Definition of Colorimeter
The body fluid such as blood (seram and Plasma) CSF and urine etc. contain several organic and Inorganic substances for example, blood contains organic substance such as Glucose, urea, uric acid, creatinine, Protein, etc. It also contains inorganic cations, such as sodium, potassium, calcium etc. and anion such as inorganic phosphorus chloride, bicarbonate, etc. Most chemical substance in the blood are equilibrium if their rate of production and loss (by degradation or excretion) are equal. The normal concentration of most chemical substance vary within narrow limits, normal range. Disease may change this equilibria in several ways. The factors which influence the chemical composition of the blood in disease may be broadly classified as physical and metabolic. The physical factor include those case of retention due to alternation or destruction of tissue or organ such as Kidneys, liver, lungs and heart. In kidney disease, accumulation of nitrogenous substance such as urea, Creatinine, etc. in blood may take place. In liver disease, bilirubin concentration of blood may increase with increase in liver enzyme such as SGPT and SGOT. In the metabolic disease, alternation in the chemistry of the blood may be included by increased or deminished formation or utilisation of various constituents. Thus in diabetes mellitus due to deficiency of insulin, accumulation of glucose in blood take place. The detection of biochemical abnormalities in tissue sample is impracticable. It is early easier to assess chemically the secondary disturbance in blood, urine, CSF and other body fluid. Owing to a number of factors such as the small concentration of many chemical substance and the difficulty of isolating them, colorimetric method are frequently used.
In colorimetry determination a specific reagent or specific reagents are are you used which react with the specific component to form a coloured complex. The concentration of the coloured complex is directly proportional to the concentration of the component in the specimen. The depth of coloured complex is measured on a photometer or spectrophotometer. The photometric reading are compared with a known primary standard or with a quality control serum sample. Photometry involves the measurement of the light transmitting power of a solution in order to determine the concentration of light absorbing material present. In order to get introduced to photochemistry.
Components of Colorimeter and Photometer
it is necessary to get familiar with the following terms and component used in colorimetry and photometry.
- Analytical procedure based upon the direct measure of colour intensity in terms of light absorption at specific wavelengths are known as photometric procedure and the instrument used is called a photometer.
- Colorimetric procedure are limited to the visible portion of the spectrum.
- Photomatric procedure, however, involve use of ultraviolet, visible and infrared portion of the spectrum.
- Sunlight is a type of radiant energy comprising ultraviolet light (UV) below 380 mm, visual light 380 -760 mm and infrared light above 760 mm.
- Light travel in waves: The quality of light is judged by the wavelength, which is the distance between the crests of the waves. It is expressed in terms of nanometre.
- Nominal wavelength: the wavelength at which peak transmittance occurs.
- Peak transmittance: The maximum percent emission at the nominal wavelength.
- Molar extinction coefficient: It is extinction (O.D. or absorbance) given by a substance concentration one mole per litre in the light path one centimetre.
- Monochromator: Any system or component that will transmit monochromatic light (light of single wavelength). In the case of a photometer, a filter is used as a monochromator which may not transmit monochromatic light but transmits a narrow band (8 to 30 nanometer) of the spectral region. However, in the case of a spectrophotometer, a prism or diffraction with a slit is used as a monochromator to transmit single wavelength.
- Light source: A bulb which emit radiant energy for the photometric determination. A tungsten lamp emits a continuous spectrum of light in the visible range (400 – 760 nm), A halogen are deuterium discharge lamp provides wavelength range 200 to 900 nanometre which covers the part of ultraviolet range, complete visible range and part of infrared range.
- Cubatte (cell): It is a round or square tube, made up of glass, quartz, silica or special type of plastic. It holds the solution meant for determination of absorbance. The width of the cubette represents the path of light, which should be preferably one centimetre.
- Photocells: These are photodetectors made up of light sensitive material such as selenium or cuprous oxide. These generate an electrical current when light transmitted by a solution falls on them. the electrical current is measured by galvanometer.
- Coloured solutions: A solution has colour because it absorbs a lesser proportion of the colour represented by it. For example, a blue solution appears blue because when white light passes through it, large proportion of blue light will be transmitted and other part of white light i. e. green, yellow, red, etc. are absorbed by the blue solution. When yellow and blue colour are transmitted, the solution will appear green.
- UV determination: The solution for UV determination are colourless but contains certain substances such as NAD, NADH, etc. These substances absorb UV light in variable proportion.The transmitted light give measure of light absorbing material present in the solution.
- Blank: Abservation as measured in photometer. involves not only the absorbance of the solute in the solution being evaluated, but also all the light molecules of the liquid through which the light passes. A reagent blank contains only reagent. A sample may be added into it without performing a reaction so that a coloured complex will not form. However if a sample is coloured than a separate sample blank is required which is preferred by adding the sample to saline or the reagent by execuding a reacting component. The blank solution is used to set the meter of the instrument to 100%. T or zero absorbance.
Basic components of a photometer
- a light source
- A lense (condenser)
- Filter
- Cuvette
- Galvanometer (read out of device).
Principles of Colorimeter, Photometer and spectrophotometer
The working of photometers and spectrophotometers is based on beer and Lambert’s law. The simplest expressions of this law are as follows.
Beers Law
It states that the optical density of a solution is directly proportional to the concentration of the solution.
Lamberts Law
It states that the optical density of a coloured solution is directly proportional to the path of light i.e., (diameter of the cuvette).
Formula for Calculation
O.D. Std
Concentration of the solution = —————— * 50
O.D. Std
