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國防醫學院 藥學系
胡明寬 教授
10/14/08 1
Introduction to Spectroscopy
10/14/08 2
How you know chemical structure?
1. To "see" a molecule?
we must use light having a wavelength smaller than the
molecule itself (roughly 1 to 15 angstrom units).
Such radiation is found in the X-ray region of the spectrum,
and the field of X-ray crystallography yields remarkably
detailed pictures of molecular structures amenable
to examination.
10/14/08 3
The Spectroscopic Techniques
• Mass (MS) Spectrometry: Sample molecules are ionized by high energy electrons.
The mass to charge ratio of these ions is measured very accurately by electrostatic
acceleration and magnetic field perturbation, providing a precise molecular weight.
Ion fragmentation patterns may be related to the structure of the molecular ion.
• Ultraviolet-Visible (UV) Spectroscopy: Absorption of this relatively high-energy
light causes electronic excitation. The easily accessible part of this region (wavelengths of
200 to 800 nm) shows absorption only if conjugated pi(π)-electron systems are present.
• Infrared (IR) Spectroscopy: Absorption of this lower energy radiation causes
vibrational and rotational excitation of groups of atoms. within the molecule. Because
of their characteristic absorptions identification of functional groups is easily accomplished.
• Nuclear Magnetic Resonance (NMR) Spectroscopy: Absorption in the
low-energy radio-frequency part of the spectrum causes excitation of nuclear spin states.
NMR spectrometers are tuned to certain nuclei (e.g. 1H, 13C, 19F & 31P). For a given
type of nucleus, high-resolution spectroscopy distinguishes and counts atoms in
different locations in the molecule.
10/14/08 4
The Nature of Electronic Excitations
hν
Io I
a transparent
material
a UV spectrum
10/14/08 5
Theoretical Introduction
ν·λ=c
c: Velocity of light, 3.0 x 1010 cm · s-1
λ: Wavelength ---- nm, 1 nm = 10-7 cm
ν: Frequency, cycles per sec (Hz)
∆E = h · ν
∆E: Energy
h: Planck’s constant, 6.63 x 10-34 J.sec or 6.63 x 10-27 erg.sec
= 1/ λ = ν / c
: Wave number, cm-1
10/14/08 6
The Visible Spectrum
10/14/08 7
What is ultraviolet (UV)?
Wavelengt
Range
h Range
Name
(nm)
UVA 315-400
UVB 280-315
UVC 200-280
Vacuum UV
100-200
(VUV)
10/14/08 8
The Electromagnetic Spectrum
10/14/08 9
UVA & UVB
UVA: a) the "tanning" rays, can penetrate deeply into
the skin resulting in damage.
b) comprises large portions of total energy of sunlight;
c) not absorbed by ozone!
UVB: a) the "burn" index, associated with sunburns.
b) highly genotoxic
Both UVA and UVB cause suntan, sunburn, and sun damage.
10/14/08 10
What is the UV Index?
UV Index Exposure
Number Level
0-2 Minimal
3-4 Low
5-6 Moderate
7-9 High
10+ Very High
10/14/08 11
UV/Vis Region of the Electromagnetic Spectrum
10/14/08 12
Principle of UV-Visible Absorption Spectra
Electromagnetic waves
hν
Absorption of
N
UV/Vis light
10/14/08 13
Electronic Transition and Radiative Processes
ψ1
absorption emission
hν hν
ψ0
∆E = E(ψ1)- E(ψ0)
=hν
10/14/08 14
Electronic transitions (bands) of MO
HOMO LUMO:
Bonding/non-bonding an empty anti-bonding
σ*
Unoccupied levels
π*
Energy nσ*
n
σσ*
π Occupied levels
σ
σπ*
σ σ* In alkanes
σ π* In carbonyl comp’ds
n π* In carbonyl comp’ds
10/14/08 16
Absorption Regions of Electron Transitions
λ
200 400 750 nm
Vacuum-uv uv vis
n π*
π π*
π π*
n σ*
σ σ*
5.0·104 cm-1 2.5 · 10 4 13.3 · 103
10/14/08 17
Terms and Symbols
10/14/08 19
Singlet/Triplet Excited States
10/14/08 20
Electronic Transitions
Orbitals States
singlet triplet
------------------------------------------
LUMO
J
ΔE S1
2k
---------------------- T1
A
HOMO
So
10/14/08 21
Instrumentation
Light source: deuterium (uv light)/tungsten
(vis light) lamps
Monochromator: a diffraction grating
Detector: photodiodes or photomultiplier tube
Double-beam instrument
Sample beam:
For vis region: cells can be glass or plastic made
For uv region: cells made of quartz must be used
Reference beam
10/14/08 22
Solvents
Criteria:
not containing conjugated systems
No solvent-solute interaction, e.g. H-bonding etc.
