cold water. Recrystallise the crude product from
ethanol/water.
Calculate the yield and collect characterisation data (1H NMR, IR, melting point).

2.3 Preparation of Dye 2 and Dye 3
Place Dye 1 (0.44 g), 1,5-diazabicyclo[4.3.0]non-5-ene DBN (0.13 g) and dichloromethane (1 mL)
in a flask equipped with a magnetic stirrer bar. Heat the reaction at 130 C for 30 min. On cooling,
the reaction should be washed with diethyl ether, isopropanol and filtered. The product, Dye 2, can be
recrystallised from chloroform/ethanol.
Place Dye 1 (0.44 g), 1,8-diazabicyclo[5.4.0]undec-7-ene DBU (0.15 g) and dichloromethane (1

mL) in a flask equipped with a magnetic stirrer bar. Heat the reaction at 130 C for 2 h. On cooling,
the reaction should be washed with diethyl ether, isopropanol and filtered. The product, Dye 3, can be
recrystallised from ethanol.
Calculate the yield and collect characterisation data (1H NMR, IR, melting point) for both dyes. The
13C NMR and mass spectrometry data are provided on LMS. Include analysis of all the characterisation
data in your report.
3 Questions
1. Provide the mechanism of the reaction to obtain Dye 1.
2. Compare the yields of the reactions for the preparation of Dye 1 in conventional conditions and in
flow. Discuss your observations. Summarise the advantages of using flow processing.
3. Provide the mechanism of the reaction to obtain Dye 2 or Dye 3. Discuss the stoichiometry of the
reagents in the reaction.
References
[1] J. Zak, D. Ron, E. Riva, H. P. Harding, B. C. S. Cross, I. R. Baxendale, Chem. Eur. J. 2012, 18,
99019910.

T3.1 - Translation of Synthesis into Flow
Dr Anastasios Polyzos and Dr Lars Goerigk

1 Introduction
In T3.0, Dye 1 was synthesised in continuous flow and Dyes 2 and 3 were made using conventional
heating. The aim of this experiment is to develop a method for converting Dye 1 to Dye 3 in flow.
By this stage, you should be familiar with the operations of the flow equipment. Consult your
demonstrator if you have any questions.
2.1 Reaction optimisation for the synthesis of Dye 3
In T3.0, the synthesis of Dye 3 from Dye 1 was essentially solvent free. Clearly, this is not pos-
sible with the flow reactor setup available. Discuss in your group possible solvent choice and reaction
conditions. Propose trial reactions. Consult with your demonstrator before proceeding.
Once appropriate conditions have been found, perform the reaction in flow. Compare your flow and
conventional reaction results.
In your report, you should include discussions on proposed trial reactions, results of trial reactions,
any reaction observations and result of the flow reaction.
273
3 Questions
The process of performing two or more flow reactions in sequence is referred to as telescoped syn-
thesis. Discuss the possibility of performing the synthesis Dye 3 from salicaldehyde without isolation
of Dye 1 in a continuous sequence in the flow reactor.

T3.2 - Photophysical Properties of
Coumarin Derivatives

1 Introduction
1.1 Aims
To be familiar with the use of different spectroscopic instruments (UV-vis and fluorescence spec-
trometer)
To learn how to characterise the photophysical properties of fluorescent molecules (molar ab-
sorptivity, quantum yield, etc.)
To learn how to correlate the information obtained from spectroscopic measurement to the Jablon-
ski diagram
To gain experience in estimating how substituents change the photophysical properties of fluores-
cent molecules
To gain experience in estimating how the change of environment (temperature, viscosity, pH, etc.)
affects the fluorescence behaviours of fluorophores
1.2 Background
Luminescence is the emission of light from certain substances and occurs from electronically excited
states. Depending on the nature of the excited state, luminescence can be divided into two categories-
fluorescence and phosphorescence. Fluorescence is generated by the relaxation of the molecule from its
singlet excited state to the ground state, while phosphorescence is the transition from triplet excited state
to the ground state.
Fluorescence usually occurs from conjugated molecules with large aromatic rings. Upon absorbing
light of a specific wavelength, these molecules can emit light of a different, typically longer wavelength.
The difference between these two wavelengths is called Stokes shift. The fluorescence intensity as
well as the emission peak position can be influenced by molecular structures and the environment en-
countered. These properties make fluorescent molecules useful as environmental probes or markers for
biomolecules. In this experiment, we will study the photophysical properties of coumarin derivatives by
using different types of electronic spectroscopy.
1.2.1 Molar absorptivity
The amount of light absorbed at a certain wavelength is expressed as the molar absorptivity (),
defined by the equation:
275
A
= (1)
(c l)
where A is absorbance, c is concentration in mol/L or M, and l is the sample pathlength in cm. The
unit for molar absorptivity is usually taken as M-1 cm-1 or L mol-1 cm-1 .
Molar absorptivity is an intrinsic physical property, characteristic of the particular substance being
observed and thus characteristic of the particular electron system in the molecule. If the coefficient
is known, it can be used to determine the concentration of the substance in solution.
1.2.2 Fluorescence Quantum Yield
When a fluorophore absorbs a photon of light, an energetically excited state is formed. The deac-
tivation process of this species (loss of energy and return from excited state to ground state) can occur
through a radiative (i.e. fluorescence) or non-radiative (e.g. internal conversion and vibration relaxation)
pathway.
The fluorescence quantum yield (F ) refers to the fraction of excited molecules that return to the
ground state with emission of fluorescence photons. In other words, F is the ratio of the number
of photon emitted to that of photon absorbed. The F value can be determined through the absolute
method using integrating sphere, or the relative method that involves the use of a well characterised
standard with known F value.
For a relative method, solutions of the standard and test samples with identical absorbance at the
same excitation wavelength can be assumed to be absorbing the same number of photons. Hence, a
simple ratio of the integrated fluorescence intensities of the two solutions will yield the ratio of the F
value. In practice, the following issues must also be taken into consideration:
The concentration effect (Working within a carefully chosen concentration range and ensures
linearity across the concentration range)
The use of different solvents for standard and test samples (The solvent refractive indices must be
included in the calculation)
The relative quantum yield of unknown sample (x ) can be calculated by using the following equa-
tion: 2
Ast Fx n
x = st 2x (2)
Ax Fst nst
where st is the quantum yield of the standard, Ast and Ax are the absorbance of the standard and
unknown samples at a certain wavelength, respectively, Fst and Fx are the area of emission spectra of
the standard and unknown samples, respectively, nst and nx are the average refractive index value of the
solvent for the standard and unknown samples, respectively.
2 Experimental procedures
General experimental considerations:
In order to minimise the re-absorption effects, absorbances in the 10 mm fluorescence cuvette

should never exceed 0.1 at and above the excitation wavelength.
All the fluorescence spectra must be recorded with constant slit widths and voltage. Changing the
parameters between samples will invalidate the quantum yield measurement.

N N
O N N
O O O
O O O O O O
Dye 1 Dye 2 Dye 3
Molecular Weight: 218.21 Molecular Weight: 294.31 Molecular Weight: 322.36
2.1 Molecular absorptivity measurement
2.1.1 Sample preparation:
To prepare 10 mL of 1 mM stock solution of each dye, weigh appropriate amount of Dye 1-3 in each
10 mL volumetric flask and add DMSO to 10 mL. Heat up the solution a little to ensure that the powders
are fully dissolved. Mix well to ensure all the powders are dissolved. Dilute 20, 40 and 80 L of the
stock solution to 10 mL of DMSO in order to make 2, 4 and 8 M of Dye 1-3.
2.1.2 Acquisition of the absorption spectrum:
Record the absorbance of your diluted dye solutions in the range of 200-500 nm using one of the
available UV-vis spectrometers
First, record the spectrum using a cuvette with just solvent to obtain the baseline
Transfer the diluted solution to the cuvette and measure the absorption spectrum
Note down the position of any peaks in the spectrum for later use
Save the data as Spreadsheet Ascii (*.csv) for further analysis
Plot a graph of absorbance vs concentration
2.2 Fluorescence quantum yield measurement
2.2.1 Fluorescence emission spectra:
Use one of the available fluorimeters to record the emission spectra of the 8 M solution of Dye
1-3 using the wavelength of maximum absorption (max ) as excitation wavelength.
Note down the position of the emission peak in the spectrum.
Choose a proper standard from Appendix I according to the absorption and emission wavelengths
The standard samples should be chosen to ensure they absorb at the excitation wavelength of
choice for the test sample and if possible, emit in a similar region to the test sample. The lists of
well characterised fluorescent standard samples are shown in the Appendix.
Record the fluorescence spectrum of all the diluted solutions of Dye 1 and also the one just solvent
without dye

Calculate and note down the integrated fluorescence intensity (that is, the area of the fluorescence
spectrum) from the fully corrected fluorescence spectrum
Plot a graph of integrated fluorescence intensity vs absorbance
Repeat the above steps for Dye 2 and 3 and the standard.
2.3 Effect of environment on the fluorescence:
2.3.1 Temperature effect:
Seal/cap the cuvette with the 8 M dye solution and measure the emission spectrum using the
Hitachi 4500 spectrofluorimeter with the temperature controller turned on.
Measure the emission spectra at increasing temperatures (every 10 C up to 60 C). The temperat-
ure can be set by the temperature controller connected to the fluorimeter. Ensure adequate time
for the solution to equilibrate.
Plot a graph of the fluorescence intensity at a specific wavelength vs temperature.
2.3.2 Viscosity effect:
Dilute 50 L of the stock solution of Dye 3 to 10 mL of glycerol - a highly viscous solvent.
Swirl the mixture to ensure the stock solution is well dispersed.
Record the emission spectrum and compare the fluorescence intensity with the one in THF
3 Questions
Is the emission maximum (em ) dependent on the excitation wavelength? Why?
Compare the absorption and emission maximum of Dye 1 and 2 and explain the shift of the spectra.
Compare the quantum yields of Dye 2 and 3 solutions and explain the difference.
Investigate the environmental effect on the fluorescence and explain how these factors change the
fluorescence behaviours
References
[1] Wurth, C.; Grabolle, M.; Pauli, J.; Spieles, M.; Resch-Genger, U. Nature Protocol 2013, 8, 1535.
http://www.horiba.com/fileadmin/uploads/Scientific/Documents/Fluorescence/quantumyieldstrad.pdf

T3.3 - Computational Study of the
Photophysical Properties of Coumarin
Derivatives

Figure 1: Structures of the three coumarin dyes.
IMPORTANT: Before you begin with this exercise, please make sure that you have obtained the exper-
imental wavelength of maximum absorbance (max ) of dye 1 in dimethylsulfoxide (DMSO)!
1 Introduction
1.1 Context and aims
Previously, you have run computational experiments in CHEM20019 and CHEM30015, where you
have explored typical Computational Chemistry concepts such as geometry optimisations, single-point
energy calculations, vibrational frequency calculations, atomic partial charges, and the visualisation of
molecular orbitals (MOs). These calculations have been all carried out for electronic ground states. In
this exercise you will apply quantum-chemical techniques to electronic excited states. In particular, you
will see how Computational Chemistry can be used to provide a better understanding of experimental
results. At this stage, this understanding will be merely of a qualitative nature, however, in actual
research, computational chemists are able to obtain results of quantitative accuracy that allow making
predictions prior to experiment.
A second important goal of this exercise will be to realise that there are many ways to perform calcu-
lations and that not every chosen method provides useful results. In fact, a good computational chemist
needs to understand the benefits and limitations of these various methods, as well as the underlying
chemistry in order to be able to assess the validity of any computational result.
279
The previous computational experiments and the two introductory lectures given in CHEM20019
have given you a basic overview of the principles of Computational Quantum Chemistry and you may
want to look this information up if necessary. In addition, references [1] and [2] are recommended for
further reading. From your previous encounter with Computational Chemistry you may recall that any
quantum-chemical calculation attempts to find an approximate solution to the electronic Schrodinger
Equation of an atomic or molecular system:
= E
H (1)
where is the molecular many-electron wave function and H the Hamiltonian - an operator acting
on to calculate the total energy, E, of the system. Commonly used approximations apply to both the
Hamiltonian and the wave function. Broadly speaking, a quantum-chemical method usually refers to
the first and the atomic-orbital basis set to the latter. Any combination of a method with a basis set is
called level of theory with the following naming convention:
METHOD/BASIS
where the slash separates the method from the basis set.
The following subsections provide brief overviews of the concepts method and basis set. Section
2 gives important technical information and details on your presentation. Section 3 contains the actual
exercises. Please read all these sections carefully before you proceed with the calculations.
1.2 Wave-function and density-functional-theory methods
We can divide most quantum-chemical methods into two categories. The first one is pure Wave
Function Theory (WFT), which works directly with the Schrodinger Equation as a starting point. In
your previous computational experiments you have used Hartree-Fock (HF) theory, which is the easi-
est representative of WFT for electronic ground states. In this exercise you will use the so-called CIS
method (Configuration Interaction Singles) that is conceptually the simplest WFT approach for elec-
tronic excited states. Both, HF and CIS are ab initio methods, which means that these methods have not
been parametrised with any empirical/experimental data, but they have been derived from first principles.
At the end of this exercise you may have a better understanding whether ab initio is automatically a
guarantee for high-quality results or not.
A conceptually different and widely used methodology in Quantum Chemistry is Density Func-
tional Theory (DFT). DFT allows calculating the total energy of a chemical system from its electron
density. According to this theory, a so-called density functional establishes a mathematical connection
between the energy and the density. One can show that the exact or true density functional would
give us the exact solution of the electronic Schrodinger Equation. Unfortunately, we do not know how
to derive this true functional and as a consequence computational chemists are faced with hundreds of
density functional approximations. In this exercise you will encounter the density functionals with the
relatively cryptic names BP86, B3LYP, and BHLYP.
DFT methods can be used both for ground and excited states. Excited-state calculations are usually
carried out with an extension of DFT called (linear-response) time-dependent density functional theory
(TD-DFT). To distinguish a DFT ground- from a DFT excited-state calculation, we normally use the
prefix (TD) before the functional name, e.g. TD-BHLYP.
1.3 Atomic-orbital basis sets
In Quantum Chemistry we usually describe the complicated many-electron wave function of our
molecule with a set of molecular orbitals (MOs). However, it is nearly impossible to find analytical

