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

Energy

Electronic structures of simple molecule

Excited state
Singlet
S1

Vibration states

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
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 (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
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: 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
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 from 320 to 2500 nm

Brightness
Line width
Background
Stability
Lifetime

( 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
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

10mx10m
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: 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

>0
<0

Bragg condition

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

Resolution of grating:

Angular resolution:

As:
So:
Therefore:

=nN

n: diffraction order
N: number of illuminated groves

d sin()+d sin()=n
n =d cos()
=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
Beam
splitter

Light source
Excitation
monochromator

8%

ht
g
i
l
of

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

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