Laboratory Manual for

CHEMISTRY
315/325/335/345

Department of Chemistry
University of British Columbia
2013-2014






















I I













































How to cite this manual:
Laboratory Manual for Chemistry 315/325/335/345; Bussiere, G., Kunz, T., Monga, V., Rogers,
C., Stoodley, R., Eds.; University of British Columbia: Vancouver, Canada, 2013

I I I

TABLE OF CONTENTS
THIRD YEAR CHEMISTRY LABORATORY LEARNING GOALS ................................. VI
LABORATORY DIRECTOR CONTACT INFORMATION .................................................................... X
YOUR PERSONAL LABORATORY SCHEDULE ............................................................................. XII
LABORATORY LOGISTICS ......................................................................................................... XIV
DURING THE LAB SESSION........................................................................................................ XVI
LAB REPORTS .......................................................................................................................... XVII
ACADEMIC HONESTY ............................................................................................................. XVIII
SAFETY INSTRUCTIONS ............................................................................................................. XIX
SAFETY EDUCATION/TRAINING PROGRAM ................................................................................ XX
WORKING SAFELY IN CHEMISTRY LABORATORIES ................................................................ XXI
EMERGENCY AND NON-EMERGENCY PHONE NUMBERS ....................................................... XXII
HAZARD SYMBOLS AND CLASSES .......................................................................................... XXIII
ANALYTICAL CHEMISTRY EXPERIMENTS .............................................. 24
ANALYTICAL CHEMISTRY LABORATORY COURSE ..................................................................... 25
ANALYTICAL GRADING STRUCTURE ........................................................................................... 26
PREPARATION FOR AND DURING THE LAB .................................................................................. 28
ANALYTICAL CHEMISTRY REPORT FORMAT .............................................................................. 29
ACCOUNTING FOR UNCERTAINTY ............................................................................................... 31
A-03 DETERMINATION OF LEAD BY ANODIC STRIPPING VOLTAMMETRY ................................ 40
A-04 DETERMINATION OF IRON AND CHROMIUM BY SPECTROPHOTOMETRY .......................... 44
A-06 DETERMINATION OF QUININE BY FLUOROMETRY AND ABSORBANCE SPECTROMETRY .. 49
A-08 DETERMINATION OF CR
3+
BY CHEMILUMINOMETRY ........................................................ 65
A-09 DETERMINATION OF CALCIUM IN THE PRESENCE OF ALUMINUM BY ATOMIC
ABSORPTION SPECTROMETRY ..................................................................................................... 72
A-11 DETERMINATION OF COPPER BY ICP-MS ISOTOPE DILUTION MS ............................... 78
A-12 DETERMINATION OF MANGANESE BY NEUTRON ACTIVATION ANALYSIS ........................ 88
A-13 DETERMINATION OF NITRITE AND NITRATE BY ION CHROMATOGRAPHY ....................... 97
A-14 DETERMINATION OF BIPHENYL AND P-TERPHENYL BY LIQUID CHROMATOGRAPHY ... 103
A-16 DETERMINATION OF NAPHTHALENE BY GC-MS ............................................................. 113
INORGANIC CHEMISTRY EXPERIMENTS ............................................... 122
EXPERIMENTS ASSIGNMENT AND LABORATORY ROUTINES .................................................... 123
INORGANIC GRADING STRUCTURE ............................................................................................ 125
INORGANIC CHEMISTRY REPORT FORMAT .............................................................................. 126
I-02A SYNTHESIS AND CHARACTERIZATION OF A DIPHOS LIGAND ......................................... 128
I-02B PREPARATION AND ANALYSIS OF SOME NI(II) DIPHOS COMPLEXES ............................ 137
I-03A PREPARATION OF TETRAETHYLTIN ................................................................................ 145
I-03B REACTION OF TETRAETHYLTIN ...................................................................................... 153
I-06 PREPARATION AND MAGNETISM OF CHROMIUM(II) ACETATE ........................................ 159
I-07 STUDY OF CU(II) AMMINE COMPLEXES ............................................................................ 165
I-10A ELECTRO-SYNTHESIS OF VANADIUM(III) ALUM ........................................................... 173
I-10B DETERMINATION OF THE COMPOSITION OF V(III) ALUM ............................................. 180
I-11 SYNTHESIS AND CHARACTERIZATION OF (CH
3
)
3
N:BF
3
................................................... 190

I V

I-12 SYNTHESIS AND CHARACTERIZATION OF A CO(III) CAGE COMPLEX .............................. 203
I-13A SYNTHESIS AND CHARACTERIZATION OF A NI(II) MACROCYCLIC COMPLEX .............. 211
I-13B ELECTROCHEMISTRY OF A NI(II) MACROCYCLIC COMPLEX ........................................ 217
I-14 SYNTHESIS AND PURIFICATION OF A PROTECTED PHOSPHINE ......................................... 229
ORGANIC CHEMISTRY EXPERIMENTS .................................................... 243
THE ORGANIC CHEMISTRY LABORATORY................................................................................ 245
LEARNING OBJECTIVES OF THE ORGANIC COURSE ................................................................. 247
O-01: ISOLATION OF PIPERINE FROM PEPPER .......................................................................... 247
O-02: THE BROMINATION OF TRANS-CINNAMIC ACID ............................................................ 252
O-03: SYNTHESIS OF METHYL TRANS-CINNAMATE ................................................................. 255
O-04: REDUCTION OF 3-NITROACETOPHENONE USING TIN AND HYDROCHLORIC ACID ...... 260
O-05: REDUCTION OF 3-NITROACETOPHENONE USING SODIUM BOROHYDRIDE ................... 263
O-06: SYNTHESIS OF N,N-DIETHYL-M-TOLUAMIDE (DEET) ................................................. 265
O-07: SYNTHESIS OF ETHYL 4-AMINOBENZOATE (BENZOCAINE) .......................................... 269
O-08: REAGENT GRADE CHOLESTEROL VIA 5,6-DIBROMOCHOLESTEROL........................ 273
O-09: THE CONVERSION OF CHOLESTEROL INTO 3 -CHLORO-5-CHOLESTENE
(CHOLESTERYL CHLORIDE)....................................................................................................... 279
O-10: THE ALKYLATION OF -DICARBONYL COMPOUNDS ...................................................... 283
O-12: THE SYNTHESIS OF A DIPEPTIDE ..................................................................................... 288
PHYSICAL CHEMISTRY EXPERIMENTS ................................................... 301
PHYSICAL CHEMISTRY REPORT FORMAT ................................................................................. 302
GENERAL HINTS AND INSTRUCTIONS FOR DOING EXPERIMENTS ............................................ 303
THERMODYNAMICS OF NON-IDEAL SYSTEMS ........................................................... 304
P-01 HEATS OF SOLUTION OF POTASSIUM NITRATE ................................................................ 308
P-02 PARTIAL MOLAR VOLUMES IN NACL SOLUTIONS ........................................................... 313
P-04 SOLID-LIQUID PHASE DIAGRAM ....................................................................................... 318
P-05 LIQUID-VAPOR EQUILIBRIUM ........................................................................................... 323
ELECTROCHEMISTRY ........................................................................................................ 327
P-06 CONDUCTANCE OF WEAK AND STRONG ELECTROLYTES ................................................ 331
P-07 ROTATIONAL-VIBRATIONAL SPECTROSCOPY OF HCL .................................................... 336
THERMODYNAMICS AND EQUILIBRIA OF ADSORPTION ON SURFACES ........... 346
P-08 SURFACE TENSION OF BUTANOL SOLUTIONS ................................................................... 349
P-10 LANGMUIR-BLODGETTRY ................................................................................................. 352
P-12 OPTICAL ROTATORY DISPERSION AND CIRCULAR DICHROISM ...................................... 362
P-13 SEDIMENTATION VELOCITY OF BOVINE SERUM ALBUMIN ............................................. 373
P-15 MOLECULAR WEIGHT OF POLYVINYL ALCOHOL BY VISCOSITY .................................... 383
P-16 DETERMINATION OF FORMATION CONSTANTS OF CA-ATP COMPLEXES ...................... 388
P-18 LIGHT SCATTERING ........................................................................................................... 398
P-19 DETERMINATION OF DIFFUSION COEFFICIENT ................................................................ 405
INTEGRATED CHEMISTRY EXPERIMENTS ............................................ 413
INTEGRATED CHEMISTRY LABORATORY EXPERIMENTS ......................................................... 414
X-01 SYNTHESIS AND OPTICAL CHARACTERIZATION OF CDSE QUANTUM DOTS................... 415
X-02 INTRODUCTION TO VACUUM SCIENCE AND MASS SPECTROMETRY ............................... 432

V

X-03 SYNTHESIS OF BIS(AMIDATE)BIS(AMIDO) TITANIUM PRECATALYST AND
HYDROAMINATION REACTION .................................................................................................. 442
X-04 LASER PHOTOIONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY ............................ 449
X-05 SYNTHESIS AND CHARACTERIZATION OF SWITCHABLE SUPERHYDROPHOBIC-
SUPERHYDROPHILIC POLYPYRROLE SURFACES ....................................................................... 462
X-06 INTRODUCTION TO ELECTROCHEMISTRY: VOLTAMMETRY OF FERRICYANIDE ............ 473
X-07 CAPILLARY ELECTROPHORESIS FOR DETERMINATION AND CHARACTERIZATION ....... 486



VI

THIRD YEAR CHEMISTRY LABORATORY LEARNING GOALS

Students that obtain a passing mark in the third-year integrated chemistry labs (CHEM 315,
CHEM 325, CHEM 335 and CHEM 345) will have developed proficiency in the following five
broad areas:

A. Laboratory Skills
A1. Work in a safe manner while in the laboratory.
Be able to identify potentially hazardous chemicals and operations before coming to the lab, then
act accordingly while doing the experiment.
A1.a: General Safety: Obey the safety rules and procedures that are outlined in the lab
manual, both general and experiment-specific, at all times. Locate reliable chemical
safety information, and from it assess the risks of exposure and the likelihood and
severity of exposure. Be able to interpret common hazard symbols (WHMIS and
NFPA). Given an experimental procedure, identify the potential chemical and
operational hazards posed. Be able to describe the actions that should be taken in the
case that a hazardous event occurs.
A1.b: Environmental Safety: Dispose of waste properly. Know the common waste
streams, which include broken glass, strongly acidic/basic aqueous solutions, aqueous
solutions that contain toxic substances, halogenated and non-halogenated organic
solvents, and unreactive solids. Be aware that incorrect disposal can lead to
unexpected reactions, injuries and/or environmental damage.
A1.c: Personal protective equipment (PPE): Wear the appropriate PPE (lab coat, safety
glasses) for the duration of the lab period. Wear any additional specialized protective
equipment (nitrile gloves, face shield, etc.) as necessary for certain experiments.

A2. Perform common laboratory procedures in a proficient and timely manner.
Be able to list general procedural steps and/or describe the purpose/use of the procedure or
process you are using.
A2.a: New skills: Learn new skills as applicable to each experiment you undertake. Be
able to follow the instructions provided in the manual to perform these new procedures
correctly with little intervention/guidance by the TA.
A2.b: Basic skills: Demonstrate a high level of proficiency in common laboratory
operations, including (but not limited to) weighing to the correct precision required for
the experiment, quantitative transfer of liquids, solids and solutions, preparation of
standard solutions, dilutions, heating (hot plate, heating mantle, oil bath), and
measuring temperature.
A2.c: Discipline-specific skills: Proficiently perform the skills taught in second-year
lab courses, such as recrystallization, reflux, distillation, titration and measurements of
volume, temperature and pressure. Be able to describe the applications and limitations
of a range of instruments and be able to select appropriate instrument(s) to satisfy a
specific scientific need.

VI I

A2.d: Clean-up: Clean all glassware and work areas (around sinks, balances, benches
and instrumentation) before you leave the lab. Report deficiencies (missing items,
empty containers, broken equipment etc.) to the in-charge person immediately.

A3. Maintain an up-to-date, detailed, research-grade laboratory notebook.
Use a laboratory notebook that has sequentially numbered, duplicate pages. Record information
in either blue or black ballpoint ink. Title and date every page. Maintain an up-to-date table of
contents. Submit the top copy of each page with your report; keep the copy page in your
notebook to retain a complete record of your experimental work. Neatly cross out errors (never
use correction fluid or tape). Cross through, but do not remove, any blank pages left between
experiments.
A3.a: Document laboratory procedures: Document all procedures, observations and
data collection at the time they occur, with sufficient detail such that a competent
scientist who is unfamiliar with the work can reproduce the experiment.
A3.b: Accurately record data and observations in an organized way: Plan for and
prepare data tables (as needed) for each experiment. Record both qualitative and
quantitative observations. Organize electronic files logically and with descriptive file
names. Record numerical data to the number of significant digits consistent with the
measuring device used. Include units consistent with the conventions of the
measurement and discipline.

A4. Apply appropriate methods of analysis to data and present raw data using meaningful
units.
Select the appropriate analysis approach for an experimental procedure, including the choice of
statistical analysis applied to the data and the presentation of the results in a graphical format
following the conventions of the discipline.

A5. Perform logical troubleshooting of laboratory procedures and instrumentation.
Based on experience in prior lab courses, anticipate potential problems and devise strategies that
can be used to overcome any problems. Use logical process(es) of elimination to narrow down
the source(s) of the problem. Attempt to solve the problem yourself, but also know when to ask
for help. Using appropriate terminology explain the problem and describe the steps you took to
solve it.

B. Written and Oral Communication
B1. Report your experiment and its results in a clear and concise manner.
Accurately summarize the principles underlying the experiment in your own words. Produce
schematics to describe experimental set-ups and processes. Present data clearly in tables and
graphs. Use units, errors and significant figures correctly.


VI I I

B2. Write in scientific style and with appropriate depth.
Write clear and well-organized laboratory reports. Discuss results in detail and explain why they
agree/disagree with theory. Write compelling scientific arguments in support of your
conclusions.

B3. Access and properly cite scientific literature.
Cite scientific literature following the conventions of peer-reviewed scientific journals; cite
within text and with an appropriate reference list. Find and cite relevant textbooks, journal
articles and websites.

B4. Build competence in scientific dialogue.
Use correct terminology. Demonstrate an appropriate level of confidence in verbal explanation
of scientific reasoning.

C. Responsibility and Professionalism
C1. Effectively prepare in advance for laboratory work.
Perform reading, calculations and background research as needed to maximize learning during
in-lab time. Be able to explain the reasoning behind every step in the experiment. Develop your
own questions to be answered during the lab.

C2. Learn from mistakes.
Use feedback from the teaching staff to avoid repeating mistakes. Anticipate problems and
challenges. Be alert to unexpected results, hypothesize the source(s), and resolve as time permits.

C3. Work as a team.
Effectively and efficiently work with a partner during in-lab time. (Note that some experiments
require only individual, not group work). Establish responsibilities and/or roles; monitor progress
and adjust as needed to successfully complete the experiment. Collaborate and communicate
effectively with your lab partner.

C4. Demonstrate high ethical standards.
Demonstrate high ethical standards in data collection, lab report writing and personal behaviour.
Recognize the point at which work should diverge from being shared with your partner to being
an individual effort. Distinguish healthy discussion and team work from plagiarism. Be aware
that report writing is an individual activity. Understand the basic principles of intellectual
property.

D. Context
This section summarizes some of the broader goals of the CHEM 3XX third-year integrated
chemistry labs.

I X

D1. Become proficient in a range of modern techniques.
Become proficient in the use of a wide range of modern techniques relevant to chemical
research. Describe the capabilities and limitations of the techniques you use.

D2. Develop awareness of the interdependence of the traditional sub-disciplines of
chemistry.
Chemical research and scholarship has traditionally fallen into a number of sub-disciplines,
including analytical, inorganic, organic and physical chemistry. The boundaries between these
sub-disciplines are however becoming less clear and future chemical innovation will require an
understanding of the potential contributions of each of the sub-disciplines to solve a given
problem. In this course you are encouraged to make links between the sub-disciplines by, for
example, carrying out one or more of the integrated experiments where appropriate.
D3. Become comfortable in an interdisciplinary research environment.
Reaching even further across disciplinary boundaries, important discoveries are increasingly
made at the interfaces between chemistry and other fields. This course aims to prepare you for
this interdisciplinary future, for example by encouraging you to appreciate that procedures are
driven by scientific need and not by available equipment or disciplinary traditions.

E. Integration and Application of Knowledge/Experience
E1. Apply critical thinking in the laboratory.
Make strategic decisions by thinking critically about observations made in the lab. Use these
observations to identify possible options, evaluate choices, and implement and justify course(s)
of action.

E2. Recognize whether results and conclusions "make sense".
Interpret various types of data to draw defendable and relevant conclusions. Continue to develop
your ability to recognize when data, conclusions, and results from calculations make sense;
estimate magnitudes of quantities and check units resulting from calculations.



X

Laboratory Director Contact Information
Analytical
Dr. Robin Stoodley
Office: C226B
Email: stoodley@chem.ubc.ca
Tel: (604) 827-5829


Inorganic
Dr. Vishakha Monga
Office: B470A
E-mail: inorglab@chem.ubc.ca
Tel: (604) 822-3678


Organic
Dr. Christine Rogers
Office: C326C
E-mail: crogers@chem.ubc.ca
Tel: (604) 827-4820


Physical
Dr. Guillaume Bussiere
Office: B450A
E-mail: bussierg@chem.ubc.ca
Tel: (604) 822-6384

















Where to find us:



XI



XI I

Your Personal Laboratory Schedule

September December 2013

Date Started Expt. Code TA Name TA Signature


















**If you are taking C315 you will only use half of these spaces.
You will fill in all of these spaces if you are taking C325.**


XI I I

Your Personal Laboratory Schedule

January April 2014

Date Started Expt. Code TA Name TA Signature


















**If you are taking C325 you will only use half of these spaces.
You will fill in all of these spaces if you are taking C345.**


XI V

Laboratory Logistics
Check-In week
**Read through the introductory material in this lab manual. Pages IX XVI.

**Arrive to your laboratory orientation session. Students who are not present during their
orientation will have their registration terminated.
According to the Faculty of Science Academic regulations:
Students registered in any Science course that has a laboratory must attend their first scheduled
laboratory class in that course. Failure to do so will result in the termination of students
registration in the course. Students who are unable to attend their first scheduled laboratory class
in a course must notify the head or the designate
1
, of the department concerned within 48 hours
of the time affixed for that class or have their registration in the course terminated.

How the labs are run
Multiple experiments run simultaneously in each lab period; students cycle through a
subset of these experiments over the course of ten weeks.
The labs are to be completed in four hours.
TAs are present; each takes care of a set of experiments.
Some labs are performed in pairs, however, each student must submit their own
unique lab report.
Safe laboratory practices are essential. Eye protection and labcoats must be worn.
Keep all instrumentation clean - especially the analytical balances. Keep the
laboratory bench clean. Your TAs will inspect your work area before you leave the
laboratory.

Missing a laboratory session
The CHEM 315/325/335/345 lab is running above capacity Make-up labs can only
be scheduled with difficulty. Make-up labs may be scheduled outside your normal lab
period or at the discretion of the lab directors.
Standard university policy for academic concessions applies. Labs missed due to
illness can be re-scheduled; a doctors note must be provided. Contact the specific lab
director in advance if you know you will miss a laboratory session.
If you miss a lab due to illness, you must hand-in any lab report that was due in the
missed session as soon as you return to attending classes. Do not wait until your next
lab session in the following week!
If you are late arriving to the lab, a penalty of 10% may be applied to your report
mark; depending on circumstances you may have to forfeit the lab with a grade of
zero.


XV

Course Designates:
Dr. Robin Stoodley [Analytical]; Dr. Vishakha Monga [Inorganic]; Dr. Christine Rogers
[Organic]; Dr. Guillaume Bussiere [Physical].

XVI

During The Lab Session
These experiments are designed to fit into the four-hour time allotted. However, you must be
familiar with the experiment and know what you are to do before you arrive in the laboratory.
Additionally, you are responsible for reading the preliminary pages of each discipline;
Analytical, Inorganic, Organic, and Physical.

The most important pre-experiment preparation is to read the manual!

WEAR YOUR LAB COAT AND EYE PROTECTION. No open-toed shoes allowed in the lab.
Tie long hair up or back you dont want it falling into your beakers!

Be respectful of everyone in the lab. Put equipment back into the appropriately-labeled lockers
and return unknown sample vials back to the dispensary. You are not the only one performing
each experiment and no one wants to clean up after you.

There is one accepted lab notebook for CHEM 315/325/335/345. It is the Hayden McNeil spiral
bound Student Laboratory Notebook. This can be purchased from the UBC bookstore.

UBC disability policy:
http://www.universitycounsel.ubc.ca/policies/policy73.html
Anyone who has a disability that requires special testing accommodation or other class
modifications should bring the request for accommodations or for changes in the accommodation
needs to the attention of appropriate personnel in a timely manner in order to allow for
arrangement of accommodations. All new and returning students who will be requesting an
accommodation are required to contact the Disability Resource Centre (DRC) at the beginning of
each term. At the beginning of each term, all students should discuss their situations with each
instructor from whom they are seeking accommodation. DRC will contact instructors prior to
this meeting if requested to do so by the student. Contact the DRC at Brock Hall 1203, 1874 East
Mall, Telephone 604-822-5844.


XVI I

Lab Reports
There is a general grading structure for lab reports submitted in CHEM 315/325/335/345. Please
see the specific report format required for each discipline.

Each 4 hours lab period corresponds with a report that is marked out of 20.
This means:
*CHEM 315/335
9 (4 hour lab periods) x 20 180 marks total

*CHEM 325/345
18 (4 hour lab periods) x 20 360 marks total

*Students registered in CHEM 325 or 345 MUST separately obtain a passing grade in each
discipline (analytical, inorganic, organic, physical, and -if selected as part of your course
schedule- integrated).

*Combined Major in Science students registered in CHEM 315 or 335 MUST separately
pass each discipline that appears in your schedule of selected experiments.

*Biochemistry students registered in CHEM 315 or 335 MUST separately pass each of
organic and physical.



XVI I I

Academic Honesty
Students are expected to follow the University of British Columbia academic integrity
guidelines, which can be found at the website:

http://www.calendar.ubc.ca/vancouver/index.cfm?tree=3,54,111,959

Please read the statement reproduced below, and note particularly the section italicized here for
emphasis, but not in the original document. Upon registering for courses, you have entered into
a contract with the university agreeing to the statement below.

Plagiarism, which is intellectual theft, occurs where an individual submits or presents the oral or
written work of another person as his or her own. Scholarship quite properly rests upon
examining and referring to the thoughts and writings of others. However, when another person's
words (i.e. phrases, sentences, or paragraphs), ideas, or entire works are used, the author must
be acknowledged in the text, in footnotes, in endnotes, or in another accepted form of academic
citation. Where direct quotations are made, they must be clearly delineated (for example, within
quotation marks or separately indented). Failure to provide proper attribution is plagiarism
because it represents someone else's work as one's own. Plagiarism should not occur in submitted
drafts or final works. A student who seeks assistance from a tutor or other scholastic aids must
ensure that the work submitted is the student's own. Students are responsible for ensuring that
any work submitted does not constitute plagiarism. Students who are in any doubt as to what
constitutes plagiarism should consult their instructor before handing in any assignments.

If you include in your laboratory reports any phrases, sentences, or other material of which you
are not the author, they must be placed in quotations and cited/referenced. Figures, graphs, etc.
of which you are not the author must be cited and referenced. There is a zero-tolerance policy for
academic dishonesty in the CHEM 315/325/335/345 laboratory. Violations of these guidelines
may result in academic discipline ranging from a zero mark for a laboratory report up to
expulsion from the university.
Remember, however, that the inclusion of material written by others often does not demonstrate
to the marker your understanding of the subject. As such, simply quoting others work is
insufficient to earn full marks.
Normally, you will submit a hardcopy (i.e. paper copy) of your lab reports. However, you may
be asked to submit an electronic copy. Electronic copies will be submitted to a service to which
UBC subscribes, called Turnitin (www.turnitin.com). This is a service that checks textual
material for originality. Failure to submit an electronic copy will minimally result in a zero
grade for that assignment. Please see your instructor for further information.





