coefficient extinction molar riboflavin

Modern Optical
Spectroscopy
http://www.scientific-web.com/en/Books/ModernOpticalSpectroscopyWithExercises.html
Introduction & the BeerLambert Law
Shu-Ping Lin, Ph.D.
Institute of Biomedical Engineering
E-mail: [email protected]
Website: http://web.nchu.edu.tw/pweb/users/splin/
Energy Absorption
The mechanism of absorption energy is different in the
Ultraviolet, Infrared, and Nuclear magnetic resonance regions.
However, the fundamental process is the absorption of certain
amount of energy.
The energy required for the transition from a state of lower
energy to a state of higher energy is directly related to the
frequency of electromagnetic radiation that causes the transition.
Wave Number (cycles/cm)
X-Ray
UV
200nm
Visible
400nm
IR
800nm
Wavelength (nm)
Spectral Distribution of Radiant Energy
Microwave
Region of the Electromagnetic Spectrum
Electromagnetic Radiation
V = Wave Number (cm-1)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
u = Frequency of Radiation (cycles/sec)
u
C
V =
=

l
The energy of photon:
h (Planck's constant) = 6.62 x 10- (Ergsec)
27
E = h u= h
C
l
u =
C
l
C = ul
Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Wave
Number V
Energy
Kcal/mol
9.4 x 107
9.4 x 103
9.4 x 101
eV
4.9 x 106
4.9 x 102
4.9 x 100
cm-1
3.3 x 1010
3.3 x 106
3.3 x 104
Wavelength
λ
cm
3 x 10-11
3 x 10-7
3 x 10-5
Frequency
υ
Type
Radiation
Type
spectroscopy
Gamma
ray
Gamma ray
emission
Hz
1021
1017
X-ray
1015
Ultra
violet
9.4 x 10-1
4.9 x 10-2
3.3 x 102
3 x 10-3
1013
9.4 x 10-3
4.9 x 10-4
3.3 x 100
3 x 10-1
1011
4.9 x 10-8
3.3 x 10-4
3 x 103
107
Infrared
Nuclear
X-ray
absorption,
emission
Electronic
(inner shell)
UV absorption
Electronic
(outer shell)
Visible
9.4 x 10-7
Type
Quantum Transition
IR absorption
Microwave
Microwave
absorption
Radio
Nuclear
magnetic
resonance
Molecular
vibration Molecular
rotation
Magnetically
induced spin
states
Dispersion of Polymagnetic Light
with a Prism
Prism - Spray out the spectrum and choose the certain wavelength
(l) that you want by slit.
Infrared
monochromatic
Ray
Polychromatic
Ray
PRISM
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
Polychromatic Ray
Monochromatic Ray
SLIT
Electronic Spectroscopy
Ultraviolet and visible
Where in the spectrum are
these transitions?
Why should we learn this stuff?
After all, nobody solves structures
with UV any longer!
Many organic molecules have chromophores that absorb UV
UV absorbance is about 1000 x easier to detect per mole than NMR
Still used in following reactions where the chromophore changes
(useful)  because timescale is so fast, and sensitivity is very high.
Kinetics, esp. in biochemistry, enzymology.
Most quantitative Analytical chemistry in organic chemistry is conducted
using HPLC with UV detectors
One wavelength may not be the best for all compound in a mixture.
Affects quantitative interpretation of HPLC peak heights
Ultra Violet Spectrometry
The absorption of ultraviolet radiation by molecules
is dependent upon the electronic structure of the
molecule.
So the ultraviolet spectrum is called electronic
spectrum.
The absorption of light energy by organic
compounds in the visible and ultraviolet region
involves the promotion of electrons in , , and norbitals from the ground state to higher energy
states. This is also called energy transition. These
higher energy states are molecular orbitals called
antibonding.
n


n *
Antibonding
n *
*
*
*
Antibonding
 *
Energy
Electronic Molecular Energy Levels
Nonbonding
Bonding
Bonding
The higher energy transitions ( *) occur a
shorter wavelength and the low energy
transitions (*, n *) occur at longer
wavelength.
Spectrophotometer
An instrument which can measure the absorbance
of a sample at any wavelength.
Light
Lens
Sample
Slit
Detector
Monochromator
Quantitative Analysis
Slits
Fluorometer
Instrument to measures the intensity of fluorescent light emitted by a
sample exposed to UV light under specific conditions.
Emit fluorescent light
as energy decreases
Antibonding
'
'
Antibonding
n->' n-> '
Nonbonding
n
 -> '

