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 (l_s = l_i) R_G, CD, ORD

Raman scattering– inelastic (l_s ¹ l_i)

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, I_o); Transmittance (T = I / I_o)

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

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

A = O.D. = e · c · l also [ E^1% · MW = 10 · e_M ]

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 ( e_{free base} > e_ss > e_ds ) 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]