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Hanoi University of Science (HUS)

Faculty of Chemistry
Department of Analytical Chemistry

Chem 223

Laboratory of Quantitative Analysis

The Advanced Study Program in Chemistry

(Collaboration with the University of Illinois at Urbana-Champaign)

Lab Manual

Hanoi, 2013

Chem 223: Laboratory Schedule

This is a 2 credit lab course for Vietnamese students who learn chemistry in English. The
laboratory course containing 15 experimental weeks, is mainly based on the lab manual
(Chem. 223) of the University of Illinois at Urbana Champaign (if the experiments are
available). However, in order to fulfill the real analytical tasks and experimental conditions in
Vietnamese laboratories, some parts of experiments were completely changed and some
experiments from other universities also have been added.

To carefully carry out experiments, the class will be divided into groups (under 20 students
each), conducting one experiment in one morning/ afternoon per week from 7.30 am to 11.30
am (or 1.30 pm to 5.30pm). For chemical “wet” analysis, each student has to conduct each
experiment individually. Working in pairs or groups is only allowed for instrumental
experiments. There will be a lecture topic for the lab on the first hour to cover all the methods
and experiments related.

The experiments will be done on the following schedule. The right column is to compare to
UIUC’s experiments.

HUS UIUC (2007)

Week Experiments Week Experiments
1 Course Introduction, Check- in, Safety, 1 Course Introduction,
Regulations (for all classes) Check- in, Safety,
(out of lab) Regulations (for all
(out of lab- lecture topic)
2 Practice with Basic Techniques 2 Practice with Basic
3 EDTA determination of Ni2+ 3 EDTA determination of
4 Acid- base titration for the determination of 4
carbonate and precipitation titration for the
determination of chloride in cheese.
5 Permanganate and dichromate titration for 5 Iodometric determination
the determination of iron in ore of Cu in brass
6 Iodometric determination of Cu in brass 6
7 Ion exchange and volumetric titration for 7 Spectrophotometry, Beer’s
the determination of Co2+, Ni2+ in a mixture law and precision
8 Potentiometric titration for determination of 8 Spectrophotometric
an acid mixture. determination of Cobalt
and Nickel
9 Spectrophotometry, Beer’s law and 9
10 Spectrophotometric determination of total 10 Kinetic method
phosphate in water determination of glucose
11 Kinetics of the oxidation ascorbic acid by 11 GC
hexacyanoferrate(III) iron and ascorbic

determination in vitamin C tablet.
12 F- AAS determination of Zn, Mn in plants 12 Designing an analytical
method for determination
Nickel in ore
13 GC/FID determination of VOCs in air 13
14 R-HPLC determination of caffeine and 14
benzoic acid in soft drink samples
15 Lab final exam on experiment 15
(1. Principles of experimental procedures and
designing an analytical method)
(2. Testing technical skills through the
analysis of real sample)

- There will be a pre-lab quiz in lab before conducting experiments from 2.00 to 2.30 pm
every Thursday. ( - You must complete the pre- lab quiz at least 60% or you will not be
admitted to lab.
- All quizzes will count (no dropping of lowest quizzes).

- Due times are absolute except for medical emergencies or deaths in your immediate family.
- Medical problems require a note from a physician or a letter from an emergency dean. Death
in the family can be validated by the emergency dean.

1. Pre-laboratory Work

You are expected to attend and prepare for the lecture by reading the Chem 223- Lab
Manual, even if your team is not scheduled for that experiment on that week.

Each new experiment will be preceded by a quiz and short lecture. You will be tested on
your reading of the Lab Manual by a short pre-lab quiz. The quiz will be graded by the
TA and returned to you. You will need to keep the pre-lab quiz for attachment to your
laboratory report for credit. Note: you need to be responsible, do not lose the pre-lab quiz!

Before each scheduled experiment for your team, ensure that you have read the Chem 223
Lab Manual and understood what you are about to carry out. You are to prepare a pre-lab
page (about 200 words) on the experiment, which will be collected when your team starts
the experiment (Write in your notebook). The page must describe in your own words the
objectives and the general plan for the experiment. This will be initialed by the TA, and
returned to you. Like the pre-lab quiz, the page must be submitted with your report for

Most of the laboratory work will be performed individually.

The TA’s signature is required in your notebook before leaving the laboratory
after the laboratory period is over.

2. Writing of Lab Reports:

Laboratory reports are submitted individually and sent to the instructors three days
before next experiment.

3. Grading of Lab Reports:

Each lab report is graded based on a maximum of 100 points. The following is a list of
the point value of each section of the lab report.

Case 1: lab reports of experiments that do not have unknowns to be analyzed.

Section PointValue
Introduction 5
Data and Observation 15
Calculation and Results 45
“For your Report” item 25
Conclusion 10
Case 2: lab reports of experiments that have unknowns to be analyzed.
Section Point Value
Introduction 5
Data and Observation 15
Calculation and Results 25
Accuracy 20
“For your Report” item 25
Conclusion 10

Points will be deducted because of the following:

Results that must be recalculated: 3 points
Reporting a value to an incorrect number of significant figures: 3 points
Late reports: 10 points per day. After one week has elapsed after the due date, the report will
not be accepted.
Reporting insufficient data: 5 points
Reissuing new unknowns: 5 points
Poor organization of lab report: up to 20 points

Grading Scale of the course

- Quizzes and your participation in lecture: 20%.
- Short pre- lab quiz and preparation of procedural outline in your notebook: 10%

- Lab report: 10%
- Lab exam - on experiment: 30%
- on principles of experimental procedure and designing an analytical
method: 30%
4. Requirements for conducting experiments in Analytical Lab
All of the following items must be complete before you can begin experiments in lab.

1.) Bring goggles, gloves

2.) No shorts or open toe shoes, all must be wearing a white blouse
3.) Bring a hardcover lab notebook, which is used to record all your data and
observations. Your job, as an analytical chemist, is to document everything you do.
4.) Read the guidelines for class and prepare a pre-lab accordingly in the
lab notebook
a. Introduction
b. Procedure outline and explaination for setting up the procuders (5.)
5.) Take the pre-lab quiz
6.) Bring your signed Laboratory Regulations sheet
8.) Completed Excel Lab

Chem 223: Experiment 1


(This experiment is completely follows the lab. “General information”. Chem 223. UIUC)


If you understand it, sign it and bring it to turn in to your instructors at the first lab.
If there is anything you do not understand, bring the UNSIGNED form to the first lab, ask the
instructor to clarify anything that you do not understand, then sign it and turn it in.

I. Chemistry Laboratory Regulations

General Regulations. The following rules, designed to preserve your health, will be strictly

1. Eye protection must be worn at all times in the laboratory. Regular glasses do not provide
sufficient protection and therefore safety goggles will be required. Never wear contact lenses in
the lab; vapors can be trapped behind the lens and cause irreparable corneal damage. Safety
goggles are available for purchase at safety- work shops. If you are caught just once not
wearing proper protective eyewear in lab, you will be dismissed for the day, and miss finishing
that lab. If you are caught twice, you will be dismissed from the lab for the remainder of the
2. No one is allowed in the laboratory at any time without proper supervision; i.e., a qualified lab
assistant or faculty member must be present.
3. No smoking, food, or beverages are allowed in the laboratory.
4. Shoes must be worn in the lab at all times. Sandals are not allowed because they do not provide
sufficient protection from falling equipment and chemicals.
5. Secure long hair behind the head to prevent it from coming near moving equipment or burners.
6. Your clothes must cover the trunk of your body and your shoulders. A lab apron or coat is also
required and available for purchase at workplace safety shops.

Safety Precautions.
1. Know the location and use of the fire extinguishers, fire blanket, eyewash, safety shower, and
first aid kit.
2. Be careful about smelling chemicals; avoid breathing any vapors. Work in a ventilation hood
when carrying out reactions involving volatile substances or gases. If you must determine the
odor of a compound, fan the vapors towards your nose, and sniff cautiously. Do not taste any
compounds; some are toxic or contain toxic impurities. Avoid prolonged exposure (inhalation
or absorption through the skin) of organic compounds. Common organic solvents such as
acetone, methanol, chloroform, carbon tetrachloride, benzene, aniline, nitrobenzene, phenol,
etc. are toxic. Use the hood when possible.
3. When heating a container such as a test tube, do not point the mouth of the container at your
neighbors or yourself. The liquid may suddenly boil or bump, causing the liquid to be blown
out of the mouth of the container.
4. Always make sure that the apparatus set-up used in an experiment does not involve a closed
system. For example, be certain that you have made provisions for the escape of air as your
equipment is heated.
5. Never pour water into concentrated acid. Always pour the acid slowly into the water with
constant stirring. Large amounts of heat are released when an acid, such as sulfuric acid,
undergoes hydration. Under certain conditions, steam is generated, blowing acidic vapor from
the container.
6. Avoid the use of strong oxidizing agents with organic compounds when cleaning glassware.
The combination of organic residue and nitric acid can be explosive.
7. Most laboratory accidents are lacerations caused by a failure to observe simple precautions
when handling glassware. Always protect your hand with a towel when cutting glass tubing or
inserting it into a stopper. When inserting glass tubing into a stopper, always lubricate the
tubing with glycerol so that it will pass easily through the stopper. Glycerol is available at the
reagent shelf or in the stockroom.
8. If you have an accident, obtain help by notifying the person in charge immediately.
9. Absolutely no pipetting by mouth; use a rubber bulb.

I have read the above regulations and safety precautions, discussed them with the professor
or TA, and retained the following copy for my own reference. Sign this copy and turn in.
Date__________ Signed __________________Print Name ____________________

II. Books on Reserve

- Brewer, S. Solving Problems in Analytical Chemistry.

- Christian, G.D. Analytical Chemistry, 6th ed., 2004
- Fritz, J.S. and D.M. Schenk, Jr. Quantitative Analytical Chemistry, 4th ed.
(especially pp. 582 & 621)
- Harris, D.C. Quantitative Chemical Analysis, 7th ed., 2007.
- Laitinen, H.A. and W.E. Harris,. Chemical Analysis, 2nd ed.
- Ramette, R.W. Chemical Equilibrium and Analysis
- Skoog, D.A. and D.M. West. Analytical Chemistry, 4th ed.
- Skoog, D.A., D.M. West, and F.J. Holler. Fundamentals of Analytical Chemistry, 5th ed.
- David Harvey. Modern analytical chemistry, 2000.
- D. Kealey & P.J. Haines. Analytical chemistry ( Instant notes). 2002

References for lab experiments cited at the beginning of most labs in this manual refer to the
following sources, which you may find helpful.

C = G. D. Christian, Analytical Chemistry, 6th Ed., John Wiley & Sons, 2004.
H7 = D. C. Harris, Quantitative Chemical Analysis, 7th ed., W. H. Freeman& Co., 2007.
S & W = D. A. Skoog and D. M. West, Analytical Chemistry, 4th Ed., Holt, Rinehart, and
Winston, Inc., 1986.
SWH = D. A. Skoog, D. M. West, F. J. Holler, Fundamentals of Analytical Chemistry, 5th
Ed.,Saunders College Publishing, 1988.
Harvey= David Harvey. Modern analytical chemistry,Mac Graw Hill 2000.
Kealey= D. Kealey & P.J. Haines. Analytical chemistry (Instant notes).BIOS Scienific
Publisher. 2002

III. General Laboratory Directions

The general purpose of this laboratory is to promote proficiency and familiarity with the
techniques of chemical analysis, especially with their particular abilities for solving real
problems and shortcomings. While the problems and methods are those of classical wet
chemistry, augmented with chromatography and spectrophotometry, the approaches of
equilibrium, kinetics, and stoichiometry are general for all chemical analyses. In lab, you will
have "hands-on" experience with a number of specific analyses. These have been selected
with 3 goals in mind:

1. Workability - you should be able to succeed.

2. Utility - These are real techniques that are frequently performed in working analytical
laboratories - for example, in clinical or agricultural, metallurgical labs... While you
may never perform another titration after you finish quant., you may well have
occasion to submit samples for analysis. The experience you gain in this course should
let you know what to expect from such an analysis - and what not to expect.
3. Representative - The 13 experiments you will do are certainly not exhaustively
representative of the techniques of analytical chemistry. However the concepts
introduced, and the kind of thinking required, are common to all analytical techniques.
It will be necessary for you to consider what interferes with a particular analysis, and
what steps may be taken to avoid interferences.

The laboratory in conjunction with the lectures, should provide a good general feeling for the
terms "accuracy" and "precision." It is important to know ahead of time what accuracy and
precision are needed for an analysis, and then select a technique - or devise one, if necessary -
- which is appropriate.

An analyst's time in industry is charged out at something like $100 an hour (that's not the
salary, unfortunately). A company would quickly lose patience with an analyst or a researcher
who does not know - almost intuitively - how much analysis is needed, and what kind! It is
hoped that you will develop some of that kind of intuition as you move through the course.

Because this kind of understanding is an important goal of this course, you should probably
expect that it will take more than extensive memorization to "ace" the course. If you do the
memorization and do a respectable job in the lab, you will probably get by just fine. However,
you will take little except your grade with you when you finish. On the other hand, if you put
forth the little extra effort necessary to understand the material, you will leave with a better
idea of how to approach problems, not only of analysis, but also of research in general.
Besides, if you understand the material, you will find you need to memorize less.

The heart of quantitative technique is to carry a sample through one or more physical and
chemical manipulations without loss of the sample or introduction of foreign materials.
Ultimately, quantitative means absolutely none of the sample is lost and no foreign material is
added. In line with this, the student must quickly develop independent judgment of his or her
work and common-sense awareness of danger spots that can cause the analysis to go astray. A
thorough knowledge of what you are doing makes this an easy task.

The following is a brief list of information to make you a better analyst.

III.A. Laboratory Safety. If you are not careful in lab, you can get hurt. Common sense and
knowledge combine to make the chances for an accident to occur very slight, but they can still
occur. At the beginning of this lab manual is a page of "Chemistry Laboratory Regulations"
which must be followed. All students will be required to sign a copy of that form to indicate
that they are familiar with and will adhere to these safety regulations.


III.B. Planning and Efficiency. It is imperative that your lab work be planned ahead of time.
There is sufficient time for each experiment, but without some semblance of order and
thorough knowledge of what you are doing you will not have enough time.

1. Read the experiment and all reference materials before coming to lab.

2. Outline the experimental procedure in your notebook before coming to lab (see the
section concerning the lab notebook).

3. Make good use of your time. When you are waiting for something to dry in the oven, or
digesting something, or waiting for something to cool, etc., start on or prepare for the
next task or experiment. Learn to do more than one thing at a time.

4. Listen for announcements from the TA. Frequently check the chalkboard and bulletin

5. Do not be afraid to ask the TAs questions about the experiment. They are there to help
you. At the same time, you should be prepared for them to come around and ask you
questions about the experiment or your technique. All TAs maintain office hours to help
with questions.

6. Label all solutions and label your weighing bottles so you do not get confused. Do not
rely on your memory. Before putting anything in the oven to dry, make sure it is clearly
labeled (don't use labeling tape) with your name and desk #.

7. There are three ovens in the lab. Each one has a different opening time. Open the ovens
only at the designated times. This is so that the ovens can maintain the desired
temperature for the required time.

8. EVERYONE OUT OF LAB BY 5:30 P.M. This means you have to stop your
experiment and clean up by 5:20 P.M., at the latest.

9. Many of the experiments require preparation during the preceding lab; thus, you should
always be at least one experiment ahead of the lab in your reading.

III.C. Neatness and Cleanliness

1. The student who is scrupulously neat and tidy usually does not mix up samples or
reagents, or spill or break or misplace items or information. Cleanliness prevents
accidents and the introduction of foreign material into your sample.

2. Glassware is clean not simply when it appears clean, but when de-ionized water drains
from the vessel as a thin, continuous film, and leaves no droplets behind (especially
the burette and pipettes). A few parts per thousand (or even parts per million) of an
impurity introduced from dirty glassware can significantly affect an analysis. In this
course, that means it may affect your grade. In the real world, it could result in an
incorrect medical diagnosis, or an incorrect forensic result. "Kitchen clean" is not good
enough for the quantitative analysis lab!

3. Do not over clean your glassware. Soluble salts can be removed with water. Soap and
cleaning solutions are for removing grease and dirt. Rinse everything well with tap water
and a couple of times with de-ionized water.

4. Do not use soap on fritted glass crucibles.

5. Do not use grease on Teflon burette stopcocks.

III.D. Laboratory Manipulations and Equipment

1. Make sure de-ionized water used to make primary standard solutions is at about 20° C.
Hot or cold water will cause an error in the actual volume your volumetric hold.
They're calibrated for 20° C solutions.

2. Do not leave tops off of desiccators. Though desiccator tops can seem difficult to
remove, when carrying a desiccator to the balance room these tops can tumble to the
floor as though they possessed some spark of life. Hold onto the top as well as the
bottom when carrying the desiccator. The desiccator lid (whether being removed or

replaced) is properly moved by sliding, rather than lifting. An airtight seal is achieved
by slight rotation and direct downward pressure on the lid.

3. A volumetric flask must be clean, but need not be dry, before use. After introducing the
solute, fill the flask about half full of solvent and mix well. Add additional solvent to
bring the liquid level almost to the mark and mix again. Use a small pipette or a squirt
bottle to add solvent to the line. If you go past the mark, start over. Stopper the flask
and invert a few times to mix. For long-term storage, cover the stopper with Parafilm.

4. To rinse a burette, close the stopcock and add 5-10 mL of solution. Rotate the burette so
the entire inside is rinsed by the solution, then drain through the tip. Repeat two more
times. Fill the burette above the zero mark. Be certain no bubbles are in the tip of the
burette; they can be removed by briefly opening the stopcock.

5. An aqueous solution in a properly cleaned burette or pipette forms a concave surface

called a meniscus. The position of the bottom of the meniscus is taken as the point of
reference when using volumetric glassware. To correctly judge the volume, your eye
must be level with the meniscus, or a parallax error will occur. When reading burettes, it
is convenient to hold a dark card behind the gradation, touching the burette immediately
below the meniscus.

6. Volume increments smaller than a normal drop (which is about 50 μL = 0.05 mL, so 20
dr = 1 mL) may be added from a burette. Open the stopcock slightly and allow a small
volume of liquid to form on the tip. Touch the tip to the wall of the titration vessel.
Combine the droplet with the bulk of solution either by rinsing the wall with solvent or
by tipping the titration vessel.

III.E. Reagents.
Reagents used in a quantitative laboratory are generally of high purity and are of utmost
importance in producing good results. Reagents left to the use of several individuals have a
good chance of becoming contaminated, so each of you has a responsibility to see that they do

1. Always check reagent bottles for formula weights and purity when using them for

2. Take only what you need.

you need into a clean container. Then remove from this the quantity desired and properly
dispose of the rest. This means that you may not use a spatula to remove reagent from
the reagent bottle.


5. A good procedure to accurately weigh solid reagent is to weigh by difference. Place an

amount of the solid reagent that is greater than needed into a clean dry container (beaker
or weighing bottle); weigh accurately. Pour some of the reagent into a second clean dry
container. Weigh the original container a second time. The difference between the first
and second weighings gives accurately the amount transferred to the second container.
Think about this carefully. You'll practice this in the Basic Techniques lab.

6. Certain chemicals must be disposed of in special containers and not down the sink.
Follow these instructions when they occur. Keeping heavy metals, strong acids, and
strong bases out of our waste stream is good citizenship and environmentalism in action.

III.F. Instruments.

1. We will use instruments such as balances, pH meters, gas chromatographs, and

spectrometers. These are complex and often delicate. They should be "tinkered" with
only by those people who have an intimate familiarity with the instruments. Please use
them only as instructed. If repair or adjustment is needed, ask the TA.
2. It is also very important to realize that the data you obtain from these instruments are not
sacred; you must know the limitations and advantages of using a particular instrument.

3. Anyone caught mistreating an instrument will be dismissed from the lab.

4. Do not turn knobs to find out what they do.

5. Never change settings on ovens. Check with the TA if something appears amiss.



III. G. Laboratory Notebook.

1. Your notebook is the only good record you have of your performance in lab.

2. You must use a bound notebook (the binding is permanently glued or stitched). This is
how it's done in industry. Loose pages, three ring notebooks, or spiral notebooks are
not acceptable. We recommend an 8.5 x 11 inch notebook with graph paper pages.

3. Your lab notebook should be neat enough that someone else can easily read and
understand it. Use plenty of room. If you cannot write legibly, then print. If you have
some physical constraint that prevents legible writing, consult your TA. \

4. Your lab notebook should have numbered pages. The first four pages should be an up-to-
date table of contents so any given experiment can be found quickly.

5. All data obtained in the laboratory should be recorded directly in the notebook at the
time the data are obtained. Recording data on loose slips of paper is forbidden. TAs are
authorized to destroy such stray papers.

6. Entries must be made in ink. A mistake is not erased, but crossed out with one line so as
to be still legible, and the correct value written above.

7. After recording a reading in your lab notebook (such as an analytical balance reading, or
a burette reading), go back and double check the reading. Check especially for gross
errors (such as 48.91 mL instead of 47.91 mL).

8. Format for Lab Notebook:

a. Reserve pages 1-4 of the notebook for a Table of Contents. It is a good idea to leave
space in the Table of Contents for analytical results for quick reference later.

b. Beginning at the top of Page 5, start Experiment 1

c. For each experiment, begin at the top of a new page. Include:

I. Title, date experiment was begun, and date finished.

II. A brief (3 to 4 sentence) statement of introduction, purpose, and principles upon

which the analysis is based. Relevant balanced chemical equations should also
appear here.

III. Outline and explaination of the procedure to be followed.

IV. A neat and complete listing of data taken during the course of the analysis, properly
labeled and identified, presented in a logical order. Remember, someone else will
read these data and you may need it for future calculations, so keep it neat, logical,
and clearly presented. You will find tabular form is best; it is strongly

V. Observations you make during the course of the experiment (color changes,
modification of procedure, any observed errors. such as titrating past the

VI. This section will include calculations and results for each sample run. It is your
responsibility (and will be to your advantage) to present results so a minimum of
effort is required to locate them (underline or enclose results in boxes). Finally,
report the mean value for the quantity of interest in the analysis, and the precision
(standard deviation) of the analytical results.
Remember significant figures.

9. Use only the right-hand pages of the lab notebook; this allows for a neater presentation
and for additions and corrections at a later date as needed on the facing blank pages.

10. Footnote any references that you used to answer questions, or for error analysis or
optional comments.

11. Sections I, II, and III above must be completed before beginning the analysis, and
Section IV must be complete except for the actual experimental data. This will prove
useful for your understanding of the work to be done and help save time while doing the
actual experiments.

12. The TA must initial your notebook on the appropriate page before you can pick
up an unknown. Your notebook will be checked to see if you are prepared for the given
experiment. You may also be asked a question to see if you are prepared. If your
notebook is not completed through the procedure outline, you will not be allowed to
proceed with the experiment.

13. Neatness and logical presentation are a must; if the TA cannot follow your work, the
grade you receive will reflect this. The TA may periodically look at your notebook and
make comments on its appearance.
14. An Example of a Procedural Outline

Gravimetric Determination of Chloride.

1. Sample preparation
a) Dry Cl unknown for 2 hours at 110ºC
b) Dissolve 3 carefully weighed Cl samples in 100-150 mL H2O + 1 mL HNO3
2. Procedure
a) Ppt. Cl as AgCl(s) by adding excess AgNO3
b) Heat, stir, and test for completeness of ppt
c) Cool solution to shift chemical equilibrium to right:

Ag+ + Cl- = AgCl(s)

3. Separation Procedure
a) Filter supernatant liquid through glass crucible
b) Wash ppt with dilute HNO3 and pour washes through crucible
c) Quantitatively transfer ppt to crucible

4. Final Prep
a) Dry crucible and ppt in oven for 1-2 hours at 110-120ºC. Cool in desiccator
b) Weigh and repeat (a) until constant weight

III.H. Lab Reports. The report you submit must be on the standard forms and be neat,
legible, and intelligible. If the report cannot be read, it will not be graded. Please read your
report before you submit it (better yet, get someone else to evaluate it); if your statements
make no sense to you they will make no sense to the TA.

Organizational Summary

I. Title of Experiment

II. Introduction
A short paragraph describing the purpose and principles of the experiment, including
relevant balanced chemical equations.

III. Data and Observations

Standardization data, sample data, or other. Include everything: drying time and
temperature, cooling time, reagent amounts, etc. Necessary data should be taken
from your lab book (immediately following procedural outline).

IV. Calculations and Results

1) For each calculation required in analysis of replicate data sets, one example should
be worked out in detail showing a listing of relationships used and one fully worked
calculation (including appropriate units). Only the results of calculations need be
shown for other samples (i.e., for the second and third samples in a triplicate

2) Report the results of the calculations in the units requested. Always report the
mean, the standard deviation, and the confidence interval for your trials.

3) Include "FOR YOUR REPORT" items from each experiment.

Lab reports of the previous experiment and prelab of the next are always due several
days before of the next lab.
Late work is not accepted.

Graded reports are returned just before you start the next lab. Nota bene: on occasion,
TAs have fallen behind in grading reports. If you don't advise the professor that grading
isn't being done in a timely manner, he or she has no way to know that you aren't getting
timely feedback. Just as we expect students to diligently perform lab work, we expect
TAs to diligently do their jobs. Complaining to your neighbor can't change anything.
Advising the faculty member in charge of the lab as to what is or isn't happening can
have useful results.

Chem 223: Experiment 2



(This experiment completely follows the lab: “Practice with basic techniques”. Chem 223. UIUC)

References: C, Chap. 2.
H4, H5 Chap. 2.
S & W, Chap. 19.
SWH, Chap. 30.
I. Purpose of the experiment
The Multi-technique Operations, Pipette Calibration and Weighing by Difference exercises
discussed on the following pages will introduce you to some basic operations you will need to
perform in later experiments. These simple measurement experiments give you practice in
pipetting, using the balance, and using your laboratory notebook. Careful attention to detail in this
experiment will inculcate good laboratory technique, which will be crucial in obtaining accurate
results in the later laboratories. Since a large fraction of your grade depends on accuracy, this lab
offers an important opportunity to practice. Think of it as spring training for analysts!
II. Before You Start the Lab

 Review the Balance Tutorial before you come to lab!

 When you arrive at the lab you will be assigned a lab bench and equipment locker, a
combination for your desk, and an equipment checklist. Check to see that you have all
the listed equipment and that it is not cracked, chipped or otherwise useless.
 Become acquainted with safety devices, such as the showers. Sign and turn in a set of
laboratory regulations, if you have not already done so.
 You will be given cards to fill out. Fill these out and return them.

 You will be assigned to a balance. Meet the other people also assigned to that balance. It
is your responsibility to keep the balance clean - you will be penalized if you abuse
it. Each section will be given a demonstration on how to use the balance. You may
not use any balance other than the one to which you are assigned without the TA’s
 You should scrupulously clean the equipment you need for next week with
detergent solution. Rinse with tap water and then three times with deionized
water. Cleaning solution should be used only if detergent solution does not do the
job. Go easy on the detergent. It does not take much.
 Clean out your desiccators, if the desiccant appears wet or is clumped (or check
with TA). To open desiccator, slide the top off. Dump the old CaCl2 into the
specially marked container in the lab. If the desiccator looks dirty, clean it and dry
it well. Remove the old petroleum jelly with a paper towel. Grease on the inside may
require acetone for removal. Add only about two 50 mL beakers full of the desiccant
(CaCl2) to your desiccator. Regrease the rim and lid and remove any excess. Keep it
III. Multi-technique Operations

III.A. Quantitative Transfer

 Using the top loading balance and a 50 mL beaker, weigh out about 2.5 g of either NaCl or KCl.
 Add about 25 mL of deionized water.
 Quantitatively transfer all of this solution to a 500.0 mL volumetric flask. This means that you
want to get all of the NaCl (or KCl) that you
put in the beaker into the volumetric flask. This
is accomplished as follows:

a. Insert a funnel into your 500.0 mL volumetric

b. Place a stirring rod on top of the beaker to guide
the liquid into the funnel as it is poured out of
the beaker.
c. Then using your polyethylene wash bottle,
(always filled with deionized water), rinse the
inside of the beaker, then the stirring rod, and
then the funnel itself such that all the rinse
water goes into the volumetric flask. This is
done with a lot of deionized water to insure
all the NaCl (or KCl) is transferred to the
volumetric flask and is not left behind, spilled
on the desk or otherwise lost.
d. Set the beaker with the stirring rod in it on the
desk and rinse what is on the tip of the funnel
Figure 1. Quantitative transfer of into your volumetric flask.
solution from a beaker to a
volumetric flask. Now, to check your technique, rinse the inside and
outside of the funnel, and the
stirring rod, and then the walls of
the beaker. Allow this rinse
solution to remain in the beaker.
Check your efficiency of transfer
now by adding one or two drops of
the 0.5 M AgNO3 solution (in a
small dark bottle that is in the lab)
to the beaker of rinse solution. DO
NOT add AgNO3 to the solution in
the volumetric flask. If the rinse
solution now turns white or cloudy,
your transfer was not quantitative.
Why does the formation of a cloudy
Figure 2. Stopcocks in the closed (left) and open
solution indicate incomplete
(right) positions.

Repeat the above experiment if you messed up the first time.

Figure 3. Rotating the burette to wet the interior surface. Figure 4. Reading the burette.

III.B. Practice using Burette.

Now, take the quantitatively transferred solution in the 500.0 mL volumetric flask, dilute to
volume with deionized water, and mix thoroughly. Practice using your burette with the solution in
your 500.0 mL flask:
 Prime and fill the burette. To prime the burette, be sure the stopcock is closed, Fig.2.
 Then pour 5 to 10 mL of the salt solution into the burette and rotate the burette (viz. Fig. 3)
to wet the walls. Allow the liquid to drain through the tip into a sink. Repeat this procedure,
and then fill the burette. Priming should be done with all delivering glassware before use.
 Wait at least thirty seconds before taking an initial reading.
Take initial reading and all burette readings using a burette
reading card. Touch the card to the burette behind the
gradations. The top of the black stripe should be below the
meniscus so that the meniscus appears dark against the
white card, cf. Fig. 4.
 Now let about 5 mL run into a 250 mL Erlenmeyer flask.
Wait at least 30 seconds and take the final reading. (The
amount of solution in the Erlenmeyer flask is equal to the
final reading minus the initial reading.) Write down the
final reading in your notebook, close it, and then ask a
neighbor or your friendly TA to take the final reading.
Compare the two readings. They should agree to within ± 0.01
mL. Do they? Notice the final digit in the burette reading is an
estimate of the distance between two lines. Always record two
decimal places from a burette reading.
Figure 5. Burette  Repeat the previous steps of making initial and final burette
transferring solution to an readings two more times.
Erlenmeyer flask.
 Refill the burette, and take a new initial reading. Now add 30 drops to the Erlenmeyer flask
with the burette, and take the final reading. Calculate the average volume of one drop.
Then repeat this, except with 40 drops and calculate the average volume of a drop. Record
these results and compare them. Now, practice adding "half-drops" to the flask. Record these
results for at least 10 half-drops, and calculate the average volume of your half-drops. In your
volumetric unknowns, you will want to try to get half-drop end points.
 Dispose of the salt solution and clean up your equipment.
IV. 10.00 mL Pipette Calibration

This calibration is done by accurately weighing the volume of the liquid dispensed by the pipette. If the
density of the liquid is accurately known at the temperature at which you are working, the volume
dispensed can be accurately determined. Deionized water is the liquid you will use.

