Spectrophotometry uses the absorption of light by chemical substances to measure concentration. A spectrophotometer directs a beam of light through a sample and measures the intensity of transmitted light, relating it to concentration through Beer's Law. It operates based on Lambert's Law stating light absorption increases with concentration and path length. Common types are single and double beam instruments, with the latter measuring sample and reference simultaneously. Components include a light source, monochromator, sample holder, and detector. Applications include quantifying analytes and studying reaction kinetics and molecular structure.

1. SPECTROPHOTOMETRY
PRESENTED BY:
KHITISH RANJAN NAIK
ROLL NO: 518 -Z-OO5
GUIDED BY:
AUROBINDO DAS

2. OUTLINES
 INTRODUCTION ON SPECTROPHOTOMETER.
 HISTORY.
 BASIC CONCEPT OF SPECTROPHOTOMETER.
PRINCIPLE.
LAMBERT’S LAW.
BEER’S LAW.
BEER –LAMBERT’S LAW.
TYPES OF SPECTROPHOTOMETER.
 INSTRUMENTATION.
 APPLICATIONS.

3. INTRODUCTION ON
SPECTROPHOTOMETER
• These are used to measure the concentration of solute in solution by
measuring the amount of light that is absorbed by the solution.
• Basically, it is used to measure the Optical Density of a given sample.

4. HISTORY
• The spectrophotometer was invented in
1940, by Arnold J. Beckman and his
colleagues at National Technology
Laboratories, the company Beckman
had started in 1935.

5. BASIC CONCEPT OF
SPECTROPHOTOMETER

6. PRINCIPLE
• Spectrophotometry is a scientific method based on the absorption of light by a substance, and
takes advantage of two laws of light absorption.
• Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship
between the absorbance and the concentration of a sample.
• Beer–Lambert law can be applied to the analysis of a mixture by Spectrophotometry, without
the need for extensive pre-processing of the sample.

7. BEER’S LAW
• When a ray of monochromatic light passes through an absorbing medium, it’s
intensity decreases with increase in the concentration of the absorbing
medium.
i.e Intensity of Light ∝ 1/concentration of absorbing medium
Io ∝ 1/c --------------------1

8. LAMBERT’S LAW
• When a ray of monochromatic light passes through an absorbing medium, it’s
intensity decreases with the increase in the length of the light path of the
absorbing medium.
i.e Intensity of light ∝ 1/ Length of light path of Absorbing Medium.
Io ∝ 1/l ----------- 2

9. BEER-LAMBERT’S LAW
• As spectrophotometry is based on the principle of Beer- Lambert’s Law, so by
combining these 2 laws we have the following statement
i.e when a ray of light passes through an absorbing medium, it’s intensity
decreases with the increase in length and concentration of absorbing medium.
Thus, from equation (1) and (2)…
Intensity of light ∝ 1/length and concentration of absorbing medium
Io ∝ 1/L*C-------------------- 3

10. TRANSMITTANCE
• It is defined as the ratio of the intensity of the transmitted
light (I) to the intensity of the incident light (Io).
T = I / Io
% Transmittance = I / Io × 100%
Absorbance is defined as the amount of light absorbed by a
sample.
Absorbance (A) is a logarithmic function of T and is expressed as:
Absorbance = Log10 1 / T = - Log10 (T) = Log10 I / Io
ABSORBANCE

11. • The absorbance is directly proportional to the concentration (c) of the solution
used in the experiment.
A ∝ c
• The absorbance is directly proportional to the length of the light path (l), which is
equal to the width of the cuvette.
A ∝ l
• Combining the two relationships,
A ∝ c l
• This proportionality can be converted into an equation by including a constant.
A = ϵ l c
From the 2 laws we conclude that;

12. TYPES OF
SPECTROPHOTOMETER
SINGLE BEAM SPECTROPHOTOMETER DOUBLE BEAM SPECTROPHOTOMETER

13. 1. SINGLE BEAM SPECTROPHOTOMETER
 It operates between 325- 1000mm wavelength.To measure the intensity of the incident light the
sample must be removed so that the reference can be placed each time as it possess only one photo cell.
 This type of spectrophotometer is usually less expensive and less complicated.
 In this type we get only 1 reading at a time.

