PowerPoint Presentation - Spectrophotometry

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Instrumental AnalysisSpectrophotometric Methods
2007
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By the end of this part of the course, you should
be able to

• Understand interaction between light and matter
• (absorbance, excitation, emission,
  luminescence,fluorescence, phosphorescence)
• Describe the main components of a
  spectrophotometer,
• (sources, monochromators, detectors,
  interferometer, grating, ATR, ICP, )
• Make calculations using Beers Law
• (analyse mixture absorption)
• Understand the mechanism and application of
  UV-Vis, FTIR, Luminescence, atomic spectroscopy

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Background knowledge
What you are expected to know before the course
Error analysis in quantitative analysis Solve
linear equations Complementary colour Exponential
and logarithm
If you have difficulty to understand above
topics, find extra reading materials!
Or discuss with me after the lecture.
What you are recommended to know before the
course
Least square fitting Basic quantum
chemistry Molecular symmetry
If you are trying to learn above topics, please
let me know.
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Todays lecture (Instruments based on light
interaction with matter)

• Properties of light
• Molecular electronic structures
• Interaction of photons with molecules
• Spectrophotometer components
• Light sources
• Single and double beam instruments
• Monochrometers
• Detectors
• Fluorescence spectroscopy

Next weeks lecture

• Fourier transformed infrared spectroscopy
• Interferometer
• Atomic spectroscopy
• Quantitative analysis
• Beers law
• Method validation
• Dilution and spike

