Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

🔬 Practical No.3:-

Determination of Paracetamol Concentration Using UV/Vis

Spectrophotometry

Objective:

To determine the concentration of an unknown paracetamol sample using UV/Visible

spectrophotometry by constructing a standard calibration curve.

Introduction:

Paracetamol (also known as acetaminophen) is a widely used analgesic and antipyretic drug. It is
a common ingredient in over-the-counter medications and is frequently analyzed in pharmaceutical
quality control.

UV/Visible (UV/Vis) spectrophotometry is a widely used analytical technique that measures the
absorbance of ultraviolet or visible light by a compound at specific wavelengths. This absorbance
is directly proportional to the concentration of the compound, as described by Beer-Lambert’s
Law:

A=εbC

Where:

 A = Absorbance
 ε = Molar absorptivity
 b = Path length of the cuvette (usually 1 cm)
 C = Concentration

A UV/Vis spectrophotometer operates by passing a beam of UV or visible light through a sample

solution and detecting how much light is absorbed. The device consists of a light source,

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

monochromator (to select specific wavelengths), a sample holder (cuvette), and a detector. The
resulting absorbance readings are used to assess the concentration of light-absorbing compounds
like paracetamol.

Paracetamol absorbs UV light strongly in the range of 243–245 nm due to its aromatic structure.
By measuring the absorbance of standard solutions and plotting a calibration curve, the
concentration of an unknown sample can be accurately determined.

Applications

 Pharmaceutical analysis: Quality control of drug formulations.

 Clinical laboratories: Monitoring drug levels in biological fluids.
 Environmental studies: Detecting pharmaceutical residues in water bodies.
 Research: Studying drug degradation or formulation stability.

🌟 Apparatus and Materials:

 UV-Visible spectrophotometer

 Quartz cuvettes

 Analytical balance

 Volumetric flasks (50 mL, 100 mL)

 Pipettes and micropipettes

 Test tubes and test tube rack

 Paracetamol reference standard

 Commercial paracetamol tablets

 Distilled water

 0.1 M NaOH solution (required for solubility)

 Filter paper and funnel

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

 Weighing paper (wax paper) or weighing boat or watch glass

 Spatula

 Mortar and pestle

Procedure:

A. Preparation of Standard Solutions

Prepare a stock solution of paracetamol:

Accurately weigh an appropriate amount of pure paracetamol (e.g., 100 µg/mL), dissolve it
in a small amount of solvent (e.g., 0.1 M NaOH or distilled water), and dilute to a known
volume in a volumetric flask.

 This creates a known, concentrated solution from which all standards will be made.

1. Dilute the stock to obtain 5 standard solutions (e.g., 10, 20, 30, 40, 50 µg/mL):
Use pipettes and volumetric flasks to prepare these solutions by dilution.

 These known concentrations are needed to construct a calibration curve.

2. Label each flask clearly:

To avoid mix-ups during measurements label each flask with correct concentration.

Table: Preparation of Stock and Standard Solutions of Paracetamol

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

B. Determine the λmax (Wavelength of Maximum Absorbance)

1. Scan the 30 µg/mL or any other standard solution from 200–400 nm using the UV/Vis
spectrophotometer.Record the absorbance spectrum.

 This helps identify the exact wavelength where paracetamol absorbs the most light.

2. Record the wavelength at which maximum absorbance occurs (λmax):

Typically around 243–245 nm.

 Using precise λmax ensures the most accurate and sensitive measurement for
concentration.

C. Measure Absorbance of Standard Solutions

1. Set the spectrophotometer to λmax between 240-250nm

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

 This ensures all measurements are made under optimal conditions.

2. Calibrate the instrument with a blank (solvent only):

Fill a cuvette with just the solvent and use it to "zero" the spectrophotometer.

 This removes background absorbance from the solvent.

3. Measure the absorbance of each standard solution:

Rinse the cuvette between samples to avoid contamination.

 This provides the data points for your calibration curve.

D. Plot Calibration Curve

1. Plot Absorbance vs. Concentration:

Use graph paper, Excel, or software provided by your instructor.

 This visually shows the relationship between absorbance and concentration.

2. Draw the best-fit line and obtain the linear equation (e.g., A = mC + b):
The slope (m) and intercept (b) will be used to calculate unknown concentrations.

E. Determine Concentration of Unknown Sample

1. Prepare the unknown sample similarly to the standards:

For example, crush a tablet, dissolve it, filter it, and dilute it to a known volume.
2. Measure its absorbance at λmax 243-245nm
Use the same procedure as for all the standard solutions prepared.
3. Use the calibration equation to calculate its concentration:

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

Conclusion:

This experiment demonstrated the use of UV/Vis spectrophotometry to determine paracetamol

concentration via a standard calibration curve. The λmax was identified, and absorbance readings
were taken for known and unknown samples. A linear relationship between absorbance and
concentration was established. The unknown sample’s concentration was accurately calculated
using the calibration equation. This method proved effective for simple, quantitative drug analysis.

Solved example

Given data:

From the table, we can observe a perfectly linear relationship:

 Slope (m) = ΔA / ΔC
 = (0.625 - 0.125) / (50 - 10)
 = 0.0125
 Intercept (b) ≈ 0 (no absorbance when concentration is zero, assuming ideal conditions)

By: Dr. Muhammad Siraj Khan Jadoon

Pharmaceutical Chemistry IIIB (Analysis) Practical Manual

Step 1: Calibration Equation

A=mC+b

=0.0125×C+0

= 0.0125x C

Step 2: Unknown Sample

Let’s say the absorbance of the unknown sample was measured at λmax and found to be:

Step 3: Rearranging to Find Concentration

_____________________________

Discussion Questions for students:

1. Why is it important to choose a solvent that does not absorb at the λmax required for our
sample?
2. What is the purpose of constructing a calibration curve?
3. Why is it important to measure the absorbance at λmax?
4. What sources of error could affect the accuracy of your results?
5. How would tablet excipients influence your measurement?
6. What alternative analytical methods can be used for given sample determination, and how
do they compare with UV/Vis spectrophotometry?
7. If your standard solutions were not mixed properly, how would that affect your calibration
curve and final result?

By: Dr. Muhammad Siraj Khan Jadoon