XI X

Safety Instructions
To avoid injury to yourself and others, you are required to follow the safety rules below. Failure
to comply with these rules while in the laboratory may result in suspension or expulsion from the
course.
1. If you have a medical problem or condition that may affect your performance or safety in
the laboratory, you must discuss it in private with the laboratory coordinator. This
information will be held in strict confidence.
2. EYE PROTECTION
a) Adequate eye protection is required for all individuals working in the laboratory. Do
not remove your eye protection until you have physically left the lab room. The
following types of eye protection are acceptable:
b) Normal prescription eyeglasses or safety glasses, either with or without safety side-
shields, as long as the lenses are shatterproof (i.e. plastic) and cover a large enough
area surrounding your eye (this usually means that the frames must be a minimum of
4 cm from top to bottom as well as from side to side).
c) Safety goggles that form a tight seal to your face. For some labs (particularly
organic), where exposure to toxic or irritating fumes is a real problem, the best form
of eye protection is safety goggles.
3. Eye injuries, whether chemical or mechanical, must always be considered serious. IN
CASE OF CHEMICAL INJURY TO THE EYE THE BEST PROCEDURE IS
IMMEDIATE PROLONGED CONTINUOUS FLUSHING WITH WATER (15 - 20
minutes) at an eyewash fountain. Eyes must be forced open to be washed well.
4. ALL-COTTON LABCOATS MUST BE WORN WHEN WORKING IN THE
LABORATORY.
5. FLUSH WITH WATER ANY PART OF YOUR BODY THAT COMES IN CONTACT
WITH ANY CHEMICAL. Use lots of water. Removal of clothing may be required.
6. Do not wear open-toed shoes or sandals. Tie-up long hair.
7. NEVER EAT OR DRINK IN THE LABORATORY.
8. Throw away cracked or chipped glassware immediately into the glass waste container.
9. Clean up chemical spills immediately. Check with the instructor for the proper procedure.
Ask your instructor about the disposal of used chemicals. Waste containers for disposal
of halogenated organic waste, non-halogenated organic waste and radioactive waste.
10. WASH YOUR HANDS WHEN LAB WORK IS FINISHED.
NOTE: The wearing of contact lenses in the chemical laboratory has historically been perceived
as a safety hazard. The concerns are: a) if chemicals are splashed into the eyes, they can become
trapped under or absorbed by the lenses; b) some chemicals can cause coagulation of the protein
in the eye within seconds; c) chemical vapours can be trapped under or dissolve into soft lenses.
It is also extremely difficult to remove lenses if something has been splashed into your eyes.
More recently, both the American Chemical Society and the (American) Occupational Health
and Safety Administration have issued statements indicating their studies suggest the wearing of
contact lenses does not pose any additional hazard and can be treated as safe if and only if safety
glasses or goggles are also worn.

XX

Safety Education/Training Program
WHMIS ("Workplace Hazardous Materials Information System") training is required to ensure
that workers are able to apply their safety knowledge to protect their health and safety.
Instruction on the content and significance of labels and MSDS, emergency procedures, and
information and procedures for safe use, handling, storage and disposal of hazardous materials
are all part of the training course.
Some products are exempted from federal WHMIS requirements for labels and MSDS if they are
already covered by other labeling legislation. These products include:

cosmetics and drugs
explosives
pesticides
radioactive substances
some consumer products

Certain products are completely exempted from both federal and provincial WHMIS
requirements. The following are included in this category.

wood and wood products
manufactured articles
tobacco and tobacco products
goods handled, offered for transport or transported pursuant to the
Transportation of Dangerous Goods Act.

Important note: WHMIS training is available to all UBC students free of charge. If you would
like to register for this training visit the following URL.

http://www.hse.ubc.ca/crs_reg/start.asp

The laboratory safety training courses are offered several times a year. It is recommended that
you enroll in this course.

Safe Handling of Materials - WHMIS Labels and the MSDS
Before using any chemical you must be aware of any hazards involved in its handling, and the
precautions which should be taken. All controlled product containers have a WHMIS label
which is a good primary source of information. The symbols used on these labels are described
below.

"Material Safety Data Sheets" (MSDS) provide detailed and comprehensive information on
material hazards. A website with a database of MSDS is:

http://ccinfoweb.ccohs.ca/msds/search.html


XXI

Working Safely in Chemistry Laboratories
It is inevitable that you will be required to handle hazardous substances outside of the fume
hoods, given the current facilities available. The following information has been provided in the
event that you have concerns about safety practices in the undergraduate chemistry laboratories.
The pre-lab talk, whether presented by a lab director or a T.A., will contain important safety
information for that laboratory period - take note! Topics such as personal protective equipment
needed (in addition to safety glasses and lab coat), the generation of corrosive gaseous by-
products that require a gas trap, safe heating of flammable liquids etc. will be highlighted. The
material presented during the pre-lab talk will, in part, reinforce the safety information readings
that you are required to do prior to coming to the lab. Make note of the physical properties of the
chemicals that you will be handling and generating, as well as specific hazards and remedies.
Material Safety Data Sheets (MSDS) are readily available online from the chemical suppliers
listed below:
Sigma-Aldrich http://www.sigmaaldrich.com/canada-english.html
Fisher Scientific http://iris.fishersci.ca/MSDS2.nsf/Search?OpenForm
TCI America http://www.tciamerica.com/
Alfa Aesar http://www.alfa.com/en/gh100w.pgm
Acros Organics http://www.acros.com/Welcome.aspx
Praxair http://www.praxair.com/na/ca/en/can.nsf
Strem http://www.strem.com/
The UBC Department of Chemistry web page also has Health and Safety links
(http://www.chem.ubc.ca/health-and-safety).
If you have safety questions during an experiment, speak with your T.A. and/or the lab director.
If the answers provided do not satisfy your concerns, contact the Chemistry Department Safety
Officer (604-827-5216, Room A237). Be prepared to provide specific information about the
circumstances, such as the date, the chemicals being used, and your particular concerns. UBC
Risk Management Services (604-822-2029) is the final level for dealing with safety issues on
campus.


XXI I

Emergency and Non-Emergency Phone Numbers

EMERGENCY NUMBERS
From cell phones From office phones

Emergency Fire, Police, Ambulance,
Hazardous materials response

911 9-911

UBC Emergency / First Aid 604-822-4444 2-4444
Campus Security 604-822-2222 2-2222
UBC Hospital Urgent Care
Department

604-822-7222 2-7222



NON-EMERGENCY NUMBERS

From cell phones From office phones

Ambulance 604-872-5151 9-604-872-5151
Campus Fire Department 604-665-6010 9-604-665-6010
Student Health Services 604-822-7011 2-7011
Chemistry Dept Safety Office 604-827-5216 7-5216
Risk Management Services 604-822-2029 2-2029







XXI I I

Hazard Symbols and Classes


24







ANALYTICAL CHEMISTRY
EXPERIMENTS












Did you know?

Payscale.com reports Canadian analytical chemists salaries based on national data. In July 2013,
the salary range for an analytical chemist with two years experience was approximately: $35,240
(10th percentile) to $69,055 (90th percentile). Vancouver has the lowest average pay compared
to other major Canadian cities.

25

Analytical Chemistry Laboratory Course
The Analytical Chemistry laboratory course is designed to introduce you to instrumental
analysis, to improve your technical lab skills and to provide you opportunities to improve your
report-writing skills. The instrumentation used in this course is complex; in addition to working
with the data that is output, you will be expect to become familiar with all aspects of the
instruments design and operation. Analytical chemistry methods such as use of calibration
curves, standard addition and internal standard must be mastered.

Learning Objectives of the Course
The goal of the Analytical Chemistry laboratories is to equip you with skills and abilities that
you will need as a practicing scientist. These include being able to design experiments, safely
undertake them, and interpret their results. Listed below are learning goals
1
to attain in the
Analytical Chemistry laboratories. The goals have been classified by type.

Safety: Identify and evaluate health, safety and environmental risks associated with your lab
work. Use best practices to minimize risks.
Teamwork: Effectively and efficiently work with a partner during in-lab time. Determine
responsibilities and/or roles; evaluate progress and adjust accordingly to finish on time.
Models: Evaluate advantages and disadvantages of different theoretical models to understand
their success at mimicking experimentally-observed behaviour. Apply your own experimental
data in support of a known model or evaluate whether or not a model sufficiently explains your
experimental data.
Experimentation: Determine a procedure including selection of instrumentation, methods, and
approaches; conduct the experiment; collect data. To an appropriate level, demonstrate and
apply trouble-shooting or problem-solving techniques.
Instrumentation: Make measurements of concentration or amount using appropriate
instrumentation, equipment, and software.
Hands-on: Demonstrate proficiency in hands-on lab skills such as pipetting, filling of
volumetric flasks, injection techniques, etc. Maintain a clear and comprehensive lab notebook.
Analyze: Improve your ability to evaluate, interpret and analyze your data. Apply calculations
to your raw data as needed; respect unit conventions and show judgment with respect to
magnitude of result. Using the data, draw conclusions and support them.
Learning from failure: Back-rationalize possible causes of unusual or incorrect data. Identify
possible solutions; implement them as time permits.
Communication: Using the format of a formal lab report, communicate clearly and concisely
the context, the background, your data, your results and the conclusions of your experiment.
Ethics: Demonstrate high ethical standards, including in data collection, writing of lab reports,
and personal behavior towards students, staff and faculty.

1
Learning goals adapted from Feisel, L.D.; Rosa, A.J. The Role of the Laboratory in
Undergraduate Engineering Education. J. Engineering Education 2005, 94, 121-130.


26

Analytical Grading Structure
Each experiment is marked out of 20. The general breakdown is:

Technique: 2 marks for working carefully and competently, and in cleaning up.
Calculations: 2 to 4 marks for numerical treatment of data. Take care to avoid errors due to
incorrect or missing dilution factors.
Data: 7 marks based upon a comparison of your result with that expected for the unknown.
Note: 1 mark deducted if your result is not given on the 1
st
page of your report; 1 mark deducted
if the result has missing or incorrect units; 1 mark deducted for missing or incorrect unknown
number. In case of calculation error(s) that impact your reported result, you may correct
your mistake and resubmit a new result. This can be done once per experiment. Guessing
is not tolerated.
Report: 7 to 9 marks based upon the report as outlined in the manual.

Late Submissions
2 marks will be deducted for each week, or part thereof, that your report is late. The absolute
final deadline for submissions is 4:00pm Friday November 29
th
2013 (Term one) and
4:00pm Friday April 8
th
2014 (Term two). No consideration will be given for reports that are
lost or submitted late as a result of computer or printer failure. Save your work frequently!
Handwritten reports are acceptable but are strongly discouraged.

Mark Normalization
Please note that the lab marks assigned by your TA may be subject to normalization. If your TA
is an unusually generous marker, the grades will be normalized downwards. If your TA is an
unusually difficult marker, the grades will be normalized up. Usually, little normalization is
required.

Notebook Guidelines and Evaluation
You will use your lab notebook to prepare for each laboratory session ahead of time and during
the lab itself. All calculations and discussion etc. will be handed in as a separate report to your
TA.

The following are some guidelines about laboratory notebooks:
Your notebook must be in the laboratory when you are.
It must be a bound notebook with numbered pages and a table of contents. Leave
space for the table of contents at the beginning of the book and update as necessary.
Entries must be made in ink, and there must be no erasures or white-outs. Draw a
single line through entries that are incorrect or otherwise not needed.
Numerical data must have units included.
Do not remove pages from your lab notebook under any circumstances.
It is a good idea to keep the left-hand pages blank for later additions or corrections or
comments.


27

A very good article on laboratory notebooks is "Keeping a Laboratory Notebook" by Eisenberg,
A. Journal of Chemical Education 1982, 59, 1045-6.

To prepare for the lab, each notebook entry should contain:
Title of Experiment and Date
Purpose of Experiment (1-2 sentences describing the purpose- make sure this is
correct!)
A brief description of what you will do in the experiment and how it will be done (~3-
5 paragraphs)
Calculations related to solution preparation


Laboratory Reports

Full lab reports are required for all experiments.
Your report should include sections of Introduction, Instrumentation/Method, Data, Calculations,
Results and Discussion. Reports are normally due one week after the experiment was performed.
Where the schedule is interrupted (e.g. by reading break) the report must be submitted at the next
scheduled lab period. Each student must prepare his or her own report independently, even when
the laboratory experiment is carried out in pairs. Reports must be submitted directly to your
teaching assistant. A penalty will be assessed for late reports.

About the Unknowns

There are two methods used to prepare the unknowns. Depending on which method is used,
your first step in the experiment is either to dilute the entire provided unknown into a volumetric
flask or to pipette 10.00mL from the unknown vial into a volumetric flask. It is very important
that you do the correct one: the lab manual will give you the correct procedure for each
experiment. In both cases, when calculating the concentration of the unknown the volume
provided can be taken as 10.00 mL. The value(s) given on the front page of your report must
be the concentration of the unknown in the original vial.



28

Preparation For and During the Lab

Write your reports Principle of Method section before coming to the laboratory. This is
intended as lab preparation, and 1 technique mark will be deducted if it has not been done. You
may revise this section after the experiment to reflect new understanding or insight gained during
the experiment. Your pre-lab preparation should also include the procedure you will follow
for preparation of standard solutions (i.e. dilution calculations).

Do some preliminary calculations and graphing on your data before you clean up and leave the
laboratory. The time to find out whether your data is satisfactory is while you are in the
laboratory and are able to repeat a questionable measurement. Dont dispose of your solutions
until you have done this!

Unexpected or unusual results should be brought to the attention of the instructor or the TA as
soon as possible.

Cleanliness is of great importance in the analytical lab. Carefully clean all glassware with tap
water, then rinse with deionized water using the squeeze bottles provided. A soap solution for
cleaning is available on the bench. Note experiment 11 is extremely sensitive to contamination:
separate instructions for acid-cleaning the plasticware used for trace-level copper analysis are
provided.


29

Analytical Chemistry Report Format

COVER PAGE: Student name, course number and section, name of partner (if applicable),
date of experiment, unknown number, the result for the concentration of your unknown (as
supplied in the vial) with the uncertainty and associated confidence level. All results should be
reported in units of g/mL.

TITLE: This should give the nature of the analytical method and of the analyte. Please avoid
simply copying the title used in the lab manual.

ABSTRACT: A brief (3-4 sentences; not more than 8 lines) description of the experiment in
your own words. Include what you did in the lab, how you did it, and what the results were.

PRINCIPLE OF METHOD: The principle of method section should be written before you attend
the laboratory. 1 technique mark will be deducted if you have not completed it before the lab
period or if it is incomplete. Once you have gained a better understanding of the experiment
by doing it, you should improve your principle of method by including your new insights.

Describe briefly the chemical and physical principles relevant to the experiment. Use diagrams,
graphs, or chemical equations where appropriate. Include any relevant laws or equations.
Describe the experimental set-up (instruments) and use a schematic diagram where applicable.
Explain the functioning of the main components of the instrument. Do not describe details of
procedure. Your goal in this section should be to convince the TA that you fully understand the
methodology used in the experiment.

RESULTS AND CALCULATIONS: A worked-example of each type of calculation should be
given. Data should be presented in tables with a table number, a title and units. Each graph
should have a graph number and a title with labels on both axes. Every graph should be on its
own page and should fill that page. Graphs and tables should be used to help the reader
understand the results; they should always be referred to in the text of the report. Rescale graph
axes to illustrate the points you wish the reader to understand. For example, if the intercept of a
line is used in your calculations, your graph should be formatted to show the intercept. Do not
put raw data into this section; use an appendix instead.

DISCUSSION: This is the most important part of your report. This section is the place where
you tell the reader what the experiment was all about and what it has shown. Examine the
analytical method critically, based on your knowledge and experience.

Discuss your results specifically. Do they agree with theory?
Describe the use of any special analytical techniques (spiking, standard addition, etc.?)
How did these techniques affect your results?
Comment on any spectra or calibration plots used. Comment on their meaning,
linearity, etc. How do they relate to quantitation of the analyte?
Discuss the numerical result(s) obtained. If you used more than one instrumental
method or data analysis treatment compare them and the results they produced.
Consider both precision and accuracy.


30

ERROR LIMITS: Give an estimate of the error limits for all reported numerical quantities (see
discussion of error analysis in your textbook). List sources of systematic error and, if possible,
estimate their limits. Estimate random error limits from the observed scatter of data. This may
be done by statistical computation (regression analysis), or graphically in the case of gross error.
Try to determine which of random or systematic errors predominates. Dont focus solely of
uncertainties in glassware volumes unless these are the main sources of error. Describe what
can be done to minimize errors. This section may be combined with the discussion if desired.

CONCLUSION: State your major findings in a few lines.

REFERENCES: Cite your sources: manuals, journal articles, textbooks, websites, etc. in ACS
format. (See policy on plagiarism)

APPENDIX (if applicable): An appendix is used to include material which is not properly
formatted: raw data, details of calculations and error propagation. If you choose to include an
appendix, you must refer to it in the body of your lab report. Improved clarity is the main reason
for including an appendix. Consider the TA who has to read your report; all else equal, unclear
lab reports receive lower grades.

As a rule of thumb for lab reports, remember that your report is a written communication from
you to show your TAs all that you know. Part of your mark will come from how well you
communicate your knowledge to your TA.



31

Accounting for Uncertainty
I. AN ERROR IS AN ERROR IS AN ERROR. We never know exactly how big an
uncertainty should be associated with a measurement; we even dont know exactly the range in
which a quantity we are trying to measure lies. Much error analysis consists of educated
guesses. Statistical analysis can help, but to use them properly we must understand that they do
not remove uncertainty, but merely let us express more clearly what the uncertainty is. For
example, the statement:
[Mn
2+
] = (1.56 "0.03) H 10
-4
g/ml
does not mean that [Mn
2+
] lies, for sure, between 1.53 and 1.59 H 10
-4
g/ml. It may mean several
things, according to the convention used by the particular writer for the meaning of 0.03. To
avoid ambiguity, state your convention along with the numerical result, for example,
[Mn
2+
] = (1.56 " 0.03) H 10
-4
g/ml (95% confidence limits).
This means: "There are 19 chances out of 20 that [Mn
2+
] lies between 1.53 and 1.59 H 10
-4

g/ml."

II. SYSTEMATIC AND RANDOM ERRORS 10 ml of a solution of Mn
2+
was diluted to 100
ml in a volumetric flask, and the manganese in the diluted solution was determined by some
analytical method. In several repeats of the same experiment, results were:
1.54, 1.58, 1.53, 1.55, 1.60 ... H 10
-5
g/ml.
Mean [Mn
2+
] (diluted solution) = 1.56 H 10
-5
g/ml.
Mean [Mn
2+
] (before dilution) = (1.56 H 10
-5
) H (100/10) g/ml = 1.56 H 10
-4
g/ml.

RANDOM error is usually estimated from statistical analysis of the scatter in results of repeat
tests, such as the set of five given above.
SYSTEMATIC error can arise in many ways, some of which may not come to mind very easily.
For example, the dilution factor 100/10 in the above calculation is very likely to conceal
systematic error. Commonly, we would use the same volumetric flask in every repeat of the
same experiment, and its volume might not be exactly 100 ml. Suppose, for simplicity, that the
10 ml sample volume were exact, but that the flask for dilution actually had a volume of 102 ml
(a great exaggeration of the likely error in a volumetric flask, just for the sake of illustration).
Then:
Mean [Mn
2+
] (before dilution) = 1.56 H 10
-5
H (102/10) = 1.59 H 10
-4
g/ml.
The previous estimate contained a systematic error of -0.03 H 10
-4
g/ml. Note that a systematic
error, being always one way, has a sign, while random error consists of scatter both positive and
negative, and is expressed as (quantity) " (random error).
We cope with systematic error by using common sense and scientific experience to list the likely
sources of it and devise procedures to eliminate as many as possible. For what sources remain,
we have to make educated guesses. A large part of error analysis necessarily consists of these
guesses.


32

Suppose, for example, that one had a couple of dozen volumetric flasks, and took a different one
off the shelf for each repeat of the experiment. Is the error in the volume of the flask now
systematic, or random? This depends on what was going on in the factory where the flasks were
made. The manufacturer may or may not have done something which made all the flasks
consistently too big. One can only guess about this kind of thing unless one has much
information about how equipment was made and calibrated, right back to the primary standards
of mass, length, time, temperature, etc. Fortunately, when experiments are being done to about
three-figure accuracy, the expected range of error in calibrations of volumetric equipment or of
the weights built into analytical balances is usually very small in comparison to other sources of
error (table 1). The purpose of the above example was not to get you locked in to worrying
about volumes, but to encourage you to be wide-ranging in your thoughts about what could have
gone wrong in your attempt to measure something quantitatively. A large part of an error
analysis should consist of your assessment, in words, of the most likely sources of large error.

TABLE 1: Reasonable Error Limits

Titre by buret (50 ml) 0.03 ml
End point detection 0.03 ml
Volumetric flask (50 ml) 0.05 ml
" (100 ml) 0.08 ml
(250 ml) 0.12 ml
Pipetting (5 ml) 0.01 ml Allow 15s drain time
(10 ml) 0.02 ml Allow 15s
" (25 ml) 0.03 ml Allow 25s
Weighing by analytical balance 0.0001g

III. STATISTICAL ANALYSIS OF RANDOM ERRORS If you set out to measure the
numerical value of some quantity, you start with the assumption that there is a "true value" to be
found. You can never determine exactly what it is, but you can try to obtain better and better
approximations, if you have enough time available. The ways to reduce errors are quite different
for systematic and random errors. For systematic errors, you have to think what they may be and
change your procedures so as to eliminate them as much as possible. For random errors, you can
reduce them by repeating the same experiment many times and averaging the results. The mean
value x of the quantity should be an increasingly close approximation to the "true value" as the
number of determinations (n) increases. But the mean becomes exact only if n is infinite.
Unfortunately, the error limits decrease with the square root of n, so that it takes an increase of a
factor of 4 in number of experiments to decrease error limits by a factor of 2.
These ideas are expressed statistically in the quantities known as variance, standard deviation,
standard deviation of the mean, and confidence limits. Variance is simply the square of the
standard deviation F, and one usually needs to deal only in terms of the latter. F is a
characteristic of the scatter about the mean value. It should not get smaller as n increases, but,
once you have done a fairly large number of determinations, should remain roughly constant
however many more you do. Standard deviation is calculated for a set of determinations x
1
,
x
2
...x
i
...x
n
of the same quantity by:
F
2
= (x
i
- x )
2
/(n - 1)
i
n
=

1

33

The precision, or reproducibility, of a measured value, is related to the standard deviation. Once
you have found a standard deviation, by the above procedure, it can in fact be used as a measure
of precision for any one determination in the series of n. But since you have made n
determinations, you can get better precision by averaging the whole set. The precision of the
mean value is given by a quantity known as the "standard deviation of the mean" which is:
F
m
= F/n
1/2
Since F is roughly independent of n, F
m
decreases as n increases; that is, one can improve
precision in the presence of random errors simply by doing the experiment more times. But it
takes four more times to halve the error limits. These limits are quoted in different ways by
different people. Some tend to use "F
m
as the error limits. These are 68% confidence limits, i.e.
there are about 2 chances out of 3 that the true value lies within the limits stated. This isn't a
remarkably good chance, and many people quote wider limits. Roughly, "2F
m
gives 95%
confidence limits, i.e. 19 chances out of 20 that the true value lies within the limits. This 95%
confidence limit is widely used in analytical chemistry.

ACCURACY, PRECISION, ACCEPTED VALUE, TRUE VALUE One never knows the true
value of any quantity; but for many quantities there is an "accepted value", being the result
obtained in the experiment which is generally judged to have been the best performed to
determine this quantity. ACCURACY of any determination means the closeness with which the
determination matches the accepted value, or, the closeness you think it has to the true value.
This must again be expressed in terms of "19 chances out of 20 ", etc., but it includes the
effects of both random and systematic errors. PRECISION, as discussed above, relates only to
random errors.

IV. QUANTITIES CALCULATED OUT OF SEVERAL MEASURED QUANTITIES, Z =
f(A, B, C). Suppose that we know the error limits (e.g. 95% confidence) for A, B and C. What
are the error limits of Z? Strictly, it depends whether the errors are systematic or random,
according to Table 2. In practice, we often don't know precisely what the mix of random and
systematic errors (think, once again, about those volumetric flasks and their manufacture).
Hence, it is common to use the method of calculation shown in Table 3, which isn't the proper
way to do it for either kind of error, but is the best we can do for an unknown set of errors. For
random errors, )A, etc., in these tables is the quantity you are putting after ", i.e. 68% or 95%
confidence limits. For systematic errors, )A has, in principle, a definite sign, but if you know
what it is the error has ceased to be an error.


34

TABLE 2: Propagation of error for systematic errors and for random errors

Computation Systematic errors Random errors
Addition or
Subtraction
Z = A + B - C
C B A Z + =
2 2 2 2
C B A Z + + =

Multiplication
or Division
Z =AB/C

C
C
B
B
A
A
Z
Z

2 2 2 2
C
C
B
B
A
A
Z
Z
|

\
|
+
|

\
|
+
|

\
|
=
|

\
|

General
Z=(A,B,C)
C
C
B
B
A
A
Z

=
f f f

2
2
2
2
2
2
2
C
C
B
B
A
A
Z

=
|
|

\
|
|
|

\
|
|
|

\
| f f f



TABLE 3: Propagation of error for an unknown mixture of systematic and random errors

Computation Errors
Addition or Subtraction
Z = A + B - C

C B A Z + + =

Multiplication or Division
Z =AB/C
C
C
B
B
A
A
Z
Z
+

General
Z=f(A,B,C)

C
C
B
B
A
A
Z

=
f f f



V. SLOPES OF STRAIGHT LINES When a graph of y versus x should be a straight line, it is
common to calculate its slope m and intercept b in the equation y = mx + b statistically, by the
method of least squares. This is a good procedure, but it has some pitfalls which should always
be borne in mind:
1. Always draw the graph, for two reasons: (a) Plotting the least squares line on the graph is a
good check against numerical error in putting data into the calculator. The line should
clearly be a very good fit to the points. (b) The words "should be a straight line" in the first
sentence above don't really mean very much. You may find that the plotted points clearly fit
a curve, and that you shouldn't be looking for a straight line.
2. In some experiments, only part of the data fit a straight line, and there is curvature in some
other region. In this case, you make a visual choice from your graph of which points to take
into account in drawing the line. There is then no point in using anything other than a visual
procedure for drawing the best line.
3. The usual method of doing the least squares calculation assumes no error in x, and random
scatter in y. For example, if you are constructing a calibration curve of spectroscopic

35

absorbance against concentration of a coloured solute, the absorbance commonly may have
errors amounting to 1%, while the standard solutions can be made up to an accuracy of 0.1%
or better. In a plot of absorbance as y versus concentration as x, the usual assumption is
legitimate. Hence: (a) Always tabulate as y the quantity which has the larger expected
random scatter. (b) If both x and y have large scatter, recognize that a more complicated
statistical analysis is called for. This type of analysis is rarely required in the Chem 3XX
laboratories.

