1. Instrumental Analysis: Spectrophotometric Methods
2007

2. 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

3. 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.

4. (Instruments based on light interaction with matter)
Today’s 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 week’s lecture:
Fourier transformed infrared spectroscopy
Interferometer
Atomic spectroscopy
Quantitative analysis
Beer’s law
Method validation
Dilution and spike

5. 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= )
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 J s)
n = wavenumber (most common units = cm-1) ~
Therefore:
Energy is inversely proportional to wavelength
but proportional to wavenumber

6. 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)

7. Regions of Electromagnetic Spectrum-the “colour” of light
Fig. 18-2

8. Electronic structures of simple molecule
Energy
Excited state
Singlet S1 T1
Excited state
Triplet
Vibration states D
Dissociated states
Bond length S0
Ground state

9. Interaction between photon and moleculeS0 S1 T1 D
S0 S1 transition S1 T1
UV-vis A F P IR S0

10. Electronic structures Singlet and triplet
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

11. 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

12. Optical spectrophotometer components
Excitation sources
Deuterium Lamp
Tungsten Lamp
Laser
X-ray tube
Mercury lamp
Xenon lamp
Silicon carbide globar
Flame
Furnaces
Plasmas
Hollow-cathode lamp UV
Detectors
PMT
CCD/CID
Photodiode
Thermocouple
MCT
Pyroelectric detector
UV-vis
Monochromators
Filters
Grating+slit
prism
X-ray, UV, vis, IR
X-ray
UV-vis
UV-vis IR
What is the advantage and disadvantage?

13. Single Beam vs. Double Beam
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 , pg. 315 "Instrument designs for photometers and spectrophotometers”

14. Light sources Brightness Line width Background
Stability
Lifetime
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 £££

15. 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 nm or 650 cm-1)
KBr (to nm or 450 cm-1)
CsI (to nm or 200 cm-1)
Best material: diamond, why?
Optical transmission coefficient
Criteria
High transmission
Chemically inert
Mechanically strong

16. Monochromators Why? 10mmx10mm 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 mm-1 for UV/vis
- can use plastic replicas in less expensive instruments
Think of diffraction on a CD
10mmx10mm

17. 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

18. Interference in diffraction
d sin(q)+d sin(f)=nl d
Bragg condition
Phase relationship
q>0
f<0 f q
n=1, 2, 3 In-phase
n=1/2, 3/2, 5/2 out-phase

19. Monochromators: reflection grating

20. 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
Resolution of grating: l
=nN
n: diffraction order
N: number of illuminated groves Dl
Angular resolution:
As: d sin(q)+d sin(f)=nl
So: n Dl=d cos(f) Df
Therefore: Df/Dl=n/[d cos(f)]
What does this mean?

21. 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!

22. 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!

23. 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?

24. 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

25. Photodiode Array Could you compare photodiode with CCD detector?
- 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?

26. 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.

27. Photodiode Array Spectrophotometers vs Dispersive Spectrophotometers
- 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:

28. Property of luminescence spectrum
Fluorescence vs phosphorescence
Phosphorescence is always at longer wavelength compared with fluorescence
Phosphorescence is narrower compared with fluorescence
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?

29. Fluorescence spectroscopy

30. Fluorescence spectroscopy
Light source
Beam
splitter
Q: why the emission is measured at 90 relative to the excitation?
Excitation
monochromator
sample
8% of light
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

31. Fluorescence spectroscopy: well defined molecules

32. 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