LABORATORY PROCEDURE

A necessary prerequisite for adequate performance in the laboratory is your

preparation prior to the laboratory period. Students must be well acquainted with the
instructions for the experiment to be carried out. You only have 3 hours to complete
the experiments! Therefore, make sure you have read the procedures and the
instrument operating instructions before coming to class. Also come to class with an
outline of exactly how you intend to prepare your standard solutions and run your
experiments. Be prepared, have a lined-up plan, do not procrastinate!
If you are unable to attend a scheduled laboratory period due to a University valid
excuse, the instructor should be notified immediately, before the lab period starts, so
that suitable alternative arrangements can be made. Please keep in mind that lab make
up is normally not allowed because of the Instruments and lab space limitations. All
students will work in groups on a given experiment during each laboratory. It is
extremely important to be as efficient as possible during each and every lab period. In
general you will finish all preparations for the experiment during the first period
(prepare stock solutions, sample solutions etc.), and conduct the instrumental analysis
during the second period.
Please come to class on time. In many cases, the experiments will take the entire 3
hours allocated! Your partner will not be allowed to work alone so be responsible with
his/her time. You will not be allowed to work if you come to the laboratory 30 min
late.
All experimental data will be recorded, as it is obtained, on a lab notebook. Upon
completion of the day’s lab work, the instructor will validate your work by placing
his/her initials in your lab-book even if the experiment has not been completed. The
original data sheets (or a photocopy) must be included in your formal report and will
be part of your grade.
SAFETY.
Remember: "SAFETY IS ALWAYS THE NUMBER ONE PRIORITY"
Should any problem arise inform the instructor immediately.
Do not put yourself in danger.
If you think something is unsafe STOP and consult the instructor before continuing.
IMPORTANT RULES
1. Please remember that we share the lab with many classes and therefore do not leave
anything out after the lab session is finished.
2. Always return all chemicals to their original place immediately after use.

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3. Keep your work area always clean, in particular when working with instruments or
in the balance room. Your work area must be clean. Point deductions will be taken off
your experiment grade if this conduct is persistent.
4. Do not leave standard solutions in volumetric flasks. Once you are done with the
preparation, transfer the solution to a labeled vial for storage. Include the Group
number and other important information in the labels; a lot of people may be doing the
same experiment at the same time. Rinse the volumetric flasks thoroughly before
returning them to the cabinet.
5. You must have a lab notebook and you must write everything in black or blue ink
(no pencils). When correcting something do not white-out, scratch the error with a
single line and write the correction above or below.
6. Remember to record the unknown sample number for each experiment.
7. Always wear a lab coat and safety glasses and please, no sandals!
8. Read the operating instructions for every instrument before you use it. If you have
any questions about anything stop and ask!
9. Be aware of the due dates for each experiment (you will be penalized for late
submissions), If you can’t make it for any reason notify the instructor in advance, not
after the fact.
10. Working in groups requires sharing of the responsibilities so do not drag your feet,
talk to your partner. If problems arise don't wait until it's too late, talk to the instructor
about it.
11. PLAGIARIZE (play-jâ-riz). to take and use another person’s ideas, writings or
inventions as one’s own.
Note that plagiarism is illegal, unethical, and you can be expelled from the University
for it. Plagiarizing old laboratory reports will not be tolerated! Any evidence of such
activities will result in no points for that exercise. Further disciplinary actions will be
taken at the discretion of the instructor.
REPORT WRITING
A Lab report is required for each one of the experiments. Reports are due at the
beginning of the next lab period after you finish an experiment. There will be a 20%
grade deduction on all late submissions and no report will be accepted after two weeks
of the experiment completion have passed. Your final report should be neatly typed or
handwritten (in ink) on A4 paper. Graphs should be plotted on the same size paper and
should be scaled so that the data occupies the majority of the plotting area. All graphs
in the final report, with the exception of your raw data, must be computer generated.
All axes should be labeled and the proper units displayed. When logarithmic scales are
used please make sure the data is accurately represented in your plot. When reporting