ERROR IN THE SLOPE, OR IN x READ OFF FOR A GIVEN y. The Skoog textbook
1
gives
formulae (in appendix 1) with which you can calculate not only the slope of a line, but also the
standard deviation (i.e. uncertainty) of that calculated slope. Treat this just like the standard
deviation of a mean: there is a 68% chance that the slope lies within " one standard deviation of
the calculated value. If you wish to use a different confidence level, scale the standard deviation
via:

Often, having calculated the line, one wants to take a particular y value (e.g. absorbance of an
unknown solution) and read the corresponding x (concentration) from the graph. Your textbook
also gives formulae to calculate the uncertainty in this x value. Note that the calculation
differs depending if your graph is of a calibration curve or a standard addition line.

VI. TESTS OF STATISTICAL SIGNIFICANCE
Significance testing is a branch of statistics that applies rigorous statistical procedures based on
probability distributions to assess the likelihood of certain hypotheses. Each test begins with a
null hypothesis and is performed to assess the probability of whether that the null hypothesis is
false. For example, if you wanted to test whether two different analytical methods give you
statistically different results, the null hypothesis is that there is no difference. You would perform
a t-test (below) to see whether there is evidence to reject the null hypothesis.

The Grubbs Test
The Grubbs test may be used if a series of replicate results has an outlier; that is, one result that
is substantially different from the others. This outlier may be due to an error in that single
measurement or due to statistical variation. If the latter, the outlier must be retained, if the
former, the outlier may be rejected. The Grubbs test determines whether the outlier is statistically
different from the others and should be rejected. As a result, this test is applied to the most
extreme value in the data set, and only one value may be rejected based on the Grubbs test. The
test cannot be used sequentially on a series of outliers. The test is:

36

Where

is the suspected outlier, is the mean of the data set including the suspected
outlier, and s is the standard deviation of the data set.
The calculated G value is then compared to the G value from a table at a selected confidence
level. If the G
calc
is smaller than the G
table
value, the data point is retained; however, if G
calc
is
greater than G
table
, the data point is considered an outlier and is therefore discarded. The mean
and standard deviations are then recalculated, excluding the now rejected outlier.

The F-test
The F-test is used solely to determine whether two variances (the square of the standard
deviations) are significantly different or whether the difference is within normal statistical
variation. In the F-test equation, the variance in the numerator is always the greater of the two
being tested (s

), so that the F-value is always greater than one. When the F-value is
calculated, it is compared to a table value at a selected confidence level. If F
calc
is greater than the
F
table
, there is reason to believe that the two variances are significantly different.

Note that result from the F-test only applies to variance of the experimental means; whether two
experimentally-derived means are significantly different is determined by the Students t test.

An application of the F-test would be when a sample is analyzed by two different methods or by
two different analysts using the same method (see case 2 below). The variances of the two results
should be compared to determine whether they differ significantly. If the two variances are not
different, then the two means can then be tested using the students t paired test (see below for
case 2: Students t paired). If the F-test shows that the two variances are different, and the two
means need to be tested using a modified Students t paired test (see below for case 2: paired
test).

The Students t-test
The Students t-test is a commonly-used statistical tool that determines whether two results are
significantly different at a certain confidence level. The t-test is used to compare results. There
are three common cases of the t-test. In all of the t-tests, a t-value is calculated. This t
calc
is then
compared to the t
table
at a desired confidence level to determine whether the two results are
significantly different.

Case 1. Comparing a Measured result with a known value
This case of t-test is utilized if an experimental result is to be tested against a known, accepted or
established value. For instance, if a new method (or new instrument or new analyst) is to be
tested, it is usually to analyze a sample that contains a known amount of analyte. Samples with
certified amount of analyte can be obtained from various sources, notably the (American)

37

National Institute of Standards and Technology, (NIST). The experimental mean ( ) then can be
compared to the known value () to determine whether the new method gave an acceptable
answer. In this case, the following equation is used to calculate the experimental t value.

In the case of an accepted value, it is assumed that the known value () is exact, so that standard
deviation (s) and number of measurements (N) are determined from the experimental data. Note
replicate measurements are required or else you wont have a value for s and N will be zero.

The t
calc
is then compared to t
table
at a desired confidence level and N-1 degrees of freedom. If the
calculated value is less than the table value, the two results are not significantly different (null
hypothesis is retained).

Case 2. Comparing Replicate Measurements.
A slightly more complex case arises if two experimental values are to be compared, for instance
if the same sample is analyzed by two different methods (or on two different instruments, etc).
The F-test must be carried out before comparing replicate measurements with the t-test because
the F-test indicates which t-test to use.

a) Case 2: Students t-paired test (two

are similar)
In this case, there are two standard deviations and two N values, and weighted averages must be
used. As shown in the following equation. The pooled standard deviation (s

) is given by the
second equation, where N is the total number of measurements combined from all methods.

Students t paired:

1. . .

1



This kind of paired test, where two analytical results are compared, arises quite often in
analytical chemistry. A classic use is when one analyst measures two different samples on one
instrument and wishes to compare the samples.



38


b) Case 2: Students t-paired test (two

are not statistically-similar)

The following equation is applied when two experimental means are to be compared, but the two
have significantly different variances (as determined from F-test).

Paired test (two

are not the same):

Where degrees of freedom =

2

For Case 2 t-tests, the t-value calculated is then compared with the table t-value at N-k degrees of
freedom (where N is the total number of measurements from all methods and k is the number of
methods used). This comparison is commonly used to determine whether two instruments or
methods or analysts give the same results, and thus determine whether two samples are identical,
for example in determining the source of an oil spill by comparing oil from a tanker with oil
from a spill.

Case 3: Paired t-test for Comparing Individual Differences
The paired t test is based on the difference (

) between a pair of results, which are obtained

from two different methods. Unlike Case 2 where the same sample is analyzed by two methods,
in Case 3, multiple different samples are analyzed by two methods (or instruments or analysts,
etc.), so that each sample is associated with two experimental results. Case 3 does not require
replicate measurements.

The difference (

) between the pair of experimental results from each sample is then used to
calculate a mean difference (

), which is utilized to calculate the t-value.

Where n is the number of samples, and

is the standard deviation based on the differences.

39

The calculated t-value is compared to the table t-value at a selected confidence level and at
degree of freedom equal to n-1.
BACKGROUND READING

1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; appendix 1.
2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010;
chapters 3 (experimental error) and 4 (statistics).


40

A-03 Determination of Lead by Anodic Stripping Voltammetry

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the method of standard addition and identify its strengths
Describe the purpose of the internal standard
Describe the processes happening at the Hg electrode during the deposition and
stripping phases of the experiment
Describe the differential pulse method and explain how it improves the signal-to-
noise ratio of the data
Recognize the safety concerns associated with using a potentiostat and in handling
nitric acid solutions and plan to use best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of lead in your unknown making appropriate use
of dilution factors, and the standard addition and internal standard approaches
Apply fundamental statistics to collected data and calculate the signal detection limit
and minimum detectable concentration
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks) through practice


BACKGROUND
Anodic stripping voltammetry is a two-step electrochemical process in which a negative
potential, measured vs. a saturated calomel reference electrode, is first applied to a hanging
mercury drop electrode (HMDE). In this step the HMDE acts as a cathode and metal analytes
from the solution plate as amalgams onto the surface of the mercury drop.
The applied potential is then scanned in a positive direction. Each metal is oxidized back into
solution at a characteristic dissolution potential. In this stripping process the mercury drop
acts as the anode. The current flowing through the HMDE is measured during the stripping
process and is the sum of the capacitive current required to charge the ionic layer adjacent to the
mercury, and the faradaic current associated with the oxidation of the analyte metal(s). A
differential pulse technique is used to subtract the capacitive component of this current. In the
differential pulse method, voltage pulses are superimposed onto the voltage ramp that is applied
to the mercury drop. The electrode current is sampled just before the pulse and again just before
the end of the pulse. The current difference representing primarily the faradaic current is
recorded vs. applied potential as a differential pulse voltammogram. The current is

41

proportional to the amount of analyte plated onto the mercury drop, and hence to its
concentration in the original solution.
An aliquot of cadmium is added to the cell at the beginning of the experiment and serves as an
internal standard to compensate for variations in plating technique. The height of the peak for
lead is compared to that for the cadmium present in constant amount throughout the experiment.
A similar comparison can be made with peak area rather than peak height.
Anomalies resulting from the emptying and filling of the cell are avoided using the standard
addition technique. A voltammogram is run of an aliquot of the unknown lead solution added to
a supporting electrolyte of dilute nitric acid. The acid maintains the pH< 2 to avoid the
adsorption of lead onto the surface of the glass cell. An aliquot of standard lead solution is then
added to the cell and a second voltammogram is run. The procedure is repeated for several
further additions of standard lead solution.

PROCEDURE
Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few
millilitres of deionized water and add the washings to the flask. Dilute to the mark with
deionized water. Mix well.

Empty the cell by aspiration without dismantling. Rinse the cell with deionized water and
empty. Rinse with supporting electrolyte and empty. Add 50 mL of supporting electrolyte to the
cell. Pipet in 1.00mL of a solution containing 1.00E-04 g/mL of cadmium.

Refer to the appropriate instructions
following, and use a PAR 174A
polarographic analyzer to record three (3)
anodic stripping voltammograms of the
blank solution containing supporting
electrolyte plus internal standard. Pipet in
0.500 mL of diluted unknown solution and
run two similar voltammograms.

Repeat for the same solution to which has
been added 0.500 mL of a standard
solution containing 1.00E-04 g/mL of Pb. Add 0.500 mL of standard solution 3 more times to
your solution (for a total of 4 additions of standard solution) and run voltammograms between
each addition. Take care to repeat each measurement twice. At the end of the experiment you
should have recorded at least 13 voltammograms: 3 (blank), 2 (unknown), and 8 (unknown +
standard). Each standard addition should be of 0.500 mL for a cumulative volume of 0.5, 1.0, 1.5
and 2.0 mL.

WARNING: Potentiostat is capable of
producing lethal current and/or voltage.
Making of electrical connections must only be
done with potentiostat OFF or with
DUMMY CELL selected.

Nitric acid solutions are corrosive and act as
strong oxidizers. Avoid skin contact. Rinse
immediately with cold water if it occurs.


42

OPERATING INSTRUCTIONS FOR THE PAR174 POLAROGRAPHIC ANALYZER
Set the following initial parameters:

Power On
Selector Off
Button Initial
Initial Potential -0.9 V
Potential Scan Rate +5 mV per sec.
Range 1.5 V
Modulation Amplitude 25 mV
Operating Mode Differential Pulse
Low Pass Filter Off
Output Offset Off
Display Direction -
Current Range 0.05 mA

Data acquisition is through custom-written program using LabVIEW. Double-click the
PAR174_echem icon on the desktop. The program has been designed to show a front panel
similar to that of the PAR174: set the virtual knobs and switches to match the settings above.
Pay particular attention to the Initial Potential and Potential Scan Rate settings as these are
used to calculate the electrode potential during the experiment.

Turn on the N
2
supply to the cell (note regulator has two knobs: one opens the cylinder, the
second allows the gas to flow into the lines). Adjust the regulator pressure to ~15 psi. Set the
electrode system to operate in the HMDE (hanging mercury drop electrode) mode and set the
drop size to "3". Leave the deaeration lever on the electrode system at "O".

Dispense two mercury drops with the "MAN" switch on the electrode system (the first drop
serves to flush the capillary). Set the stirrer speed to "2", start the timer, and allow the solution
to mix for 1 minute. Start deposition by setting the SELECTOR switch to EXTERNAL CELL
thus applying a -0.90 V potential to the mercury drop. After 100 sec. plating, turn off the stirrer
then wait 20 sec. to allow the diffusion layer near the drop to stabilize (i.e. for mixing to cease).

To record the voltammogram, simultaneously press the SCAN button on the potentiostat and
start the LabVIEW software (arrow button in top left). The scan is complete once you have
scanned to -0.30V. In LabVIEW, depress the rectangular STOP button to end data acquisition
and save your datafile. If you instead click the stopsign button, your data will not be saved. Set
the SELECTOR switch on the potentiostat to OFF and depress the INITIAL button. Change the
filename or filename number in LabVIEW in readiness for the next run.
To repeat the run it is not necessary to remove the solution from the cell. Dispense a fresh
mercury drop, make any necessary changes to the instrument or software settings, and repeat the
plating and the scan as before.

43


After the last run empty the cell by aspiration. Rinse with deionized water and refill with
supporting electrolyte. Leave the cell in this condition with a mercury drop attached to the
capillary. Determine the concentration in g/mL of lead in your original unknown solution as
received in the vial.

IN YOUR REPORT
Describe the principles and application of both the standard addition and the internal standard
methods. Consider how the use of the internal standard corrects for plating variations how
should you process your data?
Describe how the differential pulse technique isolates only the portion of the signal due to the
analyte.

BACKGROUND READING

1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 25D-E.
2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010;
chapters 17-5 and 5-2.
3. Bard, A.J.; Faulkner, L.R. Electrochemical Methods: fundamentals and applications; Wiley:
New York, 2001; chapters 6 and 7. (QD553 .B37 2001 in IKBLC)


44

A-04 Determination of Iron and Chromium by Spectrophotometry
LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the multiwavelength linear regression analysis method
Describe Beers law and its limitations
Describe the advantages and disadvantages of single-beam and double-beam
spectrophotometers
Describe the advantages and disadvantages of using photodiode array and
photomultiplier tube detectors
Recognize the safety concerns associated with the use of UV light sources and plan to
use best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of iron and chromium in your unknown
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks) through practice


BACKGROUND
Solutions containing mixtures of two non-interacting species can be determined
spectrophotometrically by multiwavelength linear regression analysis (MLRA) provided the
analytes have highly-overlapped UV-visible spectra. Absorbances are measured at several
wavelengths in the overlapping spectral region for the unknown mixture and the standard
solutions containing the individual species. By Beer's law the absorbances of the standards, A
s1

and A
s2
, are:
A
s1

=
1
bc
s1
(1)
and
A
s2

=
2
bc
s2
(2)

Where b is the cell path length, and
1
and
2
, and c
s1

and c
s2

are the molar extinction coefficients
and molar concentrations, respectively, of each species. The absorbance A
m
of the mixture is:

A
m
=
1
bc
1
+
2
bc
2
(3)

Where c
1
and c
2
are the molar concentrations of the components. Substitution for
1

and
2

from
eq'ns (1) and (2) into eq'n (3) gives:

45

A
m
/A
s1
= c
1
/c
s1
+ (c
2
/c
s2
)(A
s2
/A
s1
) (4)
Thus a plot of A
m
/A
s1
vs. A
s2
/A
s1
gives a straight line. The concentrations of species 1 and 2 are
calculated from the intercept and slope respectively.
The method is used for the simultaneous determination of iron and chromium. Acid solutions of
the unknown mixture and the individual standards have been prepared for you. Absorbances are
measured in the spectrally overlapped 240 - 370 nm region.
You will make duplicate analyses are made with a PE124 double beam spectrophotometer and a
Hewlett-Packard 8452A diode array spectrophotometer. The PE124 measures the ratio of the
intensity of light transmitted by the sample solution to that transmitted by the reference solution.
In this way it compensates for the spectral characteristics of the lamp and the detector. In this
experiment absorbances will be recorded at 10 nm (minimum) intervals.
The HP8452A spectrophotometer uses an array of 328 light detecting diodes to simultaneously
monitor light intensities at 2 nm intervals over the complete spectral range. As this is a single
beam instrument, a lamp spectrum is first measured with a blank solution in the cell
compartment. The blank spectrum is stored by computer, then the spectrum of the sample
solution is recorded and the absorbances are calculated as a function of wavelength. A complete
spectrum is recorded and plotted in less than a second.

Note: your accuracy in this experiment depends strongly on the quality of your solution
preparation.

PROCEDURE
Transfer all of your unknown solution to a 100mL volumetric flask. Note that the unknown
provided is made up in 0.1M H
2
SO
4
. Rinse the vial with a few mL of deionized water and add
the washings to the flask. Dilute to the mark with water. Mix well.

Using the iron stock solution (2.00E-04 g/mL iron in 0.1M H
2
SO
4
), prepare 100mL of 2.00E-05
g/mL Fe solution. Using the chromium stock solution (1.93E-04 g/mL in 0.1M H
2
SO
4
),
similarly prepare 100mL of 1.93E-05 g/mL Cr solution. Prepare a blank solution of 0.01M
H
2
SO
4
solution using the 0.2M H
2
SO
4
stock.

Use the Perkin Elmer 124 spectrophotometer to measure the absorbances of the standard
solutions and of the mixture. Take readings at least every 10 nm from 240 - 370 nm. Recording
more measurements should result in increased accuracy.

Turn on POWER SUPPLY. Wait 15 seconds. Turn on the D
2
lamp. Set the
VISIBLE-ULTRAVIOLET lever (left side of instrument) to ULTRAVIOLET. Also
power on the instrument itself (Operation knob on front panel).
Set wavelength anywhere between 240 nm and 370 nm.
WARNING: UV light can be harmful because it is energetic enough to break bonds and
thus harm tissue (skin, eyes, etc). The best protection is avoidance. Polycarbonate safety
glasses block most UV.


46

Switch ABS/%T to %T. Insert a 0%T shutter into the SAMP. cell holder and use
ZERO CHECK (right side of instrument) to zero the meter.
NOTE: HANDLE THE CELLS CAREFULLY they cost $500(!) per pair because
they are spectrally-matched. The solutions contain sulfuric acid. Take care to avoid
spills when filling the cells and inserting them into the cell compartment of the
spectrophotometer.
Two-thirds fill two cells with blank solution and insert into the SAMP. and REF. cell
holders. When inserting the cell, always keep the same orientation and try to position
the cell reproducibly.
Orient the clear windows towards the right and left. Set ABS/%T to ABS. Set
absorbance SCALE to 0-1. Zero the meter with the front panel ZERO knob. Do not
change this setting for the rest of the experiment. Measure the absorbances of the
blank solution at 10 nm intervals between 240 and 370 nm.
Replace the blank solution in the sample cell with iron standard, chromium standard,
or unknown mixture, and measure the absorbance every 10 nm between 240 and 370
nm. Calculate the concentrations of iron and chromium in your original unknown
solution as contained in the vial. Repeat the experiment using a Hewlett Packard
8452A diode array spectrophotometer to measure the absorbances of the standard
solutions and of the unknown mixture.

HP 8452A Instrument control and acquisition of data:

Make sure there are no cuvettes in the cell holders, then turn on the spectrophotometer power
(switch at rear of instrument). The spectrophotometer must be powered on before initializing the
computer software.

Wait until the LAMP indicator on the instrument confirms the lamp has been ignited,
do not turn the deuterium lamp on and off unnecessarily! This shortens the life of the
lamp (~$1000)!

Allow 15 min. for the lamp to warm-up and its intensity to stabilize before making
measurements.

Double click the Olis GlobalWorks icon to load the software, then select HP Diode
Array under the Data collection tab, click open.

Check that these parameters are set:
Under Live Display tab, set wavelengths to Scan from 240 nm to 410 nm
Under Repeated Scans tab, set number of scans to 1
Rinse and fill the cuvette (2/3 full) with blank solution. When inserting the cell,
always keep the same orientation and try to position the cell reproducibly. Click

47

Collect Ref. The background will be subtracted automatically from all subsequent
spectra.

Rinse and fill the cell (2/3 full) with sample solution. Insert the cuvette into the cell
compartment using the same orientation as for the blank. Click Collect Data.


Click yes to transfer data into GlobalWorks. To change the file
names, double click the Name option in the lower right window as
shown here at right:

If graph displays a 3D view of the data: from the tool-bar menu,
select View and then 2D view (Double click graphs to expand).
Run similar scans for the remaining standard solution and the
unknown. To collect data for the next sample, click the HP Diode
Array on the right hand side of the screen and then collect data.
The graphs for subsequent runs will be displayed under the
Experiment window as shown below:


To overlay sample spectra, open one graph from under the experiment window, click the graph
area so that the graph turns yellow. Then, select Edit from the menu bar and then Copy slice.
Open the second graph, click graph area, select Edit and Paste slice. Select View and then 2D
View. The overlay graph will be created as a new file.

Double click on overlay graph area to expand graph, select File, Print to print overlay spectra.

To save overlay graph and data in GlobalWorks, click the overlay graph, File and Save dataset
as into the folder CHEM 3XX A4 DATA located on the desktop. Save the file in the format:
StudentNames_Sample name. NOTE: only the overlay data and graph will be saved!

Right click the overlay graph, select Export data to Excel. The first column displays the
wavelengths and the column to the right display absorbance values (all overlay data are
exported). Name the Excel file with student name and sample identification.

Processing data with Microsoft Excel:
Note: You are assumed to be familiar with the use of Microsoft Excel for data analysis and
plotting graphs. Helpful tutorials can be found on the internet if you need a refresher.


48

The spreadsheet will display columns of data labelled 0, 1, 2, and 3. The leftmost should
be readily identifiable as your wavelength in nm. The other three represent absorbance of your
samples. The identity of each is determined by the order in which you copied/pasted the data
before exporting it to Excel. If you have ANY doubt about which is which, you should
export the spectra to Excel individually. In this case, you should first label the absorbance
columns in the three files, and then combine the three spectra into one spreadsheet.

Choose which absorbances will be A
s1
and which will be A
s2
and set up a column in which
A
s2
/A
s1
is calculated for each wavelength. Set up another column in which A
m
/A
s1
is calculated.
Use the Excel plotting functions to plot graphs of the spectral overlay and A
m
/A
s1
vs. A
s2
/A
s1
in
the 240 nm to 370 nm spectral region. Use the linear regression function to calculate the slope,
intercept, and the associated 95% C.L. values.

Calculate the concentrations of iron and chromium in your original unknown solution as
contained in the vial. Note a common mistake is to mix up which species you gave the
designation 1 and which 2; this reverses the results for Fe and Cr.

IN YOUR REPORT
Compare the results obtained with the two instruments. In comparing your results, make sure to
use the appropriate statistical tests to determine whether the results agree. Discuss the
advantages and disadvantages of each instrument. Identify one result for the iron analysis and
one for the chromium analysis to be marked. This could be the result from the HP instrument,
or from the PE instrument, or an average of the two. Rationalize your choice.

BACKGROUND READING

1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapters 13 and 14.
2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010;
chapters 17, 18 and 19.
3. Blanco, M.; Iturriaga, H.; Maspoch, S.; Tarin, P. J. Chem. Ed. 1989, 66(2), 178.
4. Perkampus, H.-H. UV-Vis spectroscopy and its applications; Springer-Verlag: New York,
1992. (QD96.U4P4713 1992 in IKBLC)


49

A-06 Determination of Quinine by Fluorometry and Absorbance Spectrometry
LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the processes of absorbance and fluorescence
Describe how absorbance and fluorescence intensity vary as a function of analyte
concentration
Describe the advantages and disadvantages of absorbance and fluorescence
measurements
Describe the differences and similarities between an absorbance spectrum, excitation
spectrum and emission spectrum
Recognize the safety concerns associated with using UV light sources and plan to use
best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of quinine in your unknown using the
calibration curve method and applying appropriate dilution factors
Describe the purpose of the various pieces of the modular fluorimeter and explain
their particular placement
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks) through practice

BACKGROUND
Quinine is an alkaloid that occurs naturally in the bark of the chinchona tree. It was widely-used
medicinally as an antimalarial agent from about 1800 to around 1940 when more effective
treatments were discovered. It was commonly administered as a solution in water, with sugar
added to mask the bitterness of the quinine. This solution became known as tonic water (a
tonic is a liquid medicine). In Canada, tonic water contains quinine as a flavouring agent with
a maximum permissible concentration of 83 ppm quinine. Owing to its cyclic aromatic moiety,
quinine is strongly fluorescent in acid solution. In this experiment quinine is analyzed both in an
unknown solution and in a sample of tonic water by absorbance spectrometry and fluorometry.
Absorbance spectrometry: Transmittance of light through a solution is defined as I/I
0
, where I
0
is the initial intensity of the light, and I is the intensity after it has passed through the solution.
The absorbance A of the solution is given by:

|
|

\
|
= =
0
I
I
log T log A
(1)

50

In practice, I
0
is usually redefined as the intensity of light passed through an optical cell
containing blank solution. Eqn (1) then gives the absorbance of an analyte without further
correction for the absorbances of the solvent or the cell. The Beer-Lambert law,
bc A = (2)
where is the molar extinction coefficient, b is the cell path length, and c is the molar
concentration of the analyte may then be applied. An unknown concentration can therefore be
interpolated from a calibration plot of absorbance vs. concentration for a series of standard
solutions.