14.  It operates between 485 -1000mm wavelength. It has 2 photocells and it splits the
lights into 2 beams. From these one beam passes through the sample and second
one is used for reference.
 This gives an advantage of taking the reference reading and sample reading
simultaneously.
2. DOUBLE BEAM SPECTROPHOTOMETER

15. INSTRUMENTATION
The spectrophotometer, in general, consists of twodevices. They are the
following :
1. SPECTROMETER :
It is used to record and measure the spectra.
2. PHOTOMETER :
It is used to measure the intensity of light.

16. COMPONENTS OF
SPECTROPHOTOMETER
 Light Source
 Collimating System
 Monochromator
 Sample compartment
 Detector
 Display Devices

17. LIGHT SOURCES
• CHARACTERISTICS OF GOOD LIGHT SOURCE:
1. High intensity
2. Wide spectral range
3. Stable output
4. Long life at reasonable cost
UV SPECTROPHOTOMETER
1. Hydrogen or Deuterium Lamp (Wavelength Range :190~420nm)
2.Mercury Lamp
3. Xenon Lamp (Wavelength Range :190~800nm)
VISIBLE SPECTROPHOTOMETER
• 1.Tungsten Lamp (Part of the UV and the whole of the Visible;Wavelength Range
:350~2500nm)

18. DEUTERIUM LAMP
UV SPECTROMETER
VISIBLE LIGHT SPECTROPHOTOMETER
MERCURY LAMP XENON LAMP
TUNGSTEN LAMP

19. COLLIMATING SYSTEM
 The radiation emitted by the source is collimated (made parallel) by lenses,
mirrors and slits.

20. MONOCHROMATOR
Accepts monochromatic input light from a lamp and outputs polychromatic light.
• CHARACTERISTICS OF A DESIRED MONOCHROMATOR
1. minimum absorption of light as it passes through the system.
2. High degree of accuracy in wavelength selection.
3. High spectral purity over a broad spectral range.
 The resolving element are of two kinds namely,
• PRISMS Simple glass prisms are used for visible range. For UV region silica, fused silica
or quartz prism is used.
• DIFFRACTION GRATINGS It is present in most advanced spectrophotometer for
selection of particular wavelength of light. Consist of series of fine , parallel refracting
surfaces which act as prism.

21. MONOCHROMATOR

22. CUVETTES
• These are specially designed to hold samples for spectroscopic experiments.
• Should be as clear as possible, without impurities that might affect the
spectroscopic reading.
FOR UV SPECTROPHOTOMETER
Quartz (crystalline silica)
FOR VISIBLE SPECTROPHOTOMETER
Glass

23. DETECTORS
• It converts radiant energy (photons) into an electrical signal.
• Three common types of detectors are used:
I. Barrier Layer Cell
II. Photo Cell Detector
III. Photomultiplier Tube Detector
Photocell Detector

24. DISPLAY DEVICES
• The data from a detector are displayed by a readout device or display devices, such
as an analog meter, a light beam reflected on a scale, or a digital display or LCD.
• The output can also be transmitted to a computer or printer.
ANALOG METER

25. APPLICATIONS
1. Concentration measurement
 Prepare sample.
 Make series of standard solutions of known concentrations.

26. 2. Detection of Impurities
 UV absorption spectroscopy is one of the best methods for determination of
impurities in organic molecules.
 Additional peaks can be observed due to impurities in the sample and it can
be compared with that of standard raw material

27. 3. Structure elucidation of organic compounds.
From the location of peaks and combination of peaks UV
spectroscopy elucidate structure of organic molecules:
 the presence or absence of unsaturation.
 the presence of aromatic ring.

28. 4. Chemical kinetics
 Kinetics of reaction can also be studied using UV spectroscopy. The UV
radiation is passed through the reaction cell and the absorbance changes
can be observed.

29. OTHER APPLICATIONS OF
SPECTROPHOTOMETER
• FORENSIC SCIENCES
• MOLECULAR BIOLOGY : IN MEASURING THE GROWTH OF MICRO-
ORGANISM.
• PATHOLOGICAL STUDIES : IN DIAGNOSING VARIOUS DISEASES BY
ANALYSING THE SAMPLES.