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tables of data please check your significant figures, they are important. All statistical
treatment of the data must be done using a computer. Your final report must consist of
the following sections:
I. Title Page: Title of the experiment, course name and number, your name and the
date.
II. Introduction: Presents a description of the parameters to be measured and the
general approach to the problem. Should also include a detailed description of the
analytical technique involved showing a thorough knowledge of the concepts
involved. This section should also state the objectives of the experiment.
III. Experimental Section:
1. Analytical procedure: This section should include a description of how the
experiment was conducted and the equipment was used. Do not copy the procedure
section from the lab manual. Write in your own words to describe how you performed
the experiment. Be sure to include any modifications or deviations from the suggested
protocols.
2. Raw Data: Record all experimental data as it is obtained. Include the original data
sheet with your report (remember an initialized copy of the data has been submitted to
the instructor).
IV. Results: Present your data in tables and graphs. Calculations and error analysis
must be shown and explained. Use SI units only. If difficulties were encountered
include a narrative description of the problem. All graphs, tables and sections must
have a title/caption and should be referenced in your text.
V. Discussion and Conclusions: Discuss your findings; make comparisons with
known values if available. Elaborate on possible sources of errors, selectivity and
sensitivity of the technique, detection limits, matrix effects, interferences, accuracy,
precision, applicability, etc. Suggest any possible improvements in the experiment and
present your summary conclusion.
VI. References: Include references using the ACS format used in the journal
Analytical Chemistry; i.e.: Number all references consecutively in parentheses and
include them at the end of your report in a section called "References". i.e.: (1) Cai,
Y.; Alzaga, R.; Bayona, J.M. Anal. Chem. 1994, 66, 1161-1167.

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EXPERIMENT 1:
UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of a
Commercial Aspirin Tablet.
Acetyl salicylic acid (ASA) is one of the oldest synthetic drugs. First synthesized in
Germany by the Bayer company and marketed under the name "Aspirin" it has
remained one of the most popular "over the counter" drugs of all time. Its main effect
is as a pain killer and fever depressant, but in addition there is strong evidence that in
low daily dosages it lowers the incidence of heart attacks. In the last few decades other
drugs such as acetaminophen (commercial trade name Panadol, also Tylenol) and
ibuprofen (trade name Advil) have taken much of the market for ASA, but ASA
remains an important and widely used medicine. Drugs, in addition to their active
compound, often contain other inactive ingredients (called excipients in the
pharmaceutical industry) such as binders, fillers, dyes, drying agents, etc. The content
of active ingredient in a tablet will always be stated on the package. In this experiment
we will determine the percent active compound in a commercial aspirin tablet. Aspirin
is the trade name for acetylsalicylic acid (ASA). The ASA in the tablet will be reacted
with Fe3+, forming an intensely violet coloured complex. The concentration of the
complex will be determined by means of spectrophotometry, using a UVVIS
spectrophotometer. Finally, we will be able to calculate the weight and the weight% of
ASA in the commercial tablet.
The determination of acetyl salicylic acid by spectrophotometry
Acetylsalicylic acid is the acetate (ethanoate) ester of salicylic acid, 2-hydroxybenzoic
acid. The “acetyl” ester is rapidly hydrolyzed to the salicylate anion in basic medium,
as shown in the following reaction.

Once the de-esterification is complete, the solution is acidified, and FeCl 3 is added.
The salycilic acid will react with the Fe3+ to form a coloured complex ion:

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Maximum absorption (minimum transmittance) is just above 500 nm, and the
concentration measurement will be done at this wavelength.
In order to calculate the concentration of the complex we would need to know the
value of ε and l. Instead, we will measure the absorbance of Fe-salicylate complex
solutions of known concentration, and plot the absorbances of a number of such
known solutions vs. the concentration. This is known as a calibration curve. The
calibration solutions are prepared by first making a solution of the Fe-salicylate
complex of known concentration. This solution is called the stock solution. Next we
make 5 standard solutions by diluting a known amount of stock solution. The
Absorbance of each of these 5 solutions is measured, and plotted vs. their
concentration resulting in a linear calibration curve of A vs C. Next we measure the
absorbance of the solution prepared from the commercial aspirin solution, and
find its concentration by comparing its absorbance value on the calibration curve.
Materials and chemicals
UV/VIS spectrophotometer and polystyrene cuvettes, hot plate, 1 10 mL graduated
cylinders, 2 100 mL volumetric flask, 6 10 mL volumetric flasks, 100 μL
micropipette, 2 125 mL Erlenmeyer flasks or 150 mL beakers, watch glass. Salicylic
acid (reagent grade), 1.0 M NaOH, 0.02M FeCl3 (buffered to pH = 1.6 with
HCl/KCl)
Commercial AspirinTM or ASA tablets for analysis (not to be used for your headache
caused by this experiment!).
Disposal
The remaining NaOH and Fe-salicylate solutions can be combined and neutralized
before disposal.
Procedure
1. Operation of the spectrophotometer.
Follow the instructions in the laboratory. Use FeCl3 as the blank solution to zero the
absorbance reading.
2. Preparation of the Fe-salicylate standard solutions and ASA unknown.
The preparation and measurements of the SA standards and ASA unknown can be
done simultaneously.

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 Salicylic Acid (SA) Aspirin (ASA)
1. Weigh approximately 0.160 g SA directly into 1. Weigh ASA tablet in beaker, record the mass,
small dry beaker. Record the mass (g). crush tablet.
2. Add 5 mL NaOH. 2. Add 5 mL NaOH.
3. Heat on hot plate until dissolved. Cool. 3. Heat on hot plate until dissolved (some fine non-
active ingredients may still be visible). Cool.
4. Transfer solution to 100. mL volumetric flask. 4. Transfer solution to 100. mL volumetric flask.
Fill to the mark with distilled water. Fill to the mark with distilled water.
This is your “stock” SA solution This is your “stock” ASA solution
5. Prepare 5 standard solutions in 10.0 mL5. Prepare one ASA solution: use micropipet to
volumetric flasks: use micropipet to add 100, add 200 μL ASA stock in 10.0 mL volumetric
200, 300, 400, and 500 μL stock SA into 10.0 flask. Fill the flask to the mark with acidified
mL volumetric flasks. Fill each flask to the mark FeCl3solution.
with acidified FeCl3solution. The solution will have a medium dark violet
Solutions will vary from light to dark violet colour.
colour.
Mark the flasks 1 (100 μL) to 5 (500 μL)
6. Measure and record the absorbance of each 6. Measure and record the absorbance at 530 nm
solution at 530 nm, using the FeCl3 solution as
blank.

Sample calculation
The following calculations are as an example only: in your report use your own mass
of SA and ASA samples, and your own Absorbance data!
1. Standard solutions and Beer’s law calibration curve
Mass of SA: 165.2 mg = 0.1652 g
Molar mass of salicylic acid (SA), HOC6H4COOH = 138.1 g/mol
(1) Moles SA = mass/molar mass = 0.1652/138.1 = 0.001197 mol SA The SA is
quantitatively transferred to a 100.0 mL volumetric flask, so:
(2) Concentration of SA stock solution = mol/0.1000L = 0.001197/0.1000 = 0.01197
mol/L
(3) Concentration of SA dilution “5” (0.5 mL stock to 10 mL) = (0.5/10)x0.01197 =
0.05x0.01197=0.0005986 mol SA/L This is (SA)
Same for concentrations of solutions “4” – “1” with 0.4, 0.3, 0.2, 0.1 mL stock
Solution 4: (0.4000/10)x0.01197 = 0.0004788 mol SA/L (SA)4
Solution 3: (0.3000/10)x0.01197 = 0.0003591 mol SA/L (SA)3

