Spectrophotometer techniques are mostly used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer.

2. Contents:
Introduction
Principle
Instrumentation
Working
Types of Spectrophotometer
Applications

3. Spectrophotometer:
Scientist Arnold J. Beckman in 1940.
Spectrophotometer consists of two instruments:
- Spectrometer producing light of any wavelength.
- Photometer measuring the intensity of light.
Spectrophotmetric technique
- Absorbance and tramission properties
- concentration of solute in solution
- Intensity of light measured

5. Principle of
Spectrophotometer
 Light passes through the solution
 Detected, measured and used to relate the light
absorbed or transmitted to the concentration of
the substance.
 Used to measure the light intensity :
- Diffracting light into spectrum of wavelength.
- Direct it to sample object
- Receiving light from object
- Charge-coupled device.
- Display graph

6. Principle of
Spectrophotometer

7.  Sample is illuminated,
-Amount of light transmitted and absorbed by
solution
 As concentration increases:
- Absorbtion increases linearly
- Transmission decrease

9. Based on Beer-Lambert Law:
“It states that the amount of light absorbed by a solution is
directly proportional to the concentration of the solution and
the length of a light path through solution.”
Linear relationship:
-Between concentration and absorbance
Beer’s law is written as :
A= ϵlc
Where
A= Amount of absorbance
ϵ = Absorption coefficient (molar extinction coefficient)
l =Path length
c = concentration

10. Instrumentation:
 Light source:
-polychromatic light
- source of different wavelength
Example:
- Tungsten lamp (at 330-900nm)
- Hydrogen and Deutrium lamp
 Monochromater:
- Select the particular wavelength
- focused and non divergent beam

11.  Dispersion devices: - causes a different wavelength of
light to be dispersion at different angles.
Types of Dispersion devices used are :
 Prism is used to isolate different wavelength.
 Filters separate different parts of the electromagnetic
spectrum by absorbing or reflecting certain wavelengths and
transmitting other wavelengths.

13.  Sample holder:
-Test tube or Cuvettes
- round, Square and rectangular
- glass , silica or plastic
- Glass cuvettes
 Detector:
- Photoelectric effect
- convert light into an electrical signal
- Example: Photomultiple tube (PMT)

14. Photomultiplier Detector

15. The PMT consists of:
 A photoemissive cathode(emitts electron when struck
by photon)
 Several dynodes(emitts several electrons from each
electron striking them)
 Anode---produce an electrical signal
 Siganl is amplified and made available for direct
display. Examples:
-phototube(UV) ,
-Photomultiplier tube(UV-Vis)
-Thermocouple( IR)

16. Display or Readout device:
Such as an analog meter, a light beam
reflected on a scale, or a digital display , Or
liquid crystal display(LCD) .The output can
also be transmitted to a computer.

17. WORKING OF THE
SPECTROPHOTOMETER

18. A sample solution is placed inside th
spectrophotometer.
A light source shines light toward the sample.
A monochromator splits the light into each color, or
rather, individual wavelengths. An adjustable slit
allows only one specific wavelength of light
through to the sample solution.
The wavelength of light hits the sample, which is
held in a little container called a cuvette.
Whatever light passes through the sample is read
and displayed on the output screen.
WORKING OF THE
SPECTROPHOTOMETER

19. Graph

20. TYPES OF
SPECTROPHOTOMETER:
 Spectrophotometer is of 2 types
- Single beam spectrophotometer
- Double beam spectrophotometer
 Based of the range of wavelength of the
light source:
- UV-visible spectrophotometer
- IR spectrophotometer

21. Single Beam
Spectrophotometer
 The light travels in one direction and the test
solution and blank are read in the same.
 All the light passes through the sample
 To measure the intensity of the incident light the
sample must be removed so that all the light
can pass through.
 This type of spectrometer is usually less
expensive and less complicated.

22. Double Beam
Spectrophotometer
 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.
 One beam is used for reference and the other for
sample reading. It eliminates the error which
occurs due to fluctuations in the light output and
the sensitivity of the detector.

24. Applications
 The determination of the concentration of colored
as well as colorless compounds
 Determination of the course of the reaction
 Compound can be identified
 For measuring DNA, RNA, and protein
concentrations in small volume samples.
 Determination of enzyme catalyzed reactions
 The visible and UV spectrophotometer may be
used to identify classes of compounds in both the
pure state and in biological preparations.

25.  Detection of concentration of substances
 Detection of impurities
 Structure elucidation of organic compounds
 Monitoring dissolved oxygen content in freshwater and
marine ecosystems
 Detection of functional groups

26. 1) aramedicsworld.com/biochemistry-practicals/demonstration-of-
spectrophotometer-principle-components-working-
applications/medical-paramedical-studynotes
2) https://steemit.com/stemng/@sheglow/spectrophotometer-
working-principle
3) https://microbenotes.com/spectrophotometer-principle-
instrumentation-applications/
4) https://byjus.com/chemistry/spectrophotometer-principle/
5) https://fac.ksu.edu.sa/sites/default/files/spctrophtometer_4.pdf
References