Spectroscopy / Fluorescence

 

1. Review

        A. Nature of light

                Electromagnetic wave – moving electric and magnetic force field

               E = hn = hc/l  (different names depending on energy or wavelength: for l in cm)

                 3000 (radio) / 0.3 (microwave) / 0.003 (IR) / 0.00003 (UV) / 0.00000003 (X-ray)

B. Effect of Light on Molecules -  vision / photosynthesis / radiation therapy

C. Effect of Molecules on “Light” –

   Absorption / Fluorescence / X-ray / Light Scattering / CD / ORD / UV / NMR

 

   Interaction of Light with Matter (induce oscillating dipoles in matter)

         a) Scattered –         (~ 10-16 sec )

                 Rayleigh scattering (ls = li)   RG,  CD,  ORD

                 Raman scattering–  inelastic (ls ¹ li)

           b) Absorption - (~ 10-15 sec ) – due to “transition” from one energy level to another

                 Since absorption is fast relative to molecular motions

                    – insensitive to molecular motions  

 

2. Absorption Spectrum – “fingerprint”

Beer-Lambert Law:           Absorbance (A); Intensity (I, Io);  Transmittance (T = I / Io)

                                         A = log (Io / I) = log (1/T)

                            Extinction Coefficient – E (1%),  eM  = Molar extinction coeff.

                    A = O.D. =  e · c · l       also     [  E1% · MW  =  10 · eM ] 

   Proteins:           A280 ;  E (1%) ~ 10 (or O.D. of 1 for 1 mg/mL)

   Nucleic Acids:  A260 ;  E (1%) ~ 200 (or O.D. of 1 for 50 mg/mL)

 

3. Environmental Effects

                lnonpolar  >  lpolar  (folding / unfolding effect)

               DNA – Helix-Coil Transitions (  efree base  >  ess  >   eds  ) follow denaturation

 

4. Instrumentation

        Light Source - Monochromator (filter, prism, grating) - Slit - Cuvette -Detector (PM tube)

 

5. Fluorescence  /  Phosphorescence

Fluorescence (~ 10-4 sec to 10-9 sec )   /   Phosphorescence  (> 10-3 sec )

           Fluorescence signal is very sensitive to environment and molecular changes.

Instrumentation:  Measure fluorescence perpendicular to light path and at different l

                            --> very low background

Resonance Energy Transfer – needs “spectral overlap”

          (Efficiency can be used to estimate distance from donor to acceptor.)

 

   R = Ro[(1-e)/e]1/6  or  FRET (Fluor. Res. Energy Transfer) Eff.  =  1/[1 + (R/Ro)6]