Fluorometry: Molecular fluorescence is a process by which molecules first absorb
electromagnetic radiation, then re-emit the absorbed energy as light. The emitted light is of
longer wavelength than the incident light, and each photon emitted goes in a random direction.
The intensity I
em
of the emitted light is given by:

( )
bc
0 em
10 1 P K I

= (3)

where K is a function of the quantum efficiency of the emission process. Equation (3) can be
expanded as the series:

( ) ( )
(

=
! 3
bc 303 . 2
! 2
bc 303 . 2
bc 303 . 2 P K I
3 2
0 em
(4)

At low concentrations eqn (4) is linearly-approximated as:


Kc bc 303 . 2 P K I
0 em
= =
(5)

Therefore the concentration of an analyte can be interpolated from a calibration plot of I
em
vs. c
for a series of standard solutions.
I
em
is usually measured at right angles to the direction of the excitation beam to minimize
detection of any scattered light from the source. Also, the light from the source is passed
through a bandpass filter or monochromator that transmits shorter wavelengths suitable for
excitation, while eliminating longer wavelengths that may be scattered and detected with the
emitted light.
Fluorometry is in principle more sensitive than absorptiometry, since the measured signal
approaches zero as the concentration of the emitting species approaches zero. At low
concentrations the emission signal can be increased by increasing the intensity of the source or
by using a more sensitive detector; for instance, a photomultiplier tube instead of a CCD array.



51

On the other hand, when measuring low concentrations by transmittance or absorbance, the
measured signal approaches a maximum as I approaches I
0
(eqn 1) and T approaches 1. It can be
shown
1
that the relative standard deviation in concentration, s
c
/c, is related to the absolute
standard deviation of transmittance, s
T
, by:


T log T
s 434 . 0
c
s
T c
=
(6)

Therefore, as T Y1, s
c
/c Y 4 . To improve the sensitivity of analysis by absorbance it is
necessary to reduce s
T
, which is a function of instrument noise, signal drift, etc.
Absorbance and fluorescence spectra: A plot of absorbance vs. wavelength is a function of the
ratio of I to I
0
. Therefore, although I and I
0
are functions of instrument parameters such as lamp
intensity and detector response, both of which vary with wavelength, the effects of the
instrument parameters cancel in the process of calculating the ratio. The resulting absorbance
spectrum is characteristic only of the absorbing species and is independent of instrument
parameters.
A fluorescence excitation spectrum is a plot of the intensity of emitted light I
em
vs. the
wavelength of the excitation light, 8
ex
. This spectrum is similar to an absorbance spectrum, and
in fact an absorbance spectrum can be used instead of an excitation spectrum to determine
wavelengths suitable for excitation.
A fluorescence emission spectrum is a plot of the intensity of emitted light I
em
vs. emission
wavelength 8
em
. Unlike plots of absorbance vs. wavelength, measurements of emission intensity
as a function of 8
em
give spectra which are characteristic not only of the analyte but also of
wavelength-dependent instrument parameters. If the detector gain is higher at a given
wavelength, the measured signal will be higher. Corrected spectra can only be constructed by
referencing the measured plots against a known emission spectrum such as the light emitted from
a black body source.
Of the two fluorimeters you will use in this experiment, one can automatically correct the spectra
using parameters determined at the factory, while the other requires manual calibration. This
manual calibration is against light from a reference source: a halogen lamp with a colour
temperature of 3500K. The light from the lamp is measured and the intensity I
W,8,meas
is stored as
a function of . The stored data is then compared with a data file of the actual spectrum of the
lamp,
, W
I
vs. , and at each wavelength a correction factor B
8

is calculated, where:

=
, W
meas , , W
I
I
B (7)
A corrected emission spectrum of the analyte, (I
em
/B
8
) vs. 8
em

is then plotted. This plot is
characteristic of the analyte, and is independent of instrument parameters.

Instrumentation: This experiment compares both absorbance spectrophotometry with
fluorescence spectrophotometry and inexpensive instrumentation with top-of-the-line
instrumentation. You will use an Ocean Optics spectrometer capable of both absorbance and
fluorescence measurements as well as the Agilent Cary Ecilpse Fluorimeter and the Agilent Cary

52

5000 UV-Vis spectrophotometer. These last two instruments are located in the departmental
shared instrument facility (SIF), room B353.
The Ocean Optics spectrometer is a good example of the current trend towards miniaturization.
Although the instrument is tiny about the size of a pack of cards, it incorporates a grating
monochromator and a linear CCD array detector. The working wavelength range of the
instrument is roughly from 250 to 910 nm and gives resolution of about 1.3 nm. The modular
design of the instrument allows you to insert different gratings that can improve the resolution
but with a loss of wavelength range. This modular design also means it can be configured to
measure absorbance or fluorescence. Light from either a deuterium/tungsten or pulsed xenon
source is monitored. An optical fiber transmits light from the source to the cell and on to the
spectrometer. For absorbance measurements the monitored light is that transmitted through the
cell, while for fluorescence measurements the light detected is that emitted at right angle to the
direction of the incident beam.
By contrast, the Cary 5000 is a double-beam spectrophotometer with two monochromators and
two detectors: a photomultiplier tube for UV-Vis detection and a lead sulfide photocell for NIR
detection. It offers vastly higher resolution (0.05 nm) and a much larger working range:
absorbance values up to 8 are measurable. The instrument covers the 190 3300 nm wavelength
range; from deep UV to near infrared. The Cary 5000 also costs about 30x more than the Ocean
Optics instrument!
The Cary Eclipse Fluorimeter is similarly top-of-the-line. It offers a broader working range,
higher sensitivity, better signal-to-noise ratio, and more user customizable parameters than the
Ocean Optics instrument. The Eclipse costs roughly 10x more.

PROCEDURE
(Before preparing your solutions, proceed to the SIF in room B354 and power on the Ocean
Optics UV source (keep shutter closed) and power on the Cary 5000 UV-Vis and Cary Eclipse
instruments. Return to C224 to make your solutions while the instruments warm-up.

Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few
millilitres of water and add the washings to the flask. Dilute to the mark with water. Mix well.
Suspend a flask containing 20 - 30 mL tonic water in an ultrasonic bath for about two to three
minutes to remove the carbonation. Dilute your degassed tonic water 25-fold using volumetric
glassware.
Using 1.00E-05 g/mL quinine stock solution prepare at least five (preferably six) calibration
standard solutions. Start with zero concentration (blank) and do not exceed 1.25E-06 g/mL
quinine for the most concentrated solution. Prepare further five-fold dilutions of your tonic
water solution and your unknown. All your final solutions (calibration, tonic water,
unknown) should be in 0.04M sulfuric acid. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume
and 50mL volumetric flasks are provided. Ensure each solution is well mixed. You should
arrive in the laboratory with a plan of how to prepare all of these solutions!



53



Bring your samples to the SIF and record the UV-Vis absorbance spectrum of your most
concentrated stock solution and of your diluted tonic water sample. Print these two spectra.
Determine the wavelength of maximum absorption for your stock solution, and then record the
absorbance at this wavelength for each of your solutions. APPENDIX I gives operating
instructions for the Cary 5000 instrument.

Return to the analytical lab to proceed with fluorescence measurements on the Ocean Optics
instrument. With the instrument configured as in Fig. 1 the optical fiber monitors light
transmitted through the sample so that transmittance and absorbance spectra can be recorded.
Refer to APPENDIX II sections B and C and measure the transmittance of the OF2U330
bandpass filter that will be used in making fluorescence measurements. Print the spectrum you
will need to comment on it in your report.
Turn off the deuterium lamp and reconfigure the spectrometer as in Fig. 2a. Refer to
APPENDIX II section D(i) to measure a reference emission spectrum of the light from a black
body source (halogen lamp). The spectrometer monitors light from the source reflected at 90 by
a glass plate.
Reconfigure the spectrometer as in Fig. 2b, then refer to APPENDIX II section D(ii) to record
corrected emission spectra of the standard solutions, the diluted unknown, and the diluted tonic
water. Print uncorrected and corrected emission spectra of the most concentrated stock solution,
and the corrected emission spectrum of the tonic water. Do not print spectra for the other
solutions. Use the cursor function to identify the wavelength of the emission maximum for
quinine and record the emission intensities of the standards and unknown solutions at this
wavelength.
Return to the SIF to make similar fluorescence measurements on the Cary Eclipse instrument.
Use the wavelength of maximum absorption that you determined previously as your excitation
wavelength. Record and print corrected fluorescence emission spectra for the most concentrated
stock solution and for the diluted tonic water sample. Record the intensity of fluorescence
emission for all samples. APPENDIX III gives operating instructions for the Cary Eclipse.
Calculate the concentrations of quinine in your original unknown solution by absorbance and
fluorescence. Decide which of your two fluorescence determinations is likely more accurate.
Comment on the reasons for your decision in your report. Comment on the meaning of your
measured concentration of quinine in tonic water.
On the coverpage of your report, include the analytical result determined by absorbance
and by fluorescence. Report only one of your two fluorescence results.
WARNING: UV light can be harmful because it is energetic enough to break bonds and
thus harm tissue (skin, eyes, etc.). The best protection is avoidance. Special UV-blocking
goggles must be worn when connecting or disconnecting fiber optic cables if the lamp is
ON.


54



IN YOUR REPORT
Explain the phenomena of absorbance and fluorescence.
Discuss the transmission spectrum of the U-330 filter and its importance in your
experiment.
Discuss the difference between the corrected and uncorrected emission spectra of
quinine.
Compare the absorbance and emission spectra of the tonic water with those of the
quinine standards.
Comment on the effect, if any, that the solution matrix may have on the analysis of
quinine in tonic water by absorbance and fluorescence.
Compare the analysis of quinine by these two methods. In comparing your results,
make sure to use the appropriate statistical tests to determine whether the results
agree.

BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 15.
2. Harris, D.A. Quantitative Chemical Analysis, 8
th
ed.; W.H. Freeman: New York, 2010;
chapters 18-5 and 18-6.
3. Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3
rd
ed.; Springer: New York, 2006.
(Available online through UBC library ebrary service)


55

APPENDIX I: Operating Instructions for the Agilent Cary 5000 UV-Vis Spectrometer
Ensure the instrument is powered on (switch on front). Open the instrument software from the
computer desktop: Open the Cary WinUV folder, then double-click the Scan icon. Choose
Setup. In the dialog box, set the following parameters:



Under the Options tab, enter: Under the Baseline tab, select:
SBW (spectral bandwidth): 5 nm Baseline Correction
Beam: Single front
Energy: 1
Slit height: full
UV/Vis source changeover: 350 nm
Then click OK

Fill a quartz cuvette two-thirds full with your blank solution and insert the cuvette into the front
position of the spectrometer, ensuring the clear windows are oriented left-right. Zero the
instrument (button on left of screen), then click Baseline to acquire a baseline. The system will

56

prompt you to insert a blank sample. Click OK to measure the baseline. Once complete,
remove the cuvette and two-thirds fill it with your most concentrated stock solution. Click
Start to begin your measurements. Note that the graph may show this absorbance as being
higher or lower than that of the baseline; this is fine as the instrument has set the reference point
to zero before measuring your sample. Data is saved automatically; use the standard Windows
print functions to print your spectrum. Repeat with your diluted tonic water solution. Close the
software after recording your spectra.
Again in the Cary WinUV folder, double-click the Concentration icon. This will launch a
program that will record absorbance at a specified wavelength, build a calibration curve and
provide (partial) results. Similar to before, click Setup and enter the parameters for your
measurements. Under the Cary tab, enter the wavelength of maximum absorption that you
determined from the scan of your standard solution. The number of replicates should be one, the
bandwidth (SBW) 5 nm, and the averaging time 1s. Other parameters should be as you set
previously. In the Standards tab, make settings similar to the ones below but provide your
own concentration values for each standard solution and adjust the units to suit. Dont forget to
include your blank solution as a standard!



In the Samples tab, enter two as the number of samples and fill in the table with names
identifying your two samples (diluted unknown and diluted tonic water solutions). Click OK.
Under Options, set the beam mode to single beam (front).

57

Click the Start button to begin the measurements. The software will prompt you to insert each
sample sequentially, will build your calibration curve and will directly output the concentration
in your diluted unknown and your diluted tonic water sample. If any of the datapoints on your
calibration curve are problematic, you can remeasure the datapoint (button at bottom-left of
screen). If it is still problematic, manual treatment of your data will be required. Print the report
the software provides you.



58

PULSED
XENON
SOURCE

DH2000
D2
SOURCE
USB2000
SPECTROMETER
TO USB PORT
OF COMPUTER
0.25m
450
MICRON
FIBER
0.25m
450
MICRON
FIBER
1 meter
450
MICRON
ATTENUATOR
LENS
LENS
CELL
APPENDIX II: Operating Instructions for the Ocean Optics USB2000 Spectrometer

A. Configuring the USB2000
(i) Configuring the spectrometer to measure
transmittance and absorbance:
The spectrometer monitors light transmitted through
the source fiber and the cell to the detector fiber.
The DH2000 deuterium source is used for
measurements in the UV spectral region. The
attenuator reduces the intensity of the transmitted
light to within the dynamic range of the instrument.
The halogen lamp of the DH2000, and the pulsed
xenon source are turned off while measuring
transmittance or absorbance.

Fig. 1: Configuration for measuring
transmittance and absorbance


(ii) Configuring the spectrometer to measure a reference spectrum of the emission from a
black body source (tungsten lamp):

Light from the DH2000 halogen source is transmitted via
450 micron fibers (red colour-code) and is reflected at
right angles by a 45 glass plate inserted into the cell
compartment. (The glass plate reflects about 4% of the
incident beam towards the spectrometer.) The
spectrometer monitors the reference light via a 600
micron fiber (brown colour-code).

The deuterium lamp of the DH2000, and the pulsed
xenon source, are turned off while the reference
spectrum is recorded.


Fig. 2a: Configuration for measuring an emission reference spectrum



ATTENUATOR
USB2000
SPECTROMETER
TO USB PORT
600
MICRON
0.25m 450
MICRON FIBER
DH2000
HALOGEN
SOURCE
LENS
PULSED
XENON
SOURCE
LENS
45 GLASS
PLATE
MIRROR
PLUG
1 meter
450
MICRON

59


(iii) Configuring the spectrometer to measure a fluorescence emission spectrum:
The spectrometer controls the triggering of the high intensity
xenon source to produce excitation pulses coincident in time
with the detection of fluorescence.

The light emitted from the sample is measured at right angles
to the direction of propagation of the incident beam. A
relatively large 600 micron fiber (brown colour-code)
increases the sensitivity of detection of the low-intensity
emitted light. The mirror plugs further increase sensitivity by
reflecting light back through the cell. The bandpass filter
minimizes detection of scattered light from the source.




Fig. 2b: Configuration for measuring fluorescence emission

B. Instrument control: OOIBase32 program
Click the OOIBase32 program. The following window will be displayed.
USB2000
SPECTROMETER
TO USB PORT
OF COMPUTER
600
MICRON
FIBER
DT1000
UV/VIS
SOURCE
MIRROR
PLUGS
PULSED
XENON
SOURCE
LENS
CELL
BANDPASS
FILTER
TRIGGER
PULSE

60


You will use the following buttons shown in the Spectrum 1 window:

Scope mode displays intensity (I
8
) vs. wavelength () without processing.

Transmittance mode calculates and displays T (eqn 8) vs. .
Absorbance mode calculates and displays A (eqn 9) vs. .
Irradiance mode calculates and displays I
em,corr
(eqn 11) vs. .
Store Reference used in the Scope mode to store I
0
for the Absorbance and
Transmittance modes, and I
em,ref

for the Irradiance (fluorescence) mode.
Store Dark Spectrum used in the Scope mode to store D

for the Absorbance,

Transmittance, and Irradiance (fluorescence) modes.
Autoscale the graph.
Manually scale the graph.
Unscale the graph.

C. Measuring Transmittance of the U330 Bandpass Filter
Wear the special UV-blocking safety goggles (in locker drawer) at all times when working with
the UV light source. Never look into the exit port of a lamp source or into the end of an optical
fiber while the source is turned on!

61

Configure the spectrometer as in Fig. 1 (APPENDIX section A). Set the Open-Close-
TTL switch at Close. Turn on the deuterium source of the DH2000 lamp supply.

Set Integ. Time to 30 msec, Average to 10 (averages 10 sets of readings), and
Boxcar to 5 (averages 125 pixels on either side of the selected wavelength).

Click

(Scope mode). Set the Open-Close-TTL switch on the lamp to Open.
Adjust the attenuator disk carefully to set a maximum signal of ~3500 counts in the 300-
400nm region. Allow the lamp to warm up for at least 10 minutes.

With an empty cell compartment (no cell), click to measure and store a reference
spectrum of I
0
vs. .
Insert a black or gray block into the cell compartment. Click to measure/store a dark
spectrum (D

vs. ). Remove the block.

Click to switch to the transmittance mode.

Insert the U330 bandpass filter into the cell compartment. Click and rescale the x-axis
to 200-800nm. Print the spectrum.
D. Measuring Fluorescence
(i) calibrating the instrument against a black body source:
Turn off the deuterium source. Configure the spectrometer as in APPENDIX section A
Fig. 2(a). Insert the 45 glass reflector into the cell compartment.

Click . Set Integ. Time to 500 msec., Average to 1, and Boxcar to 4.

Turn on the Halogen source of the DH2000 lamp supply. The beam from the source is
reflected 90 by the glass plate and the attenuated light is monitored by the spectrometer via the
600 micron fiber. Install the cell compartment cover to prevent detection of ambient light.
Carefully adjust the attenuator to reset the maximum intensity to ~3500 counts.

Switch Average to 4.

Click to measure and store a reference spectrum of
meas , , W
I

vs. .

Insert a black or gray block into the cell compartment. Wait until the baseline is flat, then
click to store a dark spectrum.

62


Turn off the DH2000 halogen source.

(ii) Measuring corrected fluorescence emission:
Configure the spectrometer with two mirror plugs installed as in Fig. 2(b). Insert the U330
bandpass filter into the filter slot. Click Shutter Open () and Strobe/Lamp Enable () to
turn on the PX-2 pulsed xenon source. (If you dont hear the pulsed source when it is turned on,
check the on/off switch on the back of the PX-2).

Half-fill the cell with your most concentrated standard solution of quinine and insert it into
the cell compartment. The fluorescence emission is visible from above the cell. Click . An
uncorrected emission spectrum will be displayed. Install the cell compartment cover. Click
to re-scale the y-axis. Print the uncorrected spectrum.

Select Spectrum, Ref. Color Temp., and set the color temperature of the reference
spectrum to 3500K. Click OK. Then click

to display the corrected emission spectrum.
Click to re-scale the y-axis. Print the corrected spectrum. Use the cursor function to
select the peak wavelength and to read the intensity of the emitted light.
Similarly measure the corrected fluorescence emissions of your remaining standard solutions,
your diluted unknown solution, and the tonic water. You do not need to print copies of the
spectra; just note the emission intensity for each solution.

The spectrometer outputs spectral data to a computer for processing. The computer processes
the data in one of four ways, depending upon the experiment:

1) Scope mode Intensity (I
8
) vs. wavelength () is displayed without any processing. The
displayed plot is characteristic of both the sample and the spectral response of the instrument.
This raw data is processed by the computer in the Transmittance, Absorbance, and Irradiance
(fluorescence) modes.

2) Transmittance mode The computer calculates and plots vs. :

% 100
D I
D I
T %
, 0

(from eqn 1) (8)

where I
0,8
is the intensity of light transmitted through the blank, I
8

is the intensity of light
transmitted through the sample, and D
8

is the dark current measured with a black block inserted
in the cell holder.


63

3) Absorbance mode The computer calculates and plots vs. :

|
|

\
|

D I
D I
log A
, 0
10
(from eqn 1) (9)
4) Irradiance (fluorescence) mode The emission from a tungsten lamp is first measured
and the software calculates and stores as a function of :

=
, W
meas , , W
I
D I
B (from eqn 7) (10)

The sample emission is then measured, and the computer calculates and plots vs. :

=
B
D I
I
meas , em
corr , em
(11)



APPENDIX III: Operating Instructions for the Agilent Cary Eclipse Fluorimeter
Ensure the instrument is powered on (switch on front). Open the instrument software from the
computer desktop: Open the Cary Eclipse folder, then double-click the Scan icon. Choose
Setup and enter the following parameters:
Data mode: Fluorescence
Scan setup: Emission
X mode: Wavelength, nm
Excitation: enter your wavelength of maximum absorption in nm
Start wavelength: 300 nm Slit width: 5 nm
Stop wavelength: 750 nm Slit width: 5 nm
Scan control: Medium.
Under the Options tab, ensure the PMT detector voltage is Low
Two-thirds fill your cuvette with diluted unknown solution and insert it in the instrument. Click
the Start button to begin the measurement. Data is saved automatically; use the standard
Windows print functions to print your spectrum. Close the software after recording your spectra.
Again in the Cary Eclipse folder, double-click the Concentration icon. Like on the Cary
5000 UV-Vis instrument, this will launch a program that will record absorbance at a specified
wavelength, build a calibration curve and provide (partial) results. Under the Cary tab, enter
your excitation wavelength as before and enter the wavelength of maximum emission from your
fluorescence scan of the diluted unknown solution. Ensure the PMT detector voltage is set to
low in the Options tab.

64

Use the Standards and Samplestabs to provide the instrument information about your
solutions as before. Click the Start button to begin the measurements. The software will
prompt you to insert each sample sequentially, will build your calibration curve and will directly
output the concentration in your diluted unknown and your diluted tonic water sample. If any of
the datapoints on your calibration curve are problematic, you can remeasure the datapoint. If it is
still problematic, manual treatment of your data will be required. Print the report the software
provides you.















A-0007 FOR BLMSC STUDENTS ONLY
A-0007 Determination of Glutamine by Capillary Electrophoresis

Learning Objectives
1. Explain the basis of analyte separation in capillary zone electrophoresis
2. Be able to predict the relative migration times of different analytes; similarly, predict how
the degree of ionization of a weak acid will affect its migration velocity.
3. Qualitatively use the Van Deemter Equation to compare the theoretical resolution of CE
to other chromatographic methods.
4. Describe the principal components of the CE instrument and outline potential
instrumental sources of error.
5. Explain the reasons for using an internal standard; describe the ideal attributes of an
appropriate internal standard.
6. Determine the mass of glutamine in your unknown using the calibration curve and
internal standard methods.
7. Apply fundamental statistics to collected data and calculate the confidence interval of
your result.

BACKGROUND
Capillary electrophoresis (CE) provides excellent resolution in the separation of biological
molecules. This experiment utilizes a basic mode of CE capillary zone electrophoresis in the
separation of a mixture of amino acids.
In the presence of an electric field, separation is achieved based on the different electrophoretic
mobilities of the analytes. The electrophoretic mobility is proportional to the charge-to-size ratio
of the analyte. The product of the applied electric field and the analytes electrophoretic mobility
equals the analytes electrophoretic velocity :
cp
. Positively-charged analytes have :
cp
in the
direction of the electric field towards the cathode while negatively-charged analytes have :
cp

against the direction of the electric field towards the anode; a higher charge-to-size ratio increases
the magnitude of :
cp
.
The applied electric field is also responsible for a common electroosmotic flow (EOF) towards the
cathode. This is caused by the movement of buffer cations which are attracted to the negative
cathode and their associated solvation shells that drag along solvent molecules. The flat flow
profile of EOF contrasts with the laminar profile of pressure-driven flow as shown in Figure 1.
The flat profile results from the buffer cations moving near the negatively-charged capillary
surface. The negative charge is due to the ionization of surface silanol groups on the inside wall of
the silica capillary; this ionization is forced by use of basic pH buffers.

Figure 1: Profiles of flat flow (a) and laminar flow (b)

The overall velocity of an analyte is the sum of its unique electrophoretic velocity and the common
EOF velocity. Because the EOF velocity is much larger than the electrophoretic velocity, all
compounds even anions migrate towards the cathode.
:
ocuII
= :
L0P
+ :
cp
(1)
Both the EOF velocity and the electrophoretic velocity are dependent on the electric field. A large
positive electric field will increase EOF velocity while increasing the separation between
positively and negatively charged analytes as the former will acquire a positive electrophoretic
velocity on top of the EOF velocity and the latter will acquire a negative electrophoretic velocity
which opposes the EOF velocity. The overall velocity can now be related to the electric field (E),
the EOF mobility (
L0P
), and the electrophoretic mobility (
cp
).
:
ocuII
= E(
L0P
+
cp
) (2)

The pH of the buffer affects the ionization of the analyte, which affects its effective electrophoretic
mobility
cp
. For instance, glutamine in its zwitterion form (abbreviated as HA) has zero net
charge and thus its electrophoretic mobility is zero, while glutamine in its anionic form (A
-
) has a
net negative charge and has an electrophoretic mobility
on
. Both forms of glutamine exist to a
significant extent when the pH is within 2 units of the pKa of the ammonium group (one can
confirm through the Henderson-Hasselbalch equation that at 2 pH units above the pKa, the ratio of
anionic glutamine to zwitterionic glutamine is 100:1).



