1 / 36

SPECTROPHOTOMETRY

BIOCHEMISTRY

Download Presentation

SPECTROPHOTOMETRY

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. SPECTROPHOTOMETRY M.PRASAD NAIDU MSC MEDICAL BIOCHEMISTRY

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

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

  4. Photon Energy packets . E = h v h = Planck’s constant ( 6.22 x 1027erg 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 ).

  5. Relationship b/n Transmittance , Absorbance Transmittance ( T ) =Is / Io % T = Is / Io x 100 A = - log10 T O D = -log10 % T

  6. 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

  7. 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 .

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

  9. Instrumentation of Spectrophotometry

  10. 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 .

  11. 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 .

  12. 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

  13. The color of filter should be complementary to the color of the solution

  14. 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 .

  15. 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 .

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

  17. 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 )

  18. 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 )

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 2 .Quantitative analysis: comparing the absorbance of a sample (unknown concentration) with standard (with known concentration)

  28. 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)

  29. 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.

  30. 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)

  31. THANK YOU

More Related