ultraviolet spectroscopy PDF/ PPT

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Description

Presentation topic —
Ultra-violate spectroscopy

Submitted by —
Moriyom Akhter
Md Shah Alam

Department of pharmacy
World university of Bangladesh

Content

» DEFINITION

*» UV RADIATION
» PRINCIPLE OF UV-VIS SPECTROMETRY
» THE ABSORPTION SPECTRUM

» TYPES OF TRANSITIONS

» ABSORBANCE LAWS

» INSTRUMENTATION

» EFFECT OF CHROMOPHORE

» WOODWARD-FEISER RULE

» SOLVENT EFFECTS

UV SPECTROSCOPY

Ultraviolet and _ visible (UV-Vis)
absorption spectroscopy iS the
measurement of the attenuation of a
beam of light after it passes through a
sample or after reflection from a sample
surface. Absorption measurements can
be at a single wavelength or over an
extended spectral range.

Why we use UV spectroscopy ?

1. Detection of functional groups.
Detection of impurities

Qualitative analysis
4. Quantitative analysis
5. Single compound without chromophore

6. Drugs with chromophoric reagent

7. It is helps to show the _ relationship
between different groups, it is useful to
detect the conjugation of the compounds

ww UN
UV RADIATION

The region beyond red is called infra-red while
that beyond violet is called as ultra —violet. The

wavelength range of uv radiation starts at blue
end of visible light(4000A) & ends at 2000A.

Visible

PRINCIPLE OF UV-VIS SPECTROMETRY

» Ultraviolet absorption spectra arise from transition of electron with ina molecule from a lower level to a
higher level.

» A molecule absorb ultraviolet radiation of frequency (9), the electron in that molecule undergo transition

from lower to higher energy level.

The energy can be calculated by the equation,

CONTINUED

E,-Eo= hd

E total=Eelectronict+Evibrotional+Erotational

The energies decreases in the following order:

Electronic 2@lVibrational 2] Rotational

CONTINUED

va
Vo A cs $ rato levels
vy
E, 4 t | ELECTRONIC EXCITED STATE

4 L
Vg

Vibrational levels

ELECTRONIC GROUND STATE

Thus the energy of the radiation in the visible range is generally: 36 to 72 kcal/mole while that
in the ultraviolet range goes as high as 143 kcal/mole

THE ABSORPTION SPECTRUM

When a sample is exposed to light energy that matches
the energy difference between a possible electronic
transition within the molecule, a fraction of the light energy
would be absorbed by the molecule and the electrons
would be promoted to the higher energy state orbital. A
spectrometer records the degree of absorption by a
sample at different wavelengths and the resulting plot of
absorbance (A) versus wavelength (A) is Known as a
spectrum.

The significant features:

Amax (wavelength at which there is a maximum
absorption)

* emax (The intensity of maximum absorption)

Absorption

=2°0.5 nm
mux

l | | | |

22 240 260 280 300

~~

wavelength in nm

fig:- UV spectrum of acetone
CONTINUED

» UV-visible spectrum of isoprene showing maximum absorption at 222 nm.

1.0 =
8 – heres = 222 AM i>
0,8+r i

a g Hc soothe
Le |
a O,6+ H
£ – Isoprene
iy 0.4f
Oo _
vm
| O.2F

ot

4 + + 7 + ‘ + 4 + ‘ + ‘ +
200 220 240 260 280 300 320 340
Az. (nm)

Every time a molecule has a bond, the atoms in a
bond have their atomic orbitals merged to form
molecular orbitals which can be occupied by
electrons of different energy levels. Ground state
molecular orbitals can be excited to anti-bonding

molecular orbitals.

These electrons when imparted with energy in the
form of light radiation get excited from the highest
occupied molecular orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO) and the
resulting species is known as the excited state or
anti-bonding state.

TYPES OF TRANSITIONS:

In U.V spectroscopy molecule undergo
electronic transition involving o, T and n
electrons.
> Four types of electronic transition are
possible.

I. O — oO” transition
li. Nn > o* transition
lili. n — Tr” transition
iv. TT — Tr” transition

1.0 — o* Transition:

» An electron in a bonding o orbital of a molecule
is excited to the corresponding anti-bonding
orbital by the absorption of radiation.