Figure 6. Filling a pipette Figure 7. Incorrect (left) and correct (right) methods of
with a mechanical suction releasing the pipette contents.

 Clean your 10.00 mL pipette so no droplets of deionized water are left on the inside surface as it
 Weigh a 30 mL glass bottle and its cap to the nearest tenth of a milligram (0.0001 g)
on the analytical balance. Use finger cots to hold the bottle and manipulate the cap.
 Fill the pipette to above the etched line with deionized water from a 250 mL beaker (note
the temperature of this water!). Use a filling bulb, viz. Fig. 6. Mouth pipetting is
forbidden! To use the suction bulb: (a) compress the bulb before you attach it to the
pipette, (b) place the compressed bulb over the end of the pipette and carefully release the
finger pressure to allow the liquid level to rise until it is somewhat above the
etched fill line, (c) rapidly remove the bulb and
simultaneously cover the end of the pipette with
the index finger of the hand holding the pipette,
cf. Fig. 7, (d) gently adjust the pressure on the
index finger to allow the liquid to fall until,
holding the pipette in a vertical position with
the etched line at eye level, the bottom of the
meniscus coincides with the etched line, (e)
touch the tip of the pipette to the side of the
beaker to remove any drop formed at the tip,
viz. Fig. 8. This manipulation is a bit tricky and
may have to be repeated several times until you
are sure you have it exactly right.
Figure 8. Removing the last drop of
liquid from a pipette by solid-solid  Once this is accomplished, touch the pipette tip to
contact. the inside of the weighing bottle, and allow the
pipette to discharge its contents into the bottle. It
is best to allow the tip to touch the inner wall of
the container near the bottom, but not in the liquid. Allow the pipette to drain by itself for
20 or 30 sec.. Do not blow out the remaining portion of water in the tip. Carefully remove
the pipette.
Note that the pipette is marked TD. What does this stand for? How would your procedure change if
your pipette were marked TC, instead?
 Cover the bottle, and weigh the bottle and its contents.
 This calibration should be done in triplicate and the three values should agree within 1 part
per thousand, i.e. 0.1%. There is no need to drain the water from the bottle; just use the final
mass of the last trial as your new initial mass for the next trial. Remember to keep the outside
of the bottle dry.
Common errors which you should be careful to avoid are: (a) warming the pipette by holding the
bulk portion in your hand, (b) failure to allow sufficient drainage time, (c) disturbing the liquid
that should remain in the tip (watch for air bubble formation!), (d) general carelessness in
handling the weighing container and top, (e) loss of water from the pipette as you move from eye
level to the weighing bottle (sudden movement will make some water squirt from the tip; slanting
the pipette a bit before moving it helps).
From the table of density vs. temperature, find the appropriate density and calculate the actual
volume your pipette holds.
Table 1. Density of Water Near 300K.
Temp. (°C) Density (g/mL) Temp. (°C) Density (g/mL)
20 0.99823 26 0.99681
21 0.99802 27 0.99654
22 0.99780 28 0.99626
23 0.99756 29 0.99597
24 0.99732 30 0.99567
25 0.99707

Obviously great care must be exercised in the manipulation of volumetric glassware. What should
you do in the following commonly encountered situations working with pipettes. (1) You notice a
small drop of liquid on the exterior surface of your pipette. (2) A small air bubble forms at the tip
of the pipette.
(3) After releasing the contents of the pipette into the receiving vessel you note that a small
drop remains on the interior surface of the pipette.
V. Weighing by Difference
Weighing by difference is both a simple concept and a simple laboratory task. However, if you
don't understand how to weigh by difference, you will do poorly on at least three labs. You
weigh by difference to avoid contaminating your precious sample. Use the data from this
section for error propagation for your lab report.
 Fill a clean dry weighing bottle about one quarter full with NaCl (or KCl).1
 Weigh the entire weighing bottle (top and bottom) and record the mass to 0.1 mg using
the analytical balance.
 Tap out about 0.8 g of solid into
your 250.0 mL volumetric flask.
Use a funnel.
 Reweigh the entire weighing
bottle again.
 Compute the change in mass. If
its change in mass is close to 0.8
g as desired, you are done. If not,
tap in some more powder and
reweigh until the difference is
Figure 9. (Left) Weighing bottle. (Right) about 0.8 g. If the mass added is
Proper handling of the weighing bottle. greater than 0.9 g, rinse your
volumetric flask well with
deionized water and start over.
 When you are done, you have the initial and final masses, to four decimal places, of
the weighing bottle containing the powder. The difference is the mass of powder in the
volumetric flask.
 Now that you’ve gone to all the trouble of weighing by difference, dilute the salt
solution and practice pipetting.
 Dilute your NaCl or KCl in your 250.0 mL volumetric flask to volume with deionized
water and mix thoroughly.
 Pour some of your salt solution into a beaker. If the beaker is wet, rinse with your
salt solution 3 times to insure that you do not dilute your salt solution.
 Prime your 20.00 mL pipette by rinsing 3 times with the salt solution in the


 Pipette 20.00 mL of salt solution into a 250.0 mL Erlenmeyer flask.  You can

dispose of the salt solution and clean up your glassware.

For this experiment only, you may dry the weigh bottle using a tissue. If you have time and
want to gain experience, use the ovens to dry to constant weight. This means the weigh bottle is
dried, cooled in a desiccator, and weighed. These three steps are repeated until nearly the same
mass is achieved twice in a row. In future experiments you must oven dry your weighing bottle.


Calculate (1) volumes dispensed from burette, (2) average volume of a drop and half-drops, (3)
mean volume (in mL) of water transferred using 10.0 mL pipette from part II, (4) standard
deviation, relative percent error and relative standard deviation (in ppt) for 10.0 mL pipette
calibration, (5) concentration (in mg/mL) of NaCl (or KCl) solution from part III, (6) error
Why is delivery glassware primed prior to use for quantitative transfer? Do volumetric flasks
need to be primed? Do volumetric flasks need to be dry before you use them? Explain.

1. Find the absolute uncertainty and percent relative uncertainty for each calculation.

Express the answers with the proper number of significant figures.

a. 6.2 (±0.2) - 4.1 (±0.1) b. 9.43 (±0.05) x 0.016 (±0.001)
c. 91.3 (±(1.0) x 40.3 (±0.2) / 21.1 (±0.2) d. [4.97 (±0.05) - 1.86 (±0.01)] / 21.1 (±0.2)

2. For the numbers 116.0, 97.9, 114.2, 106.8 and 108.3, find the mean, standard deviation, relative
standard deviation (in ppt), and 90% confidence interval for the mean.
3. What volume of 0.0110 M NaOH is required to completely titrate a 10.00 mL aliquot of a 0.0240 M
4. If it takes 28.41 mL of 0.0510 M NaOH to titrate 20.00 mL of HCl, what is the concentration of the
5. How many milliliters of 0.100 M KI are needed to react with 40.0 mL of 0.0400 M Hg2(NO3)2 if the
reaction is Hg22+ + 2I-  Hg2I2(s)?
6. A cyanide solution with a volume of 12.73 mL was treated with 25.00 mL of Ni 2+ solution
(containing excess Ni2+) to convert the cyanide to tetracyanonickelate(II):
4CN- + Ni2+  Ni(CN)42-
The excess Ni2+ was then titrated with 10.15 mL of 0.01307 M ethylenediaminetetraacetic
acid (EDTA). One mole of this reagent reacts with 1 mol of Ni2+:
Ni2+ + EDTA4-  Ni(EDTA)2-
Ni(CN)42- does not react with EDTA. If 39.35 mL of the 0.01307 M EDTA was required to
react with 30.10 mL of the original Ni2+ solution, calculate the molarity of CN- in the 12.73
mL cyanide sample.
Chem 223: Experiment 3

Complexometric Determination of Nickel

(This experimental procedure of this experiment is mainly based on the lab. “Volumetric
determination of Nickel”. Chem 223. UIUC)

References: 1. C. Chaps 7, 9 (223-235), 3 (pp. 79-86)

2. Harris, 7th ed. Chap. 12
3. S&W Chaps 6, 10, pp581-583
4. SWH, Chaps 4, 11, pp. 771-773

I. Purpose of the experiment

A volumetric titration is one in which the volume measurement capabilities of the burette are
exploited to determine a highly precise value of the mass of one reagent needed to react
stoichiometrically and completely with an unknown. The particular type of titration being
examined in this lab is a complexometric titration in which the titrant, EDTA, is a ligand that can
react completely with the analyte, Ni(II), with a well-defined stoichimetry. This lab also
introduced introduces the concept of a secondary standard, since the EDTA is standardized
against CaCO3 to allow calculation of the amount of nickel in the solution

II. Introduction
The use of ethylenediaminetetraacetic acid (EDTA) as a titrant for the determination of metal ion
concentrations is a common analytical method.


>N - CH2 - CH2 - N<


EDTA is a tetraprotic hexaprotic acid (pK1 = 0.0, pK2 =1.5, pK3 = 2.0, pK2 pK4 = 2.8, pK3
pK5 = 6.2, pK4 pK6 = 10.3), since each of the carboxyl groups and H+ on the amines is ionizable.
The completely deprotonated (dissociated) EDTA molecule is capable of forming up to 6
separate coordination bonds with a single metal ion; this is accomplished by donation of six
separate lone pairs of electrons on the dissociated EDTA molecule to empty orbitals existing on
the metal ion.

The structure of EDTA and the magnesium-EDTA complex (without the hydrogen atoms) is
shown below:
The resulting product of this reaction is a metal-chelate complex with one-to-one stoichiometry;
the reaction is described as:

Y4- + Mn+ ----> MYn-4

where Y represents the completely dissociated EDTA molecule and M represents the metal ion.
The reaction is usually rapid and, under the correct conditions, quantitative (conversion of 99.9%
of the metal ions into a metal-chelate complex is achieved at the equivalence point). The
condition of greatest concern is the pH of the solution containing the metal ion. Since EDTA is a
weak, polyprotic acid and can only react effectively with a metal ion when it is completely
dissociated, the pH of the metal ion solution directly affects whether the titration can be
performed quantitatively. This is reflected in the conditional formation constant for the
titration; a conditional formation constant for an EDTA titration is the product of the intrinsic
formation constant (the equilibrium constant for the condition where all EDTA is completely
dissociated) and the fraction of EDTA which is completely dissociated. This fraction is
determined by the pH of the solution containing the metal ion. (For a more detailed of conditional
formation constants see your text book.)

A note about indicators: Another complicating factor in this analysis is the choice of an
indicator; the indicators for EDTA titrations are chemically similar to EDTA itself. They are
polyprotic acids which can form complexes with the analyte and these complexes impart a color
to the solution.

HxInd + M M(Ind) + x H+
(Color #1)

Ideally, only a small fraction (> 0.1%) of the metal is complexed by the indicator, so that a
quantitative amount of the metal remains free to react with EDTA as it is added during the
progress of the titration; once sufficient EDTA has been added to reach the equivalence point, the
first excess drops of EDTA react with the metal-indicator complex to liberate the indicator and
produce a color change.
M(Ind) + Y + x H+  MY + HxInd
(Color #2)

To achieve this ideal behavior, the formation constant should be large enough to prevent reaction
between the EDTA and the metal-indicator complex before the equivalence point is reached, yet
significantly smaller than the formation constant for the metal-EDTA complex so that the EDTA
may displace the indicator from its complex with the metal after only adding a drop or two of
excess EDTA solution.

In this analysis, the standardization of the EDTA titrant solution against the Ca2+ solution is
carried out at pH = 109.7 and uses the indicator Erichrome Black T, which is added as a solid Comment [AS1]: We have found that at pH 10,
CaO precipitates. pH 9.7 works better!
powder (1g Erichrome Black T in 100 g NaCl). The initial solution color is wine-red and the
endpoint is marked by a sharp color change to a deep blue.

The titration of the sample solution for both Ni content is carried out at the same pH, but uses
Murexide as an indicator, since Ni forms complexes with Erichrome Black T which are too
stable to be decomposed by EDTA. Additionally, the pH 10 9.7 buffer (an NH3/NH4+ solution)
must be added carefully, so that excess NH3 does not interfere with the formation of the Formatted: Font: 12 pt, Subscript
complexes between Murexide and the analyte metals. The initial color is yellow to greenish-
yellow and the endpoint is marked by a change to a violet color. This change is slightly less sharp
than the Ca/EDTA titration using Erichrome Black T, with an orange color being seen just prior
to the endpoint. Once this orange color appears, the titration should be performed very slowly to
avoid overshooting the endpoint volume. Unfortunately, this lower pH reduces the stability
constant for the Ni-Murexide complex as well; as a result the titration does not produce a sharp
endpoint, but starts out as orangish-yellow and progresses gradually through a series of peachy-
orange colors until a reddish-violet color is reached at the endpoint. Since no sharp color change Comment [AS2]: What lower pH? This
paragraph looks like it is all at pH 9.7. My
is observed, the best strategy for obtaining a reproducible endpoint is to have a titration solution experience suggests leaving out everything from
at the endpoint color for comparison while carrying out each individual titration. "Unfortunately" through the end of the paragraph. Is
your experience different?

III. Reagents and materials

A. Reagents and Solutions available:

 Dry 1-2 g of CaCO3 for 90 min at 110°C in a weigh bottle. Cool to room temperature in
a desiccator before using.

 Concentrated HCl (12M)

 Eriochrome black Black T ( ground 1:10 with NaCl).

 Murexide indicator: Dissolve 150 mg dye in 100 g absolute ethylene glycol. Aqueous
solutions of the indicator are not stable for more than 1 day. Another way to prepare this
indicator is to grind 0.20 g of murexide to a fine powder with 20 g of potassium nitrate.

 unknown EDTA solution which was prepared from Na2H2Y.2H2O

 a 0.8 g/L (~ 4 mM) solution of MgCl2·6H2O

 pH 10 buffer ( 2000 mL has been prepared by dissolving 140.0 g of NH4Cl in 650 mL

of deionized water, adding 1136 mL of conc. ammonia and diluting to 2000 mL.)

 Five 250 mL volumetric flasks and five 250 mL beakers for preparing Ca2+ solution by
group of 4 students.

 Each 100.0 mL volumetric flask containing unknown sample of Nickel have been
prepared for each student. To save time, the sample has already been dissolved for

B. Solutions to be prepared:
1. Preparation of Standard Solution of Ca2+.

 Weigh (to the nearest 0.1 mg on the analytical balance - an opportunity to weigh by
difference) about 0.15 - 0.25 g of primary standard calcium carbonate.
 Transfer the solid directly from your weigh bottle to a 250.0 mL volumetric flask, then
rinse the neck of the flask with about 20 mL of water.
 Calculate the amount of and add 6 M hydrochloric acid (dilute the 12 M HCl in the hood
to 6 M) dropwise until effervescence ceases and the solution is clear. Be careful to add
only enough acid to complete the evolution of CO2. It may not dissolve the solid
completely, but it should dissolve most of the CaCO3. A large excess of acid may cause
pH problems in the titrations that use this solution.

Dilute with water to the mark, mix the solution thoroughly, and label. I.B.

2. Sample Unknown.
 When you get the unknown solution from TA, dilute the contents to the 100.0 mL line
with deionized water. When diluting to the mark on the neck of the flask, add the last
few drops carefully with a small pipette. If you go past the mark, you have ruined your
unknown, and you'll need to ask the TA for a new one.

IV. Experimental procedure

A. Standardization of EDTA Solution.

• Pipette a 10.00 mL aliquot of the standard calcium solution into a 250.0 mL Erlenmeyer
flask and add about 10 mL of an ammonia-ammonium chloride buffer solution pH10pH9.7.
Then add a few crystals of the Eriochrome Black T indicator. The solution should look violet
(or pink-violet). If the solution, instead, appears to be a pale peach-pink, the problem is that
the pH is too low; add 5 mL more buffer.

• Next add a 5.00 mL aliquot (use a pipette) of a 0.8 g/L (~ 4 mM) solution of MgCl2. The
Mg2+ ion is needed to get a sharp color change in this titration.
• First, do a “dry-run titration” in which you deliberately over-titrate, i.e. blow past the
endpoint, so you can see the color changes associated with the endpoint. There is no need to
carefully record titrant volumes, since the purpose of this step is to calibrate yourself to the
color changes you want to observe.

• Now carefully titrate with the EDTA solution to the point where the color changes from
pink violet to blue.2. The endpoint is where the solution has just turned blue and no tinge of
pink remains.
• At the endpoint, you have added enough EDTA to complex not only the Ca2+ but also the
Mg2+. You need to do another titration to determine this indicator blank (the amount of
titrant added in excess of that needed to just titrate the Ca2+.) To determine this indicator
blank: place in a 250 mL Erlenmeyer flask ca. 10 mL water, 10 mL of ammonia-ammonium
chloride buffer solution pH 10, Eriochrome Black T indicator and 5.00 mL (use a pipette) of
the Mg2+ solution. Titrate to the same blue endpoint as before.
• Repeat both the Ca2+ titration and the indicator blank titration at least two additional times
to get good, average values and enough data so you can calculate precision. The average Formatted: Superscript
volume used for the indicator blank titration needs to be subtracted from the volumes used in
the Ca2+ titration to determine the correct amount of EDTA required to react with the Ca2+
standard solution.

B. Determination of the Nickel Unknown via Direct Titration

• Pipette a 10.00 mL aliquot of your unknown nickel solution into a 250 mL Erlenmeyer flask
and add about 10 mL of an ammonia-ammonium chloride buffer solution pH 10. Then add
Murexide indicator and about 20 mL of DI H2O. Do not add any MgCl2.

• Titrate carefully with the EDTA solution to the point where the color changes from yellow
to purple. The color will be yellow before the endpoint and change to purple at and beyond
the endpoint. Take as the endpoint the first definite purple color (no yellow remaining). For
best results, a titration should use more than 10 mL, but not so much that you have to use
more than one burette-full. Adjust the size of the aliquot on Ni2+ as necessary to stay within
this range.
purple. The color will be yellow before the endpoint and change to purple at and beyond the
endpoint. Take as the endpoint the first definite purple color (no yellow remaining). For best
results, a titration should use more than 10 mL, but not so much that you have to use more
than one burette-full. Adjust the size of the aliquot on Ni2+ as necessary to stay within this

• Repeat the titration with at least two additional aliquots of the nickel solution. Each time,
check that the pH is in the desired range.

C. Determination of the Nickel Unknown via Back Titration.

Please take care to add the reagents in the order specified here.

- Pipette a 10.00 mL aliquot of your unknown nickel solution into a 250 mL Erlenmeyer flask
and add about 10 mL of the ammonia ammonium chloride buffer solution, pH10pH9.7.
- Add a volume of EDTA solution that is 5-6 mL greater than what you would expect to be
needed to exactly react with the nickel; record this volume added accurately (to 0.01 mL).
After the EDTA has been added, then add Eriochrome Black T indicator1. Do not add any
- Prepare a total of four of these samples.3 Drain the EDTA solution from the burette and rinse
well with water; drain; rinse the burette with a small amount of standard calcium solution;
drain; fill the burette with the standard calcium solution. Formatted: Highlight

- Titrate the excess EDTA in each of your samples by using the standard calcium solution
titrant. The endpoint will now be the color change from blue to violet. The color will be blue
before the endpoint and change to violet at and beyond the endpoint. Take as the endpoint the
first permanent appearance of violet.


1. Add sufficient indicator (2-3 drops if using indicator solution or size as green bean seed if
using indicator solid ) to give a pale but distinct color to the solution. The solution should
not be so deeply colored that it is difficult to see through it.

2. Always titrate with a piece of plain, white paper beneath the flask so you can see color
changes more clearly.

3. It is advisable to titrate standards and unknowns during the same lab period because the
EDTA solution is somewhat unstable.

4. You need at least three assay values. To be safe, use one extra aliquot.
Formatted: Font: (Default) TimesNewRoman,
English (U.S.)
Graphical summary of the 4 types of titrations in this lab. Thin lines: concentration not known at the time
of titration. Thick lines: standard (primary is Ca2+, secondary is EDTA).


Calculate (1) molarity of your Ca2+ stock solution, (2) molarity of your EDTA solution (mean
value),(3) total mass (mean value) of the Ni(II) in your sample (100 mL) in milligrams. Report a
value for the direct titration, the back titration, and the average of the two.

Report the standard deviation of your measurements (both direct and back titration): (1) in
absolute terms (see Appendix I). (2) as a relative standard deviation in parts per thousand. (3)
calculate a 95% confidence interval for your results (use the t statistic; see Appendix I).

Test for a statistically significant different in precision (F-test; see Appendix I) between your
direct and back titration results at the 95% confidence level. Test for a statistically significant
difference between the mean values (t-test) for your direct titration and back titration results at
the 95% confidence level.

Estimate the uncertainty involved in each step of the direct titration (weight of standard, dilution
of standard, titration volume, etc.). Use propagation of error techniques (see Appendix (() to
arrive at the resultant uncertainty in your answers. How does the magnitude of this predicted
value compare to your observed standard deviation?

As an example of uncertainty estimation, you might feel very good about your ability to locate
the endpoint, so assign it a ± 0.02 mL uncertainty. Or, you might feel your technique is shaky, so
assign a ±0.05 mL uncertainty to it. Often, the volume uncertainty in a piece of glassware is
stated right on it. Analytical chemistry textbooks also list uncertainties for all types of glassware.
Draw the structure (indicate all atoms) of the disodium salt of EDTA (abbreviated Na2H2Y where
EDTA is Y4-). When Na2H2Y is dissolved in water, what immediately happens to the two sodium
What are four essential requirements for a volumetric analysis (also called a titrimetric

Why is MgCl2 added to the solution for standardization of EDTA?

Can you simultaneously determine Nickel and copper in a mixture using murexide indicator? If
you can, describe the procedure.

Why was the Ca2+ stock solution prepared from CaCO3 plus HCl instead of from solid CaCl2?

What is the purpose of the HCl? Why do you add HCl until effervescence ceases?

What compound is the primary standard for this determination?

Chemistry 223: Experiment 4

Acid- Base and Precipitation Titration

References: 1. C, Chap. 7 .
2. H7, Chapter 11,
3. Kellner, Chap 4
4. SWH, Chaps. 10,11,12,13
5. Harvey, C5 and C7
6. Kealey, pp. 139-143
This experiment is partly based on the experiment - chem. 213 – Winova State University
and Chemistry 3.1- University of Canterbury

A. Determination of total alkalinity of soda ash

I. Introduction:
Crude sodium carbonate, Na2CO3, is commonly called soda ash. It is frequently used as a
commercial neutralizing agent. Besides the carbonate, small amounts of sodium hydroxide,
NaOH, and sodium hydrogen carbonate, NaHCO3, may also be present. Titrating with
standard acid, usually HCl, makes it possible to determine the total alkalinity of the soda ash.
It is common practice to report the total alkalinity as percent sodium carbonate or sodium
oxide, Na2O. Since samples are frequently non-homogeneous, the method of aliquot portions
is usually employed. Instead of weighing out three separate samples of soda ash, one
accurately weighs out a larger amount. This is then transferred into a volumetric flask,
dissolved in water and then diluted to an accurately known volume. From this solution are
then taken samples or aliquots on which the titration is carried out.

One-tenth molar hydrochloric acid is standardized against primary standard sodium carbonate.
Phenolphthalein is used to approximate the halfway point of the titration, and then either
modified methyl orange indicator or bromcresol green indicator is used to detect the final end
point; the last indicator is used with boiling of the solution near the end point to remove CO2.
Altematively, the hydrochloric acid is standardized against standardized sodium hidroxide
hydroxide using phenophthalein indicator. The soda ash sample is titrated with the
hydrochloric acid solution, with the addition of 2 mol hydrogen per mole Na2C03Na2CO3. Comment [AS1]: Here and several other places,
the numeral 0 and the letter O are not the same!
CO32- + 2H+  H2C03H2CO3 Formatted: Character scale: 100%

H2C03 H2CO3  H20 H2O + CO2  Formatted: Character scale: 100%

Formatted: Character scale: 100%

II. Solutions and Chemicals

 0.2% phenolphthalein in 90% ethanol
 modified methyl orange (mixture of methyl orange and xylene cylanole FF available
from Eastman Kodak Co.)
 0.1 % bromcresol green in 0.001 M NaOH, Formatted: Character scale: 100%

 primary standard Na2C03 Na2CO3 was dried at 160°C for 2 h or more. Cool at least 30
min in a desiccator before weighing.
 standardized 0.1 M NaOH. Formatted: Character scale: 100%

To be prepared. 0.1 M HCl. Concentrated hydrochloric acid has a density of 1.18 g/mL and
contains 37% by weight HCl. Hence, about 4 mL concentrated acid should be diluted to 500
mL to make 0.1 M acid. Measure about 0.5 mL more than this amount in a 10-mL graduated Formatted: Character scale: 100%
cylinder and pour into water in a 500-mL glass-stoppered bottle that is filled to the shoulder
with distilled water. Shake until the solution is homogeneous.
III. Experimental Procedure

Standardize the HCl by either method 1 or method 2 below.

1. Standardization of HCl against Na2CO3 . Weigh accurately (to 0.1 mg) the weighing bottle
containing the dried sodium carbonate. Keep the stopper in place while weighing. Transfer
quantitatively (for four-figure accuracy) about 0.2 g to a clean 250-mL Erlenmeyer flask. .
(This procedure is called "weighing by difference" and is necessary be cause Na2C03 Na2CO3 Formatted: Character scale: 100%
is hygroscopic.) Weigh out a second and third portion in the same way. Add about 50 mL
distilled water and one to two drops phenolphthalein indicator solution to each flask.

The Na2C03 Na2CO3 will be titrated first to a phenolphthalein end point (pink to colorless; 1
H+ added: CO32- + H+  HC03 HCO3 -) to approximate where the final end point should be Formatted: Character scale: 100%
(modified methyl orange or bromcresol green indicator; 1 more H+ added: HC03 HCO3 - + H+

H2C03H2CO3). Rinse the buret three times with small portions (about 5 mL each) of the
approximately 0.1 M acid prepared above, then fill and adjust to near the zero mark. Record Formatted: Character scale: 100%
the volume to the nearest 0.02 mL. Titrate the first sample of the sodium carbonate, adding
the acid no faster than 0.5 mL per second, swirling the flask constantly until the pink color
disappears. At this point, about half the total volume of acid necessary has been added
(actually a slight excess). Use this number to estimate where the final end point will occur,
keeping in mind that slightly less acid should be required than has been added. Continue the Formatted: Character scale: 100%
titration by either method (a) or method (b) below.

(a) Modified methyl orange indicator. Before beginning, check with your instructor which
indicator you are to use. Add two to three drops of modified methyl orange indicator and
titrate until the indicator color changes from green to gray. The proper color can be more
easily discerned by comparing with the color of two drops of the indicator in a solution
prepared by adding 0.20 g potassium acid phthalate in 100 mL distilled water. The pH of this
solution is 4.0, the same as the end-point pH in the presence of CO2. Use the same technique
for the standardization and the unknown titrations.

(b) Bromcresol green indicator (alternative method). Add two to three drops bromcresol
green. The color change for this indicator is from blue through pale green to yellow. Titrate to
a blue-green color (just before the end point); interrupt the titration at this point and boil the Formatted: Character scale: 100%
solution carefully for 2 to 3 min to drive off the carbon dioxide. The color should revert to
blue. Cool the solution to room temperature and continue the titration to the pale-green color. Formatted: Character scale: 100%
This marks the final end point .

Titrate the other two samples in the same manner as the first and calculate the molarity of the Formatted: Character scale: 100%
HCl from the weights of Na2C03 taken, remembering that each carbonate has reacted with
two protons. Use the mean of the three determinations for calculations involving the un - Formatted: Character scale: 100%

2. Standardization of HCl against standardized 0.1 M NaOH (alternate procedure. Rinse your Formatted: Character scale: 100%
25-mL pipet with 0.1 M HCl, and add with the pipet 25 mL to a 250-mL Erlenmeyer flask.
Add two to three drops phenolphthlein indicator solution. Rinse your buret with three small
portions of the 0.1 N NaOH solution prepared and standardized in Experiment 6. Fill and Formatted: Character scale: 100%
adjust to near zero. The NaOH is placed in the buret rather than in the flask to protect it more
from atmospheric CO2. Record the volume to 0.02 mL. Titrate the acid until a faint pink color
is obtained that lasts for at least 30 s. Repeat the titration two more times, using your 50-mL
pipet for the second and third titrations if less than 25 mL NaOH was required in the first.
Calculate the molarity of the HCI.

3. Determination of sodium carbonate in soda ash. Accurately weigh out by difference three
samples of about 0.025 to 0.035 g each, dissolve each in about 60 mL water, and, titrate with Formatted: Character scale: 100%
0.1 1 M HCl, following the same procedure with respect to indicators and end point used in Comment [AS2]: Use a non-breaking space
the standardiiation procedure: against Na2C03 or, if not used, the one specified by your (CTRL-Shift-space) to keep digits adjacent to units.

instructor. Formatted: Character scale: 100%

Formatted: Character scale: 100%
B. Determination of Chloride in cheese Cheese by Precipitation Titration with
Silver Nitrate - Volhard's Method

I. Introduction

The salt sodium chloride is added during the manufacture of cheddar cheese. The content of
NaCl ranges from 0.22 to 4.76% in cheese. In this method, the cheese is ‘digested’ to release
this salt to obtain the concentration of chloride ions. To carry out this digestion, the cheese is
reacted with nitric acid and potassium permanganate. The chloride ions are then ‘free’ to form
a precipitate with the added silver ions.

