Master Program in Industrial Pharmaceutical

Technology

Analytical Techniques (MIPT 735)

Lab Report # 1
UV-Visible Spectrophotometry

Mayson Bali 1185273

Tujan Liddawieh 1185176

Dr. Feras Qanaze

Objectives:
  To develop suitable spectrophotometric method for assay of Paracetamol tablet.
 To measure unknown concentrations of paracetamol by using calibration with
standards.
 To understand spectrophotometry techniques in pharmaceutical analysis.

Experimental:
1- Materials

 Paracetamol standard.

 Paracetamol tablets containing 500 mg Paracetamol.

 Analytical grade methanol and water.

2- Instrumentation
UV-Visible double beam spectrophotometer with matched quartz cells (1 cm).

3- Diluent preparation

 Methanol and water (15:85, v/v) used as a diluent.

 We prepared one litter diluent phase containing 300ml methanol and 700ml
water.
 Mixed well and filtered

4- Standard preparation(0.01 mg/ml or 10 ppm)

1. Introduce 10 mg paracetamol in 100ml volumetric flask, dissolve in 15 ml

methanol, and shake well.
2. Then add 85 ml water to adjust the volume up to 100 ml (100 ppm).
3. From that, dilute 5 ml to 50 ml with diluent.
4. Prepare four different standard solutions.

Dilution NO Concentration up to 50 ml w/v (mg/ml) Concentration

of standard
1 1ppm 0.02 0.0002 mg/ml
2 5ppm 0.1
3 10ppm 0.2
4 30ppm 0.6
 5- Sample (Test) preparation (0.01 mg/ml or 10 ppm)

 Four tablets were weighed and powdered using mortar and pestle.
 Powdered tablet equivalent to 100 mg of paracetamol was weighed and taken into
100 ml volumetric flask then 15 ml of methanol was added and shaken well to
dissolve it after that 85 ml of water was added to adjust the volume up to 100 ml.
 From that, 1 ml of solution was withdrawn and taken in 100 ml volumetric flask. The
volume was adjusted with diluent up to 100 ml.
Tablet NO Tablet weight (g) Average WT(g) WT equivalent to
100 mg of
paracetamol
1 0.5651 0.567 0.1134 g
2 0.564
3 0.5678
4 0.5687

 The concentration of the sample

- 100mg paracetamol upt to 100ml
- 1st dilution 1ml of stock sample up to 100ml
- 2nd dilution 1ml of 1st dilution up to 25ml
- Concentration of sample =
100 mg
∗1ml
100 ml
∗1 ml .
100 ml mg
=0.0004 =0.4 ppm
25 ml ml
Selection of wavelength
 Scan standard solution in UV spectrophotometer between 200 nm to 400 nm on
spectrum mode, using diluents as a blank.
 Paracetamol shows λmax at 243.

Procedure and calculations:

1- Measure the absorbance of the standard and sample solutions at wavelength of 243
nm using diluent as blank.
2- Calculate the Assay (%) of paracetamol in the samples as follows AS/Astd. 100%
 The absorbance of standard and sample solutions were measured at 243 nm,
using diluent as a blank.

 Absorbance of sample= 0.036

 Absorbance of standard= 0.019
 Assay (%) of paracetamol is:
absorbance of sample AS∗conc . of standard
∗100 %
absorbance of standard Astd .∗conc . of sample

AS 0.036 0.0002
X 100 %= X X 100 %=94.74 %
Astd . 0.019 0.0004
Determination of paracetamol by using calibration curve
 The Measurements of the absorbance of the standard solutions at wavelength of 243
nm using diluent as blank.
Concentration (mgl100 ml) Concentration ppm Absorbance at 243 nm
Blank = 0 0 0
STD₁= 0.0002 0.2 0.019
STD₂= 0.001 1 0.071
STD₃= 0.002 2 0.145
STD₄= 0.006 6 0.417
Sample= 0.0004 0.4 0.036
The assay%= 94.7%

Figure 2: the calibration curve between conc. and absorbance for standard solution.

 Check the linearity of the calibration curve (do regression analysis: regression
equation, and correlation coefficient 0.998.

The calibration curve is linear, the equation is:

y=0.0691 x +0.0032. R2=0.9997.
Questions:

1- Can we use the same method for determination of paracetamol in combination

of aspirin? Explain, suggest another method?
 N0 we cant
 UV- spectrophotometer method is non-selective and cannot detect nor distinguish
between two substances.
 In this case, we can use a reverse-phase HPLC method.

2- What is λmax? Why we use it for quantitative determination using UV-vis.

Spectrophotometry?
 Lambda max (λmax): The wavelength at which a substance has its strongest
photon absorption (highest point along the spectrum's y-axis. We can
determine λmax by plotting absorbance vs. wavelength in graph. 
 The value of λmax is important for several reasons:
1. This wavelength is characteristic of each compound.
2. It provides information on the electronic structure of the analyte.
3. It ensures highest sensitivity and minimize deviations from Beer's
Law. 

