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Instrumental Analysis:
Spectrophotometric Methods
2007
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 Beer’s Law
(analyse mixture absorption)
•Understand the mechanism and application of UV-Vis, FTIR, Luminescence,
atomic spectroscopy
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.
Today’s lecture:
(Instruments based on light interaction with matter)
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Properties of light
Molecular electronic structures
Interaction of photons with molecules
Spectrophotometer components
• Light sources
• Single and double beam instruments
• Monochrometers
• Detectors
Fluorescence spectroscopy
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Next week’s lecture:
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Fourier transformed infrared spectroscopy
• Interferometer
Atomic spectroscopy
Quantitative analysis
• Beer’s law
• Method validation
• Dilution and spike
Review on properties of light:photon
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 >1)
in a vacuum: c=2.998 x 108 m s-1
(n=1 exactly, in air n=1.0002926)
c/n= nl
And of course, the relationship between energy and frequency:
~
E = hn = hc/l = hc n
h = Planck’s constant (6.626 x 10-34 J s)
n =~wavenumber (most common units = cm-1)
Therefore:
Energy is inversely proportional to wavelength
but proportional to wavenumber
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)
Regions of Electromagnetic Spectrum-the “colour” of light
Fig. 18-2
Energy
Electronic structures of simple molecule
Vibration states
Excited state
Singlet
S1
T1 Excited state
Triplet
D
Dissociated states
S0
Ground state
Bond length
Interaction between photon and molecule
S0 S1 transition
S1
T1
UV-vis
S1
T1
A
D
F
P
IR
S0
S0
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 (Beer’s law, UV-vis)
Frank Condon condition and The Stokes' shift
Radionless relaxation and vibration relaxation
Luminescence-fluorescence/phosphorescence
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
Optical spectrophotometer components
Excitation sources
Deuterium Lamp
Tungsten Lamp
Laser
X-ray tube
UV
UV-vis
X-ray, UV, vis, IR
X-ray
Mercury lamp
UV-vis
Xenon lamp
UV-vis
Silicon carbide globar
Flame
IR
Detectors
Monochromators
Filters
Grating+slit
prism
PMT
CCD/CID
Photodiode
Thermocouple
MCT
Pyroelectric detector
Furnaces
Plasmas
Hollow-cathode lamp
What is the advantage and disadvantage?
Design of optical spectrophotometers
Single Beam vs. Double Beam
Q: what’s 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."
(a)
(c)
(b)
Fig. 13-12, pg. 315 "Instrument designs for photometers and spectrophotometers”
Light sources
What is the important properties of a source?
Brightness
Line width
Background
Stability
Lifetime
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
( How much in cm-1, J, Hz and eV?)
- produces l from 320 to 2500 nm
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 £££
Sample a source containers:
for UV: quartz (won’t 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
Monochromators
Early spectrophotometers used prisms
- quartz for UV
Why?
- glass for vis and IR
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
10mmx10mm
http://www.veeco.com/library/nanotheater_detail.php?type=
application&id=331&app_id=34
http://www.ii.com/images/prism.jpg
http://www.mrfiber.com/images/
cddiffract.jpg
Monochromators: cont’d
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
Interference in diffraction
d sin(q)+d sin(f)=nl
d
q>0
f<0
q
Bragg condition
f
Phase relationship
n=1, 2, 3 In-phase
n=1/2, 3/2, 5/2 out-phase
Monochromators: reflection grating
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:
Dl
Angular resolution:
As:
So:
Therefore:
=nN
n: diffraction order
N: number of illuminated groves
d sin(q)+d sin(f)=nl
n Dl=d cos(f) Df
Df/Dl=n/[d cos(f)]
What does this mean?
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 signal:noise
Small slit width: less energy reaching the
detector BUT better resolution!
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!
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?
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
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?
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.
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/
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
1. absorption is mirrored relative to emission
2. Absorption is always on the shorter wavelength compared to emission
3. Absorption vibrational progression reflects vibrational level in the electronic excited
states, while the emission vibrational progression reflects vibrational level in the
electronic ground states
4. l0 transition of absorption is not overlap with the l0 of emission
Why?
Fluorescence spectroscopy
Fluorescence spectroscopy
Beam
splitter
Light source
Excitation
monochromator
sample
Q: why the emission is
measured at 90 relative to
the excitation?
Emission
Monochromator
Reference
diode
PMT
Amplifier
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
Fluorescence spectroscopy: well defined molecules
Summary of spectrophotometric techniques
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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
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Describe and assess advantages and disadvantages of various monochromators e.g.
Prism, diffraction gratings
Explain how to asses the quality of grating
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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