1 / 27

125:583 Biointerfacial Characterization Introduction to Spectroscopy Sep 28, 2006 Oct 12, 16, 2006

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

meryle
Télécharger la présentation

125:583 Biointerfacial Characterization Introduction to Spectroscopy Sep 28, 2006 Oct 12, 16, 2006

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 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

  2. LECTURE #1: Introduction

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. FTIR Surface Spectroscopy • Infrared Spectroscopy Theory • IR spectrometers Grating systems • Interferometers (FTIR) • Surface Spectroscopy Methods • Examples

  10. 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)

  11. 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

  12. LECTURE # 2:Instruments and surface spectroscopyOctober 12, 2006

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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)

  19. LECTURE #3: ApplicationsOctober 16, 2006

  20. Example 1: FTIR for biointerfacial characterization Attaching linker for biomolecule (e.g. antibody) immobilization on Silicon substrate MPS models a tiny antibody!

  21. Step 2: Formation of Urea linkage during PMPI attachment

  22. Step 3: Formation of succinimide (evidence for thioether bonding) during MPS attachment

  23. Example 2: Fibrinogen immobilization Primary structure: Peptide (Amino acid) chain Secondary structure: alpha helices, beta pleats or folds Tertiary: Domains as shown above

  24. 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

  25. 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.

  26. 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

  27. Determination of fibronectin structure from the Amide I spectrum -sheet -turn

More Related