Figure 2: Second ionization of glutamine occurs at the amino group

Since only the anionic form contributes to the analytes effective electrophoretic mobility, then

cp
must be equal to the fraction of the anionic form of analyte multiplied by the anions
electrophoretic mobility
on
.

cp
=
on
_
|A
-
]
|EA] + |A
-
]
_ (S)





Procedure
NOTE: THE PROCEDURES MUST BE FOLLOWED CLOSELY IN ORDER TO COMPLETE
THE EXPERIMENT IN THE ALLOTTED TIME.

In the SIF (room B354), begin setting up the CE instrumentation (Part A of the Appendix). The
table below shows the contents of each vial that will be in the vial tray.

Vial Solution
1 1M NaOH
2 Methanol
3 Deionized water
4 20mM pH 9.30 borate buffer
5 20mM pH 9.30 borate buffer
6 Deionized water
7 Solution 1 (blank)
8 Solution 2
9 Solution 3
10 Solution 4
11 Solution 5
12 Solution 6
13 Solution 7 (unknown)
20 Waste (empty vial)
Table 1: CE Vial Contents

Use separate pipettes to fill up the vials containing sodium hydroxide, deionized water, and
buffer. Make sure vial 20 (waste vial) is empty. Then, load these vials (vials 1, 3, 4, 5, 6, 20) into
the vial tray. Set up the instrument method described in Part B of the Appendix. Note that the
method CHEM3XX_CE.M should have all the correct parameters in instrument method, so
you only need to make sure it has not been altered. Use the Single Sample method described in
Part C of the Appendix to run the deionized water sample. The data from this run is not
analytically useful, but the run serves to ensure the instrument is flushed and prepared for your
blank run (solution 1). The deionized water run will take approximately 15 minutes. During this
time you will prepare your standards and unknown.

Prepare 0.00375 g/mL asparagine and 0.0001 g/mL caffeine in the same 100 mL volumetric
flask. Prepare 0.0075 g/mL glutamine solution in a separate 100 mL volumetric flask to the
highest level of accuracy. The best way to accomplish this is to weigh by difference the solid
glutamine and dissolve it in ~75 mL deionized water in a beaker. Use the sonicator to ensure
complete dissolution and quantitatively transfer the solution to the volumetric flask before
making to the mark.

Prepare the seven solutions indicated below in 25 mL volumetric flasks. Dilute to 25 mL with
deionized water. For the unknown solution (solution 7), it is recommended to dissolve the
unknown in ~15 mL deionized water to allow room for the 5 mL of asparagine/caffeine solution.



Solution asparagine/caffeine
standard (mL)
glutamine
standard (mL)
unknown (g)
1 (blank) 5 0 0
2 5 1 0
3 5 2 0
4 5 3 0
5 5 4 0
6 5 5 0
7 5 0 x
Table 2: Preparation of standards and unknown

Bring Solutions 1 to 7 to the SIF. Use a 1 mL syringe to inject Solution 1 through a 0.45 m
Durapore membrane filter into a capillary vial (Vial 7). Ideally, the vial should be about 7/8 full
(around 0.6 mL). Load Vial 7 into the vial tray. In Sample Info (see Part C of Appendix), change
the Vial Location to Vial 7 and rename the sample. Use Single Sample to run your first standard.

While the sample is running, finish injecting your other samples into their respective capillary
vials (see Table 1) using the same injection technique described for Solution 1. That is, you will
inject Solution 2 through the filter into Vial 8, and so on. To avoid cross-contamination, when
injecting a new sample, flush the filter unit at least twice with your new sample before filling the
vial: simply inject 1 mL of the new solution through the filter into a waste beaker. As soon as a
Single Sample run finishes, load the next sample vial into the vial tray, adjust the parameters in
Sample Info, and run the sample.

Once all the standards have been measured and you are in the process of running the unknown
sample, check the electropherograms for consistency and confirm that you can plot a linear
calibration curve from the data.

Print out the overlay of the integration results for the standard solutions and unknown. See Part
D of the Appendix.


Calculations
Determine the mass of the unknown glutamine in milligrams and report it on the first page of
your report. Include the uncertainty and confidence level when reporting your result.

In Your Report
A full formal report is required for this experiment. Your report should include the following, but
is not limited to:

A description of the method of separation in capillary electrophoresis: use the learning
objectives as a guideline for what you should discuss
An explanation of why the internal standard is important to the method and how the internal
standard was used in treating the data

Include answers to the following questions in your report:

1. Explain the relative migration rates of the compounds used in this experiment. What would
the relative migration rates be if pH 6.2 phosphate buffer is used?
2. Outline the appropriate conditions to determine the first ionization constant of glutamine
(pKa of the carboxylic acid group). Hint: think about the buffer and capillary.

References
1. Skoog, D.A., Holler J.F., Crouch S.R. Principle of Instrumental Analysis 6th Ed.
Thompson Brooks: Belmont, 2007, Chapter 30.
2. Harris, D.C. Quantitative Chemical Analysis 7th Ed.W.H. Freeman and Co: New York,
2007, Chapter 26.
3. Solow, M. Weak Acid pKa Determination Using Capillary Zone Electrophoresis. J. Chem.
Ed. 2006, 83 (8), p 1194-1195.


Appendix
A. Setup
Turn on the computer and log in. Turn on the power button on the lower left of the Agilent
Technology 7100 CE Instrument. In the Start Menu, open the program Instrument 1 Online.
You will be greeted with this screen.



Figure 3: Method and Run Control - Instrument Control

Load the CHEM3XX_CE.M method. Note the file extension must be .M.
In the lower left corner, you will see that you are in the Method and Run Control mode. The Run
Method Task (single vial icon in top left corner) should be selected. Under the Instrument
Control tab, you will have access to most of the commands related to initializing the instrument
(On/Off), flushing the capillary, and running a single sample; once a sample is running, the
sample identity is displayed at the bottom (circled in Figure 3), and the time remaining can be
monitored. Turn on the UV lamp by clicking on the power icon within the DAD section.

The inlet and outlet vials (circled in Figure 3) displayed on the instrument control interface refer
to the vials in which the two electrodes are introduced. To remove the inlet (or outlet) vial
from the instrument, the slot in the vial tray corresponding to the vial number must be
empty and there must be another vial available for the electrode to dip into. Right click on the
picture of the inlet vial and select Set Inlet Vial. A pop-up will then allow you to choose
another vial as the inlet vial. For example, by replacing Vial 3 with Vial 2 as the inlet vial, Vial 3
will now be in the vial tray while the electrode will be dipped in Vial 2. The outlet vial can be
switched in the same manner.

Right clicking on the pressure gauge (circled in Figure 3) gives options for injection and
flushing. The flushing option is useful towards the end of the experiment. Before flushing the
capillary, make sure the outlet vial is set to Vial 20 (waste) and the inlet vial is set to the solution
that you wish to flush the capillary with. To begin flushing, right click on the pressure gauge and
select Flush. A pop-up will allow you to set the flushing time.

B. Instrument Method
Under the Instrument tab, click Set up Instrument Method. Under the DAD tab, make sure all
the settings are the same as that shown in Figure 4. The must haves are:

Signal: 210 nm with a bandwidth of 5 nm
Stoptime: As CE
Spectrum Range from: 190.0 nm to 400.0 nm
UV Lamp required


Figure 4: Setup Method DAD









Under the CE tab, the parameters on the left panel are:
Inlet Home: 04 Buffer
Outlet Home: 05 Buffer
Cassette Temperature: 20.0 C
Enable High Voltage
Voltage: 25.0 KV
Current: 300 A
Power: 6.0 W
Low Current alarm limit: Off
Stoptime: 5:00 min
Posttime: Off

Under the CE tab, the right panel displays each step in a Single Sample run. Each run will start
with preconditioning which consists of flushing the capillary for 4 minutes with buffer. This step
is represented by a box under Preconditioning (see Figure 5). Steps can be created or removed
using the Insert and Delete buttons. The parameters of each step can be set: the inlet, outlet,
and duration can then be specified. For the injection step, the drop down menu on the left should
display Apply Pressure; additional parameters include applying a pressure of 50 mbar for 5 s; the
inlet is Injection Vial and the outlet is Outlet Home Vial. Following injection is postconditioning,
which consists of two steps: the capillary is flushed with NaOH for 3 minutes, and then with
deionized water for 3 minutes.


Figure 5: Preconditioning, Injection, and Postconditioning Steps

C. Single Sample
Under the Run Control tab, click Sample Info. Fill out the file name and type CHEM3XX_CE as
the subdirectory for the file to be saved. Then fill out the sample name and enter the vial location
in Location. Figure 6 below shows the Sample Info for a deionized water run (vial location 6).
To run the sample, either click Run Method or on the main screen click Single Sample.

Figure 6: Single Sample - Sample Info

To use the single sample command for other samples, simply change the sample name and
specify the vial location.

D. Data Analysis
During the run, Instrument 1 offline can be opened by clicking on the Data Analysis tab near the
bottom left. Under the File tab, click Load Signal. On the right of the Load Signal window, the
CHEM3XX_CE folder where your files are saved can be accessed through the path:
C:\CHEM32\1\DATA\CHEM3XX_CE. On the left, the data files contained in the selected folder
are displayed.

Click on Signal Details (near the bottom right of the Load Signal window); make sure that the
Signal Description only contains the signal at 210nm. To remove other signals from Signal
Description, select the entire row corresponding to the signal by clicking on the left margin of
the row, and click Delete Row. To add a signal to Signal Description, select the desired signal
from the drop down menu under Available Signals, and click Add to Method. In the Load Signal
window, then select your data file from the left and click OK to load. If multiple signals are
being displayed (for example the current, voltage, or power signals) then go back to File -> Load
Signal -> Signal Details, delete all signals and reload the file. All available signals are displayed.
Once again, go to File -> Load Signal -> Signal Details and add only the 210 nm signal to the
method. Upon reloading the file, only the 210 nm signal will be displayed.


Figure 7: Load Signal - Signal Details

When all the electropherograms are overlaid, the automatic integration takes only a few seconds
to integrate all the peaks. Once the signals for the blank, standard solutions, and unknown have
been loaded, check the boxes next to all the runs as shown in Figure 8.


Figure 8: Selecting Runs to Overlay

Finally, in the area below Sample Name, double-click on the blank run. A pop-up will display
a warning that too many signals are selected for overlay and will ask if you want to continue;
click Yes to continue. All the selected signals will now be stacked one on top of the other as seen
in Figure 9.

Figure 9: Overlay of Signals

Under the Integration tab, click the peak sign that says Auto Integrate. If necessary, use the
manual integration tools (circled in Figure 9) to redraw or remove peaks.

In File, go to Print, and click Integration Results. On the print-out, there will be no spectrum but
only the tabulated integration results of each signal. Save your integration results by printing
them to a PDF file, locating the file in the CHEM3XX_CE folder. Be cautious in interpreting
the integration results; they are not always printed in an intuitive order.

Print the electropherogram for one calibration solution and for the unknown. An overlay of these
is acceptable.

Before closing the program, the capillary needs to be flushed with methanol for 5 minutes
followed by 5 minutes of deionized water. Note that methanol is volatile and you may have to
refill Vial 2 using the HPLC grade methanol in the SIF. After the flush, set the inlet vial as Vial 3
and the outlet vial as Vial 6. Close Instrument 1 Offline, then close Instrument 1 Online, and
finally shut off the instrument power.

65

A-08 Determination of Cr
3+
by Chemiluminometry

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Briefly describe the process of chemiluminescence
Identify which of your reactants will be limiting the emitted light intensity
Describe the advantages and disadvantages of the manual injection technique
compared to the flow injection method
Describe the principles and use of a photomultiplier tube (PMT) detector
Recognize the safety concerns associated with using high-voltage power sources and
plan to use best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of Cr
3+
in your unknown using the calibration
curve method and applying appropriate dilution factors
Draw appropriate baselines for peak integration and height
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice


BACKGROUND
The oxidation of luminol by hydrogen peroxide in basic solution is a chemiluminescent reaction.
The reaction is enhanced by chromium (III) but not by chromium (VI). Hence,
chemiluminometry can distinguish between different chemical forms of chromium, this being a
valuable feature in some biological applications. While the chromium is often referred to as a
catalyst, the enhancement effect is not true catalysis in that the Cr (III) is consumed in the
reaction. Many other metals enhance the oxidation, but interference by these species can be
suppressed by the addition of ethylenediamine tetraacetic acid (EDTA). The inactive EDTA
complexes form rapidly with the interfering metals, but only slowly with chromium. While your
unknown does not deliberately contain metals other than chromium, this masking technique will
prevent trace metal ions present in the deionized water from interfering with the analysis.



Figure 1: Oxidation of luminol in basic solution
Reaction times are short under the conditions of this experiment, and light is emitted for only a
few seconds. Therefore the light must be measured
sensitive to such factors as reagent concentrations, temperatur
technique is critical, particularly during injection. The analysis will be done using both manual
injection and flow injection analysis (FIA) techniques. In the former case optimal signal
noise ratios are obtained by injec
while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol
and Cr (III). The reason for the difference is not obvious, but is probably related to the
of the reaction and the very different rates of injection
PROCEDURE

Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of
water and add the washings to the flask. Dilute to the mark with water. Mix well.

Using the 5.00E-06 g/mL chromium (III) stock s
calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E
chromium for the most concentrated solution. Prepare a further five
unknown. Ensure each solution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume
and 50mL volumetric flasks are provided.


PART I: Analysis of Chromium by Manual Injection
The manual chemiluminometer consists of a cell compartment equipped with a septum through
which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected
with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is
digitized, stored, and processed by computer.

Power on the computer and then open the Acquisition for Windows (AFW) program. Use the
mouse to activate the buttons of the following tool bar.

WARNING: The high voltage power supplies used are capable of producing lethal
currents/voltage. Making of electrical connections must only be done with HV
supply OFF.

66

: Oxidation of luminol in basic solution
Reaction times are short under the conditions of this experiment, and light is emitted for only a
few seconds. Therefore the light must be measured as the reactants are mixed. The reaction is
sensitive to such factors as reagent concentrations, temperature, and mixing. Reproducible
technique is critical, particularly during injection. The analysis will be done using both manual
injection and flow injection analysis (FIA) techniques. In the former case optimal signal
noise ratios are obtained by injecting the Cr (III) samples into a mixture of peroxide and luminol,
while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol
and Cr (III). The reason for the difference is not obvious, but is probably related to the
of the reaction and the very different rates of injection used in the two techniques.
Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of
water and add the washings to the flask. Dilute to the mark with water. Mix well.
06 g/mL chromium (III) stock solution, prepare at least five (preferably six)
calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E
chromium for the most concentrated solution. Prepare a further five-fold dilution of your
ution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume
and 50mL volumetric flasks are provided.
PART I: Analysis of Chromium by Manual Injection
The manual chemiluminometer consists of a cell compartment equipped with a septum through
which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected
with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is
digitized, stored, and processed by computer.
computer and then open the Acquisition for Windows (AFW) program. Use the
mouse to activate the buttons of the following tool bar.
The high voltage power supplies used are capable of producing lethal
currents/voltage. Making of electrical connections must only be done with HV

Reaction times are short under the conditions of this experiment, and light is emitted for only a
the reactants are mixed. The reaction is
e, and mixing. Reproducible
technique is critical, particularly during injection. The analysis will be done using both manual
injection and flow injection analysis (FIA) techniques. In the former case optimal signal-to-
ting the Cr (III) samples into a mixture of peroxide and luminol,
while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol
and Cr (III). The reason for the difference is not obvious, but is probably related to the kinetics
used in the two techniques.
Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of
water and add the washings to the flask. Dilute to the mark with water. Mix well.
olution, prepare at least five (preferably six)
calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E-07 g/mL
fold dilution of your
ution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume
The manual chemiluminometer consists of a cell compartment equipped with a septum through
which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected
with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is
computer and then open the Acquisition for Windows (AFW) program. Use the
The high voltage power supplies used are capable of producing lethal
currents/voltage. Making of electrical connections must only be done with HV power

67


Click File then New to open a new file. Click Acquire, then Configure Run and set the
following run parameters:
Append to Current File (acquires data for several runs into one file)
Rate 120.00
Additional Run Time 0.10 (entered through the keyboard)
Temporary File Enable

Click SAVE then OK. It should not be necessary to reset the configuration for the rest of the
experiment. Power on the chemiluminescence amplifier and set the selector switch to MAN to
monitor the photomultiplier tube of the manual apparatus. Turn on the magnetic stirrer.

Five duplicate manual injections will be made for each standard solution and the unknown. Use
the following procedure to charge the cell with peroxide and luminol solutions, and make test
injections with the most concentrated chromium standard solution.


Turn on the high-voltage dc power supply with the button marked MAINS. Adjust the
voltage to ~700 volts. Before proceeding, check that the Output ON/OFF button of the
supply is OFF (ie. there should be no -ve sign before the displayed voltage setting and the
HV ON light should not be lit.) Remove the cell compartment cover. Flush the cell with
water, then empty by aspiration. Add 1 mL of 0.0002 M luminol solution and 0.5 mL of 0.12
M hydrogen peroxide solution. Replace the cell compartment cover! The PMT can be
damaged by exposure to room light.

Switch the Output of the power supply to ON so that -700 volts is applied to the PMT
cathode. A -ve sign should appear before the displayed voltage.

To check that the baseline voltage is ~0.5 to 1.0 V, click Acquire, then Manual Zero.
(The system records positive voltages only, and the offset ensures that the baseline will not
drift to a negative value.) Close the Manual Zero window.
Click and enter the relevant information into the Header Labels window. Click SAVE
and OK.
Fill a syringe with 5.00E-07 g/mL chromium solution (your most concentrated standard
solution). Adjust the volume to 0.4 mL. Insert the syringe into the injection port, click OK

68

to initiate acquisition, and inject the solution after 1 to 2 seconds. NOTE! Reproducible
injection technique is important! The injection itself should be as rapid as possible.

Switch the Output of the power supply to OFF, remove the cover of the cell compartment,
and empty the cell by aspiration. Rinse the cell, then recharge with peroxide and luminol.

If the peak height for the first injection (using the most concentrated standard solution) is
above 9 volts, reduce the PMT high voltage and repeat the injection. If the peak is less than
7 volts, increase the high voltage (1000 volts maximum!). Once the voltage has been set,
do not change it for the rest of the analysis by manual injection.
Click to initiate the acquisition routine for the next run. Acquire data for 5 injections
of each standard solution, the diluted unknown, and the blank. Save the file.
If the software should happen to crash, advise your TA and/or instructor before attempting to
restart the software.

Processing the Data:
Although the peaks can be identified automatically by computer (try it - just push the
button), the selected baselines will often be unsatisfactory. It is difficult to select a single set of
criteria that will adequately define the wide range of peak shapes common in this experiment.
It is likely that each displayed peak should be individually rescaled and processed manually. To
rescale, hold down the left button of the mouse, and drag the cursor diagonally to draw a
rectangle around the area to be displayed. Release the button. Click the button. Position
the cursor at the start of the base of the peak, hold down the left key of the mouse, and draw the
baseline. Release the button. Use the scroll bar to scan the data to the next peak. Draw a
baseline for each peak on the displayed file. Turn off the button and click to reset the
original display.
Click to print the peaks. Click to display the data table, and to print the table.

From the tabulated values of peak heights and areas, calculate the concentration of chromium
(g/mL) in your original unknown solution as received in the vial. (Note: Inspect the data
carefully before deciding upon the best method of calculation.)

PART II: Assay of Chromium by FIA
In FIA systems, sample or reagent "plugs" of 20 to 150L volume are sequentially transferred
from an injection loop by a carrier flow. The plugs are then taken through narrow-bore tubing to
a flow-through cell. During delivery the plugs may undergo reaction with reagents in the carrier
stream, or may combine with separate reagent streams. Various analytical instruments

69

(spectrometers, fluorometers, potentiometers, conductivity bridges etc.) can serve as detectors to
monitor the cell. The FIA system used in this experiment consists of the following components.
a peristaltic pump that maintains constant flow rates of solutions through four narrow bore
tubes,
an 8-port automatic valve that allows for multiple injections of 25L plugs of hydrogen
peroxide from an injection loop into a carrier stream of water,
a spiral flow-through cell in which the analyte is mixed with the reagents,
a photomultiplier tube (and amplifier) that monitors the light emitted from the cell,
a computer, equipped with an analog-to-digital converter, that acquires and processes the
amplified signal from the photomultiplier tube.

The system status, in each of the possible valve positions, is as follows:
LOAD POSITION:
The injection loop is filled
with peroxide. Excess is
pumped to waste.

Sample is mixed with
luminol. The mixture is
merged with the carrier stream
(water) at the cell.

INJECT POSITION:
The water stream is
redirected through the
injection loop, carrying the
peroxide plug to the cell where
it reacts with the mixture of
luminol and sample.

H
2
O
2
stock solution is
pumped directly to waste.
Initial settings for Alitea C-
4V pump: Obtain help in fitting the four silicone tubes around the roller drum of the Alitea
peristaltic pump, then make the following settings on the pump:
POWER ON/OFF ON
CLOCKWISE/COUNTERCLOCKWISE CW
SPEED 500
LUMINOL
SAMPLE
H2O2
WATER
INJ.
LOOP
PUMP
WASTE WASTE
VALVE
LOAD
POSITION
DETECTOR
(PMT)
0.8ml/min
0.8ml/min
0.8ml/min
1.3ml/min

LUMINOL
SAMPLE
H2O2
WATER
INJ.
LOOP
PUMP
WASTE WASTE
VALVE
INJECT
POSITION
DETECTOR
(PMT)
hv
0.8ml/min
0.8ml/min
0.8ml/min
1.3ml/min


70

START/STOP STOP

Initial settings for 8-port valve controller:
POWER I/O I (i.e. ON)
T1 (load time) 6.0 (~30 SECONDS)
T2 (inject time) 2.0 (~10 SECONDS)
POSITION A
CONTROL AUTO
START/RESET RESET

Computer and amplifier: Set the chemiluminescence amplifier to monitor the PMT of the FIA
system. A single acquisition file of 30 minutes should be sufficient to acquire the data for all
solutions.
Click Acquire, then Configure Run and set the following run parameters:
Append to Current File disable
Rate 30.00
Run Time 30
Temporary File Enable

Dilute 30 mL of 0.12 M hydrogen peroxide to about 100 mL with deionized water. Immerse
the inlet ends of the appropriate silicone tubes into three 125 mL Erlenmeyer flasks
containing 100 mL respectively of water, 0.0002 M luminol, and diluted hydrogen peroxide.

Insert the end of the tube labeled "sample" into the volumetric flask containing the most
concentrated Cr(III) standard. Insert the two outlet tubes into a waste container.

Press "START" on the pump. To ensure that the system is pumping satisfactorily,
momentarily lift each tube from the solution. An air bubble should pump through the tube.
Switch the valve controller to "RESET". The valve will rotate repeatedly through load/inject
cycles. (The green LED indicates when the valve is in the "inject" position). Switch the
valve to "START".

Switch on the dc power supply so that about 600 volts is applied to the PMT cathode. Click
to start the acquisition of data. Adjust the dc voltage until the displayed peaks are about
90% of full scale. Do not change this setting for the rest of the experiment. Once 5
satisfactory peaks have been recorded, quickly and smoothly remove the "sample" tube and
insert it into the volumetric flask containing the next standard solution. Record five
satisfactory peaks for each standard solution, the blank, and the unknown.

71


When finished, insert all inlet tubes into a flask of deionized water and flush the system
thoroughly. Obtain help in releasing the silicone tubes from the pump and shutting down the
system.

Process the peak data as before. From the tabulated values of peak height and peak area,
calculate the concentration in g/mL of chromium in your original unknown solution as received
in the vial.

IN YOUR REPORT
Report both the [Cr] determined by FIA and by manual injection technique on the front
cover of your report. Compare the FIA results with those using the manual injection. In
comparing your results, make sure to use the appropriate statistical tests to determine whether the
results agree.

Using the data from the five blank runs, calculate the signal detection limit and the minimum
detectable concentration for both the manual and FIA techniques. Ensure that the units of these
two values are given correctly.


