photometry.pptx

principles of photometry, colorimetry and spectrophotometry

1. PRINCIPLES OF PHOTOMETRY AND COLORIMETRY SPECTROPHOTOMETRY ; SPECTRUM, TRANSMITTANCE, ABSORBANCE, AND LAMBERT BEER LAW

2. Laboratory Techniques Basic practices used in clinical and molecular diagnostic laboratories includes :- Optical Chromatographic Electrochemical Electrophoretic Mass spectrometric Enzymatic Immunoassay techniques

3. Optical Techniques It involves the measurement of radiant energy emitted, transmitted, absorbed, scattered or reflected under controlled conditions. 1. Photometry- The measurement of light. 2. Spectrophotometery - Measurement of intensity of light at selected wavelengths 3. Colorimetery – Measurement of intensity of color in a solution. Optical techniques are based on two laws :- Beer’s Law Lambert’s Law

4. Transmittance : T he passing of light through a sample. Absorbance : A mount of light absorbed by a sample (the amount of light that does not pass through or reflect off a sample ). %Transmittance: T he manner in which a spectrophotometer reports the amount of light that passes through a sample. Absorbance units: A unit of light absorbance determined by the decrease in the amount of light in a light beam. Absorbance spectrum: A graph of a sample’s absorbance at different wavelengths. Lambda max: T he wavelength that gives the highest absorbance value for a sample. Terms:/Parameters

5. Light Light :- Light is electromagnetic radiation includes radiant energy with wavelength from 10 nm to 1000 km. In laboratory techniques radiant energy of ultraviolet and visible spectrum (i.e. 180-800 nm) is used. 180 200 380 440 500 580 600 620 750 ---I---I------------------I ------ I ------ I -------- I ----- I ------ I ----------- I----------- Ultraviolet---------I violet I blue I green I yellow I orange I red I infrared I ----------visible to human eye-----------I -Energy of the light is inversely proportional to the wavelength. -Human eye is able to detect radiant energy with wavelength b/t 380 and 750 nm.

6. Spectrum of Light

7. Transmittance Photometric instruments measure light energy from solution as transmittance (T). The ratio of intensity of emergent light (E) to incident light (I) is termed as transmittance (T). T= E/I. A portion of incident light may be reflected by the surface or may be absorbed by the cell wall or solvent. These factors are eliminated by using reference cell identical to the sample cell except compound of interest omitted from solvent. In practice light beam is blocked and detector signal set to zero transmittance, then a reference cell is inserted and detector signal adjusted to 100% transmittance. Transmittance is inversely proportional to concentration.

8. Absorbance Absorbance (A) is directly proportional to the concentration. Amount of light absorbed (A) as the incident light passes through the sample is :- A = - log X E/I = - log T The plot of concentration versus transmittance is not linear, but graph of concentration versus Absorbance (A) is linear. Since it is in logarithmic scale, values too high or low not acceptable for accurate results. The amount of light absorbed or transmitted is related mathematically to the concentration of analyte by Beer’s Law .

9. Beer’s Law BEER’S LAW The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. A α C

10. Lambert’s Law When a ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the light path through light absorbing material increases. The amount of light absorbed by a solution depends on the length of column or the depth of liquid. A α L.

11. Beer-Lambert’s Law By combining the two laws :- The Absorbance (A) of a solution is directly proportional to the concentration of the absorbing substance in the solution and the path length. Combining the two laws A α C x L OR A= K x C x L = constant X concentration X length 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

12. 11/2/2022 11:16 AM A T A S KxC T x L KxC S x L = A T A S C T C S = C T = A T A S X C S A S = KxC S x L A T = KxC T x L

13. 11/2/2022 11:16 AM Absorbance of test Absorbance of standard Conc. of standard X = Absorbance of test Absorbance of standard Conc. of Std X 100 X = Xml Concentration of test solution Concentration of test /100ml Concentration of test /100m = (A) of test - (A) of blank (A) of standard – (A) of blank X Conc. of Std X 100 Volume of test

