UCB008 - APPLIEDCHEMISTRY SCBC-TIET

Molecular Spectroscopy

UV-Visible Spectroscopy – Electronic Transitions

 UV-Visible Spectroscopy (or) SCBC-TIET

Electronic Spectroscopy

Antibonding
Molecular
ABMO
Orbital (ABMO)
hn
E Atomic Atomic UV-Vis
radiations
orbital orbital
Bonding BMO
Molecular Excited State
Orbital (BMO)
Ground State
 UV-Visible Spectroscopy (or)
Electronic Spectroscopy

SCBC-TIET

EM Radiations of UV–visible region Covalent bonds in organic molecule

+
compound having multiple bond

s bond p bond
Absorption of radiation

Transition of valence electron from
ground to excited state
s s* p p*
(bonding) (anti-bonding) (bonding) (anti-bonding)
• Compounds containing hetero atom have non-
bonding electrons
• A electronic transition is always from bonding
molecular orbital (BMO) to anti-bonding molecular
orbital (ABMO)
 Types of Electronic Transitions
s * (bonding) SCBC-TIET

s s *
p* (bonding)
ns*
n  p* s  p*
Energy

p s *
n (non-bonding)
p  p*
p(bonding)

s (bonding)
 Energy Requirement for Electronic Transitions
SCBC-TIET

s-s* > n-s * ~ p- p* > n-p*

~150nm ~170-190nm 280nm

s-s* - saturated hydrocarbons e.g. ethane

n- s * - saturated compounds containing hetro atom having unshared pair

of electrons. e.g. saturated halides, alcohols, ethers, aldehydes, amines,
etc.

p-p* - compounds having double or triple bond and aromatics e.g.

butadiene, benzene, etc.

n-p* - unsaturated compounds containing hetro atom having unshared

pair of electrons e.g. carboxylic acids, aldehydes, ketones, etc.
 Electronic Transitions
SCBC-TIET

• An allowed electronic transition involves orbitals having same symmetry

e.g., s  s* and p  p * transitions.

• Allowed transitions have higher extinction coefficient (emax) values of

greater than 104.
• e.g., p  p * transition of 1-3 butadiene absorbs at 270 nm (emax : 21000)

• Transitions between orbitals having different symmetry are classified as

symmetry forbidden transitions.
• e.g., s  p * and p  s* transitions are possible only theoretically .

• The forbidden transition namely n  p * have very low extinction coefficient

(emax) values.
• e.g., n  p * transition of carbonyl compounds, at 280 nm, has emax 15.
 Electronic Transitions
lmax
SCBC-TIET

Chromophore Example Excitation

(nm)
e
C=C Ethene pp* 171 15,000

C≡C 1-Hexyne pp* 180 10,000

np* 290 15
C=O Ethanal
pp* 180 10,000

np* 275 17
N=O Nitromethane
pp* 200 5,000

C-X Methyl bromide ns* 205 200

X=Br; X=I Methyl Iodide ns* 255 360
 s-s* transitions SCBC-TIET

Far UV UV V I B G Y O R
s -s *
~150nm 100 200 380 760
nm nm nm nm
Vacuum ultraviolet
region

• s -s * requires photons of 150 nm.

• Conventional UV-visible spectrophotometer works in the range of
200 nm to 760 nm.
• This instrument cannot be used below 200 nm as oxygen in air
strongly absorbs in far UV region.
• Thus, s -s * transitions cannot be observed using
conventional UV-visible spectrophotometer.
 s-s* transitions
SCBC-TIET

• Analysis in far UV region requires vacuum ultraviolet

spectrophotometer which is cost intensive.

• Vacuum UV spectrophotometer is used mainly for
determining bond energies.
• This equipment is not used for organic structural
determination.
 Chromophore SCBC-TIET

• Isolated covalently bonded groups that shows characteristic

absorption in UV-Visible region and even small structural changes
brings about change in wavelength of absorption.

or

• The functional groups containing multiple bonds capable of absorbing

radiations above 200 nm due to n-p* and p-p* transitions.