This method uses a back titration with potassium thiocyanate, and is applied to determine the
concentration of chloride ions in a solution. Before the titration, an excess volume of a silver
nitrate solution is added to the solution containing chloride ions, forming a precipitate of
silver chloride. The term ‘excess‘ is used as the moles of silver nitrate added are known to
exceed the moles of sodium chloride present in the sample so that all the chloride ions present
will react.

Ag+(aq) + Cl-(aq) --> AgCl(s)

The indicator Fe3+ (ferric ion) is then added and the solution is titrated with the potassium
thiocyanate solution. The titrate remains pale yellow as the excess (unreacted) silver ions
react with the thiocyanate ions to form a silver thiocyanate precipitate.

Ag+(aq) + SCN -(aq) --> AgSCN(s)

Once all the silver ions have reacted, the slightest excess of thiocyanate reacts with Fe3+ to
form a dark red complex.

Fe3+(aq) + SCN -(aq) --> [FeSCN]2+(aq)

The concentration of chloride ions is determined by subtracting the titration findings of the
moles of silver ions that reacted with the thiocyanate from the total moles of silver nitrate
added to the solution.

This method is used when the pH of the solution after the sample has been prepared is acidic.
If the pH is neutral or basic, Mohr’s method or the gravimetric method should be used. The

method is illustrated below by using the procedure to determine the concentration of chloride
(from sodium chloride) in cheese.

II. Solutions and Chemicals

Provided. Primary standard AgN03: Obtain about 3 g primary standard AgN03 in a

weighing bottle from your instructor. Dry in the oven at 1000C for 1 to 2 h, but Formatted: Character scale: 100%
no longer. Cool in a desiccator for 30 to 40 min. This will be weighed by the Formatted: Character scale: 100%
direct method.

 Ferric alum indicator [KFe(S04SO4)2.12H2O, saturated solution],

 6 M HN03 HNO3 (free of oxides of nitrogen).

 Nitric acid free from lower oxides of nitrogen should be colorless and can be Formatted: Character scale: 100%
prepared if necessary, by boiling 1: 1 HN03 HNO3 until N02 NO2 is expelled.

 Potassium permanganate solution:(5%) Add 1.5 g KMnO4 to 30 mL of Formatted: Character scale: 100%

distilled water. Formatted: Character scale: 100%

 Ferric ammonium sulfate solution:( :(saturated) Add 8 g of
 NH 4 Fe(SO4 )2.12H 2O to 20 mL of distilled water and add a few Formatted: Indent: Left: 2.52 cm, No bullets
or numbering
drops of concentrated nitric acid.
Formatted: Character scale: 100%

 0.1 M KSCN solution. Weigh approximately 5.0 g KSCN, dissolve in water in

a 500-mL or l-L bottle and add 500 mL distilled water. This solution will be
standardized against primary standard AgN03AgNO3.

 Chess Cheese sample

III. Experimental Procedure

1. Standardization of KSCN solution.

Place a clean, dry weighing dish on the balance, add 0.70 to 0.75 g AgN03 AgNO3 to the
weighing dish and determine its weight to the nearest 0.1 mg. Transfer quantitatively to a
clean 250-mL wide-mouth Erlenmeyer flask. Keep away from strong light as much as Formatted: Character scale: 100%
possible until ready to titrate. Add 50 mL distilled water to the flask ready for titration, 50
mL of 6 M nitric acid free from lower oxides of nitrogen, and 2 mL ferric alum indicator. Formatted: Character scale: 100%

(Lower oxides of nitrogen form nitroso complexes with Fe3+ that are red in color and will
interfere with the end point.)

Fill your 50-mL buret with the KSCN solution, record the initial volume to the nearest
0.01 mL, and titrate with constant vigorous agitation until a faint reddish-brown color
appears in the solution; this is more easily seen if the precipitate is allowed to settle after
each addition near the end point. It will be helpful to compare the color with a solution
made by adding 5 mL of 6 M nitric acid and 2 mL ferric alum to 75 mL water. The color Formatted: Character scale: 100%
must be permanent after strong shaking. Titrate the other two AgN03 samples in the same
manner and calculate the molarity of the KSCN solution from each titration. Use the mean
of the three determinations.

2. Sample Preparation

1. Cut or grate the cheese into fine pieces and accurately weigh (to the nearest 0.1 mg)
about 1 g into a 250 mL conical flask.

2. Precisely add 10.00 mL of 0.1 mol/L silver nitrate solution (by pipette if possible), 10
mL of concentrated nitric acid (very carefully - see safety notes), 410 mL of distilled Formatted: Character scale: 100%
water and a few boiling chips, and heat the solution to boiling.

3. As the solution boils, add 1mL of 5% potassium permanganate solution. This addition Formatted: Character scale: 100%
will cause a very smelly reaction and should be done in a fume hood. Keep boiling until
the purple color disappears, then add another 1 mL of potassium permanganate solution.
Continue this process until 5 mL of potassium permanganate solution has been added
and the cheese particles are completely digested (or as close as possible). To find out Comment [AS3]: Starting here, something looks
"funny" about formatting on my screen. I don't know
when digestion is complete, remove the flask from heat and allow it to stand for a few what is wrong, but the text may not be proportionally
moments. Undigested cheese particles will float upon the surface of the clear liquid, spaced.
while the white precipitate of silver chloride will sink to the bottom. If there is still too Formatted: Character scale: 100%
much undigested cheese, the boiling and addition of 1 mL of potassium permanganate Formatted: Character scale: 100%
should be continued, checking each time until there is a satisfactory level of digestion.

4. Cool the solution and filter it. Wash the solid residue with a few mL of distilled water.

5. Make the filtrate up to 100 mL in a volumetric flask.

3. Titration

1. Use a pipet to get 10.00 mL of the cheese extract solution and pour it into a conical flask. Formatted: Character scale: 100%

2. Add 1 mL of saturated ferric ammonium sulfate solution as indicator.

3. Titrate the unreacted silver ions with the 0.05 mol L−1 potassium thiocyanate solution. The Formatted: Character scale: 100%
end point is the first appearance of a dark red colour due to the ferric thiocyanate complex .

4. Repeat the titration with 10.00 mL samples of the cheese extract solution until you obtain
concordant results (titres agreeing within 0.1 mL).

Additional Notes:

1. Silver nitrate solution will stain clothes and skin. Any spills should be rinsed with water

2. Residues containing silver ions and precipitate are usually saved for later recovery of silver
metal. Check this with your teacher.

3. A ‘blank’ titration substituting sucrose (sugar) for the cheese should be carried out to see if
there are any ‘contaminating’ chloride ions present in the reagent solutions used. If any are
found, the figure should be subtracted from the titration results.

4. For greatest accuracy it is a good idea to standardize your thiocyanate solution by titrating
several samples against your standardized silver nitrate solution (once again using ferric
ammonium sulfate indicator). The concentration of SCN- determined by this titration should
then be used in all calculations.

Comment [AS4]: Never have an orphan heading

FOR YOUR REPORT at the end of a page. If there isn't something else after
the heading, move it to the next page.

A. From your experimental data calculate, for each aliquot taken, the percentage of Na2CO3
in the unknown. Your report must include the following data.
1. Unknown number

2. The mass of each sample anhydrous sodium carbonate

3. Volume of HCl for each sample of anhydrous sodium carbonate

4. Average molarity of the HCl used

5. The mass of your unknown sample.

6. Aliquot volume of unknown solution to be titrated

7. For each aliquot taken give the net volume of acid used to the bromocresol green endpoint
(the corrected volume which will be used to calculate the percent sodium carbonate).

8. Percentage of Na2CO3 in the soda ash for each aliquot titrated

9. Average percentage of Na2CO3 in the soda ash

10. Average deviation from the mean of the individual values of percent Na2CO3.

11. Pages in your lab notebook containing the pertinent data

B. Result Calculations on Precipitation titration Formatted: Character scale: 100%

Formatted: Character scale: 100%
1. Determine the average volume of potassium thiocyanate used from your concordant titres.

2. Calculate the moles of potassium thiocyanate used. Formatted: Character scale: 100%

3. Use the equation of the reaction between silver ions and thiocyanate ions Formatted: Character scale: 100%

(Ag+ (aq) + SCN-(aq) → AgSCN) to calculate the moles of unreacted silver nitrate in 100 mL Formatted: Character scale: 100%
of cheese extract, and multiply the figure by five to determine the total moles of unreacted
silver nitrate (the excess) in the 500 mL volumetric flask.

4. Calculate the moles of silver nitrate in the 50 mL of solution that was added during the
sample preparation to the cheese.

5. Calculate the total moles of silver nitrate that reacted with the salt from the cheese by
subtracting the moles of unreacted silver nitrate (the excess) from the total moles of silver
nitrate added to the cheese.
6. Use the equation of the reaction between the silver ions and the chloride ions to calculate Formatted: Character scale: 100%
the moles of sodium chloride in the sample of cheese.

Ag+ + Cl- → AgCl

(aq) (aq) (s)

7. Calculate the concentration of sodium chloride in the cheese as grams of salt per 100 g Formatted: Character scale: 100%
cheese (% salt).

Chem 223: Experiment 5

Redox Oxidation with Potassium Permanganate and

Dichromate- Determination of Iron in an Ore.

References: Harries: Chap. 16.

Harvey. 9D
Kealy: pp. 90-94

This experiment is partly based on the experiment 7- Chem 1502-Villanova University

and Expt. 31- Chem 10 – UWI- Mona University.

A. Redox Titration with Potassium Permanganate.

I. Introduction:
In this experiment, the percentage of iron in an unknown iron oxide sample is determined by
titration with a standard solution of potassium permanganate.
The overall reaction is:
MnO4+ 8 H+ + 5 Fe2+ Mn2+ + 5 Fe3+ + 4 H2O
To ensure that all of the iron in the unknown is in the Fe2+ state prior to titration, the unknown
is treated with tin(II) chloride, which reduces any small amount of Fe3+ that might be present:
Sn2+ + 2 Fe3+ Sn4+ + 2 Fe2+
The excess Sn2+ is then removed by reaction with mercury(II) chloride, which produces
insoluble mercury(I) chloride:
Sn2+ + 2 HgCl2 Sn4+ + Hg2Cl2(s) + 2 Cl
The potassium permanganate is previously standardized against sodium oxalate in acid
2 MnO4 + 5 H2C2O4 + 6 H+ 2 Mn2+ + 10 CO2 + 8 H2O
Potassium permanganate acts as its own indicator both in the standardization and in the
iron determination.
II. Reagents
Provided: * Prepared 0.02 M KMnO4 solution as follow: Dissolve about 3.2 g of KMnO4 in
1 L of deionized water using a large beaker. Cover that beaker with a watch-glass and
heat to boiling (using a hot plate in the hood first) to destroy reducible substances (traces
of organics remaining in deionized water). Keep the solution at a gentle boil for about 1
hr. Let the solution stand overnight. Remove MnO2 by filtering through a filter crucible.
Transfer the solution to a clean amber glass-stoppered bottle; store in the dark when not
in use. If the solution is kept in the dark and is not acidified, its concentration will
remain stable for several weeks.
Important: clean the filter and beakers using a solution of sodium bisulfite (use about 1g
Comment [AS1]: If it were "the" amber bottle, it
would be mixed with the standard KMnO4, obviously
in 400 mL of water. Store this solution in the an amber bottle). not what is meant.
 Prepare 1 L of 1 M H2SO4 by slowly adding 60 mL of concentrated acid to about 700
mL of water then diluting to 1 L.
 Dry about 1.5 g of primary-standard sodium oxylate, Na2C2O4, at about 110 °C for at
least 1 hr . Cool in the desiccator.
 Dry the unknown iron ore at 1100C for at least 3 hr . Keep it in the desiccator.
 A fresh solution of the SnCl2 reducing agent needs to be prepared each lab session.
Dissolve 6 g of SnCl2· H2O in 10 mL of concentrated HCl, using a 100 mL volumetric
flask. After the SnCl2 dissolves, dilute to volume. Keep this solution tightly sealed to
prevent air oxidation.
 Mercury(II) chloride, 5% (w/v).: Dissolve 50 g of HgCl2 in 1 L of distilled water.
 Zimmerman-Reinhard reagent or "preventive" solution: Dissolve 300 g of MnSO4. 4
H2O in 1 L of water. Cautiously add 400 mL of concentrated H2SO4, 400 mL of 85%
H3PO4, and dilute to 3 L.
III. Experimental procedure
1. Standardization of potassium permanganate solution:
The standardization and the determination of the unknown should each be based on at
least three titrations. After your first determination, it is preferable to use the procedure
described in Note.
Procedure: Weigh about 0.2 g Na2C2O4 (to the nearest 0.1 mg) and put a sample into a
400 mL beaker. Dissolve each sample in about 250 mL of 1 M H2SO4. Heat each solution
to 80 - 90 °C, and titrate with KMnO4 while stirring with a thermometer. The pink color
imparted by one addition should be permitted to disappear before further titrant is added.
The temperature should not drop below 60 °C. The end point is marked by the appearance
of a faint pink color that persists at least 30 s. Determine a blank by titrating an equal
volume of the 1 M H2SO4. Correct the titration data for the blank, and calculate the
concentration of the permanganate solution.

1. Add the permanganate directly into the oxylate solution (not down the walls of the
beaker). Promptly wash down any KMnO4 that spatters on the walls of the beaker into
the bulk of the liquid using a wash bottle.

2. Finely divided MnO2 will form if the KMnO4 is added too rapidly and will cause the
solution to acquire a faint brown discoloring. This is not a serious problem iof sufficient
oxylate remains to reduce the MnO2 to Mn2+; simply discontinue the titration until the Formatted: Subscript
brown color disappears.

3. The surface of the permanganate solution rather that the bottom of the meniscus can
be used to measure titrant volumes. While you may use either the meniscus or the top of
the solution, you must be consistent, always making the same choice.

4. Partial decomposition of the permanganate to MnO2 may occur if it remains in

the buret for a long time. Clean the buret with a dilute sodium bisulfite solution.

2. The determination of iron in an ore:

Use a first sample size of about 0.5 g if your unknown is < 30% iron and 0.4 g if > 30%
Sample preparation. Weigh a sample into a 100 mL glass beaker. Add 10 mL of
concentrated HCl. Cover the flask with a small watch glass. Heat the solution in the hood
at just below boiling until the sample is dissolved, about 5-10 min. A white residue of
hydrated silica may remainn. . All sample solution should be transferred to the 100 mL
volumetric flask (solution A). This solution is also used for the redox titration with
Reduction with tin(II) chloride. Take 10.00 mL of solution A to the 250 ml conical flask
(individually through this step to avoid air-oxidation of iron(II)). Adjust the sample size to
about 15 mL by dilution. Heat the solution nearly boiling. Add sufficient KMnO4 solution
to impart a faint yellow color to the solution (thus einsuring that there is not a large excess
of tin(II)). And then add 10 ml of 6 N HCl followed by Now add ingthe SnCl2 drop by
drop until the color changes from yellow to colorless or a very light green; then add two
more drops. Cool to room temperature, and rapidly add 10 mL of 5% HgCl2 solution. A
small amount of silky white Hg2Cl2 should precipitate. An absence of precipitate indicates
that insufficient SnCl2 was used and the reduction of iron(III) was incomplete. A gray
residue indicates the presence of elemental mercury resulting from the use of too much
SnCl2. If no precipitate forms or if the precipitate is gray or black, the trial must be
Titration. Wait 2 to 3 minutes after adding the HgCl2. Then add 10 mL of Zimmerman-
Reinhard reagent (Caution! This is a caustic mixture containing concentrated sulfuric acid
and phosphoric acid.) and 100 mL of water. Titrate immediately with standard KMnO4 to
the first faint pink that persists for 15 to 20s. Do not add the KMnO4 rapidly at any time.
Correct the titrant volume for the blank if it has been determined.

B. Determination of iron using potassium dichromate: Redox indicators

I. Introduction

As an oxidant, dichromate has some advantages over permanganate, but, as it is less powerful,
its use is much more limited. Furthermore, Cr(VI) is thought to be a carcinogen, so any use of
dichromate is discouraged so that the Cr(VI) burden in the environment is minimized. It is
obtainable in a state of high purity and can be used as a primary standard. Solutions of
dichromate in water are stable indefinitely. The half reaction for the dichromate system is:

Cr2O72- + 14H+ + 6e- → 2Cr3+ + 7H2O E° = 1.33 V

The most important application of dichromate is in its reaction with iron(II) in which it is
often preferred to permanganate.

The relevant half reaction is :

Fe2+ → Fe3+ + e- E° = -0.77 V

and the total reaction is:

Cr2O72- + 6 Fe2+ + 14H+ → 2Cr3+ + 6 Fe3+ + 7H2O E° = 0.56 V

Unlike permanganate, dichromate titrations require an indicator. There are three indicators
that may be used for the titration of Fe2+ with K2Cr2O7. These are diphenylamine,
diphenylbenzidine and diphenylamine sulfonate. The colour change for all three indicators is
green to violet and the standard electrode potentials are all ca 0.78 V. According to Kolthoff
and Sandell, this should lie between the electrode potentials of the two reduction reactions.
This not being the case, phosphoric acid is added to reduce the electrode potential for the Fe3+
→ Fe2+ reaction by stabilizing the ferric ion.
As the titration proceeds, the sample solution will turn green due to the presence of Cr . The
endpoint is reached when the very fine yellow color (at the beginning of titration curve) of
the Cr titrant appears (at the end of titration curve).

II. Reagents:
Provided: - Potassium dichromate can be used as a primary standard if it is dried in an oven at
150- 200 C for two hours to remove any bound water.
- Zinc metal; conc. H2SO4; conc. H3PO4
To be prepared: Prepare a standard dichromate solution by dissolving an accurately weighed
sample of about 0.4 g in water and make up to 250 mL in a volumetric flask. Calculate the
concentration of dichromate solution in mol.L-1. (FW of K2Cr2O7 = 294.19).

III. Experimental procedure

The reduction step:

Add about 1 g of zinc metal in 10.00 mL of solution A, together with 5 ml of 6 NM HCl,

replacing the watch glass immediately. You should carry out titrations with 3 portions of
solution A. Do not add all of the zinc at once since this may reduce the Fe3+ to metallic
iron. Heat the flask as necessary while swirling it gently, to maintain a vigorous but not
violent reaction until all of the zinc is consumed. A few flecks of non-reactive black
residue may be ignored. Add a second 1 g of zinc in 3 portions and heat the solution as

Repeat the addition of zinc in smaller portions until reduction is complete. Usually this
means that the solution is clear with no trace of yellow. This reaction is, in essence, a
titration of the Fe3+ with Zn(s). Adding too much extra zinc metal will greatly increase the
time required to dissolve all of the excess zinc. (In concentrated Fe2+ solutions, a very
light green hue of iron(II) may be apparent in a solution that is completely reduced; a
solution that appears green with a trace of yellow will require a small additional portion of
zinc. In addition, a solution that is too low in volume may appear to show a yellow-green
color — add some distilled water and see if the color of the solution turns clear, a sign that
all of the iron(III) has been reduced.)

When the solution appears to be completely reduced, add another 0.1 g of zinc and heat
nearly to boiling for about 5 min. (Approximately 3 g of zinc will suffice for reduction of
0.4 g samples dissolved as described above. The final portion of zinc should not be added
until one is ready to complete that particular titration, since iron(II) undergoes air
oxidation.) If any zinc remains undissolved, add 5 mL of concentrated H2SO4 in 20 mL of
water, and swirl the flask until the remaining zinc is dissolved. Very fine bubbles of H2
will emanate from any undissolved zinc as opposed to the larger bubbles of a boiling

solution. Set your reaction mixture off the hotplate and allow the boiling to stop to check
for any "fizzing" unreacted zinc.

Titration with K2Cr2O7

After waiting only about 2 to 3 minutes, add to the pre-reduced solution, about 5ml of
concentrated sulfuric acid and 7ml of syrupy phosphoric acid (measured in a graduated
cylinder). Dilute with distilled water to bring the volume to about 125ml. Again cool the
solution to room temperature by running the outside of the flask under cold water. Add 8
drops of diphenylamine sulfonate indicator and slowly titrate with your standard K2Cr2O7
solution from a blue-green, through a greyish tinge to the first permanent violet, which is
the end point. The titration should be conducted dropwise when the grey tinge is noted
because the oxidation of the indicator is somewhat slow at this point. Do the a trial run
first to see the end point and determine the sample weight.

Then do three good trials. The average relative standard deviation should not exceed 0.02 Comment [AS2]: Relative errors are unitless!

There is a significant indicator blank for this titration. To run a blank, all the reagents
except for the iron unknown should be added to a 250ml Erlenmeyer flask and the volume
should then be brought up to the approximate volume at the end point using distilled
Remember the actual end point change is slow. The blank should be no more than 1 to 4
drops (about 0.20ml) and its value needs to be subtracted from the volume of K2Cr2O7
required to titrate each iron sample. Do the blank twice.
NOTE: Solutions containing Cr or Hg should not be poured down the sink, since these metals
are highly toxic. Dispose of these solutions in the waste containers that are provided.


1. How else could you determine the end points in dichromate and permanganate
2. A solid is known to contain iron(III). Outline how you could determine the iron
content using a redox titration.
3. In another research, the scientists use the above methods to determine iron in natural
magma using concentrates concentrated sulphuric and hydrofluoric acids for sample
digestion. Explain the role of HF in this experiment. And wWhat kind of beaker
should be used?
4. 4. Report the % Fe found in the unknown for each determination, the mean value,
standard deviation, and relative standard deviation

Chemistry 223: Experiment 6


References: 1. C, Chap. 10 (especially pp. 263-264 and 267-272) and pp.583-584.

2. H7, Chapter 16, especially section 16-7.
3. S & W, Chaps. 11, 12 (especially pp. 312-17, 320-23), pp.593-96.
4. SWH, Chaps. 13, 14 (especially pp. 339-344), 31 (pp. 781-784).
This experimental procedure is mainly based on the lab. Iodometric determination of copper
in brass. Chem. 223- UIUC

I. Purpose
Brass is an alloy consisting principally of copper, zinc, lead, and tin. In addition, several other
elements, iron and nickel, for example, may be present in minor amounts. The iodometric
method is convenient for estimating the copper content of such alloys.
A solid brass sample is dissolved in acid. The Cu2+ is reacted with I- to form CuI(s) and I2. Formatted: Superscript
The iodine generated is determined by titration with standardized thiosulfate, thus allowing
calculation of the copper content of the brass.

II. Introduction:

In acid solution practically all oxidizing agents will oxidize iodide ion to iodine
quantitatively. The iodine formed in the reaction can then be titrated by means of a standard
sodium thiosulfate solution. This type of indirect titration is given the general name of

Iodometric methods of analysis have a wide applicability for the following reasons:

1. Potassium iodide, KI, is readily available in high purity. Formatted: Space Before: 0 pt, After: 6 pt

2. A good indicator, starch, is available to signal the equivalence point in the reaction
between iodine and thiosulfate. Starch turns blue-black in the presence of iodine.
Therefore, when the blue-black color disappears, the iodine has been completely
reduced to the iodide ion.
3. Iodometric reactions are rapid and quantitative.
1.4.A precise and stable reducing agent, sodium thiosulfate (Na2S2O3), is available to react
with the iodine.

2. The amount of iodine liberated in the reaction between iodide ion and an oxidizing Formatted: Space Before: 0 pt, After: 6 pt,
No bullets or numbering
agent is a measure of the quantity of oxidizing agent originally present in the solution. The
amount of standard sodium thiosulfate solution required to titrate the liberated iodine is then
equivalent to the amount of oxidizing agent. Iodometric methods can be used for the
quantitative determination of strong oxidizing agents such as potassium dichromate,
permanganate, hydrogen peroxide, cupric ion and oxygen.

As has been mentioned above, the endpoint in a titration of iodine with thiosulfate is signaled
by the color change of the starch indicator. When starch is heated in water, various
decomposition products are formed, among which is beta-amylose which forms a deep blue-
black complex with iodine. The sensitivity of the indicator is increased by the presence of
iodide ion in solution. However, if the starch indicator solution is added in the presence of a
high concentration of iodine, the disappearance of the blue-black color is very gradual. For
use in indirect methods, the indicator is therefore added at a point when virtually all of the
iodine has been reduced to iodide ion, causing the disappearance of the color to be more rapid
and sudden. The starch indicator solution must be freshly prepared since it will decompose
and its sensitivity is decreased. However, a properly prepared solution will keep for a period
of a few weeks. A preservative such as a small amount of mercuric ions may be added to
inhibit the decomposition.

Solutions of sodium thiosulfate are made up to an approximate concentration by dissolving

the sodium salt in water that has previously been boiled. Boiling the water is necessary to
destroy micro-organisms which metabolize the thiosulfate ion. A small amount of Na2CO3 is
added to the solution in order to bring the pH to about 9. The solution is standardized by
taking a known amount of oxidizing agent, treating it with excess iodide ion and then titrating
the liberated iodine with the solution to be standardized. Oxidizing agents such as potassium
dichromate, bromate, iodate or cupric ion can be employed for this procedure. You will be
using potassium iodate, KIO3, as your primary standard. The reaction between IO3- and I- is
given as

6H++IO3-+5I---- >3I2+3H2O

Reactions Involved in Iodometric Processes

Iodometric methods depend on the following equilibrium:
I2 + I-<===> I3-
Since the solubility of I2 in water is quite low, the formation of the tri-iodide ion, I3-, allows us
to obtain useful concentrations of I2 in aqueous solutions. The equilibrium constant for this
reaction is approximately 700. For this reason iodometric methods are carried out in the
presence of excess iodide ion.
The reaction between iodine and the thiosulfate ion is:
I2 + 2S2O32- <===> 2I- + S4O62-

This reaction proceeds quantitatively in neutral or slightly acidic solutions. In strongly

alkaline or acidic solutions the oxidation of the thiosulfate does not proceed by a single
reaction. In the former, the thiosulfate ion is oxidized to sulfate as well as to the tetrathionate.
In the latter, the thiosulfuric acid formed undergoes an internal oxidation-reduction reaction to
sulfurous acid and sulfur. Both of these reactions lead to errors since the stoichiometry of the
reactions differs from that shown above for the thiosulfate as a reducing agent. The control of
pH is clearly important. In many cases the liberated iodine is titrated in the mildly acidic
solution employed for the reaction of a strong oxidizing agent and iodide ion. In these cases
the titration of the liberated iodine must be completed quickly in order to eliminate undue
exposure to the atmosphere since an acid medium constitutes an optimum condition for
atmospheric oxidation of the excess iodide ion.
The basic reaction in the determination of copper using the iodometric method is represented
by the equation:

2Cu2+ + 4I- <===> 2CuI(s) + I2

This is a rapid, quantitative reaction in slightly acidic solutions, if there is a large excess of
iodide ion present and if the copper is in the form of a simple ion rather than a complex one.
The iodine that is liberated can be titrated in the usual manner with standard thiosulfate
solution. The reaction involving cupric ion and iodide takes place quantitatively since the
cuprous ion formed as result of the reduction is removed from the solution as a precipitate of
cuprous iodide.

Iron interferes since iron(III) ions will oxidize iodide. Since the iron will be found in the +3
oxidation state as a result of the dissolution of the brass sample, a means of preventing this
interference is necessary. This can be accomplished by converting the iron(III) to a soluble
iron(III) phosphate complex using phosphoric acid. At a pH of 3.0-4.0 the iron phosphate
complex is not reduced by iodide ion. If arsenic and antimony are present, they will provide
no interference at this pH if they are in their higher oxidation states.

Brass formulations also may contain up to 39% Zn, 2.5% Sn and 8.5% Pb. When dissolved in
concentrated nitric acid, the zinc and the lead become Pb2+ and Zn2+ . These do not interfere
with the analysis of copper because they are not reduced to the Pb+ and Zn+ states by the
action of iodide ion under the conditions of this experiment. The tin is oxidized to Sn4+ by the
concentrated nitric acid and after dilution and adjustment of pH this form becomes SnO2
which is insoluble and may be observed as an inert white precipitate at the bottom of your
flask. Under these conditions the tin does not interfere with the analysis.

A cursory examination of electrode potentials suggests that an analysis based upon reduction
of copper(II) by iodide would not be feasible.

Cu2+ + e- ⇔⇔Cu+ E0 = 0.15 V

I2 + 2e- ⇔ 2I- E0 = 0.54 V Formatted: Font: 12 pt

In fact, however, the reduction is quantitative in the presence of a reasonable excess of iodide
by virtue of the low solubility of copper(I) iodide. Thus, when the more appropriate half-

Cu2+ + I- + e- ⇔ CuI(s) E0 = 0.86 V Formatted: Font: 12 pt

is employed, it becomes apparent that the equilibrium

2Cu2+ + 4 I- ⇔ 2CuI(s) + I2 E0 = 0.32 V Formatted: Font: 12 pt

is reasonably favorable. Here, the iodide ion serves not only as a reducing agent for
copper(II) ion, but also as a precipitant for copper(I).

III. Reagents: Comment [AS1]: I would start this section on a

new page unless more than 2 lines of text show up on
Provided: 1-2 g KIO3 was dried at 110°C for 2 hours. (Don't mix up the KIO3 (iodate) and KI this page.

(iodide); they both look the same.)

