Dentistry Department; 2nd grade Dijlah University college Biochemistry Lab. 2 Ass. Lec. Anas Hashim Spectrophotometer An instrument designed to detect the amount of radiant light energy absorbed by molecules in specified wavelength, this method is used as a quantitative way to determine concentration of an absorbing species (unknown) in solutions, using the Beer-Lambert law. The spectrophotometer allows selection of wavelength pass through the solution. Usually the wavelength (measured in nanometer nm) has chosen which corresponds to the absorption maximum of the solute. Colorimetry: methods for quantitative analysis of blood, urine and other biological materials based upon the production of a coloured compounds in solution, the intensity of which is used as a measure of concentration. Beer's-Lambert law: Lambert Beer's law means how light is absorbed by matter. The absorbance of light is directly proportional to the thickness of the media through which the light is being transmitted multiplied by the concentration of absorbing chromophore: A = ɛbc where: A absorbance ɛ wavelength-dependent molar absorptivity coefficient b length of light path c concentration of the solute 1 Dentistry Department; 2nd grade Dijlah University college when we compare the concentration of an unknown test solution with a standard solution measured in the same way we use the following relation: A test = ɛbC test A standard = ɛbC standard If the blank reading is significant then : Absorbance (A): the spectrophotometer reading for the quantity of the light that absorbed by the sample molecules at a specified wavelength . A = - log T = log (1/T) = log (100/%T) = log (100) – log T = 2 - log %T %T per cent transmittance Transmittance (T): is defined as the ratio of transmitted light to incident light : T=I/Io Where: 2 Dentistry Department; 2nd grade I Dijlah University college light intensity after it passes through the sample and Io the initial light intensity. The relation between A and T is: A = -log T = - log (I / I o( Design There are two major classes of devices: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. A single-beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double-beam instruments are easier and more stable. Major components of spectrophotometer 1. Light source: two kinds of lamps, a Deuterium for measurement in the ultraviolet range and a tungsten lamp for measurement in the 3 Dentistry Department; 2nd grade Dijlah University college visible and near-infrared ranges, are used as the light sources of a spectrophotometer. 2. Wavelength selector Monochromator: All monochromators contain the following component parts; An entrance slit A collimating lens A dispersing device (usually a prism or a grating) A focusing lens An exit slit 3. Sample cell: a container that contains a sample is usually called "cell"; three types are available: plastic, glass and quartz cells. Since light in the ultraviolet range with a wavelength of 340 nm or less hardly passes through a glass cell, it is used for measurement in the visible range of 340 nm or more. On the other hand, although a quartz cell allows passage of light in the entire wavelength in the ultraviolet and visible ranges, it is mainly used for the measurement in the ultraviolet range due to its high price. 4. Detector: measure the light intensity. 5. Signal processor and readout Solution required for photometric measurements: 1. Test: made from any unknown specimen (blood, plasma and serum) 2. Blank: contain all reagents used except the substance. 3. Standard: made from a known quantity of the substance. 4. Control: make in enzyme assay. 5. Quality control: made from pooled serum of known chemical constituents. 4 Dentistry Department; 2nd grade Dijlah University college Calibration (standard) curve A calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. The operator prepares a series of standards across a range of concentrations near the expected concentration of analyte in the unknown. The concentrations of the standards must lie within the working range of the technique they are using. Analyzing each of these standards using the chosen technique will produce a series of measurements. For most analyses a plot of instrument response vs. concentration will show a linear relationship. The operator can measure the response of the unknown and, using the calibration curve, can interpolate to find the concentration of analyte. 5
© Copyright 2024