3- If paracetamol solution with 1 ppm concentration was found to have a very low
absorbance? Comment on the method linearity?

 The UV-Vis instrument that only works linear within a certain range. A
meaningful absorbance reading should be between A=0.1 and A=1.0. The
concentration has to be low enough, so that the maximum absorbance does
not exceed A=1.
 Linearity is determined by the regression analysis R2, if when plotting the
calibration curve with this low absorbance and R 2 still approximate to 0.99 it
is considered linear with this range.

4- Can we use this method for determination of paracetamol in a pharmaceutical

tablet that is degraded (with p-aminophenol degradation product)? Explain.

 No we cannot, it will not give peaks for other existing materials.

 UV- spectrophotometer method is non-selective and cannot detect nor
distinguish between two substances.
 as the assay is calculated it could show that it is not in the accepted range of
the API, p-aminophenol has an absorbance that will interfere with the
paracetamol absorbance peak and misleading results will be obtained.
 In this case, we can use a reverse-phase HPLC method.
5- Why methanol is used in diluent? Can we use only water as diluent? Explain

 For best solution stability results at different time intervals for test preparation. It was
concluded that the test preparation solution was found stable up to 8 hr. at room
temperature
 To improve its solubility because the solubility of paracetamol in water is much lower
than in other polar solvents such as the methanol.
 In addition, this is done because the samples are used in HPLC which it should have
organic solvent for specificity.

6- Comment on the following cases:

a) In an experiment for paracetamol determination, assay of paracetamol
was found to be 99.5 ± 15 (RSD = 15%), is it accurate, precise, knowing
that the true value of assay is 100%

 For absorbance precision: standard deviation must not exceed 0.5 %

or 0.5 % multiplied by A for absorbance values above 1.0 A, so we
can say the experiment is not precise due to the RSD is high which
indicates that the data are not close to each other.
 For absorbance accuracy: The assay of paracetamol was found to be
99.5 of the spiked drug were obtained indicating that the method
was accurate.

b) In an experiment for paracetamol determination, assay of paracetamol

was found to be 89.1 ± 0.1 (RSD = 0.11%), is it accurate, precise,
knowing that the true value of assay is 100%

 For absorbance precision: standard deviation must not exceed 0.5 %

or 0.5 % multiplied by A for absorbance values above 1.0 A, so we
can say the experiment is precise due to the RSD is low which
indicates that the data are close to each other.
 For absorbance accuracy: The assay of paracetamol was found to be
89.1 of the spiked drug were obtained indicating that the method was
not accurate.
c) What is the reason behind such results in a and b.

 Accuracy is a general term that describes the agreement between a

measurement and a true value. Precision is a general term that
describes how close measured values are to each other, if they are
clustered or spread out.
 Accuracy affected by both random and systematic errors, while
precision affected by random errors.
 Systemic errors such as instrument calibration errors, environmental
factors such as temperature and humidity, which can lead to
wavelength-dependent responsivity changes in UV meters. In
addition, UV radiation itself induces aging of the optical elements of
meters.
 Random errors such as sample preparation, sample-surface cleaning,
blank measurements and buffers.
 Compare between single point assay and calibration curve?

 Calibration curve method: The absorbance’s of a number of standard

solutions of the reference substance at concentrations encompassing
the sample concentrations are measured and a calibration graph is
constructed.
 The single point standardization: Procedure involves the
measurement of the absorbance of a sample solution and of a
standard solution of the reference substance. The concentration of the
substances in the sample is calculated from the proportional
relationship that exists between absorbance and concentration. C
test=(A test X C std)/A std where, C test and C std are the
concentrations in the sample and standard solutions respectively and
A test and A std are the absorbance’s of the sample and standard
solutions respectively.

Conclusion
 
We concluded that UV Spectrophotometry can be used for quantitative
determination of materials and the analytical method gave specific, precise,
linear and accurate results. In addition, we were able to achieve our
objectives with this simple method.

References
[1] S. Behera, S. Ghanty, F. Ahmad, S. Santra, and S. Banerjee, “Analytical &
Bioanalytical Techniques UV-Visible Spectrophotometric Method Development and
Validation of Assay of Paracetamol Tablet Formulation,” vol. 3, no. 6, 2012.
[2] C. M. El Maraghy and N. T. Lamie, “Three smart spectrophotometric methods for
resolution of severely overlapped binary mixture of Ibuprofen and Paracetamol in
pharmaceutical dosage form,” BMC Chem., pp. 1–8, 2019.
[3] F. Al-rimawi, “College of Pharmacy Instrumental analysis Lab manual Second
semester 2016 / 2017,” pp. 1–81, 2017.
[4] V. D. Hoang, D. Thi, H. Ly, N. H. Tho, H. Minh, and T. Nguyen, “UV
Spectrophotometric Simultaneous Determination of Paracetamol and Ibuprofen in
Combined Tablets by Derivative and Wavelet Transforms,” vol. 2014, 2014.