ACADEMIC SESSION 2021/2022

SEMESTER I
FAR 313/4 PHARMACEUTICAL ANALYSIS

PRACTICAL 4

ULTRAVIOLET AND VISIBLE SPECTROMETRY - ASSAY

OF THEOPHYLLINE IN AMINOPHYLLINE TABLET

GROUP 20

NAME MATRIC NO
CHAI YI NING 146302
KURNIATI BINTI SUR’AN 146440
NUR ANIS ADILLA BINTI CHE YAHYA 146831
NURUL SYAZANA BINTI MOHAMAD ZAHARI (LEADER) 146344
ONG SHENG CONG 145401

LECTURER: DR WAQAS AHMAD

DATE OF SUBMISSION: 26th NOVEMBER 2021

ACCESS LINK TO EXPORTED CHAT:

https://docs.google.com/document/d/1ZziCENnmuUAapntzdMzHlXgxqsJxYg15STNaDvoU
iB8/edit?usp=sharing
AIM

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

analysis.
2. To obtain the percentage of theophylline in aminophylline tablet.

INTRODUCTION

Ultraviolet and visible spectroscopy is a method to measure the absorption of

compounds within the wavelength region of 200 nm to 800 nm. A spectrophotometer consists
of a light source, a monochromator, a cell (usually 1 cm length) and a photoelectric detector. It
will display the absorption spectrum of compounds plotted in a graph of absorbance versus
wavelength. From the absorption spectrum, we can obtain the wavelength for maximum
absorption (λmax) and the molar absorptivity (Ɛmax). All molecules absorb and emit light
differently based on the structure of the compound. Therefore, different compounds will show
different absorption spectrums thus identification of a substance is also possible. Aside from
qualitative analysis, quantitative analysis also can be determined using this method.

Readings from the absorption spectrum are used to calculate the transmittance,
absorbance, and concentration. Transmittance, T is the ratio of the intensity of emergent
radiation, I to the intensity of the incident radiation, I0 while absorbance, A is reciprocal of
logarithm T. At a certain wavelength, the absorbance is proportional to the concentration, c,
the path length of the solution, l and constant k (Assoc Prof Dr Nornisa Mohamed, 2019). These
relations are shown as below:

𝐼
𝑇 =
𝐼

1
𝐴 = 𝑙𝑜𝑔
𝑇

𝐴 = klc

The analysis is dependent on the complexity of the sample. When only one of the
components absorbed the radiation, λmax is used to analyse the wavelength and the
concentration of the sample is determined based on the calibration curve. For a multicomponent
sample, separation should first be conducted before measuring using spectrophotometry while
simultaneous spectrophotometry is used for special cases where the measurements are analysed
at two or more suitable wavelengths.

In the experiment, 100 mg of aminophylline containing 75 mg of theophylline is

dissolved in 0.1 M sodium hydroxide and water. Aminophylline is used to treat obstructive
airway disease associated with chronic asthma and other chronic lung diseases such as chronic
bronchitis (FDA, 2021). Aminophylline consists of 2:1 of theophylline and ethylenediamine
compound with the molecular formula (C7H8N4O2)2,C2H8N2,2H2O. The major component,
theophylline is a xanthine derivative known as dimethylxanthine that has two methyl groups at
positions 1 and 3. Based on British Pharmacopoeia, aminophylline is consist of 84 to 87.4
percent of theophylline (Pharmacopoeia, 2020). Therefore, hypothetically, the result for tablet
assay should be within the percentage range. Wavelength control with holmium oxide and
absorbance control with potassium dichromate is performed before the assay of aminophylline
tablet is conducted to ensure the reliability of the result using the instrument.

APPARATUS AND MATERIALS

Apparatus

1. UV/visible spectrometer, double beam

2. Beaker (250mL)
3. Volumetric flask (50mL, 100mL, 250mL)
4. Graduated pipette (5mL)
Materials

1. Plain water
2. Potassium dichromate, A.R. (100mg)
3. NaOH solution, 0.1M (50mL)
4. H SO solution, 0.005M (5mL)
5. Theophylline (20mg)
6. Aminophylline tablets (2 tablets)
METHODS

Preparation of different concentrations of theophylline solutions

1. The theophylline stock solution was prepared by dissolving 12 mg of theophylline in

10 mL of 0.1 M sodium hydroxide solution and the solution was then further diluted to
100 ml with distilled water. The percentage of theophylline stock solution (%w/v) was
calculated.
2. 1-, 2-, 3-, 4-, and 5 ml of theophylline solution were taken out and each of them was
diluted to 50 mL with 0.01 M sodium hydroxide.

