125 583 biointerfacial characterization introduction to spectroscopy sep 28 2006 oct 12 16 2006 n.
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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 yves@agere.rutgers.edu Prabhas Moghe

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125 583 biointerfacial characterization introduction to spectroscopy sep 28 2006 oct 12 16 2006
125:583Biointerfacial CharacterizationIntroduction to SpectroscopySep 28, 2006Oct 12, 16, 2006

Yves Chabal

Departments of Chemistry and Chemical Biology, and Biomedical Engineering

Nanophysics Lab, Room 205 yves@agere.rutgers.edu

Prabhas Moghe

Departments of Chemical Engineering, and Biomedical Engineering

spectroscopy
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

slide4

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

Atoms/molecules

Valence electrons

Core electrons

Materials responseto 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

  • 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

Libration

(hindered rotations)

Molecular

vibrations

Electronic

Absorption

Valence band and shallow electronic levels (atoms)

Deep electronic core levels (atoms)

Infrared,

Raman,

EELS

UV absorption

UV photoemission

Electron loss

Microwave,

THz

Visible

Fluorescence

Luminescence

X-ray photoemission

(XPS, ESCA)

Auger Electron (AES)

Techniques and information content
photoelectron spectroscopy

Photons in

Electrons out

Photoelectron Spectroscopy

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
Optical Spectroscopy

Photons in Photons out

  • Large penetration into solid
  • Low energy photons  Non destructive
  • Can interact linearly (absorption)
  • or non-linearly (Raman, harmonic generation)

Photons out

slide9

FTIR Surface Spectroscopy

  • Infrared Spectroscopy Theory
  • IR spectrometers Grating systems
  • Interferometers (FTIR)
  • Surface Spectroscopy Methods
  • Examples
classical theory for linear absorption
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

asym. stretching

as(CH2)

sym. stretching

s(CH2)

Stretching modes -CH2-

Bending modes -CH2-

x

rocking

(CH2)

scissoring

s(CH2)

wagging

(CH2)

twisting

(CH2)

Examples
grating or prism spectrometer
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
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

Interferogram

FT

Mirror displacement

Waveforms

For a broad spectral range (white light),

The interferogram is most peaked at zpd

Fourier-Transform Infrared spectroscopy

Spectrum

wavenumber

400 cm-1 - 4000 cm-1

25000 nm - 2500 nm

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

surface and interface spectroscopy

Final state

SiH+Si

SiH added

SiO2 removed

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)

Initial state (reference)

SiO2+Si

Si(111)

Si(111)

etching

Reprocessing:

Subtraction of reference spectrum from final state spectrum

maximizing surface interaction

IR in

IR out

n and k large

Reflection

IR in

IR

out

Transmission

Multiple internal Reflections

Evanescent field ~ 1-10 m

IR in

IR out

Maximizing Surface Interaction
  • For highly absorbing or reflecting (metal) substrates
  • grazing incidence reflection

tan (B) = ñ 

k small

  • 2. For weakly absorbing substrates
  • “Brewster” incidence transmission

tan (B) = n

n large (2-4)

k very small

int

  • 3. For transparent substrates
  • Multiple internal reflections

int ~ 45o

Need double-sided polish + bevels at sides

In-situ possible for liquid environments

slide18

contact

IR out

IR in

IR in

IR out

liquid in

liquid out

electrodes

IR in

IR out

Buried interface

Attenuated Total Reflection (ATR)

  • Multiple internal reflection:
  • In-situ wet chemistry/electrochemistry
  • Multiple internal transmission:
  • (Handbook of Vibrational Spectroscopy, Wiley, Vol.1, p. 1117, 2002)
example 1 ftir for biointerfacial characterization
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
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

Hydrophilic amino acids

Fibrinogen structure and composition

http://www.people.virginia.edu/~rjh9u/gif/aminacid.gif

Primary structure: Peptide (Amino acid) chain

Secondary structure: alpha helices, beta pleats or folds

Tertiary: Domains as shown above

fibrinogen size and structure
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
  • 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

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

17 A

11 A

300 A

600 A

CHICKEN FIBRINOGEN:

Molecular Weight 54193

Number of Residues 491

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.

slide26

R-CO-NH2

Amide II band

C-NH2

Amide I band

C=O

Functional chemical group

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

Germanium

Tripod attachment

Use hydrolysis of SiCl3-(CH2)16-COCl