(exact) solutions for the MOs and one has to rely on approximations. As you have learned in your first-
year lectures, we can describe an MO as a linear combination of atomic orbitals (AOs); see, for instance,
the well-known MO diagram for the H2 molecule. This approach is called Linear Combination of
Atomic Orbitals (LCAO). A given collection of AOs in the LCAO approach is called an AO basis
set. The more AOs we use in the LCAO approach, i.e. the larger our basis set, the more reliable and
accurate the underlying approximation, albeit at the cost of computational efficiency (computer time and
memory requirements). Given current technical limitations, most of the calculations in this exercise will
be carried out with the relatively small basis set 3-21G. However, in one of the exercises you will also
explore the basis-set dependence on your results with the STO-3G and 6-31G(d) basis sets. STO-3G
is significantly smaller than 3-21G, while 6-31G(d) is larger. Routine applications in actual research
employ even larger basis sets than those. Any good computational study needs to analyse the basis-set
dependence of the results.
1.4 Examples for various levels of theory
Here are some examples for possible levels of theory that you may use:
The BP86/3-21G level of theory indicates an electronic ground-state calculation with the BP86
density functional and the 3-21G basis set.
The CIS/STO-3G level of theory indicates an excited state wave-function calculation with the CIS
method and the STO-3G basis set.
The TD-B3LYP/6-31G(d) level of theory indicates an excited-state TD-DFT calculation with the
B3LYP density functional and the 6-31G(d) basis set.
2 General considerations
2.1 Using Gaussian09 with WEBMO
You will run all calculations with the program Gaussian09. To facilitate usage of this program you
will use the already familiar browser-based WEBMO interface http://webmo.chemistry.unimelb.edu.au/
with the same username and password as before. Basic usage of WEBMO (how to draw molecules, how
to prepare and run basic calculations, and how to analyse them) has been described in the computational
experiment in CHEM30015. Herein, only specific instructions are given.
2.2 Importing xyz files into WEBMO
This exercise computationally analyses the three dyes that you have worked with before (Fig. 1).
Usually, a computational treatment of these molecules would begin with a construction of these mo-
lecules in WEBMOs molecular editor, followed by a quantum-chemical geometry optimisation to ob-
tain the most stable (lowest-energy) structure. Due to limited resources, you will not need to carry out
this procedure for dyes 1-3. Instead, you can obtain already optimised structures from the LMS or the
Prac server. These structures have been optimised at the BP86/3-21G level of theory and they have been
saved in separate files in xyz format. To use these structures in your calculations, follow these steps:
i. Download the three xyz files from the LMS or Prac server and save them to your local computer. If
you are interested in what an xyz file looks like, you can open it with a simple text editor. The first
line of the file contains the number of atoms followed by a blank or a comment line. The subsequent
lines contain the Cartesian coordinates in Angstrom for each of the atoms.
ii. Create a new job in WEBMO; the familiar molecular editor opens. However, instead of drawing the
molecules from scratch, click on the option Import Molecule in the lower panel of the page.

iii. A small window (Import Molecule) like the one shown opens. In the drop-down menu, choose
XYZ Format and tick the box Generate Bonds. The last step is very important or otherwise the
molecular editor will not recognise this structure as a molecule. Finally, you can select your xyz file
under Browse.
iv. Click on Import Molecule. The molecular editor now displays the molecule. For most exercises,
you do not have to make any modifications to it. Simply click on the continue arrow in the
lower right-hand corner of the page to prepare your calculation. If you do not want to change the
molecule it is crucial that you AVOID the Comprehensive Cleanup buttons that you have used in
your previous computational exercises.
2.3 Assessing the error of a computational method
In any good computational study, it is important to assess and understand the error of the applied
level of theory. For this purpose we usually compare our result with either a highly accurate wave-
function method or, as in this case, with experimental values. In your discussion of the results please
calculate the error as:
Error = Theory - Experiment
This will give you the signed error. In some cases, it will be sufficient to simply discuss the absolute
or unsigned error.
2.4 Working with energy units
Quantum-chemical programs calculate energies in atomic or Hartree units (symbol Eh ). 1 Eh is

a relatively large quantity, which is why energies in Hartree should have 5 to 6 significant figures.
Energy differences between two molecules or electronic states are usually converted from Hartree to
chemically more useful units, such as kJ/mol, kcal/mol, eV etc. Electron volts (eV) are often used for
HOMO-LUMO gaps or electronic excitation energies. 1 Eh equals 27.21 eV, and energies in eV have
two significant figures. Electronic excitations are also discussed with respect to the wavelength (in nm)
of the absorbed photon. Wavelengths in nm have no significant figures and they should be rounded up
or down to the next integer value.
2.5 Comments on how to discuss the results in your talk
You should include a very brief introduction at the beginning of your discussion, as well as a short
conclusion at the end. The introduction is followed by a very short section outlining general computa-
tional details, such as the program that you have used and the class of methods that you have applied
(DFT, TD-DFT etc.). The exercises can be discussed separately. Briefly describe the tasks that you have

carried out (such as the chosen level of theory and type of calculation) followed by a discussion of the
results. You should support this discussion with tables and/or figures where appropriate. You can also
include screenshots from WEBMO when necessary, e.g. pictures of MOs.
3 Instructions and Questions
Optimisation of derivatives of dye 1
Figure 2: Derivative of dye 1 with a functional group R that is either an amino, hydroxyl or a nitro
group.
Before you start with the actual calculations in question 3.1, it is highly recommended that you first
prepare and submit some time-intensive calculations in preparation for exercise 3.3. In that particular
exercise, you will analyse three derivatives of dye 1 with different functional groups R in Fig. 2.
Instructions:
i) Import the xyz file for dye 1 and substitute the hydrogen atom in the position of the R group in
Fig. 2 with an amino group. It is best to select the H atom in the R position and then to change
the atom type to N. Next, draw two H atoms and connect them to the N atom. Do NOT click on
Comprehensive Cleanup. This would distort the atomic positions throughout the molecule, thus
shifting their positions away from the already optimised ideal ones, and unnecessarily prolong the
computation. Also, do not symmetrise the molecule. As an additional exercise (you do not need to
address this in the talk) you may want to think about what symmetry you expect the molecule to
have. Click on the lower right-hand arrow to continue.
Hint: Try to draw the amino group in a chemically reasonable way to save computer time.
ii) A geometry optimisation needs to be performed next. As most atoms in the molecule have been pre-
optimised, this calculation should be computationally more economical than starting from scratch.
In the Gaussian configuration menu that has just opened, choose Geometry Optimisation under
Calculation. Under Theory choose Other and type BP86 into the small pop-up window.
Chose 3-21G as the basis set and choose the correct charge and multiplicity.

iii) Run the optimisation. It may last a while, depending on how well you have drawn the input struc-
ture.
iv) Repeat these steps and run optimisations with a hydroxyl and a nitro group as the respective sub-
stituents.
v) The structures will be needed for exercise 3.3. You do not need to discuss exercise 3.0 separately
in your talk. It suffices to describe the optimisation in one or two sentences in the discussion of
exercise 3.3.
3.1 Studies on dyes 1, 2, and 3:
a) Identification of an optimal level of theory

Instructions:
i) Import the structure of dye 1 and click on continue in the molecular editor.
ii) The following menu allows you to choose the type of calculation. WEBMO has a pre-defined
routine that allows the calculation of UV/Vis spectra. However, we will NOT use this. Instead,
we will use the option Themed Exp: TD-B3LYP under Calculation. This is a routine
specifically defined for this exercise and it will calculate the first excited state with the TD-
B3LYP method.
iii) Select the 3-21G basis set and specify molecular charge and multiplicity.
iv) So far you have set up a calculation to treat dye1 in the gas phase. However, measurements have
been carried out in a solvent. Therefore, you need to specify a solvent model for the calculation.
This can be done by clicking on the Advanced tab. This menu allows you to specify additional
options. You see that there is an option called Solvent with predefined options, however, we
will NOT make use of this in this exercise. Instead, we will add the required information manu-
ally in the section Additional Keywords near the bottom of the menu. In that section we can
provide Gaussian09 with additional options. The measurements have been carried out in DMSO.
The correct keyword for the DMSO solvent model is: scrf(PCM,solvent=Dimethylsulfoxide)
v) Submit the calculation by clicking on the arrow in the lower right-hand corner.

vi) Repeat the previous steps with the options Themed Exp: TD-BHLYP and Themed Exp:
CIS. All other options must be exactly the same as for the TD-B3LYP calculation.
vii) Once a calculation has finished, click on the respective link in the job manager to analyse the
output. Scroll down to the section Excited States where you can find information on the first
excited state.
Question:
Compare the calculated excitations with the experimentally measured max value. Calculate the errors
and determine which level of theory is closer to experiment. Use this level of theory in subsequent
TD-DFT treatments unless stated otherwise. This level of theory will be called optimal level of
theory in the following. However, be aware that this level of theory may only be optimal in the
context of this exercise.
b) Nature of the calculated excitation

Instructions:
The lowest-lying excitation in dye 1 is a HOMO-LUMO transition. In this exercise we will invest-
igate the nature of this transition. For that purpose, we will need to visualise the relevant MOs first.
Prepare a new calculation for dye 1. This time select Molecular Orbitals under Calculation.
Under Theory look for the name of the optimal method that you have identified in part a). Choose
the same basis set, charge and multiplicity as in part a). Finally, add the keywords for the DMSO
solvent model and run the calculation. Write down the HOMO and LUMO energies in Hartree for
later, visualise the HOMO and LUMO, and take screenshots for the talk.
Question:
What type of excitation is the lowest lying transition in dye 1? Examples for possible transitions
in molecules are n-*, -*, -*, n-*, -*, etc. Hint: Visualise the HOMO and LUMO and
identify if they have n- (nonbonding; lone pair), -, or -character. For instance, if you see that the
HOMO has -character, and the LUMO *-character, then you have identified a-* transition.
c) Basis-set dependence
Instructions:
Calculate the lowest-lying electronic excitation energies in dye 1 with the optimal method identified
part a), but with the STO-3G and 6-31G(d) basis sets. The DMSO solvent model needs to be used
again.
Questions:
1) Compare the excitation energies and errors with respect to experiment and with the energy that
you have obtained with the 3-21G basis set. What is the likely source of the error for STO-3G?
Given what has been said about the LCAO approximation and basis sets in the Introduction, what
do the observed trends tell you about our optimal approach? Would you say it is reliable and
robust?
2) Compare the computer times for your TD-DFT calculations performed with the three different
basis sets and discuss them. Relate them to what you know about these basis sets.
d) Calculations of dyes 2 and 3

Instructions:
Carry out excitation-energy calculations for dyes 2 and 3 with the optimal level of theory and the
other options described in part a). Also calculate the MOs in the same way as described in part b).
Questions:

1) Discuss the errors with respect to experiment for dyes 2 and 3, and compare these with the error
for dye 1. Suggest how we may reduce the error.
Hint: Think about the trends you have observed in part c).
2) Compare the HOMO-LUMO gaps for dyes 1, 2, and 3. Identify any correlations between the
HOMO-LUMO gaps and the measured absorption maxima. Do the HOMO-LUMO gaps allow
for a qualitative interpretation of the experimental values?
3) Compare the computing times for your TD-DFT calculations of dyes 1, 2, 3 with the 3-21G
basis set. Plot time against number of atoms. Do you see a linear dependence? Based on your
observations, try to discuss the following statement: doubling the number of atoms in the system
increases the computer time by a factor of 2. Is this statement true? Imagine you were to apply
the same quantum-chemical method to a very large system with hundreds of atoms. What do your
observations tell you about the likely success of such a strategy? Hint: Pay close attention to the
change in number of atoms and increase in computer time.
3.2 Solvent effects in dye 1:
Instructions:
i) Run calculations on dye 1 with the optimal approach, but instead of DMSO, use water as the
solvent.
ii) Repeat step i) for cyclohexane.
iii) Repeat the calculation with the same settings as before, but this time leave the Additional
keywords section empty. This will prepare an excited-state calculation of dye 1 in the gas
phase.
Question:
For the discussion, please familiarise yourself with the terms blue shift and red shift. Analyse
the different excitation energies and errors. What are the various trends for the solvent calculations
in DMSO, water and cyclohexane with respect to the gas-phase result? Use the terms blue and red
shift in your discussion. Compare your trends with the polarities of the various solvents. Is there a
qualitative correlation?
3.3 Substituent effects in dye 1:
Instructions:
i) Check that the geometry optimisations that you have run at the very beginning have finished.
Use the optimised geometries as inputs for new calculations. Alternatively you can also export
and save the geometries as xyz files and then reimport these in new jobs (see Figure below).
ii) For all three optimised derivatives, calculate the lowest-lying excitation energies with the op-
timal level of theory identified in exercise 3.1.
Question:
Compare the three calculated absorption wavelengths with those for dye 1 and rank the molecules
accordingly. Based on your results, predict which substituent has the largest effect on the excitation
energies and which the least. What do you know about the functional group that has the largest effect?

3.4 Additional problem
Question:
Imagine you heave measured an absorption wavelength of 600 nm, but your calculated absorption
wavelength is 500 nm. The unsigned error is therefore 100 nm. What are the excitation energies (in eV)
associated to those wavelengths and how large is the unsigned error in eV?
The difference between the wavelengths of 300 nm and 200 nm is also 100nm. What is the energy
difference in eV? Compare the two energy differences that you have calculated; why do we have to be
careful when we discuss errors in nm? While you can easily check your results online with an energy
unit converter, demonstrate in the presentation how you calculate the energies in eV!
Hint: Start with the famous Planck-Einstein Relation/Planck Equation that links the energy of a
photon to its frequency.
References
Both books are available as e-books through the University Library.
[1] Bachrach, S. M. Computational Organic Chemistry, 2nd ed.; John Wiley & Sons: Hoboken, 2014.
[2] Jensen, F. Introduction to Computational Chemistry, 2nd ed.; John Wiley & Sons: Chichester, 2007.