• Examples: C=C, C≡C, N=O, N=N, NO2, C=O, C=N, C≡N, C=C,C=S,
-CONH2, -COOH, etc
 SCBC-TIET

Chromophore

• Non-conjugated alkenes show an absorption below 200 nm and

therefore cannot be observed by conventional UV spectrophotometer.

• If these transitions are to be observed, vacuum UV

spectrophotometer is required.

• Non-conjugated carbonyl group compound give a weak absorption

band in the 200 - 300 nm due to n-p* transition.
 Effect of conjugation on l m a x SCBC-TIET

Compound l m a x (nm)
H2C=CH2 180
217
O
220

220

255
Me
HO3S N N N
Me
460
 Effect of conjugation on l m a x SCBC-TIET

CH2 CH3
H 2C H 3C

1,5 - hexadiene : λmax = 178 nm 2,4 - hexadiene : λmax = 227 nm

When double bonds are conjugated in a compound, λmax is shifted to

longer wavelength.
 Effect of conjugation on l m a x
SCBC-TIET

O
C
H 2C CH2 H3C CH3
Ethylene has λmax = 171 nm Acetone has λmax = 279 nm

Conjugation of C=C and carbonyl group shifts the λmax of both

groups to longer wavelength.
O

H2C C
CH3
Crotonaldehyde has λmax = 290 nm
 Auxochrome
SCBC-TIET

• The functional group with non-bonding electrons, that does not absorb
radiation in UV/visible region, when attached to a chromophore
increases the wavelength and intensity of absorption.
OR
• Any group which does not itself act as chromophore but its presence
brings about a shift of absorption bands towards red end of the
spectrum

Examples: -OH, -OR, -NH2 , -NHR, -NR2, -SH etc.

 Auxochrome SCBC-TIET

• Auxochrome when combined with chromophore, modifies the position

of absorption band relative to the parent chromophore
Chromophore + Auxochrome

New chromophore
having higher l max

Examples: -OH, -OR, -NH2 , -NHR, -NR2, -SH etc.

• Auxochrome extends the conjugation of a chromophore by sharing

its lone pair of electrons
 Auxochrome SCBC-TIET

e.g. Benzene λmax = 255 nm

OH

Phenol λmax = 270 nm

NH2

Aniline λmax = 280 nm

 Shifts and Effects SCBC-TIET

Hyperchromic effect
Absorbance ( A )

Red shift
Blue shift
(Bathochromic
(Hypsochromic
shift)
shift)

Hypochromic effect

λmax
Wavelength ( λ )
 SCBC-TIET

Bathochromic Shift (Red Shift)

• When absorption maximum (emax) is shifted towards longer wavelength,
it is known as bathochromic shift or red shift.

• The effect is due to presence of an auxochrome or by the change of

solvent.

• For example, an auxochrome group like –OH, -OCH3 causes

absorption of compound at longer wavelength.
 SCBC-TIET

Bathochromic Shift (Red Shift)

• p-nitrophenol, in alkaline medium, shows red shift, because negatively
charged oxygen delocalizes more effectively than the unshared pair of
electron.
- -
O + O O + O
N N

-
OH

A lk a lin e
m e d iu m -
OH O

λmax = 255 nm λmax = 265 nm

p-nitrophenol p-nitro-phenoxide
 Hypsochromic Shift (Blue Shift)
SCBC-TIET

• When absorption maximum (emax) is shifted towards shorter

wavelength, it is known as Hypsochromic shift or blue shift.
• The effect is due to removal of conjugation or by the change of
solvent.
• Aniline shows blue shift in acidic medium because it loses
conjugation.
+ -
NH2 + NH 3 Cl
H

A c id ic Conjugation decreases
due to protonation
m e d iu m

λmax = 280 nm λmax = 265 nm

Aniline Anilinium chloride
 Hyperchromic effect SCBC-TIET

• When intensity of absorption maximum (emax) of a compound

increases, it is known as hyperchromic effect.

N
A = ec x N CH3
λmax = 257 nm λmax = 260 nm
ε = 2750 ε = 3560
Pyridine 2-methyl pyridine

• Inclusion of an auxochrome also increases intensity of absorption.

