Absorption Spectroscopy

• Uv visible spectroscopy

March 30, 2022 nizam 1

 Origin and theory of uv visible
spectra
• 1. electronic transitions
• ---- different type of electrons
• ---- ground state + excited state

March 30, 2022 nizam 2

March 30, 2022 nizam 3
 BEER’S -LAMBERT’S LAW

March 30, 2022 nizam 4

Beer’s law ( related to concentration of absorbing species)

“The intensity of a beam of monochromatic light decreases exponentially with

increase in the concentration of absorbing species arithmetically
or
( absorbance is directly proportional to Concentration)

March 30, 2022 nizam 5

Lambert’s law (related to thickness/path length of absorbing species)

“The intensity of monochromatic light decreases with increases in thickness of

the medium.

March 30, 2022 nizam 6

LAWS GOVERNING ABSORPTION OF RADIATION

The two laws related to the absorption of radiation are:

• Beer’s law ( related to concentration of absorbing species)

• Lambert’s law (related to thickness/path length of absorbing species)

These two laws are applicable under the following condition:

Io = Ia + It
Io = Intensity of incident light
Ia = Intensity of absorbed light
It =Intensity of transmitted light and
No reflection/scattering of light takes place
March 30, 2022 nizam 7
 (Equation of beer – Lambert’s law)
A= ε ct

Where:

A – Absorbance or optical density or extinction co- efficient.

ε – Molecular extinction coefficient ( Absorbance of a specified
concentration in a cell of specific path length A(1% 1cm)
c – Concentration of the drug (mol/lit)
t – Path length (normally 10mm or 1cm)

March 30, 2022 nizam 8

ε – Molecular extinction coefficient ( Absorbance of a specified
concentration in a cell of specific path length)
A(1%1cm)

ε= A1% 1cm X MOLECULAR WEIGHT

10

March 30, 2022 nizam 9

 APPLICATION

• 1. Apply to medium containing more than one kind of species

( no interaction)
• 2. only for dilute solution

• LIMITATIONS

• 1. NOT APPLICABLE FOR SUSPENSION

• 2. NOT FOR COAGULATED MATTER
• 3. NO IDEA ABOUT THE WAVELENGTH

March 30, 2022 nizam 10

 • Real limitation to Beer’s law ( limit to concentration)

• At higher concentration ---------- distance between the molecule of

absorbing solutes was more hence the ability of absorbing molecule
to absorb the light affected there by it affect the relationship
between conc v/s Abs
• ( PREVENTED BY DILUTION)

• Ex: Electrolytes ( more No of ions take part)

March 30, 2022 nizam 11

 Typical Beer’s Law Plot
1.2
1
y = 0.02x
0.8
0.6
A

0.4
0.2
0
0.0 20.0 40.0 60.0
concentration (uM)

March 30, 2022 nizam 12

 Deviations from Beer’s law

March 30, 2022 nizam 13

• In beer’s law it states that if we plot absorbance (A) against
• concentration (C) a straight line passing through origin is obtained,
• but usually a deviation from a linear relationship between concentration
and absorption and an apparent failure of beer’s law.

There are two type of deviation :

• POSITIVE DEVIATION:
When a small change in concentration produces a greater change in
absorbance.

• NEGATIVE DEVIATION:
When a large change in concentration produces a smaller change in
absorbance.

March 30, 2022 nizam 14

REASONS FOR DEVATION FROM BEER’S LAMBERT LAW

• Instrumental deviation
• Chemical Deviations (Physicochemical change in solution)
•
Instrumental deviation:

Factors like stray radiation, improper slit width, fluctuation in single beam
and when monochromatic light is not used can influence the deviation.

Chemical Deviations ( Physicochemical change in solution):

Factors like association, dissociation, ionization (change in pH), faulty

development of colour (incompletion of reaction) refractive index at high
concentration, can influence such deviation.

March 30, 2022 nizam 15

Example:
• Association:

Methylene blue at a concentration 10 -5 M exists as monomer and has

λmax of 660nm. But methylene blue at concentration above 10-4 M exist
as a dimer or trimer , but has a λmax of 600.
• Dissociation:

Potassium dichromate at high concentration exist as a orange solution

(λmax of 450nm).but on dilution, dichromate ions are dissociated into
chromate ions which is yellow in colored (λmax of 410nm)

Cr2O7 2- + H2O → 2H+ + 2CrO4

( orange ) (yellow)

March 30, 2022 nizam 16

• If coloured solute ionizes , dissociates in solution
• Benzyl alcohol in chloroform exists in a polymeric equilibrium;

4C6H5CH2OH ↔ (C6H5CH2OH)4.

