The document summarizes the principles and components of a UV-Visible spectrophotometer. It discusses how Isaac Newton and Joseph von Fraunhofer made early contributions to spectroscopy. The key components of a UV-Vis spectrophotometer include a light source, monochromator, sample holder, detector, and signal processor. The monochromator separates light into wavelengths and the detector measures light intensity. UV-Vis spectroscopy provides information on functional groups and chemical structure by measuring absorption of light across wavelengths.

1. UV-VISIBLE SPECTROPHOTOMETER
LEKSHMI M.R
MPhil. Scholar
Department Of Chemistry, University of Kerala

2. Isaac Newton Joseph Von Fraunhofer
Spectroscopy began with Isaac Newton's optics
experiments (1666–1672). Newton applied the word
"spectrum" to describethe rainbow of colors
During the early 1800s, Joseph von Fraunhofer made
experimental advances with dispersive spectrometers
that enabled spectroscopy to become a more precise and
quantitative scientific technique.
I N T R O D U C T I O N
The history of Spectroscopy began in the 17th century.

3. I N T R O D U C T I O N
 When continuous radiation passes through a transparent
material, a portion of the radiation may be absorbed.
 If that occurs, the residual radiation, when it is passed through a
prism, yields a spectrum with gaps in it, called an
Absorption spectrum.
 As a result of energy absorption, atoms or molecules pass from
a state of low energy (Ground State) to a state of higher
energy(Excited State).
 In the case of Ultraviolet and Visible spectroscopy, the
transitions that result in the absorption of electromagnetic
radiation in this region of the spectrum are transitions between
electronic energy levels.

4. UV-Visible Spectroscopy
Used for analyzing liquids,
gases and solids through the use
of radiant energy in the far and
near ultraviolet, visible regions
of the electromagnetic spectrum.
Operates by passing a beam of light
through a sample and measuring
wavelength of light reaching a detector.
The wavelength gives valuable
information about the chemical structure
and the intensity is related to the number
of molecules, means quantity or
concentration
Analytical information can be
revealed in terms of transmittance,
absorbance or reflectance of energy
in the wavelength range .
W H Y ?H O W ?W H A T ?
O V E R V I E W

5. P R I N C I P L E
B e e r- L a m b e r t ’s L a w
The following relationship is established when light with
intensity Io is directed at a material and light with intensity I
is transmitted.
A =
UV-Visible Spectroscopy

6. O V E R V I E W
P o s s i b l e E l e c t ro n i c T r a n s i t i o n s a re
Energy




n



n
n





In alkanes
In carbonyl compounds
In alkenes, alkynes, azo compounds, and other
unsaturated compounds.
In O, N, S, halogens
In carbonyls

7. O V E R V I E W
 UV spectra of organic compounds are generally collected from 200-700 nm.
 Hence electronic transitions require either a lone pair or a π-bond from which
the electron can be promoted.
 That is, UV Spectra are generally only of interest if the
system is unsaturated and conjugated.
Transition Chromophore λmax
Alkanes 150 nm
Carbonyls 170 nm
Unsaturated Compounds 180 nm
O, N, S, Halogens 190 nm
Carbonyls 300 nm
 
 
n 
n
√ - if conjugated!
√ - if conjugated!
 

8. O V E R V I E W
What is the
information you
get from UV
Spectroscopy???
The Wavelength of the
absorbed light will provide
the information on the
energy gap, which is related
to functional group.

9.  Spectrophotometer is a kind of spectrometer, which measures the
transmittance or absorbance of a sample as a function of wavelength.
 When light of certain intensity and frequency range is passed through the sample
unlike a spectrometer which is any instrument that can measure the properties of
light over a range of wavelengths, a spectrophotometer measures only the
intensity of light as a function of its wavelength.
 Invented by Arnold O. Beckman in 1940, the spectrophotometer was
created with the aid of his colleagues at his company National Technical
Laboratories.
 This would come as a solution to the previously created spectrophotometers
which were unable to absorb the ultraviolet correctly.
Arnold O. Beckman
H I S T O R Y

10. EXPERIMENTAL SETUP
 Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
The typical ultraviolet-visible spectrophotometer consists of a light
source, a monochromator, a sample cell and a detector.
The source of radiation for the
UV region is a deuterium lamp,
which emits in 160-375nm range.
10 10
U LT R AV I O L E T S O U R C E S V I S I B L E S O U R C E S
The source of radiation for the
visible region is usually a tungsten-
halogen lamp which emits in 350-
2500nm.
Deuterium Lamp Tungsten-halogen Lamp

11. The main function of the monochromator is to disperse the beam of light
obtained from the primary source into its components..
 Entrance slit
 Collimators (Mirrors
and Lenses)
 Dispersing element
 Focusing Mirror
 Exit slit
11 11
M o n o c h ro m a t o r s
I t c o n s i s t s o f f i v e b a s i c c o m p o n e n t s .
 Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
EXPERIMENTAL SETUP

12. The radiation emitted from the primary source,which is
polychromatic is collimated (made parallel) by lenses, mirrors and
slits.
 Focuses the light passing
through the entrance slit in
parallel rays onto the
dispersing element.
 The focusing mirror collects
the dispersed light and
reforms the image of the
entrance slit on the exit slit.
12 12
C o l l i m a t i n g s y s t e m
C O L L I M AT O R SSLITS
 Narrow openings between two
metal jaws(slit jaws) through
which radiation passes.
 The entrance slit actually serves
as a radiation source as its
image is focused on the exit slit
placed on the focal plane of
monochromator.
 Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
EXPERIMENTAL SETUP

13.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
It is a device used to isolate the radiation of the desired wavelength
from the wavelengths of a continuous spectra.
 Prisms
 Gratings
13
D i s p e r s i o n e l e m e n t EXPERIMENTAL SETUP

14.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
 The cells or cuvettes are used for handling liquid samples and it may
either be rectangular or cylindrical in nature.
 For study in UV region; the cells are prepared from quartz or fused
silica whereas color corrected fused glass is used for visible region.
 The surfaces of absorption cells must be kept
scrupulously clean. No fingerprints or blotches should be present on
cells.
 Cleaning is carried out bywashing with distilled water or with dilute
alcohol, acetone.
EXPERIMENTAL SETUP

15.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
Detectors
 Radiation detectors are essentially transducers which convert radiant
energy into electrical energy.
 An ideal transducer should have high sensitivity, high signal-to-
noise ratio, and a constant response to radiation over a
wide range of wavelengths.
 Radiation detectors used in the UV-Visible region are generally
known as photon transducers, as they generate an electrical signal in
the form of photocurrent by absorbing the photons.
EXPERIMENTAL SETUP

16.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
Photon Transducers
They include a variety of devices such as photovoltaic cells,
phototubes, photomultiplier tubes, silicon photodiode and
photodiode array detectors, and charge transfer transducers.
EXPERIMENTAL SETUP
Photomultiplier tube (PMT) is similar to the phototube but
has the advantage of very high sensitivity due to built-in amplification of
current, and hence, useful for measuring even radiation of low intensity.
 This consists of an evacuated glass tube into which are sealed the
cathode and anode and an additional intervening electrodes known as
dynodes. As the radiation strikes the cathode electrons are liberated
and the applied potential difference accelerates the electrons towards
the first dynode. Each successive dynode is at higher electrical
potential acts as amplifier.

17.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
EXPERIMENTAL SETUP

18.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
Silicon Photodiode consists of a p-n
junction made of a strip of p-type silicon in contact
with an n-type silicon chip and they are
semiconductors that charge their charged voltage
upon being striked by the radiation, the voltage is
converted to current and it is measured.
Photodiode array detector consists of
an array or a large number (1000 or more) silicon
diodes formed on a single silicon chip and
connected through an integrated circuit.
EXPERIMENTAL SETUP

19. EXPERIMENTAL SETUP
 Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.

20.  Light Sources
 Monochromator
 Sample holder
 Detector
 Signal processor and
readout.
Signal processors and display units
 They are electronic devices which generally amplify the electric
signal generated by the transducer.
 They may also alter the signal from DC to AC or reverse, filter
unwanted components and perform mathematical operations.
 They are coupled to display or readout devices, for example,
digital meters, Oscilloscopes, recorders, etc
EXPERIMENTAL SETUP

21. T Y P E S
Single-Beam: There is only one light
beam or optical path from the source
through to the detector. It measures the
absorbance of the reference first, followed
by the sample.
Double-Beam: The light from the source, after
passing through the monochromator, is split
into two separate beams-one for the sample
and the other for the reference, and compares
the light intensity between two light paths.
Spectrophotometers

22. I N S T R U M E N T AT I O N

23. I N S T R U M E N T AT I O N
Double Beam Spectrometer

24. I N S T R U M E N T AT I O N
Double Beam Spectrometer

25. I N S T R U M E N T AT I O NSPECIFICATIONS
Parameter LAMBDA 365
Monochromator
Blazed holographic
grating with 1200
gr/mm
Czerny-Turner with 0.2
m focal length
Detector
Si diodes, Sample and
Reference
Photometric System Double Beam Optics
Wavelength Range 190-1100 nm
Spectral Bandwidth 0.5, 1, 2, 5, 20 nm

26. Diffuse Reflectance Spectroscopy(DRS) O V E R V I E W
 Reflectance spectroscopy is very closely related to UV/Vis
spectroscopy, in that both of these techniques use visible light
to excite valence electrons to empty orbitals.
 The difference in these techniques is that in UV/Vis
spectroscopy one measures the relative change of
transmittance of light as it passes through a solution, whereas
in diffuse reflectance, one measures the relative change in the
amount of reflected light off from a surface.

27. D I F F E R E N C E S UV-Vis spectroscopy refers the absorption or
transmission spectra. In general, DRS refers
to diffuse reflection spectra (spectroscopy).
 By UV-Vis, we refer just the UV and Visible
spectral range. DRS can also be measured in
this range.
 Generally in UV-Vis spectroscopy we record
absorption or transmission spectrum in UV-
Vis range.
 Used to characterise samples in thin film
form or in liquid form, where there is not
much dispersion.
 In the case of granular/powder or thin films
of high surface roughness, the reflection is
not specular, and hence we can not measure
the transmitted intensity (it is too low) to
get the absorption of the sample.
 Therefore, for powders or thin films of high
surface roughness, we use Diffuse
Reflectance Spectroscopy,

28. M E A S U R E M E N T

29. A P P L I C AT I O N S
 Qualitative & QuantitativeAnalysis:
 It is used for characterizing aromatic compounds and conjugated olefins.
 It can be used to find out molar concentration of the solute under study.
 Detection of impurities:
 It is one of the important method to detect impurities in organic
solvents.
Forensic Toxicology.
Detectors in Chromatography.
Elucidation of structure of molecules in combination with IR and NMR
data.
Fat Quality determination.
Determination of metal contaminants.

30. 1 . L a m p m a n , G a r y M ; P a v i a , D o n a l d L ; K r i z , G e o r g e S ; a n d Vy v y a n ,
J a m e s R . ( 2 0 1 0 ) . S p e c t r o s c o p y ( 4 t h e d . ) . B r o o k s / C o l e , C e n g a g e
L e a r n i n g . p p . 3 7 9 - 4 1 2 .
2 . W i l l a r d , H o b a r t H ; M e r r i t t , L y n n e L ; a n d D e a n , J o h n A . ( 1 9 6 5 ) .
I n s t r u m e n t a l M e t h o d s o f A n a l y s i s ( 4 t h e d . ) . D . Va n N o s t a r d
C o m p a n y , I N C . p p . 3 3 - 7 3 .
3 . W i l l i a m K e m p . ( 1 9 8 7 ) . O r g a n i c S p e c t r o s c o p y ( 2 n d e d . ) . E n g l i s h
L a n g u a g e B o o k S o c i e t y / M a c m i l l a n . p p . 1 8 8 - 2 0 6 .
4 . S k o o g , D o u g l a s A ; F. J a m e s ; C r o u c h , S t a n l e y
R . ( 2 0 0 7 ) . P r i n c i p l e s o f I n s t r u m e n t a l A n a l y s i s ( 6 t h e d . ) . B e l m o n t ,
C A : T h o m s o n B r o o k s / C o l e . p p . 1 6 9 - 1 7 3 .
5 . h t t p s : / / w w w . s s i . s h i m a d z u . c o m / p r o d u c t s / u v - v i s -
s p e c t r o p h o t o m e t e r s / d i f f u s e - r e f l e c t a n c e - m e a s u r e m e n t . h t m l .
6 . R . J . A n d e r s o n ; D . J . B e n d e l l a n d P. W.
G r o u n d w a t e r . ( 2 0 0 4 ) . O r g a n i c S p e c t r o s c o p i c A n a l y s i s . R o y a l
S o c i e t y o f C h e m i s t r y . p p . 7 - 1 9 .
7 . B . S i v a s a n k a r . ( 2 0 1 2 ) . I n s t r u m e n t a l M e t h o d s o f A n a l y s i s . O x f o r d
U n i v e r s i t y P r e s s . p p . 1 9 3 - 2 0 5 .
8 . h t t p s : / / e n . w i k i p e d i a . o r g / w i k i / S p e c t r o p h o t o m e t r y .
R E F E R E N C E S

31. t h a n k y o u