Experiments

To Accompany
Exploring Chemical Analysis, 5th Edition (2012)
Daniel C. Harris

0. Green Analytical Chemistry 5

1. Calibration of Volumetric Glassware 13
2 Gravimetric Determination of Calcium as CaC2O4 . H2O 16
3. Gravimetric Determination of Iron as Fe2O3 18
4. Penny Statistics 21
5. Statistical Evaluation of Acid-Base Indicators 29
6. Preparing Standard Acid and Base 34
7. Using a pH Electrode for an Acid-Base Titration 37
8. Analysis of a Mixture of Carbonate and Bicarbonate 40
9. Analysis of an Acid-Base Titration Curve: The Gran Plot 42
10. Fitting a Titration Curve with Excel Solver® 47
11. Kjeldahl Nitrogen Analysis 55
12. EDTA Titration of Ca2+ and Mg2+ in Natural Waters 58
13. Synthesis and Analysis of Ammonium Decavanadate 60
14. Iodimetric Titration of Vitamin C 64
15. Preparation and Iodometric Analysis of High-Temperature Superconductor 66
16. Potentiometric Halide Titration with Ag+ 71
17. Measuring Ammonia in an Aquarium with an Ion-Selective Electrode 75
18. Electrogravimetric Analysis of Copper 76
19. Measuring Vitamin C in Fruit Juice by Voltammetry with Standard Addition 78
20. Polarographic Measurement of an Equilibrium Constant 81
21. Coulometric Titration of Cyclohexene with Bromine 83
22. Spectrophotometric Determination of Iron in Vitamin Tablets 87
23. Microscale Spectrophotometric Measurement of Iron in Foods by Standard Addition 89
24. Spectrophotometric Determination of Nitrite in Aquarium Water 92
25. Spectrophotometric Measurement of an Equilibrium Constant: The Scatchard Plot 93
26. Spectrophotometric Analysis of a Mixture: Caffeine and Benzoic Acid in Soft Drink 97

1
27. Mn2+ Standardization by EDTA Titration 100
28. Measuring Manganese in Steel by Spectrophotometry with Standard Addition 102
29. Measuring Manganese in Steel by Atomic Absorption Using a Calibration Curve 106
30. Properties of an Ion-Exchange Resin 109
31. Analysis of Sulfur in Coal by Ion Chromatography 113
32. Measuring Carbon Monoxide in Automobile Exhaust by Gas Chromatography 115
33. Amino Acid Analysis by Capillary Electrophoresis 117
34. DNA Composition by High-Performance Liquid Chromatography 122
35. Analysis of Analgesic Tablets by High-Performance Liquid Chromatography 125
36. Anion Content of Drinking Water by Capillary Electrophoresis 127
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Oil 130

2
Experiments Exploring Chemical Analysis

Experiments
The following experiments illustrate major analytical techniques described in the textbook,
Exploring Chemical Analysis. Procedures are organized roughly in the same order as topics in
the text. You are invited to download these instructions and reproduce them freely. Lists of
many additional experiments published in the Journal of Chemical Education are also given at
the web site for Exploring Chemical Analysis. Some experiments refer to standards or unknowns
from Thorn Smith. 1
Although these procedures are safe when carried out with reasonable care, all chemical
experiments are potentially hazardous. Any solution that fumes (such as concentrated HCl) and
all nonaqueous solvents should be handled in a fume hood. Pipetting should never be done by
mouth. Spills on your body should be flooded immediately with water and washed with soap and
water. Your instructor should be notified for possible further action. Spills on the benchtop
should be cleaned immediately. Toxic chemicals should not be flushed down the drain. Your
instructor should establish a procedure for disposing of each chemical that you use.
When equipment permits, it is environmentally friendly and economical to reduce the
scale of an experiment. For example, 50-mL burets can be replaced by 10-mL burets at some
loss in analytical precision. An even smaller scale is possible with microburets made from 2-mL
pipets. 2 Micropipets and small volumetric flasks can replace large glass pipets and large
volumetric flasks (again at a sacrifice of precision). With properly chosen electrodes,

1 Thorn Smith Inc., 7755 Narrow Gauge Road, Beulah, MI 49617. Phone: 231-882-4672;
www.thornsmithlabs.com. The following analyzed unknowns are available: Al-Mg alloy (for Al, Mg), Al-Zn
alloy (for Al, Zn), Sb ore (for Sb), brasses (for Sn, Cu, Pb, Zn), calcium carbonate (for Ca), cast iron (for P, Mn,
S, Si, C), cement (for Si, Fe, Al, Ca, Mg, S, ignition loss), Cr ore (for Cr), Cu ore (for Cu), copper oxide (for Cu),
ferrous ammonium sulfate (for Fe), Fe ore (for Fe), iron oxide (for Fe), limestone (for Ca, Mg, Si, ignition loss),
magnesium sulfate (for Mg), Mn ore (for Mn), Monel metal (for Si, Cu, Ni), nickel silver (for Cu, Ni, Zn), nickel
oxide (for Ni), phosphate rock (for P), potassium hydrogen phthalate (for H+), silver alloys (for Ag, Cu, Zn, Ni),
soda ash (for Na2CO3), soluble antimony (for Sb), soluble choride, soluble oxalate, soluble phosphate, soluble
sulfate, steels (for C, Mn, P, S, Si, Ni, Cr, Mo), and Zn ore (for Zn). Primary standards are also available:
potassium hydrogen phthalate (to standardize NaOH), As2O3 (to standardize I2), CaCO3 (to standardize EDTA),
Fe(NH4)2(SO4)2 (to standardize dichromate or permanganate) , K2Cr2O7 (to standardize thiosulfate), AgNO3,
Na2CO3 (to standardize acid), NaCl (to standardize AgNO3), and Na2C2O4 (to standardize KMnO4).
2 M. M. Singh, C. McGowan, Z. Szafran, and R. M. Pike, J. Chem. Ed. 1998, 75, 371; ibid. 2000, 77, 625.

3
Experiments Exploring Chemical Analysis

potentiometric titrations can be performed on a scale of a few milliliters, instead of tens of

milliliters, by using a syringe or a micropipet to deliver titrant. Instructions here were written for
the commonly available, large-size lab equipment, but your modification to a smaller scale is
encouraged.

4
0. Green Analytical Chemistry Exploring Chemical Analysis

0. Green Analytical Chemistry 3

“Green chemistry” is a set of ideas designed to protect our environment and conserve our
resources. These ideas can be traced back to the book Green Chemistry: Theory and Practice 4
published in 1998 by Paul Anastas and John Warner. They defined green chemistry as “the use
of a set of principles that reduces or eliminates the use or generation of hazardous substances in
the design, manufacture, and application of chemical products.” Green chemistry is a way of
thinking about chemistry. It is not a separate chemical discipline like analytical, organic, or
physical. In fact, it is hoped that the current generation of chemistry students taking this course
will have this mode of thinking become so natural to them that we will no longer have to think
about “green chemistry,” but just “chemistry.”
How, then, does analytical chemistry fit into this way of thinking? What is green
analytical chemistry? To answer these questions, we must first reconsider what is analytical
chemistry and what is green chemistry.
During this course, you will learn a series of methods to qualitatively and quantitatively
characterize a sample or chemical system. Knowledge of these methods and the chemical
principles behind them is an asset valued by many industries. On the other hand, analytical
chemists may be viewed as a necessary evil, for example, to analyze samples in order to comply
with Food and Drug Administration or other regulatory agency requirements. Analytical
chemistry is something that is tolerated, but the analysis costs money, rather than adding to a
company’s profits. Consider the following example: You work for a detergent manufacturer and
a product development chemist comes into your lab with a bottle of detergent, asking you to
determine the silicone level in this product. While you could easily do what you are told, upon
questioning you find out that the product development chemist really doesn’t want to know the
silicone concentration. What he really wants to know is why the product isn’t performing the
way it should (maybe it foams too much or doesn’t clean effectively) and he thinks it’s related to
the silicone concentration. A good analytical chemist does not merely provide data, but rather
supplies information and knowledge upon which educated decisions can be based.

3 This section was contributed by Douglas E. Raynie, Department of Chemistry and Biochemistry, South Dakota
State University, Brookings SD 57007; [email protected].
4 P. T. Anastas and J. C. Warner, Green Chemistry: Theory and Practice (New York: Oxford University Press,
1998).

5
0. Green Analytical Chemistry Exploring Chemical Analysis

Analytical chemists are problem solvers. We use our knowledge to become partners with
our customers to answer their questions. In the detergent analysis example, one analytical
chemist might have run the silicone analysis and then several other analyses in order to
eventually solve the problem. But the analytical chemist who was a true scientific partner in the
process understood the problem and used his knowledge of chemistry to develop a more refined
hypothesis and streamline the analyses need to answer the ultimate question more easily. This
role of the analytical chemist was summarized by Prof. Herb Laitinin who editorialized,
“Analysis of a sample is not the true aim of analytical chemistry…the real purpose of the analysis
is to solve a problem.” 5 Development of problem-solving skills comes with a combination of
analytical knowledge and experience. In summary, the analytical chemist is a problem solver
and solving problems involves a means of thinking about chemistry.

What is Green Chemistry?

We’ve defined green chemistry as something that is applied over the entire lifespan of a product.
Hidden within this definition is a combination of environmental and economic concerns. That is,
a product may have environmental benefits, but if it cannot compete in the marketplace, it is
doomed to failure. Green chemistry is guided by the set of 12 principles developed by Anastas
and Warner4 and shown in Table 1.
If green chemistry and concern for the environment are the right things to do, why haven’t
we been doing this all along? Think about organic chemistry. It isn’t always easy to choose the
right reagents to put the correct functional groups in the right places for a complex synthesis.
Now consider that you are working as a product development chemist in industry. Not only must
you synthesize the compound with properties desired by your company, but you must do it in a
manner that can be passed on to a process engineer who can develop the process with high yield,
speed, safety, and minimal cost. Now add environmental concerns on top of all of that and the
lament of a certain amphibian character, “It’s not easy being green,” becomes more obvious. In
summary, green chemistry involves a way of thinking about chemistry in an environmentally and
economically sound manner.

5 H. A. Laitinen, Anal. Chem., 1966, 38, 1441.

6
0. Green Analytical Chemistry Exploring Chemical Analysis

Table 1. The 12 Principles of Green Chemistry4

1. Prevention
It is better to prevent waste than to treat or clean up waste after it has been created.
2. Atom Economy
Synthetic methods should be designed to maximize the incorporation of all materials used in
the process into the final product.
3. Less Hazardous Chemical Syntheses
Wherever practicable, synthetic methods should be designed to use and generate substances
that possess little or no toxicity to human health and the environment.
4. Designing Safer Chemicals
Chemical products should be designed to effect their desired function while minimizing their
toxicity.
5. Safer Solvents and Auxiliaries
The use of auxiliary substances (e.g., solvents, separation agents, etc.) should be made
unnecessary wherever possible and innocuous when used.
6. Design for Energy Efficiency
Energy requirements of chemical processes should be recognized for their environmental and
economic impacts and should be minimized. If possible, synthetic methods should be
conducted at ambient temperature and pressure.
7. Use of Renewable Feedstocks
A raw material or feedstock should be renewable rather than depleting whenever technically
and economically practicable.
8 Reduce Derivatives
Unnecessary derivatization (use of blocking groups, protection/ deprotection, temporary
modification of physical/chemical processes) should be minimized or avoided if possible,
because such steps require additional reagents and can generate waste.
9. Catalysis
Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
10. Design for Degradation
Chemical products should be designed so that at the end of their function they break down
into innocuous degradation products and do not persist in the environment.
11. Real-time analysis for Pollution Prevention
Analytical methodologies need to be further developed to allow for real-time, in-process
monitoring and control prior to the formation of hazardous substances.
12. Inherently Safer Chemistry for Accident Prevention
Substances and the form of a substance used in a chemical process should be chosen to
minimize the potential for chemical accidents, including releases, explosions, and fires.

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0. Green Analytical Chemistry Exploring Chemical Analysis

What is Green Analytical Chemistry?

Reviewing the previous summary statement, we can correctly guess that green analytical
chemistry is a marriage of thought processes — a unique way of thinking about how to do
chemistry. Performing chemical analyses in a green manner does not mean that we must make
compromises to the requisite accuracy, precision, and other analytical demands. Inserting green
values into the laboratory is about several factors related to the 12 green chemistry principles,
including waste, energy, toxicity, and others. For example, the hazard associated with a given
chemical is related to both our exposure to the chemical and the inherent risk possessed by the
chemical. While we can wear personal protective equipment, like safety glasses, lab coats, and
latex gloves, or work in a fume hood, we cannot change the inherent risk associated with a
compound. So perhaps we can change the reagent. We’ve been doing this for years. Benzene
and carbon tetrachloride were banished from the lab since the mid-1970s. But our practices must
continue to evolve as our knowledge about chemicals and their inherent risk progresses.
At least half of the 12 principles of green chemistry apply to the analytical laboratory:
Principle 1: Prevent Waste. While the wastes generated in chemical production
overshadow the amount of waste coming from a typical analytical laboratory, individual
analytical procedures may involve as much as one liter of organic solvent to extract the analytes
from the sample. When used in an analytical procedure, even water, which everyone would
agree is an environmentally benign solvent, is converted to wastewater which must be treated
appropriately. So the professional analyst must stay abreast of developments that can minimize
waste generation.
Principle 5: Safer Solvents and Auxiliaries. The inherent risk associated with a chemical
must be considered and alternatives explored. The risk may be toxicological, but may also be
flammability, corrosiveness, or other attributes. If alternatives do not exist, appropriate exposure
controls are needed.
Principle 6: Energy Efficiency. Several common analytical procedures can be considered
energy intensive. For example, not only are large amounts of solvents used in extractions, in
most cases the solvent must then be evaporated to concentrate the solutes for analysis.
Instrumental procedures can require high temperatures or have high power demands. Sometimes
trade-offs must be considered. Does it require less energy to keep a drying oven turned on
overnight and maintain a constant temperature or to turn off the oven and require a rapid heat up
just prior to use? If water is used as a nontoxic solvent, do we pay a price in the energy needed to

8
0. Green Analytical Chemistry Exploring Chemical Analysis

evaporate water compared with many organic solvents? Later, we will present a set of metrics to
judge these compromises.
Principle 8: Reduce Derivatives. Many times chemical derivatives are employed to
enhance the solubility or detectability of sample components. For example, the determination of
fats in foods requires conversion of fatty acids into methyl esters for gas chromatography.
However, technological advances can provide similar analytical results while avoiding the need
for derivatization. For example, modern high-performance liquid chromatography can provide
results equivalent to previous generations of gas chromatography.
Principle 11: Real-time Analysis. This principle is analytical chemistry!!! The field of
process analytical chemistry, involving in-line, on-line, or at-line procedures, is a valuable tool in
chemical processing. By monitoring the creation of a by-product, the poisoning of a catalyst,
reaction pH, or other properties, feedback is provided to allow a reaction or process to stay in
control, avoiding potential process disruptions.
Principle 12: Safer Chemistry. The flammability of solvents, oxidative capability of
reagents, and similar factors should be considered in developing analytical methods.
Several of the experiments in this laboratory manual illustrate green chemistry. For
example, Experiment 11 (Kjeldahl Nitrogen Analysis) uses selenium-coated boiling chips, rather
than a mercury compound, as a catalyst (Principle 9); Experiment 15 (Preparation and Iodometric
Analysis of a High-Temperature Semiconductor) reminds us that high-temperature
superconductors could help us toward energy sustainability (Principle 6); and Experiments 31
(Analysis of Sulfur in Coal by Ion Chromatography) and 32 (Measuring Carbon Monoxide in
Automobile Exhaust by Gas Chromatography) illustrate the role of analytical chemistry in
pollution prevention (Principles 1 and 11). Experiment 37, Green Chemistry: Liquid Carbon
Dioxide Extraction of Lemon Peel Oil, demonstrates the potential of supercritical fluid extraction
to address solvent alternatives (Principles 1 and 5). We challenge students to discuss your
experiments relative to the 12 green chemistry principles as you write your reports.
Now that we’ve gotten some idea of what green analytical chemistry is, let’s discuss some
specific green analytical considerations. Some review articles 6, 7, 8 present more detailed
discussions of advances in the field of analytical chemistry that exhibit green chemistry

6 P.T. Anastas, Crit. Rev. Anal. Chem. 1999, 29, 167.

7 J. Namiesnik, J. Sep. Sci. 2001, 24, 151.
8 L.H. Keith, L.U. Gron, and J.L. Young, Chem. Rev. 2007, 107, 2695.

9
0. Green Analytical Chemistry Exploring Chemical Analysis

attributes. However, general considerations for the practice of green analytical chemistry can be
summarized:
Planning. Proper planning allows the maximum amount of information to be obtained
from the minimal number of analyses. A branch of statistics called Design of Experiments can
be used to guide planning laboratory procedures. The developing field of chemometrics allows
us to uncover relationships between sample sets that might be hidden if conventional thinking is
used.
Sampling. Attention to proper sampling is an often overlooked portion of chemical
analysis, but perhaps the most important step in the entire analysis. A common misconception is
that an analytical method can be made green simply by being performed at the micro-scale.
While this is true, one must take care to ensure that the appropriate (minimum) sample size is
used. The sample must be collected to be statistically representative of the system under study
and homogenized to reduce error.
Direct Analysis. Methods which do not require sample work-up prior to the measurement
step have several advantages, including greenness. Techniques employing ion-selective
electrodes, reflectance spectroscopy, or surface analysis can often provide chemical information
without using additional analytical reagents.
Sample Preparation. Organic extractions and acid digestions generate the greatest
amount of waste (while using hazardous solvents or acids) in many procedures. This field has
received considerable attention in modern times. Acid digestions can be made safer through the
application of microwave irradiation. Alternative solvent processes using water, ionic liquids, or
careful manipulation of heat and energy can greatly enhance the sample preparation process.
Chromatography. Separation of analyte from other sample components is necessary in
most analytical procedures. Chromatography can involve copious amounts of solvents, sorbents,
and other chemicals. Microcolumn chromatography reduces solvent consumption, minimizes the
use of chromatographic sorbents, and provides superior performance.
Data Reduction. Statistical manipulations can mine information from analytical results.
Many times unambiguous compound identification is not needed; rather knowledge of trends is
sufficient.
Field Analysis and Process Analysis. Taking the analysis to the sample generally
provides several green advantages. Near instantaneous feedback can minimize the likelihood of
a process upset. Similarly, the backhoe operator at an environmental clean-up site would like

10
0. Green Analytical Chemistry Exploring Chemical Analysis

instantaneous notification that the site is clean so she can quit digging, rather than waiting for
results to come back from the laboratory. One contribution of analytical chemistry to the
environmental movement has been the identification of hazards within the environment. Careful
execution of field and process analysis may advance analytical chemistry from an identification
and compliance model to a hazard prevention model.

How Do We Decide if a Method is Green?

A good place to start is to compare an analytical method against the 12 principles of green
chemistry. However, keep in mind that compromises are made along the way. One method
might use less solvent, while another is less energy intensive. No procedure is truly green; rather,
one method may be considered greener than another.
One set of metrics used to assess the green profile of a method is associated with the
National Environmental Monitoring Index (NEMI) database (www.nemi.gov). The NEMI
database was compiled by researchers from several federal agencies, the American Chemical
Society Green Chemistry Institute, and others. This free, on-line database lists regulatory and
nonregulatory methods and procedures for analysis of water, sediment, air, and tissues. It
presents information on cost and performance of each method. Presented with each method is a
green profile, developed in collaboration with 25 environmental methods experts from five
regulatory agencies and private labs. An example of this profile is shown in Figure 1 for the
Environmental Protection Agency method for the determination of total water hardness by EDTA
titration (similar to Experiment 12).

Figure 1. Green Profile for EPA Method 130.2:

Total Hardness of Water by Titrimetry

The symbol for the green profile is simple, visual, and allows an individual to make a
value judgment against the profile criteria. Here’s a description of the symbol criteria:
Hazardous: A method fails this criterion if it uses chemicals in either of two important
databases, the Resource Conservation and Recovery Act D, F, P, or U lists 9 or the Environmental

9 http://www.epa.gov/epawaste/hazard/wastetypes/pdfs/listing-ref.pdf

11
0. Green Analytical Chemistry Exploring Chemical Analysis

Protection Agency Toxic Release Inventory. 10 About half of the methods in the NEMI database
fail this criterion.
Waste Amount: A method fails this criterion if the amount of waste generated is greater
than 50 grams. This 50 grams includes the mass of sample, as well as all chemicals used in the
procedure, but does not include calibration standards, cleaning (unless significant amounts of
chemicals are stipulated in cleaning steps), and related factors. The waste criterion is perhaps the
most rigorous. When applied to 560 methods on the NEMI database, approximately 2/3 failed
this criterion. Failures generally were due to solvent use for organic analysis and mineral acid
use for inorganics.
Corrosive: A method fails this criterion if the pH during the analysis is less than 2 or
greater than 12. The failure rate for this criterion was about 20% for the NEMI methods.
PBT (Persistent, Bioaccumulative, or Toxic): A method fails this criterion if it uses
chemicals considered to be persistent in the environment, bioaccumulative, or toxic as defined by
the EPA Toxic Release Inventory. Only about 5% of the NEMI methods did not meet this
criterion and each of the methods that failed the PBT criterion also failed for hazard. Most
commonly, these methods included lead or mercury compounds.
For the EPA water hardness method (EPA Method 130.2) illustrated in Figure 1, we see
that the method is considered green by hazard and PBT criteria, but not by waste and corrosion
criteria. During the analysis, the pH is less than 2 and 66 grams of waste are generated.

Summary

Both analytical chemistry and green chemistry are based on a way of thinking about how you
perform experiments. Green analytical chemistry is a convergence of these thought processes.
Good analytical chemistry is inherently green chemistry.
For each experiment in this laboratory manual, a green profile is displayed. A suggested
component of your report should be a discussion of why a particular experiment met or failed the
individual green profile criteria. Suggest ways that the experiment may be made greener. If you
follow through with this exercise, these thought processes should become increasingly natural to
you. If you develop this mode of thinking so that it becomes second nature, you will be poised to
take on leadership responsibilities in your generation of professional chemists.

10 http://www.epa.gov/tri/trichemicals/pbt%20chemicals/pbt_chem_list.htm

12
1. Calibration of Volumetric Glassware Exploring Chemical Analysis

1. Calibration of Volumetric Glassware

An important trait of a good analyst is the ability to extract the best possible
data from his or her equipment. For this purpose, it is desirable to calibrate
your own volumetric glassware (burets, pipets, flasks, etc.) to measure the
exact volumes delivered or contained. This experiment also promotes
Green Profile improved technique in handling volumetric glassware.
See Section 0

Calibrating a 50-mL Buret

This procedure tells how to construct a graph such as Figure 3-2 in the textbook to convert the
measured volume delivered by a buret to the true volume delivered at 20˚C.

1. Fill the buret with distilled water and force any air bubbles out the tip. See whether the buret
drains without leaving drops on its walls. If drops are left, clean the buret with soap and
water or soak it with cleaning solution. 11 Adjust the meniscus to be at or slightly below 0.00
mL, and touch the buret tip to a beaker to remove the suspended drop of water. Allow the
buret to stand for 5 min while you weigh a 125-mL flask fitted with a rubber stopper. (Hold
the flask with a paper towel to prevent fingerprints from changing its mass.) If the level of the
liquid in the buret has changed, tighten the stopcock and repeat the procedure. Record the
level of the liquid.
2. Drain approximately 10 mL of water at a rate < 20 mL/min into the weighed flask, and cap it
tightly to prevent evaporation. Allow 30 s for the film of liquid on the walls to descend
before you read the buret. Estimate all readings to the nearest 0.01 mL. Weigh the flask
again to determine the mass of water delivered.
3. Drain the buret from 10 to 20 mL, and measure the mass of water delivered. Repeat the
procedure for 30, 40, and 50 mL. Then do the entire procedure (10, 20, 30, 40, 50 mL) a
second time.
4. Use Table 2-5 in the textbook to convert the mass of water to the volume delivered. Repeat
any set of duplicate buret corrections that do not agree to within 0.04 mL. Prepare a

11 Prepare cleaning solution by dissolving 36 g of ammonium peroxydisulfate, (NH ) S O , in a loosely stoppered

42 2 8
2.2-L ("one gallon") bottle of 98 wt% sulfuric acid. Add ammonium peroxydisulfate every few weeks to
maintain the oxidizing strength.

13
1. Calibration of Volumetric Glassware Exploring Chemical Analysis

calibration graph like Figure 3-2 in the textbook, showing the correction factor at each 10-mL
interval.

EXAMPLE Buret Calibration

When draining the buret at 24˚C, you observe the following values:

Final reading 10.01 10.08 mL

Initial reading 0.03 0.04
Difference 9.98 10.04 mL
Mass 9.984 10.056 g
Actual volume delivered 10.02 10.09 mL
Correction +0.04 +0.05 mL
Average correction +0.045 mL

To calculate the actual volume delivered when 9.984 g of water is delivered at 24˚C, use
the conversion factor 1.003 8 mL/g in Table 2-5 of the textbook. We find that 9.984 g occupies
(9.984 g)(1.003 8 mL/g) = 10.02 mL. The average correction for both sets of data is +0.045 mL.
To obtain the correction for a volume greater than 10 mL, add successive masses of water
collected in the flask. Suppose that the following masses were measured:

Volume interval (mL) Mass delivered (g)

0.03–10.01 9.984
10.01–19.90 9.835
19.90–30.06 10.071
Sum 30.03 mL 29.890 g

The total volume of water delivered is (29.890 g)(1.003 8 mL/g) = 30.00 mL. Because the
indicated volume is 30.03 mL, the buret correction at 30 mL is –0.03 mL.

What does this mean? Suppose that Figure 3-2 in the textbook applies to your buret. If
you begin a titration at 0.04 mL and end at 29.00 mL, you would deliver 28.96 mL if the buret

14
1. Calibration of Volumetric Glassware Exploring Chemical Analysis

were perfect. Figure 3-2 tells you that the buret delivers 0.03 mL less than the indicated amount;
so only 28.93 mL was actually delivered. To use the calibration curve, either begin all titrations
near 0.00 mL or correct both the initial and the final readings. Use the calibration curve
whenever you use your buret.

Calibrating Other Glassware

Other volumetric glassware can also be calibrated by measuring the mass of water they
contain or deliver. Glass transfer pipets and plastic micropipets can be calibrated by weighing
the water delivered from them. A volumetric flask can be calibrated by weighing it empty and
then weighing it filled to the mark with distilled water. Perform each procedure at least twice.
Compare your results with the tolerances listed in tables in Chapter 2 of the textbook.

15
2. Gravimetric Determination of Calcium as CaC2O4 . H2O Exploring Chemical Analysis

2. Gravimetric Determination of Calcium as CaC2O4 . H2O 12

Calcium ion can be analyzed by precipitation with oxalate in basic solution

to form CaC2O4 . H2O. The precipitate is soluble in acidic solution because
the oxalate anion is a weak base. Large, easily filtered, relatively pure
crystals of product will be obtained if the precipitation is carried out slowly.
Slow precipitation is achieved by dissolving Ca2+ and C2O2-
4 in acidic
Green Profile
See Section 0 solution and gradually raising the pH by thermal decomposition of urea:
O

C heat +
H2N NH2 + 3H2O → CO2 + 2NH4 + 2OH-
Urea

Reagents

Ammonium oxalate solution: Make 1 L of solution containing 40 g of (NH4)2C2O4 plus 25 mL

of 12 M HCl. Each student will need 80 mL of this solution.
Methyl red indicator: Dissolve 20 mg of the indicator in 60 mL of ethanol and add 40 mL H2O.
0.1 M HCl: (225 mL/student) Dilute 8.3 mL of 37% HCl up to 1 L.
Urea: 45 g/student.
Unknowns: Prepare 1 L of solution containing 15–18 g of CaCO3 plus 38 mL of 12 M HCl.
Each student will need 100 mL of this solution. Alternatively, solid unknowns are available
from Thorn Smith.1

Procedure

1. Dry three medium-porosity, sintered-glass funnels for 1–2 h at 105˚C. Cool them in a
desiccator for 30 min and weigh them. Repeat the procedure with 30-min heating periods
until successive weighings agree to within 0.3 mg. Use a paper towel or tongs, not your
fingers, to handle the funnels. Alternatively, a 900-W kitchen microwave oven dries the
crucible to constant mass in two heating periods of 4 min and 2 min (with 15 min allowed for

12 C. H. Hendrickson and P. R. Robinson, J. Chem. Ed. 1979, 56, 341.

16
2. Gravimetric Determination of Calcium as CaC2O4 . H2O Exploring Chemical Analysis

cooldown after each cycle). 13 You will need to experiment with your oven to find
appropriate heating times.
2. Use a few small portions of unknown to rinse a 25-mL transfer pipet, and discard the
washings. Use a rubber bulb, not your mouth, to provide suction. Transfer exactly 25 mL of
unknown to each of three 250- to 400-mL beakers, and dilute each with ~75 mL of 0.1 M
HCl. Add 5 drops of methyl red indicator solution to each beaker. This indicator is red
below pH 4.8 and yellow above pH 6.0.
3. Add ~25 mL of ammonium oxalate solution to each beaker while stirring with a glass rod.
Remove the rod and rinse it into the beaker with small portions of distilled water. Add ~15 g
of solid urea to each sample, cover it with a watchglass, and boil gently for ~30 min until the
indicator turns yellow.
4. Filter each hot solution through a weighed funnel, using suction (Figure 2-12 in the
textbook). Add ~3 mL of ice-cold water to the beaker, and use a rubber policeman to help
transfer the remaining solid to the funnel. Repeat this procedure with small portions of ice-
cold water until all of the precipitate has been transferred to the funnel. Finally, use two 10-
mL portions of ice-cold water to rinse each beaker, and pour the washings over the
precipitate.
5. Dry the precipitate, first with aspirator suction for 1 min, then in an oven at 105˚C for 1–2 h.
Bring each filter to constant mass. The product is somewhat hygroscopic, so only one filter
at a time should be removed from the desiccator, and weighings should be done rapidly.
Alternatively, the precipitate can be dried in a microwave oven once for 4 min, followed by
several 2-min periods, with cooling for 15 min before weighing. This drying procedure does
not remove the water of crystallization.
6. Calculate the average molarity of Ca2+ in the unknown solution or the average weight
percent of Ca in the unknown solid. Report the standard deviation and relative standard
deviation (s / x = standard deviation/average).