Sharma YR, Elementary Organic Spectroscopy, S. Chand & Co.

 Hypochromic effect SCBC-TIET

• When intensity of absorption maximum (emax) of a compound decreases,

it is known as hypochromic effect.

CH3
e= 19000 e= 10250
λmax = 250 nm λmax = 237 nm
Naphthalene 2-methyl naphthalene

• emax decreases due to the distortion caused by the methyl group.

Sharma YR, Elementary Organic Spectroscopy, S. Chand & Co.

 SCBC-TIET

How conjugation causes bathochromic shift….

• More the number of double bonds in conjugation, longer wavelength
photon is required for transition.

• Thus, energy requirement for electronic transition decreases.

• Compounds having > 8 double bonds in conjugation will appear
coloured to human eye.

• Energy requirement for pp* transitions decreases as the

conjugation increases and can reach the visible region of the spectrum
which make the compounds coloured.
• The added conjugation in naphthalene, anthracene and tetracene causes
bathochromic shifts of these absorption bands
• Naphthalene and anthracene are colorless, but tetracene is orange
 Color of Phenolphthalein

Conjugation broken Completely Conjugated

at carbon
 SCBC-TIET

How conjugation causes bathochromic shift….

• An electronic transition is from bonding molecular orbital to anti-

bonding molecular orbital.

• Energetically favored electron promotion will be from the highest

occupied molecular orbital (HOMO) to the lowest unoccupied
molecular orbital (LUMO).

• This results in excited state.

 Relative energies of orbitals - electronic spectroscopy
SCBC-TIET

s* s*

p4* Lowest Unoccupied

Molecular Orbital
p* p*
p3* (LUMO)
Energy

n n Highest Occupied
Molecular Orbital
p2 (HOMO)
p p
p1

s s
C-C C=C C=O C=C-C=C
s - s* p- p* p- p*
n-p* p - p*
2 3
Kemp, W., Organic Spectroscopy, Palgrave Publ.
Effect of conjugation on relative energies of molecular orbitals
SCBC-TIET
 How conjugation causes bathochromic shift….
SCBC-TIET

• When two double bonds are conjugated, the four p-

atomic orbitals combine to generate four p -molecular
orbitals (two are bonding and two are antibonding).
• When two double bonds are in conjugation the
energy level of HOMO is raised and that of LUMO is
lowered.

• The energetically most favorablep- p* excitation

occurs from the highest energy bonding pi-orbital
(HOMO – p2) to the lowest energy antibonding pi-
orbital (LUMO – p3*).
• In a similar manner, the three double bonds of a
conjugated triene create six p - molecular orbitals,
half bonding and half antibonding.
 Colour in organic compounds
SCBC-TIET
Number of conjugated
double bonds

5 colourless
Naphthalene

7 colourless

Anthracene

9 orange

Tetracene
 Colour in phenolphthalein
SCBC-TIET

Colourless 2 1 Pink
2 1
4 4
l max = 552 nm
3 6
3
5

7 8
colourless pink
 Colours in nature
SCBC-TIET

620 nm

Lycopene : 443-502 nm
446
475
421

Lutein : 421-475 nm

45 2

490 nm

b-carotene : 452 nm
 UV-Vis SCBC-TIET

Spectrophotometer
• Light source
– Deuterium lamp – UV radiation
– Tungsten filament lamp – Visible radiation
• Sample containers
– Cuvettes
• Plastic
• Glass
• Quartz
 Sample SCBC-TIET

Handling
Solvent - Should not absorb in the region under investigation
Solvent
1 mg sample 100ml

Sample solution
1 cm

Solvents – water, ethanol, n-hexane, cyclo-hexane, benzene, methyl

alcohol, diethyl ether etc.
 Sample SCBC-TIET

Handling

Sample solution
1 cm

• Cuvettes are used for keeping the sample solution in the instrument.
• Thickness of sample solution in cuvette – 1cm
• Cuvette material should not absorb in the region under investigation.
• Glass/plastic – used only for visible region as they absorb UV radiation
• Quartz – used for UV-visible region
Qualitative analysis using UV-visible spectrophotometer SCBC-TIET

