colorimetry spectrophotometry by dr.Tasnim

colorimetry....spectrophotometry

1. COLORIMETRY & SPECTROPHOTOMETRY Dr.Tasnim Ara Jhilky MD(Part-1) Department of Biochemistry Sir Solimullah Medical College

2. Introduction Photometry is the most common analytical technique used in the biochemical laboratory. It is designed to measure the intensity of a beam of light. Photometric principles are applied to the several kinds of analytical techniques: (a) where absorbed or transmitted light is measured: Colorimetry Spectrophotometry Atomic absorption, and T urbidometry (b) where emitted light is measured: Flame photometry

3. Introduction (cont.) The components of most photoelectric colorimeters are basically the same and the basic method of operation is also similar for all the instruments. In analytical chemistry, Colorimetry is a technique “used to determine the concentration of colored compounds (analytes) in sample solution” at visible spectrum of light (400 – 700 nm).

4. Properties of Light Light is the visible spectrum of electromagnetic radiation,emitted in the form of waves of different wave lengths ranging from380nm to 750nm.

5. Electromagnetic Spectrum

6. Colors & Wavelengths COLOR WAVELENGTH ( λ in nm) Ultraviolet < 380 Violet 380 – 435 Blue 436 – 480 Greenish-blue 481 – 490 Bluish-green 491 – 500 Green 501 – 560 Yellowish-green 561 – 580 Yellow 581 – 595 Orange 596 – 650 Red 651 – 780 Near Infrared > 780 Visible Light

7. Principle of Photometry Substance to be measured by photometry must be colored to begin with or can be made to produce color derivatives by using certain reagents and reactions. Intensity of colour produced is propotional to the concentration of the colour producing subs.present in solution. Colored subs.absorbs light of a particular wave length and the extent of light absorption depends on the conc .of color producing subs.in solution.

8. Principle (con.)…… A characteristic wavelength of absorption spectrum is isolated from light passing it through filter monochromator Solution with colored subs. is kept in a cuvet & allowed the subs. to absorb light. Degree of light absorption by a solute of unknown conc. Is propotional to degree of light absorption by same solute in a solution of known conc. Subs.of unknown conc. is measured by comparing with same subs .in another solu . Of known conc.

9. Colorimetry Principle . Colored solutions have the property of absorbing certain wavelength of light when a monochromatic light is passed through them . . The amount of light absorbed or transmitted by a colored solution is in accordance with two laws: Beer’s law Lambert’s law

10. Beer’s law : This law states that,the intensity of transmitted light decreases exponentially with the increase in concentration of colored substance in the solution. i.e . the amount of light absorbed by a colored solution is directly proportional to the conc. Of substance in the solution. A α C

11. Beer’s law Beer’s law

12. Lambert’s law : This law states that,the intensity of transmitted light decreases exponentially with increase in length of light pathway. ( diameter of the cuvette) i.e. the amount of light absorbed by a colored solution is directly propotional to the length of light path. A α L

13. Transmittance Transmittance: It is the ratio of intensity of transmitted light (It) to the intensity of incident light (lo) across a solution. It is expressed as % Transmittance (T) = It/ 1o Transmittance is inversely and logarithmically proportional to the concentration.i.e T α log1/C.

14. Absorbance Optical density: it is the amount of light absorbed by the colored substance. OD may be defined as the logarithmic ratio of incident light to that of transmitted light. So A = log ( I/T ) ;= log 10 ( 100/T); = 2 - log 10 T Absorbace is directly and linearly propotional to con.

15. Relationship between absorbance and transmittance 2 - log 10 T I O I E OD %T OD=

16. Combined Beer’s- Lambert’s law Combining the two laws: A α C x L A = K x C x L Let A T =absorbance of the test solution C T =concentration of the test solution A S =absorbance of the standard solution C S =concentration of the standard solution

17. A T A S K x C T x L K x C S x L = A T A S C T C S = C T = A T A S x C S A S = K x C S x L A T = K x C T x L

18. C T = A T A S x C S Concentration of TEST sol. Absorbance of TEST Absorbance of STANDARD Con. of STANDARD x = Concentration of TEST/100ml Absorbance of TEST Absorbance of STANDARD Concn of Std X 100 x = X ml = OD T OD S x C S

19. Standard (calibration curve) The standard curve is prepared to check whether the method of assaying a particular substance follows Beer’s Law, i.e. whether the absorbance of the substance increases in a linear way with its concentration. The standard curve is constructed by plotting a vertical axis (y – axis, ordinate) for optical densities (absorbance) and a horizontal axis (x – axis, abscissa) the concentration of standard solution. The concentration of the test/unknown can be measured from the graph (standard curve).

20. Standard Curve / Calibration curve

21. Preparation of solution for investigation In colorimetric estimation it is necessary to prepare 3 solutions: 8/10/2016 11:37 PM

22. Complimentary color Wavelength between 400nm to 700 nm form the visible spectrum of light Light passed through a solution which selectivity absorbs radiation at fixed wave lengths,then the color of the transmitted light is complementary to that of the absored light.

23. Colors and complimentary colors of visible spectrum Color of the solution/ solution color transmitted Filter used/ color absorbed Wavelength (nm) Yellow Blue 450 – 479 Red Green 505 – 534 Blue yellow 640 – 689 Green Red 620 - 689

24. Colorimeter

25. Components of Colorimetry L ight source: T he light source is usually a tungten lamp, for wavelength in the visible range (320 – 700nm) and a deutarium or hydrogen lamps for ultraviolet light (below 350nm). T ungsten lamp  Visible range D eutarium/hydrogen lamp (preferred)  UV Rays B lack body radiators (Nerst glower)  Infrared radiations

26. Monochromators/Filters This device used for spectral isolation(light of single wavelength) this means of selecting a sufficiently narrow wave band. Filter SBW about 50nm (wide band pass monochromator ) Prism SBW is 5-10 or <5nm (narrow band pass monochromator ) Diffraction grating SBW is 15-20nm.

