Instrumental Analysis Spectroscopy - ppt12

Instrumentation and
Interpretation of Spectra
Tarekegn Berhanu (Ph. D)
1. Absorption molecular spectrophotometric

techniques
Introduction to spectroscopy
Light interacting with matter as an analytical tool
The Electromagnetic Spectrum

The visible spectrum constitutes but a small part of the total
radiation spectrum. Most of the radiation that surrounds us
cannot be seen, but can be detected by dedicated sensing
instruments. This electromagnetic spectrum ranges from
very short wavelengths (including gamma and x-rays) to very
long wavelengths (including microwaves and broadcast radio
waves).
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)
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/Beers Law
Atomic Absorption
UV-vis region
Quantitative analysis
Beers 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
EM regions related to different Analytical Techniques

(a) The visible spectrum constitutes a small portion of the complete
electromagnetic radiation spectrum that extends from the ultra-short
wave gamma rays at one end to that of the radio-waves at the other
(400-700 nm),
(b) The wave length scale is nonlinear,
(c) -Rays Region : Mossbauer Spectroscopy (due to absorption) and Ray Spectroscopy (due to emission) are used as analytical means.
(d) Inner-shell Electrons : X-Ray absorption spectroscopy (due to
absorption) and X-Ray Fluorescence spectroscopy (XRF) (due to
emission) are employed as analytical means.
(e) From Vacuum-UV to Infra-Red Region : UV-VIS, IR-spectroscopy,
spectrophotometry, atomic absorption spectroscopy (AAS) (due to
absorption) and atomic emission spectroscopy (AES, ESS, ICP) ; atomic
fluorescence spectroscopy (AFS) (due to emission) are used as analytical
techniques.
(f) Microwave Region : Microwave spectroscopy and electron spin
resonance (ESR) (due to absorption) are employed as analytical
methods.
(g) Radiowave Region : Nuclear Magnetic Resonance (NMR) (due to
6
absorption) is used as analytical method.
The UV Absorption process

The lowest energy transition (and most often obs. by UV) is
typically that of an electron in the Highest Occupied Molecular
Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital
(LUMO)
* 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
corresponding to a transition from ground to excited state.
Energies correspond to a 1-photon of 300 nm light are ca. 95
The Spectroscopic Process

1. In UV spectroscopy, the sample is irradiated with the
broad spectrum of the UV radiation
2. If a particular electronic transition matches the energy of
a certain band of UV, it will be absorbed
3. The remaining UV light passes through the sample and is
observed
4. From this residual radiation a spectrum is obtained with
gaps at these discrete energies this is called an
absorption spectrum
Electronic Transition
In all compounds other than alkanes, the electrons may undergo sev
Transitions of different energies. Some of the most important transiti
10
Selection Rules
1. Not all transitions that are possible are observed
2. For an electron to transition, certain quantum
mechanical constraints apply these are called
selection rules
3. For example, an electron cannot change its spin
quantum number during a transition these are
forbidden
Other examples include:
the number of electrons that can be
excited at one time
symmetry properties of the molecule
symmetry of the electronic states
4. To further complicate matters, forbidden
transitions are sometimes observed (albeit at
low intensity) due to other factors
11
MOLAR ABSORPTIVITY
The molar absorptivity is mostly controlled by two vital

factors, namely :
(i)polarity of the excited state, and (ii) probability of the
electronic transition. So as to materialize an interaction, a photon
should evidently strike a molecule very closely within the space
of the molecular dimensions.
The probability of the electronic transition, designated as g,
shall be responsible for the target hits that may ultimately lead to
absorption. However, the molar absorptivity may be expressed as
follows :
12
SPECTRAL PRESENTATION
Absorption spectra may be presented in a number of
fashions as depicted in Figure 21.2, namely :
(a) Wavelength Vs Absorbance,
(b) Wavelength Vs Molar Absorptivity, and
(c) Wavelength Vs Transmittance.
A few important features related to spectral presentation are enumerated
below :
(a) In order to simplify the conversion of spectra in qualitative
identification the spectral data should be plotted either as log A or as log
Vs wavelength, thereby giving rise to the following expression
where, b = Cell-length, and c = Sample concentration
13
14
Absorption Bands
1. four different types of absorption bands have so far gained
cognizance in the spectra of organic compounds, which are
namely : K-bands ; R-bands ; B-bands ; and E-bands.
These bands will be discussed briefly here with regard to the
structural features.
(a) K-bands : They normally arise from - structures and
result from * transitions. These are invariably
characterized by high molar absorptivity.
Examples :
(i) A diene : C = CC = C to C+C = CC ; where K-band is due
to the resonance transition,
(ii) Vinyl benzene or acetophenone : i.e., aromatic compounds
having chromophoric substitution.
(b) R-bands : They usually arise from n * transitions.
They seldom display very noticeable results in aliphatic
compounds, but marked and pronounced bathochromic shifts (i.e.,
shifting of absorption towards longer wavelengthsas in extended
open-chain-conjugated systems) do take place whenSH, OH
and NH2 replace hydrogen atom in unsaturated groups. Thus,15R-
Absorption Bands ..
(c) B-bands : These are rather weak-type of absorption
bands. They are
characteristic of both heteroatomic and aromatic
molecules and may also
consist of fine vibrational sub-bands.
(d) E-bands : They usually result from oscillations of
electrons in aromatic-ring
systems,
Conjugated Systems :
It is quite evident that the conjugated systems might fail to
display the expected conjugated bands due to the following two
reasons, namely :
(a) Orbitals of adjacent multiple bonds are at right angles instead
of being parallel, and
(b) Resonating dipolar structures cannot be envisaged.
The resulting spectrum may seem to appear as a mere
superimposition of the spectra of the individual chromophoric
groups
16
UV Spectroscopy
When the energy levels are superimposed, the effect can be readily
explained any transition has the possibility of being observed
Disassociation
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
V2
V1 R1 - Rn
E1
Vo
R1 - Rn
Disassociation
Energy
R1 - Rn
V4
R1 - Rn
V3
R1 - Rn
E0
V2
V1 R1 - Rn
Vo
R1 - Rn
17
UV Spectroscopy
II.
Instrumentation and Spectra

