CHE 314
TEFO OLEFILE
201502191
EXPERIMENT 7: THE SIMULTANEOUS
DETERMINATION OF CAFFEINE AND
ACETYLSALICYCLIC ACID IN AN ANALGESIC BY
ULTRAVIOLET SPECTROPHOTOMETRY
WEEK 4
DATE OF SUBMISSION:27 MARCH 2018
GROUP: TUESDAY 3-6PM
AIM
The aim of this experiment was to simultaneously determine the concentrations of caffeine and
acetylsalicylic acid in an analgesic by ultraviolet spectrophotometry.
ABSTRACT
The aim of this experiment was to simultaneously determine the concentrations of caffeine and
acetylsalicylic acid in an analgesic by ultraviolet spectrophotometry. The stock solutions of
caffeine,acetylsalicylic acid,sample A and B were prepared in different volumetric flasks and the
ultraviolet spectrophotometry was used to analyse the solutionsPercentage mass of caffeine was
found to be 0.22 % from sample A and percentage mass of acetylsalicylic acid was found to be
0.73 % while the absorbance at wavelengths of 245 nm and 270 nm were found to be 2233.6 M-
1
cm- and 9523.8 M-1cm- for caffeine and for acetylsalicylic was found to be 4315.4 M -1cm- and
1933.3 M-1cm- for 255 and 270nm wavelength.
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.(Beckett,1988)Caffeine is a central nervous system (CNS) stimulant of
the methylxanthine class It is the world's most widely consumed psychoactive drug. Unlike
many other psychoactive substances, it is legal and unregulated in nearly all parts of the world.
There are several known mechanisms of action to explain the effects of caffeine. The most
prominent is that it reversibly blocks the action of adenosine on its receptor and consequently
prevents the onset of drowsiness induced by adenosine. Caffeine also stimulates certain portions
of the autonomic nervous system. (Malenka,2009) Spectrophotometry is an instrument used for
the simultaneous analysis of a mixture. It measure the amount of light absorbed by a chemical
substances by measuring light intensity as a beam of light that passes through sample solution.
The basic principle is that each compound absorbs or transmits light over a certain range of
wavelength which make Beer's law to be used at the wavelength which is a characteristic of each
component to determine the concentration of that component. Ultraviolet-visible absorption
bands of polyatomic species are usually broad making it to be difficult to determine wavelength
for each component at which no other component absorbs radiation. A wavelength that is chosen
for an analysis at which more than one solution component absorbs radiation, then the
absorbance of that solution at that wavelength is the sum of the absorbance of the solution
components. (Shoemaker,2009)The following equation is for a sample which contains two
absorbing components,
Aλ1, sample = Aλ1,1 + Aλ1,2
In which
Aλ1,1= is the absorbance at wavelength 1 of component 1
Aλ1,2= is the Absorbance of component 2.
This will yield the following equation when substituted into Beer’s Law
Aλ1, sample = ελ1,1bC1 + ελ1,2bC2
Where Aλ1 is obtained from the spectrum of the sample.
ελ1,1, ελ1,2 and b can be measured independently.
In a two component mixture, absorbance measurements done at a second wavelength λ2 and the
equation below can be written (Srivastava, 2011).
Aλ2, sample = ελ2, 1bC1 + ελ2, 2bC2
Spectrophotometry is an instrument used for the simultaneous analysis of a mixture. It measure
the amount of light absorbed by a chemical substances by measuring light intensity as a beam of
light that passes through sample solution. (Negi,2004)
REAGENTS AND APPARATUS
Reagents Apparatus
i.2 x 1cm cuvettes (cells) i.Acetylsalicylic acid (reagents grade)
ii.1 ml pipette, 2 ml pipette ii.Caffeine (Reagents grade)
iii.Scanning UV-Vis spectrophotometer iii.Unknown containing a mixture of
iv.14 x 50 mL volumetric flasks, 3 x 250 mL acetylsalicylic acid and caffeine
volumetric flask
PROCEDURE
0.117 g of caffeine was weighed was placed in a labeled 250 mL volumetric flask together with
200 mL of 0.1 HCl. This was allowed to dissolve and was diluted to the mark with HCl. Pipettes
were used to deliver 0.5, 1, 1.5, 2.0 and 2.5 ml of 2.41 x 10 -3M caffeine stock solution into the
volumetric flasks labelled C1, C2, C3, C4, and C5 respectively. This was diluted to the mark
with HCl. 0.12 g of acetylsalicylic acid was placed in a labeled 250 ml volumetric flask with 100
ml of HCl added to the flask and the solid was dissolved. This was diluted to the mark with HCl.
