125:583 biointerfacial characterization introduction to spectroscopy sep 28, 2006 oct 12, 16, 2006...
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125:583Biointerfacial Characterization
Introduction to Spectroscopy
Sep 28, 2006Oct 12, 16, 2006
Yves ChabalDepartments of Chemistry and Chemical Biology, and Biomedical Engineering
Nanophysics Lab, Room 205 [email protected]
Prabhas MogheDepartments of Chemical Engineering, and Biomedical Engineering
Spectroscopy
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
Spectrum: A plot of the intensity as a function light or particle energy (frequency, wavelength)
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.
Basics of Light, E&M Spectrum, and X-rays
Materials responseto radiation or particles
Valence electrons
Core electrons
Atoms/molecules
• E&M radiation interacts with materials because electrons and molecules in materials are polarizable:
•(refraction, absorption)ñ= n+ i k
n = refraction, k = absorption
• Ions, electrons and atoms incident on materials can interact with materials becausethey are either charged or can scatter from atomic cores
Techniques and information content
MolecularLibration
(hindered rotations)
Molecularvibrations
Electronic Absorption
Valence band and shallow electronic
levels (atoms)
Deep electronic core levels
(atoms)
Microwave,THz
Infrared,Raman,EELS
VisibleFluorescenceLuminescence
UV absorptionUV photoemission
Electron lossX-ray photoemission
(XPS, ESCA)Auger Electron (AES)
Photoelectron Spectroscopy
Photons in Electrons out
Core electrons
Vacuum level
Valence 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
Photons out
• Large penetration into solid• Low energy photons Non destructive • Can interact linearly (absorption) or non-linearly (Raman, harmonic generation)
FTIR Surface Spectroscopy
• Infrared Spectroscopy Theory
• IR spectrometers Grating systemsInterferometers (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)
scissoring
s(CH2)
rocking
(CH2)wagging
(CH2)
twisting
(CH2)
Bending modes -CH2- x
Grating or prism spectrometer
Selects one wavelength (energy) at a time, requiring rotation to scan the spectrumArray 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
Source
Interferometers
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
http://www.wooster.edu/chemistry/is/brubaker/ir/ir_works_modern.html
Michelson Interferometer
Detect IR intensity as a function of mirror displacement: INTERFEROGRAM
(broadband)
Fourier-Transform Infrared spectroscopy
For a single frequency (i.e. laser light), the signal on the detector (interferogram) is a sine wave
As more frequencies are added, the interferogrambecomes a more complex function, with the largest
amplitude at the zero path difference (zpd)
500 1000 1500 2000 2500 3000 3500
0
5
10
15
20
25
Absorbance
Wavenumber (cm-1)
Spectrum
For a broad spectral range (white light),The interferogram is most peaked at zpd
FT
Interferogram
Waveforms Mirror displacement
wavenumber
http://www.wooster.edu/chemistry/is/brubaker/ir/ir_works_modern.html
400 cm-1 - 4000 cm-1
25000 nm - 2500 nmcf== λ 1~
Surface and Interface Spectroscopy
500 1000 1500 2000 2500 3000 3500
0
5
10
15
20
25
Absorbance
Wavenumber (cm-1)
Initial state (reference)
SiO2+Si
500 1000 1500 2000 2500 3000 3500
0
5
10
15
20
25
Absorbance
Wavenumber (cm-1)
SiH+Si
Final state
500 1000 1500 2000 2500 3000 3500 4000
-0.006
-0.004
-0.002
0.000
0.002
0.004
0.006
Absorbance
Wavenumber (cm-1)
SiH added
SiO2 removed
etching
Si(111) Si(111)
Reprocessing:Subtraction of reference spectrum from final state spectrum
IR wavelength (~ m) is much larger than surface dimensions (nm) Need to Eliminate all other contributions to spectrum (selecting a reference system)
Maximizing Surface Interaction
IR in
IR out
Transmission
Need double-sided polish + bevels at sidesIn-situ possible for liquid environments
IRout
IR in
Multiple internal Reflections
Evanescent field ~ 1-10 m
1. For highly absorbing or reflecting (metal) substrates
grazing incidence reflectiontan (B) = ñ
2. For weakly absorbing substrates “Brewster” incidence transmission tan (B) = n
3. For transparent substrates Multiple internal reflections int ~ 45o
IR in IR out
Reflectionn and k large
k small
n large (2-4)k very small
int
Attenuated Total Reflection (ATR)
IR in IR out
• Multiple internal reflection:
IR in IR outBuried interface
• Multiple internal transmission:(Handbook of Vibrational Spectroscopy, Wiley, Vol.1, p. 1117, 2002)
liquid inliquid out
electrodes
contact
IR outIR in
• In-situ wet chemistry/electrochemistry
Example 1: FTIR for biointerfacial characterization
Attaching linker for biomolecule (e.g. antibody) immobilization on Silicon substrate
MPS models a tiny antibody!
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
http://www.people.virginia.edu/~rjh9u/gif/aminacid.gif
Hydrophobic
Amino acids
Hydrophilic amino acids
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
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.)
Fibrinogen on mica Fibrinogen on graphite
17 A
300 A
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.
11 A
600 A
AFM
IR bands present in all protein backbones• 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
• CH stretch
• NH stretch
Minor Axis60 – 90 A Peptide chain in solution
(R1, R2, R3, R4: Amino Acid Residues)http://bio.winona.msus.edu/berg/ChemStructures/Polypep2.gif
Major Axis
CHICKEN FIBRINOGEN:
Molecular Weight 54193
Number of Residues 491
Germanium
Tripod attachment
Functional chemical group (olefins, esters, ethers, nitriles, thioethers, thioesters) acids or alcohols
Use hydrolysis of SiCl3-(CH2)16-COCl
Amide I bandC=O
Amide II bandC-NH2
R-CO-NH2