BACKGROUND READING
1. Hanson, E.H.; Ruzicka, J. J. Chem. Ed. 1979, 156(10), 677.
(Hanson and Ruzicka are the co-inventors of the FIA technique).
2. Ruzicka, J.; Hansen, E.H. Flow Injection Analysis, Wiley: New York, 1988.
(QD79.I5 R89 1988 in IKBLC)
3. Wade, A.P.; Shiundu, P.M.; Wentzell, P.D. Anal. Chim. Acta 1990, 237, 361.
4. Ruzicka, J.; Hansen, E.H. Anal. Chem. 2000, 72(5), 212A.
5. Seitz, W.R.; Suydam, W.W.; Hercules, D.M. Anal. Chem. 1972, 44, 957.


72

A-09 Determination of Calcium in the Presence of Aluminum by Atomic Absorption
Spectrometry

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the processes occurring between sample introduction and analyte
atomization in the flame
Describe the effect of aluminum on the calcium signal strength and how addition of
strontium can overcome the interference.
Describe the advantages and disadvantages of the calibration curve method compared
to the standard addition method
Recognize the safety concerns associated with igniting, using and extinguishing an
air-acetylene flame and plan to use best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of Ca
2+
in your unknown using the calibration
curve method and standard addition method applying appropriate dilution factors
Explain the flame colour changes that occur upon introduction of different solutions
including the variation in colour with height in the flame
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through substantial practice


BACKGROUND
Unlike the absorption and emission spectra of molecules, the absorption and emission spectra of
atoms are characterized by very narrow bandwidth. From an analysis perspective, these narrow
bandwidths both complicate the analysis by requiring precise instrumental control over
wavelength and also improve the analysis by providing built-in specificity: it is unlikely that
any two atomic lines overlap. Instead of suffering spectral interferences, these analyses are more
susceptible to chemical interference. In this experiment, we examine the specific interference of
aluminum when trying to determine calcium, and demonstrate one particular technique for
minimizing the effect of any aluminum present.

To begin, we first need to think about one of the key steps in atomic spectrometry: production of
the analyte in atomic (i.e. not molecular) form. The atomic absorption of calcium is normally
measured by means of a reducing (fuel-rich) air-acetylene flame. In an oxidizing (oxidant-rich)
flame calcium is partially converted to oxide and, there being fewer free atoms, the sensitivity is

73

diminished. The measurement is subject to chemical interference, as opposed to spectral
interference, from several cations and anions. For example, aluminum depresses the calcium
signal because of the formation of the refractory compound calcium aluminate which passes
through the flame as a solid without dissociation into atoms. This interference can be
substantially removed by the addition of a strontium salt as a so-called releasing agent. In
sufficient quantity the strontium ties up the aluminum as refractory strontium aluminate, and the
calcium then exists in the flame mainly in the atomic state. Strontium also controls the
ionization of calcium in the flame. A correction must be made for the calcium usually found as
an impurity in strontium salts.

Residual interference can be compensated by the technique of standard addition, whereby
measurements are conducted on a series of solutions, each of which is prepared from a fixed
volume of unknown solution together with one of a range of volumes of standard solution.
Provided the resulting plot of absorbance versus concentration of added standard is linear, the
concentration of the unknown solution can be obtained by extrapolation to the concentration
axis. The standard addition technique should be applied with discretion. It sometimes cannot
cope alone with gross chemical interference (why not?), so should preferably be combined with
the releasing technique.

PROCEDURE
Your unknown solution contains aluminum as well as calcium. Add all of your unknown
solution to a 250 mL volumetric flask. Rinse the vial with a few millilitres of water and add the
washings to the flask. Dilute to the mark with water. Mix well. The first runs will demonstrate
the interference of aluminium and the releasing action of strontium:

Part 1 Qualitative Analysis

Run 1 Determining the signal strength
with calcium alone:
To a 50 mL volumetric flasks add 5.00
mL of a solution containing 4.00E-05
g/mL calcium. Dilute to the mark with
water. Measure the calcium absorbance
using water as a reference.


Run 2 - Interference by aluminum:
To a 50 mL flask add 5.00 mL of a solution containing 4.00E-05 g/mL calcium and 1 mL of
4.00E-04 g/mL aluminum, dilute to the mark. Put 1 mL of 4.00E-04 g/mL aluminum in a 50 mL
flask dilute to the mark to use as a blank. Measure the absorbance of calcium using the blank
solution (not water) as the reference.

WARNING: Ignition, use and extinguishing
the air-acetylene flame is a potential-explosive
action. Strict protocols are given in the lab
manual and must be followed. As always, lab
coat and eye protection must be worn.

With some AA instruments, the flame is large
and poorly-shielded. Care should be taken to
avoid accidental contact with the flame or
nearby hot surfaces.
Atomic emission of some elements produces
UV light. The instrument operator should
avoid staring at the flame.


74

Run 3 - Releasing action of strontium:
Proceed as in Run 2 but add 5 mL of 1% strontium chloride solution to each flask before
diluting. Use blank solution as the reference.
Refer to the appendix and use the Perkin-Elmer 305A atomic absorption spectrophotometer to
measure the absorbance of calcium in an air-acetylene flame for each of the calcium-containing
solutions. Record the absorbance for each run.

Part 2 Quantitative analysis
Clean the 50 mL flasks and prepare the following three sets of solutions. The measured
absorbances will be used to determine calcium in the unknown sample. In run 4 calcium will be
determined using the releasing technique alone, in run 5 it will be determined using the
combined releasing and standard addition techniques, and in run 6 it will be determined using the
standard addition technique alone.
Run 4 - releasing technique:
Using the 4.00E-05 g/mL calcium stock solution, prepare at least five (preferably six) calibration
solutions. Start with zero concentration (blank) and do not exceed 4.5E-06 g/mL calcium for the
most concentrated solution. Prepare a further five-fold dilution of your unknown. In each
volumetric flask, also add 5 mL of 1% (w/w) strontium chloride solution. Do not add
aluminum solution. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric
flasks are provided. Ensure each solution is well mixed.
Measure the absorbance of calcium using the blank solution as the reference. Plot your
calibration curve and by interpolation determine the concentration in g/mL of calcium in your
original unknown solution.

Run 5 - combined releasing and standard addition techniques:
To each of six 50 mL volumetric flasks add 10.00 mL of diluted unknown solution. Add 4.00E-
5 g/mL calcium stock solution sufficient to have solutions with added calcium in the range of
zero to 2.00E-4 g. To each flask add 5 mL of 1% strontium chloride, but no aluminum solution.
Prepare a blank solution by diluting 5 mL of 1% strontium chloride solution to 50 mL.
Measure the absorbance of calcium using your blank solution for the reference. Plot your
standard addition line and by extrapolation determine the concentration in g/mL of calcium in
your original unknown solution.

Run 6 - standard addition technique:
Finally, for comparison, apply the standard addition technique without the releasing technique as
follows. Proceed as in the last run, but omit the addition of strontium chloride. Use deionized
water as the reference.




75

IN YOUR REPORT

Of the three determinations of unknown choose the best and report only one calcium
concentration, in g/mL, on the first page of your lab report. Compare and discuss the results
obtained with the different methods. In comparing your results, make sure to use the appropriate
statistical tests to determine whether the results agree. Explain fully why the measured calcium
absorbance differed between runs 1 and 2. Describe the role strontium played in determining
your results for run 3.

BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 9.
2. Harris, D.A. Quantitative Chemical Analysis, 8
th
ed.; W.H. Freeman: New York, 2010;
chapters 21-1, 21-2 and 21-5.




76

APPENDIX I: Use of Perkin-Elmer 305A Spectrophotometer

CAUTION: INCORRECT USE OF THE AIR-ACETYLENE BURNER COULD RESULT
IN A FLASH-BACK EXPLOSION. Consult your TA before proceeding and note the
following safety precautions:
The drain tubing loop must always be filled with water
The air supply must always be turned on before the acetylene supply is turned on
The air supply must always be turned OFF after the acetylene supply is turned off

Operating Instructions:

1) Ensure that the AIR/ACETYLENE burner is installed.

2) Ensure that the loop in the plastic drain tubing is filled with water and the end of the tube is
inserted in the drain reservoir (on the floor).

3) Turn SOURCE fully counterclockwise. Push POWER on to power the instrument and the
exhaust fan.

4) Lighting the flame:
Ensure that the air compressor is switched on (ask for help if it is not).
Switch on AIR using the black valve. Monitor the pressure gauge and set the air
pressure to 30 psi with the silver knob.
Monitor the flow meter and set the air flow rate to 9 (middle of ball).
Open the acetylene cylinder and if necessary, set the supply pressure to 12 15 psi.
Open the FUEL SHUT OFF valve. Monitor the pressure gauge and adjust fuel
pressure to 8 psi with the silver knob.
Monitor the flow meter and set the fuel flow rate to 9 (middle of ball).
Light the flame from below with the ignitor (wear leather gloves). Allow the burner
chamber to warm-up for 10 minutes.



77

5) Instrument settings:
Monitor the ammeter (current meter) inside the lamp compartment and use SOURCE
to set the lamp operating current at 10 mA.
Set FILTER to OFF (button not lit)
Set PHASE to NORMAL.
Turn EM CHOPPER to OFF.
Set DAMPING at 2.
Set SLIT at 4.
Set MODE to ABS.
Set RANGE to VIS.
Set the WAVELENGTH initially at 211.3 (this sets at about 422.6 nm), then make
a fine adjustment (up or down) until the ENERGY meter indicates a maximum
reading.
Adjust the GAIN until the needle of the ENERGY meter is in the dark red zone.

6) Reading the absorbance: (NOTE aspirate deionized water into the burner between
measurements.)
Turn the EXPANSION knob fully counterclockwise.
Switch the DIRECT/NULL knob to NULL.
Use the absorbance knob to zero the digital readout of ABSORBANCE. Aspirate the
appropriate reference solution into the burner. Use the ZERO knob to set the meter
needle to zero.
While aspirating the test sample into the burner, use the absorbance knob to reset the
meter needle at zero. Record the absorbance reading from the digital meter. After
reading the absorbances of the solutions in each set, aspirate the blank solution and
re-check the zero setting. For each set of solutions plot the absorbance readings as
they are taken.

7) Shut-down procedure: (OBTAIN ASSISTANCE)
Turn the FUEL SHUT OFF lever down.
Close the valve on the acetylene cylinder. Open the FUEL SHUT OFF lever to bleed
the lines. Once the pressure gauge reads zero, close the fuel shut off lever.
Turn the black gas control lever from AIR to OFF.
Turn the POWER to OFF
Turn the SOURCE and GAIN knobs fully counterclockwise.
Turn off the air compressor only if no-one else is using any the flame AA
instruments.

78

A-11 Determination of Copper by ICP-MS Isotope Dilution MS

LEARNING OBJECTIVES
Before commencing the experiment, you should be able to:

Describe the way in which the argon plasma is generated and sustained
Describe the processes that occur when sample is introduced into the plasma
Describe the components of the vacuum system that allow for sample introduction at
atmospheric pressure and detection under high vacuum.
Explain the advantages and disadvantages of the ratio method compared to the
isotope dilution method.
Recognize the safety concerns associated with nitric acid use and plan to use best
practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of Cu in your unknown using the ratio method
and isotope dilution method applying appropriate dilution factors
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice


BACKGROUND
Inductively-coupled plasma mass spectrometry (ICP-MS) is a highly-sensitive
analytical method suitable for the determination of trace levels of multiple
elements or isotopes. The combination of high-sensitivity and rapid
determination of multiple elements makes the ICP-MS a valuable instrument in
analytical labs. Theyre also valuable to the manufacturers; a new ICP-MS
costs ~$250,000!

In the instrument, the sample is nebulized into an argon plasma at a temperature of ~6000C.
Analyte isotopes are converted to charged ions typically M
+
and are introduced through a
partially-evacuated interface into a mass spectrometer operating under high vacuum. Ion
intensity (counts/sec) is output as a function of mass-to-charge ratio, m/z (or AMU for ions with
a charge of +1). Analyte concentrations are typically in the sub-ppb to low-ppm range. At these
concentrations good experimental technique is critical. Background corrections are particularly
important, as are the methods of treating uncertainties in the instrumental measurements. Many
of these uncertainties in ICP-MS can be minimized through use of an elegant technique called
isotope dilution mass spectrometry (IDMS).

79


Most elements from natural sources exist as mixtures of isotopes having well-defined relative
abundances (see appended Table 1), although a few notable exceptions such as Pb have isotopic
abundances that vary depending upon the source of the element. (Isotopes of Pb are end-
products of the radioactive decay of uranium and thorium.) In IDMS, a spike enriched in one
of the isotopes of the analyte element is added to the unknown sample. The mass spectrometer
measures the enriched isotope and one other isotope of the analyte element. The concentration
of the element present in the original unknown is calculated from the mass spectrum, and the
published values of the isotopic compositions of the original analyte (Table 1) and the spike.
The spike serves both as an ideal internal standard (why is it ideal?), and as an added standard
of the analyte.

Copper from natural sources contains two isotopes,
63
Cu and
65
Cu. In the IDMS analysis of
copper, a mass spectrum is recorded of a solution containing the Cu analyte (natural isotopic
abundance) to which has been added an aliquot of copper standard enriched in
65
Cu. Schematic
representations of the mass spectra of the unknown, the spike solution, and the mixture are
shown in Fig. 1.

The fractional isotopic abundances
( )
u
63
Cu
f and
( )
u
65
Cu
f of the Cu in the original unknown are
given in table 1. The fractional abundances
( )
s
63
Cu
f and
( )
s
65
Cu
f of the copper in the
65
Cu-enriched
spiking solution are furnished by the supplier or can be calculated from your data. (Note that all
isotopic abundances f are in units of mole fraction, and not weight fraction; these differ since the
mass of any one isotope of copper is distinct from the copper atomic mass found on the periodic
table.)













i
o
n

i
n
t
e
n
s
i
t
y

mass number

FIG. 1a: Unknown
Cu (natural relative
isotopic abundance).

i
o
n

i
n
t
e
n
s
i
t
y

mass number

FIG. 1b: Spike
solution isotopically
enriched in
65
Cu.

mass number

i
o
n

i
n
t
e
n
s
i
t
y

FIG. 1c: Unknown Cu
spiked with isotopically
enriched
65
Cu.


80

The sensitivity of the instrument towards
63
Cu and
65
Cu should be expected to be similar but not
equal. Variation in the efficiency of the plasma in producing the ionized atoms, as well as
differences in quadrupole and detector response mean that the sensitivities are likely slightly
different. The counting efficiency k (1) evaluates this experimentally from the mass spectrum
of the copper in the original, natural source, unknown.

( )
( )
( )
( )
u
65
u
63
u
65
u
63
Cu
u
Cu
u
Cu
Cu
f Cu moles
f Cu moles
k
counts
counts
= (1)
and therefore:
( ) ( )
( ) ( )
u
65
u
63
u
65
u
63
Cu Cu
Cu Cu
f f
counts counts
k = (2)

where
( )
u
63
Cu
f and
( )
u
65
Cu
f are the abundances listed in table 1. k is used in later calculations as a
normalizing factor.

From Fig.1c the count ratio R of
63
Cu to
65
Cu for the spiked unknown solution is:


( )
( )
( ) ( )
( ) ( )
s
65
u
65
s
63
u
63
s u
65
s u
63
Cu
s
Cu
u
Cu
s
Cu
u
Cu
Cu
f Cu moles f Cu moles
f Cu moles f Cu moles
k
counts
counts
R
+
+
= =
+
+
(3)

where the amounts in moles referred to correspond to amounts in the 50ml flask. Solving for the
amount of copper (sum of isotopes) in the unknown gives:

( ) ( )
( ) ( )
u u
s s
Cu Cu
Cu Cu
s
u
f f
k
R
f
k
R
f Cu moles
Cu moles
63 65
65 63

= (4)

In addition to the isotope dilution method above, analysis by ICP-MS can be accomplished by
measurement of an unknown and a standard much like most other instrumental methods. Here,
the isotopic nature of the sample (natural abundance or enriched) must be the same for both
unknown and standard. Concentration in the unknown is calculated from the ratio of the counts
for a chosen isotope. This method is useful for multi-element analysis, by which as many as 81
different elements can be quantified from a single mass spectrum. In this experiment Cu will be
analyzed by quantification against a known Cu standard of natural isotopic abundance and by
IDMS.


81

NOTE: Students will do this experiment individually, not in pairs. Each student will receive a
different unknown solution.

PROCEDURE



At the start of the laboratory period, check that the ELAN 6000 ICPMS is pumped down. If it is
not, refer to the APPENDIX and get help from the TA to initiate the startup procedure. DO NOT
ignite the plasma at this time.

To five 50mL PMP volumetric flasks add the following volumes of unknown, a solution
containing 5.00E-07g/mL Cu (natural isotopic abundance), and a solution containing 5E-07g/mL
of Cu isotopically enriched in
65
Cu. (NOTE: record the standardized concentration of this latter
solution!) Dilute to the mark with 1% EG HNO
3
.


NOTE 1a: Plasticware rather than glassware must be used for these trace level analyses.
Contaminants adhering to the surface of glass, even after thorough cleaning, give
unacceptably high background levels of the analytes. The polymethyl-pentene (PMP)
plasticware used in this experiment must be rinsed at the beginning of each period, first with
a few mL of 3% AR nitric acid provided especially for cleaning do NOT use the
environmental grade acid for cleaning and then several times with deionized water. The
same sample of acid can be cycled between several pieces of plasticware. The AR nitric acid
is not suitable for preparing your solutions!

NOTE 1b: The solutions used in this experiment are very expensive and must not be
contaminated. The ultrapure acid costs $500/liter, and the stock 10g/mL
65
Cu spiking
solution is $6/mL (or $600,000 per gram of
65
Cu).
A small amount of the
65
Cu spiking solution will be provided in a plastic bottle from which
aliquots can be pipetted directly using a dedicated micropipetter. Therefore it is NOT
necessary to rinse the micropipetter with the spiking solution.
Add 1% HNO
3
to the volumetric flasks from a minimum volume of acid contained in a clean
250mL PMP beaker. Never return excess to the bottle.
Prepare the following solutions with a micropipetter using new disposable plastic tips. A
plastic dropper is provided for diluting solutions to the mark with 1% HNO
3
.
WARNING: Nitric acid solutions are corrosive and act as strong oxidizers. Avoid skin
contact. Rinse immediately with cold water if it occurs.


82

Solution
Vol. unknown
(mL)
Vol. 5E-07g/mL Cu
(natural isotopic
ratio) (mL)*
Vol. 5E-07g/mL
65
Cu-enriched
standard (mL)*
Add 1% HNO
3

to dilute to
(mL)
1 0 0 0 50.00
2 2.00 0 0 50.00
3 0 0 2.00 50.00
4 2.00 0 2.00 50.00
5 0 2.00 0 50.00

* NOTE: Record the exact concentration of the stock solution of
65
Cu-enriched standard
indicated on the bottle. Both Cu stock solutions contain 1% nitric acid.
Refer to the Appendix for operating instructions for the ELAN 6000 ICP-MS and obtain five sets
of counts at mass numbers of 63 and 65 for each of the solutions.

CALCULATIONS
Quote all final results in units of ppb.
4. Use the data for solution 1 to determine background corrections for all calculations.
5. Use the data for solution 3 to check the purity of the
65
Cu-enriched spiking solution.
Compare your calculated value to the suppliers purity certificate posted in the lab.
6. Calculate k (Eqn 2) from the mass spectral data for copper of natural isotopic abundance
(solutions 2 or 5).
7. Calculate R (Eqn 3), the count ratio for spiked unknown (solution 4).
8. Calculate the concentration of Cu in your original unknown by IDMS (Eqn 4) using k, R, the
concentration of the
65
Cu spiking solution and the published molar isotopic abundances of
63
Cu and
65
Cu in the unknown and spiking solutions.
9. Calculate the concentration of Cu in your original unknown by ratio. Use the count data for
either
63
Cu or
65
Cu in solutions 2 and 5. Remember that solutions 2 and 5 both contain Cu of
natural isotopic abundance.
IN YOUR REPORT
Report both your IDMS and ratio method result on the cover of your report. Use units of
ppb.
Justify your choice (
63
Cu or
65
Cu) used in the calculation by ratio. Refer to Table 1 and explain
why
63
Cu and
101
Ru are the isotopes recommended for analyses of Cu and Ru respectively by this
second method. Compare this result with that calculated by IDMS. In comparing your results,
make sure to use the appropriate statistical tests to determine whether the results agree.

83


BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 11 (especially 11C).
2. Platzner, I.T.; Habfast, K.; Walder, A.J.; Goetz, A. Modern Isotope Ratio Mass
Spectrometry; Wiley: New York, 1997. (QD96.M3 P57 1997 in IKBLC ASRS)



84

APPENDIX: Operating Instructions for the ELAN 6000 ICP-MS
Do not try to use this instrument without the help of a teaching assistant! It is left on at all
times with the mass spectrometer pumped down and thermally-stabilized. Do not ignite the
plasma until you have prepared your solutions. The plasma consumes argon at a rate of ~17 to
20 liters/min.
The monitor display may appear in a number of configurations depending upon the status of the
instrument. However the following toolbar will appear at the top of the display:



Use only the buttons described on the following pages. Some of the other buttons are for tuning
and optimizing the mass spectrometer section of the instrument. If used incorrectly, they will
detune the instrument thereby affecting adversely your data. Click

to open the instrument
control window. If necessary, you may have to click the Front Panel tab to display the
following window:





85

The displayed graphic includes the pumps, the mass spectrometer housing, the nebulizer, and the
argon gas lines. Be sure to include in your report a short description of the function of these
components as well as the torch, the RF load coil positioned around the nozzle of the torch, the
interface between the torch and the mass spectrometer, and the mass spectrometer itself.

The sample solutions will be pumped into the nebulizer at a rate of 0.8 to 0.9 mL/min using a
peristaltic pump. The inlet tube to the nebulizer is labeled with black retaining tags, and the
outlet tube is labeled with white tags. The outlet tube is slightly larger than the inlet tube to
ensure that test solutions will not accumulate in the nebulizer housing. Insert the tube with the
black tag into a 100mL beaker of 1% EG nitric acid. The tube with the white tag should drain
into a beaker or flask. Set the pump at ~ -15.0 to give an optimal CCW flow rate of 0.8 to
0.9 mL/min. Acid should be pumped into the nebulizer housing whenever solutions are not
being measured.

When you are ready to make measurements, click Start under Plasma to initiate the ignition
sequence (~1 minute). The plasma can be viewed through the window on the top right of the
instrument. Allow the plasma to stabilize for about 15 minutes before making measurements.

Recording the mass spectra:
Several files are required for the operation of the ELAN 6000, and the choice of these files will
depend upon the experiment. The files define the tuning and optimization of the instrument, the
method of acquiring data, the output of reports, etc. A Workspace file saves and recovers all
the files needed for a given experiment. Click File, Open Workspace, and open Chem 3XX-
425 Quantification.wrk. If a box indicating No analytes to measure is displayed, click OK.
Click to open the method file which defines the measurement parameters. Click the
Processing then Timing tabs, and check and/or set the following:

Sweeps/Reading 10
Readings/Replicate 1
Replicates 5

Check that the two isotopes of copper, and no other isotopes, have been selected in the Analytes
table. To remove isotopes from the list, click the numbered cell to the left of the isotope, then
click Edit, Cut Row at the top of the window. To add isotopes to the list, right click on any
cell in the Analyte column thereby displaying a periodic table. Single click on elements to
select them, and double click to deselect them. The software defaults to selecting the most
commonly used isotope for each element. However this IDMS analysis requires that both
isotopes of Cu be measured. Hold down CTRL SHIFT, click on both isotopes, and click OK.
The Scan mode will default to Peak Hopping in order that data will be measured at the
center of the peak for each isotope and not between the peaks. Close the Data Only Method
window.

86

Click then the Manual tab to open the Sample window. Click Details and enter Cu
and your initials in the Sample ID box, and the sample description (blank, unknown,
spike etc.) and your initials in the Description box. This information will be printed on the
final report.
Click

to open the Realtime window in which the data will be displayed during the run.
Click Analytes to confirm the two isotopes of Cu have been selected. If not, click Add All.
Click OK. The box at the top left of the Realtime window should be set to Numeric, and not
Spectral or Signal.
Rinse the peristaltic pump inlet tube with deionized water, and then insert it into the flask
containing your blank solution. Pump blank through the system for 2 minutes, and then click the
Analyze Sample button to acquire data into the Realtime window. Do not use the Analyze
Blank function when measuring the blank. Click to display the collected data. Print the
raw data.

Pump 1% environmental grade HNO
3
through the system between samples. Similarly collect
data for all five solutions. Remember to make appropriate entries into the Description box in
the Sample window before running each sample. Once all samples have been measured, rinse
the plasticware thoroughly with deionized water, then with your remaining blank solution (1%
nitric acid). Flush again with DI water.

To turn off the plasma click the Stop button under Plasma. Do NOT turn off the vacuum or
the argon supply! Use the peristaltic pump to flush the system with 1% nitric acid for
10 minutes, then stop the pump and release the silicone tubes from the drum.




Table 1 is on the following page.