Bonding
 ->'
Energy

Bonding
Ground state
Electron's molecular energy levels
UV Light Source
Monochromator
90° C
Sample
Detector
Monochromator
Spectrophotometry
Key Concepts
• Lambert’s Law of Absorption
• Beer’s Law
• Beer-Lambert Law
• Absorption Cross-Sections
• Photometric quantities
• Spectrophotometer
• The Cary 50 Spectrophotometer
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Lambert‟s Law of Absorption
Lambert described how intensity changes with distance
in an absorbing medium.
The intensity I0 if a beam of light decreases exponentially as it passes though
a uniform absorbing medium with the linear decay constant α.
Restatement: In a uniform absorbing medium, the intensity of a
beam of light decreases by the same proportion for equal path
lengths traveled.
The linear decay constant α is a characteristic of the medium. It has units of
reciprocal length. α is the path length over which the intensity is attenuated
to 1/e.
l
α
I0
I = I 0e- l
I
I ( x) = I 0e- x
I(x)
The distance traveled through the medium is
called the path length.
d I = - I d x
I = I 0e
x
Johann Heinrich Lambert
1728-1777
- x
dI
= - I
dx
I
- x
=e
I0
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Lambert’s Law of Absorption
(base 10)
Typically base 10 is used in photometry.
I = I 0e
- x
= I 010
-kx
k =  ln 10
I
- x
-kx
= e = 10
I0
k is the path length over which the intensity is attenuated to 1/10.
I
= 10 - k x
I0
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Lambert’s Law Example
If one slab of absorbing material of thickness l reduces the intensity of a beam of light
to half.
l
α
I0
I
1
= 10 - k l =
I0
2
I
Then two slabs of the same absorbing material will then reduce the
intensity of a beam of light to one quarter.
l
l
α
I0
α
2
I
1
1
= 10 - k 2l =   =
I0
4
2
I
And three slabs will reduce the intensity of a beam of light to one eight.
l
I0
l
α
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l
α
α
3
I
I
1 1
- k 3l
= 10
=  =
I0
2 8
Beer’s Law
Beer found that Lambert’s linear decay constant k for
a solution of an absorbing substance is linearly related
to its concentration c by a constant, the absorptivity ε,
a characteristic of the absorbing substance.
Restatement: The linear decay constant k is linear in
concentration c with a constant of proportionality ε.
(August Beer, 1825-1863)
k = c
Typical units are: k cm−1; c M (moles/liter); ε M−1cm−1
A colored absorber has an absorptivity that is dependent on wavelength of the
light ε(λ).
The absorptivity is the fundamental property of a substance. This is the
property that contains the observable spectroscopic information that can be
linked to quantum mechanics (also see absorption cross section.)
Photometric Quantities
In photometry we measure the intensity of light and characterize its
change by and object or substance. This change is typically expresses
as percent transmittance or absorbance.
Transmittance (T)
Frequently when your primary
interest is the light beam
Absorbance (A)
I
T=
I0
usually given in percent
(AKA optical density, O.D.)
Used almost exclusively when your interest
concerns the properties of the material
 I 
A = - log   = - log T
 I0 
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by convention, base 10 logs are used
Beer – Lambert Law
Light
I0
I
Glass cell filled with
concentration of solution (C)
As the cell thickness increases, the transmitted
intensity of light of I decreases.
T- Transmittance
T=
I
I0
I0 - Original light intensity
I- Transmitted light intensity
I
I0
% Transmittance = 100 x
Absorbance (A) = Log
I
1
T
= Log
I0
I
= 2 - Log%T
Log I0 is proportional to C (concentration of solution)
and is also proportional to L (length of light path through
the solution).
Beer-Lambert Law
＃Beers Law states that absorbance is proportional
to concentration over a certain concentration range
Lambert‟s and Beer‟s Laws are combined to describe
the attenuation of light by a solution. It is easy to
see how the two standard photometric quantities
can be written in terms of this law.
I = I 010
Transmittance
I
T=
I0
T = 10 - c x
- c l
http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law
Absorbance
 I 
A = - log   = - log T
 I0 
A =  cl
A = absorbance
 = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1)
c = concentration (M or mol L-1)
l = path length (cm) (width of cuvette)
Beer-Lambert Law