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Review on properties of lightphoton
Light is energy in the form of electromagenetic
field
Wavelength (l) Crest-to-crest distance between
waves Frequency (n) Number of complete
oscillations that the wave makes each
second units number of oscillations/sec or
s-1 or Hertz (Hz)
Light travelling speed in
other media c/n (n refractive index,
generally gt1) in a vacuum c2.998 x 108 m
s-1 (n1 exactly, in air n1.0002926) c/n
nl
Therefore Energy is inversely proportional
to wavelength but proportional to wavenumber
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Frequency Scanning Techniques a few definitions
Emission method source of light is sample
Absorption method intensities of a source with
and without the sample in place are compared
Spectrum a plot of intensity vs.
frequency/wavelength
In quantitative analysis common to work at 1
wavelength running a spectrum is an important
initial step (to select best conditions)
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Regions of Electromagnetic Spectrum-the colour
of light
Fig. 18-2
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Electronic structures of simple molecule
Energy
Excited state Singlet
S1
Excited state Triplet
T1
Vibration states
D
Dissociated states
Bond length
S0
Ground state
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Interaction between photon and molecule
S1
T1
UV-vis
F
A
P
IR
S0
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Key concept from energy diagram
Electronic structures Singlet and triplet Bond
length for ground and excited states Vibrational
structures-infrared absorption/transmission
(FTIR) Internal conversion Intersystem
crossing Photon adsorption excitation (Beers
law, UV-vis) Frank Condon condition and The
Stokes' shift Radionless relaxation and
vibration relaxation Luminescence-fluorescence/ph
osphorescence
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Type of optical spectroscopy
UV-vis absorption spectroscopy (UV-Vis) FT-IR
absorption/transmission spectroscopy
(FTIR) Atomic absorption spectroscopy
(AAS) Atomic fluorescence spectroscopy
(AFS) X-ray fluorescence spectroscopy (XFS)
What you will learn
The excitation mechanism
Monochromator design
Instrument principle
Quantitative methods
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Optical spectrophotometer components
UV
UV-vis
X-ray, UV, vis, IR
X-ray
UV-vis
UV-vis
IR
What is the advantage and disadvantage?
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Design of optical spectrophotometers
Single Beam vs. Double Beam
Q whats the advantage of double beam
spectrophotometer?
(a) single-beam design (b) dual channel design
with beams separated in space but simultaneous in
time (c) double-beam design in which beams
alternate between two channels."
Fig. 13-12, pg. 315 "Instrument designs for
photometers and spectrophotometers
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Light sources
Brightness Line width Background Stability Lifetim
e
What is the important properties of a source?
Black-body radiation for vis and IR but not UV -
a tungsten lamp is an excellent source of
black-body radiation - operates at 3000 K -
produces l from 320 to 2500 nm
( How much in cm-1, J, Hz and eV?)
For UV - a common lamp is a deuterium arc
lamp - electric discharge causes D2 to
dissociate and emit UV radiation (160 325
nm) - other good sources are Xe (250 1000
nm) Hg (280 1400 nm)
Lasers - high power - very good for studying
reactions - narrow line width - coherence -
can fine-tune the desired wavelength (but choice
of wavelength is limited) - expensive
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Sample a source containersfor UV quartz
(wont block out the light) for vis glass l
800nm (red) to l 400 nm (violet) for IR
NaCl (to or 15384 nm or 650 cm-1) KBr (to
22222 nm or 450 cm-1) CsI (to 50000 nm or
200 cm-1)
Best material diamond, why?
Optical transmission coefficient
Criteria
High transmission Chemically inert Mechanically
strong
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Monochromators
Early spectrophotometers used prisms - quartz
for UV - glass for vis and IR
Why?
These are now superseded by Diffraction
gratings - made by drawing lines on a glass
with a diamond stylus ca. 20 grooves mm-1 for
far IR ca. 6000 mm-1 for UV/vis - can use
plastic replicas in less expensive
instruments Think of diffraction on a CD
http//www.ii.com/images/prism.jpg
http//www.mrfiber.com/images/cddiffract.jpg
10mmx10mm
http//www.veeco.com/library/nanotheater_detail.ph
p?typeapplicationid331app_id34
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Monochromators contd
What is the purpose of concave mirrors?
Polychromatic radiation enters
The light is collimated the first concave mirror
Reflection grating diffracts different
wavelengths at different angles
Second concave mirror focuses each wavelength at
different point of focal plane
Orientation of the reflection grating directs
only one narrow band of wavelengths to exit slit
http//oco.jpl.nasa.gov/images/grating_spec-br.jpg
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Interference in diffraction
d sin(q)d sin(f)nl
d
Bragg condition
Phase relationship
qgt0 flt0
f
q
n1, 2, 3 In-phase
n1/2, 3/2, 5/2 out-phase
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Monochromators reflection grating
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Monochromators reflection grating
Each wavelength is diffracted off the grating at
a different angle
Angle of deviation of diffracted beam is
wavelength dependent ? diffraction grating
separates the incident beam into its constituent
wavelengths components
Groove dimensions and spacings are on the order
of the wavelength in question
In order for the emerging light to be of any use,
the emerging light beams must be in phase with
each other
l
Resolution of grating
n diffraction order N number of illuminated
groves
nN
Dl
Angular resolution
As d sin(q)d sin(f)nl
So n Dld cos(f) Df
Therefore Df/Dln/d cos(f)
What does this mean?
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Monochromators slit
Bottom line - it is usually possible to arrange
slits and mirrors so that the first order (n 1)
reflection is separated - a waveband of ca. 0.2
nm is obtainable
However, the slit width determines the resolution
and signal to noise ratio Large slit width more
energy reaching the detector ? higher
signalnoise Small slit width less energy
reaching the detector BUT better resolution!
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Detectors
Radiation-----charger converter
Choice of detector depends upon what wavelength
you are studying Want the best response for the
wavelength (or wavelength range) that you are
studying In a single-beam spectrophotometer, the
100 transmittance control must be adjusted each
time the wavelength is changed In a double-beam
spectrophotometer, this is done for you!
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Photomultiplier-single channel, but very high
sensitivity
- Light falls on a photosensitive alloy (Cs3Sb,
K2CsSb, Na2KSb)
- Electrons from surface are accelerated towards
secondary electrodes called dynodes and gain
enough energy to remove further electrons
(typically 4-12, to 50 with GaP).
- For 9 stages giving 4 electrons for 1, the
amplification is 49 or 2.6 x 105)
- The output is fed to an amplifier which
generates a signal
- To minimise noise it is necessary to operate at
the lowest possible voltage
What decide the sensitive wavelength?
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Photodiode Array-multiplex, but low sensitivity
Good for quick (fraction of a second) scanning of
a full spectrum Uses semiconductor
material Remember n-type silicon has a
conduction electron P or As doped p-type
silicon has a hole or electron vacancy Al or
B doped
A diode is a pn junction under forward bias,
current flows from n-Si to p-Si under reverse
bias, no current flows boundary is called a
depletion layer or region
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Photodiode Array
- Electrons excited by light partially discharge
the condenser
- Current which is necessary to restore the
charge can be detected
- The more radiation that strikes, the less
charge remains
- Less sensitive than photomultipliers ? several
placed on placed on single crystal
- Different wavelengths can be directed to
different diodes
- Good for 500 to 1100 nm
- For some crystals (i.e. HgCdTe) the response
time is about 50 ns
Could you compare photodiode with CCD detector?
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Photodiode Array Spectrophotometer
- For photodiode array spectrophotometers, a
white light passes through sample
- The grating polychromator disperses the light
into the component wavelengths
- All wavelengths are measured simultaneously
- Resolution depends upon the distance between
the diodes and amount of dispersion
No moving parts! Simple mechanical and optical
design, very compact.
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Photodiode Array Spectrophotometers vs
Dispersive Spectrophotometers
Dispersive Spectrophotometer - only a narrow
band of wavelengths reaches the detector at a
time - slow spectral acquisition (ca. 1 min) -
several moving parts (gratings, filters, mirrors,
etc.) - resolution ca. 0.1 nm - produces less
stray light ? greater dynamic range for measuring
high absorbance - sensitive to stray light from
outside sources i.e. room light
Photodiode Array Spectrophotometer - no moving
parts ? rugged - faster spectral acquisition
(ca. 1 sec) - not dramatically affect by room
light
What are the components 1 to 10?
From http//www.oceanoptics.com/
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Property of luminescence spectrum
Fluorescence vs phosphorescence