0.1 0.1 M Sodium Thiosulfate solution: Heat 1/2 liter of distilled H20 to boiling. Formatted: Font: 12 pt, Subscript
Boil at least 5 min. Allow to cool, then add 12.5 g Na2S2O3 • 5H20 and about 0.05 g Formatted: Indent: Left: 1.27 cm, No bullets
Na2CO3. Stir until solution is complete. Transfer to a CLEAN, glass-stoppered bottle. or numbering

Store in the DARK (Cover the bottle with foil). This solution must be made up a week
before you plan to use it.
Conc. HNO3 is about 16 M and 6 M HNO3 ; 85% H3PO4. Formatted: Font: 12 pt, Subscript
Formatted: Font: 12 pt, Subscript
To be Prepared:
 7.5×10-3 M Potassium Iodate Standard Solution: Weigh into your 250.0 mL
volumetric flask 0.36-0.44 g (to the nearest 0.1 mg) of dried KIO3. Dilute to volume
with deionized water. Store in the DARK.
 Preparation of Sample Unknown:
a. Weigh out 0.4-0.5 g (weighed exactly) samples of brass into 100 mL glass beaker
Samples are easy to spill. BE CAREFUL!! You will not be given more. (Some students
will destroy one sample in the fuming step, and others will waste a sample in the
titration. This titration is an easy one to see, but once it is overshot the sample is wasted.
Be careful!)
b. Add 2.5 mL of 6 M HNO3 to each.
c. Warm the solutions in the hood until all dark particles dissolve. A white precipitate is
d. Transfer all the solution to the 100 mL volumetric flask, wash the beaker woith
distilled water, dilute the solution to the mark (A solution) .
e. Take 10.00 mL of A solution to 250 ml conical flask , add 4mL of 4M H2SO4 and
evaporate on a hot plate until white fumes of SO3 form, then stop heating [1]. Be
careful; some samples will have a solid precipitate at this point and bumping
(sudden, violent boiling) can easily occur in this viscous solution. Do not evaporate
to dryness. Allow to cool, then carefully add about 10 mL deionized H20H2O.
f. Boil 1-2 minutes, then allow to cool again.
g. With good mixing, add 1:1 NH3 ( = one part conc. NH3 + one part water) slowly until Formatted: Subscript
the blue-purple color persists. Add only a SLIGHT excess of NH3. A slight excess of
NH3 can be detected by its odor. Stop when this odor is just detectable. It may take
10-20 mL of the NH3 solution.
h. Add 1.0 mL of 85% H3PO4.
i. Test the pH of each solution and add more NH3 or H3PO4 to adjust to between pH 3
and pH 4. Check with a pH meter [2]
The standardization and titration of the samples should be done the same day.
III. Experimental Procedure:
1. Standardization of Na2S2O3 solution: Pipette 50.00 mL aliquots of the KIO3 solution into
four 250 mL Erlenmeyer flasks. (The fourth aliquot might be used to make a quick
titration to get an idea of what the endpoint looks like, and to see how many mL it takes to
get to the endpoint.) Then, treating each flask separately:
a. Add about 1g of iodate-free KI.
b. When dissolution is complete, add 10 mL 1.0 M HCl. (Conc. HCl is 12 M.)
c. Titrate immediately with the thiosulfate solution until the color becomes pale yellow.
Then add 5 mL starch indicator and titrate to the disappearance of the starch-blue
color. (See comment concerning starch under Item 1 b, above.) After the starch is
added, swirl the contents of the flask. As you approach the endpoint, continue
swirling. Wait about 10 seconds with swirling to make sure the reaction is complete
before adding the next drop. At the endpoint, the solution turns from blue-black to
colorless in about 1/2 drop.

Blank Determination

Potassium iodide may contain appreciable amounts of iodate ion which in acid solution
will react with iodide and yield iodine. The liberated iodine would react with thiosulfate
and thereby cause the apparent molarity of the thiosulfate to be too low. The following
procedure allows for the determination of a blank correction which will properly correct
for any iodate that might be present. Prepare a solution of exactly 2.00 g of KI dissolved
in 50 mL of distilled water and then acidify the solution with 5 mL of 3 M sulfuric acid
and then immediately add 5 mL of starch indicator. If a blue-black color appears right
after mixing, use the thiosulfate solution in the burette to determine the volume of
solution required to cause the color- to disappear. This volume must be subtracted from
the standardization and analyses volumes. If the potassium iodide is completely iodate-
free no color will of course develop and no blank correction is necessary.

2. Titration of Cu-Brass Solutions: Titrate samples successively.

a. Dissolve about 2 g KI (iodate free) in 5 mL deionized water, and add this to the
sample. The solution should now be yellow-brown; if it is green, the pH is wrong
and needs to be adjusted to between 3 and 4. Titrate immediately with the thiosulfate
solution until the iodine color (yellow) is no longer distinct.
b. Add 3 mL starch solution. (The color of the starch-iodine complex is quite dependent
upon the source of starch and the age of the starch preparation. A correctly prepared
indicator solution should give a blue iodine-starch complex. Partially decomposed
starch or solutions containing amylopectins which have not been hydrolyzed will
give a reddish-blue colored complex which does not equilibrate with the I2 solution
concentration rapidly. The effect occurs if the starch is added too soon before the
endpoint.) Titrate until the starch-blue color begins to fade. Add 1 g of potassium
thiocyanate, KSCN, and complete the titration. (I2 tends to adsorb strongly on the
solid CuI precipitate. Thiocyanate is added to displace the adsorbed I2 making it Formatted: Font: 12 pt, Subscript
accessible to the starch colloidal particles, hence yielding a sharper endpoint.)
The endpoint is reached when the starch-blue color disappears. Do not titrate further Formatted: Indent: Hanging: 0.16 cm
even if the solution turns blue again several minutes after stopping at the endpoint.
c. Repeat with remaining sample solution .

[1] Concentrated acids are dangerous. Transport the acid from the dispensing station to your
work area in the small polyethylene containers you have; keep the caps screwed on. If
you spill any of the concentrated acid, wipe it up immediately. If you get the acid on
yourself, flood the area with water immediately, and contact the TA for assistance.
[2] Use of the pH meter:
a. Store electrode in saturated KCl or pH 4 buffer when not in use.
b. Rinse the electrode into a waste beaker (not the storage buffer!) with distilled
c. Put the electrode into fresh calibration buffer to ensure that the instrument is
calibrated. Once the meter is stable, this need be done only once every 5 minutes,
not between every use.
d. Rinse the electrode into the waste beaker. Wick excess H2O from the electrode
with a Kimwipe.
e. Lower the electrode into the unknown, NOT touching the bottom or sides of the
beaker or flask. Once the reading has stabilized, record the reading in your lab
book. If you are trying to adjust the sample to a specific pH, remove the electrode,
add acid or base, swirl (being careful not to bump the electrode), return the
electrode to the solution, read the equilibrium reading, and repeat until satisfied.
Be careful not to lose drops of solution!
f. Rinse the electrode INTO YOUR SAMPLE FLASK with distilled H2O (so as not
to lose precious sample!).
g. Return the electrode to water or buffer, step a. above.


Report your results as mass % Cu in the brass. Also report your relative standard
deviation in ppt for both the standardization and the sample analysis.
1. Why did you do each of the following?
a. prepared the thiosulfate solution several days in advance of its use as a titrant
solution and stored this solution in the dark.
b. boiled the water for the thiosulfate for at least five minutes when other experiments
in this course only required that the water be brought to a boil.
c. used three different acids (HNO3, H2SO4, H3PO4)? Formatted: Font: 12 pt
Formatted: Font: 12 pt
d. necessary to boil the water used to prepare the thiosulfate solution
Formatted: Font: 12 pt
e. added sodium carbonate to the thiosulfate. Formatted: Font: 12 pt
2. In this experiment you titrate I2 produced in the reduction of Cu2+ by I-, using S2O3 2-. Why Formatted: Font: 12 pt
not use S2O3 2- for direct reduction of the Cu2+?
3. What compound is the primary standard for this determination?

4. Why is HCl added to the IO3- mixture and why must the solution be titrated immediately?

5. Why is the solution containing the dissolved brass sample heated to expel SO3 fumes?

6. Why is H3PO4 added to the brass sample?

7. What is the purpose of the KSCN that is added just before the endpoint in the titration?

8. Why is the solution containing the dissolved brass made basic with concentrated NH3 and
then again acidified with H2SO4?

9. What is the formula of the tetrammine copper(II) complex?

10. Why do Zn2+ and Pb2+ not interfere in this procedure?

11. What sort of complications would arise if the iodine-thiosulfate titration were carried out
in a highly acidic solution?

12. If the solution were highly basic, how would the iodine thiosulfate reaction be influenced?

13. Why is the starch indicator not added at the beginning of the tritration?

Notes: Sources of Error

The following are the most important sources of error in the iodometric method:

1. Loss of iodine by evaporation from the solution. This can be minimized by having a large
excess of iodide in order to keep the iodine tied up as tri-iodide ion. It should also be apparent
that the titrations involving iodine must be made in cold solutions in order to minimize loss
through evaporation.

2. Atmospheric oxidation of iodide ion in acidic solution. In acid solution, prompt titration of
the liberated iodine is necessary in order to prevent oxidation.

3. Starch solutions that are no longer fresh or improperly prepared. The indicator will then not
behave properly at the endpoint and a quantitative determination is not possible.
Chem 223 : Experiment 7

Ion – exchange and Titration method Method for the

determination Determination of Cobalt and Nickel


1. C, Chaps. 13 (pp. 371-376), 16 (pp. 451-455).

2. H7, Chaps. 19, 23, and 25
3. S & W, Chaps. 15 (especially pp. 443-49), 17 (pp. 487-89).
4. SWH, Chaps. 18, 20 (pp. 505-518), 25 (pp. 652-653), 29 (pp. 715-716).
5. J. S. Fritz & G. H. Schenk, Jr., Quanta. Analyt. Chem., 4th ed., (pp. 582 & 621).
6. J. A. Dalziel and A. K. Slawinski, Talanta 15, 367-372 (1968).
7. Bjerrum, A. S. Halonin, and L. H. Skibsted, Acta Chem. Scand. 29A, 326-332
8. Chem 222. Truman State University
The procedure of separation of Co2+ and Ni2+ in this experiment is mainly based on the lab.
“Spectrophotometric determination of cobalt and nickel” Chem. 223. UIUC

I. Purpose of the experiment

Determination of multiple components in a single unknown is frequently required. An analyst

has a choice of attempting a simultaneous determination or performing a preliminary
separation. Each approach has its advantages and drawbacks. Because few techniques are
truly specific, any simultaneous determination usually suffers from some mutual interference
between the combined components, with resulting loss of accuracy and/or precision.
Frequently, however, the resulting uncertainties are less than those introduced by the extra
sample handling required for preliminary separation. Furthermore, simultaneous
determinations are often faster, and therefore more efficient, than those relying on separations.
Generally, the choice of a preferred method must be based on the analyst's assessment of the
balance among these factors.

In this experiment, the amounts of Co(II) and Ni(II) in a mixture will be determined by two
different approaches. First of all, the Co(II) and Ni(II) will be isolated by ion exchange and
the separated components assayed. After that Co2+ will be complexometrically titrated using
replacement method and Ni2+ will be determined by gravimetric analysis.

II. Introduction

Ion Exchange Chromatography relies on charge-charge interactions between the solutes in

your sample and the charges immobilized on the resin of your choice. Ion exchange
chromatography can be subdivided into cation exchange chromatography, in which positively
charged ions bind to a negatively charged resin; and anion exchange chromatography, in
which the binding ions are negative, and the immobilized functional group is positive. Once
the solutes are bound, the column is washed to equilibrate it in your starting buffer, which
should be of low ionic strength, then the bound molecules are eluted off using a gradient of a
second buffer which steadily increases the ionic strength of the eluent solution. Alternatively,
the pH of the eluent can be modified as to give your solues or the matrix a charge at which
they will not interact and your molecule of interest elutes from the resin. If you know the pH
you want to run at and need to decide what type of ion exchange. If it is negatively charged at
the pH you wish, use an anion exchanger; if it is positive, use a cation exchanger. In many
cases it may be more advantageous to actually select conditions at which your solutes will
flow through while the contaminants will bind. This mode of binding is often referred to as
"flow through mode". This is a particularly good mode to use in the case of anion exchange.
In this experiment, the separation employs an ion exchange column, and depends on the
selective formation of an anionic complex of cobalt(II). In 9 M HCl the principal equilibrium
forms of the two metal ions are CoCl42- and Ni2+ (or NiCl+). Formatted: Superscript

The Co(II) ions are exchanged onto the resin:

2 Res-NR3 +Cl- + CoCl42-→ (Res-NR3+)2 CoCl42- + 2Cl-

And the Ni(II) passes through the resin. The R group is often -CH3. In 4 M HCl the CoCl42-
ion breaks down into cationic forms of cobalt(II) and all the cobalt(II) passes through the

Determinations of the separated components (and of the ions in the mixture) will employ
volumetric and gvarimetric method. For the determination of Cobalt or Nickel, the EDTA
with substitution titration (PAN as an indicator) will be applied and dimethylglyoxime uses as
precipitant for the determination of nickel.

Nickel(II) forms a precipitate with the organic compound dimethylglyoxime, C4H6(NOH)2.

The formation of the red chelate occurs quantitatively in a solution in which the pH is
buffered in the range of 5 to 9. The chelation reaction that occurs is illustrated below.

Adding tartarate or citrate ions before the precipitation of the red nickel complex prevents
interference from Cr, Fe and other metals. These anions selectively form tightly bound soluble
complexes with the metals and prevent the formation of insoluble metal hydroxides in the
buffered solution.
An alcoholic solution of dimethyglyoxime (DMG) is used as the precipitating reagent during
the experiment because DMG is only slightly soluble in water (0.063 g in 100 mL at 25°°C).
It is therefore crucial to avoid the addition of too large an excess of the reagent because it may
crystallize out with the chelate. It is also important to know that the complex itself is slightly
soluble to some extent in alcoholic solutions. By keeping the volume added of the chelating
reagent small, the errors from these sources are minimized. The amount of the reagent added
is also governed by the presence of other metals such as cobalt, which form soluble
complexes with the reagent. If a high quantity of these ions is present, a greater amount of
DMG must be added.

III. Reagents

Provided: • Prepare an ion exchange column as demonstrated by the teaching staff. Use
a glass wool plug in the bottom then add a slurry of resin (Dowex-1X8, 20-50 mesh), until
the height of the resin column is 15 cm. Never allow the liquid to drop below the resin

• Next, add another glass wool plug to the top of the level of glass wool column. Formatted: Indent: First line: 0.63 cm

Keep about 5 cm of liquid above the resin column initially, and keep the liquid just above
the glass wool at all other times. Don't let any air into the resin.

Sample Unknown.

 Obtain an unknown from the TA in a clean 25.0 mL volumetric flask. Add 18 mL of

12 M HCl, then dilute to volume with deionized water and mix thoroughly.

WARNING - Glassware used for this experiment must be scrupulously clean! The various
dilutions are tricky to keep track of, and are a common cause of error.

IV. Experimental Procedure

A. Ion- exchange Separation

• After the column is made up, it should be "charged" by passing 9 M HCl through the
column. See the following figure for details, and follow the directions there very carefully.
• With a pipette, add 5.00
mL of the unknown onto
the column.1Allow the level
of the liquid to fall to just
the top of the glass wool
before adding the HCl

• Elute the nickel from the

column with 9 M HCl using
the portions and flow rates
specified below. During
elution of the nickel, the
cobalt complex will appear
as a green or blue stationary
band on the column. Collect
the NiCl+ complex in a 250
mL beaker.

• Transfer the collected

nickel solution
quantitatively to a 50.0 0mL
volumetric flask and dilute
to volume with deionized
water. (A solution).

• After all the nickel is

seen to be eluted,2 stop the
flow of eluent, and replace
the collection beaker with
another 250 mL beaker.

Figure 1. Details of the anion exchange process.

• Elute the cobalt with 4 M HCl using the portions and flow rates as indicated in the figure. As
the HCl is diluted on the column, the blue CoCl42- complex will break down to form a pink
color as Cl- is replaced by water in the complex.

• After all the cobalt is eluted,3 stop the flow and transfer the collected solution to a 500 mL
volumetric flask. Dilute to volume with deionized water (sample B).

• Rinse the column with 75 mL of DI H2O in 15 mL portions. Remove the glass wool, and
dump the resin in the container provided.

The following dilutions should be made during this lab period in preparation for the second
week of this experiment. (Use pipettes and volumetric flasks for all dilutions.)
B. Gravimetric determination of Nickel Comment [AS1]: I'm not sure if this will work. I
don't have specific experience with samples that are
appropriate for the spectrophotometric determination
Pipetting 10.00 mL of (A) sample to 250 mL beaker. Add 10 ml. 20% ammonium doing DMG precipitation.
chloride solution, add 20 ml. 10% tartaric acid, dilute to 200 ml., heat to nearly boiling,
cautiously add ammonium hydroxide (1:1) from a buret until the solution is slightly
alkaline. Take back to feebly acid by addition of a few ml. HCl. The reason the
precipitation is from acid solution is that a more easily filterable precipitate will result.
The changes in color will indicate the acidity to alkalinity of the solution, but this should
be checked against a sensitive indicator paper (not litmus). If iron hydroxide appears when
the solution is made alkaline, insufficient tartaric acid is present.

Add 15 ml. of 1% solution (alcoholic) of dimethylglyoxime drop-wise. (A buret is an easy

way to do this.) Add 1:1 ammonium hydroxide until the solution is just alkaline (do NOT
use litmus) then add 2 or 3 drops in excess. (A buret is a convenient method for the
addition of ammonium hydroxide.) A dense red ppt. should form. Stir vigorously. Place
on a hot plate for 15-20 minutes, stir occasionally, finally check alkalinity and filter
through a constant weight sintered glass crucible. Wash well with hot water containing 2
or 3 drops of ammonium hydroxide. Dry at 110o - 120o C for 2 hours. Weigh as
C8H14H4O4Ni containing 20.32% Ni.

TO GET AN ITEM TO CONSTANT WEIGHT: Constant weight is defined as that state

when a substance will not change its weight on heating. This is achieved by heating the
object. Weighing the cooled object and then heating again, cooling again, and weighing a
second time. If the two weights agree within 0.5 mg, the object is said to be at constant
weight. If not then the heating, cooling and weighing process is continued until two
successive weighings do agree within 0.5 mg. Use the final weight in the calculations.

C.Volumetric determination of Cobalt Comment [AS2]: This won't work. Diluting the
Co unknown (and Ni unknown) as the Illinois lab
suggests (to 1 L) leaves concentration around 1 mM
Method A: Direct titration or less. The procedure is optimized for
spectrophotometry. Diluting to 100 mL, or 250 mL if
you must, is more likely to give useful results.
1. Add 10 mL of 4 M sodium acetate to each 10.00 mL of unknown Co2+ solution (B
solution) and using a pH meter, adjust the pH to 5.8 with 3 M sodium hydroxide.

2. Heat the solution to approximately 950 C using the hot plate/stirrer (DO NOT BOIL!).

3. Add 5 drops of xylenol orange indicator (0.2 g/100 mL, 50% alcohol) and a stirring bar
to the solution, and titrate immediately with EDTA solution. Maintain the solution
temperature in the range of 85-950 C. The endpoint is a sudden color change from violet
to yellow-pink.

Method B: Replacement Titration

1. Add 10 mL of 4 M sodium acetate to each 10.00 mL of unknown Co2+ solution (B

solution) and using a pH meter, adjust the pH to 5.8 with 3 M sodium hydroxide.

2. Heat solution to the boiling using the hot plate/stirrer (DO NOT BOIL!)

3. Add 3 drops of copper sulfate-EDTA solution ( CuY) and 8 drops of PAN indicator
solution. Titrate to a green-yellow color with standard solution.

1. Once you load the sample on the resin, the elution must be completed within the same lab

2. Use dimethylglyoxime test solution with l drop of eluent to test for completion of Ni2+
elution. Add a couple of drops of NH4OH to the test solution. Look for red Ni(DMG)2. In a
separate test, you may also use a drop of the yellow ligand solution (which you will use more
of in week 2). Ni2+ is indicated by a color change from yellow, to pink or red.

3. Use thiocyanate test solution with 10 drops of ethanol and l drop of eluent to test for
completion of Co2+ elution. A blue color signifies Co2+ is present. In a separate test, you may
also use a drop of the yellow ligand solution (which you will use more of in week 2). Co2+ is
indicated by a color change from yellow, to pink or red.

4. Use a graduate cylinder to deliver the buffer solution, a volumetric pipette to deliver the
complexing agent, and the graduated (Mohr) pipette for the metals and unknown (columns 2,
3, and 4, of all three tables).


Calculate the Ni(II) and Co(II) concentrations in unknown sample giving by TA .

What are some of the advantages of method A and B used in this experiment? Which would
you expect to be more precise?

Could KCl be used instead of HCl (as a source of chloride ion) for elution? How about LiCl?

Why is solution # used as a blank instead of deionized water?

What is the difference between ion- exchange chromatography and ion chromatography? Try
to figure out the principle of ion chromatography for determination of Cobalt and nickel in a

What are the advantage(s) and limitation(s) of using gravimetric analysis?

What are gravimetric interferences in determining Nickel by dimethylglyoxime.

Suggest alternative methods for the determination of nickel in steel. Explain in detail the
principle of ONE alternative method.
Chem 223: Experiment 8


• SWH. pp. 212-218;224-229; 402-439;

 Harries. Chapter 15
This experiment is revised from Expt. 5- Chem 5.310. Massachusetts institute Institute
of technologyTechnology.


In this experiment the quantitative composition of a solution, which is a mixture of a

monoprotic strong acid and a weaker, triprotic, acid, will be determined by
potentiometric methods. This experiment will introduce you to quantitative volumetric
analysis and potentiometric titrations.

(A) A carbonate-free sodium hydroxide solution is prepared and standardized

against pure potassium hydrogen phthalate (KHP), and is then used in a
potentiometric titration of the acid mixture.

(B) Three or four 1.0 mL aliquots of your acid will be used for potentiometric
titrations. Each titration must be continued through two equivalence points.


Strong acids, like hydrochloric acid, are completely dissociated in water, but weak acids,
like acetic acid, are only partially dissociated. The extent of dissociation can be
calculated from the value of the equilibrium constant and the amounts of weak acid and
strong base added to the solution.

The relative acidities of acids and bases are commonly expressed in terms of pKa = - log10
Ka, where Ka is the dissociation constant for the reaction

HA → H+ + A-In Formatted: Subscript

the following derivation, aA- , [A-] , and  A - represent, respectively, the activity, the
molar concentration, and the activity coefficient of the conjugate base, A-.

Since pKa ≡ - log Ka

then pKa = - [log (aH+) + log (aA-) - log (aHA)]

since pH ≡ - log (aH+)

then pKa = pH + log (aHA) - log (aA-)

Making the approximation that

Making the approximation that

This equation is referred to as the Henderson-Hasselbalch equation. It is very useful in

the buffering region of the titration of a weak acid. The pKa is -logKa at the ionic strength Formatted: Not Superscript/ Subscript
of the solution. Accordingly, the value of Ka obtained will deviate slightly from values
listed in standard references since they report the thermodynamic value at zero ionic

Figure 1. Typical Plot of a Potentiometric Titration to Determine the Equivalence point

and pKa value (monoprotic acid).

Note that the pKa is the pH at which the activities of the acid HA and its conjugate base
A- are equal.

For a triprotic acid the successive dissociation constants are defined by:

H3A = H2A- + H+ +
K1 = [H2A-] [H+]/ [H3A] Formatted: Portuguese (Brazil)

H2A- = HA2- + H+ +
K2 = [HA2-] [H+] / [H2A-]

HA2- = A3- + H+ +
K3 = [A3-] [H+] / [HA2-]

The normality of an acid solution is the number of equivalents per liter (moles per liter)
of protons that it can dissociate. The normality of a solution of a base is equal to the
number of equivalents of acid it can neutralize. The normality of a solution of a triprotic
acid is three times its molarity. In discussing titrations of acid and base solutions, it is
often convenient to think in terms of milli-equivalents.



 The TAs will prepare a solution of approximately 0.05 M NaOH as follows: Put 2.00
00 L of distilled water into the one gallon polyethylene bottle.2 Measure out 5.5 mL of Formatted: Font: 12 pt, Superscript
50% w/w NaOH* in a 10 mL graduate cylinder and pour into the water in the bottle.3
Both the solution bottle and 50% NaOH bottle should be closed immediately with
their polyethylene screw caps. Mix the dilute sodium hydroxide solution very
thoroughly by vigorous shaking with repeated inversions for at least a minute.

 100 mg of reagent grade potassium acid phthalate* (KHP) was placed into a dry
weighing bottle, and placed in a 110 ºC drying oven for 1.5 hour or overnight.5 At the
end of the drying period, remove the weighing bottle from the oven and let cool in a
small desiccator charged with calcium chloride*. Leave the stopper off of the
weighing bottle until the first time the desiccator is opened after the KHP has cooled.

 phenolphthalein indicator,1%: Dissolve 1.0 g of phenolphthalein in 100 mL of

alcohol (pH 8.0–10.0).


1. Standardization of Approximately 0.05 M sodium hydroxide

Weigh (to nearest 0.0001 g) by difference at least three samples of KHP into numbered
or otherwise identified 250-mL Erlenmeyer flasks. Sample weights should be 30 mg for
the primary standard.

Dissolve the KHP samples by swirling in 7 mL of water. Warming may be necessary. It

is essential that the samples dissolve completely; even a few small particles remaining
can cause a serious titration error.

Add 1 drop of phenolphthalein* indicator and titrate with the sodium hydroxide
solution from a buret.

The titration may be carried out rapidly at first, but the endpoint should be approached
carefully. With low-carbonate NaOH, the endpoint should be sharp and easily located
to within a fraction of a drop.6 Try to obtain the same intensity of pink color at the
endpoint for all your titrations. At the endpoint, the ideal indicator color is a barely
detectable shade of pale pink which persists for 30 seconds or more.

Run a sufficient number of titrations to assure a precise and presumably accurate

standardization. The standardization titration should be repeatable to within 2 to 3%
when volumes of about 1.5 mL of base are used. If three titrations do not result in the
desired precision, it will be necessary to conduct additional titrations.

2. Titration of a Mixture of Hydrochloric Acid* and Phosphoric Acid*

You will be given a mixture of these acids.* Yourr goal is to report the molarity of the
hydrochloric acid and the molarity of the phosphoric acid in the mixture, with their
uncertainties. You will also determine the value of pK2 for the phosphoric acid.

Using a VOLUMETRIC PIPET, pipet 1.00 mL of the acid mixture into a 30-50 mLMl
beaker and add 19.0 mL using graduated pipets of distilled H2O.

Place a magnetic stir bar in the beaker.

Rinse the pH electrode with distilled water into an empty beaker. and p Position the
electrode and 10 mL buret filled with sodium hydroxide as indicated in Figure 3. Be
sure that the pH electrode is not in a position to be damaged by the magnetic stir bar.
No air bubbles should be trapped under the polyethylene shield of the electrode. Also,
make sure that no air bubble is trapped in the tip of your buret.

Insulate the beaker from the magnetic stirrer with a layer of folded paper towel to
prevent warming of the solution by the magnetic stirrer.

Figure 3. Titration apparatus

With continuous stirring, add small increments of the approximately 0.05 M standard
solution of NaOH from the buret. Give the solution and pH meter time to equilibrate.
Read and record in the notebook the pH of the solution after addition of each portion of
the NaOH solution. Initially, the change of pH upon the addition of titrant will be
minimal. However, as the first equivalence point is approached, pH increments will
increase more rapidly, and only dropwise increments of NaOH should be added until it

is apparent that the first equivalence point has been passed.

Periodically the inside of the beaker may be washed down with distilled H2O from your
water bottle.

Warning: Be sure no drops are left on the tip of the buret when you are reading the pH.
The drop is part of the measured volume. You will not have good correlation between
volume and pH if part of the volume measured is left out. This is especially

important in the regions of rapid pH change.

Do not take pH readings above pH 11.5, because high pH damages the glass Comment [AS1]: You might think about getting
some of the ISFET electrodes rather than glass
electrode. electrodes. They're rugged and they CAN be used at
high pH.
After the first potentiometric titration, remove the electrode from the solution, wash it
with distilled water and allow the electrode to stand in a beaker of distilled water for at
least 15 minutes before proceeding with the second titration. It is a good procedure to
check the calibration of the meter against a standard aqueous buffer of pH 7.0 before
beginning another titration.

While waiting, plot your data by hand or use your PC. Review your titration curve with
your TA. Discuss any changes which should be made for subsequent titrations.


Analyze the data from your titrations with Microsoft Excel as it is described in the lecture

All the questions below should be discussed in your report.

1. Plot your experimental data as pH versus volume of standard NaOH added. Use a full
sheet of graph paper for maximum accuracy and draw a smooth curve through the points.

2. For each titration identify the two equivalence points and draw vertical lines to
determine the corresponding volumes of NaOH. The portion of the titration curve
between the two equivalence points should follow the Henderson-Hasselbalch equation.

3. Calculate the pKa2 for H3PO4, and test the validity of the Henderson-Hasselbalch
equation by calculating several other points on this part of the titration curve.

a. How does your pKa2 value compare with the literature value?

b. Your titration does not yield values of pKa1 and pKa3 but something can be said
about these pKa values from your experimental data. What is it?

c. Since the titration will most likely not continue through pKa3, how does its value
depend the shape of the curve at the point where the titration ends if pKa3 is
approximated by extrapolation?

4. From the sample volume and the distances to the first and second equivalence points,
calculate the MOLARITIES of hydrochloric and phosphoric acids in the sample. Give
uncertainties for the calculated molarities. How well do your two or three titrations

Chem 223: Experiment 8

Spectrophotometry, Beer's Law, and Precision

(This experiment is completely based on the lab. Spectrophotometry, Beer's Law, and
Precision. Chem. 223- UIUC)

SAFETY FIRST! Remember to bring AND WEAR your goggles. "But we aren't doing any
reactions this week, and the chemicals are largely innocuous." "They're hot and steam up."
"They're uncomfortable." "I forgot." "I look like I came from Mars." We've heard 'em all.
Doesn't matter. You'll be in a lab. Get to the point that it's a reflex:


Simple way to tell if you've been careful this week: if you don't have blue fingers, you did
fine. (Methylene blue is used to stain tissue during surgery, so it's safe, just ugly.)


Analytical Chemistry is the science that identifies the components of a portion of the physical
world, quantifies them, and characterizes their interactions. Doing this requires:

• A problem whose solution requires knowledge of chemical composition or dynamics.

• One or more means of measurement
• An understanding of the chemistry and physics that effects the measurement
• A means to characterize the quality of the measurement (both precision and accuracy)
• A theoretical or practical way to use the results of the measurement to solve a problem.

Our purpose here is to demonstrate a common means of measurement (spectrophotometry), to

show examples where the measurement tool works, to show examples where the measurement
tool fails, and to show examples where the chemistry interferes with the measurement. All
this will be done under circumstances where the operator's manual dexterity is of minimal or
no influence on the results.

In later labs, your skill will contribute to the precision of the experiment. Many past students
have felt that imprecision was some mark of shame or that "skill is proportional to 1/standard
deviation." There are times when this is true, but in this experiment you will see differences in
uncertainty caused by chemistry, sample, and instrument. What you should learn is:

• Non-zero uncertainty is a fundamental property of nature.