Beer‟s law is valid at low concentrations, but
breaks down at higher concentrations
For linearity, A < 1
1
Beer-Lambert Law


If your unknown has a higher
concentration than your
highest standard, you have to
ASSUME that linearity still
holds (NOT GOOD for
quantitative analysis)
Unknowns should ideally fall
within the standard range
Quantitative Analysis

A<1

If A > 1:
 Dilute the sample
 Use a narrower cuvette




(cuvettes are usually 1 mm, 1 cm or 10 cm)
Plot the data (A v C) to produce a calibration
„curve‟
Obtain equation of straight line (y=mx) from line
of „best fit‟
Use equation to calculate the concentration of
the unknown(s)
25
Quantitative Analysis
Absorbance ( no units)
Calibration curve showing absorbance as
a function of metal concentration
1.2
y = 0.9982x
R2 = 0.9996
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
1.2
Concentration (mg L-1)
26
Steps in Developing a
Spectrometric Analytical Method
2. Obtain a monochromatic
wavelength for the maximum
absorption wavelength.
3. Calculate the concentration of
your sample using Beer
Lambert Equation: A = ECL
Absorbance
1. Run the sample for spectrum
2.0
0.0
200
250
300
350
400
Wavelength (nm)
450
Spectrometer Reading
A
C
Slope of Standard Curve =
x
1.0
x
0.5
x
1
4
2
3
Concentration (mg/ml)
5
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where
becomes non-linear.
Spectrometric Analysis Using
Standard Curve
1.2
0.8
0.4
3
1
2
Concentration (g/l) glucose
4
Avoid very high or low absorbencies when drawing a
standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
straight line
Homeworks
1.
Calculate the Molar Extinction Coefficient ε at 351
nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from
the following data which were obtained in 1 cm cell.
Solution
C x 105 M
A
2.23
100
27
B
1.90
100
32
2.
Io
I
The molar extinction coefficient (ε) of compound
riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance
reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is
the concentration of compound riboflavin in sample?
3.
The concentration of compound Y was 2 x 10-4
moles/liter and the absorption of the solution at 300 nm using 1
cm quartz cell was 0.4. What is the molar extinction coefficient
of compound Y?
4.
Calculate the molar extinction coefficient ε at 351
nm for aquocobalamin in 0.1 M phosphate buffer. pH =7.0 from
the following data which were obtained in 1 cm cell.
Solution
C x 105 M
A
2.0
I0
I
100
30
Cross-Sections and Absorptivity
the connection to single particles and
molecules
The absorption of light by particles (and single molecules) is characterized
by an absorption cross section C. In this model the particle is replaced by
a perfectly absorbing sphere with a cross sectional area C. This cross
section is a property of the particle and is not related to its geometric
cross sectional area. The concentration of particles per unit volume is N.
 = NC
k = NC ln 10
typical units are: C cm2; N cm−3
cross section can be directly related to the
 -3 liter  The
molar absorptivity. NA is Avagadro‟s number.
 = N A C ln 10  10
3  units are: C cm2; N cm−3; N mole−1; ε
A
cm  −1 −1

M cm
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Efficiency
The absorption efficiency Q of a particle is the ratio of its
absorption cross section C to its geometric cross section Cgeo.
Absorption efficiency is dimensionless.
C
Q=
C geo
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Extension to Scattering and
Extinction
Attenuation of light by absorption and scattering both obey
Lambert‟s Law. Thus we can extend our treatment of absorption
to scattering and extinction. (Recall that extinction is the effect of
absorption + scattering.)
Cext = Cabs  C sca
Qext = Qabs  Qsca
The scattering efficiency can be much larger than unity.
Extinction paradox: Qext = 2 (Qabs = 1; Qsca = 1)
for an perfectly absorbing particle very large compared
to the wavelength of light.
 ext =  abs   sca
A =  ext cx =  abs   sca )cx
Note:
•All of these quantities are in general wavelength dependent.
•Our discussion has not included the mechanism (cause) of absorption and
scattering.
•There are many different mechanisms that cause of absorption and
scattering.
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Instrumentation

Spectrometer: measures I vs λ.
Simply measures the spectrum of the light (e.g. emission
spectroscopy).