A PPENDICES
Appendix A - Techniques for Synthesis
1 Reactions under Nitrogen
Reactions that involve intermediates that are sensitive to oxygen and/or moisture are best run under
nitrogen; such reactions include Stobbe condensations and Grignard reactions.
In situations where the consequences of air contact are severe, pure nitrogen is passed into the
reaction vessel via a syringe needle that pierces a serum camp (surplus nitrogen is vented through another
syringe needle), or via tubing connected to the glassware by a standard adaptor vented via a bubbler
to the atmosphere.
A high nitrogen flow is to be avoided for two reasons:
1. The nitrogen in the synthetic laboratory is not 100.00% pure. A high flow will expose the reactants
to larger quantities of the contaminants (O2 , H2 O) during the reaction than a low flow.
2. A high flow is not only unnecessary and undesirable but also wastes nitrogen.
2 Safe Handling of Sodium
The alkali metals are very reactive. They combine rapidly with oxygen and with moisture - if
dropped into water, a piece of an alkali metal usually inflames and explodes violently. They also react
violently with chlorinated solvents such as CHCl3 and CCl4 .
Although sodium reacts explosively with water its reaction with alcohols is much more controlled;
with methanol and, to a lesser degree, with ethanol the reaction is vigorous but not dangerously so. The
higher alcohols react much more slowly, so much so that the higher sodium alkoxides are best prepared
with sodium hydride rather than with sodium itself.
Sodium is supplied as lumps in a jar of paraffin oil or xylene. The function of the hydrocarbon is
to minimise contact of the sodium with the atmosphere; this tactic is not entirely successful, and the
sodium (a silvery metal) becomes coated with a layer of oxides, hydroxide and carbonate which must be
removed before the sodium is used.
Transfer a lump of sodium to a small dry beaker containing sufficient petrol (bp 60-80 ) to cover the
lump. Take the beaker to a balance bench. Weigh a small dry conical flask containing sufficient petrol
to cover the sodium you are going to weigh out. Using a large spatula, cut off the crust to expose the
bright sodium metal. Cut off pieces of clean sodium and transfer them into the weighed flask until the
required amount of sodium has been transferred.
Return the unused part of the sodium lump (or lumps) to the storage jar. The larger scraps of crust
and sodium are placed in the scrap jar provided. Add a few mL of meths to the petrol in the beaker to
destroy any remaining flecks of sodium - leave the beaker in a fume hood overnight just to make sure.
(Once you have transferred the weighed sodium to your reaction mixture you should treat the petrol
in the small conical flask in the same way.)
291
A PPENDIX A CHEM30015: Advanced Practical Chemistry
3 Liquid-liquid Extraction (and Washing)
Efficient extraction requires thorough mixing of the organic and aqueous phases.
CAUTION: The mixing must be gentle at first and the pressure within the separating funnel must
be released frequently (the solvents usually employed ether, pentane, CH2 Cl2 , CHCl3 - - have low
boiling points). Obviously, the aqueous phase must be cold before extraction is attempted. This precau-
tion applies particularly when you are extracting an acid from an organic solvent with aqueous sodium
bicarbonate or sodium carbonate. Only when little gas is being formed should the mixture be shaken
thoroughly.
For a given volume of organic solvent it is best to carry out several successive extractions with
portions of the solvent rather than use all of the solvent in a single extraction. E.g., three successive
extractions with 33 ml of solvent (3 33 ml) is more efficient than one extraction with 100 ml. WHY?
A 10 10 ml extraction would be even more efficient but normally would be too time- consuming to
be profitable. (In cases where simple extraction is inherently inefficient continuous extraction techniques
are available.)
Hint: When the lower phase is to be extracted or washed again, borrow another one or two separ-
ating funnels so that you can run the lower phase directly into a separating funnel containing the next
extraction solvent or wash solution. This will save a lot of the traditional transfers of liquids to and from
beakers or conical flasks, and the consequent rinsings.
Emulsions. Emulsions are frequently encountered during extraction; particularly when the aqueous
phase has a low ionic strength. Very often experimental instructions call for washing an organic solution
with water to remove inorganic acids or bases. You will use saturated aqueous sodium chloride (often
called brine) instead of pure water. The sodium chloride not only reduces the incidence and severity
emulsions but also:
(a) reduces the solubility of organic compounds in the aqueous phase,
(b) reduces the water content of the organic phase, and so facilitates the final step - that of drying the
organic phase.
If in spite of using brine a stable emulsion is formed, try mixing in some filter aid (a form of
silica) or macerated filter paper then filtering the emulsion; flocculent solid impurities often stabilise
emulsions, and the additives gather the tiny particles and so make their removal by filtration easier.
When the product of an organometallic reaction (e.g., a Grignard reaction) is being worked up, metal
oxides can cause stable emulsions; these emulsions usually can be broken by adding potassium sodium
tartrate or a little EDTA (WHY?).
Another Hint: The tap and stem of the separating funnel usually will be very wet with the organic
phase. Therefore it is usually best, when the washings have been completed, to transfer the organic
phase (if it is the upper layer) by decanting it through the neck of the funnel (dried with tissue) into the
drying flask. Do not forget to rinse the funnel with one or two small portions of the organic solvent.
If, despite your best efforts, drops of aqueous phase are visible in the drying flask, they can be
removed with a Pasteur pipette.
4 Drying Organic Solutions
Removal of dissolved water from organic solutions is usually highly desirable because recovery of
the solute by evaporating the solvent generally leaves much of the water contaminating the residue of
solute. This doesnt matter much if the solute is to be recrystallised from a hydroxylic solvent (e.g.,

CHEM30015: Advanced Practical Chemistry A PPENDIX A
methanol) but can be a nuisance if water is insoluble in the recrystallisation solvent. If the solute is to
be purified by distillation, then drying the solution is essential because the water tends to stick to the
glass of the condenser, and will contaminate the desired distillate. Further, there will be some loss of
compounds susceptible to hydrolysis during the prolonged heating involved in distillation.
The most generally useful way of drying an organic solution is to add a substance that absorbs water
a drying agent. Most drying agents are salts that form hydrates.
4.1 Commonly-used drying agents:
CaCl2 Complexes with most functional groups, and is used only for drying hydrocarbons, and alkyl
and aryl halides. It has a high capacity for water (forms CaCl2 6 H2 O) but is rather slow in action.
CaSO4 Is generally useful but, although it can dry a solution quickly and thoroughly, its capacity is
low (forms 2 CaSO4 H2 O). It is best used after a preliminary drying with a high capacity agent.
MgSO4 H2 O Is a good general-purpose agent. It is rapid in action and has a high capacity (up to
MgSO4 7 H2 O).
K2 CO3 Has a fairly good capacity for water but is somewhat slow in action. It should not be used for
solutions containing solutes that can react with bases (e.g., alkyl halides, esters, aldehydes, or ketones).
Anhydrous Na2 SO4 is a very popular drying agent but is in fact not very effective.
By far the best general purpose drying agents are Molecular sieves. They have high capacities, rapid
in action (especially as powders rather than pellets), and dry solutions very thoroughly (much more so
than the other agents listed here). They should not be used with acids (eg, acetic acid) as these react
with the sieves destroying their activity and contaminating the liquid. Unfortunately, molecular sieves
are rather too expensive for use in class.
It is not easy to prescribe the quantity of drying agent to be used, or the time of contact because there
are too many variables: the nature of the solvent, the nature and concentration of the solute, the particle
size and porosity of the drying agent (the rate of drying will depend critically on the surface area of the
drying agent exposed to the solution), the temperature.
The greater the amount of drying agent used the quicker and more thorough will be the drying. But
then large volumes of solvent will be required for washing when the drying agent is filtered off; and this
could re-introduce moisture to the solution.
4.2 Recommended procedure
For about 100 ml of solution (free from visible drops of water at the bottom of, or adhering to the
wall of, the container): Add 1 to 2 g of drying agent, swirl the suspension for half a minute then observe
the appearance of the drying agent: usually the particles of drying agent become coated with an aqueous
solution of the drying agent which causes the particles to stick together. Continue adding portions of
1 to 2 g of drying agent (swirl the suspension after each addition) until the particles no longer clump
soggily together. Then add drying agent roughly equal to twice the total amount already added. Swirl
the suspension thoroughly then seal the flask with aluminium foil. Allowing the suspension to stand for
up to half an hour with occasional swirling is quite adequate, and, of course, the mixture can be left for
much longer if there is no time to complete the next operation in the same session.
4.3 Removal of the drying agent
Do not filter the suspension through a fluted filter paper (the text-book method). Instead, pour
the solution and drying agent into a filter column (there are two in your set of glassware) packed with a
short (2 cm) column of oven-dried cotton wool. This method has the advantages of speed and minimal

exposure to atmospheric moisture. Also, oven-dried cotton wool is itself quite a good drying agent.
Rinse the flask containing the remaining drying agent several times with small volumes of solvent, and
pour these down the column.
4.4 Removal of solvent
Rotary evaporators are very useful, time-saving, pieces of equipment for solvent removal. (Do not
grease the joint of the rotary evaporator.) However, volatile solutes can easily be lost because their
vapours are carried off in the stream of solvent vapour. This can be a problem even with solvents, like
ether, having very low boiling points. If your compound has a boiling point below 170 or is a globular
molecule of low polarity (e.g., camphor),remove the ether by distillation through a fractionating column.
To minimise the chance of bumping, the round-bottom flask should be no more than one- third
full of solution. Never use conical flasks (unless they are small (10 ml)) because the flat bottoms of
conical flasks are liable to implode.
5 Distillation
Distillation is normal method of separating liquids from dissolved solids, and from other liquids hav-
ing sufficiently different boiling points. The vapour over a liquid mixture is richer in the lower-boiling
component; two boiling-point/composition phase diagrams are shown in Figure 1. In the simplest form
of distillation, the vapours rising from a boiling liquid are collected by passage into a condenser sloped
so that the condensate (distillate) runs down into a receiver. If the we start with a roughly 1:1 mixture
that behaves according to the phase diagram in Figure 1(a) it can be seen that the distillate will contain
about 75% of the lower-boiling component A; this is not a useful enrichment for simple distillation. The
situation is worse than this because, as distillation progresses, the boiling mixture becomes enriched
in B, and the distillate correspondingly becomes progressively poorer in A. A more favourable phase
behaviour is shown in Figure 1(b); in this case a separation of a component of satisfactory purity could
be achieved if it was by far the major component of the mixture, and if the distillation was conducted
carefully. Fortunately, there are many mixtures that have phase diagrams better than Figure 1(b).
Figure 1: Examples of boiling-point/composition phase diagrams.
In general, and if the product is required in high purity, it is necessary to resort to fractional distilla-
tion.
In fractional distillation the vapours of a boiling mixture pass up a column having a large internal
surface area provided by various sorts of packing materials or, in the most efficient columns, a spinning
band of wire mesh. In all of these fractionating columns the rising vapours are close contact with

the falling film of condensed liquid. Under these conditions molecules pass between vapour and liquid
phases many times during their passage up the column. In effect the mixture is being subjected to a series
of simple distillations as it passes through the column. Only simple distillations will be encountered in
this course (though notes on fractional distillation are included).
The most commonly used apparatus for simple distillation is shown in Figure 2. It consists of a
boiling flask (stilpot), a stilhead, a condenser, a receiver adaptor, and a receiver. The stilhead shown is
thesimplest form of the Claisen stilhead; often the vertical part containing the thermometers extended
and indented to provide some fractionation (though this is minimal). The stilhead should be lagged to
minimise heat loss. The receiver adaptor shown is also the simplest of its kind; for vacuum distillations
especially, a three-cone adaptor is used so rotation (about the axis of the condenser) allows each of
the three receiving flasks in turn to collect different fractions all without interrupting the vacuum. The
adaptor side-arm prevents a build-up of pressure during atmospheric-pressure distillations, and provides
a connection to a pump for reduced-pressure (vacuum) distillations.
Not shown in the figure are the wire hooks and rubber bands used to hold the pieces of glassware
together, and the clamps that support the assembly.
Figure 2: A diagram of distillation equipment.
General comments concerning distillation technique
1. Except for Kugel distillation (described below) a stirrer/hotplate is used to heat the distill and and
to provide the agitation necessary to promote smooth boiling. [Boiling chips lose their ability after
a while, and lose it quickly under vacuum]. The heat is supplied to the stilpot (distillation flask)
by means of an air-bath - a two-part aluminium box that is assembled round the stilpot on top of
the hotplate.
(Air bath temperatures can be adjusted much more rapidly than is practical with the traditional
oil-bath, and air-baths are much cleaner and do not fume - and of course they cannot ignite.)
Assemble the distillation apparatus starting with the distillation flask (stilpot) resting on the centre
of the stirrer hot-plate (so that the flask will not fall off the distillation column or stilhead and so
that there will be reasonable thermal contact between the hot-plate and the contents of the flask).
2. Note the weights of the receiving vessels before you perform the distillation.

3. The bulb of the thermometer must be completely bathed in the vapour of the distillate otherwise
the temperature registered by the thermometer will be too low and will fluctuate. Lag the stilhead
to prevent excessive heat loss. The rate of distillation during collection of a major fraction should
be maintained at about one drop per two seconds. A faster rate of distillation gives inefficient
separation of the components of the distilland, while slower rates will result in incomplete contact
of the stilhead thermometer with the distilling vapours - and the distillation might take longer than
is convenient.
(Why should distillation be as slow as possible?)
Distillation of a mixture containing two major components should give a plot of temperature vs
quantity-of-distillate of the type shown in Figure 3. The temperature of the air bath can be raised
quite rapidly until the distillate is refluxing in the column. Thereafter the temperature should be
kept slowly rising during the distillation to maintain the specified distillation rate.
It is not possible to specify the optimum rate of increase of the temperature of the air-bath; the
rate must depend on the individual distillation apparatus and on the composition of the mixture to
be distilled. (Note: the bath temperature will be much higher than the boiling point of the mixture
in the stilpot.)
This kind of profile (Figure 3) will be obtained only if the phase diagram is like Figure 1(b) or
better, or if an efficient fractional distillation is conducted.
Figure 3: An example plot of temperature vs quantity-of-distillate.
4. Flooding. (This applies particularly to fractional distillations.) If there is too much heat loss
through the wall of the fractionating column and/or stillhead, condensation will be so rapid that
the condensing liquid will not be able to drain quickly down into the flask but will be buoyed
up by the vapour rising through the column. Formation of this mobile volume of condensate is
known as flooding. Because the flooding liquid is being rapidly stirred by the rising vapour,
the composition of the liquid is the same throughout the flooded zone. Therefore there is no
fractionation in the flooded zone of the column and consequently the efficiency of the column is
impaired. The excessive heat loss can be prevented by passing a small current through the electric
heating coil of the column jacket.
The current is controlled with a Variac; it will never be necessary to go beyond the 60 mark on
the Variac used to control the current.
5. Vacuum Distillation For most applications ground-glass joints should be greased to avoid the
joints jamming and to minimise the escape of vapours between the ground-glass surfaces.
Obviously, particular care must be taken in greasing the ground-glass joints of apparatus to be
used in vacuum distillation in order to prevent the development of air leaks while at the time not
allowing the grease to contaminate the distillate. Joints that will become hot should be greased
with Apiezon-T or Apiezon-H not with the softer Apiezon-L or -M greases or petroleum