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Solution 2: (0.2000/10)x0.01197 = 0.0002394 mol SA/L (SA)2
Solution 1: (0.1000/10)x0.01197 = 0.0001197 mol SA/L (SA)1
You have recorded the absorbances, A of the 5 solutions (with the FeCl 3 subtracted
already by zeroing the instrument with the FeCl3 solution in the cuvette).
Record the absorbance values for solutions 1-5 in the table (below), and graph A vs
concentration for these 5 standard solutions.
Make your horizontal scale the concentration (e.g. from 0 to 0.0006 M with divisions
of 0.0001, and your vertical scale the absorbance, e.g. 0 to 1 with divisions of 0.1
2. Analysis of unknown Aspirin tablet
Note that now your initial mass is the mass of the aspirin (or ASA) tablet, which is not
pure salicylic acid! Molar mass ASA = 180.0 g/mol
Again, the numbers here are as an example only, use your own weight of the ASA
tablet, and ASA UNKNOWN Absorbance
(1) Mass of aspirin tablet = 0.354 g
(2) Absorbance (A) of “ASA UNKNOWN” = 0.866
(3) Concentration of ASA in unknown as determined from the calibration curve
below: CASA, unknown = 0.000528 (5.28 x 10-4) mol/L (see below)
(4) Concentration of “ASA UNKNOWN”: prepared by dilution 0.3000 mL to 10.0
mL, so the concentration in the “STOCK UNKNOWN” was 10/0.3, therefore
CASA stock = (10/0.3)xCASA,unknown = 10x0.000528/0.3000= 0.0176 mol/L
The aspirin tablet was dissolved and hydrolyzed and then diluted to 100 mL,
therefore:
moles ASA in 100 mL unknown = Cstock unknown(mol/L)x0.100(L) =
0.0176x0.100= 0.00176 mol ASA
Mass of acetylsalicylate (ASA) = mol x molar mass = 0.00176x180 = 0.317 g ASA or
317 mg.
% ASA in commercial tablet = g ASA/mass tablet = 0.317/0.354 = 89.5%
Calibration Curve:
Make graph of Absorbance A (absorbance units, y-axis) vs. concentration C (mol/L,
x-axis)

Concentration, Absorbance
SA Solution (examples ASA solution
M
From (3) above only) 7 Concentration
Absorbance (from graph)
1 0.000112 0.198
0.866 0.000528
2 0.000239 0.404
3 0.000359 0.586
For this example, the graph of A vs. M would be as follows:

The concentration of the ASA sample now can be read directly from the graph (the
value of M at A = 0.802) If you use Excel to make the graph (the program used in this
example), you can use the equation for the trendline (linear option) to find M from the
equation.
Safety precautions
1.0M NaOH is caustic. Avoid contact with skin, wear gloves and safety glasses at all
times. Be particularly careful when heating the ASA-NaOH mixture on the hot plate,
cover with watch glass to prevent spattering.

