Introduction to Spectroscopy

Dr Wan Nor Roslam Wan Isahak

Department of Chemical and Process Engineering,

Faculty of Engineering and Built Environment,
UKM
 Spectroscopy
Spectrum: A plot of the intensity as a function light or particle energy (frequency,
wavelength)

Spectroscopy: Using a probe (radiation, ions or electrons) and sorting its

content into energy bins to identify the materials response in each region of the
spectrum

Recall that any material system made up of atoms, molecules and electrons
responds to external stimuli such as light or particles over a wide range of
energies in a distinct manner
Spectroscopy
Types of spectroscopy:
(a) Continuous spectroscopy
(b) Absorption spectroscopy
(c) Emission spectroscopy
 Emission Process
Electrons ground level
Energy emission

Absorb energy

High energy level

What kind of states?
electronic UV-Vis
vibrational infrared

rotational microwave

nuclear spin radiofrequency

Equipment
Left: Equipment diagram
Right: Schematic diagram
Basics of Light, E&M Spectrum,
and X-rays
Light can take on many forms. Radio waves, microwaves, infrared, visible, ultraviolet, X-ray and gamma
radiation are all different forms of light.

The energy of the photon tells what kind of light it is. Radio waves are composed of low energy photons. Optical
photons--the only photons perceived by the human eye--are a million times more energetic than the typical
radio photon. The energies of X-ray photons range from hundreds to thousands of times higher than that of
optical photons.

The speed of the particles when they collide or vibrate sets a limit on the energy of the photon. The speed is
also a measure of temperature. (On a hot day, the particles in the air are moving faster than on a cold day.)

Very low temperatures (hundreds of degrees below zero Celsius) produce low energy radio and microwave
photons, whereas cool bodies like ours (about 30 degrees Celsius) produce infrared radiation. Very high
temperatures (millions of degrees Celsius) produce X-rays.
 Materials response
to radiation or particles

• Electromagnetic radiation interacts with materials because

electrons and molecules in materials are polarizable:
•(refraction, absorption)
ñ= n+ i k
n = refraction, k = absorption

Atoms/molecules
Valence electrons

Core electrons

• Ions, electrons and atoms incident on materials can interact with materials because
they are either charged or can scatter from atomic cores
 Techniques and information content

Molecular Molecular Electronic Valence band and Deep electronic

Libration vibrations Absorption shallow electronic core levels
(hindered rotations) levels (atoms) (atoms)

Infrared, UV absorption
Raman, UV photoemission
EELS Electron loss
Microwave, Visible X-ray photoemission
THz Fluorescence (XPS, ESCA)
Luminescence Auger Electron (AES)
Common Spectroscopic Methods Based on
Electromagnetic Radiation
Type of Usual Usual Wave Type of
Spectroscopy Wavelength number Quantum
Range Range, cm-1 Transition
Gamma-ray 0.005-1.4 Å _ Nuclear
emission

X-ray absorption, 0.1-100 Å _ Inner electron

emission,
fluorescence, and
diffraction
Vacuum ultraviolet 10-180 nm 1x106 to 5x104 Bonding electrons
absorption
Ultraviolet visible 180 -780 nm 5x104 to 1.3x104 Bonding electrons
absorption,
emission,
fluorescence
Infrared absorption 0.78-300 mm 1.3x104 to 3.3x101 Rotation/vibration
and Raman of molecules
scattering
Microwave 0.75-3.75 mm 13-27 Rotation of
absorption molecules
Electron spin 3 cm 0.33 Spin of electrons in
resonance a magnetic field

Nuclear magnetic 0.6-10 m 1.7x10-2 to 1x103 Spin of nuclei in a

resonance magnetic field
Photoelectron Spectroscopy

Photons in Electrons out

Vacuum level

Valence
electrons

Core electrons

• X-ray (photon) penetration into solid is large (~ microns)

• Electron escape from solid is only from shallow region (~ 5-10 Å)

because of short mean free path of electrons with energies
between 10 and 1000 eV

 XPS is only sensitive to surface and near surface region

Optical Spectroscopy
Photons in Photons out

• Large penetration into solid

• Low energy photons  Non destructive Photons out
• Can interact linearly (absorption)
or non-linearly (Raman, harmonic generation)
 FTIR Surface Spectroscopy

• Infrared Spectroscopy Theory

• IR spectrometers Grating systems

Interferometers (FTIR)

• Surface Spectroscopy Methods

• Examples
Classical theory for linear absorption

• The electronic interactions between atoms in molecules or solids provide a binding force and a
restoring force often compared to springs. Therefore each system (molecule, solid) displays
characteristic vibrations (normal modes) associated with bond stretching and bond bending
motions.

• The frequency of the radiation identical to the frequency of these characteristic vibrations is
absorbed

• Absorption of infrared radiation by a vibrating molecule can only take place if the vibration
produces an alternating electric field (changing dipole moment)

e.g. O–C–O symmetric stretch (IR inactive)

O–C–O asymmetric stretch (IR active)

O–C–O bending mode (IR active)

Examples

  
Stretching modes -CH2- Bending modes -CH2-
x

asym. sym.
stretching stretching scissoring rocking wagging twisting
as(CH2) s(CH2) s(CH2) (CH2) (CH2) (CH2)
 Grating or prism spectrometer

Source

Selects one wavelength (energy) at a time, requiring rotation to scan the spectrum
Array detectors allow detection of a restricted range of wavelengths
 Good to study single vibrational line (e.g. time resolved spectroscopy)

Higher resolution requires narrowing slits  Inefficient for high resolution spectroscopy