• Many factors contribute to uncertainty.
• Understanding the sources of uncertainty can lead to their minimization.
• Learning analytical chemistry means learning the "tools of the trade" PLUS developing "lab
technique" PLUS learning how to integrate many pieces of information into the solution of a Formatted: Indent: Left: 0 cm, Hanging:
0.32 cm
single problem.
Important Advice

Bring a thumb drive to lab. Save ALL your data files both to the hard disk (during the
experiment, immediately after obtaining raw data) and to your thumb drive BEFORE leaving
lab. The computers are NOT on the network; you can NOT get at your data (without which
you can't write your report) unless you remember to save your data!

One of the hardest parts of doing analytical chemistry is that we must deal with the world as a
SYSTEM (many things interacting and happening at once), yet we have been taught to deal
with learning and living as COMPONENTS (single activities, single calculations, individual
controls or pieces of apparatus).

Think of driving a carmotor bike. You don't think about each spark plug firing or turning the
wheel 2.78º to the right or pushing on the gas pedal bythrottle to get an additional 10-3 Formatted: Superscript
Newtons to accelerate. You Just Drive. You are taking a systems approach, with a lot of
activity implicit in what you do. But when you learned to drive, you DID pay attention to
pressure on the gas or the angle at which you steered. A mechanic pays careful attention to the
details of e.g. timing. Similarly, when you're doing error propagation or taking a spectrum,
you're dealing with a component. The big picture is that there's some problem involving a
chemical system you're trying to solve. For this lab, the components connect like this:

We'll discuss the theory of the components here, you'll make measurements and do
computations, and then in the questions at the end, we'll tie it all together in a systematic way.
Fig. 1: UV-1601 PC Photomultiplier Spectrophotometer

I. Beer's Law

See Harris, Seventh Edition, Chapter 18, or similar material in any other quantitative
analysis text written since the Second World War).

Suppose we have light of a wavelength λ (that's a Greek lambda; you can find it as the lower-
case l in the symbol font on nearly any computer) such that a molecule can absorb the light. If
a homogeneous solution of the analyte is illuminated by a source containing light of
wavelength λ, only a fraction of the light will be transmitted through the solution i.e.
illuminating the solution with light of intensity I0 (Watts cm-2) results in intensity I<I0 being Formatted: Subscript
transmitted. Only the transmitted light can reach the detector. Formatted: Superscript

Without worrying about the electronics and optics involved, we can say that we MEASURE
Transmittance T = I/I0. If there are no molecules to absorb the light, T = 1. If the solution
contains some absorbing molecules, 0<T<1.

The negative base 10 logarithm of transmittance is called absorbance, A = - log10T. A is a

computed quantity, not a directly measured quantity. Beer's Law says that, absent
complications (some of which we'll discuss),

A = εbC (1)
where A = absorbance
ε = molar absorptivity in liter mole-1 cm-1
b = path length over which absorbance is observed (typically 1 cm in a square cuvette,
but it can range from 1 nm for a molecular monolayer to light-years in astronomy)
C = concentration of the analyte in mole/L

This is a deceptively simple result. If one knows the molar absorptivity and path length, an
absorbance measurement directly gives concentration. Experimentally, if one has a standard
solution of known concentration Cs, then measuring its absorbance As should give the product
εb. The problem is that absorbances are additive. If there is any hint of absorbance from a
background substance, then A =(εbC)analyte + Abackground. This behavior is commonly referred
to as a linear working curve, since a plot of A vs C is just a straight line, with slope εb and
intercept Abackground. Such working curves are the most common relationships used in doing
concentration measurement in chemical analysis. Almost always, one measures BOTH slope
AND intercept. Without measuring that Abackground = 0, don't assume it or you're likely to get
yourself into a bind. The reason we're doing spectrophotometry as the first lab is to get used to
the idea that there are such things as working curves. Note that to find the slope and intercept,
you need a set of standards. This sets up a problem: how do you know the composition of the
standards and the source of absorbance in the blank? We'll answer those questions later in the

II.Deviations from Beer's Law

There are many situations where there appear to be deviations from Beer's Law. In most
cases, it's not Beer's Law that's the problem – it's that one or more assumptions that went into
Beer's Law don't apply.
For example, if there are many substances that absorb at the same wavelength,

A    bC (2)
i i i

where the index i refers to different species in solution. For example, what happens if
substances can form new compounds or complexes at high concentrations? Suppose a
compound M can pair with another molecule of M to form M2.

M+M ⇔ M2
We have no way to know the molar absorptivity of M2. What we do know is that there has to
be conservation of matter, so if CM is the total concentration of M prepared initially,
CM = [M] + 2[M2]. We also know from the law of mass action that

(3) Comment [AS1]: Use the Equation Editor to reset

these equations. This is Really Ugly. True of all
other blurry equations; if you want the original Word
file so you can just copy and paste the equations
rather than images of the equations, just ask.
In this lab, we use the well-known example of methylene blue which, in water, forms such
dimer adducts. Since we have two unknowns, the concentrations of the monomeric and
dimeric species, we will need measurements at two wavelengths to figure out the molar
absorptivities and the equilibrium constant. At low concentrations, practically no dimer will
form. Furthermore, we can make solutions in which we know CM, the total concentration of
methylene blue moieties in solution (some of which will be present as monomer and some as


Note carefully that the molar absorptivity of the dimer, M2, at wavelength λ is different than
that of the monomer, because it is a different molecule. Substituting the equilibrium constant
into Equation 4, we obtain

What we know experimentally is CM (that's what we can make up with a balance and a
volumetric flask – NOT [M]). So to express the dependence of absorbance on an
experimentally-known variable, we use CM = [M] + 2[M2] = [M] + 2K[M]2

This is a quadratic equation in the variable [M], which has the solution:


Substituting this into Equation 5 yields


That formula should bother you. If you go back to the original expression relating CM and
[M], setting K=0 gives (unsurprisingly) [M] = CM. But in the full formula, CM shows up under
a square root sign! How do we bail out of this problem? Using the nearly universal solution to
messy math problems: turn the expression into a Taylor series and look for limiting cases.

(Recall "h.o.t." doesn't mean what you say after biting into habanero peppers, but rather
"higher order terms"). Demonstration: 1.11/2 ≈ 1.04880, but 1 + 0.1/2 = 1.05, which isn't too
far off. So if K is small,

(If you think this is reasonable, you're happy with the primary mathematical trick used in
physical chemistry!)

An equation such as Equation 7 is true, but awkward. Dealing with it now is getting ahead of
ourselves; it will be the middle of the course before you have enough experience with
equilibria, Excel, and data reduction to finish processing the data. For right now, it's enough to
know that this is one example of how Beer's Law can work, but a working curve can appear to
be nonlinear.

Why else might working curves be non-linear? Two reasons are other chemical equilibria
involving the measured species, e.g. a protonoation/deprotonation reaction or instrumental
effects. An example of the latter is spectrometer resolution, which can be a problem. If the
absorption band is narrow compared to the spectrometer resolution, even a very concentrated
solution of the analyte won't absorb all the light before reaching the detector. Here's a sketch.
In the case of a very narrow spectral profile, it doesn't matter how high the molar absorptivity
is; almost all the light will reach the detector. It's just like putting a single piece of black
thread in front of a bay window – the black thread doesn't transmit any light, but the room
isn't darkened. If we make a Beer's Law plot for the ideal situation (wide absorbance spectrum
compared to the resolution of the instrument) we get the straight line in the following plot,
while the narrow spectrum above (line width less than 2 nm, while the instrument resolution
is 2 nm) gives the dashed line. At low concentrations, the slope of the Beer's Law plot for the
narrow line is 0.84 that of the broad line plot. At high concentration, the slope has fallen to
0.78. If you use a ruler on the plot, you can see that the shallower-sloped line is curved.

III. Error Propagation

See Harris, Chapter 3, Section 5 and Appendix C for general error propagation formulas.
Alternatively, see Appendix II of this lab manual. The error illustrated at the end of section II
above is an example of determinate error. It results from a systematic problem with the
measurement itself, namely, inadequate spectrometer resolution. It could be completely
eliminated by the expedient of acquiring the spectrum with another spectrometer with smaller
spectral resolution. There is another kind of error, indeterminate error, which is random, and
can never be completely eliminated from your measurements. This section addresses the
question: If I know the uncertainties in all but one quantity involved in an experiment, what
is the uncertainty in the remaining quantity? Error/fluctuation/distribution/variance/variability
of a quantity on successive measurements are fundamental characteristics of nature and useful
characteristics we can study.

Suppose that we are interested in a measured value Z, which is a function of other variables,
Z = f(A, B, …) then


where δZ represents the uncertainty in Z and similarly for A and B. Since all random errors, if
averaged, have a zero mean, just averaging this equation would reduce to 0=0, which is not
very helpful. So instead, we square both sides, THEN average (because the square of real
numbers is positive or 0, never negative).

The function's variance, δZ2, is either positive or zero provided the δAδB averages to 0, as
it usually does. We can average the variance and take the square root to obtain the root mean
square error, <δZ2>1/2, or the
relative root mean square error, <δZ2>1/2/Z, where the angle brackets, < >, denote that an
average has been taken. These are more commonly known, respectively, as the standard
deviation and the relative standard deviation.

IV. Sources of Random Error in the Spectrophotometer

A potential source of systematic error is small differences of baseline between different

spectra. In order to minimize baseline errors it is preferable that neither sample nor reference
cell should be moved between measurements of spectra. In practice this means either using a
flow-cell or a fibre-optic probe or building a titration cell for a particular spectrophotometer.
If measurements are to be made in alkaline solutions then the necessity of excluding
atmospheric CO2 indicates that a closed titration system must be used.

Baseline error is also affected by whether the spectrophotometer is a single- or double-beam

device. Instruments based on diode-array detectors are usually single-beam devices, so the
background has to be measured on the same cell as the sample spectra.

The type of random error depends principally on the nature of the detector. Older instruments
used a photomultiplier detector. The error of this type of detector increases with the intensity
of the light falling on it. In Hyperquad there is a module for determining an absorbance error
function which is based on the use of repeated scans of a standard spectrum.

In the instance shown below the data were obtained with an instrument that has a
photomultiplier detector, using a holmium glass filter as sample. There is a general increase in
error as absorbance increases but the trend is irregular because of correlation of errors
between absorbance values. They appear to belong to different sets according to whether
absorbance is increasing or decreasing.
Modern instruments use semiconductor detectors such as a diode array or charge-coupled
detector (CCD). The error associated with these detectors tends to be constant and
independent of wavelength. Here is an example from a diode-array spectrometer, also using
the holmium filter. The error is virtually constant.

What sources of random error could there possibly be from a high-class diode array
spectrometer such as the HP8452A? Here are some possibilities.
• • Digitization noise. Going from the raw diode charge signal to a number involves a Formatted: Indent: Left: 0 cm, Bulleted +
Level: 1 + Aligned at: 0.63 cm + Indent at:
device called an analog-to-digital converter. It has only 216 = 65536 different numbers it 1.27 cm
can record. Thus, there's a limit to how finely we can measure raw intensity. Formatted: Superscript
• • Dark noise. Even when no light hits the photodiodes, there's an uncertainty in the signal
• • Dark current. Some charge leaks off the diode as a function of time.
• • Readout pattern noise. Because of the way the diode array integrated circuit is made,
alternate diodes have different readout characteristics.
• • Light source flicker. Fluctuations in the light source.
• • Shot noise. For N discrete, random events such as detecting a photon, the uncertainty is
N1/2. Formatted: Superscript
• • Sample cell positioning error. The cell never seats exactly the same way twice, so the
path length
 and refraction give slightly shifted values each time the cuvette is reseated.. Formatted: Indent: Left: 0 cm, Bulleted +
Level: 1 + Aligned at: 0.63 cm + Indent at:
• • Wavelength drift from temperature changes in the spectrometer. 1.27 cm
• • Density changes in the sample due to temperature changes. Formatted: Indent: Left: 0 cm, Bulleted +
There are additional factors, but this is a sufficient start. Notice that none of these say Level: 1 + Aligned at: 0.63 cm + Indent at:
1.27 cm
anything about changes in the sample! Any random variation in the sample adds to the noise.

Before You Come to Lab

Read the ENTIRE lab. BRING YOUR SAFETY GLASSES. Wearing safety glasses in lab will be
strictly enforced. Bring a floppy disk or thumb drive so you can copy your data.

I. Using the UV 1601 PC Shimadzu Spectrophotometer

I.A. Turning On and Warming Up the Instrument.

When you come to lab, the computer and instrument will likely be turned on. If not, push the
power switch on the lower left side of the Shimadzu UV-1601 PC spectrophotometer to turn
on the instrument. Turn the spectrometer on BEFORE turning the computer on. Turn the
spectrometer OFF at the end of the lab. It takes approximately 20 minutes for the
spectrometers to warm up to the point where their output is stable.

I.B. Using the Software.

The computers are not on the network – just turn them on, when you get to the sign-on screen,
leave the user as "Administrator" and the password blank. On the Windows desktop, double –
click the UVProbe icon, followed by OK button. The UV Probe software will load and the
spectrum window will appear.

The TAs will provide additional instruction from here. For a preview, see the tutorial on the
software menus hot-linked in the on-line lab book

I.C. Handling the Glassware

Think of the walls of the cuvette as windows. If you set 100% T when the windows are clean,
what happens if the windows become dirty? Less light gets through,. Fingerprints can't be
differentiated from anything else that causes absorbance. It is CRITICAL that cuvettes be
scrupulously clean. For quartz cuvettes, one typically wipes the cuvette with a lintless wipe
(Kimwipes® are a sufficiently common brand that "wiping with a Kimwipe" has become the
pseudogeneric that "make a Xerox copy" has become for photocopying) just before inserting a
cuvette into the spectrophotometer. However, for plastic cuvettes such as you will use, wiping
tends to scratch the outside surface, making things worse than if you hadn't done anything at
all. So when you remove a cuvette from the box,

• Only touch the top at first.

• Locate the frosted sides – henceforth only touch those frosted sides or the very top,
far from where light will go through the cuvette.
• Load the cuvette with solution, buffer, or water, being careful not to overfill.
• Insert the cuvette into the spectrometer with the cuvette clamp lever in the UP
position. Make sure the light goes through the clear sides, not the frosted sides, of the
• Lower the lever to lock the cuvette in place.

NEVER mouth pipette. EVER. Suction to fill the pipette must ALWAYS be provided by a
mechanical device. That way, if solution squirts out the top of the pipette, you may
contaminate the pipette bulb but you won't hurt yourself. DO NOT THROW PIPETTES
INTO THE TRASH CANS. There are separate receptacles for glass such as disposable
pipettes. Plastic cuvettes can go in the trash cans after they've been rinsed.


II.A. Cuvette Transparency.

While the plastic cuvettes you'll be using aren't terribly expensive, they don't transmit light at
wavelengths shorter than 300 nm. Shorter wavelengths can be observed using quartz cuvettes.
These can run over $100 apiece and should be treated accordingly. To see how transparent an
empty cuvette is,
• Blank the instrument with only air in the optical path. That is, make sure there's
nothing in the cuvette holder. Put the lever DOWN so that the optical path is
unobstructed. Take a blank spectrum.
• Raise the lever, put the clean, dry cuvette into the holder, push it into place, lock it by
lowering the lever.
• Take a spectrum. In your lab report, describe what you see. You should report
apparent absorbance vs. wavelength and describe how much light gets through the
cuvette in the UV, visible, and near IR regions of the spectrum. You can tell by eye
that the cuvette is clear in the visible.

Why does the spectrometer indicate that less than 100% of the light gets through in this
wavelength region?

• Squirt some distilled water into the cuvette (if any slops over the side, you'll have to
get another cuvette and go back to step 2) You only need to have enough to make the
top of the liquid be above the observation axis hole). Take another spectrum.

What has changed? Why?

• Usually, one takes a blank spectrum with buffer or solvent in the cuvette. With water
still in the cuvette, re-blank the instrument.

Explain what is going on in the UV region of the spectrum. Describe physically what is
happening and explain the implications for making measurements on substances with
absorbance only in the wavelength range 200-300 nm.

II. B. Reproducibility of Absorbance Measurements

• Remeasure 100%T to see how reproducible this setting is.
• Put blank 30% methanol in a cuvette and measure absorbance for this combination vs.
water only. Does the methanol absorb? (note: methanol is poisonous. Do not drink,
avoid breathing fumes)
• Reblank system using the blank solution and cuvette.
• Take the cuvette out and put it back in.

Is there a measureable change? Why or why not?

There is an ambiguity in that last step. To find out if the change is due to moving the cuvette
or the precision of the absorbance measurement itself:
• Leave the cuvette in place and measure the absorbance of the buffer 15 times at 500
nm. Compute the mean and standard deviation of these measurements. In a perfect
world, you would get A = 0.0000 with a standard deviation of 0.0000. You won't.
• Make another 15 measurements, but this time lift the lever, remove the cuvette, put it
back into the holder, lower the lever, and make the absorbance measurement. Again,
compute mean and standard deviation.

IN YOUR LAB REPORT: are the mean readings with and without physically removing and
replacing the cuvette statistically different? See Harris, 7th edition, section 4-3 on Student's t.
The question here is identical in character to Case 3, P. 62. Are the standard deviations
different? Does removing and reseating the cuvette reduce experimental precision? From what
you have seen here about the precision of measuring A=0 and what you saw for wavelengths
less than 300 nm for the empty cuvette, suggest the range of absorbances one can reliably
measure with these spectrophotometers.

III.Working Curve for Crystal Violet

III.A. Make the working curve.

You are provided with standard solutions of crystal violet, otherwise known as Gentian
Violet, in DI H2O. Concentrations are given in terms of the mass of crystal violet per mL
solvent in parts per million, ppm, i.e. μg/mL. We define concentration on a weight-per-
volume basis so we don't need to assume the density of the solution is 1 g/mL (in fact, we
don't care about density at all).

In any order you like (as long as you carefully record the concentration to which each
spectrum corresponds), obtain and store spectra for the following solutions:
Blank – 3 replicate measurements
1.70 μμg/mL = 1.70 ppm crystal violet
3.40 ppm crystal violet
5.70 ppm crystal violet
7.60 ppm crystal violet
8.60 ppm crystal violet

III.B. Baseline correction

- Open the spectrophotometer sample- chamber cove. Make sure that neither the
sample beam nor the reference beam is obstructed, and that there is no sample
in the sample compartment.
- Add 2 mL of the solvent (methanol 30%) to a quart cuvette and place the
cuvette in the sample compartment. (Caution: Quartz cuvettes are very
expensive! Only touch the frosted, not the clear surfaces. When preparing a
solution for analysis, be careful not to contaminate the sample nor the
equipment with acetone, which has a large absorbance maximum at 268 nm).
Close the sample- chamber cover.
- Click the connect button, followed by the baseline button. The baseline
parameter window will appear. Enter the appropriate wavelength limits. Then
click the OK button to acquire a background spectrum.

III.C. Method preparation

* Spectrum method:
- Select the File > Open menu item. Them choose the desires method ( e.g.
crystal_violet.smd) and click the Open button. The spectrum Method window
will appear
- Set the following parameters in the measurement subwindow
a. Wavelenght Range: Choose the appropriate wavelength limits.
b. Scan speed: medium
c. Sampling Interval: 1.0
d. Scan mode: Single.
- Click on the Instrument parameters tab. Then choose the Absorbance
Measuring mode. Click the OK button to send the method parameters to the
 Photometric method ( see detail in appendix)
- Select the File > Open menu item. Them choose the photometric method and
click the Open button. The photometric Method window will appear
- Set the parameters in the measurement subwindow.

III.D. Sample preparation

- Open the sample- chamber cover and remove the cuvette. Empty the cuvette.
Then use a solution of the sample dissolved in the solvent to rinse the cuvette
twice. Use about ½ mL for each rinse.
- Once the cuvette is rinsed, add appropriately 2 mL of the sample solution and
place the cuvette in the sample compartment. Close the sample-chamber cover.

IV. Data Acquisition

IV. A. For recording spectrum:
- Click the overlay tab the place the spectrum in Overlay mode. Click the minimum
absorbance value on the Y- axis and set it to 0. then click the maximum absorbance
value on the y- axis and set it to 2. ( Note: If the acquired spectrum do not display
properly on the graph, right- click the mouse and choose the auto Scale function to
adjust the graph’s coordinate.)
Click the Start button to begin the scan. Watch the spectrum trace in the graph pane to
see that the absorbance values do not exceed the upper limit during the acquisition.
The new Data Set window will appear the scan is complete. Enter an appropriate File
-Click the Print button to print the spectrum
- open the sample- chamber cover, remove the sample from the instrument and close
the sample- chamber cover.
- Clean the cuvette thoroughly (without acetone).

IVB. For calibration method

- Click to read the standard and view a calibration curve
- Click to raead unknown samples

-Clean the cuvette thoroughly (without acetone). and return it to the instructor

IN YOUR REPORT: Identify the wavelength of maximum absorbance. Use this wavelength
to plot a working curve (absorbance as a function of concentration). Find the slope and
intercept of the working curve using least-squares regression procedure given Section VI.
Find the standard deviation of the slope and standard deviation of the intercept. What's going
on at wavelengths shorter than 300 nm? What if you chose a wavelength other than 588 nm
for your working curve? Would it affect accuracy? Precision?
What if you averaged absorbance over a range of wavelengths – would that affect accuracy?

You will notice "funny" regions in the spectrum near 656 nm and near 486 nm. Recall that the
light source in the spectrometer is a deuterium lamp. Atomic hydrogen (whether H, D, or T)
emits and absorbs light in the "Balmer series" that includes Hα (656 nm) and Hβ (486 nm).
For details, see the discussion of the Bohr atom in your general chemistry text.

From your spectrum, find the absorbance at the wavelength you used for making your
working curve and, from the equation you fitted to your working curve data, compute the
concentration and uncertainty in the concentration of the unknown.

V. Chemical and Spectral Interference

• Recheck 100% T
• Take a fresh spectrum of crystal violet
• Take a spectrum of crystal violet containing 12.5 ppm Bromophenol Blue

IN YOUR REPORT explain what the presence of Bromophenol Blue does to the crystal violet
spectrum and what effect this would have on a quantitative analysis. Estimate the smallest
concentration of crystal violet which could be reliably determined in the presence of 12.5 ppm
Bromophenol Blue.

IMPORTANT INSIGHT: methods for determination only work if there are no interferences.
All the sample preparation and separation are for the purpose of providing a clean sample,
free of interferences, so that accurate determination is possible.

VI. Methylene Blue

Not only can methylene blue form dimers, but also trimers. In non-aqueous solvents, there's
an anhydrous form that has a different spectrum than the aquated species. We've chosen a
solvent where the anhydrous form doesn't appear, 30% methanol. In this section of the lab,
you'll see how the shape of a spectrum changes with concentration.
• Blank the spectrophotometer again using 30% methanol
• Record spectra for all the concentrations of methylene blue in water/30% methanol
provided. You will have a set of about 15 spectra.
• Make working curves from the data at 648 nm, 656 nm, and 664 nm
Methylene Blue

IN YOUR REPORT: Is the curvature you see due to the instrument? How do you know? If it's
not due to the instrument, what might cause the curvature? If you wanted to use the
spectrometer to assay methylene blue, what wavelength would you choose and why? What's
the lowest concentration at which you get an absorbance that is unambiguously greater than
that of just 30% methanol/water? That's your detection limit. What's the highest concentration
you can measure? The difference between the high and low concentrations is the WORKING
RANGE of the method. The RATIO of the highest usefully measurable concentration to the
detection limit is the DYNAMIC RANGE. Report this number.

If there is curvature in a working curve that can not be attributed to instrumental behavior,
then either the methylene blue is reacting or complexing with the solvent or the methylene
blue is reacting with itself to form dimers or higher complexes. For now, you should conclude
which circumstances lead to ideal methylene blue behavior (Beer's Law is followed and the
working curve is straight) and the circumstances which lead to Beer's Law not being followed
(working curve is non-linear).

Questions to Answer at the End of Your Report

• For some of the experiments, you set 0 absorbance/100% transmittance using the same
cuvette in which you made the sample measurement. In others, you set the baseline
using a different cuvette. Does this make a difference in accuracy? In precision?
• In every case, we told you (or hinted strongly) what wavelength to use. What
characteristic did you notice that led us to choose those wavelengths?
• When there was an interference because another compound absorbed at the chosen
wavelength, how would the computed concentration for an unknown change if you
didn't account for the interference, i.e., if you made a working curve with the
interference absent, but unknown to you the interference was present in the unknown, so
simply used the original working curve? Would you overestimate, underestimate, or
correctly estimate the concentration of the unknown? Explain.
• How can you tell if a supposedly linear working curve really is linear? Use your own
data and appropriate curve fitting to provide examples for your discussion.

Things we didn't talk about but you might worry about in the future

• You didn't make up any of the solutions. You'll learn to do this in the next lab or two.
• You didn't prepare the samples starting from solids, so you didn't weigh anything, nor
did you have to deal with complete dissolution, filtering, pH adjustment, or any other
wet chemistry.
• The operation of the spectrometer wasn't explained very much. You'll learn about this
in lecture.
• Noise sources in the spectrometer weren't discussed in detail. The best textbook on
spectrometer noise is J. D. Ingle and S. R. Crouch, Spectrochemical Analysis. There are
newer journal articles (see particularly the work of Joel Tellinghuisen), but Ingle and
Crouch is the gold standard.
• We ignored systematic errors that can occur if the spectrometer's resolution is poor
compared to the width of the spectral features being observed. In the Real World, you
can't do that, or the working curve may be highly nonlinear.
• We assumed that light could only be transmitted or absorbed. It can also be emitted,
reflected, or scattered, and each of these can be used either to characterize samples or
can interfere with sample characterization. Imagine trying to do an absorbance
measurement in fog – the fog would act as a scatterer, not an absorber (since water is
transparent in the visible, but you know you can't see through a fog).
• When is spectrophotometry useful for solving problems, and when is some other tool
preferable? When can it be used alone, and when must it be combined with other
techniques? Until we've studied more techniques, these questions can't be answered.

Additional References
 Beer's Law: Harris, Seventh Edition, Chapter 18, or similar material in any other
quantitative analysis text written since the Second World War)
 Harris 7th Ed., Chapter 4, Sections 7-9 for the derivation of regression formulas
 Harris 7th Ed., Chapter 3, Sections 4-5 and Appendix C for general error propagation
 E. B. Sandell, Colorimetric Determination of Traces of Metals, 3rd Ed., Interscience
(New York, 1959).
 J. O. Rawlings, Applied Regression Analysis, Wadsworth and Brooks/Cole (Belmont,
CA, 1988).
 Z. Zhao and E. R. Malinowski, "Determination of the Hydration of Methylene Blue
Aggregates and Their
 Dissociation Constants Using Visible Spectroscopy," Appl. Spectrosc. 53, 1567-1574
 B. D.McCullough and Berry Wilson, "On the Accuracy of Statistical Procedures in
Microsoft Excel 97,"
 Computational Statistics and Data Analysis, 31, 27-37 (1999).
Chem 223: Experiment 10

Spectrophotometric Determination of Total Phosphate in waterWater Formatted: Centered

1. Harris. 3rd Edition: Ch. 18, Sect. 2-4
2. Harris. 2nd Edition: Ch. 17, Sect. 2-4
3. Shimadzu Application News- Spectrophotometric analysis. No A 354
4. J, Murphy and J. P. Riley. J. Mar. Biol. Ass. U.K. (1958) 37, 9-14
5. Standard method for the examination of water and waste water (1997). 4500-
P- Phosphorous.
6. C. C. Kircher and S. R. Crouch, "KINETICS OF THE FORMATION AND Formatted: Indent: Left: 2.22 cm
242-248 (1983).

I. Purpose of the experiment:

Phosphorus, widely found in various products such as fertilizer, agricultural pesticides, dyes,
processed food, and alkaline detergent, has greatly enriched our lives. However, it also
contaminates the environmental waters by flowing into rivers and lakes along with residential
and industrial waste water, or agricultural runoff. Phosphorous and nitrogen, which enhance
the growth of plankton in lakes, are used as indices of eutrophication. Consequently,
measurement of phosphorus concentration is important for water quality management.
Phosphorus occurs in 3 compounds in natural waters:
• inorganic, dissolved ortho-phosphate
• dissolved organic phosphorus compounds
• particulate phosphorus (bound in biomass or attached to particles),
which add up to the total of phosphorus content P-Total, an important parameter in
monitoring wastewater treatment plant effluents.
IN In this experiment, students will become familiar with the two standard method
approaches and additional method for the determination of anions using spectrophotometric

II. Introduction

There are two common spectrophotometric methods available for determining phosphate or
phosphorus concentrations:
• Molybdenum blue method
• Vanadate/molybdate method (yellow method)
Both techniques are based on the measurement of ortho-phosphate. Digestion of both
dissolved organic as well as particulate phosphorus compounds is therefore mandatory for
determining the total P content. In addition, an unfiltered sample must be acquired in order to
include all solid matters in the digestion process. Digestion is usually performed by heating
the sample with peroxodisulfate and sulfuric acid.

Inorganic phosphorus as orthophosphate, PO43-, is analyzed by reacting the phosphate with

ammonium molybdate under acid conditions to form a heteropoly acid, molybdophosphoric
acid. In the presence of vanadium, yellow vanadomolybdophosphoric acid ( max=690 nm) is
formed. The intensity of the yellow color is proportional to the phosphate concentration
present in the unknown sample. The analysis will be performed on a double-beam

In order to improve the sensitivity of thefor phosphate, ascorbic acid is added to reduce
Mo(VI) to Mo(V) and blue vanadomolybdophosphoric acid ( max= 880 nm) is formed. In
retreatment of the unknown samples, in addition to the above, a high-pressure steam sterilizer
is used to decompose substances in the solution that contain phosphorus (P) under high
temperature and high pressure, so that all the phosphorus is in the form of phosphate ions.
(This process is unnecessary for the phosphorus standard solutions used to generate the
calibration curve.) “Total phosphorus” expresses the total amount of phosphorus (P)
contained in the compounds in an aqueous solution, and is measured and expressed in the
form of phosphate ions.
In the experimental procedure, positive interference is caused by silica and arsenic only if the
sample is heated. Negative interferences are caused by arsenate, fluoride, thorium, bismuth,
sulfide, thiosulfate, thiocyanate, or excess molybdate. Blue color is caused by ferrous iron but
this does not affect results if the Fe(II) is less than 100 mg/L. If nitric acid is used in the test,
chloride interferes at 75 mg/L.