1. Phosphorescence is always at longer wavelength
   compared with fluorescence
2. Phosphorescence is narrower compared with
   fluorescence
3. Phosphorescence is weaker compared with
   fluorescence

Why?
Absorption vs emission

• absorption is mirrored relative to emission
• Absorption is always on the shorter wavelength
  compared to emission
• Absorption vibrational progression reflects
  vibrational level in the electronic excited
  states, while the emission vibrational
  progression reflects vibrational level in the
  electronic ground states
• l0 transition of absorption is not overlap with
  the l0 of emission

Why?
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Fluorescence spectroscopy
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Fluorescence spectroscopy
Beam splitter
Light source
Q why the emission is measured at 90 relative to
the excitation?
Excitation monochromator
sample
8 of light
Emission Monochromator
Reference diode
Amplifier
PMT
Computer
Emission spectrum hold the excitation
wavelength steady and measure the emission at
various wavelengths Excitation spectrum vary
the excitation wavelength and vary the wavelength
measured for the emitted light
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Fluorescence spectroscopy well defined molecules
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Summary of spectrophotometric techniques

• Describe the main components of a
  spectrophotometer and distinguish between single
  double beam instruments
• Describe suitable sources for ultraviolet
  (UV)/visible (vis), infra red (IR) and atomic
  absorption (AA) instruments

• Describe and assess advantages and disadvantages
  of various monochromators e.g. Prism, diffraction
  gratings
• Explain how to asses the quality of grating

• Explain how photomultipliers and diode detectors
  work
• Explain the advantage of multiplex detecting
• Describe the luminescence spectroscopy and energy
  transfer process
• Compare the emission and absorption spectrum