spectro photometry

SPECTROPHOTOMETRYSPECTROPHOTOMETRY

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 = Planck’s constant ( 6.22 x 10

27 erg sec )

Frequency ( v ) Number of wave passing through a

fixed point per second . v = c / λ c = speed of light in vaccum

(3x1010cms/sec )

Wave length (λ ) Distance between two peaks as the

light travels in wave like manner . Expressed in nanometers ( nm ).

Transmittance ( T ) =Is / Io

% T = Is / Io x 100

A = - log10 T

O D = -log10 % 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 : - simplestThin 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 The color of filter should be complementary to the color of the solutionto 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 ------1000—2000 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 :H2O: 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

Monochromater

Filters

Prism Diffraction gratings

Cuvetts Glass

Silica / Quartz

BorosilicateGlass

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 devicesElectrical 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 parametersTo verify that a spectrophotometer is

performing satisfactorily or not .

Parameters tested 1. Wave length accuracy2. Special band width3. 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)