Stabilize either ground state or excited state of
absorbed wavelength of uv light
Table 1.7. Solvent Cutoffs
CH3CN 190 nm n-Hexane 201 nm
CHCl3 240 CH3OH 205
C6H12 195 isooctane 195
1,4-dioxane 215 H2O 190
10/14/08
95% EtOH 205 Me3PO4 210 23
Light Absorption and the Spectrum
hν
Io I
a transparent
material
10/14/08 24
A Typical Spectrometer
10/14/08 25
Absorption
Transmittance: T = I/Io
Absorbance: A = Log Io/I
if no absorption, T = 1; A = 0
A = Log Io/I = ε · c · d
A = acd
c = g/L
a ---- absorptivity
ε = a Μ (M = m.w.)
10/14/08 27
Excitation for Double Bond(s)
10/14/08 28
Excitation for Double Bond(s)
10/14/08 29
Excitation for Double Bond(s)
10/14/08 30
Absorption Intensity
10/14/08 33
Substituent Effects
Hyperchromic
Hypsochromic Bathochromic
ε (blue shift) (red shift)
Hypochromic
400 800 nm
blue red
10/14/08 34
The Importance of Conjugation
10/14/08 35
The Importance of Conjugation
Chromo Excitatio λ , nm
Example max ε Solvent
phore n
C=C Ethene π π* 171 15,000 hexane
C≡C 1-Hexyne π π* 180 10,000 hexane
n π* 290 15 hexane
C=O Ethanal
ππ* 180 10,000 hexane
Nitromet- n π* 275 17 ethanol
N=O
hane ππ* 200 5,000 ethanol
Methyl
C--X
bromide n σ* 205 200 hexane
X=Br
Methyl n σ* 255 360 hexane
X=I
Iodide
10/14/08 36
The Importance of Conjugation
10/14/08 37
Electronic Transitions
10/14/08 39
Characteristic Absorptions
10/14/08 40
Characteristic Absorptions
10/14/08 41
Characteristic Absorptions
λ = 178 nm λ = 205 nm
εmax = 17000 εmax = 2100
Homoannular Heteroannular
10/14/08 43
The position of absorption of homo- (Ho)
and hetero (He)-annular systems.
λ = total
Characteristic Absorptions
Poor correlations are obtained for the data of Table 7.5 are
applied to cross-conjugated polyene systems.
10/14/08 45
Summary of UV/Visible Absorptions
Cholesta-3,5-diene Cholesta-2,4-diene
1
1 2 2
3 3
Carbonyl compounds
Two principal UV transitions: π*
The allowed π π* Forbidden
280 nm
The forbidden n π* Allowed
n
190 nm
π
10/14/08 48
Hypsochromic effects of lone-pair
anxochromes on n π* of carbonyls
Solvent corrections
H2O -8 dioxane +5
MeOH, EtOH 0 Et2O +7
CHCl3 +1 hydrocarbons +11
10/14/08 50
Summary of UV-Visible Absorptions
alpha
Carbonyl compounds- rules of enones beta
O
10/14/08 51
The spectra of polyene aldehydes
10/14/08 52
The orbitals of an enone system
10/14/08 53
Summary of UV-Visible Absorptions
Carbonyl compounds
Oximes come near parent ketone.
Semicarbazones, + 30 ~ 40
2,4-DNP’s: saturated 360 ~ 365, unsaturated, 375 ~ 385.
O O
OH O
O
O O
O O O
O O O
280 (1.4) 298 (1.5) 280 (1.3)
466 (1.5) 380 (1.0) 420 (1.0)
10/14/08 55
Summary of UV-Visible Absorptions
Benzene Derivatives
tBu OH
10/14/08 56
Summary of UV-Visible Absorptions
O NH2 NH3+
10/14/08 57
Summary of UV-Visible Absorptions
O CH3 C6H5
NO2 C6H5 O
252 (4.00) 246 (4.3) 244 (4.08) 240 (4.11) 252 (4.3)
270 (2.90) 282 (2.65) 278 (3.04) 325 (2.26)
330 (2.10) 319 (1.70)
10/14/08 58
What to look for in a UV spectrum
A single band of low to medium intensity (ε = 100 ~ 10,000) at < 220 nm: an
n σ* (amines, alc. Ethers, thiols --)
A single band of low intensity (ε = 10 ~ 100) at 250 ~ 360 nm and no major
peak at shorter λ (200 ~ 250 nm): an n π* (C=O, C=N, N=N, -NO2,
-COOR, -CONH2 --) & check IR, NMR.