A. Instrumentation
1. The construction of a traditional UV-VIS spectrometer is very
similar to an IR, as similar functions sample handling,
irradiation, detection and output are required
2.
Here is a simple schematic that covers most modern UV

spectrometers:
I0
I0
detector
monochromator/
beam splitter optics
I0
sample
UV-VIS sources
reference
log(I0/I) = A
200
700
, nm
18
UV Spectroscopy
II.

A. Instrumentation
3. Two sources are required to scan the entire UV-VIS band:
Deuterium lamp covers the UV 200-330
Tungsten lamp covers 330-700
4.
As with the dispersive IR, the lamps illuminate the entire band of
UV or visible light; the monochromator (grating or prism)
gradually changes the small bands of radiation sent to the beam
splitter
5.
The beam splitter sends a separate band to a cell containing the

sample solution and a reference solution
6.
The detector measures the difference between the transmitted

light through the sample (I) vs. the incident light (I 0) and sends
this information to the recorder
19
19
UV Spectroscopy
A. Instrumentation
7. As with dispersive IR, time is required to cover the entire UV-VIS
band due to the mechanism of changing wavelengths
8.
A recent improvement is the diode-array spectrophotometer here a prism (dispersion device) breaks apart the full spectrum
transmitted through the sample
9.
Each individual band of UV is detected by a individual diodes on

a silicon wafer simultaneously the obvious limitation is the size
of the diode, so some loss of resolution over traditional
instruments is observed
Diode array
UV-VIS sources
sample
II.
Polychromator
entrance slit and dispersion device
20
UV Spectroscopy
II.

B. Sample Handling
1. Virtually all UV spectra are recorded solution-phase
2.
Cells can be made of plastic, glass or quartz
3.
Only quartz is transparent in the full 200-700 nm range; plastic

and glass are only suitable for visible spectra
4.
Concentration (we will cover shortly) is empirically determined
A typical sample cell (commonly called a cuvet):
21
II. Instrumentation and Spectra

Spectrometric instruments have a common
set of
general features.
Often, one technique is distinguished from
another by
differences in these features. Here we look at
specific
features for the UV/Visible experiment.
Sources: D2 lamp, W filament (halogen
lamp), and Xe
arc lamp.
Wavelength Selectors: Filters and
22
Monochromators.
UV Spectroscopy
II.

C. Solvents
5. Solvents must be transparent in the region to be observed; the
wavelength where a solvent is no longer transparent is referred
to as the cutoff
6.
Since spectra are only obtained up to 200 nm, solvents typically

only need to lack conjugated systems or carbonyls
Common solvents and cutoffs:
acetonitrile
190
chloroform
240
cyclohexane
195
1,4-dioxane
215
95% ethanol
205
n-hexane
methanol
isooctane
water
190
201
205
195
23

Solvents can interact with the analyte molecules and
shift
absorbance peaks and intensities.
Red Shift (Bathochromic) Peaks shift to longer
wavelength.
Blue Shift (Hypsochromic) Peaks shift to
shorter wavelength.
n * generally blue shifted by solvent;
solvation of and
hydrogen
bonding to the lone pair. Large shifts (up to 30 nm).
Both n* and * red shifted; attractive
polarization forces,
increase with increasing solvent polarity. Small
shifts (less than 5
24
nm).
UV Spectroscopy
Solvents
7. Additionally solvents must preserve the fine structure (where it
is actually observed in UV!) where possible
8.
H-bonding further complicates the effect of vibrational and

rotational energy levels on electronic transitions, dipole-dipole
interacts less so
9.
The more non-polar the solvent, the better (this is not always
possible)
25
UV Spectroscopy
II.

D. The Spectrum
1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV,
200-700 for UV-VIS determinations
2.
Due to the lack of any fine structure, spectra are rarely shown in
their raw form, rather, the peak maxima are simply reported as
a numerical list of lamba max values or max
NH2
max =
O
206 nm
252
317
376
26
UV Spectroscopy
II.

D. The Spectrum
1. The y-axis of the spectrum is in absorbance, A
2.
From the spectrometers point of view, absorbance is the inverse

of transmittance: A = log10 (I0/I)
3.
From an experimental point of view, three other considerations

must be made:
i. a longer path length, l through the sample will cause
more UV light to be absorbed linear effect
ii.
the greater the concentration, c of the sample, the

more UV light will be absorbed linear effect
iii. some electronic transitions are more effective at the

absorption of photon than others molar absorptivity,
this may vary by orders of magnitude

27
UV Spectroscopy
II.

D. The Spectrum
4. These effects are combined into the Beer-Lambert Law:
cl
A=
i.
for most UV spectrometers, l would remain constant

(standard cells are typically 1 cm in path length)
ii.
concentration is typically varied depending on the strength

of absorption observed or expected typically dilute sub .
001 M
iii. molar absorptivities vary by orders of magnitude:

values of 104-106 104-106 are termed high intensity
absorptions
values of 103-104 are termed low intensity absorptions
values of 0 to 103 are the absorptions of forbidden
transitions
A is unitless, so the units for are cm-1 M-1 and are rarely
expressed
UV Spectroscopy
II.

