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What is spectrophotometer ?
- A device used for measuring transmittance and
absorbance of a sample as a function of wavelength of
Components of spectrophotometer→
- Wavelength selector Device
- Sample Container
- Read out
Characterstics of an ideal source-
- Gives constant intensity
- Have low noise
- Prolonged stability
Types of UV-Visible sources-
- Deuterium Arc Lamp
- Tungsten lamp
- Xenon Arc Lamp
DEUTERIUM ARC LAMP
- It is a low pressure gas discharge light source.
- Works on the principle of molecular emission.
- Wavelength range→ (190-420)nm
- Life time approx 2000hrs.
-Yields good intensity continuum
-Provides intensity in UV region and useful intensity in the visible
-Produce low noise
-Don’t have prolonged stability
-Works on the principle of black body radiation.
-It is a spatially and temporarily incoherent source
-Wavelength range – (350 – 3000) nm
-Lifetime approx 10000 hrs
-Yields good intensity continuum.
-Provides intensity over a part of the UV region and over the entire
part of the visible region.
-Produce low noise.
-Have poor assembly which can cause mechanical damage
through impact or vibration.
-Evaporation and condensation of tungsten filament causes
blackening of glass surface.
-Generates light by passing current through a gas (Xe) under
very high pressure.
-Wavelength range – (200 – 1000) nm.
-Lifetime – (500 – 4000) hrs.
XENON ARC LAMP
-Yields good intensity continuum.
-Provides intensity over the entire UV and visible region.
-Ruggedness – good to very good.
-Audible noise – higher.
-Requirement of high voltage
-Generate significant heat and
need external cooling.
Tungsten lamp is a better source than deuterium and xenon arc
lamp because –
-Produce less noise
-It have less cost
• Function – to produce a beam of monochromatic (single
wavelength) radiation that can be selected from a wide range
• Components –
- Entrance slit .
- Collimating device (to produce parallel light ).
- Dispersing device (grating , prism).
- A focusing lens or mirror .
- An exit slit .
- By rotating prism or
grating we can get
the light of desired
Comparison between prism
and grating –
- Dispersion given by prism is
angularly non linear and temprature
sensitive while the dispersion given
by grating is angularly linear and
temprature insensitive .
- Dispersion is high in grating as
compaired to prism .
Filters are of four types :-
a) Absorption filters
b) Cut-off filters
c) Interference filters
d) Interference Wedges
Absorption Filters –
• They are made up of coloured glass
or dye .
• Transmit only a specific band
• They are inexpensive.
• Transmission is only about (10-20)%.
• Transmission is about 100%.
• Used to achieve only a specific band
• They are used in combination with
absorption filters to decrease the
bandwidth of the absorption filters.
Interference filters –
• Works on the concept of wave interference.
• The thickness of the dielectric and reflectivity of the metallic
films are carefully selected because these factors control the
• The transmitted radiation will have very narrow bandwidth.
• A wedge of dielectric of different thickness can transmit a wide
range of wavelengths.
• By choosing the correct position on the wedges , variable
bandwidths of about 20 nm can isolated.
Comparison between interference filters and
• The interference filters gives superior bandwidth over absorption
• Greater the bandwidth the lower the transmittance % through that
- Sample containers are generally made up of
plastic, quarts and glass.
• Plastic cuvette- used for fast spectroscopic
assays, where speed is more important than
• Glass cuvette- used in the wavelength range
of visible light.
- transmission range 340-2000nm
• Quartz cuvettes- they are of two types-
→ UV quartz- most common material.
- transmission range 190-2500 nm
→ IR quartz- rarely used
- transmission range 190-3500 nm
- Smallest capacity holding- 70 microliter
- Largest capacity holding- 2.5ml or larger
→A detector converts a light signal into an electrical signal. Ideally, it
should give a linear response over a wide range with low noise and
high sensitivity. Spectrophotometers normally contain either a
photomultiplier tube detector or a photodiode detector.
- Incident photon causes release of
– Photocurrent α Plight
– Not best for low-light scenarios
– Ejected photoelectron strikes dynode, secondary e- released
– Voltage accelerates e- to next dynode and so on
• big voltage divider
– Result is large charge packet hitting anode
Photodiodes and Photodiode arrays :–
Reverse biased junction
– Photons produce e-- hole pairs α current
– Current α Plight
• less sensitive
• Assembly of individual photodiodes on a Si chip
• Each diode can be addressed individually
Single beam spectrophotometer
• Low in cost.
• High energy throughput due to non splitting of source beam.
• Have high sensitivity.
• Instability due to lack of compensation for disturbance like
electronic circuit fluctuation, voltage fluctuation, mechanical
components instability or drift in energy of light sources.
Double beam spectrophotometer
• Dual-beam instruments contain more optical components which
reduces throughput and sensitivity.
• The more complex mechanical design of the dual-beam
spectrophotometer may result in poorer reliability.
• Instability factors due to lamp drift, stray light, voltage
fluctuations do not affect the measurement.
1. Detection of Impurities- UV absorption spectroscopy is one of the
best methods for determination of impurities in organic molecules.
2. Structure elucidation of organic compounds- UV spectroscopy is
useful in the structure elucidation of organic molecules, the presence
or absence of unsaturation, the presence of hetero atoms.
3. Quantitative analysis- UV absorption spectroscopy can be used for
the quantitative determination of compounds that absorb UV
4. Qualitative analysis- UV absorption spectroscopy can characterize
those types of compounds which absorbs UV radiation. Identification
is done by comparing the absorption spectrum with the spectra of
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. By also measuring the
absorbance at specific wavelength, the impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its
presence can be easily detected by its absorption at 255
I wish to express my sincere gratitude to Mrs. Jyoti Panday for
providing me an opportunity to do my presentation work on “UV
I sincerely thank Mrs. Jyoti Panday for their guidance and
encouragement in carrying out this presentation work. I also wish to
express my gratitude to my class friends of BBAU, who rendered
their help during the period of my presentation work.