SPECTROPHOTOMETRY versatile and widely used analytical tool

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Presentation transcript:

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)

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

chromophores exhibit unique absorption spectra absorption maxima (max)

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

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

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)

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

Variations in Spectrophotometry