Five 100 ml volumetric flask were labeled A1, A2, A3, A4, and A5. Pipettes were used to deliver
0.5, 1.0, 1.5, 2.0, 2.5 mL of acetylsalicylic acid stock solution into each of the volumetric flasks
and diluted to the mark. 0.5 mL of the stock solution was delivered using a pipettes and 1.0 mL
of the acetylsalicylic acid stock solution to a 100 mL volumetric flask. This were filled to the
mark with HCl. 0.1094g (sample A) and 0.1017 g (sample B) of analgesic were weighed in the
sample capsule and dissolved in HCl. This was quantitatively transferred into a labeled 250 mL
volumetric flask and filled to the mark with HCl. 0.5 mL from stock was transferred to 1 100 mL
volumetric flask using a pipette and filled to the mark with HCl.
On a single piece of chart paper the spectra between 220 and 230 nm of pure HCl, of the solution
of caffeine and acetylsalicylic acid and a solution of 0.5 mL sample solution in 100 mL
volumetric flask on a second piece of chart paper the spectra of the five caffeine standards was
recorded and the spectra labelled. On a third piece of paper the spectra of the five acetylsalicylic
acid solutions
RESULTS
Standard solutions of Concentration X 10-5 Absorbances
caffeine
(mol/L) Wavelength Wavelength 2
1
(270nm)
(245nm)
C1 1.205 0.025 0.065
C2 2.41 0.030 0.093
C3 3.615 0.05 0.125
C4 4.82 0.05 0.141
C5 6.025 0.139 0.269
Table 1; Absorbance and concentrations of caffeine in standard solutions of caffeine
ii.
Standard solutions of Concentration × 10-5(mol/L) Absorbance
acetylsalicylic acid
Wavelength Wavelength
(230nm) (270 nm)
A1 1.05 0.0025 0.027
A2 2.1 0.0030 0.046
A3 3.15 0.0075 0.070
A4 4.2 0.01 0.089
A5 5.25 0.02 0.107
Table 2; Absorbance and concentrations of acetylsalicylic acid in standard solutions of
acetylsalicylic
GRAPH OF ABSORBANCE VS CONCENTRATION M FOR
CAFFEINE
0.3
f(x) = 4315.35269709544 x + 0.00839999999999999
R² = 0.977048043013239
0.25
0.2
Absorbance
0.15
f(x) = 2233.55068168346 x + 0.00902857142857144
R² = 0.947006467311263
0.1
0.05
0
0 0.00001 0.00002 0.00003 0.00004 0.00005 0.00006 0.00007
Concentration M
KEY
Y=2233.6x+0.009 -----------------Wavelength1(245nm)
Y=4315.4x+0.0084----------------Wavelength2(270nm)
GRAPH OF ABSORBANCE VS CONCENTRATION FOR
ACETYLISALIC ACID
0.6
f(x) = 9523.80952380953 x + 0.0291999999999999
0.5 R² = 0.997577880905162
0.4
Absorbance
0.3
0.2
0.1 f(x) = 1933.33333333333 x + 0.00690000000000001
R² = 0.997603369807301
0
0.000005 0.00001 0.000015 0.00002 0.000025 0.00003 0.000035 0.00004 0.000045 0.00005 0.000055
Concentration M
KEY
Y=9523.8x+0.0292-----------------Wavelength(255nm)
Y=1933.3x+0.0069-----------------Wavelength(270nm)
Calculations for molar absorptivity’s
Absorbance = εCl
Cell path length = 1.0 cm hence slope = εC = ε which is molar absorptivity.
