EXPERIMENT 12
UV/VIS Spectroscopy and Spectrophotometry:
Spectrophotometric Analysis of a Commercial Aspirin Tablet
Outcomes
After completing this experiment, the student should be able to:
1.
2.
3.
4.
Prepare standard solutions.
Construct calibration curve based on Beer’s Law.
Use Beer’s Law to determine molar absorptivity.
Explain the fundamental principal behind spectrophotometric analysis.
Introduction
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 UV/VIS 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.
O
O
C
CH3
+ 3OH
_
O
_
_
O
OH
_
+ CH3COO + 2H2O
O
O
Acetyl salicylic acid
Salicylate dianion acetate anion
Once the de-esterification is complete, the solution is acidified, and FeCl3 is added. The salycilic acid
will react with the Fe3+ to form a coloured complex ion:
OH
O
O
O
_
+
Fe(H2O)63+
+
+
Fe(H2O)4
O
+H2O +H3O+
O
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The spectrum of the Fe salicylate complex was shown in figure 1, plotted as T vs. wavelength. Note
that the lowest transmission (i.e., the largest absorption of light) is in the blue green and yellow areas
of the spectrum, resulting in the violet colour. 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 Fesalicylate 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 azsbsorbance of the solution prepared from the commercial aspirin solution,
and find its concentration by comparing its absorbance value on the calibration curve.
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 g/mol
(1) Moles SA = mass/molar mass = 0.1652/138 = 0.001197 mol SA
The SA is quantitatively transferred to a 100 mL volumetric flask, so:
(2) Concentration of SA stock solution = mol/0.100L = 0.001197/0.100 = 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)5
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
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 FeCl3 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.
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
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(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%
ASA stated on the box is 325 mg. Of course this is a fictitious example, we would expect most
students to come within 10 mg (3%) of the 300 mg, possibly even better!
Calibration Curve:
Make graph of Absorbance A (absorbance units, y-axis) vs. concentration C (mol/L, x-axis)
SA Solution
Concentration, M
From (3) above
Absorbance
(examples only)
1
0.0001197
0.198
Absorbance
Concentration
(from graph)
2
0.0002394
0.404
0.866
0.000528
3
0.0003591
0.586
4
0.0004788
0.784
5
0.0005986
0.982
ASA solution
For this example, the graph of A vs. M would be as follows:
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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.
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, 1 mL graduated pipet or 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.
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 mL
volumetric flasks: use micropipet to add 100,
200, 300, 400, and 500 μL stock SA into 10.0 mL
volumetric flasks. Fill each flask to the mark with
acidified FeCl3solution.
Solutions will vary from light to dark violet
colour.
Mark the flasks 1 (100 μL) to 5 (500 μL)
5. Prepare one ASA solution: use micropipet to
add 300 μL ASA stock in 10.0 mL volumetric
flask. Fill the flask to the mark with acidified
FeCl3solution.
The solution will have a medium dark violet
colour.
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.
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Experimental General Chemistry 1
Experiment 12: Analysis of a Commercial Aspirin or ASA Tablet
Laboratory Data Sheet
Name: ______________________________________________ Section:
________
Brand name of commercial tablet: ________________________________
Calculations.
1. Standard solutions and Beer’s law calibration curve
(1) Mass of SA
= __________ g
Molar mass of salicylic acid (SA), HOC6H4COOH
= __________ g/mol
(2) Moles SA = mass/molar mass
= __________mol SA
The SA is hydrolyzed and quantitatively transferred to a 100 mL volumetric flask, so:
(3) Concentration of SA stock solution = mol/0.100L = (2)/0.100 = __________ mol/L
(4) Concentration of SA solution “5” (0.5 mL stock to 10 mL)
= (0.5/10)x(stock solution concentration)
= __________ mol/L
A5 = ____
SA solution “4” (0.4 mL stock to 10 mL)
= __________ mol/L
A4 = ____
SA solution “3” (0.3 mL stock to 10 mL)
= __________ mol/L
A3 = ____
SA solution “2” (0.2 mL stock to 10 mL)
= __________ mol/L
A2 = ____
SA solution “1” (0.1 mL stock to 10 mL)
= __________ mol/L
A1 = ____
Enter these concentrations and absorbance in an Excel sheet and create a calibration curve. Attach
this calibration curve with this report.
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!
(1) Mass of tablet
= __________ g Aspirin
(2) Absorbance of “ASA UNKNOWN”
= __________
(3) Concentration of ASA in unknown diluted solution as determined from the calibration curve,
CASA, unknown
= __________ mol/L
(4) Concentration of “ASA UNKNOWN”: prepared by dilution 0.3 mL to 10 mL, so the
concentration in the “STOCK UNKNOWN” was 10/0.3, therefore
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CASA stock unknown = (10/0.3)xCASA,unknown
= __________ mol/L
The aspirin tablet was dissolved and hydrolyzed and then diluted to 100 mL, so moles ASA in
unknown = Cstock unknown(mol/L)x0.100(L)
= __________ mol ASA
Molar mass of ASA = 180 g/mol
Mass of acetylsalicylate (ASA) = mol x molar mass
= __________ g ASA
% ASA in commercial tablet = g ASA/mass tablet x 100%
= __________ %
Make graph of Absorbance A (absorbance units) vs. concentration C (mol/L)
You may want to enter concentrations and absorbances of solutions 1-5 in a table in Excel and graph
the calibration curve in Excel !
Compare your result to the mg/tablet noted on the label of the box!
Conclusion and Comments:
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