about instrument (spectrophotometer)

1. SPECTROPHOTOMETER
Present by :
JINAL mehta
T . Y. bsc

2. DEFINTION :
A Spectrophotometer is an
instrument that measures the amount of light
adsorbed by a sample.
OR
The Spectrophotometer technique is
to measures light intensity as a function of
wavelength it.

3. HISTORY
 The Spectrophotometer invented by ARNOLD O.
BECKMAN in 1940 , It was created with the aid of his
colleagues at his company NATIONAL TECHNICAL
LEBORATORIES which would become BACKMAN
COULTER.

4. Beer’s Law
 TRANSMITTANCE:
The relative amount of light passing
through the sample is known as the
TRANSMITTANCE (T) .
𝑇 =
𝐼
𝐼0
Percent transmittance :
0
0 𝑇 = 100 × 𝐼
𝐼0

5.  ADSORBANCE :
It is relative amount of light
absorbed by the sample & it related to
Transmittance.
• It cannot be measured directly by a
spectrophotometer but rather is mathematically
derived from %T .
• ABSORBANCE is sometimes called OPTICAL
DENSITY (OD) .

6.  Absorbance is useful since it is directly related
to the analyte concentration , cell leanth &
molar absorptivity
 THESE LOW IS KNOWN AS BEER’S LOW :
𝐴 = 𝜀𝑙𝑐

7. COMPONENTS OF
SPECTROPHOTOMETER
1) SOURCE
2) WAVELENGTH SELECTOR DEVICE
3) SAMPLE CONTAINAR
4) DETECTOR
5) READ OUT

8. BLOCK DIAGRAM
SOURCE
WAVELENGTH
SELECTOR
DEVICE
SAMPLE
DETECTOR
READOU
T

10. 1. SOURCES
 Characteristics of an ideal source :
• Gives constant intensity
• Have low noise
• Prolonged stability
 Types of UV-Visible source :
A. Deuterium ARC Lamp
B. Tungsten Lamp
C. Xenon Arc Lamp

11. A. Deuterium ARC Lamps :
It is a low pressure gas discharge light source.
 Works on the principle of molecular emission.
 Wavelength range 190nm – 420nm .
 Life time approx 2000hrs .

12.  ADVANTAGES :
i. Yield good intensity continuum
ii. Provides intensity in UV region & useful
intensity in the visible region
iii. Produce low noise
 DISADVANTAGES :
i. Don’t have prolonged stability

13. B. Tungsten Halogen Lamps :
Works on the principle of black body
radiation.
 It is a spatially & temporarily incoherent source.
 Wavelength range 350nm – 300nm.
 Life time approx 10000hrs.

14. ADVANTAGES :
i. Yields good intensity continuum.
ii. Provides intensity over a part of the UV
region & over the entire part of the
visible region.
iii. Produce low noise.
 DISADVANTAGES :
i. Have poor assembly which can cause
mechanical damage through impact or
vibration.
ii. Evaporation & condensation of tungsten
filament causes blackening of glass surface.

15. C. XENON ARC LAMPS :
i. Generates light by passing current through a
gas (Xe) under very high pressure.
ii. Wavelength range 200nm – 1000nm.
iii. Life time 500hrs – 4000hrs.

16.  ADVANTAGES :
i. Yields good intensity continuum.
ii. Provides intensity over the entire UV &
visible region.
iii. Ruggedness good or very good.
 DISADVANTAGES :
i. Audible noise – higher.
ii. Requirement of high voltage for initiation.
iii. Generate significant heat & need external
cooling.

17. CONCLUSION
 Tungsten lamp is a better source than deuterium
& xenon arc lamp because :
i. Produce less noise
ii. It have less cost
iii. Greater lifetime

18. MONOCHROMATOR
2. WAVELENGHT SELECTOR DEVICE

19. A. MONOCHROMATOR
 FUNCTION :
To produce a beam of monochromatic
(single wavelength) radiation that can be
selected from a wide range of wavelengths.
 COMPONENTS :
I. Entrance slit.
II. Collimating device (to produce parallel
light)
III. Dispersing devices ( grating , prism ,
filter)
IV. A focusing lens or mirror
V. An exit slit

20.  By rotating prism or
grating we can get the
light of desired
wavelength.

21. COMPARISON BETWEEN PRISM & GRATING
 Dispersion given by PRISM is
angularly non linear &
temperature sensitive .
 Dispersion given by GRATING
is angularly linear &
temperature insensitive.
 Dispersion is high in grating as
compared to prism.

22. FILTERS
 There are 4 types of filter :
i. ABSORPTION FILTERS
ii. CUT – OFF FILTERS
iii. INTERFERENCE FILTER
iv. INTERFERENCE WEDGES

23. i. ABSORPTION FILTERS
 They are made up of colored GLASS or
DYE.
 Transmit only a specific band of
wavelength.
 They are inexpensive.
 Transmission is only about 10 – 20 % .

24. ii. CUT - OFF FILTERS
 Transmission is about
100%.
 Used to achieve only a
specific band of
wavelength.
 They are used in
combination with
absorption filters to
decrease the bandwidth of

25. iii. INTERFERENCE FILTERS
 Works on the concept of wave interference.
 The thickness of dielectric &
reflectivity of the metallic film
are carefully selected because
these factors control the
transmitted wavelength.
 The transmitted radiation
will have very narrow
bandwidth.

