Spectroscopy

Importance of Structure Determination:

1. To determine the structure of a new natural product.

2. To determine the structure of a chemical reaction product.

• In the past, chemists have to use systematic chemical reactions

(e.g. oxidation, reduction, substitution, addition or elimination)

• Disadvantages of these reactions are:

1. Time-consuming.
2. Require experimental skills.
3. Require deductive reasoning.
4. A large amount of sample also required, which will never be
recovered.
 Now:
• Structure is determined now using sensitive non-destructive
spectroscopic instruments.
• Advantages:
• enabling adequate data acquiring using minimal time
• Micro-gram sample quantities.
• The sample is easily recovered.
• Spectroscopy: a technique by which we study the interaction
of electromagnetic radiation with molecules.
• Examples of electromagnetic radiation: X-rays, UV, visible
light, IR, microwave & radio waves.
The Electromagnetic spectrum:

• The electromagnetic spectrum is divided into zones according to the

wave length () and the frequency () as shown in the following figure.

- +
Energy E
- +
Frequency 
+ -
Wavelength 

Wave length
(meter)

NMR visible IR UV
1-5 m 2.5-16 μ 200-380 nm

Visible
380-780 nm
Spectroscopic Methods :

Spectroscopic Methods:
1.Ultra Violet, UV.
2.Nuclear Magnetic Resonance, NMR
3.Infrared, IR
4.Mass spectrometry, MS
Ultraviolet and Visible
Spectroscopy
(Electronic Spectroscopy)
Definition of UV-spectroscopy:

• A physical means used to detect conjugated systems.

• Known also as Electronic Spectroscopy → because of the

promotion of electrons from ground state to excited state as a
result of the absorption of UV/visible radiation by a molecule.

• The UV region of the electromagnetic spectrum ranging from

200-380 nm.

• Visible region lies between 380-780 nm.

Definitions:

 Chromophore: A chromophore is any

functional group that absorbs UV light,
e.g. C=C, -C=O, -N=N, and -C≡N.
 Bathochromic shift: A shift to red or
longer wavelength and lower energy (red
shift).
 Hypsochromic shift: A shift to shorter
wavelength or towards the blue and
higher energy (Blue shift).
 Cont. Definitions:

 Auxochrome: Is an auxiliary functional

group that interacts with a chromophore,
causing a bathochromic shift, -OH & NH2.
 Hyperchromic effect: results in increase of
molecule absorptivity (or absorption
intensity).
 Hypochromic effect: results in decrease of
molecule absorptivity (or absorption
intensity).
 Estimation of oxygenation
pattern of Flavonoids by UV-
shift reagents
 UV-VIS
UV-VIS Spectra
Spectra of flavonoids:

λmax (MeOH) of different classes

of flavonoids:
O B
A
• Most of the flavonoids exhibit
Band II Band I
two major absorption maxima: Benzoyl
O
Cinnamoyl
– One is due to the absorption of
the benzoyl system in ring A
(Band II),
– The other band is due to the
absorption of cinnamoyl
system in ring B (band I).
Values of Band I and II, UV methanolic spectrum

skeleton Band I Band II

Flavones 300 – 350 nm 250 - 270 nm
Flavonols 350 – 370 nm 250 – 270 nm
Isoflavones 300 – 340 nm 245 – 270 nm
Flavanones 310 – 330 nm 270 – 295 nm

2` 3`
8 1 O
O 1`
B 4`
O B
O B 7
2
A
A B A
A 3 6` 5`
6 OH
5 4
O
O O
O
Flavone Flavonol Isof lavone Flavanone

In all types of flavonoids Band II absorption is ≈ 250-280 nm

Stucture Elucidation of flavonoids by UV-Vis using shift reagents

2. Shift reagents:
1- NaOMe (Sod. Methoxide 2.5%):
 Is a strong base ionizes all free OH groups in the flavonoid nucleus.
 Role: to detect free 4` OH group.
 It causes large bathochromic shift (red shift) in Band I (about 40-70 nm).
 Note: it is mainly used for free 4`-OH group.