jelly, which will certainly run out of hot joints. Joints that will remain cool should be greased with
petroleum jelly, not with the very expensive Apiezon greases.
Put small blobs of grease around the widest part of the cone (Figure 4). Put the cone and socket
together, then gently rotate cone and socket in opposite directions to smear the grease into a
complete and clear band. Some of the grease will be squeezed out of the joint to form a reservoir
that will repair any breakdown of the seal within the joint. If too much grease is used some will
be squeezed out at the narrow end of the joint and will contaminate the distillate.
Figure 4:
6. The liquid in the still-pot must be kept boiling smoothly because super-heating and the subsequent
violent boiling will cause temperature fluctuation at the still-head, poor fractionation, and even
splashing of undistilled liquid into the receivers. For vacuum distillation boiling chips are virtually
useless as a means of preventing superheating. Instead, agitation of the boiling liquid is achieved
by vigorous magnetic stirring.
7. Do not heat the distillation flask until the pressure has ceased to fall: otherwise the contents of the
flask are likely to boil over into the receivers.
8. If you have been using an oil pump: the shut-down procedure is described in the section on Rotary
Oil Pumps.
Kugel Distillation
A major part of material loss in distillation is the distillate adhering to the surface of the distillation
pathway - column, stilhead, condenser and receiver adaptor. This loss can be unacceptably large in
small-scale distillation because there is a limit to how far the dimensions of the apparatus can be scaled
down. The limit is set by the surface tensions of the distillate: if the distillation pathway is too narrow
it becomes flooded throughout and much of the distillate will not drain into the receiver. Use of an
effective fractionating column (necessarily of high surface area) is virtually precluded.Usually a small-
scale distillation apparatus must have as short a distillation pathway as possible to minimise the internal
surface area. It follows that there can be little fractionation; the distillation serves to remove very
volatile and highly involatile impurities from the required material. The best way to effect this type
of purification is to use bulb-to-bulb distillation (Kugel distillation). As the name suggests the crude
liquid is placed in a r-b flask connected directly to a bulb that serves as condenser and receiver: the bulb
may be connected to another in case re-distillation is required (Figure 5). The necessary agitation of the
hot crude liquid is provided by rotating the train of bulbs about its axis.
Method is as follows: Transfer the crude liquid to the stillpot using the minimum of warm volatile
solvent (e.g., ether, petrol) for rinsing: you can boil off some of the rinsing solvent by heating on a
steam bath. assemble the stillpot and receiver(s) [pre-weigh the receivers]; grease the joints carefully
in the usual way using Apiezon T or H only for those joints that will be heated. Set the bulbs rotating
then gradually apply the vacuum. Increase the temperature of the oven gradually to allow the volatile
impurities to evaporate completely form the bulbs and to avoid spattering of crude liquid. The oven

Figure 5: Appartus for Kugel distillation
temperature required for distillation at a convenient rate is often much higher than the expected boiling
point. If it is desirable, the distillate can be redistilled by moving the bulb train so that the first receiver-
bulb is in the oven. When the distillation is judged to be complete, dismantle the bulb train taking care to
keep the receiver horizontal so that the distillate is not spilled or contaminated by the grease on the glass
joints. Remove as much as possible of the grease from the joints then weigh the receiver. Distillate that
is to be stored is transferred with a Pasteur pipette that has about 1 cm of tip bent (in a Bunsen flame) at
an angle of about 45 . Use the Kugelhohr in conjunction with a Pressure-Temperature Nomograph.
Rotary Pumps
For distillation at very low pressures, rotary oil pumps are used. The demonstrators in the laboratory
will explain the operation and use of the mobile units. These are expensive items of equipment and are
easily damaged by ill-use - be careful with them.
There are two important points that should be noted. Firstly, each pump is fitted with a cold trap, the
purpose of which is to trap any volatile compounds that would otherwise enter the pump. This serves to
preserve the pump oil in a usable condition, giving rise to higher efficiencies and also tends to remove
any residual condensable material in the system thus allowing lower working pressures. The trap must
be cooled in liquid nitrogen, whenever the pump is in use.
The second point is that even with the cold trap some vapours pass into the pump and as they are
compressed they condense and form an emulsion with or dissolve in the oil. This leads to a deterioration

Figure 6: Visit the site http://www.sigmaaldrich.com/chemistry/solvents/learning-center/nomo-

assets.html for interactive pressure temperature Nomograph

in the lubricating and sealing properties of the oil as well as leading to internal corrosion of the pump.
In practice this problem is overcome by passing air through the oil; this process is called ballasting.
To turn off the pump:- Close the tap on the line to the distillation apparatus. Gently disconnect the
pressure tubing from the distillation apparatus.
Lower the Dewar of liquid nitrogen away from the trap. Admit air into the pump and immediately
switch off the pump motor. As soon as the trap is warm enough to handle, it is dismantled and cleaned.
6 Recrystallisation
6.1 Introduction
One of the most necessary and useful techniques to be mastered by an organic chemist is that of
recrystallisation. Many organic compounds are solids, and thus solids are frequently encountered as
reaction products. As they are likely to contain impurities they need to be purified. Recrystallisation
is an important method of doing so. Essentially, the process is one in which the crystal structure is
completely disrupted, either by fusion (melting) or by dissolution of the solid, and then crystals are
allowed to re-grow so that impurities are left in either the melt or the solution.
Purification occurs because foreign molecules can seldom fit into the crystal structure of another
compound. The fusion method is seldom used because the crystals are usually formed in the presence of
a rather viscous oil (containing the impurities) from which they are difficult to separate. In solution re-
crystallisation, a solvent is generally chosen in which the impurities are more soluble than the substance
being purified. Crystals grown from such a solution will be of much greater purity than the original
solid if the technique was performed properly and if the impurities are present only to the extent of a few
percent. If impurities comprise more than a few percent of the original solid, two or more successive
recrystallisations may be necessary.
Solid recrystallisation as a technique involves several steps. These are:
1. Selection of the appropriate solvent.
2. Dissolution of the solid to be purified in the solvent at or near its boiling point.
3. Filtration of the hot solution to remove insoluble impurities.
4. Crystallisation from the solution as it cools.
5. Separating the crystals s from the supernatant solution.
6. Washing the crystals to remove the adhering solution
7. Drying the crystals.
6.2 Selecting a Solvent
A solvent must satisfy certain criteria in order to be used as a crystallisation solvent.
1. Its temperature coefficients for the solute and impurities should be favourable: that is, the com-
pound being purified should ideally be quite soluble in the hot solvent but somewhat insoluble in
the cold (this will minimise losses), and the impurities should remain at least moderately soluble
in the cold solvent. Another possibility here is that the impurities be insoluble in the hot solution,
from which they may be filtered.

2. The boiling point of the solvent should be low enough so that it can be easily removed from the
crystals in the final drying step.
3. It is generally preferable that the boiling point of the solvent be lower than the melting point of
the solute.
4. The solvent should not react chemically with the compound being purified.
If the compound has been previously studied, consultation of the chemical literature will generally
give information concerning a suitable solvent. If not, it will be necessary to resort to trial and error
methods using small amounts of material. Some general solubility principles should be kept in mind if
this needs to be done. Normally polar compounds are insoluble in nonpolar solvent and soluble in polar
solvent. These solubility relationships are frequently summarised with the phrase like dissolves like.
Therefore, a highly polar compound is unlikely to be soluble in a hot nonpolar solvent but may well
be too soluble in a cold very polar solvent, so that a solvent of intermediate polarity will be optimum.
Occasionally, mixed solvents are found to work well.
6.3 Instructions
The steps followed during a recrystallisation are described in the following order:
1. Solution (preparation of a hot, saturated, solution of a crude product in a suitable solvent).
2. Hot filtration - if necessary.
3. Crystallisation.
4. Filtration (of the purer product).
5. Drying (removal of remaining solvent).
These steps are described in more detail below.
6.3.1 Solution
Note:
(a) When toxic solvents (e.g., CH2 Cl2 , CHCl3 , CCl4 , benzene, toluene) are being used, this and the
subsequent operations are conducted in a fume hood.
(b) In previous years students have been trained to the habit of heating solvents before their addition
to the suspension or solution of the material to be recrystallised. This habit must be eradicated
because it is:
(i) Wasteful: the unused solvent is too contaminated with moisture and perhaps even with chem-
icals to be returned to the stock bottles.
(ii) Dangerous: the habit increases the amount of toxic vapour in the laboratory atmosphere, and
increases the risk of fire.
(iii) Totally unnecessary: Take the bottle of solvent to the place where the recrystallisation is
being conducted. Pour the solvent directly from the bottle or transfer it with a Pasteur pipette
whichever is appropriate for the scale of the recrystallisation or the safety of the operation
(obviously there is a risk of a large fire if a large quantity of flammable solvent is handled on
or near a hot-plate).

(c) The stopper of the solvent bottle must be replaced between transfers of solvent, and the bottle must
be returned to its place on the storage shelves as soon as possible.
Save a few crystals of the impure solid; they may be needed later to induce crystallisation if problems
are encountered at that step. The solid to be purified is placed in an appropriately sized conical flask,
along with a few millilitres of desired solvent. With constant stirring1 the mixture is then heated to
boiling using either a hot-plate (magnetic stirring can be used) or steam bath. A Bunsen burner may be
used only if the solvent is water. More solvent is added in small portions to the boiling mixture until
just enough boiling solvent is present to dissolve the solid. When it appears that additional solvent is
not dissolving any more of the solid, particularly when only a relatively small quantity of solid remains,
enough solvent has probably been added. The remaining solid is likely to consist of insoluble impurities
and may be removed in the hot filtration step (below).
Many students obtain poor results in their recrystallisation, because, when trying to dissolve the last
traces of solid, quantities of solvent are added that are far in excess of that needed. During the solution
of the impure solid, time should be allowed between each small addition of fresh solvent, for some solids
dissolve only slowly.
When the crude product is being dissolved impurities may collect as an oily smear on the bottom of
the flask. This is good. Filtration is unnecessary; merely decant the clear solution into another flask for
crystallisation. Heat the smear with a little more solvent then decant into the first solution: repeat this
extraction until the smear seems not to diminish in size.
When mixed solvents are used, they of course must be miscible. The crystals being purified should
be soluble in one of the solvents, but insoluble or only slightly soluble in the second. The crystals are
first dissolved in that pure, boiling solvent in which they are soluble filter off insoluble impurities if any).
The second solvent is then added to the boiling solution until the solution turns cloudy. The second
solvent decreases the dissolving ability of the solvent medium; when the solubility limit is reached, the
solute begins to come out of solution resulting in a cloudy appearance. [However, addition of the poor
solvent sometimes causes precipitation of small quantities of impurities before the solution becomes
saturated with respect to the desired compound. When this occurs, give the solution a quick filtration:
use charcoal to bind the precipitate as it is usually very fine and may pass through the filter.] Finally,
more of the first solvent is added dropwise until the solution just becomes clear again.
Sometimes the solute is very slow to crystallise from a mixed-solvent system even though the solu-
tion is highly supersaturated. If, by the time the volume of the second solvent has reached approximately
ten times the volume of the first, no cloudiness has developed, do not add any more of the second solvent,
but set the solution aside to crystallise.
6.3.2 Hot filtration
Gravity filtration. Use a glass funnel and fluted filter paper for filtration into a conical flask contain-
ing some boiling recrystallisation solvent. The condensing vapours will heat the funnel and the filter
paper preventing crystallisation. Cover the funnel with a clock glass to keep the filter hot, to minimise
solvent loss,and to minimise the condensation and absorption of moisture.
Choose a filter funnel and paper of appropriate size. If you have, say, 100 ml of solution to filter,
you dont want to be using a fluted filter paper that can only hold 5-10 ml at a time. The funnel must be
of a size that the fluted filter paper doesnt stick up above the rim of the funnel.
For low-boiling solvents a steam bath may be used (but beware of water condensing into your ma-
terials); for solvents higher than 80 an electric hot-plate must be used. The rate of heating should be
adjusted so that the solvent just refluxes on the clock glass.
1 Stirring will help to prevent bumping of the boiling mixture. When relatively large quantities of undissolved solids are
present, boiling stones are not very efficient.

An alternative method is suction filtration. Suction filtration of a hot saturated solution can give
problems. Under reduced pressure some of the hot solvent will be lost through rapid evaporation.
Loss of the solvent and the consequent cooling can cause the solute to crystallise in the filter clogging
the filter paper and the funnel perforations. The obvious way to avoid such an aggravating event is to
use rather more hot solvent than is necessary to dissolve the solute. This compensates for the loss by
evaporation. The amount of excess solvent required is a matter of guesswork. Use a large funnel so that
filtration is rapid. While the filter pump is operating, wet the filter paper with a little solvent and press
the paper against the perforated plate of the funnel with fingers or thumb to ensure that the circumference
of the paper is in contact with the plate. If this is not done some of the insoluble material is likely to
escape into the filtrate.
If decolourising carbon is being used, filter the solution through a paper made for fine crystalline
solids; see the back of a pack of Whatman filter papers. Very finely divided solids, like a small proportion
of decolourising carbon, are not always completely removed by filtration through Whatman #1 paper.
Gravity filtrations through fine crystalline papers are slow compared with filtration through #1 papers.
6.3.3 Crystallisation
The hot filtrate is allowed to cool slowly by standing at room temperature. Crystallisation should
then ensue. Rapid cooling by immersion in water, etc., is undesirable because the crystals formed
will tend to be quite small. Their large surface areas may then facilitate adsorption of impurities from
the solution. Generally the solution should not be agitated while cooling, since this will also lead to
formation of small crystals. However, formation of very large crystals may cause occlusion (trapping)
of solution within the crystals. Such crystals are difficult to dry and will, when dried, have deposits of
impurities in them. If large crystals seem to be forming, agitation may be used to lower the average size.
Rapid cooling increases the chance of the product separating as an oil (see below). If crystallisation does
not occur after cooling, the supersaturated solution can usually be made to yield crystals by seeding. A
tiny crystal of the original solid is added to the cooled solution. Crystals will usually form quite rapidly.
Another method that may be used to induce crystal formation is to scratch the inside surface of the flask
at or just above the surface of the solution with a glass rod.
Oiling out: Not infrequently, the solute will separate from the solution as an oil rather than as
crystals. This must occur when the limit of solubility of the solute is reached at a temperature that is
above the melting point of the substance. This should be avoided, and usually can be, by diluting the
solution with more solvent, reheating, and again allowing to cool. It quite frequently happens that a
product precipitates as an oil (solution becomes cloudy) at temperatures well below its m.p.. When this
occurs, add a little more solvent to clarify the solution, and seed crystals and swirl the solution.
Repeat the process if necessary until the solution shows no tendency to go cloudy.
When the suspension of crystals has cooled to room temperature it should be chilled in ice (or a
refrigerator) to make separation of the product more complete.
6.3.4 Filtration
The cool mixture of crystals and solution is now filtered by using a Buchner funnel and a vacuum
filter flask attached to an aspirator. Before filtration, the filter paper should be wetted with the solvent
and pressed down with finger or thumb in order to seal it to the funnel.
A stirring rod or spatula may be used as an aid to transferring the crystals to the funnel. The last
quantity of crystals may be transferred by washing them out of the flask with some of the filtrate (mother
liquor). The crystals should then be washed by releasing the pressure and just covering the crystals
with cold fresh solvent. After a few seconds this solvent is removed by suction. The purpose of this
washing step is to remove the solution with which the crystals are wet. This must be done for successful