87


88

A-12 Determination of Manganese by Neutron Activation Analysis

LEARNING OBJECTIVES
Before commencing the experiment, you should be able to:

Describe how
55
Mn is activated to form
56
Mn
Describe the radioactive decay of
56
Mn
Describe how radioactive decay events are detected by the scintillation counter
Describe how you will use statistical analysis of the radioactive decay from a tritium
sample to assess whether the scintillation counter is producing expected results
Recognize the safety concerns associated handling of radioactive samples and plan to
use best practices

On completion of the experiment and lab report, you should be able to:

Correctly apply correction factors to your data to give a dataset that directly relates
concentration and decay counts
Correctly calculate the concentration of Mn in your unknown using a calibration
curve applying appropriate dilution factors
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice

BACKGROUND
Chemical analysis by neutron activation exploits the radioactive properties of materials in order
to determine their amount. Radioactive decay events are counted, and this count related to the
amount of material present. Alas, there are complicating factors: 1) Although larger amounts of
radioactive material will give more decay events, we must also consider that radioactive decay is
a random process. If the number of decay events in a given period of time is measured several
times, the number of decays will generally vary between measurements. To deal with this,
careful application of statistical analysis is needed and this is described in detail later. 2) Not all
materials are naturally radioactive, limiting the number of analytes which can be determined by
counting decay events. One can widen the range of possible analytes by making them
radioactive. This process is termed activation.

In this experiment, you will activate your aqueous unknown sample of natural, non-radioactive
manganese (
55
Mn) into radioactive
56
Mn by bombarding it with neutrons. The radioactivity of
the freshly-prepared
56
Mn sample is then measured via the Cerenkov effect, in which beta
particles emitted by the
56
Mn generate pulses of light as they travel through a solvent solution.
The light pulses are detected and counted with a scintillation counter.


89

Dealing with the random nature of radioactive decay:

56
Mn decays via the release of a beta particle and subsequent emission of gamma photon(s).
Like other nuclear processes, the time-sequence of these releases is random in nature. In a series
of equal time intervals, different numbers of photons are emitted. The frequency of occurrence
of particular counts during the time intervals follows a Poisson distribution,

P e
x
x
x
=


!


where P
x

is the probability of obtaining a count of x, and is the mean count after some number
of measurements. Increasing the number of measurements will give increasingly closer to the
true value. For any finite number of measurements, the best analytical data possible is a best
value for the mean count and some associated uncertainty. This best value of decay counts
will be used to quantify the amount of
56
Mn present. We must now consider how we will verify
that a Poisson distribution is experimentally-achieved and how we will characterize the
uncertainty in the mean count.

We note that since x, but not necessarily , is an integer, we can represent measured data as a
histogram and observe whether the histogram appears similar to a model discontinuous Poisson
distribution. If the number of repeated measurements is small (<10), the histogram is
assymetrical. However, if the number of repeated measurements is larger (>10), the shape of the
histogram is symmetrical and resembles a Gaussian curve. Taking the standard deviation of this
Gaussian curve allows us to define the uncertainty in our mean count.

Whether or not we observe a Poisson distribution of the measured data depends on two factors: if
the underlying process is random in nature and if the measurement method is not biased. We
know that the radioactive decay of
56
Mn is a random process, but we should check that our
measurement method is suitable. Is the counting efficiency of the instrument stable over some
given period of time? Does the fluctuation in the decay count rate seem too variable? Too
consistent? Any of these would point to instrumental bias in counting the decay events. To
quantify the terms too variable and too consistent we use a chi-squared test (aside: chi
rhymes with guy).

We make use of a fundamental property of the Poisson distribution model: that the average value
and the variance (square of the standard deviation) are equal. That is, the standard deviation is
given by
1/2
.

As long as we have a high the conformity of experimental observations to a Poisson
distribution (taking into account the limited number of observations, N) can be tested by
statistical means. We compare the average and the standard deviation of the measured data, but
taking the ratio of the two to calculate the coefficient of variation:

1
2

x
S
x


90

For data which is perfectly Poissonian, the ratio should be exactly 1. If the ratio is much larger
than one, the data are over-dispersed (more variable than expected), and if the ratio is much
smaller than one, the data are under-dispersed (less variable than expected). We use this to test
the goodness-of-fit of the data to a Poisson model. We calculate D, the index of dispersion:

D N
S
x
x
= ( ) 1
2


This is just N-1 times the coefficient of variation. As statisticians like to do, we hypothesize:

2
1

N
D


Which says that under a Poisson model, the value D should follow the Chi-squared distribution
for N-1 degrees of freedom. This Chi-squared distribution characterizes the variability we expect
to see in D for a given number of measurements. If the data are perfectly Poissonian, the two
terms should be equal. Given real experimental data, you are unlikely to find this equality. The
question is then: How close do the index of dispersion and the Chi-squared distribution need to
be to indicate that the data are likely Poissonian? First, we need to choose a confidence level. In
keeping with the usual convention in analytical chemistry, we choose 95%. Then we compare
our calculated value of D with critical values for Chi-squared. This is very similar to comparing
a calculated t value against a tabulated t value in the Student t-test. However, in this case we use
a so-called two-sided test. That is, we are interested to know if our data are either over-
dispersed or under-dispersed. This means that we compare our D value to both a lower critical
value and an upper critical value. Values at the 95% confidence level are tabulated on the
following page.

If the calculated value of D lies between the lower and upper critical value, our hypothesis is true
and we accept that our data fit a Poisson model. If D is either below the lower or above the
upper value, the test shows that our data do not fit the Poisson model at the 95% confidence level
(i.e. there is still a 5% chance the data actually is Poissonian). If your data do not appear
Poissonian, faulty equipment or technique may be the problem. In this experiment, verification
that the scintillation counter is unbiased is done by repeatedly measuring the decay of a tritium
sample and calculating D.


91

Degrees of Freedom
Critical Values for chi-square distribution at
95% CL
(n-1) Lower Upper
15 6.262 27.488
16 6.908 28.845
17 7.564 30.191
18 8.231 31.526
19 8.907 32.852
20 9.591 34.170
21 10.283 35.479
22 10.982 36.781
23 11.689 38.076
24 12.401 39.364
25 13.120 40.646
26 13.884 41.923
27 14.573 43.195
28 15.308 44.461
29 16.047 45.722
30 16.791 46.979
31 17.539 48.232
32 18.291 49.480
33 19.047 50.725
34 19.806 51.966
35 20.569 53.203
PROCEDURE
Prepare solutions quickly. From the time of introduction of samples into the neutron flux, the
experiment takes approximately two and a half hours. You must arrive in the laboratory with a
plan for how you will make your solutions (see below).

Add all of your 10 mL unknown solution to a 100 mL volumetric flask. Rinse the vial with a
few milliliters of deionized water and add the washings to the flask. Dilute to the mark with
deionized water. Mix well.

Using the 2.43E-02 g/mL manganese stock solution, prepare at least five (preferably six)
calibration solutions. Start with zero concentration (blank) and do not exceed 5.00E-03 g/mL
manganese in the most concentrated solution. Prepare a further 2.5-fold dilution of your
unknown. In each volumetric flask, also add 5 mL of methanol, which acts as an antifreeze

92

agent. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 25mL volumetric flasks are
provided. Ensure each solution is well mixed.

Transfer 10.00 mL of solution from each flask to seven plastic scintillation vials.

Before irradiating your samples, familiarize yourself with the proper procedures outlined
in Appendix 1. Use the Am-Be neutron source to irradiate the samples for about 5000 seconds.



During the irradiation period use the Perkin Elmer TriCarb 2910 scintillation counter to perform
a Poisson test. Measure the activity of the tritium standard in a series of 25 time intervals each of
0.5 minutes (see appendix 2).

Note that the default output for this instrument is in units of counts per minute. You must convert
the data to the actual number of counts in 0.5 minutes. For the following statistical analysis the
distribution of counts obtained in one minute intervals is not the same as that for 2 x (the counts
obtained in 0.5 minute intervals).

You can open the datafile output in spreadsheet software. Calculate the sample mean and the
sample standard deviation s
x
. s
x

should be about equal to x
1/2
.

Calculate the index of dispersion,
D. For a Poisson distribution, D should be about N 1 = 24. If the distribution is apparently
Poissonian, continue with the next part of the experiment. If not, investigate the possible causes.
Consider the impact of chosen confidence limits.

Remove the vials from the neutron source after 5000 seconds. By automatic operation of the
scintillation counter (see appendix 2) measure the Cerenkov radiation of the vials using counting
periods of 10 minutes. The instrument will print out the elapsed times automatically. Once
counting is complete, dispose of all samples into the holding tank provided.

Note that the instrument scales the data to counts/min (CPM). Correct each sample count for
background. Use the total elapsed time at which each sample was counted to correct for the
exponential decay that occurred between the counting of the samples. The resulting counts are
proportional not only to the concentration of manganese, but also to the neutron flux density. As
flux density
w
varies slightly with the geometry of each activation well, it is necessary to
normalize the counts to a common flux density . The ratios of the well flux densities as
compared to the average density are listed in the following table:
WARNING: Radioisotopes, such as those created in this experiment,
demand specific safety protocols. Their hazards and thus protocols vary
with the nature of the radioactive decay.
56
Mn emits beta particles, a form
of ionizing (thus tissue damaging) radiation.

Beta particles are easily blocked, thus the greatest safety concern is to skin
exposure. Wash off with soap and cold water in the event of skin exposure.
Wear gloves (provided) whenever handling
56
Mn samples. Inform your
TA and instructor immediately if spillage occurs.


93

Well
No.

w

1 1.01
3
0.00
7

2 0.98
9
0.00
9

3 0.97
7
0.01
3

4 0.97
5
0.01
5

5 1.05
8
0.01
3

6 0.97
8
0.01
1

7 1.00
9
0.01
3


Normalize the counts for each sample, then determine the concentration in g/mL of manganese
in your original unknown solution as received in the vial.

IN YOUR REPORT
What element is the decay product of
56
Mn? Include all the nuclear reaction equations for the
processes necessary to this experiment. (Example: radium decay can be written as:
226
Ra
222
Rn +
4
)

Describe how the scintillation counter works; what is it that is actually counted?
Describe how the Am-Be source produces neutrons.

BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 32.
2. Ehman, W.D.; Vance, D.E. Radiochemistry and Nuclear Methods of Analysis. In
Chemical Analysis, Vol. 116, Winefordner, J. D.; Kolthoff I. M., Eds.; Wiley: New
York, 1991.
3. LAnnunziata, M.F. Ed. Handbook of Radioactivity Analysis; Academic Press: San
Diego, 1998. (QC795 .H36 1998 in IKBLC)





94

APPENDIX I: Sample Irradiation with the Am-Be Source
The samples are activated with an americium-beryllium neutron source of 1 curie activity. This
is an "alpha neutron" source in which americium is the alpha emitter, and beryllium is the target
material from which neutrons are emitted. Paraffin oil and wax are used to shield the laboratory
from the source.

Note: In the following procedure it is important to record the number of the well used for the
activation of each sample. Attach a Plexiglas rod with a Velcro tip to the lid of each sample
vial. Carefully align one sample vial in the top of each activation well, then simultaneously
lower the vials into the neutron flux. Start a timer and activate the samples for at least 5000
seconds.

Handle the activated samples with rubber gloves. After the samples have been activated,
simultaneously lift the vials from the wells with the Plexiglas rods. Remove the rods and insert
the vials into consecutive holes of the conveyor of the scintillation counter. Count the samples
sequentially as described in Appendix 2.

Once all samples have been counted, pour the activated solutions into the waste bottle provided.
The isotopes will be allowed to decay for at least 10 half-lives before disposal of the solutions.
"Wipe tests" are legally required on a daily basis to check laboratory work areas for
contamination by radioactive species. The activation products of the 1-curie neutron source used
in this laboratory do not produce detectable levels of high energy gamma radiation. However a
simple test can still be made for contamination by activation products such as
56
Mn.

Upon completion of your experiment wipe the working area with a "Kim Wipe" tissue dampened
with methanol/water. Place the test tissue into a scintillation vial, and add water and methanol.
Similarly prepare a blank sample vial containing a fresh tissue, methanol, and water. Measure
the Cerenkov radiation of the vials using a 1 minute counting period. Counts of less than 100 per
minute above background are considered to be indicative of low-level (acceptable)
contamination. Report the test results on the posted table.

APPENDIX II: Operating Instructions for the Perkin Elmer Tri-Carb 2910 Scintillation
Counter
Note: this instrument is quite new; it was purchased in October 2010 at a total cost of about
$75,000. It is used for both teaching and research within the department.

Like most other counters, this instrument is capable of handling a large number of samples
simultaneously, but counting must be done sequentially. Cassettes holding sample vials are
moved around the deck of the counter. Each cassette is associated with an assay (a set of
counting instructions) by the use of a protocol flag. The flag is a black plastic label that sits on
the left side of the cassette, as shown in Fig. 2. The flag can be set so that the instrument counts
the samples or so that the instrument bypasses counting that cassette. To set the flag for
counting, gently push the tab to the left.


95


Figure 2. PE TriCarb Counter Cassette

COUNTING TRITIUM DECAY
Place the tritium (
3
H) vial into the leftmost position in the cassette, then open the lid of the
instrument and place the cassette on the right-hand side, parallel to the front of the instrument.
Ensure the flag is set for counting. On the left-side of the main software screen, choose the flag
number of your cassette, then right-click and choose Associate Assay. Select
3H_Chem_3XX.isa and click OK. This assay will count the sample for 0.5 minute intervals, 25
times. Data will be printed and also saved to a datafile. To start the counting process, click the
green flag button . If you need to stop the counting prematurely, click the checkered flag
button.

AUTOMATED COUNTING OF MANGANESE SAMPLES

Once the
3
H counting is finished, remove the tritium sample vial from the cassette and place it in
the middle storage section of the instrument.
Load your samples sequentially into the cassette, starting with the least concentrated sample in
the leftmost position. Your unknown sample should be in the middle of the group and your
blank sample in the rightmost position. Set the cassette flag for counting by sliding it gently to
the left.
An assay has been developed for you to use for counting manganese decay, but you should open
it to check that the parameters havent been changed accidentally. Click File then Open Assay,
followed by choosing 56Mn_Chem_3XX.isa. The assay type should be CPM. Click Count
conditions and then verify the following settings:





96


Radionuclide: Mn56
Count Mode: High Sensitivity
Pre-count Delay 0.00 min
Count Time 10.00 min

Quench Indicator SIS
Assay Count Cycles 1
Repeat Sample Count 1
#Vials/Sample 1

2 Sigma % Terminator should be set to Any region and 4.50%. This sets the counting time to
be a maximum of ten minutes but it will stop counting early if the two standard deviation
uncertainty of the measurement falls below 4.50%.

Click Count Corrections and verify the following settings
Static Controller Coincidence Time 18ns
Luminescence Correction Delay before Burst 75ns
Apply Half-life Correction should be OFF (box not checked)

Click OK after verifying these parameters. If any parameters need to be changed, consult your
TA or the lab director.

On the left side of the main screen, choose the flag number of your cassette, then right-click and
choose Associate Assay. Select 56Mn_Chem_3XX.isa and click OK.

Once your sample vials have been placed in the cassette and the tab set, use the software to
initiate counting: on the left side of the main screen, choose the flag number of your cassette,
then click the green flag button in the top left corner to start the measurements. Data will be
printed and also saved to a datafile. If you need to stop the counting prematurely, click the
checkered flag button.


97

A-13 Determination of Nitrite and Nitrate by Ion Chromatography

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the processes that allow ion separation in ion chromatography
Describe the components of the instrument including the pumping system, the column
and the detector system
Explain the advantages and disadvantages of using peak height compared to peak area
as your analytical signal
Recognize the general lab safety concerns and plan to use best practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of NO
2
-
and NO
3
-
in your unknown using the
calibration curve method after applying appropriate dilution factors
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice


BACKGROUND
Ions in solution may be separated and quantified using ion exchange chromatography, an HPLC
technique in which the stationary phase is a weak ion exchange resin. Resins made up of
negatively-charged groups (for cation exchange) and of positively-charged groups (for anion
exchange) have been developed. In this experiment the column packing is a low-capacity
surface-sulfonated styrene-divinylbenzene resin to which have been bonded aminated latex
beads. The eluant is a carbonate/bicarbonate buffer. The anions of the analyte and the
bicarbonate buffer are in equilibrium with the aminated sites of the resin as follows:
resin-N
+
HCO
3
-
+ Na
+
X
-
resin-N
+
X
-
+ Na
+
HCO
3
-

where X
-
is an analyte anion. Separation of the analyte anions depends upon their relative
affinities for the aminated sites.

Ions are conveniently detected using a conductivity detector. To enhance the measurement it is
necessary to suppress the high background conductivity of the carbonate/bicarbonate eluant. A
cation exchange device between the separator column and the detector converts the bicarbonate
anions of the buffer to carbonic acid, a weak electrolyte of low conductivity (<20S), and the
analyte anions to strong acids of high conductivity (i.e. sulfuric acid, nitric acid etc.). Whereas

98

conventional ion exchange columns become saturated and require periodic regeneration, the ion
chromatograph is equipped with a membrane suppressor that is regenerated continuously. The
eluant and sample anions flow in one direction over a cation-permeable membrane, while a
regenerating solution of sulfuric acid flows continuously and simultaneously in the opposite
direction over the opposite side of the membrane.

The concentrations of nitrite and nitrate anions in solution are quantified simultaneously using
this technique. Other analytical methods for determining nitrogen speciation typically involve
the elemental analysis of total nitrogen followed by a separate analysis of one of the anions, and
the determination of the second anion by difference. The advantages of the analysis by ion
chromatography soon become apparent.

PROCEDURE
Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few
millilitres of deionized water and add the washings to the flask. Dilute to the mark with
deionized water. Mix well.

To seven 50 mL volumetric flasks add solutions containing 1.00E-04 g/mL nitrite, 2.00E-04
g/mL nitrate, and diluted unknown as follows:

Flask
mL NO
2
-

(1.00 E-04 g/mL)
mL NO
3
-

(2.00 E-04 g/mL)
mL
diluted
unknown
Dilute to
(mL)
1 0 0 0 50.00
2 1.00 1.00 0 50.00
3 2.00 2.00 0 50.00
4 3.00 3.00 0 50.00
5 4.00 4.00 0 50.00
6 5.00 5.00 0 50.00
7 0 0 10.00 50.00

Refer to Appendix I for operating instructions for the Metrohm Ion Chromatograph.




WARNING: Sulfuric acid solution is used to regenerate the suppressor. Sulfuric
acid is corrosive and acts as a strong oxidizer. Avoid skin contact. Rinse
immediately with cold water if it occurs.


99

IN YOUR REPORT
Compare the result for peak height vs. peak area; justify in the report your choice of peak area or
height result. Report only one result for [nitrate] and one for [nitrite] on the first page. In
comparing your results, make sure to use the appropriate statistical tests to determine whether the
results agree. Discuss the principles of ion chromatography thoroughly.

BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 28 (especially 28F).
2. Harris, D.A. Quantitative Chemical Analysis, 8
th
ed.; W.H. Freeman: New York, 2010;
chapters 25-1 and 25-2.
3. Fritz, J.S. Ion Chromatography, Wiley-VCH: New York, 2000. (QD79.C453 G52 2000
in IKBLC)
4. Haddad, P.R. Ion Chromatography: Principles and Applications, Elsevier: New York,
1990. (QD79.C453 H33 1990 in IKBLC)






















100

APPENDIX I: Operating Instructions for the Metrohm 883 Ion Chromatograph
Overview:
Transfer ~15 mL of the diluted unknown solution (i.e. flask 7 solution) into a 25 mL beaker.
Follow the instructions below and start by running chromatograms of the following solutions:
the diluted unknown,
the diluted unknown solution to which has been added ~1 to 2 mL of a
solution containing 1.00 E-04 g/mL nitrite, and
the diluted unknown to which has been added ~1 to 2 mL of a solution
containing 2.00 E-04 g/mL nitrate.

This technique is called spiking and is used to identify which chromatographic peak is due to
the NO
2
-

and which due to the NO
3
-
.
Run a chromatogram of each standard solution (flasks 1 to 6) and three duplicate chromatograms
of the unknown solution (two additional runs if your first one looked ok). Integrate the peaks in
each chromatogram and tabulate in your lab notebook data for each run. Data should include:
retention time, peak height and peak area for each of nitrate and nitrite.
Use both the peak height and peak area data to calculate the concentrations in g/mL of nitrite and
nitrate in the original sample vial. (Note: Inspect the plots carefully before deciding upon the
best method of calculation.)

Instrument Use
This instrument is fully-computer controlled. To begin, ensure the instrument itself is powered
on by verifying a green light is present at bottom left of the instrument front panel. If not, the
power switch is located on the rear. Load the MagicIC software from the desktop icon. Start
the instrument hardware by clicking the green Start HW button as shown in the Figure 1
below. This starts the flow of both eluent and acid-regenerant solution. The conductivity should
settle to a baseline of between 15-20 :S/cm with dips-and-rebounds every 10 minutes of
inactivity.

Under the Single Determination tab, select CHEM3XX_A13 as the method to use. This
loads basic experiment parameters: eluent flow rate, column type, etc. In the Remark field,
enter your initials. Provide a sample name in the Ident field. At the front of the instrument,
remove the tubing from the volumetric flask of water, wipe it with a clean kim-wipe and then
insert the tubing into your beaker of sample.


101

Figure 1. MagicIC Software Layout
Click the Start button. You now have 1 minute to fill the sample loop of tubing with the
solution from your beaker. Do to this, pull gently on the syringe. The sample loop has a volume
of 20:l, so drawing up 2 ml into the syringe is adequate to rinse and fill the tubing and the loop.
Do not allow the end of the tubing to come out of your solution. Remove pressure from the
syringe but leave the syringe in place. At the end of the 1 minute period, the instrument will
automatically re-route eluent flow through the sample loop, pushing the sample onto the column.
Each run will take approximately 15 minutes.

To evaluate the data, select the Database icon at left. Choose the datafile you wish to process;
the most recently saved file is at the bottom of the list. Select Determinations then
Reprocess from the top menu. The reprocessing window opens with four panels. The bottom
right panel shows the chromatogram. To zoom into the data, drag a box around the area of
interest. To zoom out, right-click and select Show all. The bottom left panel describes the
parameters that will be used in the processing. Select Integration and set Smoothing to 20
and Sensitivity to 30. These are reasonable starting values for integrating your
chromatographic peaks; you may need to adjust these later. Click the Components button and
enter as many components (named as 1,2,3,) as you have peaks. Enter the retention time for
each peak. Click Update to integrate your chromatogram. The table at top right displays peak
data for each component: retention time, peak height, and peak area.

Print two chromatograms:
1) An overlay of each of your standard solution runs. To do this, select all the relevant datafiles,
then choose Determinations, Overlay curves then select Print PDF. Once the pdf file is

102

created, you can print a hardcopy. If you are having trouble selecting datafiles, CONTROL +
click on the time the determination started.
2) A chromatogram overlaying your three determinations of your unknown. Proceed as above,
printing one copy of the pdf file that is generated.

Shutting Down the System:
After your determinations are complete:
Flush the sample introduction tubing by starting a run of deionized water. Fill the
sample loop as before. After the instrument has injected the sample, you can stop the
run.
Leave the instrument on for at least 10 minutes before shutting down the hardware
(red Stop HW button on the workspace screen).
After shutting down the hardware, power it off and shut-down the computer and
monitor.




103

A-14 Determination of Biphenyl and p-Terphenyl by Liquid Chromatography

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the processes that allow analyte separation in reverse phase liquid
chromatography
Describe the components of the instrument including the pumping system, solvent
choice, the column and the detector system
Explain the advantages and disadvantages of using peak height compared to peak area
as your analytical signal
Recognize the safety concerns associated with methanol use and plan to use best
practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of biphenyl and p-terphenyl in your unknown
using the calibration curve method after applying appropriate dilution factors
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice


BACKGROUND
Involatile, non-polar compounds such as aromatic hydrocarbons can be determined by reverse-
phase high performance liquid chromatography (RP-HPLC). In reversed phase chromatography
the stationary phase is non-polar (the typical column material consists of silica gel with
octadecylsilane (-Si-C
18
H
37
) chemically bonded to the surface) and the mobile phase is relatively
polar. The eluents in this experiment will consist of mixtures of methanol and water.

The retention times for species eluting from the column depend upon the nature of the
compounds, the column packing, and the eluent composition. Elution with a single solvent
composition ("isocratic elution") may adequately separate certain species while leaving others
unresolved. This is an example of the so-called general elution problem. In liquid
chromatography, peak shape and separation can be improved significantly by changing or
"ramping" the composition of the solvent during the course of the elution, a technique called
gradient elution. Similar gradient-based techniques have been developed to address the
general elution problem in other forms of chromatography.

A Hewlett-Packard model 1100 liquid chromatograph will be used to analyze biphenyl and p-
terphenyl in a complex solution matrix. Chromatograms prepared under isocratic conditions, one

104

with 90:10 (% volume) methanol:water and the second with 75:25 methanol:water, will be
compared. Chromatographic conditions will then be optimized using gradient elution.
Peaks of the analytes in the unknown mixture are identified by "spiking" the mixture with
aliquots of the pure analytes in turn, and repeating the runs. Initially detection is by UV
absorption at 254 nm. Once the peaks have been identified, the detector is programmed to
monitor each analyte at its absorbance maximum (278 nm for p-terphenyl and 248 nm for
biphenyl).