• Dissociation of the polymer increases with dilution. The monomer

• absorbs at 2.750 to 2.765 µ. shows negative deviation Where as the

polymer absorbs at 3.0 µ. gives positive deviation

March 30, 2022 nizam 17

• 3) Deviation may also occur due to presence of impurities that

• fluorescence or absorb at the absorption wavelength the interference

• introduces an error in the measurement of absorption or radiation

• penetrating the sample .

March 30, 2022 nizam 18

5) If width of slit is not proper and therefore it allows undesirable radiations to
fall on the sample . These undesirable radiation might be absorbed by the
impurities present in sample and there by change in absorbance.

6)

In case of suspension Beer’s law can’t be applied, but can be

estimated calorimetrically with different known concentration
reference curve.

March 30, 2022 nizam 19

 INSTRUMENTAL DEVIATION FROM BEER’S LAW

• 1. polychromatic light 2. Stray light 3. slit width 4. fluctuation

• 1.Beer’s law requires monochromatic radiation .The possibility of error

due to practical impossibility of obtaining a monochromatic radiation
may be minimized by selection of spectral region,

March 30, 2022 nizam 20

• 2. Stray light : any radiation which reaches the detector other than
monochromatic light ( Detector measures transmitted + stray light)
• 3. slit width : (Used to convert polychromatic to monochromatic light ) if slit
width Increases spectral band width increases
• ( reduces the monochromaticity of the light )

• Hence it is necessary to practice to prepare a calibrated curve for the

absorbance concentration relationship at chosen wavelength.

March 30, 2022 nizam 21

• Thank you

March 30, 2022 nizam 22

 SOME OF THE IMPORTANT TERMS USED IN SPECTROSCOPY

CHROMOPHORE

A chromophore is the part (or moiety) of a molecule

responsible for its color.

• When a molecule absorbs certain wavelengths of visible light

and transmits or reflects light, the molecule has a color.

• A chromophore is a region in a molecule where the energy

difference between two different molecular orbits falls within
the range of the visible spectrum. Visible light that hits the
chromophore can thus be absorbed by exciting an electron
from its ground state into an excited state

March 30, 2022 nizam 23

TYPES OF CHROMOPHORES.

Independent Chromophores :

• when a single chromophore is sufficient to impart

colour to the compound.
• For example, azo group, -N = N- , nitroso group, -
NO, and 0- and p- quinonid group etc are
independent chomophores.

Dependent Chromophores :

• When more than one chromophore is required to

produce colour in the chromogen.
• For example, > C=O group, >C=C < group etc.
March 30, 2022 nizam 24
AUXOCHROME

• This is a group of atoms attached to a

chromophore which modifies the ability of that
chromophore to absorb light.

• Example: -OH, - NH2 , Aldehyde

March 30, 2022 nizam 25

BATHOCHROMIC SHIF T/ RED SHIFT
 It is a change of spectral band position in the absorption,
reflectance, transmittance, or emission spectrum of a
molecule to a longer wavelength (lower frequency).

 This can occur because of a change in environmental

conditions

 A series of structurally related molecules in a

substitution series can also show a bathochromic shift.

 Bathochromic shift is a phenomenon seen in molecular

spectra, not atomic spectra.

nizam March 30, 2022 26

HYPSOCHROMIC SHIFT / BLUE SHIFT

 It is a change of spectral band position in the absorption,

reflectance, transmittance, or emission spectrum of a molecule
to a shorter wavelength (higher frequency).

 This can occur because of a change in environmental conditions

 Hypsochromic shift is a phenomenon seen in molecular spectra,

not atomic spectra

nizam March 30, 2022 27

HYPERCHROMIC EFFECT

 An increase in absorption intensity

 If structural modification leads to an increase in the molar

extinction coefficient for a particular chromophoric group it is
said to have brought about a hyperchromic effect.