III. Chemical Reagents

1. Mixed Color Reagent (vanadate molybdate color reagent): Dissolve 3.60 grams of
sodium vanadate in 800 ml of cold water, then add 48.0 grams of sodium molybdate
dehydrate. Filter if necessary and dilute to 1 liter.
2. Ammonium molybdate - ascorbic acid mixed solution: Mix thoroughly 125 mL of 2.5M Formatted: Indent: Left: 0.11 cm, Hanging:
0.53 cm, Tab stops: 0.63 cm, List tab + Not at
sulphuric acid and 37.5 mL of 4% ammonium molybdate solution. Add 75 ml of ascorbic 0.74 cm
acid solution and dilute to 250 mL. The mixed reagent does not keep welel and should be
prepare within an hour of using. Comment [AS1]: This is interesting. When I did
my undergrad. thesis, we let the molybdate solution
3. potassium Potassium peroxodisulfate solution (40g/L) "age" for at least 3 days before using it. MoO3
4. water Water sample with unknown phosphate precipitates, then redissolves as Mo2O7=, or so we
thought. It's easier to keep it reproducible if the aged
solution is used, at least in my experience.
To be prepared: All glassware used in this experiment should be washed with phosphate free
MICRO cleaner, then rinsed with: DI water, 0.1 M HCl (prepared by mixing about 4 mL
of 6 M HCl with about 250 mL water), and again with DI water.

Preparation of standard phosphate stock solution

(1) Weigh about 0.1 gram of KH2PO4 to the nearest 0.0001 g and dissolve in 500.0 mL
water (volumetric flask).
(2) Calculate the ppm P in this stock solution to the appropriate number of significant

IV. Experimental Procedure

IV.A. Determination of phosphate in yellow heteropoly acid ( using calibration curve)

1. Preparation of standards for the calibration curve:

(1) Pipet 0.25; 0.5; 1.25, and 2.5 mL aliquots of the phosphate stock into separate 25.0 mL
volumetric flasks then dilute to the mark with DI water.
(2) Calculate the ppm P in each standard to the appropriate number of significant figures.

2. Preparation of yellow vanadomolybdophosphoric acid

(1) Pipet 25 mL aliquots of each standard, the unknown sample (group members will Comment [AS2]: You can't use all of the standard
when pipetting. Either pipet <25 mL or make 100
analyze their own samples), and DI water into separate 50 mL volumetric flasks. The mL of standard.
flask containing the DI water serves as the blank.
(2) Pipet 10 mL of the vanadate molybdate color reagent into each flask, including the
(3) Dilute each flask to the mark with DI water, mix well, then wait ten minutes for color
development to occur.
Caution: the color reagent contains NH4VO3 , a suspected mutagen. Wear gloves
when pipeting the color reagent.

3. Spectrophotometric analysis.

(1) Be sure the UV 1601 spectrophotometer is turned on at the beginning of the lab period
to provide enough time for warm up.
(2) In the “Scan" mode, under “Setup”, set the parameters as per Teaching Assistant’s
instructions. Zero the instrument with blank in both cuvettes, and then obtain a spectrum
from 700 nm to 300 nm of the most concentrated standard. Find the wavelength
corresponding to the maximum absorbance. Obtain a printout of the spectrum.
(3) In the "Concentration" mode set the wavelength to the maximum wavelength
recorded in step (2). Set all of the other parameters in Setup according to the Teaching
Assistant’s instructions. The exact concentrations of the standards will have to be
measured. Choose the “linear direct” fit type for the least square analysis. This will force
the curve through zero. Zero the instrument as in step (2). Then, run the standards and
(4) Print out the results and calibration curve.

4. Data analysis

The computer analyzed the data from the standards using a "Beer's Law" fit, i.e. no
intercept. The computer uses the absorbance for the unknown sample to calculate the
concentration of phosphorus in the unknown. You need to do your own linear regression
analysis using the absorbance data for the standards and their concentrations. After you
calculate the slope (m) and the intercept (b), calculate the phosphorus concentration in the Formatted: Font: Italic
unknown. Thus, you should have two calibration curves and two values for the Formatted: Font: Italic
phosphorus concentration: one from the computer and the other from your own linear least
square analysis.

Students should also measure the blank samples 10 times and find out the LOD and LOQ
based on the 3 rule.

5. Determining Dynamic Range

Students need to establish the limit of linearity (LOL) in concentration. This quantitation
could be performed using the UV-1601 should be determined based on the calibration curve
generated from measurement of the phosphorus standard solutions (the estimated results from
the calibration curve result were at about 0.05mg/mL- 3mg/mL; the slit width was set to 5 nm,
and a 10 mm square cell was used). In this case, unknown samples were not measured.
Formatted: Font: Not Bold
IV.B. Determination of Total phosphate in blue heteropoly acid (using thea second
additional standard method) Formatted: Font: Not Bold
Formatted: Font: Not Bold
(1) Take six decomposition flasks
(2) Transfer V(ml) (V< 40 ml) of unknown sample to each decomposition flask,
(3) Transfer phosphorus standard solution (P: 5ppm) of V’ mL (0mL, 0.5 mL, 1.25 mL,
2.5 mL, 5.0 mL and 10 mL respectively) to each decomposition flask, and add
(50-V-V’) mL of deionized water to these decomposition flasks, respectively.
(4) Add 10mL of potassium peroxodisulfate (40g/L), stopper and mix.
(5) Place in a high-pressure steam sterilizer, heat to about 120°C and continue
decomposition heating for 30minutes.
(6) Remove the decomposition flask, let it cool, transfer 25mL of supernatant to a stopper-
equipped test tube.
(7) Add 2mL of ammonium molybdate – ascorbic acid mixed solution, shake and set aside
for about 15 minutes at 20 - 40°C.
(8) Transfer a portion of solution to an absorption cell, and measure absorbance near
(9) As a blank, use 25mL of water, perform steps (6) and (7), measure absorbance, and
use this value to correct the absorbance values of the samples (subtract the blank
(10) The concentration value is determined by applying 6/5 (correction from (3)) to the
concentration obtained from the additional calibration curve.


- Report all the requirements in each experiment.
- Think about the interferences to the determination of phosphate in 2 methods.
- Figure out the main advantages of blue heteropoly acid compared to yellow
- List the substances can be used for reduction of yellow heteropoly acid to blue one
Chem 223 : ExperimentLab 1110


GLUCOSEascobic acid in grape

References: 1. C, Chap. 18, pp. 625-627.

2. SWH Chap. 22.
3. Harvey. Chapter 13.
3. Experiment 1: Chemical Kinetis- Masshchuset Institute of Technology.
( 5.311 Introductory Chemical Experimentation) 4. N. Burger; V.
Karas- Gasparec. Talantoa, Vol. 20(8). pp. 782-785, (1973)
15. Steinfeld, J.I., J.S. Francisco, and W.L. Hase, Chemical Kinetics and Formatted: Font: 12 pt
Dynamics, 2nd ed. (Prentice-Hall, Upper Formatted: Font: 12 pt
Saddle River, N.J., 1999), Chapter 1. Formatted: Font: (Default) Times New
This experiment is mainly revised from experiment 1: Chemical Kinetics Chem 5.311 Roman, 12 pt
Introductory Chemical Experimentation - Massachusetts Institute of Technology) Formatted: Font: 12 pt
Formatted: Font: (Default) Times New
Roman, 12 pt

This is an integrated experiment that includes topics from inorganic, organic, analytical, Formatted: Font color: Auto
physical, and computational chemistry. It is designed to introduce you to the basics of:

- how to acquire experimental kinetic data for a chemical reaction;

- how to perform data manipulation in order to extract information such as reaction

order and rate constants from experimental kinetic data

- In the present case, how adding a salt modifies the environment.

Pieced together, correct information will elucidate the reaction mechanism. Also, the
numerical results allow a convincing check of the validity of the mechanistic assumptions.
This experiment will contribute to improving your lab technique in the following areas:

- precise volumetric and gravimetric measurements

- correct handling of the UV-VIS instrument

- use of Microsoft Excel Solver that provides graphical and numerical output resulting
from the experimental data.

The rate of a chemical reaction is usually a function of the concentration of species involved
in that reaction. As a result, measurement of reaction rate can be used to quantitative


The reaction between hexacyanoferrate(III) and ascorbic acid has been used for visual,
potentiometric, amperometric and photometric titrations of both ascorbic acid and
hexacyanoferrate(III). This reaction has been used as the basis for spectrophotometric
determinations of microquantities of the reactants by using the total catalytic decomposition
of the hexacyanoferrate(III) formed in the process. Since acid and alkaline aqueous solutions
of hexacyanoferrate(III) show a rather intense absorption maximum in the visible spectral
region at 420 nm ( = 101 ×x l03 l.L mole-1 . cm-1) where the reaction products
[hexacyanoferrate(II) and dehydroascorbic acid] do not absorb, it is obvious that a
determination of ascorbic acid with hexacyanoferrate(II1III) is feasible by measuring the
absorbance of the reaction mixture at 420 nm against a reagent blank or water.

IIA. Chemical Kinetics: Formatted: Font: Bold

One of the main goals in chemical kinetics is to understand the steps by which a
reaction takes place. This series of steps is the reaction mechanism. Understanding the
mechanism allows us to find ways to facilitate the reaction.

„,,The reaction rate is defined as the change in concentration of a reactant or product, [A],
per unit of time t.…
Formatted: Underline
(3.1) Formatted: Space Before: 12 pt

1. Rate laws may be expressed in a differential form or an integrated form.

a) The differential rate law, often simply called the rate law, shows how the rate of
a reaction depends on the concentration of reactant and product species:

Rate = ƒ(concentrations) Formatted: Space Before: 12 pt, After: 12 pt,

Line spacing: single

b) The integrated rate law shows how the concentration of the species in the
reaction depends on time.

Concentration = f(time) Formatted: Indent: Left: 0.63 cm, Hanging:

0.63 cm, Space Before: 12 pt, After: 12 pt,
Line spacing: single
The integrated rate law may be derived from the differential rate law, but closed-form
aanalytic solutions cannot always be found.

2. Form of the Rate Law.

The first step in understanding how a chemical reaction occurs is to determine the form of the Formatted: Left, Space Before: 12 pt, After:
12 pt
r rate law. Consider the reaction aA + bB. The rate of this reaction may be given by:
Formatted: Space Before: 12 pt, After: 12 pt

where k is the kinetic constant or rate coefficient and n is the order of the reaction with Formatted: Indent: Left: 0 cm, Space Before:
12 pt, After: 12 pt, Line spacing: single
respect to A. For n = 1 the reaction is first order in A. For n = 2 the reaction is second
order in A, and so forth.

II.B. Ascorbic acid oxidation. Formatted: No underline

L-Ascorbic acid (vitamin C) is widely used in chemical and biological systems as a

reducing agent, mostly in aqueous solution. Depending on the nature of the oxidant and the
acidity of the reaction medium either
the ascorbic acid (H2Asc), the ascorbate anion (HAsc ) or the ascorbate dianion (Asc2- Formatted: Subscript
) is the kinetically important species. Among the oxidants which have been investigated are
metal ion-complexes, excited states of metal complexes and phenothiazene radicals.
The experiment described here involves the oxidation of the ascorbic acid (C6H8O6) by Formatted: Subscript
hexacyanoferrate(III) ion to dehydroascorbic acid (C6H6O6). Formatted: Left, Space Before: 12 pt, After:
12 pt
Formatted: Subscript
Formatted: Subscript
Formatted: Subscript
Formatted: Subscript
Formatted: Subscript

The overall stoichiometry is:

Formatted: Portuguese (Brazil), Subscript
H2 Asc + 2Fe(CN) ® Asc + 2 Fe(CN)64-+ 2H+ (3.3)

66 Formatted: Indent: Left: 2.61 cm, Hanging:

12.63 cm, Space Before: 12 pt, After: 12 pt

(H2Asc = ascorbic acid; Asc = dehydroascorbic acid) Formatted: Subscript

The empirical rate law, at constant pH, is: Formatted: Left, Space Before: 12 pt, After:
12 pt

A mechanism that is consistent with the kinetic data comprises four steps: Formatted: Left, Space Before: 12 pt, After:
12 pt





Formatted: Font: GOFGNC+TimesNewRoman,

- Superscript
The ascorbate ion (HAsc -) is formed by ionization of the ascorbic acid in a very fast process
Formatted: Font: GOFGNC+TimesNewRoman,
(3.5a). The rate determining step (3.5b) consists of an electron transfer from the ascorbate Bold
(HAsc- ) to the hexacyanoferrate(III) anion. The third step (3.5c) is a fast ionization process. Formatted: Indent: First line: 0 cm, Space
During the final step (3.5d) the second electron is transferred, as a fast process, from the Before: 12 pt, After: 12 pt, Line spacing:
ascorbate free radical ( Asc- . ) to hexacyanoferrate(III) anion.
Comment [AS1]: Move to top of next page
- 3-
Formatted: Font: Bold
Note that the rate determining step is an ionic reaction between HAsc and [Fe(CN)6] in
Formatted: Font: Bold, No underline
which each of the reacting species carries a negative electrical charge. Changing the ionic
strength of the solution will influence the measured rate constant, a phenomenon known as the Formatted: Font: Bold, No underline

"Salt Effect". This will be explained in more detail below. Formatted: Font: Bold, No underline, Subscript
Formatted: Font: Bold
II.C. Determination of the concentration of unreacted [Fe(CN)6] with time Formatted: Font: Bold, No underline
Formatted: Font: Bold
The kinetics of the oxidation of ascorbic acid can be followed by determining the Formatted: Indent: First line: 0.63 cm
concentration of unreacted [Fe(CN)6] with time. The concentration is assessed by its Formatted: Subscript
absorbance at 420 nm. According to the Lambert-Beer law, the amount of light transmitted by
an absorbing sample is given by

absorbing sample is given by

Comment [AS2]: Should be base 10, not base e

where the absorbance A is proportional to the concentration (c, in mol/L) of the solute, the
length of the path the light travels through the sample (l, in cm), and the constant of
-1 -1
proportionality, , called molar absorptivity coefficient (L mol cm ) or molar extinction

Aqueous solutions of hexacyanoferrate(III) are yellow, showing absorption of light in the Formatted: Indent: First line: 0.63 cm
blue-violet range, while ascorbic acid, dehydroascorbic acid and hexacyanoferrate(II) are
colorless, and therefore they do not interfere in the spectroscopic determination of
Fe(CN)6 .
The unreacted fraction of [Fe(CN)6] remaining at time t is At/Ao, where At is the absorbance Formatted: Subscript
3- Formatted: Subscript
due to Fe(CN)6 at time t and Ao is the absorbance at zero time. The concentration of
Formatted: Subscript

[Fe(CN)6] at time t is given by:

where [Fe(CN)6 ]o is the initial concentration of hexacyanoferrate(III). Formatted: Subscript

T he concentration of ascorbic acid at time t is given by:

In this experiment you will collect kinetic data on the oxidation of ascorbic acid by
K3[Fe(CN)6] occurring in four different environments differing in the ionic strength (using . Formatted: Subscript
Varying the concentration of added NNaNO3) produces the differences in the . Two important Formatted: Subscript
variables, temperature (room temperature or other constant temperature), and pH (acidity) are Formatted: Font color: Auto
kept constant throughout the experiment. Formatted: Subscript

II.DC. Kinetic determination of ascorbic acid Comment [AS3]: Move to top of next page

Initial Rate Method (or tangent method)

From the trials of absorbance at 420 nm was recorded as a function of time against the reagent
blank prepared similarly. The initial rate of the reaction (ν) at different concentrations was
obtained from the slope of the tangent to the absorbance-time curve. The calibration curve
was constructed by plotting the logarithm of the initial rate of reaction (log ν) vs the logarithm
of the molar concentration of the (log C). The amount of the ascorbic acid was obtained either
from the calibration graph or the regression equation.

Fixed-Time Method

In the fixed time method, the absorbance at 420 nm of each sample solution was measured at
a preselected fixed time against a reagent blank prepared similarly. The calibration curve was
constructed by plotting the absorbance against the final concentration of the drug. The amount
of the drug in each sample was computed either from calibration curve or regression equation.

III. REAGENTS Formatted: Font: Bold

- Provided : Solution containing 0.001% disodium EDTA dihydrate and; 0.010 M HNO3 Formatted: English (U.S.)
Formatted: Font: Bold, Not Italic
McIlvaine buffer solution (0.1 M citric acid + 0.2 M NaH2PO4) at pH 5.2, Formatted: Font: Not Italic

-3 Formatted: Font: Not Italic, English (U.S.)

Solution A: One pair will prepare a 1.0 x 10 M K3[Fe(CN)6] solution in a 250- Formatted: English (U.S.)
mL volumetric flask. This solution will be used as a stock solution both for day
Formatted: English (U.S.)
one dilutions to verify Beer„s Law and to determine molar absorptivity and for
Formatted: Font: Not Bold, Subscript
day two/three kinetics experiments.
Formatted: English (U.S.)
- To be prepared: Formatted: Subscript
Formatted: Subscript
- Solution B, C, D, ED, F: Comment [AS4]: Note rejected change
Formatted: Bullets and Numbering
- TA. Suggested temperatures: 10, 20, 30, 40, 50 °C. (Note: high humidity may
lead to

fogged cell surfaces at lower temperatures.)

Four pairs will each prepare one of the following four solutions made by
adding the appropriate quantity of solid NaNO3 to the 50-mL volumetric flask
specified volumetric flask and diluting to the mark with stock solution A in Formatted: Font: Not Bold
order to obtain concentration of NaNO3 are 0.02M, 0.05 M, 0.10 M and 0.20
M respectively.

Solution F: At the beginning of each "kinetic day"experiment, the fifth pair of

students will prepare a 2.5x10 M ascorbic acid solution by adding the
appropriate amount of solid ascorbic acid to a 250-mL volumetric flask. The
volumetric flask is next filled with a stock solution consisting of 0.010 M
HNO3 and 0.001% disodium EDTA dihydrate . Since ascorbic acid reacts Formatted: Subscript
slowly with dissolved oxygen, the solution must be prepared and used the same

A vitamin C tablet solution was prepared by the following procedure: 10 Formatted: Font: Bold
tablets of the proprietary drug to be investigate were accurately weighed,
crushed and powdered,. A mount of this powder equivalent to 0.200 g was
dissolved in 50 ml water. This was then left fotr 10 min for all gases evolution
to susbside, filtered, sashed and 2.0 mmoles of sulfuric acid addesd before Comment [AS5]: ?? I have no guess what this
word was supposed to be.
finally being made up to volume in a 100 ml volumetric flask with water.

All chemicals used were of analytical-reagent grade. Formatted: Font: Bold, Italic

McIlvaine buffer, pH = 5.2, was prepared by mixing 107.2 ml of O2M disodium Formatted: Font: Bold
hydrogen phosphate and

92.8 ml of 0.1 M citric acid.

Potassium hexacyanoferrate(II1) solution, 5 x 10m3M, was prepared from Merck

reagent. The solution

remains stable for a few weeks if protected from light.

Ascorbic acid solution, approximately 1 x 10W3M (OG180 g/100 ml), was prepared
from the Riedel reagent

(99.7%) and standardized against O.lM sodium hydroxide.” Only fresh solutions were
used for the spectrophotometric

measurements, because of their low stability.

The analysed tablets and injections containing ascorbic acid were obtained on the local
market. Their

solutions were also prepared immediately before use, and 2 ml of the sample solution
contained about 200 ,ag

of ascorbic acid.

All glassware was washed with redistilled water and all the solutions and subsequent
dilutions were made

with redistilled water because of the decomposition of ascorbic acid by traces of metallic


To 2 ml of McIlvaine buffer (pH 52) add 1 ml of 5 x lo-)M hexacyanoferrate(II1)

and 2 ml of


Teamwork is an important part of research. In order to minimize the amount of chemicals

required and reduce the amount of waste generated, solutions will be prepared by pairs of
students and shared by the whole group. This way the importance of teamwork and shared
responsibility is emphasized while keeping waste to a minimum.

IV.A. Day 1: The Lambert-Beer equation and determination of e

- UsingGoals Formatted: No underline

Formatted: Font: Not Bold, No underline
The UV-1601 PC Shimadzu to record · Become familiar with the Cary 100 spectrometer by
recording the UV-VIS spectrum (l =
360-550 nm) of each of the five solutions. Determine the lmax for K3[Fe(CN)6] in aqueous Formatted: Subscript
Formatted: Subscript
solution. Formatted: Subscript

- · Determine the validity of Lambert-Beer law by taking five UV spectra for known
K3[Fe(CN)6] solutions. Plot Absorbance at 420 nm versus concentration. The slope obtained Formatted: Subscript
by least square curve fit is your e value. Report this value to your TA. · Become familiar Formatted: Subscript
with Microsoft Excel Solver.


On the UV-VIS spectrophotometer‟s computer select “General Scanning”. Check that

the parameter file is set up correctly, and then take a background reading using a cell filled
with distilled water. Step-by-step instructions for running the Cary100 spectrometer to
obtain a UV-VIS Spectrum are provided in Appendix 1. Be sure the outer walls of the
cuvette are dry and clean by wiping them with a Kim-Wipe.

Check the validity of the Lambert-Beer equation over a range of concentrations for
K3[Fe(CN)6] by recording the absorption maximum for K3[Fe(CN)6] solutions at
differing concentrations. Each pair of students will determine the absorbance of solution
A, followed

by the absorbance resulting from 1/2, 1/3, 1/4 and 1/5 dilutions of solution A. There
are different ways of carrying out the dilutions, the more careful the measurements
the higher quality the data.

Plot absorbance (A ) versus concentration (c). Don't forget to include the (0, 0) data

in your calculations. If the Lambert-Beer equation is valid over this range of K3[Fe(CN)6]
concentrations, a straight line with slope (e x l) should result (the length, l, of the UV cell
is 1 cm). Use this e value throughout all your kinetic curve fitting calculations. THIS

IV.BDay 2: Kinetics

Goals Formatted: Space Before: 12 pt, After: 12 pt

-· Make the ascorbic acid solution and K3[Fe(CN)6]-NaNO3 solutions: Formatted: Subscript
Formatted: Subscript
. · Run the kinetics trials. Each pair of students runs all four trials. · Get a printout with the Formatted: Subscript
graph A = f(t) and the corresponding table for each kinetics trial. · Edit your kinetics data (4
files) into your Microsoft Excel. Do some (or all) of the data



On the UV-VIS spectrophotometer‟s computer select “Kinetics”. Check that the parameter
file is set correctly, and then take a background reading using a cell filled with distilled water.
Details of running the Cary UV 1601 PC spectrometer for kinetics runs are provided in
Appendix 2. Formatted: Highlight
- When ready to start the kinetics trials, pipet 3 mL of one of the K3[Fe(CN)6]-NaNO3 Formatted: Subscript
solutions (solutions B,C,D,E) into a small Erlenmeyer flask, and add 3 mL of the ascorbic Formatted: Subscript
acid-HNO3-EDTA solution (solution F). Formatted: Subscript
Formatted: Indent: Left: 0 cm, First line: 0
***Swirling the solution of reactants for a few seconds improves mixing*** cm, Space Before: 12 pt, After: 12 pt, Line
spacing: single
Pour the solution into a UV-VIS cell. Dry and clean the outside walls of the cuvette by Formatted: Subscript
wiping it with a Kim-Wipe and place the cuvette in the spectrophotometer. Record the Formatted: Indent: Left: 0 cm, First line: 0
absorbance at 1 minute intervals for 20 minutes. All four runs may be set up cm, Space Before: 12 pt, After: 12 pt, Line
spacing: single
simultaneously and allowed to thermally equilibrate before data collection begins.

Transfer the data to your Microsoft Excel Solver. Formatted: Indent: Left: 0 cm, Space Before:
12 pt, After: 12 pt

Get a printout with the graph A = f(t) and the corresponding table for each kinetics trial. Formatted: Indent: First line: 0 cm, Space
Before: 12 pt, After: 12 pt

IV.C.Kinetics continued Formatted: Space Before: 12 pt, After: 12 pt,

Line spacing: single

· Bring your results. Consult your TA about the general appearance of your results. Decide

whether any experiments should be repeated. · Make FRESH ascorbic acid solution if kinetic Formatted: Indent: Left: 0 cm, Space Before:
12 pt, After: 12 pt
trials need to be re-run. · Run or re-run any remaining kinetics trials.
Formatted: Space Before: 12 pt, After: 12 pt
Quantitative Formatted: Indent: Left: 0 cm, Space Before:
12 pt, After: 12 pt
IV.C.1. Spectrophotometrics Formatted: Font: Italic, Underline

-3 Formatted: Font: (Default) Times New

Method 1: To 2 ml of McIlvaine buffer (pH 5.2) add 1 ml of 5 x l0 o-)M Roman, 12 pt
hexacyanoferrate(III1) and 2 ml of sample Formatted: Font: (Default) Times New
Roman, 12 pt, Superscript
solution containing about 200 peg of ascorbic acid. Mix the solution, place the sample in the Formatted: Font: (Default) Times New
„check‟ position Roman, 12 pt
Formatted: Font: (Default) Times New
Roman, 12 pt
and the reagent blank in the „measure‟ position of the sspectrophotometer and compare at 420
nm. This Formatted: Font: (Default) Times New
Roman, 12 pt, Superscript

arrangement gives absorbances which are a direct measure of the hexacyanoferrate(III1) Formatted: Font: (Default) Times New
Roman, 12 pt
reduced by the
Formatted: Font: (Default) Times New
Roman, 12 pt
ascorbic acid. Find the amount of ascorbic acid by reference to a calibration curve or a
Formatted: Font: (Default) Times New
calibration equation. Roman, 12 pt
Formatted: Font: (Default) Times New
Measure immediately after mixing the reactants or within a period of 3 hr. Roman, 12 pt
Formatted: Font: (Default) Times New
Prepare the calibration curve from a series of standard solutions containing between 45 and Roman, 12 pt
360 pg of Formatted: Font: (Default) Times New
Roman, 12 pt
ascorbic acid in 2 ml and proceed as described above. Formatted: Font: (Default) Times New
Roman, 12 pt, Superscript
Method 2: The reagents: 2.0x10-3M Fe (III) chloride salt (1mL), 2.0.x10-3M potassium Formatted: Font: (Default) Times New
Roman, 12 pt
hexacyanoferrate (III) solution (1mL), 0.1M KCl (1mL), 0.01M HCl (1mL) and the samples
(1mL) were mixed and fill up to 50mL in calibrated flasks with distilled water. After 10 Formatted: Font: (Default) Times New
Roman, 12 pt, Superscript
minutes absorbance of the complex was read at the 700nm. The calibration curve was linear
Formatted: Font: (Default) Times New
over the range 0.0880-0.7040mg/L (5x10-5-4x10-4M) with a correlation coefficient of 0.9994 Roman, 12 pt
Formatted: Font: (Default) Times New
IV.C.2. Kinetic spectrophotometry Roman, 12 pt
- On the other experiment, start kinetic trials with the concentration of ascorbic acid
range from 45 to 360 g of ascorbic and try to find the unknown concentration using fixed
time method or tangent method.


The rate constants are calculated for different ionic strengths by fitting the
experimental A = ƒ(t) curves to the calculated A = ƒ(t) function, assuming that the ascorbic
acid oxidation with hexacyanoferrate(III) follows the second order reaction kinetics as
described by equation (3.4).

Consider the general chemical equation of the type:

bB + cC → products

The integrated rate equation1 is:

In our case B = ascorbic acid, C = K3[Fe(CN)6], b = 1 and c = 2. By using equations (3.7) and
(3.8), equation (3.9) becomes:

where A is the current absorbance at time t, Ao is the value of absorbance at t = 0, Af is the

final absorbance (t →∞), cf is the final concentration, and kobs is the observed rate constant.
The curve fitting with Microsoft Excel Solver will provide the corresponding kobs, and Af for
each kinetic run.

Applying the steady-state approximation2 to the elementary steps of the reaction

mechanism (equations 3.5), and assuming k−1 [H +]>> k2[Fe(CN)63−], the theoretical rate law

The real rate constant (kreal = k2) can be determined from the observed rate constant (kobs) if
the concentration of H+ due to nitric acid and the dissociation constant for ascorbic acid (Ka
= 6.76x10-5 = k1/k-1) are known:
The measured effect of ionic strength on the real rate constant, known as the primary
salt effect, is then compared to the theoretical equations predicting the ionic strength effects3.
Let us begin with a few words on the concept of ionic strength. Ionic strength was defined by
G. N. Lewis as:

Here zi is the charge of the ion i and ci its concentration, while the summation encompasses
all the ions in the solution. For a simple solution of a univalent electrolyte, the ionic strength
is equal to the molar concentration, but is greater than the concentration when the salt
contains ions of higher valencies.

According to the Debye-Hückel theory for aqueous solutions of electrolytes5, the rate
constant at ionic strength I (kreal) is given by:

where ko is the rate constant when the extraneous ionic strength is absent. The charges of
the two reacting species (see the reaction mechanism scheme) are Z1 and Z2, respectively. The
equation applies to dilute solutions (c < 0.2 M).


1. Draw the plot log kreal versus I ½ / (I½ + 1). Calculate ko and Z1Z2. Compare the calculated
Z1Z2 with the expected value predicted by the reaction mechanism. If there are discrepancies,
discuss the reason.

2. What is the effect of increased ionic strength upon the experimental reaction rate of
ascorbic acid oxidation by hexacyanoferrate(III)?

3. What would be the added salt effect upon reaction rate between ions with opposite charges?

4. If one species is neutral, what would be the effect of the added salt upon the reaction rate?

5. What would be the effect of changing the temperature of the solution on the reaction rate?

6. What would be the effect of changing the pH at constant ionic strength?

7. Report the ascorbic content in vitamin C tablet.

Chemistry 223: Experiment 12

Determination of lead and zinc in water samples by F- AAS

References: 1. C, Chapter. 20 . Comment [AS1]: ?? If no specific citation,
2. H7, Chapter 21,
3. Kellner, Chap 8
4. SWH, Chaps. 24
5. Standard method for examination of water and waste water. EPA.

I. Purpose of the experiment

Atomic line absorption spectra are used for analysing analyzing various metals. Atomic
absorption spectrophotometry ("AAS") offers sensitivity, selectivity and simplicity and was
developed by CSIRO Melbourne scientists around 1955. "AAS" is an important tool in
analytical laboratories. Using this technique, students will be determine Pb and Zn in water
samples by flame atomic absorbance spectroscopy. Students will also be familiar with
QA/QC for the analytical procedure through the example of analyzing Pb and Zn in soluble
species and total concentrations in water samples.