E. Practical application of UV spectroscopy
1. UV was the first organic spectral method, however, it is rarely
used as a primary method for structure determination
2.
It is most useful in combination with NMR and IR data to

elucidate unique electronic features that may be ambiguous in
those methods
3.
It can be used to assay (via max and molar absorptivity) the

proper irradiation wavelengths for photochemical experiments,
or the design of UV resistant paints and coatings
4.
The most ubiquitous use of UV is as a detection device for HPLC;

since UV is utilized for solution phase samples vs. a reference
solvent this is easily incorporated into LC design
UV is to HPLC what mass spectrometry (MS) will be to GC
29
UV Spectroscopy
III. Chromophores
A. Definition
1. Remember the electrons present in organic molecules are
involved in covalent bonds or lone pairs of electrons on atoms
such as O or N
2.
Since similar functional groups will have electrons capable of

discrete classes of transitions, the characteristic energy of these
energies is more representative of the functional group than the
electrons themselves
3.
A functional group capable of having characteristic electronic

transitions is called a chromophore (color loving)
4.
Structural or electronic changes in the chromophore can be

quantified and used to predict shifts in the observed electronic
transitions
30
UV Spectroscopy
III. Chromophores
B. Organic Chromophores
1. Alkanes only posses -bonds and no lone pairs of electrons, so
only the high energy * transition is observed in the far UV
This transition is destructive to the molecule, causing cleavage
of the -bond
C
31
UV Spectroscopy
III. Chromophores
2. Alcohols, ethers, amines and sulfur compounds in the cases of
simple, aliphatic examples of these compounds the n * is the
most often observed transition; like the alkane * it is most
often at shorter than 200 nm
Note how this transition occurs from the HOMO to the LUMO
CN
nN sp
CN
N
anitbonding
orbital
N
32
UV Spectroscopy
III. Chromophores
3. Alkenes and Alkynes in the case of isolated examples of these
compounds the * is observed at 175 and 170 nm,
respectively
Even though this transition is of lower energy than *, it is
still in the far UV however, the transition energy is sensitive to
substitution
33
UV Spectroscopy
III. Chromophores
4. Carbonyls unsaturated systems incorporating N or O can
undergo
n * transitions (~285 nm) in addition to *
Despite the fact this transition is forbidden by the selection rules
( = 15), it is the most often observed and studied transition for
carbonyls
This transition is also sensitive to substituents on the carbonyl
Similar to alkenes and alkynes, non-substituted carbonyls
undergo the * transition in the vacuum UV (188 nm, =
900); sensitive to substitution effects
34
UV Spectroscopy
III. Chromophores
4. Carbonyls n * transitions (~285 nm); * (188 nm)
It has been
determined from
spectral studies, that
carbonyl oxygen
more approximates
sp rather than sp2 !
CO transitions omitted for clarity
35
UV Spectroscopy
III. Chromophores
C. Substituent Effects
General from our brief study of these general chromophores, only
the weak n * transition occurs in the routinely observed UV
The attachment of substituent groups (other than H) can shift
the energy of the transition
Substituents that increase the intensity and often wavelength of
an absorption are called auxochromes
Common auxochromes include alkyl, hydroxyl, alkoxy and amino
groups and the halogens
UV Spectroscopy
III. Chromophores
General Substituents may have any of four effects on a
chromophore
i. Bathochromic shift (red shift) a shift to longer ; lower
energy
ii.
Hypsochromic shift (blue shift) shift to shorter ; higher

energy
Hyperchromic
iii. Hyperchromic effect an increase in intensity

iv. Hypochromic effect a decrease in intensity
Bathochromic
Hypochromic
200 nm
Hypsochromic
700 nm
37
UV Spectroscopy
III. Chromophores
1. Conjugation most efficient means of bringing about a
bathochromic and hyperchromic shift of an unsaturated
chromophore:
nm
H2C
max
CH2
175
15,000
217
21,000
258
35,000
465
125,000
-carotene
O
n * 280
* 189
12
900
n * 280
* 213
27
7,100
38
UV Spectroscopy
III. Chromophores
1. Conjugation Alkenes
The observed shifts from conjugation imply that an increase in
conjugation decreases the energy required for electronic
excitation
From molecular orbital (MO) theory two atomic p orbitals, 1 and
2 from two sp2 hybrid carbons combine to form two MOs 1 and
2* in ethylene
39
UV Spectroscopy
III. Chromophores
When we consider butadiene, we are now mixing 4 p orbitals
giving 4 MOs of an energetically symmetrical distribution
compared to ethylene
E for the HOMO LUMO transition is reduced

