UV-Vis Spectroscopy

國防醫學院 藥學系
胡明寬 教授

10/14/08 1
 Introduction to Spectroscopy

Most of what we know about the structure of atoms

and molecules comes from studying their interaction
with light (electromagnetic radiation).

The Light of Knowledge

A given frequency or wavelength of light is associated with
a "light quanta" of energy we now call a photon.

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.

2. To “get” structure information?

The spectroscopic techniques described below (MS, UV, IR, &
NMR) do not provide a three-dimensional picture of a molecule,
but instead yield information about certain characteristic
features.

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.

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 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

The portion of the UV/visible region most useful for analysis of

organic compounds is having a wavelength range from 200 to
780 nm, with a corresponding frequency range from (------) to (-----) Hz.

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 What is ultraviolet (UV)?
Wavelengt
Range
h Range
Name
(nm)
UVA 315-400
UVB 280-315
UVC 200-280
Vacuum UV
100-200
(VUV)

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 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?

•The UV Index is a forecast, of the amount of

ultraviolet expected to reach the Earth's surface
when the sun is highest in the sky.
•The Index predicts UV levels on a 0-10+ scale.

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

λ (nm) 10 200 400 750

x-rays Far Near v b g y o r IR

uv uv vis (2.5 µM ~ 25 µM)

(cm-1) 106 5•104 2.5•104 1.3•104

Wavenumber  (cm-1) = 104 / µm (wavelength)

10/14/08 12
 Principle of UV-Visible Absorption Spectra

Electromagnetic waves
hν

Absorption of
N
UV/Vis light

UV-A: 400 – 320 nm Excitation of (valence)

UV-B: 320 – 280 nm electrons
UV-C: 280 – 10 nm

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 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 nσ*
n
σσ*
π Occupied levels

σ
σπ*

Figure 1.6 Molecular orbitals and electron transitions

10/14/08 15
Most common transitions

σ σ* In alkanes
σ π* In carbonyl comp’ds

π π* In alkenes, carbonyl comp’ds, alkynes,

Energy azo comp’ds, etc.

n σ* In oxygen, nitrogen, sulfur, and

halogen comp’ds, etc.

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

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 Terms and Symbols

Table 1. Important terms and symbols employed in

absorption measurement

Term and symbol Definition Alt’ve name and symbols

Radiant power, P, Po (in joules) radiation intensity, I, Io
Absorbance, A, log Po/P optical density, D;
extinction, E
Transmittance, T P/Po transmission, T
Path length in cm, b -
Molar conc of solute, c -
Absorptivity, a A/bc extinction coefficient, k
Molar absorptivity, ε A/bc molar extinction coefficient
10/14/08 18
 Molar Absorptivity
 Formerly known as the molar extinction
coefficient
 Values ranges: 0 ~ 106
 High-intensity absorptions: > 104
 Low-intensity absorptions: < 103
 e.g. forbidden transitions, σπ*
 Beer-Lambert Law

10/14/08 19
 Singlet/Triplet Excited States

(for a free radical)

(one unpaired e)

Ground Ground Excited Excited

Doublet state Singlet state Singlet state Triplet state
(due to 2 magnetic
Quantum numbers, (Spin paired) (Spin unpaired)
+1/2, -1/2)
(two unpaired electrons like oxygen having 3 magnetic quantum numbers,
-1, 0, +1; so called triplet states)

10/14/08 20
 Electronic Transitions

Figure 1.3 Energy scheme for the electronic transition

between HOMO and LUMO

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

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 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
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95% EtOH 205 Me3PO4 210 23
 Light Absorption and the Spectrum

hν
Io I

a transparent
material

If sample does not absorb light: I=Io

If I < Io : the difference can be plotted on
a graph versus wavelength, as
shown on the right

10/14/08 24
 A Typical Spectrometer

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 Absorption

Transmittance: T = I/Io
Absorbance: A = Log Io/I
if no absorption, T = 1; A = 0

Most spectrometers display absorbance on vertical axis,

and the commonly observed range from 0 (100% T)
to 2 (1% T)

