Spectro Photometry

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  • SPECTROPHOTOMETRYM.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 (3x1010cms/sec ) Wave length ( ) 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 % TRelationship 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 lampLaser sources :-

    These provide intense light and narrow wave length .

  • 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 .

  • 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 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 :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 FilamentHydrogen /Deuterium Tungsten / Halogen Monochromater Filters Prism Diffraction gratings Cuvetts Glass Silica / QuartzBorosilicateGlass 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 Photomultiplier tube 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 parameters

    To verify that a spectrophotometer is performing satisfactorily or not .

    Parameters tested Wave length accuracySpecial band widthStray light Linearity Photometric accuracy

  • Deviation from Beer Lambert s LawReasons

    -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 absorbanceEg; 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)