Preparation of sample tablet.

1. 2 aminophylline tablets were weighed and recorded. The tablets were grinded until
becoming a fine powder.
2. 100 mg of aminophylline tablet powder was shaken with 25 ml of 0.1 M sodium
hydroxide and 60 mL of water for 10 minutes.
3. Water was added to make up to 250 ml solution, and the solution was mixed and
filtered.
4. 5 ml of the filtrate was diluted with 0.01 M sodium hydroxide to 250 mL.

A. Wavelength Control

1. The wavelength scale of spectrophotometer was examined using the maximum

absorption of holmium oxide.
2. Holmium oxide was inserted into spectrophotometer and it was allowed to run. The
graph was generated and the peaks were observed and compared to the theoretical peaks
which were 241.2 nm, 287.2 nm, 361.5 nm, and 536.3 nm.

B. Absorbance control

1. An exactly known amount of potassium dichromate (which has been dried at 180°C)
was weighed using electrical weighing balance and dissolved in 0.005 M of sulfuric
acid, H2SO4 until the final volume, 1000 mL.
2. The final concentration was obtained and recorded.
3. The absorbance of potassium dichromate solution was measured with 0.005 M H 2SO4
as reference at the following wavelengths. The values of A (1%, 1 cm) were calculated
and compared with the following values:
 Wavelength A (1 %, 1 cm) Maximum tolerance
235 124.5 122.9 to 126.2
257 144 142.4 to 145.7
313 48.6 47.0 to 50.3
350 106.6 104.9 to 108.2

C. Theophylline assay in aminophylline tablet

1. Spectrum and calibration curve of theophylline

1. Two cuvettes of 0.01 M sodium hydroxide solution were placed into spectrophotometer
and the spectrophotometer was calibrated by auto-zeroing.
2. One of the cuvettes was removed and replaced with the third theophylline solution (with
3 mL theophylline stock solution). The absorbance for the third solution was measured
using a double beam spectrometer in the wavelength range of 220 nm to 360 nm. The
absorbance for each 20 nm range was obtained. When it was close to the maximum and
minimum of the spectrum, the reading was made for every 5 nm. The graph of
absorbance versus wavelength was plotted to get the spectrum.
3. The wavelength at the maximum absorbance was chosen for the analysis.
4. Then, the absorbance for all the prepared solutions were measured using the double
beam instrument.
5. The absorbance versus concentration was plotted to obtain the calibration curve A (1
percent, 1 cm) was calculated.

2. Assay of the tablet

1. The solution containing the sample tablet from the preparation before was poured into
the cuvette and was analysed. The absorbance was also recorded.
2. Then, the concentration of theophylline in one aminophylline tablet was calculated
using two methods, which the reference to the calibration curve or by using the A (1
percent, 1 cm) determined from the section above.
RESULT

A. Wavelength Control

Figure 1: Wavelength readings of holmium oxide solution.

Theoretical Observed Difference between observed and

wavelength (nm) wavelength (nm) theoretical wavelength (nm)
241.2 - -
287.2 287.41 + 0.21
461.5 360.92 - 0.58
536.3 536.38 + 0.08
Table 1: The observed wavelength of holmium oxide solution and the difference compared to
its theoretical wavelength at maximum absorption.
Permitted tolerance is ± 1 nm for ultraviolet region and ± 3 nm for visible region.
B. Absorbance Control

Figure 2: Wavelength and absorbance readings of potassium dichromate solution.