Hence Molar absorptivity = slope
The following table give molar absorbance obtained from the graphs
Wavelength Molar absorbance
For caffeine
i.245nm 2233.6
ii.270nm 4315.4
For acetylsalicylic
i.255nm 9523.8
ii.270nm 1933.3
ABSORBANCE
A = ԑ λcaff x 1 x C caff +ԑ λasa x 1 x Casa
C caff = 1.205 x10-5 M
Casa = 1.05 x 10-5 M
At wavelength 1
ԑ caff = 2233.6 M-1.cm-1
ԑ asa = 9523.8 M-1.cm-1
therefore
A = 2233.6 x 1 cm x (1.205 x 10-5) + 9523.8 x 1 cm x (1.05 x 10-5)
A = 0.127
At wavelength 2
ԑ caff = 4315.4 M-1.cm-1
ԑ asa = 1933.3 M-1.cm-1
therefore
A = 4315 x 1 cmx (1.205 x 10-5) + 1933.3 x 1 cm x (1.05 x 10-5)
A = 0.0723
CONCENTRATIONS OF CAFFEINE AND ACETYLSALICYLIC IN SAMPLE A
Asa stands for acetylsalicylic acid
caff stands for caffeine
therefore A λsample = ԑ λasa x 1 x Casa + ԑ λcaff x 1 x C caff
Equation 1, At λ1 0.115 = 2233.6 M-1 cm-1 x 1 cm x Casa + 9523.8 M-1 cm-1 x 1 cm x C caff
Equation 2, At λ2 0.016 = 4315.4 M-1 cm-1 x 1cm x Casa + 1933.8 M-1 cm-1 x 1cm x C caff
Multiplying equation 2 by 2233.6/4315.4 and subtracting it from equation 1 we get;
C caff = 1.12 x 10-5 M
Substituting C caff in equation 1;
C asa = 4.06 x 10-5 M
PERCENTAGE OF ACETYLSALICYLIC ACID AND CAFFEINE IN SOLID SAMPLE
Mass of sample = 0.1 g
Concentration of caffeine and acetylsalicylic acid in stock solution
M1V1 = M2V2
Caffeine
1.12 x 10-5 M x 0.100 L / 0.250
= 4.48 x 10-6 M
Moles = concentration x volume
Moles = 4.48 x 10-6 M x 0.250 L
= 1.12x 10-6 moles
Mass = molar mass x moles
= 194.1906 g mol-1 x 1.12 x 10-6
= 2.17 x 10-4 g
Acetylsalicylic Acid
Mass=7.31 x 10-4
Percentage of caffeine in solid sample
2.17 x 10-4 g / 0.1 g x 100 = 0.22%
Percentage of acetylsalicylic acid in solid sample
7.31 x 10-4 g / 0.1 g x 100 = 0.73 %
DISCUSSION
Caffeine and acetylsalicylic acid where determined simultaneous by ultraviolet
spectrophotometry. Sample A and sample B solution were used in the experiment. Standards
solutions of caffeine and acetylsalicylic acid were used to obtain the molar absorptivity of both
caffeine and acetylsalicylic acid at wavelengths of 225 nm and 270 nm which were found to be
2233.6 M-1cm- and 9523.8 M-1cm- for caffeine at 225 and 270nm respectively.For acetylsalicylic
it was found to be 4315.4 M-1cm- and 1933.3 M-1cm- for 225 and 270nm wavelength. . In sample
A, % mass of caffeine was found to be 0.22% and % mass of acetylsalicylic acid was found to be
0.73 %.
The limitations in the Beer’s law indicate possible sources of errors in the experiment which
could be personal or experimental errors. Such errors include; deviations in absorptive
coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules
in close proximity scattering of light due to particulates in the sample, fluorescence or
phosphorescence of the sample, changes in refractive index at high analyte concentration, shifts
in chemical equilibrium as a function of concentration, non-monochromatic radiation and stray
light through reflections and refractions (Negi,2004). Deviations can be minimized by using a
relatively flat part of the absorption spectrum such as the maximum of an absorption band.
Furthermore some errors could have been be caused by inappropriate handling of the cuvette and
not wiping it clearly which left some fingerprints that might have altered with the absorption of
radiation and caused the deviations.Which could have be minimized by wiping the curvets
thoroughly with the use of a soft tissue before placing it in the spectrophotometer.
CONCLUTION
Percentage mass of caffeine was found to be 0.22 % from sample A and percentage mass of
acetylsalicylic acid was found to be 0.73 % while the absorbance at wavelengths of 245 nm and
270 nm were found to be 2233.6 M -1cm- and 9523.8 M-1cm- for caffeine and for acetylsalicylic
was found to be 4315.4 M-1cm- and 1933.3 M-1cm- for 255 and 270nm wavelength.
REFERENCES
1.Beckett, A.H., Stenlake, J.B. “Practical Pharmaceutical Chemistry: Part II”; A and C Black.,
London, pp 275. 1988
2.Malenka RC, Nestler EJ, Hyman SE "Chapter 15: Reinforcement and Addictive Disorders". In
Sydor A, Brown RY. Molecular Neuropharmacology: A Foundation for Clinical
Neuroscience (2nd ed.). New York: McGraw-Hill Medical. p. 375.2009
3. Negi A.S and Anand S.C Textbook of Analytical Spectroscopy’s Chemistry 6th ed.,New Age
International. New Delhi, p102.2004
4..Shoemaker, D. P. Experiments in Analytical Chemistry. New York: McGraw Hill Higher
Education.p354.2009
