Beer's Lamberts Law

1. Sadhana Gupta
Lecturer Bhilai Mahila Mahavidyalaya

2. To perform various laboratory techniques-
a) Centrifugation .
b) Chromatography-TLC , ion exchange
Chromatography.
c) Spectrophotometery.
d) Electrophoresis.
e) ELISA.
f) SDS PAGE
g) Southern Blotting
h) PCR.

3. AIM: To verify Lambert – beer ‘s law using spectrophotometer.
REQUIREMENTS –Spectrophotometer, cuvettes, test tubes ,pipettes or
graduated cylinders , beakers 1% methylene blue solution ,distilled water, test
tube rack ,stirring rod , tissue paper.
Principle- It is based in Beer-Lambert Law, which state that -
“for a given material sample path length and concentration of the sample are
directly proportional to the absorbance of the light.”
The Beer-Lambert law is expressed as:
A = εLc
where,
A is the amount of light absorbed for a particular wavelength by the sample
ε is the molar extinction coefficient
L is the distance covered by the light through the solution
c is the concentration of the absorbing species

4. An overview of beer’s lamberts law

5. Procedure-
1. Prepare 1% methylene blue solution in a glass beaker .
2. Take five test tube and prepare a series from 2-10 ml and make up
it with distilled water ,and prepare a blank and unknown
separately.
3. Take the blank and transfer it to cuvette and set zero using it.
4. Measure the absorbance of the sample solutions from 2-10 ml at
600nm on spectrophotometer.
5. Measure the concentration of the unknown sample by plotting a
graph between concentration and absorbance.
Graph

6. Observation table-
s.n Sample solution Distilled water Conc. Absorbance at 600nm
1 blank 10ml - -
2 2ml 8ml 0.002
3 4ml 6ml 0.004
4 6ml 4ml 0.006
5 8ml 2ml 0.008
6 10ml - 0.0100
7 unknown - x

7. Result- A straight line graph is obt1ained which proves beer’s
lamberts law.
Precautions-
1) Always use clean and dry cuvette.
2) Wipe the cuvette with clean tissue paper.
3)Set zero each time before taking the reading.