26. iv. INTERFERENCE WEDGES
 A wedges of dielectric of different thickness can
transmit a wide range wavelengths.
 By choosing the correct position on the wedges ,
variable bandwidths of about 20nm can isolated.

27. • Combinations of LENSES , SLITS & MIRRIORS.
• Variable slits also permit adjustments in the
total radiant energy reaching the detector.
• The Ebert & Czerny-Turner monochromators &
there variations are combinations of prisms or
grating & focusing devices.
FOCUSING DEVIECE

28. OPTICAL MATERIALS :
TYPES OF RAYS
1. X-rays-
Ultraviolet(UV)
2. Visible
3. Near infrared
4. Infrared (IR)
MIRROR / LENSE
MAERIAL
Aluminum / Fused
silica
Aluminum / Glass
Gold
Copper or Gold
/Glass

29. 3. SAMPLE CONTAINER
 Sample container are generally made up of
PLASTIC ; GLASS ; QUAETS .
 PLASTIC CUVETTE :
Used for fast spectroscopic
assays , where speed is more important than
high accuracy.
 GLASS CUVETTE :
Used in the wavelength range
of visible light , Transmission range 340nm –
2000nm.

30. UV quarts cuvettes
• Most common material.
• Transmission range 190nm –
2500nm
IR quarts cuvettes
• Rarely used
• Transmission range 190nm –
3500nm
Smallest capacity holding : 70 microliter
Largest capacity holding : 2.5ml OR Larger

31. 4. DETECTORS
 A detector converts a light signal into an
electrical signal.
 Ideally , it should give a linear response
over a wide range with low noise & high
sensitivity.
 Spectrophotometer normally contain either
a photomultiplier tube detector or a
photodiode detector.

32.  Phototube :
Incident photon causes release of an
electron.
• Photocurrent Alpha Plight.
• Not best for low-light scenarios.

33.  Photodiodes & Photodiode arrays :
 Blue enhanced for spectral
range from 350nm –
1100nm
 Designed for low noise.
 Medium bandwidth
application.
 Application include
i. Low light level
measurement
ii. Particle counting
iii. Chemical & analytical

34. 5. DISPLAY DEVICES
 The data from a detector are displayed by a
readout device such as an ANALOG METER ; A
LIGHT BEAM REFLECTED ON A SCALE ; A
DIGITAL DISPLAY ; LIQUID CRYSTAL
DISPLAY .
 The output can also be transmitted to a computer
or printer .

35. TYPES OF
SPECTROPHOTOMETER
 There are three types :
1. SINGLE BEAM
SPECTROPHOTOMETER
2. DOUBLE BEAM
SPECTROPHOTOMETER
3. SPLIT BEAM
SPECTROPHOTOMETER

36. 1. SINGLE BEAM SPECTROPHOTOMETER
Sample & blank are alliteratively
measured in same sample chamber

37. SINGLE BEAM
SPECTROPHOTOMETER
ADVANTAGES
 They are less expensive
 High energy throughput
due to non-splitting of
source beam results in high
sensitivity of detection
DISADVANTAGES
 Instability due to lack of
compensation for
disturbances like electronic
circuit fluctuations , voltage
fluctuations , mechanical
component’s instability or
drift in energy of light
sources. Such drifts result
in abnormal fluctuations in
the results.

38. 2. DOUBLE BEAM SPECTROPHOTOMETER
Continuously compares sample &
blank automatically corrects for
changes in electronic signal or light
intensity of source .

39. ADVANTAGES
• Modern improvements in optics
permit high level of automation
& offer the same or even better
level of detection as compared to
earlier single beam systems.
• Instability factors due to lamp
drift , stray light , voltage
fluctuations do not affect the
measurement in real-time.
• Little or no lamp warm up time
is required , this not only
improves throughput of results
but also conserves lamp life .
DISADVANTAGES
• The cost factor is more than
offset by the advantages
offered by modern double
beam systems

40. 3. SPLIT BEAM SPECTROPHOTOMETER
 Similar to the double beam spectrophotometer
but it is uses a beam splitter instead of a chopper
to send light along the blank.
 Sample paths simultaneously to two separate but
identical detectors.

41. USES OF SPECTROPHOTOMETER
1. Measure the concentration of the solution :
A spectrophotometer optically
determines the absorbance or transmission of
characteristic wavelengths of radiant energy
by a chemical species in solution. Each
molecule absorbs light at certain wavelengths
in a unique spectral pattern because of the
number & arrangement of this characteristic
functional groups , such as double bonds
between carbon atoms.

42. 2. Identify organic compounds by determining the
absorption maximum
3. Used for color determination within the
Spectral range

43. APPLICATION
1. Detection of impurities :
UV absorption spectrophotometer is one
of the best methods for determination of impurities
in organic molecules.
2. Structure elucidation of organic
compounds :
UV spectrophotometer is useful in the
structure elucidation of organic molecules , the
presence or absence of unsaturation , the presence of
hetero atoms .

44. 3. Quantitative analysis :
UV absorption spectrophotometer
can be used for the quantitative determination of
compounds that absorb UV radiation .
4. Qualitative analysis :
UV absorption
spectrophotometer can characterize those types of
compounds which absorbs UV radiation.
Identification is done by comparing the absorption
spectrum of known compounds .