Colorimetry and spectroscopy

A presentation outlining the method of colorimetry & spectroscopy. Also detailed information regarding spectrophotometer, calculation of absorbance and transmittance according to Beer & Lambert's law

1. COLORIMETRY AND SPECTROSCOPY DR. KARUN KUMAR JR-I DEPT. OF PHARMACOLOGY

2. WHAT IS COLOR ? Visual perceptual property corresponding in humans to the categories red, blue , yellow, green and others

3. TYPES OF COLORS Cannot be mixed Mixing primary color Mixing a primary and secondary color

4. COLOR WHEEL

5. CIE ( Commission International de L'Eclairage ) COLOR MODEL

6. Hue, Saturation, Value (HSV) Color Model

7. BASIC CONCEPTS Energy is transmitted via electromagnetic waves Wavelength  The distance b/w 2 wave crests Visible light  390 – 780 nm region of the e.m . spectrum UV rad.  180 – 390 nm

8. Wavelength (nm) Region name Observed <380 Ultraviolet Invisible 380-440 Visible Violet 440-500 Visible Blue 500-580 Visible Green 580-600 Visible Yellow 600-620 Visible Orange 620-750 Visible Red 800-2500 Near-infrared Not visible

9. RELATIONSHIP BETWEEN ABSORBANCE & TRANSMITTANCE Absorbance/Optical density :- Amount of light absorbed Transmittance :- Amount of light transmitted

11. A = -log = -log 10 T  

12. PRINCIPLE BEER’S LAW :- Conc. Of a substance amount of light absorbed OR Conc. Of a substance T  Transmitted light  

13. LAMBERT ’S LAW :- Amount of light absorbed depends on the thickness of the medium Combining the 2 laws, I = I o ct  Molar extinction coefficient C  Conc. Of absorbing substance (g/dl) t  Thickness of medium through which light passes  

14. Combining the 2 laws, K → Constant for colored solution; A T → Absorbance of test solution; C T → Conc. of test soln.; A S → Absorbance of standard soln.; C S → Conc. Of std.soln .; L  Light path(in cm)  

15. ; since L is same C T X X 100 (Conc. in 100 ml of test sample [% conc .]; 𝓍 is ml of sample taken)  

16. COLORIMETER Instrument used for the measurement of coloured substances Works in the visible range ( 380 – 750 nm) of the e.m . spectrum of light

17. COMPONENTS OF COLORIMETER Lamp (Light source) Provides light in the visible range of the spectrum Usually tungsten lamp is used

18. 2. Adjustable slit 3. Condensing lens Provides parallel beam of light 4. Filter Provides desired monochromatic light (of single wavelength)

19. Color of filter is complementary to the color of the solution Filter Colour of solution Blue Red Purple Green Yellow Violet Orange Blue green

20. 5. Cuvette (Sample holder) Glass tube which holds solution to be analyzed in a colorimeter 6. Photodetector Produces current in response to the light impinging upon it

21. SELECTION OF FILTER Colour of the filter should be complementary to the colour of the solution to give maximum absorbance

22. PREPARATION OF SOLUTION In colorimetric estimation, 3 solutions should be prepared :- Blank (B) Standard (S) Test (T)

23. BLANK It is prepared to eliminate the effect of light absorption due to the reagents used 2 types of blank are used :- Water blank Reagent blank

24. STANDARD SOLUTION Solution of known concentration of the substance in pure form to be estimated Both O.D. and concentration of standard is known to calculate concentration of unknown

25. TEST SOLUTION It is made by treating a specific volume of the test sample with reagents as mentioned in the procedure

26. STEPS IN OPERATION Glass filter is placed in the filter slot 3/4 th of cuvette is filled with distilled water and placed in the cuvette slot Instrument is switched ‘on’ and allowed to warm-up for 4-5 minutes

27. Button is adjusted using ‘coarse’ and ‘fine’ knobs to give zero optical activity in the galvanometer Blank solution is placed in an identical cuvette and the OD is read (‘B’) Blank solution is transferred to the original test tube

28. Test solution is taken in the same cuvette and O.D. is read (‘T’) Test solution is transferred back to the original test tube Standard solution is taken in same cuvette and O.D. is read (‘S’) Standard solution is transferred back to the test tube Cuvette is washed

29. CALCULATION X X 100   Cs  Conc. Of standard V  Volume of test sample NOTE :- Satisfactory results are obtained only when the O.D. values are in the range 0.1 – 0.7

30. APPLICATIONS OF COLORIMETER Estimation of biochemical compounds (such as glucose, urea, creatinine, uric acid, bilirubin, lipids, total proteins, enzymes [ALT, AST, ALP], minerals [Ca, P] in blood, plasma, serum, CSF, urine, etc.

