versatile and widely used analytical tool

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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)

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E energy h Planck's constant c speed of
light ? wavelength ? frequency
E hc/? h? (photon)
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• chromophores exhibit unique absorption spectra

• absorption maxima (?max)

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Beer-Lambert Law
I Io10-edc I light intensity e extinction
coefficient d thickness c concentration
Absorption (A) -log(I/Io) edc
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Spectrophotometer
-log(I/Io) A
photo multiplier tube (photo electric cell)
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Beer-Lambert Law
A edc, or c A/ed c concentration d
thickness (1 cm) e extinction coefficient
Molar Extinction Coefficient e A of 1 M of
pure compound (liter/molecm) E1 A of 1
(w/v) solution (under 'standard' conditions)
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Factors Affecting Absorption

• pH (ionization of chromophore)
• redox state
• polarity or solvent effects
• orientation effects

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Variations in Spectrophotometry