• l m a x of organic compounds can be determined using UV-visible spectra

• This value can be used to detect the extent of conjugation
• … and absence/presence of the chromophore in an organic compound.

l max
Applications SCBC-TIET

• Qualitative Analysis:
• Extent of conjugation in polyenes can be estimated.
• Shift in the absorption, due to addition of unsaturation in a compound,
towards longer wavelength can be detected.
• Presence or absence of a chromophore can be detected.
• If UV-visible spectrum of a compound has no peaks above 200 nm, it
shows the absence of conjugation, carbonyl group and aromatic ring.
• It is used for characterizing aromatic compounds and conjugated
olefins.
• Distinction between conjugated and non-conjugated compounds
(CH3)2C=CH-CO-CH3 CH2=C(CH3)-CH2-CO-CH3
A B
Applications SCBC-TIET

• Qualitative Analysis:
• Geometric isomers can be distinguished

H
C=C C=C
H H
H
cis-stilbene trans-stilbene
l m a x : 283 nm l max : 295 nm

• Trans isomer always absorb at higher wavelength

Applications SCBC-TIET

• Qualitative Analysis:
• Identification of a compound in different solvents- If structure of a
compound changes in different solvents, the change can be detected
by uv-vis spectra.
CCl3CHO.H2O

Hexane Water

290nm No absorption at 290nm (absence of C=O)

CCl3C=O.H2O CCl3C OH
OH
H H

Sharma YR, 2010, Elementary Organic Spectroscopy, S.Chand & Co.

Quantitative analysis of an unknown sample solution using UV- SCBC-TIET

visible spectrophotometer
• Series of standard solutions having known concentration of analyte X are
prepared.
• One of these solutions is analyzed by the spectrophotometer and its l m a x
is determined.
• Absorbance of all the standard solutions are analyzed at the determined
l ma x.
• A calibration curve (absorbance vs concentration) is prepared for the
standard samples.
• Signal intensity of unknown sample of analyte X is taken.
• The concentration of the analyte X is then determined based on the
absorbance value from the calibration curve.
Quantitative analysis of an unknown sample solution using UV-
visible spectrophotometer SCBC-TIET

Step 1: Series of standard solutions having known concentration of analyte

X are prepared.
Quantitative analysis of an unknown sample solution using UV-
visible spectrophotometer SCBC-TIET

Step 2: Solvent is taken as blank solution for which absorbance is measured

and absorbance value is set to Zero.

Io

Blank Solution
Quantitative analysis of an unknown sample solution using UV-
visible spectrophotometer SCBC-TIET

Step 3: One of the standard solutions is analyzed by the UV-visible

spectrophotometer and its l m a x is determined.

l max

Instrument is calibrated with blank, periodically, when wavelength is changed,

in case of single beam spectrophotometer.
Quantitative analysis of an unknown sample solution using UV-
visible spectrophotometer SCBC-TIET

Step 4: Absorbance of all the standard solutions are analyzed at the

determined l ma x.
Step 5: A calibration curve (absorbance vs concentration) is prepared by
analyzing the standard samples.

3.0
2.5
Absorbance

2.0
1.5
1.0
0.5
0
0 1 2 3 4 5
Concentration (mg/ml)
Quantitative analysis of an unknown sample solution using UV-
visible spectrophotometer SCBC-TIET

Step 6: Absorbance value of the solution of

analyte X (unknown) is determined at
the same l m a x .
Step 7: The concentration of the analyte X is then
determined based on the absorbance value from
the calibration curve by extrapolating towards
concentration in X axis. - concentration of analyte X
2.5 3.0 - e.g., ~3.66 mg/ml in this case
2.0
Absorbance

1.5
1.0
0.5
0
0 1 2 3 4 5
Concentration (mg/ml)
 Applications
SCBC-TIET

• Quantitative Analysis:
• It can be used to find out molar concentration of the solute under study.

3.0
2.5
Absorbance

2.0
1.5
1.0
0.5
0
0 1 2 3 4 5
Concentration (mg/ml)