λmax: The wavelength of maximum absorbance

10/14/08 26
 Beer-Lambert law

A = Log Io/I = ε · c · d

A: absorbance (no dimensions)

d: thickness in cm
c: concentration, mol · L –1
ε = molar absorptivity, cm2 · mmol-1 (L mol -1 cm -1)

A = acd

c = g/L
a ---- absorptivity
ε = a Μ (M = m.w.)
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 Excitation for Double Bond(s)

Examples of π  π* Excitation (1)

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 Excitation for Double Bond(s)

Examples of π  π* Excitation (2)

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 Excitation for Double Bond(s)

Examples of π  π* Excitation (3)

10/14/08 30
 Absorption Intensity

The oscillator strength f is a measure of the intensity of a transition.

The higher the transition probability is, the short the radiation
lifetime τo of an excited state.

The intensity of a band is judged by εmax

τo = 1/ (104 · εmax)

ε ≤ 100 transition: forbidden

100 < ε < 1000 weakly allowed
1000 < ε < 100 000 allowed
ε ≥ 1000 000 strong allowed
10/14/08 31
 Substituent Effects

The position of absorptions depend on steric, inductive, and

resonance effects --- the latter also affected by the chromophore
in large conjugated systems.
Chromophore: A covalently unsaturated group responsible for
electronic absorption (e.g. C=C, C=O, & NO2).
Auxochrome: A saturated group with non-bonded electrons which,
when attached to a chromophore, alter both the
wavelength and the intensity of the absorption.
Bathochromic effects: a shift to longer wavelengths (red shift)
Hypochromic effects: a shift to short wavelengths (blue shift)
Hyperchromic effects: an increase in intensity
Hypochromic effects: a decrease in intensity
10/14/08 32
 Terminology for Absorption Shifts

Nature of Shift Descriptive Term

To Longer Wavelength Bathochromic

To Shorter Wavelength Hypsochromic

To Greater Absorbance Hyperchromic

To Lower Absorbance Hypochromic

10/14/08 33
 Substituent Effects
Hyperchromic

Hypsochromic Bathochromic
ε (blue shift) (red shift)

Hypochromic

400 800 nm
blue red
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 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
ππ* 180 10,000 hexane
Nitromet- n π* 275 17 ethanol
N=O
hane ππ* 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

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 Electronic Transitions

n  π* (R bands): e.g. C=O, NO2

Forbidden, low εmax (< 100).
Increase in solvent polarity  blue shift

π  π* (K bands): e.g. in conjugated π systems, e.g.

butadiene, mesityl chloride.
Most seen in aromatic molecules
usually high εmax (>10,000).
In general, unresponsive to solvent polarity.
π  π* (B bands): benzenoid bands.
benzene shows broad absorption band, multiple peaks.
10/14/08 230 ~ 270 nm, εmax at ~ 255 nm. 38
 Electronic Transitions

E bands (ethylenic bands):

E1 and E2 bands of benzene near 180 and 200 nm.
In auxochromic substitution shift to near-UV region.
Mostly, E bands εmax = 2000 ~ 14,000.

10/14/08 39
 Characteristic Absorptions

• Compounds containing only σ bond.

σ  σ*; at far (vacuum)-UV region
energy required: 185 kcal/mol

2. Saturated compounds containing n electrons.

heteroatoms appear in structures, e.g. O, N, S, X (Halogen)
n  σ*; requires less energy than σ  σ*
Alc or ethers absorb at shorter than 185 nm, the mostly were
used as the solvent.

10/14/08 40
 Characteristic Absorptions

3. Compounds containing π and n electron chromophores

(a) n  σ* (b) n  π* (c) π  π*
3a. Ethylenic chromophore
RCH=CHR’: in vapor phase: 165 nm, 200 nm
Me-S-CH=CHR: bathochromic shift to ~ 228 nm.
(attach a heteroatom)
two or more ethylenic linkages:
intensity increase but at the same absorb positions.
Conjugated dienes, e.g. 1,3-butadiene:
π2  π3 * : batochromic shift compared to π  π*

10/14/08 41
 Characteristic Absorptions

Chromophoric groups and their interactions

Conjugated chromophores are of special importance for
uv/vis spectroscopy

λ = 178 nm λ = 205 nm
εmax = 17000 εmax = 2100

A bathochromic and hyperchromic effects is in general

observed when atoms or groups with n-orbitals
(-OH, OR, NH2, -NHR, -NR2, -SH, -SR, -Hal etc)
Auxochromic group is used.
10/14/08 42
 Characteristic Absorptions

Homoannular and Heteroannular systems:

Homoannular Heteroannular

Heteroannular and acyclic dienes: more εmax values (8000 ~20,000).

