Uv vis spectroscopy

1. Dr. Priyanka S. Chaudhari

2. Instrumentation
The essential components of spectrophotometer
are:
1. Suitable source of electromagnetic radiation
2. Lenses, mirror, slits which collimate &focus
the beam on the sample
3. Filter or monochromator
4. Sample containers
5. A detector
6. Recorder or display

4. Detector:
 The light or the intensity of transmitted radiation by a
sample is collected on a detector device.
 Amount of transmitted radiation is measured.
 Most modern detectors generate an electrical current
after receiving the radiation. The generated current is
often amplified & passed on to a meter, a galvanometer
or a recorder.
 The detectors are of many types. They give different
signals.

5. Some important requirements are:
 It should give quantitative response
 High sensitivity & low noise level.
 Short response time
 It should provide signal or response quantitative in
wide spectrum of radiation received

8. 1. Barrier layer cells/Photovoltaic cell:
This detector is most simple & sturdy in
nature.
It is simple to construct & easy to operate.
It consist of a metal base plate like iron or
aluminium which acts as one electrode.
On its surface a thin layer of a
semiconductor metal like selenium is
deposited.
Then the surface of selenium is covered by a
very thin layer of silver or gold which acts as a
second collector electrode.

9.  When the radiation is incident upon the surface of
selenium, electrons are generated at the selenium-
silver interface.
 These electrons are collected by the silver. The
accumulation of electrons on the silver surfaces creates
an electric voltage difference between the silver surface
& the basis of the cell.
 If the external circuit has a low resistance, a
photocurrent will flow.
 If this cell is connected to a galvanometer, a current
will flow which will vary with the intensity of the
incident light.

11. Detector
Barrier Layer/Photovoltaic

12. Principle of Barrier
Layer/Photovoltaic Detector
 This device measures the intensity of photons
by means of the voltage developed across the
semiconductor layer.
 Electrons, ejected by photons from the
semiconductor, are collected by the silver layer.
 The potential depends on the number of
photons hitting the detector.

13. Advantage:
 Rugged & do not require external power
supply.
 Low cost
 These are sensitive over the whole visible
region.
 But less sensitive in the blue region than in
green & yellow.

14. 2. Photo-tubes:
 These tubes are also known as photoemissive
tubes.
 It consists of spherical shape vacuum glass
bulb.
 It contains photoemissive cathode & an anode.
 The inner surface of semi-cylindrical cathode
mounted inside the bulb is coated with photo-
sensitive material like cesium or potassium
oxide/silver oxide
 A metal ring inserted near the centre of bulb
acts as an anode.

16.  When radiation is incident upon surface of
photosensitive cathode, photoelectrons are emitted.
These are attracted & collected by anode. These
causes current to flow. This current is amplified &
measured.
 The response of phototube is dependent on the
material used for coating & wavelength of light
striking it.
 These are more sensitive than photovoltaic cell.
 Current produced is small & hence requires
amplification.

17. Detector
Phototube

18. The chart recorder
Chart recorders usually plot absorbance against
wavelength.
The output might look like this:

19. Detectors:
The following detectors are commonly used in UV/Vis
spectroscopy:
3. Photomultiplier tube:
 A PMT consists of a photocathode and a series of dynodes (9-
16) in an evacuated glass enclosure.
 Light that strikes the photo cathode causes the ejection of
electrons due to the photoelectric effect.
 The electrons are accelerated towards a series of additional
electrodes called dynodes. (highly sensitive)(90v more positive)
 These electrodes are each maintained at a more positive
potential.
 When the electrons strike the first dynode, more electrons are
emitted by the surface of dynode, these emitted electrons are
then attracted by a second dynode where similar type of electron
emission takes place.

20. Additional electrons are generated at each dynode. This
cascading effect creates 105 to 107 electrons for each photon
hitting the first cathode depending on the number of
dynodes and the accelerating voltage.
The process is repeated over all the dynodes present in the
PMT until a shower of electrons reaches the collector.
By this time, each original photon has produced 106 - 107
electrons
This amplified signal is finally collected at the anode,
photocurrent is produced. where it can be measured.
The light received by cathode releases electrons which
through series of dynodes produce more electrons.
The PMT is extremely sensitive & fast in response
Transmit time between absorption of the photon & arrival of
electrons is in range of 10-100 µsec.

