SCHOOL OF PHARMACEUTICAL SCIENCE

FAR 313/4: Pharmaceutical Analysis

Experiment 4
Ultraviolet and Visible Spectrophotometry
Assay of Theophylline in Aminophylline Tablet
Group B 13
Name

Matric No.

TAN LI MAY
TAN SIU SIEN
TAN YI YEN
TAY SUE CHYEN

Lecturer

114563
114564
114565
114566

: DR. LAI CHOON SHEEN

Date of Submission : 26th NOVEMBER 2014

1.0 Aims

1. To study the utilization of ultraviolet and visible spectrophotometry in quantitative analysis.

2. To calculate the amount of theophylline in one aminophylline tablet.
2.0 Introduction
Ultraviolet and visible spectroscopy refers to absorption spectroscopy in the ultraviolet-visible
spectral region (William Reusch, 2013). It is a method used to measure the absorption of substance radiation
in the wavelength region between 200nm and 800nm. Ultraviolet absorption spectrum arises from transition
of electrons within a molecule or an iron from lower to higher electronic energy levels and ultraviolet
emission spectrum arises when the electrons move back to their ground energy state. Ultraviolet
spectroscopy is a very efficient tool for qualitative as well as quantitative analyses of substances which
absorb these radiations. It is also frequently used for detection of impurities, molecular weight
determinations and the determination of dissociation constants of acids and bases.
A spectrophotometer is employed in ultraviolet-visible spectroscopy to measure the amount of light
that a sample absorbs. It consists of a light source, a diffraction grating, filter, mirrors, lens and detectors. A
spectrophotometer can be either single beam or double beam. Single beam instrument is used for assay and
this method is limited to quantitative analysis that is done at a fixed wavelength. On the other hand, double
beam instrument is convenient to use to obtain a complete spectrum. Double beam instruments allow
scientists to simultaneously measure transmissions through the target sample and solvent. This method is
used in this experiment to compare the absorbance values between the sample and the reference. The
instrument operates by passing a beam of light through a sample and measuring the intensity of light
reaching the detector.
The beam of light consists of a stream of photons. When a photon encounters a testing substance,
there is a chance it will absorb the photon. This absorption reduces the number of photons in the beam of
light, thereby reducing the intensity of the light beam. The testing substance is normally dissolved in a
solvent which is not absorbing the radiation. The samples for UV/Vis spectrophotometry are most often
liquids, although the absorbance of gases and even of solids can also be measured. Samples are typically
placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with
an internal width of 1 cm. (This width becomes the path length, l, in the Beer-Lambert law).
The amount of light that penetrates a solution is known as transmittance. Transmittance can be
expressed as the ratio of the intensity of the transmitted light, I, to the initial intensity of the light beam, I o,

as expressed by the formula: T = I / Io where the logarithm for the reciprocal of T is called the absorbance,
A, which is expressed as: A = log (1/T).
The principal behind spectrophotometer is the absorption law, the Beer-Lambert law. It
quantitatively shows how the amount of attenuation depends on the concentration of the absorbing molecule
and the path length over which absorption occurs. According to Beer-Lambert law, absorbance A is linearly
related to the concentration of the absorbing species (c) and the path length (l) of the absorbing medium, at
certain wavelength: A = klc
where k is the constant (molar absorptivity), l is path length of solution and c is concentration of the
absorbing species. This formula states that the light absorbed by a solution depends on the absorbing ability
of the solute, the distance travelled by the light through the solution and the concentration of the solution. A
calibration curve is then plotted using a series of increasing concentration of the standard. We can then
measure the absorbance of the unknown from the spectrophotometer and use the calibration curve to
determine the concentration of the unknown.