Homoannular dienes: εmax values (5000 ~8,000).

10/14/08 43
 The position of absorption of homo- (Ho)
and hetero (He)-annular systems.

Base value for He-diene 214

Base value for Ho-diene 253
Increment for
double bonds extending conj. + 30
alkyl or ring residue +5
exocyclic double bond +5
Polar g’ps: OAc +0
OAlk +6
SAlk + 30
Cl, Br +5
N(Alk)2 + 60
Solvent correction +0
10/14/08 44

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

Woodward-Fieser rules for Conjugated Dienes

Heteroannular (transoid) Homoannular (cisoid)

214 (3.9 ~ 4.3) 253 (3.7 ~ 3.9)
Group increments
-R +5 -Br, -Cl +5
-O2CR 0 -NR2 +60 H2C=C=C=CH2
-OR +6 -C=C* +30 241 (4.3)
-SR +30 exocyclic
C=C +5 RC≡C-C≡CR
*For extended conjugation only. 240 (2.5)
Cross conjugation doesn’t work well
10/14/08 46
 Characteristic Absorptions

Cholesta-3,5-diene Cholesta-2,4-diene

1
1 2 2
3 3

214 (base) 253 (base)

+ 15 (3 ring residues)
+ 5 (1 exocyclic)
Cal: 234 nm
Obs. 235 nm, εmax =19,000
10/14/08
+ 15 (3 ring residues)
+ 5 (1 exocyclic C=C)
Cal: 273 nm
Obs. 275 nm, εmax =10,000
47
 Summary of UV-Visible Absorptions

Carbonyl compounds
Two principal UV transitions: π*
The allowed π  π* Forbidden
280 nm
The forbidden n  π* Allowed
n
190 nm
π

• n  π* is observed just above the usual cutoff points of solvents

• Substitution on C=O by auxochrome with a lone pair of electrons, such as
-NR2, -OH, -OR, -NH2 or –X  hypsochromic effects on n  π*

10/14/08 48
 Hypsochromic effects of lone-pair
anxochromes on n  π* of carbonyls

λ max ε max Solvent

O
H3C H 293 nm 12 hexane
O
3 15 hexane
H3C CH3
O 5 53 hexane
H3C Cl
O
H3C NH2 8 − water
O
H3C OCH2CH3 10 60 water
O
H3C OH 204 41 ethanol
10/14/08 49
 Carbonyl compounds
 Woodward’s rule for enones
 220 ~ 250 nm for simple enones with a π  π* transition and
Shift to more intense absorption (ε = 8000 ~ 20000)

 Base: 215 (acyclic or six-ring)

 Extended conj +30 cyclopentenone -10
 Homodiene +39 aldehyde -5
 Exocyclic C=C +5 acid, ester -20

 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

Base: 215 (acyclic or six-ring) beta

Substituent α β γ

-R +10 +10 +18

-OH +35 +30 +50
-OR +35 +30 +17
-OAc +6 +6 +6
-Cl +15 +12
-Br +25 +30
-NR2 +95
-SR +85

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

244 (4.3) 260 (4.0) 260 (3.3)

279 (2.8) 369 (3.1) 400 (3.0)
430 (1.3) 560 (1.6)
10/14/08 54
 Summary of UV-Visible Absorptions

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

198 (3.9) 208 (3.78) 211 (3.79)

255 (2.36) 257 (2.23) 270 (3.16)

10/14/08 56
 Summary of UV-Visible Absorptions

O NH2 NH3+

235 (3.97) 230 (3.93) 203 (3.88)

287 (3.42) 280 (3.16) 254 (2.5)

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

•Peptidyl Prolyl cis-trans Isomerase (PPIase)

•A binding protein of immunosuppressant,
cyclosporine A
O
O O
X N X N

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

 Assay conditions: Enz-Inh 4 h preincubation in

Buffer (890 mL), then adding substrate (10 mL) and
chymotrypsin (100 mL).