27. Monochromators Early colorimeters used Absorption filters (i.e. glass filter, Gelatin filter) that transmitted a wide segment of spectrum (50nm or more). Newer instrument use Interface filters that consist of thin layer of magnesium fluoride crystals with a semitransparent coating of silver on each side. Monochromator consists of: Entrance slit Absorption/ interface filter and Prisms or diffraction grating for wavelength selection Exit slit

28. Sample Holder/ Cuvette Cuvettes are rectangular cell , square cell or circular one. Made up of optical glass for visible wavelength (quartz or fused silica for UV). Common one is square , rectangular to avoid refraction artifacts. Optical path (length) of cuvette is always1cm. Capacity may be 3ml/2ml/1ml depending upon the thickness of the wall of the cuvette. For accurate and precise reading cuvette must be transparent, clean, devoid of any scratches and there should be no bubble adhering to the inner surface of the filled cuvette.

29. Photosensitive detectors Detectors are the transducers, which convert light energy to electrical enagery . A detector should be possess follwing characteristics: S hould be sensitive,stable S hould have linear response,short response. Different detectors used are: Barrier layer cells (photocells) – simpliest Photoconductive cells (photodiodes) – newest

30. Read out devices The detector response can be measured by any of the following devices: Galvanometer Ammeter Recorder Digital readout . The signal may be transmitted to computer or print out devices.

31. Criteria for satisfactory colorimetric estimations Stability of color Intensity of color The color of the solution should be intense. Clarity of the solution Substance under investigation should be completely soluble. Specificity Color produced should be specific for the desired constituent . Validity of Beer’s law The intensity of color should be proportional to concentration.

32. Applications Of Colorimeter Estimation of biochemical compounds in blood, plasma , serum, CSF, urine, etc .: Glucose Urea Creatinine Uric Acid Bilirubin Lipids Total Proteins Enzymes [e.g. ALT , AST, ALP] Minerals [ Calcium, Phosphorus etc.] etc….

33. Spectrophotometry : Instruments & Applications 37

34. Principle of Spectrophotometer Solutes in a solution show characteristic absorption spectrum in UV or visible or infrared region of electromagnetic radiation. Characteristic absorption spectrum can be isolated by passing the electromagnetic radiation through a prism monochromator . Degree of absorption of electromagnetic radiation depends on the condensation of solute in solution.

35. The Spectrophotometer

36. The Spectrophotometer

37. Introduction Spectrophotometer: Single-beam b) Double-beam [4] 41

38. Instruments Light source: provide a sufficient of light which is suitable for marking a measurement. T he light source typically yields a high output of polychromatic light over a wide range of the spectrum.[4] 42

39. Monochromator : Accepts polychromatic input light from a lamp and outputs monochromatic light. Monochromator consists of these parts: Entrance slit Collimating lens or mirror Dispersion element Focusing lens or mirror Exit slit [6] 43 Common monochromators : Filter Prism Diffraction grating Interference filter

40. Instruments Dispersion devices: A special plate with hundreds of parallel grooved lines. The grooved lines act to separate the white light into the visible light spectrum. 44 The more lines the smaller the wavelength resolution.[5]

41. Instruments Focusing devices: Combinations of lenses, slits, and mirrors. relay and focus light through the instrument.[2] 45

42. Instruments Cuvettes : designed to hold samples for spectroscopic experiments. made of Plastic, glass or optical grade quartz should be as clear as possible, without impurities that might affect a spectroscopic reading.[2] 46

43. Instruments Detectors : Convert radiant energy (photons) into an electrical signal. The photocell and phototube are the simplest photodetectors , producing current proportional to the intensity of the light striking Them .[1],[2] 47

44. Instruments Display devices: The data from a detector are displayed by a readout device, such as an analog meter, a light beam reflected on a scale, or a digital display , or LCD . The output can also be transmitted to a computer or printer. [3] 48

45. Applications Concentration measurement Prepare samples Make series of standard solutions of known concentrations [4] 49

46. Applications Set spectrophotometer to the λ of maximum light absorption Measure the absorption of the unknown, and from the standard plot, read the related concentration[4] 50

47. Applications 2 . Detection of Impurities UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. [7] 51 Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material.

48. Applications 3 . Structure elucidation of organic compounds. From the location of peaks and combination of peaks UV spectroscopy  elucidate structure of organic molecules: the presence or absence of unsaturation,  the presence of hetero atoms.[7] 52

49. Advantages: Ensure higher degree of spectral purity. Minimum stray light into the exit beam (wave length outside the desired light is called stray light). Greater accuracy. More sensitivity, specificity and precision.

50. Source of errors in spectrophotometer Stray light Low resolution of light source Lacking linearity Variation in temp. Low sample volume

51. Difference between colorimeter and spectrophotometer Traits Colorimeter Spectrophotometer Monochromator Filter Prism Spectral bandwidth Broad band Narrow band Spectral purity Less More Spectral isolation Filter has to be changed Desired wave length can be adjusted Stray light More Minimum Accuracy Less More Sample Larger volume needed Small volume needed Cost Cheaper More costly Light source Visible range of light used Beyond visible range of light used