13 R. Q. Thompson and M. Ghadiali, J. Chem. Ed. 1993, 70, 170.

17
3. Gravimetric Determination of Iron as Fe2O3 Exploring Chemical Analysis

3. Gravimetric Determination of Iron as Fe2O3 14

A sample containing iron can be analyzed by precipitation of the hydrous

oxide from basic solution, followed by ignition to produce Fe2O3:

Base
Fe3+ + (2 + x)H2O FeOOH . xH2O(s) + 3H+

Green Profile Hydrous ferric oxide

See Section 0 900ºC
FeOOH . xH2O Fe2O3(s)

The gelatinous hydrous oxide can occlude impurities. Therefore, the initial precipitate is
dissolved in acid and reprecipitated. Because the concentration of impurities is lower during the
second precipitation, occlusion is diminished. Solid unknowns can be prepared from reagent
ferrous ammonium sulfate or purchased from Thorn Smith.1

Procedure

1. Bring three porcelain crucibles and caps to constant mass by heating to redness for 15 min
over a burner (Figure 1). Cool for 30 min in a desiccator and weigh each crucible. Repeat
this procedure until successive weighings agree within 0.3 mg. Be sure that all oxidizable
substances on the entire surface of each crucible have burned off.

Figure 1. Positioning a crucible above a burner.

14 D. A. Skoog and D. M. West, Fundamentals of Analytical Chemistry, 3d ed. (New York: Holt, Rinehart and
Winston, 1976).

18
3. Gravimetric Determination of Iron as Fe2O3 Exploring Chemical Analysis

2. Accurately weigh three samples of unknown containing enough Fe to produce ~0.3 g of

Fe2O3. Dissolve each sample in 10 mL of 3 M HCl (with heating, if necessary). If there are
insoluble impurities, filter through qualitative filter paper and wash the filter well with
distilled water. Add 5 mL of 6 M HNO3 to the filtrate, and boil for a few minutes to ensure
that all iron is oxidized to Fe(III).
3. Dilute the sample to 200 mL with distilled water and add 3 M ammonia 15 with constant
stirring until the solution is basic (as determined with litmus paper or pH indicator paper).
Digest the precipitate by boiling for 5 min and allow the precipitate to settle.
4. Decant the supernatant liquid through coarse, ashless filter paper (Whatman 41 or Schleicher
and Schuell Black Ribbon, as in Figures 2-13 and 2-14 in the textbook). Do not pour liquid
higher than 1 cm from the top of the funnel. Proceed to step 5 if a reprecipitation is desired.
Wash the precipitate repeatedly with hot 1 wt% NH NO until little or no Cl- is detected in
4 3
the filtered supernate. (Test for Cl- by acidifying a few milliliters of filtrate with 1 mL of
dilute HNO and adding a few drops of 0.1 M AgNO . If precipitate is observed, Cl- is
3 3
present.) Finally, transfer the solid to the filter with the aid of a rubber policeman and more
hot liquid. Proceed to step 6 if a reprecipitation is not used.
5. Wash the gelatinous mass twice with 30 mL of boiling 1 wt% aqueous NH4NO3, decanting
the supernate through the filter. Then put the filter paper back into the beaker with the
precipitate, add 5 mL of 12 M HCl to dissolve the iron, and tear the filter paper into small
pieces with a glass rod. Add ammonia with stirring and reprecipitate the iron. Decant
through a funnel fitted with a fresh sheet of ashless filter paper. Wash the solid repeatedly
with hot 1 wt% NH4NO3 until little or no Cl- is detected in the filtered supernate. Then
transfer all the solid to the filter with the aid of a rubber policeman and more hot liquid.
6. Allow the filter to drain overnight, if possible, protected from dust. Carefully lift the paper
out of the funnel, fold it (Figure 2), and transfer it to a porcelain crucible that has been
brought to constant mass.

15 Basic reagents should not be stored in glass bottles because they slowly dissolve glass. If ammonia from a glass
bottle is used, it may contain silica particles and should be freshly filtered.

19
3. Gravimetric Determination of Iron as Fe2O3 Exploring Chemical Analysis

Figure 2. Folding filter

paper and placing it inside
1. Flatten 2. Fold in 3. Fold over
paper edges top a crucible for ignition.
Continue folding paper so
entire package fits at the
bottom of the crucible. Be
careful not to puncture the
paper.

4. Place inside crucible

with point pushed
against bottom

7. Dry the crucible cautiously with a small flame, as shown in Figure 1. The flame should be
directed at the top of the container, and the lid should be off. Avoid spattering. After the
filter paper and precipitate are dry, char the filter paper by increasing the flame temperature.
The crucible should have free access to air to avoid reduction of iron by carbon. (The lid
should be kept handy to smother the crucible if the paper inflames.) Any carbon left on the
crucible or lid should be burned away by directing the burner flame at it. Use tongs to
manipulate the crucible. Finally, ignite the product for 15 min with the full heat of the burner
directed at the base of the crucible where the Fe2O3 is located.
8. Cool the crucible briefly in air and then in a desiccator for 30 min. Weigh the crucible and
the lid, reignite, and bring to constant mass (within 0.3 mg) with repeated heatings.
9. Calculate the weight percent of iron in each sample, the average, the standard deviation, and
the relative standard deviation (s/ x ).

20
4. Penny Statistics Exploring Chemical Analysis

4. Penny Statistics 16

U.S. pennies minted after 1982 have a Zn core with a Cu overlayer. Prior to
1982, pennies were made of brass, with a uniform composition (95 wt% Cu /
5 wt% Zn). In 1982, both the heavier brass coins and the lighter zinc coins
were made. In this experiment, your class will weigh many coins and pool
the data to answer the following questions: (1) Do pennies from different
Green Profile
See Section 0 years have the same mass? (2) Do pennies from different mints have the
same mass? (3) Do the masses follow a Gaussian distribution?

Gathering Data

Each student should collect and weigh enough pennies to the nearest milligram to provide a data
set for the entire class containing 300 to 500 brass coins and a similar number of zinc coins.
Instructions are given for a spreadsheet, but the same operations can be carried out with a
calculator. Compile all the class data in a spreadsheet. Each column should list the masses of
pennies from only one calendar year. Use the spreadsheet “sort” function to sort each column so
that the lightest mass is at the top of the column and the heaviest is at the bottom. (To sort a
column, click on the column heading to select the entire column, go to the DATA menu, select the
SORT tool, and follow the directions that come up.) There will be two columns for 1982, in
which both types of coins were made. Select a year other than 1982 for which you have many
coins and divide the coins into those made in Denver (with a “D” beneath the year) and those
minted in Philadelphia (with no mark beneath the year).

Discrepant Data

At the bottom of each column, compute the mean and standard deviation. Retain at least one
extra digit beyond the milligram place to avoid round-off errors in your calculations.
Damaged or corroded coins may have masses different from those of the general
population. Discard grossly discrepant masses lying ≥4 standard deviations from the mean in any
one year. (For example, if one column has an average mass of 3.000 g and a standard deviation

16 T. H. Richardson, J. Chem. Ed. 1991, 68, 310. In a related experiment, students measure the mass of copper in
the penny as a function of the year of minting: R. J. Stolzberg, J. Chem. Ed. 1998, 75, 1453.

21
4. Penny Statistics Exploring Chemical Analysis

of 0.030 g, the 4-standard-deviation limit is ±(4 × 0.030) = ±0.120 g. A mass that is ≤2.880 or
≥3.120 g should be discarded.) After rejecting discrepant data, recompute the average and
standard deviation for each column.

Confidence Intervals and t Test

Select the two years (≥1982) in which the zinc coins have the highest and lowest average masses.
For each of the two years, compute the 95% confidence interval. Use the t test to compare the
two mean values at the 95% confidence level. Are the two average masses significantly
different? Try the same for two years of brass coins (≤ 1982). Try the same for the one year
whose coins you segregated into those from Philadelphia and those from Denver. Do the two
mints produce coins with the same mass?

Distribution of Masses

List the masses of all pennies made in or after 1983 in a single column, sorted from lowest to
highest mass. There should be at least 300 masses listed. Divide the data into 0.01-g intervals
(e.g., 2.480 to 2.489 g) and prepare a bar graph, like that shown in Figure 1. Find the mean (x- ),
median, and standard deviation (s) for all coins in the graph. For random (Gaussian) data, only 3
out of 1 000 measurements should lie outside of x- ± 3s. Indicate which bars (if any) lie beyond
± 3s. In Figure 1 two bars at the right are outside of x- ± 3s.

Least-Squares Analysis: Do Pennies Have the Same Mass Each Year?

Prepare a graph like Figure 2 in which the ordinate (y-axis) is the mass of zinc pennies minted
each year since 1982 and the abscissa (x-axis) is the year. For simplicity, let 1982 be year 1,
1983 be year 2, and so on. If the mass of a penny increases systematically from year to year, then
the least-squares line through the data will have a positive slope. If the mass decreases, the slope
will be negative. If the mass is constant, the slope will be 0. Even if the mass is really constant,
your selection of coins is random and the slope is not exactly 0.
We want to know whether the slope is significantly different from zero. Suppose that you
have data for 14 years. Enter all of the data into two columns of the least-squares spreadsheet in
Figure 4-9 in the textbook. Column B (xi) is the year (1 to 14) and column C (yi) is the mass of
each penny. Your table will have several hundred entries. (If you are not using a)

22
4. Penny Statistics Exploring Chemical Analysis

95
100
Dat a f or 613 C ompos ite
Pennies (1982-1992)

83
Av erage = 2. 507 g

75
St andard Dev iat ion = 0. 031 g
80
Median = 2. 506 g

69

65
Calc ulat ed G aus s ian Curv e
Number of Coins

60 Obs erv ed Num ber

48
40
31
29

26
3 Standard

24
Deviati on
18

Limi t
20
12

10
9
4

4
3

3
1

1
0
2 .41 5
2 .42 5
2 .43 5
2 .44 5
2 .45 5
2 .46 5
2 .47 5
2 .48 5
2 .49 5
2 .50 5
2 .51 5

2 .52 5
2 .53 5
2 .54 5
2 .55 5
2 .56 5
2 .57 5
2 .58 5
2 .59 5
2 .60 5
2 .61 5
Mas s (g)

Figure 1. Distribution of penny masses from the years 1982 to 1992 measured in Dan's house by
Jimmy Kusznir and Doug Harris in December 1992.

spreadsheet, just tabulate the average mass for each year. Your table will have only 14 entries.
Calculate the slope (m) and intercept (b) of the best straight line through all points and find the
uncertainties in slope (sm) and intercept (sb).
Use Student's t to find the 95% confidence interval for the slope:

confidence interval for slope = m ± tsm (1)

where Student’s t is for n – 2 degrees of freedom. For example, if you have n = 300 pennies,
n – 2 = 298, and it would be reasonable to use the value of t (= 1.960) at the bottom of the table
for n = ∞. If the least-squares slope is m ± sm = –2.78 ± 0.40 mg/year, then the 95% confidence
interval is m ± tsm = –2.78 ± (1.960)(0.40) = –2.78 ± 0.78 mg/year.

23
4. Penny Statistics Exploring Chemical Analysis

2.65

Slope = -2.78 ± 0.40 mg/yr

Intercept = 2.523 2 ± 0.0002 7 g

2.60
2.6

2.55
Mass (g)

2.50
2.5

1992
2.45
1982

2.40
2.4
0 2 4 6 8 10 12
Year

Figure 2. Penny mass versus year for 612 coins. Because the slope of the least-squares line is
significantly less than 0, we conclude that the average mass of older pennies is greater than the
average mass of newer pennies.

The 95% confidence interval is –2.78 ± 0.78 = –3.56 to –2.00 mg/year. We are 95%
confident that the true slope is in this range and is, therefore, not 0. We conclude that older zinc
pennies are heavier than newer zinc pennies.

Does the Distribution of Masses Follow a Gaussian Distribution?

The smooth Gaussian curve superimposed on the data in Figure 1 was calculated from the
formula

number of coins/100 –(x – x-)2/2s2

ycalc = e (2)
s 2π

24
4. Penny Statistics Exploring Chemical Analysis

Now we carry out a χ2 test (pronounced “ki squared”) to see if the observed distribution
(the bars in Figure 1) agrees with the Gaussian curve. The statistic χ2 is given by

∑
(yobs – ycalc)2
χ2 = ycalc (3)

where yobs is the height of a bar on the chart, ycalc is the ordinate of the Gaussian curve
(Equation 2), and the sum extends over all bars in the graph. Calculations for the data in
Figure 1 are shown in Table 1.

Table 1. Calculation of χ2 for Figure 1

Observed Calculated
number ordinate of
Mass (g) of coins Gaussian curve
(yobs – ycalc)2
(x) (yobs) (ycalc) yobs – ycalc ycalc

2.415 3 1.060 1.940 3.550

2.425 4 2.566 1.434 0.801
2.435 1 5.611 –4.611 3.789
2.445 12 11.083 0.917 0.076
2.455 18 19.776 –1.776 0.159
2.465 29 31.875 –2.875 0.259
2.475 31 46.409 –15.409 5.116
2.485 69 61.039 7.961 1.038
2.495 75 72.519 2.481 0.085
2.505 95 77.829 17.171 3.788
2.515 83 75.453 7.547 0.755
2.525 65 66.077 –1.077 0.176
2.535 48 52.272 –4.272 0.349
2.545 26 37.354 –11.354 3.451
2.555 24 24.112 –0.112 0.001
2.565 9 14.060 –5.060 1.821
2.575 10 7.406 2.594 0.909
2.585 3 3.524 –0.524 0.078
2.595 4 1.515 2.485 4.076
2.605 3 0.588 2.412 9.894
2.615 1 0.206 0.794 3.060
χ2 (all 21 points) = 43.231
χ2 (19 points — omitting bottom two points) = 30.277

25
4. Penny Statistics Exploring Chemical Analysis

Table 2. Critical values of χ2 that will be exceeded in 5% of experiments*

Degrees Critical Degrees Critical Degrees Critical

of freedom value of freedom value of freedom value

1 3.84 11 19.7 21 32.7

2 5.99 12 21.0 22 33.9
3 7.81 13 22.4 23 35.2
4 9.49 14 23.7 24 36.4
5 11.1 15 25.0 25 37.7
6 12.6 16 26.3 26 38.9
7 14.1 17 27.6 27 40.1
8 15.5 18 28.9 28 41.3
9 16.9 19 30.1 29 42.6
10 18.3 20 31.4 30 43.8

*Example: The value of χ2 from 15 observations is 17.2. This value is less than 23.7 listed for 14 (= 15 – 1)
degrees of freedom. Because χ2 does not exceed the critical value, the observed distribution is consistent with the
theoretical distribution.

At the bottom of Table 1 we see that χ2 for all 21 bars is 43.231. In Table 2 we find a
critical value of 31.4 for 20 degrees of freedom (degrees of freedom = one less than number of
categories). Because χ2 from Equation 3 exceeds the critical value, we conclude that the
distribution is not Gaussian.
It would be reasonable to omit the smallest bars at the edge of the graph from the
calculation of χ2 because these bars contain the fewest observations but make large contributions
to χ2. Suppose that we reject bars lying >3 standard deviations from the mean. This removes the
two bars at the right side of Figure 1 which give the last two entries in Table 1. Omitting these
two points gives χ2 = 30.277, which is still greater than the critical value of 28.9 for 18 degrees
of freedom in Table 2. Our conclusion is that at the 95% confidence level the observed
distribution in Figure 1 is not quite Gaussian. It is possible that exceptionally light coins are
nicked and exceptionally heavy coins are dirty or corroded. You would need to inspect these
coins to verify this hypothesis.

26
4. Penny Statistics Exploring Chemical Analysis

Reporting Your Results

Gathering data:
1. Attach a table of masses, with one column sorted by mass for each year.
2. Divide 1982 into two columns, one for light (zinc) and one for heavy (brass) pennies.
3. One year should be divided into one column from Denver and one from Philadelphia.
Discrepant data:
1. List the mean (x- ) and standard deviation (s) for each column.
2. Discard data that lie outside of x- ± 4s and recompute x- and s.
Confidence intervals and t test:
1. For the year ≥ 1982 with highest average mass:
95% confidence interval (= x- ± ts/ n ) = _______________
For the year ≥ 1982 with lowest average mass:
95% confidence interval = _______________
Comparison of means with t-test:
tcalculated = __________
ttable = ____________
Is the difference significant? _____________
2. For the year ≤ 1982 with highest average mass:
95% confidence interval = _______________
For the year ≤ 1982 with lowest average mass:
95% confidence interval = _______________
Comparison of means with t test:
tcalculated = __________
ttable = ____________
Is the difference significant? _____________
3. For Philadelphia versus Denver coins in one year:
Philadelphia 95% confidence interval = _______________
Denver 95% confidence interval = _______________
Comparison of means with t test:
tcalculated = __________
ttable = ____________
Is the difference significant? _____________

27
4. Penny Statistics Exploring Chemical Analysis

Gaussian distribution of masses:

Prepare a graph analogous to Figure 1 with labels showing the ±3s limits.
Least-squares Analysis:
Prepare a graph analogous to Figure 2 and find the least-squares slope and intercept and their
standard deviations.
m ± sm = _________________
t95% confidence = ________ t99% confidence = ________
95% confidence: m ± tsm = ______________________.
Does interval include zero? ____________
99% confidence: m ± tsm = ______________________.
Does interval include 0? ____________
Is there a systematic increase or decrease of penny mass with year? ____________
χ2 test:
Write Equation 2 for the smooth Gaussian curve that fits your bar graph.
Construct a table analogous to Table 1 to compute χ2 for the complete data set.
Computed value of χ2 = ____________ Degrees of freedom = ____________
Critical value of χ2 = ____________
Do the data follow a Gaussian distribution? ____________
Now omit bars on the graph that are greater than 3 standard deviations away from the mean
and calculate a new value of χ2 with the reduced data set.
Computed value of χ2 = ____________ Degrees of freedom = ____________
Critical value of χ2 = ____________
Does the reduced data set follow a Gaussian distribution? ____________

28
5. Statistical Evaluation of Acid-Base Indicators Exploring Chemical Analysis

5. Statistical Evaluation of Acid-Base Indicators 17

This experiment introduces you to the use of indicators and to the statistical
concepts of mean, standard deviation, Q test, and t test. You will compare the
accuracy of different indicators in locating the end point in the titration of the
base “tris” with hydrochloric acid:

Green Profile
See Section 0
(HOCH2)3CNH2 + H+ → (HOCH2)3CNH+3
Tris(hydroxymethyl)aminomethane
“tris”

Reagents

~0.1 M HCl: Each student needs ~500 mL of unstandardized solution, all from a single batch that
will be analyzed by the whole class.
Tris: Solid, primary standard powder should be available (~4 g/student).
Indicators should be available in dropper bottles:
Bromothymol blue (BB): Dissolve 0.100 g in 16.0 mL 0.010 0 M NaOH and add 225 mL H2O
Methyl red (MR): Dissolve 20 mg in 60 mL of ethanol and add 40 mL H2O
Bromocresol green (BG): Dissolve 0.100 g in 14.3 mL 0.010 0 M NaOH and add 225 mL H2O
Methyl orange (MO): Dissolve 10 mg in 100 mL H2O
Erythrosine (E): Dissolve 100 mg disodium erythrosine in 100 mL H2O

Color changes to use for the titration of tris with HCl are
BB: blue (pH 7.6) → yellow (pH 6.0) (end point is disappearance of green)
MR: yellow (pH 6.0) → red (pH 4.8) (end point is disappearance of orange)
BG: blue (pH 5.4) → yellow (pH 3.8) (end point is green)
MO: yellow (pH 4.4) → red (pH 3.1) (end point is first appearance of orange)
E: red (pH 3.6) → orange (pH 2.2) (end point is first appearance of orange)

Procedure

Each student should perform the following procedure with one indicator. Different students
should be assigned different indicators so that at least four students evaluate each indicator.

17 D. T. Harvey, J. Chem. Ed. 1991, 68, 329.

29
5. Statistical Evaluation of Acid-Base Indicators Exploring Chemical Analysis

1. Calculate the molecular mass of tris and the mass required to react with 35 mL of 0.10 M
HCl. Weigh this much tris into a 125-mL flask.
2. It is good practice to rinse a buret with a new solution to wash away traces of previous
reagents. Wash your 50-mL buret with three 10-mL portions of 0.1 M HCl and discard the
washings. Tilt and rotate the buret so that the liquid washes the walls, and drain the liquid
through the stopcock. Then fill the buret with 0.1 M HCl to near the 0-mL mark, allow a
minute for the liquid to settle, and record the reading to the nearest 0.01 mL.
3. The first titration will be rapid, to allow you to find the approximate end point of the titration.
Add ~20 mL of HCl from the buret to the flask and swirl to dissolve the tris. Add 2–4 drops
of indicator and titrate with ~1-mL aliquots of HCl to find the end point.
4. From the first titration, calculate how much tris is required to cause each succeeding titration
to require 35–40 mL of HCl. Weigh this much tris into a clean flask. Refill your buret to
near 0 mL and record the reading. Repeat the titration in step 3, but use 1 drop at a time near
the end point. When you are very near the end point, use less than a drop at a time. To do
this, carefully suspend a fraction of a drop from the buret tip and touch it to the inside wall of
the flask. Carefully tilt the flask so that the bulk solution overtakes the droplet and then swirl
the flask to mix the solution. Record the total volume of HCl required to reach the end point
to the nearest 0.01 mL. Calculate the true mass of tris with the buoyancy equation 2-1 in the
textbook (density of tris = 1.327 g/mL). Calculate the molarity of HCl.
5. Repeat the titration to obtain at least six accurate measurements of the HCl molarity.
6. Use the Grubbs test in Section 4-5 in the textbook to decide whether any results should be
discarded. Report your retained values, their mean, their standard deviation, and the relative
standard deviation (s/ x ).

Data Analysis

Pool the data from your class to fill in Table 1, which shows two possible results. The quantity
sx is the standard deviation of all results reported by many students. The pooled standard
deviation, sp, is derived from the standard deviations reported by each student. If two students
see the end point differently, each result might be very reproducible, but the reported molarities
will be different. Together, they will generate a large value of sx (because their results are so
different), but a small value of sp (because each one was reproducible).

30
5. Statistical Evaluation of Acid-Base Indicators Exploring Chemical Analysis

Table 1. Pooled data

Number of Number of Mean HCl Relative standard
measurements students molarity (M)a deviation (%)
Indicator (n) (S) (x) sx (M)b 100 sx / x sp (M)c

BB 28 5 0.095 65 0.002 25 2.35 0.001 09

MR
BG 29 4 0.086 41 0.001 13 1.31 0.000 99
MO
E
a. Computed from all values that were not discarded with the Q test.
b. sx = standard deviation of all n measurements (degrees of freedom = n – 1)
c. sp = pooled standard deviation for S students (degrees of freedom = n – S). Computed with the equation
2 2 2
s1 (n1 − 1) + s2 (n2 − 1) + s3 (n3 − 1) + …
sp =
n−S

where there is one term in the numerator for each student using that indicator.

Select the pair of indicators giving average HCl molarities that are farthest apart. Use the
t test (Equation 4-5 in the textbook) to decide whether the average molarities are significantly
different from each other at the 95% confidence level. When you calculate the pooled standard
deviation for Equation 4-5, the values of s1 and s2 in Equation 4-6 in the textbook are the values
of sx (not sp) in Table 1.
A condition for using Equations 4-5 and 4-6 is that the standard deviations for the two
sets of measurements should not be “significantly different” from each other. The F test tells us
whether two standard deviations are “significantly” different from each other. F is the quotient
of the squares of the standard deviations:

s21
Fcalculated = 2 (1)
s2

We always put the larger standard deviation in the numerator so that F ≥ 1. If Fcalculated > Ftable
in Table 2, then the difference is significant.
Use the F test in Equation 1 to decide whether or not the standard deviations for the two
indicators giving the largest difference in mean HCl molarity are significantly different.

31
5. Statistical Evaluation of Acid-Base Indicators Exploring Chemical Analysis

Table 2. Critical values of F = s21 / s22 at 95% confidence level

Degrees of freedom for s1

Degrees
of freedom
for s2 2 3 4 5 6 7 8 9 10 12 15 20 30 ∞

2 19.0 19.2 19.2 19.3 19.3 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.5 19.5

3 9.55 9.28 9.12 9.01 8.94 8.89 8.84 8.81 8.79 8.74 8.70 8.66 8.62 8.53

4 6.94 6.59 6.39 6.26 6.16 6.09 6.04 6.00 5.96 5.91 5.86 5.80 5.75 5.63

5 5.79 5.41 5.19 5.05 4.95 4.88 4.82 4.77 4.74 4.68 4.62 4.56 4.50 4.36

6 5.14 4.76 4.53 4.39 4.28 4.21 4.15 4.10 4.06 4.00 3.94 3.87 3.81 3.67

7 4.74 4.35 4.12 3.97 3.87 3.79 3.73 3.68 3.64 3.58 3.51 3.44 3.38 3.23

8 4.46 4.07 3.84 3.69 3.58 3.50 3.44 3.39 3.35 3.28 3.22 3.15 3.08 2.93

9 4.26 3.86 3.63 3.48 3.37 3.29 3.23 3.18 3.14 3.07 3.01 2.94 2.86 2.71

10 4.10 3.71 3.48 3.33 3.22 3.14 3.07 3.02 2.98 2.91 2.84 2.77 2.70 2.54

11 3.98 3.59 3.36 3.20 3.10 3.01 2.95 2.90 2.85 2.79 2.72 2.65 2.57 2.40

12 3.88 3.49 3.26 3.11 3.00 2.91 2.85 2.80 2.75 2.69 2.62 2.54 2.47 2.30

13 3.81 3.41 3.18 3.02 2.92 2.83 2.77 2.71 2.67 2.60 2.53 2.46 2.38 2.21

14 3.74 3.34 3.11 2.96 2.85 2.76 2.70 2.65 2.60 2.53 2.46 2.39 2.31 2.13

15 3.68 3.29 3.06 2.90 2.79 2.71 2.64 2.59 2.54 2.48 2.40 2.33 2.25 2.07

16 3.63 3.24 3.01 2.85 2.74 2.66 2.59 2.54 2.49 2.42 2.35 2.28 2.19 2.01

17 3.59 3.20 2.96 2.81 2.70 2.61 2.55 2.49 2.45 2.38 2.31 2.23 2.15 1.96

18 3.56 3.16 2.93 2.77 2.66 2.58 2.51 2.46 2.41 2.34 2.27 2.19 2.11 1.92

19 3.52 3.13 2.90 2.74 2.63 2.54 2.48 2.42 2.38 2.31 2.23 2.16 2.07 1.88

20 3.49 3.10 2.87 2.71 2.60 2.51 2.45 2.39 2.35 2.28 2.20 2.12 2.04 1.84

30 3.32 2.92 2.69 2.53 2.42 2.33 2.27 2.21 2.16 2.09 2.01 1.93 1.84 1.62

∞ 3.00 2.60 2.37 2.21 2.10 2.01 1.94 1.88 1.83 1.75 1.67 1.57 1.46 1.00

32
5. Statistical Evaluation of Acid-Base Indicators Exploring Chemical Analysis

If they are significantly different, use Equations 2 and 3 below in place of Equations 4-5 and 4-6
of the textbook for the t test.
.

|x-1 – x- 2| E

tcalculated = EA

2 2
E A (2)
s1/n1 + s1/n2
A A A A

 (s1/n1 + s2/n2)2 
2 2
Degrees of freedom =   A A EA A

– 2 (3)
(s21/n1)2 (s22/n2)2
EA E A

 n1 + 1 + n2 + 1 
A A A A

Equation 3 usually gives a non-integer result, which you can round to the nearest integer
Select the pair of indicators giving the second most different molarities in Table 1 and use
the t test again to see whether or not this second pair of results is significantly different.

Reporting Your Results

Your Individual Data

Mass of True mass HCl Calculated

tris from corrected for volume HCl
Trial balance (g) buoyancy (g) (mL) molarity (M)
1
2
3
4
5
6

On the basis of the Q test, should any molarity be discarded? If so, which one?
Mean value of retained results:
Standard deviation:
Relative standard deviation (%):
Pooled Class Data
1. Attach your copy of Table 1 with all entries filled in.
2. Compare the two most different molarities in Table 1. (Show your F and t tests and state
your conclusion.)
3. Compare the two second most different molarities in Table 1.

33
6. Preparing Standard Acid and Base Exploring Chemical Analysis

6. Preparing Standard Acid and Base

Hydrochloric acid and sodium hydroxide are the most common

strong acids and bases used in the laboratory. Both reagents need to
be standardized to learn their exact concentrations. Section 10-5 in
the textbook provides background information for the procedures
described below. Unknown samples of sodium carbonate or
Green Profile potassium hydrogen phthalate (available from Thorn Smith1) can
See Section 0
be analyzed by the procedures described in this section.

Reagents

50 wt% NaOH: (3 mL/student) Dissolve 50 g of reagent-grade NaOH in 50 mL of

distilled water and allow the suspension to settle overnight. Na2CO3 is insoluble in
the solution and precipitates. Store the solution in a tightly sealed polyethylene
bottle and handle it gently to avoid stirring the precipitate when liquid is withdrawn.
Phenolphthalein indicator: Dissolve 50 mg in 50 mL of ethanol and add 50 mL of
distilled water.
Bromocresol green indicator: Dissolve 100 mg in 14.3 mL of 0.01 M NaOH and add
225 mL distilled water.
Concentrated (37 wt%) HCl: 10 mL/student.
Primary standards: Potassium hydrogen phthalate (~2.5 g/student) and sodium
carbonate (~1.0 g/student).
0.05 M NaCl: 50 mL/student.

Standardizing NaOH

1. Dry primary standard grade potassium hydrogen phthalate for 1 h at 105˚C and store it
in a capped bottle in a desiccator.