PROCEDURE

NOTE: Dispose of all methanol waste into the appropriately-labeled red plastic container in
the fume hood.

Using a dry 10 mL pipette cleaned with methanol pipette 10.00 mL from your unknown
vial to a 50 mL volumetric flask. Dilute to the mark with methanol. To save time, proceed
immediately to the following preliminary tests. The standard solutions described on the
following pages can be prepared while the chromatograms are being run.

NOTE: You MUST pipette 10.00 mL of the unknown solution do not use all of it!

Refer to Appendix I for operating instructions for the pumps, detectors, and data acquisition
systems.
Make the appropriate instrument settings (Appendix I(a)) and run a chromatogram of the
unknown solution under isocratic conditions using 90:10 methanol:water as the eluent and a
detection wavelength of 254 nm.
Allow at least 5 minutes for all components to elute. Run a second chromatogram using
75:25 methanol:water. Allow at least 15 minutes for all components to elute.

Refer to these chromatograms and program the pump to deliver an elution gradient as follows
(Appendix I(a)(ii)):

deliver 75:25 methanol:water for sufficient time to allow the first group of two or
three major peaks to elute. (The first minor peak(s) are from the solvent.)
increase ("ramp") the methanol content of the eluent during the next 2 minutes to a
final composition of 90:10 methanol:water.
deliver 90:10 methanol:water for sufficient time to allow the last peak(s) to elute.
WARNING: Methanol is toxic primarily by ingestion and highly flammable. Keep
bottles capped when not in use. Avoid skin contact and rinse with cold water if it occurs.
Waste methanol must be placed in red solvent waste cans.

Biphenyl and p-terphenyl are both skin/eye irritants and toxic by ingestion. Avoid skin
contact with both. Wash with soap and cold water if it occurs. Both are harmful to
aquatic life.

105


Run a gradient chromatogram of the unknown test sample under these conditions.

Spike about 10 mL of the diluted unknown solution with ~1 mL of 2.00 E-05 g/mL biphenyl
solution. Run a gradient chromatogram. Spike the same solution with 1 mL of 1.00 E-05 g/mL
p-terphenyl solution. Run a gradient chromatogram. Compare the chromatograms and identify
the peaks corresponding to the analytes. Finally, program the detector (Appendix I(b)(ii) or
II(b)(ii)) to monitor each analyte at its optimum wavelength.

Using the 2.00E-05 g/mL biphenyl and 1.00E-05 g/mL p-terphenyl stock solutions, prepare at
least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not
exceed 2.30E-06 g/mL biphenyl and 1.25E-06 g/mL p-terphenyl in the most concentrated
solution. All solutions should be prepared in HPLC-grade methanol. Pipets of 1, 1.5, 2, 2.5,
3, 4, 5 and 10 mL volume and 50mL volumetric flasks are provided. Ensure each solution is
well mixed.

Use the programmed instrument settings to run gradient chromatograms of the diluted unknown
and solutions 1 to 6. Plot calibration curves of peak height and peak area vs. concentration of
each standard. Calculate the concentrations of biphenyl and p-terphenyl in the original vial.

IN YOUR REPORT
Compare the result for peak height vs. peak area, report only one value on the first page, justify
in the report. In comparing your results, make sure to use the appropriate statistical tests to
determine whether the results agree. Discuss the principles of reverse phase high pressure liquid
chromatography thoroughly.

BACKGROUND READING
1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 28.
2. Harris, D.A. Quantitative Chemical Analysis, 8
th
ed.; W.H. Freeman: New York, 2010;
chapters 24.
3. Ahuja, S. Trace and Ultratrace Analysis by HPLC, Wiley: New York, 1991.
(QD79.C454 A38 1991 in IKBLC)
4. Hanai, T. HPLC: A Practical Guide, Royal Society of Chemistry: London, 1999.
(QD79.C454 H356 1999 in IKBLC)


106

APPENDIX I: Operating Instructions for the HP1100 HPLC System

(a) Isocratic Elution with Detection at a Single Wavelength:

The HP1100 system consists of four modules the variable wavelength detector (VWD),
the pump, the vacuum degassing module, and the solvent module. The modules are
stacked vertically with the VWD on the bottom.
Check that reservoir A is at least half-filled with deionized water, and reservoir B is at
least half-filled with HPLC-grade methanol. If solvents must be added, get assistance!
The solvents must be specially-filtered.
Turn on the power switches at the bottom left of the detector module and the pump. The
vacuum degasser is left on permanently.
Turn on the computer. Ignore the request for a password (press Cancel). Double click
the LC ChemStation icon to initialize the software. Check that the Method and Run
Control window is opened and the following graphical representation of the instrument
is displayed. If not, click View then select Method and Run Control and turn on
System Diagram.




The injector is at the left, followed by the pump, the column, the detector, and the acquired
signal. Note: the order of this representation is incorrect! (In what way?) The remaining
symbols represent data processing that will not be done in this experiment. In the following
procedures the pump will be controlled by clicking ( ) with the mouse. Click ( )
to control the detector. The buttons will be used to ignite the deuterium lamp of the
detector and turn on/off the pump.



107

(i) Setting the pump:
Click ( ), then Set up Pump to display the following window:



In the Control box:
set Flow to 1.000 mL/min.
set StopTime to no Limit. At this setting the chromatographic runs will
proceed until they are stopped manually.
set PostTime to Off. The post time allows the eluent composition to
restabilize after a gradient run. It is not required for isocratic elutions.

In the Solvents box set the percent of solvent B: (methanol) at 90% or 75%. The percent of
solvent A (water) will be set automatically.
The Timetable box will be used later for defining elution gradients. For isocratic runs there
should be no entries in the Timetable box.
Click OK.
(ii) Setting the detector:
Click ( ), then Set up VWD signal to display the following window:

108



In the Signal box check that the Wavelength is set to 254 nm.
In the Time box set the Stoptime to as Pump, no Limit, and the Posttime to Off. These
settings of the detector will not conflict with the time settings of the pump and will be used for
the duration of the experiment.
The Timetable box will be used later to program the detector to monitor each analyte at its
optimum wavelength.
Click OK.

(iii) Running the chromatogram:

To ignite the lamp and start the pump, click the on button at the right of the Method & Run
Control screen. Do not switch the lamp off until the end of the experiment! Wait a few minutes
for the displayed baseline to stabilize.
Click RunControl at the top of the Method & Run Control window, then Sample Info and
make the appropriate entries into the following table:

109















At the start of the experiment enter:
Operator Name:
your name
Subdirectory:
C3XX
Prefix:
your initials
Counter:
000000

Before running each new sample enter Sample Name and any Comments. Click OK.
Rotate the injection valve lever to the 2:00 o'clock position. Use the 50 L syringe to flush the
valve 2 or 3 times. With the syringe needle still inserted in the valve, rotate the lever to the stop
(at the 4:00 o'clock position). Remove the syringe.

When a run is in progress, the chromatogram will be plotted in the signal box at the bottom left
of the Method & Run Control screen. Click

in the signal box to open a larger view of the
chromatogram. Click the buttons in the bottom left corner of this new display to rescale the
peaks. Once all peaks have eluted, click RunControl then Stop Run/Inject/Sequence to stop
the run.
Print the displayed chromatogram. Close the display of the chromatogram.



110

(b) Gradient Elution with Optimized Detection:

(i) Programming the pump and running the chromatogram:
In the next steps the pump will be programmed to elute 75:25 methanol:water until the first
group of two or three peaks has just eluted. The eluent composition will then be ramped to
90:10 methanol:water over a 2 minute interval. This composition will be maintained until the
last peak has eluted.
Click ( ), then Set up Pump to display the window shown in
Appendix II(a)(i).
In the Solvents window set initial composition of solvent B: (methanol) to 75%.
Click Append in the Timetable window. This opens the first row of the program
timetable for the pump. Enter 0 min in the Time column. Enter 75% in the %B
column. This timetable entry will set the initial eluent composition for all runs to 75:25
methanol:water.
Click Append to open the second row of the timetable. Refer to the test isocratic
chromatogram that was run using 75:25 methanol:water. Our goal is to set the instrument
to begin the gradient just after the first group of peaks has eluted from the column. You
might expect that the best time to input is the time the absorbance returns to the baseline
after the first group of peaks, but this is not the case. All chromatography instruments
have a dead time the time required to pump through a non-retained species. In
particular here, there is a finite time required for compounds to pass from the end of the
column to the detector. You can subtract ~30s from the time that your absorbance returns
to the baseline and use this as an estimate of the time those compounds eluted from the
column.
Enter the time determined above. The gradient will be programmed to start at this time.
Re-enter the initial % B (probably 75% methanol).
Click Append. Enter the time at which the gradient will end, approximately 2 minutes
after the elution time of the first group of peaks. Enter the desired % B at the end of the
gradient (probably 90% methanol). Click OK.

Set the detector to monitor 254 nm or, if you have determined the elution order of the analytes,
set it to monitor each analyte at its
max
(Appendix I(b)(ii)).

Rotate the injection valve lever to the 2:00 o'clock position. Use the 50 L syringe to flush the
valve 2 or 3 times. With the syringe needle still inserted in the valve, rotate the lever to the stop
(at the 4:00 o'clock position). Remove the syringe.
Once all peaks have eluted, click RunControl then Stop Run/Inject/Sequence to stop the run.
Print the displayed chromatogram.
After the first gradient run click ( ), then Set up Pump. Re-program the StopTime
to terminate subsequent runs automatically after all peaks have eluted. Set PostTime to 3 min to

111

allot 3 minutes at the end of each run for the eluent to restabilize at its initial composition in
readiness for the next run.

(ii) programming the detector:
Once the peaks for the analytes have been identified by spiking, and their retention times are
known, the detector can be programmed to switch wavelengths at timed intervals so that each
analyte is monitored at its optimum wavelength.
Click ( ), then Set up VWD signal to display the window shown in
Appendix II(a)(ii).
Refer to the test chromatograms of the spiked unknown sample. In the Signal box set
the default Wavelength to the
max
of the analyte of interest that will elute first.
In the Timetable box click Append to open the first row of the timetable. Enter 0 in
the Time column. In the Wavelength column enter the
max
of the analyte that will elute
first. Turn on ( ) the Balance option to zero the baseline.
Click Append to open the second row of the timetable. Enter a time between the elution
times of the two analytes of interest. Take care to program the wavelength to switch at a
time when the signal is at baseline and is not indicating a peak. In the Wavelength
column enter the
max
of the second analyte of interest.
Click OK. This completes the program.

(c) Manual Reprocessing of Data:

The computer selects peak baselines according to preset criteria, and calculates heights and areas
based upon these baselines. These criteria may not be optimal for all peaks. It may be better to
set the baselines of some peaks manually.
Check the baseline processing of each chromatogram. If any require re-drawing, select View
from the menu bar, then Data Analysis. Click Files, then Load Signal. Your files are in the
[C3XX] directory. Check the file number that is printed on the top of the selected
chromatogram, and open the data file.

Get help to enable the manual re-draw button , as it was disabled when the program was
started (an unavoidable nuisance!) Click then hold down the left button of the mouse and
draw the baseline.
Record the recalculated values for peak height and area on your original chromatograms.



112

(d) Shutting Down the HP1100:
Use the syringe to flush the injector 3 or 4 times with HPLC methanol.
Click View then select Method and Run Control to return to the main screen. Once
all samples have eluted from the column, press the Off button to shut off the pump
and the deuterium lamp.
Turn off the main power switches on the detector and pump. Do not turn off the
power to the vacuum degassing unit!

113

A-16 Determination of Naphthalene by GC-MS

LEARNING OBJECTIVES

Before commencing the experiment, you should be able to:

Describe the processes that allow analyte separation in gas chromatography
Describe the components of the instrument including the injector, carrier gas, the
column, vacuum system and detector system
Explain the advantages and disadvantages of using peak height compared to peak area
as your analytical signal
Describe the reasons an internal standard is required for this GCMS method and
explain the principles of choosing an appropriate internal standard
Recognize the safety concerns associated with methanol use and plan to use best
practices.

On completion of the experiment and lab report, you should be able to:

Correctly calculate the concentration of naphthalene in your unknown using the
calibration curve and internal standard methods after applying appropriate dilution
factors
Apply fundamental statistics to collected data and calculate the confidence interval of
your result
Improve your accuracy and precision of liquid handling (pipeting, filling of
volumetric flasks, etc.) through practice


BACKGROUND
GC-MS is a combined analytical technique whereby components in complex mixtures are
separated chromatographically, then are detected by mass spectrometry. The separated
compounds elute directly into the mass spectrometer where they are ionized, with the ions of the
molecules and molecular fragments being counted and stored as a function of mass-to-charge
ratio (m/z). Entire mass spectra are acquired at a rate of about two per second. The MS data can
be re-processed and output in the following ways:

reconstructed ion chromatogram (RIC) a plot of the summed totals of the counts
for all ions in each mass spectrum vs. time. RICs are equivalent to chromatograms
output by conventional detectors such as FIDs or TCDs.
extracted ion chromatogram (EIC) plots of counts vs. time using data selected at
m/z ratios unique to particular analyte ions. EIC plots exclude the data for most non-
analytes, thereby improving significantly the signal-to-noise ratios. EIC plots can also

114

resolve by mass those components that are not otherwise chromatographically
resolved. This allows the use of deuterated isotopomers as ideal internal standards.
mass spectra plots of counts vs. m/z ratios of the ions of the eluted compounds.
Mass spectra provide qualitative information about the analytes. Molecular structures
can be determined directly from the m/z count patterns of the ions of the molecules
and molecular fragments, or by comparison of the mass spectra with data stored in a
library.

Note: Your Principle of Method should include a functional description of the gas
chromatograph and its interfacing with an MS detector. However, an outline of the ion trap mass
spectrometer itself is provided here:
















The Varian GCMS used in this experiment is equipped with a capillary chromatographic column
internally coated with 5% phenylpolysiloxane and 95% dimethylpolysiloxane (the stationary
phase). The carrier gas is helium. Analytes elute directly from the column into the cavity of an
ion trap mass spectrometer. This cavity is defined by a ring electrode and two cap electrodes. A
70eV electron beam from a heated filament ionizes the molecules. The ions initially are trapped
in the cavity by the field of a radio frequency (RF) storage voltage applied to the ring
electrode. The RF voltage is then ramped such that the ions, in order of increasing m/z ratio,
oscillate axially towards the cap electrodes. At critical RF voltages the ion trajectories exceed
the trapping field, and the ions are ejected sequentially in order of increasing m/z ratio
through holes in the bottom end cap where they are detected by an electron multiplier and are
counted as a function of m/z.

Ion traps can be programmed to isolate ions of a single selected mass (m/z). These ions in turn
can be further fragmented by collision induced dissociation (CID) to produce a second set of
fragment ions which can be counted and stored as a second mass spectrum. This procedure,
known as MS-MS, can be repeated sequentially. The analysis of successive mass spectra
provides additional structural information about the original molecule.



115


PROCEDURE
Before the laboratory period you should record into your notebook the structures and molecular
weights of naphthalene and its fully-deuterated isotopomer, naphthalene-d
8
, which will be used
as an internal standard. Without this information you cannot process your data.


Using 1.00E-05 g/mL naphthalene stock solution prepare at least five (preferably six) calibration
standard solutions. Start with zero concentration (blank) and do not exceed 1.20E-06 g/mL
naphthalene in the most concentrated solution. Dilute your unknown solution five-fold. Add
sufficient 5.00E-06 g/mL naphthalene-d
8
stock solution to make each solution (standards
and unknown) 5.00E-07 g/mL in the deuterated compound. Dilute each solution to the mark
with methanol. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric flasks
are provided. Ensure each solution is well mixed.

NOTE: You MUST pipette 10.00 mL of the unknown solution do not use all of it!

Refer to appendix 1. Inject 1 L samples and run chromatograms of the blank solution, the
standards, and the unknown. Once all chromatograms have been run, refer to TREATMENT
OF DATA and locate the peak(s) for naphthalene and naphthalene-d
8
. Construct EIC plots for
naphthalene and naphthalene-d
8
in the standard solutions and the unknown. Determine the areas
of the EIC plot peaks. Using naphthalene-d
8
as the internal standard, calculate the concentration
of naphthalene in the original unknown solution. Use the software library search function to
check the mass spectra corresponding to the peaks in the RIC chromatogram of the unknown
sample.

IN YOUR REPORT
Thoroughly describe the principles and operation of both the separation-side of the instrument
(the GC) and the detection-side (the MS). Discuss the internal standard method, including why
deuterated isotopomers are ideal internal standards for GC-MS. Identify the contaminants in
your unknown and comment upon as many eluted species as possible. Suggest source(s) for the
species corresponding to background peaks. Why does the naphthalene mass spectrum show a
strong line for the molecular ion?
Note that naphthalene is spelled with a phth.

WARNING: Methanol is toxic primarily by ingestion and highly flammable. Keep
bottles capped when not in use. Avoid skin contact and rinse with cold water if it occurs.
Waste methanol must be placed in red solvent waste cans.
Naphthalene is a skin/eye irritant and toxic (known carcinogen). Harmful to aquatic life.
Avoid skin contact; wash with soap and cold water if exposure occurs.


116

BACKGROUND READING

1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6
th
ed.;
Thomson Brooks/Cole: Belmont, CA, 2007; chapter 20 and 27.
2. Harris, D.A. Quantitative Chemical Analysis, 8
th
ed.; W.H. Freeman: New York, 2010;
chapters 21 and 23.
3. Grob, R.L. Ed. Modern Practice of Gas Chromatography, 4
th
ed.; Wiley: New York.
2004. (QD79.C45 M63 2004 in IKBLC).
4. Watson, J.T. Introduction to Mass Spectrometry: instrumentation, applications and
strategies for data interpretation, 4
th
ed. Wiley: Hoboken, NJ. 2007. (QC454.M3 W38
2007 in IKBLC)
5. Hubschmann, H.-J. Handbook of GC/MS: fundamentals and applications. Wiley-VCH:
New York. 2001. (QD272.C44 H83 2001 in IKBLC)



117

APPENDIX: Operation of the Varian Saturn GC-MS

The instrument is turned on at all times. Initially the System Control window should be
displayed with the DailyChecks method running as follows:


NOTE: Care must be taken in using the GC-MS. MS detection is extremely sensitive, and a
gas leak could damage the detector. Before using the instrument ask the TA or the lab
director to do a routine check of the instrument. Use only the method file that has been set
up for your experiment. This ensures the detector will be turned on only after air and solvent
have eluted from the column. The method also sets a mass range that avoids detection of
these peaks, and sets proper temperatures for the injector, oven, and detector.


118

The CHEM 3XX A16.mth file: sets the following automated sequence for separating and
detecting naphthalene and the other compounds in the solution matrix:

Initial settings the GC oven is set at 55 C. The injector operates in splitless mode with 1
mL/min of helium flowing through the injector into the column.
0 to 0.7 minutes the automated sequence is initiated when the sample is injected. For the first
0.7 minutes the sample is vaporized in the 250 C injector. The semi-volatile analytes
concentrate at the head of the 55 C column.
0.7 minutes the injector switches to the split mode allowing ~80 mL/min of helium to flush
residual solvent and analyte from the injector to a split vent. The 1 mL/min He flow through
the column is maintained. This is known as an 80:1 split ratio.
0.7 to 4 minutes the oven temperature starts ramping at a rate of 14 C/min. The relatively
low boiling solvent elutes first as the oven temperature increases.
4 minutes the mass spectrometer is turned on only after air and solvent have fully eluted from
the column. m/z ratios from 70 to 250 are monitored.
4 to 11.7 minutes as the temperature increases, analytes elute sequentially and are detected.
An RIC chromatogram is plotted.
11.7 minutes the oven temperature reaches 210 C and the run terminates. The mass
spectrometer is switched off and the oven temperature starts cooling to 55 C in preparation for
the next run.


You will use only two buttons on the main toolbar at the top of the display:
Selects the System Control window shown above. This window is used to check the
status of the instrument and to control the instrument during chromatographic runs in accordance
with appropriate method files.

Selects the MS Data Review window. Data can be re-processed after the runs have been
completed. Mass spectra can be recalled, and analytes can be identified through library searches.
Chromatograms can be constructed using the data for selected ions (EIC plots), and
chromatographic peak areas can be determined. In the System Control window click File,
Activate Method, select the CHEM 3XX A16.mth method file, and click Open.


Click Windows then 3800.44-Equilibrating to monitor temperature stabilization of the GC.
Wait until 3800.44-Ready is displayed. (If 2000.40-Not Ready is still displayed, this
indicates the mass spectrometer has not yet been turned on.)


119

Click Inject, Inject Single Sample. Enter the sample name in the Sample Name box in
the Inject Single Sample dialog window. Click the Inject button and wait for the status
indicator to change from NOT READY to WAITING.

Thoroughly flush a 10 L syringe several times with the sample solution. Remove the needle
from the solution, fully depress the plunger, and then draw in 1 L of air. Put the needle tip into
the solution and withdraw the plunger to the 2 L mark. Finally draw in a further 1 L of air.
The syringe barrel now contains 1 L of sample solution separated from the plunger and the
needle tip by two 1 L plugs of air.
IMPORTANT! Proper injection technique is critical, and it varies with the particular analyte.
Solutions of naphthalene in a complex matrix should be injected at a slow consistent rate. Over
an interval of ~2 seconds insert the needle into the front injector, then depress the plunger at a
consistent rate over a time interval of ~2 seconds. The automated sequence will start. The
chromatogram and/or the mass spectrum can be monitored in real time during the run. Choose
Chromatogram and Spectra in the box at the left of the screen. Click Hide Keypad to
expand the display. The run will be monitored as follows:



While the run proceeds, use the cursor to expand the display of any peaks of interest in the
chromatogram. (You can use the key to return the display of the full chromatogram.) Click
then click with the cursor on the chromatographic peak. The corresponding mass spectrum

120

will be displayed. Locate the peak(s) for naphthalene and deuterated naphthalene keeping in
mind their molecular weights. The run will terminate automatically once the data has been
acquired and saved. Do not print the chromatogram at this time. Click Show Keypad or you
will not be able to start another run.

Similarly run chromatograms of the standards and the unknown solution. When all runs have
been completed, click File, Activate Method, and activate the DailyChecks.mth file.
Leave the instrument in this condition.


TREATMENT OF DATA
Click in the main tool bar to open the MS Data Review window. In the Select Plot(s)
window open the data file for the RIC chromatogram of the unknown solution. Use the cursor to
highlight and expand the first few peaks. Click with the cursor to select and display mass spectra
at two or three points on each peak. Significant differences between mass spectra measured at
different points on a chromatographic peak are indicative of co-eluting compounds with slightly
different retention times. Similarly rescale and check the other peaks in the chromatogram.
Locate the peak(s) for naphthalene and naphthalene-d
8
. The mass spectrum for each of these
compounds should include a strong line at the m/z value corresponding to the molecular ion,
where m/z = MW/(1
+
). (Not all mass spectra include strong lines for the molecular ions. Some
compounds are highly fragmented during ionization, whereby the strongest spectral lines
correspond to the m/z values of the fragment ions.)

To construct a EIC plot for naphthalene, click

in the Spectra window. Enter the m/z ratio of
the strongest line in the mass spectrum (assuming the m/z value is unique to naphthalene). Click
Plot. The EIC plot will be displayed below the original RIC chromatogram. Similarly
construct a EIC plot for naphthalene-d
8
using the m/z value of the strongest line in its mass
spectrum. The RIC chromatogram and the two EIC plots will be displayed as a vertical stack.
Click Quantitation, Integrate All Plots in Time Range, then click Print,

to print a
peak area table. Close the report window. Click

(Print all chromatogram and spectrum
plots). In the Plot Arrangement box select the plots to be stacked vertically and printed in
landscape format. Click Print, to print the plot. Similarly construct and print a set of EIC
plots and a peak area table for each of the standard solutions.

Tabulate all results. Using the data for naphthalene-d
8
as an internal standard, calculate the
concentration of naphthalene in the original unknown solution.

Re-open and compare the RIC chromatograms of the unknown and the blank. Determine which
peaks represent background and which represent contaminant species in the unknown. Click on
each peak to display the mass spectra of the eluted compounds. Click on the mass spectra to run
library searches of the compounds. For each mass spectrum ten possible matches are displayed,
as well as a statistical evaluation of the quality of each match. A correlation >900 is considered

121

to be excellent. Click in the MS Data Review toolbar to Print the Active Chromatogram
and Spectrum Plot. In the Plot Arrangement box select the plots to be stacked vertically and
printed in landscape format. Click Print to print the report. On the printout identify as
many of the contaminant species as possible. Can you suggest the source(s) of some of these
compounds? (Keep in mind that detection by mass spectrometry is VERY sensitive.)
Furthermore, can you suggest the source(s) of the species corresponding to the peaks shown in
the chromatogram of your blank?