SPECTROPHOTOMETRY

M.PRASAD NAIDU
MSC MEDICAL BIOCHEMISTRY



SPECTROPHOTOMETRY :-

Defined as the measurement
of intensity of light at selected wave
length .

Basic principles of light



Light has dual characteristics

Photon ( energy )
Wave form
Photon
Energy packets .
E = h v h = Plancks constant ( 6.22 x 10

27

erg sec )
Frequency ( v )
Number of wave passing through a
fixed point per second .
v = c / c = speed of light in vaccum

(3x10
10
cms/sec )
Wave length ( )
Distance between two peaks as the
light travels in wave like manner .
Expressed in nanometers ( nm ).


Transmittance ( T ) =I
s
/ I
o

% T = I
s
/ I
o
x 100

A = - log
10
T

O D = -log
10
% T

Relationship b/n Transmittance , Absorbance

The Laws of Absorption
1. Beer s Law :-

States that concentration of a substance
is

directly proportional to the amount of
light

absorbed or inversely proportional to the

logarithm of the transmitted light


2. Lambert s Law :-

States that the amount of light
absorbed is

proportion to the thickness of absorbing
material

and is independent of the intensity of the
incident

light .
A = abc


A = Absorbance
a = proportionality
constant
(absorptivity)
b = Light path in
cms
c = concentration
of
the absorbing
compound in
g/L
Instrumentation of Spectrophotometry
Light Source

Incandescent :-
visible spectrum------Tungsten light
bulb
uv spectrum --------Hydrogen &
deuterium
lamps
atomic absorption-----Hollow
cathode lamp
Laser sources :-
These provide intense light
and narrow
wave length .



Monochromater ( Spectral isolation
)
System for isolating radiant energy
of a desired wave length .

1. Filters

2. Prisms

3. Diffraction gratings

Slits may be inserted before &
after the monochromater device to
render light rays parallel or to isolate
narrow portion of the light beam .
Filters : -
simplest
Thin layer of colored glass
Operates by absorbing light in all other
region except for one ,which they reflect
.
Resolve polychromatic light into a
relatively wide band width ( 50 nm )
Used only in colorimeter

Disadvantage
Low transmittance ( 5 20 % )
Normal T = 20 -80 % A = 0.1 -
0.7
The color of filter should be complementary
to the color of the solution
Filter color Wavelength
(nm )
Color of
solution
Violet 420 Brown
Blue 470 Yellowish
brown
Green 520 Pink
Yellow 580 Purple
Red 680 Green \ Blue
Prisms :-

Separates white light into a continues
spectrum by refraction ----shorter wave
length are refracted more than longer
wave length .

this results in non linear with the
longer wave length closer together .
Diffraction gratings:-

Prepared by depositing a thin layer of
aluminium copper alloy on the surface
of a flat glass plate .Then ruling many
parallel grooves into the metal coating .

These are then used as moulds to
prepare less expensive replicas for
instrumental use .

Better gratings ------10002000 lines
/mm .

Cuvetts

Absorption cells
Shapes ---round
square
rectangle
Material ---glass
silica (quartz )
plastic
All have constant path length ---1cm

Precautions
Cuvetts must be clean & optically clear

Etching / deposition on the surface
effects absorbance value

Cuvetts are cleaned by copious rinsing
with distilled water

Wash with mild detergent or soak in a
mixture containing HCl :H
2
O: Ethanol ( 1:
3 : 4 )
Cont----
Alkaline solution not left standing for
prolonged period as it dissolves glass
and produces etching

Never soak in dichromate cleaning
solution as it is hazardous and tends to
adsorb onto and discolor the glass

Invisible scratches , finger prints or
residual traces of previously measured
substance may interfere with
absorbance ( uv-vis spectrophotometry
)


Cont d ---

A good practice is to fill all Cuvetts with

distilled water and measure the
absorbance for

each against a reference blank over the

wavelength to be used .

This value should be essentially ZERO
Colorimeter
UV Visible
Light
Filament Hydrogen /
Deuterium
Tungsten /
Halogen
Monochro
mater

Filters
Prism Diffraction
gratings
Cuvetts
Glass
Silica /
Quartz
Borosilicate
Glass

Cheaper

Costly

Sensitive
Photo detectors

Converts light into an electric signal
that is proportional to the number of
photons striking its photosensitive
surface .

Commonly used are
1. Photomultiplier tube
2. Solid state detectors
-Photodiodes
-Charge couple detectors
Read out devices
Electrical energy from a detector is
displayed on meter or read out system .

Direct reading system
no further amplification .

Digital read out device
Provides visual numerical display
of absorbance or converted values of
concentration

Performance parameters

To verify that a spectrophotometer is
performing satisfactorily or not .

Parameters tested
1. Wave length accuracy
2. Special band width
3. Stray light
4. Linearity
5. Photometric accuracy

Deviation from Beer Lambert
s Law
Reasons

-High sample concentration
Specimens may polymerize or ionize
Coagulate to form turbid solution (
higher
absorbance )

-Instrumentation limitations
Imperfect monochromacy
Stray lights
Power fluctuations
- Temperature effects
Changes in temp changes the
degree of solubility ,dissociation
/association properties of the solute
,hydration etc.

So absorbance measurement must
always be done at constant temp .

- Sample instability
Color developed may be unstable
Application of UV-VIS
Spectrophotometer

1. Qualitative analysis
-identify compounds in pure state \in
biological preparation
-by plotting a absorption graph
-curves are specific to a compound
eg:-
- Nucleic acid 254 nm
-Proteins 280 nm
Absorption of compounds in different region
gives some hint of its structure

220 280nm No absorption aliphatic
alicyclic
hydrocarbons

220 -250nm absorption + two
unsaturated
linkages
Benzidine
derivative

250 300nm absorption + more than two
double bonds

2 .Quantitative analysis:

comparing the absorbance of a sample
(unknown concentration) with standard
(with known concentration)
3. Enzyme assay:
Determination of enzyme activity
change in absorbance
Eg; LDH
Lactate + NAD
+
Pyruvate + NADH+H
+

( 340nm)
Coupled enzyme assay:
Eg; PK
PEP + ADP Pyruvate + ATP

LDH
Pyruvate + NADH+H
+
Lactate +
NAD
+

( 340nm)

4. Study of cis trans isomerism:

- Differs in spatial arrangement of groups
around the plane. So absorption spectra
also differs

- Trans ------- more elongated -----
maximum absorption at longer wave
length.

5 .Control of purification:
- Impurities in a compound easily
detected

Eg; carbon disulphide in carbon
tetrachloride
(impurity, 318nm)

Benzene impurity in commercial
alcohol
(280nm)
(210nm)