34
6. Preparing Standard Acid and Base Exploring Chemical Analysis

2. Boil 1 L of distilled water for 5 min to expel CO2. Pour the water into a polyethylene
bottle, which should be tightly capped whenever possible. Calculate the volume of 50
wt% NaOH needed to prepare 1 L of 0.1 M NaOH. (The density of 50 wt% NaOH is
1.50 g per milliliter of solution.) Use a graduated cylinder to transfer this much
NaOH to the bottle of water. (CAUTION: 50 wt% NaOH eats people. Flood any
spills on your skin with water.) Mix well and cool the solution to room temperature
(preferably overnight).
3. Weigh four samples of solid potassium hydrogen phthalate and dissolve each in ~25
mL of distilled water in a 125-mL flask. Each sample should contain enough solid to
react with ~25 mL of 0.1 M NaOH. Add 3 drops of phenolphthalein to each flask and
titrate one rapidly to find the end point. The buret should have a loosely fitted cap to
minimize entry of CO2 from the air.
4. Calculate the volume of NaOH required for each of the other three samples and titrate
them carefully. During each titration, periodically tilt and rotate the flask to wash all
liquid from the walls into the bulk solution. Near the end, deliver less than 1 drop of
titrant at a time. To do so, carefully suspend a fraction of a drop from the buret tip,
touch it to the inside wall of the flask, wash it into the bulk solution by careful tilting,
and swirl the solution. The end point is the first appearance of faint pink color that
persists for 15 s. (The color will slowly fade as CO2 from the air dissolves in the
solution.)
5. Calculate the average molarity (–
x ), the standard deviation (s), and the percent relative
E

A A

standard deviation (= 100 × s/–

x ). If you were careful, the relative standard deviation
E

A A

should be < 0.2%.

Standardizing HCl

1. Use the table inside the cover of the textbook to calculate the volume of ~37 wt% HCl
that should be added to 1 L of distilled water to produce 0.1 M HCl and prepare this
solution.
2. Dry primary standard grade sodium carbonate for 1 h at 105˚C and cool it in a
desiccator.

35
6. Preparing Standard Acid and Base Exploring Chemical Analysis

3. Weigh four samples, each containing enough Na2CO3 to react with ~25 mL of 0.1 M
HCl and place each in a 125-mL flask. When you are ready to titrate each one,
dissolve it in ~25 mL of distilled water. Add 3 drops of bromocresol green indicator
and titrate one rapidly to a green color to find the approximate end point.

2HCl + Na2CO3 → CO2 + 2NaCl + H2O

FM 105.99

4. Carefully titrate each sample until it turns from blue into green. Then boil the
solution to expel CO2. The color should return to blue. Carefully add HCl from the
buret until the solution turns green again and report the volume of acid at this point.
5. Perform one blank titration of 50 mL of 0.05 M NaCl containing 3 drops of indicator.
Subtract the volume of HCl needed for the blank from that required to titrate Na2CO3.
6. Calculate the mean HCl molarity, standard deviation, and percent relative standard
deviation.

36
7. Using a pH Electrode for an Acid-Base Titration Exploring Chemical Analysis

7. Using a pH Electrode for an Acid-Base Titration

In this experiment you will use a pH electrode to follow the course of

an acid-base titration. You will observe how pH changes slowly
during most of the reaction and rapidly near the equivalence point.
You will compute the first and second derivatives of the titration
curve to locate the end point. From the mass of unknown acid or
Green Profile
See Section 0 base and the moles of titrant, you can calculate the molecular mass of
the unknown. Section 10-4 of the textbook provides background for
this experiment. Experiment 10 teaches you how to use Excel SOLVER to fit a theoretical
titration curve to the data obtained in Experiment 7.

Reagents

Standard 0.1 M NaOH and standard 0.1 M HCl: From Experiment 6.

Bromocresol green indicator: Dissolve 0.100 g of the indicator in 14.3 mL 0.010 0 M
NaOH and add 225 mL H2O.
Phenolphthalein indicator: Dissolve 50 mg of the indicator in 50 mL of ethanol and add
50 mL H2O.
pH calibration buffers: pH 7 and pH 4. Use commercial standards.
Unknowns: Unknowns should be stored in a desiccator by your instructor.
Suggested acid unknowns: potassium hydrogen phthalate (Table 10-3, FM 204.22),
2-(N-morpholino)ethanesulfonic acid (MES, Table 9-2, FM 195.24), imidazole
hydrochloride (Table 9-2, FM 104.54, hygroscopic), potassium hydrogen iodate
(Table 10-3, FM 389.91).
Suggested base unknowns: tris (Table 10-3, FM 121.14), imidazole (FM 68.08),
disodium hydrogen phosphate (Na2HPO4, FM 141.96), sodium glycinate (may be
found in chemical catalogs as glycine, sodium salt hydrate, H2NCH2CO2Na.xH2O,
FM 97.05 + x(18.015)). For sodium glycinate, one objective of the titration is to find
the number of waters of hydration from the molecular mass.

Procedure

1. Your instructor will recommend a mass of unknown (5–8 mmol) for you to weigh

37
7. Using a pH Electrode for an Acid-Base Titration Exploring Chemical Analysis

accurately and dissolve in distilled water in a 250-mL volumetric flask. Dilute to the
mark and mix well.
2. Following instructions for your particular pH meter, calibrate a meter and glass
electrode, using buffers with pH values near 7 and 4. Rinse the electrode well with
distilled water and blot it dry with a tissue before immersing in any new solution.
3. The first titration is intended to be rough, so that you will know the approximate end
point in the next titration. For the rough titration, pipet 25.0 mL of unknown into a
125-mL flask. If you are titrating an unknown acid, add 3 drops of phenolphthalein
indicator and titrate with standard 0.1 M NaOH to the pink end point, using a 50-mL
buret. If you are titrating an unknown base, add 3 drops of bromocresol green
indicator and titrate with standard 0.1 M HCl to the green end point. Add 0.5 mL of
titrant at a time so that you can estimate the equivalence volume to within 0.5 mL.
Near the end point, the indicator temporarily changes color as titrant is added. If you
recognize this, you can slow down the rate of addition and estimate the end point to
within a few drops.
4. Now comes the careful titration. Pipet 100.0 mL of unknown solution into a 250-mL
beaker containing a magnetic stirring bar. Position the electrode in the liquid so that
the stirring bar will not strike the electrode. If a combination electrode is used, the
small hole near the bottom on the side must be immersed in the solution. This hole is
the salt bridge to the reference electrode. Allow the electrode to equilibrate for 1 min
with stirring and record the pH.
5. Add 1 drop of indicator and begin the titration. The equivalence volume will be four
times greater than it was in step 3. Add ~1.5-mL aliquots of titrant and record the
exact volume, the pH, and the color 30 s after each addition. When you are within 2
mL of the equivalence point, add titrant in 2-drop increments. When you are within 1
mL, add titrant in 1-drop increments. Continue with 1-drop increments until you are
0.5 mL past the equivalence point. The equivalence point has the most rapid change
in pH. Add five more 1.5-mL aliquots of titrant and record the pH after each.

38
7. Using a pH Electrode for an Acid-Base Titration Exploring Chemical Analysis

Figure 1. Locating the maximum position of the first derivative of a titration curve.

Data Analysis

1. Construct a graph of pH versus titrant volume. Mark on your graph where the
indicator color change(s) was observed.
2. Following the example in Figures 10-4 and 10-5 of the textbook, compute the first
derivative (the slope, ∆pH/∆V) for each data point within ±1 mL of the equivalence
volume. From your graph, estimate the equivalence volume as accurately as you can,
as shown in Figure 1.
3. Following the example in Figure 10-5, compute the second derivative (the slope of
the slope, ∆(slope)/∆V). Prepare a graph like Figure 10-6 in the textbook and locate
the equivalence volume as accurately as you can.
4. Go back to your graph from step 1 and mark where the indicator color changes were
observed. Compare the indicator end point to the end point estimated from the first
and second derivatives.
5. From the equivalence volume and the mass of unknown, calculate the molecular mass
of the unknown.

39
8. Analysis of a Mixture of Carbonate and Bicarbonate Exploring Chemical Analysis

8. Analysis of a Mixture of Carbonate and Bicarbonate

This procedure involves two titrations. First, total alkalinity

(= [HCO3-)] + 2[CO2-
A
E

3 ]) is measured by titrating the mixture with

A
E

standard HCl to a bromocresol green end point:

HCO3- + H+ → H2CO3
A
E

Green Profile
3 + 2H → H2CO3
CO2- + E

See Section 0 A A

A separate aliquot of unknown is treated with excess standard NaOH to convert HCO3- to A
E

CO2-
3: A
E

HCO3- + OH- → CO2-

A

3 + H2O
E

A A
E

Then all the carbonate is precipitated with BaCl2:

3 → BaCO3(s)
Ba2+ + CO2- E

A A

The excess NaOH is immediately titrated with standard HCl to determine how much
HCO3- was present. From the total alkalinity and bicarbonate concentration, you can
A
E

calculate the original carbonate concentration.

Reagents

Standard 0.1 M NaOH and standard 0.1 M HCl: From Experiment 6.

CO2-free water: Boil 500 mL of distilled water to expel CO2 and pour the water into a 500-mL
plastic bottle. Screw the cap on tightly and allow the water to cool to room temperature.
Keep tightly capped when not in use.
10 wt% aqueous BaCl2: 35 mL/student.
Bromocresol green and phenolphthalein indicators: See Experiment 7 for recipes.
Unknowns: Solid unknowns (2.5 g/student) can be prepared from reagent-grade sodium
carbonate or potassium carbonate and bicarbonate. Unknowns should be stored in a
desiccator and should not be heated. Heating at 50˚–100˚C converts NaHCO3 to Na2CO3.

40
8. Analysis of a Mixture of Carbonate and Bicarbonate Exploring Chemical Analysis

Procedure

1. Accurately weigh 2.0–2.5 g of unknown into a 250-mL volumetric flask by weighing the
sample in a capped weighing bottle, delivering some to a funnel in the volumetric flask, and
reweighing the bottle. Continue this process until the desired mass of reagent has been
transferred to the funnel. Rinse the funnel repeatedly with small portions of CO2-free water
to dissolve the sample. Remove the funnel, dilute to the mark, and mix well.
2. Total alkalinity: Pipet a 25.00-mL aliquot of unknown solution into a 250-mL flask and
titrate with standard 0.1 M HCl, using bromocresol green indicator as in Experiment 6 for
standardizing HCl. Repeat this procedure with two more 25.00-mL aliquots.
3. Bicarbonate content: Pipet 25.00 mL of unknown and 50.00 mL of standard 0.1 M NaOH
into a 250-mL flask. Swirl and add 10 mL of 10 wt% BaCl2, using a graduated cylinder.
Swirl again to precipitate BaCO3, add 2 drops of phenolphthalein indicator, and immediately
titrate with standard 0.1 M HCl. Repeat this procedure with two more 25.00-mL samples of
unknown.
4. From the results of step 2, calculate the total alkalinity and its standard deviation. From the
results of step 3, calculate the bicarbonate concentration and its standard deviation. Using the
standard deviations as estimates of uncertainty, calculate the concentration (and uncertainty)
of carbonate in the sample. Express the composition of the solid unknown in a form such as
63.4 (±0.5) wt% K2CO3 and 36.6 (±0.2) wt% NaHCO3.

41
9. Analysis of an Acid-Base Titration Curve: The Gran Plot Exploring Chemical Analysis

9. Analysis of an Acid-Base Titration Curve: The Gran Plot

In this experiment, you will titrate a sample of pure potassium hydrogen

phthalate (Table 10-3) with standard NaOH. A Gran plot will be used to
find the equivalence point and Ka. Activity coefficients are used in the
calculations of this experiment.

Green Profile
See Section 0
Using a Gran Plot to Find the End Point of a Titration

A problem with using derivatives to find the end point of a titration is that titration data are least
accurate right near the end point because buffering is minimal and electrode response is sluggish.
A Gran plot uses data from before the end point (typically from 0.8 Ve or 0.9 Ve up to Ve) to
locate the end point.
Consider the titration of a weak acid, HA:

[H+]γH+[A-]γA-
HA H+ + A- Ka = A

[HA] γHA E A (1)

It will be necessary to include activity coefficients in this discussion because a pH electrode

responds to hydrogen ion activity, not concentration.
At any point between the initial point and the end point of the titration, it is usually a
good approximation to say that each mole of NaOH converts 1 mol of HA into 1 mol of A-. If
we have titrated Va mL of HA (whose formal concentration is Fa) with Vb mL of NaOH (whose
formal concentration is Fb), we can write

moles of OH- delivered VbFb

[A-] = A

total volume = Vb + Va
E A A E

original moles of HA – moles of OH- VaFa – VbFb

[HA] = A

total volume = Va + Vb E A A E

Substitution of these values of [A-] and [HA] into the equilibrium constant gives

[H+]γH+VbFbγA-
Ka = (V F – V F ) γ
A E

a a b b HA

42
9. Analysis of an Acid-Base Titration Curve: The Gran Plot Exploring Chemical Analysis

which can be rearranged to

γHA VaFa – VbFb

Vb[H+]γH+ = γ - Ka   (2)
 Fb 
A E A E

A A

A

10-pH
A E

The term on the left is Vb . 10-pH, because [H+]γH+ = 10-pH. The term in parentheses on the right
is
VaFa – VbFb VaFa
A

Fb = Fb – Vb = Ve – Vb
E A A E A

Equation 2 can, therefore, be written in the form

γHA
Gran plot equation: Vb . 10-pH = Ka (Ve – Vb) (3)
γA-
A E A

A graph of Vb . 10-pH versus Vb is called a Gran plot. If γHA/γA- is constant, the graph is
a straight line with a slope of –KaγHA/γA- and an x-intercept of Ve. Figure 1 shows a Gran plot
for the titration in Figure 10-4 of the textbook. Any units can be used for Vb, but the same units
should be used on both axes. In Figure 1, Vb is expressed in microliters on both axes.

Figure 1. Gran plot for the first

equivalence point of Figure 10-4 of
the textbook. The last 10–20% of
volume prior to Ve is normally used
for a Gran plot.

43
9. Analysis of an Acid-Base Titration Curve: The Gran Plot Exploring Chemical Analysis

The beauty of a Gran plot is that it enables us to use data taken before the end point to
find the end point. The slope of the Gran plot enables us to find Ka. Although we derived the
Gran function for a monoprotic acid, the same plot (Vb . 10-pH versus Vb) applies to polyprotic
acids (such as H6A in Figure 10-4 of the textbook).
The Gran function, Vb . 10-pH, does not actually go to 0, because 10-pH is never 0. The
curve must be extrapolated to find Ve. The reason the function does not reach 0 is that we have
used the approximation that every mole of OH- generates 1 mol of A-, which is not true as Vb
approaches Ve. Only the linear portion of the Gran plot is used.
Another source of curvature in the Gran plot is changing ionic strength, which causes
γHA/γA- to vary. In Figure 10-4 of the textbook, this variation was avoided by maintaining
nearly constant ionic strength with NaNO3. Even without added salt, the last 10–20% of data
before Ve gives a fairly straight line because the quotient γHA/γA- does not change very much.
The Gran plot in the acidic region gives accurate results even if there is CO2 dissolved in the
strong base titrant. The Gran plot in the basic region can be used to measure CO2 in the strong
base.
For completeness, we note that if weak base, B, is titrated with a strong acid, the Gran
function is

1 γB 
Va . 10+pH = K .  (Ve – Va) (4)
 a γBH+ 
A E A

where Va is the volume of strong acid and Ka is the acid dissociation constant of BH+. A graph
of Va . 10+pH versus Va should be a straight line with a slope of –γB / (γBH+Ka) and an
x-intercept of Ve.

Procedure (An Easy Matter)

1. Dry about 1.5 g of potassium hydrogen phthalate at 105˚C for 1 h and cool it in a desiccator
for 20 min. Accurately weigh out ~1.5 g and dissolve it in water in a 250-mL volumetric
flask. Dilute to the mark and mix well.
2. Following the instructions for your particular pH meter, calibrate a meter and glass electrode
using buffers with pH values near 7 and 4. Rinse the electrode well with distilled water and
blot it dry with a tissue before immersing in a solution.

44
9. Analysis of an Acid-Base Titration Curve: The Gran Plot Exploring Chemical Analysis

3. Pipet 100.0 mL of phthalate solution into a 250-mL beaker containing a magnetic stirring bar.
Position the electrode in the liquid so that the stirring bar will not strike the electrode. The
small hole near the bottom on the side of the combination pH electrode must be immersed in
the solution. This hole is the reference electrode salt bridge. Allow the electrode to
equilibrate for 1 min (with stirring) and record the pH.
4. Add 1 drop of phenolphthalein indicator (recipe in Experiment 7) and titrate the solution with
standard ~0.1 M NaOH. Until you are within 4 mL of the theoretical equivalence point, add
base in ~1.5-mL aliquots, recording the volume and pH 30 s after each addition. Thereafter,
use 0.4-mL aliquots until you are within 1 mL of the equivalence point. After that, add base
1 drop at a time until you have passed the pink end point by a few tenths of a milliliter.
(Record the volume at which the pink color is observed.) Then add five more 1-mL aliquots.
5. Construct a graph of pH versus Vb (volume of added base). Locate the equivalence volume
(Ve) as the point of maximum slope or zero second derivative, as described in Section 10-4.
Compare this with the theoretical and phenolphthalein end points.

Calculations (The Work Begins!)

1. Construct a Gran plot (a graph of Vb10-pH versus Vb) by using the data collected between
0.9Ve and Ve. Draw a line through the linear portion of this curve and extrapolate it to the
abscissa to find Ve. Use this value of Ve in the calculations below. Compare this value with
those found with phenolphthalein and estimated from the graph of pH versus Vb.
2. Compute the slope of the Gran plot and use Equation 3 to find Ka for potassium hydrogen
phthalate as follows: The slope of the Gran plot is –KaγHA/γA- = –KaγHP-/γP2-. In this
equation, HP- is monohydrogen phthalate (the weak acid) and P2- is phthalate anion (the
weak base). Because the ionic strength changes slightly as the titration proceeds, so also do
the activity coefficients. Calculate the ionic strength at 0.95Ve, and use this “average” ionic
strength to find the activity coefficients.

EXAMPLE Calculating Activity Coefficients

Find γHP- and γP2- at 0.95 Ve in the titration of 100.0 mL of 0.020 0 M potassium hydrogen
phthalate with 0.100 M NaOH.

45
9. Analysis of an Acid-Base Titration Curve: The Gran Plot Exploring Chemical Analysis

Solution The equivalence point is 20.0 mL, so 0.95Ve, = 19.0 mL. The concentrations of H+
and OH- are negligible compared with those of K+, Na+, HP-, and P2-, whose concentrations are

100 mL
[K+] = 119 mL (0.020 0 M) = 0.016 8 M
 
A E A

Dilution Initial
factor concentration

 19 mL 
[Na+] = 119 mL (0.100 M) = 0.016 0 M
 
A E A

100 mL
[HP-] = (0.050) 119 mL (0.020 0 M) = 0.000 84 M
 
A E A

Fraction Dilution Initial

remaining factor concentration

100 mL
[P2-] = (0.95) 119 mL (0.020 0 M) = 0.0160 M
 
A E A

The ionic strength is

µ =
1
2
Σcizi2
1
= [ (0.016 8) . 12 + (0.016 0) . 12 + (0.000 84) . 12 + (0.016 0) . 22 ] = 0.048 8 M
2

To estimate γP2- and γHP- at µ = 0.048 8 M, interpolate in Table 12-1. In this table, we find that
the hydrated radius of P2- [phthalate, C6H4(CO-2 )2] is 600 pm. The size of HP- is not listed, but
we will suppose that it is also 600 pm. An ion with charge ±2 and a size of 600 pm has γ = 0.485
at µ = 0.05 M and γ = 0.675 at µ = 0.01 M. Interpolating between these values, we estimate γP2-
= 0.49 when µ = 0.048 8 M. Similarly, we estimate γHP- = 0.84 at this same ionic strength.

3. From the measured slope of the Gran plot and the values of γHP- and γP2-, calculate pKa.
Then choose an experimental point near ⅓Ve and one near ⅔Ve. Use Equation 1 to find pKa
with each point. (You will have to calculate [P2-], [HP-], γP2-, and γHP- at each point.)
Compare the average value of pKa from your experiment with pK2 for phthalic acid listed in
the Appendix of the textbook.

46
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

10. Fitting a Titration Curve with Excel Solver® 18 17F

Excel Solver provides tremendous power for curve fitting and solving
equations. Practicing scientists and engineers make frequent use of this
capability, which you will never regret making the effort to learn. Study
Section 10-7 of the textbook before working on this experiment.
We will fit the theoretical titration curve to the potentiometric data
Green Profile
See Section 0 (pH versus volume of titrant) that you obtained in Experiments 7 or 9. The
results of your titration can be plotted in a graph such as Figure 1.

12
Figure 1. pH measured during the titration
10 of acetic acid (HA) with standard NaOH.
8 The objective of Experiment 10 is to fit a
pH

theoretical titration curve to the measured

6
points and extract the concentration of HA
4 and its acid dissociation constant, Ka, from
2
the data.
0 1 2 3 4 5 6 7 8 9 10
Vb (mL)

For the titration of a weak acid with standard NaOH

HA + NaOH → Na+ + A- + H2O

the theoretical titration curve is described by Equation 10-14 in the textbook:

[H+] – [OH-]
αA- –Ca
Fraction of titration for CbVb
φ = CV = (10-14)
weak acid by strong base: [H ] – [OH-]
+
A E A

a a
1+ Cb

where φ is the fraction of the way to the end point, Cb is the concentration of standard base, Vb is
the volume of base added at a given point in the titration, Ca is the (unknown) initial
concentration of weak acid, Va is the initial volume of acid being titrated, αA- is the fraction of
weak acid in the form A-, [H+] is the concentration of H+ at a point in the titration, and [OH-] is

18 J. Burnett and W. A. Burns, “Using a Spreadsheet to Fit Experimental pH Titration Data to a Theoretical
Expression: Estimation of Analyte Concentration and Ka,” J. Chem. Ed. 2006, 83, 1190.

47
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

the concentration of OH- at that same point. Equation 10-14 neglects activity coefficients. The
expression for αA- is

Fraction of weak acid Ka

αA- = (10-12)
in the form A-: [H+] + Ka
A E A A E A

where Ka is the acid dissociation constant for HA. By finding the curve that best fits the titration
data, we will find the best values of Ca and Ka. You could analyze the titration of a weak base
with standard HCl by using Equations 10-15 and 10-16 in the textbook.

Procedure

1. Create a spreadsheet like Figure 2. Type the constants (Cb, Va, etc.) in cells A3:A8. Enter
the known concentration of standard base in cell B3 and the initial volume of HA in cell B4.
Enter 1e-14 for Kw in cell B8. Leave cells B5:B7 blank for the moment
2. Enter experimental volumes of base (Vb,obs) and pH in columns A and B (beginning in cells
A12 and B12 in Figure 2). Prepare a graph of the experimental points, as in Figure 1.
3. Estimate the equivalence volume, Ve. In Figure 1, the steep part of the titration curve is near
Vb = 8 mL. In the spreadsheet, we find the steepest rise in pH between 8.01 and 8.31 mL. So
we can estimate Ve ≈ 8.1 mL for this example. (The equivalence point in your titration will
be at some other volume of base.) From the initial volume of acid (Va = 200 mL) and the
concentration of standard base (Cb = 0.4905 M), we can estimate the concentration of acid:

VeCb (8.1 mL)(0.4905 M)

VaCa = VeCb ⇒ Ca = A

Va ≈ A

(200 mL)
A = 0.196 M
E E
A

In Figure 2, we enter the estimated value Ca = 0.196 M in cell B5. You will enter the
estimated value of Ca for your titration.
4. For any titration, pH = pKa when Vb = ½Ve. If Ve ≈ 8.1 mL, then ½Ve ≈ 4.05 mL. Inspecting
the data in the spreadsheet, we see that pH ≈ 4.85 near Vb ≈ 4.05 mL. In Figure 2, the
estimated value of 4.85 for pKa is entered in cell B6. You will enter the estimated pKa for
your titration. The spreadsheet calculates Ka = 10-pKa in cell B7. Eventually, we will use
Excel SOLVER to find the best values for Ca and pKa to fit the titration data.

48
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

5. Compute [H+] = 10-pHobs in column C and [OH-] = Kw/[H+] in column D. Compute αA-
with Equation 10-12 in column E. Compute φ in column F with the ugly expression at the
right side of Equation 10-14. Spreadsheet formulas are given in the upper part of Figure 2.
6. From φ in column F, calculate the volume of base, Vb,calc, in column G.

CbVb,calc φCaVa
φ = A

CaVa A ⇒ Vb,calc = A

Cb A

E E

7. The calculated titration curve is a graph of observed pH versus Vb,calc. We have not yet
optimized the values of Ca and pKa, but we have pretty good estimates that should give a
reasonable fit to the experimental data. To superimpose the calculated titration curve on the
data in Figure 1, click on the graph in your spreadsheet. In Excel 2007, in the Chart Tools
ribbon go to Design and then Select Data. Click Add. Name the new data “Calc”. The x
values are Vb,calc in column G. The y values are pH in column B. Click OK and the
calculated points appear on the graph. To change the calculated points to a smooth curve,
click on one of the calculated points so they are all highlighted. Select Chart Tools, Format,
Format Selection. For Marker Options, click None to remove the markers. For Line Color,
select Solid Line and give it a color. For Line Style, select a Width of 0.75 points. Click
Close and you should see the smooth calculated curve.
For earlier versions of Excel, go to the Chart menu, select Source Data. Select the Series
tab and click Add. Name the new data “Calc”. The x values are Vb,calc in column G. The y
values are pH in column B. Click OK and the calculated points appear on the graph. To
change the calculated points to a smooth curve, select the chart symbol of Calc in the legend
of the graph. Double click the chart symbol and the Format Legend Key window appears.
For Line, select Automatic. For Marker, select None. Click OK and you should see the
smooth calculated curve.
8. Before finding the best values of Ca and pKa to fit the data, we are going to give each
experimental point a weight in column H. The greater the weight, the more importance we
attach to an experimental point in the least-squares fitting procedure. We could have
weighted all points equally with a weight of 1. An empirical weight that attaches more

49
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

A B C D E F G H I
1 Titration of Acetic Acid with NaOH Σ(wt*residual2) =
2 [H+]: C11 = 10^-B11 0.265
-
3 Cb = 0.4905 M [OH ]: D11 = $B$8/C11 = sum(I11:I42)
-
4 Va = 200 mL Alpha(A ): E11 = $B$7/(C11+$B$7)
5 Ca = 0.01990 M Phi: F11 = (E11-(C11-D11)/$B$5)/(1+(C11-D11)/$B$3)
6 pKa = 4.850 Vb,calc: G11 = F11*$B$5*$B$4/$B$3
7 Ka = 1.41E-05 = 10^-B6 Weight: H11 = (B12-B11)/(A12-A11)
8 Kw = 1.00E-14 wt*(Vb,obs - Vb,calc)2: I11 = H11*(A11-G11)^2
9
10 Vb,obs wt*residual2 =
- -
11 (mL) pHobs [H+] [OH ] Alpha(A ) Phi Vb,calc Weight wt*(Vb,obs - Vb,calc)
2

12 0.21 3.48 3.3E-04 3.0E-11 0.041 0.024 0.20 0.80 1.39E-04

13 0.51 3.72 1.9E-04 5.2E-11 0.069 0.059 0.48 0.50 3.89E-04
14 0.81 3.87 1.3E-04 7.4E-11 0.095 0.088 0.71 0.47 4.31E-03
15 1.11 4.01 9.8E-05 1.0E-10 0.126 0.121 0.98 0.47 7.33E-03
16 1.41 4.15 7.1E-05 1.4E-10 0.166 0.163 1.32 0.33 2.66E-03
17 1.71 4.25 5.6E-05 1.8E-10 0.201 0.198 1.61 0.33 3.61E-03
18 2.01 4.35 4.5E-05 2.2E-10 0.240 0.238 1.93 0.23 1.45E-03
19 2.31 4.42 3.8E-05 2.6E-10 0.271 0.269 2.18 0.27 4.34E-03
20 2.61 4.50 3.2E-05 3.2E-10 0.309 0.307 2.49 0.27 3.69E-03
21 2.91 4.58 2.6E-05 3.8E-10 0.349 0.348 2.82 0.30 2.21E-03
22 3.21 4.67 2.1E-05 4.7E-10 0.398 0.397 3.22 0.17 1.44E-05
23 3.51 4.72 1.9E-05 5.2E-10 0.426 0.425 3.45 0.20 8.07E-04
24 3.81 4.78 1.7E-05 6.0E-10 0.460 0.459 3.72 0.23 1.73E-03
25 4.11 4.85 1.4E-05 7.1E-10 0.500 0.499 4.05 0.23 8.06E-04
26 4.41 4.92 1.2E-05 8.3E-10 0.540 0.540 4.38 0.20 2.01E-04
27 4.71 4.98 1.0E-05 9.5E-10 0.574 0.574 4.66 0.23 6.95E-04
28 5.01 5.05 8.9E-06 1.1E-09 0.613 0.613 4.97 0.23 3.48E-04
29 5.31 5.12 7.6E-06 1.3E-09 0.651 0.650 5.28 0.30 3.48E-04
30 5.61 5.21 6.2E-06 1.6E-09 0.696 0.696 5.65 0.27 3.44E-04
31 5.91 5.29 5.1E-06 1.9E-09 0.734 0.733 5.95 0.30 4.96E-04
32 6.21 5.38 4.2E-06 2.4E-09 0.772 0.772 6.26 0.37 1.05E-03
33 6.51 5.49 3.2E-06 3.1E-09 0.814 0.813 6.60 0.40 3.27E-03
34 6.81 5.61 2.5E-06 4.1E-09 0.852 0.852 6.91 0.50 5.18E-03
35 7.11 5.76 1.7E-06 5.8E-09 0.890 0.890 7.22 0.70 9.18E-03
36 7.41 5.97 1.1E-06 9.3E-09 0.929 0.929 7.54 1.03 1.79E-02
37 7.71 6.28 5.2E-07 1.9E-08 0.964 0.964 7.82 3.17 4.06E-02
38 8.01 7.23 5.9E-08 1.7E-07 0.996 0.996 8.08 9.70 4.83E-02
39 8.31 10.14 7.2E-11 1.4E-04 1.000 1.007 8.17 2.37 4.46E-02
40 8.61 10.85 1.4E-11 7.1E-04 1.000 1.037 8.41 1.17 4.44E-02
41 8.91 11.20 6.3E-12 1.6E-03 1.000 1.083 8.79 0.63 9.30E-03
42 9.21 11.39 4.1E-12 2.5E-03 1.000 1.129 9.16 0.50 1.20E-03
43 9.51 11.54 2.9E-12 3.5E-03 1.000 1.183 9.60 0.50 3.68E-03

Figure 2. Initial spreadsheet for fitting Equation 10-14 to experimental points for titration of HA
with standard NaOH.