Wavelength (nm) Absorbance Absorptivity (1%, 1cm)

observed Maximum
Given Observed Given Calculated
(A) tolerance
235 234.76 0.76498 124.5 124.5 122.9 to 125.2
257 256.63 0.88763 144.0 144.5 142.4 to 145.7
313 312.98 0.29505 48.6 48.0 47.0 to 50.3
350 350.85 0.65780 106.6 107.1 104.9 to 108.2
Table 2: The absorbance reading and calculated molar absorptivity of potassium
dichromate solution.
C. Theophylline Assay in Aminophylline Tablet
1. Spectrum & calibration curve of theophylline

Figure 3: Spectrum and calibration curve of theophylline

Volume of Concentration of
Preparation theophylline stock theophylline Absorbance, A
solution used (ml) (%w/v)
1 1.0 2.4 × 10-4 0.2613
2 2.0 4.8 × 10-4 0.3117
3 3.0 7.2 × 10-4 0.4871
4 4.0 9.6 × 10-4 0.6370
5 5.0 1.2 × 10-3 0.8027
Table 3: Absorbance at different concentrations of theophylline.
Based on table 3, the graph of absorbance against concentration of theophylline is plotted.

Graph 1 : Graph of absorbance against concentration of theophylline (%w/v).

2. Assay of the tablet

Total weight of 2 aminophylline tablet (mg) 528

528
Average weight of a tablet (mg) = 264
2
Absorbance of unknown (theophylline tablet) 0.4326
sample
Amount of powder used to prepare solution 100
(mg)
Using reference to Using the A (1%, 1 cm)
calibration curve determined from section 1
Amount of theophylline in one tablet (g)
0.1998 0.2109

Percentage of theophylline contains in one 75.7 79.9

tablet (%)
Table 4: Table on weight, absorbance and total of theophylline of sample
DISCUSSION

A. Wavelength Control

Wavelength control is an important calibration process to determine the accuracy and

reproducibility in measurement (Allen, 2007). For the purpose of calibration of this equipment,
holmium oxide is generally used as a wavelength standard. Unlike atomic emission lamps,
holmium does not cause split positioning error which makes it a better option to verify the
wavelength scale of ultraviolet (UV) or visible spectrophotometers. On top of that, it has some
good characteristics such as easy to use, compact and stable over a long period of time (Allen,
2007).

In this experiment, holmium oxide is chosen to validate the wavelength scale of the
instrument whether it is within the range of the manufacturer’s tolerance. Since the range of
wavelength is set at 250 nm to 600 nm, the expected values of wavelength peaks for holmium
oxide are 287.2 nm, 361.5 nm and 536.3 nm. The wavelength scale obtained is accurate
provided the readings are within  1 nm of the expected range for UV region and  3 nm of the
expected range of the visible region.

According to the results shown in table 1, the difference between the wavelengths peak
observed and the expected values of 287.2 nm, 361.5 nm and 536.3 nm are + 0.21 nm, – 0.58
nm and +0.08 nm respectively. Although there are some extend of deviation from the specific
wavelengths standard of holmium oxide provided, the results obtained are still in the permitted
tolerance of ± 1 nm for UV region. Thus, this confirms that this spectrophotometer is
functioning well and eligible for the next parts of the experiment.

In the calibration step, the cuvette is rinsed with distilled water after each trial and the
outer part is wiped carefully before continuing with the next measurement to prevent the
solution droplets and fingerprint that may affect the results obtained.
B. Absorbance Control

Apart from wavelength control, absorbance control is an alternative method that can be
used to determine the reliability as well as the photometric or absorbance accuracy of the
spectrophotometer (Pepenene and Coetzee, 2013). Potassium dichromate solution is an acidic
media that are commonly used to assess the photometric accuracy of the spectrophotometer
because this solution produces an observable and clear spectrum that can be easily
distinguished (Analytics, 2020). In this experiment, we measured the absorbance of potassium
dichromate solution with 0.005M H SO solution as the reference. The concentration of the
potassium dichromate solution used is 61.42 mg/L. The A (1%, 1cm) values are calculated
using the measured absorbance values of the reference standard material. A comparison
between the A values calculated and the A values of the established standard can then be done
to determine whether the calculated A values fall within the range of maximum tolerance.

