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Instrumental Analysis Laboratory

College of Charleston UV-VIS SPECTROMETRY


UV-VIS Spectrometry
Multivariate Linear Regression
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SPECTROPHOTOMETRIC MEASUREMENTS

Adaptedfrom: SalicylateDetectionbyComplexationwithIron(III)andOpticalAbsorbance
SpectroscopyAnUndergraduateQuantitativeAnalysisExperiment,J.Chem.Ed.,2008,85,16581659.

J. T. Mitchell-Koch, Department of Chemistry, Emporia State University, Emporia, KS, K. R. Reid and M. E.
Meyerhoff, Department of Chemistry, University of Michigan, Ann Arbor, MI

INTRODUCTION

For chemical species that appear to have color, it is a logical assumption that the intensity of the
color is proportional to the concentration of the species in solution. We see color as a
complement of the visible wavelength being absorbed by the sample. Things that appear red
absorb blue visible light and reflect other visible colors to our eyes. Conversely, things that
appear blue are absorbing red light. Absorption of light or more precisely electromagnetic
radiation is related to available energy levels in the molecule or ion. A molecule in its "ground
state" or lowest energy level can absorb energy to jump to an "excited state" or higher energy
state. The amount of energy and therefore the wavelength of radiation involved in this transition
is a function of the electronic structure of the molecule or ion.

The eye can only see a limited range of electromagnetic radiation, from approximately 400 to
700 nm. However, molecules, atom, and ions are capable of absorbing many different energies
of radiation ranging from ultraviolet (UV) to microwaves depending on the specific energy
levels being excited. For some types of energy changes, the wavelengths of light are very
specific for certain types of chemical structure resulting in a method of qualitatively identifying
chemical species. Other types of energy absorption may be less qualitative since it may relate
only to bond types. In both cases however, our initial premise that intensity of absorption is
related to concentration can be used for quantitative analysis.

Since our vision if not quantitatively calibrated, an electronic instrument called a
spectrophotometer is used to precisely measure light intensities at given energy (wavelength)
settings. A spectrophotometer is an instrument that measures the amount of transmission of light
through a substance. The drawing below illustrates a simple spectrophotometer system
consisting of a light (energy) source, a monochromator to select a given energy range, a sample,
and a light intensity detector.

Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
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When light is absorbed by a sample, the radiant power or intensity of the light beam decreases.
Radiant power, I, refers to the energy per second per unit area of the beam. In the figure, light
passes through a monochromator that selects one wavelength. Light of this wavelength, with
radiant power I
0
, passes through a sample of pathlength b. The radiant power of the beam
emerging from the other side of the sample is I. Mathematically, the amount of light that is
absorbed (A) is given by
0
I
A ln
I
| |
=
|
\ .

Note that if no light is absorbed, A = 0 and if all the light is absorbed ( I = 0) then A = . The
amount of light absorbed by the sample should be proportional to the probability that the
molecule or ion will absorb the electromagnetic radiation (a), the number of absorbing molecules
or ions per unit volume that the light beam passes through (C), and the length of the light path
(b). This relationship is quantified in the Beer-Lambert (or Beer's) Law which is
A = a b C
Note that this equation is in the form of Y = m X + b where the intercept, b, is zero when X or
the concentration, C, is zero. If we measure a series of solutions of known C at a given
wavelength in a cuvet or sample cell with a constant pathlength, b, then we can determine the
proportionality constant, m, which is a b. This procedure generates a "calibration curve" which
allows the determination of an unknown concentration, C
unk
, from the measurement of the
absorbance of the unknown, A
unk
. Determination of the slope, m, and intercept, b, of the
calibration curve gives
unk
unk
A
C
m

=
b

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
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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
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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
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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
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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
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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
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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

1 0.100 0.900 3.000 9.00 0.90


2 0.200 0.800 3.000 4.00 0.80
3 0.250 0.750 3.000 3.00 0.75
4 0.330 0.670 3.000 1.99 0.67
5 0.400 0.600 3.000 1.50 0.60
6 0.500 0.500 3.000 1.00 0.50
7 0.600 0.400 3.000 0.67 0.40
8 0.670 0.330 3.000 0.50 0.34
9 0.750 0.250 3.000 0.33 0.25
10 0.800 0.200 3.000 0.25 0.20
11 0.900 0.100 3.000 0.11 0.10
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 9 of 11
4. Using the 60 mM HNO
3
as your blank, collect the absorbance values each solution.

