The document explains the function and principles of spectrophotometers, instruments that measure light intensity based on wavelength to determine the concentration of substances in a solution. It covers types of spectrophotometers, including UV-Vis and infrared, their components, and the application of Beer's and Lambert's laws in quantitative analysis. Additionally, it discusses the various detectors and methodologies used in both qualitative and quantitative assessments in spectroscopy.

2. MEANING OF SPECTROPHOTOMETER
INTRODUCTION
PRINCIPLE
TYPES OF INSTRUMENT
TYPES OF SPECTROPHOTOMETER
UV-VIS SPECTOPHOTOMETER
INFRA RED SPECTOPHOTOMETER
APPLICATION
REFERENCE

3. spectrophotometer
spectrum
photometry
colorimetry

4.  INTRODUCTION
 Spectrophotometer :An instrument that measures the
amount of light that passes through (is transmitted
through) a sample.
 It is a photometer (a device for measuring light
intensity) that can measure intensity as a function of
the color (or more specifically the wavelength).
 This means it use light in the visible and adjacent
(near ultraviolet and near infrared) range.

5. Principle:
when a beam of monochromatic light passed through a
solution it may transmitted as a such or some of may be
absorb.
Proportional the transmitted light can be represented by
intensity of the incident radiation.
T=I/I₀
Absorbance(A) of light through a solution in inversely
proportional to log₁₀ of %T
A=Log(₁∕T)
=Log(I/I₀)
Where I=intensity of transmitted light
I₀=intensity of incident light

6. The quantitative determination of compounds by spectrometeric
techniqe is based on two law.
1 Lamberts Law
2 Beers Law
1 Lamberts law- its state that light absorbed by solution is
directly proportional to length of the light through the solution.
hence
A=log(Io/I)= Є.C
where A= absorbance
Є=molar absorptivity coefficient
l= path length of the sample (usually 1cm)

7. 2- Beers law-its state that the amount of light absorbed is
directly proportional to concentration of absorbing solute in the
solution.
Thus
A=log(Io/I)= Є.C
where C=concentration of solution moles liter-1
combining equation
A=log(Io/I)= Є.C.l
it a standard cuvette with light path of 1.cm is used.
A=log(Io/I)= Є.C
Limitation of Beers Lamberts Law:
1. when different forms of the absorbing molecules are in
equilibrium as in keto –enol tautomers.
2. when fluorescent compounds are present.
3. when solute and solvent forms complex though some sort of
association.

8. 1.Single beam-A single beam
spectrophotometer measures the light
intensity of the beam and after a test sample
is inserted.
2.Double beam- A double beam
spectrophotometer compares the light
intensity b/w two path.one path containing a
reference sample & the other is test sample.

10. Ultraviolet (UV)-Vis
Spectrophotometers
Uses ultraviolet light of wave lengths
from 200 nm to 350 nm
 Infra Red (Ir)- covers the range of the
electromagnetic spectrum between
0.78 and 1000 m, m. In the context
of infra red spectroscopy
wavelength is measured in "wave
numbers", which have the units cm-1. The
most useful I.R. region lies between
4000 - 670cm-1.

11. UV-visible spectroscopy involve the
spectroscopy of photons in the UV-visible
region. This means it use light in the visible
and adjacent (near UV and near
Infrared)range.
It uses light over the ultraviolet range (185-
400 nm) and visible range(400-700 nm) of
electromagnetic radiation spectrum.

12. Spectrophotometer have the following
basic components.
1-Source
2.Filter and monochromators
3. Cell / sample holder
4. Detector

13. 1-Source:- A continuous source of radiant energy
covering the region of spectrum in which the
instrument is designed to work.
UV- Hydrogen Deuterium lamp
Visible- Tungsten lamp
2. Filter and monochromators:- A light filter is a
device that allow light of the required wavelength to
pass but absorbs light of the other wavelength wholly
and partially.
Filter-:filter are of two types
1 Absorption filter
2 Interference filter

14. Colour of filter Approximate range of
absorption band in mu
Yellow 450
Orange 500
Red 575
Purple 450 – 650
Blue 480
Green 400 – 475 , 575 - 700

15. Monochromators: The monochromator is to
dispersing the radiation according to the wavelength.
a. dispersing element (a prism or grating)
b. Slits-: there are two types of slit
Entrance slit
Exit slit
3. Cell / sample holder :-
The cell holding the sample
(usually a solution should be transparent to the
wavelength region being recorded.

16. 4. Detector-: In order to detect radiation , three types
of photosensitive device are-
a. Photovoltaic cell
b. Phototube
c. Photomultiplier
Photovoltaic cell Phototube

17. Several electrons
Light beam
Anode

18. Power supply:-
The power supply serves as triple function-
a. It decreases the line voltage to the instrument
operating level with a transformer.
b. It convert A.C to D.C with a rectifier if direct is
required by the instrument .
c. It smooths out any ripple which may occur in the
line voltages in order to deliver a constant voltage
to the source lamp and instrument.

20. IR spectroscopy is one of the analytical technique for
chemical identification .
It is used to identify particular bond functional group
in organic molecules by measuring a substance’s
absorption of infra-red radiation at different
frequency.
The main parts of IR spectrophotometer are:
 IR radiation source
 Monochromators
 Sample cell & Smpling
 Detectors

21.  IR radiation sources
 IR instruments require a source of radiant energy
which emit IR radiation which must be steady,
intense enough for detection and extend over the
desired wavelength. Various sources of IR
radiations are as follows.
a) Nernst glower
b) Incandescent lamp
c) Mercury arc
d) Tungsten lamp
e) Glober source
f) Nichrome wire

22. Filter &Monochromators – Various types of monochromators
are prism, gratings and filters. Prisms are made of Potassium
bromide, Sodium chloride or Cesium iodide. Filters are made up
of Lithium Fluoride and Diffraction gratings are made up of
alkali halides.
sample cells and sampling of substances
It has been used for the characterization of solid, liquid or gas
samples.
Detectors – Detectors are used to measure the intensity of
unabsorbed infrared radiation. Detectors like thermocouples,
Bolometers, thermisters, Golay cell, and pyro-electric detectors
are used.
Recorders – Recorders are used to record the IR spectrum.

24.  Qualitative analysis
 Quantitative analysis
 Study of cis trams isomerism
 Structure elucidation of organic compound
 Chemical kinetics Kinetics of reaction can also
be studied using UV spectroscopy.
 Molecular weight determination•Molecular
weights of compounds can be measured
spectrophotometrically by preparing the suitable
derivatives of these compounds.
 Extent of conjugation

25. o Instrumentation by UPADHAYA & UPADHYAYA NATH
o Instrumentation by CHATWAL & CHATWAL
o Instrumental methods of analysis by B.K.SHARMA
o Microbiology by R.P.SINGH
o Microbiology by DUBEY & MAHESHWARI

26. Thank
you