25. The UV Visible spectrophotometer-
A spectrophotometer is an instrument for measuring the
transmittance or absorbance of a sample as a function of
the wavelength of electromagnetic radiation.
The key components of a spectrophotometer are:
- Light source
- Monochromator
- Sample holder
- Detector
- Amplifier

26. 2 Major types of spectrophotometers :
1. Single beam spectrometers
2. Double beam spectrometers

27.  Single-Beam: There is only one light beam or
optical path from the source through to the
detector.

28. Spectrophotometer
 Spectrophotometer consists of two instruments,
namely
1) a spectrometer for producing light of any selected
color (wavelength), and
2)a photometer for measuring the intensityof light.
The instruments are arranged so that liquid in a
cuvette can be placed between the spectrometer beam
and the photometer.
The amount of light passing through thetube is
measured by the photometer.
The photometer delivers a voltage signal to adisplay
device, normally a galvanometer. The signal changes as
the amount of lightabsorbed by the liquid changes.

29. The spectrometer-
Because only small numbers of absorbing molecules are required, it is
convenient to have the sample in solution (ideally the solvent should not
absorb in the ultraviolet/ visible range however, this is rarely the case).
In conventional spectrometers electromagnetic radiation is passed
through the sample which is held in a small square-section cell (usually 1
cm wide internally). Radiation across the whole of the ultraviolet/visible
range is scanned over a period of approximately 30 s, and radiation of
the same frequency and intensity is simultaneously passed through a
reference cell containing only the solvent.
Photocells then detect the radiation transmitted and the spectrometer
records the absorption by comparing the difference between the
intensity of the radiation passing through the sample and the reference
cells
(Fig).
In the latest spectrometers radiation across the whole range is monitored
simultaneously

30. Working :
 UV radiation is given off by the source.
 A convex lense gathers the beam of radiation & focuses it on inlet slit. The inlet
slit permits light from the source to pass but blocks out stray radiation.
Light then reaches the monochromator which splits it up according to
wavelength & transmits a narrow band of light.
The exit slit is positioned to allow light of required wavelength to pass
through. Radiation at all other wavelength is blocked out.
 Selected radiation then pass through the sample cell to the detector.
 The absorbance of a sample is determined by measuring the intensity of light
reaching the detector.
By comparing the intensity of light reaching the detector before & after passing
through the sample it is possible to measure how much radiation is absorbes by
the sample at the particular wavelength used. The output of the detecotr is
usually recorded on graph paper.
As discussed above, most spectrophotometers contain two source lamps, a
deuterium lamp and a tungsten lamp, and use either photomultiplier tubes or,
more recently, photodiodes as detectors.

33. Drawback
 it measures the total amount of light reaching the detector
rather than percentage absorbed.
Light may be lost at reflecting surfaces or may be absorbed
by the solvent

34. 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.

35. Double Beam Spectrophotometers in space:
Working:
The radiation from the source is allowed to pass via a mirror
system to the monochromator unit.
The monochromator allows a narrow range of wavelength to
pass through an exit slit.
The radiation coming out of monochromator through exit
slit is split into two equal intensity by beam splitter.
One beam, the sample beam (coloured magenta), passes
through a small transparent container (cuvette) containing a
solution of the compound being studied.
The other beam, the reference (coloured blue), passes
through an identical cuvette containing only the solvent.

36.  The reference beam intensity is taken as 100%
Transmission (or 0 Absorbance), and the
measurement displayed is the ratio of the two
beam intensities.
 The intensities of these light beams are then
measured by photo detectors and compared.
 The intensity of the reference beam, which should
have suffered little or no light absorption but the
same reflection losses as the sample beam, is
defined as I0.
 The intensity of the sample beam is defined as I.
Within a short period of time, the spectrometer
can automatically scan across the chosen
wavelength range.

