Download uv-vis spectroscopy

UV-VIS SPECTROSCOPY
Light interacting with matter as an
analytical tool
Electronic Excitation by UV/Vis Spectroscopy :
X-ray:
core electron
excitation
UV:
valance
electronic
excitation
IR:
molecular
vibrations
Radio waves:
Nuclear spin states
(in a magnetic field)
Where in the spectrum are these
transitions?
Spectroscopic Techniques and
Chemistry they Probe
UV-vis
UV-vis region
bonding electrons
Atomic Absorption
UV-vis region
atomic transitions (val. e-)
FT-IR
IR/Microwave
vibrations, rotations
Raman
IR/UV
vibrations
FT-NMR
Radio waves
nuclear spin states
X-Ray Spectroscopy
X-rays
inner electrons, elemental
X-ray Crystallography
X-rays
3-D structure
Spectroscopic Techniques and Common Uses
UV-vis
UV-vis region
Quantitative
analysis/Beer’s Law
Atomic Absorption
UV-vis region
Quantitative analysis
Beer’s Law
FT-IR
IR/Microwave
Functional Group Analysis
Functional Group
Analysis/quant
Raman
IR/UV
FT-NMR
Radio waves
X-Ray Spectroscopy
X-rays
Elemental Analysis
X-ray Crystallography
X-rays
3-D structure Anaylysis
Structure determination
Different Spectroscopies
• UV-vis – electronic states of valence e/dorbital transitions for solvated transition
metals
• Fluorescence – emission of UV/vis by certain
molecules
• FT-IR – vibrational transitions of molecules
• FT-NMR – nuclear spin transitions
• X-Ray Spectroscopy – electronic transitions
of core electrons
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 so 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
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  for a chromophore containing a phenol, carboxylic acid, etc.
“hypsochromic” shift
“bathochromic” shift

The UV Absorption process
•  * and   * transitions: high-energy,
accessible in vacuum UV (max <150 nm). Not usually
observed in molecular UV-Vis.
•n  * and   * transitions: non-bonding electrons
(lone pairs), wavelength (max) 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 max = 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*
n
π
π
-*; max=218
=11,000
π*
π*
π*
n
π
π
π*
Example
for a
π*
simple
π*
enone
n
π
π
n-*; max=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 absorbtivities  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
maxwith 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
UV bands are much
broader than the photonic
transition event. This is
because vibration levels
are superimposed on UV.
0.0
200
400
Wavelength, , generally in nanometers (nm)
800
Solvents for UV (showing high
energy cutoffs)
Water
205
THF
220
CH3CN
210
CH2Cl2
235
C6H12
210
CHCl3
245
Ether
210
CCl4
265
EtOH
210
benzene
280
Hexane
210
Acetone
300
MeOH
210
Dioxane
220
Various buffers for
HPLC, check before
using.
Organic compounds (many of
them) have UV spectra
One thing is clear
Uvs can be very non-specific
Its hard to interpret except at a
cursory level, and 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 Absorptivity (l/mol-c m)
Electronic Spectra
50243
40194
30146
20097
10049
nacindolA
0
220
Wavelength
230
240
250
260
270
280
290
300
Electronic Spectra
Molar Absorptivity (l/mol-c m)
51972
41578
Semi-empirical (MOPAC) at
AM1, then ZINDO for
config. interaction level 14
Bandwidth set to 3200 cm-1
31183
20789
10394
Nacetylindo
0
220
Wavelength
230
240
250
260
270
280
290
300
The orbitals involved
Electronic Spectra
Molar Absorptivity (l/mol-cm)
55487
44390
33292
22195
11097
0
200
210
220
230
240
250
260
270
280
290
Showing
atoms whose
MO’s
contribute
Nacetylindol
most to the
Wavelength
(nm)
bands
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.
Quantitative Spectroscopy
• Beer’s Law
Al1 = el1bc
e is molar absorptivity (unique for a
given compound at l1)
b is path length
c concentration
Beer’s Law
slit
cuvette
source
detector
•
•
•
•
A = -logT = log(P0/P) = ebc
T = Psolution/Psolvent = P/P0
Works for monochromatic light
Compound x has a unique e at different
wavelengths
Characteristics of
Beer’s Law Plots
• One wavelength
• Good plots have a range of
absorbances from 0.010 to 1.000
• Absorbances over 1.000 are not that
valid and should be avoided
• 2 orders of magnitude
Standard Practice
• Prepare standards of known
concentration
• Measure absorbance at max
• Plot A vs. concentration
• Obtain slope
• Use slope (and intercept) to determine
the concentration of the analyte in the
unknown
A
Typical Beer’s Law Plot
1.2
1
0.8
0.6
0.4
0.2
0
y = 0.02x
0.0
20.0
40.0
concentration (uM)
60.0
UV-Vis Spectroscopy
• UV- organic molecules
– Outer electron bonding transitions
– conjugation
• Visible – metal/ligands in solution
– d-orbital transitions
• Instrumentation
Characteristics of UV-Vis spectra of
Organic Molecules
• Absorb mostly in UV unless highly
conjugated
• Spectra are broad, usually to broad for
qualitative identification purposes
• Excellent for quantitative Beer’s Lawtype analyses
• The most common detector for an
HPLC
Molecules have quantized energy levels:
ex. electronic energy levels.
energy
energy
hv
}
= hv
Q: Where do these quantized energy levels come from?
A: The electronic configurations associated with bonding.
Each electronic energy level
(configuration) has
associated with it the many
vibrational energy levels we
examined with IR.
Broad spectra
• Overlapping vibrational and rotational
peaks
• Solvent effects
Molecular Orbital Theory
• Fig 18-10
*
*
2p
n
2p


