Spectrophotometry (the measurement of light absorption or transmission) is one of the most valuable analytical techniques available to biochemists. Unknown compounds may be identified by their characteristic
absorption spectra in the ultraviolet, visible or infrared regions of the electromagnetic spectrum. Concentrations of unknown compounds in solutions may be determined by measuring the light absorption at one or more wavelengths. Enzyme catalysed reactions can be followed by spectrophotometrically measuring the appearance of a product or disappearance of the substrate.Spectrophotometric measurements in clinical
laboratory, most commonly, use the absorption of light in the visible and the ultraviolet region. Since absorption of visible light is responsible for the colour of the solutions, the measurement of intensity of coloured solutions is commonly known as Colourimetry.
1. Principle: All spectrophotometric measurements are based upon the Lambert-Beer’s law.
The Beer –Lambert Law When a monochromatic light of initial intensity Io passes through a solution in a transparent vessel, some of the light is absorbed so that the intensity of the transmitted light I is less than Io .There is some loss of light intensity from scattering by particles in the solution and reflection at the interfaces, but mainly from absorption by the solution. The relationship between I and Io depends on the path length of the absorbing medium, l, and the concentration of the absorbing solution,c. These factors are related in the laws of Lambert and Beer .
Lambert’s law: When a ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the absorbing medium increases.
Beer’s law : When a monochromatic light passes through an absorbing medium its intensity decreases exponentially as the concentration of the absorbing medium increases
Transmittance: The ratio of intensities is known as the transmittance (T) and this is usually expressed as percentage
Percent T = I/I0
Log I0 /I is known as the Optical Density (OD) of the solution. Therefore:
Optical Density (OD) = KxCxL
Optical Density of a solution (also known as Absorbance or Extinction) is directly proportional to the concentration of the substance and the depth of the solution through which the light passes.
Parts of Photoelectric colourimeter: The instrument measures the intensity of the incident as well as the
transmitted light and hence the light absorbed by a given solution. Therefore, the instrument is also
called as absorptiometer.
a. Components
i. Light source: Most common light source used is the tungsten iodide lamp for visible light spectrum and halogen/deuterium lamp for the ultraviolet spectrum.
ii. Monochromators: Selected filters or prism/ diffraction gratings are placed in the path
of the light to select light of a specific colour or wavelength.
iii. Sample cell (cuvette): It can be round or square. The light path must be kept constant in order to have the Lambert-Beer’s law obeyed.
iv. Photodetector: The purpose of photodetector (Photocathode) is to convert the transmitted radiant energy into an equivalent amount of electric energy.
v. Readouts: These are broadly classified as meters, recorders or digital signals from the detector.
Photometric analysis: There are four general steps in carrying out a photometric analysis:
a. separation of the substance from the complex mixture- for e.g., estimation of blood glucose requires the precipitation of lipids and proteins by deproteinising agents which otherwise interfere with the colour reaction of glucose
b. quantitative conversion to a coloured or light absorbing substancefor e.g., after deproteinisation as mentioned above for glucose estimation, the supernatant is made to react with orthotoluidine
reagent to give a greenish blue coloured complex
c. measurement of light absorption- for e.g., the colour intensity of the above mentioned complex is measured by using a red filter.
d. calculation of the concentration of the substance - for e.g., by comparing the extinction with that of the standard solution of the same substance of known concentration.
Concentration
of unknown=absorbance of unknown/absorbance of known x Concentration of known
(standard) (standard)
Applications of spectrophotometry
Colorimetry and spectrophotometry have widest application in biological sciences. These techniques are used for
a. The determination of glucose, proteins, lipids, nucleic acid etc
b.The determination of turbidity of solutions( bacterial cell mass)
c. The determination of absorption spectrum of a compound
d. The determination of purity of compound by knowing the molar extinction coefficient which is maximum for a pure compound
absorption spectra in the ultraviolet, visible or infrared regions of the electromagnetic spectrum. Concentrations of unknown compounds in solutions may be determined by measuring the light absorption at one or more wavelengths. Enzyme catalysed reactions can be followed by spectrophotometrically measuring the appearance of a product or disappearance of the substrate.Spectrophotometric measurements in clinical
laboratory, most commonly, use the absorption of light in the visible and the ultraviolet region. Since absorption of visible light is responsible for the colour of the solutions, the measurement of intensity of coloured solutions is commonly known as Colourimetry.
1. Principle: All spectrophotometric measurements are based upon the Lambert-Beer’s law.
The Beer –Lambert Law When a monochromatic light of initial intensity Io passes through a solution in a transparent vessel, some of the light is absorbed so that the intensity of the transmitted light I is less than Io .There is some loss of light intensity from scattering by particles in the solution and reflection at the interfaces, but mainly from absorption by the solution. The relationship between I and Io depends on the path length of the absorbing medium, l, and the concentration of the absorbing solution,c. These factors are related in the laws of Lambert and Beer .
Lambert’s law: When a ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as the length of the absorbing medium increases.
Beer’s law : When a monochromatic light passes through an absorbing medium its intensity decreases exponentially as the concentration of the absorbing medium increases
Percent T = I/I0
Log I0 /I is known as the Optical Density (OD) of the solution. Therefore:
Optical Density (OD) = KxCxL
Optical Density of a solution (also known as Absorbance or Extinction) is directly proportional to the concentration of the substance and the depth of the solution through which the light passes.
Parts of Photoelectric colourimeter: The instrument measures the intensity of the incident as well as the
transmitted light and hence the light absorbed by a given solution. Therefore, the instrument is also
called as absorptiometer.
a. Components
i. Light source: Most common light source used is the tungsten iodide lamp for visible light spectrum and halogen/deuterium lamp for the ultraviolet spectrum.
ii. Monochromators: Selected filters or prism/ diffraction gratings are placed in the path
of the light to select light of a specific colour or wavelength.
iii. Sample cell (cuvette): It can be round or square. The light path must be kept constant in order to have the Lambert-Beer’s law obeyed.
iv. Photodetector: The purpose of photodetector (Photocathode) is to convert the transmitted radiant energy into an equivalent amount of electric energy.
v. Readouts: These are broadly classified as meters, recorders or digital signals from the detector.
a. separation of the substance from the complex mixture- for e.g., estimation of blood glucose requires the precipitation of lipids and proteins by deproteinising agents which otherwise interfere with the colour reaction of glucose
b. quantitative conversion to a coloured or light absorbing substancefor e.g., after deproteinisation as mentioned above for glucose estimation, the supernatant is made to react with orthotoluidine
reagent to give a greenish blue coloured complex
c. measurement of light absorption- for e.g., the colour intensity of the above mentioned complex is measured by using a red filter.
d. calculation of the concentration of the substance - for e.g., by comparing the extinction with that of the standard solution of the same substance of known concentration.
Concentration
of unknown=absorbance of unknown/absorbance of known x Concentration of known
(standard) (standard)
Applications of spectrophotometry
Colorimetry and spectrophotometry have widest application in biological sciences. These techniques are used for
a. The determination of glucose, proteins, lipids, nucleic acid etc
b.The determination of turbidity of solutions( bacterial cell mass)
c. The determination of absorption spectrum of a compound
d. The determination of purity of compound by knowing the molar extinction coefficient which is maximum for a pure compound
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