~ Toinduce a o— o* transition it required LARGE
ENERGY.

> Ex: Methane

» Methane contain only single C-H bonds it undergo
only o— o* transition only, it gives absorption
maximum at 125nm.

ll. N— o* transition:

In this type saturated compounds containing atoms
with unshared electron pairs are undergo n — o*
transition.

It require less energy than the o — o* type.

Most of the absorption peaks appearing below
200nm.

In the presence of polar solvents the absorption
maximum tend to shift shorter wavelength

Ex: Water , ethanol.
» In this the peaks in U.V region relatively small.
Ex: Methlychloried , Oxygen, Nitrogen.

lin — 1* & 1 — T* transitions

Most organic compounds are undergo transitions
for n> Tl and TT > Tr* transition.

~» Because energies required for processes bring
the absorption peaks into spectral region.

» Both transition require the presence of an
unsaturated functional group to the [] orbitals.

. Ex: For 1 11* I> Alkenes, carbonyl
compounds, alkynes

a Forn — Tr I> carbonyl compounds.

o* (anti-bonding)

m™* (anti-bonding)

n (non-bonding)

mt (bonding)

o (bonding)

Four types of transitions
ABSORBANCE LAWS

BEER’S LAW
“ The intensity of a beam of monochromatic light
decrease exponentially with the increase in concentration
of the absorbing substance” .

Arithmetically;
-dI/ deal
I= lo. e Ke ————————– eq (1)

LAMBERT’S LAW

“ When a beam of light is allowed to pass through a
transparent medium, the rate of decrease of
intensity with the thickness of medium is directly
proportional to the intensity of the light”

mathematically;
-dI/ dt a1
-In. | = kt+b —————- eq(2)
the combination of eq 1 & 2 we will get

A= Ket
A= Ect (K=€)
LIMITATION OF LAWS

~ The real limitation of the beer’s law is successfully

in describing the absorption behavior of dilute
solution only.

> In this regarding it may be considered as a
limiting law.

* As degree of interaction depends upon the
contraction, the occurrence of this phenomenon
causes deviations from linear relationship
between absorbance and contraction.

INSTRUMENTATION

Components of spectrophotometer
Source

+ Monochromator

41 Sample compartment
4 Detector

+ Recorder

INSTRUMENTATION

v
RADIANT WAVELENGTH PHOTO-
= >

Fig.-block diagram of instrumentation of UV-spectrophotometer

>a

amplifier

v

Monochromator Detector

Sample

Exit slit

Read out

Dispersion
device

Entrance
slit

RADIATION SOURCE

It is important that the power of the radiation source does
not change abruptly over its wavelength range. The
electrical excitation of deuterium or hydrogen at low
pressure produces a continuous UV spectrum.

Both Deuterium and Hydrogen lamps emit radiation in
the range 160 – 375 nm.

Problem-

» Due to evaporation of tungsten life period decreases.
> It is overcome by using tungsten-halogen lamp.

» Halogen gas prevents evaporation of tungsten.

RADIATION SOURCE

For ultra violet region-

Hydrogen discharge lamp

>» consist of two electrode contain in deuterium filled silica
envelop.

UV-Vis spectrophotometer have both deuterium & tungsten
lamps.

4 Selection of lamp is made by moving lamp mounting or
mirror to cause the light fall on Monochromator.

Deuterium lamps:-

» Radiation emitted is 3-5 times more than the hydrogen
discharge lamps.

Xenon discharge lamp:-

» Xenon stored under pressure in 10-30 atmosphere.

FILTERS OR MONOCHROMATORS

All Monochromators contain the following component parts;
¢ An entrance slit
* A collimating lens
* A dispersing device (a prism or a grating)
* A focusing lens
* An exit slit

az Reflection,
Entrance grating Exit
slit slit

U Filters —
a)Glass filters- Made from pieces of colored glass which

transmit limited wave length range of spectrum. Wide band
width 150nm.

b)Gelatin filters- Consist of mixture of dyes placed in gelatin
& sandwiched between glass plates. Band width 25nm.

c)Inter ferometric filters- Band width 15nm

UPrisms-
-Prism bends the monochromatic light.
-Amount of deviation depends on wavelength
– They produce non linear dispersion.

Focusing Collimating
Lens Lens

—Sunlight

Fig.-mechanism of working of prism.