purification; the solution of course, contains the impurities. If the solvent was properly chosen, the
crystals should now be pure, except for solvent.
After filtering the crystals a second crop of crystals can usually be obtained from the filtrate by
further cooling or by concentrating the solution by evaporating away some of the solvent and cooling.
The crystals obtained as a second (or even third) crop may not be as pure as the first. Melting points of
the crystals obtained in various crops can be used as criteria of their purity.
6.3.5 Drying
In general the best way to complete the drying of recrystallised material is to spread it between sheets
of clean filter paper and leave it exposed to the air overnight (Hygroscopic compounds are dried in a
vacuum dessicator or by warming in vacuo. Some compounds sublime quite readily in the atmosphere
but can be dried by sublimation.)
6.4 Melting Points (melting point ranges)
1. Pack no more than 2mm depth of powdered sample into the mp tube.
2. Raise the temperature rapidly to within 10-20 of the expected mp then adjust the heating rate so
that the temperature rises at no more than 2 /minute. Only then place the packed mp tube in the
mp apparatus. This procedure minimises prolonged exposure of the compound to heat, and is, of
course, necessary for compounds that decompose at an appreciable rate near their melting points.
If the melting point is not known to you, pack two mp tubes so that an approximate mp can be
determined using a rather rapid temperature rise (10 /min).
3. A few degrees below the mp the sample will shrink and may appear soggy; the sample is sintering.
By all means record the onset of sintering, but sintering is not considered to be melting.
The beginning of melting is defined by the appearance of the first distinct droplet of liquid. The
end of the melting point range is the disappearance of the last crystals (Fig. 7).
Figure 7: Determination of melting point.

4. Each of the temperatures delimiting the mp range should be quoted only to the nearest 0.5.
Even this claim to precision is rather illusory because the mp apparatus is not perfectly calibrated
and, more significantly your judgements of the end and (especially) the beginning of melting are
subjective.
6.5 Analytical T.L.C.
1. Use the t.l.c. plates as soon as possible after removal from the storage dessicator. Prolonged
exposure to the gel to the atmosphere results in absorption of moisture and consequently loss of
activity.
2. If too much of the solutes are spotted onto the plates they will appear as large smears instead of
spots after development and the chromatograms will be useless. If such smears are obtained, try
the chromatography again using much less of the sample solution.
3. apillary movement of eluant up the edges of the plate can distort the chromatogram and even make
it useless. Below the spots but above the level of eluants in the developing jar, nick the edges of
the gel (as shown in Fig. 8) with a finger nail or a spatula - scrape outward towards the edge of the
plate.
Figure 8: Analytical T.L.C.
4. Keep the developing jar covered. Exposure of the contents of the jar to the air must be minimal
to prevent excessive evaporation of eluant from, and condensation of moisture on, the developing
plate.
5. The solvent front will take 20 to 25 minutes to travel to 0.5 cm from the top of the plate.
7 Infra-Red Spectra
7.1 Solids
The preferred method is to use the KBr disc technique (see CHEM20019 manual).
7.2 Liquids
Simply place a drop of the liquids between the discs in the same way as a mull: this is called the
liquid film method.
Note: Make sure your compound are free from water: moist samples will cause pitting of the discs.

Do not touch the flats of the discs with your fingers. It is very difficult to remove all traces of water
from compounds and apparatus, therefore, a weak band in the O-H region of the spectrum does not
necessarily imply the presence of a hydroxyl group in your compound.
8 Laboratory Hints
8.1 Magnetic stirring
Stirrer bars should be of an appropriate size. For reaction mixtures in conical flasks, the bar should
be as large as possible without it banging into the side of the flask at high stirring speed (especially
if a pleated (creased) flask is being used). It is particularly important to use large stirrer bars for
heterogeneous mixtures. On the other hand, the stirrer bars for agitating a liquid being distilled from a
round-bottom flask should be small; 1 to 0.5 cm is quite adequate.
Vigorous stirring means as fast as possible without causing the spinning bar to become unstable or
causing the mixture to splash out of the flask.
8.2 Ice baths and Ice-Salt baths
These cooling baths should contain liquid water to give good thermal contact with the vessel being
chilled; the level of the water should be at least equal to that of the liquid in the vessel to maximise the
rate of heat transfer. The slurry of ice and water should be stirred frequently in order to disperse the
layer of relatively warm water in contact with the vessel; and, in the case of ice-salt baths, to mix the
brine with the floating ice. When prolonged chilling is required, and magnetic stirring is not employed,
use the foam-insulated baths rather than the aluminium bowls. If an aluminium bowl must be used,
remove much of the melt water by syphoning or suction with a water pump (not the vacuum line) before
replenishing the ice (and salt).
8.3 Cleaning Glassware
Organic residues can usually be removed by rinsing with small portions of methylated spirit (meths);
if cold meths is not very effective, try hot meths. Should meths prove inadequate try acetone. Residues
that resist acetone can often be loosened by overnight contact with a 3:1 mixture of acetone:Teepol.
The last resort is conc. H2 SO4 (cold or, if necessary, hot); before using conc. H2 SO4 drain out any
organic solvent, rinse with water, then drain thoroughly. (Dispose of the used H2 SO4 by pouring it
gradually and with stirring into a large volume of water.) Greases (petroleum jelly, Apiezon) are best
removed with non-polar solvents (petrol or toluene); remove as much as possible of the grease with
tissue then complete the cleansing by wiping with tissue dampened with petrol.
Inorganic residues: Mg 2+ use dil. HCl.
Al 3+ use 1:1 conc. HCl:water
MnO2 use aqueous NaHSO3 .
Cu(I) and Cu(II) use dil. HNO3
8.4 Protecting reaction mixtures from moisture
When Qfit apparatus is being heated on a steam bath, steam will condense on the outside of the
condenser and trickle down onto the Qfit joint. The water will penetrate the joint unless it is greased
(see Figure 4).

For short reaction times up to half an hour or so, the open tops of condensers and dropping funnels
can be blocked to the diffusion of moisture by a plug of oven-dried cotton wool or by a tight cover of Al
foil. For longer periods the precautions must be more elaborate; there are several satisfactory methods:
(In order of preference)
(a) Maintain the mixture under a nitrogen blanket if dry nitrogen is available.
(b) Use a diffusion tube. This is a length (30 cm)of plastic capillary tubing fitted with a Qfit adaptor
so that it can be plugged into the top of, say, a condenser.
(c) Use a Qfit drying tube packed with oven-dried cotton wool or with anhydrous calcium chloride
(held in place with wads of cotton wool). Calcium chloride can be messy as it is deliquescent.
Note that the problem of protecting a mixture from the air becomes more serious the smaller the
volume of the mixture (why?).

Appendix B - Crystallography and
Instrumentation
1 Introduction to X-ray Crystallography
1.1 Background
The discovery of the diffraction of X-rays by crystals and the interpretation of the resulting diffrac-
tion patterns in terms of the atomic arrangements within crystals represents one of the great scientific
breakthroughs of the 20th century. The determination of the structure of DNA is just one of a number of
triumphs of X-ray diffraction. The following quote from W. L. Braggs Nobel Prize address delivered
over 80 years ago provides us with an indication of why the technique is so important to chemists.
The examination of crystal structure, with the aid of X-rays has given us for the first time an insight
into the actual arrangement of the atoms in solid bodies. The study of structure by means of a microscope
is limited by the coarseness of the light which illuminates the object, for we can never hope to see details
smaller than the wavelength of the light.
By using X-rays with their very short wavelengths, this limit of minuteness has at one step been
decreased ten thousand times, for the wavelength of the X-rays is of a smaller order than the dimensions
of the atomic structure.
We are actually looking into the interior of the molecule and the atom with this fine-grained form of
light.
The difference between using light and using X-rays is perhaps not as simple as Braggs comments
suggest, since there is no lens for X-rays which would allow us to focus an image. The use of X- rays
to see atomic arrangements exploits the periodic internal structure of crystals i.e. the same unit is
repeated many, many times within the crystal. The interpretation of the scattered X-rays is also made
simpler by the use of monochromatic (single wavelength) radiation.
While there have been spectacular advancements in the type of equipment used to measure diffrac-
tion data from crystals, the essential features of the set-up devised by Friedrich and Knippings in the
early part of the 20th century are present in modern diffractometers.
The technique generally involves directing a monochromatic beam of X-rays towards a crystal. The
X-rays are scattered by the crystal and the intensity and position of the diffracted beam is measured
(Fig. 1. By changing the orientation of the crystal, new diffracted beams are produced. Aided by
computers, the manipulation of the diffraction data, using sophisticated mathematics, allows the determ-
ination of the crystal structure.
1.2 Theory
1.2.1 The Unit Cell
The unit cell is a selected small regular figure, a parallelapiped, which by continuous replication and
translation generates the whole crystal (Fig. 2. The unit cell may be defined by the 3 cell lengths, a, b,
c and the angles between the edges, , , (the angles between the b and c, the a and c and the a and
b axes respectively). In this exercise we will be considering a tetragonal unit cell. In a tetragonal unit
309
A PPENDIX B CHEM30015: Advanced Practical Chemistry
Figure 1: Friedrich and Knippings experimental setup for single crystal X-ray diffraction.
cell the , and angles are all 90 and the a and b cell lengths are equal. The corners of the unit cell
represent points that may be considered as lattice points within the crystal. Note that all lattice points
are equivalent. You may have noticed that the crystal shape is consistent with the tetragonal unit cell.
Figure 2: The unit cell.
1.2.2 Miller indices
In X-ray crystallography it is useful to consider planes defined by sets of lattice points. As an

example, consider a lattice derived from a unit cell with lengths a, b and c, viewed down the c axis.
The lines in the figure represent planes which are parallel to the c axis. Families of parallel planes may
be indicated by Miller indices. Planes belonging to three such families are indicated in Figure 3. In
order to determine the Miller indices of the planes it is useful to consider the Weiss system of indexing.
Firstly, consider the family of planes represented by the 3 lines that run horizontally. The three planes
shown, intersect the b axis at 6b, 7b and 8b. Therefore the difference between the points of intersection
along the b axis is 1b. The fact that the planes are parallel with both the a and c axes means that the
planes intersect these axes at all points (a and c). This family of parallel planes (of which only 3 are
shown) may be denoted as (, 1, ) in the Weiss system. The symbol is inconvenient and one way
of dealing with this is to use Miller indices which may be obtained by taking reciprocals of the Weiss
numbers with fractions cleared. Thus, the Miller indices of this family of planes is (0, 1, 0). The first
number is the h value, the second number the k value and third number the l value.
Consider now the planes indicated by the lines which pass from top left to bottom right. The Weiss
numbers are 1, 1, and the Miller indices are (1, 1, 0). Finally the Weiss numbers corresponding to
the remaining set of planes are (-2, 1, ) which gives reciprocal values of (-1/2, 1, 0) corresponding to
Miller indices of (-1, 2, 0).
An important point to note is that the smaller the value of one of the indices the closer it is to being
parallel to the axis direction to which it corresponds. The smaller the number of h in the index (hkl) the

CHEM30015: Advanced Practical Chemistry A PPENDIX B
Figure 3: Miller indices.
closer it is to being parallel to the a axis. The same applies to k with the b axis and l with the c axis.
When any of the indices (h, k or l) are 0 then the family of planes is parallel to the corresponding axis.
In crystallography, the separation between neighbouring parallel planes is represented by the symbol
d. In an orthogonal system the separation of hkl planes is related to the indices and cell lengths and is
given by the equation:
2
1/dhkl = (h/a)2 + (k/b)2 + (l/c)2 (1)
1.2.3 Braggs Law
When X-rays strike a crystal, reflection from lattice planes can occur. The reflected waves interfere
with each other in a destructive or constructive manner. The figure below represents a set of conditions
required for constructive interference. If the difference in path length of the two waves is equal to an
integral number of wavelengths then the waves reinforce each other following reflection and an X-ray
beam will emerge from the crystal corresponding to this reflection.
The conditions required for reflection are represented by Braggs Law:
n = 2d sin (2)
where: n = an integer (the order of the reflection), = the wavelength of the radiation, d = the interplanar
separation and = the angle of incidence of the reflection.
In Figure 4, the lower wave travels a distance (CB + BD) further than the top wave. Using trigono-
metry it can be seen that this distance is equal to 2d sin , which in this case, is equal to two times the
wavelength. In this case, constructive interference occurs and a reflection will be observed.
Through the use of equations 1 and 2 we can calculate the angle of a reflection if we know the
Miller indices, the unit cell lengths and wavelength of the radiation. Each reflection may be labelled
according to the Miller indices of the family planes from which it is reflected. Thus the reflection from
the family of planes with Miller indices 2 3 4 is the reflection 2 3 4.
1.2.4 Data collection using a modern diffractometer
At first glance modern diffractometers closely resemble the experimental set-up of Friedrich and
Knippings (Fig. 5. X-rays produced in an X-ray tube by firing electrons at a metal surface are directed
towards a crystal and the diffraction spots (or reflections) are recorded on a screen, which is sensitive

Figure 4: Braggs law.
to X-rays. After exposure to the X-ray beam for a period of generally 10 to 30 seconds the crystal is
rotated very slightly (typically by 0.3 ) and another image is recorded. The process of rotation brings
new families of planes into the reflecting condition (i.e. Braggs law is satisfied). After 1400 rotations
(typically) the data set is said to be complete. Generally in this process tens of thousands of reflections
will have been recorded however depending upon the symmetry of the unit cell many of these reflections
will be equivalent and much of the data is redundant.
Figure 5: Modern CCD diffractometer.
With the assistance of some sophisticated software, the unit cell can be determined and the reflections
can be indexed i.e. assigned hkl values. We are then left with a reflection file, which list the indices of
the reflections along with the corresponding intensity (or more correctly a value related to the intensity)
and the value (standard deviation) associated with this value. The whole process can be completed
within six hours. Just a few years ago the same process would take many days and sometimes weeks.
Inspection of the diffraction data provides important clues as to the symmetry within the crystal i.e.
the space group. Associated with each space group is a list of symmetry operations which represent the
symmetry of the unit cell.

