Infra-Red and Raman Spectroscopy

Both measure vibrational energy levels of chemical bonds

I. Infra-Red

A. Wavelength/Energy Bands

  1. near IR: 37 - 10 kcal/mole -- no instrumentation available
  2. middle IR: 10-1 kcal/mole--this is the energy we're concentrating on
  3. far IR: 1-0.1 kcal/mole

B. Terminology

  1. wavenumber ==> n = 1/l l in µ (microns)
  2. Vibration modes
    1. Stretching: symmetric and asymmetric
    2. Bending
    3. Internal rotation about single bonds

  3. Energy values are quantized ==> in order to absorb radiation in going from one energy state to another, there must be a change in dipole moment ==> Selection Rule ==> Infra-Red Active
    Symmetric molecules:
    H-H; NN; Br-Br not IR active because dipole does not change with stretching
  4. 6.

C. The larger the change in Dipole Moment, the stronger the absorption (and vice versa)

  1. Triatomic
  2. Hooke's Law: classical physical description of energy involved; described as two masses connected by a spring

n = vibrational frequency in cm-1; c = velocity of light; m1 & m2 - masses of atoms; f = force constant

Bond Type

f (Force Constant )


5 x 105 dyne/cm


10 x 105 dyne/cm


15 x 105 dyne/cm

n related inversely to atomic masses
for C-H f = 5 x 10
5 dyne/cm ==> n = 3022 cm-1 which is approximately correct

D. Instrumentation

  1. Dispersive--same as for UV/Vis spectrometer with different optic materials which are IR transparent
    Media--generally avoid H
    2O which has a rich IR spectrum that might obscur
    1. solution -- Cl4, CHCl3
    2. solid -- 1% in KBr --> transparent pellet or as film dried between polished disks of salt
    3. gas -- sample as gas
    4. liquid -- liquid sample between 2 salt disks
  2. Fourier Transform IR -- FTIR

    Advantages over dispersive

    • all l's are measured simultaneously
    • complete spectrum obtained rapidly
    • average many scans
    • FTIR achieves same S/N as dispersive in much less time
    • He-Ne laser calibrates frequency scale --> v. accurate
    • negligible stray light
    • resolution the same at all l (dispersive resolution varies with l )
    • no grating or filter changes during scanning

D. Uses

  1. Identification of substances
  2. Determination of molecular structure
  3. Determination of purity, quantitative analysis, product control
  4. Reaction kinetics
  5. Quantitative Analysis -- Beer's Law

E. Protein Structure--measure secondary structure by looking at peptide backbone frequencies--Amid bonds.

  1. Amide II band is very weak
  2. Amide I and Amide III are strong and depend on the dihedral angles Y and f ==> secondary structure

wavenumber range



Secondary Structure


Amide I


beta sheet


Amide I


random coil


Amide I


alpha helix


Amide III


alpha helix


Amide III


random coil


Amide III


beta sheet

F. Dichroism

  1. Illuminate with Plane Polarized IR--bonds oriented parallel to Electric Vector absorb prefferentially
  2. Orient sample--e.g. membranes or filamentous molecules by drying
  3. Analysis of Amide I and III band dichroism gives not only measures amount of secondary structure but also its orientation; e.g. tilt of transmembrane alpha helices with respect to the plane of the membrane

II Raman

A. Measures scattered photons which have transferred part of their energy to excite the moledule to a higher vibrational energy level

  1. This occurs with very low probability; thus the proportion of scattered photons which have lost energy is low (only 1-2%) and the Raman spectrum is very weak ==> requires very intense radiation (i.e. laser) and concentrated samples.
  2. With even lower probability a photon may gain the amount of energy transferred when it interacts with a molecule in a higher vibrational energy level; this probability is lower because fewer molecules are in higher vibrational energy levels
  3. Light is in the visible spectrum and the exact wavelength doesn't matter, but scattered light samples vibrational energy levels ==> information similar to that provided by IR
  4. Samples can be dissolved in H2O since the Raman spectrum will not be obscurred by H2O absorption as in the IR.
  5. Instrumentation:
    1. High powered monochromatic light source--laser--shined on sample
    2. light scattered at 90° with respect to incident light is collected by a curved mirror and focused onto the monochromator
    3. mochromator selects each wavelength of light and measures intensity; note that the wavelength of scattered light is in the same region as the incident light--e.g. visible--but the Raman spectrum is plotted as frequency shifts from the incident light and, hence, looks like an IR spectrum
  6. Experimental limitations
    1. causes heating-->need efficient cooling
    2. sample shouldn't absorb light
    3. fluorescence must be low ==> pulse laser at times <<fluorescece lifetime

B. The shifts in frequence observed in Raman Spectroscopy do not always correspond to observed IR absorption bands because the only requirement for a Raman band is that the chemical bond must be polarizable while IR absorption requires a change in dipole. Thus, symmetric molecules have Raman spectra but not many IR bands

C. Applications

  1. Protein Secondary Structure--as described above for IR including dichroism
  2. The Raman MicroProbe: Raman spectrometer coupled to a microscope
    1. Beam: 1 µm2 examines a surface
    2. Point Mode: look at a single 1 µm2 region and record a Raman spectrum
    3. Global Mode: look at a larger area

D. Resonance Raman: looks at virtual states very close to an electronic state ==> Raman spectrum becomes very intense