50
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

A B C D E F G H I
1 Titration of Acetic Acid with NaOH Σ(wt*residual2) =
2 [H+]: C11 = 10^-B11 0.240
-
3 Cb = 0.4905 M [OH ]: D11 = $B$8/C11 = sum(I11:I42)
-
4 Va = 200 mL Alpha(A ): E11 = $B$7/(C11+$B$7)
5 Ca = 0.01981 M Phi: F11 = (E11-(C11-D11)/$B$5)/(1+(C11-D11)/$B$3)
6 pKa = 4.840 Vb,calc: G11 = F11*$B$5*$B$4/$B$3
7 Ka = 1.44E-05 = 10^-B6 Weight: H11 = (B12-B11)/(A12-A11)
8 Kw = 1.00E-14 wt*(Vb,obs - Vb,calc)2: I11 = H11*(A11-G11)^2
9
10 Vb,obs wt*residual2 =
- -
11 (mL) pHobs [H+] [OH ] Alpha(A ) Phi Vb,calc Weight wt*(Vb,obs - Vb,calc)
2

12 0.21 3.48 3.3E-04 3.0E-11 0.042 0.025 0.20 0.80 4.57E-05

13 0.51 3.72 1.9E-04 5.2E-11 0.070 0.061 0.49 0.50 1.76E-04
14 0.81 3.87 1.3E-04 7.4E-11 0.097 0.090 0.73 0.47 3.29E-03
15 1.11 4.01 9.8E-05 1.0E-10 0.129 0.124 1.00 0.47 5.64E-03
16 1.41 4.15 7.1E-05 1.4E-10 0.169 0.166 1.34 0.33 1.65E-03
17 1.71 4.25 5.6E-05 1.8E-10 0.204 0.201 1.63 0.33 2.27E-03
18 2.01 4.35 4.5E-05 2.2E-10 0.244 0.242 1.96 0.23 6.99E-04
19 2.31 4.42 3.8E-05 2.6E-10 0.275 0.273 2.21 0.27 2.77E-03
20 2.61 4.50 3.2E-05 3.2E-10 0.314 0.312 2.52 0.27 2.18E-03
21 2.91 4.58 2.6E-05 3.8E-10 0.354 0.353 2.85 0.30 9.96E-04
22 3.21 4.67 2.1E-05 4.7E-10 0.403 0.402 3.25 0.17 2.39E-04
23 3.51 4.72 1.9E-05 5.2E-10 0.431 0.430 3.47 0.20 2.47E-04
24 3.81 4.78 1.7E-05 6.0E-10 0.465 0.464 3.75 0.23 7.91E-04
25 4.11 4.85 1.4E-05 7.1E-10 0.506 0.505 4.08 0.23 2.41E-04
26 4.41 4.92 1.2E-05 8.3E-10 0.546 0.545 4.40 0.20 9.31E-06
27 4.71 4.98 1.0E-05 9.5E-10 0.580 0.579 4.68 0.23 2.34E-04
28 5.01 5.05 8.9E-06 1.1E-09 0.618 0.618 4.99 0.23 8.00E-05
29 5.31 5.12 7.6E-06 1.3E-09 0.656 0.655 5.29 0.30 8.78E-05
30 5.61 5.21 6.2E-06 1.6E-09 0.701 0.700 5.66 0.27 6.23E-04
31 5.91 5.29 5.1E-06 1.9E-09 0.738 0.738 5.96 0.30 7.15E-04
32 6.21 5.38 4.2E-06 2.4E-09 0.776 0.776 6.27 0.37 1.18E-03
33 6.51 5.49 3.2E-06 3.1E-09 0.817 0.817 6.60 0.40 3.08E-03
34 6.81 5.61 2.5E-06 4.1E-09 0.855 0.855 6.90 0.50 4.35E-03
35 7.11 5.76 1.7E-06 5.8E-09 0.893 0.893 7.21 0.70 6.92E-03
36 7.41 5.97 1.1E-06 9.3E-09 0.931 0.931 7.52 1.03 1.24E-02
37 7.71 6.28 5.2E-07 1.9E-08 0.965 0.965 7.79 3.17 2.25E-02
38 8.01 7.23 5.9E-08 1.7E-07 0.996 0.996 8.04 9.70 1.19E-02
39 8.31 10.14 7.2E-11 1.4E-04 1.000 1.007 8.14 2.37 7.14E-02
40 8.61 10.85 1.4E-11 7.1E-04 1.000 1.037 8.38 1.17 6.25E-02
41 8.91 11.20 6.3E-12 1.6E-03 1.000 1.084 8.75 0.63 1.58E-02
42 9.21 11.39 4.1E-12 2.5E-03 1.000 1.130 9.12 0.50 3.67E-03
43 9.51 11.54 2.9E-12 3.5E-03 1.000 1.183 9.56 0.50 1.21E-03

Figure 3. Spreadsheet after executing SOLVER to optimize the values of Ca and pKa to minimize
the weighted sum of the squares of the residuals in cell I2.

51
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

significance to points near Ve is the derivative ∆pH / ∆Vb,obs. 19 For the first weight in cell
18F

H12, the formula is

∆pH B13 – B12 3.72 – 3.48

weight = = A13 – A12 = 0.51 – 0.21 = 0.80
∆Vb
A A A A A A

E E

This procedure finds a weight for every point except the last one in cell H43, because there is
no data in cells A44 and B44 with which to compute a derivative. For simplicity, assign the
last weight in cell H43 equal to the weight computed in cell H42.
9. In least squares curve fitting, we seek to minimize the sum of squares of the difference
between an observed and a calculated quantity. In your experiment, you measured Vb,obs and
calculated Vb,calc. We want to minimize the sum (Vb,obs – Vb,calc)2. To find Ve (and,
therefore Ca) more accurately, we attach higher weights to points near Ve. Therefore, we will
minimize the sum Σ[weight*(Vb,obs – Vb,calc)2]. In column I of the spreadsheet compute the
product weight*(Vb,obs – Vb,calc)2 for each row. In cell I2, compute the sum of weighted
squares of the residuals:

sum = Σ[weight*(Vb,obs – Vb,calc)2] ⇒ I2 = SUM(I12:I43)

10. Now we are ready for a least-squares optimization of Ca and pKa to find the theoretical
titration curve that best fits the experimental points. In Excel 2007, in the Data ribbon, select
Solver. (If you don’t see Solver in the Analysis section of the ribbon, click the Microsoft
Office button at the top left of the spreadsheet. Click Excel Options and select Add-Ins.
Highlight Solver Add-In, click OK, and Solver is loaded.) In the Data ribbon, click on
Solver. In the Solver window, Set Target Cell $I$2 Equal To Min By Changing Cells
$B$5,$B$6.

19 The weighting function ∆pH / ∆V 18

b,obs is recommended by Burnett and Burns to improve the accuracy of
finding Ve and, hence, Ca. In general, the accuracy of measuring pH is poorest near Ve, so it could be argued
that these points should be given the least weight, not the most weight. However, points near Ve are most
important for finding Ve accurately, so we weight them the highest. The Gran plot in the preceding experiment is
the best way to find Ve without having to rely on less accurate data near Ve.

52
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

Click the Solve button at the upper right. Solver takes a few seconds to vary the quantities Ca
and pKa in cells B5 and B6 to minimize the sum in cell I2. The result is shown in Figure 3.
The sum in cell I2 is reduced from 0.428 in Figure 2 to 0.240. The optimum values Ca =
0.01981 M and pKa = 4.840 appear in cells B5 and B6 in Figure 3.
To use Solver in earlier versions of Excel, go to the Tools menu, select Solver, and follow
the same instructions given above. (If you don’t see Solver in the Tools menu, select Add-Ins
and click on Solver in the Add-Ins window. Click OK and Solver appears in the Tools
menu.)
11. Superimpose the optimum computed curve on your experimental points to obtain a graph like
Figure 4.

12
Obs
11
Calc
10
Figure 4. Calculated titration
9
curve (smooth line) superimposed
8 on measured points after
optimizing the fit with SOLVER.
pH

7
6
5
4

3
2
0 1 2 3 4 5 6 7 8 9 10
Vb (mL)

53
10. Fitting a Titration Curve with Excel SOLVER Exploring Chemical Analysis

Try different starting values of Ca and pKa in cells B5 and B6 to see how well SOLVER
can optimize two parameters at once. You will find that if the initial values of Ca and pKa
are not close to the correct values, SOLVER might not be able to find an answer. In this
experiment, it was easy to find values of Ca and pKa that are close to the true values by
inspection of the experimental titration data.
In case you could not find good starting estimates for Ca and pKa, try to optimize one
variable at a time. In the Solver window, Set Target Cell $I$2 Equal To Min By Changing
Cells $B$5. Once Solver has found the best value in cell B5, Set Target Cell $I$2 Equal To
Min By Changing Cells $B$6. With these two individually optimized values in cells B5 and
B6, you can optimize them together by Set Target Cell $I$2 Equal To Min By Changing
Cells $B$5,$B$6.
From this exercise, turn in a graph such as Figure 4 and a spreadsheet such as Figure 3.
Report the optimum values of Ca and pKa that fit your experimental data.

54
11. Kjeldahl Nitrogen Analysis Exploring Chemical Analysis

11. Kjeldahl Nitrogen Analysis

The Kjeldahl nitrogen analysis is widely used to measure the nitrogen content
of pure organic compounds and complex substances such as milk, cereal, and
flour. Digestion in boiling H2SO4 with a catalyst converts organic nitrogen
into NH+4. The solution is then made basic, and the liberated NH3 is distilled
A

E
A

into a known amount of HCl (Reactions 10-5 to 10-7 in the textbook).

Green Profile
See Section 0 Unreacted HCl is titrated with NaOH to determine how much HCl was
consumed by NH3. Because the solution to be titrated contains both HCl and
NH+4, we choose an indicator that permits titration of HCl without beginning to titrate NH+4.
A

E
A A

E
A

Bromocresol green, with a transition range of pH 3.8 to 5.4, fulfills this purpose.
The Kjeldahl digestion captures amine (–NR2) or amide (–C[=O]NR2) nitrogens (where
R can be H or an organic group), but not oxidized nitrogen such as nitro (–NO2) or azo (–N=N–)
groups, which must be reduced first to amines or amides.

Reagents

Standard NaOH and standard HCl: From Experiment 6.

Bromocresol green and phenolphthalein indicators: Recipes in Experiment 7.
Potassium sulfate: 10 g/student.
Selenium-coated boiling chips: Hengar selenium-coated granules are convenient. Alternative
catalysts are 0.1 g of Se, 0.2 g of CuSeO3, or a crystal of CuSO4.
Concentrated (98 wt%) H2SO4: 25 mL/student
Unknowns: Unknowns are pure acetanilide, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES buffer, Table 9-2 in the textbook), tris(hydroxymethyl)aminomethane (tris
buffer, Table 9-2), or the p-toluenesulfonic acid salts of ammonia, glycine, or nicotinic acid.
Each student needs enough unknown to produce 2–3 mmol of NH3.

Digestion

1. Dry your unknown at 105˚C for 45 min and accurately weigh an amount that will produce
2–3 mmol of NH3. Place the sample in a dry 500-mL Kjeldahl flask (Figure 10-8 in the
textbook), so that as little as possible sticks to the walls. Add 10 g of K2SO4 (to raise the

55
11. Kjeldahl Nitrogen Analysis Exploring Chemical Analysis

boiling temperature) and three selenium-coated boiling chips. Pour in 25 mL of 98 wt%

H2SO4, washing down any solid from the walls. (CAUTION: Concentrated H2SO4 eats
people. If you get any on your skin, flood it immediately with water, followed by soap and
water.)
2. In a fume hood, clamp the flask at a 30˚ angle away from you. Heat gently with a burner
until foaming ceases and the solution becomes homogeneous. Continue boiling gently for an
additional 30 min.
3. Cool the flask for 30 min in the air, and then in an ice bath for 15 min. Slowly, and with
constant stirring, add 50 mL of ice-cold, distilled water. Dissolve any solids that crystallize.
Transfer the liquid to the 500-mL 3-neck distillation flask in Figure 1. Wash the Kjeldahl
flask five times with 10-mL portions of distilled water, and pour the washings into the
distillation flask.

Distillation

1. Set up the apparatus in Figure 1 and tighten the connections well. Pipet 50.00 mL of standard
0.1 M HCl into the receiving beaker and clamp the funnel in place below the liquid level.
2. Add 5–10 drops of phenolphthalein indicator to the three-neck flask in Figure 1 and secure
the stoppers. Pour 60 mL of 50 wt% NaOH into the adding funnel and drip this into the
distillation flask over a period of 1 min until the indicator turns pink. (CAUTION: 50 wt%
NaOH eats people. Flood any spills on your skin with water.) Do not let the last 1 mL
through the stopcock, so that gas cannot escape from the flask. Close the stopcock and heat
the flask gently until two-thirds of the liquid has distilled.
3. Remove the funnel from the receiving beaker before removing the burner from the flask (to
avoid sucking distillate back into the condenser). Rinse the funnel well with distilled water
and catch the rinses in the beaker. Add 6 drops of bromocresol green indicator solution to the
beaker and carefully titrate to the blue end point with standard 0.1 M NaOH. You are
looking for the first appearance of light blue color. (Practice titrations with HCl and NaOH
beforehand to familiarize yourself with the end point.)
4. Calculate the wt% of nitrogen in the unknown.

56
11. Kjeldahl Nitrogen Analysis Exploring Chemical Analysis

Figure 1. Apparatus for

Kjeldahl distillation.

57
12. EDTA Titration of Ca2+ and Mg2+ in Natural Waters Exploring Chemical Analysis

12. EDTA Titration of Ca2+ and Mg2+ in Natural Waters

The most common multivalent metal ions in natural waters are Ca2+ and
Mg2+. In this experiment, we will find the total concentration of metal ions
that can react with EDTA, and we will assume that this equals the
concentration of Ca2+ and Mg2+. In a second experiment, Ca2+ is analyzed
separately after precipitating Mg(OH)2 with strong base.
Green Profile
See Section 0
Reagents

EDTA: Na2H2EDTA . 2H2O (FM 374.24), 0.6 g/student.

Buffer (pH 10): Add 142 mL of 28 wt% aqueous NH3 to 17.5 g of NH4Cl and dilute to 250 mL
with distilled water.
Eriochrome black T indicator: Dissolve 0.2 g of the solid indicator in 15 mL of triethanolamine
plus 5 mL of absolute ethanol. (Alternatively, Calmagite could be used by dissolving 0.05 g
in 100 mL of water. The color changes are the same for both indicators.)
Hydroxynaphthol blue indicator: 0.5 g/student.
Unknowns: Collect water from streams or lakes or from the ocean. To minimize bacterial
growth, plastic jugs should be filled to the top and tightly sealed. Refrigeration is
recommended.
50 wt% NaOH: Dissolve 100 g of NaOH in 100 g of H2O in a 250-mL plastic bottle. Store
tightly capped. When you remove solution with a pipet, try not to disturb the solid Na2CO3
precipitate.

Procedure

1. Dry Na2H2EDTA . 2H2O (FM 372.24) at 80˚C for 1 h and cool in the desiccator. Accurately
weigh out ~0.6 g and dissolve it with heating in 400 mL of water in a 500-mL volumetric
flask. Cool to room temperature, dilute to the mark, and mix well.
2. Pipet a sample of unknown into a 250-mL flask. A 1.000-mL sample of seawater or a 50.00-
mL sample of tap water is usually reasonable. If you use 1.000 mL of seawater, add 50 mL
of distilled water. To each sample, add 3 mL of pH 10 buffer and 6 drops of Eriochrome
black T indicator. Titrate with EDTA from a 50-mL buret and note when the color changes

58
12. EDTA Titration of Ca2+ and Mg2+ in Natural Waters Exploring Chemical Analysis

from wine red to blue. Practice finding the end point several times by adding a little tap
water and titrating with more EDTA. Save a solution at the end point to use as a color
comparison for other titrations.
3. Repeat the titration with three samples to find an accurate value of the total Ca2+ + Mg2+
concentration. Perform a blank titration with 50 mL of distilled water and subtract the value
of the blank from each result.
4. For the determination of Ca2+, pipet four samples of unknown into clean flasks (adding 50
mL of distilled water if you use 1.000 mL of seawater). Add 30 drops of 50 wt% NaOH to
each solution and swirl for 2 min to precipitate Mg(OH)2 (which may not be visible). Add
~0.1 g of solid hydroxynaphthol blue to each flask. (This indicator is used because it remains
blue at higher pH than does Eriochrome black T.) Titrate one sample rapidly to find the end
point; practice finding it several times, if necessary.
5. Titrate the other three samples carefully. After reaching the blue end point, allow each
sample to sit for 5 min with occasional swirling so that any Ca(OH)2 precipitate may
redissolve. Then titrate back to the blue end point. (Repeat this procedure if the blue color
turns to red upon standing.) Perform a blank titration with 50 mL of distilled water.
6. Calculate the total concentration of Ca2+ and Mg2+, as well as the individual concentrations
of each ion. Calculate the relative standard deviation of replicate titrations.

59
13. Synthesis and Analysis of Ammonium Decavanadate Exploring Chemical Analysis

13. Synthesis and Analysis of Ammonium Decavanadate 20 19F

The species of aqueous V5+ depend on both pH and concentration (Figure 1).
3– 4– 4– 5– +
VO 4 A
E

A V2O 7
A
E

A V4O12A
E

A HV10O28 A
E

A VO2 A
E

In strong In strong
base NH +4 , acid
acetic acid,
alcohol
Green Profile
See Section 0
(NH4)6V10O28 . 6H2O
FM 1 173.7

The decavanadate ion (V10 O6-

28 ), which we will isolate in this experiment as the ammonium salt,
consists of 10 VO6 octahedra sharing edges with one another (Figure 2).

Figure 1. Phase diagram for aqueous vanadium(V) as a function of total vanadium concentration
and pH. [From J. W. Larson, J. Chem. Eng. Data 1995, 40, 1276.] The region marked
“precipitate” probably refers to a vanadium hydroxide.

20 G. G. Long, R. L. Stanfield, and F. C. Hentz, Jr., J. Chem. Ed. 1979, 56, 195.

60
13. Synthesis and Analysis of Ammonium Decavanadate Exploring Chemical Analysis

Figure 2. Structure of
V10 O6-28 anion, consisting
of VO6 octahedra sharing
edges with one another.
There is a vanadium atom
at the center of each
octahedron.

After preparing this salt, we will determine the vanadium content by a redox titration and
NH +4 by the Kjeldahl method. In the redox titration, V5+ will first be reduced to V4+ with
sulfurous acid and then titrated with standard permanganate.

V10 O6-
28 + H2SO3 → VO2+ + SO2

VO2+ + MnO4- A E A → VO+2 + Mn2+

A E A

blue purple yellow colorless

Reagents

Ammonium metavanadate (NH4VO3): 3 g/student.

50 vol% aqueous acetic acid: 4 mL/student.
95 % ethanol: 200 mL/student.
KMnO4: 1.6 g/student or prepare 0.02 M KMnO4 (~300 mL/student) for use by the class.
Sodium Oxalate (Na2C2O4): 1 g/student.
0.9 M H2SO4: (1 L/student) Slowly add 50 mL of concentrated (96–98 wt%) H2SO4 to 900 mL
of H2O and dilute to ~1 L.
1.5 M H2SO4: (100 mL/student) Slowly add 83 mL of concentrated (96–98 wt%) H2SO4 to
900 mL of H2O and dilute to ~1 L.
Sodium hydrogen sulfite (NaHSO3, also called sodium bisulfite): 2 g/student.
Standard 0.1 M HCl: (75 mL/student) From Experiment 6.
Standard 0.1 M NaOH: (75 mL/student) From Experiment 6.
Phenolphthalein indicator and bromocresol green indicators: Recipes in Experiment 7.
50 wt% NaOH: 60 mL/student. Mix 100 g NaOH with 100 mL H2O and dissolve.

61
13. Synthesis and Analysis of Ammonium Decavanadate Exploring Chemical Analysis

Synthesis

1. Heat 3.0 g of ammonium metavanadate (NH4VO3) in 100 mL of water with constant stirring
(but not boiling) until most or all of the solid has dissolved. Filter the solution and add 4 mL
of 50 vol% aqueous acetic acid with stirring.
2. Add 150 mL of 95% ethanol with stirring and then cool the solution in a refrigerator or ice
bath.
3. After maintaining a temperature of 0˚–l0˚C for 15 min, filter the orange product with suction
and wash with two 15-mL portions of ice-cold 95% ethanol.
4. Dry the product in the air (protected from dust) for 2 days. Typical yield is 2.0–2.5 g.

Analysis of Vanadium with KMnO4

Preparation and Standardization of KMnO4 21 (See Section 6-3 in the textbook)

20F

1. Prepare a 0.02 M permanganate solution by dissolving 1.6 g of KMnO4 in 500 mL of

distilled water. Boil gently for 1 h, cover, and allow the solution to cool overnight. Filter
through a clean, fine sintered-glass funnel, discarding the first 20 mL of filtrate. Store the
solution in a clean glass amber bottle. Do not let the solution touch the cap.
2. Dry sodium oxalate (Na2C2O4) at 105˚C for 1 h, cool in a desiccator, and weigh three
~0.25-g samples into 500-mL flasks or 400-mL beakers. To each, add 250 mL of 0.9 M
H2SO4 that has been recently boiled and cooled to room temperature. Stir with a
thermometer to dissolve the sample, and add 90–95% of the theoretical amount of KMnO4
solution needed for the titration. (This can be calculated from the mass of KMnO4 used to
prepare the permanganate solution. The chemical reaction is given by Equation 6-1 in the
textbook.)
3. Leave the solution at room temperature until it is colorless. Then heat it to 55˚–60˚C and
complete the titration by adding KMnO4 until the first pale pink color persists. Proceed
slowly near the end, allowing 30 s for each drop to lose its color.
4. As a blank, titrate 250 mL of 0.9 M H2SO4 to the same pale pink color. Subtract the blank
volume from each titration volume. Compute the average molarity of KMnO4.

21 R. M. Fowler and H. A. Bright, J. Res. National Bureau of Standards 1935, 15, 493.

62
13. Synthesis and Analysis of Ammonium Decavanadate Exploring Chemical Analysis

Vanadium Analysis

1. Accurately weigh two 0.3-g samples of ammonium decavanadate into 250-mL flasks and
dissolve each in 40 mL of 1.5 M H2SO4 (with warming, if necessary).
2. In a fume hood, add 50 mL of water and 1 g of NaHSO3 to each and dissolve with swirling.
After 5 min, boil the solution gently for 15 min to remove SO2.
3. Titrate the warm solution with standard 0.02 M KMnO4 from a 50-mL buret. The end point
is taken when the yellow color of VO +2 takes on a dark shade (from excess M nO-4 ) that
persists for 15 s.
4. Calculate the average wt% of vanadium in the ammonium decavanadate and compare your
result to the theoretical value.

Analysis of Ammonium Ion by Kjeldahl Distillation

1. Set up the apparatus in Figure 1 of Experiment 11 and press the stoppers to make airtight
connections. Pipet 50.00 mL of standard 0.1 M HCl into the receiving beaker and clamp the
funnel in place below the liquid level.
2. Transfer 0.6 g of accurately weighed ammonium decavanadate to the three-neck flask and
add 200 mL of water. Add 5–10 drops of phenolphthalein indicator and secure the stoppers.
Pour 60 mL of 50 wt% NaOH into the adding funnel and drip this into the distillation flask
over a period of 1 min until the indicator turns pink. (Caution: 50 wt% NaOH eats people.
Flood any spills on your skin with water.) Do not let the last 1 mL through the stopcock, so
that gas cannot escape from the flask. Close the stopcock and heat the flask gently until
two-thirds of the liquid has distilled.
3. Remove the funnel from the receiving beaker before removing the burner from the flask (to
avoid sucking distillate back into the condenser). Rinse the funnel well with distilled water
and catch the rinses in the beaker. Add 6 drops of bromocresol green indicator solution to the
beaker and carefully titrate to the blue end point with standard 0. 1 M NaOH. You are
looking for the first appearance of light blue color. (Several practice titrations with HCl and
NaOH will familiarize you with the end point.)
4. Calculate the weight percent of nitrogen in the ammonium decavanadate.

63
14. Iodimetric Titration of Vitamin C Exploring Chemical Analysis

14. Iodimetric Titration of Vitamin C 22 21F

Ascorbic acid (vitamin C) is a mild reducing agent that reacts rapidly with
triiodide (See Section 16-3 and Box 16-2 in the textbook). In this
experiment, we will generate a known excess of I3- by the reaction of iodate
A
E

with iodide (Reaction 16-20), allow the reaction with ascorbic acid to

Green Profile
proceed, and then back titrate the excess I3- with thiosulfate (Reaction 16-21
A
E

See Section 0 and Color Plate 12).

Reagents

Starch indicator: Make a paste of 5 g of soluble starch and 5 mg of Hg2I2 in 50 mL of distilled

water. Pour the paste into 500 mL of boiling distilled water and boil until it is clear.
Sodium thiosulfate: 9 g Na2S2O3 . 5H2O/student.
Sodium carbonate: 50 mg Na2CO3/student.
Potassium iodate: 1 g KIO3/student.
Potassium iodide: 12 g KI/student.
0.5 M H2SO4: 30 mL/student.
Vitamin C: Dietary supplement containing 100 mg of vitamin C per tablet is suitable. Each
student needs six tablets.
0.3 M H2SO4: 180 mL/student.

Preparation and Standardization of Thiosulfate Solution

1. Prepare starch indicator by making a paste of 5 g of soluble starch and 5 mg of HgI2 in 50 mL

of water. Pour the paste into 500 mL of boiling water and boil until it is clear.
2. Prepare 0.07 M Na2S2O3 23 by dissolving ~8.7 g of Na2S2O3.5H2O in 500 mL of freshly
2F

boiled water containing 0.05 g of Na2CO3. Store this solution in a tightly capped amber
bottle. Prepare ~0.01 M KIO3 by accurately weighing ~1g of solid reagent and dissolving it

22 D. N. Bailey, J. Chem. Ed. 1974, 51, 488.

23 An alternative to standardizing Na S O solution is to prepare anhydrous primary standard Na S O by
2 2 3 2 2 3
.
refluxing 21 g of Na2S2O3 5H2O with 100 mL of methanol for 20 min. Then filter the anhydrous salt, wash with
20 mL of methanol, and dry at 70˚C for 30 min. [A. A. Woolf, Anal. Chem. 1982, 54, 2134.]

64
14. Iodimetric Titration of Vitamin C Exploring Chemical Analysis

in a 500-mL volumetric flask. From the mass of KIO3 (FM 214.00), compute the molarity of
the solution.
3. Standardize the thiosulfate solution as follows: Pipet 50.00 mL of KIO3 solution into a flask.
Add 2 g of solid KI and 10 mL of 0.5 M H2SO4. Immediately titrate with thiosulfate until
the solution has lost almost all its color (pale yellow). Then add 2 mL of starch indicator and
complete the titration. Repeat the titration with two additional 50.00-mL volumes of KIO3
solution. From the stoichiometries of Reactions 16-20 and 16-21, compute the average
molarity of thiosulfate and the relative standard deviation.

Analysis of Vitamin C

Commercial vitamin C containing 100 mg per tablet can be used. Perform the following analysis
three times, and find the mean value (and relative standard deviation) for the number of
milligrams of vitamin C per tablet.

1. Dissolve two tablets in 60 mL of 0.3 M H2SO4, using a glass rod to help break the solid.
(Some solid binding material will not dissolve.)
2. Add 2 g of solid KI and 50.00 mL of standard KIO3. Then titrate with standard thiosulfate as
above. Add 2 mL of starch indicator just before the end point.

65
15. Preparation and Iodometric Analysis Exploring Chemical Analysis
of a High-Temperature Superconductor

15. Preparation and Iodometric Analysis of a High-Temperature

Superconductor 24 23F

In this experiment you will determine the oxygen content of yttrium barium
copper oxide (YBa2Cu3Ox). This material is an example of a
nonstoichiometric solid, in which the value of x is variable, but near 7. Some
of the copper in the formula YBa2Cu3O7 is in the unusual high-oxidation
state, Cu3+. This experiment describes the synthesis of YBa2Cu3Ox, (which
Green Profile
See Section 0 can also be purchased) and then gives two alternative procedures to measure
the Cu3+ content. The iodometric method is based on the reactions in
Problem 16-17 in the textbook. The more elegant citrate-complexed copper titration 25 24F

eliminates many experimental errors associated with the simpler iodometric method and gives
more accurate and precise results.

Preparation of YBa2Cu3Ox

1. Place in a mortar 0.750 g of Y2O3, 2.622 g of BaCO3, and 1.581 g of CuO (atomic ratio
Y:Ba:Cu = 1:2:3). Grind the mixture well with a pestle for 20 min and transfer the powder to
a porcelain crucible or boat. Heat in the air in a furnace at 920˚–930˚C for 12 h or longer.
Turn off the furnace and allow the sample to cool slowly in the furnace. This slow cooling
step is critical for achieving an oxygen content in the range x = 6.5–7 in the formula
YBa2Cu3Ox. The crucible may be removed when the temperature is below 100˚C.
2. Dislodge the black solid mass gently from the crucible and grind it to a fine powder with a
mortar and pestle. It can now be used for this experiment, but better quality material is
produced if the powder is heated again to 920˚–930˚C and cooled slowly as in step 1. If the
powder from step 1 is green instead of black, raise the temperature of the furnace by 20˚C
and repeat step 1. The final product must be black, or it is not the correct compound.

24 D. C. Harris, M. E. Hills, and T. A. Hewston, J. Chem. Ed. 1987, 64, 847. Alternative syntheses of YBa Cu O
2 3 x
are described by C. D. Cogdell, D. G. Wayment, D. J. Casadonte, Jr., and K. A. Kubat-Martin, J. Chem. Ed.
1995, 72, 840 and P. I. Djurovich and R. J. Watts, J. Chem. Ed. 1993, 70, 497.
25 E. H. Appelman, L. R. Morss, A. M. Kini, U. Geiser, A. Umezawa, G. W. Crabtree, and K. D. Carlson, Inorg.
Chem. 1987, 26, 3237.