There were 4 peaks observed at the wavelengths of 234.76nm, 256.63nm, 312.98nm

and 350.85nm. At these observed wavelengths, the observed absorbance values were recorded.
Then, the A values for the wavelengths were calculated using the observed absorbance values.
By applying Beer-Lambert Law, the molar absorptivity calculated for wavelengths of
234.76nm, 256.73nm, 312.98nm and 350.85nm are 124.5 Lg-1 cm-1. 144.5 Lg-1 cm-1, 48.0 Lg-
1 cm-1 and 107.1 Lg-1 cm-1 respectively. Upon comparison with the maximum tolerance
provided in FAR 313/4 Pharmaceutical Analysis Lab Manual, all A values obtained fell within
the range. For example, at the wavelength of 350.85nm, the A value calculated is 107.1 and
hence it fell within the range of maximum tolerance which is 104.9 to 108.2. Therefore, based
on the data obtained, we summarize that the spectrophotometer is well functioning as the
photometric accuracy of the spectrophotometer is achieved.
C. Theophylline Assay in Aminophylline Tablet
1. Spectrum and calibration curve of theophylline

Spectrophotometry is one of the most widely used qualitative and quantitative analysis
methods, particularly for determining the concentration of specific substances. The
spectrophotometry technique is used in this experiment to determine the concentration of
theophylline in one aminophylline tablet. The sample solution is expected to absorb a certain
amount of light or photons when a beam of light passes through it. As a result, the concentration
of theophylline can be determined by measuring the intensity of light detected (Anon, 2020).

Diagram 1: The chemical structure of theophylline

The aromatic structure, double bond, and diketone group in the theophylline all contain
chromophores that allow light to be absorbed (Pubchem, 2020). The chemical group in the
molecule known as the chromophore is an essential moiety that causes the molecule to change
conformation when exposed to light. The electrons will be excited from the ground state to the
excited state as a result of the light energy gained. Furthermore, when light strikes the
compound, the light energy is converted into an electron being promoted from a bonding or
non-bonding orbital to one of the empty anti-bonding orbitals. Hence, the energy difference
between two molecular orbitals falls in the range of the visible spectrum in the chromophore
region (Shukla et al., 2017).

When performing drug assays using UV/VIS spectrophotometer, a calibration curve of

theophylline is required to determine the concentration of theophylline in aminophylline tablet.
Hence, it is necessary to prepare a range of concentrations of an analyte standard sample and
measure the absorbance of each solution. So, five different volumes of 1.0 mL, 2.0 mL, 3.0
mL, 4.0 mL and 5.0 mL of theophylline stock solution were diluted in 50 mL of sodium
hydroxide solvent respectively. As the solvent itself may have some absorbance value, the auto
zero procedure was performed before testing the various concentrations of the sample. Hence,
the absorbance sodium hydroxide solution was measured first, serving as a blank or the
reference solution. The blank must match the composition of the solvent or medium in which
the sample will be analysed (Cairns, 2003). On the other hand, λmax, also known as maximum
absorbance, is the wavelength at which the absorption of a substance reaches its highest peak.
From the spectrum obtained, the maximum absorbance was 274.89 nm due to the presence of
conjugated double bond (for the n → π* transition) in theophylline. This maximum absorbance
occurs at the concentration of theophylline in the 3rd preparation. The spectrophotometer is
then set to transmit light at this specific wavelength to measure the absorbance of the remaining
solutions.

Diagram 2: The graph of absorbance vs concentration is directly proportional

under ideal conditions, according to Beer’s Law.

According to the Beer-Lambert law, there is a direct linear relationship between the
absorbance and the concentration of a sample for dilute solutions (John, 2020). Theoretically,
by referring to the Beer-Lambert equation, the A (1%, 1cm) value should be calculated from
the slope of the curve. This is accurate when the equation obtained is a straight best-fit line that
starts from the origin (CL, 2020). Ideally, there should be no y-intercept as there should be no
absorbance occurring at the zero concentration of solution. From this experiment, a line of best
of positive slope was produced when a graph of absorbance against concentration of
theophylline standard solution was plotted. The linear line shows that the absorbance is directly
proportional to the concentration of theophylline standard solution. Hence, as the concentration
of theophylline standard solution increases, the absorbance increases.
 In addition, from the graph above, an equation, y = 586.71x + 0.0775, where y =
absorbance and x = concentration of theophylline standard solution, was generated to calculate
A (1%, 1 cm) value. However, the line did not intercept the y-axis at the origin due to
inaccuracy or interference in one absorbance reading that made the line deviate slightly from
the origin. Furthermore, there is a possibility where the standard solutions were not prepared
properly, hence the presence of impurities or contaminants might affect the readings.
Therefore, this calculation is not suitable to be used for our results as it does not obey the Beer-
Lambert law. Instead, A (1%, 1 cm) or k can be calculated using the values in preparation 3
since it has the maximum absorbance, based on the formula of Beer-Lambert law as follow:

A = klc

Where,

A = absorbance,

k = rate constant,

l = path length = 1 cm

c = concentration of theophylline.

