Photometry by Dr. Anurag Yadav

INTRODUCTION TO PHOTOMETRY AND SPECTROPHOTOMETER DETAIL

1. P HOTOMETRY Presenter: Dr. Anurag Yadav Moderator : Mr. Arun kumar

2. What is light? How light interact with matter. Is form of energy E= hv V=1/ λ Energy is inversely related to wavelength. Matter react to the light either by -Absorbing - Emiting

3. Emission process When electron return to ground state, the energy is dissipated as radiant energy. Used in flame photometry & flurometric methods.

4. Absorption spectroscopy I o impinging on & passing through a square cell that contain solution of compound that absorb radiant energy of certain wavelength. The intensity I s is less than Io. The transmittance in solution is defined as proportion of the incident light that is transmitted T = I s /I o

5. As the conc of the compound in solution increases more the light is absorbed by the solution & less is the light is transmitted.

6. T = I E /I o % T= I E /I o X 100% A = - log T A = log 1/T To convert T to % A = log 1/T X 100%/100%= log 100%/%T Rearranging A = log 100%- log%T Equation A = 2 – log %T

7. A α C Laws of light absorption:

8. Beer’s law Beer’s law

9. A α L

10. LAMBERT’S LAW

11. Bcz of linear relationship btwn absorbance and concentration, it is possible to relate unknown conc to single std by a simple proportional equation; A s C s A u C u C u A u x C s A s = =

12. C T = A T A S X C S Concentration of TEST solution Absorbance of TEST Absorbance of STANDARD Concn of STANDARD X = Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100 X = Xml

13. Concentration of TEST /100ml Absorbance of TEST Absorbance of STANDARD X = Xml Concn of Std X 100 Concentration of TEST /100ml O.D of ‘T’- O.D of ‘B’ O.D of ‘S’- O.D of ‘B’ X = Volume of ‘T’ Amount of ‘S’ X 100 Concentration of TEST /100ml T - B S - B X = Volume of ‘T’ Amount of ‘S’ X 100

14. LIMITATIONS: Very elevated conc cant be measured. Incident radiant energy is not monochromatic. Solvent absorption is significant compared to solute absorbance. Radiant energy is transmitted by other mechanism ( stray light). The sides of the cell are not parallel.

15. Note on Stray Light: Is radiant energy that reaches the detector at wavelength other than those indicated by monochromator setting. All radiant energy that reaches detector with/without having passed through the sample is recorded. As the amount of the stray light increases, deviation from the Beer’s Law also increases.

16. Can be due to Light leaks – excluded by covering cell compartment Fluorescence - that increases signal to the detector and causes apparent decrease in A Most spectrophotometers are equipped with stray light filters

17. SPECTROPHOTOMETER : Definitions PHOTOMETER: If a filter is used as a wavelength selector, monochromatic light at only discrete wavelength is avialable & the instrument is called photometer. SPECTROPHOTOMETER: If a monochromater is used( prism/grating) as a wavelength selector, the inst can provide monochromatic light over a continous range of wavelengths & is called spectrophotometer.

18. SPECTROPHOTOMETER: TYPES: 1. Single beam spectrophotometer 2. Double beam in space spectrophotometer 3. Double beam in time spectrophotometer 4. Multichannel COMPONENTS:

19. SOURCE: Tungsten filament lamps – continous spectrum Tungsten iodide lamps – visible & near UV Hydrogen & deuterium discharge lamps – cont UV Mercury vapour lamps – Discontinous /line spectrum Light emitting diode(LED’s) – 2types of semiconductors 2. ENTRANCE SLIT : Focuses light on grating/prism, where it can be dispersed with minimum stray light.

20. 3. WAVELENGTH SELECTOR : For isolation of a required wavelength/range of wavelength. 2types- a. Filters b. Monochromators FILTERS : Consists of only a material that selectively transmits the desired wavelength & absorbs the rest. a. Those selective transmission characteristics- glass & Wratten filter b. Those based on the principle of inteference . Simple wide wavelength Non adjustable

21. 2. MONOCHROMATORS : A grating/prism disperses radiant energy from the source lamp into a spectrum from which the desired wavelength is isolated by mechanical slits. Prism - Nonlinear dispersion Grating - Linear dispersion PRISMS: Less linear over lower wavelength over 550nm Give only 1 order of emerging spectrum thus provide higher optical efficiency Therefore 3 wavelength checks are required

22. b . GRATING : a. Linear dispersion b. Therefore only 2wavelength checks required to certify accuracy 4. EXIT SLIT : Determines the band width of light that will be selected from the dispersed spectrum. 1000-2000line/mm

23. 5. CUVETTES/CELL : a. Receptacle for sample b. Optical property depends on composition. c. Calibrated to path length 1cm 6. PHOTODETECTORS: A device that converts light into an electric signal that is proportional to the number of photons striking its photosensitive surface.

24. 6. PHOTODETECTORS: a. Photomultiplier tubes b. Photodiodes c. Charged coupled devices a. Photomultiplier tubes: An electron tube that is capable of significantly amplifying a current. Ideal detector : high sensitivity, high signal/noise, constant response for λ s, and fast response time.