37. During a wavelength scan, intensity changes and fluctuations are
equally sensed by the two detectors and normalized out by the
division of I by I0.
However, even if both cuvettes contain the same solution, these
two intensities may not be exactly the same, for example because
of different detector efficiencies or spatial beam drifts.
This leads to a small background spectrum, which can even be
negative in some frequency ranges. Like with a single beam
spectrometer (no reference beam) it is thus important to first
record the background spectrum with only solvent in the sample
cell.
This spectrum must then be subtracted from the one recorded
with the sample solution.

39. A simple double beam spectrometer in time:

40. The light coming from the diffraction grating and slit will hit the
rotating disc and one of three things can happen.
If it hits the transparent section, it will go straight through and
pass through the cell containing the sample. It is then bounced
by a mirror onto a second rotating disc.
This disc is rotating such that when the light arrives from the
first disc, it meets the mirrored section of the second disc. That
bounces it onto the detector.
It is following the red path in the diagram

41. If the light meets the first disc at the black section, it is blocked -
and for a very short while no light passes through the
spectrometer. This just allows the computer to make allowance
for any current generated by the detector in the absence of any
light.
If the original beam of light from the slit hits the mirrored
section of the first rotating disc, it is bounced down along the
green path. After the mirror, it passes through a reference cell.
Finally the light gets to the second disc which is rotating in such
a way that it meets the transparent section. It goes straight
through to the detector.

42. Dual-beam design:
In a conventional single-beam spectrophotometer, the blank and
the sample are measured consecutively, with an interval of
several seconds for a single wavelength measurement and up to
several minutes for a full spectrum measurement with a
conventional instrument.
Lamp drift can result in significant errors over long time
intervals.
The dual-beam spectrophotometer was developed to
compensate for these changes in lamp intensity between
measurements on blank and sample cuvettes.
In this configuration, a chopper is placed in the optical path,
near the light source. The chopper switches the light path
between a reference optical path and a sample optical path to the
detector.
It rotates at a speed such that the alternate measurements of
blank and sample occur several times per second, thus correcting
for medium- and long-term changes in lamp intensity (drift).

43. Figure shows a schematic of a dual-beam
spectrophotometer.

44. Some double-beam instruments have two detectors
(photodiodes), and the sample and reference beam are
measured at the same time.
In other instruments, the two beams pass through a beam
chopper, which blocks one beam at a time. The detector
alternates between measuring the sample beam and the
reference beam in synchronism with the chopper.
There may also be one or more dark intervals in the
chopper cycle. In this case, the measured beam intensities
may be corrected by subtracting the intensity measured in
the dark interval before the ratio is taken.
Specialized instruments have also been made. These
include attaching spectrophotometers to telescopes to
measure the spectra of astronomical features. UV-visible
microspectrophotometers consist of a UV-visible
microscope integrated with a UV-visible spectrophotometer.

46. If you pass white light through a coloured substance, some of the light gets
absorbed. A solution containing hydrated copper(II) ions, for example, looks
pale blue because the solution absorbs light from the red end of the spectrum.
The remaining wavelengths in the light combine in the eye and brain to give the
appearance of cyan (pale blue).
Some colourless substances also absorb light - but in the ultra-violet region.
Since we can't see UV light, we don't notice this absorption.
Different substances absorb different wavelengths of light, and this can be used
to help to identify the substance - the presence of particular metal ions, for
example, or of particular functional groups in organic compounds.
The amount of absorption is also dependent on the concentration of the
substance if it is in solution. Measurement of the amount of absorption can be
used to find concentrations of very dilute solutions.
An absorption spectrometer measures the way that the light absorbed by a
compound varies across the UV and visible spectrum.[[

47. Data acquisition
The earliest instruments simply directly connected the
amplified detector signal to a chart recorder.
Today, all experimental settings are controlled by a
computer and the detector signals are digitized,
processed and stored. Nevertheless it is important that
you note parameters which you set via the instrument
software (slit width, scan range, scan speed, single
beam/dual beam…) into your laboratory journal, along
with the name of the file containing the data (and its
path).
Otherwise it can become very difficult to find or
reproduce a measurement after other users have
changed these settings