2 Use of Group Theory to Predict Infrared Spectra
In order for the absorption of radiation to stimulate a molecular vibration there must be a change in
dipole moment during the course of the molecular vibration, in other words a dipole change accompanies
the vibrations that lead to absorption bands in the IR spectrum. By the use of simple group theoretical
techniques we can identify the number and form of the vibrations responsible. One approach used to
perform this feat is demonstrated with reference to the platinum complexes cis and trans platin. First we
must determine the point group symmetry of each isomer. This is achieved by finding key elements of
symmetry as outlined below:
(i) Is the molecule a member of a special group? (is there more than one axis of rotation
of order greater than two?). E.g. regular tetrahedron (Td ) or octahedron (Oh ).
(ii) Find the highest order rotational axis (Cn ) and call it z. Thus in CO2 for example,
this is an infinite fold axis (C ) along the internuclear axes.
a.) If n=1:
and the molecule has a centre of symmetry . . . . . . . . . . . . . . . . . . . . . . . then Ci
and the molecule has a plane of symmetry . . . . . . . . . . . . . . . . . . . . . . . . then Cs
and the has molecule no symmetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . then Cl
b.) If n2:
and there are nC2 axes perpendicular to Cn . . . . . . . . . . . . . . . . . then dihedral
(iii) If dihedral:
and the molecule has a horizontal plane . . . . . . . . . . . . . . . . . . . . . . . . . . . . then Dnh

and the molecule has n vertical planes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . then Dn
(iv) If not dihedral:
and there is a S2n axis coincident with Cn . . . . . . . . . . . . . . . . then S2n (very rare)
and the molecule has a horizontal plane . . . . . . . . . . . . . . . . . . . . . . . . . . . . then Cnh
and the molecule has n vertical planes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . then C2v
and none of the above three . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . then Cn
For example, show that the cis and trans isomers of Pt(NH3 )2 Cl2 (Fig. 6) conform to the C2v and
D2h point groups.
Figure 6: cis and trans isomers of Pt(NH3 )2 Cl2 .
We can now use the symmetry properties of the molecule to predict a number of its physical prop-
erties, for example which of the molecular vibrations will be infrared active. In order to do this we
label the bonds and examine each symmetry element in turn, noting the number of bonds unaltered by

the operation. Thus for the two equivalent platinum-chlorine bonds in cis platin;
(i) the identity, E, leaves both bonds unchanged i.e. 2 unmoved bonds the C2 rotation inter-converts
bonds i.e. 0 unmoved bonds
(ii) the mirror plane which encompasses the atoms (v0 ) leaves both bonds unchanged i.e. 2 unmoved
bonds
(iii) the mirror plane bisecting the two chlorine atoms (v ) inter-converts bonds i.e. 0 unmoved bonds
For n bond stretches we must have a character of n for the identity element of the reducible represent-
ation. The task at hand involves identifying which combination of irreducible representations together
gives the reducible representation (red ) under consideration. While in some cases this may be obvious
and obtained by inspection, the general solution to the problem is to use the reducing formula. By
application of this formula any reducible representation of a point group may be decomposed into its
irreducible representation:
Reducing formula: ai = 1h R g i (R)T (R)
where ai = the number of times the irreducible representation i occurs in the reducible representation, red
h = the order (total number of symmetry operations) of the point group (h = 4 for C2v )
R = the symmetry element (e.g. E, C2 (z), v , v0 in C2v )
g = number of such elements for a particular column
i (R) = Character of the operation R in the irreducible representation i
T (R) = Character of the operation R in the reducible representation red .
This appears complex but is actually very simple. Thus for cis platin;

and hence for the Pt Cl stretches red = A1 + B2
But how do we know whether these vibrations are infrared active?

Remember that there must be a change in dipole moment during the course of the vibration in order
to observe it in the infrared spectrum. If we look in the character tables we see that at the far right of
each row there is a column which describes the symmetry of x, y, or z vector properties within the space
group. Thus for the A1 row this column contains a z, while for the B2 row it contains an y. This tells
us that during these vibrations there is a change in dipole moment along the z-, and y-axes respectively.
Hence, both the symmetric A1 and asymmetric B2 Pt-Cl stretching vibrations of Pt(NH3 )2 Cl2 are
infrared active.

3 Notes on the treatment of elemental analyses
Experimentally found percentages for various elements are given for each of the products in the
Oxo-Mo folder (obtained from the service room). These may be used to deduce the molecular formula
of an unknown product as follows:
First determine the ratios of the numbers of different atoms implied by the found individual per-
centages then on this basis propose a composition for the particular product.
Calculate the theoretical percentages for your proposed composition.
Compare the experimental percentages with the calculated percentages for each element.
The above is the way elemental analyses are treated in research and in the literature.
Initially ignore the numbers given in the Theory columns in the sections of the Oxo-Mo folder
dealing with elemental analyses as indicated above these are numbers you should calculate yourself
from the composition you propose.
Example and comments:
Suppose a salt containing Co, Cl, N and H gives an analysis with x1 % Co, x2 % Cl, x3 % N and x4 %
H. The ratio of the numbers of different atoms will be:
Co, Cl and so on.
The empirical formula (in terms of the elements for which you have an analysis value) can be ob-
tained by dividing each of these numbers by the smallest calculated value. If you obtain whole numbers
(or close enough to whole numbers) then fine, if you obtain a number, or numbers, close to half integers
then multiply all the numbers by two, etc.
Since the experimentally determined percentages are always subject to error (due either to the ac-
curacy and precision of the measurement or, more likely, due to contamination of the sample) then there
will be times when the interpretation of the analysis is ambiguous. In these cases you will need to use
your chemical experience and knowledge to interpret the analytical results.

4 Brief notes on conductance
Conductance, G, is defined as the reciprocal of resistance: G = 1/R

The units for G are reciprocal ohms, mhos or Siemens (S) which are equivalent.
For a conductor of length l and constant cross sectional area A:
A A
G or G = constant
l l
This constant is characteristic of a particular material, independent of the size and shape, and is
called the specific conductance, (kappa).
Gl
=
A
The units of are S cm-1 if G is in S and l and A are in cm and cm2 , respectively. We shall adhere
to these old-fashioned units in cm, but in SI units is measured in S m-1 .
When the sample is a solution between two electrodes increases linearly with increasing concen-
tration, c, or
= c
where the constant is called the molar conductivity.
The molar conductivity, , of a solute in a solution is therefore given by:

=
c
where the units for are in S cm2 mol-1 , and c is the concentration in moles per cm3 .
NOTE: the appropriate units for c are mol cm-3 NOT mol L-3 . (In SI units molar conductivities are
measured in S m2 mol-1 ).
In this exercise the microelectrode assembly has been pre-calibrated to read out the specific conduct-
ance in S cm-1 .
Glossary
Non SI Unit SI Unit
G Conductance ohm1 = mho = Siemens (S) ohm1 = mho = Siemens (S)
R Resistance ohms ohms
l Length cm m
A Cross sectional area cm2 m2
C Concentration mol cm3 mol cm3
Specific conductance S cm1 S cm1
Molar conductivity S cm2 mol1 S cm2 mol1

5 NMR spectra of two inequivalent interacting spin 1/2 nuclei
In the absence of spin-spin coupling the two nuclei will have resonances with chemical shifts des-
ignated by A and B . The value of the chemical shift will depend on the magnetic environments of the
two nuclei. The difference in frequency between A and B , AB , for such a case is shown in Figure 7
opposite. If the values of A and B are given in terms of ppm then the value of AB (which is an
energy difference in Hz) will be equal to the difference in chemical shifts (in ppm) multiplied by the
spectrometer frequency.
Figure 7:
Spin-spin coupling results from magnetic interaction between two nuclei, for example the resonance
frequency of nuclei A will be slightly different according to whether nuclei B have spins aligned with,
or opposed to, the applied magnetic field. The magnitude of this interaction depends on the strength of
the magnetic interaction and its value is given the symbol JAB , where JAB is expressed in Hz. Provided
that AB JAB then the pattern of bands shown in Figure 8 is obtained. Notice that the resonances of
both interacting nuclei are split by exactly the same amount and the splitting is symmetrical about the
uncoupled values of A and B . Also note that the relative intensities of the four lines are also the same.
If these conditions apply the spectrum is said to be first order.
Figure 8:
If the condition, AB JAB , is not satisfied then the situation is more complex and the spectrum is
said to be second order (Fig 9). The most obvious manifestation of second order effects is a deviation in
the relative intensities of the coupled resonances from a 1:1:1:1 pattern however, the coupled resonances
are no longer split symmetrically about the uncoupled resonance.
Analysis of a situation such as this proceeds as follows:

Figure 9:
1. Label the four resonances 1 through 4 starting at the resonance with the highest chemical shift.
The coupling constant, JAB , will be given by the difference in chemical shift between peaks 1 and
2 or between peaks 3 and 4 (i.e. JAB = 1-2 = 3-4). Notice that this would give you a coupling
constant in ppm and this will need to be converted into Hz.
2. Calculate the value of AB . This may be obtained from the following expression:
q
1 3 = 2 4 = 2AB + JAB2
3. Calculate the values of A and B . These are obtained with reference to the mid-point of the
spectrum (MPS); that is the arithmetic mean of the four resonance frequencies. Thus:
1 1
A = MPS AB , and B = MPS + AB
2 2
Note that you will have to be careful with units (i.e. you will have to convert between ppm and
Hz).
4. The relative intensities of the lines can be measured directly from the spectra. These values should
be compared with the values obtained from the following expressions:
JAB JAB
i1 = i4 = 1 q , and i2 = i3 = 1 + q
2AB + JAB
2 2AB + JAB
2
Notes:
1 Hz = 1 cps = 1 s1
106
(ppm) =
spectrometer frequency (Hz)
Resonances having a larger chemical shift are deshielded and have a smaller value of in Hz.

6 Operation of a Vacuum Line
Vacuum lines are used in a number of experiments in this practical course and some familiarity
with the operation of pumps that can evacuate a line is therefore desirable. For detailed notes on the
performance and capabilities of the pumps in the laboratory, see the Technical Officer. Dushman1 and
Sanderson2 are useful general references on pumps and vacuum techniques.
To obtain a high vacuum, a dual pumping system is generally employed. An Edwards ES- 330
rotary pump can usually evacuate a line to 103 torr. A diffusion pump with a 4 inch throat and using a
high molecular weight oil (MW 300 to 500) normally evacuates a line from 103 to 107 or 108 torr.
Consequently the pumps are used in tandem, with the rotary pump backing the diffusion pump.
Figure 10 illustrates a typical design of a mechanical oil pump and Figure 11 is a schematic drawing
of a multistage, metal diffusion pump.
Figure 10: Mechanical rotary oil pump.
A diffusion pump should never be allowed to evacuate a line with pressure greater than 101 torr
since the oil cracks and eventually bakes onto the diffusion stack. Furthermore, some cracked oil is
expelled via the rotary pump, releasing carcinogenic fumes in the laboratory.
A typical pump arrangement is shown in Figure 12.
The following instructions will help you to operate such a vacuum line:
1. Starting up
1. With valves A, B, C and D closed, turn on the rotary pump.
2. Open valves B and C.
3. When the pressure has fallen below 0.1 torr, open A and close B.
4. Turn on the diffusion pump.

1 Dushman, S., Scientific Foundations of Vacuum Technique, Wiley, 2nd ed., 1962.
2 Sanderson, R.T., Vacuum Manipulation of Volatile Compounds, Wiley, 1948.

Figure 11: Multi-stage oil diffusion pump. Arrows indicate flow of oil vapour.
Figure 12: Typical pump arrangement.
The diffusion pump takes approximately twenty minutes to reach operating temperature following
which the pressure should fall below 103 torr.
Always isolate the diffusion pump by closing A and C and opening B when the system pressure is
likely to rise above 0.1 torr, for example when purging the system after an experimental run.

2. Closing down
1. Evacuate the system to 0.1 torr or better.
2. Close valves A, B and C and then turn off both pumps. Air should not be admitted to the diffusion
pump while it is hot, regardless of whether it is switched on or off.
3. Do not forget to use valve D to admit air to the rotary pump, when switching it off, in order to
prevent oil from entering the vacuum line!

Appendix C - Error Analysis
The assessment of the numerical reliability of the result of an experiment is an important skill in
Physical Chemistry practical work. The principles of the analysis of the error in a derived quantity are
set out below, based on [1, 2].
1 Average and Standard Deviation
When a quantity whose true value is x0 is measured to be x, the error in the measurement is x =
(x x0 ) and the relative or fractional error in x is defined as:

x
(x) =
x
Usually, since x0 is being determined, x is unknown and its magnitude must be estimated. The best
estimates of x0 and x are obtained by measuring x repeatedly (say, N times) and calculating the average,
and the standard deviation, sx :
x,
x
x =
N
2
(x x)
s2x =
(N 1)
sx is an indication of how well x approximates x0 - the probabilities that x0 lies within the ranges
sx 2sx
x and x are dependent on the size of the sample. For example, if N is large (> 30),
N N
these probabilities are 68% and 95% respectively; for N = 3, they are approximately 60% and 85%
respectively. It is acceptable to quote values of the mean and standard deviation without commenting on
probabilities.
2 Propagation of Error: Some Useful Results
The error in a quantity y derived entirely from a measured quantity x by use of the relation y = y(x)
can most simply be estimated in terms of relative errors. Since:
dy
y = x (1)
dx
it follows that
dy x
(y) = (x) (2)
dx y
Thus, for constants and ,
If y = x,
then (y) = (x) (3)
323
A PPENDIX C CHEM30015: Advanced Practical Chemistry
If y = /x,
then (y) = (x) (4)
If y = x2 ,
x
then (y) = 2x (x) = 2(x) (5)
y
If y = exp x,
x
then (y) = ( exp x) (x) = x (x) (6)
y
If y = ln x,
x (x)
then (y) = (x) = (7)
x ln x ln x
Etc.
When y is derived from two measured quantities x and z by use of the relation y = y(x, z) then:

y y
y x + z (8)
x z
which leads to
y x y z
(y) = (x) + (z) (9)
x y z y
Since x and z may actually be positive or negative, they may add or partly cancel each other; hence
(y) in equation 9 is the maximum relative error. A more realistic estimate of the error may be obtained;
the square of equation 8 is:
2 2
2 y 2 y 2 y y
(y) (x) + (z) + 2 xz (10)
x z x z
The first two terms will always be positive but the last term will sometimes be positive, sometimes
negative and sometimes zero and so will approach zero in the average value of y2 . Hence the average
error in y is given by:
2 2
2 y 2 y
(y) (x) + (z)2 (11)
x z
and it follows that: 2 2
2 y x y z
(y) = (x) + (z) (12)
x y z y
Note that the magnitude of the average error is less than that of the maximum error, given by equa-
tion 9, which was used in first- and second-year courses.
Thus,
If y = xz
then 2 (y) = 2 (x) + 2 (z) (13)
If y = x/z
then 2 (y) = 2 (x) + 2 (z) (14)
If y = xz2
then 2 (y) = 2 (x) + 42 (z) (15)
Etc.

