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 yves@agere.rutgers.edu

Prabhas MogheDepartments of Chemical Engineering, and Biomedical Engineering

LECTURE #1: Introduction

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

LECTURE # 2: Instruments and surface spectroscopy

October 12, 2006

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

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

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

Determination of fibronectin structure from the Amide I spectrum

-sheet

-turn