Instructor Notes

Lab Documentation/Supporting Materials for, "Salicylate Detection by Complexation and

Optical Absorbance Spectroscopy: An Undergraduate Quantitative Analysis Experiment"
Instructor Notes:

The equipment requirement for this exercise is very flexible. The experiment may be
conducted with scanning/diode array instruments or single wavelength
spectrophotometers. If only single wavelength instruments are available, it is best to
provide students with the maximum absorbance wavelength (max = 535 nm).
We typically provide students with the ferric nitrate solution and the dilute nitric acid. As
an option, students may prepare the ferric nitrate. However, as a safety precaution, it is
advisable to provide students with the dilute acid instead of having them dilute the
concentrated form.
Error analysis for Part A can be as simple as determining the standard deviation of three
samples. Instructors have the option to include the error introduced by the calibration
curve. A prelab powerpoint presentation that is included in these materials walks
students through this process. For the MS Excel analysis, utilize Tools:Data
Analysis:Regression to give the output shown in the supporting MS Excel File.
A sample grading rubric is also included in these materials to assist instructors in
assessing the quality of the lab reports.

Reagent/Equipment List:
1.
2.
3.
4.
5.

Spectrophotometers
Cuvettes for the spectrophotometers
Solid sodium salicylate (CAS#: 54-21-7)
Volumetric flasks (10, 50, and 100 mL)
Clean & Clear salicylic acid acne medication (several products will work, the best results
are obtained with solutions that are colorless in the visible region of the spectrum.
6. 10 mM ferric nitrate (CAS #: 7782-61-8) dissolved in 0.06 M nitric acid (CAS #: 769737-2)
7. 0.06 M nitric acid (CAS #: 7697-37-2)
8. Test tubes/vials that hold at least 15 mL
9. 10.00 mL volumetric pipets
10. 100-1000 L micropipettes, or microliter syringes

Student Instructions
Visible Spectrophotometry: Determination of Salicylate via Reaction with Fe(III)
Background
Spectroscopic analysis is a critical tool in the identification and quantitation of different
molecules. This experiment introduces you to the use of electronic absorption spectroscopy in
the visible region of the spectrum for the determination of salicylate. There are several uses for
salicylate and it is therefore included in many everyday products. Salicylic acid is the major
metabolite of aspirin and is commonly found in medications that treat acne, warts and other
similar ailments. When acetylsalicylic acid (aspirin) is taken for a headache or inflammation, it is
rapidly hydrolyzed in the stomach. The products of this reaction are salicylic acid and acetic
acid. The former is readily absorbed into the blood stream and is then able to act as an analgesic
agent.
In acne treatment, the salicylic acid decreases the shedding of skin cells from hair
follicles. These cells are typically responsible for clogging pores and causing pimples. Salicylic
acid also has a keratolytic (peeling) effect, which causes dead cells to be shed more easily. This
facilitates in the removal of a thin layer of skin and promotes the unclogging of pores. More
concentrated solutions of salicylic acid are used in wart treatment to help soften the wart and to
stimulate an immune response toward the human papillomavirus, responsible for causing wart
formation.
Due to the many medical applications of salicylic acid, the development of analytical
techniques for its quantification is important. Indeed, there are a number of methods that have
been employed, including, gas-liquid chromatography (GLC), ultraviolet spectroscopy, and
fluorescence spectroscopy. The most widely used methods in clinical laboratories, however, use
colorimetric or visible spectrophotometry. A version of this method will be applied throughout
the experimental procedure to first quantitate salicylate in a commercial product (face wash), and
also in an unknown solution that you will be given. The second part of the procedure uses
spectrophotometry to investigate the chemical nature of the reaction that yields the colored
product you analyze.
Measurement Principles
Beer's Law states that the absorbance of a compound is directly proportional to its
concentration (A=bc). This linear relationship allows us to first construct a calibration curve by
collecting the absorbance values for samples of known concentration at a given wavelength,
preferably the max, the wavelength where maximum absorption occurs. The resulting equation
for the linear regression then lets us determine the concentration of an unknown sample by
determining its absorbance at the same wavelength.
Salicylate and salicylic acid do not absorb visible light, creating an experimental
challenge. Upon reaction with iron (III) ions, however, a highly colored species results:
O
OH

(Fe3+)y(Sal)x

+ y Fe3+

iron-salicylate complex
Highly Colored

OH

salicylic acid (sal)

The complex can be easily detected with a simple spectrophotometer and thus, you will be able
to quantify salicylate in unknown samples. Under the acidic experimental conditions all
salicylate will be protonated as shown in the chemical equation above.
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Student Instructions
The chemical equation shown above contains the coefficients and subscripts x and y. In
the second portion of this experiment, you will use the method of continuous variation (also
called Job's method) to determine these quantities for the predominant complex. For this
procedure, several solutions containing different quantities of salicylate and Fe3+ will be
prepared. While the amount of each reactant is varied, the total moles of both reagents will
remain constant. The solution that yields the greatest absorbance at max indicates the
predominant stoichiometry of the iron-salicylate complex.
Safety Hazards
General laboratory safety rules should be followed. Nitric acid is corrosive, and spills
should be cleaned up immediately.
Procedure
In this experiment, the concentration of salicylate present in an over the counter acne
medication/face wash and in an unknown sample will be determined by spectrophotometry.
Salicylate itself absorbs ultra-violet radiation and is therefore difficult to measure directly with
simple instrumentation. One method adopted for the measurement of salicylate in clinical
situations involves mixing samples containing salicylate with an excess of ferric ions (FeIII)
under acidic conditions. The resulting complex absorbs strongly in the visible region of the
spectrum and can be easily determined spectrophotometrically. The first section of the
experiment involves using this salicylate-iron complex for the determination of salicylate
concentration in an acne medication and an unknown sample. This will be possible by first
generating a calibration curve for salicylate from several standard solutions of different
concentration. In the second section the nature of the salicylate-iron complex will be
investigated by using the method of continuous variation. This procedure involves varying the
amount of each reagent added (salicylate and FeIII) while keeping the total number of moles
constant. The mixture yielding the maximum absorbance corresponds to the predominant
stoichiometry of the complex formation.
Part A: Spectrophotometric Determination of Salicylate in Acne Medication
1. Prepare five standard solutions of sodium salicylate in deionized water. For this task, use
a 100 mL volumetric flask to prepare an initial stock solution of 100 mM (0.1M) of
sodium salicylate (you should weigh out sodium salicylate so you will know the exact
molarity after you dilute the volumetric flask to mark). By dilution in appropriate
volumetric flasks, prepare standards of 20 mM, 40 mM, 60 mM and 80 mM (10 mL
volumetric flasks will give enough of these standards for this experiment). Prepare 100
mL of a 10 mM standard also in water. The 10 mM solution is also required in Part B
and making a larger quantity now will save time!! (NOTE: Part B requires that this
standard be exactly 10 mM!!) Sodium salicylate has a formula weight of 160.11
g/mole. It is important to record the accurate mass of sodium salicylate that you used to
prepare the stock solution, so that you will know the exact molarity of these standards.
2. Obtain a sample of acne face wash solution as well as an unknown salicylate sample
(solution). Be sure to record the code of your unknown for your lab report.
3. In separate test tubes, pipet exactly 100 L of each standard, the acne face wash, and
your unknown. Repeat this procedure two more times for the acne wash and unknown
for a total of three samples for each (to allow for a standard deviation to be generated). It
is important to label these test tubes for identification of each solution. Add 10.00 mL of
the acidic 10 mM ferric nitrate solution (stock in lab) to each test tube. Be sure to mix
these solutions well!
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Student Instructions