3.0 Procedure
A) Wavelength Control
1. The wavelength scale of a spectrometer was examined using maximum absorption by holmium oxide.
2. The wavelengths detected must be close to the values of 241.2 nm, 287.2 nm, 361.5 nm and 536.3
nm.
3. The permitted tolerance was 1 nm for wavelengths falling within the ultraviolet region (200 nm
400 nm) and 3 nm for those falling within the visible light region (> 400 nm).
B) Absorbance Control
1. An amount of potassium dichromate (57.0 mg 63.0 mg) which had been dried at 180 0C was
weighed accurately and dissolved in 0.005 M sulphuric acid up to 1000 mL.
2. The absorbance of potassium dichromate solution was measured with 0.005 M sulphuric acid as the
reference (blank) at the wavelengths of 235 nm, 257 nm, 313 nm and 350 nm.
3. The values of A (1 %, 1 cm) were calculated and compared with the following values of maximum
tolerance. At least three of the four A values corresponding to each of the four wavelengths must be
within the maximum tolerance range stipulated:
Wavelength (nm)

A (1 percent, 1 cm)

Maximum tolerance

235
257
313
350

124.5
144.0
48.6
106.6

122.9 126.2
142.4 145.7
47.0 50.3
104.9 108.2

C) Theophylline Assay in Aminophylline Tablet

I. Spectrum and Calibration Curve of Theophylline
1. Theophylline stock solution was prepared by weighing exactly 12 mg of theophylline and
dissolved in 10 mL of 0.1 M sodium hydroxide solution and diluted to 100 mL with water. The %
w/v was calculated.
2. Each portion of 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of the stock solution was diluted to 50 mL with
0.01 M sodium hydroxide.
3. The absorbance of the third solution (3 mL) was measured using a single beam spectrometer in the
wavelength range of 220 nm to 360 nm, with 0.01 M sodium hydroxide as the blank.
4. The wavelength at the maximum absorbance was chosen. The spectrum of absorbance versus
wavelength was plotted automatically.
5. The absorbance of the other four solutions (1 mL, 2 mL, 4 mL and 5 mL) was measured using the
single beam spectrometer (at the wavelength chosen in Step 4). The spectrum of absorbance versus
wavelength was plotted automatically to yield the calibration curve. From the slope of the
calibration curve, A (1 percent, 1 cm) was calculated.
II. Assay of the Tablet
1. Two aminophylline tablets were weighed using an analytical balance and ground evenly until fine
powder was formed.
2. 70 mg of the fine powder was shaken with 25 mL 0.1 M sodium hydroxide and 60 mL of distilled
water in a volumetric flask for 10 minutes.
3. Distilled water was added up to 250 mL after which the solution contents were mixed and filtered.
4. 5 mL of the filtrate was diluted with 0.01 M sodium hydroxide to 250 mL in a volumetric flask.
5. The absorbance of this solution was measured at the analysis wavelength chosen in Step 4 of Part
I.
6. The concentration of theophylline in the aminophylline tablet was thus calculated with reference to
the calibration curve (or using A previously determined).
7. The amount of theophylline in one tablet was calculated.
4.0 Results and Calculation
A. Wavelength Control

The wavelength scale of a spectrometer was examined using maximum absorbance by holmium oxide. The
table below shows the observed wavelength of holmium oxide compared to its specified wavelength at
maximum absorption.
Table 1: The observed wavelengths of holmium oxide compared to specified wavelengths

Specified wavelength (nm)

Observed wavelength (nm)

Difference (nm)

241.2

287.2

287.16

-0.04

361.5

360.84

-0.66

536.3

536.36

-0.06

Permitted tolerance = 1 nm for ultraviolet region (200 - 400nm)

= 3 nm for visible region (> 400 nm)

B. Absorbance Control
The absorbance and molar absorptivity of potassium dichromate solution in sulphuric acid with 0.005 M
sulphuric acid as the reference was measured. The table below shows the absorbance and molar absorptivity
of potassium dichromate at the observed wavelength compared to the specified wavelength.