The calculated A (1%, 1 cm) obtained is 676.5278 Lg-1cm-1 and this value will be used in the
calculation of the next part.

2. Assay of the tablet

There are two methods of using spectroscopic measurements in drug analysis, the
absolute and the comparative methods of assay. The Beer–Lambert equation is commonly used
in the absolute technique of assay in the United Kingdom and Europe. From the graph plotted
in Part C (1), the graph is allowed to be used for calibration. A solution with an unknown
concentration is prepared in the same way as the standards, and its absorbance is measured at
the same wavelength. Subsequently, the Beer–Lambert equation is solved for c, the drug
concentration, in this manner. For this reason, A values are quoted in drug monographs by the
British Pharmacopoeia and the European Pharmacopoeia (Cairns, 2003). In Part C (2), two
aminophylline tablets were grinded and the powder formed were diluted with 0.01M of NaOH
and distilled water. The stock solution underwent a series of dilution before being assayed on
the spectrophotometry.

The absorbance of the solution of unknown theophylline concentration is 0.4326.

According to British Pharmacopoeia (BP) 2020, one aminophylline tablet should contain
between 84.0 % to 87.4 % of theophylline (Pharmacopoeia, 2020). In our experiment, the
percentage of theophylline contained in one tablet of aminophylline using reference to
calibration curve and by using the A (1%, 1 cm) determined from Part C (1) are 75.7 % and
79.9 % respectively. These values do not fulfil the criteria mentioned in the BP. This deviation
of the result from what is stated in the BP might be due to some experimental errors in the
procedure such as during grinding and dilution. Improper grinding may lead to an imbalanced
portion of theophylline, which in turn will contribute to the variance in its composition
throughout the test. Besides, the scattering of light caused by non-homogeneity of the sample
might occur especially if the sample dissolves partially in the chosen solvent, or if there are air
bubbles adhering to the sample cuvette, as well as any other unwanted material on the outside
of the cuvette will affect the accuracy of the absorbance measurements. Lastly, parallax error
might occur if the observer’s eyes are not perpendicular to the scale.

PRECAUTIONS

1. Before putting the cuvette into the spectrophotometry, the outer walls of the cuvette
must be wiped to prevent it from fingerprints and solution droplets that will
influence the absorbance of the equipment.
2. The cuvette must be rinsed with distilled water after each measurement in the
spectrophotometer and followed by the solution to be measured next to prevent the
dilution of the measured solution.
3. The cuvette should be filled with sample solution with 4/5 of the cuvette.
4. The transparent side of the cuvette should face the light source of UV visible
spectrophotometry.
5. The eyes must be perpendicular to the meniscus during preparation of samples and
standards to avoid parallax error.
6. The calibration of equipment must be within the tolerated range given before
starting the experiment.
 7. Theophylline powder should be dissolved completely to ensure the accuracy of the
concentration in the solution.
8. The tablet must be grinded into fine powder to accelerate the dissolution of the
tablet in the solvent and prevent scattering of lights caused by non-homogeneity of
the sample.
9. The powder in the solvent must be shaken continuously until all the fine particles
are completely dissolved in the solution.

CONCLUSION

1. The difference between the wavelength peaks obtained and the expected values
given are + 0.21 nm, – 0.58 nm and +0.08 nm respectively.
2. Both wavelength control and absorbance control are significant to evaluate the
performance, accuracy and reliability of spectrophotometer. In this experiment, the
outcomes from both methods are within the range of maximum tolerance and this
shows that the spectrophotometer is functioning well and capable to assess the
compounds accurately.
3. The percentage of theophylline in aminophylline tablet obtained using reference to
the calibration curve and by using the A (1%, 1 cm) determined from Part C (1) are
75.7% and 79.9% respectively. According to BP, one aminophylline tablet should
contain between 84.0 % to 87.4 % of theophylline. The deviation of the result
findings from the percentage range mentioned in the BP might be due to
experimental errors.
REFERENCES

ALLEN, D. W. 2007. Holmium Oxide Glass Wavelength Standards. J Res Natl Inst Stand
Technol, 112, 303-6.