II. Introduction

Flame atomic absorption spectroscopy (F-AAS) is a very common technique for detecting
metals and metalloids in solid and aqueous samples. It is very reliable and simple to use. The
technique is based on the fact that ground state metals absorb light at specific wavelengths.
Metal ions in a solution are converted to atomic state by means of a flame. Light of the
appropriate wavelength is supplied and the amount of light absorbed can be measured against
a standard curve.

AAS quantitatively measures the concentrations of elements present in a liquid sample. It

utilises utilizes the principle that elements in the gas phase absorb light at very specific
wavelengths which gives the technique excellent specificity and detection limits.

The sample may be an aqueous or organic solution, indeed it may even be solid provided it
can be dissolved successfully. The liquid is drawn in to a flame where it is ionised in the gas

Light of a specific wavelength appropriate to the element being analysed analyzed is shone
through the flame, the absorption is proportional to the concentration of the element.
Quantification is achieved by preparing standards of the element.

The technique of (FAAS) requires a liquid sample to be aspirated, aerosolized, and mixed
with combustible gases, such as acetylene and air or acetylene and nitrous oxide. The mixture
is ignited in a flame whose temperature ranges from 2100 to 2800 oC. During combustion,
atoms of the element of interest in the sample are reduced to free, unexcited ground state
atoms, which absorb light at characteristic wavelengths. The characteristic wavelengths are
element specific and accurate to 0.01-0.1nm. To provide element specific wavelengths, a light
beam from a lamp whose cathode is made of the element being determined is passed through
the flame. A device such as a photonmultiplier can detect the amount of reduction of the light
intensity due to absorption by the analyte, and this can be directly related to the amount of the
element in the sample.
Flame atomic absorption hardware is divided into six fundamental groups that have two major
functions: generating atomic signals and signal processing. Signal processing is a growing
additional feature to be integrated or externally fitted to the instrument. The instrument parts
are shown in figure 1 and 2.

Fig. 1: Atomic Spectrometer novAA-400 AnalytikJena

Figure 2. Schematic of basic instrumental parts of atomic absorption spectrometer

A cathode lamp (1), shown in figure 3, is a stable light source, which is necessary to emit the
sharp characteristic spectrum of the element to be determined. A different cathode lamp is
needed for each element, although there are some lamps that can be used to determine three or
four different elements if the cathode contains all of them. Each time a lamp is changed,
proper alignment is needed in order to get as much light as possible through the flame, where
the analyte is being atomized, and into the monochromator.

Fig.3: Hollow cathode lamp

The atom cell (2), shown in figure 4, is the part with two major functions: nebulization of
sample solution into a fine aerosol solution, and dissociation of the analyte elements into free
gaseous ground state form. Not all the analyte goes through the flame, part of it is disposed as
shown in the figure.

Fig. 4: Atom cell

As the sample passes through the flame, the beam of light passes through it into the
monochromator (3). The monochromator isolates the specific spectrum line emitted by the

light source through spectral dispersion, and focuses it upon a photomultiplier detector (4),
whose function is to convert the light signal into an electrical signal.

The processing of electrical signal is fulfilled by a signal amplifier (5). The signal could be
displayed for readout (6), or further fed into a data station (7) for printout by the requested

F-AAS is also based on transmitting light through the sample and measuring the fraction of
light absorbed. Mathematically, the measurement can be expressed as:

T(λ) = P(λ) / P0(λ) (1)

In Eq. 1, T is termed the transmittance, P denotes the light intensity transmitted through the

sample, and P0 is the light intensity incident on the sample. The P0 value is measured by use
of a blank solution that contains the sample matrix but contains no analyte. The values T, P,
and P0 are all a function of λ, the wavelength of the light. The measured transmittance can be

related to the analyte concentration through the Beer-Lambert law:

A(λ) = -log10(T(λ)) = εbc (2)

In Eq. 2, A is termed the absorbance, c is the analyte concentration, and ε (absorptivity) and b
(optical path length) are constants that define the linear relationship between A and c.

Absorptivity is an intrinsic property of the analyte, and the path length is a characteristic of
the instrumental measurement. The value of εb is determined by a calibration procedure that
employs a set of known standards that bracket the desired concentration range.

III. Reagents, apparatus and materials.

Apparatus: - All sample plastic containers must be prewashed with detergents, acids, and
water. Both plastic and glass containers are suitable.

- Atomic Spectrometer novAA-400 AnalytikJena.

- Watch glasses or equivalent.

Provided Reagents: - Reagent Water shall be interference free. All references to water in the
method refer to reagent water unless otherwise specified.

- Nitric acid (concentrated), HNO3. Acid should be analyzed to determine level of impurities.
If method blank is < MDL, then acid can be used.

- Hydrochloric acid (concentrated), HCl. Acid should be analyzed to determine level of

impurities. If method blank is < MDL, then acid can be used.

- Water Sampling:

+Total recoverable metals - All samples must be acidified at the time of collection with
HNO3 (5 mL/L).

+ Dissolved metals - All samples must be filtered through a 0.45-m filter and then
acidified at the time of collection with HNO3 (5 mL/L).

To be prepared:

Concentrations of the metal ion standard solutions need to cover a range of 0 to 25 mg/L (ie. 0
to 25 ppm). Some metal ion stock solutions (250 mg/L or 1000 mg/L) will already have been
prepared for you and simply require dilution with 0.002M to the appropriate final

Note: High levels of silicon, copper, or phosphate may interfere to the determination of Zn.
Addition of strontium (1,500 mg/L) removes the copper and phosphate interference. Zinc is a
universal contaminant, and great care should be taken to avoid contamination.

Sample preparation:

* Dissolved metals - The sample is filtered through a 0.45-m filter at the time of collection
and the liquid phase is then acidified at the time of collection with nitric acid. Samples for
dissolved metals do not need to be digested as long as the acid concentrations have been
adjusted to the same concentration as in the standards.

* Total recoverable metals: The entire sample is acidified at the time of collection with nitric
acid. At the time of analysis the sample is heated with acid and substantially reduced in
volume. The digestate is filtered and diluted to volume, and is then ready for analysis.

Interferences: The analyst should be cautioned that this digestion procedure may not be
sufficiently vigorous to destroy some metal complexes.

Precipitation will cause a lowering of the silver concentration and therefore an inaccurate
Together with unknown sample, add the amount of several ppm of mixture containing heavy
metals to the samples and digest follow the above procedure to evaluate the recovery rate.

IV. Experimental Procedure

1. Sample preparation for F- AAS analysis

- Transfer a 100-mL aliquot of well-mixed sample to a beaker.

- For metals that are to be analyzed, add 2 mL of concentrated HNO3 and 5 mL of

concentrated HCl. The sample is covered with a ribbed watch glass or other suitable covers
and heated on a steam bath, hot plate or other heating source at 90 to 950C until the volume
has been reduced to 15-20 mL.

CAUTION: Do not boil. Antimony is easily lost by volatilization from hydrochloric acid

- Remove the beaker and allow to cool. Wash down the beaker walls and watch glass with
water and, when necessary, filter or centrifuge the sample to remove silicates and other
insoluble material that could clog the nebulizer.

Filtration should be done only if there is concern that insoluble materials may clog the
nebulizer; this additional step is liable to cause sample contamination unless the filter and
filtering apparatus are thoroughly cleaned and pre-rinsed with dilute HNO3.

- Adjust the final volume to 100 mL with reagent water.

2. Quality control

- For each analytical batch of samples processed, blanks should be carried throughout the
entire sample preparation and analytical process. These blanks will be useful in determining if
samples are being contaminated.

- Replicate samples should be processed on a routine basis. A replicate sample is a sample

brought through the whole sample preparation and analytical process. Replicate samples will
be used to determine precision. The sample load will dictate the frequency, but 5% is

- Spiked samples or standard reference materials should be employed to determine accuracy

by calculating recovery rate. A spiked sample should be included with each batch.

3. Determination of Pb and Zn (direct aspiration)

3.1. Instrument parameters (general):

Parameters Determination of Pb Determination of Zn

Lead hollow cathode lamp Zinc hollow cathode lamp
Fuel Acetylene.
Wavelength 283.3 nm 213.9 nm.
Fuel Acetylene Acetylene
Oxidant Air Air
Type of flame Oxidizing (fuel lean) Oxidizing (fuel lean)
Background correction Required Required

3.2. Calibration standards

Calibration standards are prepared by single or multiple dilutions of the stock metal solutions.
Prepare a reagent blank and at least 3 calibration standards in graduated amount in the
appropriate range of the linear part of the curve. The calibration standards must contain the
same acid concentration as in the samples following processing. For precipitation samples,
that would be 1% (v/v) HNO3 and for suspended particulate matter10% (v/v) HNO3. The
calibration standard should be transferred to polyethylene bottles.

- For determination of Pb: Optimum concentration range: 1-20 mg/L ;

Sensitivity: 0.5 mg/L.
Detection limit: 0.1 mg/L.

- For determination of Zn: Optimum concentration range: 0.05-1 mg/L

Sensitivity: 0.02 mg/L.

Detection limit: try to calculate from measuring the procedural
blank at least 8 times.

3.3. Blank solutions

Three different blank solutions are required; calibration blank, procedural blank and rinse

 Calibration blank is used for establishing the calibration curve. The calibration blank
consists of the same concentration(s) of the acid(s) used to prepare the final dilution of
the calibration standards. In addition, an appropriate concentration of internal standard
is added.

 Procedural blank (or reagent blank) is used to monitor for possible contaminations
resulting from the sample preparation procedure. The procedural blank must be carried
through the same procedure and contain the same volume of reagents as the sample
solution. In addition an appropriate concentration of internal standard is added.

 Rinse blank is consist of 1-2 % (v/v) HNO3 and is used to flush the sample
introduction system between standards and samples.

3.4. Instrumental procedure

The operating procedure will vary between instrument brands, so the instrument manual
should be followed carefully. The position of observation and the fuel:oxidant ratio must be
optimised. Some general guidelines are outlined below:

 Light the hollow cathode lamp or electrode discharge lamp and D2-lamp if such
background correction is used. Set the lamp current to the value specified by the

 Position the monochromator at wavelength 283.3 for Pb and 213.9 for Zn and choose
slit with 0.7 and slit height “high”.

 Carefully balance the intensity of the hollow cathode lamp and the D2-lamp if such
background correction is used.

 Align the burner head to assure that the centre of the light beam passes over the burner

 Light the flame and regulate the flow of fuel and oxidant to produce an oxidising
flame (lean blue).

 Aspirate calibration blank and establish a zero point.

 Aspirate standard solutions and construct a calibration curve.

 Aspirate samples.

Aspirate distilled water after each standard or sample.

3.5. Instrument performance

The “characteristic concentration” (sometimes called sensitivity) is defined as the

concentration of an element (mg l-1) that will absorb 1 % of the incoming radiation. This
equals a signal of 0.0044 absorbance units (AU). The “characteristic concentration” is
instrument dependent and is calculated as follows:

C = (S * 0.0044 AU) / measured absorbance

C: Characteristic concentration (mg l-1)
S: Concentration of measured standard (mg l-1)

Knowing the “characteristic concentration” allows the analyst to check if the instrument is
correctly optimised and performing up to specifications.

3.6. Sequence of analysis

 Aspirate calibration blank and establish a blank level

 Aspirate calibration blank and standard solutions and construct a calibration curve.
Use at least 3 standard solutions in addition to the calibration blank to cover the linear
range. Every point at the calibration curve should, if possible, be based on replicate
analysis. Distilled water should be aspirated after each standard and sample.

 A quality control standard should be analysed to verify the calibration.

 A calibration blank should be analysed to check for memory effects.

 Aspirate unknown samples.

 Aspirate a quality control standard for every 10th sample to check for drift.

 Samples that are found to have concentration higher than the highest standard should
be diluted and reanalysed.


1. Report the experimental results. Summarize the calculations that produced your calibration

2. Calculate the exact concentrations of your calibration standards. Be sure to use the
appropriate number of significant figures. data. Report values to the correct number of
significant figures.

4. Make calibration models for each set of data. Use least-squares procedures to determine the
slope and intercept of each calibration model. Report the value of r2 and the standard error of

4. Include separate plots of absorbance (with error bars) vs. concentration (ppm) for each
calibration model.

5. Use your calibration model together with your dilution scheme to compute the weight
percent of iron in your original solid sample. Report as weight percent Fe2O3. Predict each
replicate of the unknown separately, then compute a mean, standard deviation, and 95%
confidence interval for the predicted amount. Repeat this calculation for both calibration

6. The limit of detection associated with each calibration can be estimated by use of the
following procedure. The intercept of the calibration model is an estimate of the absorbance
when no metal is present in the sample (i.e., the blank signal). The standard error of estimate
is an estimate of the measurement noise (i.e., in the absence of systematic error, the variation
about the least-squares line should be due solely to instrumental noise). A widely used
definition of limit of detection is the concentration corresponding to an absorbance value
equal to the blank signal plus three times the noise level. Thus, ALOD = b + 3s (3)

where ALOD is the absorbance corresponding to the limit of detection, b is the intercept of the
calibration model, and s is the computed standard error of estimate. The limit of detection,
CLOD, can then be estimated as CLOD = (ALOD - b)/m (4)

where ALOD are as defined above and m is the computed least-squares slope of the calibration
model. Equations 3 and 4 can be combined to yield

CLOD = 3s/m (5)

Use your calibration data to estimate the limit of detection for each method.

7. In your discussion section, compare the atomic and molecular spectroscopic methods

8. Tabulate the mean, standard deviation, and 95% confidence interval for your absorbance-
Report the data of the calibrations with linear regression.

- Calculate the detection limit of Zn determination.

- Calculate the Pb, Zn content in samples by using the calibration regression. Also report
uncertainty of the sample’s data from calibration curve.


- Explain how metal atoms are produced and excited in HCL?

-Flame AA has poor sampling efficiency and sensitivity. Explain why?

- Spectral overlap is insignificant in AAS method. Explain!

- Discuss the major factors involved in the detection to use flame of electrothermal
atomization in AAS.

- F-AAS is known as a technique with few problems related to interference effects. Do you
know what are they?

- Why do hydride techniques for As and Se and the cold vapor techniques for Hg yield better
AA detection limit than conventional flame AAS with solution nebulization?

Formatted: Font: Bold
Chem 223: Experiment 13

Formatted: Font: Times New Roman Bold,

GC/FID Determination of Toluene in Air Character scale: 100%

1. Harries. Chap. 24
2. Kealey. Pp. 137-154
3. Harvey. 12D
This experimental procedure is based on the experiment on website:

I. Purpose of the experiment

In this experiment you will measure the volume fraction concentration (in parts per billion by
volume, ppbv) of toluene in air samples that you will collect. The samples will be collected and
stored in Teflon bags which will be returned to the laboratory for analysis. The method of
analysis is gas chromatography with cryogenic preconcentration and flame ionization detection
(GC/FID). The quantitation method will be based on an internal standard using m-xylene and a
standard addition of toluene. Several air samples will be analyzed.

Your group will first go out into the “real world” and draw a few (three to four) air samples, and
bring them back to the laboratory for analysis. You should try to collect samples that will show a
variation in hydrocarbon pollution content. Take notes on where you took the samples and the
relevant atmospheric conditions.

You’ll run two gas chromatograms per sample (raw and spiked, both with the internal standard)
except for the first sample, where you will also run an experiment without cryogenic
preconcentration. The non-preconcentrated run can be compared to the same sample with
preconcentration to determine the extent of the preconcentration. The time per analysis (each
chromatographic analysis will be referred to as a “run”) is over 20 minutes, so you need to be on
the ball to get three or more air samples done during the lab period.

You will be pumping the air samples through a small (~ 100 μL) metal sample loop that is
immersed in liquid nitrogen (the cryogen) for a pre-planned length of time, usually about 5
minutes. The N2 doesn’t affect the majority components of air (nitrogen and oxygen) under our
conditions, so they flow on through to the air pump. The hydrocarbons (and water and carbon
dioxide) are trapped in the sampling loop during the passage. Theoretically, the longer you run the
air through the cryogenically cooled loop, the more hydrocarbon you will end up with in the loop;
but in the interest of time, we won’t test that idea.

After the preconcentration (or shortly after the flow is established in the non-preconcentrated
sample) a sample valve similar to those used in liquid chromatography is used to inject the
volatile contents of the sample loop onto the column for separation and eventual detection and
measurement by a flame ionization detector (FID).

Gas chromatography - specifically gas-liquid chromatography - involves a sample being
vapourised and injected onto the head of the chromatographic column. The sample is
transported through the column by the flow of inert, gaseous mobile phase. The column itself
contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
The picture and schematic diagram of a gas chromatography are following.

Fig. GC ( Shimadzu) basic system

Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon,
and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector
which is used. The carrier gas system also contains a molecular sieve to remove water and other

Sample injection port

For optimum column efficiency, the sample should not be too large, and should be introduced
onto the column as a "plug" of vapour - slow injection of large samples causes band broadening
and loss of resolution. The most common injection method is where a microsyringe is used to
inject sample through a rubber septum into a flash vapouriser port at the head of the column.
The temperature of the sample port is usually about 50C higher than the boiling point of the
least volatile component of the sample. For packed columns, sample size ranges from tenths of
a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample,
typically around 10-3 L. For capillary GC, split/splitless injection is used. Have a look at this
diagram of a split/splitless injector;
The injector can be used in one of two modes; split or splitless. The injector contains a heated
chamber containing a glass liner into which the sample is injected through the septum. The
carrier gas enters the chamber and can leave by three routes (when the injector is in split mode).
The sample vapourizses to form a mixture of carrier gas, vapourizsed solvent and vapourizsed Comment [AS1]: Changed from the British
spelling to the American spelling
solutes. A proportion of this mixture passes onto the column, but most exits through the split
outlet. The septum purge outlet prevents septum bleed components from entering the column.

There are two general types of column, packed and capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in
length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of
two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT).
Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary
phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of
support material such as diatomaceous earth, onto which the stationary phase has been adsorbed.
SCOT columns are generally less efficient than WCOT columns. Both types of capillary
column are more efficient than packed columns.
In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT)
These have much thinner walls than the glass capillary columns, and are given strength by the
polyimide coating. These columns are flexible and can be wound into coils. They have the
advantages of physical strength, flexibility and low reactivity.

Column temperature
For precise work, column temperature must be controlled to within tenths of a degree. The
optimum column temperature is dependant upon the boiling point of the sample. As a rule of
thumb, a temperature slightly above the average boiling point of the sample results in an elution
time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times.
If a sample has a wide boiling range, then temperature programming can be useful. The column
temperature is increased (either continuously or in steps) as separation proceeds.

There are many detectors which can be used in gas chromatography. Different detectors will
give different types of selectivity. A non-selective detector responds to all compounds except
the carrier gas, a selective detector responds to a range of compounds with a common physical
or chemical property and a specific detector responds to a single chemical compound. Detectors
can also be grouped into concentration dependant detectors and mass flow dependant detectors.
The signal from a concentration dependant detector is related to the concentration of solute in
the detector, and does not usually destroy the sample Dilution of with make-up gas will lower
the detectors response. Mass flow dependant detectors usually destroy the sample, and the
signal is related to the rate at which solute molecules enter the detector. The response of a mass
flow dependant detector is unaffected by make-up gas. Have a look at this tabular summary of
common GC detectors:
Support Dynamic
Detector Type Selectivity Detectability
gases range
Flame ionization Hydrogen
Mass flow Most organic cpds. 100 pg 107 Formatted: Font: 12 pt
(FID) and air
Thermal conductivity
Concentration Reference Universal 1 ng 107 Formatted: Font: 12 pt
Halides, nitrates,
Electron capture nitriles, peroxides,
Concentration Make-up 50 fg 105 Formatted: Font: 12 pt
(ECD) anhydrides,
Hydrogen Nitrogen,
Nitrogen-phosphorus Mass flow 10 pg 106 Formatted: Font: 12 pt
and air phosphorus
Hydrogen phosphorus, tin,
Flame photometric and air boron, arsenic,
Mass flow 100 pg 103 Formatted: Font: 12 pt
(FPD) possibly germanium,
oxygen selenium,
aromatics, ketones,
Concentration Make-up esters, aldehydes, 2 pg 107 Formatted: Font: 12 pt
Halide, nitrogen,
Hall electrolytic Hydrogen,
Mass flow nitrosamine,
conductivity oxygen

The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds
burning in the flame produce ions and electrons which can conduct electricity through the flame.
A large electrical potential is applied at the burner tip, and a collector electrode is located above
the flame. The current resulting from the pyrolysis of any organic compounds is measured.
FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that
changes in mobile phase flow rate do not affect the detector's response. The FID is a useful
general detector for the analysis of organic compounds; it has high sensitivity, a large linear
response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the

III. Reagent and Apparatus:

- Shimadzu Gas Chromatograph with FID detection and air sample loop injection Tedlar (a type
of Teflon) gas sampling bags with valve and septum port “Bucket brigade” air sampling apparatus
Dewar and liquid nitrogen 1.0mL Pressure-Loc Gas Syringe.

Standards Available:

~100ppmv m-xylene air standard bag

~100ppmv toluene air standard bag

(Ask your TA for the data needed to calculate the exact concentration of the standards.)

IV. Experimental Procedure:

A. Air sampling
Formatted: Indent: Left: 0 cm, Hanging:
• Have the TA show you how to use the sampling bucket and Tedlar bags. 0.48 cm

• Go out into the “field” and take a few air samples - i.e., fill up three or four Tedlar bags with
air. Air samples taken in this way are referred to as “grab” samples. Try and fill the bags as
completely as possible.

• To make things interesting, try taking samples from areas where you expect there to be a lot of
hydrocarbons (high traffic) and only a little bit of pollution (nearly the city-wide background.)
Keep records of where you took each sample from and other observations (e.g., heavy traffic,
wind from South, temperature, barometric pressure, raining…)

• Bring all samples back to the laboratory for analysis.

B. Internal Standard/Standard Addition Procedure

For each air sample, we will run one chromatogram of the raw sample with m-
xylene and one of a toluene spiked sample with the internal standard. (We’re making the
assumption here that m-xylene isn’t present in ambient air.). To add m-xylene or toluene to the air

• Insert the 1.0 mL graduated gas syringe into the standard bag through the septum in the Tedlar
bag (the tiny hole on the side of the valve/inlet for the bag).

• Press the green button to open the syringe valve.

• Rinse the syringe a few times and then pull a 1.0mL sample of the gas standard.

• Press the red button to close the valve.

• Pull the syringe out of the standard bag and insert it through the septum port in the sample gas

• Press the green button.

• Inject the spike and remove the syringe.

• Mix the sample thoroughly by “squishing” the bag for a few minutes at least. {It’s boring and
noisy, but if you don’t work at it, you will not end up with good results because the diffusional
mixing of gases at atmospheric pressure is very slow.}

• Measure the volume of the bag and calculate the concentration of m-xylene or toluene. (We are
currently measuring volume using a water displacement method. See the Appendix for

• Put the internal standard in all of the sample bags before you start the analyses.

C. Analysis:

• Make sure that the computer and the GC are on and that the FID is lit and has
• Get a transfer dewar full of liquid nitrogen from the big stainless steel storage dewar. (If you
haven’t done this before, ask the TA for help.)

For the non-preconcentrated run

Formatted: Font: Not Bold
• Choose the Tedlar bag that you think will have the highest toluene concentration and insert the
valve stem into the flexible tubing coming out of the center of the sampling valve on the front
of the GC. (It’s a tight fit, but you don’t have to have more than a few mm on to make a seal.)

• Make sure the sampling valve is in the LOAD position, turn on the air pump, open the valve on
the bag (push it in), and allow the sample to flow through the loop for a few seconds.

• Switch the sampling valve to the INJECT position and press the START button on the GC.
Within a few seconds, a red vertical line should appear on the continuous output screen on the
computer, signifying the beginning of a chromatogram.
Formatted: Font: Not Bold
• Close the valve (pull it out) on the Tedlar bag and remove it from the flexible tubing.

• After ~ 1 minute, return the sampling valve to the LOAD position to get ready
for the next run. {You can leave the air pump on all the time, to help flush the last
sample from the sampling loop before the next chromatogram.}

• The run takes 18.75 minutes and the toluene and m-xylene elute well before the end, but you
should always let the chromatograph run for the full time to drive higher molecular weight
compounds from the column.

• When the chromatogram finishes, the status bar on the computer will change from BLUE
(running) to RED (not ready) and the temperature of the oven will start to go down. The
chromatogram will briefly appear on the computer screen (then the screen will go back the
running signal display) and the Report Screen will refresh. {At this point, you can print the
report by selecting Tabulate Print on the top of the Report Screen.}

For ALL preconcentrated runs (i.e., all the rest of the analyses)

• Attach the Tedlar bag’s valve to the flexible tubing on the pump.

• Make sure the sampling valve is in the LOAD position and turn on the air pump, open the
valve on the bag (push it in), and allow the sample to flow through the loop for a few seconds.

• Next you will simultaneously place a Styrofoam cup full of liquid nitrogen around the sample
loop, immersing the bottom half of the coils in the cryogen, and start a timer. {The length of
time that the cryogen is on the loop determines the extent of preconcentration, and thus should
be as consistent between a sample and its spiked version as possible. You could change the
trapping time between different samples if more preconcentration was desired, but this is
usually not necessary and can cause confusion.}
Read through the next three steps as they are all time critical

• At the end of the predetermined trapping time, usually around 5 minutes, remove the cup
with the liquid nitrogen cryogen and wait for ~3 seconds to allow liquid oxygen to escape
from the sampling loop

• then switch the sampling valve to the INJECT position

• then place a cup full of room temperature (or warmer) water around the loop and press the
START button on the GC. {The water heats the loop and rapidly drives the volatile contents
out of the loop and onto the GC column.}

• Close the valve on the gas sampling bag, remove it from the flexible tubing, and measure the
new air bag volume.

After ~ 1 minute, return the sampling valve to the LOAD position to get ready for the next run.

Again, when the chromatogram finishes, it will briefly appear on the computer screen (then the
screen will go back the running signal display) and the Report Screen will refresh. {At this
point, the chromatogram and peak integrations should print automatically. If not, you can print
this information by selecting Tabulate Print on the top of the Report Screen. Don’t forget to
print before the next chromatogram finishes, or you will lose your data.}

After the chromatogram of the raw sample (with m-xylene) is done, and you’ve measured the
volume of the air in the bag, and you have the report printout in hand, spike the sample with
toluene. {This last part seems silly, since it would be better to measure the volume of the bag and
spike it immediately after the “raw” injection so that the spike would have more time to mix into
the air in the bag. But you can’t “unspike” a bag, and if anything goes wrong during a run and
you don’t get the data from the “raw” sample, there is no point in running the spiked version. Up
to you, but this is the safest way.}

Analyze the spiked sample as you did with the raw one {start over again at For ALL
preconcentrated runs…} and obtain the report printout. Locate the toluene and m-xylene peaks
in the “raw” and spiked runs. (The relative peak area of toluene should have increased.) {Also,
retention times should be nearly the same, but the toluene peak isn’t the only one that changes}
and obtain the peak areas. Calculate the toluene concentration of the air sample using the
volume of the standard and sample bags and the ideal gas law.


In writing this report, you should include the following:

1. Present the relevant peak areas, concentrations, and quantitative results (in ppbv toluene) in
tabular form.

2. Be sure to explain in the Introduction how toluene concentration was calculated.

3. Comment on the extent of preconcentration, based on your sample run without the cryogen -
quantitatively if possible (i.e., you have to have seen something to figure out how much it
increases - otherwise the answer might just be “a lot”.)

4. Provide a map of your sampling locations and say whether your results were consistent with
your expectations during sampling.

5. In addition to quoting the volume fraction concentration for the “unknown” air samples, also
give the ppmvC (volume fraction carbon.) This is the relevant parameter in calculating the
ozone producing potential of VOCs.


1. The GC you used was equipped with a thermal conductivity detector. What are the strengths
and weaknesses of this kind of detector?

2. Why is temperature programming sometimes useful in gas chromatography?

3. What changes would you expect in retention time, peak shape, and resolution if

a. You decreased the column temperature between replicate injections of the same mixture?

b. 3. You increased the mobile phase flow rate between replicate injections of the same
Chem 223: Experiment 14

Determination of caffeine in soft drinks by High Performance Liquid

Chromatography (HPLC).


1. Harris, 7th ed. Chap. 25

2. Harvey: 578-589
3. Kealey: pp. 155-164
4. Experimental procedure. The University of Adelaine. Australia. Department of Chemistry.
5. Experiment 5. University Kuala Lumpur. Malaysian Institute of Chemical And
BioEngineering Technology
6. Experiment 5. Chem 426. Washington State University.
7. Expriment . Chem 427. Portland State University

I. Purpose of the experiment

The traditional method for the determination of caffeine is via extraction with
spectrophotometric quantification. Use of the liquid chromatography permits a fast and
easy separation of caffeine from other substances such as tannic acid, caffeine acid, and
sucrose found in these beverages. The amount present in soft drinks is controlled by the
manufacturer, and can be obtained upon request.

The following experiment illustrates the utility of high performance liquid chromatography
(HPLC) as an analytical tool for the determination the amounts of caffeine in various
commercially available soft drinks.

From the resulting chromatograms, measurements of retention time tR and peak areas
are made. If the flow rate and pump pressure are held constant throughout the entire
experiment, tR may be used as qualitative measure and the peak area as a quantitative
measure. A calibration curve for peak area vs. concentration of the caffeine standards
can then be employed to determine the concentration of caffeine in the beverages.

II. Introduction

Chromatographic methods are based on differential affinities of solutes for two phases, a
stationary phase and a mobile phase. Molecular interactions leading to these affinities are
polar forces (dipole-dipole and H-bonding) and dispersion forces (induced dipoles). Thus,
in a chromatographic system, solute molecules will be attracted to the phase of similar

Mobile phases in chromatography are either liquid or gaseous. Gas chromatography (GC)
is the only technique that uses a gas as a mobile phase, while many techniques employ
liquids (liquid chromatography, LC).