*
2s
2s

Ethane
C C
*
*
hv



H
C C
H
H
HH
H
*
max = 135 nm (a high energy transition)
Absorptions having max < 200 nm are difficult to observe because
everything (including quartz glass and air) absorbs in this spectral
region.
C C
*
*
*
*
= hv
=hc/
hv





*
Example: ethylene absorbs at longer wavelengths:
max = 165 nm = 10,000
C O
*
*
*
*
n
hv
n




n
*
The n to pi* transition is at even lower wavelengths but is not
as strong as pi to pi* transitions. It is said to be “forbidden.”
Example:
Acetone:
n* max = 188 nm ; = 1860
n* max = 279 nm ; = 15
C C
*
135 nm
C C
*
165 nm
n*
183 nm
weak
*
n*
n*
150 nm
188 nm
279 nm
weak
H
C O
C O
180 nm
C O
A
279 nm

Conjugated systems:
C
C
LUMO
HOMO
Preferred transition is between Highest Occupied Molecular Orbital
(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).
Note: Additional conjugation (double bonds) lowers the HOMOLUMO energy gap:
Example:
1,3 butadiene:
max = 217 nm ; = 21,000
1,3,5-hexatriene
max = 258 nm ; = 35,000
Similar structures have similar UV spectra:
O
O
O
max = 238, 305 nm
max = 240, 311 nm
max = 173, 192 nm
Lycopene:
max = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm
max(Actual) = 474.
Polyenes, and Unsaturated
Carbonyl groups;
an Empirical triumph
R.B. Woodward, L.F. Fieser and others
Predict max for π* in extended conjugation
systems to within ca. 2-3 nm.
Homoannular, base 253 nm
Acyclic, base 217 nm
heteroannular, base 214 nm
Attached group
increment, nm
Extend conjugation
+30
Addn exocyclic DB
+5
Alkyl
+5
O-Acyl
0
S-alkyl
+30
O-alkyl
+6
NR2
+60
Cl, Br
+5
Similar for Enones
b
b
O

X=H 207
O
O
x
227
202
215
Base Values, add these increments…
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
NR2
Dioxane
-5
S-alkyl
Et2O
-7
+15/+25
+12/+30
X=R 215
X=OH 193
X=OR 193
Hydrcrbn -11
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
15
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…
max
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, 2
nd
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)
Substituent effects don’t really add up
Can’t tell any thing about substitution geometry
Exception to this is when adjacent substituents can
interact, e.g hydrogen bonding.
E.g the secondary benzene band at 254 shifts to
303 in salicylic acid
In p-hydroxybenzoic acid, it is at the phenol or
benzoic acid frequency
Heterocycles
Nitrogen heterocycles are pretty similar to the benzenoid
anaologs that are isoelectronic.
Can study protonation, complex formation (charge transfer
bands)
Quantitative
analysis
Great for nonaqueous titrations
Example here
gives detn of
endpoint for
bromcresol green
Isosbestic points
Single clear point, can exclude
intermediate state, exclude light
scattering and Beer’s law applies
Binding studies
Form I to form II
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)
Metal ion transitions
E
Degenerate
D-orbitals
of naked Co
D-orbitals
of hydrated Co2+
Octahedral Configuration
Octahedral Geometry
H2O
H2O
H2O
Co2+
H2O
H2O
H2O
Instrumentation
• Fixed wavelength instruments
• Scanning instruments
• Diode Array Instruments
Fixed Wavelength Instrument
• LED serve as source
• Pseudo-monochromatic light source
• No monochrometer necessary/ wavelength selection
occurs by turning on the appropriate LED
• 4 LEDs to choose from
sample
beam of light
LEDs
photodyode
Scanning Instrument
Scanning Instrument
monochromator
Tungsten
Filament (vis)
slit
slit
Deuterium lamp
Filament (UV)
Photomultiplier
tube
cuvette
sources
• Tungten lamp (350-2500 nm)
• Deuterium (200-400 nm)
• Xenon Arc lamps (200-1000 nm)
Monochromator
• Braggs law, nl = d(sin i + sin r)
• Angular dispersion, dr/d = n / d(cos r)
• Resolution, R = /=nN, resolution is
extended by concave mirrors to refocus
the divergent beam at the exit slit
Sample holder
• Visible; can be plastic or glass
• UV; you must use quartz
Single beam vs. double beam
• Source flicker
Diode array Instrument
mirror
Diode array detector
328 individual detectors
Tungsten
Filament (vis)
slit
slit
cuvette
Deuterium lamp
Filament (UV)
monochromator
Advantages/disadvantages
• Scanning instrument
– High spectral resolution (63000), /
– Long data acquisition time (several
minutes)
– Low throughput
• Diode array
– Fast acquisition time (a couple of
seconds), compatible with on-line
separations
– High throughput (no slits)
– Low resolution (2 nm)
HPLC-UV
HPLC
Pump
Mobile
phase
Sample
loop
6-port
valve
HPLC
column
UV
detector
syringe
Solvent
waste