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

Figure 1. A simple Jablonski diagram showing some important radiative

and non-radiative processes (Abs.: absorption, Fl.: fluorescence, Ph.:
phosphorescence, VR: vibrational relaxation, IC: internal conversion,
10/14/08
ISC: intersystem crossing) 64
Spin Multiplicity 自旋重態數

 The number of possible orientations,

calculated as 2S + 1, of the spin angular
momentum corresponding to a given total
spin quantum number (S, 自旋量子數 ), for the
same spatial electronic wavefunction. A state
of singlet multiplicity has S = 0 and 2S + 1 =
1. A doublet state has S = 1/2, 2S + 1 = 2,
triplet multiplicity has S = 1, 2S + 1 = 3, etc.

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)

 (5) S1 + Q  products (quenching)

 (6) T1  S0 + hν (phosphorescence)
ph

10/14/08 66
 Summary of UV-Visible Absorptions

Aromatics

255 (2.36) 314 (2.4) 380 (3.95)

295 265 (3.6) 270 (4.7)

345 324 (4.0) 347 (3.6)
338 (2.3) 656 (2.5)
10/14/08 67
 Summary of UV-Visible Absorptions

N N
N N
N N N

252 (3.3) 246 (3.1) 246 (3.1) 261 (3.8)

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

210 (3.5) 211 (3.6) 227 (3.3) 250 (3.6)

272 (1.7) 287 (4.2)

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)

RONO tBuNO PhNO

350 (2.7) 300 (2.0) 280 (3.95)

665 (1.3) 755 (1.7)

MeN=NMe (t) PhN=NPh (t) R2CN2

319 (4.3) 400 (0.5)
347 (0.7)
10/14/08
445 (2.5) 70
 Summary of UV-Visible Absorptions

Sulfur and Halogen-containing Functions

RSR RSSR R2SO R2SO2

210-215 (3.4) 204 (3.3) 210-215 (3.2) end

235-240sh 251 (2.6)

RBr RI

end 255-260 (2.7)

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 Instruments for UV/Visible and IR

A. Instrument components
(1) sources
(2) wavelength selectors
(3) sample containers
(4) radiation detectors
(5) signal processors and readout devices

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 Instruments for UV/Visible and IR

• Sources: a continuous source is required

A. D- and H-lamps at low pressure

§ provide full continuous spectrum at 160 ~ 375 nm
at > 400 nm, the lamps produce emission lines.

§ quartz window is employed in D- or H-lamp, because

glass absorbs strongly at wavelength < 350 nm.

10/14/08 73
 Instruments for UV/Visible and IR

B. Tungsten Filament lamps.

Contain a small quantity of iodine and a quartz envelop
that houses the tungsten filament wavelength
regions: 350 ~ 2500 nm.

C. Xenon Arc lamps.

Produces intense radiation.
regions: 250 ~ 600 nm.

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 Instruments for UV/Visible and IR

(2). Sample containers.

Cuvettes or cells: quartz or fused silica is required for
work below 350 nm.
Silica glasses: used in 350 ~ 2000 nm.
Plastic containers: applied in the visible region.

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 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:

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 Instruments for UV/Visible and IR

The most commonly used (no absorbing) solvents are

water, ethanol, hexane and cyclohexane.

The absorbance of a sample will be proportional to

its molar concentration in the sample cuvette,
a corrected absorption value known as the
molar absorptivity is used.

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 Chirooptical Methods

A chiral compound rotates the plane of linearly polarized light.

α = [α] T · l · c
λ

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

[α]T specific rotation in degrees

λ

“+” dextrorotatory; “-” levorotatory

l thickness in cm
10/14/08
c concentration in mol/l 78