Spectroscopy

Spectroscopy

1. Light is an electric and magnetic field and has wave like and particle like properties (quanta)

   • Speed(c)=3 billion m/s
     • Frequency=c/wavelength

2. Energy=Planks constant(6.62*10^-32Js)* Frequency

   • Longer wavelength means lower energy
     • WAVE NUMBER(cm-1)= 1/wavelength(m or nm)
   • Infared has lower energy than ultraviolet
   • Ground state to excited state= E1-->E2, this has discrete intervals (at some frequencies there is no absorption)
   • There are different energy levels
     • Vibrational
       • There is stretching and bending
       • This is in the infared part of the spectrum
     • Rotational
       • Single covalent bonds can rotate
       • Energy increments here are smaller than vibrational so correspond to microwave part
     • electron spin orientation
       • Microwaves also change electron spin
     • Nuclear spin orientation
       • Related to radio waves

3. x-rays cause electrons to jump from ground state and is then ejected

4. INFARED

   • The C=O may stretch and bend ( stretching occurs at 1600cm-1 and bending at 800cm-1), these may cause differences in bond length and angle. There are also twisting vibrations (happens between torsion angles-phi/psi)
   • The more bonds there are the lower the frequency, so a double bond has a low frequency
   • An infared plot is absorbance versus frequency/ wave number
     • Water gives a strong absorption so need to be removed it is replaced by deuterated water which replaces H with D which is much heavier. You can also just do it in an non aqueous solution (dry films) or a KBr disk
       • As it can study non aqueous solutions insoluble membrane proteins can also be studied
       • The protein should be 10mg/ml, 10 microlitre path length
       • Can identify is prion protein has been mutated as the mutated form is a insoluble and has a b-sheet structure opposed to it's original a-helix
   • Most infrared of a protein comes from its backbone producing different AMIDE BANDS which are associated with the hydrogen bonds
     • Amide band 1-associated with stetching of c=o -1690 to1600cm-1
     • Amide band 2- due to N-H (1575-1480cm-1) bending and C-H stretching (1575-1480cm-1)
     • The peak postions depend on the 2 structure
       • a-helix-1648-1657cm-1
       • b-sheet:1640-1620cm-1)
     • Amide A- due to N-H stretch (3300-3100cm-1)
     • Amide bands measure the secondary structure , FTIR measures the different structures.
       • The IR spectra has a helix accuracy of +/- 4% and b-sheet accuracy of +/-8%
   • The picture shows a Michelson interferometer ot FTIR
     • The crystal will transmit half the light and reflect the other half- this is the BEAM SPLITTER. This is parallel to a mirror. There is also a moving mirror where the trasmitted beam shines. The beams reflected by the mirrors combine to make a RESULTANT BEAM
       • If the 2 beams are in phase it will make constructive interferance , if they're not it will be destructive

5. UV also called Electron absorption spectroscopy

   • A SPECTROPHOTOMETER measures UV. A deutrium light source is needed for UV, a tungsten halogen lamp is needed for UV in the vsible range. A MONOCHROMATER scans the wavelengths from the lamp to select ONE, this is a DIFFRACTION GRATING.Transmitted light then goes to the photodetector which coverts light intensity to an elctrical signals by amplifying it
     • The cuvette with the sample should be made of quarts glass if absorbing UV
     • Incident light is light before it hits the sample
     • Transmittance=Transmitted light/Incident light
       • Absorbance=log10 1/transmittance
         • If the absorbance is below 1 it is very dark. If the absorbance is between 1 and 1.5 it's a good sample
         • BEER LAMBERT LAW: Extinction coefficient(M-1cm-1)* concentration(Molar) *Path length (usually 1cm)
           • The extinction coefficient can usually be found as the slope on a graph of absorbance against concentration
             • The exctinction coefficent at 280nm= total of them for tryptophan, Tyrosine and disulphide bonds

6. Fluorescence

   • Light is absorbed and then emmitted
     • Electrons go from an excited to ground state
     • The emmisions have longer wavelengths than excitations and the energy gap is smaller
   • PHOSPHORESCENCE=emission from a triplet excited state,

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