Spectrophotometer: measures I/I0 vs λ.
Measures how the sample changes the spectrum of the light (e.g.
transmission, reflection, scattering, fluorescence).
All spectrophotometers contain a spectrometer.
 -meter: the detector is electronic
 -graph: light intensity recorded on film
 photometer: measures I/I0 without λ selection.
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The Spectrophotometer
Measures absorbance as a function of wavelength
Components: light source, monochromator, sample cell, detector,
optical system.
sample cell
slit
diffraction grating
monochromator
light source
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detector
Cary 50 UV-Vis Spectrophotometer
Computer controlled
acquisition
of absorption spectra
monochromator
balance the forces:
detector
sample
light
source
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Can you find the
diffraction grating and the
slit?
www.varianinc.com
Making a Measurement
with the Cary 50




First, measure the baseline using a blank sample. This is raw I0.
The blank sample is the cuvette with deionized water
(everything but your nanoparticles). This corrects for any
absorption due to the cuvette, water, and variations of the light
intensity of the light source, monochromator, etc.
Second, measure the zero by inserting the beam block. This
corrects the instrument for the detector background.
Third, measure your sample. This is the raw I. The Cary 50
automatically calculates the corrected intensities (I and I0) by
subtracting the zero from each of the raw intensities.
Subsequent measurements do not require re-measuring the
blank and zero, simply repeat step 3.
I
raw I - zero
T= =
I 0 raw I 0 - zero
A = - log T
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Applications of
Spectrophotometry






Spectroscopy
Chemical Analysis:
trace analysis, pH, forensic, in situ
monitoring, remote monitoring, geology, astronomy, ....
Particle size
Thin film characterization
Color matching
Optics
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Sample Cells (Cuvettes)
UV Spectrophotometer
Quartz (crystalline silica): works in the UV region below 350 nm,
can be employed across the whole UV-Visible wavelength range,
190-1,000 nm
 Visible Spectrophotometer
Optical glass: employed in the region above 300nm
Moulded plastic cells: used in the visible region above 350 nm,
low cost
 Common size of cells is the 1cm rectangular cell,
which has an optical path length of 1cm and path width
of 1 cm. Typically, these cells hold 2-3 ml of sample.
When sample volume is an important issue  Consider
micro cells
Light Sources
UV Spectrophotometer
1.
Hydrogen Gas Lamp
2.
Mercury Lamp
Visible Spectrophotometer
1.
Tungsten Lamp
Uses for UV, continued
Knowing UV can help you know when to be skeptical of quant results.
Need to calibrate response factors
Assessing purity of a major peak in HPLC is improved by “diode array”
data, taking UV spectra at time points across a peak. Any differences
could suggest a unresolved component. “Peak Homogeneity” is key for
purity analysis.
Sensitivity makes HPLC sensitive
e.g. validation of cleaning procedure for a production vessel
But you would need to know what compounds could and could
not be detected by UV detector! (Structure!!!)
One of the best ways for identifying the presence of acidic or basic
groups, due to big shifts in l for a chromophore containing a phenol,
carboxylic acid, etc.
“hypsochromic” shift
“bathochromic” shift
l
The UV Absorption process
•  * and   * transitions: high-energy, accessible
in vacuum UV (lmax <150 nm). Not usually observed in
molecular UV-Vis.
•n  * and   * transitions: non-bonding electrons
(lone pairs), wavelength (lmax) in the 150-250 nm region.
•n  * and   * transitions: most common
transitions observed in organic molecular UV-Vis, observed
in compounds with lone pairs and multiple bonds with lmax
= 200-600 nm.
•Any of these require that incoming photons match in
energy the gap corrresponding to a transition from ground
to excited state.
•Energies correspond to a 1-photon of 300 nm light are ca.
95 kcal/mol
What are the
nature of these
absorptions?
π*
π*
π*
π*
π*
π*
n
π
π
n
π
π
-*; lmax=218
=11,000
π*
π*
π*
n
π
π
Example
for a
simple
enone
n-*; lmax=320
=100
Example:   * transitions responsible for ethylene UV absorption at ~170
nm calculated with ZINDO semi-empirical excited-states methods (Gaussian
03W):
h 170nm photon
HOMO u bonding molecular orbital
LUMO g antibonding molecular orbital
How Do UV spectrometers work?
Rotates, to achieve scan
Matched quartz cuvettes
Sample in solution at ca. 10-5
M.
System protects PM tube from
stray light
D2 lamp-UV
Tungsten lamp-Vis
Double Beam makes it a
difference technique
Two photomultiplier
inputs, differential
voltage drives amplifier.
Diode Array Detectors
Diode array
alternative puts
grating, array of
photosens.
Semiconductors
after the light goes
through the
sample. Advantage,
speed, sensitivity,
The Multiplex
advantage
Model from Agilent literature. Imagine replacing
“cell” with a microflow cell for HPLC!
Disadvantage,
resolution is 1 nm,
vs 0.1 nm for
normal UV
Experimental details
What compounds show UV spectra?
Generally think of any unsaturated compounds as good
candidates. Conjugated double bonds are strong absorbers
Just heteroatoms are not enough but C=O are reliable
Most compounds have “end absorbance” at lower frequency.
Unfortunately solvent cutoffs preclude observation.
You will find molar absorptivities  in L•cm/mol, tabulated.
Transition metal complexes, inorganics
Solvent must be UV grade (great sensitivity to impurities with
double bonds)
The NIST databases have UV spectra for many compounds
An Electronic Spectrum
Make solution of
concentration low
enough that A≤ 1
(Ensures Linear Beer‟s
law behavior)
1.0
lmaxwith certain
extinction 
UV
Visible
Even though a dual
beam goes through a
solvent blank, choose
solvents that are UV
transparent.
Absorbance
Can extract the  value
if conc. (M) and b (cm)
are known
0.0
200
UV bands are much
broader than the
photonic transition
event. This is because
vibration levels are
superimposed on UV.
400
Wavelength, l, generally in nanometers (nm)
800
Solvents for UV (showing high
energy cutoffs)
Water
205
THF
CH2Cl2
CH3CN 210
220
235
C6H12
210
CHCl3
245
Ether
210
CCl4
265
EtOH
210
benzene 280
Acetone 300
Hexane 210
MeOH
Dioxane 220
210
Various buffers for HPLC,
check before using.
Chemical Structure & UV
Absorption
Chromophoric Group ---- The groupings of the
molecules which contain the electronic system
which is giving rise to absorption in the ultraviolet region.
Chromophoric Structure
Group
Structure
nm
Carbonyl
>C=O
280
Azo
-N = N-
262
Nitro
-N=O
270
Thioketone
-C =S
330
Nitrite
-NO2
230
Conjugated Diene
-C=C-C=C-
233
Conjugated Triene
-C=C-C=C-C=C-
268
Conjugated Tetraene
-C=C-C=C-C=C-C=C-
315
Benzene
261
Organic compounds (many of them)
have UV spectra
One thing is clear
UVs can be very non-specific
It is hard to interpret except at a
cursory level, and hard to say that the
spectrum is consistent with the
structure
Each band can be a superposition of
many transitions
Generally we don‟t assign the
particular transitions.
From Skoog and West et al. Ch 14
An Example--Pulegone
Frequently
plotted as log
of molar
extinction