HYPOCHROMIC EFFECT

 An decrease in absorption intensity

 If structural modification leads to a decrease in the molar

extinction coefficient for a particular chromophoric group it is
said to have brought about a hypochromic effect.

nizam March 30, 2022 28

 Chromophores:
Substituent Effects

General – Substituents may have any of four effects on

a chromophore

Bathochromic shift (red shift) – a shift to longer

wave length; lower energy

Hypsochromic shift (blue shift) – shift to shorter

wave length ; higher energy
Hyperchromic effect – an increase in intensity
(Absorbance increases)

Hypochromic effect – a decrease in intensity

(Absorbance decreases)
March 30, 2022 nizam 29
 Hyperchromic


abs Hypsochromic Bathochromic


Hypochromic
200 nm
800 nm
conc

March 30, 2022 nizam 30

• Terminology for Absorption Shifts

• Nature of Shift Descriptive Term

• To Lower Absorbance Hypochromic
• To Greater Absorbance Hyperchromic
• To Shorter Wavelength Hypsochromic
• To Longer Wavelength Bathochromic

March 30, 2022 nizam 31

 Effect of solvents
• # good solvents < 210 nm
• # 95% ethanol used as polar solvent
• # selection: depends on transparency
• # shifts may due to : type of solvent + solute
• # polarity: influence: position + intetensity
• #polar compounds: λmax shifts: change in polarity
• #non polar : λmax same : change in polarity

• Polar solvents: water, alcohol, esters, ketone

• Non polar: n-hexane, ccl4, cs2
March 30, 2022 nizam 32
• EX: I2 in non polar solvents = purple colour
• I2 in polar solvents = brown colour ( blue shift)
( dipole –dipole moment)

• Effect of solvent
• * nonpolar substance in nonpolar : RI
• * polar substance in polar : dipole interaction

March 30, 2022 nizam 33

• in non bonding electrons
• n-π * + n-σ * : very sensitive to polarity
• Ex: c=o
• in bonding electrons
• π – π*: lesser extent: red shift or batho:
increasing polarity

March 30, 2022 nizam 34

Beer’s law
“The intensity of a beam of monochromatic light decreases
exponentially with increase in the concentration of
absorbing species arithmetically

Accordingly, - dI / dc α I
(The decrease in the intensity of incident light (I) with concentration c
is proportional to the intensity of incident
light (I))
-dI / dc = kI
(removing and introducing the constant of proportionality ‘k’)
-dI / I = k dc (rearranging terms)

-In I = kc + b ……Equation (1)

(on integration , b is constant of integration)
When concentration = 0, there is no absorbance. Hence I= Io
Substituting in equation 1, -I n Io
March 30, 2022
= k*0 + b 35
-In Io = b nizam
Substituting the value of b, in equation 1,

-In I = kc –InIo

In Io – In I = kc

In Io/I = kc (since log A-log B = log A/B)

Io / I = e kc (removing natural logarithm)

I / Io = e –kc (making inverse on both sides)

I = Io e -kc ….Equation (2) (equation of Beer’s law)

March 30, 2022 nizam 36
Lambert’s law

“The rate of decrease of intensity (monochromatic light) with

the thickness of the medium is directly proportional to the
intensity of incident light”

i.e. –dI / dt α I

This equation can be simplified similar to equation 2 to get

the following equation (by replacing ‘c’ with ‘t’)

I = Io e –kt ….. Equation (3)

[equation of Lambert’s law]
March 30, 2022 nizam 37
BEER – LAMBERT,S LAW

Equations (2) and (3) can be combined to get

I= Io e –kct
I = Io 10 –kct
(converting natural algorithm to base 10 & K = k * 0.4343)

I / Io = 10 –kct (rearranging terms)

Io / I = 10 kct (inverse on both side Log

Io / I = kct (taking log on both sides) ….. Equation 4

It can be learnt that transmittance (T) = I / Io and Absorbance

(A) = log 1 / T

Hence A = log 1 / I/ Io

A = log Io /I ……. Equation 5

March 30, 2022 nizam 38
 Using Equation 4 & 5 ,

Since A= log Io /I

and log Io /I = Kct

we can infer that

A= Kct (instead of K, we can use ε)

A= ε ct (Equation of beer – Lambert’s law)

Where:

A – Absorbance or optical density or extinction co- efficient.

ε – Molecular extinction coefficient
c – Concentration of the drug (mol/lit)
t – Path length (normally 10mm or 1cm)
March 30, 2022 nizam 39
• Contd…..

March 30, 2022 nizam 40