COLORIMETER & LAMBERTS – BEER’S LAW.pptx

colorimeter is used in lab diagnostic medicine and is based on lamberts and beer s law. these ppts gives a clear view regarding the mentioned subject.

1. COLORIMETER & LAMBERTS – BEER’S LAW

2. 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

3. 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 . Terms:/Parameters

4. Spectrum of Light

5. 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.

6. 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 .

7. 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

8. 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.

9. 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

10. 12/8/2013 10:07 PM 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

11. 12/8/2013 10:07 PM 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

12. 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

13. 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.

14. 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

15. Components of the Colorimeter 12/8/2013 10:04 PM Tungsten lamp Slit Condensing lens Filter Cuvette Photocell Galvanometer

16. 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

17. 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.