The document explains spectrophotometry, a method for quantitatively measuring the absorption of light by compounds, directly related to their concentration. It describes the working and components of spectrophotometers, which can be single or double beam, and categorized by the light wavelength they utilize (visible, UV, and IR). Additionally, it covers the Beer-Lambert Law, standardization processes, and various applications of spectrophotometry.

1. PRESENTED BY :
Sushma P. R
2nd M. Sc. Biotech
Brindavan college
PRESENTED TO :
Mrs. Divya
Dept. of Chemistry
Brindavan College

2. INTRODUCTION
 Compounds absorbs light radiation of a specific
wavelength.
 The light absorbed by the sample is directly proportional
to the concentration of sample in the solution.
 As concentration increases , absorption increases
exponentially.

3. SPECTRPHOTOMETRY
 A method in which the absorption or transmission
properties of a material is quantitatively measured as
a function of wavelength.
 The basic principle behind this method is that :
“Each compound absorbs or transmits light over a
certain range of wavelength”

4.  A spectrophotometer is an instrument that measures
the amount of photons absorbed by a sample after it
is passed through its solution.
 With the spectrophotometer , the amount of a
known chemical substance can be determined by
measuring the absorbance.

5. Spectrophotometer can be classified into two different
types :
 SINGLE BEAM SPECTRMETER :
To measure the intensity of the incident light the
sample must be removed so that the reference can be
placed each time. This type of spectrometer is usually
less expensive and less complicated.

6.  DOUBLE BEAM SPECTOMETER:
In this type, before it reaches the sample, the light
source is split into two separate beams. From these one
passes through the sample and second one is used for
reference. This gives an advantage because the
reference reading and sample reading can take place at
the same time.

7. Based on the wavelength of light used it can be classified into :
 VISIBLE SPECTROMETER
Uses visible range (400 – 700nm) of electromagnetic radiation
spectrum.
Visible spectrophotometers vary in accuracy. Plastic and
glass cuvettes can be used for visible light spectroscopy.
 UV SPECTOMETER
Uses light over the UV range (180 - 400 nm).
UV spectroscopy is used for fluids, and even solids. Cuvettes, only
made of quartz, are used for placing the samples.
 IR SPECTROPHOTOMETER
Uses light over infra red range (700 -15000) of electromagnetic
radiation spectra.

8. DEVICE AND MECHANISM
 The spectrophotometer, in general, consists of two
devices. They are the following :
1. SPECTROMETER :
A device that produce, typically disperse and measure
the light.
2. PHOTOMETER :
Indicates the photoelectric dictator that measures the
light.

9.  The spectrometer consists of the following parts :
1. Light source : it produce a desired range of
wavelength of light.
2. Collimator : transmits a straight beam of light.
3. Monochromator : split the light into its
component wavelength.
4. Wavelength selector : transmits only the desired
wavelength.

10.  The photometer detects the light absorbed by the
sample as the light from the slit is passed through
the solution. And then it sends signal to the
galvanometer or digital display.

12. BEER – LAMBERT LAW
It states that the absorbance of light by a material in a
solution is directly proportional to its concentration in that
solution.
A = ϵlc
Where,
A – absorbance
ϵ - molar absorptivity
l – length of solution
c – concentration

13.  STANDARDIZATION GRAPH
Standards (solutions of known concentration) of the
compound of interest are made, treated, and their
absorbances (ABS) and concentration values are used to
create a Standardization Graph.

14.  Absorption spectra :
A spectrum of electromagnetic radiation
transmitted through a substance, showing dark lines
or bands due to absorption at specific wavelengths.

16. APPLICATIONS
1. Concentration measurement
2. Detection of impurities
3. Chemical kinetics
4. Detection of functional group
5. Molecular weight determination

17. REFERENCES
 Fundamentals of UV –Visible spectrophotometry ,
Tony Owen, 1996
 UNIT : spectrophotometry : Clinical chemistry lab
manual
 www.wikipedia.com
 www.colorado.edu
 www.chemwiki.com
 www.chemguide.co.uk
 www.slideshare.com