Determination of (a) Wavelength of maximum absorbance (λmax) and (b) Absorptivity coefficient of Paracetamol.

1. Name of the experiment: Determination of
(a) Wavelength of maximum absorbance (λmax) and
(b) Absorptivity coefficient of Paracetamol.
(a) wavelength of maximum absorbance (λmax)
The extent to which a sample absorbs light depends upon the wavelength of light. The
wavelength at which a substance shows maximum absorbance is called absorption maximum
or λmax.
The value of λmax is important for several reasons.
 This wavelength is characteristic of each compound
 It provides information on the electronic structure of the analyte
 It ensures highest sensitivity and minimize deviations from Beer's Law.
We can determine λmax by plotting absorbance vs wavelength in graph.
Preparation of stock solution:
1. 5 mg paracetamol powder were weighed by electronic balance
2. This powder was taken in a 50 ml volumetric flask and was filled with distilled water
upto the mark.
3. The flask was shacked well until the paracetamol powder was dissolved. This solution
was the stock solution.
Preparation of working stock:
Take 10 ml of stock solution and dilute up to 100ml with distilled water.

2. Procedure:
1. Set the spectrophotometer wavelength to 200 nm and with a cuvette containing distilled
water to set the instrument reference level.
2. Place the cuvette containing the prepared dilution in the sample compartment. Record
the absorbance.
3. Repeat steps 2 at wavelength increments of 200 nm up to 400 nm and record absorbance
at each wavelength setting.
4. Plot the results as absorbance against wavelength.
5. From the graph note the wavelength of maximum absorbance for this solution.
Data Table:
Wavelength (λ) Absorbance
235 0.399
240 0.592
245 0.574
250 0.442
255 0.307
260 0.228
265 0.148
270 0.115
Result:

3. (a) Absorptivity coefficient
Absorptivity coefficient, also known as the molar absorptivity coefficient, is a measure of how
well a chemical species (chemically identical molecules) absorbs a given wavelength of light.
It is the absorbance of a substance placed in 1cm cuvette cell when the concentration is 1 molar.
According to Beer-Lambert Law:
When working in concentration units of molarity, the Beer-Lambert law is written as:
A = ε * b * c
Where ε is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.
Therefore, if we know A at the λmax, c, and b (always equal to 1 cm), we can calculate ε by
rearranging the Beer-Lambert equation as follows:
ε=A/cb
Reagents:
 Standard paracetamol powder
 Distilled water
Apparatus:
 UV Spectrophotometer
 Test tube
 Pipette
 Volumetric flask
 Electronic balance
A = a * b * c where a = the absorptivity of the substance
b = path length
c = concentration of the substance

4. Procedure:
Preparation of working standard:
1. 2 test tubes were taken, and were marked serially from 1-2 and were kept in a test tube
holder.
2. Test tube marked 1 was taken and was added with 5 ml stock solution (Previously
prepared) and 100 ml distilled water.
3. Test tube marked 2 was taken and was added with 10 ml stock solution and 100 ml
distilled water.
Test Tube No Stock Solution (ml) Distilled Water (ml) Concentration(µg/ml)
1 5 100 5
2 10 100 .01
Calibration of UV Spectrophotometer:
1. Turn on the UV spectrophotometer and wait for the calibration to be completed
2. Fill 2/3rd of the cell with distilled water, set the wavelength at 249 nm and press the
auto-zero button
Determination of absorbance for the working standard and the sample solution:
1. Take test tube 1 and fill the cell with it
2. Place the cell into the sample holder and measure the absorbance
3. Same way, determine the absorbance for test tube 2 and 3.
Data table:
Concentration (µg/ml) Absorbance
0 0
5 0.592
10 0.930

5. Calculation:
From Beer-Lambert equation as follows:
For test tube 1, ε1=A1/c1b
=0.592/ (5×1)
=0.1184 cm-1ml µg-1
For test tube 2, ε2=A2/c2b
=0.930/ (10×1)
=0.093 cm-1ml µg-1
Therefore, Absorptivity coefficient, ε = (ε1+ ε2)/2
= (0.1184+0.093)/2
= 0.1057 cm-1ml µg-1
Result: