Colormetric analysis refers to the technique used to determine the concentration of a particular chemical compound or element in a solution using a color reagent. This method is applicable in the analysis of both organic and inorganic compounds. It is widely applied in medical laboratories and industries. It is the main method used to analyze water samples in industrial water treatment.
The most essential equipment is a color reagent, a colormeter and several cuvettes. Advancement in technology has led to the automation of the process by using an AutoAnalyzer or by adopting the Flow injection analysis. There are cases which require enzymatic reactions while others involve non-enzymatic reactions. The most commonly used method in medical laboratories is enzymatic analysis because it is reliable and produces accurate results.
The necessary and sufficient conditions for enzymatic reactions to occur include the availability of a buffer solution and a suitable temperature of 37 degrees Celsius. These are the conditions for optimum enzyme functioning . These reactions involve the use of substrate specific enzymes which stimulate the reactions to form visible colored complexes. Glucose oxidase is needed to stimulate the reaction of oxygen, glucose and water to produce hydrogen peroxide and a colored complex called gluconate. Cholesterol oxidase speeds up the reaction of cholesterol and oxygen forming cholestenone complex and hydrogen peroxide.
The non-enzymatic methods are mostly used in quantitative analysis of compounds containing creatinine, iron and calcium. The chemicals used in this process react with these elements to form the required colored complexes. Some chain reactions involve enzymatic and non-enzymatic reactions. A common example is the reaction of urea and water which is catalyzed with enzyme urease yielding ammonium carbonate. The resultant ammonium carbonate is reacted with phenol in the absence of any enzyme to form a complex.
Ultraviolet methods involve the use of ultraviolet light to determine the quantity of the colored products of the reaction. The measurement of these substances is done using a spectrophotometer. The sample is placed inside the spectrophotometer and subjected to ultraviolet light of wavelength 340 nm. The principle involved is that the different compounds in the solution absorb ultraviolet light at different rates. The absorbance of each element is directly proportional to the concentration of the element in the solution.
The concentration of the substances in the solution is determined with the help of a standard sample whose concentration is already known. The standard sample is compared with the test sample and if the two have similar absorbance then the concentration is also the same. However, it is impractical to have standard samples for all values generated by a test sample; a standard curve gives all possible values.
A standard curve is constructed by plotting absorbance against the amount of the substance or the concentration. These values are obtained by measuring the absorbance of different concentrations of a given sample. Each time the absorbance reading on the spectrophotometer is taken the corresponding concentration value is obtained from the standard curve.
Colormetric analysis also involves the calibration of the particular spectrophotometer used. This is done using a reference tube containing water to avoid absorbance from taking place. Values which cannot be read from the standard curve are obtained through extrapolation of the curve by assuming a linear relationship.
The most essential equipment is a color reagent, a colormeter and several cuvettes. Advancement in technology has led to the automation of the process by using an AutoAnalyzer or by adopting the Flow injection analysis. There are cases which require enzymatic reactions while others involve non-enzymatic reactions. The most commonly used method in medical laboratories is enzymatic analysis because it is reliable and produces accurate results.
The necessary and sufficient conditions for enzymatic reactions to occur include the availability of a buffer solution and a suitable temperature of 37 degrees Celsius. These are the conditions for optimum enzyme functioning . These reactions involve the use of substrate specific enzymes which stimulate the reactions to form visible colored complexes. Glucose oxidase is needed to stimulate the reaction of oxygen, glucose and water to produce hydrogen peroxide and a colored complex called gluconate. Cholesterol oxidase speeds up the reaction of cholesterol and oxygen forming cholestenone complex and hydrogen peroxide.
The non-enzymatic methods are mostly used in quantitative analysis of compounds containing creatinine, iron and calcium. The chemicals used in this process react with these elements to form the required colored complexes. Some chain reactions involve enzymatic and non-enzymatic reactions. A common example is the reaction of urea and water which is catalyzed with enzyme urease yielding ammonium carbonate. The resultant ammonium carbonate is reacted with phenol in the absence of any enzyme to form a complex.
Ultraviolet methods involve the use of ultraviolet light to determine the quantity of the colored products of the reaction. The measurement of these substances is done using a spectrophotometer. The sample is placed inside the spectrophotometer and subjected to ultraviolet light of wavelength 340 nm. The principle involved is that the different compounds in the solution absorb ultraviolet light at different rates. The absorbance of each element is directly proportional to the concentration of the element in the solution.
The concentration of the substances in the solution is determined with the help of a standard sample whose concentration is already known. The standard sample is compared with the test sample and if the two have similar absorbance then the concentration is also the same. However, it is impractical to have standard samples for all values generated by a test sample; a standard curve gives all possible values.
A standard curve is constructed by plotting absorbance against the amount of the substance or the concentration. These values are obtained by measuring the absorbance of different concentrations of a given sample. Each time the absorbance reading on the spectrophotometer is taken the corresponding concentration value is obtained from the standard curve.
Colormetric analysis also involves the calibration of the particular spectrophotometer used. This is done using a reference tube containing water to avoid absorbance from taking place. Values which cannot be read from the standard curve are obtained through extrapolation of the curve by assuming a linear relationship.
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