High Performance Liquid Chromatography (HPLC) is a modern (late 60's) modification

of the classical open column techniques that established chromatography as the ultimate
separation technique. The name is derived from the fact that much higher column
efficiencies are possible when the particle size of the stationary phase is small (3-10 mm
in HPLC versus 40 mm conventional open column LC). As a result of these smaller
particles, large back-pressures require the mobile phase to be pumped through the
column under high pressures. HPLC has several advantages over GC:

1. HPLC is not limited to volatile compounds.

2. A greater control and wider selection of stationary and mobile phases.
3. Many detectors are non-destructive, allowing further analysis of eluted

The basic liquid chromatograph consists of four elements. These are: a pump to move
the mobile phase, and injector to deliver the sample onto the column, a column to
separate the sample, and a detector to visualize the eluted compounds. The basic HPLC
system is illuminated in fig. 1
Fig. 1 : The basic HPLC system


In HPLC, narrow columns with internal diameters 2-80 mm are used. These columns are
packed with particles having an average diameter of less than 50 microns (50 x 10-6m). Such
columns require high pressures (1000-6000 psi) to maintain a convenient flow of the eluting
solvent, usually in the range 0.1-10 mL/min, but occasionally higher. Resolution is
considerably superior to that achieved with an ordinary column, in part because of the tight
packing of the stationary phase, which reduces lateral diffusion, and because of the large
surface area of the packing.

Compared with classical column chromatography, where the columns are gravity fed and a
separation can take hours or even days, HPLC can offer analysis times of 5-30 min.

There are two main classes of column: "normal" and "reversed" phase.

Normal phase columns are most usually packed with silica gel; they work in the
partition/adsorption mode in the same manner as a normal silica gel column in conventional
Reversed phase chromatography, which is the most common form of HPLC, is a type of
partition chromatography. Frequently, reversed phase columns are packed with a chemically
bonded octadecylsilyl coated silica; such columns are referred to as C-18 and are very non-
polar. Other popular bonded phase columns have octasilyl, cyanopropyl, or phenylsilyl

The eluent used with reversed phase columns is relatively polar, e.g. MeOH/H2O. Unlike
normal phase chromatography, the more polar components of a mixture elute first, since these
partition is relatively unfavorable on the highly non-polar packing. Increasing the polarity of
the solvent increases the retention time of a particular component. The situation is the reverse
of normal adsorption chromatography:

Normal vs. Reversed Phase

Normal Reverse

Packing polarity High Low

Solvent polarity Low High

Elution order Non-polar first, then Polar first, then non-

polar polar
Effect of increasing solvent Decreases retention Increases retention
polarity time time

The other major improvement over column chromatography concerns the detection methods
which can be used. These methods are highly automated and extremely sensitive.

Important parameters associated with the chromatographic process are measured directly
from the chromatogram for example, resolution, R, describes the degree of separation of
successive solute peaks. A value of 1.5 indicates complete (baseline) resolution, while an
R value of 1.0 is considered adequate for analytical purposes. The separation factor
describes the relative retention of two components for separation to occur, a must be
greater than 1. The capacity factor, k', gives an indication of the relative amount of a
particular solute in each phase (stationary and mobile). It is of the utmost importance,
since the observed retention characteristics of a solute depend on it's distribution between Comment [AS1]: This is one of the most common
spelling errors. "It's" is a contraction for "it is." "Its"
these phases, and, therefore, it's relative affinity towards each. The capacity factor is (no apostrophe) means "pertaining to the item being
proportional to the volumes of liquid and mobile phases, and is an indication of the discussed."
increase in retention volume (or time) for a given solute relative to that of the mobile

The number of theoretical plates, N, is a measure of the efficiency of the chromatographic

process. Obviously, if the individual molecules of a particular solute spread out over a
wide as they travel through the column, separation will be difficult, and the separation
process inefficient. Peak width is proportional to this spreading, and is a reflection of
the number of theoretical equilibrium steps of the solute between the two phases that
have occurred within the column. The higher the value of N (or the narrower the peaks),
the more efficient the chromatographic process.
III. Reagents and instrument. Comment [AS2]: Never end a page with a
heading! Move it to the next page.

- Solutions provided:
- Chemicals: Caffeine reagent grade for standards.

Fig. 2: Molecular structure of caffeine

(1,3,7- trimetylxanthine)

- Solutions available:

+ Solvent for mobile phase: 20% Methanol:80% water (pH 3.50)

+ Caffeine stock Standard (1000ppm): Accurately weigh out 0.10 gram (to
0.0001g) of caffeine, quantitatively transfer into a 100 mL volumetric flask,
dissolve in about 50 mL methanol, fill to the mark with distilled water, and
mix thoroughly.

+ Internal standard stock solution (1000ppm): Accurately weigh out 0.10

gram ( to 0.0001g) of m-methoxybenzioc acid (MW=152.15),
quantitatively transfer into a 100 mL volumetric flask, dissolve in about
50 mL of methanol fill to the mark with distilled water, and mix

-Solution to be prepared:

+ Preparing the working standard solution

Place 0; 0.5; 1.0; 1.5; 2.0 and 2.5 mL of the caffeine stock solution into a series
of six clean and dry 25-mL volumetric flasks. Add 2.5 mL of the internal
standard stock solution to each of the six flasks. Dilute to the mark with the
previously prepared solution of 20% methanol: 80% H 2O solvent, adjusted to
about pH 3.50 with glacial acetic acid. This is the same solvent to be used as
the mobile phase.

+ Preparing the unknowns: For carbonated beverages, you must first remove the
carbonation. You can leave the cap off for a couple of days or put a small amount
of the beverage in a beaker and carefully bring it to a boil on a hotplate. Bring the
unknown back to room temperature before you proceed with the filtering. Remove
all suspended particulates (solids) before you inject into the LC system.

Pour 10 to 15 mL of each soft drink samples into a small clean, dry beaker. To
decarbonate the beverage, transfer into another dry beaker back and forth until no
more the bubbling is observed. The soda is now adequately decarbonated. Into a
clean, dry 25-mL volumetric flask pipette 8 mL of decarbonated Pepsi-Cola, and
into a second 25-mL flask, pipette 8 mL of decarbonated Coca-Cola. Dilute each
volumetric flask to the mark with 20 methanol: 80% water solvent adjusted to pH

-Apparatus and equipment:

+ Volumetric flasks, five 100 mL, four 20 mL, Pipettes, 2 mL, 4 mL, 8 mL (one of

+ The Shimadzu HPLC instrument comprises four main components: the injector LC-
10Advp, the solvent delivery system , column furnace CTO 10AS and the PDA
(Photodiode array) SPD-M10A ( D2 and W lamp). Cartridge: Supelco LiChrospher
RP-18 (6.1 x 250 mm) (a reversed phase column).

IV. Experimental Procedure

Prior to injection of the standards into the column run the mobile phase (20% methanol:
80% H2O, pH 3.50) to equilibrate the column for 5 to 10 minutes. Simultaneously
monitor the detector response to insure that there are no substances left on the column
from previous experiments.

Shake the five caffeine solutions adequately for proper dissolution and then degas
each for 5 min before injection into the chromatograph. Set the pump flow rate at 2.3
3 mL/min.

A. Operation of the HPLC - General Procedure

The greatest enemy of HPLC is fine particulate matter, which can damage the pumping
system and irreversibly block the column. Therefore, all solvents have been filtered
through fine membranes (0.4-0.5 micron) and all solutions to be injected MUST be
prepared either with filtered solvent, or filtered as specified later in these notes.

B. Start-up Procedure

First, ensure that there is sufficient filtered solvent in the reservoir for your run.

Using the screw on the right hand side of the instrument, pressure the column to 800 psi
(beginning of the yellow region). Switch the solvent selector on the inlet manifold at the
front of the pump to the running solvent. Switch on the power to the pump and slowly
increase the flow rate to 3 mL/min. Switch on the U.V. detector and once the absorbance
reading has settled (10 min), set the zero to 0.01 AU.

C. To inject a sample: Switch the injector lever (top) to "load". Switch the lower lever to
vertical and remove the plug (store in hole in injector switch). Wipe the needle with a
clean tissue and insert into the injector. Inject the sample into the loop with even
pressure (excess of solvent in the loop will be pushed out of the vent tube on the right of
the injector). Replace the injector plug and move the low lever to the horizontal position.
Smoothly switch the injector lever to "inject", and at the same time press "inject" on the
data module to start the data collection. The data module plots a real time chromatogram,
and at the end of the run time (15 min) replots the chromatogram with details of
retention time (RT), peak area (A or H) and relative areas of the peaks (conc.). Although
the integration is not affected if a plotted peak is off scale, the chromatogram can be
replotted at a different attenuation by resetting the ATN (powers of 2, the bigger the
attenuation setting the smaller the peaks will look, normally set at 30) and then recalling
the plot (Recall). The plot is stored until the next injection. "Feed" moves the paper
forward for the next plot.

D. Shut down procedure

After the last run, flush the column with 50 mL of 20% methanol: 80% water
(not at pH 3.50).

Increase the flow to 3mL/min slowly and flush the column with solvent for 10 min. Stop
the flow and switch off the pump. Remove the plots from the Data module and switch
off at the front of the unit. Switch off the U.V. detector. Depressure the column by
unscrewing the pressure screw on the right of the instrument until 4 threads are showing.

Notes: Normal running pressure for this experiment is between 1500-2200 psi. If a high
pressure shutdown occurs (>2500 psi), consult a demonstrator. Look for leaks at
connections through the system; if there are any, consult a demonstrator.

Listen to the pump during the experiment; if there are any unusual noises, consult a
demonstrator. Look at the outlet flow; it should be a thin stream. If there is no flow,
consult a demonstrator.

Analysis of data

i. Standard caffeine samples are used to identify the caffeine peak and the retention time
of caffeine is recorded. From 20 ppm to the 100 ppm, the peak is increased.

ii. The retention time has been used to determine the present of caffeine in the soda

iii. The peak ratios between the standard and internal standard are measured to
quantitatively determine the amount of caffeine in the sample. Then the standard curve
is constructed.

iv. The peak ratios between the standard and internal standard in the soda sample
chromatograph has been measured. The concentration and peak ratio area has been
used to determine the concentration of the caffeine in the soda sample.

v. Express this concentration also in grams/litrer.


(a) Data should be presented in the form of a Table which is easily read and which clearly
shows how the values were obtained.

(b) Calculations should be clearly shown with an explanation for each step.

(c) Treatment of Data: The tR can be used as a diagnostic tool to determine

qualitatively the presence of a substance in a chromatographic mixture. In a tabular
form, record for the caffeine standards, the concentration, retention times, and peak
areas. Also, calculate the mean and standard deviation of the retention times. Plot
the peak area vs. the concentration in mg/mL for the caffeine standards. From the average
retention time for the caffeine standard identify the caffeine peak in the coffee, tea,
and beverages.

(d) Calculate the average and standard deviation of the peak areas and the concentration
of caffeine in mg/mL in the samples. Include corrections for dilution in the

More Questions

1. Explain the rationale for using a reverse phase column for the determination of

2. Would the construction of a calibration curve based on peak high (rather than area) give
accurate results for the determination of caffeine in this experiment?

3. Could an ion-exchange column be used for the determination of caffeine? Explain.

4. Choose a caffeine peak and determine:

(i) Capacity factor,

(ii) Number of theoretical plates,
(iii) Asymmetric factor
Operating Instructions for Perkin-Elmer AAnalyst 300 Atomic Absorption
Using the Instrument for Flame Experiments:

• Turn on the computer

• Turn on the instrument; switch is on the right hand side. Do not turn on the furnace
(left hand side).
• Turn on the fan switch (on the wall behind the computer).
• Click on AA Winlab.
o Software will check for connection.
• Make sure the technique says Flame in the bottom left corner.
• Click on “Use custom design workspace”. Choose Chem223.flm
o 3 windows will appear: Results, Peaks, and Manual Analysis
• In the toolbar click on Lamps
o Select the lamp you want to use by clicking in the setup column. A green
light will appear.
� Note: Cu and Zn are on the same lamp, so you must simply type the
element you want to analyze. The software will change the wavelength
and slit width accordingly.
• Close the lamp window.
• Make sure the method you are using corresponds to the element you want to analyze.
(Top right corner) If not, click on method and select the appropriate method. All
names are in the format: Ch223_element.
• Click on the flame icon in the toolbar. Make sure the aspiration tube is in reagent-
grade water.
• Open the AIR tank
• Open the ACETYLENE tank
o Order for tank is not so important with this instrument because it
has interlocks, but it is always a good habit to open oxidant first and
then fuel.
This reduces the possibility of explosions in instruments without
• When interlocks turns green, turn flame ON.
o The instrument has an igniter incorporated.
• Place the aspiration tube in your blank.
• Press Analyze Blank
o This will measure the absorbance of your blank and then automatically
autozero the reading. This has to be done before each different element, but
not in between samples analyzed for the same element.
• In the Manual Analysis window fill in:
o Sample ID
o Results data Set Name : Click browse and Type the name of your data file in
Result Name.
� Use format name223 then write a description so we know what can
be erased later.
o Check the Save Data box. Do not check the Print Log box.
o Leave Sample Information file as “Untitled”
• Place the aspiration tube in your sample and press Analyze Sample.
o The method is set so that a triplicate measurement of your data is acquired,
leave the tube in your sample until the instrument is done and the result in the
result window shows a mean value.
• Repeat as often as necessary, make sure to use an informative sample ID, so that you
know which sample it was when you go back to write your report.
• If you wish to make measurements with more than one element, repeat the same
o Change the Lamp.
o Change the method.
o Start data acquisition by running a blank.
• When you are finished with all your measurements:
o Place the aspiration tube in distilled water, leave flame on for a minimum of 5
• Collecting data in an Excel file:
o Go to File, Utilities, Reformat.
o Click Open Design, choose Chem325.
o Browse to find your Data Set Name.
o Click Save Results.
o Your file is now in the chem325 folder on the desktop.
• Shut Down Procedure
o Click Flame Icon.
o Turn Flame off.
o Wait for shutdown procedure to be completed.
o Turn ACETYLENE off at the cylinder.
o Turn AIR off at the cylinder.
o Press Bleed Gases.
o Turn off spectrometer.
o Shut down Software.
o Wait 5 minutes before turning fan off.
Appendix on Linear Regress in Excel

There are at least two ways to do regression fitting in Excel, There's the manual way and the built-in
automatic way. The reason not to be fully-reliant on the automatic way is that it's part of a "Data
Analysis" add-in (that, confusingly, shows up under "Tools" on the menu in versions of Excel prior to
2007, but is on the "Data" tab thereafter). Further, some Apple/Macintosh versions of Excel lack the
"Data Analysis" add-in completely.

Let yi be the absorbances you measured and xi be the concentrations of the solutions. If a working curve
is linear i.e. if Beer's Law holds, there's no interference, or the concentration of an interference is constant
including in the blank, then

yi  b  mxi  yi (10)

where b = extrapolated blank when xi = 0, m = slope of the working curve, the change in absorbance per
unit concentration, yi = the measurement error in yi. The mean of yi is presumed to be 0. We assume
 or else independent of concentration so that the contribution from errors in
that the error in xi is negligible
sample preparation can be lumped in with yi.

See Harris, Chapter 5, Section 1 (or analogous sections of Harvey), to see where the math comes from. It
turns out that

y i  bN  m x i
i1 i1


 x i y i  b x i  m x i 2
 i1 i1 i1

where N is the number of independent data points. If you make replica measurements at a particular
concentration, each measurement is independent, so if you make triplicate measurements on each of 5
solutions, N = 3*5 = 15.What happened to the errors, yi? They average to 0, as does the product xiyi.

Finding N is easy – just count how many measurements you made. Then make a table, where column A
has all the concentrations and column B has the corresponding absorbance values. Total the entries in
column A to get  x i Total the entries in column B to get 
y i . In column C, find the square of each
entry in column A (for example, in cell C5, type =A5*A5). Add that column up to get x 2
i In column
D, find the product of xi and yi, e.g. D5 would read = A5*B5. Now you have all the sums, and you have
two equations
 with two unknowns. It's very handy  to have as an intermediate quantity D defined as:
N  N 2 
D  N  x   x i 
i (12)
i1 i1 

Then b and m can be found by:



y x x y x
i i i i

b i1 i1 i1 i1

N xi yi   yi  xi
 m i1 i1 i1
In each case, the sums are quantities
0.5 you have already computed, so they can
 be used directly. Always check that the
least squares fitted line goes through the

center of your data by using the m and b

you calculated to plot the fitted line. If
the line doesn't go through the data,
check to ensure that you did the
0.1 arithmetic correctly. Something else
you should always do with any fit is to
0.0 plot the residuals vs. the independent
variable, i.e. yi – yfit, where yi is the
experimentally determined value of y at

xi, and yfit is the y value predicted by the
50 100 150 200 model at xi. Figure 4 shows an example
( of a fit to a linear absorbance working
Figure 4 curve with the residuals plotted
separately at the bottom. If examination
of the residual plot reveals a trend, this is a good indication that the model (straight line in this example)
is not adequate to describe the experimental behavior.

How do the uncertainties in b and m determine the uncertainties in a concentration estimated from
measuring an absorbance, i.e. what's the uncertainty in x for a single measurement y? From Harris, we
need an intermediate quantity:

y i  mxi  b

sy  i1
N 2

Then the standard deviation of the slope and intercept are given by,

 N
sm  sy (15a)
1/ 2
 N 2 
  xi 
 sb  s y  i 1  (15b)
 D 
 
 

We now know the uncertainty in the slope, the intercept, and in an individual measurement of the
absorbance or analytical signal (that's sy). So if we now measure a signal y, we can use propagation of
error calculations to
compute the uncertainty
in the apparent
concentration x. Error
propagation is discussed
in section VII.

For the less adventurous

if you put your working
curve data in columns A
and B of an Excel
spreadsheet, then pop up
the Regression window,
you set up the fitting
problem as shown in Fig.

Figure 5
The least squares
regression is done
automatically, the
residuals are computed -
slopes, intercepts,
residuals. After a little
moving around of graphs
and adjusting their size, it
looks like Fig. 6.

Excel spreadsheet terms

are defined below.

Multiple R The
coefficient i.e.
the degree to
which the model Figure 6
fits the data. +1
= perfect match. –1 = anti-correlated, i.e. as the independent variable increases, the dependent
variable decreases. 0 = uncorrelated i.e. there's no functional relationship between the
independent and dependent variables.
R Square The square of the correlation coefficient.

Adjusted R Square The correlation coefficient, scaled for the effect of non-infinite degrees of
freedom. Radj  1
1 R N 1 where p' is the number of parameters fit (2 in the case of a

N  p
straight line on a Cartesian plane)
Standard Error Related to standard deviation. The sum of the squares of the distance from the regressed
data to the fitted line.
Observations Number of data points, what we've called N up to now.

ANOVA Acronym for Analysis of Variance

Regression Description of the results of the analysis of variance
Residual How much of the raw data is NOT described by the model
Total Sum of Regression and Residual factors
Df Degrees of freedom – how many parameters can one legitimately extract from the data?
SS Sum of squares
MS Modified sum of squares i.e. sum of squares divided by degrees of freedom
m 2
x i  x

F The statistical significance of a non-zero slope. The bigger, the better: F  1 i1
Significance F The statistical significance of the F statistic. The smaller, the better.
Coefficients Coefficients of the model (intercept and slope for the straight line)
Intercept Self-explanatory
X Variable 1 Slope 
Standard Error Uncertainty in the coefficients
t Statistic Student's t for the hypothesis that the intercept is non-zero.
Upper/Lower Limits Confidence intervals for each computed value.
Residual Output A table giving the computed value for each of the dependent variables given the
input values of the dependent variables, and the residual i.e. observed - predicted

This is more than you really need. What you are looking for:

 Is there a pattern to the residuals, indicating that the model is inadequate? The residual graph
can give you a visual idea of this (if it's a smooth curve, rather than random, there's a
problem). r2 is a numerical means of judging. Anything less than 0.999 says there's some
other significant source of variance that slope and intercept don't account for.
 What are the uncertainties in the model parameters (slope and intercept in this case)?

Chemistry 223
Designing an Analytical Method

Fall, 2009


All semester, indeed in most labs you've ever done, you have been told how to do experiments ("Heat to
60°C," "Weigh 0.2 g."). The practice of quantitative analysis requires the analyst to DEVELOP methods
as well as to APPLY them. To give you a flavor of how to develop a method, this lab will guide you to
write a procedure for the analysis of a nickel ore sample. You will turn in a lab report after one week,
explaining how you will solve the problem of analyzing the ore sample. You will also carry out the
procedure you devise; you will then write a report after two more weeks on the nickel determination you
have carried out. If you devise a good method, your results will work well. Make bad choices, and the
precision will be poor. YOU make the decisions, YOU reap the rewards! The TAs will grade your first
(method design) lab report for clarity, logic, and accuracy. You will find for yourself when you're part way
through following your own instructions whether you did an adequate job.

While there will be a post-lab quiz for Week One (developing your procedure), there will NOT be post-lab
quizzes the remaining weeks of the semester. Since no two groups will use identical procedures,
devising a fair quiz is difficult. Furthermore, the reason we gave post-lab quizzes was so you wouldn't
quit thinking about the lab after you did it. On this lab, your ingenuity and focus will so heavily inform the
results that additional paperwork seems superfluous. Similarly, while there's a prelab quiz for Week One
and for Week Two, there is no pre-lab quiz for the last week of the lab. The course "curve" (the
guarantees for minimum number of points to achieve a particular letter grade) reflect this.

The parts of the lab are:

I. List the Universe of Possible Methods

II. Estimate the Amount of Analyte
III. Catalog Expected Interferences
IV. Narrow the Possible Techniques to Those Consistent with Expected Amounts and Interferences
V. Write Out a First Estimate of How to Carry Out Each Technique (Including Equilibrium or Kinetics of
Reactions as Appropriate). Decide on Sample Pretreatment
VI. Compute Masses, Volumes, and Other Signals Expected
VII. Estimate Errors
VIII. Discard Methods with Inadequate Detection Limit, Dynamic Range, or Precision
IX. Among Remaining Methods, Choose Based on Any Additional Available Criteria

In weeks 2 and 3, you'll make use of the results of the above body of work to analyze an unknown.

Students will work in groups. The 3 people on one side of a lab bench will form a single team. If there
are only 2 people on a bench, they can work as a pair or absorb a single person from another bench. If
there is only 1 person on a bench, they should preferably join a pair of people in need of a third. If (and
only if!) the number of students in a lab section is 3N+1, N an integer (so that there's no way for everyone
to be in 3's and there are no spontaneous groups of 2), one of the 3-somes should split so that the final
grouping of 3+1 becomes 2+2. In contrast to all the other labs this semester, each group will turn in a
single report and get a single grade. You will find that splitting up the work so that each group member
spearheads computations on one method will be the most effective way to proceed.

The sample you will plan to analyze is a mineral object, thought to be a siderite meteorite.
Such meteorites are composed mostly of iron and magnesium but also contain 1-10%
nickel. The metals are present as carbonates, phosphides, and sulfides.

I. List the Universe of Possible Methods

Methods we have NOT used previously, and that are NOT available to you (but are commonly used in
well-instrumented labs) are:
a) Inductively-coupled Plasma Mass Spectrometry
b) Inductively-coupled Plasma Emission Spectrometry
c) Flame or Furnace Atomic Absorption Spectrometry
d) X-ray Fluorescence
We list these so that, if you're ever faced with this sort of analysis in the Real World, you'll consider them.

Methods that we have used previously or that are discussed sufficiently in Harris to be worthy of your
consideration include:
A. EDTA Titration (forward or reverse – whichever works better)
B. Quinoxoline Complexation with Spectrophotometric Determination (using pre-separation to
remove interferences)
C. Quinoxoline Complexation with Spectrophotometric Determination (using multiple absorbance
measurements at multiple wavelengths and standards for any interfering species to determine the
amounts of both nickel and interferents)
D. Dimethylglyoxime Precipitation and Determination by Gravimetry

II. Estimate the Amount of Analyte

Suppose the meteorite weighs 15 g. You don't want to destroy the whole meteorite in doing the analysis.
You can chip ~ 1 g from the meteorite and grind it up.

FOR A 5 g SAMPLE: compute the range of Ni masses expected and the range in the number of moles

Minimum expected mass in sample _______ g Maximum expected mass in sample _______ g.

Minimum expected moles ____________. Maximum expected moles ____________.

III. Catalog Expected Interferences

The interferences are Fe, Mg, metal oxides and hydroxides, and possibly CO3=, P3-, or S=. For each:
- List possible redox states that must be considered.
- Are the species soluble in acid? In base? What nickel salts are highly soluble? Are Fe and Mg
salts of the same anions soluble?
- Look up the solubility product of FeS, MgS, NiS, FeCO3, Fe3P2, Ni3P2, Mg3P2.
- Why can we ignore most Fe(II) compounds?

Compound Ksp If Ksp not found, why not?

Other Fe(III) oxy-

Look up the pKa's of the relevant acids: H2S, H2CO3, PH3. Note that both hydrogen sulfide and phosphine
are HIGHLY toxic.

From what you have determined so far: RECOMMEND whether to dissolve the specimen in acid, base, or
neutral water AND explain why. We'll figure out which acid or which base (if one of those is what you
choose) later. Also, recommend whether we should try to maintain the oxidation state of all the elements,
or if we should try to oxidize some or reduce some.

IV. Narrow the Possible Techniques to Those Consistent with Expected Amounts and Interferences

Clearly, the 5 g of sample is going to have to be diluted into a reasonable volume of solvent or buffer. We
don't yet know what that volume will be. Thus, let's compute what amounts and concentrations will be
present for various amounts of dilution. For amounts in aliquots, assume the amount of analyte is mid-
way between minimum and maximum expected amounts.

Volume of diluted Molarity for Molarity for Approximate Approximate

Sample Minimum [Ni2+] Maximum [Ni2+] moles in 5 mL moles in 25 mL
aliquot aliquot
100 mL
250 mL
1000 mL

V. Write Out a First Estimate of How to Carry Out Each Technique (Including Equilibrium or Kinetics of
Reactions as Appropriate). Decide on Sample Pretreatment.

This is where each member of the group should take the lead in looking at one of the methods. Assign
one member to look at EDTA titration, one at DMG gravimetry, and one at quinoxoline complexation.

For each of the techniques in I.A. – I.D., once Ni2+ is in solution, write out:
- the reactions that Ni2+ undergoes.
- any redox reactions needed to remove interferents
- any pKa's for reactants that haven't yet been tabulated
- the equilibrium constants for all reactions
- the range of pH in which one wishes to work and what happens if the determination is attempted
outside that range.
- what buffer would be best to use when carrying out the method
- what effect that buffer would have on the other components

Now that you have all the chemistry listed, suggest a procedure for sample preparation. Your explanation
will start out with grinding the sample. Should you dry the sample? If so, at what temperature? If it's too
low, you won't succeed in drying it, and if too high, you might oxidize some of the sulfides or phosphides,
leading to an erroneously high measured [Ni2+]. Next, specify how much sample to weigh (yes, I know it's
~ 5g. Specify an acceptable range. Is it 5.0000±0.0001 g? 4.8 to 5.2 g, measured to 2 places? To 4
places?). Specify how to dissolve the specimen. What acid, base, buffer, or other solvent should you
use? What volume? What temperature? After the dissolution step, what additional materials need to be
added, in what quantity, to carry out what steps? Hint: how will you get rid of the sulfide so you don't form
pyrite (fool's gold, FeS) as a precipitate in basic solution? Should the operations be carried out on the
bench top or under the hood? See how the steps that you've been told to follow all semester are
determined in light of the chemistry and safety?

VI. Compute Masses, Volumes, and Other Signals Expected

Now for the fun! For each of the techniques in I.A. – I.D., what behavior would we expect? Choose the
volume into which to dilute the specimen you dissolved in step V. Specify whether to dilute with water or
with buffer (and, if buffer, what buffer?). Use the table in step IV to help you out.

FOR TITRATION METHODS: give an approximate value for the optimum concentration of the titrant. For
a 25 mL aliquot, compute the volume of titrant required to reach the equivalence point. Compute the
titration error (overshoot or undershoot when the indicator will recognize that you've hit the endpoint).
Simulate the titration curve; will there be a sharp enough inflection to see an endpoint?

FOR SPECTROPHOTOMETRIC METHODS: give an approximate value for the absorbance of a 5 mL

aliquot processed with the appropriate complexing agent. What concentration of complexing agent
should be used? Is the complexing agent soluble in the buffer of choice at the necessary concentration?
If the absorbance is too high, by what additional factor should the sample be diluted? If the absorbance is
too low, would you be better off with an initial dissolution volume, and is that volume practical?

FOR GRAVIMETRIC METHODS: specify the approximate concentration of the precipitating agent (Harris
explains this, P. 631-635). Specify the volume of aliquot to take and the volume of precipitating agent to
add. Estimate the mass of precipitate and the solubility error i.e. the fraction of the analyte still in solution
because Ksp isn't 0.

VII. Estimate Errors

Do a propagation of error calculation for each method. You will have a deterministic error for the
gravimetric and titrimetric methods because of indicator/endpoint error. Background will cause error in
the spectrophotometric method IF the complexing agent reacts with any of the interfering species AND
you haven't separated the interferents. If the interferents are present, you'll have to run working curves
for the interferents so you can compensate for their presence. All methods will have random errors.
From your experience this semester, estimate the magnitudes of expected errors. Each member of your
group will do a propagation of error computation for the method on which they are taking the lead.

VIII. Discard Methods with Inadequate Detection Limit, Dynamic Range, or Precision

Now compare the methods. Are there any where the signal is so low or the error so high (over 1%) that
the method can't be used? Are there any where the signal is so high that a greater dilution should be
used? Explain your reasoning.

IX. Among Remaining Methods, Choose Based on Any Additional Available Criteria

If you find there are no methods that will work, so state (Hint: at least one will work.).

If you find there is only one method that will work, so state.

If you find there is more than one method that will work, choose which one YOU would use and justify
your choice.

Your combined report MUST list the names of those who are collaborating.

"Nickel is green in solution. Why not measure the absorbance directly?" The molar absorptivity of
Ni(H2O)62+ at 388 nm is 5.3 L mol-1 cm-1. [Wannowius, K. J.; Krimm, K.; Elias, H., "Visible spectra of the
nickel ethylenediamine complex species Ni(en)32+, Ni(en)2(H2O)22+ and Ni(en)(H2O)42+ from kinetic
studies," Inorganica Chimica Acta, 127(2), L43-L44 (1987).] Since the minimum absorbance one can see
is about 0.001, how does the detection limit compare to the numbers you found in Section IV? Can one
quantitate at this low level (think error propagation)? What concentration would be needed to allow
spectrophotometric precision of 1%? Is this obtainable from the meteorite sample?

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