UV/Vis (Ultraviolet and
Visible) Spectroscopy
Agilent 8453 Diode Array UV-Vis Spectrophotometer Varian Cary 5000 UV-Vis-NIR Spectrophotometer
To Dos
Read Chapters 13 & 14.
Complete the end-of-chapter problems,
13-1, 13-2, 13-3,13-6 and 13-8.
Complete the end-of-chapter problems,
14-2, 14-5, 14-6, 14-7, 14-8, 14-9,
14-10, 14-12, 14-14, 14-16, 14-19 and
14-20.
Wavelength and Spectroscopy
What Molecular Changes Cause UV/Vis
Absorptions?
UV/Vis spectroscopy is used to detect the
presence of certain functional groups called
= 200 ~ 700 nm chromophores, especially in conjugate
systemssuch as aromatics, dienes, polyenes,
and , -unsaturated ketones
Electronic excitation
Absorption of UV or visible light occurs only when the energy of incident radiation
is the same as that of an electronic transition in the molecule.
Electronic Transitions
Only n * and * transitions usually show up
in 200 - 700 nm.
Only limited set of organic functional groups show UV/Vis
absorptions.
Instrument (double beam)
Typical = 200 ~ 700 nm
The Beer s Law
Iref Iref
A = log ( ) > 1, thus A is aways positive.
Isample Isample
Iref
=1 A = 0
Isample
A = x c x l Iref
= 10 A = 1
Isample
: molar absorptivity constant
Iref
or extinction coefficient = 100 A = 2
(l mol-1 cm-1) Isample
c : concentration (mol/l) Iref
l : path length (cm)
= 1000 A = 3
Isample
Instrument (Diode Array)
Rugged, fast, and reliable
Solvents and Cuvettes for UV/Vis Spectroscopy
Typical UV/Vis Spectra
O
CH3
Franck-Condon Principle
Nuclear motion is negligible during the time required
for an electronic transition.
Excitation can occur from the ground state to any of the excited
state vibrational levels.
A B
The lines overlap and a continuous broad band is observed.
Difference Spectroscopy
UV absorption spectra and temperature
-difference spectra of HO5dCyt in 0.02 M
NaOH/NaHCO3 buffer at pH 10.5 at 25C
(A, dashed line), 75C (A, solid line) and
75 25C (B). Comparable spectra of
dCyt in 0.02 M NaOH/NaHCO3 buffer
at pH 7 at 25C (C, dashed line), 75C
(C, solid line) and 75 25C (D).
Isosbestic Points
HA = A- HA A-
Isosbestic points are usually a diagnostic for the presence of only two
absorbing species.
Spectral Shift
blue shift red shift
Polymeric Nucleic Acids Have an Increased Molar Absorptivity When They
are Converted to Smaller Units
Common Organic Chromophores
conjugated
and non-conjugated
The Carbonyl Groups
O O
max 270 ~ 300 nm and
C C
max 10 ~ 100, n * R R1 R H
!*
c o n
The two orbitals, n and *, are orthogonal to each other and promotion of
electron requires significant changes in geometry. The electronic transition
must be coupled with vibration that allows some overlap between these orbitals.
Other Carbonyl Groups
!max "max !*
O O
C (hexane) 274 15
H3C CH3
R C
O
n
C
X
H 3C Cl (hexane) 235 53
O
C
H3C NH2 (water) 214 -
!*
O
C (water) 204 60 C=O
H3C OEt
n (X)
n
O
C (ethanolr) 204 41
H3C OH
Different Carbonyl
Compounds and Their UV/Vis
Solvent Effects
C OI H O
R
!*
n
Aldehydes and Ketons
Unconjugated Alkenes
Conjugated Alkenes
Conjugation and Spectral Shifts
-Carotene
Diene Isomers
max = 239 nm
7 6 5 4 3 2 1 0
PPM
max = 278 nm 7 6 5 4
PPM
3 2 1 0
Empirical Calculation of max for
Conjugated Alkenes
Empirical Calculation - Examples
Empirical Calculation More Examples
Steric Hindrance and Spectral Shifts
CH 3
H 3C
CH 3
CH3 CH3 CH3
H3 C CH 2 H3 C CH 2
!max 225 231
"max 6.400 10,000
CH 3
H 3C
CH 3
CH3
Twisted diene low conjugation max and max
H3 C CH 2
Conjugated Carbonyls
alkyl or ring residues
Empirical
Calculation
Empirical Calculation - Examples
Exocyclic Alkene Issue
215
12 -alkyl
3 x 18 + alkyl
2x5 exocyclic C=C
O 2 x 30 extra conjugation
351 nm
Observed 348 nm
Homoconjugation
Homoconjugation- More Examples
Solvent Effect
In hexane In ethanol
max = 295 max = 255
max = 50 max = 12,500
215
12 (-alkyl)
30 (-OH)
257nm
Charge Transfer(CT) Bands
h!
A+D A D A D
CT complex
Intramolecular CT Bands
!-
O
!+
Benzene Chromophore
n *
Solvent Effects
Benzene Bands Designation
Substituent Effects
Benzene Derivatives - Monosubstituted
pH Sensitive Derivatives
O O
OH-
OH O
OH OH- O
pH Titration of Phenol
Isosbestic point
pKa Measurement
Aromatic Amino Acids
R
O
H3N
O
R= Phe
OH Tyr
H
N
Trp
Benzene Derivatives -
Disubstituted
The mono-substituted moiety with the largest red shift will
dominate the absorption of a di-substituted benzene.
Para-substituted Benzenes
If one substituent is e-withdrawing and the other is
e-donating, a significant red-shift is observed due to the
extended conjugation.
Disubstituted Anilines
Disubstituted Benzaldehydes
Disubstituted Benzoic Acids
Fused Ring Systems
Heterocyclic Aromatics
Steric Effect Cis & Trans
Steric Effect E & Z
Z
E
Z
E E
Z
!max 328 313 299
"max 56,200 30,600 29,500
Steric Effect - Biphenyls
Empirical Calculation Benzoyl Derivatives
Empirical Calculation Examples
Br 246 (ketone) 230 (acid)
+ 3 (o-ring) HO CO2H + 7 x 2 (m-OH)
+ 2 (m-Br) + 25 (p-OH)
HO
!max = 251nm !max = 269nm
O OH obs = 270 nm
obs = 253 nm
Tautomerization
O OH
239 (14,100)
N N
H
OH
260 (2,200)
N
257 (2,750)
N
Porphyrin Chromophore
Exciton Coupling
UV/Vis Analysis
A single band (e = 10 ~ 100) in 250 ~ 300 nm, no major absorption in
200 ~ 250 nm.
n *, C=O (ketone and aldehyde), C=N. N=N, -NO2 etc
Two bands (e = 1,000 ~ 10,000) > 200 nm.
*, aromatics
Bands (e = 10,000 ~ 20,000) > 210 nm.
*, conjugated C=C and C=O
Compounds that are highly colored
A long-chain conjugated system
A polycyclic aromatic system
pH-depended absorption
Ionizable group attached to chromophore