25. b. PHOTODIODES : Semiconductors that change their charged voltage upon being struck by light. Change is converted to current & measured. c. Charged coupled devices : Solid-phase devices that are made of small silicon cells. Electron released is captured and quantified. 7. READOUT DEVICES: Electric energy from detector is displayed on a meter or display system

26. DOUBLE BEAM SPECTROPHOTOMETER: Designed to compensate for possible variations in intensity of light source. Accomplished by splitting the light beam

27. DOUBLE BEAM SPECTROPHOTOMETER: ADVANTAGES OF A DOUBLE-BEAM OVER A SINGLE-BEAM INSTRUMENT: Compensate for variations in the source intensity. Compensate for drift in the detector and amplifier. Compensate for variation in intensity as a function of wavelength

28. MULTICHANNEL INSTRUMENTS Able to “scan ” an entire spectrum in ~ 0.1 sec high throughput of radiant energy due to the minimal optics use a deuterium lamp source for a spectral range of 200nm - 820 nm and have a spectral bandwidth of 2 nm.

29. COMPARISION COLORIMETERS Light measurement only in visible region Filters Can choose only a bandwidth of wavelength Only coloured solutions measured Absorbance- less accurate SPECTROPHOTOMETERS UV, visible, IR Diffraction gratings, prisms Can choose exact wavelength, Colourless solution can also be measured Absorbance –more accurate Kinetic studies and spectrum can be better studied

30. Wavelength accuracy : mercury vapor lamp, dueterium lamp,(strong emmision lines), holmium oxide (strong absorbtion lines). Linearity of detector response : solutions of varying concentrations of compound(Beer’s law) Eg : oxyhb at 415nm, cobalt ammonium sulphate at 512nm Stray radiation : by LiCO3 below 250nm, NaBr below 240nm Photometric accuracy : pottasium dichromate soln , cobalt ammonium sulphate soln Quality control check for spectrophotometer

31. NIST formerly NBS provide the SRM –useful for calibration/verification of performance of the instrument Eg - SRM 930e –verifies and calibrates T and A – visible range of spectrophotometer. IRMM –provide reference material for verification of performance of the instrument. listed in the IRMM BCR ref material catalogue. Quality control check for spectrophotometer

32. satisfactory if its close to λ max of chromogen and if its reproducible. H and Du lamps have built in sources of checking accuracy Prisms(2) and gratings(3) – continuous choice of λ . Rare earth glass filters like holmium oxide and didymium - narrow and wide spectral band widths 1. Wavelength calibration

33. Holmium oxide – 280-360nm , show sharp absorption peaks at defined λ

34. Another method is by use of solutions. Disadvantage absorption peaks are broad and causes spectral shifts due to contamination aging or preparation errors

35. Hg vapor lamp that shows a no of sharp, well defined emissions lines bet 250 - 580nm . Can be calculated from manufactures specifications. Interference filters – 1-2 nm are available and can be used to check spectral bandwidth of 8nm or more 2. Spectral bandwidth

36. Increases at extreme ends of spectral range where detector response is lowest. Methods to detect filters or solu that’s highly transmitting over a portion of the spectrum but opaque below an abrupt cutoff λ Egs Li 2 CO 3 below 250nm NaBr – below 240nm Acetone- below 320nm 3.Stray radiation

37. Stray light can also be due to Light leaks – excluded by covering cell compartment Fluorescence - that increases signal to the detector and causes apparent decrease in A Most spectrophotometers are equipped with stray light filters

38. Blue filters - used with Tungsten lamps for λ below 400nm Red filters- λ range 650 – 800nm Eg - spectrophotometer set to 350nm -stray light - visible range absorbed by the blue filter transmits UV portion of spectrum

39. If solu / filters –transmitting no radiant energy at measurement λ measured T = amount of stray light T X 100% = % of stray radiation . If stray light > 1% - instrument malfunction Liquid cut off filters- UV range where there is more stray light problem UV stray light filters

40. Spectrophotometer should exhibit a linear relationship between radiant energy absorbed and readout. Solid glass may be used for the above Common method use of solu of varying conc of compound following beer’s law DISADV – dilution errors, stability problems, shifts in Ph,temp effects. 4. Linearity

41. Absorbance std – constant stable A with no variation to spectral band width / light beam. NIST –set of 3 neutral density glass filters with known A at 4 λ for each filter. They are not always stable- need recalibration by NIST periodically Standards for checking accuracy - potassium dichromate -cobalt amm sulfate -nitrate solu 5. Photometric accuracy

42. Background interferences – min by including blank or taking A at 2-3 λ . Bichromatic -A is measured at 2 λ 1. corresponds to peak A 2. at a point at the base of the peak serves as baseline. Diff in A is related to conc – gives a blank ref point for each sample. Another method to correct background interference- measure A at 2 λ equidistant from peak and latter is averaged to get a baseline and that’s subtracted from the peak A – CORRECTED A 6. Multiple wavelength readings

43. In cases of spectral overlap – extinction coeff of each component at each λ should be known. Eg – in blood Hb (red Hb ,oxy Hb , carboxyHb ,meth Hb , sulfHb ) ext coeff is known the matrix eq can be set up to calculate each component – principle used in COOXIMETERS

44. Visible Spectrophotometer Application - Niacin, Pyridoxine, Vitamin B12, Metal Determination (Fe), Fat-quality Determination, Enzyme Activity (glucose oxidase ) UV Spectrophotometer Application -Protein, Amino Acids (aromatic), Pantothenic Acid, Glucose Determination, enzyme Activity ( Hexokinase ) Applictions of Spectrophotometer

45. Thank You