EXPERIMENT 2: Spectrometric analysis of complex mixture

Introduction
The analysis of a mixture of compounds is often difficult by optical methods if the
UV-visible spectra overlap appreciably. For example, both potassium dichromate and
potassium permanganate absorb strongly in the visible and ultraviolet region. Their
spectra overlap sufficiently so that the presence of one interferes with the quantitative
analysis of the other. Accurate analysis of the mixture is, however, possible by
employing multiwavelength techniques.
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If a solution is a binary mixture of two components in which the optical spectra only
partially overlap, only two wavelengths are necessary for a complete analysis. The
determination of the molar absorptivities of both pure components at both
wavelengths followed by the measurement of the absorbance of the mixture leads to 2
equations with 2 unknowns – an algebraically simple system of equations to solve. In
the analysis of a ternary system, 3 wavelengths are necessary. As the number of
components in the mixture increases, it becomes increasingly difficult to analyze for
the concentration of the components.
Multicomponent Analysis (MCA) allows an analyst to analyze for many components
in a mixture as long as the components of the mixture are known and spectra of each
pure component can be obtained. MCA can be performed "by hand" but lends itself
well to automation by computer. The spectrum of each component with known
concentration is obtained and stored in computer memory. Being digitally stored, the
molar absorptivity at each wavelength interval over a wide range of wavelengths is
determined by the program for each component.
Finally, the spectrum of the solution of the complex mixture is sampled and stored.
The program then, using least squares fitting or other error minimization technique,
reconstructs the spectrum of the sample from the spectra of the pure components. The
calculated concentrations of each component necessary to reconstruct the sample
spectrum are reported by the computer in the units selected by the operator. In the
analysis to be performed, the amount of KMnO4 and K2Cr2O7 in a binary mixture will
be determined by two-wavelength analysis. This type of analysis could be used in, for
example, the simultaneous analysis of Mn and Cr in steel.
Theory
In the absence of molecular interactions of the absorbing species in a mixture, the
observed absorption spectrum at a given wavelength for the mixture is the sum of the
absorbance contribution of each species. Thus, the observed spectrum of a mixture of
the two species in Figure 1 would be a wavelength-by-wavelength sum of the
absorbances due to each species. This being the case, it is possible to analyze for both
species in solution almost simultaneously by measuring the absorbance of the mixture
at two wavelengths (1 and 2 in Figure 1). If the molar absorptivity of each
component is known, then the concentration of each component can be determined
algebraically. The following is a derivation of the Beer's law analysis of a two-
component mixture. A 1.0 cm pathlength sample cell will be assumed for all
equations.
The Beer-Lambert law for a single component is:
A = lC (1)
Where A is the observed absorbance for concentration C,  is the molar absorptivity,
and l is the pathlength of the sample cell. The quantity l is a constant at a given
wavelength and can be denoted as k.
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For a solution containing two absorbing species, the absorbance at a given wavelength
is
A = AA + AB (2)
Which can be rewritten

A = kCA + kCB (3)

Since this is an equation with 2 unknowns (C A and CB) it is necessary to take

measurements at 2 wavelengths.

A = kCA + kCB (4)

A = kCA + kCB (5)

Then by the solution of two simultaneous equations, the concentrations of species A
and B can be calculated. The values of k are determined from the slopes of the
calibration plots prepared at the wavelengths of interest. It should be clear that the
spectra must have sufficient "non-overlap" so that only one species is the primary
contributor to the observed absorbance at a given wavelength. It should also be
evident that better accuracy would be achieved if multiple wavelengths were chosen
and concentrations determined by multiple rather than two equations.
Procedure
Preparation of Standard Solutions

Prepare 100 mL of 3-7 x 10 -3 M K2Cr2O7 by a suitable dilution of a stock solution.

The concentration of this dilution should be known to the best possible precision. Use
0.5 M H2SO4 as the diluent.

Prepare 100 mL of 3-7 x 10-3 M KMnO4 by a suitable dilution of a stock solution

as above. Use 0.5 M H2SO4 as diluent also. The standard KMnO4 should
contain~0.2% KIO4 added as a stabilizer; no additional precautions need be
taken to prevent decomposition.

Prepare four dilutions of the standard solutions. Into four 100 mL volumetric flasks,
pipette 2, 5, 7, and 10 mL of the K 2Cr2O7. Dilute with 0.5 M H2SO4 to the mark.
Repeat with the KMnO4 standard solution. Calculate the concentrations to the
appropriate precision.
Analysis of Standards
Rinse and fill one cuvette of a matched pair with 0.5 M H 2SO4 (Reference). Rinse and
fill the other with the most concentrated dilution of K2Cr2O7 (Sample). Measure and
plot the absorption spectrum from 200-700 nm against the reference.

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Rinse and fill the sample cuvette with the most concentrated dilution of KMnO 4.
Measure and plot the absorption spectrum on the same page with the same scale over
the same wavelength range (Disable autoscaling if necessary).
Select two wavelengths for analysis. Scan the remaining standards and obtain
absorbance readings for each dilution at both wavelengths. Plot the absorbance vs.
concentration (Beer's law plot) at both wavelengths for both solutions. Calculate by
the method of least-squares, the molar absorptivities, , of both anions at both
wavelengths.

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