SPECTROPHOTOMETRY

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SPECTROPHOTOMETRY

• M.PRASAD NAIDU
• MSC MEDICAL BIOCHEMISTRY

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

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• Basic principles of light
• Light has dual characteristics
• Photon ( energy )
• Wave form

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• 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 (3x1010cms/sec )
• Wave length (? )
• Distance between two peaks as the
  light travels in wave like manner .
• Expressed in nanometers ( nm ).

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Relationship b/n Transmittance , Absorbance

• Transmittance ( T ) Is / Io
• T Is / Io x 100
• A - log10 T
• O D -log10 T

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• 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

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• 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 .

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• A abc
• A
  Absorbance
• a
  proportionality
• 
  constant (absorptivity)
• b Light
  path in cms
• c
  concentration of
• the
  absorbing
• 
  compound in g/L

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• Instrumentation of Spectrophotometry

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• 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 .

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

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• 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

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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
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• 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 .

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• 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 .

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

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• 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 )

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• 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 )

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• 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

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Colorimeter UV Visible
Light Filament Hydrogen / Deuterium Tungsten / Halogen
Monochromater Filters Prism Diffraction gratings
Cuvetts Glass Silica / Quartz Borosilicate Glass
Cheaper Costly Sensitive
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• Photo detectors
• Converts light into an electric signal that is
  proportional to the number of photons striking
  its photosensitive surface .
• Commonly used are
• Photomultiplier tube
• Solid state detectors
• -Photodiodes
• -Charge couple detectors

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• 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

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• Performance parameters
• To verify that a spectrophotometer is performing
  satisfactorily or not .
• Parameters tested
• Wave length accuracy
• Special band width
• Stray light
• Linearity
• Photometric accuracy

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• 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

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• 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

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• 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

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• 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

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• 2 .Quantitative analysis
• comparing the absorbance of a sample (unknown
  concentration) with standard (with known
  concentration)

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• 3. Enzyme assay
• Determination of enzyme activity change in
  absorbance
• Eg LDH
• Lactate NAD Pyruvate NADHH
• 
  ( 340nm)
• Coupled enzyme assay
• Eg PK
• PEP ADP Pyruvate ATP
• LDH
• Pyruvate NADHH
  Lactate NAD
• ( 340nm)

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• 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.

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• 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)

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