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Simultaneous determination of phenacetin and caffeine using

SIMULTANEOUS DETERMINATION OF PHENACETIN AND CAFFEINE USING A
DOUBLE BEAM SPECTROMETER
To simultaneously determine the concentrations of phenacetin and caffeine in an unknown aqueous solution using scanning UV-visible spectrophotometryat two wavelengths.
Introduction: This experiment is a portion of the standard analysis for the three activecomponents in (Aspirin/Phenacetin/Caffeine) tablets. However, these tablets are nolonger marketed, since newer analgesic preparations do not produce the side effectssometimes observed with phenacetin.
In the older standard method, am APC tablet is ground up and extractd with aqueous base to dissolve the three active ingredients. The compound structures are as follows: In basic solution, aspirin exists as the carboxylate anion (RCOO-) and is removed fromthe aqueous tablet extract by passing thru an anion exchange column, which retains theaspirin quantitatively. Phenacetin and Caffeine remain in the aqueous extract, and theirconcentrations can be determined spectrophotometrically, since both have strong UVabsorption spectra.
The analyses cannot be performed by simply reading an absorbance at one wavelength for phenacetin and at another for caffeine, because their UV spectraoverlap, and thus each component interferes with the other's determination, as can beseen in Figure 1.
However, it is still possible to analyze both components in the same soluiton by measuring absorbances at two different wavelengths where the components havedifferent absorptivities. Beer's Law states that A = abC, where A is the absorbancemeasured on the spectrophotometer (in "absorbance units", AU), a is the absorptivity,b is the cell pathlength (in this case, 1.00cm), and C is the concentration in mg/mL.
The total absorbance of a solution at any wavelength is the sum of the individual absorbances due to each component in the solution (see Figure 1), so for phenacetin(P) and caffeine (C): The absorptivities (a) are constants at a specific wavelength and under the condition ofthe experiment, and their values are determined by measuring individual phenacetinand caffeine standards of known concentration.
If Atotal is measured at two different wavelengths, then two equations in two unknowns can be written and solved for Cp and Cc, Chemicals/Solutions Required:Phenacetin Stock Solution (ca. 0.20 mg/mL, exact value given on the label).
Caffeine Stock Solution (ca. 0.50 mg/mL, exact value given on the label).)Unknown Solution (obtained from the Instructor or teaching assistant).
Recording Double-beam UV-Visable SpectrophotometerGA/12 Glassware KitQuartz Cells, 1.00cm path (Very expensive, about $ 120 per pair - handle with care. Be sure cells are quartz, not glass.
Using volumetric glassware, dilute 25.0mL of the 0.20 mg/mL Phenacetin StockSolution to 100mL with distilled water, to give an approximate concentration of 0.05 _ _mg/mL. Further dilute as shown to give three solutions for absorbance measurements: Calculate the exact final concentrations from the concentration of the PhenacetinStock Solution.
Using volumetric glassware, dilute 10.0mL of the 0.50 mg/mL Caffeine Stock Solutionto 100mL with distilled water to give an aproximate concentration of 0.05 _ _ mg/mL.
Further dilute as shown to give three solutions for absorbance measurements: Calculate the exact final concentrations of these dilute caffeine Stock Solution.
The dual-beam Spectrophotometer should be allowed to warm up for about 10 mins. Check to see if it has previously been turned on by the You will not be using the Computer Data Station to control the scan, record and save the spectra. Instead the spectrometer will be used manually and the spectra outputed on a recorder.
To operate the scanning spectrometer see the instructions: "Perkin ElmerLamba 3B Spectrometer" kept by the instrument.
Use the following instrumental settings.
c. Use recorder settings of 1000 mv range and a scan rate of 120 nm/min on the recorder.
d. Use the 1.00 absorbance full scale range.
3. On one set of axes. (Absorbance versus wavelength) record the ultraviolet
absorption spectra for 0.010mg/mL phenacetin solution. If this spectra is, off-
scale, input more appropriate values for its axes and have the spectrometer
record a new spectra. On the same axes, record the spectra for the 0.0050 and
the 0.0075mg/mL phenacetin solutions, make sure you record the absorbance
limits.

4. On a new set of axes (Absorbance versus wavelength) record the ultravioletabsorption spectra for the 0.010 mg/mL caffeine solution. If this spectra is off-scale, input more appropriate values for the axes and have the spectrometer record the new spectra on this second set of axes, record the spectra for the0.0050 and 0.0025 mg/mL caffeine solutions.
5. On a new set of axes record the ultraviolet absorption spectra for thecombined caffeine and planacetin unknown solution. It is best if the same axesunits are used for all the solution of parts 3, 4, and 5.
a. From the spectrum of phenacetin, find its wavelenght maximum, which should be near 240 nm (wavelength 1). From the recorded spectra, obtain the absorbances ofthe three phenacetin standards at this wavelength. Also obtain the absorbances of thethree caffeine standards at this wavelength.
b. From the spectrum of caffeine, find its wavelength maximum, which should be near 270 nm (wavelength 2). From the recorder spectra, read the absorbances of thethree caffeine standards and the three phenacetin standards at this wavelength.
c. Construct four Beer's Law plots (absorbance vs. concentration). Use linear regression to obtain the best line for each plot. The slope of each plot, obtained bylinear regression calculation, will equal the absorptivity, a. What are the units of a? For the unknown solution, read the absorbances A1, and A2 at wavelengths 1 and 2.
Set up two simultaneous equations, having Cp and Cc as unknowns: Solve the simultaneous equations for Cp and Cc. The following procedure isrecommended: First, simplify the terms by multiplying the "a" and "b" constantsas follows to obtain: (Note: This does not change the numerical values of the terms, since b = 1 cm,but b must be included in the computation, since it carries one of the units (cm)which will yield the correct units (mg/mL) in your result.) Multiply all terms in the first equation by the value of "f", and all terms in the
second equation by the value of "d". Substract the second equation from the
first, eliminating Cc. Report the value for Cp.
Substitute the result for Cp into the first equation, and solve for Cc.
Report the value for Cc.
An important part of analytical chemistry is sample prepartion, the conversion ofa real-world sample into a form suitable for analysis. Due to time constraintsand the current unavailability of APC tablets, an actual tablet sample will not beprepared and analyzed, but the associated calculations should be continued asfollows: Assume your unknown solution was prepared as follows: One APC tablet was ground and extracted with 10 mL of 0.lN NaOH. The 10 mL waspassed thru an anion exchange column and the effluent from the column wascollected in a 200mL volumetic flask and diluted to volume with distilled water.
Ten mL of this solution was diluted to 100 mL, then 11.0 mL of this dilution wasfurther diluted to 100 mL to give your unknown solution. (The objective of allthese dilutions is to get a solution having an absorbance in the range of 0.2 to0.7, so it can be read on the spectrophotometer.) A convenient way to visualize this and similar problems is with a dilution scheme.
The scheme for the tablet preparation is: (Horizontal steps retain the entire sample, but dilute it. Vertical steps fractionatethe sample.) Calculate the number of mg of phenacetin and of caffeine in the orginal tablet.
In tabular form your report should include the absorptivities of each compound ateach wavelength used, the concentrations of P and C in the unknown solution,and the mg of P and C in the hypothetical APC tablet.

Source: http://chemweb.chem.uconn.edu/teaching/chem-232/Laboratory_Manual/GA12_uvvis_phen%26caff_rev1_9.pdf

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