4. Using the acidic 10 mM ferric nitrate solution as your blank (100%T), collect optical
absorbance spectra for all of the solutions. Collect an absorbance spectrum (400-700 nm)
for your most concentrated standard to determine max (remember to save the file!). If the
maximum absorbance is greater than 1, check your max by collecting a spectrum with a
less concentrated standard. Once you determine max, set the Spec 20 to this wavelength
and analyze your standards and unknowns. Record the absorbance values for all
solutions in your notebook. Be sure that all solutions yield an absorbance of less that 1.0
at the max . Absorbance greater than this cannot be accurately determined with the
spectrophotometer. Make sure you rinse the cuvette out between each measurement and
then rinse with small amount of standard/sample solution first, before filling the cuvette
with the given test solution.
Reminder: It is ideal to generate your calibration curve during your data collection. This
will allow time to repeat any measurements during the same lab period. Doing this will
likely improve your accuracy.
Part B: Determination of Reaction Stoichiometry: An Application of the Method of
Continuous Variation
1. Obtain ~20 mL 10 mM acidic ferric nitrate as well as ~50 mL of dilute nitric acid (60
mM). You will also need the 10 mM sodium salicylate solution prepared in Part A. This
salicylate standard must be exactly 10 mM for the volumes in Table 1 to work properly.
2. Prepare solutions for spectrophotometric analysis by pipetting the appropriate amount of
each solution into a vial (total 1 mL). Use the amounts from the Table 1 below. Label all
your vials!!! Add 4.00 mL of dilute nitric acid to each mixture of salicylate and ferric
nitrate to make a total volume of 5.00 mL.
3. Using the dilute nitric acid as your blank, collect the absorbance values at max for each
solution.
Table 1: Solution Composition for Method of Continuous Variation
Volume of 10 mM
salicylate/mL
0.100
0.200
0.250
0.330
0.400
0.500
0.600
0.670
0.750
0.800
0.900

Volume of 10 mM
ferric nitrate/mL
0.900
0.800
0.750
0.670
0.600
0.500
0.400
0.330
0.250
0.200
0.100

Mole Ratio
Fe(NO3)3:salicylate
9.00:1.00
4.00:1.00
3.00:1.00
2.00:1.00
1.50:1.00
1.00:1.00
1.00:1.50
1.00:2.00
1.00:3.00
1.00:4.00
1.00:9.00

Mole Fraction of
Fe(NO3)3
0.900
0.800
0.750
0.667
0.600
0.500
0.400
0.333
0.250
0.200
0.100

Data Handling
1. The objective for the first section of the experiment is to determine the concentration
of salicylate in unknown samples. Construction of a calibration curve from the
absorbance data collected from the salicylate standard solutions is the first necessary
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Student Instructions
step towards this goal. In Excel, plot the absorbance value of each standard (at the
max) versus the standard's concentration. The data should show a linear relationship.
Generate a linear regression line and equation for this line. Only include data points
with an absorbance less than 1.
2. Determine the concentration of salicylate for each of your samples of acne medication
and unknowns from the linear regression line. Find the average and standard deviation
for each determination. In order to compare your data with the value printed on the
bottle's label, convert your face wash data to units of weight percent. Enter this data
into the list of class data for further analysis.
3. The data collected in the second part of this experiment will allow you to examine the
nature of the reaction between FeIII and salicylate. While the mole ratios of reagents
were varied in each mixture, the total number of moles remained the same. Therefore,
the mixture that yields the greatest absorbance represents the predominant reaction
stoichiometry. In order to find which stoichiometry is favored by this complex, plot
the absorbance value of each solution (at the max) versus the mole fraction of iron.
Lab Data Report
1. Provide a plot of the salicylate-Fe(III) complex spectrum for the most concentrated
standard solution indicating the max.
2. Show the plot of the calibration curve for salicylate (including equation of line with R2
value).
3. Show the "Job plot" of absorbance (at max) versus mole fraction of iron (III) that you
obtained in part B of the experiment. From this data, indicate what you believe is the
stoichiometry of the reaction.
4. Report the mean concentration of salicylate in acne face wash (in units of weight
percent) with confidence interval (95%) that you found. Compare your average value
to class average using 95% confidence range treating the class average as the true
value.
5. Report the average concentration of salicylate in your unknown sample (in units of
molarity, or millimolar) with associated confidence interval. Make sure you indicate
your sample code.

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Student Instructions

Visible Spectrophotometry Gradesheet

Part

Description

Points

Introduction and
Presentation

Concise and clear

documentation in
professional style

10

Part A:
Salicylate
Determination

Overlay of Fe-salicylate
complex spectra for
standards and the
corresponding calibration
curve

10

Report mean value for

salicylate concentration in
Clean & Clear Face Wash
(in units of weight percent).
-- 95% Confidence Interval
-- Comparison with Class
Data

20

Report identity and mean

salicylate concentration
present in unknown sample
(in units of molarity or
millimolar).
-- 95% Confidence Interval

30

Part B:
Job Plot

Job Plot
-- Determination of reaction
stoichiometry

25

Data entry

If entered

5
100

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Points

Total

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