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 all the research is used in medicine.
The essence of the colorimetric method
There are many types of tests that determine the chemical composition of substances. 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 lies in the fact 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 certain 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 squeezetannins to distinguish iron from copper in solution. It was Boyle who noticed that the greater the concentration of iron in the solution, the richer the tone of the latter.
Colorimetry
Colorimetry is a method for determining the amount of a substance in solutions. The analysis is based on the Bouguer-Lambert-Beer law: when a beam of light enters a dense absorbing layer of a substance, its intensity weakens.
The colorimetric method for determining the concentration of a substance can use a visual comparison or comparison using special instruments to measure color intensity. The comparison is made 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 reference solution. When using photocolorimeters and spectrophotometers, distilled water is used as a reference solution. The devices measure the intensity of the current, which depends on the intensity of the light.
- Compensatory. The method is based on bringing the color of the studied sample to the reference one. The result is achieved by adding a solvent or increasing the height of the layer of the stained medium.
The use of a spectrophotometer, monochromator and other precision instruments further simplifies the already simple method of research and allows you to increase the accuracy of the results. These devices are able to measure the amount of light transmission and determine the wavelength.
Where analysis is applied
Colorimetric method of analysis 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 you need to quickly determine the amount of a colored substance, but it is so small that it is impossible to apply the analytical method.
Colorimetry is widely used in medicine for clinical biochemical research and other areas:
- pharmaceutical industry;
- food and alcohol industry;
- agronomy (determination of soil quality).
Pros and cons
The colorimetric method, like any other, has pros and cons.
The advantages include the following:
- ease of analysis, minimum preparatory procedures;
- possibility of testing even with a small amount of test material;
- there is no need for expensive equipment, although modern devices that are not cheap have been increasingly used lately. But they provide the most accurate result.
Disadvantages: low accuracy of analysis compared to analytical methods.
Biuret method for protein determination
Protein in blood serum is an indicator reflecting the state of homeostasis. An increase in the concentration of a macromolecular compound in the blood is called hyperproteinemia, a low content is called hypoproteinemia.
Proteins inblood serum have a different composition, structure, properties, perform different functions. They are divided into enzymes, hormones, immunoglobulins and others. All groups of proteins have a number of common characteristics, on the basis of which methods have been developed for the determination of macromolecular 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 are quite simple. The most common is the biuret method. The essence of the method: proteins in an alkaline environment react with copper sulfate and form purple compounds. The protein content in the blood is determined by the saturation of the staining. 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 biomaterial sampling;
- last weeks of pregnancy and lactation;
- the use of the drugs Corticotropin, Miscleron, Clofibrate increases the protein content in the blood, and the use of Pyrazinamide and estrogen - to reduce the concentration;
- wrong hand position during biomaterial sampling.
Determination 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 ensures the transport of oxygen to the tissues. Fordetermination of iron by the colorimetric method, most often, a solution of bathophenanthroline (0.02%) is used as the main reagent. Biomaterial - serum without traces of hemolysis.
The essence of the method: the interaction of ferrous ions and sulfated bathophenanthroline forms a colored complex, the saturation of which is determined photometrically. To obtain a clear solution, it is necessary to follow the rules for sampling the biomaterial, but to accurately determine the optical densities of iron-ligand complexes, iron is released from hemoglobin using hydroxylamine and detergents (sodium dodecyl sulfate). The test result determines the presence and degree of pathology. Normal 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 dietary intake, or poor absorption from the gastrointestinal tract.
Cholesterol test
Cholesterol is an organic substance found in the cell wall of many organisms, including humans. It is necessary for the production of cholecalciferol and steroid hormones. A cholesterol level of 3.37-5.2 mmol/L is considered normal. Elevated content is one of the main causes of atherosclerosis.
Colorimetric methods for determining cholesterol allow you to recognize vascular diseases at an early stage. According to pathoanatomical results in lethal outcome of ischemia, the concentration of lipophilic alcohol in patientswas 6.5-7.8 mmol/L.
The principle of the colorimetric method is that cholesterol is oxidized by 3beta-hydroxy-steroid oxidoreductase, releasing hydrogen peroxide, which converts p-aminoatipyrine into a colored compound. The content of cholesterol is determined by the saturation of its color.
When conducting a test in a child, it must be taken into account 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 by 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 the analysis of certain substances in the presence of other substances similar in composition. The most common methods based on the use of the enzyme - glucose oxidase. The test is used to determine the concentration of glucose in the blood. The accuracy of the test allows it to be used to adjust the dosage of hypoglycemic agents in patients with type 2 diabetes.
Glucose oxidant method is considered one of the best quantitative methods for determining glucose. Both blood (capillary) and serum can be used as a biomaterial, but plasma is more preferable because it has a lower hematocrit content, which negatively affects the accuracy of the result.
The kinetic method of photometry is mainly used. Its essence lies in the fact thatthe established ratio of glucose oxidase and peroxidase, for a certain amount of time after the start of the reaction, the rate of formation of a colored compound will be commensurate with 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 - expensive measuring instruments from German or Swedish manufacturers are required for the test.
Conclusion
The colorimetric method is accurate and easy to use. Its use in medicine makes it possible to identify various pathological changes in the body at an early stage. With the introduction of new technologies, the method is being improved and is becoming more and more popular.