SAMPLE CONTAINERS OR SAMPLE CELLS

A variety of sample cells available for UV region. The
choice of sample cell is based on

a) the path length, shape, size

b) the transmission characteristics at the desired
wavelength

c) the relative expense

» The cell holding the sample should be transparent to the
wavelength region to be recorded. Quartz or fused silica
cuvettes are required for spectroscopy in the UV region.
Silicate glasses can be used for the manufacture of
cuvettes for use between 350 and 2000nm. The
thickness of the cell is generally 1 cm. cells may be
tangular in shape or cylindrical with flat ends.

DETECTORS

Three common types of detectors are used

|. Barrier layer cell

Il. Photo cell detector

Ill. Photomultiplier , Photo voltaic cells
barrier layer cells

It consist of flat Cu or Fe electrode on which semiconductor such
as selenium is deposited. on the selenium a thin layer of silver or
gold is sputtered over the surface.

Av
CSC) Semitransparent

oo Silver

S @) – —Semiconductor

CONTINUED

Photomultiplier tube

It is generally used as detector in UV- spectrophotometer It is the
combination of photodiode & electron multiplier.

It consist of evacuated tube contains photo- cathode. 9-16 electrodes
known as dynodes.

Photosensitive Cathode

i ©
Anode CG)

i

DESCRIPTION OF UV- SPECTROPHOTOMETER

Advantage of double beam spectrophotometer:- It is not necessary to
continually replace the blank with the sample or to adjust the auto zero.

The ratio of the powers of the sample & reference is constantly obtained.

It has rapid scanning over the wide wavelength region because of the
above two factors.

UV-VIS sources lo a
an ry OO
=
g » —. Oo
/ ; :
eS
monochromator/
beam splitter optics Loe
— o
O
o

single beam spectrophotometer

Entrance slit

Lens
Collimating lens
Lamp

Grating

eK. Wavelength
{ control cam
knob
Exit slit
“Meter Cuvetite

Phototube

Double beam colorimeter

Photo-

Reference

a :

% T

Display device

CHROMOPHORE

> Any Functional group which is
responsible for impairing colour to the
compound is called as chromophore.
=x: NO2
» Covalently unsaturated groups
responsible for the impairing of the
colures.
Ex: C=C, C=O
> Two types of chromophore
a) Independent

ee Op ho re
ment) dependent chromophore

SIMPLE CHROMOPHORIC GROUPS

Groups
C-C 1350
C=C 1900
C=O 1900
2800
O-H 1850
NOz 2800

1950
2500

C6Hs5( PHENYL)

AUXOCHROME

It is the group which itself does not act as a
chromophore but when attached to chromophore it shifts
the absorption maximum towards longer wavelength
along with an increase in intensity of adsorption.

Ex: -OH, -NH2, -OR groups
For example when the auxochrome —NH2 is attached to

the benzene ring, it absorption changes from Amax 255
to 280nm.

TYPES
Two types

a. Bathochromic groups
b. Hypsochromic group
BATHOCHROMIC GROUPS

Those groups which deepen the colour of
chromogen are called bathochromic groups.

Deepening of colour means displacement
to longer wavelength.

yellow— orange — red — purple >
violet— blue — green

HYPSOCHROMIC GROUPS

Those groups which diminish or lighten the colour of the chromogen
are called hypsochromic groups.

~ They cause displacement to shorter wavelength.
Ex:- acetylation of -OH or —NH2 groups, -OCOCH3 and —
NHCOCH3

Hyperchromic

La
< Hypsochromic Bathochromic

700 nm

WOODWARD-FEISER RULE

» It is used for calculating the absorption maxima

» Woodward (1941) gives certain rule for correlating Amax with the
molecular structure

This rule for calculating Amax in conjugated dienes, trienes, polyenes.

* Homoannular dienes:-
cyclic dienes having conjugated double bonds in the same ring.

e.g.

CONTINUED

» Hateroannuler dienes

e.g. Heteroannuler dienes

» Endocyclic double bonds
it is the double bond present in ring as shown.

BQQocyclic double bond

Exocyclic double bonds

double bond in which one of the double bonded
atom is the part of ring system.