31. PHOTOMETRY Measurement of luminous intensity of light SPECTROPHOTOMETRY Measurement of intensity of light at selected wavelengths

32. Modern instruments isolate a narrow range of spectrum Those that use filters  filter photometers Those that use prisms/gratings  spectrophotometer  

33. SPECTROPHOTOMETER It is an instrument used to measure light transmitted by a solution in a cuvette Operation is based on Beer – Lambert’s law

34. CLASSIFICATION Single-beam spectrophotometer Double-beam (DB) spectrophotometer DB In-space spectroph . DB In-time spectroph .

35. COMPONENTS OF SPECTROPHOTOMETER Light source Incandescent lamps i ) Visible spectrum  tungsten light bulb ii) UV spectrum  Hydrogen and deuterium lamps are used ( 2 H is more stable) LASER–Argon fluoride, He-Cd, Nitrogen, Helium-neon, etc.

36. 2. Monochromators Used for isolation of the required range of wavelengths They consist of filters, diffraction gratings, or prisms in combination with entrance and exit slits

37. Entrance slit excludes unwanted or stray light Exit slit permits only a narrow beam of the spectrum to pass through the cuvette Efficiency of monochromator depends on range of wavelengths it can produce

38. This range of wavelengths is known as BAND PASS or BANDPASS WIDTH A narrow range of wavelengths is the most desirable (since Beer’s law is dependent on monochromatic light)

39. Filters Wide- bandpass filters (Glass filters) Narrow- bandpass filters Sharp-cutoff filters Spectral purity of a filter is described in terms of its spectral bandwidth

40. It is a measure of the spectral purity of the instrument The narrower the bandpass , the purer the light is BANDPASS

41. Prisms & gratings produce narrower bandpass widths (<5 nm) Interference filters produce wide bandpass widths (10 – 20 nm) Glass filters produce more wider bandpass widths (>30 nm)

42. It can be defined as the range of wavelengths, which a monochromato r can isolate b/w 2 points of a spectral scan where the transmittance is ½ of peak transmittance

43. A narrow bandpass width is required to measure the sharpness of the spectral peaks & avoid deviations from Beer’s law

44. B) DIFFRACTION GRATING Prepared by depositing a thin layer of Aluminium -Copper alloy on the surface of a flat glass plate, then ruling many small parallel grooves into the metal coating

45. They are of 2 types :- Transmittance grating  Made of glass & transmits light Reflection grating  Made of Aluminium & acts as mirrors

46. Resolution of spectrum obtained depends on the number of grooves on the polished surface

47. Incident light strikes the grooves on the reflection grating Many tiny spectra are formed (one from each groove) Wave fronts are formed from these spectra ( Wavefronts that are in phase, reinforce one another whereas those out of phase, cancel each other) A linear, parallel spectrum is formed

48. C) PRISM It disperses polychromatic radiation (white light) into a continuous spectrum by refraction Shorter wavelengths (violet) are refracted more than longer wavelengths (red)

49. A continuous, nonparallel spectrum is produced By choosing an appropriate exit slit width, a particular wavelength can be isolated & allowed to reach the cuvette

50. Glass prisms are used in visible region Quartz/fused silica is used in the UV region Dispersion qualities of glass prisms are better than those of quartz

51. FIBER OPTICS Fiber optics a/k/a light pipes are bundles of thin, transparent fibers of glass, quartz, or plastic that are enclosed in material of a lower refractive index

52. CUVET It is a small vessel used to hold a liquid sample It may be round, square or rectangular & is constructed from glass, silica (quartz), or plastic

53. PHOTODETECTOR Device that coverts light  electrical signal Photomultiplier tube (PMT) is commonly used for measuring light intensity in the UV & visible regions of the spectrum

54. READOUT DEVICE Electrical energy from the detector is displayed on a digital readout device It operates on the principle of selective illumination of portions of a bank of LEDs controlled by voltage signal

55. COMPUTER With a resident computer & software :- Output from a calibrator is digitally stored Digital signals from blanks are subtracted from calibrators & unknowns

56. 3. Concentration of unknowns is automatically calculated Computers & their resident software are also used to convert kinetic data into concentration or enzyme activity

57. PERFORMANCE PARAMETERS NIST provides several SRMs for spectrophotometry IRMM, a metrology institute that belongs to EC, also provides reference materials

58. LIFE IS C O L O R F U L 

59. THANK YOU