40
UV Spectroscopy
III. Chromophores
Extending this effect out to longer conjugated systems the
energy gap becomes progressively smaller:
Energy
Lower energy =
Longer wavelengths
ethylene
butadiene
hexatriene
octatetraene
41
UV Spectroscopy
III. Chromophores
Similarly, the lone pairs of electrons on N, O, S, X can extend
conjugated systems auxochromes
Here we create 3 MOs this interaction is not as strong as that
of a conjugated -system
A
Energy
nA
42
UV Spectroscopy
III. Chromophores
Methyl groups also cause a bathochromic shift, even though
they are devoid of - or n-electrons
This effect is thought to be through what is termed
hyperconjugation or sigma bond resonance
H
C
C
H
H
43
UV Spectroscopy
Next time We will find that the effect of substituent groups can be reliably
quantified from empirical observation of known conjugated structures
and applied to new systems
This quantification is referred to as the Woodward-Fieser Rules which we
will apply to three specific chromophores:
1. Conjugated dienes
2. Conjugated dienones
3. Aromatic systems
max = 239 nm
44
UV Spectroscopy
IV. Structure Determination
A. Dienes
1. General Features
For acyclic butadiene, two conformers are possible s-cis and strans
s-trans
s-cis
The s-cis conformer is at an overall higher potential energy than

the s-trans; therefore the HOMO electrons of the conjugated
system have less of a jump to the LUMO lower energy, longer
wavelength
45
UV Spectroscopy
A. Dienes
1. General Features
Two possible * transitions can occur for butadiene
and 2 4*

s-trans
175 nm forb.
217 nm
175 nm
253 nm
s-cis
The 2 4* transition is not typically observed:
The energy of this transition places it outside the

region typically observed 175 nm
For the more favorable s-trans conformation, this

transition is forbidden
The 2 3* transition is observed as an intense absorption
46
UV Spectroscopy
A. Dienes
1. General Features
The 2 3* transition is observed as an intense absorption (
= 20,000+) based at 217 nm within the observed region of the
UV
While this band is insensitive to solvent (as would be expected)
it is subject to the bathochromic and hyperchromic effects of
alkyl substituents as well as further conjugation
Consider:
max = 217
nm
253
220
227
227
256
263
47
UV Spectroscopy
A. Dienes
2. Woodward-Fieser Rules
Woodward and the Fiesers performed extensive studies of
terpene and steroidal alkenes and noted similar substituents and
structural features would predictably lead to an empirical
prediction of the wavelength for the lowest energy *
electronic transition
This work was distilled by Scott in 1964 into an extensive
treatise on the Woodward-Fieser rules in combination with
comprehensive tables and examples (A.I. Scott, Interpretation
of the Ultraviolet Spectra of Natural Products, Pergamon, NY,
1964)
A more modern interpretation was compiled by Rao in 1975
(C.N.R. Rao, Ultraviolet and Visible Spectroscopy, 3rd Ed.,
Butterworths, London, 1975)
48
49
UV Spectroscopy
A. Dienes
2. Woodward-Fieser Rules - Dienes
The rules begin with a base value for max of the chromophore
being observed:
acyclic butadiene = 217 nm
The incremental contribution of substituents is added to this
base value from the group tables:
Group
Extended conjugation
Increment
+30
Each exo-cyclic C=C
+5
Alkyl
+5
-OCOCH3
+0
-OR
+6
-SR
+30
-Cl, -Br
-NR2
+5
+60
50
UV Spectroscopy
A. Dienes
2. Woodward-Fieser Rules - Dienes
For example:
Isoprene - acyclic butadiene = 217 nm
one alkyl subs.
+5
nm
Experimental value
Allylidenecyclohexane
- acyclic butadiene =
one exocyclic C=C
2 alkyl subs.
Experimental value
222 nm
220 nm
217 nm
+ 5 nm
+10 nm
232 nm
237 nm
51
UV Spectroscopy
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
In the pre-NMR era of organic spectral determination, the power
of the method for discerning isomers is readily apparent
Consider abietic vs. levopimaric acid:
C OH
O
abietic acid
C OH
O
levopimaric acid
52
UV Spectroscopy
A. Dienes
3. Woodward-Fieser Rules Cyclic Dienes
Be careful with your assignments three common errors:
R
This compound has three exocyclic

double bonds; the indicated bond is
exocyclic to two rings
This is not a heteroannular diene; you

would use the base value for an acyclic
diene
Likewise, this is not a homooannular
diene; you would use the base value for
an acyclic diene
53
UV Spectroscopy
B. Enones
1. General Features
Carbonyls, as we have discussed have two primary electronic
transitions:
Remember, the * transition is

allowed and gives a high , but lies
outside the routine range of UV
observation
The n * transition is forbidden
and gives a very low e, but can
routinely be observed
54
UV Spectroscopy
B. Enones
1. General Features
For auxochromic substitution on the carbonyl, pronounced
hypsochromic shifts are observed for the n * transition (max):
O
H
293 nm
O
CH3
279
Cl
235
NH2
214
O
O
O
OH
204
204
This is explained by the inductive

withdrawal of electrons by O, N or
halogen from the carbonyl carbon this
causes the n-electrons on the carbonyl
oxygen to be held more firmly
It is important to note this is different
from the auxochromic effect on *
which extends conjugation and causes
a bathochromic shift
In most cases, this bathochromic shift is
not enough to bring the *
transition into the observed range
55
UV Spectroscopy
B. Enones
1. General Features
Conversely, if the C=O system is conjugated both the n *
and * bands are bathochromically shifted
Here, several effects must be noted:
i. the effect is more pronounced for *
ii.
if the conjugated chain is long enough, the much

higher intensity * band will overlap and drown
out the n * band
iii. the shift of the n * transition is not as predictable

For these reasons, empirical Woodward-Fieser rules for
conjugated enones are for the higher intensity, allowed *
transition
UV Spectroscopy
B. Enones
1. General Features
These effects are apparent from the MO diagram for a
conjugated enone:
57
UV Spectroscopy

B. Enones
C C C
2. Woodward-Fieser Rules - Enones
O

C C C C C
Group
Increment
6-membered ring or acyclic enone
Base 215
nm
5-membered ring parent enone
Base 202
nm
Acyclic dienone
Base 245
nm
Double bond extending conjugation
30
Alkyl group or ring residue
and higher
10, 12, 18
-OH
and higher
35, 30, 18
-OR
35, 30, 17,