Data Analysis Part A:

The data collected in this part of this experiment will allow you to examine the nature of the
reaction between Fe(III) 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.

Part B: Spectrophotometric Determination of Salicylate in Acne Medication

1. Prepare five standard solutions of sodium salicylate in deionized water. For this task, we use
an initial stock solution of 100 mM (0.1M) of sodium salicylate. By dilution in appropriate a 10
mL volumetric flask; 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). 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. Dilute them to volume in the 10 mL volumetric flask with distilled water.

Table 2: Solution Preparation for Calibration Curve
Solution
mL 100 mM sodium
salicylate/10 mL
volumetric
Concentration of
sodium salicylate
1 2.00 20mM
2 4.00 40mM
3 6.00 60mM
4 8.00 80mM

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 50 L of each standard, including the 100 mM sodium
salicylate, the acne face wash, and your unknown. It is important to label these test tubes for
identification of each solution. Add 5.00 mL the acidic 10 mM ferric nitrate solution (stock in
lab) to each test tube. Be sure to mix these solutions well!

4. Using the acidic 10 mM ferric nitrate solution as your blank (100%T), collect optical
absorbance spectra for all of the solutions

Data Analysis Part B:

1. The objective for this section of the experiment is to determine the concentration of salicylate
in unknown samples. Construction of a calibration curve from the absorbance data collected
Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 10 of 11
from the salicylate standard solutions is the first necessary step towards this goal. In Excel, plot
the absorbance value of each standard (at the versus the standard's concentration. The data
should show a linear relationship. Generate a linear regression line and equation for this line.

2. Determine the concentration of salicylate for each of your samples of acne medication and
unknowns from the linear regression line. Convert your face wash data to units of weight
percent assuming the density of the solution to be 1.00 gm/mL.

Data Processing in Excel:

After the spectra are run the data files can be imported into EXCEL

. The data will be in the


form of wavelength and absorbance data for each spectrum. Copy this data into EXCEL using
the text file input command and tab-delimited data. Column A is always constant contains the
wavelength of the spectrum and column B should be the absorbance. Move the cursor to column
C and import the next set of data. Make sure that the data is aligned correctly according to
wavelength and then copy only the absorbance data column B). After importing the spectral
data, your spreadsheet should now contain the wavelengths in column A and columns of
absorbances. Be sure to label the data in each column. You can simplify the spreadsheet by
deleting all wavelengths from 187 to 300 and 650 to 900 nm. Plot the absorbances versus
wavelength for your method of continuous variation and analytical data. This is simplified in
Excel by finding the wavelength and absorbances that you want to copy and the using the
TRANSPOSE option in the Past e Speci al command.


Instrumental Analysis Laboratory
College of Charleston UV-VIS SPECTROMETRY
UV-VIS Spectrometry
Multivariate Linear Regression
Page 11 of 11
Your report should contain the following:

1.ProvideaplotofthesalicylateFe(III)complexspectrafromthemethodof
continuousvariations.

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.
2. Ferguson, G. K. J. Chem. Educ. 1998, 75, 467469.
3. Hein, J.; Jeannot, M. J. Chem. Educ. 2001, 78, 224225.
4. Simonson, L. A. J. Chem. Educ. 2001, 78, 1387.
5. Yang, S.-P.; Tsai, R.-Y. J. Chem. Educ. 2006, 83, 906909.
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,
354, 274278.
9. Rogic, D. J. Mol. Struc. 1993, 294, 255258.
10. Lange, W. E.; Bell, S. A. J. Pharm. Sci. 1966, 55, 386389.
11. Saltzman, A. J. Biol. Chem. 1948, 174, 399404.
12. Annino, J. S.; Giese, R. W. Clinical Chemistry: Principles and Procedures, 4th ed.; Little,
Brown and Co.: Boston, 1976; pp 355357.

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