Two bands of medium intensity (ε = 100 ~ 10,000) with λmax> 200 nm: an
aromatic system; if substitution at aryl ring: ε > 10,000
Bands of high intensity (ε = 10000 ~ 20,000) at > 210 nm: α,β-unsaturated
ketones, a diene, or a polyene; for dienes: using Woodward-Fieser rules.
Simple ketones, acids, esters, amides --: n π* (> 300 nm, low
intensity) & π π* (< 250 nm, high intensity); for enonr system:
using Woodward’s rules.
Compounds with highly colored: containing long-chain conjugated
system or a polycyclic
10/14/08 59
Cyclophilin
O
AA-cis-Pro AA-trans-Pro
10/14/08 60
Materials for PPI assay
Substrate: Suc-Ala-Ala-Pro-Phe-pNA in 0.47 M LiCl
in THF (The cis Ala-Pro conformer: ~ 40%)
Emzyme: cyclophilin (5 mM, 40 mL)
Buffer: (1 mL, 50 mM Herpes-NaOH, 100 mM NaCl,
pH 8.0 at 0 ℃)
Inhibitors: CsA & its Analogs
10/14/08 61
PPIase Assay
chymotrypsin
∆
Suc-AA-cis-PF-pNA Suc-AA-trans-PF-pNA
PPIase Suc-AA-trans-PF-OH
E.Scis E.Strans +
p-nitroaniline
(uv, 390 nm)
H2 N NO2
10/14/08 62
Endogenous Proteasome Assay
Certain 20s proteasomes showing
chymotrypsin-like activity
Substrate: Suc-LeuLeuValTyr-AMC
Inhibitor: N-Ac-LeuLeuNor-H (ALLN)
Detected by fluorescence spectrometer: 340 nm (ex),
535 nm (emi)
Enzyme activities: nmol AMC/min/mg protein
CH3
E
Suc-LLVY-AMC Suc-LLVY + AMC
H2N O O
7-amino-4-methylcoumarin
10/14/08 63
Fluorescence
10/14/08 65
Emission Process
(1) S1 S0 + hνf (fluorescence)
(2) S1 S0 (internal conversion)
(3) S1 triplet states (T1 or T2) (intersystem
crossing)
(4) S1 fragments (dissociation)
(6) T1 S0 + hν (phosphorescence)
ph
10/14/08 66
Summary of UV-Visible Absorptions
Aromatics
N N
N N
N N N
N
N N
275 (3.6) 265 (3.6) 250 (5.0)
313 (2.5) 313 (2.5) 355 (4.0)
10/14/08 68
Summary of UV-Visible Absorptions
N
O N S
H N
H
205 (3.8) 211 (4.0) 235 (3.7) 207 (3.7)
N
N CHO
N O O N
H H O
10/14/08 69
Summary of UV-Visible Absorptions
Nitrogen-containing Functions
Et3N RCN RNO2 RONO2
222 (2.2) 210 (4.2)
287 (1.3) 340 (2.1) 275 (2.0) 265sh (2.0)
RBr RI
A. Instrument components
(1) sources
(2) wavelength selectors
(3) sample containers
(4) radiation detectors
(5) signal processors and readout devices
10/14/08 72
Instruments for UV/Visible and IR
10/14/08 73
Instruments for UV/Visible and IR
10/14/08 74
Instruments for UV/Visible and IR
10/14/08 75
Instruments for UV/Visible and IR
• Single-beam instrument:
the steps for measurement:
(1) the 0% T setting with a shutter in place
(2) the 100% T adjustment with the solvent in light path
(3) the measurement of % T with the sample in place.
B. Double-beam instruments:
C. Diode array:
10/14/08 76
Instruments for UV/Visible and IR
10/14/08 77
Chirooptical Methods
α in degrees
l layer thickness in dm
c concentration in g · ml-1
For comparison of various optical compounds:
[Φ]T = 100 α / l · c = [α]T · MW / 100
λ λ