CHEM30015: Advanced Practical Chemistry A PPENDIX C
Note: When the relation is just a linear sum; y = x + z, then:

2 2 2 2
2 x z x z
(y) = (x) + (z) = + (16)
y y y y
so that
(y)2 = 2 (x)2 + 2 (z)2 (17)
and it is simpler to work with absolute rather than relative errors. (Note again that this expression gives
a smaller error than that used in first- and second-year courses).
3 Lines of Best Fit
When there is some correlation between two experimentally determined quantities, x and y, it is
common practice to plot each value of yi versus xi i.e. as a set of data (xi , yi ). In the case where there is a
linear correlation between x and y, we can determine the line of best fit visually. Each experimental point
should be plotted with error bars showing the maximum error in abscissa and ordinate (as described in
the 200 level notes). Depending on the deviation (spread) of the data from the line-of-best-fit and the
error bars associated with each yi , the slope (and intercept) so determined will necessarily vary, as shown
in Figure 1. Any reported value derived from the slope or intercept should include the error associated
with this uncertainty.
Figure 1: The importance of error bars.
Random errors arising from the measurement of x and y are expressed as the deviation of the plotted
data from a line of best fit to the data assuming some mathematical function. Any systematic error (i.e.
an error which contributes equally to all points measured) will not be expressed in the scatter of the
points.
In many circumstances, and when the number of data pairs is sufficiently large, the method of least-
squares can be used. In the least-squares method, any random uncertainty in x is assumed to be negligible
and a line of best fit, e.g. y = ax + b, is chosen so as to minimise the squares of the deviations between
the measured values, yi , and those calculated from the line, y(xi ) = axi + b. The standard deviations of

the gradient, a, and intercept, b, of the line can be calculated and these express all random uncertainties
involved in the measurement of y.
There are two widely used methods of least-squares fitting, namely, linear and non-linear least
squares, and there are many commercial computer programs that enable such curve fitting. Programs
such as pro Fit or KaleidaGraph, available on many of the computers in the computer room, can be
used to calculate the line of best fit for a set of data, and provide the standard deviations in the gradient
and intercept, as well as parameters that reflect the goodness-of-fit of the model function to the exper-
imental data (e.g. the correlation coefficient see below). Note: Excel is not particularly good for curve
fitting purposes, so pro Fit is preferred.
3.1 Linear Least Squares
The method of linear least squares can be used with any function that can be expressed in a form
where the pairs of variables (xi , yi ) can be related linearly somehow to one another. For example, if the
model relationship between yi and xi is: yi = A expkxi , this can be linearised to: ln(yi ) = ln(A) kxi .
Polynomials, of the form y(x) = a1 + a2 x + a3 x2 + a4 x3 + ... + Am xm1 can also be analysed in this way
[3].
The linear least squares method is discussed in the 200-level Practical notes, and summarised here.
The principle of least squares asserts that the best representative line through n points (xi , yi ) is that
for which the sum of the squares of the residuals (the difference between the experimental and calculated
values) is a minimum. For the expression: yi = a + bxi this implies:
n
S = (a + bxi yi )2 (18)
i=1
be a minimum. The necessary conditions for such a minimum are:
S S
=0 and =0 (19)
a b
These two equations may be solved simultaneously for a and b to yield:
(n xy x y) ((x x)(y
y))
b= = 2
(20)
n x2 ( x)2 ((x x)
)
x2 y x xy

a= = y bx (21)
n x2 ( x)2
where the summations are carried out from 1 to n. Equations for the standard deviations (and standard
error) in a and b can also be derived. Similar expressions can be derived for other appropriate functions
such as polynomials. The commercial programs available to you provide the line of best fit to data using
linear least squares for a limited number of functions, along with standard deviations associated with the
parameters recovered from the fit.
3.2 Non-Linear Least Squares
There are many functions that cannot be linearised in which case the linear least squares method
cannot be used. In many such situations, however, the method of non-linear least squares can be used,
making it a more versatile approach. This method generally relies on an iterative procedure whereby

the difference between the calculated and experimental values is minimised whilst the parameters are
varied. Usually the value of a parameter, often called chi squared (2 ), is minimised.
n2
[yi yi ]2
2 = (22)
i=n1 2i
where 2i is the variance, and n1 and n2 are the range of data points required to be analysed (a useful
advantage in large data sets). The iterative process begins with the supply (by the user) of sensible initial
guesses of the value of each parameter that is to be adjusted. These values are included into the trial
function, and the resultant computed values of the function, yi , are compared to the experimental yi
and a value of 2 is determined. The parameters are varied and the computed result compared with the
previous value of 2 . If the 2 has been reduced, the parameters are adjusted further in that direction,
otherwise alternative values of the parameters are tried. This process is repeated until a pre-determined
level of (small) change in 2 is achieved, at which point the best-fit parameters are assumed to have
been determined. The minimisation of the value of 2 is in as many dimensions as there are adjustable
parameters i.e. for the function: yi = a + bxi , 2 must be minimised in two dimensional space e.g. as
shown in Figure 2.
Figure 2: 2 hypersurface for minimisation in 2 dimensions (i.e. 2 adjustable parameters) [3].
There are numerous methods that can be used to control the adjustment of the parameters while
minimising 2 e.g. grid search, simplex or more commonly the Marquardt method; these are discussed
elsewhere [4]. Again, each method provides estimates of the standard deviation of each of the recovered
parameters, which is related to the degree of certainty that one can place in the recovered value. These
standard deviations should be quoted when a parameter is reported.
3.3 Goodness-of-Fit Parameters
3.3.1 Correlation Coefficients
When least square fitting methods are used, it is important to reflect on the quality of the fit that
has been achieved. There are a number of criteria that can be used to assess the quality of a fit. A
shallow minimum in the 2 hypersurface will result in greater uncertainty in the values of the recovered
parameters (a1 and a2 in the example shown in Figure 2). In non-linear least squares fitting the value of
the 2 achieved (or the reduced 2r 1) can be used as a measure of the goodness-of-fit.
Linear least squares fitting programs usually return the correlation coefficient, r, as an indicator of
the goodness of fit of the line to the experimental points. The correlation coefficient is defined for a set

of pairs of measurements. Let us use (xi ,yi ) to denote the ith pair and suppose there are n pairs in total.
Then:
(xi x)(y
i y)
r= p (23)
2 (yi y)
(xi x) 2
where x and y are the averages of the x and y measurements and denotes summation over all n
observations. It has the same sign as the gradient and as the fit improves, |r| 1.
It should be highlighted that the statistical concept of a correlation coefficient, r, is frequently mis-
used in interpreting analytical data. It has been pointed out: It is a mathematical fact that r lies between
+1 and -1, and is +1 (-1) when the points lie exactly on a straight line of positive (negative) slope. It is
this which gives rise to the idea that r being near 1 indicates a linear relationship between the x and
y measurements. [5] Claims of the type the correlation coefficient was near one, indicating linearity
are common but are at best misleading and can lead to important features of a set of data being over-
looked. A large value of r does not necessarily indicate an ideal linear relationship between two
measurements; this is demonstrated graphically for three cases in Figure 3. [5]
Figure 3: Three examples of fits to data resulting in high correlation coefficients (0.96, 0.94 and 0.97
respectively) despite clearly poor fits. The first two clearly do not represent a linear relationship between
y and x, and the third illustrates a scatterplot whose high correlation is due entirely to one outlying point.
[5]
4 Example: Oxidation of Metals
The following analysis of typical results from an experiment in which silver is oxidised to silver
iodide in iodine/hexane solution provides a useful illustration of the general procedure. In this exper-
iment, pieces of silver have been oxidised in the iodine/hexane solution for a reaction time, t, and the
silver iodide analysed by electrolytic reduction at a known current, I, for a measured time, . The extent
of the reaction, X moles of AgI per m2 , is found to depend on t 1/2 . The rate constant is determined
from the gradient of the X 2 versus t plot at two temperatures, 22.9 and 35.1 C, and the activation energy
calculated.

4.1 Measured quantities from which the results are derived
Quantities which are measured for each point on the graph:
1. Reaction time, t/s, estimated to be accurate to 2 sec.,
2. Electrolysis time, /s, to 6 s,
3. Electrolysis current, I/A, to 0.002 mA,

in none of which is there expected to be any significant systematic error, and quantities which are
measured once for all points:
4. Area of one side of plate, A /m2 , given to 0.03 cm2 ,
5. Reaction temperature, estimated to be accurate to 0.2 C.
4.2 Values of rate constants and their reliability
The linear dependence of X 2 on t has been established at 22.9 C by using the program LINEAR
which gave the rate constant k1 (gradient) to be 8.59 108 mol2 m4 s1 with a standard deviation of
0.13 108 .
Now X was calculated from:
I
X=
2FA
where F is the Faraday constant and A is 3.69 cm2 . The error in k1 , provided by LINEAR, accounts
for the effects of random errors in X 2 which cause the points to deviate from a straight line. These errors
are derived from random errors in I and . An additional uncertainty in X is caused by the error in A
which does not contribute to the scatter of points because it is constant for all X values. Since k = X 2 /t,
the overall error in k1 is then calculated using equations 14 and 15:
2 (k1 ) = 2 (gradient) + 42 (A) = (0.13/8.59)2 + 4 (0.03/3.69)2
i.e. (k1 ) = 2.2 102 or k1 = (2.2 102 )(8.59 108 ) = 0.19 108 .
Hence, k1 = (8.6 0.2) 108 mol2 m4 s1 at 22.9 C.
Similarly, corresponding calculations for the rate constant at the second temperature lead to: k2 =
(13.0 0.3) 108 mol2 m4 s1 at 35.1 C.
4.3 Value of the activation energy Ea and its reliability
Ea is calculated from the rate constants k1 and k2 at temperatures T1 and T2 respectively:

R ln kk21 8.314 ln 13.0

Ea = = 8.6 = 25.70 103 Jmol1 (24)
1 1 1 1
T2 T1 (273.2+35.1) (273.2+22.9)

k2
relative error in k1
k2
Now, firstly, the relative error in ln k1 is given by equation 7 as:
ln kk12
which, by equation 14, is:
1/2
h
0.3 2
i1/2
0.2 2
+
2
(k2 ) + 2 (k1 ) 13.0 8.6
= 13.0
= 0.079
ln kk21 ln 8.6

Secondly, since the error in each temperature is 0.2 K, the relative error in each T 1 is, by equation 4,
0.2/T , so that the absolute error in each T 1 is 0.2/T 2 .
Thus, by equation 17, (error)2 in [T21 T11 ] is:
2 2
0.2 0.2
+ = 9.63 1012 ,
(308.3)2 (296.1)2
and the relative error in [T21 T11 ] is:

(9.63 1012 )1/2
= 0.023.

(308.3)1 (296.1)1

Hence, by equation 14, the relative error in Ea is:
[(0.079)2 + (0.023)2 ]1/2 = 0.082,
so that the absolute error in Ea is:
(25.70 103 )(0.082) = 2.11 103 J mol1 .
Therefore Ea = 25.70 103 2.11 103 and the final value for the activation energy can be ex-
pressed, to indicate its reliability, as:
Ea = 26 2 kJ mol1 .
References
[1] A. Findlay, Practical Physical Chemistry, ed., J. A. Kitchener, Longmans, London, 1954, Ch. 1.
[2] R. E. Walpole, Introduction to Statistics, Macmillan, New York, 1969.
[3] P. R. Bevington and D. K. Robinson, Data Reduction and Error Analysis for the Physical Sciences,
McGraw-Hill, New York, 3rd ed., 2003.
[4] W. H. Press, B. P. Flannery, S. A. Teukolsky and W. T. Vettering, Numerical Recipes: The Art of
Scientific Computing, Cambridge University Press, Sydney, 1987.
[5] Analytical Measurement Committee, Uses (Proper and Improper) of Correlation Coefficients,
Analyst, 1988, 113, 1469-1471.

Appendix D - Introduction to pro Fit
This is a quick introduction to pro Fit (version 6.1.11). You may have completed this in 2nd year, but
it is included here as it may contain tips that you might find useful in analysing your data.
The program pro Fit is a data plotting and fitting program installed on the Macintosh computers
in the Multimedia Room. Of course, you are welcome to use any program you are familiar with such
as Microsoft Excel for plotting data, however, data fitting is often not readily accomplished with such
programs. On the other hand, pro Fit is designed for such a purpose, and we encourage you to explore
its capabilities. Note that the current limitation is that the licenses are only installed on the Multimedia
Room computers, and so it cant be used from outside this room.
First Steps
Open pro Fit by clicking on the pro Fit icon on the computer data bar. The following error message
may appear. If it does, click on Work without preferences.
In the following we describe a typical session with pro Fit. We will plot and analyse the rate con-
stants of a chemical reaction as a function of temperature. Rate constants for the decomposition of
acetaldehyde at different temperatures are given in the table below.
T/K k/(L mol1 s1 )

700 0.011
730 0.035
760 0.105
790 0.343
810 0.789
840 2.17
910 20.0
1000 145
To enter these data, you have to open a new data window:
Choose New Data from the File menu.
An empty data window appears:
331
A PPENDIX D CHEM30015: Advanced Practical Chemistry
The data are arranged in horizontal rows and vertical columns. The topmost cell of each column
shows the name of the column (by default Column 1, Column 2, etc.). The cells below contain the
data of each column. Now you must enter the data into this window:
Click on the first empty cell of column 1 and enter the first temperature, 700.
Fill the first column with the temperatures (in K) and the second column with the rate constants in
L.mol1 .s1 . The first temperature is 700.
Click on the first cell of column 2 and enter the rate constant, 0.011.
Repeat the last 2 steps to enter the remaining data in the following rows.
Fill in the temperature and rate constant data as given in the table above. Note that you can use the
arrow keys, the Tab key, and the return/enter key to move from one cell to another.
Enter the column titles, temperature (K) and rate constant (L/mol/s).
Click the column titles Column 1 or Column 2 and enter the new names. Double-click the column
number above the titles to change the column format. Drag the line between the columns to adjust their
width.
Save the data as acetaldehyde data
Your window should now be similar to the one below.
Now that you have entered the data, you can display it graphically:
Choose Data y(x) from the Plot menu.
The following dialog box appears.

