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However, many systems involve more than one colored component. If these components act
independently, then Beers Law is still applicable but more than one wavelength must be used
for the analysis. The figure below illustrates a two component spectrum.
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 3 of 11
The maximum absorbance of component 1 occurs at wavelength 1 while the maximum
absorbance of component 2 occurs at wavelength 2. The dotted line represents a solution that is
a mixture of components 1 and 2.
In this experiment, we will use a fiber optic diode array spectrophotometer. A schematic
diagram of this instrument is shown below.
The advantage of this instrument is that all wavelengths are recorded at once. Therefore we can
signal-average to reduce noise and apply other digital spectral smoothing techniques. The
spectrometer uses a high-pressure deuterium lamp to produce ultraviolet radiation but the
instrument is less sensitive in the UV region as compared to the visible region where the spectral
source is a incandescent tungsten lamp.
In this experiment, we will only use spectral data between 450 and 650 nm although every
spectrum records data from 187 to 900 nm. We will analyze the quantitative data using linear
regression which in this case is applying Beers Law the component in our sample.
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
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 4 of 11
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=abc). 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:
Salicylic acid (sal) iron(iii)-salicylate complex
highly colored
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.
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 Fe
3+
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.
OH
O
OH
+ X Y Fe
3+
(Fe )
3+
y
(Sal)
x
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 5 of 11
Safety Hazards
General laboratory safety rules should be followed. Nitric acid is corrosive, and spills should be
cleaned up immediately.
INSTRUCTIONS UV-VIS SPECTROPHOTOMETRY EXPERIMENT
Be sure to clean up the area when finished
Run the SpectraSuite program from the Desktop. Make sure the detector is hooked up to the
USB port and the lamp module is on. The instructor will show how to run the dark current
(electronic diode noise) and the use water or the 10 mM Fe(NO
3
)
3
solutions in a cuvet as the
reference spectrum. You may have to adjust the integration time, number of runs, and boxcar
smoothing to obtain the optimum spectra. Check with the instructor on how this can be done.
Save each spectrum as a tab-separated variable *.txt file and save. You will need a flash drive
to copy the files.
With the spectrometer on, block the light beam with a plug and save the dark current by pressing
the gray bulb.
Then place your blank in the beam and adjust scans to average to 3 and the boxcar integrator to
5. Adjust the integration time until the high point of the spectrum stays at or near 4000 counts all
across the spectral region that you are interested in.
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 6 of 11
The block the source again and record the dark current under the new conditions. Never select
anything from the top level commands. To save the spectrum, always select the file disk circled
in the picture.
The A or absorbance button should be lit and scale set to absorbance. You can select the little
magnifying glass highlighted in the last picture to set the viewing wavelength and absorbance
units.
To save the file, select the file disk, choose Pr ocessed Spect r um, Tab Del i mi t ed- No
Header , and then choose the Br owse button.
Create a file with the first spectrum to be saved, open the folder and then type in the file name
and select Save.
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 7 of 11
Select Save again.
The Save command will gray out and the choose Cl ose. You are now ready to insert the cuvet
with your next sample.
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, Fe(III)
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 Fe(III), while keeping the total number of moles
constant. The mixture yielding the maximum absorbance corresponds to the predominant
stoichiometry of the complex formation.
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 8 of 11
Solutions Needed for the Experiment:
Solution Composition Notes
1.
0.1 M (100 mM)
sodium salicylate)
Weigh out 16.01 of sodium salicylate (MM - 160.11
g/mole) and dilute to 1.0 L with distilled water.
0.010 M (10 mM)
sodium salicylate
Dilute the 100 mM sodium salicylate 1:10.
10 mM Fe
3+
Dilute 5.987 mL of the stock 404 gm/L Fe(NO
3
)
3
stock to
1.0 L with 0.060 M HNO
3
60 mM HNO
3
Dilute 3.797 mL of concentrated HNO
3
(15.8 M) to 1.0 L.
Part A: 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 (60mM) in
small beakers. You will also need the 10 mM sodium salicylate solution. Return any unused
solution to the bottle.
2. Prepare solutions for spectrophotometric analysis by pipetting the appropriate amount of each
solution into a small test tube. Use the amounts from the Table 1 below. Label all your vials.
3. Add 3.00 mL of 60 mM HNO
3
to make each test tube to a total volume of 4.00 mL. Mix
thoroughly and add to a plastic cuvet.
Table 1: Solution Composition for Method of Continuous Variation
Solution
Volume
10mM
salicylate
(mL)
Volume
10mM
ferric
nitrate
(mL)
Volume
60mM
nitric
acid
Mole
Ratio
Fe(NO
3
)
3
:
salicylate
Mole
Fraction
Fe(NO
3
)
3
2.Showthe"JobPlot"ofabsorbance(at
max
)versusmolefractionofiron(III)that
youobtainedinpartAoftheexperiment.Fromthisdata,indicatewhatyou
believeisthestoichiometryofthereaction.
3.ProvideaplotofthesalicylateFe(III)complexspectraforyourcalibrationcurves
(includingyourunknownspectra.)
4.Showtheplotofthecalibrationcurveforsalicylate(includingequationoflinewith
R
2
value).
5.Reportthemeanconcentrationofsalicylateinacnefacewash(inunitsofweight
percent)thatyoufound.
6.Reporttheaverageconcentrationofsalicylateinyourunknownsample(inunitsof
molarity,ormillimolarity.)Makesureyouindicateyourunknownsamplecode..
REFERENCES:
1. Lab Handout.
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6. Cavanaugh, M. A.; Bambenek, M. A. J. Chem. Educ. 1978, 55, 464.
7. Lane, S. R.; Stewart, J. T. J. Chem. Educ. 1974, 51, 588589.
8. Battezzati, A.; Fiorillo, G.; Spadafranca, A.; Bertoli, S.; Testolin, G. Anal. Biochem. 2006,
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9. Rogic, D. J. Mol. Struc. 1993, 294, 255258.
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11. Saltzman, A. J. Biol. Chem. 1948, 174, 399404.
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