Table 2: Absorbance control by using potassium dichromate

Wavelength, nm

Absorbance
(A)

A (1%,1cm)

Specifi

Observe

Specific

Experimenta

Maximum Tolerance

235.0

234.50

0.73785

124.5

122.95

122.9 to 126.2

257.0

256.27

0.85881

144.0

143.10

142.4 to 145.7

313.0

312.57

0.28776

48.6

47.95

47.0 to 50.3

350.0

350.24

0.63772

106.0

106.26

104.9 to 108.2

Absorbance, A = klc
where A

= absorbance

= rate constant

l
c

= path length of the solution = 1 cm

= concentration of the potassium dichromate solution in g/L or % w/v

= 60.014 mg/L

Calculation

= 0.060014 g/ 1000 mL
= 0.0060014 % w/v

When wavelength = 234.50 nm, A = 0.73785

A (1%, 1 cm) = A / lc
= 0.73785 / (1 x 0.0060014)
= 122.95
When wavelength = 256.27 nm, A = 0.85881
A (1%, 1cm) = A / lc

= 0.85881/ (1 x 0.0060014)
= 143.10
When wavelength = 312.57 nm, A = 0.28776
A(1%, 1cm) = A / lc
= 0.28776 / (1 x 0.0060014)
= 47.95
When wavelength = 350.24 nm, A = 0.63772
A(1%, 1cm) = A / lc
= 0.63772/ (1 x 0.0060014)
= 106.26

C. Theophylline Assay in Aminophylline Tablet

I. Spectrum and calibration curve of theophylline
Weight of theophylline = 0.0124 g
Concentration of the theophylline stock solution = (0.0124 g/100mL) x 100%
= 0.0124 %w/v
M1V1= M2V2
where M1 = Concentration of theophylline stock solution = 0.0124 % w/v
V1 = Volume of theophylline stock solution
M2 = Concentration of theophylline solution after diluted
V2 = Final volume of theophylline solution after diluted
1 mL of stock solution was diluted to 50mL with 0.01M sodium hydroxide
Thus, (0.0124) (1) = M2 (50)
M2 = 2.48 x 10-4 % w/v
2 mL of stock solution was diluted to 50mL with 0.01M sodium hydroxide
Thus, (0.0124) (2) = M2 (50)

M2 = 4.96 x 10-4 % w/v

3 mL of stock solution was diluted to 50mL with 0.01M sodium hydroxide
Thus, (0.0124) (3) = M2 (50)
M2 = 7.44 x 10-4 % w/v
4 mL of stock solution was diluted to 50mL with 0.01M sodium hydroxide
Thus, (0.0123) (4) = M2 (50)
M2 = 9.92 x 10-4 % w/v
5 mL of stock solution was diluted to 50mL with 0.01M sodium hydroxide
Thus, (0.0124) (5) = M2 (50)
M2 = 12.4 x 10-4 % w/v
The third serial dilution solution is used to determine the wavelength at which maximum absorbance occurs.
From the graph of absorbance against wavelength, maximum absorbance of third serial dilution occurs at
274.64 nm.

Table 3: The absorbance is measured at certain concentration of theophylline solution

Preparation

Volume of Theophylline stock solution, mL

%w/v of Theophylline

Absorbance, A

1.0

2.48 x 10-4

0.1472

2.0

-4

0.2883

-4

4.96 x 10

3.0

7.44 x 10

0.4230

4.0

9.92 x 10-4

0.5582

5.0

12.4 x 10-4

0.7140

Graph of Absorbance versus Concentration of Theophylline

0.8
0.7

f(x) = 0.06x + 0
R = 1

0.6
0.5

Absorbance, A

0.4
0.3
0.2
0.1
0

10

12

14

Concentration of Theophylline ( 10-4 % w/v)

By referring to Beer-Lambert equation,

Absorbance, A = klc
Since l = 1cm, A = kc,
Since the equation of the curve is y = mx + 0
A = kc + 0(2)
By comparing equation (1) & (2), k can be m (the gradient of the slope).
The linear equation obtained from the calibration curve is y = 0.0569x + 0.0024,
Thus, k= 0.569 x10-4 Lg-1cm-1
2. Assay of the Tablet
The combined weight of two tablets = 0.51 g.
Average weight of one tablet = 0.51 g / 2 = 0.255 g.
Amount of powder used to prepare the solution = 0.07 g.
Absorbance of the unknown sample = 0.3503 A

(1)

Based on the calibration curve plotted, we obtain a straight line of y = 0.0569x + 0.0024
The slope, m = kl
Since A = klc, where kl is 0.0569, A= 0.3503
c = A/kl
= 0.3503/0.0569
= 6.16 x 10-4 %w/v
Therefore, the concentration of theophylline in the sample = 6.16 x 10-4 %w/v