So at 240
nm,
pulegone
has a molar
extinction of
7.24 x 103
Antilog of 3.86
O
Can we calculate UVs?
Molar Abs orptivity (l/mol-cm)
Electronic Spectra
50243
40194
30146
20097
10049
nac indolA
0
220
Wavelength
230
240
250
260
270
280
290
300
Electronic Spectra
Molar Abs orptivity (l/mol-cm)
51972
41578
Semi-empirical (MOPAC) at AM1,
then ZINDO for config. interaction
level 14
31183
20789
Bandwidth set to 3200 cm-1
10394
Nac etylindo
0
220
Wavelength
230
240
250
260
270
280
290
300
The orbitals involved
Electronic Spectra
Molar Absorptivity (l/mol-cm)
55487
44390
Showing atoms
whose MO‟s
contribute most
to the bands
33292
22195
11097
Nacetylindol
0
200
Wavelength (nm)
210
220
230
240
250
260
270
280
290
300
The Quantitative Picture
• Transmittance:
T = P/P0
• Absorbance:
A = -log10 T = log10 P0/P
P0
(power in)
P
(power out)
B(path through sample)
• The Beer-Lambert Law (a.k.a. Beer‟s Law):
A = bc
Where the absorbance A has no units, since A = log10 P0 / P
 is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample in cm
c is the concentration of the compound in solution, expressed in mol L-1 (or M,
molarity)
Beer-Lambert Law
Linear absorbance with increased concentration-directly proportional
Makes UV useful for quantitative analysis and in
HPLC detectors
Above a certain concentration the linearity curves
down, loses direct proportionality--Due to molecular
associations at higher concentrations. Must
demonstrate linearity in validating response in an
analytical procedure.
Polyenes, and Unsaturated
Carbonyl groups; an Empirical
triumph
R.B. Woodward, L.F. Fieser and others
Predict lmax for π* in extended conjugation systems to within
ca. 2-3 nm.
Attached group
increment, nm
Extend conjugation +30
Homoannular, base 253
nm
Addn exocyclic DB +5
Acyclic, base 217 nm
O-Acyl
0
S-alkyl
+30
O-alkyl
+6
NR2
+60
Cl, Br
+5
heteroannular, base 214
nm
Alkyl
+5
Similar for Enones
b
b
O
O
O
227
202
215
Base Values, add these increments…