WOODWARD’’S-FIESER RULE FOR CONJUGATED DIENES

» a)Parent values-

1. acyclic & Hateroannuler conjugated dienes 215 nm
2.Homoannular conjugated dienes 253 nm
3.Acyclic trienes 245 nm

» — b)Increments-

1.Each alkyl substituent or ring residue 5nm
2.Exocyclic double bond 5 nm
3.Double bond extending conjugation 30 nm
4.auxochromes-

» -OR 6nm

» -SR 30 nm

» -Cl , Br 5 nm

> -NR2 60 nm

> -OCOCH3 Onm

1,4- dimethyl cyclohex-1,3,-diene

H3C CHs

Parent value for Homoannular dienes = 253 nm
Two alkyl substituent’s 2.5 = 10nm
Two ring residues 2° 5 =10 nm

Calculated value = 273 nm

SOLVENT EFFECTS – INTENSITY

Solvents can induce significant changes in the intensity of peaks.

Hyperchromic — Increase in absorption intensity.

Hypochromic — Decrease in absorption intensity.

Absorption characteristics of 2-methylpyridine
Solvent hes Enax , Eo
Hexane 260 2000 | ——<” |
Chloroform 263 4500 — fi
Ethanol 260 4000
Water 260 4000
Ethanol – HCI (1:1 5200

SOLVENT EFFECTS

> TT -> TI* transitions leads to more polar excited state that is more easily
stabilized by polar solvent associations (H-bonds). The tl state is more polar
and stabilized more in polar solvent relative to nonpolar one, thus in going

from nonpolar to polar solvent there is a red shift or bathochromic shift
(increase in A_,, decrease in AE).

> For n -> 11 transition, the n state is much more easily stabilized by polar
solvent effects (H-bonds and association), so in going from nonpolar to polar
solvent there is a blue shift or hypsochromic shift (decrease in A,,,, increase in

AE).

{_ joo

9 > — went
Nonpolor Solvent Potar Solve

APPLICATIONS:

A. APPLICATIONS IN ORGANIC COMPOUNDS
1.It is helps to show the relationship between different groups, it is useful to

detect the conjugation of the compounds

2.Detection of geometrical isomers, In case of geometrical isomers compounds,
that trans isomers exhibits Amax at slightly longer wavelength and have larger
extinction coefficient then the cis isomers .

3.Detection of functional groups, it is possible to detect the presence of certain
functional groups with the help of UV Spectrum.

GENERAL APPLICATIONS:

1.Qualitative analysis, UV absorption spectroscopy can characterizes those type of
compounds which absorb UV radiation. Identification is done by comparing the
absorption spectrum with the spectra of known compound.

2. Itis useful in Quantitative analysis of the compounds.

3. Detection of impurities, UV absorption spectroscopy is the one of the best
bad for detecting impurities in organic compounds.

a

Tautomeric equilibrium, UV spectroscopy can be used to determine the
percentage of various keto and enol forms present in tautomeric equilibrium.

5. Chemical kinetics, UV spectroscopy can be used to study the kinetics of
reactions.

6. Molecular weight determination, molecular weights of compounds can be
measured by spectroscopy.

7. Analysis of inorganic compounds.

8. Measuring concentration of solution, absorption band can also used to
determine the concentration of compounds in a solution.

9. Inorganic chemistry, absorption spectra have been used in connection with
many problems in inorganic chemistry.

10. It is useful to determine the structure of the chloral.

QUALITATIVE ANALYSIS
Pharmacopoeial identification of drug
(1) By using absorbance & wavelength
(2) By taking absorption ratio
(3) Limit test (b)Structural analysis

Quantitative analysis
Quantitative analysis A)By using beer’s law and using absorptivity value By using
reference standard Multiple standard method

B)Single compound analysis direct analysis Using separation method After

extraction after chromatographic separation Using column chromatography Using
HPLC

Indirect analysis

a)Single compound without chromophore

b) Drugs with chromophoric reagent

1.For analyte which absorb weakly in UV region
2.For avoiding interference

3.Ilmprove selectivity of assay
4. Determination of composition of complex Mole ratio method Continuous

variation method ( job curve method )
5 . Study of kinetics

Disadvantages:

>» Samples should be in solution. Mixture of substances poses difficult to
analyse and requires prior separation.
> Interference from the sample’s matrix makes the measurement difficult .