Amount of theophylline in 250 mL solution

= 6.16 x10-4 / 100 mL x 250 mL = 1.539 x 10-3 g
Amount of theophylline before dilution
= 1.539 x 10-3 g / 5 mL x 250 mL = 0.077 g
Therefore, in 0.070 g of the powder, there is 0.077 g of theophylline.
The amount of theophylline in one tablet
= 0.077 g / 0.070 g x 0.255 g = 0.2805 g
Thus, the percentage of theophylline in one tablet
= 0.2805 g / 0.255 g x 100% = 110%
By referring to the British Pharmaceutical Codex 1973, the amount or percentage of theophylline in
aminophylline tablet is about 78% 84%. However, the amount that we obtained from the calculation in the
experiment is only 110%. This significant deviation might be due to some errors occurred during the
experiment.
5.0 Discussion
A. Wavelength control
Before we start this experiment, we are required to examine the wavelength scale of the spectrometer
first. The purpose is to ensure that the spectrometer is working accordingly. Hence, we will use a reference
whereby the wavelength of it is already known.

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In this experiment, holmium oxide was used as a wavelength reference which allows us to verify the
ultraviolet wavelength scale of the spectrometer. Holmium oxide is used because it is the reference material
that is standardized throughout the worldwide. Hereby, the wavelength scale of the spectrometer was
examined by scanning the entire range of wavelength from 200nm to 600nm through the maximum
absorption with holmium oxide. After all, the wavelength obtained from the spectrometer is then compared
with the theoretical wavelength.
In the spectrum observed, there are only 3 peaks that could be seen. We could not observe the
presence of any peaks for the specified wavelength, 241.2 nm. However, for the 3 other wavelengths that
obtained by us were almost the same as the specified wavelength. The permitted tolerance is 1 nm for
ultraviolet region (200-400nm) and 3 nm for the visible region (>400 nm). Based on the calculation, we
can see that all the differences between the specified wavelength and observed wavelength calculated were
below the 1nm tolerance. Thus, this shows that the wavelength scale of the spectrophotometer used was
accurate and within permitted standard range.
B. Absorbance control
Besides, before start with the analysis of theophylline, it is also a must to evaluate the absorbance
control using a standard solution. This is because it will reflect the tolerance and reliability of the instrument
or method used which will indirectly affect the accuracy of the results that we are going to obtain.
For the absorbance accuracy testing, either solutions or glass/quartz filters can be used. In this part,
we used potassium dichromate solution with 0.005M H2SO4 as a reference. The peak absorbance value of
potassium dichromate solution is determined in corresponds to respective wavelength, measured within the
range of 220nm-360nm wavelength. Then, the absorbance value obtained is used to calculate the A, (1%,
1cm) value which is compared with the given maximum tolerance range.
From the graph and calculated results, all the 4 observed wavelengths are almost the same as the
specified wavelengths. Moreover, all the A (1%, 1cm) values that calculated are also falling in the maximum
tolerance range given. Thus, this indicated that the instrument used is reliable and is well calibrated.
C. Theophylline assay in aminophylline table
1. Spectrum and calibration curve of theophylline

11

Concentration of theophylline in aminophylline can be determined by a calibration curve of

theophylline. From the graph of absorbance versus wavelength, the analysis wavelength can be showed
through the maximum wavelength. In our experiment, absorbance of the third solution, 7.44 x 10-4 % w/v is
measured first using double beam spectrometer to get the wavelength at the maximum absorbance while
0.01 M sodium hydroxide (NaOH) is used as a blank. This is because this wavelength (max) provides a
more sensitive and selective result. Lower concentrations of theophylline solution produce too low peaks
while higher concentrations of theophylline solution produce too high peaks. The maximum wavelength
(max) obtained is 274.64 nm. The presence of the conjugated double bond (n * transition) in the
theophylline molecule gives rise to the observed peak.