CHEM30015: Advanced Practical Chemistry A PPENDIX D
Choose rate constant (L/mol/s) as the Y-column. In this dialog box you can enter the range of
the plot, the columns to be plotted, and some other options. In this introductory session we keep the
settings as they are.
Click the OK button.
A drawing window appears, showing a graph of the data.
Now you can edit this graph by using some of pro Fits drawing features.
Double-click the vertical axis to change its range
(Double-click the vertical axis itself, not the numbers to the left of it.) A dialog box appears, present-
ing some options for the axis:

You can change a variety of options here. Most often you will use the edit fields First and Last.
They define the range of the axis. Another important field is Distance, which defines the distance
between the major ticks on the axis.
Enter 0 for First and 200 for Last, then click OK.
The vertical axis of the graph starts now with 0 and ends with 200. Double-click other parts of the
graph or its legend to see other dialog boxes, where you can change more attributes. Try double-clicking
the horizontal axis, the centre of the plot, or the dot in the legend. You can also double-click any text in
the drawing to change it. Or you can choose any of the drawing tools at the left border of the window to
add lines, polygons, text, etc. Try it out!
Finding a mathematical model to describe temperature variation of the rate constant
The temperature variation of the rate constant for a chemical reaction can often be represented by
the Arrhenius expression:
k(T ) = AeEa /RT
where k(T ) is the rate constant at temperature T , A is the pre-exponential factor, Ea is the activation
energy, and R is the gas constant (8.314 J K1 mol1 ). The equation can be rearranged:
Ea
ln[k(T )] = ln[A]
RT
That is, if you plot ln[k(T )] versus 1/T you should obtain a straight line whose slope is Ea /R.
Lets rearrange the data so that it can be represented in this way.
From the Calc (Calculation) menu choose Data Transform
A dialogue box will appear, resembling the one below.

The X value should correspond to temperature (K), while the Y value should be Column 3.
Select the Various functions option and from the sub-menu select 1/X.
Click OK
Now the data window should look like this:
Now the data in Column 3 correspond to 1/T .

From the Calc menu choose Data Transform again
The dialogue box appears again. This time select rate constant (L/mol/s) for X, while the Y
value should be Column 4. Choose ln(X) from the Various functions sub-menu.
Click OK.

Now, after titling Column 3 as 1/T and Column 4 as ln(k) the data window should look like this:
We can plot and analyse the data to work out the Arrhenius parameters (A and Ea ). First, lets plot
the transformed data.
Choose Data y(x) from the Plot menu.
The following dialog box appears.
Select (1/T ) for the Coordinate input in the Data box, and ln(k) for the Magnitude output.
Select the New window option, and tick Auto range for both input and output.
Click OK.
A drawing window appears, showing a graph of the ln(k) versus 1/T data.

Now we can fit a straight line to the data.
From the Func (Function) menu choose Polynomial
A parameter window will appear.
Change the degree of the polynomial to 1 (corresponding to a straight line) and hit the return
button. The parameter window should now look like this:
Note that the const and a1 are in bold. This means that they will be used for fitting. (If necessary,
you can deactivate them by clicking on them).
At this stage it is worthwhile introducing the Preview window. The preview window is not an
actual plot or drawing, it is only a window to preview data and fits before plotting them.
From the Windows menu select Preview
Click the data button in the preview window and select (1/T ) for the X column and ln(k) for
the Y column. The preview window should look like the above. You can see that the data points are
approximately linear.
Here you will see the usefulness of the preview window. Bring up the Parameters window again
(from the Windows menu). Make sure that the Use for fitting button is ticked. Set a1 = 2.2e+4 and
const = 27. The straight line corresponding to the fit should now be roughly parallel to the data. Change
the values of a1 and const until the fitting function provides a good straight line fit. The preview
window is useful for determining acceptable initial parameters for a fit or whether a particular fitting
function will be an appropriate model for the experimental data. There are other ways of manipulating
the parameters from the preview window that you can try out.
From the Calc menu choose Fit

Check that the input and output data columns are set respectively to 1/T and ln(k).
Click the Fit button.
In the Preview window you will now see the both the data and the fitted straight line.
To view the results of the fit, from the Windows menu choose Results. The slope of the line
corresponds to a1 = -2.2651x104 while the error in the slope, a1 , is 341. As the slope = a1 = -Ea /R
we get Ea = 188,3202835 J/mol = 188.3202.835 kJ/mol. Considering the error, the answer is more
appropriately written: Ea = 188.32.8 kJ/mol or, more correctly, Ea = 1883 kJ/mol.

Now you can plot the fitted straight line along with the data.
From the Plot menu choose Function f(x)
Make sure that Plot into current graph and Use fitted parameters are ticked.
Click OK.

In the drawing window, the fitted line is shown on the graph of the ln(k) versus 1/T data.
Another way of fitting the data
It is possible to fit the original data with the Arrhenius expression directly rather than recasting it
so that it can be plotted as a straight line. To do this we need to define a new function representing the
Arrhenius expression:
k(T ) = AeEa /RT
From the File menu choose New Function

A box in which we define the function will appear.
Type in:
y:=a[1]*exp(-a[2]/(8.314*x));
Note the form of your fitting function and its parameters. In the function k(T ) corresponds to y
and T to x, while a[1] corresponds to A and a[2] to Ea . It is crucial to get the syntax absolutely correct,
including the ; mark at the end of the line!

Click the To menu button. This will compile the function and identify if there are any syntax
problems. Correct the function if required and repeat the To menu command.
Rename the function Arrhenius instead of User Function and save it as Arrhenius.fn.
Click the To menu button again.

Choose Parameters from the Windows menu.
Set a[1] to 1.000e+11 and a[2] to 1.5000e+5
Now fit the original data using the new function.

Choose Fit from the Calc menu.

Make sure that the input data column is temperature (K) and the the output data column is rate
constant (L/mol/s).
Click Fit.
The results window should appear. If it does not, go to Window then click on Results.
You can see that the fitted value for the activation energy (parameter a[2]) is 1682 kJ/mol while
the pre-exponential factor, A, is (8.8 2.0) 1010 L/mol/s.
A Word of Caution:
The results of fitting a function to data can depend upon the initial parameters. Sometimes, for
inappropriate parameters, fitting is not possible.
To see this:
Select Parameters from the Window menu.
Change both a[1] and a[2] to 0.
Choose Fit from the Calc menu again, then press the Fit button.
The following warning appears:
To give yourself the best chance of fitting the data it is best to choose starting parameters that are
not too far from the correct ones. The Preview window can help you choose appropriate starting
parameters, because you can vary the parameters while viewing the function.

To think about...
Why does the Ea value obtained by fitting the data directly (1682 kJ/mol) differ from the value that
you obtain if ln(k) versus 1/T is fitted by a straight line (Ea = 1883 kJ/mol). The reason lies in the fact
that the fitting routine assumes that the errors in the X and Y values are constant. In fact, if the errors
in the rate constant k(T ), do not vary with temperature, then the errors in ln(k(T )) should decrease as
1/k(T ). The consequence is that the lower T data are weighted more highly in the linear ln(k) versus
1/T fits than in the direct k versus T fits. You can see this when the two functions are compared with the
experimental data in the figure below. The function with the directly fitted parameters better reproduces
the high temperature data.
Statistics with pro Fit
If you have a data table and wish to find the mean, sum, standard deviation, etc. of numbers in a
column, then select the column (click the mouse right at the top of the column).
Firstly, enter the numbers 1 to 10 into a column, then select that column.
Next, go to the Calc menu and choose Statistics. Here you can choose which statistical calcula-
tions you want performed. For now, we will do all of them.

Make sure that you have chosen the right column, highlight the cells, then click Results Window.
The results will appear in the Results window.
Tips for pro Fit
If you need to do something check out the Help menu. There youll find information on defining
functions, plotting, data manipulation, fitting, the Preview window and more.

Appendix E - How to Prepare a Sample
for Analysis by NMR Spectroscopy
Contact: Dr Paul Donnelly

The School Chemistry has invested in a new high-resolution benchtop NMR spectrometer for use
by undergraduate chemistry students. The 43 MHz Magritek Ultra Compact Spectrometer was commis-
sioned on 24 January 2013.
The spectrometer is expensive and it is important that is used correctly. It is essential that samples
for NMR analysis are prepared in the correct manner (see below). Please ask your demonstrator to show
you how to use the spectrometer before acquiring a spectrum.
Sample Preparation
1. Obtain a clean and dry NMR tube from the laboratory staff. Check that the tube is not broken or
cracked and that the lid is intact.
2. Weigh out 50 mg of your pure and dry compound into a sample vial.
3. Dissolve the sample in 0.5 mL of deuterated solvent. For most of the experiments in the 3rd year
course the solvent will be either CDCl3 or d6 -DMSO. Consult your demonstrator to find out the
best solvent for your sample.
4. Filter the sample into the clean and dry NMR tube through a glass Pasteur pipette with cotton-wool
inserted (see Figure 1).
5. Put the lid on the tube. Wipe the outside of the tube with a tissue to remove grease and fingerprints
from the tube.
6. It is important not invert the tube or shake the tube with the lid on. Sometimes the solvents used
can dissolve the plastic used in the lids.
7. Consult your demonstrator to show you how to acquire the spectrum.
8. Consult your demonstrator to show you how to clean the NMR tube.
9. The iNMR software on the iMACs in the Multimedia room can be used to analyse NMR data, but
iNMR cannot read the file format of the Magritek propriety software directly. It must first be saved
in Mestrelab Nova format, which iNMR can read (see Sioe See Volaric).
345
A PPENDIX E CHEM30015: Advanced Practical Chemistry
Figure 1: A glass Pasteur pipette with cotton wool is used to filter the solution into the NMR tube.

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School of Chemistry

Safety in the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Appendix A - Techniques for Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Appendix B - Crystallography and Instrumentation . . . . . . . . . . . . . . . . . . 309

Appendix C - Error Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Appendix D - Introduction to pro Fit . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Appendix E - How to Prepare a Sample for Analysis by NMR Spectroscopy . . . . 345

3 Course Structure and Organisation of the Laboratory

4 16th February 2017

1. Synthesis & Characterisation 1 (SC1)

Table 1: The core block themes and their abbreviations.

4 Laboratory Attendance Book

16th February 2017 5

5.1 Prac. Server

MAC: Use command-K ( -K) or WindowConnect to server.

5.2 Other Requirements

6 16th February 2017

A maximum of 3 sessions of medically certified absences is permitted. More than 3 absences

Assessment of students technical competence, background knowledge, reporting and interpretative

Table 2: Marking scheme for the course.

16th February 2017 7

Requirements for all assessments types:

8 16th February 2017

11 Prac. booking system

16th February 2017 9

10 16th February 2017

16th February 2017

16 K2 SC1.4 K2 SC2.2 K2 ESC1 K2 K2 K2 K2 Easter K2 TE: Solar cell T2.0

26 SC2.3 CC TE: Solar cell T2.0

Figure 1: Schedule of experiments for 2017.

Read this section carefully

Tongs should be used where appropriate, when handling hot vessels.

Long hair must be fastened securely.

Eating, drinking, smoking or chewing of gum is not permitted in the laboratory.

Accidents and First Aid

14 16th February 2017

Do not use any type of chemical fire extinguisher on a person.

When the alarm sounds:

Listen to evacuation instructions.

16th February 2017 15

Care of benches and apparatus

Pre-lab preparation and general considerations

16 16th February 2017

16th February 2017 17

Experiments in Synthesis and Characterisation 1

SC1.1 - Preparation of N-Benzylphthalimide . . . . . . . . . . . . . . . . . . . . . . . 27

SC1.2 - 2-Phenyl-4-benzylidene-5-oxazolone: Azlactone formation . . . . . . . . 29

SC1.3 - Synthesis of a para-Substituted Cinnamic Acid Using a Palladium-Catalysed

SC1.4 - Flow Synthesis of a Bicyclic Lactone . . . . . . . . . . . . . . . . . . . . . . . 35

Experiments in Synthesis and Characterisation 2

SC2.1 - Synthesis of 5,5-Diphenylhydantoin (DPH) . . . . . . . . . . . . . . . . . . . 41

SC2.2 - Synthesis of 3-Carbethoxycoumarin . . . . . . . . . . . . . . . . . . . . . . . 43

SC2.3 - Thiamine-Catalysed Synthesis of Benzoin . . . . . . . . . . . . . . . . . . . . 45

SC2.4 - Synthesis of 4-Phenylbenzoic acid by Palladium-catalysed Cross Coupling 47

Extended Synthesis and Characterisation of Organic Com-

ESC1 - Preparation of a Bicyclo[2.2.1]heptene by Cycloaddition . . . . . . . . . . . 51

ESC2 - Grignard Synthesis of Triphenylmethanol . . . . . . . . . . . . . . . . . . . . 53

ESC3 - Synthesis of Methyl 4-Nitrocinnamate Using the Wittig Reaction . . . . . . 57

ESC4 - Synthesis of (3-Ethyl-pentane-3-ol) . . . . . . . . . . . . . . . . . . . . . . . . 61

K1 - Excited State Reaction Kinetics: Pyrene Excimer Formation . . . . . . . . . . 65

K3 - Kinetics of the Acid Catalysed Aquation of Fe(II)-Tris(chelates) . . . . . . . . 83

PC1 - Dynamic Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

PC - Introduction to Investigations into Surface Tension . . . . . . . . . . . . . . . . 97

PC2 - Surface Tension Determination Pendant Drop . . . . . . . . . . . . . . . . . . 99