66
15. Preparation and Iodometric Analysis Exploring Chemical Analysis
of a High-Temperature Superconductor

lodometric Analysis

1. Sodium Thiosulfate and Starch Indicator. Prepare 0.03 M Na2S2O3 as described in

Experiment 14, using 3.7 g of Na2S2O3.5H2O instead of 8.7 g. The starch indicator solution
is the same one used in Experiment 14.
2. Standard Cu2+. Weigh accurately 0.5–0.6 g of reagent Cu wire into a 100-mL volumetric
flask. In a fume hood, add 6 mL of distilled water and 3 mL of 70 wt% nitric acid, and boil
gently on a hot plate until the solid has dissolved. Add 10 mL of distilled water and boil
gently. Add 1.0 g of urea or 0.5 g of sulfamic acid and boil for 1 min to destroy HNO2 and
oxides of nitrogen that would interfere with the iodometric titration. Cool to room
temperature and dilute to the mark with 1.0 M HCl.
3. Standardization of Na2S2O3 With Cu2+ . The titration should be carried out as rapidly as
possible under a brisk flow of N2, because I- is oxidized to I2 in acid solution by atmospheric
oxygen. Use a 180-mL tall-form beaker (or a 150-mL standard beaker) with a loosely fitting
two-hole cork at the top. One hole serves as the inert gas inlet and the other is for the buret.
Pipet 10.00 mL of standard Cu2+ into the beaker and flush with N2. Remove the cork just
long enough to pour in 10 mL of distilled water containing 1.0–1.5 g of KI (freshly dissolved)
and begin magnetic stirring. In addition to the dark color of iodine in the solution, suspended
solid CuI will be present. Titrate with Na2S2O3 solution from a 50-mL buret, adding 2 drops
of starch solution just before the last trace of I2 color disappears. If starch is added too soon,
there can be irreversible attachment of I2 to the starch and the end point is harder to detect.
You may want to practice this titration several times to learn to distinguish the colors of I2
and I2/starch from the color of suspended CuI(s). (Alternatively, Pt and calomel electrodes
can be used instead of starch to eliminate subjective judgment of color in finding the end
point. 26) Repeat this standardization two more times and use the average Na2S2O3 molarity
25F

from the three determinations.

4. Superconductor Experiment A. Dissolve an accurately weighed 150- to 200-mg sample of
powdered YBa2Cu3Ox in 10 mL of 1.0 M HClO4 in a titration beaker in a fume hood.
(Perchloric acid is recommended because it is inert to reaction with superconductor, which
might oxidize HCl to Cl2. We have used HCl instead of HClO4 with no significant
interference in the analysis. Solutions of HClO4 should not be boiled to dryness because of

26 P. Phinyocheep and I. M. Tang, J. Chem. Ed. 1994, 71, A115.

67
15. Preparation and Iodometric Analysis Exploring Chemical Analysis
of a High-Temperature Superconductor

their explosion hazard.) Boil gently for 10 min, so that Reaction A in Problem 16-17 goes to
completion. Cool to room temperature, cap with the two-hole-stopper–buret assembly, and
begin N2 flow. Dissolve 1.0–1.5 g of KI in 10 mL of distilled water and immediately add the
solution to the beaker. Titrate rapidly with magnetic stirring as described in step 3. Repeat
this procedure two more times.
5. Superconductor Experiment B. Place an accurately weighed 150- to 200-mg sample of
powdered YBa2Cu3Ox in the titration beaker and begin N2 flow. Dissolve 1.0–1.5 g of KI in
10 mL of 1.0 M HClO4 and immediately add the solution to the titration beaker. Stir
magnetically for 1 min, so that the Reactions B and C of Problem 16-17 occur. Add 10 mL
of water and rapidly complete the titration. Repeat this procedure two more times.

Calculations

1. Suppose that mass, mA, is analyzed in Experiment A and the volume, VA, of standard
thiosulfate is required for titration. Let the corresponding quantities in Experiment B be mB
and VB. Let the average oxidation state of Cu in the superconductor be 2 + p. Show that p is
given by

VB / mB − VA / mA
p =
VA / mA (1)

and x in the formula YBa2Cu3Ox, is related to p as follows:

7 3
x = + (2 + p) (2)
2 2

For example, if the superconductor contains one Cu3+ and two Cu2+, the average oxidation
state of copper is 7/3 and the value of p is 1/3. Setting p = 1/3 in Equation 2 gives x = 7.
Equation 1 does not depend on the metal stoichiometry being exactly Y:Ba:Cu 1:2:3, but
Equation 2 does require this exact stoichiometry.
2. Use the average results of Experiments A and B to calculate the values of p and x in
Equations 1 and 2.
3. Suppose that the uncertainty in mass of superconductor analyzed is 1 in the last decimal
place. Calculate the standard deviations for steps 3, 4, and 5 of the iodometric analysis.

68
15. Preparation and Iodometric Analysis Exploring Chemical Analysis
of a High-Temperature Superconductor

Using these standard deviations as uncertainties in volume, calculate the uncertainties in the
values of p and x in Equations 1 and 2.

Citrate-Complexed Copper Titration25

This procedure directly measures Cu3+. The sample is first dissolved in a closed container with
4.4 M HBr, in which Cu3+ oxidizes Br- to Br3- :

11 - 1
Cu3+ + Br → CuBr42- + Br3- (3)
2 2

The solution is transferred to a vessel containing excess I-, excess citrate, and enough NH3 to
neutralize most of the acid. Cu2+ is complexed by citrate and is not further reduced to CuI(s).
(This eliminates the problem in the iodometric titration of performing a titration in the presence
of colored solid.) Br3- from Reaction 3 oxidizes I- to I -3 :

Br3- + 3I- → 3Br- + I 3

-
(4)

-
and the I 3 produced in Reaction 4 is titrated with thiosulfate.
Our experience with the iodometric procedure is that the precision in oxygen content of
YBa2Cu3Ox, is ±0.04 in the value of x. The uncertainty is reduced to ±0.01 by the citrate-
complexed copper procedure.

Procedure

1. Prepare and standardize sodium thiosulfate solution as described in steps 1–3 of the
iodometric analysis in the previous section.
2. Place an accurately weighed 20- to 50-mg sample of superconductor in a 4-mL screw-cap vial
with a Teflon cap liner and add 2.00 mL of ice-cold 4.4 M HBr by pipet. (The HBr is
prepared by diluting 50 mL of 48 wt% HBr to 100 mL.) Cap tightly and gently agitate the
vial for 15 min as it warms to room temperature. (We use a motor to rotate the sample
slowly for 15 min.)
3. Cool the solution back to 0˚C and carefully transfer it to the titration beaker (used in the
thiosulfate standardization) containing an ice-cold, freshly prepared solution made from 0.7 g
of KI, 20 mL of water, 5 mL of 1.0 M trisodium citrate, and approximately 0.5 mL of 28 wt%

69
15. Preparation and Iodometric Analysis Exploring Chemical Analysis
of a High-Temperature Superconductor

NH3. The exact amount of NH3 should be enough to neutralize all but 1 mmol of acid
present in the sample. When calculating the acid content, remember that each mole of
YBa2Cu3Ox consumes 2x moles of HBr. (You can estimate that x is close to 7.) Wash the
vial with three 1-mL aliquots of 2 M HBr to complete the quantitative transfer to the beaker.
4. Add 0.1 mL of 1 wt% starch solution and titrate with 0.1 M standard Na2S2O3 under a brisk
flow of N2, using a 250-mL Hamilton syringe to deliver titrant. The end point is marked by a
change from dark blue (I2-starch) to the light blue-green of the Cu2+-citrate complex.
5. Run a blank reaction with CuSO4 in place of superconductor. The moles of Cu in the blank
should be the same as the moles of Cu in the superconductor. In a typical experiment, 30 mg
of YBa2Cu3O6.88 required approximately 350 µL of Na2S2O3 and the blank required 10 µL
of Na2S2O3 If time permits, run two more blanks. Subtract the average blank from the titrant
in step 4.
6. Repeat the analysis with two more samples of superconductor.

Calculations

1. From the thiosulfate required to titrate I -3 released in Reaction 4, find the average moles of
Cu3+ per gram of superconductor (1 mol S2 O2- 3+
3 = 1 mol Cu ) and the standard deviation for
your three samples.
2. Defining R as (mol Cu3+ )/(g superconductor), show that z in the formula YBa2Cu3O7–z is

1 – 666.20 R
z = 2 – 15.999 R
A A

E
(5)

where 666.20 is the formula mass of YBa2Cu3O7 and 15.999 4 is the atomic mass of O.
3. Using your average value of R and using its standard deviation as an estimate of uncertainty,
calculate the average value of z and its uncertainty. Find the average value of x and its
uncertainty in the formula YBa2Cu3Ox. If you are really daring, use Equation C-1 in
Appendix C of the textbook D. C. Harris, Quantitative Chemical Analysis, 7th ed. (New
York: Freeman, 2007) for propagation of uncertainty in Equation 5.

70
16. Potentiometric Halide Titration with Ag+ Exploring Chemical Analysis

16. Potentiometric Halide Titration with Ag+

Mixtures of halides can be titrated with AgNO3 solution as described in

Section 6-5 in the textbook. In this experiment, you will use the apparatus in
Figure 6-4 of the textbook to monitor the activity of Ag+ as the titration
proceeds. The theory of the potentiometric measurement is described in
Section 15-1 of the textbook.
Green Profile
See Section 0 Each student is given a vial containing 0.22–0.44 g of KCl plus 0.50–
1.00 g of KI (both weighed accurately). The object is to determine the
quantity of each salt in the mixture. A 0.4 M bisulfate buffer (pH 2) should be available in the
lab. It is prepared by titrating 1 M H2SO4 with 1 M NaOH to a pH near 2.0.

Reagents

Unknowns: Each student receives a vial containing 0.22–0.44 g of KCl plus 0.50–1.00 g of KI
(weighed accurately). The object is to determine the quantity of each salt in the mixture.
Buffer (pH 2): (6 mL/student) Titrate 1 M H2SO4 with 1 M NaOH to a pH near 2.0.
Silver nitrate: 1.2 g AgNO3/student.

Procedure

1. Pour your unknown carefully into a 50- or 100-mL beaker. Dissolve the solid in ~20 mL of
water and pour the solution into a 100-mL volumetric flask. Rinse the sample vial and
beaker five times with small portions of H2O and transfer the washings to the flask. Dilute to
the mark and mix well.
2. Dry 1.2 g of AgNO3 (FM 169.87) at 105˚C for 1 h and cool in a dessicator for 30 min with
minimal exposure to light. Some discoloration is normal (and tolerable in this experiment)
but should be minimized. Accurately weigh 1.2 g and dissolve it in a 100-mL volumetric
flask.
3. Set up the apparatus in Figure 6-4 of the textbook. The silver electrode is simply a 3-cm
length of silver wire soldered to copper wire. (Fancier electrodes can be prepared by housing
the connection in a glass tube sealed with epoxy at the lower end. Only the silver should
protrude from the epoxy.) The copper wire is fitted with a jack that goes to the reference

71
16. Potentiometric Halide Titration with Ag+ Exploring Chemical Analysis

socket of a pH meter. The reference electrode for this titration is a glass pH electrode
connected to its usual socket on the meter. If a combination pH electrode is employed, the
reference jack of the combination electrode is not used. The silver electrode should be taped
to the inside of the 100-mL beaker so that the Ag/Cu junction remains dry for the entire
titration. The stirring bar should not hit either electrode.
4. Pipet 25.00 mL of unknown into the beaker, add 3 mL of pH 2 buffer, and begin magnetic
stirring. Record the initial level of AgNO3 in a 50-mL buret and add ~1 mL of titrant to the
beaker. Turn the pH meter to the millivolt scale and record the volume and voltage. It is
convenient (but is not essential) to set the initial reading to +800 mV by adjusting the meter.
5. Titrate the solution with ~1-mL aliquots until 50 mL of titrant have been added or until you
can see two abrupt voltage changes. You need not allow more than 15–30 s for each point.
Record the volume and voltage at each point. Make a graph of millivolts versus milliliters to
find the approximate positions (±1 mL) of the two end points.
6. Turn the pH meter to standby, remove the beaker, rinse the electrodes well with distilled
water, and blot them dry with a tissue. (Silver halide adhering to the glass electrode can be
removed by soaking in concentrated sodium thiosulfate solution. This thorough cleaning is
not necessary between steps 6 and 7 in this experiment. The silver halides in the titration
beaker can be saved and converted back to pure AgNO3. 27) Clean the beaker and set up the
26F

titration apparatus again. The beaker need not be dry.

7. Perform an accurate titration using 1-drop aliquots near the end points (and 1-mL aliquots
elsewhere). You need not allow more than 30 s per point for equilibration.
8. Prepare a graph of millivolts versus milliliters and locate the end points as in Figure 6-5 of
the textbook. The I- end point is taken as the intersection of the two dashed lines in the inset
of Figure 6-5. The Cl- end point is the inflection point (the steepest point) at the second
break. Calculate milligrams of KI and milligrams of KCl in your solid unknown.

Analyzing Cl- in Streams, Lakes, or Salt Water

A variation of the preceding procedure could make a class project in environmental analysis. For
example, you could study changes in streams as a function of the season or recent rainfall. You

27 E. Thall, J. Chem. Ed. 1981, 58, 561.

72
16. Potentiometric Halide Titration with Ag+ Exploring Chemical Analysis

could study stratification (layering) of water in lakes. The measurement responds to all ions that
precipitate with Ag+, of which Cl- is, by far, the dominant ion in natural waters.
You can use the electrodes in Figure 6-4 of the textbook, or you can construct the rugged
combination electrode shown in Figure 1. 28 The indicator electrode in Figure 1 is a bare silver
27F

wire in contact with analyte solution. The inner (reference) chamber of the combination
electrode contains a copper wire dipped into CuSO4 solution. The inner electrode maintains a

Figure 1. Combination electrode

constructed from 8-mm-outer-
diameter glass tubing. Strip the
insulation off the ends of a two-
wire copper cable. One bare Cu
wire extends into the glass tube.
A silver wire soldered to the other
Cu wire is left outside the glass
tube. The assembly is held
together at the top with heat-
shrink tubing. When ready for
use, insert a cotton thread into the
bottom of the tube and fit a 14/20
serum cap moistened with 0.1 M
CuSO4 over the end so that part
of the thread is inside and part is
outside. After adding 0.1 M
CuSO4 to the inside of the tube,
the electrode is ready for use.

constant potential because the concentration of Cu2+ in the solution is constant. The solution
makes electrical contact at the septum, where a piece of thread leaves enough space for solution
to slowly drain from the electrode into the external sample solution.

28 G. Lisensky and K. Reynolds, J. Chem. Ed. 1991, 68, 334; R. Ramette, Chemical Equilibrium (Reading, MA:
Addison-Wesley, 1981), p. 649.

73
16. Potentiometric Halide Titration with Ag+ Exploring Chemical Analysis

Procedure

1. Collect water from streams or lakes or from the ocean. To minimize bacterial growth, plastic
jugs should be filled to the top and tightly sealed. Refrigeration is recommended.
2. Prepare 4 mM AgNO3 solution with an accurately known concentration, as in step 2 at the
beginning of this experiment. One student can prepare enough reagent for five people by
using 0.68 g of AgNO3 in a 1-L volumetric flask.
3. Measure with a graduated cylinder 100 mL of a natural water sample and pour it into a 250-
mL beaker. Position the combination electrode from Figure 1 or the pair of electrodes from
Figure 6-4 of the textbook in the beaker so that a stirring bar will not hit the electrode.
4. Carry out a rough titration by adding 1.5-mL increments of titrant to the unknown and
reading the voltage after 30 s to the nearest millivolt. Prepare a graph of voltage versus
volume of titrant to locate the end point, which will not be as abrupt as those in Figure 6-5 of
the textbook. If necessary, adjust the volume of unknown in future steps so that the end point
comes at 20–40 mL. If you need less unknown, make up the difference with distilled water.
5. Carry out a more careful titration with fresh unknown. Add three-quarters of the titrant
volume required to reach the equivalence point all at once. Then add ~0.4-mL increments
(8 drops from a 50-mL buret) of titrant until you are 5 mL past the equivalence point. Allow
30 s (or longer, if necessary) for the voltage to stabilize after each addition. The end point is
the steepest part of the curve, which can be estimated by the method shown in Figure 1 of
Experiment 7.
6. Calculate the molarity and parts per million (µg/mL) of Cl- in the unknown. Use data from
several students with the same sample to find the mean and standard deviation of ppm Cl-.

74
17. Measuring Ammonia in an Aquarium Exploring Chemical Analysis
with an Ion-Selective Electrode

17. Measuring Ammonia in an Aquarium with an Ion-Selective Electrode

Box 6-1 in the textbook provides background for this experiment.

Reagents

Standard ammonia solution: Dissolve ~0.382 g of NH4Cl (FM 53.49) in a

1-L volumetric flask to obtain a solution containing ~100 µg of nitrogen
Green Profile
See Section 0 per milliliter. From the mass of NH4Cl that you weighed, calculate the
nitrogen concentration in µg/mL.

Procedure 29, 30
28F
29F

1. Using appropriate dilutions of your standard ammonia solution, prepare standards containing
3, 1, 0.3, and 0.1 µg N/mL in 100-mL volumetric flasks.
2. Pour the 100-mL standard solution containing 0.1 µg N/mL into a clean, dry 150-mL beaker.
Immerse an ammonia ion-selective electrode and a reference electrode in the solution and
begin magnetic stirring. Do not stir the solution so rapidly that air bubbles are drawn into the
liquid. Add 1.0 mL of 10 M NaOH (to convert NH+4 into NH3) and record the voltage to the
A E A

nearest millivolt when the reading stabilizes. Do not allow more than 5 min before reading
the voltage, because the temperature of the solution will increase from the stirring and the
reading will change.
3. Repeat the same procedure for the other three standards. Prepare a graph of mV versus
log [N], where [N] is the concentration of nitrogen in µg/mL. You should observe a
reasonable straight line.
4. Measure 100 mL of freshly collected aquarium water in a graduated cylinder. Pour it into a
clean, dry 150-mL beaker and repeat the process in step 2.
5. From the least-squares slope and intercept of the calibration curve from step 3, and the
potential measured in step 4, compute the concentration of ammonia nitrogen in the aquarium
water.
6. Repeat steps 4 and 5 once more with a fresh sample to obtain a duplicate measurement.

29 K. D. Hughes, Anal. Chem. 1993, 65, 883A.

30 Standard Methods for the Examination of Water and Wastewater, 20th ed. (Washington, DC: American Public
Health Association, 1998).

75
18. Electrogravimetric Analysis of Copper Exploring Chemical Analysis

18. Electrogravimetric Analysis of Copper

Most copper-containing compounds can be electrolyzed in acidic solution,

with quantitative deposition of Cu at the cathode. Section 17-1 of the
textbook discusses this technique.
Students may analyze a preparation of their own (such as copper
acetylsalicylate 31) or be given unknowns prepared from CuSO4.5H2O or
30F

Green Profile
See Section 0 metallic Cu. In the latter case, dissolve the metal in 8 M HNO3, boil to
remove HNO2, neutralize with ammonia, and barely acidify the solution with
dilute H2SO4 (using litmus paper to test for acidity). Samples must be free of chloride and
nitrous acid. 32 Copper oxide unknowns (soluble in acid) are available from Thorn Smith.1
31F

The apparatus in Figure 17-1 of the textbook uses any 6–12 V direct-current power
supply. A tall-form 150-mL beaker is the reaction vessel.

Procedure

1. Handle the Pt gauze cathode with a tissue, touching only the thick stem, not the wire gauze.
Immerse the electrode in hot 8 M HNO3 to remove previous deposits, rinse with water and
alcohol, dry at 105˚C for 5 min, cool for 5 min, and weigh accurately. If the electrode
contains any grease, it can be heated to red heat over a burner after the treatment above. 33 32F

2. The sample should contain 0.2–0.3 g of Cu in 100 mL. Add 3 mL of 98 wt% H2SO4 and
2 mL of freshly boiled 8 M HNO3. Position the cathode so that the top 5 mm are above the
liquid level after magnetic stirring is begun. Adjust the current to 2 A, which should require
3–4 V. When the blue color of Cu(II) has disappeared, add some distilled water so that new
Pt surface is exposed to the solution. If no further deposition of Cu occurs on the fresh

31 E. Dudek, J. Chem. Ed. 1977, 54, 329.

32 J. F. Owen, C. S. Patterson, and G. S. Rice, Anal. Chem. 1983, 55, 990, describe simple procedures for the
removal of chloride from Cu, Ni, and Co samples prior to electrogravimetric analysis.
33 Some metals, such as Zn, Ga, and Bi, form alloys with Pt and should not be deposited directly on the Pt surface.
The electrode should be coated first with Cu, dried, and then used. Alternatively, Ag can be used in place of Pt
for depositing these metals. Platinum anodes are attacked by Cl2 formed by electrolysis of Cl- solutions. To
prevent chlorine attack, 1–3 g of a hydrazinium salt (per 100 mL of solution) can be used as an anodic
depolarizer, because hydrazine is more readily oxidized than Cl-: N H → N + 4H+ + 4e-.
2 4 2

76
18. Electrogravimetric Analysis of Copper Exploring Chemical Analysis

surface in 15 min at a current of 0.5 A, the electrolysis is complete. If deposition is observed,

continue electrolysis and test the reaction for completeness again.
3. Without turning off the power, lower the beaker while washing the electrode with a squirt
bottle. Then the current can be turned off. (If current is disconnected before removing the
cathode from the liquid and rinsing off the acid, some Cu could redissolve.) Wash the
cathode gently with water and alcohol, dry at 105˚C for 3 min, cool in a desiccator for 5 min,
and weigh.
4. Report the mass of Cu in the unknown.

77
19. Measuring Vitamin C in Fruit Juice by Voltammetry Exploring Chemical Analysis
with Standard Addition

19. Measuring Vitamin C in Fruit Juice by Voltammetry with Standard

Addition

This experiment is described in Section 17-3 of the textbook and standard

addition is discussed in Section 5-3. The three-electrode cell in Figure 17-9
of the textbook employs a wax-impregnated graphite working electrode, an
Ag | AgCl reference electrode, 34 and a graphite auxiliary electrode. The
3F

electrochemistry at the working electrode is the oxidation of ascorbic acid

Green Profile
See Section 0 (vitamin C) in Reaction 17-9.

Reagents

Nitric acid: ~3 M HNO3.

Standard ascorbic acid: This standard should be freshly prepared each lab period. Follow these
directions if standard additions are to be made in 1-mL aliquots: Accurately weigh ~0.5 g of
ascorbic acid (FM 176.12) into a 100-mL volumetric flask. Add ~50 mL of water and swirl
to dissolve. Add ~1 mL of ~3 M HNO3, swirl, and dilute to 100 mL with water to obtain
~0.03 ascorbic acid standard. Nitric acid slows the reaction of ascorbic acid with oxygen. If
standard additions are to be made in ~100 µL aliquots, prepare the ~0.3 M standard in a 10-
mL volumetric flask from 0.5 g ascorbic acid (accurately weighed) and ~100 µL of ~3 M
HNO3.
Unknowns: ~100 mL of fruit juice for each student or group of students working together.
Possible unknowns include powdered drink mixes, canned drinks, different brands of orange
juice, or vegetable juices.

Preparation of Working Electrode

Many instructors will want to prepare the inexpensive auxiliary and working electrodes in
advance and provide them to students. The auxiliary electrode is made of very pure graphite,

34 A recipe to prepare your own Ag | AgCl reference electrode is given by G. A. East and M. A. del Valle, J. Chem.
Ed. 2000, 77, 97.

78
19. Measuring Vitamin C in Fruit Juice by Voltammetry Exploring Chemical Analysis
with Standard Addition

which is a form of carbon. 35 It is used as received. The working electrode, called a wax-
34F

impregnated graphite electrode, is made of the same pure graphite sharpened to a point in a
pencil sharpener. Soak the sharpened rod for 3 h in molten paraffin at 100°C to fill up pores with
the electrically inert wax. Then use forceps to withdraw the rod slowly from the wax bath and
allow an electrically insulating layer of paraffin to form on the outside of the rod. Slice the sharp
tip with a razor blade to expose a ~1/2-mm-diameter section of graphite and gently grind the tip
with fine sand paper to leave a small, flat exposed graphite surface, as shown in the inset of
Figure 17-9 of the textbook. Prior to beginning the experiment, clean the graphite tip by rubbing
it on filter paper with the electrode perpendicular to the paper. (If the electrode becomes fouled,
cut off the tip with a razor to expose fresh surface.)

Procedure

1. Use small quantities of unknown fruit juice to rinse the inside of a pipet and discard the
washings. Then pipet 50 mL of unknown juice into a clean, dry 100-mL beaker containing a
stirring bar.
2. Clean the tip of the working electrode by rubbing it perpendicular to a piece of filter paper on
a flat surface.
3. Set up the apparatus in Figure 17-9 of the textbook on top of a magnetic stirrer. Use clamps
with insulated claws to hold the electrodes in place. (If the claws are bare metal, use tubing
or tape to insulate them.) The potentiostat varies the voltage between the working and
reference electrodes and measures the current between the working and auxiliary electrodes.
The voltammogram (a graph of current versus potential) is recorded on a computer or strip
chart.
4. Following instructions for your potentiostat, apply the following sequence of voltages:
a. Condition the working electrode at –1.5 V (versus Ag | AgCl ) for 2 min with stirring.
This voltage reduces and removes organic material from the tip of the electrode.
b. Change the potential to –0.4 V and continue stirring for 30 s. Dislodge any bubbles of
gas from the tip of the electrode by gentle tapping. Discontinue stirring and let the
solution become calm for an additional 30 s.

35 Graphite rods (6.15 mm diameter × 102 mm long) can be purchased from Alpha/Aesar, catalog number 40766,
99.9995% carbon. http://www.alfa.com.

79
19. Measuring Vitamin C in Fruit Juice by Voltammetry Exploring Chemical Analysis
with Standard Addition

c. Scan the potential from –0.4 V to + 1.2 V at a rate of +33 mV/s while measuring current
to record a voltammogram such as the lowest trace in Figure 17-10 of the textbook.
5. Standard addition. Using a transfer pipet or a microliter pipet, add 1.00 mL of ~0.03 M
standard ascorbic acid (or 100 µL of ~0.3 M standard) into the beaker and turn on the stirrer.
Repeat the sequence in step 4 to condition the electrode and record a new voltammogram.
6. Add another increment of standard and record another voltammogram by repeating the
sequence in step 4. The objective is to increase the current from that of the unknown by a
factor of 1.5 to 3 by using at least four standard additions. The total volume of added
standard should not exceed ~10 mL. Depending on your unknown, it may be necessary to
adjust the concentration of standard or volume of each increment to achieve this goal.

Data Analysis

1. Following the example in Figure 17-10 of the textbook, extrapolate the baseline from the
region where there is no reaction (–0.4 to 0 V) into the region of the current plateau (~0.8 V).
Measure the peak current on each curve with respect to the baseline. The very shallow peak
shifts to higher potential as more acid is added.
2. Prepare a graph of Equation 5-8 of the textbook, such as Figure 5-6. The spreadsheet in
Figure 5-5 is handy for this purpose.
3. Use the method of least squares to find the equation of the straight line and compute the
x-intercept (where y = 0), which is the concentration of ascorbic acid in the original unknown.
4. Estimate the uncertainty in the x-intercept with the following equation:

sy 1 y- 2
Standard deviation of x-intercept = m n +
m2 Σ(xi – x- )2
A E

where sy is the standard deviation of y (Equation 4-15), m is the slope of the least-squares line
(Equation 4-12), n is the number of data points (including the unknown plus the standard
additions), y- is the average value of y for all data points, and x- is the average value of x for all
A
E

A A
E

data points. The sum inside the square root extends over all data points. If you measured the
unknown plus five standard additions, n = 6 and the sum includes six terms.

80
20. Polarographic Measurement of an Equilibrium Constant Exploring Chemical Analysis

20. Polarographic Measurement of an Equilibrium Constant 36 35F

In this experiment, you will find the overall formation constant (βp) and
stoichiometry for the reaction of oxalate with Pb2+:

2- 2-2 p [Pb(C 2 O 4 ) 2p− 2 p ]

Pb2+ + pC2 O4 Pb(C2O4 ) p βp =
[Pb 2 + ][C 2 O 24 − ] p
Green Profile
See Section 0
where p is a stoichiometry coefficient. We will do this by measuring the
polarographic half-wave potential for solutions containing Pb2+ and various amounts of oxalate.
Polarography is described in Section 17-4 of the textbook. The change of half-wave potential,
∆E1/2 [= E1/2(observed) – E1/2(for Pb2+ without oxalate)] is expected to obey the equation

RT pRT 2-
∆E1/2 = – nF lnβp – nF ln[C2 O4 ]
A E A A E A (1)

where R is the gas constant, F is the Faraday constant, and T is temperature in kelvins. You
should measure the lab temperature at the time of the experiment or use a thermostatically
controlled cell.
An electrode reaction is considered to be reversible when it is fast enough to maintain
equilibrium at the electrode surface. The shape of a reversible polarographic wave is given by

RT  I 
E = E1/2 – nF lnI – I (2)
d 
A E A

where I is current and Id is diffusion current (the current at the plateau of the wave).

Procedure

1. Pipet 1.00 mL of 0.020 M Pb(NO3)2 into each of five 50-mL volumetric flasks labeled A–E
and add 1 drop of 1 wt% Triton X-100 to each. Then add the following solutions and dilute
to the mark with water. The KNO3 may be delivered carefully with a graduated cylinder.
The oxalate should be pipetted.

36 W. C. Hoyle and T. M. Thorpe, J. Chem. Ed. 1978, 55, A229.

81
20. Polarographic Measurement of an Equilibrium Constant Exploring Chemical Analysis

A: Add nothing else. Dilute to the mark with 1.20 M KNO3.

B: Add 5.00 mL of 1.00 M K2C2O4, and 37.5 mL of 1.20 M KNO3.
C: Add 10.00 mL of 1.00 M K2C2O, and 25.0 mL of 1.20 M KNO3.
D: Add 15.00 mL of 1.00 M K2C2O4 and 12.5 mL of 1.20 M KNO3.
E: Add 20.00 mL of 1.00 M K2C2O4.
2. Transfer each solution to a polarographic cell, deoxygenate with bubbling N2 for 10 min, and
record the polarogram from –0.20 to –0.95 V (versus S.C.E.). Measure the residual current,
using the same settings and a solution containing just 1.20 M KNO3 (plus 1 drop of 1 wt%
Triton X-100). Record each polarogram on a scale sufficiently expanded to allow accurate
measurements.
3. For each polarogram, make a graph of E versus ln[I/(Id – I)], using 6–8 points for each graph.
Be sure to subtract the residual current at each potential. According to Equation 2, E = E1/2
when ln[I/(Id – I)]] = 0. Use this condition to locate E1/2 on each graph.
4. Make a graph of ∆E1/2 versus ln[C 2O2-
4 ]. From the slope, use Equation 1 to find p, the
stoichiometry coefficient. Then use the intercept to find the value of βp. Use the method of
least squares to find the standard deviations of the slope and intercept. From the standard
deviations, find the uncertainties in p and βp and express each with the correct number of
significant figures.

