12 UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of a Commercial Aspirin Tablet
Outcomes After completing this experiment, the student should be able t o: 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 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 i s the trade name for acetylsalicylic acetylsalicylic 3+ acid (ASA). The ASA in the tablet will be reacted with Fe , forming an intensely violet coloured complex. The concentration of the complex will be determined by means of spectrophotometry, spectrophotometry, using a UV/VIS spectrophotometer. Finally, we will be able to calculate the weight and the weight% of ASA in the commercial tablet. Th e dete deterr mi nati on of acetyl acetyl sali cyli c aci aci d b by y spe spectr ctr ophotometr ophotometr y
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
_ _ + CH3COO + 2H2O
O
OH 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
67
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 fol lowin g calcul ations are as an example only: in your report (() use your own mass of SA and ASA samples, and your own Absorbance data! 1. Standar d soluti ons 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 u nkn own Aspir in 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. Agai n, the numbers her e are as an example only, use your own weight of the ASA tablet, and ASA UN KN OWN Absorbance
(1) Mass of aspirin tablet
= 0.354 g 66
(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 t o 100 mL, therefore: moles ASA in 100 mL unknown = C stock ASA
unknown (mol/L)x0.100(L)
= 0.0176x0.100= 0.00176 mol
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:
6
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.
Oper ation of the spectrophotometer.
Follow the instructions in the laboratory. Use FeCl3 as the blank solution to zero the absorbance reading. 2.
Prepar ation of the F e-sali cylate standard soluti ons and ASA unk nown
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 small dry beaker. Record the mass (g).
1. Weigh ASA tablet in beaker, record the mass, 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. Fill to the mark with distilled water. This is your “stock” SA solution
4. Transfer solution to 100 mL volumetric flask. Fill to the mark with distilled water. This is your “stock” ASA soluti on
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 solution at 530 nm, using the FeCl 3 solution as blank.
6. Measure and record the absorbance at 530 nm
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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 Molar mass of salicylic acid (SA),
= __________ g 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 u nkn own Aspir in 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 dil uted 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 8
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:
