1. SPECTROPHOTOMETRY versatile and widely used analytical tool
based on how substances affect radiation (i.e., light)
advantages:
often non-destructive
can be selective
short time interval of measurement (10-14 s)

2. E = hc/ = h (photon) E = energy h = Planck's constant
c = speed of light
 = wavelength
 = frequency
E = hc/
= h
(photon)

4. chromophores exhibit unique absorption spectra
absorption maxima (max)

5. Beer-Lambert Law I = Io10-edc Io I Absorption (A) = -log(I/Io) = edc
I = light intensity
e = extinction coefficient
d = thickness
c = concentration
Absorption (A) = -log(I/Io) = edc

6. Spectrophotometer -log(I/Io) = A
photo multiplier tube (photo electric cell)

7. Beer-Lambert Law A = edc, or c = A/ed Io I
c = concentration
d = thickness (1 cm)
e = extinction coefficient
ß d à Io I
Molar Extinction Coefficient
e = A of 1 M of pure compound*
(liter/mole•cm)
E1% = A of 1% (w/v) solution*
*(under 'standard' conditions)

9. Factors Affecting Absorption
pH (ionization of chromophore)
redox state
polarity or solvent effects
orientation effects
Hypochromism of Nucleic Acids
free
nucleotides
single
stranded
double
>

10. Variations in Spectrophotometry