2` 3`
8 1
1`
7
O 2 B 4`

A 3 6` 5`
6 OH
5 4

O
\\ 2- NaOAc: (Sod. acetate - anhydrous powder):
Is a weak base than NaOMe, i.e: It ionizes only the less
acidic OH groups in the skeleton (i.e 7-OH).
Because the ionization of 7-OH is mainly affect Band II, so
the skeleton contains free OH group at position 7 exhibit a
diagnostic bathochromic shift (red shift) “5-20 nm” in
band II.
2` 3`
8 1
1`
7
O 2 B 4`

A 3 6` 5`
6 OH
5 4
O
3- Na- acetate/ boric acid: (powders)
 In presence of Na acetate, boric acid will chelate with the dihydroxy
groups in all position except at C5, 6;(Steric hindrance).
 Presence of Ortho-dihydroxy group in ring B, shows
bathochromic shift (12-30 nm) in band I in presence of NaOAc/
boric acid

OH
O B
3`
OH
O O
4`

6
5
O
 4- AlCl3
 Flavonoids containing free -OH group(s) at C-3 and/or C-5
Or
 Ortho-dihydroxy groups at C-3`/C-4` and C-6 /C-7 form
chelate complexes with AlCl3, causes a bathochromic shift
(red shift) > 30 nm.
O Al

O O O
Al
O
O
O O
+2 Al
Al
5- AlCl3/ HCl:
 Complexes formed between AlCl3 and orthodihydroxy
groups in ring A or B are decomposed in presence of acid
(HCl) Except that formed between C4 ketone group and C3
or C5 (stable complex)

 So, if upon addition of HCl;

 Hypsochromic shift in Band I on addition of HCl
indicates orthodihydroxy group in ring B.
 Hypsochromic shift in Band II on addition of HCl
indicates orthodihydroxy group in ring A.
 3` Al3+
OH 4` O
OH O
acid labile
OH O OH O

3 AlCl3
OH O
OH O4
O O
5 Al 3+
Al 3+
acid stable

OH
OH
HCl
OH O

O
O
O
Al 3+
Al 3+

acid stable
 Problem
Using the following UV data deduce the structure (oxygenation
:pattern) of the flavonoid

λmax (nm)
+NaOAc +AlCl3/
MeOH +NaOMe +NaOAc +AlCl3
/H3BO3 HCl
Band

λmax λmax λmax λmax λmax λmax

II 269 275 284 269 276 277

I 342 395 345 343 345 345

I = +53 nm I =No signif. No No signif. No signif.
II =(+15 nm) signif.
4` OH 7` OH
INFRA-RED SPECTROSCOPY
Introduction

• IR-spectroscopy is a rapid and simple method for

obtaining preliminary information on the structure of
an organic molecule.

• IR-Spectroscopy is commonly known as Vibrational

Spectroscopy

Energy Transitions in IR → vibrational

 Sampling
1. Gas sample: the gas is introduced into a gas cell.
2. Liquids and solutions: thin film squeezed between 2 IR-transparent
windows (e.g. NaCl flats, polytetrafluoroethylene cell)
3. Solids:
a) KBr disc: grind the sample with highly pure and dry KBr in a small
mortar, compress manually or in a special "die" into a transparent
disc.
b) Liquid paraffin (Nujol) mull: the solid sample is ground with a drop
of the oil then squeeze between transparent windows as in liquid
sample. In this case, bands corresponding to nujol must be
excluded.

c) Solid film: The solution of the sample is evaporated on the surface

of the flat NaCl disc e.g. polymers and fatty materials.
Typical Infrared Absorption Regions
BASE VALUES These are
the minimum
(+/- 10 cm-1) number of
values to
memorize.
O-H 3600
N-H 3400
C-H 3000

C N 2250
C C 2150
C=O 1725
C=C 1650
C O ~1100 large range
 Typical Infrared Absorption
Regions
WAVELENGTH (m)
C-H
2.5 4 5 5.5 6.1 6.5 15.4

O-H C-H C N C=O C=N C-Cl

Very C-O
N-H C C few C=C
C-N
X=C=Y bands C-C
(C,O,N,S) N=O N=O *

4000 2500 2000 1800 1650 1550 650

FREQUENCY (cm-1)
We will look at
this area first
 ALKANE
Hexane

Aliphatic CH bending
vibrations

CH
stretching
vibrations
CH3 CH2 CH2 CH2 CH2 CH3
1-Hexyne
ALKYNE

=
C=C

CH2, CH3

= C-H HC C CH2 CH2 CH2 CH3

=C-H
 CARBOXYLIC ACID

Butanoic Acid
Neat solution

O-H
H-bond

C-O
CH2 O

C-H C=O CH3 CH2 CH2 C OH

 Carbonyl Compounds

•Aldehydes
•Ketones
•Esters
•Amides
•Acid Chlorides