82
21. Coulometric Titration of Cyclohexene with Bromine Exploring Chemical Analysis

21. Coulometric Titration of Cyclohexene with Bromine 37 36F

Coulometry is described in Section 17-1 of the textbook. In this experiment,

cyclohexene will be titrated with Br2 generated by electrolytic oxidation of
Br-:

2Br- → Br2 + 2e- (1)

Green Profile
See Section 0 Br

Br2 + (2)

Br
Cyclohexene trans-1,2-Dibromocyclohexane

The initial solution contains an unknown quantity of cyclohexene and a large amount of Br-.
When Reaction 1 has generated just enough Br2 to react with all the cyclohexene, the moles of
electrons liberated in Reaction 1 are equal to twice the moles of Br2 and therefore twice the
moles of cyclohexene.
The reaction is carried out at a constant current with the apparatus in Figure 1. Br2
generated at the Pt anode at the left immediately reacts with cyclohexene. When cyclohexene is
consumed, the concentration of Br2 suddenly rises, signaling the end of the reaction.
The equation relating moles of electrons to the electric current and the time is

Relation between electrons, I.t

Moles of e- = F (3)
current, and time:
A E A

where I is the current in amperes (= coulombs/s = C/s), t is the time in seconds, and F is the
Faraday constant (96 485 C/mol).
The rise in Br2 concentration is detected by measuring the current between the two
detector electrodes at the right in Figure 1. A voltage of 0.25 V applied between these two
electrodes is not enough to electrolyze any solute, so only a tiny current of <1 µA flows through
the microammeter. At the equivalence point, cyclohexene is consumed, [Br2] suddenly
increases, and detector current flows by virtue of the reactions:

Detector anode: 2Br- → Br2 + 2e-

Detector cathode: Br + 2e- → 2Br-
2

37 D. H. Evans, J. Chem. Ed. 1968, 45, 88.

83
21. Coulometric Titration of Cyclohexene with Bromine Exploring Chemical Analysis

Figure 1. Apparatus for coulometric titration of cyclohexene with Br2.

In our procedure, enough Br2 is first generated in the absence of cyclohexene to give a
detector current of 20.0 µA. When cyclohexene is added, the current decreases to a tiny value
because Br2 is consumed. Br2 is then generated by the coulometric circuit, and the end point is
taken when the detector again reaches 20.0 µA. Because the reaction is begun with Br2 present,
impurities that can react with Br2 before analyte is added are eliminated.
The electrolysis current (not to be confused with the detector current) for the
Br2-generating electrodes can be controlled by a hand-operated switch. As the detector current
approaches 20.0 µA, you close the switch for shorter and shorter intervals. This practice is
analogous to adding titrant dropwise from a buret near the end of a titration. The switch in the
coulometer circuit serves as a “stopcock” for addition of Br2 to the reaction.

84
21. Coulometric Titration of Cyclohexene with Bromine Exploring Chemical Analysis

EXAMPLE Coulometric Titration

A 2.000-mL volume containing 0.611 3 mg of cyclohexene/mL is to be titrated with a constant
current of 4.825 mA. How much time will required for complete titration?

Solution The quantity of cyclohexene is

(2.000 mL)(0.611 3 mg/mL)

A

E(82.146 mg/mmol) = 0.014 88 mmol

E A

In Reactions 1 and 2, each mole of cyclohexene requires 1 mol of Br2, which requires 2 mol of
electrons. For 0.014 88 mmol of cyclohexene to react, 0.029 76 mmol of electrons must flow.
From Equation 3,

I.t (moles of e-)F (0.029 76 × 10-3 mol)(96 485 C/mol)

Moles of e- = F ⇒ t = = = 595.1 s
I (4.825 × 10-3 C/s)
A E A A E A A E A

E
E

It will require just under 10 min to complete the reaction.

For this experiment, you can use a commercial coulometer or the circuits in Figure 2. 38 37F

Start timing with a stopwatch when the generator switch is closed.

Figure 2. Circuits for coulometric titrations. (a) Generator circuit. (b) Detector circuit.

38 A constant-current circuit for coulometer generator electrodes is given by J. Swim, E. Earps, L. M. Reed, and D.
Paul, J. Chem. Ed. 1996, 73, 679. An operational amplifier circuit for the detector and a circuit for controlled-
potential coulometry are given by E. Grimsrud and J. Amend, J. Chem. Ed. 1979, 56, 131.

85
21. Coulometric Titration of Cyclohexene with Bromine Exploring Chemical Analysis

Procedure

1. The electrolyte is a 60:26:14 (vol/vol/vol) mixture of acetic acid, methanol, and water. The
solution contains 0.15 M KBr and 0.1 g of mercuric acetate per 100 mL. (The latter catalyzes
the reaction between Br2 and cyclohexene.) The electrodes should be covered with
electrolyte. Begin vigorous magnetic stirring (without spattering) and adjust the voltage of
the detector circuit to 0.25 V.
2. Generate Br2 with the generator circuit until the detector current is 20.0 µA. The generator
current should be ~5–10 mA.
3. Pipet 2–5 mL of unknown (containing 1–5 mg of cyclohexene in methanol) into the flask and
set the clock or coulometer to 0. The detector current should drop to near 0 because the
cyclohexene consumes the Br2.
4. Turn the generator circuit on and begin timing. While the reaction is in progress, measure the
voltage (E) across the precision resistor (R = 100.0 ± 0.1 Ω) to find the exact current (I)
flowing through the cell (I = E/R). Continue the electrolysis until the detector current rises to
20.0 µA. Then stop the coulometer and record the time.
5. Repeat the procedure two more times and find the average molarity (and relative standard
deviation) of cyclohexene.
6. When you are finished, be sure all switches are off. Soak the generator electrodes in 8 M
HNO3 to dissolve Hg that is deposited during the electrolysis.

86
22. Spectrophotometric Determination of Iron in Vitamin Tablets Exploring Chemical Analysis

22. Spectrophotometric Determination of Iron in Vitamin Tablets 39 38F

In this procedure, iron from a vitamin supplement tablet is dissolved in acid,

reduced to Fe2+ with hydroquinone, and complexed with o-phenanthroline to
form an intensely colored complex (Color Plate 13 in the textbook).

Green Profile 2Fe3+ + HO OH 2Fe2+ + O O + 2H+

See Section 0

Hydroquinone Quinone
`` FM 110.11

N N
3 + Fe 2+
Fe2+
N N

3
o-Phenanthroline λmax = 510 nm
FM 180.21 Red complex
Reagents

Hydroquinone: (20 mL/student) Freshly prepared solution containing 10 g/L in distilled water.
Store in an amber bottle.
Trisodium citrate: (20 mL/student) 25 g/L Na2citrate . 2H2O (FM 294.10) in distilled water.
o-Phenanthroline: (25 mL/student) Dissolve 2.5 g in 100 mL of ethanol and add 900 mL of
distilled water. Store in an amber bottle.
6 M HCl: (25 mL/student) Dilute 124 mL of 37 wt% HCl up to 250 mL with distilled water.
Standard Fe (0.04 mg Fe/mL): (35 mL/student) Dissolve 0.281 g of reagent-grade
Fe(NH4)2(SO4)2 . 6H2O (FM 392.14) in distilled water in a 1-L volumetric flask containing
1 mL of 98 wt% H2SO4.

Procedure

1. Place one tablet of the iron-containing vitamin in a 125-mL flask or 100-mL beaker and boil
gently (in a fume hood) with 25 mL of 6 M HCl for 15 min. Filter the solution directly into a

39 R. C. Atkins, J. Chem. Ed. 1975, 52, 550

87
22. Spectrophotometric Determination of Iron in Vitamin Tablets Exploring Chemical Analysis

100-mL volumetric flask. Wash the beaker and filter several times with small portions of
water to complete a quantitative transfer. Allow the solution to cool, dilute to the mark and
mix well. Dilute 5.00 mL of this solution to 100.0 mL in a fresh volumetric flask. If the
label indicates that the tablet contains <15 mg of Fe, use 10.00 mL instead of 5.00 mL.
2. Pipet 10.00 mL of standard Fe solution into a beaker and measure the pH (with pH paper or a
glass electrode). Add sodium citrate solution 1 drop at a time until a pH of ~3.5 is reached.
Count the drops needed. (It will require about 30 drops.)
3. Pipet a fresh 10.00-mL aliquot of Fe standard into a 100-mL volumetric flask and add the
same number of drops of citrate solution as required in step 2. Add 2.00 mL of hydroquinone
solution and 3.00 mL of o-phenanthroline solution, dilute to the mark with water, and mix
well.
4. Prepare three more solutions from 5.00, 2.00, and 1.00 mL of Fe standard and prepare a blank
containing no Fe. Use sodium citrate solution in proportion to the volume of Fe solution. (If
10 mL of Fe requires 30 drops of citrate solution, 5 mL of Fe requires 15 drops of citrate
solution.)
5. Determine how many drops of citrate solution are needed to bring 10.00 mL of the iron tablet
solution from step 1 to pH 3.5. This will require about 3.5 or 7 mL of citrate, depending on
whether 5 or 10 mL of unknown was diluted in the second part of step 1.
6. Transfer 10.00 mL of solution from step 1 to a 100-mL volumetric flask. Add the required
amount of citrate solution determined in step 5. Then add 2.00 mL of hydroquinone solution
and 3.0 mL of o-phenanthroline solution. Dilute to the mark and mix well.
7. Allow the solutions to stand for at least 10 min. Then measure the absorbance of each
solution at 510 nm in a 1-cm cell. (The color is stable, so all solutions may be prepared and
all the absorbances measured at once.) Use distilled water in the reference cuvet and subtract
the absorbance of the blank from the absorbance of the Fe standards.
8. Make a graph of absorbance versus micrograms of Fe in the standards. Find the slope and
intercept (and standard deviations) by the method of least squares. Calculate the molarity of
Fe(o-phenanthroline )23 + in each solution and find the average molar absorptivity (ε in Beer's
law) from the four absorbances. (Remember that all the iron has been converted to the
phenanthroline complex.)
9. Using the calibration curve, find the number of milligrams of Fe in the tablet. Use Equation
4-16 in the textbook to find the uncertainty in the number of milligrams of Fe.

88
23. Microscale Spectrophotometric Measurement of Iron in Foods Exploring Chemical Analysis
by Standard Addition

23. Microscale Spectrophotometric Measurement of Iron in Foods by

Standard Addition 40 39F

This microscale experiment uses the same chemistry as that of Experiment

22 to measure iron in foods such as broccoli, peas, cauliflower, spinach,
beans, and nuts. A possible project is to compare processed (canned or
frozen) vegetables to fresh vegetables. Instructions are provided for small
volumes, but the experiment can be scaled up to fit available equipment.
Green Profile
See Section 0 Section 5-3 in the textbook describes the method of standard addition.

Reagents

2.0 M HCl: 15 mL/student. Dilute 165 mL of concentrated (37 wt%) HCl up to 1 L.

Hydroquinone: 4 mL/student; prepared as in Experiment 22.
Trisodium citrate dehydrate: 4 g/student.
o-Phenanthroline: 4 mL/student; prepared as in Experiment 22.
Standard Fe (40 µg Fe/mL): 4 mL/student; prepared as in Experiment 22.
6 M HCl: Dilute 500 mL of 37 wt% HCl up to 1 L with distilled water. Store in a bottle and
reuse many times for soaking crucibles.

Procedure

1. Fill a clean porcelain crucible with 6 M HCl in the hood and allow it to stand for 1 h to
remove traces of iron from previous use. Rinse well with distilled water and dry. After
weighing the empty crucible, add 5–6 g of finely chopped food sample and weigh again to
obtain the mass of food. (Some foods, like frozen peas, should not be chopped because they
will lose their normal liquid content.)
2. This step could require 3 h, during which you can be doing other lab work. Carefully heat the
crucible with a Bunsen burner in a hood (Experiment 3, Figure 1). Use a low, flame to dry
the sample, being careful to avoid spattering. Increase the flame temperature to char the
sample. Keep the crucible lid and tongs nearby. If the sample bursts into flames, use tongs
to place the lid on the crucible to smother the flame. After charring, use the hottest possible

40 Idea based on P. E. Adams, J. Chem. Ed. 1995, 72, 649.

89
23. Microscale Spectrophotometric Measurement of Iron in Foods Exploring Chemical Analysis
by Standard Addition

flame to ignite the black solid, converting it to white ash. The bottom of crucible should be
red hot during ignition. Continue ignition until all traces of black disappear.
3. After cooling the crucible to room temperature, add 10.00 mL of 2.0 M HCl by pipet and
swirl gently for 5 min to dissolve the ash. Filter the mixture through a small filter and collect
the filtrate in a vial or small flask. You need to recover >8 mL for the analysis.
4. Weigh 0.71 g of trisodium citrate dehydrate into each of four 10-mL volumetric flasks.
Using a 2-mL volumetric pipet or a 1-mL micropipet, add 2.00 mL of ash solution to each
flask. Add 4 mL of distilled water and swirl to dissolve the citrate. The solution will have a
pH near 3.6. Using a micropipet, add 0.20 mL of hydroquinone solution and 0.30 mL of
phenanthroline solution to each flask.
5. Label the volumetric flasks 0 through 3. Add no Fe standard to flask 0. Using a micropipet,
add 0.250 mL of Fe standard to flask 1. Add 0.500 mL of Fe standard to flask 2 and 0.750
mL to flask 3. The four flasks now contain 0, 1, 2, and 3 µg Fe/mL, in addition to Fe from the
food. Dilute each to the mark with distilled water, mix well, and allow 15 min to develop
full color.
6. Prepare a blank by mixing 0.71 g of trisodium citrate dehydrate, 2.00 mL of 2.0 M HCl, 0.20
mL of hydroquinone solution, and 0.30 mL of phenanthroline solution and diluting to 10 mL.
The blank does not require a volumetric flask.
7. Measure the absorbance of each solution at 512 nm in a 1-cm cell with distilled water in the
reference cell. Before each measurement, remove all liquid from the cuvet with a Pasteur
pipet. Then use ~1 mL of your next solution (delivered with a clean, dry Pasteur pipet) to
wash the cuvet. Remove and discard the washing. Repeat the washing once more with fresh
solution and discard the washing. Finally, add your new solution to the cuvet for measuring
absorbance.
8. Subtract the absorbance of the blank from each reading and make a graph like that in Figure
5-5 in the textbook to find the Fe content of the unknown solution. Note that when all
solutions have the same final volume (as they do in this experiment), the functions to plot in
the standard addition graph are the absorbance on the y-axis and the final concentration of
added standard Fe on the x-axis. Calculate the wt% of Fe in the food.
9. Estimate the uncertainty in wt% Fe from the uncertainty in the x-intercept of the least-squares
line in the standard addition graph. If you fit four points (unknown plus three standards), the
uncertainty in the x-intercept (the standard deviation of [X]f) is

90
23. Microscale Spectrophotometric Measurement of Iron in Foods Exploring Chemical Analysis
by Standard Addition

Standard deviation sy
A

of intercept = E A A n(intercept)2 – 2(intercept)Σxi + Σ(x2i )

E E A (1)
m D

where sy is the standard deviation of y (Equation 4-15 in the textbook), m is the slope of the least-

squares line (Equation 4-12), D is given by Equation 4-14, n is the number of points on the
graph, including the unknown (4 in this experiment), intercept is the x-intercept, and xi are the

values of x for the 4 points.

91
24. Spectrophotometric Determination of Nitrite in Aquarium Water Exploring Chemical Analysis

24. Spectrophotometric Determination of Nitrite in Aquarium Water

Background for this experiment29,30 is found in Box 6-1 and Section 18-4.

Green Profile
See Section 0

Reagents

Sodium nitrite: 1 g NaNO2 (FM 69.00)/student.

Sodium oxalate: 4 g Na2C2O4 (FM 134.00)/student.
Potassium permanganate: 1 g KMnO4 (FM 158.03)/student.
0.9 M H2SO4: 1 L/student.
Concentrated (96 wt%) H2SO4: 20 mL/student.
Color-forming reagent: (Reaction 18-7; 15 mL/student) Mix 1.0 g of sulfanilamide, 0.10 g of
N-(1-naphthyl)ethylenediamine dihydrochloride, and 10 mL of 85 wt% H3PO4 and dilute
with distilled water to 100 mL. Store in a dark bottle in the refrigerator.

Procedure

1. Prepare and standardize 0.01 M KMnO4 solution as described in Experiment 13. Reduce the
quantities of KMnO4 and Na2C2O4 by a factor of 2.
2. Prepare 0.018 M NaNO2 by dissolving 0.62 g of NaNO2 in 500 mL of distilled water.
3. Standardize the NaNO2 as described in Section 6-3 of the textbook.
4. Withdraw ~30 mL of aquarium water and filter it to remove suspended solids. Analyze
duplicate 10.00-mL aliquots of freshly drawn aquarium water by the procedure in Section
18-4 of the textbook. Use three or four standard points for the calibration curve such that the
unknown lies within the range of the standards (Figure 18-11 of the textbook).

92
25. Spectrophotometric Measurement of an Equilibrium Constant: Exploring Chemical Analysis
The Scatchard Plot

25. Spectrophotometric Measurement of an Equilibrium Constant: The

Scatchard Plot

In this experiment, we will use the Scatchard plot described below to find the
equilibrium constant for the formation of a complex between iodine and
pyridine in cyclohexane: 41 40F

Green Profile I2 + N I2 N
See Section 0
Iodine-pyridine
complex

Both I2 and I2 . pyridine absorb visible radiation, but pyridine is colorless. Analysis of the
spectral changes associated with variation of pyridine concentration (with a constant total
concentration of iodine) allows us to evaluate K for the reaction. The experiment is best
performed with a recording spectrophotometer, but single-wavelength measurements can be
used.

Scatchard Plot

Consider the equilibrium in which the species P and X react to form PX.

P+X PX (1)

Neglecting activity coefficients, we can write

[PX]
K = [P][X]
A E A (2)

Consider a series of solutions in which increments of X are added to a constant amount of P.

Letting Po be the total concentration of P (in the forms P and PX), we can write

[P] = Po – [PX]

Now the equilibrium expression, Equation 2, can be rearranged as follows:

41 For literature values of the equilibrium constant for the reaction between I and pyridine, see S. S. Barton and R.
2
H. Pottier, J. Chem. Soc. Perkin Trans. II 1984, 731.

93
25. Spectrophotometric Measurement of an Equilibrium Constant: Exploring Chemical Analysis
The Scatchard Plot

[PX]
A

[X] = K[P] = K(Po – [PX])

E A (3)

A graph of [PX]/[X] versus [PX] will have a slope of –K and is called a Scatchard plot. It is
widely used in biochemistry to measure equilibrium constants.
If we know [PX], we can find [X] with the mass balance

Xo = [total X] = [PX] + [X]

To measure [PX], we might use spectrophotometric absorbance. Suppose that P and PX each
have some absorbance at wavelength λ, but X has no absorbance at this wavelength. For
simplicity, let all measurements be made in a cell of pathlength 1.000 cm so that we can omit b
(= 1.000 cm) when writing Beer's law.
The absorbance at some wavelength is the sum of absorbances of PX and P:

A = εPX[PX] + εP[P]

Substituting [P] = Po – [PX], we can write

A = εPX[PX] + εPPo – εP[PX]

Ao

But εPPo is Ao, the initial absorbance before any X is added. Therefore,

∆A
A = [PX](εPX – εP) + Ao ⇒ [PX] = (4)
∆ε
A A

where ∆ε = εPX – εP and ∆A (= A – Ao) is the observed absorbance after each addition of X
minus the initial absorbance.
Substituting [PX] from Equation 4 into Equation 3 gives

∆A
Scatchard equation: [X] = K∆εPo – K∆A
A E A (5)

A graph of ∆A/[X] versus ∆A should be a straight line with a slope of –K. Absorbance measured
while P is titrated with X can be used to find K for the reaction of X with P.

94
25. Spectrophotometric Measurement of an Equilibrium Constant: Exploring Chemical Analysis
The Scatchard Plot

Procedure

All operations should be carried out in a fume hood, including pouring solutions into and out of
the spectrophotometer cell. Only a capped cuvet containing the solution whose spectrum is to be
measured should be taken from the hood. Do not spill solvent on your hands or breathe the
vapors. Used solutions should be discarded in a waste container in the hood, not down the drain.

1. The following stock solutions should be available in the lab:

a. 0.050–0.055 M pyridine in cyclohexane (40 mL for each student, concentration known
accurately).
b. 0.012 0–0.012 5 M I2 in cyclohexane (10 mL for each student, concentration known
accurately).
2. Pipet the following volumes of stock solutions into six 25-mL volumetric flasks labeled
A–F, dilute to the mark with cyclohexane, and mix well.

Pyridine stock I2 stock

Flask solution (mL) solution (mL)
A 0 1.00
B 1.00 1.00
C 2.00 1.00
D 4.00 1.00
E 5.00 1.00
F 10.00 1.00

3. Using glass or quartz cells, record a baseline between 350 and 600 nm with solvent in both
the sample and the reference cells. Subtract the absorbance of the baseline from all future
absorbances. If possible, record all spectra, including the baseline, on one sheet of chart
paper. (If a fixed-wavelength instrument is used, first find the positions of the two absorbance
maxima in solution E. Then make all measurements at these two wavelengths.)
4. Record the spectrum of each solution A–F or measure the absorbance at each maximum if a
fixed-wavelength instrument is used.

95
25. Spectrophotometric Measurement of an Equilibrium Constant: Exploring Chemical Analysis
The Scatchard Plot

Data Analysis

1. Measure the absorbance at the wavelengths of the two maxima in each spectrum. Be sure to
subtract the absorbance of the blank from each.
2. The analysis of this problem follows that of Reaction 1, in which P is iodine and X is
pyridine. As a first approximation, assume that the concentration of free pyridine equals the
total concentration of pyridine in the solution (because [pyridine] >> [I2]). Prepare a graph of
∆A/[free pyridine] versus ∆A (a Scatchard plot, Equation 5), using the absorbance at the
I2 . pyridine maximum.
3. From the slope of the graph, find the equilibrium constant by using Equation 5. From the
intercept, find ∆ε (= εPX – εX).
4. Now refine the values of K and ∆ε. Use ∆ε to find εPX. Then use the absorbance at the
wavelength of the I2 . pyridine maximum to find the concentration of bound and free pyridine
in each solution. Make a new graph of ∆A /[free pyridine] versus ∆A, using the new values of
[free pyridine]. Find a new value of K and ∆ε. If justified, perform another cycle of
refinement.
5. Using the values of free pyridine concentration from your last refinement and the values of
absorbance at the I2 maximum, prepare another Scatchard plot and see if you get the same
value of K.
6. Explain why an isosbestic point is observed in this experiment.

96
26. Spectrophotometric Analysis of a Mixture: Exploring Chemical Analysis
Caffeine and Benzoic Acid in a Soft Drink

26. Spectrophotometric Analysis of a Mixture: Caffeine and Benzoic Acid in

a Soft Drink42 41F

In this experiment we use ultraviolet absorbance (Figure 1) to measure two

major species in soft drinks. Caffeine is added as a stimulant and sodium
benzoate is a preservative.

O CH3
Green Profile H3C N
See Section 0 N CO2H
O N N
CH3
Caffeine Benzoic acid (pKa = 4.20)
FM 182.18 FM 122.12

All solutions will contain 0.010 M HCl, so the sodium benzoate is protonated to make benzoic
acid. Caffeine has no appreciable basicity, so it is neutral at pH 2.

Figure 1. Ultraviolet
absorption of benzoic
acid, caffeine, and a
1:50 dilution of
Mountain Dew soft
drink. All solutions
λ' contain 0.010 M HCl.

λ"

42 V. L. McDevitt, A. Rodriquez and K. R. Williams, J. Chem. Ed. 1998, 75, 625.

97
26. Spectrophotometric Analysis of a Mixture: Exploring Chemical Analysis
Caffeine and Benzoic Acid in a Soft Drink

We restrict ourselves to non-diet soft drinks because the sugar substitute aspartame in diet
soda has some ultraviolet absorbance that slightly interferes in the present experiment. We also
avoid darkly colored drinks because the colorants have ultraviolet absorbance. Mountain Dew,
Mello Yello, and, probably, other lightly colored drinks are suitable for this experiment. There is
undoubtedly some ultraviolet absorbance from colorants in these beverages that contributes to
systematic error to this experiment.
The procedure we describe includes the construction of calibration curves. The
experiment could be shortened by recording just one spectrum of caffeine (20 mg/L) and one of
benzoic acid (10 mg/L) and assuming that Beer's law is obeyed. The experiment could be
expanded to use high-performance liquid chromatography (HPLC) and/or capillary electro-
phoresis to obtain independent measurements of caffeine and benzoic acid (and aspartame in diet
drinks).42

Reagents

Stock solutions: An accurately known solution containing ~100 mg benzoic acid/L in water and
another containing ~200 mg caffeine/L should be available.
0.10 M HCl: Dilute 8.2 mL of 37 wt% HCl to 1 L.

Procedure

1. Calibration standards: Prepare benzoic acid solutions containing 2, 4, 6, 8 and 10 mg/L in

0.010 M HCl. To prepare a 2 mg/L solution, mix 2.00 mL of benzoic acid standard plus 10.0
mL of 0.10 M HCl in a 100-mL volumetric flask and dilute to the mark with water. Use 4, 6,
8 and 10 mL of benzoic acid to prepare the other standards. In a similar manner, prepare
caffeine standards containing 4, 8, 12, 16 and 20 mg/L in 0.010 M HCl.
2. Soft drink: Warm ~20 mL of soft drink in a beaker on a hot plate to expel CO2 and filter the
warm liquid through filter paper to remove any particles. After cooling to room temperature,
pipet 4.00 mL into a 100-mL volumetric flask. Add 10.0 mL of 0.10 M HCl and dilute to the
mark. Prepare a second sample containing 2.00 mL of soft drink instead of 4.00 mL.
3. Verifying Beer's law: Record an ultraviolet baseline from 350 to 210 nm with water in the
sample and reference cuvets (1.000 cm pathlength). Record the ultraviolet spectrum of each
of the 10 standards with water in the reference cuvet. Note the wavelength of peak

98
26. Spectrophotometric Analysis of a Mixture: Exploring Chemical Analysis
Caffeine and Benzoic Acid in a Soft Drink

absorbance for benzoic acid (λ') and the wavelength for the peak absorbance of caffeine (λ").
Measure the absorbance of each standard at both wavelengths and subtract the baseline
absorbance (if your instrument does not do this automatically). Prepare a calibration graph of
absorbance versus concentration (M) for each compound at each of the two wavelengths.
Each graph should go through 0. The least-squares slope of the graph is the molar
absorptivity at that wavelength.
4. Unknowns: Measure the ultraviolet absorption spectrum of the 2:100 and 4:100 dilutions of
the soft drink. With the absorbance at the wavelengths λ' and λ", use Equations 19-5 in the
textbook to find the concentrations of benzoic acid and caffeine in the original soft drink.
Report results from both dilute solutions.
5. Synthetic unknown: If your instructor chooses, measure the spectrum of a synthetic,
unknown mixture of benzoic acid and caffeine prepared by the instructor. Use Equations
19-5 in the textbook to find the concentration of each component in the synthetic unknown.

99
27. Mn2+ Standardization by EDTA Titration Exploring Chemical Analysis

27. Mn2+ Standardization by EDTA Titration

Experiments 27-29 illustrate a sequence in which students (1) prepare and

standardize a Mn2+ solution by EDTA titration and then (2) use this standard
in the analysis of Mn in steel by two different instrumental techniques. 43 42F

Green Profile
See Section 0

Reagents

MnSO4 . H2O: (1 g/student) This material is not a primary standard.

EDTA: Na2H2EDTA . 2H2O, 1 g/student.
0.50 M NH3/1.1 M NH+4 buffer (pH 9.7): Mix 6.69 g of NH4Cl (FM 53.49) plus 16.86 g of 28%
A
E

aqueous NH3 (FM 17.03) with enough water to give a total volume of 250 mL.
Hydroxylamine hydrochloride: (NH3OH+Cl-, FM 69.49) 1 g/student. (CAUTION: Do not
breathe dust from NH3OH+Cl-. Avoid contact with skin and eyes.)
Calmagite indicator: Dissolve 0.05 g in 100 mL H2O.

Procedure

1. Standard 0.005 M EDTA: Dry Na2H2EDTA . 2H2O (FM 372.25) at 80˚C for 1 h and cool in
a desiccator. Accurately weigh out ~0.93 g and dissolve it with heating in 400 mL of
distilled water in a 500-mL volumetric flask. Cool to room temperature, dilute to the mark,
and mix well.
2. Mn2+ stock solution: Prepare a solution containing ~1.0 mg Mn/mL (~0.018 M) by
dissolving ~0.77 g MnSO4.H2O (FM 169.01) in a clean plastic screw cap bottle with 250 mL
water delivered from a graduated cylinder. Masses and volumes need not be accurate
because you will standardize this solution.
3. Rinse a clean 50-mL pipet several times with small volumes of Mn2+ stock solution and
discard the washings into a chemical waste container. Then pipet 50.00 mL of Mn2+ stock
solution into a 250-mL volumetric flask. Add 0.80 g of solid hydroxylamine hydrochloride

43 Adapted from San Jose State University Laboratory Manual for a curriculum described by S. P. Perone, J. Pesek,
C. Stone, and P. Englert, J. Chem. Ed. 1998, 75, 1444.

100
27. Mn2+ Standardization by EDTA Titration Exploring Chemical Analysis

to the flask, and swirl to dissolve the solid. Add ~150 mL of water and swirl to mix the
contents. Dilute to the mark with water, set the cap firmly in place, and invert 20 times to
mix the solution. This solution contains ~0.0036 M Mn2+. The reducing agent,
hydroxylamine, maintains manganese in the +2 state.
4. Rinse a 50-mL pipet several times with small volumes of the diluted Mn2+ solution from step
3. Pipet 50 mL of the diluted Mn2+ solution into a 250-mL Erlenmeyer flask Add 10 mL of
pH 9.7 buffer (by graduated cylinder), and add 3-5 drops of calmagite indicator. The pH of
the solution will be near 9.2. Titrate with standard EDTA from a 50-mL buret and note the
end point when the color changes from wine red to blue.
5. Repeat step 4 twice more to obtain a total of three replicate titrations. The Erlenmeyer flask
must be clean, but it need not be dry for each new titration.
6. From the molarity and volume of standard EDTA required for titration, calculate the molarity
and standard deviation of the original ~0.018 M MnSO4 stock solution. Express your answer
with an appropriate number of significant digits.