31
-Cl
15, 12
-Br
25, 30
95
-O(C=O)R
-NR2
Exocyclic double bond
58
UV Spectroscopy
B. Enones
Aldehydes, esters and carboxylic acids have different base
values than ketones
Unsaturated system
Base Value
Aldehyde
208
With or alkyl groups
220
230
With alkyl groups
242
Acid or ester
208
217
Group value exocyclic double

bond
+5
Group value endocyclic bond

in 5 or 7 membered ring
+5
59
UV Spectroscopy
B. Enones
Unlike conjugated alkenes, solvent does have an effect on max
These effects are also described by the Woodward-Fieser rules
Solvent correction
Water
Increment
+8
Ethanol, methanol
Chloroform
-1
Dioxane
-5
Ether
-7
Hydrocarbon
-11
60
UV Spectroscopy
B. Enones
Some examples keep in mind these are more complex than
dienes
cyclic enone =
215
O
nm
2 x - alkyl subs.
(2 x 12)+24 nm
239 nm
Experimental value
238 nm
O
nm
+ 5 nm
cyclic enone =
215 nm
extended conj.
+30 nm
-ring residue
+12 nm
-ring residue
+18
exocyclic double bond
280 nm
61
UV Spectroscopy
B. Enones
Take home problem can these two isomers be discerned by UVspec
O
O
Eremophilone
allo-Eremophilone
62
UV Spectroscopy
C. Aromatic Compounds
1. General Features
Although aromatic rings are among the most widely studied and
observed chromophores, the absorptions that arise from the
various electronic transitions are complex
On first inspection, benzene has six -MOs, 3 filled , 3 unfilled
*
63
UV Spectroscopy
1. General Features
One would expect there to be four possible HOMO-LUMO *
transitions at observable wavelengths (conjugation)
Due to symmetry concerns and selection rules, the actual
transition energy states of benzene are illustrated at the right:
u
200 nm
(forbidden
)
260 nm
(forbidden
)
Expected
Actual
u
u
180 nm
(allowed)
g
64
UV Spectroscopy
1. General Features
The allowed transition ( = 47,000) is not in the routine range of
UV obs. at 180 nm, and is referred to as the primary band
The forbidden transition ( = 7400) is observed if substituent
effects shift it into the obs. region; this is referred to as the
second primary band
At 260 nm is another forbidden
transition ( = 230), referred to
as the secondary band.
This transition is fleetingly allowed
due to the disruption of symmetry
by the vibrational energy states,
the overlap of which is observed
in what is called fine structure
65
UV Spectroscopy
1. General Features
Substitution, auxochromic, conjugation and solvent effects can
cause shifts in wavelength and intensity of aromatic systems
similar to dienes and enones
However, these shifts are difficult to predict the formulation of
empirical rules is for the most part is not efficient (there are
more exceptions than rules)
There are some general qualitative observations that can be
made by classifying substituent groups --
66
UV Spectroscopy
2. Substituent Effects
a. Substituents with Unshared Electrons
If the group attached to the ring bears n electrons,
they can induce a shift in the primary and secondary
absorption bands
Non-bonding electrons extend the -system through

resonance lowering the energy of transition *
More available n-pairs of electrons give greater shifts

G
67
UV Spectroscopy
The presence of n-electrons gives the possibility of n
* transitions
If this occurs, the electron now removed from G,

becomes an extra electron in the anti-bonding *
orbital of the ring
This state is referred to as a charge-transfer excited

state
*
*
68
UV Spectroscopy
pH can change the nature of the substituent group
deprotonation of oxygen gives more available n-pairs,
lowering transition energy
protonation of nitrogen eliminates the n-pair,
raising transition energy
Primary
Secondary
Substitue
nt
max
max
-H
203.5
7,400
254
204
-OH
211
6,200
270
1,450
-O-
235
9,400
287
2,600
-NH2
230
8,600
280
1,430
-NH3+
203
7,500
254
169
-C(O)OH
230
11,600
273
970
-C(O)O-
224
8,700
268
560
69
UV Spectroscopy
b. Substituents Capable of -conjugation
When the substituent is a -chromophore, it can
interact with the benzene -system
With benzoic acids, this causes an appreciable shift in

the primary and secondary bands
For the benzoate ion, the effect of extra n-electrons

from the anion reduces the effect slightly
Primary
Secondary
Substitue
nt
max
max
-C(O)OH
230
11,600
273
970
-C(O)O-
224
8,700
268
560
70
UV Spectroscopy
d. Di-substituted and multiple group effects
With di-substituted aromatics, it is necessary to
consider both groups
If both groups are electron donating or withdrawing,

the effect is similar to the effect of the stronger of the
two groups as if it were a mono-substituted ring
If one group is electron withdrawing and one group

electron donating and they are para- to one another,
the magnitude of the shift is greater than the sum of
both the group effects
Consider p-nitroaniline:
O
H2N
O
H2N
N
O
71
UV Spectroscopy
If the two electonically dissimilar groups are ortho- or
meta- to one another, the effect is usually the sum of
the two individual effects (meta- no resonance; orthosteric hind.)
For the case of substituted benzoyl derivatives, an

empirical correlation of structure with observed max
has been developed
This is slightly less accurate than the Woodward-Fieser

rules, but can usually predict within an error of 5 nm
O
R
G
72
UV Spectroscopy
Parent Chromophore
max
R = alkyl or ring
residue
246
R=H
250
R = OH or O-Alkyl
230
Substituent increment
Alkyl or ring residue
10
-O-Alkyl, -OH, -O-Ring
25
-O-
11
20
78
-Cl
10
-Br
15
-NH2
13
13
58
-NHC(O)CH3
20
20
45
-NHCH3
-N(CH3)2
73
20
20
85
73
UV Spectroscopy
d. Polynuclear aromatics
When the number of fused aromatic rings increases,
the for the primary and secondary bands also
increase
For heteroaromatic systems spectra become complex

with the addition of the n * transition and ring size
74
effects and are unique to each case
What happens after absorbing UV-Vis radiation?