Requires calibration
 Interferometers
Detect IR intensity as a function of
mirror displacement: INTERFEROGRAM

Michelson
Interferometer
(broadband)

http://www.wooster.edu/chemistry/is/brubaker/ir/ir_works_modern.html

All wavelengths are measured simultaneously (Felgett advantage)

 Faster and more efficient

No need for narrow slits (resolution determined by mirror travel)

 higher optical throughput (Jacquinot advantage)

Internally calibrated by He-Ne laser control of moving mirror (Connes advantage)

Ideal to examine broad spectral regions and weak absorptions with high resolution
Fourier-Transform Infrared spectroscopy

As more frequencies are added, the interferogram

becomes a more complex function, with the largest
For a single frequency (i.e. laser light), the signal
amplitude at the zero path difference (zpd)
on the detector (interferogram) is a sine wave
Spectrum
25
Interferogram
20

Absorbance
15

FT 10
Mirror displacement
Waveforms 5

500 1000 1500 2000 2500 3000 3500

For a broad spectral range (white light), Wavenumber (cm )

-1

The interferogram is most peaked at zpd wavenumber

4000 cmf-1
~ -1- 1
400 cm

25000 nm - 2500  nm
c
 Surface and Interface Spectroscopy
IR wavelength (~ mm) is much larger than surface dimensions (nm)
 Need to Eliminate all other contributions to spectrum (selecting a reference system)
Initial state (reference) Final state

25 25

20 SiO2+Si 20
SiH+Si

Absorbance
Absorbance

15 15

10 10

5
Si(111) 5 Si(111)
0 0

500 1000 1500 2000 2500 3000 3500

etching 500 1000 1500 2000 2500 3000 3500
-1
Wavenumber (cm )
-1 Wavenumber (cm )

0.006

Reprocessing: 0.004
SiH added
Subtraction of reference 0.002
Absorbance

spectrum from final state

0.000
spectrum
-0.002

-0.004
SiO2 removed
-0.006

500 1000 1500 2000 2500 3000 3500 4000

-1
Wavenumber (cm )
 Maximizing Surface Interaction
IR in IR out
1. For highly absorbing or 
reflecting (metal) substrates
 grazing incidence reflection n and k large
tan (B) = ñ  Reflection

IR in
 k small
2. For weakly absorbing substrates
 “Brewster” incidence transmission IR out
tan (B) = n Transmission

n large (2-4)
k very small
IR in int IR
3. For transparent substrates out

 Multiple internal reflections

int ~ 45o
Need double-sided polish + bevels at sides
Multiple internal Reflections
In-situ possible for liquid environments Evanescent field ~ 1-10 mm
 Attenuated Total Reflection
(ATR)
• Multiple internal reflection:

IR in IR out

• In-situ wet chemistry/electrochemistry

contact

IR in IR out

liquid out liquid in

electrodes

• Multiple internal transmission:

(Handbook of Vibrational Spectroscopy, Wiley, Vol.1, p. 1117, 2002)

IR in IR out
Buried interface
 Fluorescence Spectroscopy
Light source, self-emission
which means the electrons
transferred to the lowest
level spontaneously

Different fluorescence:
(a) different meta-stable
states
(b) different various
vibrational states of the
ground state
Time-resolved fluorescence
spectroscopy
It provides fluorescence intensity decay in terms of lifetimes
Advantages:
enhance the discrimination among fluorophores (overlapping
emission spectra )
sensitive to various parameters of the biological
microenvironment
Examples
 Example 1: FTIR for
biointerfacial characterization
Attaching linker for biomolecule (e.g. antibody) immobilization on Silicon substrate
MPS models a tiny antibody!
Step 2: Formation of Urea linkage
during PMPI attachment
Step 3: Formation of succinimide (evidence
for thioether bonding) during MPS
attachment
Example 2: Fibrinogen immobilization

Primary structure: Peptide (Amino acid) chain

Secondary structure: alpha helices, beta pleats or folds
Tertiary: Domains as shown above
 Fibrinogen: size and structure
Size estimates

Minor Axis
60 – 90 A
Peptide chain in solution
(R1, R2, R3, R4: Amino Acid Residues)
http://bio.winona.msus.edu/berg/ChemStructures/Polypep2.gif

Major Axis
IR bands present in all protein backbones
http://homepages.uc.edu/~retzings/fibrin2.htm (Hall CE, Slayter HS: The fibrinogen
molecule: Its size, shape and mode of polymerization. J Biophys Biochem Cytol • Amide I band: C=O stretch
5:11-15, 1959. Weisel JW, Stauffacher CV, Bullitt E, Cohen C: A model for
fibrinogen: domains and sequence. Science 230:1388-1391, 1985.) • Amide II band: N-H deformation coupled to C-N stretch
AFM • Amide IV band: coupled C-N and C-O stretch
• CH stretch
17 A 11 A
• NH stretch

300 A 600 A
CHICKEN FIBRINOGEN:
Fibrinogen on mica Fibrinogen on graphite Molecular Weight 54193
Marchin K. L. and Berrie C.L., Conformational changes in the plasma protein fibrinogen upon Number of Residues 491
adsorption to graphite and mica investigated by atomic force microscopy, Langmuir 19 (2003) p.9883.
 R-CO-NH2
Amide I band Amide II band
C=O C-NH2

Functional chemical group

(olefins, esters, ethers, nitriles, thioethers,
thioesters) acids or alcohols

Germanium

Tripod attachment

Use hydrolysis of SiCl3-(CH2)16-COCl