101
28. Measuring Manganese in Steel by Exploring Chemical Analysis
Spectrophotometry with Standard Addition

28. Measuring Manganese in Steel by Spectrophotometry with Standard

Addition

Experiments 27-29 illustrate a sequence in which students (1) prepare and

standardize a Mn2+ solution and then (2) use this standard in the analysis of
Mn in steel by two different instrumental techniques.43 In this experiment,
steel is dissolved in acid and its Mn is oxidized to the violet-colored
permanganate ion, whose absorbance is measured with a spectrophotometer:
Green Profile
See Section 0
2Mn2+ + 5IO4- A E + 3H2O →
A 2MnO4- A E + 5IO3- + 6H+
A A E A

Periodate Permanganate Iodate

(colorless) (colorless) (violet) (colorless)
λmax ≈ 525 nm

Steel is an alloy of iron that typically contains ~0.5 wt% Mn plus numerous other
elements. When steel is dissolved in hot nitric acid, the iron is converted to Fe(III).
Spectrophotometric interference in the measurement of MnO4- by Fe(III) is minimized by adding
A
E

H3PO4 to form a nearly colorless complex with Fe(III). Interference by most other colored
impurities is eliminated by subtracting the absorbance of a reagent blank from that of the
unknown. Appreciable Cr in the steel will interfere with the present procedure. Carbon from the
steel is eliminated by oxidation with peroxydisulfate (S2O2-
8 ): A
E

8 + 2H2O →
C(s) + 2S2O2- CO2(g) + 4SO2- +
A E A

4 + 4H A E A

Reagents

3 M Nitric acid: (150 mL/student) Dilute 190 mL of 70 wt% HNO3 to 1 L with water.
0.05 M Nitric acid: (300 mL/student) Dilute 3.2 mL of 70 wt% HNO3 to 1 L with water.
Ammonium hydrogen sulfite: (0.5 mL/student) 45 wt% NH4HSO3 in water.
Potassium periodate (KIO4): 1.5 g/student
Unknowns: Steel, ~2 g/student. Analyzed samples are available from Thorn Smith.1

102
28. Measuring Manganese in Steel by Exploring Chemical Analysis
Spectrophotometry with Standard Addition

Procedure

1. Steel can be used as received or, if it appears to be coated with oil or grease, it should be
rinsed with acetone and dried at 110°C for 5 min, and cooled in a desiccator.
2. Weigh duplicate samples of steel to the nearest 0.1 mg into 250-mL beakers. The mass of
steel should be chosen to contain ~2–4 mg of Mn. For example, if the steel contains 0.5 wt%
Mn, a 0.6-g sample will contain 3 mg of Mn. Your instructor should give you guidance on
how much steel to use.
3. Dissolve each steel sample separately in 50 mL of 3 M HNO3 by gently boiling in the hood,
while covered with a watchglass. If undissolved particles remain, stop boiling after 1 h.
Replace the HNO3 as it evaporates.
4. Standard Mn2+ (~0.1 mg Mn/mL): While the steel is dissolving, pipet 10.00 mL of standard
Mn2+ (~1 mg Mn/mL) from Experiment 27 into a 100-mL volumetric flask, dilute to the
mark with water, and mix well. You will use this solution in Experiments 28 and 29. Keep it
stoppered, and wrap the stopper with Parafilm or tape to minimize evaporation.
5. Cool the beakers from step 3 for 5 min. Then carefully add ~1.0 g of (NH4)2S2O8 or K2S2O8
and boil for 15 min to oxidize carbon to CO2.
6. If traces of pink color (MnO4- ) or brown precipitate (MnO2(s)) are observed, add 6 drops of
A E A

45 wt% NH4HSO3 and boil for 5 min to reduce all manganese to Mn(II):

2MnO4- A E + 5HSO3-
A A E + H+
A → 2Mn2+ + 5SO2-
4 + 3H2O
A E A

MnO2(s) + HSO3- A E + H+
A → Mn2+ + SO2-
4 + H2O
A E A

(The purpose of removing colored species at this time is that the solution from step 6 is
eventually going to serve as a colorimetric reagent blank.)
7. After cooling the solutions to near room temperature, filter each solution quantitatively
through #41 filter paper into a 250-mL volumetric flask. (If gelatinous precipitate is present,
use #42 filter paper.) To complete a “quantitative” transfer, wash the beaker many times with
small volumes of hot 0.05 M HNO3 and pass the washings through the filter to wash liquid
from the precipitate into the volumetric flask. Finally, allow the volumetric flasks to cool to
room temperature, dilute to the mark with water, and mix well.
8. Transfer ~100 mL of solution from each 250-mL volumetric flask to clean, dry Erlenmeyer
flasks and stopper the flasks tightly. Label these solutions A and B and save them for atomic

103
28. Measuring Manganese in Steel by Exploring Chemical Analysis
Spectrophotometry with Standard Addition

absorption analysis in Experiment 29. To help prevent evaporation, it is a good idea to seal
around the stoppers with a few layers of Parafilm or tape.
9. Carry out the following spectrophotometric analysis with one of the unknown steel solutions
prepared in step 7:
a. Pipet 25.00 mL of liquid from the 250-mL volumetric flask in step 7 into each of three
clean, dry 100-mL beakers designated “blank,” “unknown,” and “standard addition.”
Add 5 mL of 85 wt% H3PO4 (from a graduated cylinder) into each beaker. Then add
standard Mn2+ (0.1 mg/mL from step 4, delivered by pipet) and solid KIO4 as follows:

Volume of Mass of
Beaker Mn2+ (mL) KIO4 (g)
Blank 0 0
Unknown 0 0.4
Standard addition 5.00 0.4

b. Boil the unknown and standard addition beakers gently for 5 min to oxidize Mn2+ to
MnO4- . Continue boiling, if necessary, until the KIO4 dissolves.
A E A

c. Quantitatively transfer the contents of each of the three beakers into 50-mL volumetric
flasks. Wash each beaker many times with small portions of water and transfer the water
to the corresponding volumetric flask. Dilute each flask to the mark with water and mix
well.
d. Fill one 1.000-cm-pathlength cuvet with unknown solution and another cuvet with blank
solution. It is always a good idea to rinse the cuvet a few times with small quantities of
the solution to be measured and discard the rinses.
e. Measure the absorbance of the unknown at 525 nm with blank solution in the reference
cuvet. For best results, measure the absorbance at several wavelengths to locate the
maximum absorbance. Use this wavelength for subsequent measurements.
f. Measure the absorbance of the standard addition with the blank solution in the reference
cuvet. The absorbance of the standard addition will be ~0.45 absorbance units greater
than the absorbance of the unknown (based on adding ~0.50 mg of standard Mn2+ to the
unknown).
10. Repeat step 9 with the other unknown steel solution from step 7.

104
28. Measuring Manganese in Steel by Exploring Chemical Analysis
Spectrophotometry with Standard Addition

Data Analysis

1. From the known concentration of the Mn standard in step 4, calculate the concentration of
added Mn in the 50-mL volumetric flask containing the standard addition.
2. All of the Mn2+ is converted to MnO4- in step 9. From the difference between the
A E A

absorbance of the standard addition and the unknown, calculate the molar absorptivity of
MnO4- . Compute the average molar absorptivity from steps 9 and 10.
A E A

3. From the absorbance of each unknown and the average molar absorptivity of MnO4-, calculate
A
E

the concentration of MnO4- in each 50-mL unknown solution.

A E A

4. Calculate the weight percent of Mn in each unknown steel sample and the percent relative
range of your results:

100 × [wt % in steel 1 – wt % in steel 2]

% relative range = A

mean wt % E

105
29. Measuring Manganese in Steel by Atomic Exploring Chemical Analysis
Absorption Using a Calibration Curve

29. Measuring Manganese in Steel by Atomic Absorption Using a

Calibration Curve

This experiment complements the results of the spectrophotometric analysis

in Experiment 28.43 In principle, the spectrophotometric analysis and the
atomic absorption analysis should give the same value for the weight percent
of Mn in the unknown steel. You will use Mn2+ that you standardized in
Experiment 27 as the standard for the atomic absorption analysis.
Green Profile
See Section 0

Reagents

0.05 M Nitric acid: (600 mL/student) Dilute 3.2 mL of 70 wt% HNO3 to 1 L with water.
Unknown steel: Solutions A and B from step 8 of Experiment 28.
Standard manganese: ~0.1 mg Mn/mL from step 4 of Experiment 28. This concentration
corresponds to ~100 µg/mL = ~100 ppm.

Calibration Curve

1. Prepare standard solutions containing ~1, 2, 3, 4 and 5 ppm Mn (= µg Mn/mL). Use your
standard solution containing ~100 ppm Mn from step 4 of Experiment 28. Pipet 1.00 mL of
the standard into a 100-mL volumetric flask and dilute to 100 mL with 0.05 M HNO3 to
prepare a 1-ppm standard. Similarly, pipet 2.00, 3.00, 4.00, and 5.00 mL into the other flasks
and dilute each to 100 mL with 0.05 M HNO3. Calculate the concentration of Mn in µg/mL
in each standard. (The purpose of the HNO3 is to provide H+ ions to compete with Mn2+
ions for binding sites on the glass surface. Without excess acid, some fraction of metal ions
from a dilute solution can be lost to the glass surface. To avoid adding impurity metal ions,
we use a dilute solution of the purest available acid.)
2. Measure the atomic absorption signal from each of the five standards in step 1. Use a Mn
hollow-cathode lamp and a wavelength of 279.48 nm. Measure each standard three times.
3. Measure the atomic absorption signal from a blank (0.05 M HNO3). We will use this signal
later to estimate the detection limit from Mn. For this purpose, measure a blank seven
separate times and compute the mean and standard deviation of the seven measurements.

106
29. Measuring Manganese in Steel by Atomic Exploring Chemical Analysis
Absorption Using a Calibration Curve

Measuring the Unknown

1. Immediately after measuring the points on the calibration curve, measure the atomic
absorption signal from unknown steel solutions A and B from step 8 of Experiment 28.
Measure the absorption of each solution three times. (If the signals from A and B do not lie
in the calibration range, dilute them as necessary so that they do lie in the calibration range.
Dilutions must be done accurately with volumetric pipets and volumetric flasks.)

Data Analysis

1. Make a calibration graph showing the blank plus 5 standards (7 blank readings and 3 × 5 = 15
standard readings, for a total of n = 22 points). Compute the least-squares slope and intercept
and their standard deviations (Section 4-6 of the textbook) and show the least-squares line on
the graph. Express the equation of the calibration curve in the form y (±sy) = [m (±sm)]x + [b
(±sb)], where y is the atomic absorbance signal and x is the concentration of Mn in ppm.
2. Use the mean value of the three readings for each unknown to calculate the concentration of
Mn solutions A and B.
3. Calculate the uncertainty in Mn concentration in each unknown from Equation 4-19 in the
textbook. Because you have measured each unknown three times, the first term in the radical
in Equation 4-19 should be 1/3. In Equation 4-19, x is the mean atomic absorption signal for
the unknown and there are 22 values of xi for the points on the standard curve.
4. From the Mn concentrations (and uncertainties) in solutions A and B, calculate the wt% Mn
(and its uncertainty) in the two replicate steel samples.
5. The uncertainty in wt% Mn is the standard deviation. Find the 95% confidence interval for
wt% Mn in each of the two steel samples that you analyzed. For example, suppose that you
find the wt% of Mn in steel to be 0.433, with a standard deviation of 0.011. (The subscripted
digits are not significant but are retained to avoid round-off errors.) The standard deviation
was derived from three replicate measurements of one solution of dissolved steel. The
equation for confidence interval is µ = x- ± ts/ n , where µ is the true mean, x- is the
A E A A E A A E A

measured mean, s is the standard deviation, n is the number of measurements (3 in this case)
and t is Student's t for 95% confidence and n – 1 = 2 degrees of freedom. In Table 4-4 of the
textbook we find t = 4.303. Therefore the 95% confidence interval is 0.433 ± ts/ n = A E A

0.433 ± (4.303)(0.011)/ 3 = 0.433 ± 0.027.

A E A

107
29. Measuring Manganese in Steel by Atomic Exploring Chemical Analysis
Absorption Using a Calibration Curve

6. Use the t test (Equation 4-4 in the textbook) to compare the two atomic absorption results to
each other. Are they significantly different at the 95% confidence level?
7. Use the mean wt% Mn for the two samples and the pooled standard deviation (Equation 4-6
in the textbook) to estimate a 95% confidence interval around the mean value. Does the
mean spectrophotometric value for wt% Mn from Experiment 28 lie within the 95%
confidence interval for the atomic absorption results? (We cannot use the t test to compare
Experiments 28 and 29 because we do not have enough samples in Experiment 28 to find a
standard deviation. Otherwise, we would use the t test.)
8. Detection limit: The detection limit of an analytical method is the minimum concentration of
analyte that can be “reliably” distinguished from 0. If you have measured points on a
calibration curve, one common definition of detection limit is

y- B + 3sB
Detection limit (ppm) = A

m
E
E

where y- B is the mean atomic absorbance reading for the blank, sB is the standard deviation
A
E

for the blank, and m is the least-squares slope of the calibration curve (absorbance/ppm). In
this experiment you measured a blank solution seven times. Use the mean and standard
deviation from these seven readings to calculate the detection limit. (If you subtracted the
mean value of the blank from each absorbance reading when you constructed the standard
curve, then y- B = 0.)
A
E

108
30. Properties of an Ion-Exchange Resin Exploring Chemical Analysis

30. Properties of an Ion-Exchange Resin 44 43F

This experiment explores the properties of a cation-exchange resin, which is

an organic polymer containing many sulfonic acid groups (—SO3H). When a
cation such as Cu2+ flows into the resin, the cation is tightly bound by
sulfonate groups, releasing one H+ for each positive charge bound to the

Green Profile
resin. Cu2+ can be displaced from the resin by a large excess of H+ or by an
See Section 0 excess of any other cation for which the resin has some affinity.

First, known quantities of NaCl, Fe(NO3)3, and NaOH will be passed through the resin in
the H+ form. The H+ released by each cation will be measured by titration with NaOH.
In the second part of the experiment, we analyze impure vanadyl sulfate (VOSO4 . 2H2O).
As supplied commercially, this salt contains VOSO4, H2SO4, and H2O. A solution will be
prepared from a known mass of reagent. The VO2+ content can be assayed spectrophotometric-
ally, and the total cation (VO2+ and H+) content can be assayed by ion exchange. Together, these
measurements enable us to establish the quantities of VOSO4, H2SO4, and H2O in the sample.

Reagents

Bio-Rad Dowex 50W-X2 (100/200 mesh) cation-exchange resin: 1.1 g/student.

0.3 M NaCl: (5–10 mL/student) Accurately weigh ~17.5 g of NaCl (FM 58.44) and dissolve it
in a 1-L volumetric flask.
0.1 M Fe(NO3)3 . 6H2O: (5–10 mL/student) Accurately weigh ~35 g of Fe(NO3)3 . 6H2O (FM
349.95) and dissolve it in a 1-L volumetric flask.

44 Part of this experiment is taken from M. W. Olson and J. M. Crawford, J. Chem. Ed. 1975, 52, 546.

109
30. Properties of an Ion-Exchange Resin Exploring Chemical Analysis

VOSO4: The commonly available grade (usually designated “purified”) is used for this
experiment. Students can make their own solutions and measure the absorbance at 750 nm,
or a bottle of stock solution (25 mL per student) can be supplied. The stock should contain 8
g/L (accurately weighed) and be labeled with the absorbance.
0.02 M NaOH: Each student should prepare an accurate 1/5 dilution of standard 0.1 M NaOH.
For example, 50.0 mL of 0.1 M NaOH can be pipetted into a 250-mL volumetric flask and
diluted to volume with distilled water.
0.1 M HCl: 50 mL/student.
Phenolphthalein indicator. Recipe in Experiment 7.

Procedure

1. Prepare a chromatography column from glass tubing that has a 0.7-cm diameter and is 15-cm
long by fitting the tubing at the bottom with a cork that has a small hole to serve as the outlet.
Place a small ball of glass wool above the cork to retain the resin. Use a glass rod to plug the
outlet and shut off the column. (Alternatively, an inexpensive column such as 0.7 × 15 cm
Econo-Column from Bio-Rad Laboratories 45 works well.) Fill the column with water, close
4F

it off, and test for leaks. Drain the water until 2 cm remains and close the column again.
2. Make a slurry of 1.1 g of Bio-Rad Dowex 50W-X2 (100/200 mesh) cation-exchange resin in
5 mL of water and pour it into the column (Figure 1). If the resin cannot be poured all at
once, allow some to settle, remove the supernatant liquid with a pipet, and pour in the rest of
the resin. If the column is stored between laboratory periods, it should be upright, capped,
and contain water above the level of the resin. (When the experiment is finished, the resin
can be collected, washed with 1 M HCl and water, and reused.)
3. The general procedure for analysis of a sample is as follows:
a. Generate the H+-saturated resin by passing ~10 mL of 1 M HCl through the column.
Apply the liquid sample to the glass wall so as not to disturb the resin.

45 Bio-Rad Laboratories, www.bio-rad.com.

110
30. Properties of an Ion-Exchange Resin Exploring Chemical Analysis

Figure 1. Pouring a slurry of

stationary phase into a
chromatography column.

b. Wash the column with ~15 mL of water. Use the first few milliliters to wash the glass
walls and allow the water to soak into the resin before continuing the washing. (Unlike
most chromatography resins, the one in this experiment retains water when allowed to run
“dry.” Ordinarily, you must not let liquid fall below the top of the solid phase in a
column.)
c. Place a clean 125-mL flask under the outlet and pipet the sample onto the column.
d. After the reagent has soaked in, wash it through with 10 mL of H2O, collecting all eluate.
e. Add 3 drops of phenolphthalein indicator to the flask and titrate with standard 0.02 M
NaOH.

111
30. Properties of an Ion-Exchange Resin Exploring Chemical Analysis

4. Analyze 2.000-mL aliquots of 0.3 M NaCl and 0.1 M Fe(NO3)3, following the procedure in
step 3. Calculate the theoretical volume of NaOH needed for each titration. If you do not
come within 2% of this volume, repeat the analysis.
5. Pass 10.0 mL of your 0.02 M NaOH through the column as directed in step 3, and titrate the
eluate. Explain what you observe.
6. Analyze 10.00 mL of VOSO4 solution as described in step 3.
7. Step 6 gives the total cation content (= VO2+ + 2H2SO4) of 10.00 mL of VOSO4 solution.
From the absorbance at 750 nm and the molar absorptivity of VO2+ (ε = 18.0 M-1.cm-1 at
750 nm), calculate the concentration of VO2+ in the solution. By difference, calculate the
concentration of H2SO4 in VOSO4 reagent. From the difference between the mass of
VOSO4 reagent that was used and the content of VOSO4 and H2SO4, calculate the mass of
H2O in the VOSO4 reagent. Express the composition of the vanadyl sulfate in the form
(VOSO4)1.00(H2SO4)x(H2O)y.

112
31. Analysis of Sulfur in Coal by Ion Chromatography Exploring Chemical Analysis

31. Analysis of Sulfur in Coal by Ion Chromatography 46 45F

When coal is burned, sulfur in the coal is converted to SO2(g), which is

further oxidized to H2SO4 in the atmosphere. Rainfall laden with H2SO4 is
harmful to plant life. Measuring the sulfur content of coal is therefore
important to efforts to limit man-made sources of acid rain. This experiment
measures sulfur in coal by first heating the coal in the presence of air in a
Green Profile
See Section 0 flux (Section 2-10 in the textbook) containing Na2CO3 and MgO, which
converts the sulfur to Na2SO4. The product is dissolved in water, and sulfate
ion is measured by ion chromatography. Although you will be given a sample of coal to analyze,
consider how you would obtain a representative sample from an entire trainload of coal being
delivered to a utility company.

Reagents

Coal: 1g/student. Coal can be obtained from electric power companies and some heavy
industries. Coal is also available as a Standard Reference Material. 47 46F

Flux: 4 g/student. 67 wt% MgO/33 wt% Na2CO3.

6 M HCl: 25 mL/student.
Phenolphthalein indicator: Recipe in Experiment 7.
Ammonium sulfate: Solid reagent for preparing standards.

Procedure

1. Grind the coal to a fine powder with a mortar and pestle. Mix 1 g of coal (accurately
weighed) with ~3 g of flux in a porcelain crucible. Mix thoroughly with a spatula and tap the
crucible to pack the powder. Gently cover the mixture with ~1g of additional flux. Cover the
crucible and place it in a muffle furnace. Then turn on the furnace and set the temperature to
800˚C and leave the sample in the furnace at 800˚C overnight. The reaction is over when all

46 E. Koubek and A. E. Stewart, J. Chem. Ed. 1992, 69, A146.

47 Standards containing 0.5–5 wt% S are available from NIST Standard Reference Materials Program, Room 204,
Building 202, Gaithersburg MD 20899-0001 (E-mail: [email protected]).

113
31. Analysis of Sulfur in Coal by Ion Chromatography Exploring Chemical Analysis

black particles have disappeared. A burner can be used in place of the furnace, but the burner
should not be left unattended overnight.
2. After cooling to room temperature, place the crucible into a 150-mL beaker and add 100 mL
of distilled water. Heat the beaker on a hot plate to just below boiling for 20 min to dissolve
as much solid as possible. Pour the liquid through filter paper in a conical funnel directly
into a 250-mL volumetric flask. Wash the crucible and beaker three times with 25-mL
portions of distilled water and pour the washings through the filter. Add 5 drops of
phenolphthalein indicator to the flask and neutralize with 6 M HCl until the pink color
disappears. Dilute to 250 mL and mix well.
3. Pipet 25.00 mL of sulfate solution from the 250-mL volumetric flask in step 2 into a
100-mL volumetric flask and dilute to volume to prepare a fourfold dilution for ion
chromatography. Inject a sample of this solution into an ion chromatograph 48 to be sure that
47F

the concentration is in a reasonable range for analysis. More or less dilution may be
necessary. Prepare the correct dilution for your equipment.
4. Assuming that the coal contains 3 wt% sulfur, calculate the concentration of SO2-
4 in the A
E

solution in step 3. Using ammonium sulfate and appropriate volumetric glassware, prepare
five standards containing 0.1, 0.5, 1.0, 1.5, and 2.0 times the calculated concentration of SO2-
4 A
E

in the unknown.
5. Analyze all solutions by ion chromatography. Prepare a calibration curve from the standards,
plotting peak area versus SO2-
4 concentration. Use the least-squares fit to find the
A
E

concentration of SO2-
4 in the unknown. Calculate the wt% of S in the coal.
A
E

48 For example, chromatography can be done with 25–50 µL of sample on a 4-mm-diameter × 250-mm-long Dionex
Ionpac AS5 analytical column and an AG5 guard column using 2.2 mM Na2CO3/2.8 mM NaHCO3 eluent at 2.0
mL/min with ion suppression. SO2-
4 is eluted near 6 min and is detected by its conductivity on a full-scale setting
of 30 microsiemens. Many combinations of column and eluent are suitable for this analysis.

114
32. Measuring Carbon Monoxide in Automobile Exhaust Exploring Chemical Analysis
by Gas Chromatography

32. Measuring Carbon Monoxide in Automobile Exhaust by Gas

Chromatography 49 48F

Carbon monoxide is a colorless, odorless, poisonous gas emitted from

automobile engines because of incomplete combustion of fuel to CO2. A
well-tuned car with a catalytic converter might emit 0.01 vol% CO, whereas
an old “clunker” could emit as much as 15 vol% CO! In this experiment,

Green Profile
you will collect samples of auto exhaust and measure the CO content by gas
See Section 0
chromatography. A possible class project is to compare different types of
cars and different states of maintenance of vehicles. CO emission is greatest within the first few
minutes after starting a cold engine. After warm-up, a well-tuned vehicle may emit too little CO
to detect with an inexpensive gas chromatograph. CO can be measured as a function of time
after start-up.
Chromatography is performed at 50˚–60˚C with a 2-m-long packed column containing
5A molecular sieves with He carrier gas and thermal conductivity detection. The column should
be flushed periodically by disconnecting it from the detector and flowing He through for 30 h.
Flushing after 50–100 injections desorbs H2O and CO2 from the sieves.

Reagents

CO gas standard: Lecture bottle containing 1 vol% CO in N2. 50 49F

Procedure

1. Attach with heavy tape a heat-resistant hose to the exhaust pipe of a car. (Caution: Avoid
breathing the exhaust.) Use the free end of the hose to collect exhaust in a heavy-walled,
0.5-L plastic zipper-type bag from the grocery. Flush the bag well with exhaust before
sealing it tightly. Allow the contents to come to room temperature for analysis.
2. Inject a 1-mL sample of air into the gas chromatograph by using a gas-tight syringe and
adjust the temperature and/or flow rate so that N2 is eluted within 2 min. You should see
peaks for O2 and N2.

49 D. Jaffe and S. Herndon, J. Chem. Ed. 1995, 72, 364.

50 Available, for example, from Scott Specialty Gases, Route 611, Plumsteadville PA 18949 (Phone:
215-766-8861; http://www.scottgas.com/).

115
32. Measuring Carbon Monoxide in Automobile Exhaust Exploring Chemical Analysis
by Gas Chromatography

Figure 1. Collecting a sample of standard gas mixture from a lecture bottle. Remove the vent
needle when withdrawing gas into the syringe. Do not open the tank valve so much that the
connections pop open. (CAUTION: Handle CO only in a hood.)

3. Inject 1.00-mL of standard 1 vol% CO in N2. One way to obtain gas from a lecture bottle is
to attach a hose to the tank (in a hood) with a serum stopper on a glass tube at the end of the
hose (Figure 1). Place a needle in the serum stopper and slowly bleed gas from the tank to
flush the hose. Insert a gas-tight syringe into the serum stopper, remove the vent needle, and
slowly withdraw gas into the syringe. Then close the tank to prevent pressure buildup in the
tubing. When you inject the standard into the chromatograph, you should see a peak for CO
with about three times the retention time of N2. Adjust the detector attenuation so the CO
peak is near full scale. Reinject the standard twice and measure the peak area each time.
4. Inject two 1.00-mL samples of auto exhaust and measure the peak area each time. Compute
the vol% of CO in the unknown from its average peak area:

vol % CO in the unknown peak area of unknown/detector attenuation

=
vol % CO in the standard peak area of standard/detector attenuation

116
33. Amino Acid Analysis by Capillary Electrophoresis Exploring Chemical Analysis

33. Amino Acid Analysis by Capillary Electrophoresis 51 50F

In this experiment you will hydrolyze a protein with HCl to break the protein
into its component amino acids. After the addition of an internal standard,
the amino acids and the internal standard will be derivatized (chemically
converted) to a form that absorbs light strongly at 420 nm.

Green Profile
See Section 0

CN

O CN-
+ H2NCHRCO2- N CHRCO2-
O

Naphthalene- Amino acid Derivatized product

2,3-dicarboxaldehyde (λmax = 420 nm)
(NDA, FM 184.19)

The mixture will then be separated by capillary electrophoresis, and the quantity of each
component will be measured relative to that of the internal standard. The goal is to find the
relative number of each amino acid in the protein.

Reagents

Protein: 6 mg/student. Use a pure protein such as lysozyme or cytochrome c. The amino acid
content should be available in the literature for you to compare with your results. 52 51F

6 M HCI: Dilute 124 mL of concentrated (37 wt%) HCl up to 250 mL with distilled water.
0.05 M NaOH: Dissolve 0.50 g of NaOH (FM 40.00) in 250 mL of distilled water.
1.5 M NH3: Dilute 26 mL of 28 wt% NH3 up to 250 mL with distilled water.

51 P. L. Weber and D. R. Buck, J. Chem. Ed. 1994, 71, 609.

52 For hen egg white lysozyme, the amino acid content is
S10T7H1G12A12(E2Q3)Y3(D8N13)V6M2I6L8F3R11K6C8W8P2
(R. E. Canfield and A. K. Liu, J. Biol. Chem. 1965, 240, 2000; D. C. Philips, Scientific American, May 1966.)
For horse cytochrome c, the composition is
S0T10H3G12A6(E9Q3)Y4(D3N5)V3M2I6L6F4R2Kl9C2W1P4
(E. Margoliash and A. Schejter, Adv. Protein Chem. 1966, 21, 114.) Both proteins are available from Sigma-
Aldrich; http://www.sigmaaldrich.com/chemistry/aldrich-chemistry/aldrich-handbook.html).

117
33. Amino Acid Analysis by Capillary Electrophoresis Exploring Chemical Analysis

10 mM KCN: Dissolve 16 mg of KCN (FM 65.12) in 25 mL of distilled water.

Borate buffer (pH 9.0): To prepare 20 mM buffer, dissolve 0.19 g of sodium tetraborate
(Na2B4O7 . 10H2O, FM 381.37) in 70 mL of distilled water. Using a pH electrode, adjust the
pH to 9.0 with 0.3 M HCl (a 1:20 dilution of 6 M HCl with distilled water) and dilute to 100
mL with distilled water.
Run buffer (20 mM borate–50 mM sodium dodecyl sulfate, pH 9.0): Prepare this as you
prepared borate buffer but add 1.44 g of CH3(CH2)11OSO3Na (FM 288.38) prior to adjusting
the pH with HCl.

Figure 1. Electropherogram of standard mixture of NDA-derivatized amino acids plus internal

standard, all at equal concentrations. Abbreviations for amino acids are given in Table 11-1 in
the textbook. The internal standard, designated X, is α-aminoadipic acid. U is an unidentified
peak. Acid hydrolysis converts Q into E and converts N into D, so Q and N are not observed.
The analysis of lysine (K) is not reliable because its NDA derivative is unstable. Cysteine (C)
and tryptophan (W) are degraded during acid hydrolysis and are not observed. Proline (P) is not
a primary amine, so it does not react with NDA to form a detectable product. [From P. L. Weber
and D. R. Buck, J. Chem. Ed. 1994, 71, 609.]