14. An instrument is calibrated by applying relationship of absorbance and concentration under specific condition using a series of reference solutions that contain increasing concentration of analyte. A linear relationship exists up to a certain concentration. When this linear relationship exists the solution is said to obey Beer’s law up to this point. Within this limitation , a calibration constant (K) may be derived and used to calculate the concentration of unknown solution by comparing with a calibrating solution. Application of Beer-Lambert’s Law

15. Colorimeter - Principle Colored solutions have the property of absorbing light of specific wavelength. When a white light passes through a coloured solution, some specific wavelengths of light are absorbed which is related to color intensity. The amount of light absorbed or transmitted by the solution is in accordance with Beer-Lambert’s law. Most of the clinical chemistry estimations are done by colorimetric methods.

16. Colorimeter Colorimeter is a instrument used for the measurement of coloured substance in solution. The instrument is operative in the visible range of the electromagnetic spectrum

17. Components of the Colorimeter 11/2/2022 11:16 AM Tungsten lamp Slit Condensing lens Filter Cuvette Photocell Galvanometer

18. Components of the Colorimeter 1. Light source: tungsten filament lamp 2. Slit : It is adjustable which allows only a beam of light to pass through. It prevents unwanted or stray light 3. Condensing lens: Light after passing through a slit falls on a condenser which gives parallel beam of light. 4. Filters : are usually made of colored glass. they absorb light of unwanted wavelength and allow only monochromatic light to pass. Filter used is always complimentary to the color of the solution. Wavelength Color of filter Color of solution 420 Violet Brown 470 Blue Yellowish blue 520 Green Pink 580 Yellow Purple 680 Red Green/blue

19. Components of the colorimeter 5. Cuvette : may be square, rectangular or round shape with fixed diameter and having uniform surface. made up of plastic ,glass material. solution in the cuvette absorbs a part of the light and the remaining is allowed to fall on the detector 6. Detector (photocell) : The detectors are photosensitive elements which converts light energy into electrical signal .the electrical signal is directly proportional to the intensity of light falling on the detector. 7. Output : the electrical signal generated in a photocell is measured by a galvanometer which displays transmission and optical density.

20. Spectrophotometer The spectrophotometer technique is to measures light intensity at selected wavelength. The spectrophotometer has all the basic components of photoelectric colorimeter with more sophistication. Wavelengths of ultraviolet region also used. Light is separated into a continuous spectrum of wavelength instead of wavelength of one color as in colorimeter. Wavelength selection is done by diffraction gratings. Spectrophotometer is 1000 times more sensitive then colorimeter.

21. Types of Spectrophotometer a) Single beam Spectrophotometer: b) Double-beam : 21

22. Components of Spectrophotometer 22 Light source (UV and visible) Optical system/Wavelength selector (Monochromator) Sample containers (cuvette) Detector Output: Signal processor and readout

23. 1. Light Source Deuterium Lamps : Continuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. (160nm- 375nm) Tungsten Filament Lamps: the most common source of visible and near infrared radiation ( at wavelength 320 to 2500 nm ) Hydrogen Gas Lamp and Mercury Lamp, Xenon ( wavelengths from 200 to 800 nm )- in UV Spectrophotometer . Silicon Carbide(SiC) Rod : Radiation wavelengths:1200-40000 nm NiChrome wire (750 nm to 20000 nm); Laser : Used when high intensity light source is required.

24. 2. Monochromator Radiant energy of desired wavelengths can be isolated by: 1. Filters:- simplest type of filter is thin layer of colored glass. 2. Prisms:- A prism separates white light into a continuous spectrum b/c shorter wavelengths bent/refracted more than longer wavelengths 3. Diffraction gratings :- is prepared by depositing a thin layer of aluminum-copper alloy on the surface of the flat glass plate, then ruling many small parallel grooves. Each line when illuminated reflects the light and give rise to tiny spectrum. 24 The more lines the smaller the wavelength resolution .

