Transcript Introduction to spectroscopy

125:583
Biointerfacial Characterization
Introduction to Spectroscopy
Sep 28, 2006
Oct 12, 16, 2006
Yves Chabal
Departments of Chemistry and Chemical Biology, and Biomedical Engineering
Nanophysics Lab, Room 205 [email protected]
Prabhas Moghe
Departments of Chemical Engineering, and Biomedical Engineering
LECTURE #1: Introduction
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
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
• E&M 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
Libration
vibrations
(hindered rotations)
Electronic
Absorption
Valence band and
shallow electronic
levels (atoms)
Infrared,
Raman,
EELS
Microwave,
THz
Deep electronic
core levels
(atoms)
UV absorption
UV photoemission
Electron loss
Visible
Fluorescence
Luminescence
X-ray photoemission
(XPS, ESCA)
Auger Electron (AES)
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
• Can interact linearly (absorption)
or non-linearly (Raman, harmonic generation)
out
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 (just like a spring pendulum)
• 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-
asym.
stretching
as(CH2)
sym.
stretching
s(CH2)
Bending modes -CH2-
scissoring
s(CH2)
rocking
(CH2)
wagging
(CH2)
x
twisting
(CH2)
LECTURE # 2:
Instruments and surface spectroscopy
October 12, 2006
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
amplitude at the zero path difference (zpd)
For a single frequency (i.e. laser light), the signal
on the detector (interferogram) is a sine wave
Spectrum
25
Interferogram
FT
Waveforms
Mirror displacement
Absorbance
20
15
10
5
0
500
For a broad spectral range (white light),
The interferogram is most peaked at zpd
http://www.wooster.edu/chemistry/is/brubaker/ir/ir_works_modern.html
1000
1500
2000
2500
3000
3500
-1
Wavenumber (cm )
wavenumber
~  1  f c
400 cm-1 - 4000 cm-1
25000 nm - 2500 nm
Surface and Interface Spectroscopy
IR wavelength (~ m) is much larger than surface dimensions (nm)
 Need to Eliminate all other contributions to spectrum (selecting a reference system)
Final state
Initial state (reference)
25
25
SiO2+Si
15
10
Si(111)
5
500
1000
1500
2000
2500
3000
15
10
Si(111)
5
0
etching
0
SiH+Si
20
Absorbance
Absorbance
20
500
3500
1000
1500
2000
2500
3000
3500
-1
Wavenumber (cm )
-1
Wavenumber (cm )
0.006
Reprocessing:
Absorbance
Subtraction of reference
spectrum from final state
spectrum
SiH added
0.004
0.002
0.000
-0.002
SiO2 removed
-0.004
-0.006
500
1000
1500
2000
2500
3000
-1
Wavenumber (cm )
3500
4000
Maximizing Surface Interaction
1. For highly absorbing or
reflecting (metal) substrates
 grazing incidence reflection
tan (B) = ñ 
IR in
IR out

n and k large
Reflection
IR in
2. For weakly absorbing substrates
 “Brewster” incidence transmission
tan (B) = n
Need double-sided polish + bevels at sides
In-situ possible for liquid environments
k small
IR out
Transmission
IR in
3. For transparent substrates
 Multiple internal reflections
int ~ 45o

int
n large (2-4)
k very small
IR
out
Multiple internal Reflections
Evanescent field ~ 1-10 m
Attenuated Total Reflection (ATR)
• Multiple internal reflection:
IR in
IR out
• In-situ wet chemistry/electrochemistry
contact
IR in
liquid out
IR out
liquid in
electrodes
• Multiple internal transmission:
(Handbook of Vibrational Spectroscopy, Wiley, Vol.1, p. 1117, 2002)
IR in
IR out
Buried interface
LECTURE #3: Applications
October 16, 2006
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 structure and composition
Hydrophobic
Amino acids
Primary structure: Peptide
(Amino acid) chain
Secondary structure: alpha
helices, beta pleats or folds
Tertiary: Domains as shown
above
http://www.people.virginia.edu/~rjh9u/gif/aminacid.gif
Hydrophilic
amino acids
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
5:11-15, 1959. Weisel JW, Stauffacher CV, Bullitt E, Cohen C: A model for
fibrinogen: domains and sequence. Science 230:1388-1391, 1985.)
AFM
• Amide I band: C=O stretch
• Amide II band: N-H deformation coupled to C-N stretch
• Amide IV band: coupled C-N and C-O stretch
17 A
• CH stretch
11 A
300 A
• NH stretch
600 A
CHICKEN FIBRINOGEN:
Fibrinogen on mica
Fibrinogen on graphite
Marchin K. L. and Berrie C.L., Conformational changes in the plasma protein fibrinogen upon
adsorption to graphite and mica investigated by atomic force microscopy, Langmuir 19 (2003) p.9883.
Molecular Weight 54193
Number of Residues 491
R-CO-NH2
Amide II band
Amide I band
C-NH2
C=O
Functional chemical group
(olefins, esters, ethers, nitriles, thioethers,
thioesters) acids or alcohols
Germanium
Tripod attachment
Use hydrolysis of SiCl3-(CH2)16-COCl
Determination of fibronectin structure from the Amide I spectrum
-sheet
-turn