It is pertinent to mention here that an excited electron normally
returns to the ground state in about 109 to 108 seconds.
Consequently, energy must now be released to compensate for
the energy absorbed by the system. In actual practice however,
the following three situations arise, namely :
Firstly, if the electron returns directly to the ground state, the
net effect would
be evolution of heat.
Secondly, if the electron returns to the ground state by
passing through a
second excited state, the net outcome would be release of
energy in the form
of heat and light.
Thirdly, if a large amount of energy is absorbed by certain
substances, bonds
may be ruptured and thereby giving rise to altogether new
compounds.
For instance : ergosterol on being subjected to UV radiation
75
yields cholecalciferols which are, in fact, altogether new
76
ASSAY METHODS
MEASUREMENT OF TRANSMITTANCE AND
ABSORBANCE
Transmittance and absorbance, as outlined in Table 13-1 (above),

cannot normally be measured in the laboratory because the analyte
solution must be held in a transparent Container or cell.
77

ABSORBANCE
FIGURE 13-1 Reflection and scattering losses with a solution

contained in a typical glass cell. Losses by reflection can occur at
all the boundaries that separate the different materials. In this
example, the light passes through the air-glass, glass-solution,
solution-glass, and glass-air interfaces.
78
Qualitative Analysis
Great deal of information from UV spectrum used
by itself
cannot be extracted
UV spectrum is most useful when at least a general
idea of the
structure is already known
There are several generalizations can serve to
guide our use of
UV data.
These generalizations will be more meaningful if
combined with
IR and NMR data
In the absence of IR and NMR data these
generalizations should
79
be taken only as guidelines
Some Important Generalizations
80
81
Some Important
Generalizations
82
83

ABSORBANCE
..
Not
all radiations are
effective
Some of ways of loss for radiation are:
- loss by reflection in passing through a sample container
containing
solvent or water.
- Attenuation of a beam may occur as a result of scattering
by large
molecules and sometimes from absorption by the
container walls (see
above figure).
To compensate for these effects, the power of the beam
transmitted by the
analyte solution is usually compared with the power of the
beam transmitted
by an identical cell containing only solvent.
An experimental transmittance and absorbance that closely
approximate the
The
terms
Po and P, refer
the power are
of radiation
after itwith
has the
true
transmittance
andto
absorbance
then obtained
passed through cells containing the solvent and the analyte
equations
84
solutions, respectively.
Quantitative Analysis
Beer- Lamber Law

Beer's law also applies to a medium containing more than
one kind of
absorbing substance. Provided that the species do not
interact, the total
absorbance for a multicomponent system is given by
where the subscripts refer to absorbing components 1,2, ... , n.
85
Ultraviolet (UV) Spectroscopy Analysing the Output
Absorbance
Beer Lambert
Law
A = .c.l
1.0
0.5
0.0
350
450
400
wavelength (nm)
Handling samples of known concentration

If you know the structure of your compound X and
you wish to acquire UV data you would do the
following.
Prepare a known concentration solution of your
sample.
Run a UV spectrum (typically from 500 down to 220
nm).
Read off the absorbance values of each of the
maxima
left). read off the wavelength values for
From the(see
spectrum
each of the maxima of the spectra (see left)
Then using the known concentration (in moles L-1 ) and the known pathlength (1
cm) calculate the molar absorptivity () for each of the maxima.
Finally quote the data as follows (for instance for the largest peak in the spectrum
to the left and assuming a concentration of 0.0001 moles L -1 ).
max = 487nm A= 0.75
= 0.75 /(0.001 x 1.0) = 7500 moles-1 L cm -1
86
Determining concentration of samples with known molar absorptivity ().

Having used the calculation in the yellow box to work out the molar absorptivity
of a compound you can now use UV to determine the concentration of compound
X in other samples (provided that these sample only contain pure X).
Simply run the UV of the unknown and take the absorbance

reading at the maxima for which you have a known value of . In
the case above this is at the peak with the highest wavelength (see
above). Having found the absorbance value and knowing and l
you can calculate c.
This is the the principle used in many experiments to determine the
concentration of a known compound in a particular test sample
for instance monitoring of drug metabolites in the urine of drug
takers; monitoring biomolecules produced in the body during
87
particular disease states
Spectrum Analysis
88
Typical Beers Law Plot
89
Other Electronic Transition absorption

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)
90
Exercises
1. During an assay of the thiamine (vitamin B1) content of a
pharmaceutical preparation, the percent transmittance scale
was accidentally read, instead of the absorbance scale of the
spectrophotometer. One sample gave a reading of 82.2% T,
and a second sample gave a reading of 50.7% T at a
wavelength of maximum absorbance. What is the ratio of
concentrations of thiamine in the two samples?
2. (a) A 3.73 10-5 M solution of Compound A from a
spectrophotometric analysis has a maximum absorbance of
0.494 at 401 nm in a 1.000-cm cell, while a reagent blank
from the same analysis has an absorbance of 0.053 at 401
nm. Find the molar absorptivity of Compound A.
(b) A 5.00 mL aliquot of unknown solution containing
Compound A was
mixed with color forming reagents and diluted to a final
volume of 250.0
mL to give an absorbance of 0.777 at 401 nm in a 1.000cm cell. The
91
reagent blank had an absorbance of 0.053. Find the
Answers
1.
2.
92
Limitations to Beer's Law