118
33. Amino Acid Analysis by Capillary Electrophoresis Exploring Chemical Analysis

Amino acids: Prepare 100 mL of standard solution containing all 15 of the amino acids in Figure
1, each at a concentration near 2.5 mM in a solvent of 0.05 M NaOH. Table 11-1 in the
textbook gives molecular masses of amino acids.
α- Aminoadipic acid internal standard: Prepare a 5 mM solution by dissolving 40 mg of
HO2C(CH2)3CH(NH2)CO2H (FM 161.16) in 50 mL of distilled water.
Naphthalene-2,3-dicarboxaldehyde (NDA): Prepare a 10 mM solution by dissolving 18 mg of
NDA in 10 mL of acetonitrile.

Hydrolysis of the Protein

1. Use a glass tube (17 × 55 mm) sealed at one end. Fit the open end with a rubber septum and
evacuate it through a needle. Dissolve 6 mg of protein in 0.5 mL of 6 M HCl in a small vial.
Purge the solution with N2 for 1 min to remove O2 and immediately transfer the liquid by
syringe into the evacuated tube. Add 0.5 mL of fresh HCl to the vial, purge, and transfer
again into the tube. Heat the part of the glass tube containing the liquid at 100˚–l10˚C in an
oil bath for 18–24 h, preferably behind a shield.
2. After cooling, remove the septum and transfer the liquid to a 25-mL round-bottom flask.
Evaporate the solution to dryness with gentle heat and suction from house vacuum or a water
aspirator. Use a trap (Figure 2-12 of the textbook) between the vacuum source and the
sample whenever you use an aspirator. Rinse the hydrolysis tube with 1 mL of distilled
water, add it to the flask, and evaporate to dryness again. Dissolve the residue in 1.0 mL of
0.05 M NaOH and filter it through a 0.45-µm pore size syringe filter. The total concentration
of all amino acids in this solution is ~50 mM.

Derivatization

3. Using a micropipet, place 345 µL of 20 mM borate buffer into a small screw-cap vial. Add
10 µL of the standard amino acid solution. Then add 10 µL of 5 mM α-aminoadipic acid (the
internal standard), 90 µL of 10 mM KCN, and 75 µL of 10 mM naphthalene-2,3-dicar-
boxaldehyde. The fluorescent yellow-green color of the amino acid-NDA product should
appear within minutes. After 25 min, add 25 µL of 1.5 M NH3 to react with excess NDA.
Wait 15 min before electrophoresis.

119
33. Amino Acid Analysis by Capillary Electrophoresis Exploring Chemical Analysis

4. Place 345 µL of 20 mM borate buffer (pH 9.0) into a small screw-cap vial. Add 10 µL of the
hydrolyzed protein solution in 0.05 M NaOH from step 2. Then add 10 µL of 5 mM
α-aminoadipic acid (the internal standard), 60 µL of 10 mM KCN, and 50 µL of 10 mM
naphthalene-2,3-dicarboxaldehyde. After 25 min, add 25 µL of 1.5 M NH3 to react with
excess NDA. After 15 min, begin electrophoresis. (Precise timing reduces variations
between the standard and the unknown.)

Electrophoresis and Analysis of Results

5. (Caution: Electrophoresis uses a dangerously high voltage of 20–24 kV. Be sure to follow
all safety procedures.) Conduct the electrophoresis in a 50-µm-inner-diameter uncoated
silica capillary with spectrophotometric detection at 420 nm. Precondition the column by
injecting 30 µL of 0.1 M NaOH and flushing 15 min later with 30 µL each of distilled water
and then run buffer. The column should be reconditioned in the same manner after every 3–4
runs.
6. Inject 3 nL of the derivatized standard amino acid mixture from step 3. The electropherogram
should look similar to Figure 1. Measure the area of each peak (or the height, if you do not
have a computer for measurement of area). Repeat the injection and measure the areas again.
7. Find the quotient
area of amino acid peak
area of internal standard peak

for each amino acid in the standard mixture. (Use peak heights if area is not available.)
Prepare a table showing the relative areas for each peak in each injection of standard and find
the average quotient for each amino acid from both runs.
8. Inject 3 nL of derivatized, hydrolyzed protein from step 4 and measure the same quotient as
measured in step 7. Repeat the injection and find the average quotient from both injections.
9. Find the concentration of each amino acid in the protein hydrolysate by using the following
equation:

concentration ratio (X/S) in unknown area ratio (X/S) in unknown

concentration ratio in standard mixture
A

E
= area ratio in standard mixture
A

E
A

E E

120
33. Amino Acid Analysis by Capillary Electrophoresis Exploring Chemical Analysis

in which X refers to an amino acid and S is the internal standard. The numerator on the right
side of the equation was found in step 8 and the denominator on the right side was found in
step 7. (Use the average values from the two sets of runs.) The denominator on the left side
is known from the masses of amino acids and internal standard weighed into the standard
solution. For each amino acid, you can now solve the equation to find the numerator on the
left side, which is the quotient

concentration of amino acid in protein hyrolysate

concentration internal standard in protein hydrolysate
A

But you know the concentration of internal standard in the protein hydrolysate from the
volume and concentration of internal standard used in step 4. Therefore, you can calculate
the concentration of each amino acid in the hydrolysate.
10. Find the mole ratio of amino acids in the protein. If there were no experimental error, you
could divide all concentrations by the lowest one. Because the lowest concentration has a
large relative error, pick an amino acid with two or three times the concentration of the least
concentrated amino acid. Define this concentration to be exactly 2 or 3. Then compute the
molarities of other amino acids relative to the chosen amino acid. Your result is a formula
such as S10.6T6.6H0.82G12.5A11.2E5.3Y≡3D19.8V6.1M2.2I5.7(L + F)11.5.

121
34. DNA Composition by High-Performance Liquid Chromatography Exploring Chemical Analysis

34. DNA Composition by High-Performance Liquid Chromatography 53 52F

This experiment illustrates quantitative analysis by high performance liquid

chromatography (HPLC). It uses only aqueous eluent, so there is no
hazardous waste to dispose.
The genetic material deoxyribonucleic acid, is a polymer made of

Green Profile four nucleotides abbreviated C, T, A, and G:

See Section 0

H
O N NH2 O N O
O OH
P O O
N H2 O3 POCH2 N
HO O

Cytosine
HO HO
Deoxyribose

2'-Deoxycytidine 5'-monophosphate (C) Thymidine 5'-monophosphate (T)

N NH2 N O

H2 O3 POCH2 O N H2 O3 POCH2 O N
N N
N N
HO HO NH2

2'-Deoxyadenosine 5'-monophosphate (A) 2'-Deoxyguanosine 5'-monophosphate (G)

In double-stranded DNA, C is hydrogen bonded to G and A is hydrogen bonded to T. Therefore

the concentrations of C and G are equal and the concentrations of A and T are equal. DNA from
different organisms has different relative amounts of (C + G) and (A + T). When DNA is
hydrolyzed by the enzyme nuclease P1, it is cleanly broken into the four nucleotides.

53 S. M. Wietstock, J. Chem. Ed. 1995, 72, 950.

122
34. DNA Composition by High-Performance Liquid Chromatography Exploring Chemical Analysis

Reagents

Standard nucleotide solution: The standard should contain accurately weighed quantities of the
nucleotide monophosphates 54 at concentrations of ~20 mM. The molecular masses of the
53F

free acids are C - 307.2, T - 322.2, A - 331.2, G - 347.2. Place the required quantities of the
solid acids in a 5-mL volumetric flask and add 2 mL of water and 1.6 mL of 0.10 M NaOH
(2 mol NaOH per mol of nucleotide). Dissolve the solid, dilute to the mark with water, mix
well, and store the standard in a refrigerator. (The volume of the standard changes when it is
cooled, but this is not important. Only the relative concentrations of nucleotides in the
standard are important in this experiment.)
Hydrolyzed DNA: The volumes of DNA and nuclease P1 solutions should be the minimum
required for the number of people doing the experiment. Prepare a solution containing 1
mg/mL of calf thymus (or other) DNA.54 Dissociate the DNA into single strands by heating
at 100°C for 10 min and then cooling immediately on ice. Prepare nuclease P154 at a final
concentration of 5 units/mL55 in 50 mM sodium acetate buffer (pH 5.3) containing 0.6 mM
54F

ZnCl2. Mix 20 µL of DNA solution with 20 µL of nuclease solution in a small vial with a
conical bottom. Heat the vial at 50°C for 1 h and analyze it immediately or store it in the
refrigerator.
HPLC Eluent: Prepare 0.010 M potassium phosphate buffer by dissolving 0.010 mol K2HPO4 in
800 mL of water, titrating with ~1 M HCl to pH 7.2, and diluting to 1.00 L.

Chromatography

1. A variety of C18-silica columns should work in this experiment. A 0.46 × 15 cm column

with 5 µm particles or a 0.46 × 25 cm column with 10 µm particles are reasonable.
Equilibrate the column with 20 empty column volumes of 0.010 M phosphate buffer (pH 7.2)
at a flow rate of 1.2 mL/min before beginning chromatography. Establish a flat baseline with
an ultraviolet detector at or near 260 nm.

54 Available from Sigma-Aldrich; http://www.sigmaaldrich.com/chemistry/aldrich-chemistry/aldrich-

handbook.html.
55 For nuclease P , one unit is defined as the amount that will liberate 1.0 µmol of acid soluble nucleotides from
1
yeast ribonucleic acid per min at pH 5.3 at 37°C. The commercial preparation has at least 200 units/mg of
nuclease P1.

123
34. DNA Composition by High-Performance Liquid Chromatography Exploring Chemical Analysis

2. Inject 10 µL of the nucleotide standard. You should observe a clean separation of all four
peaks (C < T < G < A) with an elution time of 5–10 min. Measure the areas of all four peaks,
preferably by computer integration. Alternatively, you can estimate peak area from the
formula: area of Gaussian peak = 1.064 × peak height × w1/2, where w1/2 is the width at half-
height (Figure 21-3 in the textbook). Express the areas of C, T, and A relative to the area of
G, which we will define as 1.000. Repeat the procedure with a second injection and measure
the relative areas. List the relative peak areas in each run and the average of the two runs.
3. Inject 10 µL of hydrolyzed DNA and measure the relative areas of the peaks. Repeat the
process a second time. List the relative areas in each run and the average of the two runs.

Calculations

1. From the average peak areas of the two standard runs, find the response factors for C, T, and
A relative to G. For example, the response factor for C is obtained from the equation

area of C  area of G 
concentration of C = F concentration of G
A E A A E

AC  AG 
= F [G]
[C]  
A E A A E

There will be similar equations for T, and A. We are using G as the internal standard.
2. From the average peak areas of the two injections of hydrolyzed DNA, find the relative
concentrations [C]/[G], [T]/[G], and [A]/[G] by using the response factors from the standard
mixture. What is the theoretical value of [C]/[G]? What is the theoretical relationship
between [T]/[G] and [A]/[G]?
3. Find the fraction of nucleotides that are C + G by evaluating the expression

[C] [G]
Fraction [C] + [G] [G] + [G] E

of C + G:
A E A

[C] + [G] + [A] + [T] = [C] [G] [A] [T]

A E A A E A

[G] + [G] + [G] + [G]

For calf thymus DNA, the literature value of the fraction of C + G is 0.42.

124
35. Analysis of Analgesic Tablets by High-Performance Liquid Chromatography Exploring Chemical Analysis

35. Analysis of Analgesic Tablets by High-Performance Liquid

Chromatography 56 5F

Nonprescription headache medications such as Excedrin or Vanquish contain

mixtures of acetaminophen and aspirin for relief and caffeine as a stimulant.
This experiment describes conditions for separating and measuring the
components by high-performance liquid chromatography (HPLC).

Green Profile Instructions are given for measuring caffeine, but any and all of the
See Section 0
components could be measured.

OH
O CH3 CO2H
H3C N
N O O

O N N CH3
H3C NH
CH3
O
Caffeine Aspirin Acetaminophen
(acetylsalicylic acid)

Reagents

HPLC solvent: Organic solvents should be handled in a fume hood. All solvents in this
experiment should be HPLC-grade. Mix 110 mL of acetonitrile, 4.0 mL of triethylamine, and
4.0 mL of acetic acid in a 2-L volumetric flask and dilute to the mark with HPLC-grade
water. Filter through a 0.45-µm filter and store in a tightly capped amber bottle.
Caffeine stock solution (100 µg/mL): Dissolve 1.000 g of caffeine in 50 mL of HPLC solvent in
a 100-mL volumetric flask with gentle heating (in the hood). Cool to room temperature and
dilute to the mark with HPLC solvent. Dilute 10.00 mL to 100 mL with HPLC solvent in a
volumetric flask to obtain 1 000 µg/mL. Dilute once again to obtain 100 µg/mL.
Acetaminophen and aspirin samples: Prepare two solutions, each containing one of the analytes
at a concentration of ~50 µg/mL in HPLC solvent. Filter through 0.22 µm nylon syringe
filters and store in capped amber bottles.

56 G. K. Ferguson, J. Chem. Ed. 1998, 75, 467.

125
35. Analysis of Analgesic Tablets by High-Performance Liquid Chromatography Exploring Chemical Analysis

Procedure

1. Caffeine quantitative analysis standards: Dilute the 100 µg/mL stock solution down to 50,
10, and 5 µg/mL with HPLC solvent. Filter ~3 mL of each solution through a 0.22-µm
syringe filter into a capped vial. Filter ~3 mL of the 100 µg/mL solution into a fourth vial.
2. Sample preparation: Grind the analgesic tablet into a fine powder with a clean mortar and
pestle. Dissolve ~0.5 g (weighed accurately) in 50 mL of HPLC solvent with gentle heating.
Cool to room temperature and dilute to volume with HPLC solvent. Dilute 10.00 mL of this
solution to 100 mL with HPLC solvent in a volumetric flask. Filter ~3 mL of the dilute
solution through a 0.22-µm syringe filter into a capped vial.
3. Chromatography conditions: Use a 2.1-mm-diameter × 10-cm-long C18-silica column with
5-µm particle size and ultraviolet detection at 254 nm. With a flow rate of 1.5 mL/min, each
run is complete in 4 min.
4. Calibration curve: Inject 10 µL of each of the caffeine standards (5, 10, 50, and 100 µg/mL)
into the HPLC and measure the peak area. Repeat this process twice more and use the
average areas from the three runs to construct a calibration curve of area versus
concentration. Compute the least-squares slope and intercept for the line through points.
5. Qualitative analysis: Record a chromatogram of 10 µL of the analgesic tablet solution. Then
mix 2 drops of the tablet solution with 2 drops of 50 µg/mL caffeine solution in a test tube or
vial. Inject 10 µL of the mixture into the chromatograph and observe which peak grows.
Repeat the process again by adding 50 µg/mL acetaminophen and 50 µg/mL aspirin and
identify which peaks in the analgesic are acetaminophen and aspirin.
6. Quantitative analysis: Inject 10 µL of the analgesic tablet solution and measure the area of
the caffeine peak. Repeat this process twice more and take the average from three injections.
Using your calibration graph, determine the concentration of caffeine in the solution and the
weight percent of caffeine in the original tablet.

126
36. Anion Content of Drinking Water by Capillary Electrophoresis Exploring Chemical Analysis

36. Anion Content of Drinking Water by Capillary Electrophoresis 57 56F

Chloride, sulfate, and nitrate are the major anions in fresh water. Fluoride is
a minor species added to some drinking water at a level near 1.6 ppm to
help prevent tooth decay. In this experiment, you will measure the three
major anions by capillary electrophoresis, which is discussed in Sections 23-
5 through 23-7 in the textbook. Possible class projects are to compare water
Green Profile
See Section 0 from different sources (homes, lakes, rivers, ocean) and various bottled
drinking waters.
Your equipment should be similar to that in Figure 23-14 in the textbook. Convenient
capillary dimensions are a diameter of 75 µm and a length of 40 cm from the inlet to the detector
(total length = 50 cm). Because the anions have little ultraviolet absorbance at wavelengths
above 200 nm, we add chromate anion (CrO2-
4 ) to the buffer and use indirect ultraviolet
A E A

detection at 254 nm. The principle of indirect detection is explained in Figure 23-17 of the
textbook.
One other significant condition for a successful separation in this experiment is to reduce
the electroosmotic flow rate to permit a better separation of the anions based on their different
electrophoretic mobilities. At pH 8, electroosmotic flow is so fast that the anions are swept from
the injector to the detector too quickly to be separated well from one another. To reduce the
electroosmotic flow, we could lower the pH to protonate some of the –O- groups on the wall.
Alternatively, what we do in this experiment is to add the cationic surfactant tetradecyl-
+
(trimethyl)ammonium ion, CH3(CH2)13N(CH3)3, which is attracted to the –O- groups on the
A E A

wall and partially neutralizes the negative charge of the wall. This cationic surfactant is
abbreviated OFM+, for “osmotic flow modifier.”

Reagents

Run buffer: 4.6 mM CrO2- + .

4 + 2.5 mM OFM at pH 8. Dissolve 1.08 g Na2CrO4 4H2O (FM
A E A

234.02) plus 25.0 mL of 100 mM tetradecyl(trimethyl)ammonium hydroxide 58 in 800 mL of

57F

57 S. Demay, A. Martin-Girardequ, and M.-F. Gonnord, J. Chem. Ed. 1999, 76, 812.
58 100 mM tetradecyl(trimethyl)ammonium hydroxide (Catalog number WAT049387) is available from Waters
Corp., 34 Maple Street, Milford, MA 01757; Phone: 1-800-478-2000; www.waters.com. The surfactant is sold
under the trade name “Osmotic Flow Modifier,” abbreviated OFM+OH-.

127
36. Anion Content of Drinking Water by Capillary Electrophoresis Exploring Chemical Analysis

HPLC-grade H2O. Place a pH electrode in the solution and add solid boric acid (H3BO3)
(with magnetic stirring) to reduce the pH to 8.0. Dilute to 1.00 L with HPLC-grade H2O,
mix well, filter through a 0.45-µm filter, and store in the refrigerator in a tightly capped
plastic bottle. Degas prior to use.
Quantitative standards: Prepare one stock solution containing 1 000 ppm Cl-, 1 000 ppm NO3- , A E A

and 1 000 ppm SO2-

4 by dissolving the following salts in 1.000 L of HPLC-grade H2O: 2.103
A
E

g KCl (FM 74.55), 1.631 g KNO3 (FM 101.10), and 1.814 g K2SO4 (FM 174.26).
(Concentration refers to the mass of the anion. For example, 1 000 ppm sulfate means 1 000
µg of SO2-
4 per mL of solution, not 1 000 µg of K2SO4.) Dilute the stock solution with
A
E

HPLC-grade H2O to make standards with concentrations of 2, 5, 10, 20, 50, and 100 ppm of
the anions. Store the solutions in tightly capped plastic bottles.
Standards for qualitative analysis: Prepare four separate 1.00-L solutions, each containing just
one anion at a concentration of ~50 ppm. To do this, dissolve ~0.105 g KCl, ~0.082 g
KNO3, ~0.091 g K2SO4, or ~0.153 g KF (FM 58.10) in 1.00 L.

Procedure

0. CAUTION: Electrophoresis uses a dangerously high voltage. Be sure to follow all safety
procedures for the instrument.
1. When preparing a capillary for its first use, wash through 1 M NaOH for 15 min, followed by
0.1 M NaOH for 15 min, followed by run buffer for 15 min. In this experiment, wash the
column with run buffer for 1 min between sample injections.
2. Identify the peaks: Inject a 50 ppm mixture of Cl-, NO3-, and SO2-
4 by applying a pressure of
A
E

A A
E

0.3 bar for 5 s. Then insert the sample end of the capillary back in run buffer and perform a
separation for 5 min at 10 kV with the capillary thermostatted near 25°C. The voltage should
be positive at the injector and negative at the detector. The detector should be set at 254 nm.
After the run, wash the column with run buffer for 1 min. Mix the 50-ppm anion mixture
with an equal volume of 50 ppm Cl- and run an electropherogram of the mixture. The Cl-
peak should be twice the size it was in the first run. Repeat the procedure with additions of
NO3-, SO2-
A A A
-
4 , and F . This process tells you which peak belongs to each anion and where to
E

E A

look for F- in drinking water.

128
36. Anion Content of Drinking Water by Capillary Electrophoresis Exploring Chemical Analysis

3. Calibration curves: Inject each of the standard mixtures from lowest concentration to
highest concentration (2, 5, 10, 20, 50, and 100 ppm) and measure the area of each peak in
each run. Repeat the sequence twice more and use the average peak area at each
concentration to construct a calibration curve for each anion. Find the least-squares straight
line to fit the graph of area versus concentration for each anion.
4. Unknowns: Make three replicate injections of each unknown water sample and measure the
areas of the peaks. Use the average area of each peak and the calibration curves to find the
concentrations of the anions in the water. If you analyze any saltwater samples, they should
be diluted by a factor of 100 with HPLC-grade water to bring the anion concentrations down
to the range of fresh waters.

129
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Exploring Chemical Analysis

37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel

Oil 59, 60
58F
59F

In analytical procedures, solvent extraction can provide the initial separation

of analyte from bulk sample. In this experiment, we extract a natural
product from a renewable feedstock using liquid carbon dioxide as a
nontoxic solvent under ambient conditions while generating minimal waste.
If you really want to calculate the waste produced in the procedure, you
Green Profile
See Section 0 would need to account for waste created in generating the energy required to
make Dry Ice (solid CO2) used in the experiment, including the amount of
Dry Ice lost between the location where it was made and your laboratory. You would also need
to account for transportation of Dry Ice from the manufacturer to your laboratory. Even “green”
procedures have hidden costs.
“Essential oils” used for flavors and fragrances are volatile organic
compounds, including monoterpenes such as limonene, commonly found in plants
such as citrus. Essential oils are often isolated by steam distillation in which citrus
is boiled in water and the oil co-distills with the water. Liquid condensed from the
steam contains both water and oil, which separate from each other. Steam
H
distillation could hydrolyze or decompose some oils. Alternatively, organic
solvents used to extract oils may be flammable, toxic, or otherwise undesirable.
D-Limonene
How can we isolate essential oils without using water or organic solvents?
Supercritical fluid extraction uses carbon dioxide at elevated temperature and pressure to
achieve a liquid or supercritical fluid state suitable for chemical extractions. In the phase
diagram for CO2 in Figure 1, the liquid-vapor boundary line ends at the critical point. At higher
temperature and pressure than the critical point, separate liquid and gaseous phases no longer
exist. Rather, there is a single supercritical phase whose properties are between those of the
liquid and gas.

59 Adapted from L. C. McKenzie, J. E. Thompson, R. Sullivan, and J. E. Hutchison, “Green Chemical Processing
in the Teaching Laboratory: A Convenient Liquid CO2 Extraction of Natural Products,” Green Chem. 2004, 6,
355-358.
60 Experiment contributed by Douglas E. Raynie, Department of Chemistry and Biochemistry, South Dakota State
University, Brookings SD 57007; [email protected].

130
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Exploring Chemical Analysis

Figure 1. Phase
diagram for CO2.

A supercritical fluid might act as a liquid solvent to dissolve solutes, yet maintains
viscosity and diffusion properties similar to those of a gas. Supercritical CO2 has advantages for
extracting essential oils from flowers, fruit rind, and other products. Supercritical CO2 is
nonflammable, relatively nontoxic, and readily available. To perform a supercritical extraction,
an instrument is needed to heat and pressurize CO2 above its critical point (31°C and 74 bar).
To mimic a true supercritical fluid extraction, we will use liquid CO2, which has
properties similar to the supercritical fluid. Dry Ice sublimes at atmospheric pressure and
temperatures above -78°C. If CO2 is sealed in a vessel during sublimation, the pressure in the
vessel increases. After the temperature and pressure have increased sufficiently, liquid CO2
forms. We can exploit the ease of CO2 liquefaction to extract essential oils from citrus rind.

Reagents and Supplies

Crushed (powdered) Dry Ice: 25 g/student

Grated lemon peel (zest): 2.5 g/student. Grate only the colored part of the lemon peel with the
smallest grating surface of a cheese grater. 61 60F

Methanol: 5 mL/student
15-mL polypropylene centrifuge tube: one/student (Corning #430052 recommended)
Copper wire: 20-cm length of 18-22 gauge wire for each student

61 Instead of lemon peel, you may chose to use orange peel. Orange oil will be about 95% D-limonene, which is
sufficiently pure to characterize by infrared spectroscoy and nuclear magnetic resonance.

131
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Exploring Chemical Analysis

500-mL plastic graduated cylinder or wide-mouth polycarbonate jar: one per student.
Alternatively, construct a watertight cylinder from a 30-cm-long clear plastic tube with a 25-
mm diameter and 3-mm wall thickness cemented to a flat plastic base with silicone calk.
Filter paper: one 1.5-cm-diameter circle of Whatman #1 filter paper per student
Thermometer: Several students can share a single thermometer to measure water at 40-50ºC.
Forceps: Several students can share a single pair of forceps capable of reaching into the cylinder
to remove the centrifuge tube.
Tweezers: Several students can share a single pair.
Pasteur pipet: One per student.

Safety Precautions

0. Pressure generated during this experiment creates a risk of flying plastic centrifuge caps and
rupture of plastic vessels. The source for this procedure59 reports that caps blew off in 4% of
all experiments, so you can EXPECT this to happen in your laboratory. Under no
circumstance should vessels other than the recommended ones be used..
1. Do not use any glass in this experiment. Substitution of glass centrifuge tubes or graduated
cylinders could result in serious injuries if shattering should occur.
2. Always wear eye protection.
3. Use gloves when handling dry ice. Contact with dry ice can damage your skin.
4. Do not liquefy CO2 more than 5 times in the same centrifuge tube. After repeated
liquefaction, the tube may become brittle and rupture.

Procedure

0. Read safety precautions and the entire procedure before beginning the experiment.
1. Verify that the cap fits tightly on the centrifuge tube. If the cap does not stop turning when
you screw it down, try a different cap or different tube. Unscrew the cap and put it aside.
2. Following Figure 2, coil the copper wire into 3 circles on one end and leave the other end
straight. The coils should fit into the centrifuge tube and rest above the tapered section of the
tube. The straight section of wire should be contained within the tube when the cap is
screwed on. Place a small circle of filter paper between the coils so that the filter paper will
support the solid to be extracted. Insert the coil with its filter paper into the centrifuge tube.

132
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Exploring Chemical Analysis

Coiled Circle of
wire filter paper Wire and filter
supported Add orange Add crushed
paper in plastic
in coil or lemon rind Dry Ice and
centrifuge tube
cap tightly
Suspend tube in plastic
cylinder of water

Figure 2. Extraction procedure.59

3. Add ~2.5 g of grated lemon peel to the tube, but do not pack tightly. The peel should rest on
the filter paper. During extraction, liquid CO2 must penetrate the loosely packed peel and
reach the bottom tip of the centrifuge tube.
4. Fill the 500-mL plastic cylinder or wide-mouth polycarbonate water bottle one-half to two-
thirds full of warm tap water at 40-50°C. Do not heat the cylinder or add hot water at any
time later in the experiment, or the cap can blow off the centrifuge tube during the extraction.
5. Wearing gloves and using a scoop or small beaker, fill the centrifuge tube with crushed Dry
Ice. Tap the tube on the lab bench and add more Dry Ice to fill the tube. Take the cap that
you have already tested for a tight fit and screw it tight. If the seal is not tight, try a different
cap or, if necessary, start again with a different tube and cap.
6. Place the centrifuge tube with the tapered end down into the cylinder or plastic jar of warm
water. Sublimation of Dry Ice will build high pressure inside the tube and liquid CO2 will
appear in about 15 seconds. Even though the cap is tight, gas will escape through the threads.
7. View the boiling CO2 in the centrifuge tube from the side of the cylinder. Do not look into
the top of the cylinder. If the cap blows, it will fly straight up and hit you in the face.
8. If liquid CO2 does not appear within a minute, the seal is not tight enough to maintain
sufficient pressure in the centrifuge tube. Take the tube out of the cylinder with forceps,

133
37. Green Chemistry: Liquid Carbon Dioxide Extraction of Lemon Peel Exploring Chemical Analysis

tighten the cap, and place it back in the warm water. If liquid CO2 does not appear after
several attempts to tighten the cap, try a different cap. If necessary, begin again with a fresh
combination of tube and cap. 62 61F

9. When the cap is tight, liquid will appear in the tube and gas will slowly escape through the
threads. Slowly rotate the cylinder so that the cold centrifuge tube does not freeze to the
cylinder wall. Do not remove the centrifuge tube from the cylinder while liquid CO2 is
present. The cylinder protects you in case the pressurized centrifuge tube explodes.
10. Liquid CO2 in the centrifuge tube boils vigorously and extracts essential oil from the lemon
peel. After a few minutes, CO2 will have escaped from the tube and no liquid will be left.
Approximately 0.1 mL of pale yellow oil should be visible at the bottom of the centrifuge
tube. If the tube is packed too tightly with lemon peel, liquid CO2 cannot reach the tip of the
tube and extracted oil will not collect at the tip.
11. When no liquid CO2 is left in the centrifuge tube, remove the tube from the cylinder with
forceps and unscrew the cap slightly to allow the remaining pressure to escape. After all
pressure has been released, it is safe to remove the cap. Using tweezers, carefully pull the
wire from the tube to remove the lemon peel.
12. Transfer the extracted oil by Pasteur pipet into a gas chromatography autosampler vial and
dilute with methanol. Use gas chromatography—mass spectrometry to compare your extract
to a commercial sample of lemon oil. We recommend a DB-5 or other 5% phenyl, nonpolar
column with a 10°C/min temperature gradient. Extracted lemon oil will have about six major
components.
13. As part of your lab report, identify the major peaks in the chromatogram from their mass
spectra. Perform a literature search on supercritical fluid extraction. (a) Find one reference
to the use of supercritical fluid extraction in the flavor and fragrance industry. (b) Aside from
essential oils, what types of compounds are commonly extracted via supercritical fluid
extraction? (c) Identify a vendor of supercritical fluid extraction equipment. 63 62F

62 In some cases, CO might not liquefy or no extract is visible. If the centrifuge tube is rinsed with a small amount
2
of methanol, enough extract to be observed via gas chromatography–mass spectrometry is usually obtained.
63 Instructors: The University of Oregon Greener Education Materials for Chemists database
(http://greenchem.uoregon.edu/Pages/Overview.php?CategoryIDString=&FullTextSearchKeywords=orange&Cat
egoriesToSearch=&NumberOfMainCategories=7&AnyAll=Any&ID=85) contains comments from people who
have used this experiment and allows you to add comments from your experience.

134