Chapter 17: Fundamentals of Spectrophotometry

HW: To be posted after class

Electromagnetic Radiation
-rays

0.001 - 0.01 nm Nuclear transitions
X-rays .01 -10 nm

Core e
-
s
Ultraviolet 10 - 400 nm

Valence e
-
s
Visible 400 - 800 nm

Valence e
-
s
Infrared 800 - 10
6
nm

Molecular Vibrations
Microwave 10
6
- 10
8
nm Molecular Rotations
Radio 10
8
- 10
11
nm Nuclear Spin

The Visible Spectrum
The visible spectrum
400 nm 750 nm
IR
UV
Energy/Photons
-rays

10
-14
J/photon
X-rays 10
-16
J/photon
Ultraviolet 10
-18
J/photon
Visible 10
-19
J/photon
Infrared 10
-20
J/photon
Microwave 10
-22
J/photon
Radio 10
-24
J/photon

Absorption Energy Levels
E
N
E
R
G
Y
Electronic level
Rotational level
Vibrational level
AE = UV/Vis photon
AE = IR photon
AE = wave photon
E
N
E
R
G
Y
Absorption Energy Levels
What happens to excited molecule?
1. UV-Vis Absorption: from ground state (S
0
) to
excited state (S
1
)
10
-15
seconds
S
0
T
1
: Spin-Forbidden (low probability)
2. Vibrational Relaxation: excited molecule bumps into
solvent, drains some E (radiationless transition)
10
-12
seconds
Cascade down manifold of excited vibrational
levels (not electronic)
3. Internal Conversion: radiationless transition between
states with the same spin quantum number (S
1
to S
0
)
Fluorescence/Phosphorescence
4. Fluorescence: the emission of radiation from excited
state to ground state (into range of vibrational states)
10
-6
to 10
-10
seconds
5. More internal conversion: non-radiant relaxation
(spreading out energy/heat)

6. Intersystem crossing: radiationless transition between
states with different spin quantum numbers (S
1
to T
1
)

7. Phosphorescence: emission of radiation from T
1
to S
1
)
> 10
-6
seconds (very long: different spins!)
Luminescence
For fluorescence the intensity is
I = KP
0
2.3cbc
Measure absolute number of photons
I is DEPENDENT on P
0

K depends upon:
efficiency of fluorescence
light collection efficiency

1. Significant overlap across
the entire spectrum.

2. Regions of no overlap

A
X
Y

A
X
Y
] Y [ b ] X [ b A
Y X m
c + c =
Know that absorbance of mixture of X and Y at
any is

Prepare standard solutions of X and Y
] Y [ b ] X [ b A
Y X m
c + c =
S X X
] X [ b A
S
c =
S Y Y
] Y [ b A
S
c =
S
X
X
b[X]
A

S
=
S
Y
Y
b[Y]
A

S
=
Case 1
S X
Y
S X
m
] X [
] X [
A
A
] Y [
] Y [
A
A
S
S
S
+
|
|
.
|

\
|
=
b[Y]
b[Y]
A
b[X]
b[X]
A
A
S
Y
S
X
m
S S
+ =
S
Y
S
X
m
[Y]
[Y] A
[X]
[X] A
A
S S
+ =
S X
Y
S X
m
] X [
] X [
A
A
] Y [
] Y [
A
A
S
S
S
+
|
|
.
|

\
|
=
y = m x + b
Case 1
How do we get enough data points to construct a line?
Case 1
S
X
m
A
A
S
S
X
Y
A
A F
i
n
d

[
Y
]

f
r
o
m

s
l
o
p
e
Find [X] from y-intercept
S
X
m
A
A
S
S
X
Y
A
A F
i
n
d

[
Y
]

f
r
o
m

s
l
o
p
e
Find [X] from y-intercept
S X
Y
S X
m
] X [
] X [
A
A
] Y [
] Y [
A
A
S
S
S
+
|
|
.
|

\
|
=
i. measure absorbance at
max
for X
ii. measure absorbance at
max
for Y

A
X
Y
'
''
] Y [ b ] X [ b A
Y X
c
'
+ c
'
=
'
] Y [ b ] X [ b A
Y X
c
' '
+ c
' '
=
' '
Case 2
Isosbestic points
Analytical useful if one analyte (X) can be converted to
another (Y) and/or vice versa.

Examples:
X = B (base) and Y = HB
+
(conjugate acid)
X = HA (acid) and Y = A
-
(conjugate base)
Characteristic feature of mixture of X and Y Abs spectra
REQUIREMENT:
Absorbance spectra of pure X and pure Y must cross
Absorbance spectra of separate solutions of
HA and A
-
prepared at same concentration
HA
A
-
] HA [ b A
465
HA
465
HA
c =
] A [ b A
465
A
465
A

c =
465
A
465
HA

c = c
Vary pH of a solution to vary principal species
pK
a
= 5.1

HA A
-
+ H
+

Will these peaks necessarily be equal?
] A [ b ] HA [ b A
465
A
465
HA
465
m

c + c =
465
A
465
HA

c = c
]) A [ ] HA ([ b A
465 465
m

+ c =
Mass balance: ([HA] + [A
-
]) = constant
The Absorbance at 465 is constant!
Other analytical uses:
Monitor progress of titration (equivalence point) by
following absorbance.
Useful in situations where analyte and product
have very different c values at a given

Example: analyte has color but product does not,
or vice versa.

Due to large number of vibrational states,
IR spectra can be quite complex
IR Absorption: real strength is ID
Functional Group Wavenumber
(cm
-1
)
Wavelength
(m)
C-H, aliphatic 3000-2850 3.3-3.5
C-H, aromatic 3150-3000 3.2-3.3
O-H 3600-3000 2.8-3.3
C=O,
aldehyde/ketone
1740-1660 5.7-6.0
-CH
2
Cl 1300-1200 7.6-8.2
C-C 850-890 13.2-14.0