Few exceptions are found to the generalization that absorbance
is linearly related
to path length.
On the other hand, deviations from the direct proportionality
between the
measured absorbance and concentration frequently occur when
b is constant.
Some of these deviations are called real deviatiolls, are
fundamental and
represent real limitations of the law.
Others are a result of how the absorbance measurements are
made (instrumental
deviations) or
a result of chemical changes that occur when the concentration
changes
(chemical deviations).
93
Real Limitations
Linearity is observed in the

low concentration
ranges(<0.01), but may not
be at higher
concentrations.
This deviation at higher
concentrations is due to
intermolecular interactions.
As the concentration
increases, the strength of
interaction increases and
causes deviations from
linearity.
The absorptivity not really
constant and independent
of concentration but is
related to the refractive
index ( ) of the solution by
the expression:
At low concentrations the
refractive index is
essentially constant-so
constant and linearity is
true
( + 2 ) 2
94
Apparent Chemical Deviations

Apparent deviations from Beer's law arise when an analyte
dissociates, associates, or reacts with a solvent to produce a
product with a different absorption spectrum than the
analyte.
A common example of this behavior is found with aqueous
solutions of acid-base indicators.
For example, the color change associated with a typical
indicator
Hln arises from shifts in the equilibrium
E.g. K2Cr2O7 solutions exist as a dichromate, chromate
equilibrium:
At max of 350 (and 450) nm and 372 nm respectively.
There is a strong dependence of position of this
equilibrium on relative pH.
Absorbance at one of these wavelengths for a given initial
concentration of [K2Cr2O7] strongly depends upon the pH.
When plotting absorbance as a function of [K 2Cr2O7], the
plot will not be linear since dilutions will affect the
95
equilibrium and thus the relative amounts of the two.
Instrument Deviation
Beer's law strictly applies only when measurements are made
with
monochromatic source radiation. In practice, polychromatic
sources that have
a continuous distribution of wavelengths are used in
conjunction with a
grating or with a filter to isolate a nearly symmetric band of
wavelengths
surrounding the wavelength to be employed
Consider a beam of radiation consisting of just two wavelengths '
and
Assuming that Beer's law applies strictly for each wavelength, we
may write for '
96
Beers law is followed

97
98
As shown in Figure 13-4, however,

the relationship between Am and
concentration no longer linear
when the molar absorptivities
differ. In addition, as the difference
between ' and increases, the
deviation from linearity increases.
This
derivation can be expanded to
include additional wavelengths: the
effect remains the same.
99
Instrumental Deviations in the Presence of

Stray Radiation
The radiation exiting from a monochromator is usually

contaminated with small
amounts of scattered or stray radiation.
This radiation, commonlv called stray light, is defined as
radiation from the
instrument that is outside the nominal wavelength band chosen
for the
determination.
This stray radiation often is the result of scattering and reflection
off the surfaces
Must
accountlenses
for theoreffect
of stray
light
the measured
of gratings,
mirrors.
filters,
andon
windows.
The
absorbance.
The measured absorbance is where P s = radiant
wavelength
of stray
power
of the
stray
light.greatly from that of the principal
radiation
often
differs
Negative deviations in the Beer's law plot observed since are
radiation and, in
the result since the measured absorbance will be smaller than
the radiation may
passed through the
Ponot
+ Phave
itaddition,
should be.
s
sample.
A m log
P+
P
s
100
Photometric errors
errors in the measurement of the transmittance;
can have a dramatic affect on the estimation of
concentration.
Normal Error Analysis
starting
with
Beer's law
1
log T
0.424 ln T
Io
log =
=
equation:
b
I
b
b
C = A/b = .
2 = C
sC
T
C = f(T).
General error equation is
s2T
C
0.434
=
T
bT
Take the derivative of both sides to get:

Substituting we get: 2 0.434 2 2 C
or
sC
sT
=
C
T ln T
sC =
bT
sT =
T ln T
s 2T
101
APPLICATIONS
Mixtures:Determining the
concentration of mixtures the
components of which absorb in the
same spectral regions is possible.
Strategy of the analysis. Total
absorption at some wavelength of
a two component mixture: Atotal,1 =
A + AN1.
Each should obey Beer's law at this
wavelength as long as
concentration is sufficiently low.
The contribution from each would
then be:
AM,1 = M,1bCM and AN1 = N,1bCN.
and
Atotal,1 = M,1bCM + N,1bCN.
Similarly at some other wavelength
we would have,
Atotal,2 = M,2bCM + N,2bCN.
.b can be determined for each
using standard solutions.
102
PHOTOMETRIC TITRATIONS
Absorbance measured
during titration of analyte.
The endpoint can be
determined by
extrapolation of the lines
that result from before and
after the endpoint.
The shape of the titration
curves depends upon the
molar absorptivities of
reactants, products and
titrants.
All absorbing species must
obey Beer's law for this
method to be successful.
103
Standard Addition Method
Standard addition method reduces problems

with matrix; analyte added to the matrix to
change the signal; signal change enables the
determination of the original concentration of
the analyte.
Another linear procedure with volume
correction:
Add volume, Vx, of the unknown solution with a
concentration cx to a series of separate
containers
Add variable amounts, Vs, of a standard solution

