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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 Beers 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.


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
Review on properties of light:photon
Light is energy in the form of electromagenetic field

Wavelength (): Crest-to-crest distance between waves


Frequency (): 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=

And of course, the relationship between energy and frequency:


E = h = hc/ = hc ~
h = Plancks constant (6.626 x 10-34 J s)
=~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
Electronic structures of simple molecule
Energy

Excited state
Singlet
S1

T1 Excited state
Vibration states

Triplet

D
Dissociated states

S0 Bond length

Ground state
Interaction between photon and molecule

S0 S1 transition
S1

T1
S1

T1
UV-vis

D
A F
P

S0
IR

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 (Beers 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 UV Detectors


Tungsten Lamp
UV-vis PMT
Laser X-ray, UV, vis, IR
Monochromators
CCD/CID
X-ray tube X-ray Filters
Mercury lamp Grating+slit Photodiode
UV-vis
prism Thermocouple
Xenon lamp UV-vis
Silicon carbide globar IR MCT
Flame Pyroelectric detector
Furnaces
Plasmas
Hollow-cathode lamp

What is the advantage and disadvantage?


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."

(a)

(c)

(b)

Fig. 13-12, pg. 315 "Instrument designs for photometers and spectrophotometers
Light sources Brightness
Line width
What is the important properties of a source? Background
Black-body radiation for vis and IR but not UV Stability
- a tungsten lamp is an excellent source of black-body radiation Lifetime
- operates at 3000 K
- produces 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
Sample a source containers:
for UV: quartz (wont block out the light)
for vis: glass [ 800nm (red) to 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
- glass for vis and IR Why?

These are now superseded by:


Diffraction gratings: http://www.ii.com/images/prism.jpg
- 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.mrfiber.com/images/
cddiffract.jpg
10mx10m

http://www.veeco.com/library/nanotheater_detail.php?
type=application&id=331&app_id=34
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
Interference in diffraction
d sin()+d sin()=n

d
Bragg condition

Phase relationship
>0
<0 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

Resolution of grating: n: diffraction order


=nN
N: number of illuminated groves

Angular resolution:
As: d sin()+d sin()=n
So: n =d cos()
Therefore: =n/[d cos()]
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

Disper sive Spectr ophotometer:


- only a narrow band of wavelengths reaches the detector at a tim e
- 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
0 transition of absorption is not overlap with the 0 of emission

Why?
Fluorescence spectroscopy
Fluorescence spectroscopy
Light source Beam
splitter Q: why the emission is
measured at 90 relative to
Excitation
monochromator
sample the excitation?

ght
li
of Emission
8% 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
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