49. Path length
/ cm 0 0.2 0.4 0.6 0.8 1.0
%T 100 50 25 12.5 6.25 3.125
Absorbance 0 0.3 0.6 0.9 1.2 1.5

69. σ-σ* Saturated
hydrocarbon
Energy require is
large
Methane - 125nm
Absorption band Below
200nm
n-σ*
Saturated
compound atom
with lone pair of
electron, O,N,S
Require less
energy than σ-σ*
Methanol - 203 nm
Ethanol - 204 nm
CCl4 - 257 nm
π-π*
Unsaturated
compounds,
alkene, alkyne,
carbonyl,nitrile
Energy require is
small, absorption at
longer wavelength
Ethylene- 175 nm
Butene- 213 nm
n-π*
Double bond with
heteroatom C=O.
N=O
Require minimum
energy. Absorption at
linger wavelength
Aldehyde
Ketone 270-300 nm
σ-π* Forbidden
transition
π-σ*
Forbidden
transition

70. Factors affecting
absorption

71.  Suitable solvent is one that does not itself absorb radiation in
region.
 Should be less polar so that it has minimum interaction with
solute molecule.
 Dilute solution prepared.
 Most commonly used 95% ethanol
 Best solvent, cheap, good dissolving power, transparent above
210 nm
 Hexane, other hydrocarbons used as less polar, have least
interaction with the molecule under investigation.
 UV- ethanol, water, cyclohexane
 Position & intensity of absorption maxima change as polarity
change
Effect of solvents:

72.  n-π* transition:
 Absorption band moves to shorter wavelength as
polarity of solvent increases.
 Ground state is more polar than excited state.
 In carbonyl group excited state, hydrogen bonding
with solvent occurs less extent.
 Absorption maxima of : acetone In hexane: 279
Water : 264

73.  π -π* transition:
 Absorption band moves to longer wavelength as
polarity of solvent increases
 The excited states of most π→π* transitions are more
polar than their ground states
 because a greater charge separation is observed in
the excited state.
 If a polar solvent is used the dipole–dipole interaction
reduces the energy of the excited state more than the
ground state, hence the absorption in a polar solvent
such as ethanol will be at a longer wavelength (lower
energy, hence lower frequency) than in a non-polar
solvent such as hexane

74.  n-σ* transition:
 Very sensitive to hydrogen bonding.
 Alcohols, amines form H bonding with solvent,
because of presence of non bonding electron on
hetero atom
 Thus transition requires greater energy.
 When a group is more polar in GS than in the ES,
increasing polarity stabilises the non bonding
electrons in GS due to H bonding, absorption shifted
to shorter wavelength.
 When a group is more polar in ES than in the GS,
increasing polarity stabilises the non bonding
electrons in ES due to H bonding, absorption shifted
to longer wavelength.

75.  In general the increasing polarity of solvents shifts -
n-π* & n-σ* bands to shorter wavelength.
π -π* bands to longer wavelength.

76.  Care must be taken when choosing a solvent, because
many solvents absorb in the ultraviolet region. The
minimum wavelengths at which some solvents are
useful are given in Table

77. Effect of conjugation :
 Conjugation of double bonds, lowers the energy required
for the transition.
 As the number of double bonds in conjugation increases
the absorption moves to longer wavelength
 e.g. Butadiene in hexane: 217 nm
 1,3,5,7 Octatriene in hexane : 296 nm
 β – carotene : 451 nm

78. Effect of conjugation:
 Ethene, containing only one double bond, has an absorption maximum at
185 nm (ε = 10 000). If the carbon chain length is increased this peak
shifts to a slightly longer wavelength because the σ bonded electrons of
the alkyl group interact with the π bond electrons in the double bond (I e
the energy of the excited state is reduced).
 The shift in wavelength is small compared with the effect of increasing
the number of double bonds, especially if the electrons in the π systems
(the double bonds) can interact with each other. The simplest example is
buta-1,3-diene, CH2=CH–CH=CH2 (Fig. 14). Buta-1,3-diene has an
absorption maximum at 220 nm,
 with an absorption coefficient of 20 000 – ie both the wavelength and the
intensity of the absorption have increased. This difference arises because
instead of the double bonds absorbing in isolation of each other the π
system extends over the length of the carbon chain – ie the system is
conjugated (or delocalised) – and lowers the energy of the excited state.