b V s c s csb
with concentration
ofVthe
x c xsame compound.
A
=
+
Dilute theseV to constantVfinal volume, V t.
T
T
Beer's law predicts the absorbance will vary

according to .
b c s
b V x c x
S = slope =
I = intercept =
VT
V
A should vary linearly
with Vs; the slope andT
interceptI should
I
V x cbe
x
=
c x = cs
S
cs
Vx S
Ratio of intercept and slope is:.
Skoog & Leary
104
STOICHIOMETRY OF COMPLEX
IONS
Ligand to metal ratio in can be

determined from absorption
measurements. Equilibrium not
affected significantly!
Assuming reactant or product absorbs
radiation, we can
determine the composition of complex ions in

solutions and
determine formation constants.
Stoichiometry: mole ratio, continuous

variation, and slope ratio methods.
One complex only!
Chapter 14 - 105
105
STOICHIOMETRY OF COMPLEX
IONS
Ligand to metal ratio in can be

determined from absorption
measurements. Equilibrium not
affected significantly!
Assuming reactant or product absorbs
radiation, we can
determine the composition of complex ions in

solutions and
determine formation constants.
Stoichiometry: mole ratio, continuous

variation, and slope ratio methods.
One complex only!
106
Mole-ratio method
Concentration of one of the components held
constant while other is varied giving a series of
[L]/[M] ratios.
The absorbance of each of these solutions is
measured and plotted against the above mole
ratio.
The ratio of ligand to metal can thus be obtained
from the plot.
Instrumental Methods of Analysis,

Ewing, p. 69.
107
MOLE RATIO METHOD (contd)

Determination Kf (ML only) non-linear portion of the plot.
Let:
Fm = [M] + [ML] = the total metal concentration at
equilibrium and
FL = [L] + [ML] = the total ligand concentration at
equilibrium
at any point on the curved part of the plot: A = Mb[M] +
MLb[ML]: assuming L = 0.
Determine b for both the metal and ligand.
Metal Let FL = 0 and [ML] = 0; AM = MbFm or Mb = AM/Fm.
Ligand:With a large excess of ligand, [ML] >> [M] and AML
= eMLbFM or eMLb = AML/FM.
Known equations:
Fm = [M] + [ML];
FL = [L] + [ML]
A = Mb[M] + MLb[ML]
108
Determine [ML],[M],[L]
SLOPE-RATIO METHOD
Makes it possible to determine ratio of ligand to
metal. Two plots performed with large excess of
either ligand or metal.
Absorbance vs. FM : large excess .ligand: [L] >>
[M]
[MnLp] = FM/n
Beer's law will be AM = b[MnLp] = bFm/n
Metal Concentration varied and plotted.
Absorbance vs. FL: large excess of metal the

[M]o >> [L]
[MnLp] = FL/p and AL = bFL/p.
Beers law : AM = b[MnLp] = bFL/n
Ligand Concentration varied and plotted.
Slopes will be b/p and b/n. The ratio of the

slopes gives the ratio of p/n.
109

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Instrumentation and

Tarekegn Berhanu (Ph. D)

1. Absorption molecular spectrophotometric

The Electromagnetic Spectrum

Electronic Excitation by UV/Vis Spectroscopy

Spectroscopic Techniques and

atomic transitions (val. e-)

nuclear spin states

inner electrons, elemental

Spectroscopic Techniques and Common Uses

Functional Group Analysis

3-D structure Anaylysis

EM regions related to different Analytical Techniques

The UV Absorption process

The Spectroscopic Process

The molar absorptivity is mostly controlled by two vital

Instrumentation and Spectra

Here is a simple schematic that covers most modern UV

Instrumentation and Spectra

The beam splitter sends a separate band to a cell containing the

The detector measures the difference between the transmitted

Each individual band of UV is detected by a individual diodes on

Instrumentation and Spectra

Cells can be made of plastic, glass or quartz

Only quartz is transparent in the full 200-700 nm range; plastic

Concentration (we will cover shortly) is empirically determined

A typical sample cell (commonly called a cuvet):

II. Instrumentation and Spectra

Instrumentation and Spectra

Since spectra are only obtained up to 200 nm, solvents typically

II. Instrumentation and Spectra

H-bonding further complicates the effect of vibrational and

Instrumentation and Spectra

Instrumentation and Spectra

From the spectrometers point of view, absorbance is the inverse

From an experimental point of view, three other considerations

the greater the concentration, c of the sample, the

iii. some electronic transitions are more effective at the

this may vary by orders of magnitude

Instrumentation and Spectra

for most UV spectrometers, l would remain constant

concentration is typically varied depending on the strength

iii. molar absorptivities vary by orders of magnitude:

Instrumentation and Spectra

It is most useful in combination with NMR and IR data to

It can be used to assay (via max and molar absorptivity) the

The most ubiquitous use of UV is as a detection device for HPLC;

Since similar functional groups will have electrons capable of

A functional group capable of having characteristic electronic

Structural or electronic changes in the chromophore can be

CO transitions omitted for clarity

Hypsochromic shift (blue shift) shift to shorter ; higher

iii. Hyperchromic effect an increase in intensity

E for the HOMO LUMO transition is reduced

The s-cis conformer is at an overall higher potential energy than

The 2 4* transition is not typically observed:

The energy of this transition places it outside the

For the more favorable s-trans conformation, this

Each exo-cyclic C=C

This compound has three exocyclic

This is not a heteroannular diene; you

Remember, the * transition is

This is explained by the inductive