ALLEN, D. W. 2007. Holmium Oxide Glass Wavelength Standards. J Res Natl Inst Stand
Technol, 112, 303-6.
ANALYTICS, H. 2020. Optical Reference Materials for UV/Vis Spectroscopy [Online].
Available:
https://www.hellma.com/fileadmin/fos/Website/Broschueren_Flyer_Handhabung/Hell
ma_Analytics/Englisch/HA_CRM_Broschure_EN_2020.pdf [Accessed 2021].
ANON 2020. Spectrophotometry

ASSOC PROF DR NORNISA MOHAMED, P. G. L. H., DR ROZIAHANIM 2019. Practical

Manual FAR313/4 Pharmaceutical Analysis, Penang, School of Pharmaceutical
Sciences.
CAIRNS, D. 2003. Essentials of pharmaceutical chemistry. London, Pharmaceutical Press.
CL, R. 2020. Graphing Example 2

FDA. 2021. Aminophylline Tablets [Online]. Available:

https://www.drugs.com/pro/aminophylline-tablets.html [Accessed 2021].
JOHN, P. 2020. Beer’s Law Definition, Equation, & Facts. Encyclopedia Britannica.
PEPENENE, R. & COETZEE, E. 2013. TRACEABILITY OF SPECTROPHOTOMETRIC
MEASUREMENTS TO THE HIGHEST STANDARD OF CALIBRATION.
PHARMACOPOEIA, B. 2020. British Pharmacopoeia 2020. London: TSO;.
PUBCHEM 2020. Theophylline.
SHUKLA, R., DUBEY, A., PANDEY, V., GOLHANI, D. & JAIN, A. 2017. Chromophore-
An Utility in UV Spectrophotometer. Inventi Rapid: Pharm Ana & Qual Assur.
APPENDICES

Calculation

A. Wavelength Control

Difference between observed and

Theoretical Observed
theoretical wavelength (nm)
wavelength (nm) wavelength (nm)
(observed - theoretical)
241.2 - -
287.2 287.41 287.41 - 287.2 = +0.21
461.5 360.92 360.92 - 461.5 = -0.58
536.3 536.38 536.38 - 536.3 = +0.08
Table 5: Calculation of the difference between observed and theoretical wavelength.

B. Absorbance Control

Formula used to calculate the absorptivity, A (1%, 1cm) of potassium dichromate solution:
A = klc
Where;
A = absorbance
k = rate constant = specific absorptivity = A (1%,1cm)
l = path length of solution (cm)
c = concentration of potassium dichromate solution used (%w/v)

Convert the concentration of potassium dichromate to (% w/v)

Concentration of potassium dichromate solution used, C = 61.428 mg/L
.
c= 𝑥 100% = 0.006143 % 𝑤/𝑣

Substitute the concentration into the formula where path length of solution in the experiment,
l = 1cm.
A = klc

k=

k=
( )( . )

k = specific absorptivity, A (1%,1cm)

𝑨
Hence, A (1%, 1cm) =
(𝟏)(𝟎.𝟎𝟎𝟔𝟏𝟒𝟑)
Calculate specific absorptivity, A (1%,1cm) for potassium dichromate.

Wavelentgh observed Absorbance Specific absorptivity, A

(nm) oberved,A (1%,1cm)
A (1%, 1cm)
=
( )( . )
234.76 0.76498 .
=
.

= 124.53 Lg-1 cm-1

A (1%, 1cm)
=
( )( . )
256.63 0.88763 .
=
.

= 144.49 Lg-1 cm-1

A (1%, 1cm)
=
( )( . )
312.98 0.29505 .
=
.

= 48.03 Lg-1 cm-1

A (1%, 1cm)
=
( )( . )
350.85 0.65780 .
=
.

= 107.08 Lg-1 cm-1

Table 6: Calculation of specific absorptivity, A (1%,1cm) for potassium dichromate.

