Lecture 3011/14/05

Spectrophotometry

Properties of Light

~hchc

hE

c

h = 6.626 x 10-34 J-s

c = 3.00 x 108 m/s

Transmittance

Absorbance TlogP

PlogA

P

PT

0

0

P = Irradiance (Intensity) = energy per second per area of light

Beer’s Law

A=bc

= extinction coefficient or molar absorptivity

b = pathlengthc = concentration

Recap

Absorbance Specific wavelengths of light UV/Vis: electronic transitionelectronic transition IR: VibrationsVibrations

Beer’s Law For Quantitation

Beer’s Law

Monochromatic light

Dilute solutions

IC: Internal conversion

ISC: Intersystem crossing

Luminescence

Fluorescence Emission of photon during transition between

S1 S0

Phosphorescence Emission of photon during transition between

T1 S0

Luminescence

More sensitive than absorption Lower energy (higher wavelength) than the

energy absorbed

IC: Internal conversion

ISC: Intersystem crossing

I = kP0c

Excitation spectrum vs. emission spectrum

Analysis of a Mixture

A = Xb[X] + Yb[Y] + Zb[Z] + . . . .

Spectra overlap

1. Constant concentration of both analytes1. Find at different

b[Y]

A ε

b[X]

Aε Y

YX

X

2. Least squares to find best values of [X] and [Y]1. Am = Xb[X] + Yb[Y]

2. Acalc = Xb[X]guess + Yb[Y]guess

Spectra not-overlapping

A’ = ’Xb[X] + ’Yb[Y] at ’

A’’ = ’’Xb[X] + ’’Yb[Y] at ’’

`

)cb()da(dc

ba

bb

bb

Ab

Ab

]Y[

bb

bb

bA

bA

]X[

''Y

''X

'Y

'X

''''X

''X

''Y

''X

'Y

'X

''Y

''

'Y

'

Recap

Absorbance Specific wavelengths of light UV/Vis: electronic transitionelectronic transition IR: VibrationsVibrations

Beer’s Law For Quantitation

Beer’s Law

Monochromatic light

Dilute solutions

IC: Internal conversion

ISC: Intersystem crossing

Luminescence

Fluorescence Emission of photon during transition between

S1 S0

Phosphorescence Emission of photon during transition between

T1 S0

Luminescence

More sensitive than absorption Lower energy (higher wavelength) than the

energy absorbed

IC: Internal conversion

ISC: Intersystem crossing

I = kP0c

Excitation spectrum vs. emission spectrum

Analysis of a Mixture

A = Xb[X] + Yb[Y] + Zb[Z] + . . . .

Spectra overlap

1. Constant concentration of both analytes1. Find at different

b[Y]

A ε

b[X]

Aε Y

YX

X

2. Least squares to find best values of [X] and [Y]1. Am = Xb[X] + Yb[Y]

2. Acalc = Xb[X]guess + Yb[Y]guess

Spectra not-overlapping

A’ = ’Xb[X] + ’Yb[Y] at ’

A’’ = ’’Xb[X] + ’’Yb[Y] at ’’

`

)cb()da(dc

ba

bb

bb

Ab

Ab

]Y[

bb

bb

bA

bA

]X[

''Y

''X

'Y

'X

''''X

''X

''Y

''X

'Y

'X

''Y

''

'Y

'

Recap

Absorbance Specific wavelengths of light UV/Vis: electronic transitionelectronic transition IR: VibrationsVibrations

Beer’s Law For Quantitation

Beer’s Law

Monochromatic light

Dilute solutions

IC: Internal conversion

ISC: Intersystem crossing

Luminescence

Fluorescence Emission of photon during transition between

S1 S0

Phosphorescence Emission of photon during transition between

T1 S0

Luminescence

More sensitive than absorption Lower energy (higher wavelength) than the

energy absorbed

IC: Internal conversion

ISC: Intersystem crossing

I = kP0c

Excitation spectrum vs. emission spectrum

Analysis of a Mixture

A = Xb[X] + Yb[Y] + Zb[Z] + . . . .

Spectra overlap

1. Constant concentration of both analytes1. Find at different

b[Y]

A ε

b[X]

Aε Y

YX

X

2. Least squares to find best values of [X] and [Y]1. Am = Xb[X] + Yb[Y]

2. Acalc = Xb[X]guess + Yb[Y]guess

Spectra not-overlapping

A’ = ’Xb[X] + ’Yb[Y] at ’

A’’ = ’’Xb[X] + ’’Yb[Y] at ’’

`

)cb()da(dc

ba

bb

bb

Ab

Ab

]Y[

bb

bb

bA

bA

]X[

''Y

''X

'Y

'X

''''X

''X

''Y

''X

'Y

'X

''Y

''

'Y

'