After the absorbance value of all five solutions of theophylline was recorded, a graph of absorbance
against the concentration of theophylline solution was plotted as a calibration curve. According to the
Beers Law, absorbance is linearly proportional to the concentration of the absorbing
species. The higher the concentration of theophylline, the greater the absorbance
obtained. Different values of absorbance were obtained due to the different light intensities that passed
through the solutions with various concentrations. The linear line of best fit which passes through the
origin was obtained from the graph. The value of k obtained from the graph is 0.569 x10-4 Lg-1cm-1

2. Assay of the tablet

To obtain the concentration of theophylline in the prepared solution, it was first diluted and run
through the spectrophotometer. The concentration of theophylline should fall within the range of the
calibration curve early plotted. The absorbance of the sample was recorded at 0.3503A and the
concentration of theophylline in the sample is 6.16 x 10-4 %w/v.
From our calculation, it shows that 0.077g of theophylline is present in the 0.07g fine powdered
aminophylline tablet added. This shows that errors have occurred during our experiment as theoretically the
amount of theophylline present should not be greater than the fine powder added. Consequently, the
percentage of theophylline in one aminophylline tablet determined from our experiment is 110% which

12

is slightly higher from the range, 78% 84% as stated in the British Pharmaceutical Codex
1973.
Theophylline is a poisonous ingredient used to prevent and treat wheezing and other breathing
difficulties caused by lung diseases such as asthma. It indirectly stimulates both 1 and 2receptors through
release of endogenous catecholamine. Thus, it is important to prevent theophylline toxicity as it will lead to
seizures and heart rhythm disturbances which are major life-threatening event.
This deviation in result might be due to some errors that have occurred during
the experiment. There might be some inaccuracies during the dilution process such
as not adding the accurate volume of sodium hydroxide solution causing the
theophylline solution becomes more concentrated. There could also be some
mistakes during the filtration of the theophylline solution such as present of
impurities and contaminants which will affect the value of absorbance obtained.
Turbid or bubbles in the solution will also diminished the accuracy of readings.
Fingerprint stains or scratches on the cuvette walls may also cause the deviation in
result as the may reflect or absorb radiation. Other than that, technical and parallax
errors also might be existed in this experiment.

There are some precaution measures that we need to consider as to minimize errors and obtain more
accurate result:
1. Calibration of glassware and instruments/devices is necessary to ensure to functionality of that devices
thus ensure the accuracy of measurement.
2. All kinds of measurement or weighing the substances should be made as accurate as possible.
3. The tablet must be grinded until it becomes a fine powder to make a better dissolution.
4. Labeling must be appropriately done in order to avoid confusion.
5. Before each measurement, the cuvet must be cleaned thoroughly. The cuvet should be properly wiped
with tissue to clean off all the dirt, particles or fingerprints to avoid interference of the measurement.
6.0 Conclusion
1. Both wavelength and absorbance control is performed to ensure the status of the spectrophotometer is
reliable and able to produce accurate results.

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2. The concentration of theophylline in the sample obtained from the calculation by using the calibration
curves equation is 6.16 x 10-4 %w/v.
3. The amount of theophylline in one aminophylline tablet obtained from the calculation by using the
calibration curves equation is 0.2805g.
4. The percentage of theophylline in one aminophylline tablet is 110%, which deviates from the range
stated in British Pharmacopeia.

7.0 References
1. William Reusch, 2013. Visible and Ultraviolet Spectroscopy. Chemistry.msu.edu. Available at:
https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/Spectrpy/UV-Vis/spectrum.htm
2. www.perkinelmer.com/.../44-136839TCH_Validating_UV_Visible.pdf
3. http://www.preservearticles.com/2014111323289/complete-information-on-ultravioletspectrophotometry.html
4. Principles of Spectrophotometry,
http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html
5. Spectrophotometry 2014. . [ONLINE] Available at:
http://www2.fiu.edu/~bch3033/bch3033l/pdf/spectra.pdf. [Accessed 16 Nov 2014].
6. Aminophylline overdose: MedlinePlus Medical Encyclopedia 2014 [ONLINE] Available
at: http://www.nlm.nih.gov/medlineplus/ency/article/002572.htm [Accesed 15 Nov 2014]