X=H 207
x
b

239
g
d,
Extnd C=C
Add exocyclic C=C
+30
+5
Homoannular diene
+39
alkyl
+10
+12
With solvent
correction of…..
OH
+35
+30
Water
+8
OAcyl
+6
+6
+6
+6
EtOH
0
O-alkyl
+35
+30
+17
+31
CHCl3
-1
Dioxane
-5
Et2O
-7
+15/+25
+12/+30
X=R 215
X=OH 193
X=OR 193
Hydrcrbn -11
NR2
S-alkyl
Cl/Br
+18
+18
+50
Some Worked Examples
O
Base value
2 x alkyl subst.
exo DB
total
Obs.
217
10
5
232
237
Base value
3 x alkyl subst.
exo DB
total
Obs.
214
30
5
234
235
Base value
2 ß alkyl subst.
total
Obs.
215
24
239
237
Distinguish Isomers!
Base value
4 x alkyl subst.
exo DB
total
Obs.
214
20
5
239
238
Base value
4 x alkyl subst.
total
Obs.
253
20
273
273
HO2C
HO2C
Generally, extending
conjugation leads to red shift
“particle in a box” QM theory; bigger box
Substituents attached to a chromophore that cause a red shift are called
“auxochromes”
Strain has an effect…
lmax
253
239
256
248
Interpretation of UV-Visible Spectra
 Transition metal
complexes; d, f
electrons.
 Lanthanide complexes
– sharp lines caused
by “screening” of the f
electrons by other
orbitals
 One advantage of this
is the use of holmium
oxide filters (sharp
lines) for wavelength
calibration of UV
spectrometers.
See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14
Benzenoid
aromatics
UV of Benzene
in heptane
From Crewes, Rodriguez, Jaspars, Organic Structure
Analysis
Group
K band ()
B band()
R band
Alkyl
208(7800)
260(220)
--
-OH
211(6200)
270(1450)
-O-
236(9400)
287(2600)
-OCH3
217(6400)
269(1500)
NH2
230(8600)
280(1400)
-F
204(6200)
254(900)
-Cl
210(7500)
257(170)
-Br
210(7500)
257(170)
-I
207(7000)
258/285(610/180)
-NH3+
203(7500)
254(160)
-C=CH2
248(15000)
282(740)
-CCH
248(17000)
278(6500
-C6H6
250(14000)
-C(=O)H
242(14000)
280(1400)
328(55)
-C(=O)R
238(13000)
276(800)
320(40)
-CO2H
226(9800)
272(850)
-CO2-
224(8700)
268(800)
-CN
224(13000)
271(1000)
-NO2
252(10000)
280(1000)
330(140)
Quantitative
analysis
Great for nonaqueous titrations
Example here gives
detn of endpoint for
bromcresol green
Binding studies
Form I to form II
Isosbestic points
Single clear point, can exclude
intermediate state, exclude
light scattering and Beer‟s law
applies
Binding of a lanthanide complex
to an oligonucleotide
More Complex Electronic Processes
• Fluorescence: absorption of
radiation to an excited state,
followed by emission of radiation to
a lower state of the same
multiplicity
• Phosphorescence: absorption of
radiation to an excited state,
followed by emission of radiation to
a lower state of different multiplicity
• Singlet state: spins are paired, no
net angular momentum (and no net
magnetic field)
• Triplet state: spins are unpaired, net
angular momentum (and net
magnetic field)
UV Spectrometer Application
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
Flurometric Application
Thiamin (365 nm, 435 nm)
Riboflavin
Vitamin A
Vitamin C
Visible Spectrometer Application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)