C. Theophylline Assay in Aminophylline Tablet

1. Spectrum and calibration curve of theophylline
Preparation of theophylline stock solution

12 mg (0.012 g) of theophylline powder is dissolved in 0.1M of sodium hydroxide solution and

diluted to 100 ml with water. The percentage of theophylline stock solution (%w/v) is as below:
.
Percentage of theophylline stock solution (%w/v) = × 100% = 0.012%
Concentration for each preparation can be calculated by using formula:
M1V1 = M2V2
Where,
M1 = Concentration of theophylline stock solution, 0.012% w/v
V1 = Volume of theophylline stock solution
M2 = Concentration of theophylline after dilution
V2 = Final volume of theophylline solution after dilution (50ml)

Volume of
Concentration of theophylline
Preparation theophylline stock
(%w/v)
solution used (ml)
(0.012 x 1.0) = M2 (50)
1 1.0
M2 = 2.4 x 10-4 %w/v
(0.012 x 2.0) = M2 (50)
2 2.0
M2 = 4.8 x 10-4 %w/v
(0.012 x 3.0) = M2 (50)
3 3.0
M2 = 7.2 x 10-4 %w/v
(0.012 x 4.0) = M2 (50)
4 4.0
M2 = 9.6 x 10-4 %w/v
(0.012 x 5.0) = M2 (50)
5 5.0
M2 = 1.2 x 10-3 %w/v
Table 7: calculation of concentration of theophylline in different preparations.
Based on table 7, the graph of absorbance against concentration of theophylline is plotted

Graph of Absorbance vs Concentration of

Theophylline standard solution (%w/v)
0.9
0.8
y = 586.71x + 0.0775
0.7 R² = 0.9767
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014
Concentration of Theophylline standard solution
(%w/v)

Graph 1 : Graph of absorbance against concentration of theophylline (%w/v).

Calculation of A (1%, 1 cm) from slope of curve using equation

According to Beer-Lambert equation,

Absorbance, A = klc
Since l = 1 cm, A = kc,
The equation of the curve is y = mx + 0 (1)
A = kc + 0 (2)
By comparing equation (1) & (2), k can be m, which is the gradient of the slope.
Based on the equation obtained from the calibration curve in Graph 1 which is y = 586.71x +
0.0775, where there is a y-intercept that deviates substantially from the origin. Thus, the
equation of the graph cannot be used in the calculation.
Therefore, A (1%, 1 cm) or k can be calculated using the values in preparation 3 since it has
the maximum wavelength.
A = klc
(0.4871) = k (1) (7.2 x 10-4)
k = 676.5278 Lg-1cm-1

∴ The A (1%, 1 cm) or k value is 676.5278 Lg-1cm-1

2. Assay of the tablet

Method 1: Calculation with reference to calibration curve

From the graph plotted (Graph 1), the equation obtained is y = 586.71x + 0.0775.
To calculate the concentration of theophylline in the sample (%w/v)
y = 586.71x + 0.0775, where y = 0.4326
(0.4326) = 586.71x + 0.0775
x = 6.0524 × 10-4 %w/v
In 100 ml, amount of theophylline is 6.0524 × 10-4 g
. ×
Amount of theophylline in 250 ml (after dilution) = × 250

= 1.5131 × 10 -3 g
. ×
Amount of theophylline in 5 ml (before dilution) = × 250

= 0.0757 g
Thus, in 0.1 g (100 mg) of aminophylline = 0.0757 g of theophylline
 .
So, the amount of theophylline in one tablet = × 0.264
.

= 0.1998 g
.
Percentage of theophylline contain in one tablet = × 100
.

= 75.7 %
Method 2: Calculation using the A (1%, 1 cm) determined from section 1
A = klc,
where, A = 0.4326
k = 676.5278
.
c= =
.

= 6.3944 × 10-4 %w/v

Concentration of theophylline in sample solution is 6.3944 × 10-4 %w/v

Thus, in 100 mL, amount of theophylline is 6.3944 × 10-4 g

. ×
Amount of theophylline in 250 ml (after dilution) = × 250

= 1.5986 × 10 -3 g
. ×
Amount of theophylline in 5 ml (before dilution) = × 250

= 0.0799 g
Thus, in 0.1 g (100 mg) of aminophylline = 0.0799 g of theophylline
.
So, the amount of theophylline in one tablet = × 0.264
.

= 0.2109 g
𝟎.𝟐𝟏𝟎𝟗
Percentage of theophylline contain in one tablet = × 𝟏𝟎𝟎
𝟎.𝟐𝟔𝟒

= 79.9 %