This document outlines a procedure to verify Beer's law using a spectrophotometer. Key steps include preparing a series of methylene blue solutions with varying concentrations, measuring their absorbance at 600nm, and plotting concentration versus absorbance. This should yield a straight line graph demonstrating that absorbance is directly proportional to concentration, as Beer's law states. Precautions are noted such as using clean cuvettes and setting the spectrophotometer to zero before each reading. The overall aim is to experimentally validate the quantitative relationship between light absorption and analyte properties.

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.