Colorimetric method - an analysis based on a comparison of the color saturation of the investigated and known substances. The results of physical and chemical tests are of great value for many branches of science, but most of the research is used in medicine.
The essence of the colorimetric method
There are many types of analyzes by which the chemical composition of substances is determined. Some of them are universal and are widely used in various fields, others are specific, with higher accuracy. The colorimetric method is universal.
The essence of the analysis is that the color saturation of a solution with an unknown concentration is compared with the color of a standard solution. During the analysis, as a result of the interaction of the investigated component with a specific reagent, a colored compound is formed. After completion of the reaction, the resulting shade is compared with the color of the solutions, the concentration of which is already known.
It is believed that the founder of colorimetry is Robert Boyle. He used a squeeze of tannins to distinguish iron from copper in a solution. It was Boyle who drew attention to the fact that the higher the concentration of iron in the solution, the richer the tone of the latter.
Colorimetry
Colorimetry is a method for determining the amount of substance in solutions. The analysis is based on the Bouguer-Lambert-Beer law: when a light beam enters a dense absorbing layer of a substance, its intensity weakens.
The colorimetric method for determining the concentration of a substance can use visual comparison or comparison using special instruments to measure color intensity. The comparison is carried out in a direct and compensatory way.
- Straight. The method involves comparing the color level of the test solution at a certain density and temperature of the liquid with a standard solution. When using photocolorimeters and spectrophotometers, distilled water is used as a reference solution. Devices measure current, which depends on the intensity of the light.
- Compensatory. The method is based on bringing the color of the studied sample to a reference. The result is achieved by adding solvent or by increasing the height of the layer of the medium to be painted.
The use of a spectrophotometer, a monochromator and other precision instruments further simplifies the already uncomplicated research method and allows to increase the accuracy of the results. These devices are capable of measuring the amount of light transmission and determining the wavelength.
Where analysis is applied
The colorimetric analysis method is used when the exact chemical composition is known, there is a reference sample for comparison, and the temperature of the test and reference sample is the same. If it is necessary to quickly determine the amount of colored substance, but it is so small that it is impossible to apply the analytical method.
Colorimetry is widely used in medicine in clinical biochemical studies and in other areas:
- pharmacological industry;
- food and alcohol industry;
- agronomy (determination of soil quality).
Advantages and disadvantages
The colorimetric method, like any other, has pros and cons.
The advantages include the following:
- simplicity of analysis, minimum preparatory procedures;
- the ability to test even with a small amount of test material;
- there is no need for expensive equipment, although recently, more and more modern devices are used, which are expensive. But they provide the most accurate result.
Disadvantages: low accuracy of analysis compared to analytical methods.
Biuret method for determining protein
Protein in the blood serum is an indicator that reflects the state of homeostasis. An increase in the concentration of high molecular weight compounds in the blood is called hyperproteinemia, a low content - hypoproteinemia.
Proteins in blood serum have different composition, structure, properties, perform different functions. They are divided into enzymes, hormones, immunoglobulins and others. For all protein groups, a number of common characteristics are inherent, on the basis of which methods have been developed for the determination of high molecular weight organic compounds in biological fluids.
Of all the methods in clinical biochemistry, colorimetric methods for determining protein are most often used. They are relatively cheap, which is important for budget organizations. and quite simple. The most common is the biuret method. The essence of the method: proteins in an alkaline medium react with copper sulfate and form violet compounds. The saturation of staining determines the protein content in the blood. The biomaterial for analysis is taken in the morning on an empty stomach.
The analysis is highly accurate, but there are factors that affect the concentration of protein in the blood:
- physical activity shortly before taking the biomaterial;
- the last weeks of pregnancy and the period of breastfeeding;
- the use of drugs "Corticotropin", "Miskleron", "Clofibrate" helps to increase the protein content in the blood, and the use of "Pyrazinamide" and estrogen - to reduce the concentration;
- improper position of the hand during the collection of biomaterial.
Definition of iron
The amount of iron in the blood is one of the main indicators in the diagnosis of various diseases. The substance is concentrated in hemoglobin, which provides oxygen transport to the tissues. For determination of iron by the colorimetric method, the solution of bathophenanthroline (0.02%) is most often used as the main reagent. Biomaterial - serum without traces of hemolysis.
The essence of the method: during the interaction of ferrous ions and sulfated bathophenanthroline, a colored complex is formed, the saturation of which is determined photometrically. In order to obtain a clear solution, it is necessary to follow the rules for biomaterial sampling, but to accurately determine the optical densities of iron-ligand complexes, iron is released from hemoglobin with hydroxylamine and detergents (sodium dodecyl sulfate). The result of the test determines the presence and degree of pathology. Normally, the iron concentration should be:
- 14.2 - 26.0 μmol / L (men);
- 10.6-21.7 μmol / L (women).
Iron deficiency is usually associated with blood loss, inadequate intake of food, or poor absorption in the digestive tract.
Determination of cholesterol
Cholesterol is an organic substance contained in the membrane of the cells of many organisms, including humans. It is necessary for the production of cholecalciferol and steroid hormones. Normal is considered to be a level of cholesterol of 3.37-5.2 mmol / L. High content is one of the main reasons for the development of atherosclerosis.
Colorimetric methods for determining cholesterol can recognize vascular diseases in the early stages. According to the pathological results with a fatal outcome of ischemia, the concentration of lipophilic alcohol in patients was 6.5-7.8 mmol / L.
The principle of the colorimetric method is that cholesterol is oxidized with 3-beta-hydroxy-steroid oxidoreductase to release hydrogen peroxide, which turns p-aminoatipyrine into a colored compound. The saturation of its color determines the content of cholesterol.
When conducting a test in a child, it should be borne in mind that the concentration of cholesterol in childhood should not exceed 4.1 mmol / L.
What is the enzymatic colorimetric method?
Enzymatic tests are based on the use of reactions with enzymes with high activity. They are widely used in analytical chemistry to determine various substances - from nitrate ions to macromolecules.
Enzymatic (enzymatic) methods are specific, allowing you to analyze some substances in the presence of other substances similar in composition. The most common methods are based on the use of an enzyme, glucose oxidase. The test is used to determine the concentration of glucose in the blood. The accuracy of the test allows you to use it to adjust the dosage of hypoglycemic agents in patients with type 2 diabetes mellitus.
The glucose oxidant method is considered one of the best quantitative methods for determining glucose. Both blood (capillary) and serum can be used as biomaterial, but plasma is more preferable because it has a reduced hematocrit content, which negatively affects the accuracy of the result.
The kinetic method of photometry is mainly used. Its essence lies in the fact that with the established ratio of glucose oxidase and peroxidase, for some amount of time after the start of the reaction, the rate of formation of the colored compound will be proportional to the level of glucose in the sample. The main advantage of the test is that the result is not affected by the presence of third-party compounds in the sample. The method also has a drawback - for the test, expensive measuring instruments from German or Swedish manufacturers are required.
Conclusion
The colorimetric method is accurate and easy to use. Its use in medicine allows the early stages to identify various pathological changes in the body. With the introduction of new technologies, the method is improved and becomes more and more popular.