25. 3. Sample Containers 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. Quartz or fused crystalline silica cuvettes for UV spectroscopy . Glass, plastic cuvettes for Visible Spectrophotometer NaCl and KBr Crystals for IR wavelengths 25

26. Detector Photodetectors : Convert radiant energy into an electrical signal that is proportional to the number of photons striking its photosensitive surface. Photomultiplier tube is most commonly used in the UV and visible region of the spectrum. It contains 1.cathode 2.light sensitive metal 3. dynodes. Photodiodes are solid state devices that are also used, made from photosensitive semiconductors silicon, gallium arsenide, indium antimonide, lead selenide and lead sulfide. In older instruments barrier layer cells (photovoltaic cells) were used 26

27. Display Devices Display devices: The data from a detector are displayed by a readout device. In the past analog meters were used. These are replaced by digital readout devices that provide a visual numeric display of absorbance or converted values of concentrations . 27

28. Working of Spectrophotometer White light radiation source that passes through a monochromator that separates the white light into all colors of the visible spectrum. After the light is separated, it passes through a filter (to block out unwanted light) and a slit (to narrow the beam of light). The light passes through the sample(cuvette) and the unabsorbed portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a readable output (absorbance )that is used in the analysis of the sample. Calibration curve is generated by measuring the absorbance of several solutions that contain known concentrations of analyte.

29. Performance Parameters Parameters of the performance of spectrophotometer to be tested : 1. wavelength accuracy- With narrow spectral bandwidth instruments a holmium oxide glass may be scanned over a range of 280-650 nm. This material shows very sharp absorbance peaks at defined wavelengths and compare the scale readings with established values. 2. Spectral bandwidth- spectral purity of a filter or monochromator is described in terms of spectral bandwidth. 3.Stray light- In practice scattering and diffraction inside monochromator introduce light of other wavelengths. Other sources are light leaks and fluorescence of the sample. To overcome this stray light filters are used. Blue filter for wavelengths <400 nm and red filter for wavelengths 650 to 800 nm. 4.Linearity- To check linearity b/w light absorbed and instrument readout didymium filter is used – has absorbance 0.09 at 550nm. 5. Photometric accuracy- Neutral density filters are used to check an instruments photometric accuracy. Solutions of potassium dichromate (k2Cr2O7) may be used.

30. SPECTROPHOTOMETRY 1. A device for measuring light intensity. 2. Measure intensity as a function of the color, or more specifically, the wavelength of light. 3. Tungsten or xenon flash lamp as the source of white light. Tungsten lamp for measurements in visible region(360-900nm) Hydrogen /deuterium lamp for UV region (200-380nm) COLORIMETRY 1. A device for measuring color intensity. 2. It determines color based on the red, blue, and green components of light absorbed by the sample. 3. Colored light beam through an optical filter, which transmits only one particular color / band of wavelengths of light to the photodectector . Spectrophotometry Versus Colorimetry

31. Spectroscopy and Spectrophotometry Light can either be transmitted or absorbed by dissolved substances Spectroscopes measure electromagnetic emission S pectrophotometers measure electromagnetic absorption Principle: Based on Beer Lambert’s LAW Spectrometer produces the light of desired wavelength and it passes through the tube and reaches photometer that measures its intensity. Then the photometer produces a voltage signal to a display device, usually a galvanometer. As the amount of light absorbed by the liquid changes; the signal also changes .

32. Reflectance Photometry In reflectance photometry diffuse reflected light is measured. The reaction mixture in a carrier is illuminated with diffuse light and intensity of reflected light from the chromogen is compared with intensity of light reflected from a reference surface. Here the geometry of the system is modified so that the light source and the detector are located next to each other on one side of the sample. It is used as measurement method with dry film chemistry system.

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